Hindawi Publishing CorporationEvidence-Based Complementary and Alternative MedicineVolume 2013 Article ID 267217 14 pageshttpdxdoiorg1011552013267217

Research ArticleInhibitive Effects of Mulberry Leaf-Related Extracts onCell Adhesion and Inflammatory Response in Human AorticEndothelial Cells

P-Y Chao1 K-H Lin2 C-C Chiu2 Y-Y Yang3 M-Y Huang4 and C-M Yang4

1 Department of Nutrition and Health Sciences Chinese Culture University Taipei 11114 Taiwan2Graduate Institute of Biotechnology Chinese Culture University Taipei 11114 Taiwan3Graduate Institute of Applied Life Science Chinese Culture University Taipei 11114 Taiwan4Research Center for Biodiversity Academia Sinica Nankang Taipei 106 Taiwan

Correspondence should be addressed to P-Y Chao pychaofacultypccuedutw

Received 2 August 2013 Revised 5 November 2013 Accepted 5 November 2013

Academic Editor Joen-Rong Sheu

Copyright copy 2013 P-Y Chao et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Effects of mulberry leaf-related extracts (MLREs) on hydrogen peroxide-induced DNA damage in human lymphocytes and oninflammatory signaling pathways in human aortic endothelial cells (HAECs) were studiedThe testedMLREswere rich in flavonolsespecially bombyx faces tea (BT) in quercetin and kaempferol Polyphenols flavonoids and anthocyanidin also abounded inBT The best trolox equivalent antioxidant capacity (TEAC) was generated from the acidic methanolic extracts of BT Acidicmethanolic and water extracts of mulberry leaf tea (MT) mulberry leaf (M) and BT significantly inhibited DNA oxidative damageto lymphocytes based on the comet assay as compared to the H

2O2-treated group TNF-120572-induced monocyte-endothelial cell

adhesionwas significantly suppressed byMLREs Additionally nuclear factor kappa B (NF-120581B) expressionwas significantly reducedby BT and MT Significant reductions were also observed in both NF-120581B and activator protein (AP)-1 DNA binding by MLREsSignificant increases in peroxisome proliferator-activated receptor (PPAR) 120572 and 120574 DNA binding by MLREs were also detected inM andMT extracts but no evidence for PPAR 120572DNA binding in 50 120583gmLMT extract was found Apparently MLREs can providedistinct cytoprotective mechanisms that may contribute to its putative beneficial effects on suppressing endothelial responses tocytokines during inflammation

1 Introduction

Atherosclerosis is a chronic inflammatory process char-acterized by increased oxidative stress [1] The resultingadhesion of monocytes to the vascular endothelium andsubsequent migration into the vessel wall are the pivotalearly events in atherogenesis [2] Inflammatory cytokinestumor necrosis factor-alpha (TNF-120572) nuclear factor kappaB (NF-120581B) and activator protein (AP)-1 are the majorredox-sensitive eukaryotic transcription factors that regulatethe expression of adhesion molecules [3 4] Because theactivation of NF-120581B and AP-1 can be inhibited to variousdegrees by different antioxidants endogenous reactive oxy-gen species (ROS)may play an important role in these redox-sensitive transcription pathways in atherogenesis [1 5] Inmacrophages nuclear receptors such as glucocorticoid estro-gen peroxisome proliferator-activated receptors (PPARs)

and liver X receptors negatively modulate inflammatoryresponses by downregulation of AP-1 and NF-120581B [6ndash8]PPARs are transcription factors activated by fatty acids andfatty acid-derived eicosanoids [9] Three isotypes (120572 120573120590and 120574) expressed in macrophages control the inflammatorystatus and regulate cholesterol metabolism [10] A number ofphytochemicals commonly used in research have antioxidantactivity that can protect cells fromROS-mediated DNA dam-age that results in mutation and subsequent carcinogenesis[11] It is evident that quercetin metabolites are distributed inhuman atherosclerotic lesions [12] The specific target shouldtherefore be taken into account when evaluating the antiox-idant activity of dietary flavonols in vivo Several herbs areconsumed to protect against common and serious diseasessuch as cardiovascular and cerebrovascular events cancerand other age-related degenerative diseases as well [13]

2 Evidence-Based Complementary and Alternative Medicine

These protective effects are considered in large part to berelated to the various antioxidants contained in them Severalstudies have shown that polyphenolic and flavonol substancesare the most common compounds in herbs with strongantioxidant activity [14ndash16] The bioactive components ofherbaceous plants may be responsible for anticancer effectsthrough growth inhibition and apoptosis in human chronicmyeloid leukemia K562 cells [17]

Morus a genus of flowering plants in the familyMoraceaecomprises 10ndash16 species of deciduous trees commonly knownas mulberries that grow wild and under cultivation in manytemperate regions of the world Mulberries are widespreadand important crops for fruit timber and silkworm feedingas well as being excellent amenity trees Morus leaves arealso recognized as excellent animal food Mulberry leafextracts contain rutin isoquercetin and various derivativesof kaempferol and quercetin glycosides that can scavenge 11-diphenyl-2-picryl-hydrazyl (DPPH) radical [18] and inhibitthe formation of conjugated dienes and thiobarbituric acid-reacting substances (TBARS) by copper-induced oxidativemodification of rabbit and human low-density lipoproteins[19] In addition to antioxidant activity mulberry leaves andextracts have antimicrobial activity [20] induce apoptosis[18] are antiinflammatory and anti-hyperglycemic attenuateatherosclerotic lesions in animal models and lower bloodlipids in mild hyperlipidemic patients [21ndash24] Although avariety of medicinal herbs including mulberry are knownto be potent sources of polyphenols and flavonols studieson the protective effects of antioxidant activity in mulberryleaf-related extracts (MLREs) especially bombyx faces teaand mulberry leaf tea on DNA damage and cell adhesionare either scarce or little known The objectives of thisstudy were to isolate identify and evaluate the antioxidantcomponents antioxidant activity and extent to which acidicmethanolic hydrolysate and water extracts of mulberry leavescan protect DNA in human lymphocytes from oxidativedamage induced by H

2O2 The effects of MLREs including

bombyx faces tea (BT)mulberry leaf tea (MT) andmulberryleaf (M) methanolic extracts on endothelial cell-monocyteadhesion and the inflammatory response were assessed inhuman aortic endothelial cells (HAEC) Moreover the effectsof MLREs on intracellular redox-sensitive transcriptionalpathways such as NF-120581B AP-1 signal transducers andactivators of transcription (STAT)3 and PPAR 120572 and 120574which may contribute to leukocyte recruitment and vascularinflammation in atherogenesis were examined by westernblot analysis and electrophoretic mobility shift assays Ourstudy explores the relationship between the compositionand content of flavonols and polyphenols having antioxidantefficiency and the prevention of DNA oxidative damageafforded by mulberry leaf-related compounds The results ofour study may help determine the antioxidant activity andanti-inflammatory effects of MLREs

2 Materials and Methods

21 Mulberry Leaf-Related Compounds Extract PreparationMulberry leaf-related compounds (MLRC) were generously

provided by the owner ofQuan-Ming SilkwormFarm locatedin Miaoli Taiwan Harvested mulberry leaves were dividedinto eight individual batches and lyophilized by freeze dryingin a Freeze Dryer (FD-5060 Panchum Scientific CorpTaipei Taiwan) grinding to powder and storing at minus80∘Cuntil use Extracts were produced from 200mg aliquotsof MLRC powder with a 50-fold volume of methanol at4∘C extract overnight and then filtered through Whatmangrade no1 qualitative filter paper Extracts were concentratedby rotary vacuum evaporator (R205 Buchi Switzerland)resuspended in 995 ethanol to 1mL and then stored atminus20∘C until HAECs treatment

The edible portion of each MLRC leaf was weighedlyophilized ground to powder and extracted by distilleddeionized (dd)H

2O [25] The extraction mixture was heated

to 90∘C in a steam bath and refluxed for 2 h cooled inthe refrigerator sonicated for 5min and diluted to 50mLwith ddH

2O to form the final extract solution Individual

water extracts were used for the comet assay Ten mL of625 aqueous methanol containing BHT (2 gL) was addedto 125 g lyophilized samples followed by adding 5mL 6MHCl to bring the total volume up to 125mL The finalmixture consisted of 12M HCl in 50 aqueous methanolAcid hydrolysatesmethanolic extracts were analyzed for theirantioxidant composition antioxidant activity and the cometassay

22 Polyphenol Assay Polyphenol content was determinedaccording to the method of Taga et al [26] Briefly standardgallic acid and an aliquot of the acidic methanolic extractwere diluted with an ethanolwater (60 40 vv) solutioncontaining 03HCl TwomLof 2Na

2CO3weremixed into

each sample of 100 120583L and allowed to equilibrate for 2minbefore adding 50 Folin-Ciocalteu reagent Absorbance at750 nm was measured at room temperature The standardcurve for gallic acid was used to calculate polyphenol levels

23 Determination of Total Flavonoids Total flavonoid con-tents were determined using the method of Ordonez et al[27] A 05mL 2 AlCl

3-ethanol solution was added to

05mL of acidic methanolic extract and absorbance wasmeasured at 420 nm after standing for 1 h at room temper-ature Extract samples were evaluated at a final concentrationof 01mgmL Total flavonoid content was calculated as aquercetin equivalent (mgg)

24 Determination of Total Flavonols Total flavonols in plantextracts were estimated using the method of Kumaran andJoel Karunakaran [28] Two mL of 2 AlCl

3ethanol and

30mL (50 gL) sodium acetate were added to 20mL acidicmethanolic extracts The absorption at 440 nm was readafter 25 h at 20∘C Sample extracts were evaluated at a finalconcentration of 01mgmL Total flavonoid content wascalculated as a quercetin equivalent (mgg)

25 Flavonols Analysis by HPLC One mL of acid hydrolysa-tes methanolic extract was filtered through 045 120583m filterprior to 20 120583L injection into a high-performance liquid

Evidence-Based Complementary and Alternative Medicine 3

chromatograph (HPLC) Samples were analyzed with a Spec-traSystem UV6000LP Photodiode Array Detection System(Thermo Separation Products San Jose USA) and an ODScolumn (250 times 46mm 5120583m YMC ODS-A YMC CoKyoto Japan)Themobile phase consisted of methanol-water(30 70 vv) with 1 formic acid and 100 methanol Thegradient was 25ndash74 methanol in 40min at a flow rateof 075mLmin the spectrum was recorded at 365 nm forflavonol determination [25]

26 ABTSHRPndashH2O2Assay of Total Antioxidant Activity

The total antioxidant capacity of hydrophilic and lipophilicantioxidants was determined using the horseradish perox-idase catalyzed oxidation of 22-azino-bis-(3-ethylbenzot-hiazoline-6-sulfonic acid) (ABTS) [29]The reaction mixturecontained 05mL of 1000 120583M ABTS (in ddH

2O) and 35mL

of 100 120583M H2O2(in 01M PBS) The reaction was started

by the addition of 05mL of 44UmL peroxidase (in 01MPBS) After 1 h 005mL of MLREs was added to the mixtureAbsorbance was measured at 730 nm after 5min Trolox wasused as a standard and the total antioxidant capacity ofmulberry extracts was measured as mM Trolox equivalent

27 Scavenging Activity on 11-Diphenyl-2-picryl-hydrazyl(DPPH) Radical The scavenging activity onDPPH radical ofMLRC acidic methanolic extracts was determined accordingto the method of Shimada et al [30] Briefly an aliquot of04 mL of acidic methanolic extracts with series dilution wasadded to 08mLof 1mMDPPH freshly prepared inmethanolmixed well and left to stand for 30min before measuringabsorbance at 517 nm The scavenging effect percentage wascalculated as [1 minus (OD

517 nm)(control OD517 nm)] times 100 TheIC50of the scavenging effect percentage was then calculated

28 Isolated Human Peripheral Blood Lymphocytes Bloodsamples were obtained from six donors four male andtwo female healthy nonsmokers that were 24 to 48 yearsold Fresh whole blood (20ndash30mL) from volunteers wastaken with informed consent and lymphocytes were isolatedusing a separation solution kit supplemented with Ficoll-Paque Plus lymphocyte isolation sterile solution (PharmaciaBiotech Sweden) [31] Cells were harvested within 1 dayof blood samples being taken and cultured with AIM Vmedium containing serum-free lymphocyte medium (GibcoInvitrogen USA) in a humidified atmosphere of 5 CO

2in

air at 37∘C for 24 h

29 Dosage Selected for the Comet Assay Previously wedemonstrated that the effect of lymphocyte exposure to 10 120583MH2O2on DNA single-strand break damage was 60-fold

greater than the control and with 994 cell viability [32]However when lymphocytes were exposed to 50 120583M H

2O2

the oxidative damage increased significantly compared to thecontrol under 982 cell viability Therefore 10120583M H

2O2

for the treatment dosage was selected in the current studyFurthermore when lymphocytes were treated with threeconcentrations (25 50 and 100 120583gmL) of acidic methanolicand water extracts from indigenous purple vegetables [33]

and herbaceous plants [34] the DNA damage was inducedby exposing the lymphocytes to 10 120583M H

2O2and shown the

protection effects Consequently these concentrations of theextracts were chosen for the effect treatments

210 Cell-Viability Testing After culturing lymphocytes wereexposed to each MLREs Each lymphocyte solution was usedat three concentrations (25 50 and 100 120583gmL) for 30min at37∘C DNA damage was induced by exposing lymphocytes toH2O2(10 120583M) for 5min on ice Treatment on ice minimized

the possibility of cellular DNA repair after H2O2injury Cells

were centrifuged at 100 g for 10minwashed and resuspendedin the samemedium as the comet assay All experiments werecarried out in triplicate Cell viability was tested using thetetrazoliumformazan (MTT) assay [35] prior to and aftertreatment with MLRC extract or H

2O2

211 DNA Single Strand Break Damage Estimation Using theComet Assay The comet assay was performed as describedin Szeto et al [36] with acidic methanolic hydrolysate andwater extracts from tested plants Cultured lymphocytes(105 cellsmL) were embedded in 75120583L of 1 low-melting-point agarose on a microscope slide (precoated with agarose)at 37∘CThe gel was allowed to set at 4∘C and cells were lysedfor a period of at least 2 h in lysis buffer at 4∘C Cells werethen alkaline-unwound following which electrophoresis wascarried out using the electrophoresis buffer at 4∘C for 15minat 25 vDC and 300mA All steps were conducted underdim light to prevent additional DNA damage Followingelectrophoresis slides were dipped into a neutralizationbuffer and stained with ethidium bromide The comet-likeimages resulting from the extension of DNA were scored asa reflection of the single strand breaks under a fluorescencemicroscope (Zeiss-Axiovert 100 Zeiss Germany) Triplicateslides were prepared for each experimental point sampleand 50 comet-like images selected at random per slide todetermine average DNA damage values A computerizedimage analysis system (VisCOMET 16 Impuls GmbH Ger-many) was employed to analyze two comet parameters DNAdamage by tail DNA percentage [(total brightness of tailareatotal brightness of total area) times 100] and tail moment(tail length times tail DNA) The inhibition percentage of tailDNA and tail moment were calculated relative to the 10 120583MH2O2treated group

212 Cell Cultures and Treatment HAECs (Clonetics Corp)were grown in Medium 200 (GIBCO Invitrogen) supple-mented with 1 low serum growth supplement (LSGSGIBCO Invitrogen) and 10 FBS (GIBCO Invitrogen) inan atmosphere of 95 air and 5 CO

2at 37∘C in plastic

flasks in an incubator (Astec Co) as described by Vielmaet al [37] The human monocytic cell line U937 (Amer-ican Type Culture Collection) was grown in a suspensionculture in RPMI-1640 (GIBCO Invitrogen) containing 10FBS (Sigma) and 1 antibiotic-antimycotic mixture (Sigma)under an atmosphere of 95 air and 5 CO

2at 37∘C

After incubation with MLREs and aspirin or TNF-120572 cellviability was assessed using the tetrazoliumformazan (MTT)

4 Evidence-Based Complementary and Alternative Medicine

assay [35] The adhesion assays were performed as describedby Zhu and Loft [38] Briefly U937 cells were labeledwith a fluorescent dye incubated with 10 120583molL 27-bis (2-carboxyethyl)-5 (6)-carboxyfluorescein acetoxymethyl esterat 37∘C for 1 h in RPMI-1640 medium and subsequentlywashed by centrifugation ConfluentHAECs in 24-well plateswere incubated with U937 cells (106 cellsmL) at 37∘C for1 h Nonadherent leukocytes were removed The numbers ofadherent leukocytes were determined by photographing andcounting four randomly chosen fields per well at 100x usinga Zeiss Axio Mager Z1 Upright Fluorescence MicroscopeExperiments were performed in triplicate and repeated threetimes HAECswere incubatedwith 25 120583g and 50 120583g ofMLREsor 10 120583M aspirin for 18 h followed by treating with 2 ngmLTNF-120572 for 6 h in theNF-120581Bp65 AP-1 and STAT-3 expressionassays

213 Nuclear Protein Isolation Protein extracts were pre-pared as described by Min et al [39] Briefly after cellactivation for the indicated times cells were washed in 1mLice-cold PBS centrifuged at 400 g for 5min resuspendedin 400 120583L ice-cold hypotonic buffer (10mM HEPES 15mMMgCl

2 01mM EDTA 10mM KCl 1mM DTT 05mM

PMSF pH79) incubated on ice for 10min vortexed and cen-trifuged at 15000 g for 30 sec The supernatant was collectedand stored at minus70∘C for cytosolic protein analysis Pelletednuclei were gently resuspended in 445 120583L ice-cold extractionbuffer (20mM HEPES pH 79 with 15mM MgCl

2 042M

NaCl 02mM EDTA and 25 glycerol) with 5 120583L of 10mMDTT and 05 120583L of Protease Inhibitor Cocktail (Sigma) incu-bated on ice for 20min vortexed and centrifuged at 15000 gfor 5min at 4∘C Aliquots of the supernatant containingnuclear proteins were frozen in liquid nitrogen and stored atminus70∘C

214 Western Blot Analysis Nuclear lysates were subjected to12 SDS-PAGE (Bio-Rad Laboratories) after which proteinswere transferred to a PVDF membrane (Bio-Rad) Mem-branes were probed with a mouse monoclonal NF-120581B p65antibody (BD Biosciences) After incubation in a secondaryantibody solution consisting of IRDye 700CW-conjugatedgoat anti-mouse IgG (LI-COR Biosciences) for 60min atroom temperature with gentle shaking the membrane waswashed four times for 5min each at room temperature in PBSwith 01 Tween-20 and while being gently shaken After afinal rinse the membrane was scanned using AlphaEaseFCimage analysis software (Alpha Innotech Corporation) toanalyze spot density The internal control was set at 100 todetermine the relative density of protein expression [40]

215 Electrophoretic Mobility Shift Assay for NF-120581B AP-1 andSTAT3 The binding reaction consisted of 1120583L 10times bindingbuffer (100mM TRIS 500mM NaCl 10mM DTT pH 75)5 120583LH

2O 2120583L 25mMDTT25Tween-20 1 120583L IRDye 700-

labeled EMSA oligonucleotides specific for NF-120581B (51015840-AGTTGA GGG GAC TTT CCC AGG C-31015840 and 31015840-CGC TTGATG ACT CAG CCG GAA-51015840) AP-1 (51015840-CGC TTG ATGACT CAG CCG GAA-31015840 and 31015840-GCG AAC TAC TGA GTC

GGC CTT-51015840) STAT3 (51015840-GAT CCT TCT GGG AAT TCCTAG ATC-31015840 and 31015840-CTA GGA AGA CCC TTA AGG ATCTAG-51015840) (Sigma) PPAR-120572 (51015840-AAA AAC TGG GCC AAAGGT CT-31015840) and PPAR-120574 (51015840-TGA AAC TAG GGT AAAGTT CA-31015840) (Sigma) 1 120583L poly (dIsdotdC) and 1 120583L nuclearextract (as prepared previously) The mixture was incubatedat room temperature for 20min in the dark After 1x OrangeLoading Dye (LI-COR Biosciences) was added the bindingreaction was loaded onto a native 4 polyacrylamide gel andseparated by electrophoresis at 90 vDC for 40min The gelswere scanned using anOdyssey Infrared Imaging System (LI-COR Biosciences) [40]

216 Statistical Analysis Data are expressed as the mean plusmnstandard deviation and statistical significance was analyzedusing one-way ANOVA followed by the Tukeyrsquos Range Testat the 005 significance level Pearsonrsquos linear correlation wasalso determined The means of three replicates are reported

3 Result

31 Antioxidant Composition and Antioxidant Activity Poly-phenols flavonoids and flavonols were abundant in BTat levels of 3465 plusmn 082mgg DW gallic acid 8483 plusmn868mgg DW quercetin equivalent and 2547 plusmn 386mggDW quercetin equivalent respectively (Table 1) Anthocy-anidins were abundant in BT and ML at levels of 14332 plusmn181 and 13582 plusmn 031 unitsgDW respectively Table 2 showsthat quercetin and kaempferol were both rich in BTat levelsof 285333 plusmn 18037 and 64667 plusmn 5033 120583ggDW respectivelywhile the levels of M were 166667 plusmn 18903 and 42667 plusmn4619 120583ggDW respectively However no myricetin or morinwas detected in the MLRC (Table 2) Thus these MLREsdisplayed variations in their antioxidant substances levels

MLREs showed antioxidant activity proving their capac-ity to scavenge the ABTS radical cation The antioxidantactivity in acidic methanolic hydrolysate sample extracts wasexpressed in trolox equivalent antioxidant capacity (TEAC)(Table 3) BT showed a significantly higher TEAC value(15514 plusmn 290 120583ggDW) than MT (10239 plusmn 095 120583ggDW)and M (7654 plusmn 161 120583ggDW) TEAC values were positivelyand significantly (119903 = 041 119875 = 0034) correlated withthe content of quercetin among MLREs (Table 4) Thusdifferent quercetin contents displayed various levels of anti-oxidant activity However M had a significantly high effi-cacy for DPPH radical scavenging activity (IC

5018883 plusmn

361 120583gmL) compared to BT (IC5066901plusmn723 120583gmL) and

MT (IC5083640 plusmn 708 120583gmL) (Table 3) Hence each tested

sample showed significant differences in scavenging of DPPHradical at IC

50

32 Effects of Acidic Methanolic and Water Extracts fromMLRC on H

2O2-Induced DNA Damage to Lymphocytes The

effects ofMLRC extracts on cell cytotoxicity were determinedby theMTTassay Lymphocyteswere exposed to each of threedifferent MLRC extracts at three concentrations (25 50 and100 120583gmL) for 30min at 37∘C DNA damage was inducedby exposing lymphocytes to H

2O2(10 120583M) for 5min on ice

Evidence-Based Complementary and Alternative Medicine 5

Table 1 The contents of various antioxidant substances in MLRC

Polyphenolmg gallic acidgDW

Flavonoids mg quercetinequivalentgDW

Flavonol mg quercetinequivalengDW

AthocyanidinunitgDW

BT 3465 plusmn 082a

8483 plusmn 868a

2547 plusmn 386a

14332 plusmn 181a

MT 2323 plusmn 054b

5599 plusmn 1055b

1326 plusmn 266b

8858 plusmn 412b

M 1987 plusmn 061c

4960 plusmn 541b

1422 plusmn 148b

13582 plusmn 031a

All values are means plusmn SD (119899 = 3)Means within a column with different superscripts (asimc) are significantly different 119875 lt 005

Table 2 The contents of flavonols in acidic methanolic extracts ofMLRC

Flavonols (120583ggDW)Samples Myricetin Morin Quercetin KaempferolBT ND ND 285333 plusmn 18037

a64667 plusmn 5033

a

MT ND ND 37333 plusmn 2309c ND

M ND ND 166667 plusmn 18903b42667 plusmn 4619

b

ND not detected

Table 3 TEAC values and the IC50 of DPPH radical scavengingactivity of acidic methanolic hydrolysates of MLRC

Sample TEAC(Trolox mM)lowast

DPPH radical scavenging activityIC50 (120583gmL)

BT 15514 plusmn 290a

66901 plusmn 723b

MT 10239 plusmn 095b

83640 plusmn 708a

M 7654 plusmn 161c

18883 plusmn 361c

lowastTEAC value at sample 100120583gmL concentration

Table 4 Correlation between antioxidant substances and TEACvalues treated with acid hydrolysates of MLRC

FlavonolsMyricetin Morin Quercetin Kaempferol

TEAClowast mdash mdash 119903 = 041

119875 = 0034

mdash

mdash no significant correlationlowastTEAC values at 100 120583gmL of sample concentration

No mulberry extracts were cytotoxic at the concentrationsused with gt98 of cells remaining viable [41] The cometassay was performed to determine the DNA damagingactivity of the plants because it is a sensitive method formonitoring single strand DNA breaks at the single celllevel Any DNA damage that occurred is represented as atail length (tail migration) of the DNA strand The effectsof pretreatment with acidic methanolic extracts of MLRCon 10 120583M H

2O2-induced DNA oxidative damage in human

lymphocytes are presented in Figure 1 indicating that therewas a protective effect of pretreatment on lymphocytes witheach of the MLRC extracts at the lowest dose (25 120583gmL)Lymphocytes pretreated with 25ndash100120583gmL extracts of MTextracts experienced a greater level of protection againstH2O2exposure than lymphocytes exposed to other tested

compounds and in a dose-dependent manner BT had betterinhibition efficacy at lower concentration (25120583gmL) andthe maximum protective effect of lymphocyte pretreatmentwas observed with pretreatment by 25 120583gmL BT exhibiting

1462 of tail DNA compared to treatment only withH2O2

MT had better inhibition efficacy (8439) than M and BTextracts at the 100 120583gmL level (Figure 1(a)) Tested plantsshowed at least 134263 of tail moment in BT extract at the25 120583gmL level while the BT extract at 100 120583gmL had thehighest tail moment (328193) compared to the rest of theacidic methanolic extract samples (Figure 1(b)) Moreoverthe trend in tail moment in all samples continued climbingup from 25ndash100 120583gmL

The effects of various water extracts of MLRC H2O2-

induced DNA damage to lymphocytes are presented inFigure 2 At lower concentrations all tested samples hadlower tail DNA indicating better inhibition efficacies(Figure 2(a)) BT and M had the best inhibition efficacieswith both exhibiting values of 1422 and 1469 tail DNAat 25 120583gmL respectively Moreover MT had a higher tailDNA than the other extracts at the same dose and thehighest tail DNA was observed in 3212 of MT at aconcentration of 100120583gmL The tail moment of all samplescontinued to increase up to the 100 120583gmL level (Figure 2(b))The minimum tail moment occurred in M at 153674 in the25 120583gmL extracts Moreover the DNA damage induced byH2O2was significantly higher than the DNA damage treated

with extracts and H2O2treated had 8726 in tail DNA and

832884 in tail moment

33 Effects of MLREs on Monocyte-Endothelial Cell Adhe-sion As shown in Figure 3 pretreatment of HAECs with100 120583gmLMLREs for 18 h significantly suppressed the adhe-sion ofU937monocytes to TNF-120572-stimulatedHAECs similarto that of 10 120583Maspirin (positive control) Data also show that25 and 50 120583gmL BT MT and M methanol extracts inhib-ited the adhesion of U937 monocytes to TNF-120572-stimulatedHAECs by 8198 and 8187 (BT) 7449 and 8331 (MT)and 8011 and 7559 (M) respectively

34 Effects of MLREs on NF-120581B Activity and MLREs Inhibitsthe Binding of NF-120581B AP-1 and STAT3 Figure 4 demon-strates that MLREs regulated the DNA-binding activity andexpressions of NF-120581B in HAECs In untreated HAECs NF-120581B p65 was only found within the cytosol however itsnuclear translocation was observed upon treatment withTNF-120572 Compared to the TNF-120572 group pretreatment withBT and MT methanol extracts significantly decreased theexpression of NF-120581B p65 in the nuclear compartment whilethe M methanol extract did not reduce the expressionof NF-120581B p65 activity Furthermore an EMSA analysiswas carried out to determine if the reduced expression of

6 Evidence-Based Complementary and Alternative Medicine

Control H2O2

BT25 BT50 BT100

MT25 MT50 MT100

M25 M50 M100

(a)

0

20

40

60

80

100

25 50 100

Tail

DN

A (

)

Mulberry extracts (120583gmL)

lowast lowastlowast

lowast

lowastlowast

alowast alowast

blowast

MTMBT

H2O2

(b)

Tail

mom

ent

0

2000

4000

6000

8000

10000

25 50 100Mulberry extracts (120583gmL)

lowastlowastlowast

ablowastclowast

alowast

alowast

alowastblowast

MTMBT

H2O2

(c)

Figure 1 Effects of various acidic methanolic extracts of MLRC on H2O2-induced DNA damage to lymphocytes The comet-like images

resulting from the extension of DNA (a) were observed using a fluorescence microscope Tail DNA (b) and tail movement (c) were exposedto test mulberry leaf-related extracts at 25 50 and 100 120583gmL and treated with H

2O2at 10120583M Values with different letters differ significantly

with regard to oxidative damage when comparing different MLRC extracts lowast119875 lt 005 refers to differences in oxidative damage as comparedwith 10 120583MH

2O2-alone treatment

Evidence-Based Complementary and Alternative Medicine 7

Control H2O2

BT25 BT50 BT100

MT25 MT50 MT100

M25 M50 M100

(a)

alowastblowast blowastblowast

blowastblowast

blowast

25 50 100Mulberry extracts (120583gmL)

MTMBT

H2O2

0

20

40

60

80

100

Tail

DN

A (

)

ablowastablowast

(b)

Tail

mom

ent

0

2000

4000

6000

8000

10000

lowastlowast lowast

alowastalowast

25 50 100Mulberry extracts (120583gmL)

MTMBT

H2O2

ablowast ablowastablowast

bclowast

(c)

Figure 2 Effects of various water extracts of MLRC on H2O2-induced DNA damage to lymphocytes The comet-like images resulting from

the extension ofDNA (a) were observed using a fluorescencemicroscope Tail DNA (b) and tailmovement (c) were exposed to testmulberryleaf-related extractsat 25 50 and 100120583gmL and treated with H

2O2at 10120583M Values with different letters differ significantly with regard to

oxidative damage when comparing different MLRC extracts lowast119875 lt 005 refers to differences in oxidative damage as compared with 10120583MH2O2-alone treatment

NF-120581B in response to treatment with MLREs resulted inreduced binding of NF-120581B to DNAThe treatment of HAECswith TNF-120572 led to increased binding of NF-120581B howeverpretreatment of HAECs with BT MT and M significantlydecreased DNA-bound NF-120581B (Figure 5(a)) The increased

binding of AP-1 with TNF-120572 resulted from the treatment ofHAECs however pretreatment of HAECs with BT MT andM significantly decreased DNA-bound AP-1 (Figure 5(b))STAT3 DNA-binding activities to its cognate recognitionsite were remarkably reduced compared to TNF-120572 treatment

8 Evidence-Based Complementary and Alternative Medicine

(A)(A)

(B)

(C) (D)

(E) (F)

(I)

(G) (H)

200120583m 200120583m

200120583m200120583m

200120583m200120583m

200120583m200120583m

200120583m

(a)

Con

trol

Asp

irin

BT25

BT50

MT2

5

MT5

0

M25

M50

Mon

ocyt

e adh

eren

ce (c

ells

HPF

)

0

20

40

60

80

100

120

140

160

180

lowast lowast lowast

lowast

lowast

lowast

lowast

TNF-120572

(b)

Figure 3 Mulberry leaf related extracts (MLREs) reduce tumor necrosis factor (TNF)-120572-induced adhesion of monocytes to human aorticendothelial cells (HAECs) (a) The number of adherent U937 cells (marked by arrow) was monitored by fluorescence microscopy undercontrol (a) 2 ngmL TNF-120572 (b) co-treated with TNF-119886 and aspirin (10120583M) (c) cotreated with TNF-120572 and BT (25 120583gmL) (d) cotreated withTNF-120572 and BT (50 120583gmL) (e) cotreated with TNF-120572 and MT (25 120583gmL) (f) cotreated with TNF-120572 and MT (50 120583gmL) (g) cotreated withTNF-120572 and M (25120583gmL) (h) and cotreated with TNF-120572 and M (50120583gmL) (i) (b) HAECs were pretreated with MLREs for 18 h followedby stimulating with TNF-120572 for 6 h Fluorescence-labeled monocytic U937 cells were added to HAECs and allowed to adhere for 2 h Data areexpressed as a percentage of TNF-120572-induced expression (mean plusmn SD) of three experiments Mean plusmn SD lowast119875 lt 005 BT MT and M at 25and 50120583gmL versus TNF-120572

119875 lt 005 control versus TNF-120572

Evidence-Based Complementary and Alternative Medicine 9

Control TNF-120572

TNF-120572

Aspirin10120583M

+ + + + + + + +

BT25 BT50 MT25 MT50 M25 M50

120583gmL 120583gmL 120583gmL 120583gmL 120583gmL 120583gmL

NF-120581B p65

120573-Actin43kDa

minus

65kDa

(a)

Con

trol

Asp

irin

BT25

BT50

MT2

5

MT5

0

M25

M50

00

02

04

06

08

10

lowast

lowast

lowast

lowastlowastlowastTN

F-120572

Ratio

(nuc

lear

120573-a

ctin

)

(b)

Figure 4 Effects of MLREs on NF-120581B activity After HAECs were pretreated with the indicated samples and incubated with TNF-120572 nuclearextracts were prepared and the expression of NF-120581B p65 was assessed by western blot analysis A representative image of three similar resultsis shown (a) Actin served as the loading control for the nuclear compartment Semiquantitative analysis of three independent experimentsare also shown (b) indicates a significant difference between the TNF-120572 treated or experimental treatment groups and the control119875 lt 005lowast indicates a significant difference between the TNF-120572 and experimental treatment groups BT MT and M at 25 and 50 120583gmL

(lane 3) in the nuclear fraction indicating that MLREs at50120583gmL inhibited STAT3-binding activities In addition theresults in Figure 5(c) (lanes 4ndash10) show that aspirinMLREs at50120583gmL and BT at 25120583gmL remarkably decreased STAT3levels compared to TNF-120572 (lane 3) however both MT andM at 25120583gmL increased STAT3-binding activities Theseresults indicate that only 50 120583gmL MLREs 25 120583gmL BTand aspirin block STAT3 activation and might downregulateTNF-120572-induced expressions of inflammatory signaling path-ways

35 MLREs Regulated the DNA-Binding Activity and Expres-sions of PPAR 120572 and PPAR 120574 in HAECs PPAR 120572 and 120574effectively bound to PPAR 120572 and 120574 oligo-IRDye 700 asindicated in lanes 3ndash10 of Figure 6 Supershifts are bands fromthe binding of each specific antibody The binding activitiesof PPAR 120572 and 120574 were detected at baseline The 25 120583gmLMT extract (lane 7) and 25 and 50 120583gmL M extracts (lanes9-10) increased activity compared to incubating with TNF-120572 alone (lane 3) However BT at both 25 and 50 120583gmLextracts showed reduced effects on PPAR 120572 and 120574 expressionMoreover the 50 120583gmLMT extract had no effect on PPAR 120572binding activity but increased the binding activity of PPAR120574 in comparison to incubation with TNF-120572 alone Thesedata suggest that MT and M both promoted PPAR 120572 and 120574

expression at least in part by increasing the DNA binding oftranscription factors PPAR 120572 and 120574

4 Discussion

41 Antioxidant Composition and Antioxidant Activity BTcontains high levels of polyphenols flavonoids flavonolsanthocyanidin quercetin and kaempferol (Tables 1 and 2)BT also had a higher TEAC value (15514mM Table 3) thanthe TEAC values of 47mM [42] and 64mM [43] reportedfor quercetin as well as 242mM reported for quercetin-3-rutinoside [44] Furthermore Gonzalez-Paramas et al [45]demonstrated that flavonol contents were significantly andhighly correlated (119903 = 08) with TEAC values Kim et al [46]reported that mulberry leaves contain abundant quercetinand inhibit lipid peroxidation at concentrations of 01 and001mgmL In our study the amount of quercetin wassignificantly correlated (119903 = 041 Table 4) with TEAC valueHowever no correlation was found among myricetin morinkaempferol and TEAC which may be due to the structurerequired to reinforce the free radical scavenging activity thatvaries with the type of free radical Naowaratwattana et al[18] reported that a 50 methanolic extract of mulberryleaf contained rutin isoquercetin and various derivatives ofkaempferol and quercetin glycosides while a water extract

10 Evidence-Based Complementary and Alternative Medicine

NF-120581BTNF-120572

TNF-120572

+

Aspirin10120583M+ +

BT25120583gmL

+

BT50120583gmL

+

MT25120583gmL

+

MT50120583gmL

+

M25

120583gmL+

M50

120583gmLControl

minus

DNAOligo

Free probe

(a)

AP-1TNF-120572

TNF-120572

+

Aspirin10120583M+ +

BT25120583gmL

+

BT50120583gmL

+

MT25120583gmL

+

MT50120583gmL

+

M25

120583gmL+

M50

120583gmLControl

minus

DNAOligo

Free probe

(b)

STAT3

TNF-120572

TNF-120572

+

Aspirin10120583M+ +

BT25120583gmL

+

BT50120583gmL

+

MT25120583gmL

+

MT50120583gmL

+

M25

120583gmL+

M50

120583gmLControl

minus

DNAOligo

Free probe

(c)

Figure 5 MLREs inhibits the binding of (a) NF-120581B (b) AP-1 and (c) STAT3 to target DNA sequence NF-120581B AP-1 and STAT3 transcriptionfactor-DNA interactions were assessed by EMSA

contained primarily chlorogenic acid and caffeoylquinic acidderivatives The IC

50of DPPH radical scavenging was 794

and 2042120583gmL for 50 methanolic and water extractsrespectively [18] In our studyM extracts contained quercetinand kaempferol (Table 2) which may account for part ofthe highly scavenged DPPH radical in comparison to therest of MLREs Khan et al [47] demonstrated that the IC

50

was 22023120583gmL which is much less efficacious than themulberry leaf acidic methanolic extract IC

50of 1888 120583gmL

(Table 3) However the IC50

of in vitro free radical scav-enging activity of mulberry leaves from Naowaboot et al[22] was 617 plusmn 21 120583gmL which is more efficacious thanthe IC

50concentration derived in the present study Taken

together the polyphenol-rich MLREs exhibited distinct cell-free antioxidant activity (ie TEAC) as validated by theirlevels of polyphenols and flavonols with distinct antioxidantactivity strongly accounting for the antioxidant activity of the

extracts The amount of quercetin was especially positivelycorrelated with the observed TEAC values

42 Estimation of DNA Single Strand Break Damage fromExposure to Acidic Methanolic and Water Extracts One ofthe antioxidant activity tests scavenging of DPPH radi-cal was selected due to its quick and simple but it is aless biologically relevant assay Previously we reported thatwith 2 120583gsdotmLminus1 of methanolic and water extracts from theindigenous vegetables [33] and herb plants [48] significantlyprolonged lag phase of low-density lipoprotein (LDL) TheLDL oxidation assay is considered to be one of the mostbiologically relevant assays [49] Both studies [33 48] alsoshowed that the concentration of the tested extracts forscavenging DPPH radical was in higher levels at 10mg oftested extracts with 2ndash53 inhibition Lower concentrationwas then chosen for the treated dosage in the present study

Evidence-Based Complementary and Alternative Medicine 11

TNF-120572

TNF-120572

+

Aspirin10120583M+ +

BT25120583gmL

+

BT50120583gmL

+

MT25120583gmL

+

MT50120583gmL

+

M25

120583gmL+

M50

120583gmLControl

minus

DNAOligo

Free probe

PPAR 120572

(a)

TNF-120572

TNF-120572

+

Aspirin10120583M+ +

BT25120583gmL

+

BT50120583gmL

+

MT25120583gmL

+

MT50120583gmL

+

M25

120583gmL+

M50

120583gmLControl

minus

DNAOligo

Free probe

PPAR120574

(b)

Figure 6 Effect of treatments on the expression of PPAR 120572 (a) and PPAR 120574 (b) in TNF-120572-stimulated HAECs by EMSA

In addition lower concentration also could avoid the toxicityeffect of higher level of extracts on lymphocytes Quercetinwas found to protect against H

2O2-induced DNA damage in

human lymphocytes at 10 120583M [50] and at 31 to 25 120583M [51]Quercetin was also reported to induce DNA strand breaks invarious cell types but only at higher doses at 100 120583Mor above[50] Duthie et al [50] proposed that the dihydroxy structureof quercetin and myricetin might be essential in protectingDNA against hydrogen peroxide No such hydroxyl groupsare present in the tocopherol molecule Noroozi et al [52]demonstrated that in addition to quercetin kaempferolcould also inhibit H

2O2-induced DNA strand breaks in

human lymphocytes In our study both BT and M were richin quercetin and kaempferol (Table 2) whichmay account forthe protective roles of both extracts in preventingDNAstrandbreaks from human lymphocytes

43 MLREs Regulated the DNA-Binding Activity and Expres-sions of NF-120581B AP-1 STAT3 and PPARs in HAECsBecause MLREs exhibited free radical scavenging the anti-inflammatory effects of MLREs on TNF-120572-induced celladhesion and adhesionmolecule expressionwere determinedin the present study Our results suggest that MLREs andaspirin significantly inhibited TNF-120572-induced monocyte-endothelial cell adhesion (Figure 3) Significant reductionsin NF-120581B expression and DNA binding by aspirin MTextract (25 120583gmL) and BT extract (both 25 and 50 120583gmL)were also observed The adhesion of monocytes to the

vascular endothelium and subsequent migration into thevessel wall are early events in atherogenesis [2] Our resultsare consistent with previous research showing that dietarypolyphenols such as catechin and quercetin significantlyreduce the binding of monocytes to HAECs [53 54] Thereduced cellular adhesion may be due to the inhibitionof cellular adhesion molecule expression as purple sweetpotato leaf extract (PSPLE) reduced both VCAM-1 andICAM-1 expression [40] Shibata et al [55] demonstratedthat pretreatment of cultured bovine aortic endothelial cells(BAECs) with mulberry leaf aqueous fractions inhibitedTNF-120572- and LPS-induced expression of Lectin-like Ox-LDLreceptor-1 (LOX-1) a cell-surface receptor for atherogenicOx-LDL (oxidized low-density lipoprotein) It also appearsto mediate Ox-LDL-induced inflammation which may becrucial in atherogenesis Furthermore mulberry leaf aqueousfractions inhibited the TNF-120572-induced activation of NF-120581Band phosphorylation of inhibitory factor of NF-120581B-alpha(I120581B-120572) Thus mulberry leaf aqueous fractions suppressTNF-120572- and LPS-induced LOX-1 gene expression mediatedby inhibiting NF-120581B activation

Chan et al [56] demonstrated that mulberry leaf extract(MLE) was rich in polyphenols MLE can effectively inhibitthe migration of vascular smooth muscle cells (VSMC) byblocking small guanosine triphosphatases (small GTPase)and AktNF-120581B signals The Rho family of small GTPasessuch as Rho Rac and Cdc42 acts as a molecular switch toregulate the actin cytoskeleton The Rho family of GTPases

12 Evidence-Based Complementary and Alternative Medicine

is also reported to be involved in the regulation of NF-120581B-dependent transcription [56] MLE has been shown to pos-sess hypoglycemic effects in an insulin-dependent diabetesmellitus (type 1 diabetes) animal model and potential hypo-glycemic compounds inmulberry leaves were suggested to be2-O-120572-D-galactopyranosyl-DNJ and fagomine [57] Konnoet al [58] further reported that mulberry latex containsalkaloidal sugar-mimic glycosidase inhibitors with antidia-betic activities such as 14-dideoxy-14-imino-D-arabinitol 1-deoxynojirimycin and 14-dideoxy-14-imino-D-ribitol Kimet al [59] recently showed that treated type 2 diabetesmellitus(T2D) with a 1 1 1 mixture of ginseng root mulberry leafwater extract and banana leaf water extract (6 gd) for 24weeks significantly decreased plasma intracellular adhesionmolecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) levels This indicates suppressed inflammatoryresponses in T2D but no significant differences in glucosehomeostasis control measurement changes Rigamonti et al[8] reported that bothPPAR120572 and 120574maybe thought of as sup-pressing the activity of NF120581B and AP-1 pathways that reduceinflammatory mediator production in the macrophages ofhumans and mice In our study MT and M promoted PPAR120572 and 120574 expression at least in part by increasing DNAbinding of transcription factors PPAR 120572 and 120574 PPAR-120572-and 120574-dependent signaling pathwaysmay also play importantroles in mediating the TNF-120572 receptor signaling pathwayby MT and M by suppressing the activity on NF-120581B andAP-1 pathways These pathways may regulate downstreamproinflammatory gene expression and adhesion molecularexpression Clinically PPAR 120574 agonists such as rosiglitazoneare widely used in the treatment of T2D [60] M and MTextracts increased PPAR 120574 activity which may partly explainwhy inflammatory responses are suppressed in T2D

Although reduced NF-120581B expression and DNA bind-ing were observed upon treatment with ML MT and BT(Figure 5) the mechanism was not assessed and is worthy offurther study In addition the present results were obtainedusing in vitro studies and may be consistent with an invivo model of atherogenesis similar to that reported byEnkhmaa et al [21] in which mulberry leaves suppressedthe development of atherosclerotic lesions Previously weproved that black beans significantly prolong LDL lag timeand decrease the atheroma regions of the aortic arch andthoracic aorta in hypercholesterolemic NZW rabbits [61]Wethen demonstrated that black soybean extract and PSPLEsignificantly decreased the adhesion of U937 monocyticcells to TNF-120572-stimulated HAECs and reduced adhesionmolecules expression (VCAM-1 and ICAM-1) as well as NF-120581B-p65 expression [37] This is particularly important giventhe opposing effects of flavonols fromMLREs obtained usingin vitro and ex vivo studies [62]

5 Conclusion

Tested MLREs were rich in flavonols and had strongantioxidant activity in protecting lymphocyte DNA fromoxidative damage In particular BT was rich in quercetinand polyphenols The antioxidant components of MLREs

might downregulate intracellular redox-dependent signalingpathways in HAECs upon TNF-120572 stimulation which mightprevent ROS-mediated endothelial cell dysfunction MLRCshave anti-inflammatory effects through the modulation ofNF-120581B AP-1 STAT3 and PPARs signaling These chemi-cals appear to provide distinct cytoprotective mechanismsthat might contribute to their putative beneficial effectsin suppressing endothelial responses to cytokines duringinflammation These results may benefit further in vivostudies to assess the therapeutic potential of phytochemicalsas anti-inflammatories for use in cytokine-induced vasculardisorders including atherosclerosis

Acknowledgment

This work was supported in part by Grants from the NationalScience Council of Taiwan (NSC 95ndash2320-B-034-001 andNSC 96ndash2320-B-034-001)

References

[1] W Palinski ldquoUnited they go conjunct regulation of aorticantioxidant enzymes during atherogenesisrdquo CirculationResearch vol 93 no 3 pp 183ndash185 2003

[2] P Libby ldquoMolecular bases of the acute coronary syndromesrdquoCirculation vol 91 no 11 pp 2844ndash2850 1995

[3] P A Baeuerle and D Baltimore ldquoNf-120581B ten years afterrdquo Cellvol 87 no 1 pp 13ndash20 1996

[4] J M Kyriakis ldquoActivation of the AP-1 transcription factor byinflammatory cytokines of the TNF familyrdquo Gene Expressionvol 7 no 4ndash6 pp 217ndash231 1999

[5] J M Muller R A Rupec and P A Baeuerle ldquoStudy of generegulation by NF-120581B and AP-1 in response to reactive oxygenintermediatesrdquoMethods vol 11 no 3 pp 301ndash312 1997

[6] G Chinetti J Fruchart and B Staels ldquoPeroxisome proliferator-activated receptors new targets for the pharmacological mod-ulation of macrophage gene expression and functionrdquo CurrentOpinion in Lipidology vol 14 no 5 pp 459ndash468 2003

[7] G Chinetti J C Fruchart and B Staels ldquoTranscriptional regu-lation of macrophage cholesterol trafficking by PPAR120572 andLXRrdquo Biochemical Society Transactions vol 34 no 6 pp 1128ndash1131 2006

[8] E Rigamonti G Chinetti-Gbaguidi and B Staels ldquoRegulationof macrophage functions by PPAR- 120572 PPAR- 120574 and LXRsin mice and menrdquo Arteriosclerosis Thrombosis and VascularBiology vol 28 no 6 pp 1050ndash1059 2008

[9] M A Bouhlel J Brozek B Derudas et al ldquoUnlike PPAR120574PPAR120572 or PPAR120573120575 activation does not promote humanmono-cyte differentiation toward alternative macrophagesrdquo Biochem-ical and Biophysical Research Communications vol 386 no 3pp 459ndash462 2009

[10] G Chinetti J-C Fruchart and B Staels ldquoPeroxisome prolife-rator-activated receptors and inflammation from basic scienceto clinical applicationsrdquo International Journal of Obesity vol 27supplement 3 pp S41ndashS45 2003

[11] Y Zhu J H-C Lin H Liao et al ldquoLDL induces transcriptionfactor activator protein-1 in human endothelial cellsrdquo Arte-riosclerosis Thrombosis and Vascular Biology vol 18 no 3 pp473ndash480 1998

Evidence-Based Complementary and Alternative Medicine 13

[12] J Terao ldquoDietary flavonoids as antioxidantsrdquo Forum of Nutri-tion vol 61 pp 87ndash94 2009

[13] Y B Feng W Q Luo and S Q Zhu ldquoExplore new clinicalapplication of Huanglian and corresponding compound pre-scriptions from their traditional userdquo Zhongguo ZhongyaoZazhi vol 33 no 10 pp 1221ndash1225 2008

[14] Y H Chu C L Chang and H F Hsu ldquoFlavonoid content ofseveral vegetables and their antioxidant activityrdquo Journal of theScience of Food andAgriculture vol 80 no 5 pp 561ndash566 2000

[15] I C Arts B van de Putte and P C Hollman ldquoCatechin con-tents of foods commonly consumed in The Netherlands 1Fruits vegetables staple foods and processed foodsrdquo Journal ofAgricultural and Food Chemistry vol 48 no 5 pp 1746ndash17512000

[16] A R Proteggente A S Pannala G Paganga et al ldquoThe anti-oxidant activity of regularly consumed fruit and vegetablesreflects their phenolic and vitamin C compositionrdquo Free RadicalResearch vol 36 no 2 pp 217ndash233 2002

[17] Y L Liu L H Tang Z Q Liang B You and S Yang ldquoGrowthinhibitory and apoptosis inducing by effects of total flavonoidsfrom Lysimachia clethroides Duby in human chronic myeloidleukemiaK562 cellsrdquo Journal of Ethnopharmacology vol 131 no1 pp 1ndash9 2010

[18] W Naowaratwattana W De-Eknamkul and E G De MejialdquoPhenolic-containing organic extracts of mulberry (Morus albaL) leaves inhibit HepG2 hepatoma cells through G2M phasearrest and inhibition of topoisomerase II120572 activityrdquo Journal ofMedicinal Food vol 13 no 5 pp 1045ndash1056 2010

[19] K Doi T Kojima and Y Fujimoto ldquoMulberry leaf extract inhi-bits the oxidative modification of rabbit and human low densitylipoproteinrdquo Biological and Pharmaceutical Bulletin vol 23 no9 pp 1066ndash1071 2000

[20] W Wang Y Zu Y Fu and T Efferth ldquoIn Vitro antioxidant andantimicrobial activity of extracts from Morus alba L leavesstems and fruitsrdquoAmerican Journal of ChineseMedicine vol 40no 2 pp 349ndash356 2012

[21] B Enkhmaa K Shiwaku T Katsube et al ldquoMulberry (Morusalba L) leaves and their major flavonol quercetin 3-(6-malon-ylglucoside) attenuate atherosclerotic lesion development inLDL receptor-deficient micerdquo Journal of Nutrition vol 135 no4 pp 729ndash734 2005

[22] J Naowaboot P Pannangpetch V Kukongviriyapan B Kongy-ingyoes and U Kukongviriyapan ldquoAntihyperglycemic antiox-idant and antiglycation activities of mulberry leaf extract instreptozotocin-induced chronic diabetic ratsrdquo Plant Foods forHuman Nutrition vol 64 no 2 pp 116ndash121 2009

[23] MM Padilha F CVilela CQ Rocha et al ldquoAntiinflammatoryproperties of Morus nigra leavesrdquo Phytotherapy Research vol24 no 10 pp 1496ndash1500 2010

[24] P Aramwit O Supasyndh T Siritienthong and N BangldquoMulberry leaf reduces oxidation and c-reactive protein levelin patients with mild dyslipidemiardquo BioMed Research Interna-tional vol 2013 Article ID 787981 7 pages 2013

[25] U Justesen P Knuthsen and T Leth ldquoQuantitative analysis offlavonols flavones and flavanones in fruits vegetables andbeverages by high-performance liquid chromatography withphoto-diode array and mass spectrometric detectionrdquo Journalof Chromatography A vol 799 no 1-2 pp 101ndash110 1998

[26] M S Taga E EMiller andD E Pratt ldquoChia seeds as a source ofnatural lipid antioxidantsrdquo Journal of the AmericanOil ChemistsrsquoSociety vol 61 no 5 pp 928ndash931 1984

[27] A A L Ordonez J D Gomez M A Vattuone and M I IslaldquoAntioxidant activities of Sechium edule (Jacq) Swartz extractsrdquoFood Chemistry vol 97 no 3 pp 452ndash458 2006

[28] A Kumaran and R Joel Karunakaran ldquoIn Vitro antioxidantactivities of methanol extracts of five Phyllanthus species fromIndiardquo LWT-Food Science and Technology vol 40 no 2 pp344ndash352 2007

[29] G Cao H M Alessio and R G Cutler ldquoOxygen-radical absor-bance capacity assay for antioxidantsrdquo Free Radical Biology andMedicine vol 14 no 3 pp 303ndash311 1993

[30] K Shimada K Fujikawa K Yahara and T Nakamura ldquoAntiox-idative properties of xanthan on the autoxidation of soybeanoil in cyclodextrin emulsionrdquo Journal of Agricultural and FoodChemistry vol 40 no 6 pp 945ndash948 1992

[31] J Cole M H L Green S E James L Henderson and H ColeldquoA further assessment of factors influencing measurements ofthioguanine-resistant mutant frequency in circulating T-lymphocytesrdquo Mutation Research vol 204 no 3 pp 493ndash5071988

[32] C-Y Hsu C-M Yang C-M Chen P Chao and S Hu ldquoEff-ects of chlorophyll-related compounds on hydrogen peroxideinduced DNA damage within human lymphocytesrdquo Journal ofAgricultural and Food Chemistry vol 53 no 7 pp 2746ndash27502005

[33] S-C Tang H-F Lo K-H Lin et al ldquoThe antioxidant capacityof extracts from Taiwan indigenous purple-leaved vegetablesrdquoJournal of the Taiwan Society for Horticultural Science vol 59no 1 pp 43ndash57 2013

[34] K-H Lin Y-Y Yang H-F Lo et al ldquoAntioxidant activity ofherbaceous plant extracts protect against hydrogen peroxide-induced DNA damage in human lymphocytesrdquo BioMed CentralResearch Notes In press

[35] A H Cory T C Owen J A Barltrop and J G Cory ldquoUse of anaqueous soluble tetrazoliumformazan assay for cell growthassays in culturerdquoCancer Communications vol 3 no 7 pp 207ndash212 1991

[36] Y T Szeto A R Collins and I F F Benzie ldquoEffects of dietaryantioxidants on DNA damage in lysed cells using a modifiedcomet assay procedurerdquoMutation Research vol 500 no 1-2 pp31ndash38 2002

[37] S Vielma G Virella A J Gorod and M F Lopes-VirellaldquoChlamydophila pneumoniae infection of human aortic endo-thelial cells induces the expression of FC 120574 receptor II (Fc120574RII)rdquoClinical Immunology vol 104 no 3 pp 265ndash273 2002

[38] C Y Zhu and S Loft ldquoEffects of Brussels sprouts extracts onhydrogen peroxide-induced DNA strand breaks in humanlymphocytesrdquo Food and Chemical Toxicology vol 39 no 12 pp1191ndash1197 2001

[39] Y D Min C-H Choi H Bark et al ldquoQuercetin inhibits exp-ression of inflammatory cytokines through attenuation of NF-120581B and p38MAPK in HMC-1 humanmast cell linerdquo Inflamma-tion Research vol 56 no 5 pp 210ndash215 2007

[40] P Y Chao Y P Huang and W B Hsieh ldquoInhibitive effect ofpurple sweet potato leaf extract and its components on celladhesion and inflammatory response in human aortic endothe-lial cellsrdquo Cell Adhesion amp Migration vol 7 no 2 pp 1ndash9 2013

[41] Y Y YangThe antioxidative capacity in herb plant extracts andtheir protection role in DNA oxidative damage of lymphocyte[MS thesis] Chinese Culture University Taipei Taiwan 2004

[42] C A Rice-Evans N J Miller and G Paganga ldquoStructure-anti-oxidant activity relationships of flavonoids and phenolic acidsrdquo

14 Evidence-Based Complementary and Alternative Medicine

Free Radical Biology and Medicine vol 20 no 7 pp 933ndash9561996

[43] M Strube G R Haenen H van den Berg and A Bast ldquoPitfallsin a method for assessment of total antioxidant capacityrdquo FreeRadical Research vol 26 no 6 pp 515ndash521 1997

[44] P G Pietta ldquoFlavonoids as antioxidantsrdquo Journal of NaturalProducts vol 63 no 7 pp 1035ndash1042 2000

[45] A M Gonzalez-Paramas S Esteban-Ruano C Santos-BuelgaSDe Pascual-Teresa and J C Rivas-Gonzalo ldquoFlavanol contentand antioxidant activity in winery byproductsrdquo Journal ofAgricultural and Food Chemistry vol 52 no 2 pp 234ndash2382004

[46] S Y Kim J J Gao and H K Kang ldquoTwo flavonoids from theleaves ofMorus alba induce differentiation of the human prom-yelocytic leukemia (HL-60) cell linerdquo Biological and Pharma-ceutical Bulletin vol 23 no 4 pp 451ndash455 2000

[47] M A Khan A A Rahman S Islam et al ldquoA comparative studyon the antioxidant activity ofmethanolic extracts from differentparts of Morus alba L (Moraceae)rdquo BMC Research vol 6 pp24ndash32 2013

[48] A-L ChungH-F Lo K-H Lin et al ldquoStudy on the antioxidantactivity in herb plant extractsrdquo Journal of the Taiwan Society ForHorticultural Science vol 59 no 2 pp 139ndash152 2013

[49] R L Prior X Wu and K Schaich ldquoStandardized methods forthe determination of antioxidant capacity and phenolics infoods and dietary supplementsrdquo Journal of Agricultural andFood Chemistry vol 53 no 10 pp 4290ndash4302 2005

[50] S J Duthie A R Collins GGDuthie andV LDobson ldquoQue-rcetin and myricetin protect against hydrogen peroxide-indu-ced DNA damage (strand breaks and oxidised pyrimidines) inhuman lymphocytesrdquo Mutation Research vol 393 no 3 pp223ndash231 1997

[51] G A Liu and R L Zheng ldquoProtection against damagedDNA inthe single cell by polyphenolsrdquo Pharmazie vol 57 no 12 pp852ndash854 2002

[52] M Noroozi W J Angerson and M E J Lean ldquoEffects of flav-onoids and vitamin C on oxidative DNA damage to humanlymphocytesrdquoAmerican Journal of Clinical Nutrition vol 67 no6 pp 1210ndash1218 1998

