Research Article Fallopia japonica , a Natural Modulator...

Research ArticleFallopia japonica a Natural Modulator Can OvercomeMultidrug Resistance in Cancer Cells

Safaa Yehia Eid12 Mahmoud Zaki El-Readi13 Mohamed Lotfy Ashour4 and Michael Wink2

1Department of Biochemistry Faculty of Medicine Umm Al-Qura University Makkah Saudi Arabia2Institute of Pharmacy and Molecular Biotechnology Heidelberg University Im Neuenheimer Feld 364 69120 Heidelberg Germany3Department of Biochemistry Faculty of Pharmacy Al-Azhar University Assiut 71524 Egypt4Department of Pharmacognosy Faculty of Pharmacy Ain Shams University Cairo Egypt

Correspondence should be addressed to Mahmoud Zaki El-Readi mzreadiuquedusa andMichael Wink winkuni-heidelbergde

Received 14 April 2015 Revised 26 June 2015 Accepted 6 July 2015

Academic Editor Jae Youl Cho

Copyright copy 2015 SafaaYehia Eid 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

Resistance of cancer cells to chemotherapy is controlled by the decrease of intracellular drug accumulation increase ofdetoxification anddiminished propensity of cancer cells to undergo apoptosis ATP-binding cassette (ABC)membrane transporterswith intracellular metabolic enzymes contribute to the complex and unresolved phenomenon of multidrug resistance (MDR)Natural products as alternative medicine have great potential to discover new MDR inhibitors with diverse modes of action Inthis study we characterized several extracts of traditional Chinese medicine (TCM) plants (119873 = 16) for their interaction with ABCtransporters cytochrome P3A4 (CYP3A4) and glutathione-S-transferase (GST) activities and their cytotoxic effect on differentcancer cell lines Fallopia japonica (FJ) (Polygonaceae) shows potent inhibitory effect on CYP3A4 P-glycoprotein activity about18-fold when compared to verapamil as positive control FJ shows significant inhibitory effect (3981) compared with the knowninhibitor ketoconazole and 100120583gmL inhibited GST activity to 14120583molminmL FJ shows moderate cytotoxicity in human Caco-2 HepG-2 and HeLa cell lines IC

50values were 63098 19880 and 31737 120583gmL respectively LC-ESI-MS were used to identify

and quantify the most abundant compounds emodin polydatin and resveratrol in the most active extract of FJ Here we presentthe prospect of using Fallopia japonica as natural products to modulate the function of ABC drug transporters We are conductingfuture study to evaluate the ability of themajor active secondarymetabolites of Fallopia japonica tomodulateMDR and their impactin case of failure of chemotherapy

1 Introduction

Increased cancer mortality and the high cost of treatmentspur a continued search for better anticancer drugs In recentdecades natural compounds have attracted considerableattention as cancer chemopreventive agents and as cancertherapeutics [1] Some of themost effective cancer treatmentsto date are natural products or compounds derived fromnatural products [2] The first natural product used as ananticancer compound was podophyllotoxin isolated fromPodophyllum peltatum in 1947 Later etoposide and teni-poside (Chemical derivatives) vinca alkaloids (vinblastineand vincristine) and paclitaxel (Taxol) were discovered asactive principle of Taxus brevifolia [3] Natural products

and their synthetic derivatives comprise over 77 (6381) ofthe approved anticancer drug candidates developed between1981 and 2006 [4] This combined percentage highlights theimportance of natural products to drug development

Traditional Chinese medicine (TCM) has a long historyof using plant combinations in alleviating and curing thesymptoms of many diseases People are used to get theirmedication directly from herbal stores or local healers inthe form of multicomponents mixtures either to augmentthe activity of each other or to reduce some of the sideeffects TCM had an important role in the 11th Five-YearPlan on National Development of Economy and Society(2006ndash2010) established by the Chinese government Foodand Drug Administration (FDA) USA has approved the uses

Hindawi Publishing CorporationEvidence-Based Complementary and Alternative MedicineVolume 2015 Article ID 868424 8 pageshttpdxdoiorg1011552015868424

2 Evidence-Based Complementary and Alternative Medicine

of the herbal mixtures therefore the sales rates of TCMproducts were elevated [5] In addition all over the worldmany pharmaceutical companies have increased interest inherbal medicine after this approval [6] On the other handthe new mechanism-based approach informs us that manydifferent events contribute to the eventual success of cancer[7] Any single drug can at best target a small number of theseevents leaving the rest to occur uninterrupted [8] Moreoverwe know that cancer cells have some ability to adapt or beresistant to therapy We can imagine that a cancer cell canadapt better to one or a few interrupted events than to many

To overcome this problem it is necessary to use multiplecompounds in combination TCM are ideally suited for thistype of application TCM drugs are active at reasonableconcentrations and yet their mild nature allows a variety oflarge combinations to be used safely [9]

Cancer cells can develop resistance not only to one drugbut also to entire classes of drugs with similar mechanismsof action After such resistance is established some cellseven become cross-resistant to drugs which are structurallyand mechanistically unrelated this phenomenon is knownas multidrug resistance (MDR) Multidrug resistance (MDR)against anticancer drugs is a major problem in chemotherapywith 30ndash80 of cancer patients developing resistance tochemotherapeutical drugs [10] Thus counteracting drugresistance is crucial to provide the best treatment Mech-anisms of drug resistance involving ATP-dependent effluxpumps belonging to ABC transporters (P-gpMDR1 MRPand BCRP) although the most thoroughly characterizedare not the only means by which drug resistance can arisewithin tumor cells Clinical studies investigating other drug-resistance mechanisms (called nonclassical MDR) are fewerin number but are not less important These nontransportmechanisms affect multiple drug classes This type of resis-tance can be caused by the altered activity of specific enzymesystems such as CYP3A4 and GST which can decreasethe cytotoxic activity of drugs in a manner independent ofintracellular drug concentrations [11ndash13] The various causesof drug resistance can work simultaneously increasing theresistance in a multifactorial manner [14] For example thesimultaneous induction of CYP3A4 GST and MDR1 wasobserved [15 16] This type of multidrug resistance can beinduced after exposure to any drug Recent evidence indicatesthat certain nuclear receptors such as pregnane X receptor(PXR) might be involved in mediating this response to envi-ronmental stress while also acting in regulating metabolicenzymes (eg CYP3A4 andGST) andABC transporters (egMDR1 and MRP) [17 18]

In living system xenobiotic metabolism involved threemain phases phase I which is responsible for the trans-formation of the substrates into polar metabolites througha hydroxylation reaction phase 2 which is related mainlyto conjugation with highly soluble glucuronides to facilitatetheir excretion and finally phase 3 which are associated withthe cleaning pump P-glycoprotein that is accountable forpumping of the metabolized substrates out of the cells

The cytochrome P450 (CYP) enzymes are the pri-mary (phase I) enzyme system involved in the oxidativemetabolism of a wide variety of xenobiotics in the body and

their elimination This metabolizing system has unsurpris-ingly been associated with a majority of the metabolism-related drug-drug interactions known to date [19] Drugmetabolism in the human liver involved more than fifteendifferent CYP enzyme isoforms however of these CYP3A4 isconsidered the most important isoform It metabolizes morethan 50 of the drugs in the liver [20]

Usually lipophilic substances undergo further conju-gation in phase II with glucuronic acid or glutathioneGST is the most famous enzyme involved in this phasewhich catalyzes the conjugation of reduced glutathione toelectrophilic centers on a wide variety of substrates Thesebiotransformations enhance the dissolution of the substratesin the cellular sap and hence increase their elimination out ofthe cells [21]

In this context and based on the fact that most of theTCM drugs are sold as over-the-counter (OTC) drugs andrare reports could be found with regards to their possibleinteractions with the metabolizing enzymes it was veryimportant to investigate the potential interference of 17 com-monly used TCM plants on P-gp as well as CYP3A4 and GSTenzymes Besides their cytotoxicity on different cell linesincluding the colorectal carcinoma (Caco-2) hepatocellularcarcinoma (HepG-2) and cervical carcinoma (HeLa) cellswas also evaluated Characterization and profiling of themost active extract of Fallopia japonica (Polygonaceae) werecarried out in order to correlate the activity with theirsecondary metabolites contents

2 Materials and Methods

21 Plant Material Identification Plant samples were ob-tained commercially and identified using DNA barcodingmethods [22] Briefly the plant DNA was isolated using thephenol chloroform extraction method A 700 bp fragmentof the ribulose-bisphosphate carboxylase gene (rbcL) wasamplified using PCR The PCR product was sequenced andthe identity of the plant species was confirmed (on eitherthe genus or the species level) by comparing the sequencewith database entries of authentic species using BioEdit Thegenetic distance of the sequenced species to the species of thedatabases was determined using MEGA 4 This part of theworkwas performed by FlorianHerrmann IPMBHeidelbergUniversity

22 Preparation of the Plant Methanolic Extracts Differ-ent plant materials (100 g) were extracted 5ndash10 times withmethanol until the colored compounds were completelyremoved from the plant powders The extracts obtained werefiltered and the total methanol extracts were dried overanhydrous sodium sulphate and evaporated until becomingdry under a vacuum at 45∘C The dried extract was resolvedin 10mL methanol Portions of the extracts were driedcompletely in vacuum and the weight of the remaining dryextracts was determined (eg in FJ 125 g100 g dry weight)Dried extracts were dissolved in DMSO for the experimentsThe experiments were carried out with freshly preparedextracts

Evidence-Based Complementary and Alternative Medicine 3

23 Cell Lines Caco-2 cells (DSMZ No ACC 169) HepG-2(DSMZ No ACC 180) and HeLa cells (DSMZ No ACC 57)were maintained in DMEM complete medium (L-glutamine10 heat-inactivated fetal bovine serum (FBS) 100UmLpenicillin and 100 120583gmL streptomycin) and in addition1mMsodiumpyruvate and 1nonessential amino acids wereadded to Caco-2 medium Cells were grown at 37∘C in ahumidified atmosphere of 5 CO

2 All experiments were

performed with cells in the logarithmic growth phase TheCaco-2 cells described were an ideal model for studyingMDR because they highly express ABC transporter proteinsincluding MDR1 (P-gp) MRP1 and BCRP

24 Cytotoxicity Assay TheMTT cytotoxicity assay is widelyused particularly in the field of drug development [23]Briefly cells were seeded in 96-well plates with a densityof 2 times 104 cellswell The cells were treated with variousconcentrations of TCM extracts (up to 4mgmL) for 24 hThen 05mgmLMTTwas added to each well and incubatedfor 4 h The formed formazan crystals were dissolved inDMSO Absorbance was detected at 570 nm using TecanSafire II (Crailsheim Germany)

25 ABC Transporter Activity ABC transporter activities ofthe TCM extracts were determined using rhodamine 123(Rho123) Rho123 is a known substrate not only for P-gpbut also for MRP1 [24] Rho123 is readily effluxed in MDR-overexpressing cancer cells Caco-2 cells cells were seededat 2 times 103 cellswell in 96-well plates and cultured understandard conditions until a confluent monolayer was formed(by day 6) as determined by light microscopy After washingcells were preincubated for 30min at 37∘C with differentconcentrations of test samples in order to determine dosedependence Rho123 (1120583gmL) was then added and the cellswere further incubated for 90min at 37∘C After washing thefluorescence of Rho123 was measured at excitationemissionwavelengths of 500535 nm using a spectrofluorometer TecanSafire II (Crailsheim Germany) The fluorescence of testsamples themselves was excluded from the calculation offluorescence intensity

To quantify and compare the results the fluorescenceintensity of treated cells was normalized by calculating therelative fluorescence intensity (inhibitory efficiency) as thepercentage of the positive (verapamil) and untreated controlInhibitory efficiency was calculated as follows

Inhibitory efficiency

=(RFUextract minus RFUuntreated control)

(RFUverapamil minus RFUuntreated control)

(1)

RFUextract = fluorescence in the presence of test extractRFUverapamil = fluorescence in the presence of verapamil andRFUuntreated control = fluorescence in the absence of the drugOnly values higher than 10 were considered significant