[53] T Koga and M Meydani ldquoEffect of plasma metabolites of (+)-catechin and quercetin on monocyte adhesion to human aorticendothelial cellsrdquoAmerican Journal of Clinical Nutrition vol 73no 5 pp 941ndash948 2001

[54] CMeng P Somers L Hoong et al ldquoDiscovery of novel pheno-lic antioxidants as inhibitors of vascular cell adhesionmolecule-1 expression for use in chronic inflammatory diseasesrdquo Journalof Medicinal Chemistry vol 47 no 25 pp 6420ndash6432 2004

[55] Y Shibata N Kume H Arai et al ldquoMulberry leaf aqueousfractions inhibit TNF-120572-induced nuclear factor 120581B (NF-120581B)activation and lectin-like oxidized LDL receptor-1 (LOX-1)expression in vascular endothelial cellsrdquoAtherosclerosis vol 193no 1 pp 20ndash27 2007

[56] K C Chan H H Ho C N Huang M Lin H Chen and CWang ldquoMulberry leaf extract inhibits vascular smooth musclecell migration involving a block of small GTPase and AktNF-kB signalsrdquo Journal of Agricultural and Food Chemistry vol 57no 19 pp 9147ndash9153 2009

[57] F Chen N Nakashima I Kimura M Kimura N Asanoand S Koya ldquoPotentiating effects on pilocarpine-induced salivasecretion by extracts and N-containing sugars derived frommulberry leaves in streptozocin- diabetic micerdquo Biological andPharmaceutical Bulletin vol 18 no 12 pp 1676ndash1680 1995

[58] K Konno H Ono M Nakamura et al ldquoMulberry latexrich in antidiabetic sugar-mimic alkaloids forces dieting oncaterpillarsrdquo Proceedings of the National Academy of Sciences ofthe United States of America vol 103 no 5 pp 1337ndash1341 2006

[59] H J Kim K H Yoon M J Kang et al ldquoA six-month sup-plementation of mulberry Korean red ginseng and banabadecreases biomarkers of systemic low-grade inflammation insubjects with impaired glucose tolerance and type 2 diabetesrdquoEvidence-based Complementary and Alternative Medicine vol2012 Article ID 735191 8 pages 2012

[60] G Nicholson and G M Hall ldquoDiabetes mellitus new drugs fora new epidemicrdquo British Journal of Anaesthesia vol 107 no 1pp 65ndash73 2011

[61] P Y Chao Y L Chen Y C Lin et al ldquoEffects of black soybeanon atherogenic prevention in hypercholesterolemic rabbits andon adhesion molecular expression in cultured HAECsrdquo Foodand Nutrition Sciences vol 4 pp 9ndash21 2013

[62] C Chen S Li C-YOChen et al ldquoConstituents in purple sweetpotato leaves inhibit in vitro angiogenesis with opposite effectsex vivordquo Nutrition vol 27 no 11-12 pp 1177ndash1182 2011

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

2 Evidence-Based Complementary and Alternative Medicine

These protective effects are considered in large part to berelated to the various antioxidants contained in them Severalstudies have shown that polyphenolic and flavonol substancesare the most common compounds in herbs with strongantioxidant activity [14ndash16] The bioactive components ofherbaceous plants may be responsible for anticancer effectsthrough growth inhibition and apoptosis in human chronicmyeloid leukemia K562 cells [17]

Morus a genus of flowering plants in the familyMoraceaecomprises 10ndash16 species of deciduous trees commonly knownas mulberries that grow wild and under cultivation in manytemperate regions of the world Mulberries are widespreadand important crops for fruit timber and silkworm feedingas well as being excellent amenity trees Morus leaves arealso recognized as excellent animal food Mulberry leafextracts contain rutin isoquercetin and various derivativesof kaempferol and quercetin glycosides that can scavenge 11-diphenyl-2-picryl-hydrazyl (DPPH) radical [18] and inhibitthe formation of conjugated dienes and thiobarbituric acid-reacting substances (TBARS) by copper-induced oxidativemodification of rabbit and human low-density lipoproteins[19] In addition to antioxidant activity mulberry leaves andextracts have antimicrobial activity [20] induce apoptosis[18] are antiinflammatory and anti-hyperglycemic attenuateatherosclerotic lesions in animal models and lower bloodlipids in mild hyperlipidemic patients [21ndash24] Although avariety of medicinal herbs including mulberry are knownto be potent sources of polyphenols and flavonols studieson the protective effects of antioxidant activity in mulberryleaf-related extracts (MLREs) especially bombyx faces teaand mulberry leaf tea on DNA damage and cell adhesionare either scarce or little known The objectives of thisstudy were to isolate identify and evaluate the antioxidantcomponents antioxidant activity and extent to which acidicmethanolic hydrolysate and water extracts of mulberry leavescan protect DNA in human lymphocytes from oxidativedamage induced by H

2O2 The effects of MLREs including

bombyx faces tea (BT)mulberry leaf tea (MT) andmulberryleaf (M) methanolic extracts on endothelial cell-monocyteadhesion and the inflammatory response were assessed inhuman aortic endothelial cells (HAEC) Moreover the effectsof MLREs on intracellular redox-sensitive transcriptionalpathways such as NF-120581B AP-1 signal transducers andactivators of transcription (STAT)3 and PPAR 120572 and 120574which may contribute to leukocyte recruitment and vascularinflammation in atherogenesis were examined by westernblot analysis and electrophoretic mobility shift assays Ourstudy explores the relationship between the compositionand content of flavonols and polyphenols having antioxidantefficiency and the prevention of DNA oxidative damageafforded by mulberry leaf-related compounds The results ofour study may help determine the antioxidant activity andanti-inflammatory effects of MLREs

2 Materials and Methods

21 Mulberry Leaf-Related Compounds Extract PreparationMulberry leaf-related compounds (MLRC) were generously

provided by the owner ofQuan-Ming SilkwormFarm locatedin Miaoli Taiwan Harvested mulberry leaves were dividedinto eight individual batches and lyophilized by freeze dryingin a Freeze Dryer (FD-5060 Panchum Scientific CorpTaipei Taiwan) grinding to powder and storing at minus80∘Cuntil use Extracts were produced from 200mg aliquotsof MLRC powder with a 50-fold volume of methanol at4∘C extract overnight and then filtered through Whatmangrade no1 qualitative filter paper Extracts were concentratedby rotary vacuum evaporator (R205 Buchi Switzerland)resuspended in 995 ethanol to 1mL and then stored atminus20∘C until HAECs treatment

The edible portion of each MLRC leaf was weighedlyophilized ground to powder and extracted by distilleddeionized (dd)H

2O [25] The extraction mixture was heated

to 90∘C in a steam bath and refluxed for 2 h cooled inthe refrigerator sonicated for 5min and diluted to 50mLwith ddH

2O to form the final extract solution Individual

water extracts were used for the comet assay Ten mL of625 aqueous methanol containing BHT (2 gL) was addedto 125 g lyophilized samples followed by adding 5mL 6MHCl to bring the total volume up to 125mL The finalmixture consisted of 12M HCl in 50 aqueous methanolAcid hydrolysatesmethanolic extracts were analyzed for theirantioxidant composition antioxidant activity and the cometassay

22 Polyphenol Assay Polyphenol content was determinedaccording to the method of Taga et al [26] Briefly standardgallic acid and an aliquot of the acidic methanolic extractwere diluted with an ethanolwater (60 40 vv) solutioncontaining 03HCl TwomLof 2Na

2CO3weremixed into

each sample of 100 120583L and allowed to equilibrate for 2minbefore adding 50 Folin-Ciocalteu reagent Absorbance at750 nm was measured at room temperature The standardcurve for gallic acid was used to calculate polyphenol levels

23 Determination of Total Flavonoids Total flavonoid con-tents were determined using the method of Ordonez et al[27] A 05mL 2 AlCl

3-ethanol solution was added to

05mL of acidic methanolic extract and absorbance wasmeasured at 420 nm after standing for 1 h at room temper-ature Extract samples were evaluated at a final concentrationof 01mgmL Total flavonoid content was calculated as aquercetin equivalent (mgg)

24 Determination of Total Flavonols Total flavonols in plantextracts were estimated using the method of Kumaran andJoel Karunakaran [28] Two mL of 2 AlCl

3ethanol and

30mL (50 gL) sodium acetate were added to 20mL acidicmethanolic extracts The absorption at 440 nm was readafter 25 h at 20∘C Sample extracts were evaluated at a finalconcentration of 01mgmL Total flavonoid content wascalculated as a quercetin equivalent (mgg)

25 Flavonols Analysis by HPLC One mL of acid hydrolysa-tes methanolic extract was filtered through 045 120583m filterprior to 20 120583L injection into a high-performance liquid

Evidence-Based Complementary and Alternative Medicine 3

chromatograph (HPLC) Samples were analyzed with a Spec-traSystem UV6000LP Photodiode Array Detection System(Thermo Separation Products San Jose USA) and an ODScolumn (250 times 46mm 5120583m YMC ODS-A YMC CoKyoto Japan)Themobile phase consisted of methanol-water(30 70 vv) with 1 formic acid and 100 methanol Thegradient was 25ndash74 methanol in 40min at a flow rateof 075mLmin the spectrum was recorded at 365 nm forflavonol determination [25]

26 ABTSHRPndashH2O2Assay of Total Antioxidant Activity

The total antioxidant capacity of hydrophilic and lipophilicantioxidants was determined using the horseradish perox-idase catalyzed oxidation of 22-azino-bis-(3-ethylbenzot-hiazoline-6-sulfonic acid) (ABTS) [29]The reaction mixturecontained 05mL of 1000 120583M ABTS (in ddH

2O) and 35mL

of 100 120583M H2O2(in 01M PBS) The reaction was started

by the addition of 05mL of 44UmL peroxidase (in 01MPBS) After 1 h 005mL of MLREs was added to the mixtureAbsorbance was measured at 730 nm after 5min Trolox wasused as a standard and the total antioxidant capacity ofmulberry extracts was measured as mM Trolox equivalent

27 Scavenging Activity on 11-Diphenyl-2-picryl-hydrazyl(DPPH) Radical The scavenging activity onDPPH radical ofMLRC acidic methanolic extracts was determined accordingto the method of Shimada et al [30] Briefly an aliquot of04 mL of acidic methanolic extracts with series dilution wasadded to 08mLof 1mMDPPH freshly prepared inmethanolmixed well and left to stand for 30min before measuringabsorbance at 517 nm The scavenging effect percentage wascalculated as [1 minus (OD

517 nm)(control OD517 nm)] times 100 TheIC50of the scavenging effect percentage was then calculated

28 Isolated Human Peripheral Blood Lymphocytes Bloodsamples were obtained from six donors four male andtwo female healthy nonsmokers that were 24 to 48 yearsold Fresh whole blood (20ndash30mL) from volunteers wastaken with informed consent and lymphocytes were isolatedusing a separation solution kit supplemented with Ficoll-Paque Plus lymphocyte isolation sterile solution (PharmaciaBiotech Sweden) [31] Cells were harvested within 1 dayof blood samples being taken and cultured with AIM Vmedium containing serum-free lymphocyte medium (GibcoInvitrogen USA) in a humidified atmosphere of 5 CO

2in

air at 37∘C for 24 h

29 Dosage Selected for the Comet Assay Previously wedemonstrated that the effect of lymphocyte exposure to 10 120583MH2O2on DNA single-strand break damage was 60-fold

greater than the control and with 994 cell viability [32]However when lymphocytes were exposed to 50 120583M H

2O2

the oxidative damage increased significantly compared to thecontrol under 982 cell viability Therefore 10120583M H

2O2

for the treatment dosage was selected in the current studyFurthermore when lymphocytes were treated with threeconcentrations (25 50 and 100 120583gmL) of acidic methanolicand water extracts from indigenous purple vegetables [33]

and herbaceous plants [34] the DNA damage was inducedby exposing the lymphocytes to 10 120583M H

2O2and shown the

protection effects Consequently these concentrations of theextracts were chosen for the effect treatments

210 Cell-Viability Testing After culturing lymphocytes wereexposed to each MLREs Each lymphocyte solution was usedat three concentrations (25 50 and 100 120583gmL) for 30min at37∘C DNA damage was induced by exposing lymphocytes toH2O2(10 120583M) for 5min on ice Treatment on ice minimized

the possibility of cellular DNA repair after H2O2injury Cells

were centrifuged at 100 g for 10minwashed and resuspendedin the samemedium as the comet assay All experiments werecarried out in triplicate Cell viability was tested using thetetrazoliumformazan (MTT) assay [35] prior to and aftertreatment with MLRC extract or H

2O2

211 DNA Single Strand Break Damage Estimation Using theComet Assay The comet assay was performed as describedin Szeto et al [36] with acidic methanolic hydrolysate andwater extracts from tested plants Cultured lymphocytes(105 cellsmL) were embedded in 75120583L of 1 low-melting-point agarose on a microscope slide (precoated with agarose)at 37∘CThe gel was allowed to set at 4∘C and cells were lysedfor a period of at least 2 h in lysis buffer at 4∘C Cells werethen alkaline-unwound following which electrophoresis wascarried out using the electrophoresis buffer at 4∘C for 15minat 25 vDC and 300mA All steps were conducted underdim light to prevent additional DNA damage Followingelectrophoresis slides were dipped into a neutralizationbuffer and stained with ethidium bromide The comet-likeimages resulting from the extension of DNA were scored asa reflection of the single strand breaks under a fluorescencemicroscope (Zeiss-Axiovert 100 Zeiss Germany) Triplicateslides were prepared for each experimental point sampleand 50 comet-like images selected at random per slide todetermine average DNA damage values A computerizedimage analysis system (VisCOMET 16 Impuls GmbH Ger-many) was employed to analyze two comet parameters DNAdamage by tail DNA percentage [(total brightness of tailareatotal brightness of total area) times 100] and tail moment(tail length times tail DNA) The inhibition percentage of tailDNA and tail moment were calculated relative to the 10 120583MH2O2treated group

212 Cell Cultures and Treatment HAECs (Clonetics Corp)were grown in Medium 200 (GIBCO Invitrogen) supple-mented with 1 low serum growth supplement (LSGSGIBCO Invitrogen) and 10 FBS (GIBCO Invitrogen) inan atmosphere of 95 air and 5 CO

2at 37∘C in plastic

flasks in an incubator (Astec Co) as described by Vielmaet al [37] The human monocytic cell line U937 (Amer-ican Type Culture Collection) was grown in a suspensionculture in RPMI-1640 (GIBCO Invitrogen) containing 10FBS (Sigma) and 1 antibiotic-antimycotic mixture (Sigma)under an atmosphere of 95 air and 5 CO

2at 37∘C

After incubation with MLREs and aspirin or TNF-120572 cellviability was assessed using the tetrazoliumformazan (MTT)

4 Evidence-Based Complementary and Alternative Medicine

assay [35] The adhesion assays were performed as describedby Zhu and Loft [38] Briefly U937 cells were labeledwith a fluorescent dye incubated with 10 120583molL 27-bis (2-carboxyethyl)-5 (6)-carboxyfluorescein acetoxymethyl esterat 37∘C for 1 h in RPMI-1640 medium and subsequentlywashed by centrifugation ConfluentHAECs in 24-well plateswere incubated with U937 cells (106 cellsmL) at 37∘C for1 h Nonadherent leukocytes were removed The numbers ofadherent leukocytes were determined by photographing andcounting four randomly chosen fields per well at 100x usinga Zeiss Axio Mager Z1 Upright Fluorescence MicroscopeExperiments were performed in triplicate and repeated threetimes HAECswere incubatedwith 25 120583g and 50 120583g ofMLREsor 10 120583M aspirin for 18 h followed by treating with 2 ngmLTNF-120572 for 6 h in theNF-120581Bp65 AP-1 and STAT-3 expressionassays

213 Nuclear Protein Isolation Protein extracts were pre-pared as described by Min et al [39] Briefly after cellactivation for the indicated times cells were washed in 1mLice-cold PBS centrifuged at 400 g for 5min resuspendedin 400 120583L ice-cold hypotonic buffer (10mM HEPES 15mMMgCl

2 01mM EDTA 10mM KCl 1mM DTT 05mM

PMSF pH79) incubated on ice for 10min vortexed and cen-trifuged at 15000 g for 30 sec The supernatant was collectedand stored at minus70∘C for cytosolic protein analysis Pelletednuclei were gently resuspended in 445 120583L ice-cold extractionbuffer (20mM HEPES pH 79 with 15mM MgCl

2 042M

NaCl 02mM EDTA and 25 glycerol) with 5 120583L of 10mMDTT and 05 120583L of Protease Inhibitor Cocktail (Sigma) incu-bated on ice for 20min vortexed and centrifuged at 15000 gfor 5min at 4∘C Aliquots of the supernatant containingnuclear proteins were frozen in liquid nitrogen and stored atminus70∘C

214 Western Blot Analysis Nuclear lysates were subjected to12 SDS-PAGE (Bio-Rad Laboratories) after which proteinswere transferred to a PVDF membrane (Bio-Rad) Mem-branes were probed with a mouse monoclonal NF-120581B p65antibody (BD Biosciences) After incubation in a secondaryantibody solution consisting of IRDye 700CW-conjugatedgoat anti-mouse IgG (LI-COR Biosciences) for 60min atroom temperature with gentle shaking the membrane waswashed four times for 5min each at room temperature in PBSwith 01 Tween-20 and while being gently shaken After afinal rinse the membrane was scanned using AlphaEaseFCimage analysis software (Alpha Innotech Corporation) toanalyze spot density The internal control was set at 100 todetermine the relative density of protein expression [40]

215 Electrophoretic Mobility Shift Assay for NF-120581B AP-1 andSTAT3 The binding reaction consisted of 1120583L 10times bindingbuffer (100mM TRIS 500mM NaCl 10mM DTT pH 75)5 120583LH

2O 2120583L 25mMDTT25Tween-20 1 120583L IRDye 700-

labeled EMSA oligonucleotides specific for NF-120581B (51015840-AGTTGA GGG GAC TTT CCC AGG C-31015840 and 31015840-CGC TTGATG ACT CAG CCG GAA-51015840) AP-1 (51015840-CGC TTG ATGACT CAG CCG GAA-31015840 and 31015840-GCG AAC TAC TGA GTC

GGC CTT-51015840) STAT3 (51015840-GAT CCT TCT GGG AAT TCCTAG ATC-31015840 and 31015840-CTA GGA AGA CCC TTA AGG ATCTAG-51015840) (Sigma) PPAR-120572 (51015840-AAA AAC TGG GCC AAAGGT CT-31015840) and PPAR-120574 (51015840-TGA AAC TAG GGT AAAGTT CA-31015840) (Sigma) 1 120583L poly (dIsdotdC) and 1 120583L nuclearextract (as prepared previously) The mixture was incubatedat room temperature for 20min in the dark After 1x OrangeLoading Dye (LI-COR Biosciences) was added the bindingreaction was loaded onto a native 4 polyacrylamide gel andseparated by electrophoresis at 90 vDC for 40min The gelswere scanned using anOdyssey Infrared Imaging System (LI-COR Biosciences) [40]

216 Statistical Analysis Data are expressed as the mean plusmnstandard deviation and statistical significance was analyzedusing one-way ANOVA followed by the Tukeyrsquos Range Testat the 005 significance level Pearsonrsquos linear correlation wasalso determined The means of three replicates are reported

3 Result

31 Antioxidant Composition and Antioxidant Activity Poly-phenols flavonoids and flavonols were abundant in BTat levels of 3465 plusmn 082mgg DW gallic acid 8483 plusmn868mgg DW quercetin equivalent and 2547 plusmn 386mggDW quercetin equivalent respectively (Table 1) Anthocy-anidins were abundant in BT and ML at levels of 14332 plusmn181 and 13582 plusmn 031 unitsgDW respectively Table 2 showsthat quercetin and kaempferol were both rich in BTat levelsof 285333 plusmn 18037 and 64667 plusmn 5033 120583ggDW respectivelywhile the levels of M were 166667 plusmn 18903 and 42667 plusmn4619 120583ggDW respectively However no myricetin or morinwas detected in the MLRC (Table 2) Thus these MLREsdisplayed variations in their antioxidant substances levels

MLREs showed antioxidant activity proving their capac-ity to scavenge the ABTS radical cation The antioxidantactivity in acidic methanolic hydrolysate sample extracts wasexpressed in trolox equivalent antioxidant capacity (TEAC)(Table 3) BT showed a significantly higher TEAC value(15514 plusmn 290 120583ggDW) than MT (10239 plusmn 095 120583ggDW)and M (7654 plusmn 161 120583ggDW) TEAC values were positivelyand significantly (119903 = 041 119875 = 0034) correlated withthe content of quercetin among MLREs (Table 4) Thusdifferent quercetin contents displayed various levels of anti-oxidant activity However M had a significantly high effi-cacy for DPPH radical scavenging activity (IC

5018883 plusmn

361 120583gmL) compared to BT (IC5066901plusmn723 120583gmL) and

MT (IC5083640 plusmn 708 120583gmL) (Table 3) Hence each tested

sample showed significant differences in scavenging of DPPHradical at IC

50

32 Effects of Acidic Methanolic and Water Extracts fromMLRC on H

2O2-Induced DNA Damage to Lymphocytes The

effects ofMLRC extracts on cell cytotoxicity were determinedby theMTTassay Lymphocyteswere exposed to each of threedifferent MLRC extracts at three concentrations (25 50 and100 120583gmL) for 30min at 37∘C DNA damage was inducedby exposing lymphocytes to H

2O2(10 120583M) for 5min on ice

Evidence-Based Complementary and Alternative Medicine 5

Table 1 The contents of various antioxidant substances in MLRC

Polyphenolmg gallic acidgDW

Flavonoids mg quercetinequivalentgDW

Flavonol mg quercetinequivalengDW

AthocyanidinunitgDW

BT 3465 plusmn 082a

8483 plusmn 868a

2547 plusmn 386a

14332 plusmn 181a

MT 2323 plusmn 054b

5599 plusmn 1055b

1326 plusmn 266b

8858 plusmn 412b

M 1987 plusmn 061c

4960 plusmn 541b

1422 plusmn 148b

13582 plusmn 031a

All values are means plusmn SD (119899 = 3)Means within a column with different superscripts (asimc) are significantly different 119875 lt 005

Table 2 The contents of flavonols in acidic methanolic extracts ofMLRC

Flavonols (120583ggDW)Samples Myricetin Morin Quercetin KaempferolBT ND ND 285333 plusmn 18037

a64667 plusmn 5033

a

MT ND ND 37333 plusmn 2309c ND

M ND ND 166667 plusmn 18903b42667 plusmn 4619

b

ND not detected

Table 3 TEAC values and the IC50 of DPPH radical scavengingactivity of acidic methanolic hydrolysates of MLRC

Sample TEAC(Trolox mM)lowast

DPPH radical scavenging activityIC50 (120583gmL)

BT 15514 plusmn 290a

66901 plusmn 723b

MT 10239 plusmn 095b

83640 plusmn 708a

M 7654 plusmn 161c

18883 plusmn 361c

lowastTEAC value at sample 100120583gmL concentration

Table 4 Correlation between antioxidant substances and TEACvalues treated with acid hydrolysates of MLRC

FlavonolsMyricetin Morin Quercetin Kaempferol

TEAClowast mdash mdash 119903 = 041

119875 = 0034

mdash

mdash no significant correlationlowastTEAC values at 100 120583gmL of sample concentration

No mulberry extracts were cytotoxic at the concentrationsused with gt98 of cells remaining viable [41] The cometassay was performed to determine the DNA damagingactivity of the plants because it is a sensitive method formonitoring single strand DNA breaks at the single celllevel Any DNA damage that occurred is represented as atail length (tail migration) of the DNA strand The effectsof pretreatment with acidic methanolic extracts of MLRCon 10 120583M H

2O2-induced DNA oxidative damage in human

lymphocytes are presented in Figure 1 indicating that therewas a protective effect of pretreatment on lymphocytes witheach of the MLRC extracts at the lowest dose (25 120583gmL)Lymphocytes pretreated with 25ndash100120583gmL extracts of MTextracts experienced a greater level of protection againstH2O2exposure than lymphocytes exposed to other tested

compounds and in a dose-dependent manner BT had betterinhibition efficacy at lower concentration (25120583gmL) andthe maximum protective effect of lymphocyte pretreatmentwas observed with pretreatment by 25 120583gmL BT exhibiting

1462 of tail DNA compared to treatment only withH2O2

MT had better inhibition efficacy (8439) than M and BTextracts at the 100 120583gmL level (Figure 1(a)) Tested plantsshowed at least 134263 of tail moment in BT extract at the25 120583gmL level while the BT extract at 100 120583gmL had thehighest tail moment (328193) compared to the rest of theacidic methanolic extract samples (Figure 1(b)) Moreoverthe trend in tail moment in all samples continued climbingup from 25ndash100 120583gmL

The effects of various water extracts of MLRC H2O2-

induced DNA damage to lymphocytes are presented inFigure 2 At lower concentrations all tested samples hadlower tail DNA indicating better inhibition efficacies(Figure 2(a)) BT and M had the best inhibition efficacieswith both exhibiting values of 1422 and 1469 tail DNAat 25 120583gmL respectively Moreover MT had a higher tailDNA than the other extracts at the same dose and thehighest tail DNA was observed in 3212 of MT at aconcentration of 100120583gmL The tail moment of all samplescontinued to increase up to the 100 120583gmL level (Figure 2(b))The minimum tail moment occurred in M at 153674 in the25 120583gmL extracts Moreover the DNA damage induced byH2O2was significantly higher than the DNA damage treated

with extracts and H2O2treated had 8726 in tail DNA and

832884 in tail moment

33 Effects of MLREs on Monocyte-Endothelial Cell Adhe-sion As shown in Figure 3 pretreatment of HAECs with100 120583gmLMLREs for 18 h significantly suppressed the adhe-sion ofU937monocytes to TNF-120572-stimulatedHAECs similarto that of 10 120583Maspirin (positive control) Data also show that25 and 50 120583gmL BT MT and M methanol extracts inhib-ited the adhesion of U937 monocytes to TNF-120572-stimulatedHAECs by 8198 and 8187 (BT) 7449 and 8331 (MT)and 8011 and 7559 (M) respectively

34 Effects of MLREs on NF-120581B Activity and MLREs Inhibitsthe Binding of NF-120581B AP-1 and STAT3 Figure 4 demon-strates that MLREs regulated the DNA-binding activity andexpressions of NF-120581B in HAECs In untreated HAECs NF-120581B p65 was only found within the cytosol however itsnuclear translocation was observed upon treatment withTNF-120572 Compared to the TNF-120572 group pretreatment withBT and MT methanol extracts significantly decreased theexpression of NF-120581B p65 in the nuclear compartment whilethe M methanol extract did not reduce the expressionof NF-120581B p65 activity Furthermore an EMSA analysiswas carried out to determine if the reduced expression of

6 Evidence-Based Complementary and Alternative Medicine

Control H2O2

BT25 BT50 BT100

MT25 MT50 MT100

M25 M50 M100

(a)

0

20

40

60

80

100

25 50 100

Tail

DN

A (

)

Mulberry extracts (120583gmL)

lowast lowastlowast

lowast

lowastlowast

alowast alowast

blowast

MTMBT

H2O2

(b)

Tail

mom

ent

0

2000

4000

6000

8000

10000

25 50 100Mulberry extracts (120583gmL)

lowastlowastlowast

ablowastclowast

alowast

alowast

alowastblowast

MTMBT

H2O2

(c)

Figure 1 Effects of various acidic methanolic extracts of MLRC on H2O2-induced DNA damage to lymphocytes The comet-like images

resulting from the extension of DNA (a) were observed using a fluorescence microscope Tail DNA (b) and tail movement (c) were exposedto test mulberry leaf-related extracts at 25 50 and 100 120583gmL and treated with H

2O2at 10120583M Values with different letters differ significantly

with regard to oxidative damage when comparing different MLRC extracts lowast119875 lt 005 refers to differences in oxidative damage as comparedwith 10 120583MH

2O2-alone treatment

Evidence-Based Complementary and Alternative Medicine 7

Control H2O2

BT25 BT50 BT100

MT25 MT50 MT100

M25 M50 M100

(a)

alowastblowast blowastblowast

blowastblowast

blowast

25 50 100Mulberry extracts (120583gmL)

MTMBT

H2O2

0

20

40

60

80

100

Tail

DN

A (

)

ablowastablowast

(b)

Tail

mom

ent

0

2000

4000

6000

8000

10000

lowastlowast lowast

alowastalowast

25 50 100Mulberry extracts (120583gmL)

MTMBT

H2O2

ablowast ablowastablowast

bclowast

(c)

Figure 2 Effects of various water extracts of MLRC on H2O2-induced DNA damage to lymphocytes The comet-like images resulting from

the extension ofDNA (a) were observed using a fluorescencemicroscope Tail DNA (b) and tailmovement (c) were exposed to testmulberryleaf-related extractsat 25 50 and 100120583gmL and treated with H

2O2at 10120583M Values with different letters differ significantly with regard to

oxidative damage when comparing different MLRC extracts lowast119875 lt 005 refers to differences in oxidative damage as compared with 10120583MH2O2-alone treatment

NF-120581B in response to treatment with MLREs resulted inreduced binding of NF-120581B to DNAThe treatment of HAECswith TNF-120572 led to increased binding of NF-120581B howeverpretreatment of HAECs with BT MT and M significantlydecreased DNA-bound NF-120581B (Figure 5(a)) The increased

binding of AP-1 with TNF-120572 resulted from the treatment ofHAECs however pretreatment of HAECs with BT MT andM significantly decreased DNA-bound AP-1 (Figure 5(b))STAT3 DNA-binding activities to its cognate recognitionsite were remarkably reduced compared to TNF-120572 treatment

8 Evidence-Based Complementary and Alternative Medicine

(A)(A)

(B)

(C) (D)

(E) (F)

(I)

(G) (H)

200120583m 200120583m

200120583m200120583m

200120583m200120583m

200120583m200120583m

200120583m

(a)

Con

trol

Asp

irin

BT25

BT50

MT2

5

MT5

0

M25

M50

Mon

ocyt

e adh

eren

ce (c

ells

HPF

)

0

20

40

60

80

100

120

140

160

180

lowast lowast lowast

lowast

lowast

lowast

lowast

TNF-120572

(b)

Figure 3 Mulberry leaf related extracts (MLREs) reduce tumor necrosis factor (TNF)-120572-induced adhesion of monocytes to human aorticendothelial cells (HAECs) (a) The number of adherent U937 cells (marked by arrow) was monitored by fluorescence microscopy undercontrol (a) 2 ngmL TNF-120572 (b) co-treated with TNF-119886 and aspirin (10120583M) (c) cotreated with TNF-120572 and BT (25 120583gmL) (d) cotreated withTNF-120572 and BT (50 120583gmL) (e) cotreated with TNF-120572 and MT (25 120583gmL) (f) cotreated with TNF-120572 and MT (50 120583gmL) (g) cotreated withTNF-120572 and M (25120583gmL) (h) and cotreated with TNF-120572 and M (50120583gmL) (i) (b) HAECs were pretreated with MLREs for 18 h followedby stimulating with TNF-120572 for 6 h Fluorescence-labeled monocytic U937 cells were added to HAECs and allowed to adhere for 2 h Data areexpressed as a percentage of TNF-120572-induced expression (mean plusmn SD) of three experiments Mean plusmn SD lowast119875 lt 005 BT MT and M at 25and 50120583gmL versus TNF-120572

119875 lt 005 control versus TNF-120572

Evidence-Based Complementary and Alternative Medicine 9

Control TNF-120572

TNF-120572

Aspirin10120583M

+ + + + + + + +

BT25 BT50 MT25 MT50 M25 M50

120583gmL 120583gmL 120583gmL 120583gmL 120583gmL 120583gmL

NF-120581B p65

120573-Actin43kDa

minus

65kDa

(a)

Con

trol

Asp

irin

BT25

BT50

MT2

5

MT5

0

M25

M50

00

02

04

06

08

10

lowast

lowast

lowast

lowastlowastlowastTN

F-120572

Ratio

(nuc

lear

120573-a

ctin

)

(b)

Figure 4 Effects of MLREs on NF-120581B activity After HAECs were pretreated with the indicated samples and incubated with TNF-120572 nuclearextracts were prepared and the expression of NF-120581B p65 was assessed by western blot analysis A representative image of three similar resultsis shown (a) Actin served as the loading control for the nuclear compartment Semiquantitative analysis of three independent experimentsare also shown (b) indicates a significant difference between the TNF-120572 treated or experimental treatment groups and the control119875 lt 005lowast indicates a significant difference between the TNF-120572 and experimental treatment groups BT MT and M at 25 and 50 120583gmL

(lane 3) in the nuclear fraction indicating that MLREs at50120583gmL inhibited STAT3-binding activities In addition theresults in Figure 5(c) (lanes 4ndash10) show that aspirinMLREs at50120583gmL and BT at 25120583gmL remarkably decreased STAT3levels compared to TNF-120572 (lane 3) however both MT andM at 25120583gmL increased STAT3-binding activities Theseresults indicate that only 50 120583gmL MLREs 25 120583gmL BTand aspirin block STAT3 activation and might downregulateTNF-120572-induced expressions of inflammatory signaling path-ways

35 MLREs Regulated the DNA-Binding Activity and Expres-sions of PPAR 120572 and PPAR 120574 in HAECs PPAR 120572 and 120574effectively bound to PPAR 120572 and 120574 oligo-IRDye 700 asindicated in lanes 3ndash10 of Figure 6 Supershifts are bands fromthe binding of each specific antibody The binding activitiesof PPAR 120572 and 120574 were detected at baseline The 25 120583gmLMT extract (lane 7) and 25 and 50 120583gmL M extracts (lanes9-10) increased activity compared to incubating with TNF-120572 alone (lane 3) However BT at both 25 and 50 120583gmLextracts showed reduced effects on PPAR 120572 and 120574 expressionMoreover the 50 120583gmLMT extract had no effect on PPAR 120572binding activity but increased the binding activity of PPAR120574 in comparison to incubation with TNF-120572 alone Thesedata suggest that MT and M both promoted PPAR 120572 and 120574

expression at least in part by increasing the DNA binding oftranscription factors PPAR 120572 and 120574

4 Discussion

41 Antioxidant Composition and Antioxidant Activity BTcontains high levels of polyphenols flavonoids flavonolsanthocyanidin quercetin and kaempferol (Tables 1 and 2)BT also had a higher TEAC value (15514mM Table 3) thanthe TEAC values of 47mM [42] and 64mM [43] reportedfor quercetin as well as 242mM reported for quercetin-3-rutinoside [44] Furthermore Gonzalez-Paramas et al [45]demonstrated that flavonol contents were significantly andhighly correlated (119903 = 08) with TEAC values Kim et al [46]reported that mulberry leaves contain abundant quercetinand inhibit lipid peroxidation at concentrations of 01 and001mgmL In our study the amount of quercetin wassignificantly correlated (119903 = 041 Table 4) with TEAC valueHowever no correlation was found among myricetin morinkaempferol and TEAC which may be due to the structurerequired to reinforce the free radical scavenging activity thatvaries with the type of free radical Naowaratwattana et al[18] reported that a 50 methanolic extract of mulberryleaf contained rutin isoquercetin and various derivatives ofkaempferol and quercetin glycosides while a water extract

10 Evidence-Based Complementary and Alternative Medicine

NF-120581BTNF-120572

TNF-120572

+

Aspirin10120583M+ +

BT25120583gmL

+

BT50120583gmL

+

MT25120583gmL

+

MT50120583gmL

+

M25

120583gmL+

M50

120583gmLControl

minus

DNAOligo

Free probe

(a)

AP-1TNF-120572

TNF-120572

+

Aspirin10120583M+ +

BT25120583gmL

+

BT50120583gmL

+

MT25120583gmL

+

MT50120583gmL

+

M25

120583gmL+

M50

120583gmLControl

minus

DNAOligo

Free probe

(b)

STAT3

TNF-120572

TNF-120572

+

Aspirin10120583M+ +

BT25120583gmL

+

BT50120583gmL

+

MT25120583gmL

+

MT50120583gmL

+

M25

120583gmL+

M50

120583gmLControl

minus

DNAOligo

Free probe

(c)

Figure 5 MLREs inhibits the binding of (a) NF-120581B (b) AP-1 and (c) STAT3 to target DNA sequence NF-120581B AP-1 and STAT3 transcriptionfactor-DNA interactions were assessed by EMSA

contained primarily chlorogenic acid and caffeoylquinic acidderivatives The IC

50of DPPH radical scavenging was 794

and 2042120583gmL for 50 methanolic and water extractsrespectively [18] In our studyM extracts contained quercetinand kaempferol (Table 2) which may account for part ofthe highly scavenged DPPH radical in comparison to therest of MLREs Khan et al [47] demonstrated that the IC

50

was 22023120583gmL which is much less efficacious than themulberry leaf acidic methanolic extract IC

50of 1888 120583gmL

(Table 3) However the IC50

of in vitro free radical scav-enging activity of mulberry leaves from Naowaboot et al[22] was 617 plusmn 21 120583gmL which is more efficacious thanthe IC

50concentration derived in the present study Taken

together the polyphenol-rich MLREs exhibited distinct cell-free antioxidant activity (ie TEAC) as validated by theirlevels of polyphenols and flavonols with distinct antioxidantactivity strongly accounting for the antioxidant activity of the

extracts The amount of quercetin was especially positivelycorrelated with the observed TEAC values

42 Estimation of DNA Single Strand Break Damage fromExposure to Acidic Methanolic and Water Extracts One ofthe antioxidant activity tests scavenging of DPPH radi-cal was selected due to its quick and simple but it is aless biologically relevant assay Previously we reported thatwith 2 120583gsdotmLminus1 of methanolic and water extracts from theindigenous vegetables [33] and herb plants [48] significantlyprolonged lag phase of low-density lipoprotein (LDL) TheLDL oxidation assay is considered to be one of the mostbiologically relevant assays [49] Both studies [33 48] alsoshowed that the concentration of the tested extracts forscavenging DPPH radical was in higher levels at 10mg oftested extracts with 2ndash53 inhibition Lower concentrationwas then chosen for the treated dosage in the present study

Evidence-Based Complementary and Alternative Medicine 11

TNF-120572

TNF-120572

+

Aspirin10120583M+ +

BT25120583gmL

+

BT50120583gmL

+

MT25120583gmL

+

MT50120583gmL

+

M25

120583gmL+

M50

120583gmLControl

minus

DNAOligo

Free probe

PPAR 120572

(a)

TNF-120572

TNF-120572

+

Aspirin10120583M+ +

BT25120583gmL

+

BT50120583gmL

+

MT25120583gmL

+

MT50120583gmL

+

M25

120583gmL+

M50

120583gmLControl

minus

DNAOligo

Free probe

PPAR120574

(b)

Figure 6 Effect of treatments on the expression of PPAR 120572 (a) and PPAR 120574 (b) in TNF-120572-stimulated HAECs by EMSA

In addition lower concentration also could avoid the toxicityeffect of higher level of extracts on lymphocytes Quercetinwas found to protect against H

2O2-induced DNA damage in

human lymphocytes at 10 120583M [50] and at 31 to 25 120583M [51]Quercetin was also reported to induce DNA strand breaks invarious cell types but only at higher doses at 100 120583Mor above[50] Duthie et al [50] proposed that the dihydroxy structureof quercetin and myricetin might be essential in protectingDNA against hydrogen peroxide No such hydroxyl groupsare present in the tocopherol molecule Noroozi et al [52]demonstrated that in addition to quercetin kaempferolcould also inhibit H

2O2-induced DNA strand breaks in

human lymphocytes In our study both BT and M were richin quercetin and kaempferol (Table 2) whichmay account forthe protective roles of both extracts in preventingDNAstrandbreaks from human lymphocytes

43 MLREs Regulated the DNA-Binding Activity and Expres-sions of NF-120581B AP-1 STAT3 and PPARs in HAECsBecause MLREs exhibited free radical scavenging the anti-inflammatory effects of MLREs on TNF-120572-induced celladhesion and adhesionmolecule expressionwere determinedin the present study Our results suggest that MLREs andaspirin significantly inhibited TNF-120572-induced monocyte-endothelial cell adhesion (Figure 3) Significant reductionsin NF-120581B expression and DNA binding by aspirin MTextract (25 120583gmL) and BT extract (both 25 and 50 120583gmL)were also observed The adhesion of monocytes to the

vascular endothelium and subsequent migration into thevessel wall are early events in atherogenesis [2] Our resultsare consistent with previous research showing that dietarypolyphenols such as catechin and quercetin significantlyreduce the binding of monocytes to HAECs [53 54] Thereduced cellular adhesion may be due to the inhibitionof cellular adhesion molecule expression as purple sweetpotato leaf extract (PSPLE) reduced both VCAM-1 andICAM-1 expression [40] Shibata et al [55] demonstratedthat pretreatment of cultured bovine aortic endothelial cells(BAECs) with mulberry leaf aqueous fractions inhibitedTNF-120572- and LPS-induced expression of Lectin-like Ox-LDLreceptor-1 (LOX-1) a cell-surface receptor for atherogenicOx-LDL (oxidized low-density lipoprotein) It also appearsto mediate Ox-LDL-induced inflammation which may becrucial in atherogenesis Furthermore mulberry leaf aqueousfractions inhibited the TNF-120572-induced activation of NF-120581Band phosphorylation of inhibitory factor of NF-120581B-alpha(I120581B-120572) Thus mulberry leaf aqueous fractions suppressTNF-120572- and LPS-induced LOX-1 gene expression mediatedby inhibiting NF-120581B activation

Chan et al [56] demonstrated that mulberry leaf extract(MLE) was rich in polyphenols MLE can effectively inhibitthe migration of vascular smooth muscle cells (VSMC) byblocking small guanosine triphosphatases (small GTPase)and AktNF-120581B signals The Rho family of small GTPasessuch as Rho Rac and Cdc42 acts as a molecular switch toregulate the actin cytoskeleton The Rho family of GTPases

12 Evidence-Based Complementary and Alternative Medicine

is also reported to be involved in the regulation of NF-120581B-dependent transcription [56] MLE has been shown to pos-sess hypoglycemic effects in an insulin-dependent diabetesmellitus (type 1 diabetes) animal model and potential hypo-glycemic compounds inmulberry leaves were suggested to be2-O-120572-D-galactopyranosyl-DNJ and fagomine [57] Konnoet al [58] further reported that mulberry latex containsalkaloidal sugar-mimic glycosidase inhibitors with antidia-betic activities such as 14-dideoxy-14-imino-D-arabinitol 1-deoxynojirimycin and 14-dideoxy-14-imino-D-ribitol Kimet al [59] recently showed that treated type 2 diabetesmellitus(T2D) with a 1 1 1 mixture of ginseng root mulberry leafwater extract and banana leaf water extract (6 gd) for 24weeks significantly decreased plasma intracellular adhesionmolecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) levels This indicates suppressed inflammatoryresponses in T2D but no significant differences in glucosehomeostasis control measurement changes Rigamonti et al[8] reported that bothPPAR120572 and 120574maybe thought of as sup-pressing the activity of NF120581B and AP-1 pathways that reduceinflammatory mediator production in the macrophages ofhumans and mice In our study MT and M promoted PPAR120572 and 120574 expression at least in part by increasing DNAbinding of transcription factors PPAR 120572 and 120574 PPAR-120572-and 120574-dependent signaling pathwaysmay also play importantroles in mediating the TNF-120572 receptor signaling pathwayby MT and M by suppressing the activity on NF-120581B andAP-1 pathways These pathways may regulate downstreamproinflammatory gene expression and adhesion molecularexpression Clinically PPAR 120574 agonists such as rosiglitazoneare widely used in the treatment of T2D [60] M and MTextracts increased PPAR 120574 activity which may partly explainwhy inflammatory responses are suppressed in T2D

Although reduced NF-120581B expression and DNA bind-ing were observed upon treatment with ML MT and BT(Figure 5) the mechanism was not assessed and is worthy offurther study In addition the present results were obtainedusing in vitro studies and may be consistent with an invivo model of atherogenesis similar to that reported byEnkhmaa et al [21] in which mulberry leaves suppressedthe development of atherosclerotic lesions Previously weproved that black beans significantly prolong LDL lag timeand decrease the atheroma regions of the aortic arch andthoracic aorta in hypercholesterolemic NZW rabbits [61]Wethen demonstrated that black soybean extract and PSPLEsignificantly decreased the adhesion of U937 monocyticcells to TNF-120572-stimulated HAECs and reduced adhesionmolecules expression (VCAM-1 and ICAM-1) as well as NF-120581B-p65 expression [37] This is particularly important giventhe opposing effects of flavonols fromMLREs obtained usingin vitro and ex vivo studies [62]

5 Conclusion

Tested MLREs were rich in flavonols and had strongantioxidant activity in protecting lymphocyte DNA fromoxidative damage In particular BT was rich in quercetinand polyphenols The antioxidant components of MLREs

might downregulate intracellular redox-dependent signalingpathways in HAECs upon TNF-120572 stimulation which mightprevent ROS-mediated endothelial cell dysfunction MLRCshave anti-inflammatory effects through the modulation ofNF-120581B AP-1 STAT3 and PPARs signaling These chemi-cals appear to provide distinct cytoprotective mechanismsthat might contribute to their putative beneficial effectsin suppressing endothelial responses to cytokines duringinflammation These results may benefit further in vivostudies to assess the therapeutic potential of phytochemicalsas anti-inflammatories for use in cytokine-induced vasculardisorders including atherosclerosis

Acknowledgment

This work was supported in part by Grants from the NationalScience Council of Taiwan (NSC 95ndash2320-B-034-001 andNSC 96ndash2320-B-034-001)

References

[1] W Palinski ldquoUnited they go conjunct regulation of aorticantioxidant enzymes during atherogenesisrdquo CirculationResearch vol 93 no 3 pp 183ndash185 2003

[2] P Libby ldquoMolecular bases of the acute coronary syndromesrdquoCirculation vol 91 no 11 pp 2844ndash2850 1995

[3] P A Baeuerle and D Baltimore ldquoNf-120581B ten years afterrdquo Cellvol 87 no 1 pp 13ndash20 1996

[4] J M Kyriakis ldquoActivation of the AP-1 transcription factor byinflammatory cytokines of the TNF familyrdquo Gene Expressionvol 7 no 4ndash6 pp 217ndash231 1999

[5] J M Muller R A Rupec and P A Baeuerle ldquoStudy of generegulation by NF-120581B and AP-1 in response to reactive oxygenintermediatesrdquoMethods vol 11 no 3 pp 301ndash312 1997

[6] G Chinetti J Fruchart and B Staels ldquoPeroxisome proliferator-activated receptors new targets for the pharmacological mod-ulation of macrophage gene expression and functionrdquo CurrentOpinion in Lipidology vol 14 no 5 pp 459ndash468 2003

[7] G Chinetti J C Fruchart and B Staels ldquoTranscriptional regu-lation of macrophage cholesterol trafficking by PPAR120572 andLXRrdquo Biochemical Society Transactions vol 34 no 6 pp 1128ndash1131 2006

[8] E Rigamonti G Chinetti-Gbaguidi and B Staels ldquoRegulationof macrophage functions by PPAR- 120572 PPAR- 120574 and LXRsin mice and menrdquo Arteriosclerosis Thrombosis and VascularBiology vol 28 no 6 pp 1050ndash1059 2008

[9] M A Bouhlel J Brozek B Derudas et al ldquoUnlike PPAR120574PPAR120572 or PPAR120573120575 activation does not promote humanmono-cyte differentiation toward alternative macrophagesrdquo Biochem-ical and Biophysical Research Communications vol 386 no 3pp 459ndash462 2009

[10] G Chinetti J-C Fruchart and B Staels ldquoPeroxisome prolife-rator-activated receptors and inflammation from basic scienceto clinical applicationsrdquo International Journal of Obesity vol 27supplement 3 pp S41ndashS45 2003

[11] Y Zhu J H-C Lin H Liao et al ldquoLDL induces transcriptionfactor activator protein-1 in human endothelial cellsrdquo Arte-riosclerosis Thrombosis and Vascular Biology vol 18 no 3 pp473ndash480 1998

Evidence-Based Complementary and Alternative Medicine 13

[12] J Terao ldquoDietary flavonoids as antioxidantsrdquo Forum of Nutri-tion vol 61 pp 87ndash94 2009

[13] Y B Feng W Q Luo and S Q Zhu ldquoExplore new clinicalapplication of Huanglian and corresponding compound pre-scriptions from their traditional userdquo Zhongguo ZhongyaoZazhi vol 33 no 10 pp 1221ndash1225 2008

[14] Y H Chu C L Chang and H F Hsu ldquoFlavonoid content ofseveral vegetables and their antioxidant activityrdquo Journal of theScience of Food andAgriculture vol 80 no 5 pp 561ndash566 2000

[15] I C Arts B van de Putte and P C Hollman ldquoCatechin con-tents of foods commonly consumed in The Netherlands 1Fruits vegetables staple foods and processed foodsrdquo Journal ofAgricultural and Food Chemistry vol 48 no 5 pp 1746ndash17512000

[16] A R Proteggente A S Pannala G Paganga et al ldquoThe anti-oxidant activity of regularly consumed fruit and vegetablesreflects their phenolic and vitamin C compositionrdquo Free RadicalResearch vol 36 no 2 pp 217ndash233 2002

[17] Y L Liu L H Tang Z Q Liang B You and S Yang ldquoGrowthinhibitory and apoptosis inducing by effects of total flavonoidsfrom Lysimachia clethroides Duby in human chronic myeloidleukemiaK562 cellsrdquo Journal of Ethnopharmacology vol 131 no1 pp 1ndash9 2010

[18] W Naowaratwattana W De-Eknamkul and E G De MejialdquoPhenolic-containing organic extracts of mulberry (Morus albaL) leaves inhibit HepG2 hepatoma cells through G2M phasearrest and inhibition of topoisomerase II120572 activityrdquo Journal ofMedicinal Food vol 13 no 5 pp 1045ndash1056 2010

[19] K Doi T Kojima and Y Fujimoto ldquoMulberry leaf extract inhi-bits the oxidative modification of rabbit and human low densitylipoproteinrdquo Biological and Pharmaceutical Bulletin vol 23 no9 pp 1066ndash1071 2000

[20] W Wang Y Zu Y Fu and T Efferth ldquoIn Vitro antioxidant andantimicrobial activity of extracts from Morus alba L leavesstems and fruitsrdquoAmerican Journal of ChineseMedicine vol 40no 2 pp 349ndash356 2012

[21] B Enkhmaa K Shiwaku T Katsube et al ldquoMulberry (Morusalba L) leaves and their major flavonol quercetin 3-(6-malon-ylglucoside) attenuate atherosclerotic lesion development inLDL receptor-deficient micerdquo Journal of Nutrition vol 135 no4 pp 729ndash734 2005

[22] J Naowaboot P Pannangpetch V Kukongviriyapan B Kongy-ingyoes and U Kukongviriyapan ldquoAntihyperglycemic antiox-idant and antiglycation activities of mulberry leaf extract instreptozotocin-induced chronic diabetic ratsrdquo Plant Foods forHuman Nutrition vol 64 no 2 pp 116ndash121 2009

[23] MM Padilha F CVilela CQ Rocha et al ldquoAntiinflammatoryproperties of Morus nigra leavesrdquo Phytotherapy Research vol24 no 10 pp 1496ndash1500 2010

[24] P Aramwit O Supasyndh T Siritienthong and N BangldquoMulberry leaf reduces oxidation and c-reactive protein levelin patients with mild dyslipidemiardquo BioMed Research Interna-tional vol 2013 Article ID 787981 7 pages 2013

[25] U Justesen P Knuthsen and T Leth ldquoQuantitative analysis offlavonols flavones and flavanones in fruits vegetables andbeverages by high-performance liquid chromatography withphoto-diode array and mass spectrometric detectionrdquo Journalof Chromatography A vol 799 no 1-2 pp 101ndash110 1998

[26] M S Taga E EMiller andD E Pratt ldquoChia seeds as a source ofnatural lipid antioxidantsrdquo Journal of the AmericanOil ChemistsrsquoSociety vol 61 no 5 pp 928ndash931 1984

[27] A A L Ordonez J D Gomez M A Vattuone and M I IslaldquoAntioxidant activities of Sechium edule (Jacq) Swartz extractsrdquoFood Chemistry vol 97 no 3 pp 452ndash458 2006

[28] A Kumaran and R Joel Karunakaran ldquoIn Vitro antioxidantactivities of methanol extracts of five Phyllanthus species fromIndiardquo LWT-Food Science and Technology vol 40 no 2 pp344ndash352 2007

[29] G Cao H M Alessio and R G Cutler ldquoOxygen-radical absor-bance capacity assay for antioxidantsrdquo Free Radical Biology andMedicine vol 14 no 3 pp 303ndash311 1993

[30] K Shimada K Fujikawa K Yahara and T Nakamura ldquoAntiox-idative properties of xanthan on the autoxidation of soybeanoil in cyclodextrin emulsionrdquo Journal of Agricultural and FoodChemistry vol 40 no 6 pp 945ndash948 1992

[31] J Cole M H L Green S E James L Henderson and H ColeldquoA further assessment of factors influencing measurements ofthioguanine-resistant mutant frequency in circulating T-lymphocytesrdquo Mutation Research vol 204 no 3 pp 493ndash5071988

[32] C-Y Hsu C-M Yang C-M Chen P Chao and S Hu ldquoEff-ects of chlorophyll-related compounds on hydrogen peroxideinduced DNA damage within human lymphocytesrdquo Journal ofAgricultural and Food Chemistry vol 53 no 7 pp 2746ndash27502005

[33] S-C Tang H-F Lo K-H Lin et al ldquoThe antioxidant capacityof extracts from Taiwan indigenous purple-leaved vegetablesrdquoJournal of the Taiwan Society for Horticultural Science vol 59no 1 pp 43ndash57 2013

[34] K-H Lin Y-Y Yang H-F Lo et al ldquoAntioxidant activity ofherbaceous plant extracts protect against hydrogen peroxide-induced DNA damage in human lymphocytesrdquo BioMed CentralResearch Notes In press

[35] A H Cory T C Owen J A Barltrop and J G Cory ldquoUse of anaqueous soluble tetrazoliumformazan assay for cell growthassays in culturerdquoCancer Communications vol 3 no 7 pp 207ndash212 1991

[36] Y T Szeto A R Collins and I F F Benzie ldquoEffects of dietaryantioxidants on DNA damage in lysed cells using a modifiedcomet assay procedurerdquoMutation Research vol 500 no 1-2 pp31ndash38 2002

[37] S Vielma G Virella A J Gorod and M F Lopes-VirellaldquoChlamydophila pneumoniae infection of human aortic endo-thelial cells induces the expression of FC 120574 receptor II (Fc120574RII)rdquoClinical Immunology vol 104 no 3 pp 265ndash273 2002

[38] C Y Zhu and S Loft ldquoEffects of Brussels sprouts extracts onhydrogen peroxide-induced DNA strand breaks in humanlymphocytesrdquo Food and Chemical Toxicology vol 39 no 12 pp1191ndash1197 2001

[39] Y D Min C-H Choi H Bark et al ldquoQuercetin inhibits exp-ression of inflammatory cytokines through attenuation of NF-120581B and p38MAPK in HMC-1 humanmast cell linerdquo Inflamma-tion Research vol 56 no 5 pp 210ndash215 2007

[40] P Y Chao Y P Huang and W B Hsieh ldquoInhibitive effect ofpurple sweet potato leaf extract and its components on celladhesion and inflammatory response in human aortic endothe-lial cellsrdquo Cell Adhesion amp Migration vol 7 no 2 pp 1ndash9 2013

[41] Y Y YangThe antioxidative capacity in herb plant extracts andtheir protection role in DNA oxidative damage of lymphocyte[MS thesis] Chinese Culture University Taipei Taiwan 2004

[42] C A Rice-Evans N J Miller and G Paganga ldquoStructure-anti-oxidant activity relationships of flavonoids and phenolic acidsrdquo

14 Evidence-Based Complementary and Alternative Medicine

Free Radical Biology and Medicine vol 20 no 7 pp 933ndash9561996

[43] M Strube G R Haenen H van den Berg and A Bast ldquoPitfallsin a method for assessment of total antioxidant capacityrdquo FreeRadical Research vol 26 no 6 pp 515ndash521 1997

[44] P G Pietta ldquoFlavonoids as antioxidantsrdquo Journal of NaturalProducts vol 63 no 7 pp 1035ndash1042 2000

[45] A M Gonzalez-Paramas S Esteban-Ruano C Santos-BuelgaSDe Pascual-Teresa and J C Rivas-Gonzalo ldquoFlavanol contentand antioxidant activity in winery byproductsrdquo Journal ofAgricultural and Food Chemistry vol 52 no 2 pp 234ndash2382004

[46] S Y Kim J J Gao and H K Kang ldquoTwo flavonoids from theleaves ofMorus alba induce differentiation of the human prom-yelocytic leukemia (HL-60) cell linerdquo Biological and Pharma-ceutical Bulletin vol 23 no 4 pp 451ndash455 2000

[47] M A Khan A A Rahman S Islam et al ldquoA comparative studyon the antioxidant activity ofmethanolic extracts from differentparts of Morus alba L (Moraceae)rdquo BMC Research vol 6 pp24ndash32 2013

[48] A-L ChungH-F Lo K-H Lin et al ldquoStudy on the antioxidantactivity in herb plant extractsrdquo Journal of the Taiwan Society ForHorticultural Science vol 59 no 2 pp 139ndash152 2013

[49] R L Prior X Wu and K Schaich ldquoStandardized methods forthe determination of antioxidant capacity and phenolics infoods and dietary supplementsrdquo Journal of Agricultural andFood Chemistry vol 53 no 10 pp 4290ndash4302 2005

[50] S J Duthie A R Collins GGDuthie andV LDobson ldquoQue-rcetin and myricetin protect against hydrogen peroxide-indu-ced DNA damage (strand breaks and oxidised pyrimidines) inhuman lymphocytesrdquo Mutation Research vol 393 no 3 pp223ndash231 1997

[51] G A Liu and R L Zheng ldquoProtection against damagedDNA inthe single cell by polyphenolsrdquo Pharmazie vol 57 no 12 pp852ndash854 2002

[52] M Noroozi W J Angerson and M E J Lean ldquoEffects of flav-onoids and vitamin C on oxidative DNA damage to humanlymphocytesrdquoAmerican Journal of Clinical Nutrition vol 67 no6 pp 1210ndash1218 1998

[53] T Koga and M Meydani ldquoEffect of plasma metabolites of (+)-catechin and quercetin on monocyte adhesion to human aorticendothelial cellsrdquoAmerican Journal of Clinical Nutrition vol 73no 5 pp 941ndash948 2001

[54] CMeng P Somers L Hoong et al ldquoDiscovery of novel pheno-lic antioxidants as inhibitors of vascular cell adhesionmolecule-1 expression for use in chronic inflammatory diseasesrdquo Journalof Medicinal Chemistry vol 47 no 25 pp 6420ndash6432 2004

[55] Y Shibata N Kume H Arai et al ldquoMulberry leaf aqueousfractions inhibit TNF-120572-induced nuclear factor 120581B (NF-120581B)activation and lectin-like oxidized LDL receptor-1 (LOX-1)expression in vascular endothelial cellsrdquoAtherosclerosis vol 193no 1 pp 20ndash27 2007

[56] K C Chan H H Ho C N Huang M Lin H Chen and CWang ldquoMulberry leaf extract inhibits vascular smooth musclecell migration involving a block of small GTPase and AktNF-kB signalsrdquo Journal of Agricultural and Food Chemistry vol 57no 19 pp 9147ndash9153 2009

[57] F Chen N Nakashima I Kimura M Kimura N Asanoand S Koya ldquoPotentiating effects on pilocarpine-induced salivasecretion by extracts and N-containing sugars derived frommulberry leaves in streptozocin- diabetic micerdquo Biological andPharmaceutical Bulletin vol 18 no 12 pp 1676ndash1680 1995

[58] K Konno H Ono M Nakamura et al ldquoMulberry latexrich in antidiabetic sugar-mimic alkaloids forces dieting oncaterpillarsrdquo Proceedings of the National Academy of Sciences ofthe United States of America vol 103 no 5 pp 1337ndash1341 2006

[59] H J Kim K H Yoon M J Kang et al ldquoA six-month sup-plementation of mulberry Korean red ginseng and banabadecreases biomarkers of systemic low-grade inflammation insubjects with impaired glucose tolerance and type 2 diabetesrdquoEvidence-based Complementary and Alternative Medicine vol2012 Article ID 735191 8 pages 2012

[60] G Nicholson and G M Hall ldquoDiabetes mellitus new drugs fora new epidemicrdquo British Journal of Anaesthesia vol 107 no 1pp 65ndash73 2011

[61] P Y Chao Y L Chen Y C Lin et al ldquoEffects of black soybeanon atherogenic prevention in hypercholesterolemic rabbits andon adhesion molecular expression in cultured HAECsrdquo Foodand Nutrition Sciences vol 4 pp 9ndash21 2013

[62] C Chen S Li C-YOChen et al ldquoConstituents in purple sweetpotato leaves inhibit in vitro angiogenesis with opposite effectsex vivordquo Nutrition vol 27 no 11-12 pp 1177ndash1182 2011

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

Evidence-Based Complementary and Alternative Medicine 3

chromatograph (HPLC) Samples were analyzed with a Spec-traSystem UV6000LP Photodiode Array Detection System(Thermo Separation Products San Jose USA) and an ODScolumn (250 times 46mm 5120583m YMC ODS-A YMC CoKyoto Japan)Themobile phase consisted of methanol-water(30 70 vv) with 1 formic acid and 100 methanol Thegradient was 25ndash74 methanol in 40min at a flow rateof 075mLmin the spectrum was recorded at 365 nm forflavonol determination [25]

26 ABTSHRPndashH2O2Assay of Total Antioxidant Activity

The total antioxidant capacity of hydrophilic and lipophilicantioxidants was determined using the horseradish perox-idase catalyzed oxidation of 22-azino-bis-(3-ethylbenzot-hiazoline-6-sulfonic acid) (ABTS) [29]The reaction mixturecontained 05mL of 1000 120583M ABTS (in ddH

2O) and 35mL

of 100 120583M H2O2(in 01M PBS) The reaction was started

by the addition of 05mL of 44UmL peroxidase (in 01MPBS) After 1 h 005mL of MLREs was added to the mixtureAbsorbance was measured at 730 nm after 5min Trolox wasused as a standard and the total antioxidant capacity ofmulberry extracts was measured as mM Trolox equivalent

27 Scavenging Activity on 11-Diphenyl-2-picryl-hydrazyl(DPPH) Radical The scavenging activity onDPPH radical ofMLRC acidic methanolic extracts was determined accordingto the method of Shimada et al [30] Briefly an aliquot of04 mL of acidic methanolic extracts with series dilution wasadded to 08mLof 1mMDPPH freshly prepared inmethanolmixed well and left to stand for 30min before measuringabsorbance at 517 nm The scavenging effect percentage wascalculated as [1 minus (OD

517 nm)(control OD517 nm)] times 100 TheIC50of the scavenging effect percentage was then calculated

28 Isolated Human Peripheral Blood Lymphocytes Bloodsamples were obtained from six donors four male andtwo female healthy nonsmokers that were 24 to 48 yearsold Fresh whole blood (20ndash30mL) from volunteers wastaken with informed consent and lymphocytes were isolatedusing a separation solution kit supplemented with Ficoll-Paque Plus lymphocyte isolation sterile solution (PharmaciaBiotech Sweden) [31] Cells were harvested within 1 dayof blood samples being taken and cultured with AIM Vmedium containing serum-free lymphocyte medium (GibcoInvitrogen USA) in a humidified atmosphere of 5 CO

2in

air at 37∘C for 24 h

29 Dosage Selected for the Comet Assay Previously wedemonstrated that the effect of lymphocyte exposure to 10 120583MH2O2on DNA single-strand break damage was 60-fold

greater than the control and with 994 cell viability [32]However when lymphocytes were exposed to 50 120583M H

2O2

the oxidative damage increased significantly compared to thecontrol under 982 cell viability Therefore 10120583M H

2O2

for the treatment dosage was selected in the current studyFurthermore when lymphocytes were treated with threeconcentrations (25 50 and 100 120583gmL) of acidic methanolicand water extracts from indigenous purple vegetables [33]

and herbaceous plants [34] the DNA damage was inducedby exposing the lymphocytes to 10 120583M H

2O2and shown the

protection effects Consequently these concentrations of theextracts were chosen for the effect treatments

210 Cell-Viability Testing After culturing lymphocytes wereexposed to each MLREs Each lymphocyte solution was usedat three concentrations (25 50 and 100 120583gmL) for 30min at37∘C DNA damage was induced by exposing lymphocytes toH2O2(10 120583M) for 5min on ice Treatment on ice minimized

the possibility of cellular DNA repair after H2O2injury Cells

were centrifuged at 100 g for 10minwashed and resuspendedin the samemedium as the comet assay All experiments werecarried out in triplicate Cell viability was tested using thetetrazoliumformazan (MTT) assay [35] prior to and aftertreatment with MLRC extract or H

2O2

211 DNA Single Strand Break Damage Estimation Using theComet Assay The comet assay was performed as describedin Szeto et al [36] with acidic methanolic hydrolysate andwater extracts from tested plants Cultured lymphocytes(105 cellsmL) were embedded in 75120583L of 1 low-melting-point agarose on a microscope slide (precoated with agarose)at 37∘CThe gel was allowed to set at 4∘C and cells were lysedfor a period of at least 2 h in lysis buffer at 4∘C Cells werethen alkaline-unwound following which electrophoresis wascarried out using the electrophoresis buffer at 4∘C for 15minat 25 vDC and 300mA All steps were conducted underdim light to prevent additional DNA damage Followingelectrophoresis slides were dipped into a neutralizationbuffer and stained with ethidium bromide The comet-likeimages resulting from the extension of DNA were scored asa reflection of the single strand breaks under a fluorescencemicroscope (Zeiss-Axiovert 100 Zeiss Germany) Triplicateslides were prepared for each experimental point sampleand 50 comet-like images selected at random per slide todetermine average DNA damage values A computerizedimage analysis system (VisCOMET 16 Impuls GmbH Ger-many) was employed to analyze two comet parameters DNAdamage by tail DNA percentage [(total brightness of tailareatotal brightness of total area) times 100] and tail moment(tail length times tail DNA) The inhibition percentage of tailDNA and tail moment were calculated relative to the 10 120583MH2O2treated group

212 Cell Cultures and Treatment HAECs (Clonetics Corp)were grown in Medium 200 (GIBCO Invitrogen) supple-mented with 1 low serum growth supplement (LSGSGIBCO Invitrogen) and 10 FBS (GIBCO Invitrogen) inan atmosphere of 95 air and 5 CO

2at 37∘C in plastic

flasks in an incubator (Astec Co) as described by Vielmaet al [37] The human monocytic cell line U937 (Amer-ican Type Culture Collection) was grown in a suspensionculture in RPMI-1640 (GIBCO Invitrogen) containing 10FBS (Sigma) and 1 antibiotic-antimycotic mixture (Sigma)under an atmosphere of 95 air and 5 CO

2at 37∘C

After incubation with MLREs and aspirin or TNF-120572 cellviability was assessed using the tetrazoliumformazan (MTT)

4 Evidence-Based Complementary and Alternative Medicine

assay [35] The adhesion assays were performed as describedby Zhu and Loft [38] Briefly U937 cells were labeledwith a fluorescent dye incubated with 10 120583molL 27-bis (2-carboxyethyl)-5 (6)-carboxyfluorescein acetoxymethyl esterat 37∘C for 1 h in RPMI-1640 medium and subsequentlywashed by centrifugation ConfluentHAECs in 24-well plateswere incubated with U937 cells (106 cellsmL) at 37∘C for1 h Nonadherent leukocytes were removed The numbers ofadherent leukocytes were determined by photographing andcounting four randomly chosen fields per well at 100x usinga Zeiss Axio Mager Z1 Upright Fluorescence MicroscopeExperiments were performed in triplicate and repeated threetimes HAECswere incubatedwith 25 120583g and 50 120583g ofMLREsor 10 120583M aspirin for 18 h followed by treating with 2 ngmLTNF-120572 for 6 h in theNF-120581Bp65 AP-1 and STAT-3 expressionassays

213 Nuclear Protein Isolation Protein extracts were pre-pared as described by Min et al [39] Briefly after cellactivation for the indicated times cells were washed in 1mLice-cold PBS centrifuged at 400 g for 5min resuspendedin 400 120583L ice-cold hypotonic buffer (10mM HEPES 15mMMgCl

2 01mM EDTA 10mM KCl 1mM DTT 05mM

PMSF pH79) incubated on ice for 10min vortexed and cen-trifuged at 15000 g for 30 sec The supernatant was collectedand stored at minus70∘C for cytosolic protein analysis Pelletednuclei were gently resuspended in 445 120583L ice-cold extractionbuffer (20mM HEPES pH 79 with 15mM MgCl

2 042M

NaCl 02mM EDTA and 25 glycerol) with 5 120583L of 10mMDTT and 05 120583L of Protease Inhibitor Cocktail (Sigma) incu-bated on ice for 20min vortexed and centrifuged at 15000 gfor 5min at 4∘C Aliquots of the supernatant containingnuclear proteins were frozen in liquid nitrogen and stored atminus70∘C

214 Western Blot Analysis Nuclear lysates were subjected to12 SDS-PAGE (Bio-Rad Laboratories) after which proteinswere transferred to a PVDF membrane (Bio-Rad) Mem-branes were probed with a mouse monoclonal NF-120581B p65antibody (BD Biosciences) After incubation in a secondaryantibody solution consisting of IRDye 700CW-conjugatedgoat anti-mouse IgG (LI-COR Biosciences) for 60min atroom temperature with gentle shaking the membrane waswashed four times for 5min each at room temperature in PBSwith 01 Tween-20 and while being gently shaken After afinal rinse the membrane was scanned using AlphaEaseFCimage analysis software (Alpha Innotech Corporation) toanalyze spot density The internal control was set at 100 todetermine the relative density of protein expression [40]

215 Electrophoretic Mobility Shift Assay for NF-120581B AP-1 andSTAT3 The binding reaction consisted of 1120583L 10times bindingbuffer (100mM TRIS 500mM NaCl 10mM DTT pH 75)5 120583LH

2O 2120583L 25mMDTT25Tween-20 1 120583L IRDye 700-

labeled EMSA oligonucleotides specific for NF-120581B (51015840-AGTTGA GGG GAC TTT CCC AGG C-31015840 and 31015840-CGC TTGATG ACT CAG CCG GAA-51015840) AP-1 (51015840-CGC TTG ATGACT CAG CCG GAA-31015840 and 31015840-GCG AAC TAC TGA GTC

GGC CTT-51015840) STAT3 (51015840-GAT CCT TCT GGG AAT TCCTAG ATC-31015840 and 31015840-CTA GGA AGA CCC TTA AGG ATCTAG-51015840) (Sigma) PPAR-120572 (51015840-AAA AAC TGG GCC AAAGGT CT-31015840) and PPAR-120574 (51015840-TGA AAC TAG GGT AAAGTT CA-31015840) (Sigma) 1 120583L poly (dIsdotdC) and 1 120583L nuclearextract (as prepared previously) The mixture was incubatedat room temperature for 20min in the dark After 1x OrangeLoading Dye (LI-COR Biosciences) was added the bindingreaction was loaded onto a native 4 polyacrylamide gel andseparated by electrophoresis at 90 vDC for 40min The gelswere scanned using anOdyssey Infrared Imaging System (LI-COR Biosciences) [40]

216 Statistical Analysis Data are expressed as the mean plusmnstandard deviation and statistical significance was analyzedusing one-way ANOVA followed by the Tukeyrsquos Range Testat the 005 significance level Pearsonrsquos linear correlation wasalso determined The means of three replicates are reported

3 Result

31 Antioxidant Composition and Antioxidant Activity Poly-phenols flavonoids and flavonols were abundant in BTat levels of 3465 plusmn 082mgg DW gallic acid 8483 plusmn868mgg DW quercetin equivalent and 2547 plusmn 386mggDW quercetin equivalent respectively (Table 1) Anthocy-anidins were abundant in BT and ML at levels of 14332 plusmn181 and 13582 plusmn 031 unitsgDW respectively Table 2 showsthat quercetin and kaempferol were both rich in BTat levelsof 285333 plusmn 18037 and 64667 plusmn 5033 120583ggDW respectivelywhile the levels of M were 166667 plusmn 18903 and 42667 plusmn4619 120583ggDW respectively However no myricetin or morinwas detected in the MLRC (Table 2) Thus these MLREsdisplayed variations in their antioxidant substances levels

MLREs showed antioxidant activity proving their capac-ity to scavenge the ABTS radical cation The antioxidantactivity in acidic methanolic hydrolysate sample extracts wasexpressed in trolox equivalent antioxidant capacity (TEAC)(Table 3) BT showed a significantly higher TEAC value(15514 plusmn 290 120583ggDW) than MT (10239 plusmn 095 120583ggDW)and M (7654 plusmn 161 120583ggDW) TEAC values were positivelyand significantly (119903 = 041 119875 = 0034) correlated withthe content of quercetin among MLREs (Table 4) Thusdifferent quercetin contents displayed various levels of anti-oxidant activity However M had a significantly high effi-cacy for DPPH radical scavenging activity (IC

5018883 plusmn

361 120583gmL) compared to BT (IC5066901plusmn723 120583gmL) and

MT (IC5083640 plusmn 708 120583gmL) (Table 3) Hence each tested

sample showed significant differences in scavenging of DPPHradical at IC

50

32 Effects of Acidic Methanolic and Water Extracts fromMLRC on H

2O2-Induced DNA Damage to Lymphocytes The

effects ofMLRC extracts on cell cytotoxicity were determinedby theMTTassay Lymphocyteswere exposed to each of threedifferent MLRC extracts at three concentrations (25 50 and100 120583gmL) for 30min at 37∘C DNA damage was inducedby exposing lymphocytes to H

2O2(10 120583M) for 5min on ice

Evidence-Based Complementary and Alternative Medicine 5

Table 1 The contents of various antioxidant substances in MLRC

Polyphenolmg gallic acidgDW

Flavonoids mg quercetinequivalentgDW

Flavonol mg quercetinequivalengDW

AthocyanidinunitgDW

BT 3465 plusmn 082a

8483 plusmn 868a

2547 plusmn 386a

14332 plusmn 181a

MT 2323 plusmn 054b

5599 plusmn 1055b

1326 plusmn 266b

8858 plusmn 412b

M 1987 plusmn 061c

4960 plusmn 541b

1422 plusmn 148b

13582 plusmn 031a

All values are means plusmn SD (119899 = 3)Means within a column with different superscripts (asimc) are significantly different 119875 lt 005

Table 2 The contents of flavonols in acidic methanolic extracts ofMLRC

Flavonols (120583ggDW)Samples Myricetin Morin Quercetin KaempferolBT ND ND 285333 plusmn 18037

a64667 plusmn 5033

a

MT ND ND 37333 plusmn 2309c ND

M ND ND 166667 plusmn 18903b42667 plusmn 4619

b

ND not detected

Table 3 TEAC values and the IC50 of DPPH radical scavengingactivity of acidic methanolic hydrolysates of MLRC

Sample TEAC(Trolox mM)lowast

DPPH radical scavenging activityIC50 (120583gmL)

BT 15514 plusmn 290a

66901 plusmn 723b

MT 10239 plusmn 095b

83640 plusmn 708a

M 7654 plusmn 161c

18883 plusmn 361c

lowastTEAC value at sample 100120583gmL concentration

Table 4 Correlation between antioxidant substances and TEACvalues treated with acid hydrolysates of MLRC

FlavonolsMyricetin Morin Quercetin Kaempferol

TEAClowast mdash mdash 119903 = 041

119875 = 0034

mdash

mdash no significant correlationlowastTEAC values at 100 120583gmL of sample concentration

No mulberry extracts were cytotoxic at the concentrationsused with gt98 of cells remaining viable [41] The cometassay was performed to determine the DNA damagingactivity of the plants because it is a sensitive method formonitoring single strand DNA breaks at the single celllevel Any DNA damage that occurred is represented as atail length (tail migration) of the DNA strand The effectsof pretreatment with acidic methanolic extracts of MLRCon 10 120583M H

2O2-induced DNA oxidative damage in human

lymphocytes are presented in Figure 1 indicating that therewas a protective effect of pretreatment on lymphocytes witheach of the MLRC extracts at the lowest dose (25 120583gmL)Lymphocytes pretreated with 25ndash100120583gmL extracts of MTextracts experienced a greater level of protection againstH2O2exposure than lymphocytes exposed to other tested

compounds and in a dose-dependent manner BT had betterinhibition efficacy at lower concentration (25120583gmL) andthe maximum protective effect of lymphocyte pretreatmentwas observed with pretreatment by 25 120583gmL BT exhibiting

1462 of tail DNA compared to treatment only withH2O2

MT had better inhibition efficacy (8439) than M and BTextracts at the 100 120583gmL level (Figure 1(a)) Tested plantsshowed at least 134263 of tail moment in BT extract at the25 120583gmL level while the BT extract at 100 120583gmL had thehighest tail moment (328193) compared to the rest of theacidic methanolic extract samples (Figure 1(b)) Moreoverthe trend in tail moment in all samples continued climbingup from 25ndash100 120583gmL

The effects of various water extracts of MLRC H2O2-

induced DNA damage to lymphocytes are presented inFigure 2 At lower concentrations all tested samples hadlower tail DNA indicating better inhibition efficacies(Figure 2(a)) BT and M had the best inhibition efficacieswith both exhibiting values of 1422 and 1469 tail DNAat 25 120583gmL respectively Moreover MT had a higher tailDNA than the other extracts at the same dose and thehighest tail DNA was observed in 3212 of MT at aconcentration of 100120583gmL The tail moment of all samplescontinued to increase up to the 100 120583gmL level (Figure 2(b))The minimum tail moment occurred in M at 153674 in the25 120583gmL extracts Moreover the DNA damage induced byH2O2was significantly higher than the DNA damage treated

with extracts and H2O2treated had 8726 in tail DNA and

832884 in tail moment

33 Effects of MLREs on Monocyte-Endothelial Cell Adhe-sion As shown in Figure 3 pretreatment of HAECs with100 120583gmLMLREs for 18 h significantly suppressed the adhe-sion ofU937monocytes to TNF-120572-stimulatedHAECs similarto that of 10 120583Maspirin (positive control) Data also show that25 and 50 120583gmL BT MT and M methanol extracts inhib-ited the adhesion of U937 monocytes to TNF-120572-stimulatedHAECs by 8198 and 8187 (BT) 7449 and 8331 (MT)and 8011 and 7559 (M) respectively

34 Effects of MLREs on NF-120581B Activity and MLREs Inhibitsthe Binding of NF-120581B AP-1 and STAT3 Figure 4 demon-strates that MLREs regulated the DNA-binding activity andexpressions of NF-120581B in HAECs In untreated HAECs NF-120581B p65 was only found within the cytosol however itsnuclear translocation was observed upon treatment withTNF-120572 Compared to the TNF-120572 group pretreatment withBT and MT methanol extracts significantly decreased theexpression of NF-120581B p65 in the nuclear compartment whilethe M methanol extract did not reduce the expressionof NF-120581B p65 activity Furthermore an EMSA analysiswas carried out to determine if the reduced expression of

6 Evidence-Based Complementary and Alternative Medicine

Control H2O2

BT25 BT50 BT100

MT25 MT50 MT100

M25 M50 M100

(a)

0

20

40

60

80

100

25 50 100

Tail

DN

A (

)

Mulberry extracts (120583gmL)

lowast lowastlowast

lowast

lowastlowast

alowast alowast

blowast

MTMBT

H2O2

(b)

Tail

mom

ent

0

2000

4000

6000

8000

10000

25 50 100Mulberry extracts (120583gmL)

lowastlowastlowast

ablowastclowast

alowast

alowast

alowastblowast

MTMBT

H2O2

(c)

Figure 1 Effects of various acidic methanolic extracts of MLRC on H2O2-induced DNA damage to lymphocytes The comet-like images

resulting from the extension of DNA (a) were observed using a fluorescence microscope Tail DNA (b) and tail movement (c) were exposedto test mulberry leaf-related extracts at 25 50 and 100 120583gmL and treated with H

2O2at 10120583M Values with different letters differ significantly

with regard to oxidative damage when comparing different MLRC extracts lowast119875 lt 005 refers to differences in oxidative damage as comparedwith 10 120583MH

2O2-alone treatment

Evidence-Based Complementary and Alternative Medicine 7

Control H2O2

BT25 BT50 BT100

MT25 MT50 MT100

M25 M50 M100

(a)

alowastblowast blowastblowast

blowastblowast

blowast

25 50 100Mulberry extracts (120583gmL)

MTMBT

H2O2

0

20

40

60

80

100

Tail

DN

A (

)

ablowastablowast

(b)

Tail

mom

ent

0

2000

4000

6000

8000

10000

lowastlowast lowast

alowastalowast

25 50 100Mulberry extracts (120583gmL)

MTMBT

H2O2

ablowast ablowastablowast

bclowast

(c)

Figure 2 Effects of various water extracts of MLRC on H2O2-induced DNA damage to lymphocytes The comet-like images resulting from

the extension ofDNA (a) were observed using a fluorescencemicroscope Tail DNA (b) and tailmovement (c) were exposed to testmulberryleaf-related extractsat 25 50 and 100120583gmL and treated with H

2O2at 10120583M Values with different letters differ significantly with regard to

oxidative damage when comparing different MLRC extracts lowast119875 lt 005 refers to differences in oxidative damage as compared with 10120583MH2O2-alone treatment

NF-120581B in response to treatment with MLREs resulted inreduced binding of NF-120581B to DNAThe treatment of HAECswith TNF-120572 led to increased binding of NF-120581B howeverpretreatment of HAECs with BT MT and M significantlydecreased DNA-bound NF-120581B (Figure 5(a)) The increased

binding of AP-1 with TNF-120572 resulted from the treatment ofHAECs however pretreatment of HAECs with BT MT andM significantly decreased DNA-bound AP-1 (Figure 5(b))STAT3 DNA-binding activities to its cognate recognitionsite were remarkably reduced compared to TNF-120572 treatment

8 Evidence-Based Complementary and Alternative Medicine

(A)(A)

(B)

(C) (D)

(E) (F)

(I)

(G) (H)

200120583m 200120583m

200120583m200120583m

200120583m200120583m

200120583m200120583m

200120583m

(a)

Con

trol

Asp

irin

BT25

BT50

MT2

5

MT5

0

M25

M50

Mon

ocyt

e adh

eren

ce (c

ells

HPF

)

0

20

40

60

80

100

120

140

160

180

lowast lowast lowast

lowast

lowast

lowast

lowast

TNF-120572

(b)

Figure 3 Mulberry leaf related extracts (MLREs) reduce tumor necrosis factor (TNF)-120572-induced adhesion of monocytes to human aorticendothelial cells (HAECs) (a) The number of adherent U937 cells (marked by arrow) was monitored by fluorescence microscopy undercontrol (a) 2 ngmL TNF-120572 (b) co-treated with TNF-119886 and aspirin (10120583M) (c) cotreated with TNF-120572 and BT (25 120583gmL) (d) cotreated withTNF-120572 and BT (50 120583gmL) (e) cotreated with TNF-120572 and MT (25 120583gmL) (f) cotreated with TNF-120572 and MT (50 120583gmL) (g) cotreated withTNF-120572 and M (25120583gmL) (h) and cotreated with TNF-120572 and M (50120583gmL) (i) (b) HAECs were pretreated with MLREs for 18 h followedby stimulating with TNF-120572 for 6 h Fluorescence-labeled monocytic U937 cells were added to HAECs and allowed to adhere for 2 h Data areexpressed as a percentage of TNF-120572-induced expression (mean plusmn SD) of three experiments Mean plusmn SD lowast119875 lt 005 BT MT and M at 25and 50120583gmL versus TNF-120572

119875 lt 005 control versus TNF-120572

Evidence-Based Complementary and Alternative Medicine 9

Control TNF-120572

TNF-120572

Aspirin10120583M

+ + + + + + + +

BT25 BT50 MT25 MT50 M25 M50

120583gmL 120583gmL 120583gmL 120583gmL 120583gmL 120583gmL

NF-120581B p65

120573-Actin43kDa

minus

65kDa

(a)

Con

trol

Asp

irin

BT25

BT50

MT2

5

MT5

0

M25

M50

00

02

04

06

08

10

lowast

lowast

lowast

lowastlowastlowastTN

F-120572

Ratio

(nuc

lear

120573-a

ctin

)

(b)

Figure 4 Effects of MLREs on NF-120581B activity After HAECs were pretreated with the indicated samples and incubated with TNF-120572 nuclearextracts were prepared and the expression of NF-120581B p65 was assessed by western blot analysis A representative image of three similar resultsis shown (a) Actin served as the loading control for the nuclear compartment Semiquantitative analysis of three independent experimentsare also shown (b) indicates a significant difference between the TNF-120572 treated or experimental treatment groups and the control119875 lt 005lowast indicates a significant difference between the TNF-120572 and experimental treatment groups BT MT and M at 25 and 50 120583gmL

(lane 3) in the nuclear fraction indicating that MLREs at50120583gmL inhibited STAT3-binding activities In addition theresults in Figure 5(c) (lanes 4ndash10) show that aspirinMLREs at50120583gmL and BT at 25120583gmL remarkably decreased STAT3levels compared to TNF-120572 (lane 3) however both MT andM at 25120583gmL increased STAT3-binding activities Theseresults indicate that only 50 120583gmL MLREs 25 120583gmL BTand aspirin block STAT3 activation and might downregulateTNF-120572-induced expressions of inflammatory signaling path-ways

35 MLREs Regulated the DNA-Binding Activity and Expres-sions of PPAR 120572 and PPAR 120574 in HAECs PPAR 120572 and 120574effectively bound to PPAR 120572 and 120574 oligo-IRDye 700 asindicated in lanes 3ndash10 of Figure 6 Supershifts are bands fromthe binding of each specific antibody The binding activitiesof PPAR 120572 and 120574 were detected at baseline The 25 120583gmLMT extract (lane 7) and 25 and 50 120583gmL M extracts (lanes9-10) increased activity compared to incubating with TNF-120572 alone (lane 3) However BT at both 25 and 50 120583gmLextracts showed reduced effects on PPAR 120572 and 120574 expressionMoreover the 50 120583gmLMT extract had no effect on PPAR 120572binding activity but increased the binding activity of PPAR120574 in comparison to incubation with TNF-120572 alone Thesedata suggest that MT and M both promoted PPAR 120572 and 120574

expression at least in part by increasing the DNA binding oftranscription factors PPAR 120572 and 120574

4 Discussion

41 Antioxidant Composition and Antioxidant Activity BTcontains high levels of polyphenols flavonoids flavonolsanthocyanidin quercetin and kaempferol (Tables 1 and 2)BT also had a higher TEAC value (15514mM Table 3) thanthe TEAC values of 47mM [42] and 64mM [43] reportedfor quercetin as well as 242mM reported for quercetin-3-rutinoside [44] Furthermore Gonzalez-Paramas et al [45]demonstrated that flavonol contents were significantly andhighly correlated (119903 = 08) with TEAC values Kim et al [46]reported that mulberry leaves contain abundant quercetinand inhibit lipid peroxidation at concentrations of 01 and001mgmL In our study the amount of quercetin wassignificantly correlated (119903 = 041 Table 4) with TEAC valueHowever no correlation was found among myricetin morinkaempferol and TEAC which may be due to the structurerequired to reinforce the free radical scavenging activity thatvaries with the type of free radical Naowaratwattana et al[18] reported that a 50 methanolic extract of mulberryleaf contained rutin isoquercetin and various derivatives ofkaempferol and quercetin glycosides while a water extract

10 Evidence-Based Complementary and Alternative Medicine

NF-120581BTNF-120572

TNF-120572

+

Aspirin10120583M+ +

BT25120583gmL

+

BT50120583gmL

+

MT25120583gmL

+

MT50120583gmL

+

M25

120583gmL+

M50

120583gmLControl

minus

DNAOligo

Free probe

(a)

AP-1TNF-120572

TNF-120572

+

Aspirin10120583M+ +

BT25120583gmL

+

BT50120583gmL

+

MT25120583gmL

+

MT50120583gmL

+

M25

120583gmL+

M50

120583gmLControl

minus

DNAOligo

Free probe

(b)

STAT3

TNF-120572

TNF-120572

+

Aspirin10120583M+ +

BT25120583gmL

+

BT50120583gmL

+

MT25120583gmL

+

MT50120583gmL

+

M25

120583gmL+

M50

120583gmLControl

minus

DNAOligo

Free probe

(c)

Figure 5 MLREs inhibits the binding of (a) NF-120581B (b) AP-1 and (c) STAT3 to target DNA sequence NF-120581B AP-1 and STAT3 transcriptionfactor-DNA interactions were assessed by EMSA

contained primarily chlorogenic acid and caffeoylquinic acidderivatives The IC

50of DPPH radical scavenging was 794

and 2042120583gmL for 50 methanolic and water extractsrespectively [18] In our studyM extracts contained quercetinand kaempferol (Table 2) which may account for part ofthe highly scavenged DPPH radical in comparison to therest of MLREs Khan et al [47] demonstrated that the IC

50

was 22023120583gmL which is much less efficacious than themulberry leaf acidic methanolic extract IC

50of 1888 120583gmL

(Table 3) However the IC50

of in vitro free radical scav-enging activity of mulberry leaves from Naowaboot et al[22] was 617 plusmn 21 120583gmL which is more efficacious thanthe IC

50concentration derived in the present study Taken

together the polyphenol-rich MLREs exhibited distinct cell-free antioxidant activity (ie TEAC) as validated by theirlevels of polyphenols and flavonols with distinct antioxidantactivity strongly accounting for the antioxidant activity of the

extracts The amount of quercetin was especially positivelycorrelated with the observed TEAC values

42 Estimation of DNA Single Strand Break Damage fromExposure to Acidic Methanolic and Water Extracts One ofthe antioxidant activity tests scavenging of DPPH radi-cal was selected due to its quick and simple but it is aless biologically relevant assay Previously we reported thatwith 2 120583gsdotmLminus1 of methanolic and water extracts from theindigenous vegetables [33] and herb plants [48] significantlyprolonged lag phase of low-density lipoprotein (LDL) TheLDL oxidation assay is considered to be one of the mostbiologically relevant assays [49] Both studies [33 48] alsoshowed that the concentration of the tested extracts forscavenging DPPH radical was in higher levels at 10mg oftested extracts with 2ndash53 inhibition Lower concentrationwas then chosen for the treated dosage in the present study

Evidence-Based Complementary and Alternative Medicine 11

TNF-120572

TNF-120572

+

Aspirin10120583M+ +

BT25120583gmL

+

BT50120583gmL

+

MT25120583gmL

+

MT50120583gmL

+

M25

120583gmL+

M50

120583gmLControl

minus

DNAOligo

Free probe

PPAR 120572

(a)

TNF-120572

TNF-120572

+

Aspirin10120583M+ +

BT25120583gmL

+

BT50120583gmL

+

MT25120583gmL

+

MT50120583gmL

+

M25

120583gmL+

M50

120583gmLControl

minus

DNAOligo

Free probe

PPAR120574

(b)

Figure 6 Effect of treatments on the expression of PPAR 120572 (a) and PPAR 120574 (b) in TNF-120572-stimulated HAECs by EMSA

In addition lower concentration also could avoid the toxicityeffect of higher level of extracts on lymphocytes Quercetinwas found to protect against H

2O2-induced DNA damage in

human lymphocytes at 10 120583M [50] and at 31 to 25 120583M [51]Quercetin was also reported to induce DNA strand breaks invarious cell types but only at higher doses at 100 120583Mor above[50] Duthie et al [50] proposed that the dihydroxy structureof quercetin and myricetin might be essential in protectingDNA against hydrogen peroxide No such hydroxyl groupsare present in the tocopherol molecule Noroozi et al [52]demonstrated that in addition to quercetin kaempferolcould also inhibit H

2O2-induced DNA strand breaks in

human lymphocytes In our study both BT and M were richin quercetin and kaempferol (Table 2) whichmay account forthe protective roles of both extracts in preventingDNAstrandbreaks from human lymphocytes

43 MLREs Regulated the DNA-Binding Activity and Expres-sions of NF-120581B AP-1 STAT3 and PPARs in HAECsBecause MLREs exhibited free radical scavenging the anti-inflammatory effects of MLREs on TNF-120572-induced celladhesion and adhesionmolecule expressionwere determinedin the present study Our results suggest that MLREs andaspirin significantly inhibited TNF-120572-induced monocyte-endothelial cell adhesion (Figure 3) Significant reductionsin NF-120581B expression and DNA binding by aspirin MTextract (25 120583gmL) and BT extract (both 25 and 50 120583gmL)were also observed The adhesion of monocytes to the

vascular endothelium and subsequent migration into thevessel wall are early events in atherogenesis [2] Our resultsare consistent with previous research showing that dietarypolyphenols such as catechin and quercetin significantlyreduce the binding of monocytes to HAECs [53 54] Thereduced cellular adhesion may be due to the inhibitionof cellular adhesion molecule expression as purple sweetpotato leaf extract (PSPLE) reduced both VCAM-1 andICAM-1 expression [40] Shibata et al [55] demonstratedthat pretreatment of cultured bovine aortic endothelial cells(BAECs) with mulberry leaf aqueous fractions inhibitedTNF-120572- and LPS-induced expression of Lectin-like Ox-LDLreceptor-1 (LOX-1) a cell-surface receptor for atherogenicOx-LDL (oxidized low-density lipoprotein) It also appearsto mediate Ox-LDL-induced inflammation which may becrucial in atherogenesis Furthermore mulberry leaf aqueousfractions inhibited the TNF-120572-induced activation of NF-120581Band phosphorylation of inhibitory factor of NF-120581B-alpha(I120581B-120572) Thus mulberry leaf aqueous fractions suppressTNF-120572- and LPS-induced LOX-1 gene expression mediatedby inhibiting NF-120581B activation

Chan et al [56] demonstrated that mulberry leaf extract(MLE) was rich in polyphenols MLE can effectively inhibitthe migration of vascular smooth muscle cells (VSMC) byblocking small guanosine triphosphatases (small GTPase)and AktNF-120581B signals The Rho family of small GTPasessuch as Rho Rac and Cdc42 acts as a molecular switch toregulate the actin cytoskeleton The Rho family of GTPases

12 Evidence-Based Complementary and Alternative Medicine

is also reported to be involved in the regulation of NF-120581B-dependent transcription [56] MLE has been shown to pos-sess hypoglycemic effects in an insulin-dependent diabetesmellitus (type 1 diabetes) animal model and potential hypo-glycemic compounds inmulberry leaves were suggested to be2-O-120572-D-galactopyranosyl-DNJ and fagomine [57] Konnoet al [58] further reported that mulberry latex containsalkaloidal sugar-mimic glycosidase inhibitors with antidia-betic activities such as 14-dideoxy-14-imino-D-arabinitol 1-deoxynojirimycin and 14-dideoxy-14-imino-D-ribitol Kimet al [59] recently showed that treated type 2 diabetesmellitus(T2D) with a 1 1 1 mixture of ginseng root mulberry leafwater extract and banana leaf water extract (6 gd) for 24weeks significantly decreased plasma intracellular adhesionmolecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) levels This indicates suppressed inflammatoryresponses in T2D but no significant differences in glucosehomeostasis control measurement changes Rigamonti et al[8] reported that bothPPAR120572 and 120574maybe thought of as sup-pressing the activity of NF120581B and AP-1 pathways that reduceinflammatory mediator production in the macrophages ofhumans and mice In our study MT and M promoted PPAR120572 and 120574 expression at least in part by increasing DNAbinding of transcription factors PPAR 120572 and 120574 PPAR-120572-and 120574-dependent signaling pathwaysmay also play importantroles in mediating the TNF-120572 receptor signaling pathwayby MT and M by suppressing the activity on NF-120581B andAP-1 pathways These pathways may regulate downstreamproinflammatory gene expression and adhesion molecularexpression Clinically PPAR 120574 agonists such as rosiglitazoneare widely used in the treatment of T2D [60] M and MTextracts increased PPAR 120574 activity which may partly explainwhy inflammatory responses are suppressed in T2D

Although reduced NF-120581B expression and DNA bind-ing were observed upon treatment with ML MT and BT(Figure 5) the mechanism was not assessed and is worthy offurther study In addition the present results were obtainedusing in vitro studies and may be consistent with an invivo model of atherogenesis similar to that reported byEnkhmaa et al [21] in which mulberry leaves suppressedthe development of atherosclerotic lesions Previously weproved that black beans significantly prolong LDL lag timeand decrease the atheroma regions of the aortic arch andthoracic aorta in hypercholesterolemic NZW rabbits [61]Wethen demonstrated that black soybean extract and PSPLEsignificantly decreased the adhesion of U937 monocyticcells to TNF-120572-stimulated HAECs and reduced adhesionmolecules expression (VCAM-1 and ICAM-1) as well as NF-120581B-p65 expression [37] This is particularly important giventhe opposing effects of flavonols fromMLREs obtained usingin vitro and ex vivo studies [62]

5 Conclusion

Tested MLREs were rich in flavonols and had strongantioxidant activity in protecting lymphocyte DNA fromoxidative damage In particular BT was rich in quercetinand polyphenols The antioxidant components of MLREs

might downregulate intracellular redox-dependent signalingpathways in HAECs upon TNF-120572 stimulation which mightprevent ROS-mediated endothelial cell dysfunction MLRCshave anti-inflammatory effects through the modulation ofNF-120581B AP-1 STAT3 and PPARs signaling These chemi-cals appear to provide distinct cytoprotective mechanismsthat might contribute to their putative beneficial effectsin suppressing endothelial responses to cytokines duringinflammation These results may benefit further in vivostudies to assess the therapeutic potential of phytochemicalsas anti-inflammatories for use in cytokine-induced vasculardisorders including atherosclerosis

Acknowledgment

This work was supported in part by Grants from the NationalScience Council of Taiwan (NSC 95ndash2320-B-034-001 andNSC 96ndash2320-B-034-001)

References

[1] W Palinski ldquoUnited they go conjunct regulation of aorticantioxidant enzymes during atherogenesisrdquo CirculationResearch vol 93 no 3 pp 183ndash185 2003

[2] P Libby ldquoMolecular bases of the acute coronary syndromesrdquoCirculation vol 91 no 11 pp 2844ndash2850 1995

[3] P A Baeuerle and D Baltimore ldquoNf-120581B ten years afterrdquo Cellvol 87 no 1 pp 13ndash20 1996

[4] J M Kyriakis ldquoActivation of the AP-1 transcription factor byinflammatory cytokines of the TNF familyrdquo Gene Expressionvol 7 no 4ndash6 pp 217ndash231 1999

[5] J M Muller R A Rupec and P A Baeuerle ldquoStudy of generegulation by NF-120581B and AP-1 in response to reactive oxygenintermediatesrdquoMethods vol 11 no 3 pp 301ndash312 1997

[6] G Chinetti J Fruchart and B Staels ldquoPeroxisome proliferator-activated receptors new targets for the pharmacological mod-ulation of macrophage gene expression and functionrdquo CurrentOpinion in Lipidology vol 14 no 5 pp 459ndash468 2003

[7] G Chinetti J C Fruchart and B Staels ldquoTranscriptional regu-lation of macrophage cholesterol trafficking by PPAR120572 andLXRrdquo Biochemical Society Transactions vol 34 no 6 pp 1128ndash1131 2006

[8] E Rigamonti G Chinetti-Gbaguidi and B Staels ldquoRegulationof macrophage functions by PPAR- 120572 PPAR- 120574 and LXRsin mice and menrdquo Arteriosclerosis Thrombosis and VascularBiology vol 28 no 6 pp 1050ndash1059 2008

[9] M A Bouhlel J Brozek B Derudas et al ldquoUnlike PPAR120574PPAR120572 or PPAR120573120575 activation does not promote humanmono-cyte differentiation toward alternative macrophagesrdquo Biochem-ical and Biophysical Research Communications vol 386 no 3pp 459ndash462 2009

[10] G Chinetti J-C Fruchart and B Staels ldquoPeroxisome prolife-rator-activated receptors and inflammation from basic scienceto clinical applicationsrdquo International Journal of Obesity vol 27supplement 3 pp S41ndashS45 2003

[11] Y Zhu J H-C Lin H Liao et al ldquoLDL induces transcriptionfactor activator protein-1 in human endothelial cellsrdquo Arte-riosclerosis Thrombosis and Vascular Biology vol 18 no 3 pp473ndash480 1998

Evidence-Based Complementary and Alternative Medicine 13

[12] J Terao ldquoDietary flavonoids as antioxidantsrdquo Forum of Nutri-tion vol 61 pp 87ndash94 2009

[13] Y B Feng W Q Luo and S Q Zhu ldquoExplore new clinicalapplication of Huanglian and corresponding compound pre-scriptions from their traditional userdquo Zhongguo ZhongyaoZazhi vol 33 no 10 pp 1221ndash1225 2008

[14] Y H Chu C L Chang and H F Hsu ldquoFlavonoid content ofseveral vegetables and their antioxidant activityrdquo Journal of theScience of Food andAgriculture vol 80 no 5 pp 561ndash566 2000

[15] I C Arts B van de Putte and P C Hollman ldquoCatechin con-tents of foods commonly consumed in The Netherlands 1Fruits vegetables staple foods and processed foodsrdquo Journal ofAgricultural and Food Chemistry vol 48 no 5 pp 1746ndash17512000

[16] A R Proteggente A S Pannala G Paganga et al ldquoThe anti-oxidant activity of regularly consumed fruit and vegetablesreflects their phenolic and vitamin C compositionrdquo Free RadicalResearch vol 36 no 2 pp 217ndash233 2002

[17] Y L Liu L H Tang Z Q Liang B You and S Yang ldquoGrowthinhibitory and apoptosis inducing by effects of total flavonoidsfrom Lysimachia clethroides Duby in human chronic myeloidleukemiaK562 cellsrdquo Journal of Ethnopharmacology vol 131 no1 pp 1ndash9 2010

[18] W Naowaratwattana W De-Eknamkul and E G De MejialdquoPhenolic-containing organic extracts of mulberry (Morus albaL) leaves inhibit HepG2 hepatoma cells through G2M phasearrest and inhibition of topoisomerase II120572 activityrdquo Journal ofMedicinal Food vol 13 no 5 pp 1045ndash1056 2010

[19] K Doi T Kojima and Y Fujimoto ldquoMulberry leaf extract inhi-bits the oxidative modification of rabbit and human low densitylipoproteinrdquo Biological and Pharmaceutical Bulletin vol 23 no9 pp 1066ndash1071 2000

[20] W Wang Y Zu Y Fu and T Efferth ldquoIn Vitro antioxidant andantimicrobial activity of extracts from Morus alba L leavesstems and fruitsrdquoAmerican Journal of ChineseMedicine vol 40no 2 pp 349ndash356 2012

[21] B Enkhmaa K Shiwaku T Katsube et al ldquoMulberry (Morusalba L) leaves and their major flavonol quercetin 3-(6-malon-ylglucoside) attenuate atherosclerotic lesion development inLDL receptor-deficient micerdquo Journal of Nutrition vol 135 no4 pp 729ndash734 2005

[22] J Naowaboot P Pannangpetch V Kukongviriyapan B Kongy-ingyoes and U Kukongviriyapan ldquoAntihyperglycemic antiox-idant and antiglycation activities of mulberry leaf extract instreptozotocin-induced chronic diabetic ratsrdquo Plant Foods forHuman Nutrition vol 64 no 2 pp 116ndash121 2009

[23] MM Padilha F CVilela CQ Rocha et al ldquoAntiinflammatoryproperties of Morus nigra leavesrdquo Phytotherapy Research vol24 no 10 pp 1496ndash1500 2010

[24] P Aramwit O Supasyndh T Siritienthong and N BangldquoMulberry leaf reduces oxidation and c-reactive protein levelin patients with mild dyslipidemiardquo BioMed Research Interna-tional vol 2013 Article ID 787981 7 pages 2013

[25] U Justesen P Knuthsen and T Leth ldquoQuantitative analysis offlavonols flavones and flavanones in fruits vegetables andbeverages by high-performance liquid chromatography withphoto-diode array and mass spectrometric detectionrdquo Journalof Chromatography A vol 799 no 1-2 pp 101ndash110 1998

[26] M S Taga E EMiller andD E Pratt ldquoChia seeds as a source ofnatural lipid antioxidantsrdquo Journal of the AmericanOil ChemistsrsquoSociety vol 61 no 5 pp 928ndash931 1984

[27] A A L Ordonez J D Gomez M A Vattuone and M I IslaldquoAntioxidant activities of Sechium edule (Jacq) Swartz extractsrdquoFood Chemistry vol 97 no 3 pp 452ndash458 2006

[28] A Kumaran and R Joel Karunakaran ldquoIn Vitro antioxidantactivities of methanol extracts of five Phyllanthus species fromIndiardquo LWT-Food Science and Technology vol 40 no 2 pp344ndash352 2007

[29] G Cao H M Alessio and R G Cutler ldquoOxygen-radical absor-bance capacity assay for antioxidantsrdquo Free Radical Biology andMedicine vol 14 no 3 pp 303ndash311 1993

[30] K Shimada K Fujikawa K Yahara and T Nakamura ldquoAntiox-idative properties of xanthan on the autoxidation of soybeanoil in cyclodextrin emulsionrdquo Journal of Agricultural and FoodChemistry vol 40 no 6 pp 945ndash948 1992

[31] J Cole M H L Green S E James L Henderson and H ColeldquoA further assessment of factors influencing measurements ofthioguanine-resistant mutant frequency in circulating T-lymphocytesrdquo Mutation Research vol 204 no 3 pp 493ndash5071988

[32] C-Y Hsu C-M Yang C-M Chen P Chao and S Hu ldquoEff-ects of chlorophyll-related compounds on hydrogen peroxideinduced DNA damage within human lymphocytesrdquo Journal ofAgricultural and Food Chemistry vol 53 no 7 pp 2746ndash27502005

[33] S-C Tang H-F Lo K-H Lin et al ldquoThe antioxidant capacityof extracts from Taiwan indigenous purple-leaved vegetablesrdquoJournal of the Taiwan Society for Horticultural Science vol 59no 1 pp 43ndash57 2013

[34] K-H Lin Y-Y Yang H-F Lo et al ldquoAntioxidant activity ofherbaceous plant extracts protect against hydrogen peroxide-induced DNA damage in human lymphocytesrdquo BioMed CentralResearch Notes In press

[35] A H Cory T C Owen J A Barltrop and J G Cory ldquoUse of anaqueous soluble tetrazoliumformazan assay for cell growthassays in culturerdquoCancer Communications vol 3 no 7 pp 207ndash212 1991

[36] Y T Szeto A R Collins and I F F Benzie ldquoEffects of dietaryantioxidants on DNA damage in lysed cells using a modifiedcomet assay procedurerdquoMutation Research vol 500 no 1-2 pp31ndash38 2002

[37] S Vielma G Virella A J Gorod and M F Lopes-VirellaldquoChlamydophila pneumoniae infection of human aortic endo-thelial cells induces the expression of FC 120574 receptor II (Fc120574RII)rdquoClinical Immunology vol 104 no 3 pp 265ndash273 2002

[38] C Y Zhu and S Loft ldquoEffects of Brussels sprouts extracts onhydrogen peroxide-induced DNA strand breaks in humanlymphocytesrdquo Food and Chemical Toxicology vol 39 no 12 pp1191ndash1197 2001

[39] Y D Min C-H Choi H Bark et al ldquoQuercetin inhibits exp-ression of inflammatory cytokines through attenuation of NF-120581B and p38MAPK in HMC-1 humanmast cell linerdquo Inflamma-tion Research vol 56 no 5 pp 210ndash215 2007

[40] P Y Chao Y P Huang and W B Hsieh ldquoInhibitive effect ofpurple sweet potato leaf extract and its components on celladhesion and inflammatory response in human aortic endothe-lial cellsrdquo Cell Adhesion amp Migration vol 7 no 2 pp 1ndash9 2013

[41] Y Y YangThe antioxidative capacity in herb plant extracts andtheir protection role in DNA oxidative damage of lymphocyte[MS thesis] Chinese Culture University Taipei Taiwan 2004

[42] C A Rice-Evans N J Miller and G Paganga ldquoStructure-anti-oxidant activity relationships of flavonoids and phenolic acidsrdquo

14 Evidence-Based Complementary and Alternative Medicine

Free Radical Biology and Medicine vol 20 no 7 pp 933ndash9561996

[43] M Strube G R Haenen H van den Berg and A Bast ldquoPitfallsin a method for assessment of total antioxidant capacityrdquo FreeRadical Research vol 26 no 6 pp 515ndash521 1997

[44] P G Pietta ldquoFlavonoids as antioxidantsrdquo Journal of NaturalProducts vol 63 no 7 pp 1035ndash1042 2000

[45] A M Gonzalez-Paramas S Esteban-Ruano C Santos-BuelgaSDe Pascual-Teresa and J C Rivas-Gonzalo ldquoFlavanol contentand antioxidant activity in winery byproductsrdquo Journal ofAgricultural and Food Chemistry vol 52 no 2 pp 234ndash2382004

[46] S Y Kim J J Gao and H K Kang ldquoTwo flavonoids from theleaves ofMorus alba induce differentiation of the human prom-yelocytic leukemia (HL-60) cell linerdquo Biological and Pharma-ceutical Bulletin vol 23 no 4 pp 451ndash455 2000

[47] M A Khan A A Rahman S Islam et al ldquoA comparative studyon the antioxidant activity ofmethanolic extracts from differentparts of Morus alba L (Moraceae)rdquo BMC Research vol 6 pp24ndash32 2013

[48] A-L ChungH-F Lo K-H Lin et al ldquoStudy on the antioxidantactivity in herb plant extractsrdquo Journal of the Taiwan Society ForHorticultural Science vol 59 no 2 pp 139ndash152 2013

[49] R L Prior X Wu and K Schaich ldquoStandardized methods forthe determination of antioxidant capacity and phenolics infoods and dietary supplementsrdquo Journal of Agricultural andFood Chemistry vol 53 no 10 pp 4290ndash4302 2005

[50] S J Duthie A R Collins GGDuthie andV LDobson ldquoQue-rcetin and myricetin protect against hydrogen peroxide-indu-ced DNA damage (strand breaks and oxidised pyrimidines) inhuman lymphocytesrdquo Mutation Research vol 393 no 3 pp223ndash231 1997

[51] G A Liu and R L Zheng ldquoProtection against damagedDNA inthe single cell by polyphenolsrdquo Pharmazie vol 57 no 12 pp852ndash854 2002

[52] M Noroozi W J Angerson and M E J Lean ldquoEffects of flav-onoids and vitamin C on oxidative DNA damage to humanlymphocytesrdquoAmerican Journal of Clinical Nutrition vol 67 no6 pp 1210ndash1218 1998

[53] T Koga and M Meydani ldquoEffect of plasma metabolites of (+)-catechin and quercetin on monocyte adhesion to human aorticendothelial cellsrdquoAmerican Journal of Clinical Nutrition vol 73no 5 pp 941ndash948 2001

[54] CMeng P Somers L Hoong et al ldquoDiscovery of novel pheno-lic antioxidants as inhibitors of vascular cell adhesionmolecule-1 expression for use in chronic inflammatory diseasesrdquo Journalof Medicinal Chemistry vol 47 no 25 pp 6420ndash6432 2004

[55] Y Shibata N Kume H Arai et al ldquoMulberry leaf aqueousfractions inhibit TNF-120572-induced nuclear factor 120581B (NF-120581B)activation and lectin-like oxidized LDL receptor-1 (LOX-1)expression in vascular endothelial cellsrdquoAtherosclerosis vol 193no 1 pp 20ndash27 2007

[56] K C Chan H H Ho C N Huang M Lin H Chen and CWang ldquoMulberry leaf extract inhibits vascular smooth musclecell migration involving a block of small GTPase and AktNF-kB signalsrdquo Journal of Agricultural and Food Chemistry vol 57no 19 pp 9147ndash9153 2009

[57] F Chen N Nakashima I Kimura M Kimura N Asanoand S Koya ldquoPotentiating effects on pilocarpine-induced salivasecretion by extracts and N-containing sugars derived frommulberry leaves in streptozocin- diabetic micerdquo Biological andPharmaceutical Bulletin vol 18 no 12 pp 1676ndash1680 1995

[58] K Konno H Ono M Nakamura et al ldquoMulberry latexrich in antidiabetic sugar-mimic alkaloids forces dieting oncaterpillarsrdquo Proceedings of the National Academy of Sciences ofthe United States of America vol 103 no 5 pp 1337ndash1341 2006

[59] H J Kim K H Yoon M J Kang et al ldquoA six-month sup-plementation of mulberry Korean red ginseng and banabadecreases biomarkers of systemic low-grade inflammation insubjects with impaired glucose tolerance and type 2 diabetesrdquoEvidence-based Complementary and Alternative Medicine vol2012 Article ID 735191 8 pages 2012

[60] G Nicholson and G M Hall ldquoDiabetes mellitus new drugs fora new epidemicrdquo British Journal of Anaesthesia vol 107 no 1pp 65ndash73 2011

[61] P Y Chao Y L Chen Y C Lin et al ldquoEffects of black soybeanon atherogenic prevention in hypercholesterolemic rabbits andon adhesion molecular expression in cultured HAECsrdquo Foodand Nutrition Sciences vol 4 pp 9ndash21 2013

[62] C Chen S Li C-YOChen et al ldquoConstituents in purple sweetpotato leaves inhibit in vitro angiogenesis with opposite effectsex vivordquo Nutrition vol 27 no 11-12 pp 1177ndash1182 2011

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

4 Evidence-Based Complementary and Alternative Medicine

assay [35] The adhesion assays were performed as describedby Zhu and Loft [38] Briefly U937 cells were labeledwith a fluorescent dye incubated with 10 120583molL 27-bis (2-carboxyethyl)-5 (6)-carboxyfluorescein acetoxymethyl esterat 37∘C for 1 h in RPMI-1640 medium and subsequentlywashed by centrifugation ConfluentHAECs in 24-well plateswere incubated with U937 cells (106 cellsmL) at 37∘C for1 h Nonadherent leukocytes were removed The numbers ofadherent leukocytes were determined by photographing andcounting four randomly chosen fields per well at 100x usinga Zeiss Axio Mager Z1 Upright Fluorescence MicroscopeExperiments were performed in triplicate and repeated threetimes HAECswere incubatedwith 25 120583g and 50 120583g ofMLREsor 10 120583M aspirin for 18 h followed by treating with 2 ngmLTNF-120572 for 6 h in theNF-120581Bp65 AP-1 and STAT-3 expressionassays

213 Nuclear Protein Isolation Protein extracts were pre-pared as described by Min et al [39] Briefly after cellactivation for the indicated times cells were washed in 1mLice-cold PBS centrifuged at 400 g for 5min resuspendedin 400 120583L ice-cold hypotonic buffer (10mM HEPES 15mMMgCl

2 01mM EDTA 10mM KCl 1mM DTT 05mM

PMSF pH79) incubated on ice for 10min vortexed and cen-trifuged at 15000 g for 30 sec The supernatant was collectedand stored at minus70∘C for cytosolic protein analysis Pelletednuclei were gently resuspended in 445 120583L ice-cold extractionbuffer (20mM HEPES pH 79 with 15mM MgCl

2 042M

NaCl 02mM EDTA and 25 glycerol) with 5 120583L of 10mMDTT and 05 120583L of Protease Inhibitor Cocktail (Sigma) incu-bated on ice for 20min vortexed and centrifuged at 15000 gfor 5min at 4∘C Aliquots of the supernatant containingnuclear proteins were frozen in liquid nitrogen and stored atminus70∘C

214 Western Blot Analysis Nuclear lysates were subjected to12 SDS-PAGE (Bio-Rad Laboratories) after which proteinswere transferred to a PVDF membrane (Bio-Rad) Mem-branes were probed with a mouse monoclonal NF-120581B p65antibody (BD Biosciences) After incubation in a secondaryantibody solution consisting of IRDye 700CW-conjugatedgoat anti-mouse IgG (LI-COR Biosciences) for 60min atroom temperature with gentle shaking the membrane waswashed four times for 5min each at room temperature in PBSwith 01 Tween-20 and while being gently shaken After afinal rinse the membrane was scanned using AlphaEaseFCimage analysis software (Alpha Innotech Corporation) toanalyze spot density The internal control was set at 100 todetermine the relative density of protein expression [40]

215 Electrophoretic Mobility Shift Assay for NF-120581B AP-1 andSTAT3 The binding reaction consisted of 1120583L 10times bindingbuffer (100mM TRIS 500mM NaCl 10mM DTT pH 75)5 120583LH

2O 2120583L 25mMDTT25Tween-20 1 120583L IRDye 700-

labeled EMSA oligonucleotides specific for NF-120581B (51015840-AGTTGA GGG GAC TTT CCC AGG C-31015840 and 31015840-CGC TTGATG ACT CAG CCG GAA-51015840) AP-1 (51015840-CGC TTG ATGACT CAG CCG GAA-31015840 and 31015840-GCG AAC TAC TGA GTC

GGC CTT-51015840) STAT3 (51015840-GAT CCT TCT GGG AAT TCCTAG ATC-31015840 and 31015840-CTA GGA AGA CCC TTA AGG ATCTAG-51015840) (Sigma) PPAR-120572 (51015840-AAA AAC TGG GCC AAAGGT CT-31015840) and PPAR-120574 (51015840-TGA AAC TAG GGT AAAGTT CA-31015840) (Sigma) 1 120583L poly (dIsdotdC) and 1 120583L nuclearextract (as prepared previously) The mixture was incubatedat room temperature for 20min in the dark After 1x OrangeLoading Dye (LI-COR Biosciences) was added the bindingreaction was loaded onto a native 4 polyacrylamide gel andseparated by electrophoresis at 90 vDC for 40min The gelswere scanned using anOdyssey Infrared Imaging System (LI-COR Biosciences) [40]

216 Statistical Analysis Data are expressed as the mean plusmnstandard deviation and statistical significance was analyzedusing one-way ANOVA followed by the Tukeyrsquos Range Testat the 005 significance level Pearsonrsquos linear correlation wasalso determined The means of three replicates are reported

3 Result

31 Antioxidant Composition and Antioxidant Activity Poly-phenols flavonoids and flavonols were abundant in BTat levels of 3465 plusmn 082mgg DW gallic acid 8483 plusmn868mgg DW quercetin equivalent and 2547 plusmn 386mggDW quercetin equivalent respectively (Table 1) Anthocy-anidins were abundant in BT and ML at levels of 14332 plusmn181 and 13582 plusmn 031 unitsgDW respectively Table 2 showsthat quercetin and kaempferol were both rich in BTat levelsof 285333 plusmn 18037 and 64667 plusmn 5033 120583ggDW respectivelywhile the levels of M were 166667 plusmn 18903 and 42667 plusmn4619 120583ggDW respectively However no myricetin or morinwas detected in the MLRC (Table 2) Thus these MLREsdisplayed variations in their antioxidant substances levels