26 CYP3A4 Activity Assay CYP450-Glo (Promega Man-nheim Germany) was used to detect the effects of the TCM

extract on recombinant human CYP3A4 according to themanufacturer instructions [25] Equal volumes (125120583L) ofdifferent sample solutions and the reaction mixture contain-ing theCYP3A4 specific substrate (200 120583Mluciferin 61015840 benzylether in phosphate buffer pH 74) and CYP3A4 (1 pmol120583L)were incubated at room temperature for 10min The addi-tion of 25120583L of the NADPH regeneration system (NADP+glucose-6-phosphate and glucose-6-phosphate dehydroge-nase in citrate buffer pH 55) initiated the enzyme reactionAfter 30min 50120583L luciferin was added 20min later theluminescence was recorded using a Tecan Safire II readerTheeffects of different extract were evaluated in triplicate relativeto blank controls containing 1 DMSO and ketoconazole(10 120583M) which was used as a positive control

27 Glutathione-S-Transferase Assay The principle of theGST activity assay is based upon the GST-catalysed reac-tion between GSH and GST substrate 1-chloro-24-dini-trobenzene (CDNB) as described by Habig et al [26] Brieflyuntreated and treated HepG-2 cell lysates were used for thisassay by preparing a sample with a total 100 120583L volume witha standard assay mixture containing 1mM CDNB 1mMreduced glutathione (GSH) and 100mM PBS (pH 65) Thereaction was monitored by spectrophotometry at 340 nmAfter calculating the GST activity unit (120583molminmL)according to manufacturer instructions the IC

50was calcu-

lated as related to the control activity

28 Characterization of the F japonica by LC-MS TheMeOHextract of 119865119869 (20mgmL) was separated by reversed-phaseHPLC by injecting 5 120583L Rheodyne system Separation wasachieved using a RP-C18e LichroCART 250-4 5120583m column(MerckDarmstadtGermany)Themobile phase consisted ofHPLC grade water with 05 formic acid (A) and acetonitrile(B) A Merck-Hitachi L-6200A system (Merck DarmstadtGermany) was used with a gradient program at a flow rateof 1mLmin as follows from 0 to 75 B in 45minutes andthen to 100 in 5minutes Mass spectrometry conditions areas follows a Quattro II system fromVGwith an ESI interfacewas used in positive ion and negative ion mode under thefollowing condition drying and nebulizing gas was nitrogen(N2) Capillary temperature was 120∘C capillary voltage was

350 kV Lens voltage was 05 kV and cone voltage was 30VFull scan mode was in the range of mz 200ndash800 for whichthe instrument was set to the following tune parametersnebulising and drying gas pressure was 350 Lh and 35Lh respectively Data were processed using MassLynx 40software (Waters)

3 Results

31 Cytotoxicity of TCM Drugs The cytotoxicity of TCMdrugs (IC

50) in human Caco-2 HepG-2 and HeLa cell

lines is shown in Table 1 The cytotoxic effect of TCMplants varies between cells and species Paris polyphylla(Melanthiaceae) demonstrated the strongest inhibition ofproliferation of these cell lines The IC

50values were 5387

4831 and 3504 120583gmL respectively whereas Cynanchum


Table 1 Cytotoxicity (IC50 values 120583gmL) of methanolic extracts from TCM plants in Caco-2 HepG-2 and HeLa cells

Plant Latin name (family) IC50 valuesCaco-2 HepG-2 HeLa

Areca catechu (Arecaceae) 139374 plusmn 12034 30866 plusmn 3975 41429 plusmn 7272Cassia tora (Fabaceae) 152001 plusmn 12343 107456 plusmn 11070 67094 plusmn 4063Chrysanthemum indicum (Asteraceae) 96523 plusmn 9087 88086 plusmn 6135 35575 plusmn 1681Cymbopogon distans (Poaceae) 59262 plusmn 1556 41052 plusmn 4019 9885 plusmn 902Cynanchum paniculatum (Apocynaceae) 259048 plusmn 29002 216754 plusmn 19984 50050 plusmn 5765Desmodium styracifolium (Fabaceae) 99854 plusmn 4789 46931 plusmn 1703 32439 plusmn 3468Kadsura longipedunculata (Schisandraceae) 143996 plusmn 6647 36854 plusmn 3009 8611 plusmn 321Mentha haplocalyx (Lamiaceae) 117012 plusmn 4011 99745 plusmn 5689 37506 plusmn 5061Ophioglossum vulgatum (Ophioglossaceae) 158414 plusmn 3808 110223 plusmn 7639 46906 plusmn 5473Paris polyphylla (Melanthiaceae) 5387 plusmn 567 4831 plusmn 443 3504 plusmn 645Patrinia scabiosaefolia (Valerianaceae) 148825 plusmn 6812 41330 plusmn 9383 15941 plusmn 856Fallopia japonica (Polygonaceae) 63098 plusmn 4021 19880 plusmn 1680 31737 plusmn 2227Sanguisorba officinalis (Rosaceae) 138069 plusmn 6077 16949 plusmn 1782 15853 plusmn 1325Saposhnikovia divaricata (Apiaceae) 242003 plusmn 21054 98034 plusmn 8976 36820 plusmn 1988Taxillus chinensis (Loranthaceae) 131034 plusmn 3121 112365 plusmn 5243 121342 plusmn 2147Viola yezoensis (Violaceae) 52023 plusmn 7043 34523 plusmn 3423 29753 plusmn 1999

paniculatum (Apocynaceae) showed the lowest cytotoxicityfor all tested cell lines IC

50values were 259048 216754 and

50050120583gmL respectively FJ shows moderate cytotoxicityIC50

values were 63098 19880 and 31737 120583gmL respec-tively Caco-2 cells were the most resistant cells against alltest extracts probability due to their high expression of MDRproteins

A significant correlation between the cytotoxicity of TCMextracts exists between HeLa and HepG-2 with 119903 = 075(119875 lt 0001) and betweenHeLa and Caco-2 cell lines 119903 = 058(119875 lt 001) (Spearman rank order correlation coefficient)Cytotoxicity of TCM extract was correlated between Caco-2 and HepG-2 119903 = 056 (119875 lt 005) These statistical datarefer to the degree of resistance in Caco-2 cell and sensi-tivity of HeLa and HepG-2 which appear highly correlatedto each other in cytotoxic response to test TCM extracts(Figures 1(a)ndash1(c))