MLREs showed antioxidant activity proving their capac-ity to scavenge the ABTS radical cation The antioxidantactivity in acidic methanolic hydrolysate sample extracts wasexpressed in trolox equivalent antioxidant capacity (TEAC)(Table 3) BT showed a significantly higher TEAC value(15514 plusmn 290 120583ggDW) than MT (10239 plusmn 095 120583ggDW)and M (7654 plusmn 161 120583ggDW) TEAC values were positivelyand significantly (119903 = 041 119875 = 0034) correlated withthe content of quercetin among MLREs (Table 4) Thusdifferent quercetin contents displayed various levels of anti-oxidant activity However M had a significantly high effi-cacy for DPPH radical scavenging activity (IC

5018883 plusmn

361 120583gmL) compared to BT (IC5066901plusmn723 120583gmL) and

MT (IC5083640 plusmn 708 120583gmL) (Table 3) Hence each tested

sample showed significant differences in scavenging of DPPHradical at IC

50

32 Effects of Acidic Methanolic and Water Extracts fromMLRC on H

2O2-Induced DNA Damage to Lymphocytes The

effects ofMLRC extracts on cell cytotoxicity were determinedby theMTTassay Lymphocyteswere exposed to each of threedifferent MLRC extracts at three concentrations (25 50 and100 120583gmL) for 30min at 37∘C DNA damage was inducedby exposing lymphocytes to H

2O2(10 120583M) for 5min on ice

Evidence-Based Complementary and Alternative Medicine 5

Table 1 The contents of various antioxidant substances in MLRC

Polyphenolmg gallic acidgDW

Flavonoids mg quercetinequivalentgDW

Flavonol mg quercetinequivalengDW

AthocyanidinunitgDW

BT 3465 plusmn 082a

8483 plusmn 868a

2547 plusmn 386a

14332 plusmn 181a

MT 2323 plusmn 054b

5599 plusmn 1055b

1326 plusmn 266b

8858 plusmn 412b

M 1987 plusmn 061c

4960 plusmn 541b

1422 plusmn 148b

13582 plusmn 031a

All values are means plusmn SD (119899 = 3)Means within a column with different superscripts (asimc) are significantly different 119875 lt 005

Table 2 The contents of flavonols in acidic methanolic extracts ofMLRC

Flavonols (120583ggDW)Samples Myricetin Morin Quercetin KaempferolBT ND ND 285333 plusmn 18037

a64667 plusmn 5033

a

MT ND ND 37333 plusmn 2309c ND

M ND ND 166667 plusmn 18903b42667 plusmn 4619

b

ND not detected

Table 3 TEAC values and the IC50 of DPPH radical scavengingactivity of acidic methanolic hydrolysates of MLRC

Sample TEAC(Trolox mM)lowast

DPPH radical scavenging activityIC50 (120583gmL)

BT 15514 plusmn 290a

66901 plusmn 723b

MT 10239 plusmn 095b

83640 plusmn 708a

M 7654 plusmn 161c

18883 plusmn 361c

lowastTEAC value at sample 100120583gmL concentration

Table 4 Correlation between antioxidant substances and TEACvalues treated with acid hydrolysates of MLRC

FlavonolsMyricetin Morin Quercetin Kaempferol

TEAClowast mdash mdash 119903 = 041

119875 = 0034

mdash

mdash no significant correlationlowastTEAC values at 100 120583gmL of sample concentration

No mulberry extracts were cytotoxic at the concentrationsused with gt98 of cells remaining viable [41] The cometassay was performed to determine the DNA damagingactivity of the plants because it is a sensitive method formonitoring single strand DNA breaks at the single celllevel Any DNA damage that occurred is represented as atail length (tail migration) of the DNA strand The effectsof pretreatment with acidic methanolic extracts of MLRCon 10 120583M H

2O2-induced DNA oxidative damage in human

lymphocytes are presented in Figure 1 indicating that therewas a protective effect of pretreatment on lymphocytes witheach of the MLRC extracts at the lowest dose (25 120583gmL)Lymphocytes pretreated with 25ndash100120583gmL extracts of MTextracts experienced a greater level of protection againstH2O2exposure than lymphocytes exposed to other tested

compounds and in a dose-dependent manner BT had betterinhibition efficacy at lower concentration (25120583gmL) andthe maximum protective effect of lymphocyte pretreatmentwas observed with pretreatment by 25 120583gmL BT exhibiting

1462 of tail DNA compared to treatment only withH2O2

MT had better inhibition efficacy (8439) than M and BTextracts at the 100 120583gmL level (Figure 1(a)) Tested plantsshowed at least 134263 of tail moment in BT extract at the25 120583gmL level while the BT extract at 100 120583gmL had thehighest tail moment (328193) compared to the rest of theacidic methanolic extract samples (Figure 1(b)) Moreoverthe trend in tail moment in all samples continued climbingup from 25ndash100 120583gmL

The effects of various water extracts of MLRC H2O2-

induced DNA damage to lymphocytes are presented inFigure 2 At lower concentrations all tested samples hadlower tail DNA indicating better inhibition efficacies(Figure 2(a)) BT and M had the best inhibition efficacieswith both exhibiting values of 1422 and 1469 tail DNAat 25 120583gmL respectively Moreover MT had a higher tailDNA than the other extracts at the same dose and thehighest tail DNA was observed in 3212 of MT at aconcentration of 100120583gmL The tail moment of all samplescontinued to increase up to the 100 120583gmL level (Figure 2(b))The minimum tail moment occurred in M at 153674 in the25 120583gmL extracts Moreover the DNA damage induced byH2O2was significantly higher than the DNA damage treated

with extracts and H2O2treated had 8726 in tail DNA and

832884 in tail moment

33 Effects of MLREs on Monocyte-Endothelial Cell Adhe-sion As shown in Figure 3 pretreatment of HAECs with100 120583gmLMLREs for 18 h significantly suppressed the adhe-sion ofU937monocytes to TNF-120572-stimulatedHAECs similarto that of 10 120583Maspirin (positive control) Data also show that25 and 50 120583gmL BT MT and M methanol extracts inhib-ited the adhesion of U937 monocytes to TNF-120572-stimulatedHAECs by 8198 and 8187 (BT) 7449 and 8331 (MT)and 8011 and 7559 (M) respectively

34 Effects of MLREs on NF-120581B Activity and MLREs Inhibitsthe Binding of NF-120581B AP-1 and STAT3 Figure 4 demon-strates that MLREs regulated the DNA-binding activity andexpressions of NF-120581B in HAECs In untreated HAECs NF-120581B p65 was only found within the cytosol however itsnuclear translocation was observed upon treatment withTNF-120572 Compared to the TNF-120572 group pretreatment withBT and MT methanol extracts significantly decreased theexpression of NF-120581B p65 in the nuclear compartment whilethe M methanol extract did not reduce the expressionof NF-120581B p65 activity Furthermore an EMSA analysiswas carried out to determine if the reduced expression of

6 Evidence-Based Complementary and Alternative Medicine

Control H2O2

BT25 BT50 BT100

MT25 MT50 MT100

M25 M50 M100

(a)

0

20

40

60

80

100

25 50 100

Tail

DN

A (

)

Mulberry extracts (120583gmL)

lowast lowastlowast

lowast

lowastlowast

alowast alowast

blowast

MTMBT

H2O2

(b)

Tail

mom

ent

0

2000

4000

6000

8000

10000

25 50 100Mulberry extracts (120583gmL)

lowastlowastlowast

ablowastclowast

alowast

alowast

alowastblowast

MTMBT

H2O2

(c)

Figure 1 Effects of various acidic methanolic extracts of MLRC on H2O2-induced DNA damage to lymphocytes The comet-like images

resulting from the extension of DNA (a) were observed using a fluorescence microscope Tail DNA (b) and tail movement (c) were exposedto test mulberry leaf-related extracts at 25 50 and 100 120583gmL and treated with H

2O2at 10120583M Values with different letters differ significantly

with regard to oxidative damage when comparing different MLRC extracts lowast119875 lt 005 refers to differences in oxidative damage as comparedwith 10 120583MH

2O2-alone treatment

Evidence-Based Complementary and Alternative Medicine 7

Control H2O2

BT25 BT50 BT100

MT25 MT50 MT100

M25 M50 M100

(a)

alowastblowast blowastblowast

blowastblowast

blowast

25 50 100Mulberry extracts (120583gmL)

MTMBT

H2O2

0

20

40

60

80

100

Tail

DN

A (

)

ablowastablowast

(b)

Tail

mom

ent

0

2000

4000

6000

8000

10000

lowastlowast lowast

alowastalowast

25 50 100Mulberry extracts (120583gmL)

MTMBT

H2O2

ablowast ablowastablowast

bclowast

(c)

Figure 2 Effects of various water extracts of MLRC on H2O2-induced DNA damage to lymphocytes The comet-like images resulting from

the extension ofDNA (a) were observed using a fluorescencemicroscope Tail DNA (b) and tailmovement (c) were exposed to testmulberryleaf-related extractsat 25 50 and 100120583gmL and treated with H

2O2at 10120583M Values with different letters differ significantly with regard to

oxidative damage when comparing different MLRC extracts lowast119875 lt 005 refers to differences in oxidative damage as compared with 10120583MH2O2-alone treatment

NF-120581B in response to treatment with MLREs resulted inreduced binding of NF-120581B to DNAThe treatment of HAECswith TNF-120572 led to increased binding of NF-120581B howeverpretreatment of HAECs with BT MT and M significantlydecreased DNA-bound NF-120581B (Figure 5(a)) The increased

binding of AP-1 with TNF-120572 resulted from the treatment ofHAECs however pretreatment of HAECs with BT MT andM significantly decreased DNA-bound AP-1 (Figure 5(b))STAT3 DNA-binding activities to its cognate recognitionsite were remarkably reduced compared to TNF-120572 treatment

8 Evidence-Based Complementary and Alternative Medicine

(A)(A)

(B)

(C) (D)

(E) (F)

(I)

(G) (H)

200120583m 200120583m

200120583m200120583m

200120583m200120583m

200120583m200120583m

200120583m

(a)

Con

trol

Asp

irin

BT25

BT50

MT2

5

MT5

0

M25

M50

Mon

ocyt

e adh

eren

ce (c

ells

HPF

)

0

20

40

60

80

100

120

140

160

180

lowast lowast lowast

lowast

lowast

lowast

lowast

TNF-120572

(b)

Figure 3 Mulberry leaf related extracts (MLREs) reduce tumor necrosis factor (TNF)-120572-induced adhesion of monocytes to human aorticendothelial cells (HAECs) (a) The number of adherent U937 cells (marked by arrow) was monitored by fluorescence microscopy undercontrol (a) 2 ngmL TNF-120572 (b) co-treated with TNF-119886 and aspirin (10120583M) (c) cotreated with TNF-120572 and BT (25 120583gmL) (d) cotreated withTNF-120572 and BT (50 120583gmL) (e) cotreated with TNF-120572 and MT (25 120583gmL) (f) cotreated with TNF-120572 and MT (50 120583gmL) (g) cotreated withTNF-120572 and M (25120583gmL) (h) and cotreated with TNF-120572 and M (50120583gmL) (i) (b) HAECs were pretreated with MLREs for 18 h followedby stimulating with TNF-120572 for 6 h Fluorescence-labeled monocytic U937 cells were added to HAECs and allowed to adhere for 2 h Data areexpressed as a percentage of TNF-120572-induced expression (mean plusmn SD) of three experiments Mean plusmn SD lowast119875 lt 005 BT MT and M at 25and 50120583gmL versus TNF-120572

119875 lt 005 control versus TNF-120572

Evidence-Based Complementary and Alternative Medicine 9

Control TNF-120572

TNF-120572

Aspirin10120583M

+ + + + + + + +

BT25 BT50 MT25 MT50 M25 M50

120583gmL 120583gmL 120583gmL 120583gmL 120583gmL 120583gmL

NF-120581B p65

120573-Actin43kDa

minus

65kDa

(a)

Con

trol

Asp

irin

BT25

BT50

MT2

5

MT5

0

M25

M50

00

02

04

06

08

10

lowast

lowast

lowast

lowastlowastlowastTN

F-120572

Ratio

(nuc

lear

120573-a

ctin

)

(b)

Figure 4 Effects of MLREs on NF-120581B activity After HAECs were pretreated with the indicated samples and incubated with TNF-120572 nuclearextracts were prepared and the expression of NF-120581B p65 was assessed by western blot analysis A representative image of three similar resultsis shown (a) Actin served as the loading control for the nuclear compartment Semiquantitative analysis of three independent experimentsare also shown (b) indicates a significant difference between the TNF-120572 treated or experimental treatment groups and the control119875 lt 005lowast indicates a significant difference between the TNF-120572 and experimental treatment groups BT MT and M at 25 and 50 120583gmL

(lane 3) in the nuclear fraction indicating that MLREs at50120583gmL inhibited STAT3-binding activities In addition theresults in Figure 5(c) (lanes 4ndash10) show that aspirinMLREs at50120583gmL and BT at 25120583gmL remarkably decreased STAT3levels compared to TNF-120572 (lane 3) however both MT andM at 25120583gmL increased STAT3-binding activities Theseresults indicate that only 50 120583gmL MLREs 25 120583gmL BTand aspirin block STAT3 activation and might downregulateTNF-120572-induced expressions of inflammatory signaling path-ways

35 MLREs Regulated the DNA-Binding Activity and Expres-sions of PPAR 120572 and PPAR 120574 in HAECs PPAR 120572 and 120574effectively bound to PPAR 120572 and 120574 oligo-IRDye 700 asindicated in lanes 3ndash10 of Figure 6 Supershifts are bands fromthe binding of each specific antibody The binding activitiesof PPAR 120572 and 120574 were detected at baseline The 25 120583gmLMT extract (lane 7) and 25 and 50 120583gmL M extracts (lanes9-10) increased activity compared to incubating with TNF-120572 alone (lane 3) However BT at both 25 and 50 120583gmLextracts showed reduced effects on PPAR 120572 and 120574 expressionMoreover the 50 120583gmLMT extract had no effect on PPAR 120572binding activity but increased the binding activity of PPAR120574 in comparison to incubation with TNF-120572 alone Thesedata suggest that MT and M both promoted PPAR 120572 and 120574

expression at least in part by increasing the DNA binding oftranscription factors PPAR 120572 and 120574

4 Discussion

41 Antioxidant Composition and Antioxidant Activity BTcontains high levels of polyphenols flavonoids flavonolsanthocyanidin quercetin and kaempferol (Tables 1 and 2)BT also had a higher TEAC value (15514mM Table 3) thanthe TEAC values of 47mM [42] and 64mM [43] reportedfor quercetin as well as 242mM reported for quercetin-3-rutinoside [44] Furthermore Gonzalez-Paramas et al [45]demonstrated that flavonol contents were significantly andhighly correlated (119903 = 08) with TEAC values Kim et al [46]reported that mulberry leaves contain abundant quercetinand inhibit lipid peroxidation at concentrations of 01 and001mgmL In our study the amount of quercetin wassignificantly correlated (119903 = 041 Table 4) with TEAC valueHowever no correlation was found among myricetin morinkaempferol and TEAC which may be due to the structurerequired to reinforce the free radical scavenging activity thatvaries with the type of free radical Naowaratwattana et al[18] reported that a 50 methanolic extract of mulberryleaf contained rutin isoquercetin and various derivatives ofkaempferol and quercetin glycosides while a water extract

10 Evidence-Based Complementary and Alternative Medicine

NF-120581BTNF-120572

TNF-120572

+

Aspirin10120583M+ +

BT25120583gmL

+

BT50120583gmL

+

MT25120583gmL

+

MT50120583gmL

+

M25

120583gmL+

M50

120583gmLControl

minus

DNAOligo

Free probe

(a)

AP-1TNF-120572

TNF-120572

+

Aspirin10120583M+ +

BT25120583gmL

+

BT50120583gmL

+

MT25120583gmL

+

MT50120583gmL

+

M25

120583gmL+

M50

120583gmLControl

minus

DNAOligo

Free probe

(b)

STAT3

TNF-120572

TNF-120572

+

Aspirin10120583M+ +

BT25120583gmL

+

BT50120583gmL

+

MT25120583gmL

+

MT50120583gmL

+

M25

120583gmL+

M50

120583gmLControl

minus

DNAOligo

Free probe

(c)

Figure 5 MLREs inhibits the binding of (a) NF-120581B (b) AP-1 and (c) STAT3 to target DNA sequence NF-120581B AP-1 and STAT3 transcriptionfactor-DNA interactions were assessed by EMSA

contained primarily chlorogenic acid and caffeoylquinic acidderivatives The IC

50of DPPH radical scavenging was 794

and 2042120583gmL for 50 methanolic and water extractsrespectively [18] In our studyM extracts contained quercetinand kaempferol (Table 2) which may account for part ofthe highly scavenged DPPH radical in comparison to therest of MLREs Khan et al [47] demonstrated that the IC

50

was 22023120583gmL which is much less efficacious than themulberry leaf acidic methanolic extract IC

50of 1888 120583gmL

(Table 3) However the IC50

of in vitro free radical scav-enging activity of mulberry leaves from Naowaboot et al[22] was 617 plusmn 21 120583gmL which is more efficacious thanthe IC

50concentration derived in the present study Taken

together the polyphenol-rich MLREs exhibited distinct cell-free antioxidant activity (ie TEAC) as validated by theirlevels of polyphenols and flavonols with distinct antioxidantactivity strongly accounting for the antioxidant activity of the

extracts The amount of quercetin was especially positivelycorrelated with the observed TEAC values

42 Estimation of DNA Single Strand Break Damage fromExposure to Acidic Methanolic and Water Extracts One ofthe antioxidant activity tests scavenging of DPPH radi-cal was selected due to its quick and simple but it is aless biologically relevant assay Previously we reported thatwith 2 120583gsdotmLminus1 of methanolic and water extracts from theindigenous vegetables [33] and herb plants [48] significantlyprolonged lag phase of low-density lipoprotein (LDL) TheLDL oxidation assay is considered to be one of the mostbiologically relevant assays [49] Both studies [33 48] alsoshowed that the concentration of the tested extracts forscavenging DPPH radical was in higher levels at 10mg oftested extracts with 2ndash53 inhibition Lower concentrationwas then chosen for the treated dosage in the present study

Evidence-Based Complementary and Alternative Medicine 11

TNF-120572

TNF-120572

+

Aspirin10120583M+ +

BT25120583gmL

+

BT50120583gmL

+

MT25120583gmL

+

MT50120583gmL

+

M25

120583gmL+

M50

120583gmLControl

minus

DNAOligo

Free probe

PPAR 120572

(a)

TNF-120572

TNF-120572

+

Aspirin10120583M+ +

BT25120583gmL

+

BT50120583gmL

+

MT25120583gmL

+

MT50120583gmL

+

M25

120583gmL+

M50

120583gmLControl

minus

DNAOligo

Free probe

PPAR120574

(b)

Figure 6 Effect of treatments on the expression of PPAR 120572 (a) and PPAR 120574 (b) in TNF-120572-stimulated HAECs by EMSA

In addition lower concentration also could avoid the toxicityeffect of higher level of extracts on lymphocytes Quercetinwas found to protect against H

2O2-induced DNA damage in

human lymphocytes at 10 120583M [50] and at 31 to 25 120583M [51]Quercetin was also reported to induce DNA strand breaks invarious cell types but only at higher doses at 100 120583Mor above[50] Duthie et al [50] proposed that the dihydroxy structureof quercetin and myricetin might be essential in protectingDNA against hydrogen peroxide No such hydroxyl groupsare present in the tocopherol molecule Noroozi et al [52]demonstrated that in addition to quercetin kaempferolcould also inhibit H

2O2-induced DNA strand breaks in

human lymphocytes In our study both BT and M were richin quercetin and kaempferol (Table 2) whichmay account forthe protective roles of both extracts in preventingDNAstrandbreaks from human lymphocytes

43 MLREs Regulated the DNA-Binding Activity and Expres-sions of NF-120581B AP-1 STAT3 and PPARs in HAECsBecause MLREs exhibited free radical scavenging the anti-inflammatory effects of MLREs on TNF-120572-induced celladhesion and adhesionmolecule expressionwere determinedin the present study Our results suggest that MLREs andaspirin significantly inhibited TNF-120572-induced monocyte-endothelial cell adhesion (Figure 3) Significant reductionsin NF-120581B expression and DNA binding by aspirin MTextract (25 120583gmL) and BT extract (both 25 and 50 120583gmL)were also observed The adhesion of monocytes to the

vascular endothelium and subsequent migration into thevessel wall are early events in atherogenesis [2] Our resultsare consistent with previous research showing that dietarypolyphenols such as catechin and quercetin significantlyreduce the binding of monocytes to HAECs [53 54] Thereduced cellular adhesion may be due to the inhibitionof cellular adhesion molecule expression as purple sweetpotato leaf extract (PSPLE) reduced both VCAM-1 andICAM-1 expression [40] Shibata et al [55] demonstratedthat pretreatment of cultured bovine aortic endothelial cells(BAECs) with mulberry leaf aqueous fractions inhibitedTNF-120572- and LPS-induced expression of Lectin-like Ox-LDLreceptor-1 (LOX-1) a cell-surface receptor for atherogenicOx-LDL (oxidized low-density lipoprotein) It also appearsto mediate Ox-LDL-induced inflammation which may becrucial in atherogenesis Furthermore mulberry leaf aqueousfractions inhibited the TNF-120572-induced activation of NF-120581Band phosphorylation of inhibitory factor of NF-120581B-alpha(I120581B-120572) Thus mulberry leaf aqueous fractions suppressTNF-120572- and LPS-induced LOX-1 gene expression mediatedby inhibiting NF-120581B activation

Chan et al [56] demonstrated that mulberry leaf extract(MLE) was rich in polyphenols MLE can effectively inhibitthe migration of vascular smooth muscle cells (VSMC) byblocking small guanosine triphosphatases (small GTPase)and AktNF-120581B signals The Rho family of small GTPasessuch as Rho Rac and Cdc42 acts as a molecular switch toregulate the actin cytoskeleton The Rho family of GTPases

12 Evidence-Based Complementary and Alternative Medicine

is also reported to be involved in the regulation of NF-120581B-dependent transcription [56] MLE has been shown to pos-sess hypoglycemic effects in an insulin-dependent diabetesmellitus (type 1 diabetes) animal model and potential hypo-glycemic compounds inmulberry leaves were suggested to be2-O-120572-D-galactopyranosyl-DNJ and fagomine [57] Konnoet al [58] further reported that mulberry latex containsalkaloidal sugar-mimic glycosidase inhibitors with antidia-betic activities such as 14-dideoxy-14-imino-D-arabinitol 1-deoxynojirimycin and 14-dideoxy-14-imino-D-ribitol Kimet al [59] recently showed that treated type 2 diabetesmellitus(T2D) with a 1 1 1 mixture of ginseng root mulberry leafwater extract and banana leaf water extract (6 gd) for 24weeks significantly decreased plasma intracellular adhesionmolecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) levels This indicates suppressed inflammatoryresponses in T2D but no significant differences in glucosehomeostasis control measurement changes Rigamonti et al[8] reported that bothPPAR120572 and 120574maybe thought of as sup-pressing the activity of NF120581B and AP-1 pathways that reduceinflammatory mediator production in the macrophages ofhumans and mice In our study MT and M promoted PPAR120572 and 120574 expression at least in part by increasing DNAbinding of transcription factors PPAR 120572 and 120574 PPAR-120572-and 120574-dependent signaling pathwaysmay also play importantroles in mediating the TNF-120572 receptor signaling pathwayby MT and M by suppressing the activity on NF-120581B andAP-1 pathways These pathways may regulate downstreamproinflammatory gene expression and adhesion molecularexpression Clinically PPAR 120574 agonists such as rosiglitazoneare widely used in the treatment of T2D [60] M and MTextracts increased PPAR 120574 activity which may partly explainwhy inflammatory responses are suppressed in T2D

Although reduced NF-120581B expression and DNA bind-ing were observed upon treatment with ML MT and BT(Figure 5) the mechanism was not assessed and is worthy offurther study In addition the present results were obtainedusing in vitro studies and may be consistent with an invivo model of atherogenesis similar to that reported byEnkhmaa et al [21] in which mulberry leaves suppressedthe development of atherosclerotic lesions Previously weproved that black beans significantly prolong LDL lag timeand decrease the atheroma regions of the aortic arch andthoracic aorta in hypercholesterolemic NZW rabbits [61]Wethen demonstrated that black soybean extract and PSPLEsignificantly decreased the adhesion of U937 monocyticcells to TNF-120572-stimulated HAECs and reduced adhesionmolecules expression (VCAM-1 and ICAM-1) as well as NF-120581B-p65 expression [37] This is particularly important giventhe opposing effects of flavonols fromMLREs obtained usingin vitro and ex vivo studies [62]

5 Conclusion

Tested MLREs were rich in flavonols and had strongantioxidant activity in protecting lymphocyte DNA fromoxidative damage In particular BT was rich in quercetinand polyphenols The antioxidant components of MLREs

might downregulate intracellular redox-dependent signalingpathways in HAECs upon TNF-120572 stimulation which mightprevent ROS-mediated endothelial cell dysfunction MLRCshave anti-inflammatory effects through the modulation ofNF-120581B AP-1 STAT3 and PPARs signaling These chemi-cals appear to provide distinct cytoprotective mechanismsthat might contribute to their putative beneficial effectsin suppressing endothelial responses to cytokines duringinflammation These results may benefit further in vivostudies to assess the therapeutic potential of phytochemicalsas anti-inflammatories for use in cytokine-induced vasculardisorders including atherosclerosis

Acknowledgment

This work was supported in part by Grants from the NationalScience Council of Taiwan (NSC 95ndash2320-B-034-001 andNSC 96ndash2320-B-034-001)

References

[1] W Palinski ldquoUnited they go conjunct regulation of aorticantioxidant enzymes during atherogenesisrdquo CirculationResearch vol 93 no 3 pp 183ndash185 2003

[2] P Libby ldquoMolecular bases of the acute coronary syndromesrdquoCirculation vol 91 no 11 pp 2844ndash2850 1995

[3] P A Baeuerle and D Baltimore ldquoNf-120581B ten years afterrdquo Cellvol 87 no 1 pp 13ndash20 1996

[4] J M Kyriakis ldquoActivation of the AP-1 transcription factor byinflammatory cytokines of the TNF familyrdquo Gene Expressionvol 7 no 4ndash6 pp 217ndash231 1999

[5] J M Muller R A Rupec and P A Baeuerle ldquoStudy of generegulation by NF-120581B and AP-1 in response to reactive oxygenintermediatesrdquoMethods vol 11 no 3 pp 301ndash312 1997

[6] G Chinetti J Fruchart and B Staels ldquoPeroxisome proliferator-activated receptors new targets for the pharmacological mod-ulation of macrophage gene expression and functionrdquo CurrentOpinion in Lipidology vol 14 no 5 pp 459ndash468 2003

[7] G Chinetti J C Fruchart and B Staels ldquoTranscriptional regu-lation of macrophage cholesterol trafficking by PPAR120572 andLXRrdquo Biochemical Society Transactions vol 34 no 6 pp 1128ndash1131 2006

[8] E Rigamonti G Chinetti-Gbaguidi and B Staels ldquoRegulationof macrophage functions by PPAR- 120572 PPAR- 120574 and LXRsin mice and menrdquo Arteriosclerosis Thrombosis and VascularBiology vol 28 no 6 pp 1050ndash1059 2008

[9] M A Bouhlel J Brozek B Derudas et al ldquoUnlike PPAR120574PPAR120572 or PPAR120573120575 activation does not promote humanmono-cyte differentiation toward alternative macrophagesrdquo Biochem-ical and Biophysical Research Communications vol 386 no 3pp 459ndash462 2009

[10] G Chinetti J-C Fruchart and B Staels ldquoPeroxisome prolife-rator-activated receptors and inflammation from basic scienceto clinical applicationsrdquo International Journal of Obesity vol 27supplement 3 pp S41ndashS45 2003

[11] Y Zhu J H-C Lin H Liao et al ldquoLDL induces transcriptionfactor activator protein-1 in human endothelial cellsrdquo Arte-riosclerosis Thrombosis and Vascular Biology vol 18 no 3 pp473ndash480 1998

Evidence-Based Complementary and Alternative Medicine 13

[12] J Terao ldquoDietary flavonoids as antioxidantsrdquo Forum of Nutri-tion vol 61 pp 87ndash94 2009

[13] Y B Feng W Q Luo and S Q Zhu ldquoExplore new clinicalapplication of Huanglian and corresponding compound pre-scriptions from their traditional userdquo Zhongguo ZhongyaoZazhi vol 33 no 10 pp 1221ndash1225 2008

[14] Y H Chu C L Chang and H F Hsu ldquoFlavonoid content ofseveral vegetables and their antioxidant activityrdquo Journal of theScience of Food andAgriculture vol 80 no 5 pp 561ndash566 2000

[15] I C Arts B van de Putte and P C Hollman ldquoCatechin con-tents of foods commonly consumed in The Netherlands 1Fruits vegetables staple foods and processed foodsrdquo Journal ofAgricultural and Food Chemistry vol 48 no 5 pp 1746ndash17512000

[16] A R Proteggente A S Pannala G Paganga et al ldquoThe anti-oxidant activity of regularly consumed fruit and vegetablesreflects their phenolic and vitamin C compositionrdquo Free RadicalResearch vol 36 no 2 pp 217ndash233 2002

[17] Y L Liu L H Tang Z Q Liang B You and S Yang ldquoGrowthinhibitory and apoptosis inducing by effects of total flavonoidsfrom Lysimachia clethroides Duby in human chronic myeloidleukemiaK562 cellsrdquo Journal of Ethnopharmacology vol 131 no1 pp 1ndash9 2010

[18] W Naowaratwattana W De-Eknamkul and E G De MejialdquoPhenolic-containing organic extracts of mulberry (Morus albaL) leaves inhibit HepG2 hepatoma cells through G2M phasearrest and inhibition of topoisomerase II120572 activityrdquo Journal ofMedicinal Food vol 13 no 5 pp 1045ndash1056 2010

[19] K Doi T Kojima and Y Fujimoto ldquoMulberry leaf extract inhi-bits the oxidative modification of rabbit and human low densitylipoproteinrdquo Biological and Pharmaceutical Bulletin vol 23 no9 pp 1066ndash1071 2000

[20] W Wang Y Zu Y Fu and T Efferth ldquoIn Vitro antioxidant andantimicrobial activity of extracts from Morus alba L leavesstems and fruitsrdquoAmerican Journal of ChineseMedicine vol 40no 2 pp 349ndash356 2012

[21] B Enkhmaa K Shiwaku T Katsube et al ldquoMulberry (Morusalba L) leaves and their major flavonol quercetin 3-(6-malon-ylglucoside) attenuate atherosclerotic lesion development inLDL receptor-deficient micerdquo Journal of Nutrition vol 135 no4 pp 729ndash734 2005

[22] J Naowaboot P Pannangpetch V Kukongviriyapan B Kongy-ingyoes and U Kukongviriyapan ldquoAntihyperglycemic antiox-idant and antiglycation activities of mulberry leaf extract instreptozotocin-induced chronic diabetic ratsrdquo Plant Foods forHuman Nutrition vol 64 no 2 pp 116ndash121 2009

[23] MM Padilha F CVilela CQ Rocha et al ldquoAntiinflammatoryproperties of Morus nigra leavesrdquo Phytotherapy Research vol24 no 10 pp 1496ndash1500 2010

[24] P Aramwit O Supasyndh T Siritienthong and N BangldquoMulberry leaf reduces oxidation and c-reactive protein levelin patients with mild dyslipidemiardquo BioMed Research Interna-tional vol 2013 Article ID 787981 7 pages 2013

[25] U Justesen P Knuthsen and T Leth ldquoQuantitative analysis offlavonols flavones and flavanones in fruits vegetables andbeverages by high-performance liquid chromatography withphoto-diode array and mass spectrometric detectionrdquo Journalof Chromatography A vol 799 no 1-2 pp 101ndash110 1998

[26] M S Taga E EMiller andD E Pratt ldquoChia seeds as a source ofnatural lipid antioxidantsrdquo Journal of the AmericanOil ChemistsrsquoSociety vol 61 no 5 pp 928ndash931 1984

[27] A A L Ordonez J D Gomez M A Vattuone and M I IslaldquoAntioxidant activities of Sechium edule (Jacq) Swartz extractsrdquoFood Chemistry vol 97 no 3 pp 452ndash458 2006

[28] A Kumaran and R Joel Karunakaran ldquoIn Vitro antioxidantactivities of methanol extracts of five Phyllanthus species fromIndiardquo LWT-Food Science and Technology vol 40 no 2 pp344ndash352 2007

[29] G Cao H M Alessio and R G Cutler ldquoOxygen-radical absor-bance capacity assay for antioxidantsrdquo Free Radical Biology andMedicine vol 14 no 3 pp 303ndash311 1993

[30] K Shimada K Fujikawa K Yahara and T Nakamura ldquoAntiox-idative properties of xanthan on the autoxidation of soybeanoil in cyclodextrin emulsionrdquo Journal of Agricultural and FoodChemistry vol 40 no 6 pp 945ndash948 1992

[31] J Cole M H L Green S E James L Henderson and H ColeldquoA further assessment of factors influencing measurements ofthioguanine-resistant mutant frequency in circulating T-lymphocytesrdquo Mutation Research vol 204 no 3 pp 493ndash5071988

[32] C-Y Hsu C-M Yang C-M Chen P Chao and S Hu ldquoEff-ects of chlorophyll-related compounds on hydrogen peroxideinduced DNA damage within human lymphocytesrdquo Journal ofAgricultural and Food Chemistry vol 53 no 7 pp 2746ndash27502005

[33] S-C Tang H-F Lo K-H Lin et al ldquoThe antioxidant capacityof extracts from Taiwan indigenous purple-leaved vegetablesrdquoJournal of the Taiwan Society for Horticultural Science vol 59no 1 pp 43ndash57 2013

[34] K-H Lin Y-Y Yang H-F Lo et al ldquoAntioxidant activity ofherbaceous plant extracts protect against hydrogen peroxide-induced DNA damage in human lymphocytesrdquo BioMed CentralResearch Notes In press

[35] A H Cory T C Owen J A Barltrop and J G Cory ldquoUse of anaqueous soluble tetrazoliumformazan assay for cell growthassays in culturerdquoCancer Communications vol 3 no 7 pp 207ndash212 1991

[36] Y T Szeto A R Collins and I F F Benzie ldquoEffects of dietaryantioxidants on DNA damage in lysed cells using a modifiedcomet assay procedurerdquoMutation Research vol 500 no 1-2 pp31ndash38 2002

[37] S Vielma G Virella A J Gorod and M F Lopes-VirellaldquoChlamydophila pneumoniae infection of human aortic endo-thelial cells induces the expression of FC 120574 receptor II (Fc120574RII)rdquoClinical Immunology vol 104 no 3 pp 265ndash273 2002

[38] C Y Zhu and S Loft ldquoEffects of Brussels sprouts extracts onhydrogen peroxide-induced DNA strand breaks in humanlymphocytesrdquo Food and Chemical Toxicology vol 39 no 12 pp1191ndash1197 2001

[39] Y D Min C-H Choi H Bark et al ldquoQuercetin inhibits exp-ression of inflammatory cytokines through attenuation of NF-120581B and p38MAPK in HMC-1 humanmast cell linerdquo Inflamma-tion Research vol 56 no 5 pp 210ndash215 2007

[40] P Y Chao Y P Huang and W B Hsieh ldquoInhibitive effect ofpurple sweet potato leaf extract and its components on celladhesion and inflammatory response in human aortic endothe-lial cellsrdquo Cell Adhesion amp Migration vol 7 no 2 pp 1ndash9 2013

[41] Y Y YangThe antioxidative capacity in herb plant extracts andtheir protection role in DNA oxidative damage of lymphocyte[MS thesis] Chinese Culture University Taipei Taiwan 2004

[42] C A Rice-Evans N J Miller and G Paganga ldquoStructure-anti-oxidant activity relationships of flavonoids and phenolic acidsrdquo

14 Evidence-Based Complementary and Alternative Medicine

Free Radical Biology and Medicine vol 20 no 7 pp 933ndash9561996

[43] M Strube G R Haenen H van den Berg and A Bast ldquoPitfallsin a method for assessment of total antioxidant capacityrdquo FreeRadical Research vol 26 no 6 pp 515ndash521 1997

[44] P G Pietta ldquoFlavonoids as antioxidantsrdquo Journal of NaturalProducts vol 63 no 7 pp 1035ndash1042 2000

[45] A M Gonzalez-Paramas S Esteban-Ruano C Santos-BuelgaSDe Pascual-Teresa and J C Rivas-Gonzalo ldquoFlavanol contentand antioxidant activity in winery byproductsrdquo Journal ofAgricultural and Food Chemistry vol 52 no 2 pp 234ndash2382004

[46] S Y Kim J J Gao and H K Kang ldquoTwo flavonoids from theleaves ofMorus alba induce differentiation of the human prom-yelocytic leukemia (HL-60) cell linerdquo Biological and Pharma-ceutical Bulletin vol 23 no 4 pp 451ndash455 2000

[47] M A Khan A A Rahman S Islam et al ldquoA comparative studyon the antioxidant activity ofmethanolic extracts from differentparts of Morus alba L (Moraceae)rdquo BMC Research vol 6 pp24ndash32 2013

[48] A-L ChungH-F Lo K-H Lin et al ldquoStudy on the antioxidantactivity in herb plant extractsrdquo Journal of the Taiwan Society ForHorticultural Science vol 59 no 2 pp 139ndash152 2013

[49] R L Prior X Wu and K Schaich ldquoStandardized methods forthe determination of antioxidant capacity and phenolics infoods and dietary supplementsrdquo Journal of Agricultural andFood Chemistry vol 53 no 10 pp 4290ndash4302 2005

[50] S J Duthie A R Collins GGDuthie andV LDobson ldquoQue-rcetin and myricetin protect against hydrogen peroxide-indu-ced DNA damage (strand breaks and oxidised pyrimidines) inhuman lymphocytesrdquo Mutation Research vol 393 no 3 pp223ndash231 1997

[51] G A Liu and R L Zheng ldquoProtection against damagedDNA inthe single cell by polyphenolsrdquo Pharmazie vol 57 no 12 pp852ndash854 2002

[52] M Noroozi W J Angerson and M E J Lean ldquoEffects of flav-onoids and vitamin C on oxidative DNA damage to humanlymphocytesrdquoAmerican Journal of Clinical Nutrition vol 67 no6 pp 1210ndash1218 1998

[53] T Koga and M Meydani ldquoEffect of plasma metabolites of (+)-catechin and quercetin on monocyte adhesion to human aorticendothelial cellsrdquoAmerican Journal of Clinical Nutrition vol 73no 5 pp 941ndash948 2001

[54] CMeng P Somers L Hoong et al ldquoDiscovery of novel pheno-lic antioxidants as inhibitors of vascular cell adhesionmolecule-1 expression for use in chronic inflammatory diseasesrdquo Journalof Medicinal Chemistry vol 47 no 25 pp 6420ndash6432 2004

[55] Y Shibata N Kume H Arai et al ldquoMulberry leaf aqueousfractions inhibit TNF-120572-induced nuclear factor 120581B (NF-120581B)activation and lectin-like oxidized LDL receptor-1 (LOX-1)expression in vascular endothelial cellsrdquoAtherosclerosis vol 193no 1 pp 20ndash27 2007

[56] K C Chan H H Ho C N Huang M Lin H Chen and CWang ldquoMulberry leaf extract inhibits vascular smooth musclecell migration involving a block of small GTPase and AktNF-kB signalsrdquo Journal of Agricultural and Food Chemistry vol 57no 19 pp 9147ndash9153 2009

[57] F Chen N Nakashima I Kimura M Kimura N Asanoand S Koya ldquoPotentiating effects on pilocarpine-induced salivasecretion by extracts and N-containing sugars derived frommulberry leaves in streptozocin- diabetic micerdquo Biological andPharmaceutical Bulletin vol 18 no 12 pp 1676ndash1680 1995

[58] K Konno H Ono M Nakamura et al ldquoMulberry latexrich in antidiabetic sugar-mimic alkaloids forces dieting oncaterpillarsrdquo Proceedings of the National Academy of Sciences ofthe United States of America vol 103 no 5 pp 1337ndash1341 2006

[59] H J Kim K H Yoon M J Kang et al ldquoA six-month sup-plementation of mulberry Korean red ginseng and banabadecreases biomarkers of systemic low-grade inflammation insubjects with impaired glucose tolerance and type 2 diabetesrdquoEvidence-based Complementary and Alternative Medicine vol2012 Article ID 735191 8 pages 2012

[60] G Nicholson and G M Hall ldquoDiabetes mellitus new drugs fora new epidemicrdquo British Journal of Anaesthesia vol 107 no 1pp 65ndash73 2011

[61] P Y Chao Y L Chen Y C Lin et al ldquoEffects of black soybeanon atherogenic prevention in hypercholesterolemic rabbits andon adhesion molecular expression in cultured HAECsrdquo Foodand Nutrition Sciences vol 4 pp 9ndash21 2013

[62] C Chen S Li C-YOChen et al ldquoConstituents in purple sweetpotato leaves inhibit in vitro angiogenesis with opposite effectsex vivordquo Nutrition vol 27 no 11-12 pp 1177ndash1182 2011

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

Evidence-Based Complementary and Alternative Medicine 5

Table 1 The contents of various antioxidant substances in MLRC

Polyphenolmg gallic acidgDW

Flavonoids mg quercetinequivalentgDW

Flavonol mg quercetinequivalengDW

AthocyanidinunitgDW

BT 3465 plusmn 082a

8483 plusmn 868a

2547 plusmn 386a

14332 plusmn 181a

MT 2323 plusmn 054b

5599 plusmn 1055b

1326 plusmn 266b

8858 plusmn 412b

M 1987 plusmn 061c

4960 plusmn 541b

1422 plusmn 148b

13582 plusmn 031a

All values are means plusmn SD (119899 = 3)Means within a column with different superscripts (asimc) are significantly different 119875 lt 005

Table 2 The contents of flavonols in acidic methanolic extracts ofMLRC

Flavonols (120583ggDW)Samples Myricetin Morin Quercetin KaempferolBT ND ND 285333 plusmn 18037

a64667 plusmn 5033

a

MT ND ND 37333 plusmn 2309c ND

M ND ND 166667 plusmn 18903b42667 plusmn 4619

b

ND not detected

Table 3 TEAC values and the IC50 of DPPH radical scavengingactivity of acidic methanolic hydrolysates of MLRC

Sample TEAC(Trolox mM)lowast

DPPH radical scavenging activityIC50 (120583gmL)

BT 15514 plusmn 290a

66901 plusmn 723b

MT 10239 plusmn 095b

83640 plusmn 708a

M 7654 plusmn 161c

18883 plusmn 361c

lowastTEAC value at sample 100120583gmL concentration

Table 4 Correlation between antioxidant substances and TEACvalues treated with acid hydrolysates of MLRC

FlavonolsMyricetin Morin Quercetin Kaempferol

TEAClowast mdash mdash 119903 = 041

119875 = 0034

mdash

mdash no significant correlationlowastTEAC values at 100 120583gmL of sample concentration

No mulberry extracts were cytotoxic at the concentrationsused with gt98 of cells remaining viable [41] The cometassay was performed to determine the DNA damagingactivity of the plants because it is a sensitive method formonitoring single strand DNA breaks at the single celllevel Any DNA damage that occurred is represented as atail length (tail migration) of the DNA strand The effectsof pretreatment with acidic methanolic extracts of MLRCon 10 120583M H

2O2-induced DNA oxidative damage in human

lymphocytes are presented in Figure 1 indicating that therewas a protective effect of pretreatment on lymphocytes witheach of the MLRC extracts at the lowest dose (25 120583gmL)Lymphocytes pretreated with 25ndash100120583gmL extracts of MTextracts experienced a greater level of protection againstH2O2exposure than lymphocytes exposed to other tested

compounds and in a dose-dependent manner BT had betterinhibition efficacy at lower concentration (25120583gmL) andthe maximum protective effect of lymphocyte pretreatmentwas observed with pretreatment by 25 120583gmL BT exhibiting

1462 of tail DNA compared to treatment only withH2O2

MT had better inhibition efficacy (8439) than M and BTextracts at the 100 120583gmL level (Figure 1(a)) Tested plantsshowed at least 134263 of tail moment in BT extract at the25 120583gmL level while the BT extract at 100 120583gmL had thehighest tail moment (328193) compared to the rest of theacidic methanolic extract samples (Figure 1(b)) Moreoverthe trend in tail moment in all samples continued climbingup from 25ndash100 120583gmL

The effects of various water extracts of MLRC H2O2-

induced DNA damage to lymphocytes are presented inFigure 2 At lower concentrations all tested samples hadlower tail DNA indicating better inhibition efficacies(Figure 2(a)) BT and M had the best inhibition efficacieswith both exhibiting values of 1422 and 1469 tail DNAat 25 120583gmL respectively Moreover MT had a higher tailDNA than the other extracts at the same dose and thehighest tail DNA was observed in 3212 of MT at aconcentration of 100120583gmL The tail moment of all samplescontinued to increase up to the 100 120583gmL level (Figure 2(b))The minimum tail moment occurred in M at 153674 in the25 120583gmL extracts Moreover the DNA damage induced byH2O2was significantly higher than the DNA damage treated

with extracts and H2O2treated had 8726 in tail DNA and

832884 in tail moment

33 Effects of MLREs on Monocyte-Endothelial Cell Adhe-sion As shown in Figure 3 pretreatment of HAECs with100 120583gmLMLREs for 18 h significantly suppressed the adhe-sion ofU937monocytes to TNF-120572-stimulatedHAECs similarto that of 10 120583Maspirin (positive control) Data also show that25 and 50 120583gmL BT MT and M methanol extracts inhib-ited the adhesion of U937 monocytes to TNF-120572-stimulatedHAECs by 8198 and 8187 (BT) 7449 and 8331 (MT)and 8011 and 7559 (M) respectively

34 Effects of MLREs on NF-120581B Activity and MLREs Inhibitsthe Binding of NF-120581B AP-1 and STAT3 Figure 4 demon-strates that MLREs regulated the DNA-binding activity andexpressions of NF-120581B in HAECs In untreated HAECs NF-120581B p65 was only found within the cytosol however itsnuclear translocation was observed upon treatment withTNF-120572 Compared to the TNF-120572 group pretreatment withBT and MT methanol extracts significantly decreased theexpression of NF-120581B p65 in the nuclear compartment whilethe M methanol extract did not reduce the expressionof NF-120581B p65 activity Furthermore an EMSA analysiswas carried out to determine if the reduced expression of

6 Evidence-Based Complementary and Alternative Medicine

Control H2O2

BT25 BT50 BT100

MT25 MT50 MT100

M25 M50 M100

(a)

0

20

40

60

80

100

25 50 100

Tail

DN

A (

)

Mulberry extracts (120583gmL)

lowast lowastlowast

lowast

lowastlowast

alowast alowast

blowast

MTMBT

H2O2

(b)

Tail

mom

ent

0

2000

4000

6000

8000

10000

25 50 100Mulberry extracts (120583gmL)

lowastlowastlowast

ablowastclowast

alowast

alowast

alowastblowast

MTMBT

H2O2

(c)

Figure 1 Effects of various acidic methanolic extracts of MLRC on H2O2-induced DNA damage to lymphocytes The comet-like images

resulting from the extension of DNA (a) were observed using a fluorescence microscope Tail DNA (b) and tail movement (c) were exposedto test mulberry leaf-related extracts at 25 50 and 100 120583gmL and treated with H

2O2at 10120583M Values with different letters differ significantly

with regard to oxidative damage when comparing different MLRC extracts lowast119875 lt 005 refers to differences in oxidative damage as comparedwith 10 120583MH

2O2-alone treatment

Evidence-Based Complementary and Alternative Medicine 7

Control H2O2

BT25 BT50 BT100

MT25 MT50 MT100

M25 M50 M100

(a)

alowastblowast blowastblowast

blowastblowast

blowast

25 50 100Mulberry extracts (120583gmL)

MTMBT

H2O2

0

20

40

60

80

100

Tail

DN

A (

)

ablowastablowast

(b)

Tail

mom

ent

0

2000

4000

6000

8000

10000

lowastlowast lowast

alowastalowast

25 50 100Mulberry extracts (120583gmL)

MTMBT

H2O2

ablowast ablowastablowast

bclowast

(c)

Figure 2 Effects of various water extracts of MLRC on H2O2-induced DNA damage to lymphocytes The comet-like images resulting from

the extension ofDNA (a) were observed using a fluorescencemicroscope Tail DNA (b) and tailmovement (c) were exposed to testmulberryleaf-related extractsat 25 50 and 100120583gmL and treated with H

2O2at 10120583M Values with different letters differ significantly with regard to

oxidative damage when comparing different MLRC extracts lowast119875 lt 005 refers to differences in oxidative damage as compared with 10120583MH2O2-alone treatment

NF-120581B in response to treatment with MLREs resulted inreduced binding of NF-120581B to DNAThe treatment of HAECswith TNF-120572 led to increased binding of NF-120581B howeverpretreatment of HAECs with BT MT and M significantlydecreased DNA-bound NF-120581B (Figure 5(a)) The increased

binding of AP-1 with TNF-120572 resulted from the treatment ofHAECs however pretreatment of HAECs with BT MT andM significantly decreased DNA-bound AP-1 (Figure 5(b))STAT3 DNA-binding activities to its cognate recognitionsite were remarkably reduced compared to TNF-120572 treatment

8 Evidence-Based Complementary and Alternative Medicine

(A)(A)

(B)

(C) (D)

(E) (F)

(I)

(G) (H)

200120583m 200120583m

200120583m200120583m

200120583m200120583m

200120583m200120583m

200120583m

(a)

Con

trol

Asp

irin

BT25

BT50

MT2

5

MT5

0

M25

M50

Mon

ocyt

e adh

eren

ce (c

ells

HPF

)

0

20

40

60

80

100

120

140

160

180

lowast lowast lowast

lowast

lowast

lowast

lowast

TNF-120572

(b)

Figure 3 Mulberry leaf related extracts (MLREs) reduce tumor necrosis factor (TNF)-120572-induced adhesion of monocytes to human aorticendothelial cells (HAECs) (a) The number of adherent U937 cells (marked by arrow) was monitored by fluorescence microscopy undercontrol (a) 2 ngmL TNF-120572 (b) co-treated with TNF-119886 and aspirin (10120583M) (c) cotreated with TNF-120572 and BT (25 120583gmL) (d) cotreated withTNF-120572 and BT (50 120583gmL) (e) cotreated with TNF-120572 and MT (25 120583gmL) (f) cotreated with TNF-120572 and MT (50 120583gmL) (g) cotreated withTNF-120572 and M (25120583gmL) (h) and cotreated with TNF-120572 and M (50120583gmL) (i) (b) HAECs were pretreated with MLREs for 18 h followedby stimulating with TNF-120572 for 6 h Fluorescence-labeled monocytic U937 cells were added to HAECs and allowed to adhere for 2 h Data areexpressed as a percentage of TNF-120572-induced expression (mean plusmn SD) of three experiments Mean plusmn SD lowast119875 lt 005 BT MT and M at 25and 50120583gmL versus TNF-120572

119875 lt 005 control versus TNF-120572

Evidence-Based Complementary and Alternative Medicine 9

Control TNF-120572

TNF-120572

Aspirin10120583M

+ + + + + + + +

BT25 BT50 MT25 MT50 M25 M50

120583gmL 120583gmL 120583gmL 120583gmL 120583gmL 120583gmL

NF-120581B p65

120573-Actin43kDa

minus

65kDa

(a)

Con

trol

Asp

irin

BT25

BT50

MT2

5

MT5

0

M25

M50

00

02

04

06

08

10

lowast

lowast

lowast

lowastlowastlowastTN

F-120572

Ratio

(nuc

lear

120573-a

ctin

)

(b)

Figure 4 Effects of MLREs on NF-120581B activity After HAECs were pretreated with the indicated samples and incubated with TNF-120572 nuclearextracts were prepared and the expression of NF-120581B p65 was assessed by western blot analysis A representative image of three similar resultsis shown (a) Actin served as the loading control for the nuclear compartment Semiquantitative analysis of three independent experimentsare also shown (b) indicates a significant difference between the TNF-120572 treated or experimental treatment groups and the control119875 lt 005lowast indicates a significant difference between the TNF-120572 and experimental treatment groups BT MT and M at 25 and 50 120583gmL

(lane 3) in the nuclear fraction indicating that MLREs at50120583gmL inhibited STAT3-binding activities In addition theresults in Figure 5(c) (lanes 4ndash10) show that aspirinMLREs at50120583gmL and BT at 25120583gmL remarkably decreased STAT3levels compared to TNF-120572 (lane 3) however both MT andM at 25120583gmL increased STAT3-binding activities Theseresults indicate that only 50 120583gmL MLREs 25 120583gmL BTand aspirin block STAT3 activation and might downregulateTNF-120572-induced expressions of inflammatory signaling path-ways

35 MLREs Regulated the DNA-Binding Activity and Expres-sions of PPAR 120572 and PPAR 120574 in HAECs PPAR 120572 and 120574effectively bound to PPAR 120572 and 120574 oligo-IRDye 700 asindicated in lanes 3ndash10 of Figure 6 Supershifts are bands fromthe binding of each specific antibody The binding activitiesof PPAR 120572 and 120574 were detected at baseline The 25 120583gmLMT extract (lane 7) and 25 and 50 120583gmL M extracts (lanes9-10) increased activity compared to incubating with TNF-120572 alone (lane 3) However BT at both 25 and 50 120583gmLextracts showed reduced effects on PPAR 120572 and 120574 expressionMoreover the 50 120583gmLMT extract had no effect on PPAR 120572binding activity but increased the binding activity of PPAR120574 in comparison to incubation with TNF-120572 alone Thesedata suggest that MT and M both promoted PPAR 120572 and 120574

expression at least in part by increasing the DNA binding oftranscription factors PPAR 120572 and 120574

4 Discussion

41 Antioxidant Composition and Antioxidant Activity BTcontains high levels of polyphenols flavonoids flavonolsanthocyanidin quercetin and kaempferol (Tables 1 and 2)BT also had a higher TEAC value (15514mM Table 3) thanthe TEAC values of 47mM [42] and 64mM [43] reportedfor quercetin as well as 242mM reported for quercetin-3-rutinoside [44] Furthermore Gonzalez-Paramas et al [45]demonstrated that flavonol contents were significantly andhighly correlated (119903 = 08) with TEAC values Kim et al [46]reported that mulberry leaves contain abundant quercetinand inhibit lipid peroxidation at concentrations of 01 and001mgmL In our study the amount of quercetin wassignificantly correlated (119903 = 041 Table 4) with TEAC valueHowever no correlation was found among myricetin morinkaempferol and TEAC which may be due to the structurerequired to reinforce the free radical scavenging activity thatvaries with the type of free radical Naowaratwattana et al[18] reported that a 50 methanolic extract of mulberryleaf contained rutin isoquercetin and various derivatives ofkaempferol and quercetin glycosides while a water extract

10 Evidence-Based Complementary and Alternative Medicine

NF-120581BTNF-120572

TNF-120572

+

Aspirin10120583M+ +

BT25120583gmL

+

BT50120583gmL

+

MT25120583gmL

+

MT50120583gmL

+

M25

120583gmL+

M50

120583gmLControl

minus

DNAOligo

Free probe

(a)

AP-1TNF-120572

TNF-120572

+

Aspirin10120583M+ +

BT25120583gmL

+

BT50120583gmL

+

MT25120583gmL

+

MT50120583gmL

+

M25

120583gmL+

M50

120583gmLControl

minus

DNAOligo

Free probe

(b)

STAT3

TNF-120572

TNF-120572

+

Aspirin10120583M+ +

BT25120583gmL

+

BT50120583gmL

+

MT25120583gmL

+

MT50120583gmL

+

M25

120583gmL+

M50

120583gmLControl

minus

DNAOligo

Free probe

(c)

Figure 5 MLREs inhibits the binding of (a) NF-120581B (b) AP-1 and (c) STAT3 to target DNA sequence NF-120581B AP-1 and STAT3 transcriptionfactor-DNA interactions were assessed by EMSA

contained primarily chlorogenic acid and caffeoylquinic acidderivatives The IC

50of DPPH radical scavenging was 794

and 2042120583gmL for 50 methanolic and water extractsrespectively [18] In our studyM extracts contained quercetinand kaempferol (Table 2) which may account for part ofthe highly scavenged DPPH radical in comparison to therest of MLREs Khan et al [47] demonstrated that the IC

50

was 22023120583gmL which is much less efficacious than themulberry leaf acidic methanolic extract IC

50of 1888 120583gmL

(Table 3) However the IC50

of in vitro free radical scav-enging activity of mulberry leaves from Naowaboot et al[22] was 617 plusmn 21 120583gmL which is more efficacious thanthe IC

50concentration derived in the present study Taken

together the polyphenol-rich MLREs exhibited distinct cell-free antioxidant activity (ie TEAC) as validated by theirlevels of polyphenols and flavonols with distinct antioxidantactivity strongly accounting for the antioxidant activity of the

extracts The amount of quercetin was especially positivelycorrelated with the observed TEAC values

42 Estimation of DNA Single Strand Break Damage fromExposure to Acidic Methanolic and Water Extracts One ofthe antioxidant activity tests scavenging of DPPH radi-cal was selected due to its quick and simple but it is aless biologically relevant assay Previously we reported thatwith 2 120583gsdotmLminus1 of methanolic and water extracts from theindigenous vegetables [33] and herb plants [48] significantlyprolonged lag phase of low-density lipoprotein (LDL) TheLDL oxidation assay is considered to be one of the mostbiologically relevant assays [49] Both studies [33 48] alsoshowed that the concentration of the tested extracts forscavenging DPPH radical was in higher levels at 10mg oftested extracts with 2ndash53 inhibition Lower concentrationwas then chosen for the treated dosage in the present study

Evidence-Based Complementary and Alternative Medicine 11

TNF-120572

TNF-120572

+

Aspirin10120583M+ +

BT25120583gmL

+

BT50120583gmL

+

MT25120583gmL

+

MT50120583gmL

+

M25

120583gmL+

M50

120583gmLControl

minus

DNAOligo

Free probe

PPAR 120572

(a)

TNF-120572

TNF-120572

+

Aspirin10120583M+ +

BT25120583gmL

+

BT50120583gmL

+

MT25120583gmL

+

MT50120583gmL

+

M25

120583gmL+

M50

120583gmLControl

minus

DNAOligo

Free probe

PPAR120574

(b)

Figure 6 Effect of treatments on the expression of PPAR 120572 (a) and PPAR 120574 (b) in TNF-120572-stimulated HAECs by EMSA

In addition lower concentration also could avoid the toxicityeffect of higher level of extracts on lymphocytes Quercetinwas found to protect against H

2O2-induced DNA damage in

human lymphocytes at 10 120583M [50] and at 31 to 25 120583M [51]Quercetin was also reported to induce DNA strand breaks invarious cell types but only at higher doses at 100 120583Mor above[50] Duthie et al [50] proposed that the dihydroxy structureof quercetin and myricetin might be essential in protectingDNA against hydrogen peroxide No such hydroxyl groupsare present in the tocopherol molecule Noroozi et al [52]demonstrated that in addition to quercetin kaempferolcould also inhibit H

2O2-induced DNA strand breaks in

human lymphocytes In our study both BT and M were richin quercetin and kaempferol (Table 2) whichmay account forthe protective roles of both extracts in preventingDNAstrandbreaks from human lymphocytes

43 MLREs Regulated the DNA-Binding Activity and Expres-sions of NF-120581B AP-1 STAT3 and PPARs in HAECsBecause MLREs exhibited free radical scavenging the anti-inflammatory effects of MLREs on TNF-120572-induced celladhesion and adhesionmolecule expressionwere determinedin the present study Our results suggest that MLREs andaspirin significantly inhibited TNF-120572-induced monocyte-endothelial cell adhesion (Figure 3) Significant reductionsin NF-120581B expression and DNA binding by aspirin MTextract (25 120583gmL) and BT extract (both 25 and 50 120583gmL)were also observed The adhesion of monocytes to the

vascular endothelium and subsequent migration into thevessel wall are early events in atherogenesis [2] Our resultsare consistent with previous research showing that dietarypolyphenols such as catechin and quercetin significantlyreduce the binding of monocytes to HAECs [53 54] Thereduced cellular adhesion may be due to the inhibitionof cellular adhesion molecule expression as purple sweetpotato leaf extract (PSPLE) reduced both VCAM-1 andICAM-1 expression [40] Shibata et al [55] demonstratedthat pretreatment of cultured bovine aortic endothelial cells(BAECs) with mulberry leaf aqueous fractions inhibitedTNF-120572- and LPS-induced expression of Lectin-like Ox-LDLreceptor-1 (LOX-1) a cell-surface receptor for atherogenicOx-LDL (oxidized low-density lipoprotein) It also appearsto mediate Ox-LDL-induced inflammation which may becrucial in atherogenesis Furthermore mulberry leaf aqueousfractions inhibited the TNF-120572-induced activation of NF-120581Band phosphorylation of inhibitory factor of NF-120581B-alpha(I120581B-120572) Thus mulberry leaf aqueous fractions suppressTNF-120572- and LPS-induced LOX-1 gene expression mediatedby inhibiting NF-120581B activation

Chan et al [56] demonstrated that mulberry leaf extract(MLE) was rich in polyphenols MLE can effectively inhibitthe migration of vascular smooth muscle cells (VSMC) byblocking small guanosine triphosphatases (small GTPase)and AktNF-120581B signals The Rho family of small GTPasessuch as Rho Rac and Cdc42 acts as a molecular switch toregulate the actin cytoskeleton The Rho family of GTPases

12 Evidence-Based Complementary and Alternative Medicine

is also reported to be involved in the regulation of NF-120581B-dependent transcription [56] MLE has been shown to pos-sess hypoglycemic effects in an insulin-dependent diabetesmellitus (type 1 diabetes) animal model and potential hypo-glycemic compounds inmulberry leaves were suggested to be2-O-120572-D-galactopyranosyl-DNJ and fagomine [57] Konnoet al [58] further reported that mulberry latex containsalkaloidal sugar-mimic glycosidase inhibitors with antidia-betic activities such as 14-dideoxy-14-imino-D-arabinitol 1-deoxynojirimycin and 14-dideoxy-14-imino-D-ribitol Kimet al [59] recently showed that treated type 2 diabetesmellitus(T2D) with a 1 1 1 mixture of ginseng root mulberry leafwater extract and banana leaf water extract (6 gd) for 24weeks significantly decreased plasma intracellular adhesionmolecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) levels This indicates suppressed inflammatoryresponses in T2D but no significant differences in glucosehomeostasis control measurement changes Rigamonti et al[8] reported that bothPPAR120572 and 120574maybe thought of as sup-pressing the activity of NF120581B and AP-1 pathways that reduceinflammatory mediator production in the macrophages ofhumans and mice In our study MT and M promoted PPAR120572 and 120574 expression at least in part by increasing DNAbinding of transcription factors PPAR 120572 and 120574 PPAR-120572-and 120574-dependent signaling pathwaysmay also play importantroles in mediating the TNF-120572 receptor signaling pathwayby MT and M by suppressing the activity on NF-120581B andAP-1 pathways These pathways may regulate downstreamproinflammatory gene expression and adhesion molecularexpression Clinically PPAR 120574 agonists such as rosiglitazoneare widely used in the treatment of T2D [60] M and MTextracts increased PPAR 120574 activity which may partly explainwhy inflammatory responses are suppressed in T2D

Although reduced NF-120581B expression and DNA bind-ing were observed upon treatment with ML MT and BT(Figure 5) the mechanism was not assessed and is worthy offurther study In addition the present results were obtainedusing in vitro studies and may be consistent with an invivo model of atherogenesis similar to that reported byEnkhmaa et al [21] in which mulberry leaves suppressedthe development of atherosclerotic lesions Previously weproved that black beans significantly prolong LDL lag timeand decrease the atheroma regions of the aortic arch andthoracic aorta in hypercholesterolemic NZW rabbits [61]Wethen demonstrated that black soybean extract and PSPLEsignificantly decreased the adhesion of U937 monocyticcells to TNF-120572-stimulated HAECs and reduced adhesionmolecules expression (VCAM-1 and ICAM-1) as well as NF-120581B-p65 expression [37] This is particularly important giventhe opposing effects of flavonols fromMLREs obtained usingin vitro and ex vivo studies [62]

5 Conclusion

Tested MLREs were rich in flavonols and had strongantioxidant activity in protecting lymphocyte DNA fromoxidative damage In particular BT was rich in quercetinand polyphenols The antioxidant components of MLREs

might downregulate intracellular redox-dependent signalingpathways in HAECs upon TNF-120572 stimulation which mightprevent ROS-mediated endothelial cell dysfunction MLRCshave anti-inflammatory effects through the modulation ofNF-120581B AP-1 STAT3 and PPARs signaling These chemi-cals appear to provide distinct cytoprotective mechanismsthat might contribute to their putative beneficial effectsin suppressing endothelial responses to cytokines duringinflammation These results may benefit further in vivostudies to assess the therapeutic potential of phytochemicalsas anti-inflammatories for use in cytokine-induced vasculardisorders including atherosclerosis

Acknowledgment

This work was supported in part by Grants from the NationalScience Council of Taiwan (NSC 95ndash2320-B-034-001 andNSC 96ndash2320-B-034-001)

References

[1] W Palinski ldquoUnited they go conjunct regulation of aorticantioxidant enzymes during atherogenesisrdquo CirculationResearch vol 93 no 3 pp 183ndash185 2003

[2] P Libby ldquoMolecular bases of the acute coronary syndromesrdquoCirculation vol 91 no 11 pp 2844ndash2850 1995

[3] P A Baeuerle and D Baltimore ldquoNf-120581B ten years afterrdquo Cellvol 87 no 1 pp 13ndash20 1996

[4] J M Kyriakis ldquoActivation of the AP-1 transcription factor byinflammatory cytokines of the TNF familyrdquo Gene Expressionvol 7 no 4ndash6 pp 217ndash231 1999

[5] J M Muller R A Rupec and P A Baeuerle ldquoStudy of generegulation by NF-120581B and AP-1 in response to reactive oxygenintermediatesrdquoMethods vol 11 no 3 pp 301ndash312 1997

[6] G Chinetti J Fruchart and B Staels ldquoPeroxisome proliferator-activated receptors new targets for the pharmacological mod-ulation of macrophage gene expression and functionrdquo CurrentOpinion in Lipidology vol 14 no 5 pp 459ndash468 2003

[7] G Chinetti J C Fruchart and B Staels ldquoTranscriptional regu-lation of macrophage cholesterol trafficking by PPAR120572 andLXRrdquo Biochemical Society Transactions vol 34 no 6 pp 1128ndash1131 2006

[8] E Rigamonti G Chinetti-Gbaguidi and B Staels ldquoRegulationof macrophage functions by PPAR- 120572 PPAR- 120574 and LXRsin mice and menrdquo Arteriosclerosis Thrombosis and VascularBiology vol 28 no 6 pp 1050ndash1059 2008

[9] M A Bouhlel J Brozek B Derudas et al ldquoUnlike PPAR120574PPAR120572 or PPAR120573120575 activation does not promote humanmono-cyte differentiation toward alternative macrophagesrdquo Biochem-ical and Biophysical Research Communications vol 386 no 3pp 459ndash462 2009

[10] G Chinetti J-C Fruchart and B Staels ldquoPeroxisome prolife-rator-activated receptors and inflammation from basic scienceto clinical applicationsrdquo International Journal of Obesity vol 27supplement 3 pp S41ndashS45 2003

[11] Y Zhu J H-C Lin H Liao et al ldquoLDL induces transcriptionfactor activator protein-1 in human endothelial cellsrdquo Arte-riosclerosis Thrombosis and Vascular Biology vol 18 no 3 pp473ndash480 1998

Evidence-Based Complementary and Alternative Medicine 13

[12] J Terao ldquoDietary flavonoids as antioxidantsrdquo Forum of Nutri-tion vol 61 pp 87ndash94 2009

[13] Y B Feng W Q Luo and S Q Zhu ldquoExplore new clinicalapplication of Huanglian and corresponding compound pre-scriptions from their traditional userdquo Zhongguo ZhongyaoZazhi vol 33 no 10 pp 1221ndash1225 2008

[14] Y H Chu C L Chang and H F Hsu ldquoFlavonoid content ofseveral vegetables and their antioxidant activityrdquo Journal of theScience of Food andAgriculture vol 80 no 5 pp 561ndash566 2000

[15] I C Arts B van de Putte and P C Hollman ldquoCatechin con-tents of foods commonly consumed in The Netherlands 1Fruits vegetables staple foods and processed foodsrdquo Journal ofAgricultural and Food Chemistry vol 48 no 5 pp 1746ndash17512000

[16] A R Proteggente A S Pannala G Paganga et al ldquoThe anti-oxidant activity of regularly consumed fruit and vegetablesreflects their phenolic and vitamin C compositionrdquo Free RadicalResearch vol 36 no 2 pp 217ndash233 2002

[17] Y L Liu L H Tang Z Q Liang B You and S Yang ldquoGrowthinhibitory and apoptosis inducing by effects of total flavonoidsfrom Lysimachia clethroides Duby in human chronic myeloidleukemiaK562 cellsrdquo Journal of Ethnopharmacology vol 131 no1 pp 1ndash9 2010

[18] W Naowaratwattana W De-Eknamkul and E G De MejialdquoPhenolic-containing organic extracts of mulberry (Morus albaL) leaves inhibit HepG2 hepatoma cells through G2M phasearrest and inhibition of topoisomerase II120572 activityrdquo Journal ofMedicinal Food vol 13 no 5 pp 1045ndash1056 2010

[19] K Doi T Kojima and Y Fujimoto ldquoMulberry leaf extract inhi-bits the oxidative modification of rabbit and human low densitylipoproteinrdquo Biological and Pharmaceutical Bulletin vol 23 no9 pp 1066ndash1071 2000

[20] W Wang Y Zu Y Fu and T Efferth ldquoIn Vitro antioxidant andantimicrobial activity of extracts from Morus alba L leavesstems and fruitsrdquoAmerican Journal of ChineseMedicine vol 40no 2 pp 349ndash356 2012

[21] B Enkhmaa K Shiwaku T Katsube et al ldquoMulberry (Morusalba L) leaves and their major flavonol quercetin 3-(6-malon-ylglucoside) attenuate atherosclerotic lesion development inLDL receptor-deficient micerdquo Journal of Nutrition vol 135 no4 pp 729ndash734 2005

[22] J Naowaboot P Pannangpetch V Kukongviriyapan B Kongy-ingyoes and U Kukongviriyapan ldquoAntihyperglycemic antiox-idant and antiglycation activities of mulberry leaf extract instreptozotocin-induced chronic diabetic ratsrdquo Plant Foods forHuman Nutrition vol 64 no 2 pp 116ndash121 2009

[23] MM Padilha F CVilela CQ Rocha et al ldquoAntiinflammatoryproperties of Morus nigra leavesrdquo Phytotherapy Research vol24 no 10 pp 1496ndash1500 2010

[24] P Aramwit O Supasyndh T Siritienthong and N BangldquoMulberry leaf reduces oxidation and c-reactive protein levelin patients with mild dyslipidemiardquo BioMed Research Interna-tional vol 2013 Article ID 787981 7 pages 2013

[25] U Justesen P Knuthsen and T Leth ldquoQuantitative analysis offlavonols flavones and flavanones in fruits vegetables andbeverages by high-performance liquid chromatography withphoto-diode array and mass spectrometric detectionrdquo Journalof Chromatography A vol 799 no 1-2 pp 101ndash110 1998

[26] M S Taga E EMiller andD E Pratt ldquoChia seeds as a source ofnatural lipid antioxidantsrdquo Journal of the AmericanOil ChemistsrsquoSociety vol 61 no 5 pp 928ndash931 1984

[27] A A L Ordonez J D Gomez M A Vattuone and M I IslaldquoAntioxidant activities of Sechium edule (Jacq) Swartz extractsrdquoFood Chemistry vol 97 no 3 pp 452ndash458 2006

[28] A Kumaran and R Joel Karunakaran ldquoIn Vitro antioxidantactivities of methanol extracts of five Phyllanthus species fromIndiardquo LWT-Food Science and Technology vol 40 no 2 pp344ndash352 2007

[29] G Cao H M Alessio and R G Cutler ldquoOxygen-radical absor-bance capacity assay for antioxidantsrdquo Free Radical Biology andMedicine vol 14 no 3 pp 303ndash311 1993

[30] K Shimada K Fujikawa K Yahara and T Nakamura ldquoAntiox-idative properties of xanthan on the autoxidation of soybeanoil in cyclodextrin emulsionrdquo Journal of Agricultural and FoodChemistry vol 40 no 6 pp 945ndash948 1992

[31] J Cole M H L Green S E James L Henderson and H ColeldquoA further assessment of factors influencing measurements ofthioguanine-resistant mutant frequency in circulating T-lymphocytesrdquo Mutation Research vol 204 no 3 pp 493ndash5071988

[32] C-Y Hsu C-M Yang C-M Chen P Chao and S Hu ldquoEff-ects of chlorophyll-related compounds on hydrogen peroxideinduced DNA damage within human lymphocytesrdquo Journal ofAgricultural and Food Chemistry vol 53 no 7 pp 2746ndash27502005

[33] S-C Tang H-F Lo K-H Lin et al ldquoThe antioxidant capacityof extracts from Taiwan indigenous purple-leaved vegetablesrdquoJournal of the Taiwan Society for Horticultural Science vol 59no 1 pp 43ndash57 2013

[34] K-H Lin Y-Y Yang H-F Lo et al ldquoAntioxidant activity ofherbaceous plant extracts protect against hydrogen peroxide-induced DNA damage in human lymphocytesrdquo BioMed CentralResearch Notes In press

[35] A H Cory T C Owen J A Barltrop and J G Cory ldquoUse of anaqueous soluble tetrazoliumformazan assay for cell growthassays in culturerdquoCancer Communications vol 3 no 7 pp 207ndash212 1991

[36] Y T Szeto A R Collins and I F F Benzie ldquoEffects of dietaryantioxidants on DNA damage in lysed cells using a modifiedcomet assay procedurerdquoMutation Research vol 500 no 1-2 pp31ndash38 2002

[37] S Vielma G Virella A J Gorod and M F Lopes-VirellaldquoChlamydophila pneumoniae infection of human aortic endo-thelial cells induces the expression of FC 120574 receptor II (Fc120574RII)rdquoClinical Immunology vol 104 no 3 pp 265ndash273 2002

[38] C Y Zhu and S Loft ldquoEffects of Brussels sprouts extracts onhydrogen peroxide-induced DNA strand breaks in humanlymphocytesrdquo Food and Chemical Toxicology vol 39 no 12 pp1191ndash1197 2001

[39] Y D Min C-H Choi H Bark et al ldquoQuercetin inhibits exp-ression of inflammatory cytokines through attenuation of NF-120581B and p38MAPK in HMC-1 humanmast cell linerdquo Inflamma-tion Research vol 56 no 5 pp 210ndash215 2007

[40] P Y Chao Y P Huang and W B Hsieh ldquoInhibitive effect ofpurple sweet potato leaf extract and its components on celladhesion and inflammatory response in human aortic endothe-lial cellsrdquo Cell Adhesion amp Migration vol 7 no 2 pp 1ndash9 2013

[41] Y Y YangThe antioxidative capacity in herb plant extracts andtheir protection role in DNA oxidative damage of lymphocyte[MS thesis] Chinese Culture University Taipei Taiwan 2004

[42] C A Rice-Evans N J Miller and G Paganga ldquoStructure-anti-oxidant activity relationships of flavonoids and phenolic acidsrdquo

14 Evidence-Based Complementary and Alternative Medicine

Free Radical Biology and Medicine vol 20 no 7 pp 933ndash9561996

[43] M Strube G R Haenen H van den Berg and A Bast ldquoPitfallsin a method for assessment of total antioxidant capacityrdquo FreeRadical Research vol 26 no 6 pp 515ndash521 1997

[44] P G Pietta ldquoFlavonoids as antioxidantsrdquo Journal of NaturalProducts vol 63 no 7 pp 1035ndash1042 2000

[45] A M Gonzalez-Paramas S Esteban-Ruano C Santos-BuelgaSDe Pascual-Teresa and J C Rivas-Gonzalo ldquoFlavanol contentand antioxidant activity in winery byproductsrdquo Journal ofAgricultural and Food Chemistry vol 52 no 2 pp 234ndash2382004

[46] S Y Kim J J Gao and H K Kang ldquoTwo flavonoids from theleaves ofMorus alba induce differentiation of the human prom-yelocytic leukemia (HL-60) cell linerdquo Biological and Pharma-ceutical Bulletin vol 23 no 4 pp 451ndash455 2000

[47] M A Khan A A Rahman S Islam et al ldquoA comparative studyon the antioxidant activity ofmethanolic extracts from differentparts of Morus alba L (Moraceae)rdquo BMC Research vol 6 pp24ndash32 2013

[48] A-L ChungH-F Lo K-H Lin et al ldquoStudy on the antioxidantactivity in herb plant extractsrdquo Journal of the Taiwan Society ForHorticultural Science vol 59 no 2 pp 139ndash152 2013

[49] R L Prior X Wu and K Schaich ldquoStandardized methods forthe determination of antioxidant capacity and phenolics infoods and dietary supplementsrdquo Journal of Agricultural andFood Chemistry vol 53 no 10 pp 4290ndash4302 2005

[50] S J Duthie A R Collins GGDuthie andV LDobson ldquoQue-rcetin and myricetin protect against hydrogen peroxide-indu-ced DNA damage (strand breaks and oxidised pyrimidines) inhuman lymphocytesrdquo Mutation Research vol 393 no 3 pp223ndash231 1997

[51] G A Liu and R L Zheng ldquoProtection against damagedDNA inthe single cell by polyphenolsrdquo Pharmazie vol 57 no 12 pp852ndash854 2002

[52] M Noroozi W J Angerson and M E J Lean ldquoEffects of flav-onoids and vitamin C on oxidative DNA damage to humanlymphocytesrdquoAmerican Journal of Clinical Nutrition vol 67 no6 pp 1210ndash1218 1998

[53] T Koga and M Meydani ldquoEffect of plasma metabolites of (+)-catechin and quercetin on monocyte adhesion to human aorticendothelial cellsrdquoAmerican Journal of Clinical Nutrition vol 73no 5 pp 941ndash948 2001

[54] CMeng P Somers L Hoong et al ldquoDiscovery of novel pheno-lic antioxidants as inhibitors of vascular cell adhesionmolecule-1 expression for use in chronic inflammatory diseasesrdquo Journalof Medicinal Chemistry vol 47 no 25 pp 6420ndash6432 2004

[55] Y Shibata N Kume H Arai et al ldquoMulberry leaf aqueousfractions inhibit TNF-120572-induced nuclear factor 120581B (NF-120581B)activation and lectin-like oxidized LDL receptor-1 (LOX-1)expression in vascular endothelial cellsrdquoAtherosclerosis vol 193no 1 pp 20ndash27 2007

[56] K C Chan H H Ho C N Huang M Lin H Chen and CWang ldquoMulberry leaf extract inhibits vascular smooth musclecell migration involving a block of small GTPase and AktNF-kB signalsrdquo Journal of Agricultural and Food Chemistry vol 57no 19 pp 9147ndash9153 2009

[57] F Chen N Nakashima I Kimura M Kimura N Asanoand S Koya ldquoPotentiating effects on pilocarpine-induced salivasecretion by extracts and N-containing sugars derived frommulberry leaves in streptozocin- diabetic micerdquo Biological andPharmaceutical Bulletin vol 18 no 12 pp 1676ndash1680 1995

[58] K Konno H Ono M Nakamura et al ldquoMulberry latexrich in antidiabetic sugar-mimic alkaloids forces dieting oncaterpillarsrdquo Proceedings of the National Academy of Sciences ofthe United States of America vol 103 no 5 pp 1337ndash1341 2006

[59] H J Kim K H Yoon M J Kang et al ldquoA six-month sup-plementation of mulberry Korean red ginseng and banabadecreases biomarkers of systemic low-grade inflammation insubjects with impaired glucose tolerance and type 2 diabetesrdquoEvidence-based Complementary and Alternative Medicine vol2012 Article ID 735191 8 pages 2012

[60] G Nicholson and G M Hall ldquoDiabetes mellitus new drugs fora new epidemicrdquo British Journal of Anaesthesia vol 107 no 1pp 65ndash73 2011

[61] P Y Chao Y L Chen Y C Lin et al ldquoEffects of black soybeanon atherogenic prevention in hypercholesterolemic rabbits andon adhesion molecular expression in cultured HAECsrdquo Foodand Nutrition Sciences vol 4 pp 9ndash21 2013

[62] C Chen S Li C-YOChen et al ldquoConstituents in purple sweetpotato leaves inhibit in vitro angiogenesis with opposite effectsex vivordquo Nutrition vol 27 no 11-12 pp 1177ndash1182 2011

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

6 Evidence-Based Complementary and Alternative Medicine

Control H2O2

BT25 BT50 BT100

MT25 MT50 MT100

M25 M50 M100

(a)

0

20

40

60

80

100

25 50 100

Tail

DN

A (

)

Mulberry extracts (120583gmL)

lowast lowastlowast

lowast

lowastlowast

alowast alowast

blowast

MTMBT

H2O2

(b)

Tail

mom

ent

0

2000

4000

6000

8000

10000

25 50 100Mulberry extracts (120583gmL)

lowastlowastlowast

ablowastclowast

alowast

alowast

alowastblowast

MTMBT

H2O2

(c)

Figure 1 Effects of various acidic methanolic extracts of MLRC on H2O2-induced DNA damage to lymphocytes The comet-like images

resulting from the extension of DNA (a) were observed using a fluorescence microscope Tail DNA (b) and tail movement (c) were exposedto test mulberry leaf-related extracts at 25 50 and 100 120583gmL and treated with H

2O2at 10120583M Values with different letters differ significantly

with regard to oxidative damage when comparing different MLRC extracts lowast119875 lt 005 refers to differences in oxidative damage as comparedwith 10 120583MH

2O2-alone treatment

Evidence-Based Complementary and Alternative Medicine 7

Control H2O2

BT25 BT50 BT100

MT25 MT50 MT100

M25 M50 M100

(a)

alowastblowast blowastblowast

blowastblowast

blowast

25 50 100Mulberry extracts (120583gmL)

MTMBT

H2O2

0

20

40

60

80

100

Tail

DN

A (

)

ablowastablowast

(b)

Tail

mom

ent

0

2000

4000

6000

8000

10000

lowastlowast lowast

alowastalowast

25 50 100Mulberry extracts (120583gmL)

MTMBT

H2O2

ablowast ablowastablowast

bclowast

(c)

Figure 2 Effects of various water extracts of MLRC on H2O2-induced DNA damage to lymphocytes The comet-like images resulting from

the extension ofDNA (a) were observed using a fluorescencemicroscope Tail DNA (b) and tailmovement (c) were exposed to testmulberryleaf-related extractsat 25 50 and 100120583gmL and treated with H

2O2at 10120583M Values with different letters differ significantly with regard to

oxidative damage when comparing different MLRC extracts lowast119875 lt 005 refers to differences in oxidative damage as compared with 10120583MH2O2-alone treatment

NF-120581B in response to treatment with MLREs resulted inreduced binding of NF-120581B to DNAThe treatment of HAECswith TNF-120572 led to increased binding of NF-120581B howeverpretreatment of HAECs with BT MT and M significantlydecreased DNA-bound NF-120581B (Figure 5(a)) The increased

binding of AP-1 with TNF-120572 resulted from the treatment ofHAECs however pretreatment of HAECs with BT MT andM significantly decreased DNA-bound AP-1 (Figure 5(b))STAT3 DNA-binding activities to its cognate recognitionsite were remarkably reduced compared to TNF-120572 treatment

8 Evidence-Based Complementary and Alternative Medicine

(A)(A)

(B)

(C) (D)

(E) (F)

(I)

(G) (H)

200120583m 200120583m

200120583m200120583m

200120583m200120583m

200120583m200120583m

200120583m

(a)

Con

trol

Asp

irin

BT25

BT50

MT2

5

MT5

0

M25

M50

Mon

ocyt

e adh

eren

ce (c

ells

HPF

)

0

20

40

60

80

100

120

140

160

180

lowast lowast lowast

lowast

lowast

lowast

lowast

TNF-120572

(b)

Figure 3 Mulberry leaf related extracts (MLREs) reduce tumor necrosis factor (TNF)-120572-induced adhesion of monocytes to human aorticendothelial cells (HAECs) (a) The number of adherent U937 cells (marked by arrow) was monitored by fluorescence microscopy undercontrol (a) 2 ngmL TNF-120572 (b) co-treated with TNF-119886 and aspirin (10120583M) (c) cotreated with TNF-120572 and BT (25 120583gmL) (d) cotreated withTNF-120572 and BT (50 120583gmL) (e) cotreated with TNF-120572 and MT (25 120583gmL) (f) cotreated with TNF-120572 and MT (50 120583gmL) (g) cotreated withTNF-120572 and M (25120583gmL) (h) and cotreated with TNF-120572 and M (50120583gmL) (i) (b) HAECs were pretreated with MLREs for 18 h followedby stimulating with TNF-120572 for 6 h Fluorescence-labeled monocytic U937 cells were added to HAECs and allowed to adhere for 2 h Data areexpressed as a percentage of TNF-120572-induced expression (mean plusmn SD) of three experiments Mean plusmn SD lowast119875 lt 005 BT MT and M at 25and 50120583gmL versus TNF-120572

119875 lt 005 control versus TNF-120572

Evidence-Based Complementary and Alternative Medicine 9

Control TNF-120572

TNF-120572

Aspirin10120583M

+ + + + + + + +

BT25 BT50 MT25 MT50 M25 M50

120583gmL 120583gmL 120583gmL 120583gmL 120583gmL 120583gmL

NF-120581B p65

120573-Actin43kDa

minus

65kDa

(a)

Con

trol

Asp

irin

BT25

BT50

MT2

5

MT5

0

M25

M50

00

02

04

06

08

10

lowast

lowast

lowast

lowastlowastlowastTN

F-120572

Ratio

(nuc

lear

120573-a

ctin

)

(b)

Figure 4 Effects of MLREs on NF-120581B activity After HAECs were pretreated with the indicated samples and incubated with TNF-120572 nuclearextracts were prepared and the expression of NF-120581B p65 was assessed by western blot analysis A representative image of three similar resultsis shown (a) Actin served as the loading control for the nuclear compartment Semiquantitative analysis of three independent experimentsare also shown (b) indicates a significant difference between the TNF-120572 treated or experimental treatment groups and the control119875 lt 005lowast indicates a significant difference between the TNF-120572 and experimental treatment groups BT MT and M at 25 and 50 120583gmL

(lane 3) in the nuclear fraction indicating that MLREs at50120583gmL inhibited STAT3-binding activities In addition theresults in Figure 5(c) (lanes 4ndash10) show that aspirinMLREs at50120583gmL and BT at 25120583gmL remarkably decreased STAT3levels compared to TNF-120572 (lane 3) however both MT andM at 25120583gmL increased STAT3-binding activities Theseresults indicate that only 50 120583gmL MLREs 25 120583gmL BTand aspirin block STAT3 activation and might downregulateTNF-120572-induced expressions of inflammatory signaling path-ways

35 MLREs Regulated the DNA-Binding Activity and Expres-sions of PPAR 120572 and PPAR 120574 in HAECs PPAR 120572 and 120574effectively bound to PPAR 120572 and 120574 oligo-IRDye 700 asindicated in lanes 3ndash10 of Figure 6 Supershifts are bands fromthe binding of each specific antibody The binding activitiesof PPAR 120572 and 120574 were detected at baseline The 25 120583gmLMT extract (lane 7) and 25 and 50 120583gmL M extracts (lanes9-10) increased activity compared to incubating with TNF-120572 alone (lane 3) However BT at both 25 and 50 120583gmLextracts showed reduced effects on PPAR 120572 and 120574 expressionMoreover the 50 120583gmLMT extract had no effect on PPAR 120572binding activity but increased the binding activity of PPAR120574 in comparison to incubation with TNF-120572 alone Thesedata suggest that MT and M both promoted PPAR 120572 and 120574

expression at least in part by increasing the DNA binding oftranscription factors PPAR 120572 and 120574

4 Discussion

41 Antioxidant Composition and Antioxidant Activity BTcontains high levels of polyphenols flavonoids flavonolsanthocyanidin quercetin and kaempferol (Tables 1 and 2)BT also had a higher TEAC value (15514mM Table 3) thanthe TEAC values of 47mM [42] and 64mM [43] reportedfor quercetin as well as 242mM reported for quercetin-3-rutinoside [44] Furthermore Gonzalez-Paramas et al [45]demonstrated that flavonol contents were significantly andhighly correlated (119903 = 08) with TEAC values Kim et al [46]reported that mulberry leaves contain abundant quercetinand inhibit lipid peroxidation at concentrations of 01 and001mgmL In our study the amount of quercetin wassignificantly correlated (119903 = 041 Table 4) with TEAC valueHowever no correlation was found among myricetin morinkaempferol and TEAC which may be due to the structurerequired to reinforce the free radical scavenging activity thatvaries with the type of free radical Naowaratwattana et al[18] reported that a 50 methanolic extract of mulberryleaf contained rutin isoquercetin and various derivatives ofkaempferol and quercetin glycosides while a water extract

10 Evidence-Based Complementary and Alternative Medicine

NF-120581BTNF-120572

TNF-120572

+

Aspirin10120583M+ +

BT25120583gmL

+

BT50120583gmL

+

MT25120583gmL

+

MT50120583gmL

+

M25

120583gmL+

M50

120583gmLControl

minus

DNAOligo

Free probe

(a)

AP-1TNF-120572

TNF-120572

+

Aspirin10120583M+ +

BT25120583gmL

+

BT50120583gmL

+

MT25120583gmL

+

MT50120583gmL

+

M25

120583gmL+

M50

120583gmLControl

minus

DNAOligo

Free probe

(b)

STAT3

TNF-120572

TNF-120572

+

Aspirin10120583M+ +

BT25120583gmL

+

BT50120583gmL

+

MT25120583gmL

+

MT50120583gmL

+

M25

120583gmL+

M50

120583gmLControl

minus

DNAOligo

Free probe

(c)

Figure 5 MLREs inhibits the binding of (a) NF-120581B (b) AP-1 and (c) STAT3 to target DNA sequence NF-120581B AP-1 and STAT3 transcriptionfactor-DNA interactions were assessed by EMSA

contained primarily chlorogenic acid and caffeoylquinic acidderivatives The IC

50of DPPH radical scavenging was 794

and 2042120583gmL for 50 methanolic and water extractsrespectively [18] In our studyM extracts contained quercetinand kaempferol (Table 2) which may account for part ofthe highly scavenged DPPH radical in comparison to therest of MLREs Khan et al [47] demonstrated that the IC

50

was 22023120583gmL which is much less efficacious than themulberry leaf acidic methanolic extract IC

50of 1888 120583gmL

(Table 3) However the IC50

of in vitro free radical scav-enging activity of mulberry leaves from Naowaboot et al[22] was 617 plusmn 21 120583gmL which is more efficacious thanthe IC

50concentration derived in the present study Taken

together the polyphenol-rich MLREs exhibited distinct cell-free antioxidant activity (ie TEAC) as validated by theirlevels of polyphenols and flavonols with distinct antioxidantactivity strongly accounting for the antioxidant activity of the

extracts The amount of quercetin was especially positivelycorrelated with the observed TEAC values

42 Estimation of DNA Single Strand Break Damage fromExposure to Acidic Methanolic and Water Extracts One ofthe antioxidant activity tests scavenging of DPPH radi-cal was selected due to its quick and simple but it is aless biologically relevant assay Previously we reported thatwith 2 120583gsdotmLminus1 of methanolic and water extracts from theindigenous vegetables [33] and herb plants [48] significantlyprolonged lag phase of low-density lipoprotein (LDL) TheLDL oxidation assay is considered to be one of the mostbiologically relevant assays [49] Both studies [33 48] alsoshowed that the concentration of the tested extracts forscavenging DPPH radical was in higher levels at 10mg oftested extracts with 2ndash53 inhibition Lower concentrationwas then chosen for the treated dosage in the present study

Evidence-Based Complementary and Alternative Medicine 11

TNF-120572

TNF-120572

+

Aspirin10120583M+ +

BT25120583gmL

+

BT50120583gmL

+

MT25120583gmL

+

MT50120583gmL

+

M25

120583gmL+

M50

120583gmLControl

minus

DNAOligo

Free probe

PPAR 120572

(a)

TNF-120572

TNF-120572

+

Aspirin10120583M+ +

BT25120583gmL

+

BT50120583gmL

+

MT25120583gmL

+

MT50120583gmL

+

M25

120583gmL+

M50

120583gmLControl

minus

DNAOligo

Free probe

PPAR120574

(b)

Figure 6 Effect of treatments on the expression of PPAR 120572 (a) and PPAR 120574 (b) in TNF-120572-stimulated HAECs by EMSA

In addition lower concentration also could avoid the toxicityeffect of higher level of extracts on lymphocytes Quercetinwas found to protect against H

2O2-induced DNA damage in

human lymphocytes at 10 120583M [50] and at 31 to 25 120583M [51]Quercetin was also reported to induce DNA strand breaks invarious cell types but only at higher doses at 100 120583Mor above[50] Duthie et al [50] proposed that the dihydroxy structureof quercetin and myricetin might be essential in protectingDNA against hydrogen peroxide No such hydroxyl groupsare present in the tocopherol molecule Noroozi et al [52]demonstrated that in addition to quercetin kaempferolcould also inhibit H

2O2-induced DNA strand breaks in

human lymphocytes In our study both BT and M were richin quercetin and kaempferol (Table 2) whichmay account forthe protective roles of both extracts in preventingDNAstrandbreaks from human lymphocytes

43 MLREs Regulated the DNA-Binding Activity and Expres-sions of NF-120581B AP-1 STAT3 and PPARs in HAECsBecause MLREs exhibited free radical scavenging the anti-inflammatory effects of MLREs on TNF-120572-induced celladhesion and adhesionmolecule expressionwere determinedin the present study Our results suggest that MLREs andaspirin significantly inhibited TNF-120572-induced monocyte-endothelial cell adhesion (Figure 3) Significant reductionsin NF-120581B expression and DNA binding by aspirin MTextract (25 120583gmL) and BT extract (both 25 and 50 120583gmL)were also observed The adhesion of monocytes to the

vascular endothelium and subsequent migration into thevessel wall are early events in atherogenesis [2] Our resultsare consistent with previous research showing that dietarypolyphenols such as catechin and quercetin significantlyreduce the binding of monocytes to HAECs [53 54] Thereduced cellular adhesion may be due to the inhibitionof cellular adhesion molecule expression as purple sweetpotato leaf extract (PSPLE) reduced both VCAM-1 andICAM-1 expression [40] Shibata et al [55] demonstratedthat pretreatment of cultured bovine aortic endothelial cells(BAECs) with mulberry leaf aqueous fractions inhibitedTNF-120572- and LPS-induced expression of Lectin-like Ox-LDLreceptor-1 (LOX-1) a cell-surface receptor for atherogenicOx-LDL (oxidized low-density lipoprotein) It also appearsto mediate Ox-LDL-induced inflammation which may becrucial in atherogenesis Furthermore mulberry leaf aqueousfractions inhibited the TNF-120572-induced activation of NF-120581Band phosphorylation of inhibitory factor of NF-120581B-alpha(I120581B-120572) Thus mulberry leaf aqueous fractions suppressTNF-120572- and LPS-induced LOX-1 gene expression mediatedby inhibiting NF-120581B activation

Chan et al [56] demonstrated that mulberry leaf extract(MLE) was rich in polyphenols MLE can effectively inhibitthe migration of vascular smooth muscle cells (VSMC) byblocking small guanosine triphosphatases (small GTPase)and AktNF-120581B signals The Rho family of small GTPasessuch as Rho Rac and Cdc42 acts as a molecular switch toregulate the actin cytoskeleton The Rho family of GTPases

12 Evidence-Based Complementary and Alternative Medicine

is also reported to be involved in the regulation of NF-120581B-dependent transcription [56] MLE has been shown to pos-sess hypoglycemic effects in an insulin-dependent diabetesmellitus (type 1 diabetes) animal model and potential hypo-glycemic compounds inmulberry leaves were suggested to be2-O-120572-D-galactopyranosyl-DNJ and fagomine [57] Konnoet al [58] further reported that mulberry latex containsalkaloidal sugar-mimic glycosidase inhibitors with antidia-betic activities such as 14-dideoxy-14-imino-D-arabinitol 1-deoxynojirimycin and 14-dideoxy-14-imino-D-ribitol Kimet al [59] recently showed that treated type 2 diabetesmellitus(T2D) with a 1 1 1 mixture of ginseng root mulberry leafwater extract and banana leaf water extract (6 gd) for 24weeks significantly decreased plasma intracellular adhesionmolecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) levels This indicates suppressed inflammatoryresponses in T2D but no significant differences in glucosehomeostasis control measurement changes Rigamonti et al[8] reported that bothPPAR120572 and 120574maybe thought of as sup-pressing the activity of NF120581B and AP-1 pathways that reduceinflammatory mediator production in the macrophages ofhumans and mice In our study MT and M promoted PPAR120572 and 120574 expression at least in part by increasing DNAbinding of transcription factors PPAR 120572 and 120574 PPAR-120572-and 120574-dependent signaling pathwaysmay also play importantroles in mediating the TNF-120572 receptor signaling pathwayby MT and M by suppressing the activity on NF-120581B andAP-1 pathways These pathways may regulate downstreamproinflammatory gene expression and adhesion molecularexpression Clinically PPAR 120574 agonists such as rosiglitazoneare widely used in the treatment of T2D [60] M and MTextracts increased PPAR 120574 activity which may partly explainwhy inflammatory responses are suppressed in T2D

Although reduced NF-120581B expression and DNA bind-ing were observed upon treatment with ML MT and BT(Figure 5) the mechanism was not assessed and is worthy offurther study In addition the present results were obtainedusing in vitro studies and may be consistent with an invivo model of atherogenesis similar to that reported byEnkhmaa et al [21] in which mulberry leaves suppressedthe development of atherosclerotic lesions Previously weproved that black beans significantly prolong LDL lag timeand decrease the atheroma regions of the aortic arch andthoracic aorta in hypercholesterolemic NZW rabbits [61]Wethen demonstrated that black soybean extract and PSPLEsignificantly decreased the adhesion of U937 monocyticcells to TNF-120572-stimulated HAECs and reduced adhesionmolecules expression (VCAM-1 and ICAM-1) as well as NF-120581B-p65 expression [37] This is particularly important giventhe opposing effects of flavonols fromMLREs obtained usingin vitro and ex vivo studies [62]

5 Conclusion

Tested MLREs were rich in flavonols and had strongantioxidant activity in protecting lymphocyte DNA fromoxidative damage In particular BT was rich in quercetinand polyphenols The antioxidant components of MLREs

might downregulate intracellular redox-dependent signalingpathways in HAECs upon TNF-120572 stimulation which mightprevent ROS-mediated endothelial cell dysfunction MLRCshave anti-inflammatory effects through the modulation ofNF-120581B AP-1 STAT3 and PPARs signaling These chemi-cals appear to provide distinct cytoprotective mechanismsthat might contribute to their putative beneficial effectsin suppressing endothelial responses to cytokines duringinflammation These results may benefit further in vivostudies to assess the therapeutic potential of phytochemicalsas anti-inflammatories for use in cytokine-induced vasculardisorders including atherosclerosis

Acknowledgment

This work was supported in part by Grants from the NationalScience Council of Taiwan (NSC 95ndash2320-B-034-001 andNSC 96ndash2320-B-034-001)

References

[1] W Palinski ldquoUnited they go conjunct regulation of aorticantioxidant enzymes during atherogenesisrdquo CirculationResearch vol 93 no 3 pp 183ndash185 2003

[2] P Libby ldquoMolecular bases of the acute coronary syndromesrdquoCirculation vol 91 no 11 pp 2844ndash2850 1995

[3] P A Baeuerle and D Baltimore ldquoNf-120581B ten years afterrdquo Cellvol 87 no 1 pp 13ndash20 1996

[4] J M Kyriakis ldquoActivation of the AP-1 transcription factor byinflammatory cytokines of the TNF familyrdquo Gene Expressionvol 7 no 4ndash6 pp 217ndash231 1999

[5] J M Muller R A Rupec and P A Baeuerle ldquoStudy of generegulation by NF-120581B and AP-1 in response to reactive oxygenintermediatesrdquoMethods vol 11 no 3 pp 301ndash312 1997

[6] G Chinetti J Fruchart and B Staels ldquoPeroxisome proliferator-activated receptors new targets for the pharmacological mod-ulation of macrophage gene expression and functionrdquo CurrentOpinion in Lipidology vol 14 no 5 pp 459ndash468 2003

[7] G Chinetti J C Fruchart and B Staels ldquoTranscriptional regu-lation of macrophage cholesterol trafficking by PPAR120572 andLXRrdquo Biochemical Society Transactions vol 34 no 6 pp 1128ndash1131 2006

[8] E Rigamonti G Chinetti-Gbaguidi and B Staels ldquoRegulationof macrophage functions by PPAR- 120572 PPAR- 120574 and LXRsin mice and menrdquo Arteriosclerosis Thrombosis and VascularBiology vol 28 no 6 pp 1050ndash1059 2008

[9] M A Bouhlel J Brozek B Derudas et al ldquoUnlike PPAR120574PPAR120572 or PPAR120573120575 activation does not promote humanmono-cyte differentiation toward alternative macrophagesrdquo Biochem-ical and Biophysical Research Communications vol 386 no 3pp 459ndash462 2009

[10] G Chinetti J-C Fruchart and B Staels ldquoPeroxisome prolife-rator-activated receptors and inflammation from basic scienceto clinical applicationsrdquo International Journal of Obesity vol 27supplement 3 pp S41ndashS45 2003

[11] Y Zhu J H-C Lin H Liao et al ldquoLDL induces transcriptionfactor activator protein-1 in human endothelial cellsrdquo Arte-riosclerosis Thrombosis and Vascular Biology vol 18 no 3 pp473ndash480 1998

Evidence-Based Complementary and Alternative Medicine 13

[12] J Terao ldquoDietary flavonoids as antioxidantsrdquo Forum of Nutri-tion vol 61 pp 87ndash94 2009

[13] Y B Feng W Q Luo and S Q Zhu ldquoExplore new clinicalapplication of Huanglian and corresponding compound pre-scriptions from their traditional userdquo Zhongguo ZhongyaoZazhi vol 33 no 10 pp 1221ndash1225 2008

[14] Y H Chu C L Chang and H F Hsu ldquoFlavonoid content ofseveral vegetables and their antioxidant activityrdquo Journal of theScience of Food andAgriculture vol 80 no 5 pp 561ndash566 2000

[15] I C Arts B van de Putte and P C Hollman ldquoCatechin con-tents of foods commonly consumed in The Netherlands 1Fruits vegetables staple foods and processed foodsrdquo Journal ofAgricultural and Food Chemistry vol 48 no 5 pp 1746ndash17512000

[16] A R Proteggente A S Pannala G Paganga et al ldquoThe anti-oxidant activity of regularly consumed fruit and vegetablesreflects their phenolic and vitamin C compositionrdquo Free RadicalResearch vol 36 no 2 pp 217ndash233 2002

[17] Y L Liu L H Tang Z Q Liang B You and S Yang ldquoGrowthinhibitory and apoptosis inducing by effects of total flavonoidsfrom Lysimachia clethroides Duby in human chronic myeloidleukemiaK562 cellsrdquo Journal of Ethnopharmacology vol 131 no1 pp 1ndash9 2010

[18] W Naowaratwattana W De-Eknamkul and E G De MejialdquoPhenolic-containing organic extracts of mulberry (Morus albaL) leaves inhibit HepG2 hepatoma cells through G2M phasearrest and inhibition of topoisomerase II120572 activityrdquo Journal ofMedicinal Food vol 13 no 5 pp 1045ndash1056 2010

[19] K Doi T Kojima and Y Fujimoto ldquoMulberry leaf extract inhi-bits the oxidative modification of rabbit and human low densitylipoproteinrdquo Biological and Pharmaceutical Bulletin vol 23 no9 pp 1066ndash1071 2000

[20] W Wang Y Zu Y Fu and T Efferth ldquoIn Vitro antioxidant andantimicrobial activity of extracts from Morus alba L leavesstems and fruitsrdquoAmerican Journal of ChineseMedicine vol 40no 2 pp 349ndash356 2012

[21] B Enkhmaa K Shiwaku T Katsube et al ldquoMulberry (Morusalba L) leaves and their major flavonol quercetin 3-(6-malon-ylglucoside) attenuate atherosclerotic lesion development inLDL receptor-deficient micerdquo Journal of Nutrition vol 135 no4 pp 729ndash734 2005

[22] J Naowaboot P Pannangpetch V Kukongviriyapan B Kongy-ingyoes and U Kukongviriyapan ldquoAntihyperglycemic antiox-idant and antiglycation activities of mulberry leaf extract instreptozotocin-induced chronic diabetic ratsrdquo Plant Foods forHuman Nutrition vol 64 no 2 pp 116ndash121 2009

[23] MM Padilha F CVilela CQ Rocha et al ldquoAntiinflammatoryproperties of Morus nigra leavesrdquo Phytotherapy Research vol24 no 10 pp 1496ndash1500 2010

[24] P Aramwit O Supasyndh T Siritienthong and N BangldquoMulberry leaf reduces oxidation and c-reactive protein levelin patients with mild dyslipidemiardquo BioMed Research Interna-tional vol 2013 Article ID 787981 7 pages 2013

[25] U Justesen P Knuthsen and T Leth ldquoQuantitative analysis offlavonols flavones and flavanones in fruits vegetables andbeverages by high-performance liquid chromatography withphoto-diode array and mass spectrometric detectionrdquo Journalof Chromatography A vol 799 no 1-2 pp 101ndash110 1998

[26] M S Taga E EMiller andD E Pratt ldquoChia seeds as a source ofnatural lipid antioxidantsrdquo Journal of the AmericanOil ChemistsrsquoSociety vol 61 no 5 pp 928ndash931 1984

[27] A A L Ordonez J D Gomez M A Vattuone and M I IslaldquoAntioxidant activities of Sechium edule (Jacq) Swartz extractsrdquoFood Chemistry vol 97 no 3 pp 452ndash458 2006

[28] A Kumaran and R Joel Karunakaran ldquoIn Vitro antioxidantactivities of methanol extracts of five Phyllanthus species fromIndiardquo LWT-Food Science and Technology vol 40 no 2 pp344ndash352 2007

[29] G Cao H M Alessio and R G Cutler ldquoOxygen-radical absor-bance capacity assay for antioxidantsrdquo Free Radical Biology andMedicine vol 14 no 3 pp 303ndash311 1993

[30] K Shimada K Fujikawa K Yahara and T Nakamura ldquoAntiox-idative properties of xanthan on the autoxidation of soybeanoil in cyclodextrin emulsionrdquo Journal of Agricultural and FoodChemistry vol 40 no 6 pp 945ndash948 1992

[31] J Cole M H L Green S E James L Henderson and H ColeldquoA further assessment of factors influencing measurements ofthioguanine-resistant mutant frequency in circulating T-lymphocytesrdquo Mutation Research vol 204 no 3 pp 493ndash5071988

[32] C-Y Hsu C-M Yang C-M Chen P Chao and S Hu ldquoEff-ects of chlorophyll-related compounds on hydrogen peroxideinduced DNA damage within human lymphocytesrdquo Journal ofAgricultural and Food Chemistry vol 53 no 7 pp 2746ndash27502005

[33] S-C Tang H-F Lo K-H Lin et al ldquoThe antioxidant capacityof extracts from Taiwan indigenous purple-leaved vegetablesrdquoJournal of the Taiwan Society for Horticultural Science vol 59no 1 pp 43ndash57 2013

[34] K-H Lin Y-Y Yang H-F Lo et al ldquoAntioxidant activity ofherbaceous plant extracts protect against hydrogen peroxide-induced DNA damage in human lymphocytesrdquo BioMed CentralResearch Notes In press

[35] A H Cory T C Owen J A Barltrop and J G Cory ldquoUse of anaqueous soluble tetrazoliumformazan assay for cell growthassays in culturerdquoCancer Communications vol 3 no 7 pp 207ndash212 1991

[36] Y T Szeto A R Collins and I F F Benzie ldquoEffects of dietaryantioxidants on DNA damage in lysed cells using a modifiedcomet assay procedurerdquoMutation Research vol 500 no 1-2 pp31ndash38 2002

[37] S Vielma G Virella A J Gorod and M F Lopes-VirellaldquoChlamydophila pneumoniae infection of human aortic endo-thelial cells induces the expression of FC 120574 receptor II (Fc120574RII)rdquoClinical Immunology vol 104 no 3 pp 265ndash273 2002

[38] C Y Zhu and S Loft ldquoEffects of Brussels sprouts extracts onhydrogen peroxide-induced DNA strand breaks in humanlymphocytesrdquo Food and Chemical Toxicology vol 39 no 12 pp1191ndash1197 2001

[39] Y D Min C-H Choi H Bark et al ldquoQuercetin inhibits exp-ression of inflammatory cytokines through attenuation of NF-120581B and p38MAPK in HMC-1 humanmast cell linerdquo Inflamma-tion Research vol 56 no 5 pp 210ndash215 2007

[40] P Y Chao Y P Huang and W B Hsieh ldquoInhibitive effect ofpurple sweet potato leaf extract and its components on celladhesion and inflammatory response in human aortic endothe-lial cellsrdquo Cell Adhesion amp Migration vol 7 no 2 pp 1ndash9 2013

[41] Y Y YangThe antioxidative capacity in herb plant extracts andtheir protection role in DNA oxidative damage of lymphocyte[MS thesis] Chinese Culture University Taipei Taiwan 2004

[42] C A Rice-Evans N J Miller and G Paganga ldquoStructure-anti-oxidant activity relationships of flavonoids and phenolic acidsrdquo

14 Evidence-Based Complementary and Alternative Medicine

Free Radical Biology and Medicine vol 20 no 7 pp 933ndash9561996

[43] M Strube G R Haenen H van den Berg and A Bast ldquoPitfallsin a method for assessment of total antioxidant capacityrdquo FreeRadical Research vol 26 no 6 pp 515ndash521 1997

[44] P G Pietta ldquoFlavonoids as antioxidantsrdquo Journal of NaturalProducts vol 63 no 7 pp 1035ndash1042 2000

[45] A M Gonzalez-Paramas S Esteban-Ruano C Santos-BuelgaSDe Pascual-Teresa and J C Rivas-Gonzalo ldquoFlavanol contentand antioxidant activity in winery byproductsrdquo Journal ofAgricultural and Food Chemistry vol 52 no 2 pp 234ndash2382004

[46] S Y Kim J J Gao and H K Kang ldquoTwo flavonoids from theleaves ofMorus alba induce differentiation of the human prom-yelocytic leukemia (HL-60) cell linerdquo Biological and Pharma-ceutical Bulletin vol 23 no 4 pp 451ndash455 2000

[47] M A Khan A A Rahman S Islam et al ldquoA comparative studyon the antioxidant activity ofmethanolic extracts from differentparts of Morus alba L (Moraceae)rdquo BMC Research vol 6 pp24ndash32 2013

[48] A-L ChungH-F Lo K-H Lin et al ldquoStudy on the antioxidantactivity in herb plant extractsrdquo Journal of the Taiwan Society ForHorticultural Science vol 59 no 2 pp 139ndash152 2013

[49] R L Prior X Wu and K Schaich ldquoStandardized methods forthe determination of antioxidant capacity and phenolics infoods and dietary supplementsrdquo Journal of Agricultural andFood Chemistry vol 53 no 10 pp 4290ndash4302 2005

[50] S J Duthie A R Collins GGDuthie andV LDobson ldquoQue-rcetin and myricetin protect against hydrogen peroxide-indu-ced DNA damage (strand breaks and oxidised pyrimidines) inhuman lymphocytesrdquo Mutation Research vol 393 no 3 pp223ndash231 1997

[51] G A Liu and R L Zheng ldquoProtection against damagedDNA inthe single cell by polyphenolsrdquo Pharmazie vol 57 no 12 pp852ndash854 2002

[52] M Noroozi W J Angerson and M E J Lean ldquoEffects of flav-onoids and vitamin C on oxidative DNA damage to humanlymphocytesrdquoAmerican Journal of Clinical Nutrition vol 67 no6 pp 1210ndash1218 1998

[53] T Koga and M Meydani ldquoEffect of plasma metabolites of (+)-catechin and quercetin on monocyte adhesion to human aorticendothelial cellsrdquoAmerican Journal of Clinical Nutrition vol 73no 5 pp 941ndash948 2001

[54] CMeng P Somers L Hoong et al ldquoDiscovery of novel pheno-lic antioxidants as inhibitors of vascular cell adhesionmolecule-1 expression for use in chronic inflammatory diseasesrdquo Journalof Medicinal Chemistry vol 47 no 25 pp 6420ndash6432 2004

[55] Y Shibata N Kume H Arai et al ldquoMulberry leaf aqueousfractions inhibit TNF-120572-induced nuclear factor 120581B (NF-120581B)activation and lectin-like oxidized LDL receptor-1 (LOX-1)expression in vascular endothelial cellsrdquoAtherosclerosis vol 193no 1 pp 20ndash27 2007

[56] K C Chan H H Ho C N Huang M Lin H Chen and CWang ldquoMulberry leaf extract inhibits vascular smooth musclecell migration involving a block of small GTPase and AktNF-kB signalsrdquo Journal of Agricultural and Food Chemistry vol 57no 19 pp 9147ndash9153 2009

[57] F Chen N Nakashima I Kimura M Kimura N Asanoand S Koya ldquoPotentiating effects on pilocarpine-induced salivasecretion by extracts and N-containing sugars derived frommulberry leaves in streptozocin- diabetic micerdquo Biological andPharmaceutical Bulletin vol 18 no 12 pp 1676ndash1680 1995

[58] K Konno H Ono M Nakamura et al ldquoMulberry latexrich in antidiabetic sugar-mimic alkaloids forces dieting oncaterpillarsrdquo Proceedings of the National Academy of Sciences ofthe United States of America vol 103 no 5 pp 1337ndash1341 2006

[59] H J Kim K H Yoon M J Kang et al ldquoA six-month sup-plementation of mulberry Korean red ginseng and banabadecreases biomarkers of systemic low-grade inflammation insubjects with impaired glucose tolerance and type 2 diabetesrdquoEvidence-based Complementary and Alternative Medicine vol2012 Article ID 735191 8 pages 2012

[60] G Nicholson and G M Hall ldquoDiabetes mellitus new drugs fora new epidemicrdquo British Journal of Anaesthesia vol 107 no 1pp 65ndash73 2011

[61] P Y Chao Y L Chen Y C Lin et al ldquoEffects of black soybeanon atherogenic prevention in hypercholesterolemic rabbits andon adhesion molecular expression in cultured HAECsrdquo Foodand Nutrition Sciences vol 4 pp 9ndash21 2013

[62] C Chen S Li C-YOChen et al ldquoConstituents in purple sweetpotato leaves inhibit in vitro angiogenesis with opposite effectsex vivordquo Nutrition vol 27 no 11-12 pp 1177ndash1182 2011

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

Evidence-Based Complementary and Alternative Medicine 7

Control H2O2

BT25 BT50 BT100

MT25 MT50 MT100

M25 M50 M100

(a)

alowastblowast blowastblowast

blowastblowast

blowast

25 50 100Mulberry extracts (120583gmL)

MTMBT

H2O2

0

20

40

60

80

100

Tail

DN

A (

)

ablowastablowast

(b)

Tail

mom

ent

0

2000

4000

6000

8000

10000

lowastlowast lowast

alowastalowast

25 50 100Mulberry extracts (120583gmL)

MTMBT

H2O2

ablowast ablowastablowast

bclowast

(c)

Figure 2 Effects of various water extracts of MLRC on H2O2-induced DNA damage to lymphocytes The comet-like images resulting from

the extension ofDNA (a) were observed using a fluorescencemicroscope Tail DNA (b) and tailmovement (c) were exposed to testmulberryleaf-related extractsat 25 50 and 100120583gmL and treated with H

2O2at 10120583M Values with different letters differ significantly with regard to

oxidative damage when comparing different MLRC extracts lowast119875 lt 005 refers to differences in oxidative damage as compared with 10120583MH2O2-alone treatment

NF-120581B in response to treatment with MLREs resulted inreduced binding of NF-120581B to DNAThe treatment of HAECswith TNF-120572 led to increased binding of NF-120581B howeverpretreatment of HAECs with BT MT and M significantlydecreased DNA-bound NF-120581B (Figure 5(a)) The increased

binding of AP-1 with TNF-120572 resulted from the treatment ofHAECs however pretreatment of HAECs with BT MT andM significantly decreased DNA-bound AP-1 (Figure 5(b))STAT3 DNA-binding activities to its cognate recognitionsite were remarkably reduced compared to TNF-120572 treatment

8 Evidence-Based Complementary and Alternative Medicine

(A)(A)

(B)

(C) (D)

(E) (F)

(I)

(G) (H)

200120583m 200120583m

200120583m200120583m

200120583m200120583m

200120583m200120583m

200120583m

(a)

Con

trol

Asp

irin

BT25

BT50

MT2

5

MT5

0

M25

M50

Mon

ocyt

e adh

eren

ce (c

ells

HPF

)

0

20

40

60

80

100

120

140

160

180

lowast lowast lowast

lowast

lowast

lowast

lowast

TNF-120572

(b)

Figure 3 Mulberry leaf related extracts (MLREs) reduce tumor necrosis factor (TNF)-120572-induced adhesion of monocytes to human aorticendothelial cells (HAECs) (a) The number of adherent U937 cells (marked by arrow) was monitored by fluorescence microscopy undercontrol (a) 2 ngmL TNF-120572 (b) co-treated with TNF-119886 and aspirin (10120583M) (c) cotreated with TNF-120572 and BT (25 120583gmL) (d) cotreated withTNF-120572 and BT (50 120583gmL) (e) cotreated with TNF-120572 and MT (25 120583gmL) (f) cotreated with TNF-120572 and MT (50 120583gmL) (g) cotreated withTNF-120572 and M (25120583gmL) (h) and cotreated with TNF-120572 and M (50120583gmL) (i) (b) HAECs were pretreated with MLREs for 18 h followedby stimulating with TNF-120572 for 6 h Fluorescence-labeled monocytic U937 cells were added to HAECs and allowed to adhere for 2 h Data areexpressed as a percentage of TNF-120572-induced expression (mean plusmn SD) of three experiments Mean plusmn SD lowast119875 lt 005 BT MT and M at 25and 50120583gmL versus TNF-120572