32 Effects of TCM Drugs on the Activities of ABC Trans-porters CYP3A4 and GST We first tested the effects of 17TCM extracts from different families on the accumulation ofrhodamine 123 in Caco-2 cells as the model for ABC trans-porters As shown in Table 2 Cymbopogon distans (Poaceae)and Ophioglossum vulgatum (Ophioglossaceae) extracts at100 120583gmL have no effect on ABC transporter function

FJ andDesmodium styracifolium (Fabaceae) increased thecellular accumulation of the fluorescent substrate about 178-fold and 171-fold respectively when compared to verapamilas positive control Sanguisorba officinalis (Rosaceae) andAreca catechu (Arecaceae) show the maximum inhibitoryeffects on the CYP3A4 enzyme at 100 120583gmL with 5996and 5694 respectively FJ shows significant inhibitoryeffect (3981) comparedwith the known inhibitor ketocona-zole which completely inhibits the enzyme activity (Table 2)

The GST specific activities of TCM plants towards1-chloro-24-dinitrobenzene substrate were measured inHepG-2 cells The majority of the test extracts showedsignificant GST inhibitory activity at the tested concentration100 120583gmL Some extracts have low level GST activity likeSaposhnikovia divaricata and Cymbopogon distans (sim7 and9 120583molminmL) respectively (Table 2) FJ whereas FJ showsa medium GST activity

Our study target was to search for natural MDRinhibitors Thus we selected the most active extract FJ forfuture study to investigate its effect onMDR as amultifactoralphenomenon in details

33 LC-ESI-MS of Fallopia japonica Plant Extract Liquidchromatography (LC) combined with electrospray ionizationmass spectrometry (ESIMS) a relatively new techniquerapidly growing in popularity has been successfully appliedto elucidate the structures of the active compounds inherbal extracts [27] We believe that this technique couldbe successfully applied to identify the peaks in the HPLCprofile of F japonica extract In this study by using LC-ESI-MS methods we identified the major constituents (includingwater-soluble and lipid-soluble compounds) in the HPLCchromatogram HPLC profile of authenticated and identifiedherbalmaterial of FJ was obtained according to the developedHPLC method described above (Figure 2)

The experiments showed that the negative ion mode ismore sensitive than the positive ion mode for identifyingwater-soluble phenolic compounds The reference standardsemodin polydatin resveratrol and rhein were analyzedby direct injection in order to optimize the electrosprayionization ESI-MS conditions

Eleven characteristic peaks presented in profile weretentatively identified by detailed studies of their ESI-MS


0

500

1000

1500

2000

2500

IC50

valu

es(120583

gm

L) o

f TCM

on

Hep

G-2

r = 056 (P lt 005)

0 500 1000 1500 2000 2500 3000IC50 values (120583gmL) of TCM on Caco-2

Caco-2 and HepG-2 correlation

(a)

0

200

400

600

800

1000

1200

1400

IC50

valu

es(120583

gm

L) o

f TCM

on

HeL

a

r = 058 (P lt 005)


Caco-2 and HeLa correlation

(b)

0

500

1000

1500

2000

2500

IC50

valu

es(120583

gm

L) o

f TCM

on

Hep

G-2

r = 075 (P lt 0001)

0 200 400 600 800 1000 1200 1400IC50 values (120583gmL) of TCM on HeLa

HeLa and HepG-2 correlation

(c)

Figure 1 Correlation of cytotoxic effects (IC50

values) of TCM plants between different cell lines cytotoxicity in Caco-2 correlated withHepG-2 (119875 lt 005) (a) and with HeLa (119875 lt 001) (b) and HeLa highly significantly correlated with HepG-2 (119875 lt 0001) (c)

1

3 54

6

7

8 910

11

12

2100

0

()

000

500

1000

1500

2000

2500

3000

3500

4000

4500

5000

5500

Time

Figure 2 HPLC profile of F japonicamethanolic extract

spectral data (Table 3) by comparison with published data[28] The identification of peaks 2 5 10 and 11 was furtherconfirmed by comparing their retention time values withthose of the standards

Emodin polydatin and resveratrol can be identifiedunambiguously by comparing theMS data (mz 269 389 and227) and the retention times (448 88 and 157min) respec-tively with those of the literature and authentic compounds[28] These compounds amount to 296 224 and 255 oftotal extract content respectively (Table 3)

4 Discussion

Multidrug resistance to chemotherapeutic drugs is a majorproblem in tumor treatment Synergistic interaction between


Table 2 Effect of TCM drugs (100120583gmL) on activities of ABC transporters GST and CYP3A4

Plant (family)Metabolic activity

MDRinhibition

GSTactivity (120583molminmL)

CYP3A4inhibition

Areca catechu (Arecaceae) 127 plusmn 013 2346 plusmn 334 5694 plusmn 435Cassia tora (Fabaceae) 884 plusmn 097 3181 plusmn 354 1638 plusmn 308Chrysanthemum indicum (Asteraceae) 983 plusmn 089 2286 plusmn 154 4618 plusmn 499Cymbopogon distans (Poaceae) 028 plusmn 003 954 plusmn 102 271 plusmn 153Cynanchum paniculatum (Apocynaceae) 1929 plusmn 432 1570 plusmn 172 2262 plusmn 362Desmodium styracifolium (Fabaceae) 17124 plusmn 1854 1093 plusmn 132 1022 plusmn 312Kadsura longipedunculata (Schisandraceae) 299 plusmn 035 1014 plusmn 139 4830 plusmn 470Mentha haplocalyx (Lamiaceae) 896 plusmn 100 1292 plusmn 181 119 plusmn 037Ophioglossum vulgatum (Ophioglossaceae) 121 plusmn 015 1391 plusmn 196 2491 plusmn 054Paris polyphylla (Melanthiaceae) 1541 plusmn 201 1888 plusmn 211 292 plusmn 118Patrinia scabiosaefolia (Valerianaceae) 632 plusmn 082 2743 plusmn 132 061 plusmn 004Fallopia japonica (Polygonaceae) 17994 plusmn 1834 1411 plusmn 123 3981 plusmn 468Sanguisorba officinalis (Rosaceae) 3582 plusmn 465 1988 plusmn 343 5996 plusmn 352Saposhnikovia divaricata (Apiaceae) 13438 plusmn 159 735 plusmn 067 821 plusmn 314Taxillus chinensis (Loranthaceae) 4360 plusmn 576 994 plusmn 19 4741 plusmn 107Viola yezoensis (Violaceae) 6577 plusmn 689 1531 plusmn 285 2497 plusmn 275MDR inhibition calculated as compared to verapamil as positive control (100)Data are means plusmn SD from three independent experiments

Table 3 Identification of secondary metabolites in methanol extract of F japonica by LC-ESIMS

Peak RT(min)

[MminusH]minus(119898119911)

Other ions(119898119911)

Compound

1 26 290 mdash Unknown 1422 88 389 mdash Polydatin 2243 12 541 mdash Polydatin gallate 34 144 431 mdash Apigenin-7-glucoside 25 157 227 mdash Resveratrol 2556 185 407 245

[MminusH-glu]minus Torachrysone-8-O-120573-glucoside 4237 197 431 mdash Emodin-8-120573-D-glucoside 1118 242 445 283

[MminusH-glu]minus Physcion-8-120573-D-glucoside 299 263 285 mdash Hydroxyemodin 1610 352 283 mdash Rhein 5211 448 269 mdash Emodin 29612 483 283 mdash Physcion 12

theMDRmechanismsABC transportersmetabolic enzymes(CYP3A4 and GST) and apoptosis was observed [29]

The general screening assays reveal the FJ extract to bethe most potent inhibitor of ABC transporters significantlyinhibiting metabolic enzymes and cell growth (Tables 1 and2) Based on the phytochemical profiling the main compo-nents of the methanol extract are resveratrol and its glycosidepolydatin emodin and its glucoside These compounds areknown inhibitors of both P-gp proteins and are able to reversemultidrug resistance in many cancer cells [30] Besidesresveratrol is a known inhibitor of different cytochromeisoforms especially CYP1A1 CYP1B1 and CYP3A4 [31 32]

Emodin and also its glucoside form are known inhibitorsof CYP1A1 and CYP1A2 [33] However nothing could befound regarding the inhibition of CYP3A4 although otherclosely related compounds such as rhein can inhibit thismetabolizing enzyme

Among the most active extracts the Radix Saposh-nikoviae (Saposhnikovia divaricataApiaceae) which is widelyused as antiphlogistic analgesic and antipyretic drug inboth Kampo and TCM medicine showed high activity Thisactivity is based mainly on the presence of highly reactivepolyacetylenes especially panaxynol [34] in addition to thehigh yield of furanocoumarins and chromones as divaricatol


[35] Presence of these highly reactive polyacetylenes withtheir reactive triple bonds can covalently bind to amino andsulphydryl groups of proteins This alkylation may lead to aconformational change and thus loss of activity [8] This typeof interaction can explain the inhibition of many enzymessuch as cytochromes and glutathione S-transferase

Another example of a plant which potentially affects theactivity of drugmetabolism especially the P-gp isDesmodiumstyracifolium (Fabaceae) The main modulatory activity canbe attributed to its flavonoidisoflavonoid contents (api-genin luteolin and genistin) triterpenoid saponinssterols(stigmasterol-3-O-120573-D-glucopyranoside 120573-daucosterol and120573-sitosterol) and alkaloids (desmodimine) [36] Theseflavonoids possess one or more phenolic hydroxyl groupsThe phenolic hydroxyl groups can partly dissociate underphysiological conditions resulting in formation of phenolateions which could interact with many enzymes unselectivelyby forming hydrogen bonds with electronegative atoms of thepeptide or ionic bonds with positively charged side chainsof basic amino acids respectively These noncovalent bondsare quite weak but they could cause a change in proteinconformation which then may lead to protein inactivation[8]

Areca catechu (Arecaceae) is the least potent inhibitor ofthe enzymatic activity in both extracts from our tested plantssamples Despite its neurotoxicity and other toxic hazards[37] the plant is still in use especially in Asia and EastAfrica as astringent and stimulant and to expel worms andnothing could be traced in literature concerning the possibleinteractions with the metabolizing enzymes The activity ofboth extracts could be ascribed to their contents of tanninswith their polyphenolic structures especially ArecatanninA1ndashA3which are abundant in both extracts which are able

to bind with the enzyme Moreover the alkaloidal contentmainly arecoline of the methanol extract could contributeto the overall activity although this action has not beenconfirmed yet to the CYP3A4 but arecoline shows a signif-icant inhibitory activity against other forms of cytochromeespecially CYP1A1 [38]

The same explanation could be also given to the San-guisorba officinalis (Rosaceae) This plant is widely used inEurope and China as astringent and antihaemorrhoidal agentand also in treatment of ulcerative colitis and many skin dis-orders [39 40] The extract contains much hydrolysable andcondensed tanninsmainly gallic and ellagic acids derivativesin addition to flavonoids (rutin) proanthocyanidins andmonodesmosidic triterpenoidal saponins Although thesesecondary metabolites show a significant inhibition of theCYP3A4 and there is one report about affecting the phar-macokinetics of ciprofloxacin [41] no reports are foundconcerning the interaction of the plant extract with thecytochromes

Chrysanthemum (Apiaceae) plants showed higher activ-ities of the extract The main activity is related to its con-tent of the triterpenoidal alcohols which are presented inhigher concentrations extracts Although these plants arewidely used in traditional medicine for treatment of feverinflammations and many infections [42] only one reportdeals with the interaction between another Chrysanthemum

spp with many cytochrome isoforms where C partheniumextracts modestly inhibited the activity of CYP1A2 CYP2C8CYP2C9 CYP2C19 and CYP3A4 [43]

In conclusion TCM plants modulate MDR through theirinteraction with P-gp CYP3A4 and GST This potent effectsof TCM extracts may be due to the synergistic interactions oftheir SMs which have diverse structure and mode of actionFallopia japonica extract shows potent inhibition of ABCtransporters and significant inhibiting metabolic enzymesand cell growth Further study is needed to evaluate the effectsof the individual active compounds of Fallopia japonica onMDR in cancer cells

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

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[1] S Nobili D Lippi E Witort et al ldquoNatural compounds forcancer treatment and preventionrdquo Pharmacological Researchvol 59 no 6 pp 365ndash378 2009