119875 lt 005 control versus TNF-120572

Evidence-Based Complementary and Alternative Medicine 9

Control TNF-120572

TNF-120572

Aspirin10120583M

+ + + + + + + +

BT25 BT50 MT25 MT50 M25 M50

120583gmL 120583gmL 120583gmL 120583gmL 120583gmL 120583gmL

NF-120581B p65

120573-Actin43kDa

minus

65kDa

(a)

Con

trol

Asp

irin

BT25

BT50

MT2

5

MT5

0

M25

M50

00

02

04

06

08

10

lowast

lowast

lowast

lowastlowastlowastTN

F-120572

Ratio

(nuc

lear

120573-a

ctin

)

(b)

Figure 4 Effects of MLREs on NF-120581B activity After HAECs were pretreated with the indicated samples and incubated with TNF-120572 nuclearextracts were prepared and the expression of NF-120581B p65 was assessed by western blot analysis A representative image of three similar resultsis shown (a) Actin served as the loading control for the nuclear compartment Semiquantitative analysis of three independent experimentsare also shown (b) indicates a significant difference between the TNF-120572 treated or experimental treatment groups and the control119875 lt 005lowast indicates a significant difference between the TNF-120572 and experimental treatment groups BT MT and M at 25 and 50 120583gmL

(lane 3) in the nuclear fraction indicating that MLREs at50120583gmL inhibited STAT3-binding activities In addition theresults in Figure 5(c) (lanes 4ndash10) show that aspirinMLREs at50120583gmL and BT at 25120583gmL remarkably decreased STAT3levels compared to TNF-120572 (lane 3) however both MT andM at 25120583gmL increased STAT3-binding activities Theseresults indicate that only 50 120583gmL MLREs 25 120583gmL BTand aspirin block STAT3 activation and might downregulateTNF-120572-induced expressions of inflammatory signaling path-ways

35 MLREs Regulated the DNA-Binding Activity and Expres-sions of PPAR 120572 and PPAR 120574 in HAECs PPAR 120572 and 120574effectively bound to PPAR 120572 and 120574 oligo-IRDye 700 asindicated in lanes 3ndash10 of Figure 6 Supershifts are bands fromthe binding of each specific antibody The binding activitiesof PPAR 120572 and 120574 were detected at baseline The 25 120583gmLMT extract (lane 7) and 25 and 50 120583gmL M extracts (lanes9-10) increased activity compared to incubating with TNF-120572 alone (lane 3) However BT at both 25 and 50 120583gmLextracts showed reduced effects on PPAR 120572 and 120574 expressionMoreover the 50 120583gmLMT extract had no effect on PPAR 120572binding activity but increased the binding activity of PPAR120574 in comparison to incubation with TNF-120572 alone Thesedata suggest that MT and M both promoted PPAR 120572 and 120574

expression at least in part by increasing the DNA binding oftranscription factors PPAR 120572 and 120574

4 Discussion

41 Antioxidant Composition and Antioxidant Activity BTcontains high levels of polyphenols flavonoids flavonolsanthocyanidin quercetin and kaempferol (Tables 1 and 2)BT also had a higher TEAC value (15514mM Table 3) thanthe TEAC values of 47mM [42] and 64mM [43] reportedfor quercetin as well as 242mM reported for quercetin-3-rutinoside [44] Furthermore Gonzalez-Paramas et al [45]demonstrated that flavonol contents were significantly andhighly correlated (119903 = 08) with TEAC values Kim et al [46]reported that mulberry leaves contain abundant quercetinand inhibit lipid peroxidation at concentrations of 01 and001mgmL In our study the amount of quercetin wassignificantly correlated (119903 = 041 Table 4) with TEAC valueHowever no correlation was found among myricetin morinkaempferol and TEAC which may be due to the structurerequired to reinforce the free radical scavenging activity thatvaries with the type of free radical Naowaratwattana et al[18] reported that a 50 methanolic extract of mulberryleaf contained rutin isoquercetin and various derivatives ofkaempferol and quercetin glycosides while a water extract

10 Evidence-Based Complementary and Alternative Medicine

NF-120581BTNF-120572

TNF-120572

+

Aspirin10120583M+ +

BT25120583gmL

+

BT50120583gmL

+

MT25120583gmL

+

MT50120583gmL

+

M25

120583gmL+

M50

120583gmLControl

minus

DNAOligo

Free probe

(a)

AP-1TNF-120572

TNF-120572

+

Aspirin10120583M+ +

BT25120583gmL

+

BT50120583gmL

+

MT25120583gmL

+

MT50120583gmL

+

M25

120583gmL+

M50

120583gmLControl

minus

DNAOligo

Free probe

(b)

STAT3

TNF-120572

TNF-120572

+

Aspirin10120583M+ +

BT25120583gmL

+

BT50120583gmL

+

MT25120583gmL

+

MT50120583gmL

+

M25

120583gmL+

M50

120583gmLControl

minus

DNAOligo

Free probe

(c)

Figure 5 MLREs inhibits the binding of (a) NF-120581B (b) AP-1 and (c) STAT3 to target DNA sequence NF-120581B AP-1 and STAT3 transcriptionfactor-DNA interactions were assessed by EMSA

contained primarily chlorogenic acid and caffeoylquinic acidderivatives The IC

50of DPPH radical scavenging was 794

and 2042120583gmL for 50 methanolic and water extractsrespectively [18] In our studyM extracts contained quercetinand kaempferol (Table 2) which may account for part ofthe highly scavenged DPPH radical in comparison to therest of MLREs Khan et al [47] demonstrated that the IC

50

was 22023120583gmL which is much less efficacious than themulberry leaf acidic methanolic extract IC

50of 1888 120583gmL

(Table 3) However the IC50

of in vitro free radical scav-enging activity of mulberry leaves from Naowaboot et al[22] was 617 plusmn 21 120583gmL which is more efficacious thanthe IC

50concentration derived in the present study Taken

together the polyphenol-rich MLREs exhibited distinct cell-free antioxidant activity (ie TEAC) as validated by theirlevels of polyphenols and flavonols with distinct antioxidantactivity strongly accounting for the antioxidant activity of the

extracts The amount of quercetin was especially positivelycorrelated with the observed TEAC values

42 Estimation of DNA Single Strand Break Damage fromExposure to Acidic Methanolic and Water Extracts One ofthe antioxidant activity tests scavenging of DPPH radi-cal was selected due to its quick and simple but it is aless biologically relevant assay Previously we reported thatwith 2 120583gsdotmLminus1 of methanolic and water extracts from theindigenous vegetables [33] and herb plants [48] significantlyprolonged lag phase of low-density lipoprotein (LDL) TheLDL oxidation assay is considered to be one of the mostbiologically relevant assays [49] Both studies [33 48] alsoshowed that the concentration of the tested extracts forscavenging DPPH radical was in higher levels at 10mg oftested extracts with 2ndash53 inhibition Lower concentrationwas then chosen for the treated dosage in the present study

Evidence-Based Complementary and Alternative Medicine 11

TNF-120572

TNF-120572

+

Aspirin10120583M+ +

BT25120583gmL

+

BT50120583gmL

+

MT25120583gmL

+

MT50120583gmL

+

M25

120583gmL+

M50

120583gmLControl

minus

DNAOligo

Free probe

PPAR 120572

(a)

TNF-120572

TNF-120572

+

Aspirin10120583M+ +

BT25120583gmL

+

BT50120583gmL

+

MT25120583gmL

+

MT50120583gmL

+

M25

120583gmL+

M50

120583gmLControl

minus

DNAOligo

Free probe

PPAR120574

(b)

Figure 6 Effect of treatments on the expression of PPAR 120572 (a) and PPAR 120574 (b) in TNF-120572-stimulated HAECs by EMSA

In addition lower concentration also could avoid the toxicityeffect of higher level of extracts on lymphocytes Quercetinwas found to protect against H

2O2-induced DNA damage in

human lymphocytes at 10 120583M [50] and at 31 to 25 120583M [51]Quercetin was also reported to induce DNA strand breaks invarious cell types but only at higher doses at 100 120583Mor above[50] Duthie et al [50] proposed that the dihydroxy structureof quercetin and myricetin might be essential in protectingDNA against hydrogen peroxide No such hydroxyl groupsare present in the tocopherol molecule Noroozi et al [52]demonstrated that in addition to quercetin kaempferolcould also inhibit H

2O2-induced DNA strand breaks in

human lymphocytes In our study both BT and M were richin quercetin and kaempferol (Table 2) whichmay account forthe protective roles of both extracts in preventingDNAstrandbreaks from human lymphocytes

43 MLREs Regulated the DNA-Binding Activity and Expres-sions of NF-120581B AP-1 STAT3 and PPARs in HAECsBecause MLREs exhibited free radical scavenging the anti-inflammatory effects of MLREs on TNF-120572-induced celladhesion and adhesionmolecule expressionwere determinedin the present study Our results suggest that MLREs andaspirin significantly inhibited TNF-120572-induced monocyte-endothelial cell adhesion (Figure 3) Significant reductionsin NF-120581B expression and DNA binding by aspirin MTextract (25 120583gmL) and BT extract (both 25 and 50 120583gmL)were also observed The adhesion of monocytes to the

vascular endothelium and subsequent migration into thevessel wall are early events in atherogenesis [2] Our resultsare consistent with previous research showing that dietarypolyphenols such as catechin and quercetin significantlyreduce the binding of monocytes to HAECs [53 54] Thereduced cellular adhesion may be due to the inhibitionof cellular adhesion molecule expression as purple sweetpotato leaf extract (PSPLE) reduced both VCAM-1 andICAM-1 expression [40] Shibata et al [55] demonstratedthat pretreatment of cultured bovine aortic endothelial cells(BAECs) with mulberry leaf aqueous fractions inhibitedTNF-120572- and LPS-induced expression of Lectin-like Ox-LDLreceptor-1 (LOX-1) a cell-surface receptor for atherogenicOx-LDL (oxidized low-density lipoprotein) It also appearsto mediate Ox-LDL-induced inflammation which may becrucial in atherogenesis Furthermore mulberry leaf aqueousfractions inhibited the TNF-120572-induced activation of NF-120581Band phosphorylation of inhibitory factor of NF-120581B-alpha(I120581B-120572) Thus mulberry leaf aqueous fractions suppressTNF-120572- and LPS-induced LOX-1 gene expression mediatedby inhibiting NF-120581B activation

Chan et al [56] demonstrated that mulberry leaf extract(MLE) was rich in polyphenols MLE can effectively inhibitthe migration of vascular smooth muscle cells (VSMC) byblocking small guanosine triphosphatases (small GTPase)and AktNF-120581B signals The Rho family of small GTPasessuch as Rho Rac and Cdc42 acts as a molecular switch toregulate the actin cytoskeleton The Rho family of GTPases

12 Evidence-Based Complementary and Alternative Medicine

is also reported to be involved in the regulation of NF-120581B-dependent transcription [56] MLE has been shown to pos-sess hypoglycemic effects in an insulin-dependent diabetesmellitus (type 1 diabetes) animal model and potential hypo-glycemic compounds inmulberry leaves were suggested to be2-O-120572-D-galactopyranosyl-DNJ and fagomine [57] Konnoet al [58] further reported that mulberry latex containsalkaloidal sugar-mimic glycosidase inhibitors with antidia-betic activities such as 14-dideoxy-14-imino-D-arabinitol 1-deoxynojirimycin and 14-dideoxy-14-imino-D-ribitol Kimet al [59] recently showed that treated type 2 diabetesmellitus(T2D) with a 1 1 1 mixture of ginseng root mulberry leafwater extract and banana leaf water extract (6 gd) for 24weeks significantly decreased plasma intracellular adhesionmolecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) levels This indicates suppressed inflammatoryresponses in T2D but no significant differences in glucosehomeostasis control measurement changes Rigamonti et al[8] reported that bothPPAR120572 and 120574maybe thought of as sup-pressing the activity of NF120581B and AP-1 pathways that reduceinflammatory mediator production in the macrophages ofhumans and mice In our study MT and M promoted PPAR120572 and 120574 expression at least in part by increasing DNAbinding of transcription factors PPAR 120572 and 120574 PPAR-120572-and 120574-dependent signaling pathwaysmay also play importantroles in mediating the TNF-120572 receptor signaling pathwayby MT and M by suppressing the activity on NF-120581B andAP-1 pathways These pathways may regulate downstreamproinflammatory gene expression and adhesion molecularexpression Clinically PPAR 120574 agonists such as rosiglitazoneare widely used in the treatment of T2D [60] M and MTextracts increased PPAR 120574 activity which may partly explainwhy inflammatory responses are suppressed in T2D

Although reduced NF-120581B expression and DNA bind-ing were observed upon treatment with ML MT and BT(Figure 5) the mechanism was not assessed and is worthy offurther study In addition the present results were obtainedusing in vitro studies and may be consistent with an invivo model of atherogenesis similar to that reported byEnkhmaa et al [21] in which mulberry leaves suppressedthe development of atherosclerotic lesions Previously weproved that black beans significantly prolong LDL lag timeand decrease the atheroma regions of the aortic arch andthoracic aorta in hypercholesterolemic NZW rabbits [61]Wethen demonstrated that black soybean extract and PSPLEsignificantly decreased the adhesion of U937 monocyticcells to TNF-120572-stimulated HAECs and reduced adhesionmolecules expression (VCAM-1 and ICAM-1) as well as NF-120581B-p65 expression [37] This is particularly important giventhe opposing effects of flavonols fromMLREs obtained usingin vitro and ex vivo studies [62]

5 Conclusion

Tested MLREs were rich in flavonols and had strongantioxidant activity in protecting lymphocyte DNA fromoxidative damage In particular BT was rich in quercetinand polyphenols The antioxidant components of MLREs

might downregulate intracellular redox-dependent signalingpathways in HAECs upon TNF-120572 stimulation which mightprevent ROS-mediated endothelial cell dysfunction MLRCshave anti-inflammatory effects through the modulation ofNF-120581B AP-1 STAT3 and PPARs signaling These chemi-cals appear to provide distinct cytoprotective mechanismsthat might contribute to their putative beneficial effectsin suppressing endothelial responses to cytokines duringinflammation These results may benefit further in vivostudies to assess the therapeutic potential of phytochemicalsas anti-inflammatories for use in cytokine-induced vasculardisorders including atherosclerosis

Acknowledgment

This work was supported in part by Grants from the NationalScience Council of Taiwan (NSC 95ndash2320-B-034-001 andNSC 96ndash2320-B-034-001)

References

[1] W Palinski ldquoUnited they go conjunct regulation of aorticantioxidant enzymes during atherogenesisrdquo CirculationResearch vol 93 no 3 pp 183ndash185 2003

[2] P Libby ldquoMolecular bases of the acute coronary syndromesrdquoCirculation vol 91 no 11 pp 2844ndash2850 1995

[3] P A Baeuerle and D Baltimore ldquoNf-120581B ten years afterrdquo Cellvol 87 no 1 pp 13ndash20 1996

[4] J M Kyriakis ldquoActivation of the AP-1 transcription factor byinflammatory cytokines of the TNF familyrdquo Gene Expressionvol 7 no 4ndash6 pp 217ndash231 1999

[5] J M Muller R A Rupec and P A Baeuerle ldquoStudy of generegulation by NF-120581B and AP-1 in response to reactive oxygenintermediatesrdquoMethods vol 11 no 3 pp 301ndash312 1997

[6] G Chinetti J Fruchart and B Staels ldquoPeroxisome proliferator-activated receptors new targets for the pharmacological mod-ulation of macrophage gene expression and functionrdquo CurrentOpinion in Lipidology vol 14 no 5 pp 459ndash468 2003

[7] G Chinetti J C Fruchart and B Staels ldquoTranscriptional regu-lation of macrophage cholesterol trafficking by PPAR120572 andLXRrdquo Biochemical Society Transactions vol 34 no 6 pp 1128ndash1131 2006

[8] E Rigamonti G Chinetti-Gbaguidi and B Staels ldquoRegulationof macrophage functions by PPAR- 120572 PPAR- 120574 and LXRsin mice and menrdquo Arteriosclerosis Thrombosis and VascularBiology vol 28 no 6 pp 1050ndash1059 2008

[9] M A Bouhlel J Brozek B Derudas et al ldquoUnlike PPAR120574PPAR120572 or PPAR120573120575 activation does not promote humanmono-cyte differentiation toward alternative macrophagesrdquo Biochem-ical and Biophysical Research Communications vol 386 no 3pp 459ndash462 2009

[10] G Chinetti J-C Fruchart and B Staels ldquoPeroxisome prolife-rator-activated receptors and inflammation from basic scienceto clinical applicationsrdquo International Journal of Obesity vol 27supplement 3 pp S41ndashS45 2003

[11] Y Zhu J H-C Lin H Liao et al ldquoLDL induces transcriptionfactor activator protein-1 in human endothelial cellsrdquo Arte-riosclerosis Thrombosis and Vascular Biology vol 18 no 3 pp473ndash480 1998

Evidence-Based Complementary and Alternative Medicine 13

[12] J Terao ldquoDietary flavonoids as antioxidantsrdquo Forum of Nutri-tion vol 61 pp 87ndash94 2009

[13] Y B Feng W Q Luo and S Q Zhu ldquoExplore new clinicalapplication of Huanglian and corresponding compound pre-scriptions from their traditional userdquo Zhongguo ZhongyaoZazhi vol 33 no 10 pp 1221ndash1225 2008

[14] Y H Chu C L Chang and H F Hsu ldquoFlavonoid content ofseveral vegetables and their antioxidant activityrdquo Journal of theScience of Food andAgriculture vol 80 no 5 pp 561ndash566 2000

[15] I C Arts B van de Putte and P C Hollman ldquoCatechin con-tents of foods commonly consumed in The Netherlands 1Fruits vegetables staple foods and processed foodsrdquo Journal ofAgricultural and Food Chemistry vol 48 no 5 pp 1746ndash17512000

[16] A R Proteggente A S Pannala G Paganga et al ldquoThe anti-oxidant activity of regularly consumed fruit and vegetablesreflects their phenolic and vitamin C compositionrdquo Free RadicalResearch vol 36 no 2 pp 217ndash233 2002

[17] Y L Liu L H Tang Z Q Liang B You and S Yang ldquoGrowthinhibitory and apoptosis inducing by effects of total flavonoidsfrom Lysimachia clethroides Duby in human chronic myeloidleukemiaK562 cellsrdquo Journal of Ethnopharmacology vol 131 no1 pp 1ndash9 2010

[18] W Naowaratwattana W De-Eknamkul and E G De MejialdquoPhenolic-containing organic extracts of mulberry (Morus albaL) leaves inhibit HepG2 hepatoma cells through G2M phasearrest and inhibition of topoisomerase II120572 activityrdquo Journal ofMedicinal Food vol 13 no 5 pp 1045ndash1056 2010

[19] K Doi T Kojima and Y Fujimoto ldquoMulberry leaf extract inhi-bits the oxidative modification of rabbit and human low densitylipoproteinrdquo Biological and Pharmaceutical Bulletin vol 23 no9 pp 1066ndash1071 2000

[20] W Wang Y Zu Y Fu and T Efferth ldquoIn Vitro antioxidant andantimicrobial activity of extracts from Morus alba L leavesstems and fruitsrdquoAmerican Journal of ChineseMedicine vol 40no 2 pp 349ndash356 2012

[21] B Enkhmaa K Shiwaku T Katsube et al ldquoMulberry (Morusalba L) leaves and their major flavonol quercetin 3-(6-malon-ylglucoside) attenuate atherosclerotic lesion development inLDL receptor-deficient micerdquo Journal of Nutrition vol 135 no4 pp 729ndash734 2005

[22] J Naowaboot P Pannangpetch V Kukongviriyapan B Kongy-ingyoes and U Kukongviriyapan ldquoAntihyperglycemic antiox-idant and antiglycation activities of mulberry leaf extract instreptozotocin-induced chronic diabetic ratsrdquo Plant Foods forHuman Nutrition vol 64 no 2 pp 116ndash121 2009

[23] MM Padilha F CVilela CQ Rocha et al ldquoAntiinflammatoryproperties of Morus nigra leavesrdquo Phytotherapy Research vol24 no 10 pp 1496ndash1500 2010

[24] P Aramwit O Supasyndh T Siritienthong and N BangldquoMulberry leaf reduces oxidation and c-reactive protein levelin patients with mild dyslipidemiardquo BioMed Research Interna-tional vol 2013 Article ID 787981 7 pages 2013

[25] U Justesen P Knuthsen and T Leth ldquoQuantitative analysis offlavonols flavones and flavanones in fruits vegetables andbeverages by high-performance liquid chromatography withphoto-diode array and mass spectrometric detectionrdquo Journalof Chromatography A vol 799 no 1-2 pp 101ndash110 1998

[26] M S Taga E EMiller andD E Pratt ldquoChia seeds as a source ofnatural lipid antioxidantsrdquo Journal of the AmericanOil ChemistsrsquoSociety vol 61 no 5 pp 928ndash931 1984

[27] A A L Ordonez J D Gomez M A Vattuone and M I IslaldquoAntioxidant activities of Sechium edule (Jacq) Swartz extractsrdquoFood Chemistry vol 97 no 3 pp 452ndash458 2006

[28] A Kumaran and R Joel Karunakaran ldquoIn Vitro antioxidantactivities of methanol extracts of five Phyllanthus species fromIndiardquo LWT-Food Science and Technology vol 40 no 2 pp344ndash352 2007

[29] G Cao H M Alessio and R G Cutler ldquoOxygen-radical absor-bance capacity assay for antioxidantsrdquo Free Radical Biology andMedicine vol 14 no 3 pp 303ndash311 1993

[30] K Shimada K Fujikawa K Yahara and T Nakamura ldquoAntiox-idative properties of xanthan on the autoxidation of soybeanoil in cyclodextrin emulsionrdquo Journal of Agricultural and FoodChemistry vol 40 no 6 pp 945ndash948 1992

[31] J Cole M H L Green S E James L Henderson and H ColeldquoA further assessment of factors influencing measurements ofthioguanine-resistant mutant frequency in circulating T-lymphocytesrdquo Mutation Research vol 204 no 3 pp 493ndash5071988

[32] C-Y Hsu C-M Yang C-M Chen P Chao and S Hu ldquoEff-ects of chlorophyll-related compounds on hydrogen peroxideinduced DNA damage within human lymphocytesrdquo Journal ofAgricultural and Food Chemistry vol 53 no 7 pp 2746ndash27502005

[33] S-C Tang H-F Lo K-H Lin et al ldquoThe antioxidant capacityof extracts from Taiwan indigenous purple-leaved vegetablesrdquoJournal of the Taiwan Society for Horticultural Science vol 59no 1 pp 43ndash57 2013

[34] K-H Lin Y-Y Yang H-F Lo et al ldquoAntioxidant activity ofherbaceous plant extracts protect against hydrogen peroxide-induced DNA damage in human lymphocytesrdquo BioMed CentralResearch Notes In press

[35] A H Cory T C Owen J A Barltrop and J G Cory ldquoUse of anaqueous soluble tetrazoliumformazan assay for cell growthassays in culturerdquoCancer Communications vol 3 no 7 pp 207ndash212 1991

[36] Y T Szeto A R Collins and I F F Benzie ldquoEffects of dietaryantioxidants on DNA damage in lysed cells using a modifiedcomet assay procedurerdquoMutation Research vol 500 no 1-2 pp31ndash38 2002

[37] S Vielma G Virella A J Gorod and M F Lopes-VirellaldquoChlamydophila pneumoniae infection of human aortic endo-thelial cells induces the expression of FC 120574 receptor II (Fc120574RII)rdquoClinical Immunology vol 104 no 3 pp 265ndash273 2002

[38] C Y Zhu and S Loft ldquoEffects of Brussels sprouts extracts onhydrogen peroxide-induced DNA strand breaks in humanlymphocytesrdquo Food and Chemical Toxicology vol 39 no 12 pp1191ndash1197 2001

[39] Y D Min C-H Choi H Bark et al ldquoQuercetin inhibits exp-ression of inflammatory cytokines through attenuation of NF-120581B and p38MAPK in HMC-1 humanmast cell linerdquo Inflamma-tion Research vol 56 no 5 pp 210ndash215 2007

[40] P Y Chao Y P Huang and W B Hsieh ldquoInhibitive effect ofpurple sweet potato leaf extract and its components on celladhesion and inflammatory response in human aortic endothe-lial cellsrdquo Cell Adhesion amp Migration vol 7 no 2 pp 1ndash9 2013

[41] Y Y YangThe antioxidative capacity in herb plant extracts andtheir protection role in DNA oxidative damage of lymphocyte[MS thesis] Chinese Culture University Taipei Taiwan 2004

[42] C A Rice-Evans N J Miller and G Paganga ldquoStructure-anti-oxidant activity relationships of flavonoids and phenolic acidsrdquo

14 Evidence-Based Complementary and Alternative Medicine

Free Radical Biology and Medicine vol 20 no 7 pp 933ndash9561996

[43] M Strube G R Haenen H van den Berg and A Bast ldquoPitfallsin a method for assessment of total antioxidant capacityrdquo FreeRadical Research vol 26 no 6 pp 515ndash521 1997

[44] P G Pietta ldquoFlavonoids as antioxidantsrdquo Journal of NaturalProducts vol 63 no 7 pp 1035ndash1042 2000

[45] A M Gonzalez-Paramas S Esteban-Ruano C Santos-BuelgaSDe Pascual-Teresa and J C Rivas-Gonzalo ldquoFlavanol contentand antioxidant activity in winery byproductsrdquo Journal ofAgricultural and Food Chemistry vol 52 no 2 pp 234ndash2382004

[46] S Y Kim J J Gao and H K Kang ldquoTwo flavonoids from theleaves ofMorus alba induce differentiation of the human prom-yelocytic leukemia (HL-60) cell linerdquo Biological and Pharma-ceutical Bulletin vol 23 no 4 pp 451ndash455 2000

[47] M A Khan A A Rahman S Islam et al ldquoA comparative studyon the antioxidant activity ofmethanolic extracts from differentparts of Morus alba L (Moraceae)rdquo BMC Research vol 6 pp24ndash32 2013

[48] A-L ChungH-F Lo K-H Lin et al ldquoStudy on the antioxidantactivity in herb plant extractsrdquo Journal of the Taiwan Society ForHorticultural Science vol 59 no 2 pp 139ndash152 2013

[49] R L Prior X Wu and K Schaich ldquoStandardized methods forthe determination of antioxidant capacity and phenolics infoods and dietary supplementsrdquo Journal of Agricultural andFood Chemistry vol 53 no 10 pp 4290ndash4302 2005

[50] S J Duthie A R Collins GGDuthie andV LDobson ldquoQue-rcetin and myricetin protect against hydrogen peroxide-indu-ced DNA damage (strand breaks and oxidised pyrimidines) inhuman lymphocytesrdquo Mutation Research vol 393 no 3 pp223ndash231 1997

[51] G A Liu and R L Zheng ldquoProtection against damagedDNA inthe single cell by polyphenolsrdquo Pharmazie vol 57 no 12 pp852ndash854 2002

[52] M Noroozi W J Angerson and M E J Lean ldquoEffects of flav-onoids and vitamin C on oxidative DNA damage to humanlymphocytesrdquoAmerican Journal of Clinical Nutrition vol 67 no6 pp 1210ndash1218 1998

[53] T Koga and M Meydani ldquoEffect of plasma metabolites of (+)-catechin and quercetin on monocyte adhesion to human aorticendothelial cellsrdquoAmerican Journal of Clinical Nutrition vol 73no 5 pp 941ndash948 2001

[54] CMeng P Somers L Hoong et al ldquoDiscovery of novel pheno-lic antioxidants as inhibitors of vascular cell adhesionmolecule-1 expression for use in chronic inflammatory diseasesrdquo Journalof Medicinal Chemistry vol 47 no 25 pp 6420ndash6432 2004

[55] Y Shibata N Kume H Arai et al ldquoMulberry leaf aqueousfractions inhibit TNF-120572-induced nuclear factor 120581B (NF-120581B)activation and lectin-like oxidized LDL receptor-1 (LOX-1)expression in vascular endothelial cellsrdquoAtherosclerosis vol 193no 1 pp 20ndash27 2007

[56] K C Chan H H Ho C N Huang M Lin H Chen and CWang ldquoMulberry leaf extract inhibits vascular smooth musclecell migration involving a block of small GTPase and AktNF-kB signalsrdquo Journal of Agricultural and Food Chemistry vol 57no 19 pp 9147ndash9153 2009

[57] F Chen N Nakashima I Kimura M Kimura N Asanoand S Koya ldquoPotentiating effects on pilocarpine-induced salivasecretion by extracts and N-containing sugars derived frommulberry leaves in streptozocin- diabetic micerdquo Biological andPharmaceutical Bulletin vol 18 no 12 pp 1676ndash1680 1995

[58] K Konno H Ono M Nakamura et al ldquoMulberry latexrich in antidiabetic sugar-mimic alkaloids forces dieting oncaterpillarsrdquo Proceedings of the National Academy of Sciences ofthe United States of America vol 103 no 5 pp 1337ndash1341 2006

[59] H J Kim K H Yoon M J Kang et al ldquoA six-month sup-plementation of mulberry Korean red ginseng and banabadecreases biomarkers of systemic low-grade inflammation insubjects with impaired glucose tolerance and type 2 diabetesrdquoEvidence-based Complementary and Alternative Medicine vol2012 Article ID 735191 8 pages 2012

[60] G Nicholson and G M Hall ldquoDiabetes mellitus new drugs fora new epidemicrdquo British Journal of Anaesthesia vol 107 no 1pp 65ndash73 2011

[61] P Y Chao Y L Chen Y C Lin et al ldquoEffects of black soybeanon atherogenic prevention in hypercholesterolemic rabbits andon adhesion molecular expression in cultured HAECsrdquo Foodand Nutrition Sciences vol 4 pp 9ndash21 2013

[62] C Chen S Li C-YOChen et al ldquoConstituents in purple sweetpotato leaves inhibit in vitro angiogenesis with opposite effectsex vivordquo Nutrition vol 27 no 11-12 pp 1177ndash1182 2011

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

8 Evidence-Based Complementary and Alternative Medicine

(A)(A)

(B)

(C) (D)

(E) (F)

(I)

(G) (H)

200120583m 200120583m

200120583m200120583m

200120583m200120583m

200120583m200120583m

200120583m

(a)

Con

trol

Asp

irin

BT25

BT50

MT2

5

MT5

0

M25

M50

Mon

ocyt

e adh

eren

ce (c

ells

HPF

)

0

20

40

60

80

100

120

140

160

180

lowast lowast lowast

lowast

lowast

lowast

lowast

TNF-120572

(b)

Figure 3 Mulberry leaf related extracts (MLREs) reduce tumor necrosis factor (TNF)-120572-induced adhesion of monocytes to human aorticendothelial cells (HAECs) (a) The number of adherent U937 cells (marked by arrow) was monitored by fluorescence microscopy undercontrol (a) 2 ngmL TNF-120572 (b) co-treated with TNF-119886 and aspirin (10120583M) (c) cotreated with TNF-120572 and BT (25 120583gmL) (d) cotreated withTNF-120572 and BT (50 120583gmL) (e) cotreated with TNF-120572 and MT (25 120583gmL) (f) cotreated with TNF-120572 and MT (50 120583gmL) (g) cotreated withTNF-120572 and M (25120583gmL) (h) and cotreated with TNF-120572 and M (50120583gmL) (i) (b) HAECs were pretreated with MLREs for 18 h followedby stimulating with TNF-120572 for 6 h Fluorescence-labeled monocytic U937 cells were added to HAECs and allowed to adhere for 2 h Data areexpressed as a percentage of TNF-120572-induced expression (mean plusmn SD) of three experiments Mean plusmn SD lowast119875 lt 005 BT MT and M at 25and 50120583gmL versus TNF-120572

119875 lt 005 control versus TNF-120572

Evidence-Based Complementary and Alternative Medicine 9

Control TNF-120572

TNF-120572

Aspirin10120583M

+ + + + + + + +

BT25 BT50 MT25 MT50 M25 M50

120583gmL 120583gmL 120583gmL 120583gmL 120583gmL 120583gmL

NF-120581B p65

120573-Actin43kDa

minus

65kDa

(a)

Con

trol

Asp

irin

BT25

BT50

MT2

5

MT5

0

M25

M50

00

02

04

06

08

10

lowast

lowast

lowast

lowastlowastlowastTN

F-120572

Ratio

(nuc

lear

120573-a

ctin

)

(b)

Figure 4 Effects of MLREs on NF-120581B activity After HAECs were pretreated with the indicated samples and incubated with TNF-120572 nuclearextracts were prepared and the expression of NF-120581B p65 was assessed by western blot analysis A representative image of three similar resultsis shown (a) Actin served as the loading control for the nuclear compartment Semiquantitative analysis of three independent experimentsare also shown (b) indicates a significant difference between the TNF-120572 treated or experimental treatment groups and the control119875 lt 005lowast indicates a significant difference between the TNF-120572 and experimental treatment groups BT MT and M at 25 and 50 120583gmL

(lane 3) in the nuclear fraction indicating that MLREs at50120583gmL inhibited STAT3-binding activities In addition theresults in Figure 5(c) (lanes 4ndash10) show that aspirinMLREs at50120583gmL and BT at 25120583gmL remarkably decreased STAT3levels compared to TNF-120572 (lane 3) however both MT andM at 25120583gmL increased STAT3-binding activities Theseresults indicate that only 50 120583gmL MLREs 25 120583gmL BTand aspirin block STAT3 activation and might downregulateTNF-120572-induced expressions of inflammatory signaling path-ways

35 MLREs Regulated the DNA-Binding Activity and Expres-sions of PPAR 120572 and PPAR 120574 in HAECs PPAR 120572 and 120574effectively bound to PPAR 120572 and 120574 oligo-IRDye 700 asindicated in lanes 3ndash10 of Figure 6 Supershifts are bands fromthe binding of each specific antibody The binding activitiesof PPAR 120572 and 120574 were detected at baseline The 25 120583gmLMT extract (lane 7) and 25 and 50 120583gmL M extracts (lanes9-10) increased activity compared to incubating with TNF-120572 alone (lane 3) However BT at both 25 and 50 120583gmLextracts showed reduced effects on PPAR 120572 and 120574 expressionMoreover the 50 120583gmLMT extract had no effect on PPAR 120572binding activity but increased the binding activity of PPAR120574 in comparison to incubation with TNF-120572 alone Thesedata suggest that MT and M both promoted PPAR 120572 and 120574

expression at least in part by increasing the DNA binding oftranscription factors PPAR 120572 and 120574

4 Discussion

41 Antioxidant Composition and Antioxidant Activity BTcontains high levels of polyphenols flavonoids flavonolsanthocyanidin quercetin and kaempferol (Tables 1 and 2)BT also had a higher TEAC value (15514mM Table 3) thanthe TEAC values of 47mM [42] and 64mM [43] reportedfor quercetin as well as 242mM reported for quercetin-3-rutinoside [44] Furthermore Gonzalez-Paramas et al [45]demonstrated that flavonol contents were significantly andhighly correlated (119903 = 08) with TEAC values Kim et al [46]reported that mulberry leaves contain abundant quercetinand inhibit lipid peroxidation at concentrations of 01 and001mgmL In our study the amount of quercetin wassignificantly correlated (119903 = 041 Table 4) with TEAC valueHowever no correlation was found among myricetin morinkaempferol and TEAC which may be due to the structurerequired to reinforce the free radical scavenging activity thatvaries with the type of free radical Naowaratwattana et al[18] reported that a 50 methanolic extract of mulberryleaf contained rutin isoquercetin and various derivatives ofkaempferol and quercetin glycosides while a water extract

10 Evidence-Based Complementary and Alternative Medicine

NF-120581BTNF-120572

TNF-120572

+

Aspirin10120583M+ +

BT25120583gmL

+

BT50120583gmL

+

MT25120583gmL

+

MT50120583gmL

+

M25

120583gmL+

M50

120583gmLControl

minus

DNAOligo

Free probe

(a)

AP-1TNF-120572

TNF-120572

+

Aspirin10120583M+ +

BT25120583gmL

+

BT50120583gmL

+

MT25120583gmL

+

MT50120583gmL

+

M25

120583gmL+

M50

120583gmLControl

minus

DNAOligo

Free probe

(b)

STAT3

TNF-120572

TNF-120572

+

Aspirin10120583M+ +

BT25120583gmL

+

BT50120583gmL

+

MT25120583gmL

+

MT50120583gmL

+

M25

120583gmL+

M50

120583gmLControl

minus

DNAOligo

Free probe

(c)

Figure 5 MLREs inhibits the binding of (a) NF-120581B (b) AP-1 and (c) STAT3 to target DNA sequence NF-120581B AP-1 and STAT3 transcriptionfactor-DNA interactions were assessed by EMSA

contained primarily chlorogenic acid and caffeoylquinic acidderivatives The IC

50of DPPH radical scavenging was 794

and 2042120583gmL for 50 methanolic and water extractsrespectively [18] In our studyM extracts contained quercetinand kaempferol (Table 2) which may account for part ofthe highly scavenged DPPH radical in comparison to therest of MLREs Khan et al [47] demonstrated that the IC

50

was 22023120583gmL which is much less efficacious than themulberry leaf acidic methanolic extract IC

50of 1888 120583gmL

(Table 3) However the IC50

of in vitro free radical scav-enging activity of mulberry leaves from Naowaboot et al[22] was 617 plusmn 21 120583gmL which is more efficacious thanthe IC

50concentration derived in the present study Taken

together the polyphenol-rich MLREs exhibited distinct cell-free antioxidant activity (ie TEAC) as validated by theirlevels of polyphenols and flavonols with distinct antioxidantactivity strongly accounting for the antioxidant activity of the

extracts The amount of quercetin was especially positivelycorrelated with the observed TEAC values

42 Estimation of DNA Single Strand Break Damage fromExposure to Acidic Methanolic and Water Extracts One ofthe antioxidant activity tests scavenging of DPPH radi-cal was selected due to its quick and simple but it is aless biologically relevant assay Previously we reported thatwith 2 120583gsdotmLminus1 of methanolic and water extracts from theindigenous vegetables [33] and herb plants [48] significantlyprolonged lag phase of low-density lipoprotein (LDL) TheLDL oxidation assay is considered to be one of the mostbiologically relevant assays [49] Both studies [33 48] alsoshowed that the concentration of the tested extracts forscavenging DPPH radical was in higher levels at 10mg oftested extracts with 2ndash53 inhibition Lower concentrationwas then chosen for the treated dosage in the present study

Evidence-Based Complementary and Alternative Medicine 11

TNF-120572

TNF-120572

+

Aspirin10120583M+ +

BT25120583gmL

+

BT50120583gmL

+

MT25120583gmL

+

MT50120583gmL

+

M25

120583gmL+

M50

120583gmLControl

minus

DNAOligo

Free probe

PPAR 120572

(a)

TNF-120572

TNF-120572

+

Aspirin10120583M+ +

BT25120583gmL

+

BT50120583gmL

+

MT25120583gmL

+

MT50120583gmL

+

M25

120583gmL+

M50

120583gmLControl

minus

DNAOligo

Free probe

PPAR120574

(b)

Figure 6 Effect of treatments on the expression of PPAR 120572 (a) and PPAR 120574 (b) in TNF-120572-stimulated HAECs by EMSA

In addition lower concentration also could avoid the toxicityeffect of higher level of extracts on lymphocytes Quercetinwas found to protect against H

2O2-induced DNA damage in

human lymphocytes at 10 120583M [50] and at 31 to 25 120583M [51]Quercetin was also reported to induce DNA strand breaks invarious cell types but only at higher doses at 100 120583Mor above[50] Duthie et al [50] proposed that the dihydroxy structureof quercetin and myricetin might be essential in protectingDNA against hydrogen peroxide No such hydroxyl groupsare present in the tocopherol molecule Noroozi et al [52]demonstrated that in addition to quercetin kaempferolcould also inhibit H

2O2-induced DNA strand breaks in

human lymphocytes In our study both BT and M were richin quercetin and kaempferol (Table 2) whichmay account forthe protective roles of both extracts in preventingDNAstrandbreaks from human lymphocytes

43 MLREs Regulated the DNA-Binding Activity and Expres-sions of NF-120581B AP-1 STAT3 and PPARs in HAECsBecause MLREs exhibited free radical scavenging the anti-inflammatory effects of MLREs on TNF-120572-induced celladhesion and adhesionmolecule expressionwere determinedin the present study Our results suggest that MLREs andaspirin significantly inhibited TNF-120572-induced monocyte-endothelial cell adhesion (Figure 3) Significant reductionsin NF-120581B expression and DNA binding by aspirin MTextract (25 120583gmL) and BT extract (both 25 and 50 120583gmL)were also observed The adhesion of monocytes to the

vascular endothelium and subsequent migration into thevessel wall are early events in atherogenesis [2] Our resultsare consistent with previous research showing that dietarypolyphenols such as catechin and quercetin significantlyreduce the binding of monocytes to HAECs [53 54] Thereduced cellular adhesion may be due to the inhibitionof cellular adhesion molecule expression as purple sweetpotato leaf extract (PSPLE) reduced both VCAM-1 andICAM-1 expression [40] Shibata et al [55] demonstratedthat pretreatment of cultured bovine aortic endothelial cells(BAECs) with mulberry leaf aqueous fractions inhibitedTNF-120572- and LPS-induced expression of Lectin-like Ox-LDLreceptor-1 (LOX-1) a cell-surface receptor for atherogenicOx-LDL (oxidized low-density lipoprotein) It also appearsto mediate Ox-LDL-induced inflammation which may becrucial in atherogenesis Furthermore mulberry leaf aqueousfractions inhibited the TNF-120572-induced activation of NF-120581Band phosphorylation of inhibitory factor of NF-120581B-alpha(I120581B-120572) Thus mulberry leaf aqueous fractions suppressTNF-120572- and LPS-induced LOX-1 gene expression mediatedby inhibiting NF-120581B activation

Chan et al [56] demonstrated that mulberry leaf extract(MLE) was rich in polyphenols MLE can effectively inhibitthe migration of vascular smooth muscle cells (VSMC) byblocking small guanosine triphosphatases (small GTPase)and AktNF-120581B signals The Rho family of small GTPasessuch as Rho Rac and Cdc42 acts as a molecular switch toregulate the actin cytoskeleton The Rho family of GTPases

12 Evidence-Based Complementary and Alternative Medicine

is also reported to be involved in the regulation of NF-120581B-dependent transcription [56] MLE has been shown to pos-sess hypoglycemic effects in an insulin-dependent diabetesmellitus (type 1 diabetes) animal model and potential hypo-glycemic compounds inmulberry leaves were suggested to be2-O-120572-D-galactopyranosyl-DNJ and fagomine [57] Konnoet al [58] further reported that mulberry latex containsalkaloidal sugar-mimic glycosidase inhibitors with antidia-betic activities such as 14-dideoxy-14-imino-D-arabinitol 1-deoxynojirimycin and 14-dideoxy-14-imino-D-ribitol Kimet al [59] recently showed that treated type 2 diabetesmellitus(T2D) with a 1 1 1 mixture of ginseng root mulberry leafwater extract and banana leaf water extract (6 gd) for 24weeks significantly decreased plasma intracellular adhesionmolecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) levels This indicates suppressed inflammatoryresponses in T2D but no significant differences in glucosehomeostasis control measurement changes Rigamonti et al[8] reported that bothPPAR120572 and 120574maybe thought of as sup-pressing the activity of NF120581B and AP-1 pathways that reduceinflammatory mediator production in the macrophages ofhumans and mice In our study MT and M promoted PPAR120572 and 120574 expression at least in part by increasing DNAbinding of transcription factors PPAR 120572 and 120574 PPAR-120572-and 120574-dependent signaling pathwaysmay also play importantroles in mediating the TNF-120572 receptor signaling pathwayby MT and M by suppressing the activity on NF-120581B andAP-1 pathways These pathways may regulate downstreamproinflammatory gene expression and adhesion molecularexpression Clinically PPAR 120574 agonists such as rosiglitazoneare widely used in the treatment of T2D [60] M and MTextracts increased PPAR 120574 activity which may partly explainwhy inflammatory responses are suppressed in T2D

Although reduced NF-120581B expression and DNA bind-ing were observed upon treatment with ML MT and BT(Figure 5) the mechanism was not assessed and is worthy offurther study In addition the present results were obtainedusing in vitro studies and may be consistent with an invivo model of atherogenesis similar to that reported byEnkhmaa et al [21] in which mulberry leaves suppressedthe development of atherosclerotic lesions Previously weproved that black beans significantly prolong LDL lag timeand decrease the atheroma regions of the aortic arch andthoracic aorta in hypercholesterolemic NZW rabbits [61]Wethen demonstrated that black soybean extract and PSPLEsignificantly decreased the adhesion of U937 monocyticcells to TNF-120572-stimulated HAECs and reduced adhesionmolecules expression (VCAM-1 and ICAM-1) as well as NF-120581B-p65 expression [37] This is particularly important giventhe opposing effects of flavonols fromMLREs obtained usingin vitro and ex vivo studies [62]

5 Conclusion

Tested MLREs were rich in flavonols and had strongantioxidant activity in protecting lymphocyte DNA fromoxidative damage In particular BT was rich in quercetinand polyphenols The antioxidant components of MLREs

might downregulate intracellular redox-dependent signalingpathways in HAECs upon TNF-120572 stimulation which mightprevent ROS-mediated endothelial cell dysfunction MLRCshave anti-inflammatory effects through the modulation ofNF-120581B AP-1 STAT3 and PPARs signaling These chemi-cals appear to provide distinct cytoprotective mechanismsthat might contribute to their putative beneficial effectsin suppressing endothelial responses to cytokines duringinflammation These results may benefit further in vivostudies to assess the therapeutic potential of phytochemicalsas anti-inflammatories for use in cytokine-induced vasculardisorders including atherosclerosis

Acknowledgment

This work was supported in part by Grants from the NationalScience Council of Taiwan (NSC 95ndash2320-B-034-001 andNSC 96ndash2320-B-034-001)

References

[1] W Palinski ldquoUnited they go conjunct regulation of aorticantioxidant enzymes during atherogenesisrdquo CirculationResearch vol 93 no 3 pp 183ndash185 2003

[2] P Libby ldquoMolecular bases of the acute coronary syndromesrdquoCirculation vol 91 no 11 pp 2844ndash2850 1995

[3] P A Baeuerle and D Baltimore ldquoNf-120581B ten years afterrdquo Cellvol 87 no 1 pp 13ndash20 1996

[4] J M Kyriakis ldquoActivation of the AP-1 transcription factor byinflammatory cytokines of the TNF familyrdquo Gene Expressionvol 7 no 4ndash6 pp 217ndash231 1999

[5] J M Muller R A Rupec and P A Baeuerle ldquoStudy of generegulation by NF-120581B and AP-1 in response to reactive oxygenintermediatesrdquoMethods vol 11 no 3 pp 301ndash312 1997

[6] G Chinetti J Fruchart and B Staels ldquoPeroxisome proliferator-activated receptors new targets for the pharmacological mod-ulation of macrophage gene expression and functionrdquo CurrentOpinion in Lipidology vol 14 no 5 pp 459ndash468 2003

[7] G Chinetti J C Fruchart and B Staels ldquoTranscriptional regu-lation of macrophage cholesterol trafficking by PPAR120572 andLXRrdquo Biochemical Society Transactions vol 34 no 6 pp 1128ndash1131 2006

[8] E Rigamonti G Chinetti-Gbaguidi and B Staels ldquoRegulationof macrophage functions by PPAR- 120572 PPAR- 120574 and LXRsin mice and menrdquo Arteriosclerosis Thrombosis and VascularBiology vol 28 no 6 pp 1050ndash1059 2008

[9] M A Bouhlel J Brozek B Derudas et al ldquoUnlike PPAR120574PPAR120572 or PPAR120573120575 activation does not promote humanmono-cyte differentiation toward alternative macrophagesrdquo Biochem-ical and Biophysical Research Communications vol 386 no 3pp 459ndash462 2009

[10] G Chinetti J-C Fruchart and B Staels ldquoPeroxisome prolife-rator-activated receptors and inflammation from basic scienceto clinical applicationsrdquo International Journal of Obesity vol 27supplement 3 pp S41ndashS45 2003

[11] Y Zhu J H-C Lin H Liao et al ldquoLDL induces transcriptionfactor activator protein-1 in human endothelial cellsrdquo Arte-riosclerosis Thrombosis and Vascular Biology vol 18 no 3 pp473ndash480 1998

Evidence-Based Complementary and Alternative Medicine 13

[12] J Terao ldquoDietary flavonoids as antioxidantsrdquo Forum of Nutri-tion vol 61 pp 87ndash94 2009

[13] Y B Feng W Q Luo and S Q Zhu ldquoExplore new clinicalapplication of Huanglian and corresponding compound pre-scriptions from their traditional userdquo Zhongguo ZhongyaoZazhi vol 33 no 10 pp 1221ndash1225 2008

[14] Y H Chu C L Chang and H F Hsu ldquoFlavonoid content ofseveral vegetables and their antioxidant activityrdquo Journal of theScience of Food andAgriculture vol 80 no 5 pp 561ndash566 2000

[15] I C Arts B van de Putte and P C Hollman ldquoCatechin con-tents of foods commonly consumed in The Netherlands 1Fruits vegetables staple foods and processed foodsrdquo Journal ofAgricultural and Food Chemistry vol 48 no 5 pp 1746ndash17512000

[16] A R Proteggente A S Pannala G Paganga et al ldquoThe anti-oxidant activity of regularly consumed fruit and vegetablesreflects their phenolic and vitamin C compositionrdquo Free RadicalResearch vol 36 no 2 pp 217ndash233 2002

[17] Y L Liu L H Tang Z Q Liang B You and S Yang ldquoGrowthinhibitory and apoptosis inducing by effects of total flavonoidsfrom Lysimachia clethroides Duby in human chronic myeloidleukemiaK562 cellsrdquo Journal of Ethnopharmacology vol 131 no1 pp 1ndash9 2010

[18] W Naowaratwattana W De-Eknamkul and E G De MejialdquoPhenolic-containing organic extracts of mulberry (Morus albaL) leaves inhibit HepG2 hepatoma cells through G2M phasearrest and inhibition of topoisomerase II120572 activityrdquo Journal ofMedicinal Food vol 13 no 5 pp 1045ndash1056 2010

[19] K Doi T Kojima and Y Fujimoto ldquoMulberry leaf extract inhi-bits the oxidative modification of rabbit and human low densitylipoproteinrdquo Biological and Pharmaceutical Bulletin vol 23 no9 pp 1066ndash1071 2000

[20] W Wang Y Zu Y Fu and T Efferth ldquoIn Vitro antioxidant andantimicrobial activity of extracts from Morus alba L leavesstems and fruitsrdquoAmerican Journal of ChineseMedicine vol 40no 2 pp 349ndash356 2012

[21] B Enkhmaa K Shiwaku T Katsube et al ldquoMulberry (Morusalba L) leaves and their major flavonol quercetin 3-(6-malon-ylglucoside) attenuate atherosclerotic lesion development inLDL receptor-deficient micerdquo Journal of Nutrition vol 135 no4 pp 729ndash734 2005

[22] J Naowaboot P Pannangpetch V Kukongviriyapan B Kongy-ingyoes and U Kukongviriyapan ldquoAntihyperglycemic antiox-idant and antiglycation activities of mulberry leaf extract instreptozotocin-induced chronic diabetic ratsrdquo Plant Foods forHuman Nutrition vol 64 no 2 pp 116ndash121 2009

[23] MM Padilha F CVilela CQ Rocha et al ldquoAntiinflammatoryproperties of Morus nigra leavesrdquo Phytotherapy Research vol24 no 10 pp 1496ndash1500 2010

[24] P Aramwit O Supasyndh T Siritienthong and N BangldquoMulberry leaf reduces oxidation and c-reactive protein levelin patients with mild dyslipidemiardquo BioMed Research Interna-tional vol 2013 Article ID 787981 7 pages 2013

[25] U Justesen P Knuthsen and T Leth ldquoQuantitative analysis offlavonols flavones and flavanones in fruits vegetables andbeverages by high-performance liquid chromatography withphoto-diode array and mass spectrometric detectionrdquo Journalof Chromatography A vol 799 no 1-2 pp 101ndash110 1998

[26] M S Taga E EMiller andD E Pratt ldquoChia seeds as a source ofnatural lipid antioxidantsrdquo Journal of the AmericanOil ChemistsrsquoSociety vol 61 no 5 pp 928ndash931 1984

[27] A A L Ordonez J D Gomez M A Vattuone and M I IslaldquoAntioxidant activities of Sechium edule (Jacq) Swartz extractsrdquoFood Chemistry vol 97 no 3 pp 452ndash458 2006

[28] A Kumaran and R Joel Karunakaran ldquoIn Vitro antioxidantactivities of methanol extracts of five Phyllanthus species fromIndiardquo LWT-Food Science and Technology vol 40 no 2 pp344ndash352 2007

[29] G Cao H M Alessio and R G Cutler ldquoOxygen-radical absor-bance capacity assay for antioxidantsrdquo Free Radical Biology andMedicine vol 14 no 3 pp 303ndash311 1993

[30] K Shimada K Fujikawa K Yahara and T Nakamura ldquoAntiox-idative properties of xanthan on the autoxidation of soybeanoil in cyclodextrin emulsionrdquo Journal of Agricultural and FoodChemistry vol 40 no 6 pp 945ndash948 1992

[31] J Cole M H L Green S E James L Henderson and H ColeldquoA further assessment of factors influencing measurements ofthioguanine-resistant mutant frequency in circulating T-lymphocytesrdquo Mutation Research vol 204 no 3 pp 493ndash5071988

[32] C-Y Hsu C-M Yang C-M Chen P Chao and S Hu ldquoEff-ects of chlorophyll-related compounds on hydrogen peroxideinduced DNA damage within human lymphocytesrdquo Journal ofAgricultural and Food Chemistry vol 53 no 7 pp 2746ndash27502005

[33] S-C Tang H-F Lo K-H Lin et al ldquoThe antioxidant capacityof extracts from Taiwan indigenous purple-leaved vegetablesrdquoJournal of the Taiwan Society for Horticultural Science vol 59no 1 pp 43ndash57 2013

[34] K-H Lin Y-Y Yang H-F Lo et al ldquoAntioxidant activity ofherbaceous plant extracts protect against hydrogen peroxide-induced DNA damage in human lymphocytesrdquo BioMed CentralResearch Notes In press

[35] A H Cory T C Owen J A Barltrop and J G Cory ldquoUse of anaqueous soluble tetrazoliumformazan assay for cell growthassays in culturerdquoCancer Communications vol 3 no 7 pp 207ndash212 1991

[36] Y T Szeto A R Collins and I F F Benzie ldquoEffects of dietaryantioxidants on DNA damage in lysed cells using a modifiedcomet assay procedurerdquoMutation Research vol 500 no 1-2 pp31ndash38 2002

[37] S Vielma G Virella A J Gorod and M F Lopes-VirellaldquoChlamydophila pneumoniae infection of human aortic endo-thelial cells induces the expression of FC 120574 receptor II (Fc120574RII)rdquoClinical Immunology vol 104 no 3 pp 265ndash273 2002

[38] C Y Zhu and S Loft ldquoEffects of Brussels sprouts extracts onhydrogen peroxide-induced DNA strand breaks in humanlymphocytesrdquo Food and Chemical Toxicology vol 39 no 12 pp1191ndash1197 2001

[39] Y D Min C-H Choi H Bark et al ldquoQuercetin inhibits exp-ression of inflammatory cytokines through attenuation of NF-120581B and p38MAPK in HMC-1 humanmast cell linerdquo Inflamma-tion Research vol 56 no 5 pp 210ndash215 2007

[40] P Y Chao Y P Huang and W B Hsieh ldquoInhibitive effect ofpurple sweet potato leaf extract and its components on celladhesion and inflammatory response in human aortic endothe-lial cellsrdquo Cell Adhesion amp Migration vol 7 no 2 pp 1ndash9 2013

[41] Y Y YangThe antioxidative capacity in herb plant extracts andtheir protection role in DNA oxidative damage of lymphocyte[MS thesis] Chinese Culture University Taipei Taiwan 2004

[42] C A Rice-Evans N J Miller and G Paganga ldquoStructure-anti-oxidant activity relationships of flavonoids and phenolic acidsrdquo

14 Evidence-Based Complementary and Alternative Medicine

Free Radical Biology and Medicine vol 20 no 7 pp 933ndash9561996

[43] M Strube G R Haenen H van den Berg and A Bast ldquoPitfallsin a method for assessment of total antioxidant capacityrdquo FreeRadical Research vol 26 no 6 pp 515ndash521 1997

[44] P G Pietta ldquoFlavonoids as antioxidantsrdquo Journal of NaturalProducts vol 63 no 7 pp 1035ndash1042 2000

[45] A M Gonzalez-Paramas S Esteban-Ruano C Santos-BuelgaSDe Pascual-Teresa and J C Rivas-Gonzalo ldquoFlavanol contentand antioxidant activity in winery byproductsrdquo Journal ofAgricultural and Food Chemistry vol 52 no 2 pp 234ndash2382004

[46] S Y Kim J J Gao and H K Kang ldquoTwo flavonoids from theleaves ofMorus alba induce differentiation of the human prom-yelocytic leukemia (HL-60) cell linerdquo Biological and Pharma-ceutical Bulletin vol 23 no 4 pp 451ndash455 2000

[47] M A Khan A A Rahman S Islam et al ldquoA comparative studyon the antioxidant activity ofmethanolic extracts from differentparts of Morus alba L (Moraceae)rdquo BMC Research vol 6 pp24ndash32 2013

[48] A-L ChungH-F Lo K-H Lin et al ldquoStudy on the antioxidantactivity in herb plant extractsrdquo Journal of the Taiwan Society ForHorticultural Science vol 59 no 2 pp 139ndash152 2013

[49] R L Prior X Wu and K Schaich ldquoStandardized methods forthe determination of antioxidant capacity and phenolics infoods and dietary supplementsrdquo Journal of Agricultural andFood Chemistry vol 53 no 10 pp 4290ndash4302 2005

[50] S J Duthie A R Collins GGDuthie andV LDobson ldquoQue-rcetin and myricetin protect against hydrogen peroxide-indu-ced DNA damage (strand breaks and oxidised pyrimidines) inhuman lymphocytesrdquo Mutation Research vol 393 no 3 pp223ndash231 1997

[51] G A Liu and R L Zheng ldquoProtection against damagedDNA inthe single cell by polyphenolsrdquo Pharmazie vol 57 no 12 pp852ndash854 2002

[52] M Noroozi W J Angerson and M E J Lean ldquoEffects of flav-onoids and vitamin C on oxidative DNA damage to humanlymphocytesrdquoAmerican Journal of Clinical Nutrition vol 67 no6 pp 1210ndash1218 1998

[53] T Koga and M Meydani ldquoEffect of plasma metabolites of (+)-catechin and quercetin on monocyte adhesion to human aorticendothelial cellsrdquoAmerican Journal of Clinical Nutrition vol 73no 5 pp 941ndash948 2001

[54] CMeng P Somers L Hoong et al ldquoDiscovery of novel pheno-lic antioxidants as inhibitors of vascular cell adhesionmolecule-1 expression for use in chronic inflammatory diseasesrdquo Journalof Medicinal Chemistry vol 47 no 25 pp 6420ndash6432 2004