[2] D A Dias S Urban and U Roessner ldquoA historical overview ofnatural products in drug discoveryrdquo Metabolites vol 2 no 4pp 303ndash336 2012

[3] S M Colegate and R J Molyneux Bioactive Natural ProductsDetection Isolation and Structural Determination CRC PressBoca Raton Fla USA 2nd edition 2008

[4] D J Newman and G M Cragg ldquoNatural products as sources ofnew drugs over the last 25 yearsrdquo Journal of Natural Productsvol 70 no 3 pp 461ndash477 2007

[5] S Wachtel-Galor and I F F Benzie ldquoHerbal medicine anintroduction to its history usage regulation current trends andresearch needsrdquo in Herbal Medicine Biomolecular and ClinicalAspects I F F Benzie and S Wachtel-Galor Eds CRC PressBoca Raton Fla USA 2011

[6] J W-H Li and J C Vederas ldquoDrug discovery and naturalproducts end of an era or an endless frontierrdquo Science vol 325no 5937 pp 161ndash165 2009

[7] V Brower ldquoBack to nature extinction of medicinal plantsthreatens drug discoveryrdquo Journal of the National Cancer Insti-tute vol 100 no 12 pp 838ndash839 2008

[8] M Wink ldquoEvolutionary advantage and molecular modes ofaction of multi-component mixtures used in phytomedicinerdquoCurrent Drug Metabolism vol 9 no 10 pp 996ndash1009 2008

[9] J BoikNatural Compounds in CancerTherapy OregonMedicalPress Princeton Minn USA 2001

[10] V S Velingkar and V D Dandekar ldquoModulation of P-glycoprotein mediated multidrug resistance (MDR) in cancerusing chemosensitizersrdquo International Journal of Pharmaceuti-cal Sciences and Research vol 2 pp 104ndash111 2010

[11] A Pluen Y Boucher S Ramanujan et al ldquoRole of tumor-hostinteractions in interstitial diffusion of macromolecules cranialvs subcutaneous tumorsrdquo Proceedings of the National Academyof Sciences of the United States of America vol 98 no 8 pp4628ndash4633 2001

[12] R K Jain ldquoDelivery of molecular and cellular medicine to solidtumorsrdquo Advanced Drug Delivery Reviews vol 46 no 1ndash3 pp149ndash168 2001


[13] Z-P Mao L-J Zhao S-H Zhou M-Q Liu W-F Tan andH-T Yao ldquoExpression and significance of glucose transporter-1 P-glycoprotein multidrug resistance-associated protein andglutathione S-transferase-120587 in laryngeal carcinomardquo OncologyLetters vol 9 no 2 pp 806ndash810 2015

[14] A Cort and T Ozben ldquoNatural product modulators to over-comemultidrug resistance in cancerrdquoNutrition and Cancer vol67 no 3 pp 411ndash423 2015

[15] E G Schuetz W T Beck and J D Schuetz ldquoModulators andsubstrates of P-glycoprotein and cytochrome P4503A coordi-nately up-regulate these proteins in human colon carcinomacellsrdquoMolecular Pharmacology vol 49 no 2 pp 311ndash318 1996

[16] C Awortwe V K Manda C Avonto et al ldquoEchinacea purpureaup-regulates CYP1A2 CYP3A4 and MDR1 gene expression byactivation of pregnane X receptor pathwayrdquoXenobiotica vol 45no 3 pp 218ndash229 2015

[17] H Jiang K Chen J He et al ldquoAssociation of pregnanex receptor with multidrug resistance-related protein 3 andits role in human colon cancer chemoresistancerdquo Journal ofGastrointestinal Surgery vol 13 no 10 pp 1831ndash1838 2009

[18] S R Pondugula P C Flannery K L Abbott et al ldquoDiindolyl-methane a naturally occurring compound induces CYP3A4andMDR1 gene expression by activating human PXRrdquo Toxicol-ogy Letters vol 232 no 3 pp 580ndash589 2015

[19] I G Denisov Y V Grinkova J L Baylon E Tajkhorshid andS G Sligar ldquoMechanism of drugndashdrug interactions mediatedby human cytochrome P450 CYP3A4 monomerrdquo Biochemistryvol 54 no 13 pp 2227ndash2239 2015

[20] F P Guengerich ldquoCytochrome P-450 3A4 regulation and rolein drug metabolismrdquo Annual Review of Pharmacology andToxicology vol 39 pp 1ndash17 1999

[21] R C Strange P W Jones and A A Fryer ldquoGlutathione S-transferase genetics and role in toxicologyrdquo Toxicology Lettersvol 112-113 pp 357ndash363 2000

[22] J J Doyle and J L Doyle ldquoIsolation of plant DNA from freshtissuerdquo Focus vol 12 pp 13ndash15 1990

[23] J Carmichael W G DeGraff A F Gazdar J D Minna and JB Mitchell ldquoEvaluation of a tetrazolium-based semiautomatedcolorimetric assay assessment of chemosensitivity testingrdquoCancer Research vol 47 no 4 pp 936ndash942 1987

[24] P R Twentyman T Rhodes and S Rayner ldquoA comparisonof rhodamine 123 accumulation and efflux in cells with P-glycoprotein-mediated and MRP-associated multidrug resis-tance phenotypesrdquo European Journal of Cancer vol 30 no 9pp 1360ndash1369 1994

[25] J J Cali DMaM Sobol et al ldquoLuminogenic cytochrome P450assaysrdquoExpert Opinion onDrugMetabolism and Toxicology vol2 no 4 pp 629ndash645 2006

[26] W H Habig M J Pabst G Fleischner Z Gatmaitan IM Arias and W B Jakoby ldquoThe identity of glutathione Stransferase B with ligandin a major binding protein of liverrdquoProceedings of the National Academy of Sciences of the UnitedStates of America vol 71 no 10 pp 3879ndash3882 1974

[27] Z Cai F S C Lee X R Wang and W J Yu ldquoA capsulereview of recent studies on the application ofmass spectrometryin the analysis of Chinese medicinal herbsrdquo Journal of MassSpectrometry vol 37 no 10 pp 1013ndash1024 2002

[28] T Yi H Zhang and Z Cai ldquoAnalysis of rhizoma Polygoni cusp-idati byHPLC andHPLC-ESIMSrdquo Phytochemical Analysis vol18 no 5 pp 387ndash392 2007

[29] S Harmsen I Meijerman J H Beijnen and J H M SchellensldquoThe role of nuclear receptors in pharmacokinetic drug-druginteractions in oncologyrdquoCancer Treatment Reviews vol 33 no4 pp 369ndash380 2007

[30] M Wink M L Ashour and M Z El-Readi ldquoSecondarymetabolites from plants inhibiting ABC transporters andreversing resistance of cancer cells and microbes to cytotoxicand antimicrobial agentsrdquo Frontiers in Microbiology vol 3article 130 2012

[31] W K Chan and A B Delucchi ldquoResveratrol a red wineconstituent is a mechanism-based inactivator of cytochromeP450 3A4rdquo Life Sciences vol 67 no 25 pp 3103ndash3112 2000

[32] T K H Chang J Chen and W B K Lee ldquoDifferentialinhibition and inactivation of human CYP1 enzymes by trans-resveratrol evidence for mechanism-based inactivation ofCYP1A2rdquo Journal of Pharmacology and ExperimentalTherapeu-tics vol 299 no 3 pp 874ndash882 2001

[33] M Bhadauria S K Nirala S Shrivastava et al ldquoEmodinreverses CCl

4induced hepatic cytochrome P450 (CYP) enzy-

matic and ultrastructural changes the in vivo evidencerdquo Hepa-tology Research vol 39 no 3 pp 290ndash300 2009

[34] C-N Wang Y-J Shiao Y-H Kuo C-C Chen and Y-L LinldquoInducible nitric oxide synthase inhibitors from Saposhnikoviadivaricata andPanax quinquefoliumrdquoPlantaMedica vol 66 no7 pp 644ndash647 2000

[35] E Okuyama T Hasegawa T Matsushita H Fujimoto MIshibashi and M Yamazaki ldquoAnalgesic components of Saposh-nikovia root (Saposhnikovia divaricata)rdquo Chemical amp Pharma-ceutical Bulletin vol 49 no 2 pp 154ndash160 2001

[36] T Kubo S Hamada T Nohara et al ldquoStudy on the constituentsof Desmodium styracifoliumrdquo Chemical and PharmaceuticalBulletin vol 37 no 8 pp 2229ndash2231 1989

[37] M Wink and B-E van Wyk Mind-Altering and PoisonousPlants of the World Timber Press Portland Ore USA 2008

[38] E E Chang Z-F Miao W-J Lee et al ldquoArecoline inhibits the2378-tetrachlorodibenzo-p-dioxin-induced cytochrome P4501A1 activation in human hepatoma cellsrdquo Journal of HazardousMaterials vol 146 no 1-2 pp 356ndash361 2007

[39] B-E Van Wyk and M Wink Medicinal Plants of the WorldAn Illustrated Scientific Guide to ImportantMedicinal Plants andTheir Uses Timber Press Portland Ore USA 1st edition 2004

[40] J-N Wu An Illustrated Chinese Materia Medica Oxford Uni-versity Press New York NY USA 2005

[41] M Zhu P Y K Wong and R C Li ldquoInfluence of Sanguisorbaofficinalis a mineral-rich plant drug on the pharmacokineticsof ciprofloxacin in the ratrdquo Journal of Antimicrobial Chemother-apy vol 44 no 1 pp 125ndash128 1999

[42] L J FundukianThe Gale Encyclopedia of Alternative MedicineGale Cengage Learning Detroit Mich USA 3rd edition 2009

[43] M Unger and A Frank ldquoSimultaneous determination of theinhibitory potency of herbal extracts on the activity of sixmajorcytochrome P450 enzymes using liquid chromatographymassspectrometry and automated online extractionrdquoRapid Commu-nications in Mass Spectrometry vol 18 no 19 pp 2273ndash22812004

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014


MEDIATORSINFLAMMATION

of


Behavioural Neurology

EndocrinologyInternational Journal of



Disease Markers


BioMed Research International

OncologyJournal of



Oxidative Medicine and Cellular Longevity


PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of



Computational and Mathematical Methods in Medicine

OphthalmologyJournal of


Diabetes ResearchJournal of



Research and TreatmentAIDS


Gastroenterology Research and Practice


Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom


of the herbal mixtures therefore the sales rates of TCMproducts were elevated [5] In addition all over the worldmany pharmaceutical companies have increased interest inherbal medicine after this approval [6] On the other handthe new mechanism-based approach informs us that manydifferent events contribute to the eventual success of cancer[7] Any single drug can at best target a small number of theseevents leaving the rest to occur uninterrupted [8] Moreoverwe know that cancer cells have some ability to adapt or beresistant to therapy We can imagine that a cancer cell canadapt better to one or a few interrupted events than to many

To overcome this problem it is necessary to use multiplecompounds in combination TCM are ideally suited for thistype of application TCM drugs are active at reasonableconcentrations and yet their mild nature allows a variety oflarge combinations to be used safely [9]

Cancer cells can develop resistance not only to one drugbut also to entire classes of drugs with similar mechanismsof action After such resistance is established some cellseven become cross-resistant to drugs which are structurallyand mechanistically unrelated this phenomenon is knownas multidrug resistance (MDR) Multidrug resistance (MDR)against anticancer drugs is a major problem in chemotherapywith 30ndash80 of cancer patients developing resistance tochemotherapeutical drugs [10] Thus counteracting drugresistance is crucial to provide the best treatment Mech-anisms of drug resistance involving ATP-dependent effluxpumps belonging to ABC transporters (P-gpMDR1 MRPand BCRP) although the most thoroughly characterizedare not the only means by which drug resistance can arisewithin tumor cells Clinical studies investigating other drug-resistance mechanisms (called nonclassical MDR) are fewerin number but are not less important These nontransportmechanisms affect multiple drug classes This type of resis-tance can be caused by the altered activity of specific enzymesystems such as CYP3A4 and GST which can decreasethe cytotoxic activity of drugs in a manner independent ofintracellular drug concentrations [11ndash13] The various causesof drug resistance can work simultaneously increasing theresistance in a multifactorial manner [14] For example thesimultaneous induction of CYP3A4 GST and MDR1 wasobserved [15 16] This type of multidrug resistance can beinduced after exposure to any drug Recent evidence indicatesthat certain nuclear receptors such as pregnane X receptor(PXR) might be involved in mediating this response to envi-ronmental stress while also acting in regulating metabolicenzymes (eg CYP3A4 andGST) andABC transporters (egMDR1 and MRP) [17 18]