[55] Y Shibata N Kume H Arai et al ldquoMulberry leaf aqueousfractions inhibit TNF-120572-induced nuclear factor 120581B (NF-120581B)activation and lectin-like oxidized LDL receptor-1 (LOX-1)expression in vascular endothelial cellsrdquoAtherosclerosis vol 193no 1 pp 20ndash27 2007

[56] K C Chan H H Ho C N Huang M Lin H Chen and CWang ldquoMulberry leaf extract inhibits vascular smooth musclecell migration involving a block of small GTPase and AktNF-kB signalsrdquo Journal of Agricultural and Food Chemistry vol 57no 19 pp 9147ndash9153 2009

[57] F Chen N Nakashima I Kimura M Kimura N Asanoand S Koya ldquoPotentiating effects on pilocarpine-induced salivasecretion by extracts and N-containing sugars derived frommulberry leaves in streptozocin- diabetic micerdquo Biological andPharmaceutical Bulletin vol 18 no 12 pp 1676ndash1680 1995

[58] K Konno H Ono M Nakamura et al ldquoMulberry latexrich in antidiabetic sugar-mimic alkaloids forces dieting oncaterpillarsrdquo Proceedings of the National Academy of Sciences ofthe United States of America vol 103 no 5 pp 1337ndash1341 2006

[59] H J Kim K H Yoon M J Kang et al ldquoA six-month sup-plementation of mulberry Korean red ginseng and banabadecreases biomarkers of systemic low-grade inflammation insubjects with impaired glucose tolerance and type 2 diabetesrdquoEvidence-based Complementary and Alternative Medicine vol2012 Article ID 735191 8 pages 2012

[60] G Nicholson and G M Hall ldquoDiabetes mellitus new drugs fora new epidemicrdquo British Journal of Anaesthesia vol 107 no 1pp 65ndash73 2011

[61] P Y Chao Y L Chen Y C Lin et al ldquoEffects of black soybeanon atherogenic prevention in hypercholesterolemic rabbits andon adhesion molecular expression in cultured HAECsrdquo Foodand Nutrition Sciences vol 4 pp 9ndash21 2013

[62] C Chen S Li C-YOChen et al ldquoConstituents in purple sweetpotato leaves inhibit in vitro angiogenesis with opposite effectsex vivordquo Nutrition vol 27 no 11-12 pp 1177ndash1182 2011

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

Evidence-Based Complementary and Alternative Medicine 9

Control TNF-120572

TNF-120572

Aspirin10120583M

+ + + + + + + +

BT25 BT50 MT25 MT50 M25 M50

120583gmL 120583gmL 120583gmL 120583gmL 120583gmL 120583gmL

NF-120581B p65

120573-Actin43kDa

minus

65kDa

(a)

Con

trol

Asp

irin

BT25

BT50

MT2

5

MT5

0

M25

M50

00

02

04

06

08

10

lowast

lowast

lowast

lowastlowastlowastTN

F-120572

Ratio

(nuc

lear

120573-a

ctin

)

(b)

Figure 4 Effects of MLREs on NF-120581B activity After HAECs were pretreated with the indicated samples and incubated with TNF-120572 nuclearextracts were prepared and the expression of NF-120581B p65 was assessed by western blot analysis A representative image of three similar resultsis shown (a) Actin served as the loading control for the nuclear compartment Semiquantitative analysis of three independent experimentsare also shown (b) indicates a significant difference between the TNF-120572 treated or experimental treatment groups and the control119875 lt 005lowast indicates a significant difference between the TNF-120572 and experimental treatment groups BT MT and M at 25 and 50 120583gmL

(lane 3) in the nuclear fraction indicating that MLREs at50120583gmL inhibited STAT3-binding activities In addition theresults in Figure 5(c) (lanes 4ndash10) show that aspirinMLREs at50120583gmL and BT at 25120583gmL remarkably decreased STAT3levels compared to TNF-120572 (lane 3) however both MT andM at 25120583gmL increased STAT3-binding activities Theseresults indicate that only 50 120583gmL MLREs 25 120583gmL BTand aspirin block STAT3 activation and might downregulateTNF-120572-induced expressions of inflammatory signaling path-ways

35 MLREs Regulated the DNA-Binding Activity and Expres-sions of PPAR 120572 and PPAR 120574 in HAECs PPAR 120572 and 120574effectively bound to PPAR 120572 and 120574 oligo-IRDye 700 asindicated in lanes 3ndash10 of Figure 6 Supershifts are bands fromthe binding of each specific antibody The binding activitiesof PPAR 120572 and 120574 were detected at baseline The 25 120583gmLMT extract (lane 7) and 25 and 50 120583gmL M extracts (lanes9-10) increased activity compared to incubating with TNF-120572 alone (lane 3) However BT at both 25 and 50 120583gmLextracts showed reduced effects on PPAR 120572 and 120574 expressionMoreover the 50 120583gmLMT extract had no effect on PPAR 120572binding activity but increased the binding activity of PPAR120574 in comparison to incubation with TNF-120572 alone Thesedata suggest that MT and M both promoted PPAR 120572 and 120574

expression at least in part by increasing the DNA binding oftranscription factors PPAR 120572 and 120574

4 Discussion

41 Antioxidant Composition and Antioxidant Activity BTcontains high levels of polyphenols flavonoids flavonolsanthocyanidin quercetin and kaempferol (Tables 1 and 2)BT also had a higher TEAC value (15514mM Table 3) thanthe TEAC values of 47mM [42] and 64mM [43] reportedfor quercetin as well as 242mM reported for quercetin-3-rutinoside [44] Furthermore Gonzalez-Paramas et al [45]demonstrated that flavonol contents were significantly andhighly correlated (119903 = 08) with TEAC values Kim et al [46]reported that mulberry leaves contain abundant quercetinand inhibit lipid peroxidation at concentrations of 01 and001mgmL In our study the amount of quercetin wassignificantly correlated (119903 = 041 Table 4) with TEAC valueHowever no correlation was found among myricetin morinkaempferol and TEAC which may be due to the structurerequired to reinforce the free radical scavenging activity thatvaries with the type of free radical Naowaratwattana et al[18] reported that a 50 methanolic extract of mulberryleaf contained rutin isoquercetin and various derivatives ofkaempferol and quercetin glycosides while a water extract

10 Evidence-Based Complementary and Alternative Medicine

NF-120581BTNF-120572

TNF-120572

+

Aspirin10120583M+ +

BT25120583gmL

+

BT50120583gmL

+

MT25120583gmL

+

MT50120583gmL

+

M25

120583gmL+

M50

120583gmLControl

minus

DNAOligo

Free probe

(a)

AP-1TNF-120572

TNF-120572

+

Aspirin10120583M+ +

BT25120583gmL

+

BT50120583gmL

+

MT25120583gmL

+

MT50120583gmL

+

M25

120583gmL+

M50

120583gmLControl

minus

DNAOligo

Free probe

(b)

STAT3

TNF-120572

TNF-120572

+

Aspirin10120583M+ +

BT25120583gmL

+

BT50120583gmL

+

MT25120583gmL

+

MT50120583gmL

+

M25

120583gmL+

M50

120583gmLControl

minus

DNAOligo

Free probe

(c)

Figure 5 MLREs inhibits the binding of (a) NF-120581B (b) AP-1 and (c) STAT3 to target DNA sequence NF-120581B AP-1 and STAT3 transcriptionfactor-DNA interactions were assessed by EMSA

contained primarily chlorogenic acid and caffeoylquinic acidderivatives The IC

50of DPPH radical scavenging was 794

and 2042120583gmL for 50 methanolic and water extractsrespectively [18] In our studyM extracts contained quercetinand kaempferol (Table 2) which may account for part ofthe highly scavenged DPPH radical in comparison to therest of MLREs Khan et al [47] demonstrated that the IC

50

was 22023120583gmL which is much less efficacious than themulberry leaf acidic methanolic extract IC

50of 1888 120583gmL

(Table 3) However the IC50

of in vitro free radical scav-enging activity of mulberry leaves from Naowaboot et al[22] was 617 plusmn 21 120583gmL which is more efficacious thanthe IC

50concentration derived in the present study Taken

together the polyphenol-rich MLREs exhibited distinct cell-free antioxidant activity (ie TEAC) as validated by theirlevels of polyphenols and flavonols with distinct antioxidantactivity strongly accounting for the antioxidant activity of the

extracts The amount of quercetin was especially positivelycorrelated with the observed TEAC values

42 Estimation of DNA Single Strand Break Damage fromExposure to Acidic Methanolic and Water Extracts One ofthe antioxidant activity tests scavenging of DPPH radi-cal was selected due to its quick and simple but it is aless biologically relevant assay Previously we reported thatwith 2 120583gsdotmLminus1 of methanolic and water extracts from theindigenous vegetables [33] and herb plants [48] significantlyprolonged lag phase of low-density lipoprotein (LDL) TheLDL oxidation assay is considered to be one of the mostbiologically relevant assays [49] Both studies [33 48] alsoshowed that the concentration of the tested extracts forscavenging DPPH radical was in higher levels at 10mg oftested extracts with 2ndash53 inhibition Lower concentrationwas then chosen for the treated dosage in the present study

Evidence-Based Complementary and Alternative Medicine 11

TNF-120572

TNF-120572

+

Aspirin10120583M+ +

BT25120583gmL

+

BT50120583gmL

+

MT25120583gmL

+

MT50120583gmL

+

M25

120583gmL+

M50

120583gmLControl

minus

DNAOligo

Free probe

PPAR 120572

(a)

TNF-120572

TNF-120572

+

Aspirin10120583M+ +

BT25120583gmL

+

BT50120583gmL

+

MT25120583gmL

+

MT50120583gmL

+

M25

120583gmL+

M50

120583gmLControl

minus

DNAOligo

Free probe

PPAR120574

(b)

Figure 6 Effect of treatments on the expression of PPAR 120572 (a) and PPAR 120574 (b) in TNF-120572-stimulated HAECs by EMSA

In addition lower concentration also could avoid the toxicityeffect of higher level of extracts on lymphocytes Quercetinwas found to protect against H

2O2-induced DNA damage in

human lymphocytes at 10 120583M [50] and at 31 to 25 120583M [51]Quercetin was also reported to induce DNA strand breaks invarious cell types but only at higher doses at 100 120583Mor above[50] Duthie et al [50] proposed that the dihydroxy structureof quercetin and myricetin might be essential in protectingDNA against hydrogen peroxide No such hydroxyl groupsare present in the tocopherol molecule Noroozi et al [52]demonstrated that in addition to quercetin kaempferolcould also inhibit H

2O2-induced DNA strand breaks in

human lymphocytes In our study both BT and M were richin quercetin and kaempferol (Table 2) whichmay account forthe protective roles of both extracts in preventingDNAstrandbreaks from human lymphocytes

43 MLREs Regulated the DNA-Binding Activity and Expres-sions of NF-120581B AP-1 STAT3 and PPARs in HAECsBecause MLREs exhibited free radical scavenging the anti-inflammatory effects of MLREs on TNF-120572-induced celladhesion and adhesionmolecule expressionwere determinedin the present study Our results suggest that MLREs andaspirin significantly inhibited TNF-120572-induced monocyte-endothelial cell adhesion (Figure 3) Significant reductionsin NF-120581B expression and DNA binding by aspirin MTextract (25 120583gmL) and BT extract (both 25 and 50 120583gmL)were also observed The adhesion of monocytes to the

vascular endothelium and subsequent migration into thevessel wall are early events in atherogenesis [2] Our resultsare consistent with previous research showing that dietarypolyphenols such as catechin and quercetin significantlyreduce the binding of monocytes to HAECs [53 54] Thereduced cellular adhesion may be due to the inhibitionof cellular adhesion molecule expression as purple sweetpotato leaf extract (PSPLE) reduced both VCAM-1 andICAM-1 expression [40] Shibata et al [55] demonstratedthat pretreatment of cultured bovine aortic endothelial cells(BAECs) with mulberry leaf aqueous fractions inhibitedTNF-120572- and LPS-induced expression of Lectin-like Ox-LDLreceptor-1 (LOX-1) a cell-surface receptor for atherogenicOx-LDL (oxidized low-density lipoprotein) It also appearsto mediate Ox-LDL-induced inflammation which may becrucial in atherogenesis Furthermore mulberry leaf aqueousfractions inhibited the TNF-120572-induced activation of NF-120581Band phosphorylation of inhibitory factor of NF-120581B-alpha(I120581B-120572) Thus mulberry leaf aqueous fractions suppressTNF-120572- and LPS-induced LOX-1 gene expression mediatedby inhibiting NF-120581B activation

Chan et al [56] demonstrated that mulberry leaf extract(MLE) was rich in polyphenols MLE can effectively inhibitthe migration of vascular smooth muscle cells (VSMC) byblocking small guanosine triphosphatases (small GTPase)and AktNF-120581B signals The Rho family of small GTPasessuch as Rho Rac and Cdc42 acts as a molecular switch toregulate the actin cytoskeleton The Rho family of GTPases

12 Evidence-Based Complementary and Alternative Medicine

is also reported to be involved in the regulation of NF-120581B-dependent transcription [56] MLE has been shown to pos-sess hypoglycemic effects in an insulin-dependent diabetesmellitus (type 1 diabetes) animal model and potential hypo-glycemic compounds inmulberry leaves were suggested to be2-O-120572-D-galactopyranosyl-DNJ and fagomine [57] Konnoet al [58] further reported that mulberry latex containsalkaloidal sugar-mimic glycosidase inhibitors with antidia-betic activities such as 14-dideoxy-14-imino-D-arabinitol 1-deoxynojirimycin and 14-dideoxy-14-imino-D-ribitol Kimet al [59] recently showed that treated type 2 diabetesmellitus(T2D) with a 1 1 1 mixture of ginseng root mulberry leafwater extract and banana leaf water extract (6 gd) for 24weeks significantly decreased plasma intracellular adhesionmolecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) levels This indicates suppressed inflammatoryresponses in T2D but no significant differences in glucosehomeostasis control measurement changes Rigamonti et al[8] reported that bothPPAR120572 and 120574maybe thought of as sup-pressing the activity of NF120581B and AP-1 pathways that reduceinflammatory mediator production in the macrophages ofhumans and mice In our study MT and M promoted PPAR120572 and 120574 expression at least in part by increasing DNAbinding of transcription factors PPAR 120572 and 120574 PPAR-120572-and 120574-dependent signaling pathwaysmay also play importantroles in mediating the TNF-120572 receptor signaling pathwayby MT and M by suppressing the activity on NF-120581B andAP-1 pathways These pathways may regulate downstreamproinflammatory gene expression and adhesion molecularexpression Clinically PPAR 120574 agonists such as rosiglitazoneare widely used in the treatment of T2D [60] M and MTextracts increased PPAR 120574 activity which may partly explainwhy inflammatory responses are suppressed in T2D

Although reduced NF-120581B expression and DNA bind-ing were observed upon treatment with ML MT and BT(Figure 5) the mechanism was not assessed and is worthy offurther study In addition the present results were obtainedusing in vitro studies and may be consistent with an invivo model of atherogenesis similar to that reported byEnkhmaa et al [21] in which mulberry leaves suppressedthe development of atherosclerotic lesions Previously weproved that black beans significantly prolong LDL lag timeand decrease the atheroma regions of the aortic arch andthoracic aorta in hypercholesterolemic NZW rabbits [61]Wethen demonstrated that black soybean extract and PSPLEsignificantly decreased the adhesion of U937 monocyticcells to TNF-120572-stimulated HAECs and reduced adhesionmolecules expression (VCAM-1 and ICAM-1) as well as NF-120581B-p65 expression [37] This is particularly important giventhe opposing effects of flavonols fromMLREs obtained usingin vitro and ex vivo studies [62]

5 Conclusion

Tested MLREs were rich in flavonols and had strongantioxidant activity in protecting lymphocyte DNA fromoxidative damage In particular BT was rich in quercetinand polyphenols The antioxidant components of MLREs

might downregulate intracellular redox-dependent signalingpathways in HAECs upon TNF-120572 stimulation which mightprevent ROS-mediated endothelial cell dysfunction MLRCshave anti-inflammatory effects through the modulation ofNF-120581B AP-1 STAT3 and PPARs signaling These chemi-cals appear to provide distinct cytoprotective mechanismsthat might contribute to their putative beneficial effectsin suppressing endothelial responses to cytokines duringinflammation These results may benefit further in vivostudies to assess the therapeutic potential of phytochemicalsas anti-inflammatories for use in cytokine-induced vasculardisorders including atherosclerosis

Acknowledgment

This work was supported in part by Grants from the NationalScience Council of Taiwan (NSC 95ndash2320-B-034-001 andNSC 96ndash2320-B-034-001)

References

[1] W Palinski ldquoUnited they go conjunct regulation of aorticantioxidant enzymes during atherogenesisrdquo CirculationResearch vol 93 no 3 pp 183ndash185 2003

[2] P Libby ldquoMolecular bases of the acute coronary syndromesrdquoCirculation vol 91 no 11 pp 2844ndash2850 1995

[3] P A Baeuerle and D Baltimore ldquoNf-120581B ten years afterrdquo Cellvol 87 no 1 pp 13ndash20 1996

[4] J M Kyriakis ldquoActivation of the AP-1 transcription factor byinflammatory cytokines of the TNF familyrdquo Gene Expressionvol 7 no 4ndash6 pp 217ndash231 1999

[5] J M Muller R A Rupec and P A Baeuerle ldquoStudy of generegulation by NF-120581B and AP-1 in response to reactive oxygenintermediatesrdquoMethods vol 11 no 3 pp 301ndash312 1997

[6] G Chinetti J Fruchart and B Staels ldquoPeroxisome proliferator-activated receptors new targets for the pharmacological mod-ulation of macrophage gene expression and functionrdquo CurrentOpinion in Lipidology vol 14 no 5 pp 459ndash468 2003

[7] G Chinetti J C Fruchart and B Staels ldquoTranscriptional regu-lation of macrophage cholesterol trafficking by PPAR120572 andLXRrdquo Biochemical Society Transactions vol 34 no 6 pp 1128ndash1131 2006

[8] E Rigamonti G Chinetti-Gbaguidi and B Staels ldquoRegulationof macrophage functions by PPAR- 120572 PPAR- 120574 and LXRsin mice and menrdquo Arteriosclerosis Thrombosis and VascularBiology vol 28 no 6 pp 1050ndash1059 2008

[9] M A Bouhlel J Brozek B Derudas et al ldquoUnlike PPAR120574PPAR120572 or PPAR120573120575 activation does not promote humanmono-cyte differentiation toward alternative macrophagesrdquo Biochem-ical and Biophysical Research Communications vol 386 no 3pp 459ndash462 2009

[10] G Chinetti J-C Fruchart and B Staels ldquoPeroxisome prolife-rator-activated receptors and inflammation from basic scienceto clinical applicationsrdquo International Journal of Obesity vol 27supplement 3 pp S41ndashS45 2003

[11] Y Zhu J H-C Lin H Liao et al ldquoLDL induces transcriptionfactor activator protein-1 in human endothelial cellsrdquo Arte-riosclerosis Thrombosis and Vascular Biology vol 18 no 3 pp473ndash480 1998

Evidence-Based Complementary and Alternative Medicine 13

[12] J Terao ldquoDietary flavonoids as antioxidantsrdquo Forum of Nutri-tion vol 61 pp 87ndash94 2009

[13] Y B Feng W Q Luo and S Q Zhu ldquoExplore new clinicalapplication of Huanglian and corresponding compound pre-scriptions from their traditional userdquo Zhongguo ZhongyaoZazhi vol 33 no 10 pp 1221ndash1225 2008

[14] Y H Chu C L Chang and H F Hsu ldquoFlavonoid content ofseveral vegetables and their antioxidant activityrdquo Journal of theScience of Food andAgriculture vol 80 no 5 pp 561ndash566 2000

[15] I C Arts B van de Putte and P C Hollman ldquoCatechin con-tents of foods commonly consumed in The Netherlands 1Fruits vegetables staple foods and processed foodsrdquo Journal ofAgricultural and Food Chemistry vol 48 no 5 pp 1746ndash17512000

[16] A R Proteggente A S Pannala G Paganga et al ldquoThe anti-oxidant activity of regularly consumed fruit and vegetablesreflects their phenolic and vitamin C compositionrdquo Free RadicalResearch vol 36 no 2 pp 217ndash233 2002

[17] Y L Liu L H Tang Z Q Liang B You and S Yang ldquoGrowthinhibitory and apoptosis inducing by effects of total flavonoidsfrom Lysimachia clethroides Duby in human chronic myeloidleukemiaK562 cellsrdquo Journal of Ethnopharmacology vol 131 no1 pp 1ndash9 2010

[18] W Naowaratwattana W De-Eknamkul and E G De MejialdquoPhenolic-containing organic extracts of mulberry (Morus albaL) leaves inhibit HepG2 hepatoma cells through G2M phasearrest and inhibition of topoisomerase II120572 activityrdquo Journal ofMedicinal Food vol 13 no 5 pp 1045ndash1056 2010

[19] K Doi T Kojima and Y Fujimoto ldquoMulberry leaf extract inhi-bits the oxidative modification of rabbit and human low densitylipoproteinrdquo Biological and Pharmaceutical Bulletin vol 23 no9 pp 1066ndash1071 2000

[20] W Wang Y Zu Y Fu and T Efferth ldquoIn Vitro antioxidant andantimicrobial activity of extracts from Morus alba L leavesstems and fruitsrdquoAmerican Journal of ChineseMedicine vol 40no 2 pp 349ndash356 2012

[21] B Enkhmaa K Shiwaku T Katsube et al ldquoMulberry (Morusalba L) leaves and their major flavonol quercetin 3-(6-malon-ylglucoside) attenuate atherosclerotic lesion development inLDL receptor-deficient micerdquo Journal of Nutrition vol 135 no4 pp 729ndash734 2005

[22] J Naowaboot P Pannangpetch V Kukongviriyapan B Kongy-ingyoes and U Kukongviriyapan ldquoAntihyperglycemic antiox-idant and antiglycation activities of mulberry leaf extract instreptozotocin-induced chronic diabetic ratsrdquo Plant Foods forHuman Nutrition vol 64 no 2 pp 116ndash121 2009

[23] MM Padilha F CVilela CQ Rocha et al ldquoAntiinflammatoryproperties of Morus nigra leavesrdquo Phytotherapy Research vol24 no 10 pp 1496ndash1500 2010

[24] P Aramwit O Supasyndh T Siritienthong and N BangldquoMulberry leaf reduces oxidation and c-reactive protein levelin patients with mild dyslipidemiardquo BioMed Research Interna-tional vol 2013 Article ID 787981 7 pages 2013

[25] U Justesen P Knuthsen and T Leth ldquoQuantitative analysis offlavonols flavones and flavanones in fruits vegetables andbeverages by high-performance liquid chromatography withphoto-diode array and mass spectrometric detectionrdquo Journalof Chromatography A vol 799 no 1-2 pp 101ndash110 1998

[26] M S Taga E EMiller andD E Pratt ldquoChia seeds as a source ofnatural lipid antioxidantsrdquo Journal of the AmericanOil ChemistsrsquoSociety vol 61 no 5 pp 928ndash931 1984

[27] A A L Ordonez J D Gomez M A Vattuone and M I IslaldquoAntioxidant activities of Sechium edule (Jacq) Swartz extractsrdquoFood Chemistry vol 97 no 3 pp 452ndash458 2006

[28] A Kumaran and R Joel Karunakaran ldquoIn Vitro antioxidantactivities of methanol extracts of five Phyllanthus species fromIndiardquo LWT-Food Science and Technology vol 40 no 2 pp344ndash352 2007

[29] G Cao H M Alessio and R G Cutler ldquoOxygen-radical absor-bance capacity assay for antioxidantsrdquo Free Radical Biology andMedicine vol 14 no 3 pp 303ndash311 1993

[30] K Shimada K Fujikawa K Yahara and T Nakamura ldquoAntiox-idative properties of xanthan on the autoxidation of soybeanoil in cyclodextrin emulsionrdquo Journal of Agricultural and FoodChemistry vol 40 no 6 pp 945ndash948 1992

[31] J Cole M H L Green S E James L Henderson and H ColeldquoA further assessment of factors influencing measurements ofthioguanine-resistant mutant frequency in circulating T-lymphocytesrdquo Mutation Research vol 204 no 3 pp 493ndash5071988

[32] C-Y Hsu C-M Yang C-M Chen P Chao and S Hu ldquoEff-ects of chlorophyll-related compounds on hydrogen peroxideinduced DNA damage within human lymphocytesrdquo Journal ofAgricultural and Food Chemistry vol 53 no 7 pp 2746ndash27502005

[33] S-C Tang H-F Lo K-H Lin et al ldquoThe antioxidant capacityof extracts from Taiwan indigenous purple-leaved vegetablesrdquoJournal of the Taiwan Society for Horticultural Science vol 59no 1 pp 43ndash57 2013

[34] K-H Lin Y-Y Yang H-F Lo et al ldquoAntioxidant activity ofherbaceous plant extracts protect against hydrogen peroxide-induced DNA damage in human lymphocytesrdquo BioMed CentralResearch Notes In press

[35] A H Cory T C Owen J A Barltrop and J G Cory ldquoUse of anaqueous soluble tetrazoliumformazan assay for cell growthassays in culturerdquoCancer Communications vol 3 no 7 pp 207ndash212 1991

[36] Y T Szeto A R Collins and I F F Benzie ldquoEffects of dietaryantioxidants on DNA damage in lysed cells using a modifiedcomet assay procedurerdquoMutation Research vol 500 no 1-2 pp31ndash38 2002

[37] S Vielma G Virella A J Gorod and M F Lopes-VirellaldquoChlamydophila pneumoniae infection of human aortic endo-thelial cells induces the expression of FC 120574 receptor II (Fc120574RII)rdquoClinical Immunology vol 104 no 3 pp 265ndash273 2002

[38] C Y Zhu and S Loft ldquoEffects of Brussels sprouts extracts onhydrogen peroxide-induced DNA strand breaks in humanlymphocytesrdquo Food and Chemical Toxicology vol 39 no 12 pp1191ndash1197 2001

[39] Y D Min C-H Choi H Bark et al ldquoQuercetin inhibits exp-ression of inflammatory cytokines through attenuation of NF-120581B and p38MAPK in HMC-1 humanmast cell linerdquo Inflamma-tion Research vol 56 no 5 pp 210ndash215 2007

[40] P Y Chao Y P Huang and W B Hsieh ldquoInhibitive effect ofpurple sweet potato leaf extract and its components on celladhesion and inflammatory response in human aortic endothe-lial cellsrdquo Cell Adhesion amp Migration vol 7 no 2 pp 1ndash9 2013

[41] Y Y YangThe antioxidative capacity in herb plant extracts andtheir protection role in DNA oxidative damage of lymphocyte[MS thesis] Chinese Culture University Taipei Taiwan 2004

[42] C A Rice-Evans N J Miller and G Paganga ldquoStructure-anti-oxidant activity relationships of flavonoids and phenolic acidsrdquo

14 Evidence-Based Complementary and Alternative Medicine

Free Radical Biology and Medicine vol 20 no 7 pp 933ndash9561996

[43] M Strube G R Haenen H van den Berg and A Bast ldquoPitfallsin a method for assessment of total antioxidant capacityrdquo FreeRadical Research vol 26 no 6 pp 515ndash521 1997

[44] P G Pietta ldquoFlavonoids as antioxidantsrdquo Journal of NaturalProducts vol 63 no 7 pp 1035ndash1042 2000

[45] A M Gonzalez-Paramas S Esteban-Ruano C Santos-BuelgaSDe Pascual-Teresa and J C Rivas-Gonzalo ldquoFlavanol contentand antioxidant activity in winery byproductsrdquo Journal ofAgricultural and Food Chemistry vol 52 no 2 pp 234ndash2382004

[46] S Y Kim J J Gao and H K Kang ldquoTwo flavonoids from theleaves ofMorus alba induce differentiation of the human prom-yelocytic leukemia (HL-60) cell linerdquo Biological and Pharma-ceutical Bulletin vol 23 no 4 pp 451ndash455 2000

[47] M A Khan A A Rahman S Islam et al ldquoA comparative studyon the antioxidant activity ofmethanolic extracts from differentparts of Morus alba L (Moraceae)rdquo BMC Research vol 6 pp24ndash32 2013

[48] A-L ChungH-F Lo K-H Lin et al ldquoStudy on the antioxidantactivity in herb plant extractsrdquo Journal of the Taiwan Society ForHorticultural Science vol 59 no 2 pp 139ndash152 2013

[49] R L Prior X Wu and K Schaich ldquoStandardized methods forthe determination of antioxidant capacity and phenolics infoods and dietary supplementsrdquo Journal of Agricultural andFood Chemistry vol 53 no 10 pp 4290ndash4302 2005

[50] S J Duthie A R Collins GGDuthie andV LDobson ldquoQue-rcetin and myricetin protect against hydrogen peroxide-indu-ced DNA damage (strand breaks and oxidised pyrimidines) inhuman lymphocytesrdquo Mutation Research vol 393 no 3 pp223ndash231 1997

[51] G A Liu and R L Zheng ldquoProtection against damagedDNA inthe single cell by polyphenolsrdquo Pharmazie vol 57 no 12 pp852ndash854 2002

[52] M Noroozi W J Angerson and M E J Lean ldquoEffects of flav-onoids and vitamin C on oxidative DNA damage to humanlymphocytesrdquoAmerican Journal of Clinical Nutrition vol 67 no6 pp 1210ndash1218 1998

[53] T Koga and M Meydani ldquoEffect of plasma metabolites of (+)-catechin and quercetin on monocyte adhesion to human aorticendothelial cellsrdquoAmerican Journal of Clinical Nutrition vol 73no 5 pp 941ndash948 2001

[54] CMeng P Somers L Hoong et al ldquoDiscovery of novel pheno-lic antioxidants as inhibitors of vascular cell adhesionmolecule-1 expression for use in chronic inflammatory diseasesrdquo Journalof Medicinal Chemistry vol 47 no 25 pp 6420ndash6432 2004

[55] Y Shibata N Kume H Arai et al ldquoMulberry leaf aqueousfractions inhibit TNF-120572-induced nuclear factor 120581B (NF-120581B)activation and lectin-like oxidized LDL receptor-1 (LOX-1)expression in vascular endothelial cellsrdquoAtherosclerosis vol 193no 1 pp 20ndash27 2007

[56] K C Chan H H Ho C N Huang M Lin H Chen and CWang ldquoMulberry leaf extract inhibits vascular smooth musclecell migration involving a block of small GTPase and AktNF-kB signalsrdquo Journal of Agricultural and Food Chemistry vol 57no 19 pp 9147ndash9153 2009

[57] F Chen N Nakashima I Kimura M Kimura N Asanoand S Koya ldquoPotentiating effects on pilocarpine-induced salivasecretion by extracts and N-containing sugars derived frommulberry leaves in streptozocin- diabetic micerdquo Biological andPharmaceutical Bulletin vol 18 no 12 pp 1676ndash1680 1995

[58] K Konno H Ono M Nakamura et al ldquoMulberry latexrich in antidiabetic sugar-mimic alkaloids forces dieting oncaterpillarsrdquo Proceedings of the National Academy of Sciences ofthe United States of America vol 103 no 5 pp 1337ndash1341 2006

[59] H J Kim K H Yoon M J Kang et al ldquoA six-month sup-plementation of mulberry Korean red ginseng and banabadecreases biomarkers of systemic low-grade inflammation insubjects with impaired glucose tolerance and type 2 diabetesrdquoEvidence-based Complementary and Alternative Medicine vol2012 Article ID 735191 8 pages 2012

[60] G Nicholson and G M Hall ldquoDiabetes mellitus new drugs fora new epidemicrdquo British Journal of Anaesthesia vol 107 no 1pp 65ndash73 2011

[61] P Y Chao Y L Chen Y C Lin et al ldquoEffects of black soybeanon atherogenic prevention in hypercholesterolemic rabbits andon adhesion molecular expression in cultured HAECsrdquo Foodand Nutrition Sciences vol 4 pp 9ndash21 2013

[62] C Chen S Li C-YOChen et al ldquoConstituents in purple sweetpotato leaves inhibit in vitro angiogenesis with opposite effectsex vivordquo Nutrition vol 27 no 11-12 pp 1177ndash1182 2011

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

10 Evidence-Based Complementary and Alternative Medicine

NF-120581BTNF-120572

TNF-120572

+

Aspirin10120583M+ +

BT25120583gmL

+

BT50120583gmL

+

MT25120583gmL

+

MT50120583gmL

+

M25

120583gmL+

M50

120583gmLControl

minus

DNAOligo

Free probe

(a)

AP-1TNF-120572

TNF-120572

+

Aspirin10120583M+ +

BT25120583gmL

+

BT50120583gmL

+

MT25120583gmL

+

MT50120583gmL

+

M25

120583gmL+

M50

120583gmLControl

minus

DNAOligo

Free probe

(b)

STAT3

TNF-120572

TNF-120572

+

Aspirin10120583M+ +

BT25120583gmL

+

BT50120583gmL

+

MT25120583gmL

+

MT50120583gmL

+

M25

120583gmL+

M50

120583gmLControl

minus

DNAOligo

Free probe

(c)

Figure 5 MLREs inhibits the binding of (a) NF-120581B (b) AP-1 and (c) STAT3 to target DNA sequence NF-120581B AP-1 and STAT3 transcriptionfactor-DNA interactions were assessed by EMSA

contained primarily chlorogenic acid and caffeoylquinic acidderivatives The IC

50of DPPH radical scavenging was 794

and 2042120583gmL for 50 methanolic and water extractsrespectively [18] In our studyM extracts contained quercetinand kaempferol (Table 2) which may account for part ofthe highly scavenged DPPH radical in comparison to therest of MLREs Khan et al [47] demonstrated that the IC

50

was 22023120583gmL which is much less efficacious than themulberry leaf acidic methanolic extract IC

50of 1888 120583gmL

(Table 3) However the IC50

of in vitro free radical scav-enging activity of mulberry leaves from Naowaboot et al[22] was 617 plusmn 21 120583gmL which is more efficacious thanthe IC

50concentration derived in the present study Taken

together the polyphenol-rich MLREs exhibited distinct cell-free antioxidant activity (ie TEAC) as validated by theirlevels of polyphenols and flavonols with distinct antioxidantactivity strongly accounting for the antioxidant activity of the

extracts The amount of quercetin was especially positivelycorrelated with the observed TEAC values

42 Estimation of DNA Single Strand Break Damage fromExposure to Acidic Methanolic and Water Extracts One ofthe antioxidant activity tests scavenging of DPPH radi-cal was selected due to its quick and simple but it is aless biologically relevant assay Previously we reported thatwith 2 120583gsdotmLminus1 of methanolic and water extracts from theindigenous vegetables [33] and herb plants [48] significantlyprolonged lag phase of low-density lipoprotein (LDL) TheLDL oxidation assay is considered to be one of the mostbiologically relevant assays [49] Both studies [33 48] alsoshowed that the concentration of the tested extracts forscavenging DPPH radical was in higher levels at 10mg oftested extracts with 2ndash53 inhibition Lower concentrationwas then chosen for the treated dosage in the present study

Evidence-Based Complementary and Alternative Medicine 11

TNF-120572

TNF-120572

+

Aspirin10120583M+ +

BT25120583gmL

+

BT50120583gmL

+

MT25120583gmL

+

MT50120583gmL

+

M25

120583gmL+

M50

120583gmLControl

minus

DNAOligo

Free probe

PPAR 120572

(a)

TNF-120572

TNF-120572

+

Aspirin10120583M+ +

BT25120583gmL

+

BT50120583gmL

+

MT25120583gmL

+

MT50120583gmL

+

M25

120583gmL+

M50

120583gmLControl

minus

DNAOligo

Free probe

PPAR120574

(b)

Figure 6 Effect of treatments on the expression of PPAR 120572 (a) and PPAR 120574 (b) in TNF-120572-stimulated HAECs by EMSA

In addition lower concentration also could avoid the toxicityeffect of higher level of extracts on lymphocytes Quercetinwas found to protect against H

2O2-induced DNA damage in

human lymphocytes at 10 120583M [50] and at 31 to 25 120583M [51]Quercetin was also reported to induce DNA strand breaks invarious cell types but only at higher doses at 100 120583Mor above[50] Duthie et al [50] proposed that the dihydroxy structureof quercetin and myricetin might be essential in protectingDNA against hydrogen peroxide No such hydroxyl groupsare present in the tocopherol molecule Noroozi et al [52]demonstrated that in addition to quercetin kaempferolcould also inhibit H

2O2-induced DNA strand breaks in

human lymphocytes In our study both BT and M were richin quercetin and kaempferol (Table 2) whichmay account forthe protective roles of both extracts in preventingDNAstrandbreaks from human lymphocytes

43 MLREs Regulated the DNA-Binding Activity and Expres-sions of NF-120581B AP-1 STAT3 and PPARs in HAECsBecause MLREs exhibited free radical scavenging the anti-inflammatory effects of MLREs on TNF-120572-induced celladhesion and adhesionmolecule expressionwere determinedin the present study Our results suggest that MLREs andaspirin significantly inhibited TNF-120572-induced monocyte-endothelial cell adhesion (Figure 3) Significant reductionsin NF-120581B expression and DNA binding by aspirin MTextract (25 120583gmL) and BT extract (both 25 and 50 120583gmL)were also observed The adhesion of monocytes to the

vascular endothelium and subsequent migration into thevessel wall are early events in atherogenesis [2] Our resultsare consistent with previous research showing that dietarypolyphenols such as catechin and quercetin significantlyreduce the binding of monocytes to HAECs [53 54] Thereduced cellular adhesion may be due to the inhibitionof cellular adhesion molecule expression as purple sweetpotato leaf extract (PSPLE) reduced both VCAM-1 andICAM-1 expression [40] Shibata et al [55] demonstratedthat pretreatment of cultured bovine aortic endothelial cells(BAECs) with mulberry leaf aqueous fractions inhibitedTNF-120572- and LPS-induced expression of Lectin-like Ox-LDLreceptor-1 (LOX-1) a cell-surface receptor for atherogenicOx-LDL (oxidized low-density lipoprotein) It also appearsto mediate Ox-LDL-induced inflammation which may becrucial in atherogenesis Furthermore mulberry leaf aqueousfractions inhibited the TNF-120572-induced activation of NF-120581Band phosphorylation of inhibitory factor of NF-120581B-alpha(I120581B-120572) Thus mulberry leaf aqueous fractions suppressTNF-120572- and LPS-induced LOX-1 gene expression mediatedby inhibiting NF-120581B activation

Chan et al [56] demonstrated that mulberry leaf extract(MLE) was rich in polyphenols MLE can effectively inhibitthe migration of vascular smooth muscle cells (VSMC) byblocking small guanosine triphosphatases (small GTPase)and AktNF-120581B signals The Rho family of small GTPasessuch as Rho Rac and Cdc42 acts as a molecular switch toregulate the actin cytoskeleton The Rho family of GTPases

12 Evidence-Based Complementary and Alternative Medicine

is also reported to be involved in the regulation of NF-120581B-dependent transcription [56] MLE has been shown to pos-sess hypoglycemic effects in an insulin-dependent diabetesmellitus (type 1 diabetes) animal model and potential hypo-glycemic compounds inmulberry leaves were suggested to be2-O-120572-D-galactopyranosyl-DNJ and fagomine [57] Konnoet al [58] further reported that mulberry latex containsalkaloidal sugar-mimic glycosidase inhibitors with antidia-betic activities such as 14-dideoxy-14-imino-D-arabinitol 1-deoxynojirimycin and 14-dideoxy-14-imino-D-ribitol Kimet al [59] recently showed that treated type 2 diabetesmellitus(T2D) with a 1 1 1 mixture of ginseng root mulberry leafwater extract and banana leaf water extract (6 gd) for 24weeks significantly decreased plasma intracellular adhesionmolecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) levels This indicates suppressed inflammatoryresponses in T2D but no significant differences in glucosehomeostasis control measurement changes Rigamonti et al[8] reported that bothPPAR120572 and 120574maybe thought of as sup-pressing the activity of NF120581B and AP-1 pathways that reduceinflammatory mediator production in the macrophages ofhumans and mice In our study MT and M promoted PPAR120572 and 120574 expression at least in part by increasing DNAbinding of transcription factors PPAR 120572 and 120574 PPAR-120572-and 120574-dependent signaling pathwaysmay also play importantroles in mediating the TNF-120572 receptor signaling pathwayby MT and M by suppressing the activity on NF-120581B andAP-1 pathways These pathways may regulate downstreamproinflammatory gene expression and adhesion molecularexpression Clinically PPAR 120574 agonists such as rosiglitazoneare widely used in the treatment of T2D [60] M and MTextracts increased PPAR 120574 activity which may partly explainwhy inflammatory responses are suppressed in T2D

Although reduced NF-120581B expression and DNA bind-ing were observed upon treatment with ML MT and BT(Figure 5) the mechanism was not assessed and is worthy offurther study In addition the present results were obtainedusing in vitro studies and may be consistent with an invivo model of atherogenesis similar to that reported byEnkhmaa et al [21] in which mulberry leaves suppressedthe development of atherosclerotic lesions Previously weproved that black beans significantly prolong LDL lag timeand decrease the atheroma regions of the aortic arch andthoracic aorta in hypercholesterolemic NZW rabbits [61]Wethen demonstrated that black soybean extract and PSPLEsignificantly decreased the adhesion of U937 monocyticcells to TNF-120572-stimulated HAECs and reduced adhesionmolecules expression (VCAM-1 and ICAM-1) as well as NF-120581B-p65 expression [37] This is particularly important giventhe opposing effects of flavonols fromMLREs obtained usingin vitro and ex vivo studies [62]

5 Conclusion

Tested MLREs were rich in flavonols and had strongantioxidant activity in protecting lymphocyte DNA fromoxidative damage In particular BT was rich in quercetinand polyphenols The antioxidant components of MLREs

might downregulate intracellular redox-dependent signalingpathways in HAECs upon TNF-120572 stimulation which mightprevent ROS-mediated endothelial cell dysfunction MLRCshave anti-inflammatory effects through the modulation ofNF-120581B AP-1 STAT3 and PPARs signaling These chemi-cals appear to provide distinct cytoprotective mechanismsthat might contribute to their putative beneficial effectsin suppressing endothelial responses to cytokines duringinflammation These results may benefit further in vivostudies to assess the therapeutic potential of phytochemicalsas anti-inflammatories for use in cytokine-induced vasculardisorders including atherosclerosis

Acknowledgment

This work was supported in part by Grants from the NationalScience Council of Taiwan (NSC 95ndash2320-B-034-001 andNSC 96ndash2320-B-034-001)

References

[1] W Palinski ldquoUnited they go conjunct regulation of aorticantioxidant enzymes during atherogenesisrdquo CirculationResearch vol 93 no 3 pp 183ndash185 2003

[2] P Libby ldquoMolecular bases of the acute coronary syndromesrdquoCirculation vol 91 no 11 pp 2844ndash2850 1995

[3] P A Baeuerle and D Baltimore ldquoNf-120581B ten years afterrdquo Cellvol 87 no 1 pp 13ndash20 1996

[4] J M Kyriakis ldquoActivation of the AP-1 transcription factor byinflammatory cytokines of the TNF familyrdquo Gene Expressionvol 7 no 4ndash6 pp 217ndash231 1999

[5] J M Muller R A Rupec and P A Baeuerle ldquoStudy of generegulation by NF-120581B and AP-1 in response to reactive oxygenintermediatesrdquoMethods vol 11 no 3 pp 301ndash312 1997

[6] G Chinetti J Fruchart and B Staels ldquoPeroxisome proliferator-activated receptors new targets for the pharmacological mod-ulation of macrophage gene expression and functionrdquo CurrentOpinion in Lipidology vol 14 no 5 pp 459ndash468 2003

[7] G Chinetti J C Fruchart and B Staels ldquoTranscriptional regu-lation of macrophage cholesterol trafficking by PPAR120572 andLXRrdquo Biochemical Society Transactions vol 34 no 6 pp 1128ndash1131 2006

[8] E Rigamonti G Chinetti-Gbaguidi and B Staels ldquoRegulationof macrophage functions by PPAR- 120572 PPAR- 120574 and LXRsin mice and menrdquo Arteriosclerosis Thrombosis and VascularBiology vol 28 no 6 pp 1050ndash1059 2008

[9] M A Bouhlel J Brozek B Derudas et al ldquoUnlike PPAR120574PPAR120572 or PPAR120573120575 activation does not promote humanmono-cyte differentiation toward alternative macrophagesrdquo Biochem-ical and Biophysical Research Communications vol 386 no 3pp 459ndash462 2009

[10] G Chinetti J-C Fruchart and B Staels ldquoPeroxisome prolife-rator-activated receptors and inflammation from basic scienceto clinical applicationsrdquo International Journal of Obesity vol 27supplement 3 pp S41ndashS45 2003

[11] Y Zhu J H-C Lin H Liao et al ldquoLDL induces transcriptionfactor activator protein-1 in human endothelial cellsrdquo Arte-riosclerosis Thrombosis and Vascular Biology vol 18 no 3 pp473ndash480 1998

Evidence-Based Complementary and Alternative Medicine 13

[12] J Terao ldquoDietary flavonoids as antioxidantsrdquo Forum of Nutri-tion vol 61 pp 87ndash94 2009

[13] Y B Feng W Q Luo and S Q Zhu ldquoExplore new clinicalapplication of Huanglian and corresponding compound pre-scriptions from their traditional userdquo Zhongguo ZhongyaoZazhi vol 33 no 10 pp 1221ndash1225 2008

[14] Y H Chu C L Chang and H F Hsu ldquoFlavonoid content ofseveral vegetables and their antioxidant activityrdquo Journal of theScience of Food andAgriculture vol 80 no 5 pp 561ndash566 2000

[15] I C Arts B van de Putte and P C Hollman ldquoCatechin con-tents of foods commonly consumed in The Netherlands 1Fruits vegetables staple foods and processed foodsrdquo Journal ofAgricultural and Food Chemistry vol 48 no 5 pp 1746ndash17512000

[16] A R Proteggente A S Pannala G Paganga et al ldquoThe anti-oxidant activity of regularly consumed fruit and vegetablesreflects their phenolic and vitamin C compositionrdquo Free RadicalResearch vol 36 no 2 pp 217ndash233 2002

[17] Y L Liu L H Tang Z Q Liang B You and S Yang ldquoGrowthinhibitory and apoptosis inducing by effects of total flavonoidsfrom Lysimachia clethroides Duby in human chronic myeloidleukemiaK562 cellsrdquo Journal of Ethnopharmacology vol 131 no1 pp 1ndash9 2010

[18] W Naowaratwattana W De-Eknamkul and E G De MejialdquoPhenolic-containing organic extracts of mulberry (Morus albaL) leaves inhibit HepG2 hepatoma cells through G2M phasearrest and inhibition of topoisomerase II120572 activityrdquo Journal ofMedicinal Food vol 13 no 5 pp 1045ndash1056 2010

[19] K Doi T Kojima and Y Fujimoto ldquoMulberry leaf extract inhi-bits the oxidative modification of rabbit and human low densitylipoproteinrdquo Biological and Pharmaceutical Bulletin vol 23 no9 pp 1066ndash1071 2000

[20] W Wang Y Zu Y Fu and T Efferth ldquoIn Vitro antioxidant andantimicrobial activity of extracts from Morus alba L leavesstems and fruitsrdquoAmerican Journal of ChineseMedicine vol 40no 2 pp 349ndash356 2012

[21] B Enkhmaa K Shiwaku T Katsube et al ldquoMulberry (Morusalba L) leaves and their major flavonol quercetin 3-(6-malon-ylglucoside) attenuate atherosclerotic lesion development inLDL receptor-deficient micerdquo Journal of Nutrition vol 135 no4 pp 729ndash734 2005

[22] J Naowaboot P Pannangpetch V Kukongviriyapan B Kongy-ingyoes and U Kukongviriyapan ldquoAntihyperglycemic antiox-idant and antiglycation activities of mulberry leaf extract instreptozotocin-induced chronic diabetic ratsrdquo Plant Foods forHuman Nutrition vol 64 no 2 pp 116ndash121 2009

[23] MM Padilha F CVilela CQ Rocha et al ldquoAntiinflammatoryproperties of Morus nigra leavesrdquo Phytotherapy Research vol24 no 10 pp 1496ndash1500 2010

[24] P Aramwit O Supasyndh T Siritienthong and N BangldquoMulberry leaf reduces oxidation and c-reactive protein levelin patients with mild dyslipidemiardquo BioMed Research Interna-tional vol 2013 Article ID 787981 7 pages 2013

[25] U Justesen P Knuthsen and T Leth ldquoQuantitative analysis offlavonols flavones and flavanones in fruits vegetables andbeverages by high-performance liquid chromatography withphoto-diode array and mass spectrometric detectionrdquo Journalof Chromatography A vol 799 no 1-2 pp 101ndash110 1998

[26] M S Taga E EMiller andD E Pratt ldquoChia seeds as a source ofnatural lipid antioxidantsrdquo Journal of the AmericanOil ChemistsrsquoSociety vol 61 no 5 pp 928ndash931 1984

[27] A A L Ordonez J D Gomez M A Vattuone and M I IslaldquoAntioxidant activities of Sechium edule (Jacq) Swartz extractsrdquoFood Chemistry vol 97 no 3 pp 452ndash458 2006

[28] A Kumaran and R Joel Karunakaran ldquoIn Vitro antioxidantactivities of methanol extracts of five Phyllanthus species fromIndiardquo LWT-Food Science and Technology vol 40 no 2 pp344ndash352 2007

[29] G Cao H M Alessio and R G Cutler ldquoOxygen-radical absor-bance capacity assay for antioxidantsrdquo Free Radical Biology andMedicine vol 14 no 3 pp 303ndash311 1993

[30] K Shimada K Fujikawa K Yahara and T Nakamura ldquoAntiox-idative properties of xanthan on the autoxidation of soybeanoil in cyclodextrin emulsionrdquo Journal of Agricultural and FoodChemistry vol 40 no 6 pp 945ndash948 1992

[31] J Cole M H L Green S E James L Henderson and H ColeldquoA further assessment of factors influencing measurements ofthioguanine-resistant mutant frequency in circulating T-lymphocytesrdquo Mutation Research vol 204 no 3 pp 493ndash5071988

[32] C-Y Hsu C-M Yang C-M Chen P Chao and S Hu ldquoEff-ects of chlorophyll-related compounds on hydrogen peroxideinduced DNA damage within human lymphocytesrdquo Journal ofAgricultural and Food Chemistry vol 53 no 7 pp 2746ndash27502005

[33] S-C Tang H-F Lo K-H Lin et al ldquoThe antioxidant capacityof extracts from Taiwan indigenous purple-leaved vegetablesrdquoJournal of the Taiwan Society for Horticultural Science vol 59no 1 pp 43ndash57 2013

[34] K-H Lin Y-Y Yang H-F Lo et al ldquoAntioxidant activity ofherbaceous plant extracts protect against hydrogen peroxide-induced DNA damage in human lymphocytesrdquo BioMed CentralResearch Notes In press

[35] A H Cory T C Owen J A Barltrop and J G Cory ldquoUse of anaqueous soluble tetrazoliumformazan assay for cell growthassays in culturerdquoCancer Communications vol 3 no 7 pp 207ndash212 1991

[36] Y T Szeto A R Collins and I F F Benzie ldquoEffects of dietaryantioxidants on DNA damage in lysed cells using a modifiedcomet assay procedurerdquoMutation Research vol 500 no 1-2 pp31ndash38 2002

[37] S Vielma G Virella A J Gorod and M F Lopes-VirellaldquoChlamydophila pneumoniae infection of human aortic endo-thelial cells induces the expression of FC 120574 receptor II (Fc120574RII)rdquoClinical Immunology vol 104 no 3 pp 265ndash273 2002

[38] C Y Zhu and S Loft ldquoEffects of Brussels sprouts extracts onhydrogen peroxide-induced DNA strand breaks in humanlymphocytesrdquo Food and Chemical Toxicology vol 39 no 12 pp1191ndash1197 2001

[39] Y D Min C-H Choi H Bark et al ldquoQuercetin inhibits exp-ression of inflammatory cytokines through attenuation of NF-120581B and p38MAPK in HMC-1 humanmast cell linerdquo Inflamma-tion Research vol 56 no 5 pp 210ndash215 2007

[40] P Y Chao Y P Huang and W B Hsieh ldquoInhibitive effect ofpurple sweet potato leaf extract and its components on celladhesion and inflammatory response in human aortic endothe-lial cellsrdquo Cell Adhesion amp Migration vol 7 no 2 pp 1ndash9 2013

[41] Y Y YangThe antioxidative capacity in herb plant extracts andtheir protection role in DNA oxidative damage of lymphocyte[MS thesis] Chinese Culture University Taipei Taiwan 2004

[42] C A Rice-Evans N J Miller and G Paganga ldquoStructure-anti-oxidant activity relationships of flavonoids and phenolic acidsrdquo

14 Evidence-Based Complementary and Alternative Medicine

Free Radical Biology and Medicine vol 20 no 7 pp 933ndash9561996

[43] M Strube G R Haenen H van den Berg and A Bast ldquoPitfallsin a method for assessment of total antioxidant capacityrdquo FreeRadical Research vol 26 no 6 pp 515ndash521 1997

[44] P G Pietta ldquoFlavonoids as antioxidantsrdquo Journal of NaturalProducts vol 63 no 7 pp 1035ndash1042 2000

[45] A M Gonzalez-Paramas S Esteban-Ruano C Santos-BuelgaSDe Pascual-Teresa and J C Rivas-Gonzalo ldquoFlavanol contentand antioxidant activity in winery byproductsrdquo Journal ofAgricultural and Food Chemistry vol 52 no 2 pp 234ndash2382004

[46] S Y Kim J J Gao and H K Kang ldquoTwo flavonoids from theleaves ofMorus alba induce differentiation of the human prom-yelocytic leukemia (HL-60) cell linerdquo Biological and Pharma-ceutical Bulletin vol 23 no 4 pp 451ndash455 2000

[47] M A Khan A A Rahman S Islam et al ldquoA comparative studyon the antioxidant activity ofmethanolic extracts from differentparts of Morus alba L (Moraceae)rdquo BMC Research vol 6 pp24ndash32 2013

[48] A-L ChungH-F Lo K-H Lin et al ldquoStudy on the antioxidantactivity in herb plant extractsrdquo Journal of the Taiwan Society ForHorticultural Science vol 59 no 2 pp 139ndash152 2013

[49] R L Prior X Wu and K Schaich ldquoStandardized methods forthe determination of antioxidant capacity and phenolics infoods and dietary supplementsrdquo Journal of Agricultural andFood Chemistry vol 53 no 10 pp 4290ndash4302 2005

[50] S J Duthie A R Collins GGDuthie andV LDobson ldquoQue-rcetin and myricetin protect against hydrogen peroxide-indu-ced DNA damage (strand breaks and oxidised pyrimidines) inhuman lymphocytesrdquo Mutation Research vol 393 no 3 pp223ndash231 1997

[51] G A Liu and R L Zheng ldquoProtection against damagedDNA inthe single cell by polyphenolsrdquo Pharmazie vol 57 no 12 pp852ndash854 2002

[52] M Noroozi W J Angerson and M E J Lean ldquoEffects of flav-onoids and vitamin C on oxidative DNA damage to humanlymphocytesrdquoAmerican Journal of Clinical Nutrition vol 67 no6 pp 1210ndash1218 1998

[53] T Koga and M Meydani ldquoEffect of plasma metabolites of (+)-catechin and quercetin on monocyte adhesion to human aorticendothelial cellsrdquoAmerican Journal of Clinical Nutrition vol 73no 5 pp 941ndash948 2001

[54] CMeng P Somers L Hoong et al ldquoDiscovery of novel pheno-lic antioxidants as inhibitors of vascular cell adhesionmolecule-1 expression for use in chronic inflammatory diseasesrdquo Journalof Medicinal Chemistry vol 47 no 25 pp 6420ndash6432 2004

[55] Y Shibata N Kume H Arai et al ldquoMulberry leaf aqueousfractions inhibit TNF-120572-induced nuclear factor 120581B (NF-120581B)activation and lectin-like oxidized LDL receptor-1 (LOX-1)expression in vascular endothelial cellsrdquoAtherosclerosis vol 193no 1 pp 20ndash27 2007

[56] K C Chan H H Ho C N Huang M Lin H Chen and CWang ldquoMulberry leaf extract inhibits vascular smooth musclecell migration involving a block of small GTPase and AktNF-kB signalsrdquo Journal of Agricultural and Food Chemistry vol 57no 19 pp 9147ndash9153 2009

[57] F Chen N Nakashima I Kimura M Kimura N Asanoand S Koya ldquoPotentiating effects on pilocarpine-induced salivasecretion by extracts and N-containing sugars derived frommulberry leaves in streptozocin- diabetic micerdquo Biological andPharmaceutical Bulletin vol 18 no 12 pp 1676ndash1680 1995

[58] K Konno H Ono M Nakamura et al ldquoMulberry latexrich in antidiabetic sugar-mimic alkaloids forces dieting oncaterpillarsrdquo Proceedings of the National Academy of Sciences ofthe United States of America vol 103 no 5 pp 1337ndash1341 2006

[59] H J Kim K H Yoon M J Kang et al ldquoA six-month sup-plementation of mulberry Korean red ginseng and banabadecreases biomarkers of systemic low-grade inflammation insubjects with impaired glucose tolerance and type 2 diabetesrdquoEvidence-based Complementary and Alternative Medicine vol2012 Article ID 735191 8 pages 2012

[60] G Nicholson and G M Hall ldquoDiabetes mellitus new drugs fora new epidemicrdquo British Journal of Anaesthesia vol 107 no 1pp 65ndash73 2011

[61] P Y Chao Y L Chen Y C Lin et al ldquoEffects of black soybeanon atherogenic prevention in hypercholesterolemic rabbits andon adhesion molecular expression in cultured HAECsrdquo Foodand Nutrition Sciences vol 4 pp 9ndash21 2013

[62] C Chen S Li C-YOChen et al ldquoConstituents in purple sweetpotato leaves inhibit in vitro angiogenesis with opposite effectsex vivordquo Nutrition vol 27 no 11-12 pp 1177ndash1182 2011

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

Evidence-Based Complementary and Alternative Medicine 11

TNF-120572

TNF-120572

+

Aspirin10120583M+ +

BT25120583gmL

+

BT50120583gmL

+

MT25120583gmL

+

MT50120583gmL

+

M25

120583gmL+

M50

120583gmLControl

minus

DNAOligo

Free probe

PPAR 120572

(a)

TNF-120572

TNF-120572

+

Aspirin10120583M+ +

BT25120583gmL

+

BT50120583gmL

+

MT25120583gmL

+

MT50120583gmL

+

M25

120583gmL+

M50

120583gmLControl

minus

DNAOligo

Free probe

PPAR120574

(b)

Figure 6 Effect of treatments on the expression of PPAR 120572 (a) and PPAR 120574 (b) in TNF-120572-stimulated HAECs by EMSA

In addition lower concentration also could avoid the toxicityeffect of higher level of extracts on lymphocytes Quercetinwas found to protect against H

2O2-induced DNA damage in

human lymphocytes at 10 120583M [50] and at 31 to 25 120583M [51]Quercetin was also reported to induce DNA strand breaks invarious cell types but only at higher doses at 100 120583Mor above[50] Duthie et al [50] proposed that the dihydroxy structureof quercetin and myricetin might be essential in protectingDNA against hydrogen peroxide No such hydroxyl groupsare present in the tocopherol molecule Noroozi et al [52]demonstrated that in addition to quercetin kaempferolcould also inhibit H