In living system xenobiotic metabolism involved threemain phases phase I which is responsible for the trans-formation of the substrates into polar metabolites througha hydroxylation reaction phase 2 which is related mainlyto conjugation with highly soluble glucuronides to facilitatetheir excretion and finally phase 3 which are associated withthe cleaning pump P-glycoprotein that is accountable forpumping of the metabolized substrates out of the cells

The cytochrome P450 (CYP) enzymes are the pri-mary (phase I) enzyme system involved in the oxidativemetabolism of a wide variety of xenobiotics in the body and

their elimination This metabolizing system has unsurpris-ingly been associated with a majority of the metabolism-related drug-drug interactions known to date [19] Drugmetabolism in the human liver involved more than fifteendifferent CYP enzyme isoforms however of these CYP3A4 isconsidered the most important isoform It metabolizes morethan 50 of the drugs in the liver [20]

Usually lipophilic substances undergo further conju-gation in phase II with glucuronic acid or glutathioneGST is the most famous enzyme involved in this phasewhich catalyzes the conjugation of reduced glutathione toelectrophilic centers on a wide variety of substrates Thesebiotransformations enhance the dissolution of the substratesin the cellular sap and hence increase their elimination out ofthe cells [21]

In this context and based on the fact that most of theTCM drugs are sold as over-the-counter (OTC) drugs andrare reports could be found with regards to their possibleinteractions with the metabolizing enzymes it was veryimportant to investigate the potential interference of 17 com-monly used TCM plants on P-gp as well as CYP3A4 and GSTenzymes Besides their cytotoxicity on different cell linesincluding the colorectal carcinoma (Caco-2) hepatocellularcarcinoma (HepG-2) and cervical carcinoma (HeLa) cellswas also evaluated Characterization and profiling of themost active extract of Fallopia japonica (Polygonaceae) werecarried out in order to correlate the activity with theirsecondary metabolites contents

2 Materials and Methods

21 Plant Material Identification Plant samples were ob-tained commercially and identified using DNA barcodingmethods [22] Briefly the plant DNA was isolated using thephenol chloroform extraction method A 700 bp fragmentof the ribulose-bisphosphate carboxylase gene (rbcL) wasamplified using PCR The PCR product was sequenced andthe identity of the plant species was confirmed (on eitherthe genus or the species level) by comparing the sequencewith database entries of authentic species using BioEdit Thegenetic distance of the sequenced species to the species of thedatabases was determined using MEGA 4 This part of theworkwas performed by FlorianHerrmann IPMBHeidelbergUniversity

22 Preparation of the Plant Methanolic Extracts Differ-ent plant materials (100 g) were extracted 5ndash10 times withmethanol until the colored compounds were completelyremoved from the plant powders The extracts obtained werefiltered and the total methanol extracts were dried overanhydrous sodium sulphate and evaporated until becomingdry under a vacuum at 45∘C The dried extract was resolvedin 10mL methanol Portions of the extracts were driedcompletely in vacuum and the weight of the remaining dryextracts was determined (eg in FJ 125 g100 g dry weight)Dried extracts were dissolved in DMSO for the experimentsThe experiments were carried out with freshly preparedextracts











(1)





50was calcu-




3 Results




50values were 5387



















0

500

1000

1500

2000

2500

IC50

valu

es(120583

gm

L) o

f TCM

on

Hep

G-2

r = 056 (P lt 005)



(a)

0

200

400

600

800

1000

1200

1400

IC50

valu

es(120583

gm

L) o

f TCM

on

HeL

a

r = 058 (P lt 005)



(b)

0

500

1000

1500

2000

2500

IC50

valu

es(120583

gm

L) o

f TCM

on

Hep

G-2

r = 075 (P lt 0001)



(c)



1

3 54

6

7

8 910

11

12

2100

0

()

000

500

1000

1500

2000

2500

3000

3500

4000

4500

5000

5500

Time




4 Discussion





MDRinhibition


CYP3A4inhibition



Peak RT(min)


Other ions(119898119911)

Compound



















References




















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


























(1)





50was calcu-




3 Results




50values were 5387



















0

500

1000

1500

2000

2500

IC50

valu

es(120583

gm

L) o

f TCM

on

Hep

G-2

r = 056 (P lt 005)



(a)

0

200

400

600

800

1000

1200

1400

IC50

valu

es(120583

gm

L) o

f TCM

on

HeL

a

r = 058 (P lt 005)



(b)

0

500

1000

1500

2000

2500

IC50

valu

es(120583

gm

L) o

f TCM

on

Hep

G-2

r = 075 (P lt 0001)



(c)



1

3 54

6

7

8 910

11

12

2100

0

()

000

500

1000

1500

2000

2500

3000

3500

4000

4500

5000

5500

Time




4 Discussion





MDRinhibition


CYP3A4inhibition



Peak RT(min)


Other ions(119898119911)

Compound



















References




















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

































0

500

1000

1500

2000

2500

IC50

valu

es(120583

gm

L) o

f TCM

on

Hep

G-2

r = 056 (P lt 005)



(a)

0

200

400

600

800

1000

1200

1400

IC50

valu

es(120583

gm

L) o

f TCM

on

HeL

a

r = 058 (P lt 005)



(b)

0

500

1000

1500

2000

2500

IC50

valu

es(120583

gm

L) o

f TCM

on

Hep

G-2

r = 075 (P lt 0001)



(c)



1

3 54

6

7

8 910

11

12

2100

0

()

000

500

1000

1500

2000

2500

3000

3500

4000

4500

5000

5500

Time




4 Discussion





MDRinhibition


CYP3A4inhibition



Peak RT(min)


Other ions(119898119911)

Compound



















References




















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of



















MDRinhibition


CYP3A4inhibition



Peak RT(min)


Other ions(119898119911)

Compound



















References




















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of



























References




















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















Research Article Fallopia japonica , a Natural Modulator...

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Transcript of Research Article Fallopia japonica , a Natural Modulator...