2O2-induced DNA strand breaks in

human lymphocytes In our study both BT and M were richin quercetin and kaempferol (Table 2) whichmay account forthe protective roles of both extracts in preventingDNAstrandbreaks from human lymphocytes

43 MLREs Regulated the DNA-Binding Activity and Expres-sions of NF-120581B AP-1 STAT3 and PPARs in HAECsBecause MLREs exhibited free radical scavenging the anti-inflammatory effects of MLREs on TNF-120572-induced celladhesion and adhesionmolecule expressionwere determinedin the present study Our results suggest that MLREs andaspirin significantly inhibited TNF-120572-induced monocyte-endothelial cell adhesion (Figure 3) Significant reductionsin NF-120581B expression and DNA binding by aspirin MTextract (25 120583gmL) and BT extract (both 25 and 50 120583gmL)were also observed The adhesion of monocytes to the

vascular endothelium and subsequent migration into thevessel wall are early events in atherogenesis [2] Our resultsare consistent with previous research showing that dietarypolyphenols such as catechin and quercetin significantlyreduce the binding of monocytes to HAECs [53 54] Thereduced cellular adhesion may be due to the inhibitionof cellular adhesion molecule expression as purple sweetpotato leaf extract (PSPLE) reduced both VCAM-1 andICAM-1 expression [40] Shibata et al [55] demonstratedthat pretreatment of cultured bovine aortic endothelial cells(BAECs) with mulberry leaf aqueous fractions inhibitedTNF-120572- and LPS-induced expression of Lectin-like Ox-LDLreceptor-1 (LOX-1) a cell-surface receptor for atherogenicOx-LDL (oxidized low-density lipoprotein) It also appearsto mediate Ox-LDL-induced inflammation which may becrucial in atherogenesis Furthermore mulberry leaf aqueousfractions inhibited the TNF-120572-induced activation of NF-120581Band phosphorylation of inhibitory factor of NF-120581B-alpha(I120581B-120572) Thus mulberry leaf aqueous fractions suppressTNF-120572- and LPS-induced LOX-1 gene expression mediatedby inhibiting NF-120581B activation

Chan et al [56] demonstrated that mulberry leaf extract(MLE) was rich in polyphenols MLE can effectively inhibitthe migration of vascular smooth muscle cells (VSMC) byblocking small guanosine triphosphatases (small GTPase)and AktNF-120581B signals The Rho family of small GTPasessuch as Rho Rac and Cdc42 acts as a molecular switch toregulate the actin cytoskeleton The Rho family of GTPases

12 Evidence-Based Complementary and Alternative Medicine

is also reported to be involved in the regulation of NF-120581B-dependent transcription [56] MLE has been shown to pos-sess hypoglycemic effects in an insulin-dependent diabetesmellitus (type 1 diabetes) animal model and potential hypo-glycemic compounds inmulberry leaves were suggested to be2-O-120572-D-galactopyranosyl-DNJ and fagomine [57] Konnoet al [58] further reported that mulberry latex containsalkaloidal sugar-mimic glycosidase inhibitors with antidia-betic activities such as 14-dideoxy-14-imino-D-arabinitol 1-deoxynojirimycin and 14-dideoxy-14-imino-D-ribitol Kimet al [59] recently showed that treated type 2 diabetesmellitus(T2D) with a 1 1 1 mixture of ginseng root mulberry leafwater extract and banana leaf water extract (6 gd) for 24weeks significantly decreased plasma intracellular adhesionmolecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) levels This indicates suppressed inflammatoryresponses in T2D but no significant differences in glucosehomeostasis control measurement changes Rigamonti et al[8] reported that bothPPAR120572 and 120574maybe thought of as sup-pressing the activity of NF120581B and AP-1 pathways that reduceinflammatory mediator production in the macrophages ofhumans and mice In our study MT and M promoted PPAR120572 and 120574 expression at least in part by increasing DNAbinding of transcription factors PPAR 120572 and 120574 PPAR-120572-and 120574-dependent signaling pathwaysmay also play importantroles in mediating the TNF-120572 receptor signaling pathwayby MT and M by suppressing the activity on NF-120581B andAP-1 pathways These pathways may regulate downstreamproinflammatory gene expression and adhesion molecularexpression Clinically PPAR 120574 agonists such as rosiglitazoneare widely used in the treatment of T2D [60] M and MTextracts increased PPAR 120574 activity which may partly explainwhy inflammatory responses are suppressed in T2D

Although reduced NF-120581B expression and DNA bind-ing were observed upon treatment with ML MT and BT(Figure 5) the mechanism was not assessed and is worthy offurther study In addition the present results were obtainedusing in vitro studies and may be consistent with an invivo model of atherogenesis similar to that reported byEnkhmaa et al [21] in which mulberry leaves suppressedthe development of atherosclerotic lesions Previously weproved that black beans significantly prolong LDL lag timeand decrease the atheroma regions of the aortic arch andthoracic aorta in hypercholesterolemic NZW rabbits [61]Wethen demonstrated that black soybean extract and PSPLEsignificantly decreased the adhesion of U937 monocyticcells to TNF-120572-stimulated HAECs and reduced adhesionmolecules expression (VCAM-1 and ICAM-1) as well as NF-120581B-p65 expression [37] This is particularly important giventhe opposing effects of flavonols fromMLREs obtained usingin vitro and ex vivo studies [62]

5 Conclusion

Tested MLREs were rich in flavonols and had strongantioxidant activity in protecting lymphocyte DNA fromoxidative damage In particular BT was rich in quercetinand polyphenols The antioxidant components of MLREs

might downregulate intracellular redox-dependent signalingpathways in HAECs upon TNF-120572 stimulation which mightprevent ROS-mediated endothelial cell dysfunction MLRCshave anti-inflammatory effects through the modulation ofNF-120581B AP-1 STAT3 and PPARs signaling These chemi-cals appear to provide distinct cytoprotective mechanismsthat might contribute to their putative beneficial effectsin suppressing endothelial responses to cytokines duringinflammation These results may benefit further in vivostudies to assess the therapeutic potential of phytochemicalsas anti-inflammatories for use in cytokine-induced vasculardisorders including atherosclerosis

Acknowledgment

This work was supported in part by Grants from the NationalScience Council of Taiwan (NSC 95ndash2320-B-034-001 andNSC 96ndash2320-B-034-001)

References

[1] W Palinski ldquoUnited they go conjunct regulation of aorticantioxidant enzymes during atherogenesisrdquo CirculationResearch vol 93 no 3 pp 183ndash185 2003

[2] P Libby ldquoMolecular bases of the acute coronary syndromesrdquoCirculation vol 91 no 11 pp 2844ndash2850 1995

[3] P A Baeuerle and D Baltimore ldquoNf-120581B ten years afterrdquo Cellvol 87 no 1 pp 13ndash20 1996

[4] J M Kyriakis ldquoActivation of the AP-1 transcription factor byinflammatory cytokines of the TNF familyrdquo Gene Expressionvol 7 no 4ndash6 pp 217ndash231 1999

[5] J M Muller R A Rupec and P A Baeuerle ldquoStudy of generegulation by NF-120581B and AP-1 in response to reactive oxygenintermediatesrdquoMethods vol 11 no 3 pp 301ndash312 1997

[6] G Chinetti J Fruchart and B Staels ldquoPeroxisome proliferator-activated receptors new targets for the pharmacological mod-ulation of macrophage gene expression and functionrdquo CurrentOpinion in Lipidology vol 14 no 5 pp 459ndash468 2003

[7] G Chinetti J C Fruchart and B Staels ldquoTranscriptional regu-lation of macrophage cholesterol trafficking by PPAR120572 andLXRrdquo Biochemical Society Transactions vol 34 no 6 pp 1128ndash1131 2006

[8] E Rigamonti G Chinetti-Gbaguidi and B Staels ldquoRegulationof macrophage functions by PPAR- 120572 PPAR- 120574 and LXRsin mice and menrdquo Arteriosclerosis Thrombosis and VascularBiology vol 28 no 6 pp 1050ndash1059 2008

[9] M A Bouhlel J Brozek B Derudas et al ldquoUnlike PPAR120574PPAR120572 or PPAR120573120575 activation does not promote humanmono-cyte differentiation toward alternative macrophagesrdquo Biochem-ical and Biophysical Research Communications vol 386 no 3pp 459ndash462 2009

[10] G Chinetti J-C Fruchart and B Staels ldquoPeroxisome prolife-rator-activated receptors and inflammation from basic scienceto clinical applicationsrdquo International Journal of Obesity vol 27supplement 3 pp S41ndashS45 2003

[11] Y Zhu J H-C Lin H Liao et al ldquoLDL induces transcriptionfactor activator protein-1 in human endothelial cellsrdquo Arte-riosclerosis Thrombosis and Vascular Biology vol 18 no 3 pp473ndash480 1998

Evidence-Based Complementary and Alternative Medicine 13

[12] J Terao ldquoDietary flavonoids as antioxidantsrdquo Forum of Nutri-tion vol 61 pp 87ndash94 2009

[13] Y B Feng W Q Luo and S Q Zhu ldquoExplore new clinicalapplication of Huanglian and corresponding compound pre-scriptions from their traditional userdquo Zhongguo ZhongyaoZazhi vol 33 no 10 pp 1221ndash1225 2008

[14] Y H Chu C L Chang and H F Hsu ldquoFlavonoid content ofseveral vegetables and their antioxidant activityrdquo Journal of theScience of Food andAgriculture vol 80 no 5 pp 561ndash566 2000

[15] I C Arts B van de Putte and P C Hollman ldquoCatechin con-tents of foods commonly consumed in The Netherlands 1Fruits vegetables staple foods and processed foodsrdquo Journal ofAgricultural and Food Chemistry vol 48 no 5 pp 1746ndash17512000

[16] A R Proteggente A S Pannala G Paganga et al ldquoThe anti-oxidant activity of regularly consumed fruit and vegetablesreflects their phenolic and vitamin C compositionrdquo Free RadicalResearch vol 36 no 2 pp 217ndash233 2002

[17] Y L Liu L H Tang Z Q Liang B You and S Yang ldquoGrowthinhibitory and apoptosis inducing by effects of total flavonoidsfrom Lysimachia clethroides Duby in human chronic myeloidleukemiaK562 cellsrdquo Journal of Ethnopharmacology vol 131 no1 pp 1ndash9 2010

[18] W Naowaratwattana W De-Eknamkul and E G De MejialdquoPhenolic-containing organic extracts of mulberry (Morus albaL) leaves inhibit HepG2 hepatoma cells through G2M phasearrest and inhibition of topoisomerase II120572 activityrdquo Journal ofMedicinal Food vol 13 no 5 pp 1045ndash1056 2010

[19] K Doi T Kojima and Y Fujimoto ldquoMulberry leaf extract inhi-bits the oxidative modification of rabbit and human low densitylipoproteinrdquo Biological and Pharmaceutical Bulletin vol 23 no9 pp 1066ndash1071 2000

[20] W Wang Y Zu Y Fu and T Efferth ldquoIn Vitro antioxidant andantimicrobial activity of extracts from Morus alba L leavesstems and fruitsrdquoAmerican Journal of ChineseMedicine vol 40no 2 pp 349ndash356 2012

[21] B Enkhmaa K Shiwaku T Katsube et al ldquoMulberry (Morusalba L) leaves and their major flavonol quercetin 3-(6-malon-ylglucoside) attenuate atherosclerotic lesion development inLDL receptor-deficient micerdquo Journal of Nutrition vol 135 no4 pp 729ndash734 2005

[22] J Naowaboot P Pannangpetch V Kukongviriyapan B Kongy-ingyoes and U Kukongviriyapan ldquoAntihyperglycemic antiox-idant and antiglycation activities of mulberry leaf extract instreptozotocin-induced chronic diabetic ratsrdquo Plant Foods forHuman Nutrition vol 64 no 2 pp 116ndash121 2009

[23] MM Padilha F CVilela CQ Rocha et al ldquoAntiinflammatoryproperties of Morus nigra leavesrdquo Phytotherapy Research vol24 no 10 pp 1496ndash1500 2010

[24] P Aramwit O Supasyndh T Siritienthong and N BangldquoMulberry leaf reduces oxidation and c-reactive protein levelin patients with mild dyslipidemiardquo BioMed Research Interna-tional vol 2013 Article ID 787981 7 pages 2013

[25] U Justesen P Knuthsen and T Leth ldquoQuantitative analysis offlavonols flavones and flavanones in fruits vegetables andbeverages by high-performance liquid chromatography withphoto-diode array and mass spectrometric detectionrdquo Journalof Chromatography A vol 799 no 1-2 pp 101ndash110 1998

[26] M S Taga E EMiller andD E Pratt ldquoChia seeds as a source ofnatural lipid antioxidantsrdquo Journal of the AmericanOil ChemistsrsquoSociety vol 61 no 5 pp 928ndash931 1984

[27] A A L Ordonez J D Gomez M A Vattuone and M I IslaldquoAntioxidant activities of Sechium edule (Jacq) Swartz extractsrdquoFood Chemistry vol 97 no 3 pp 452ndash458 2006

[28] A Kumaran and R Joel Karunakaran ldquoIn Vitro antioxidantactivities of methanol extracts of five Phyllanthus species fromIndiardquo LWT-Food Science and Technology vol 40 no 2 pp344ndash352 2007

[29] G Cao H M Alessio and R G Cutler ldquoOxygen-radical absor-bance capacity assay for antioxidantsrdquo Free Radical Biology andMedicine vol 14 no 3 pp 303ndash311 1993

[30] K Shimada K Fujikawa K Yahara and T Nakamura ldquoAntiox-idative properties of xanthan on the autoxidation of soybeanoil in cyclodextrin emulsionrdquo Journal of Agricultural and FoodChemistry vol 40 no 6 pp 945ndash948 1992

[31] J Cole M H L Green S E James L Henderson and H ColeldquoA further assessment of factors influencing measurements ofthioguanine-resistant mutant frequency in circulating T-lymphocytesrdquo Mutation Research vol 204 no 3 pp 493ndash5071988

[32] C-Y Hsu C-M Yang C-M Chen P Chao and S Hu ldquoEff-ects of chlorophyll-related compounds on hydrogen peroxideinduced DNA damage within human lymphocytesrdquo Journal ofAgricultural and Food Chemistry vol 53 no 7 pp 2746ndash27502005

[33] S-C Tang H-F Lo K-H Lin et al ldquoThe antioxidant capacityof extracts from Taiwan indigenous purple-leaved vegetablesrdquoJournal of the Taiwan Society for Horticultural Science vol 59no 1 pp 43ndash57 2013

[34] K-H Lin Y-Y Yang H-F Lo et al ldquoAntioxidant activity ofherbaceous plant extracts protect against hydrogen peroxide-induced DNA damage in human lymphocytesrdquo BioMed CentralResearch Notes In press

[35] A H Cory T C Owen J A Barltrop and J G Cory ldquoUse of anaqueous soluble tetrazoliumformazan assay for cell growthassays in culturerdquoCancer Communications vol 3 no 7 pp 207ndash212 1991

[36] Y T Szeto A R Collins and I F F Benzie ldquoEffects of dietaryantioxidants on DNA damage in lysed cells using a modifiedcomet assay procedurerdquoMutation Research vol 500 no 1-2 pp31ndash38 2002

[37] S Vielma G Virella A J Gorod and M F Lopes-VirellaldquoChlamydophila pneumoniae infection of human aortic endo-thelial cells induces the expression of FC 120574 receptor II (Fc120574RII)rdquoClinical Immunology vol 104 no 3 pp 265ndash273 2002

[38] C Y Zhu and S Loft ldquoEffects of Brussels sprouts extracts onhydrogen peroxide-induced DNA strand breaks in humanlymphocytesrdquo Food and Chemical Toxicology vol 39 no 12 pp1191ndash1197 2001

[39] Y D Min C-H Choi H Bark et al ldquoQuercetin inhibits exp-ression of inflammatory cytokines through attenuation of NF-120581B and p38MAPK in HMC-1 humanmast cell linerdquo Inflamma-tion Research vol 56 no 5 pp 210ndash215 2007

[40] P Y Chao Y P Huang and W B Hsieh ldquoInhibitive effect ofpurple sweet potato leaf extract and its components on celladhesion and inflammatory response in human aortic endothe-lial cellsrdquo Cell Adhesion amp Migration vol 7 no 2 pp 1ndash9 2013

[41] Y Y YangThe antioxidative capacity in herb plant extracts andtheir protection role in DNA oxidative damage of lymphocyte[MS thesis] Chinese Culture University Taipei Taiwan 2004

[42] C A Rice-Evans N J Miller and G Paganga ldquoStructure-anti-oxidant activity relationships of flavonoids and phenolic acidsrdquo

14 Evidence-Based Complementary and Alternative Medicine

Free Radical Biology and Medicine vol 20 no 7 pp 933ndash9561996

[43] M Strube G R Haenen H van den Berg and A Bast ldquoPitfallsin a method for assessment of total antioxidant capacityrdquo FreeRadical Research vol 26 no 6 pp 515ndash521 1997

[44] P G Pietta ldquoFlavonoids as antioxidantsrdquo Journal of NaturalProducts vol 63 no 7 pp 1035ndash1042 2000

[45] A M Gonzalez-Paramas S Esteban-Ruano C Santos-BuelgaSDe Pascual-Teresa and J C Rivas-Gonzalo ldquoFlavanol contentand antioxidant activity in winery byproductsrdquo Journal ofAgricultural and Food Chemistry vol 52 no 2 pp 234ndash2382004

[46] S Y Kim J J Gao and H K Kang ldquoTwo flavonoids from theleaves ofMorus alba induce differentiation of the human prom-yelocytic leukemia (HL-60) cell linerdquo Biological and Pharma-ceutical Bulletin vol 23 no 4 pp 451ndash455 2000

[47] M A Khan A A Rahman S Islam et al ldquoA comparative studyon the antioxidant activity ofmethanolic extracts from differentparts of Morus alba L (Moraceae)rdquo BMC Research vol 6 pp24ndash32 2013

[48] A-L ChungH-F Lo K-H Lin et al ldquoStudy on the antioxidantactivity in herb plant extractsrdquo Journal of the Taiwan Society ForHorticultural Science vol 59 no 2 pp 139ndash152 2013

[49] R L Prior X Wu and K Schaich ldquoStandardized methods forthe determination of antioxidant capacity and phenolics infoods and dietary supplementsrdquo Journal of Agricultural andFood Chemistry vol 53 no 10 pp 4290ndash4302 2005

[50] S J Duthie A R Collins GGDuthie andV LDobson ldquoQue-rcetin and myricetin protect against hydrogen peroxide-indu-ced DNA damage (strand breaks and oxidised pyrimidines) inhuman lymphocytesrdquo Mutation Research vol 393 no 3 pp223ndash231 1997

[51] G A Liu and R L Zheng ldquoProtection against damagedDNA inthe single cell by polyphenolsrdquo Pharmazie vol 57 no 12 pp852ndash854 2002

[52] M Noroozi W J Angerson and M E J Lean ldquoEffects of flav-onoids and vitamin C on oxidative DNA damage to humanlymphocytesrdquoAmerican Journal of Clinical Nutrition vol 67 no6 pp 1210ndash1218 1998

[53] T Koga and M Meydani ldquoEffect of plasma metabolites of (+)-catechin and quercetin on monocyte adhesion to human aorticendothelial cellsrdquoAmerican Journal of Clinical Nutrition vol 73no 5 pp 941ndash948 2001

[54] CMeng P Somers L Hoong et al ldquoDiscovery of novel pheno-lic antioxidants as inhibitors of vascular cell adhesionmolecule-1 expression for use in chronic inflammatory diseasesrdquo Journalof Medicinal Chemistry vol 47 no 25 pp 6420ndash6432 2004

[55] Y Shibata N Kume H Arai et al ldquoMulberry leaf aqueousfractions inhibit TNF-120572-induced nuclear factor 120581B (NF-120581B)activation and lectin-like oxidized LDL receptor-1 (LOX-1)expression in vascular endothelial cellsrdquoAtherosclerosis vol 193no 1 pp 20ndash27 2007

[56] K C Chan H H Ho C N Huang M Lin H Chen and CWang ldquoMulberry leaf extract inhibits vascular smooth musclecell migration involving a block of small GTPase and AktNF-kB signalsrdquo Journal of Agricultural and Food Chemistry vol 57no 19 pp 9147ndash9153 2009

[57] F Chen N Nakashima I Kimura M Kimura N Asanoand S Koya ldquoPotentiating effects on pilocarpine-induced salivasecretion by extracts and N-containing sugars derived frommulberry leaves in streptozocin- diabetic micerdquo Biological andPharmaceutical Bulletin vol 18 no 12 pp 1676ndash1680 1995

[58] K Konno H Ono M Nakamura et al ldquoMulberry latexrich in antidiabetic sugar-mimic alkaloids forces dieting oncaterpillarsrdquo Proceedings of the National Academy of Sciences ofthe United States of America vol 103 no 5 pp 1337ndash1341 2006

[59] H J Kim K H Yoon M J Kang et al ldquoA six-month sup-plementation of mulberry Korean red ginseng and banabadecreases biomarkers of systemic low-grade inflammation insubjects with impaired glucose tolerance and type 2 diabetesrdquoEvidence-based Complementary and Alternative Medicine vol2012 Article ID 735191 8 pages 2012

[60] G Nicholson and G M Hall ldquoDiabetes mellitus new drugs fora new epidemicrdquo British Journal of Anaesthesia vol 107 no 1pp 65ndash73 2011

[61] P Y Chao Y L Chen Y C Lin et al ldquoEffects of black soybeanon atherogenic prevention in hypercholesterolemic rabbits andon adhesion molecular expression in cultured HAECsrdquo Foodand Nutrition Sciences vol 4 pp 9ndash21 2013

[62] C Chen S Li C-YOChen et al ldquoConstituents in purple sweetpotato leaves inhibit in vitro angiogenesis with opposite effectsex vivordquo Nutrition vol 27 no 11-12 pp 1177ndash1182 2011

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

12 Evidence-Based Complementary and Alternative Medicine

is also reported to be involved in the regulation of NF-120581B-dependent transcription [56] MLE has been shown to pos-sess hypoglycemic effects in an insulin-dependent diabetesmellitus (type 1 diabetes) animal model and potential hypo-glycemic compounds inmulberry leaves were suggested to be2-O-120572-D-galactopyranosyl-DNJ and fagomine [57] Konnoet al [58] further reported that mulberry latex containsalkaloidal sugar-mimic glycosidase inhibitors with antidia-betic activities such as 14-dideoxy-14-imino-D-arabinitol 1-deoxynojirimycin and 14-dideoxy-14-imino-D-ribitol Kimet al [59] recently showed that treated type 2 diabetesmellitus(T2D) with a 1 1 1 mixture of ginseng root mulberry leafwater extract and banana leaf water extract (6 gd) for 24weeks significantly decreased plasma intracellular adhesionmolecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) levels This indicates suppressed inflammatoryresponses in T2D but no significant differences in glucosehomeostasis control measurement changes Rigamonti et al[8] reported that bothPPAR120572 and 120574maybe thought of as sup-pressing the activity of NF120581B and AP-1 pathways that reduceinflammatory mediator production in the macrophages ofhumans and mice In our study MT and M promoted PPAR120572 and 120574 expression at least in part by increasing DNAbinding of transcription factors PPAR 120572 and 120574 PPAR-120572-and 120574-dependent signaling pathwaysmay also play importantroles in mediating the TNF-120572 receptor signaling pathwayby MT and M by suppressing the activity on NF-120581B andAP-1 pathways These pathways may regulate downstreamproinflammatory gene expression and adhesion molecularexpression Clinically PPAR 120574 agonists such as rosiglitazoneare widely used in the treatment of T2D [60] M and MTextracts increased PPAR 120574 activity which may partly explainwhy inflammatory responses are suppressed in T2D

Although reduced NF-120581B expression and DNA bind-ing were observed upon treatment with ML MT and BT(Figure 5) the mechanism was not assessed and is worthy offurther study In addition the present results were obtainedusing in vitro studies and may be consistent with an invivo model of atherogenesis similar to that reported byEnkhmaa et al [21] in which mulberry leaves suppressedthe development of atherosclerotic lesions Previously weproved that black beans significantly prolong LDL lag timeand decrease the atheroma regions of the aortic arch andthoracic aorta in hypercholesterolemic NZW rabbits [61]Wethen demonstrated that black soybean extract and PSPLEsignificantly decreased the adhesion of U937 monocyticcells to TNF-120572-stimulated HAECs and reduced adhesionmolecules expression (VCAM-1 and ICAM-1) as well as NF-120581B-p65 expression [37] This is particularly important giventhe opposing effects of flavonols fromMLREs obtained usingin vitro and ex vivo studies [62]

5 Conclusion

Tested MLREs were rich in flavonols and had strongantioxidant activity in protecting lymphocyte DNA fromoxidative damage In particular BT was rich in quercetinand polyphenols The antioxidant components of MLREs

might downregulate intracellular redox-dependent signalingpathways in HAECs upon TNF-120572 stimulation which mightprevent ROS-mediated endothelial cell dysfunction MLRCshave anti-inflammatory effects through the modulation ofNF-120581B AP-1 STAT3 and PPARs signaling These chemi-cals appear to provide distinct cytoprotective mechanismsthat might contribute to their putative beneficial effectsin suppressing endothelial responses to cytokines duringinflammation These results may benefit further in vivostudies to assess the therapeutic potential of phytochemicalsas anti-inflammatories for use in cytokine-induced vasculardisorders including atherosclerosis

Acknowledgment

This work was supported in part by Grants from the NationalScience Council of Taiwan (NSC 95ndash2320-B-034-001 andNSC 96ndash2320-B-034-001)

References

[1] W Palinski ldquoUnited they go conjunct regulation of aorticantioxidant enzymes during atherogenesisrdquo CirculationResearch vol 93 no 3 pp 183ndash185 2003

[2] P Libby ldquoMolecular bases of the acute coronary syndromesrdquoCirculation vol 91 no 11 pp 2844ndash2850 1995

[3] P A Baeuerle and D Baltimore ldquoNf-120581B ten years afterrdquo Cellvol 87 no 1 pp 13ndash20 1996

[4] J M Kyriakis ldquoActivation of the AP-1 transcription factor byinflammatory cytokines of the TNF familyrdquo Gene Expressionvol 7 no 4ndash6 pp 217ndash231 1999

[5] J M Muller R A Rupec and P A Baeuerle ldquoStudy of generegulation by NF-120581B and AP-1 in response to reactive oxygenintermediatesrdquoMethods vol 11 no 3 pp 301ndash312 1997

[6] G Chinetti J Fruchart and B Staels ldquoPeroxisome proliferator-activated receptors new targets for the pharmacological mod-ulation of macrophage gene expression and functionrdquo CurrentOpinion in Lipidology vol 14 no 5 pp 459ndash468 2003

[7] G Chinetti J C Fruchart and B Staels ldquoTranscriptional regu-lation of macrophage cholesterol trafficking by PPAR120572 andLXRrdquo Biochemical Society Transactions vol 34 no 6 pp 1128ndash1131 2006

[8] E Rigamonti G Chinetti-Gbaguidi and B Staels ldquoRegulationof macrophage functions by PPAR- 120572 PPAR- 120574 and LXRsin mice and menrdquo Arteriosclerosis Thrombosis and VascularBiology vol 28 no 6 pp 1050ndash1059 2008

[9] M A Bouhlel J Brozek B Derudas et al ldquoUnlike PPAR120574PPAR120572 or PPAR120573120575 activation does not promote humanmono-cyte differentiation toward alternative macrophagesrdquo Biochem-ical and Biophysical Research Communications vol 386 no 3pp 459ndash462 2009

[10] G Chinetti J-C Fruchart and B Staels ldquoPeroxisome prolife-rator-activated receptors and inflammation from basic scienceto clinical applicationsrdquo International Journal of Obesity vol 27supplement 3 pp S41ndashS45 2003

[11] Y Zhu J H-C Lin H Liao et al ldquoLDL induces transcriptionfactor activator protein-1 in human endothelial cellsrdquo Arte-riosclerosis Thrombosis and Vascular Biology vol 18 no 3 pp473ndash480 1998

Evidence-Based Complementary and Alternative Medicine 13

[12] J Terao ldquoDietary flavonoids as antioxidantsrdquo Forum of Nutri-tion vol 61 pp 87ndash94 2009

[13] Y B Feng W Q Luo and S Q Zhu ldquoExplore new clinicalapplication of Huanglian and corresponding compound pre-scriptions from their traditional userdquo Zhongguo ZhongyaoZazhi vol 33 no 10 pp 1221ndash1225 2008

[14] Y H Chu C L Chang and H F Hsu ldquoFlavonoid content ofseveral vegetables and their antioxidant activityrdquo Journal of theScience of Food andAgriculture vol 80 no 5 pp 561ndash566 2000

[15] I C Arts B van de Putte and P C Hollman ldquoCatechin con-tents of foods commonly consumed in The Netherlands 1Fruits vegetables staple foods and processed foodsrdquo Journal ofAgricultural and Food Chemistry vol 48 no 5 pp 1746ndash17512000

[16] A R Proteggente A S Pannala G Paganga et al ldquoThe anti-oxidant activity of regularly consumed fruit and vegetablesreflects their phenolic and vitamin C compositionrdquo Free RadicalResearch vol 36 no 2 pp 217ndash233 2002

[17] Y L Liu L H Tang Z Q Liang B You and S Yang ldquoGrowthinhibitory and apoptosis inducing by effects of total flavonoidsfrom Lysimachia clethroides Duby in human chronic myeloidleukemiaK562 cellsrdquo Journal of Ethnopharmacology vol 131 no1 pp 1ndash9 2010

[18] W Naowaratwattana W De-Eknamkul and E G De MejialdquoPhenolic-containing organic extracts of mulberry (Morus albaL) leaves inhibit HepG2 hepatoma cells through G2M phasearrest and inhibition of topoisomerase II120572 activityrdquo Journal ofMedicinal Food vol 13 no 5 pp 1045ndash1056 2010

[19] K Doi T Kojima and Y Fujimoto ldquoMulberry leaf extract inhi-bits the oxidative modification of rabbit and human low densitylipoproteinrdquo Biological and Pharmaceutical Bulletin vol 23 no9 pp 1066ndash1071 2000

[20] W Wang Y Zu Y Fu and T Efferth ldquoIn Vitro antioxidant andantimicrobial activity of extracts from Morus alba L leavesstems and fruitsrdquoAmerican Journal of ChineseMedicine vol 40no 2 pp 349ndash356 2012

[21] B Enkhmaa K Shiwaku T Katsube et al ldquoMulberry (Morusalba L) leaves and their major flavonol quercetin 3-(6-malon-ylglucoside) attenuate atherosclerotic lesion development inLDL receptor-deficient micerdquo Journal of Nutrition vol 135 no4 pp 729ndash734 2005

[22] J Naowaboot P Pannangpetch V Kukongviriyapan B Kongy-ingyoes and U Kukongviriyapan ldquoAntihyperglycemic antiox-idant and antiglycation activities of mulberry leaf extract instreptozotocin-induced chronic diabetic ratsrdquo Plant Foods forHuman Nutrition vol 64 no 2 pp 116ndash121 2009

[23] MM Padilha F CVilela CQ Rocha et al ldquoAntiinflammatoryproperties of Morus nigra leavesrdquo Phytotherapy Research vol24 no 10 pp 1496ndash1500 2010

[24] P Aramwit O Supasyndh T Siritienthong and N BangldquoMulberry leaf reduces oxidation and c-reactive protein levelin patients with mild dyslipidemiardquo BioMed Research Interna-tional vol 2013 Article ID 787981 7 pages 2013

[25] U Justesen P Knuthsen and T Leth ldquoQuantitative analysis offlavonols flavones and flavanones in fruits vegetables andbeverages by high-performance liquid chromatography withphoto-diode array and mass spectrometric detectionrdquo Journalof Chromatography A vol 799 no 1-2 pp 101ndash110 1998

[26] M S Taga E EMiller andD E Pratt ldquoChia seeds as a source ofnatural lipid antioxidantsrdquo Journal of the AmericanOil ChemistsrsquoSociety vol 61 no 5 pp 928ndash931 1984

[27] A A L Ordonez J D Gomez M A Vattuone and M I IslaldquoAntioxidant activities of Sechium edule (Jacq) Swartz extractsrdquoFood Chemistry vol 97 no 3 pp 452ndash458 2006

[28] A Kumaran and R Joel Karunakaran ldquoIn Vitro antioxidantactivities of methanol extracts of five Phyllanthus species fromIndiardquo LWT-Food Science and Technology vol 40 no 2 pp344ndash352 2007

[29] G Cao H M Alessio and R G Cutler ldquoOxygen-radical absor-bance capacity assay for antioxidantsrdquo Free Radical Biology andMedicine vol 14 no 3 pp 303ndash311 1993

[30] K Shimada K Fujikawa K Yahara and T Nakamura ldquoAntiox-idative properties of xanthan on the autoxidation of soybeanoil in cyclodextrin emulsionrdquo Journal of Agricultural and FoodChemistry vol 40 no 6 pp 945ndash948 1992

[31] J Cole M H L Green S E James L Henderson and H ColeldquoA further assessment of factors influencing measurements ofthioguanine-resistant mutant frequency in circulating T-lymphocytesrdquo Mutation Research vol 204 no 3 pp 493ndash5071988

[32] C-Y Hsu C-M Yang C-M Chen P Chao and S Hu ldquoEff-ects of chlorophyll-related compounds on hydrogen peroxideinduced DNA damage within human lymphocytesrdquo Journal ofAgricultural and Food Chemistry vol 53 no 7 pp 2746ndash27502005

[33] S-C Tang H-F Lo K-H Lin et al ldquoThe antioxidant capacityof extracts from Taiwan indigenous purple-leaved vegetablesrdquoJournal of the Taiwan Society for Horticultural Science vol 59no 1 pp 43ndash57 2013

[34] K-H Lin Y-Y Yang H-F Lo et al ldquoAntioxidant activity ofherbaceous plant extracts protect against hydrogen peroxide-induced DNA damage in human lymphocytesrdquo BioMed CentralResearch Notes In press

[35] A H Cory T C Owen J A Barltrop and J G Cory ldquoUse of anaqueous soluble tetrazoliumformazan assay for cell growthassays in culturerdquoCancer Communications vol 3 no 7 pp 207ndash212 1991

[36] Y T Szeto A R Collins and I F F Benzie ldquoEffects of dietaryantioxidants on DNA damage in lysed cells using a modifiedcomet assay procedurerdquoMutation Research vol 500 no 1-2 pp31ndash38 2002

[37] S Vielma G Virella A J Gorod and M F Lopes-VirellaldquoChlamydophila pneumoniae infection of human aortic endo-thelial cells induces the expression of FC 120574 receptor II (Fc120574RII)rdquoClinical Immunology vol 104 no 3 pp 265ndash273 2002

[38] C Y Zhu and S Loft ldquoEffects of Brussels sprouts extracts onhydrogen peroxide-induced DNA strand breaks in humanlymphocytesrdquo Food and Chemical Toxicology vol 39 no 12 pp1191ndash1197 2001

[39] Y D Min C-H Choi H Bark et al ldquoQuercetin inhibits exp-ression of inflammatory cytokines through attenuation of NF-120581B and p38MAPK in HMC-1 humanmast cell linerdquo Inflamma-tion Research vol 56 no 5 pp 210ndash215 2007

[40] P Y Chao Y P Huang and W B Hsieh ldquoInhibitive effect ofpurple sweet potato leaf extract and its components on celladhesion and inflammatory response in human aortic endothe-lial cellsrdquo Cell Adhesion amp Migration vol 7 no 2 pp 1ndash9 2013

[41] Y Y YangThe antioxidative capacity in herb plant extracts andtheir protection role in DNA oxidative damage of lymphocyte[MS thesis] Chinese Culture University Taipei Taiwan 2004

[42] C A Rice-Evans N J Miller and G Paganga ldquoStructure-anti-oxidant activity relationships of flavonoids and phenolic acidsrdquo

14 Evidence-Based Complementary and Alternative Medicine

Free Radical Biology and Medicine vol 20 no 7 pp 933ndash9561996

[43] M Strube G R Haenen H van den Berg and A Bast ldquoPitfallsin a method for assessment of total antioxidant capacityrdquo FreeRadical Research vol 26 no 6 pp 515ndash521 1997

[44] P G Pietta ldquoFlavonoids as antioxidantsrdquo Journal of NaturalProducts vol 63 no 7 pp 1035ndash1042 2000

[45] A M Gonzalez-Paramas S Esteban-Ruano C Santos-BuelgaSDe Pascual-Teresa and J C Rivas-Gonzalo ldquoFlavanol contentand antioxidant activity in winery byproductsrdquo Journal ofAgricultural and Food Chemistry vol 52 no 2 pp 234ndash2382004

[46] S Y Kim J J Gao and H K Kang ldquoTwo flavonoids from theleaves ofMorus alba induce differentiation of the human prom-yelocytic leukemia (HL-60) cell linerdquo Biological and Pharma-ceutical Bulletin vol 23 no 4 pp 451ndash455 2000

[47] M A Khan A A Rahman S Islam et al ldquoA comparative studyon the antioxidant activity ofmethanolic extracts from differentparts of Morus alba L (Moraceae)rdquo BMC Research vol 6 pp24ndash32 2013

[48] A-L ChungH-F Lo K-H Lin et al ldquoStudy on the antioxidantactivity in herb plant extractsrdquo Journal of the Taiwan Society ForHorticultural Science vol 59 no 2 pp 139ndash152 2013

[49] R L Prior X Wu and K Schaich ldquoStandardized methods forthe determination of antioxidant capacity and phenolics infoods and dietary supplementsrdquo Journal of Agricultural andFood Chemistry vol 53 no 10 pp 4290ndash4302 2005

[50] S J Duthie A R Collins GGDuthie andV LDobson ldquoQue-rcetin and myricetin protect against hydrogen peroxide-indu-ced DNA damage (strand breaks and oxidised pyrimidines) inhuman lymphocytesrdquo Mutation Research vol 393 no 3 pp223ndash231 1997

[51] G A Liu and R L Zheng ldquoProtection against damagedDNA inthe single cell by polyphenolsrdquo Pharmazie vol 57 no 12 pp852ndash854 2002

[52] M Noroozi W J Angerson and M E J Lean ldquoEffects of flav-onoids and vitamin C on oxidative DNA damage to humanlymphocytesrdquoAmerican Journal of Clinical Nutrition vol 67 no6 pp 1210ndash1218 1998

[53] T Koga and M Meydani ldquoEffect of plasma metabolites of (+)-catechin and quercetin on monocyte adhesion to human aorticendothelial cellsrdquoAmerican Journal of Clinical Nutrition vol 73no 5 pp 941ndash948 2001

[54] CMeng P Somers L Hoong et al ldquoDiscovery of novel pheno-lic antioxidants as inhibitors of vascular cell adhesionmolecule-1 expression for use in chronic inflammatory diseasesrdquo Journalof Medicinal Chemistry vol 47 no 25 pp 6420ndash6432 2004

[55] Y Shibata N Kume H Arai et al ldquoMulberry leaf aqueousfractions inhibit TNF-120572-induced nuclear factor 120581B (NF-120581B)activation and lectin-like oxidized LDL receptor-1 (LOX-1)expression in vascular endothelial cellsrdquoAtherosclerosis vol 193no 1 pp 20ndash27 2007

[56] K C Chan H H Ho C N Huang M Lin H Chen and CWang ldquoMulberry leaf extract inhibits vascular smooth musclecell migration involving a block of small GTPase and AktNF-kB signalsrdquo Journal of Agricultural and Food Chemistry vol 57no 19 pp 9147ndash9153 2009

[57] F Chen N Nakashima I Kimura M Kimura N Asanoand S Koya ldquoPotentiating effects on pilocarpine-induced salivasecretion by extracts and N-containing sugars derived frommulberry leaves in streptozocin- diabetic micerdquo Biological andPharmaceutical Bulletin vol 18 no 12 pp 1676ndash1680 1995

[58] K Konno H Ono M Nakamura et al ldquoMulberry latexrich in antidiabetic sugar-mimic alkaloids forces dieting oncaterpillarsrdquo Proceedings of the National Academy of Sciences ofthe United States of America vol 103 no 5 pp 1337ndash1341 2006

[59] H J Kim K H Yoon M J Kang et al ldquoA six-month sup-plementation of mulberry Korean red ginseng and banabadecreases biomarkers of systemic low-grade inflammation insubjects with impaired glucose tolerance and type 2 diabetesrdquoEvidence-based Complementary and Alternative Medicine vol2012 Article ID 735191 8 pages 2012

[60] G Nicholson and G M Hall ldquoDiabetes mellitus new drugs fora new epidemicrdquo British Journal of Anaesthesia vol 107 no 1pp 65ndash73 2011

[61] P Y Chao Y L Chen Y C Lin et al ldquoEffects of black soybeanon atherogenic prevention in hypercholesterolemic rabbits andon adhesion molecular expression in cultured HAECsrdquo Foodand Nutrition Sciences vol 4 pp 9ndash21 2013

[62] C Chen S Li C-YOChen et al ldquoConstituents in purple sweetpotato leaves inhibit in vitro angiogenesis with opposite effectsex vivordquo Nutrition vol 27 no 11-12 pp 1177ndash1182 2011

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

Evidence-Based Complementary and Alternative Medicine 13

[12] J Terao ldquoDietary flavonoids as antioxidantsrdquo Forum of Nutri-tion vol 61 pp 87ndash94 2009

[13] Y B Feng W Q Luo and S Q Zhu ldquoExplore new clinicalapplication of Huanglian and corresponding compound pre-scriptions from their traditional userdquo Zhongguo ZhongyaoZazhi vol 33 no 10 pp 1221ndash1225 2008

[14] Y H Chu C L Chang and H F Hsu ldquoFlavonoid content ofseveral vegetables and their antioxidant activityrdquo Journal of theScience of Food andAgriculture vol 80 no 5 pp 561ndash566 2000

[15] I C Arts B van de Putte and P C Hollman ldquoCatechin con-tents of foods commonly consumed in The Netherlands 1Fruits vegetables staple foods and processed foodsrdquo Journal ofAgricultural and Food Chemistry vol 48 no 5 pp 1746ndash17512000

[16] A R Proteggente A S Pannala G Paganga et al ldquoThe anti-oxidant activity of regularly consumed fruit and vegetablesreflects their phenolic and vitamin C compositionrdquo Free RadicalResearch vol 36 no 2 pp 217ndash233 2002

[17] Y L Liu L H Tang Z Q Liang B You and S Yang ldquoGrowthinhibitory and apoptosis inducing by effects of total flavonoidsfrom Lysimachia clethroides Duby in human chronic myeloidleukemiaK562 cellsrdquo Journal of Ethnopharmacology vol 131 no1 pp 1ndash9 2010

[18] W Naowaratwattana W De-Eknamkul and E G De MejialdquoPhenolic-containing organic extracts of mulberry (Morus albaL) leaves inhibit HepG2 hepatoma cells through G2M phasearrest and inhibition of topoisomerase II120572 activityrdquo Journal ofMedicinal Food vol 13 no 5 pp 1045ndash1056 2010

[19] K Doi T Kojima and Y Fujimoto ldquoMulberry leaf extract inhi-bits the oxidative modification of rabbit and human low densitylipoproteinrdquo Biological and Pharmaceutical Bulletin vol 23 no9 pp 1066ndash1071 2000

[20] W Wang Y Zu Y Fu and T Efferth ldquoIn Vitro antioxidant andantimicrobial activity of extracts from Morus alba L leavesstems and fruitsrdquoAmerican Journal of ChineseMedicine vol 40no 2 pp 349ndash356 2012

[21] B Enkhmaa K Shiwaku T Katsube et al ldquoMulberry (Morusalba L) leaves and their major flavonol quercetin 3-(6-malon-ylglucoside) attenuate atherosclerotic lesion development inLDL receptor-deficient micerdquo Journal of Nutrition vol 135 no4 pp 729ndash734 2005

[22] J Naowaboot P Pannangpetch V Kukongviriyapan B Kongy-ingyoes and U Kukongviriyapan ldquoAntihyperglycemic antiox-idant and antiglycation activities of mulberry leaf extract instreptozotocin-induced chronic diabetic ratsrdquo Plant Foods forHuman Nutrition vol 64 no 2 pp 116ndash121 2009

[23] MM Padilha F CVilela CQ Rocha et al ldquoAntiinflammatoryproperties of Morus nigra leavesrdquo Phytotherapy Research vol24 no 10 pp 1496ndash1500 2010

[24] P Aramwit O Supasyndh T Siritienthong and N BangldquoMulberry leaf reduces oxidation and c-reactive protein levelin patients with mild dyslipidemiardquo BioMed Research Interna-tional vol 2013 Article ID 787981 7 pages 2013

[25] U Justesen P Knuthsen and T Leth ldquoQuantitative analysis offlavonols flavones and flavanones in fruits vegetables andbeverages by high-performance liquid chromatography withphoto-diode array and mass spectrometric detectionrdquo Journalof Chromatography A vol 799 no 1-2 pp 101ndash110 1998

[26] M S Taga E EMiller andD E Pratt ldquoChia seeds as a source ofnatural lipid antioxidantsrdquo Journal of the AmericanOil ChemistsrsquoSociety vol 61 no 5 pp 928ndash931 1984

[27] A A L Ordonez J D Gomez M A Vattuone and M I IslaldquoAntioxidant activities of Sechium edule (Jacq) Swartz extractsrdquoFood Chemistry vol 97 no 3 pp 452ndash458 2006

[28] A Kumaran and R Joel Karunakaran ldquoIn Vitro antioxidantactivities of methanol extracts of five Phyllanthus species fromIndiardquo LWT-Food Science and Technology vol 40 no 2 pp344ndash352 2007

[29] G Cao H M Alessio and R G Cutler ldquoOxygen-radical absor-bance capacity assay for antioxidantsrdquo Free Radical Biology andMedicine vol 14 no 3 pp 303ndash311 1993

[30] K Shimada K Fujikawa K Yahara and T Nakamura ldquoAntiox-idative properties of xanthan on the autoxidation of soybeanoil in cyclodextrin emulsionrdquo Journal of Agricultural and FoodChemistry vol 40 no 6 pp 945ndash948 1992

[31] J Cole M H L Green S E James L Henderson and H ColeldquoA further assessment of factors influencing measurements ofthioguanine-resistant mutant frequency in circulating T-lymphocytesrdquo Mutation Research vol 204 no 3 pp 493ndash5071988

[32] C-Y Hsu C-M Yang C-M Chen P Chao and S Hu ldquoEff-ects of chlorophyll-related compounds on hydrogen peroxideinduced DNA damage within human lymphocytesrdquo Journal ofAgricultural and Food Chemistry vol 53 no 7 pp 2746ndash27502005

[33] S-C Tang H-F Lo K-H Lin et al ldquoThe antioxidant capacityof extracts from Taiwan indigenous purple-leaved vegetablesrdquoJournal of the Taiwan Society for Horticultural Science vol 59no 1 pp 43ndash57 2013

[34] K-H Lin Y-Y Yang H-F Lo et al ldquoAntioxidant activity ofherbaceous plant extracts protect against hydrogen peroxide-induced DNA damage in human lymphocytesrdquo BioMed CentralResearch Notes In press

[35] A H Cory T C Owen J A Barltrop and J G Cory ldquoUse of anaqueous soluble tetrazoliumformazan assay for cell growthassays in culturerdquoCancer Communications vol 3 no 7 pp 207ndash212 1991

[36] Y T Szeto A R Collins and I F F Benzie ldquoEffects of dietaryantioxidants on DNA damage in lysed cells using a modifiedcomet assay procedurerdquoMutation Research vol 500 no 1-2 pp31ndash38 2002

[37] S Vielma G Virella A J Gorod and M F Lopes-VirellaldquoChlamydophila pneumoniae infection of human aortic endo-thelial cells induces the expression of FC 120574 receptor II (Fc120574RII)rdquoClinical Immunology vol 104 no 3 pp 265ndash273 2002

[38] C Y Zhu and S Loft ldquoEffects of Brussels sprouts extracts onhydrogen peroxide-induced DNA strand breaks in humanlymphocytesrdquo Food and Chemical Toxicology vol 39 no 12 pp1191ndash1197 2001

[39] Y D Min C-H Choi H Bark et al ldquoQuercetin inhibits exp-ression of inflammatory cytokines through attenuation of NF-120581B and p38MAPK in HMC-1 humanmast cell linerdquo Inflamma-tion Research vol 56 no 5 pp 210ndash215 2007

[40] P Y Chao Y P Huang and W B Hsieh ldquoInhibitive effect ofpurple sweet potato leaf extract and its components on celladhesion and inflammatory response in human aortic endothe-lial cellsrdquo Cell Adhesion amp Migration vol 7 no 2 pp 1ndash9 2013

[41] Y Y YangThe antioxidative capacity in herb plant extracts andtheir protection role in DNA oxidative damage of lymphocyte[MS thesis] Chinese Culture University Taipei Taiwan 2004

[42] C A Rice-Evans N J Miller and G Paganga ldquoStructure-anti-oxidant activity relationships of flavonoids and phenolic acidsrdquo

14 Evidence-Based Complementary and Alternative Medicine

Free Radical Biology and Medicine vol 20 no 7 pp 933ndash9561996

[43] M Strube G R Haenen H van den Berg and A Bast ldquoPitfallsin a method for assessment of total antioxidant capacityrdquo FreeRadical Research vol 26 no 6 pp 515ndash521 1997

[44] P G Pietta ldquoFlavonoids as antioxidantsrdquo Journal of NaturalProducts vol 63 no 7 pp 1035ndash1042 2000

[45] A M Gonzalez-Paramas S Esteban-Ruano C Santos-BuelgaSDe Pascual-Teresa and J C Rivas-Gonzalo ldquoFlavanol contentand antioxidant activity in winery byproductsrdquo Journal ofAgricultural and Food Chemistry vol 52 no 2 pp 234ndash2382004

[46] S Y Kim J J Gao and H K Kang ldquoTwo flavonoids from theleaves ofMorus alba induce differentiation of the human prom-yelocytic leukemia (HL-60) cell linerdquo Biological and Pharma-ceutical Bulletin vol 23 no 4 pp 451ndash455 2000

[47] M A Khan A A Rahman S Islam et al ldquoA comparative studyon the antioxidant activity ofmethanolic extracts from differentparts of Morus alba L (Moraceae)rdquo BMC Research vol 6 pp24ndash32 2013

[48] A-L ChungH-F Lo K-H Lin et al ldquoStudy on the antioxidantactivity in herb plant extractsrdquo Journal of the Taiwan Society ForHorticultural Science vol 59 no 2 pp 139ndash152 2013

[49] R L Prior X Wu and K Schaich ldquoStandardized methods forthe determination of antioxidant capacity and phenolics infoods and dietary supplementsrdquo Journal of Agricultural andFood Chemistry vol 53 no 10 pp 4290ndash4302 2005

[50] S J Duthie A R Collins GGDuthie andV LDobson ldquoQue-rcetin and myricetin protect against hydrogen peroxide-indu-ced DNA damage (strand breaks and oxidised pyrimidines) inhuman lymphocytesrdquo Mutation Research vol 393 no 3 pp223ndash231 1997

[51] G A Liu and R L Zheng ldquoProtection against damagedDNA inthe single cell by polyphenolsrdquo Pharmazie vol 57 no 12 pp852ndash854 2002

[52] M Noroozi W J Angerson and M E J Lean ldquoEffects of flav-onoids and vitamin C on oxidative DNA damage to humanlymphocytesrdquoAmerican Journal of Clinical Nutrition vol 67 no6 pp 1210ndash1218 1998

[53] T Koga and M Meydani ldquoEffect of plasma metabolites of (+)-catechin and quercetin on monocyte adhesion to human aorticendothelial cellsrdquoAmerican Journal of Clinical Nutrition vol 73no 5 pp 941ndash948 2001

[54] CMeng P Somers L Hoong et al ldquoDiscovery of novel pheno-lic antioxidants as inhibitors of vascular cell adhesionmolecule-1 expression for use in chronic inflammatory diseasesrdquo Journalof Medicinal Chemistry vol 47 no 25 pp 6420ndash6432 2004

[55] Y Shibata N Kume H Arai et al ldquoMulberry leaf aqueousfractions inhibit TNF-120572-induced nuclear factor 120581B (NF-120581B)activation and lectin-like oxidized LDL receptor-1 (LOX-1)expression in vascular endothelial cellsrdquoAtherosclerosis vol 193no 1 pp 20ndash27 2007

[56] K C Chan H H Ho C N Huang M Lin H Chen and CWang ldquoMulberry leaf extract inhibits vascular smooth musclecell migration involving a block of small GTPase and AktNF-kB signalsrdquo Journal of Agricultural and Food Chemistry vol 57no 19 pp 9147ndash9153 2009

[57] F Chen N Nakashima I Kimura M Kimura N Asanoand S Koya ldquoPotentiating effects on pilocarpine-induced salivasecretion by extracts and N-containing sugars derived frommulberry leaves in streptozocin- diabetic micerdquo Biological andPharmaceutical Bulletin vol 18 no 12 pp 1676ndash1680 1995

[58] K Konno H Ono M Nakamura et al ldquoMulberry latexrich in antidiabetic sugar-mimic alkaloids forces dieting oncaterpillarsrdquo Proceedings of the National Academy of Sciences ofthe United States of America vol 103 no 5 pp 1337ndash1341 2006

[59] H J Kim K H Yoon M J Kang et al ldquoA six-month sup-plementation of mulberry Korean red ginseng and banabadecreases biomarkers of systemic low-grade inflammation insubjects with impaired glucose tolerance and type 2 diabetesrdquoEvidence-based Complementary and Alternative Medicine vol2012 Article ID 735191 8 pages 2012

[60] G Nicholson and G M Hall ldquoDiabetes mellitus new drugs fora new epidemicrdquo British Journal of Anaesthesia vol 107 no 1pp 65ndash73 2011

[61] P Y Chao Y L Chen Y C Lin et al ldquoEffects of black soybeanon atherogenic prevention in hypercholesterolemic rabbits andon adhesion molecular expression in cultured HAECsrdquo Foodand Nutrition Sciences vol 4 pp 9ndash21 2013

[62] C Chen S Li C-YOChen et al ldquoConstituents in purple sweetpotato leaves inhibit in vitro angiogenesis with opposite effectsex vivordquo Nutrition vol 27 no 11-12 pp 1177ndash1182 2011

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

14 Evidence-Based Complementary and Alternative Medicine

Free Radical Biology and Medicine vol 20 no 7 pp 933ndash9561996

[43] M Strube G R Haenen H van den Berg and A Bast ldquoPitfallsin a method for assessment of total antioxidant capacityrdquo FreeRadical Research vol 26 no 6 pp 515ndash521 1997

[44] P G Pietta ldquoFlavonoids as antioxidantsrdquo Journal of NaturalProducts vol 63 no 7 pp 1035ndash1042 2000

[45] A M Gonzalez-Paramas S Esteban-Ruano C Santos-BuelgaSDe Pascual-Teresa and J C Rivas-Gonzalo ldquoFlavanol contentand antioxidant activity in winery byproductsrdquo Journal ofAgricultural and Food Chemistry vol 52 no 2 pp 234ndash2382004

[46] S Y Kim J J Gao and H K Kang ldquoTwo flavonoids from theleaves ofMorus alba induce differentiation of the human prom-yelocytic leukemia (HL-60) cell linerdquo Biological and Pharma-ceutical Bulletin vol 23 no 4 pp 451ndash455 2000

[47] M A Khan A A Rahman S Islam et al ldquoA comparative studyon the antioxidant activity ofmethanolic extracts from differentparts of Morus alba L (Moraceae)rdquo BMC Research vol 6 pp24ndash32 2013

[48] A-L ChungH-F Lo K-H Lin et al ldquoStudy on the antioxidantactivity in herb plant extractsrdquo Journal of the Taiwan Society ForHorticultural Science vol 59 no 2 pp 139ndash152 2013

[49] R L Prior X Wu and K Schaich ldquoStandardized methods forthe determination of antioxidant capacity and phenolics infoods and dietary supplementsrdquo Journal of Agricultural andFood Chemistry vol 53 no 10 pp 4290ndash4302 2005

[50] S J Duthie A R Collins GGDuthie andV LDobson ldquoQue-rcetin and myricetin protect against hydrogen peroxide-indu-ced DNA damage (strand breaks and oxidised pyrimidines) inhuman lymphocytesrdquo Mutation Research vol 393 no 3 pp223ndash231 1997

[51] G A Liu and R L Zheng ldquoProtection against damagedDNA inthe single cell by polyphenolsrdquo Pharmazie vol 57 no 12 pp852ndash854 2002

[52] M Noroozi W J Angerson and M E J Lean ldquoEffects of flav-onoids and vitamin C on oxidative DNA damage to humanlymphocytesrdquoAmerican Journal of Clinical Nutrition vol 67 no6 pp 1210ndash1218 1998

[53] T Koga and M Meydani ldquoEffect of plasma metabolites of (+)-catechin and quercetin on monocyte adhesion to human aorticendothelial cellsrdquoAmerican Journal of Clinical Nutrition vol 73no 5 pp 941ndash948 2001

[54] CMeng P Somers L Hoong et al ldquoDiscovery of novel pheno-lic antioxidants as inhibitors of vascular cell adhesionmolecule-1 expression for use in chronic inflammatory diseasesrdquo Journalof Medicinal Chemistry vol 47 no 25 pp 6420ndash6432 2004

[55] Y Shibata N Kume H Arai et al ldquoMulberry leaf aqueousfractions inhibit TNF-120572-induced nuclear factor 120581B (NF-120581B)activation and lectin-like oxidized LDL receptor-1 (LOX-1)expression in vascular endothelial cellsrdquoAtherosclerosis vol 193no 1 pp 20ndash27 2007

[56] K C Chan H H Ho C N Huang M Lin H Chen and CWang ldquoMulberry leaf extract inhibits vascular smooth musclecell migration involving a block of small GTPase and AktNF-kB signalsrdquo Journal of Agricultural and Food Chemistry vol 57no 19 pp 9147ndash9153 2009

[57] F Chen N Nakashima I Kimura M Kimura N Asanoand S Koya ldquoPotentiating effects on pilocarpine-induced salivasecretion by extracts and N-containing sugars derived frommulberry leaves in streptozocin- diabetic micerdquo Biological andPharmaceutical Bulletin vol 18 no 12 pp 1676ndash1680 1995

[58] K Konno H Ono M Nakamura et al ldquoMulberry latexrich in antidiabetic sugar-mimic alkaloids forces dieting oncaterpillarsrdquo Proceedings of the National Academy of Sciences ofthe United States of America vol 103 no 5 pp 1337ndash1341 2006

[59] H J Kim K H Yoon M J Kang et al ldquoA six-month sup-plementation of mulberry Korean red ginseng and banabadecreases biomarkers of systemic low-grade inflammation insubjects with impaired glucose tolerance and type 2 diabetesrdquoEvidence-based Complementary and Alternative Medicine vol2012 Article ID 735191 8 pages 2012

[60] G Nicholson and G M Hall ldquoDiabetes mellitus new drugs fora new epidemicrdquo British Journal of Anaesthesia vol 107 no 1pp 65ndash73 2011

[61] P Y Chao Y L Chen Y C Lin et al ldquoEffects of black soybeanon atherogenic prevention in hypercholesterolemic rabbits andon adhesion molecular expression in cultured HAECsrdquo Foodand Nutrition Sciences vol 4 pp 9ndash21 2013

[62] C Chen S Li C-YOChen et al ldquoConstituents in purple sweetpotato leaves inhibit in vitro angiogenesis with opposite effectsex vivordquo Nutrition vol 27 no 11-12 pp 1177ndash1182 2011

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom