Research Article Evaluation of Plant Phenolic Metabolites as...

Research ArticleEvaluation of Plant Phenolic Metabolites as a Source ofAlzheimerrsquos Drug Leads

Yara Hassaan1 Heba Handoussa1 Ahmed H El-Khatib2 Michael W Linscheid2

Nesrine El Sayed34 and Nahla Ayoub56

1 Department of Pharmaceutical Biology Faculty of Pharmacy and Biotechnology German University in Cairo Cairo 11835 Egypt2 Laboratory of Analytical and Environmental Chemistry Department of ChemistryHumboldt-Universitat zu Berlin 12489 Berlin Germany

3Department of Pharmacology and Toxicology Faculty of Pharmacy Cairo University Cairo 11562 Egypt4Department of Pharmacology and Toxicology Faculty of Pharmacy amp Biotechnology German University in Cairo Cairo 11835 Egypt5 Department of Pharmaceutical Biology Faculty of Pharmacy Ain-Shams University Cairo 11613 Egypt6Department of Pharmaceutical Biology Faculty of Pharmacy British University in Cairo Cairo 11613 Egypt

Correspondence should be addressed to Nahla Ayoub nahlaayoubyahoode

Received 27 February 2014 Revised 26 April 2014 Accepted 28 April 2014 Published 29 May 2014

Academic Editor Cristiano Capurso

Copyright copy 2014 Yara Hassaan 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

Epidemiological studies have proven an association between consumption of polyphenols and prevention of Alzheimerrsquos diseasethe most common form of dementia characterized by extracellular deposition of amyloid beta plaques The aim of this study ispharmacological screening of the aqueous alcohol extract of Markhamia platycalyx leaves Schotia brachypetala leaves and stalksand piceatannol compared to aqueous alcohol extract ofCamellia sinensis leaves as potential Alzheimerrsquos disease drugs LC-HRESI(-ve)-MSn was performed to identify phenolicsrsquo profile of Schotia brachypetala stalks aqueous alcohol extract and revealed tenphenolic compounds as first report daidzein naringin procyanidin isomers procyanidin dimer gallate quercetin 3-O-rhamnosidequercetin 3-O-glucuronide quercetin hexose gallic acid quercetin hexose protocatechuic acid and ellagic acid Alzheimerrsquos diseasewas induced by a single intraperitoneal injection of LPS Adult male Swiss albino mice were divided into groups of 8ndash10 mice eachreceiving treatment for six days In vivo behavioral tests (Y maze and object recognition) and in vitro estimation of amyloid beta 42by ELISA showed significant differences between results of treated and nontreated animals

1 Introduction

Alzheimerrsquos disease (AD) is the most prevalent neurodegen-erative disease in the elderly population which eventuallyleads to complete incapacity and death of patients It is char-acterized by the progressive and selective loss of neurons andsynapses extracellular deposition of amyloid beta plaquesand formation of intracellular neurofibrillary tangles com-posed of hyperphosphorylated tau proteinmainly in the brainregions that are involved in cognitive processes [1] Naturaloccurring dietary polyphenolics have received considerablerecent attention as alternative candidates for Alzheimerrsquosdisease therapy [2] Epidemiological studies have provenan association between the consumption of polyphenolicrich foods or beverages and the prevention of neurological

diseases including Alzheimerrsquos disease [2] The ability ofpolyphenols to cross the intestinal wall of mammals [3]and the ability of some to cross the blood brain barrier [4]confers their biological properties Moreover one of themajor properties of this group of compounds is their abilityto interact with peptides and proteins such interaction canprove valuable at the biological level in general and especiallyin Alzheimerrsquos disease [5] Green tea EGCG inhibits 120573-amyloid induced memory dysfunction evaluated by passiveavoidance and water maze tests [6] Piceatannol showedneuroprotective effect against toxicity induced by amyloidbeta peptides in rat hippocampal cell cultures Also decreasedamyloid beta 1ndash42 induced fluorescence in a TH-t fluores-cence assay [7] Bark extracts of Schotia brachypetala exhib-ited pronounced monoamine oxidase-B inhibition activity

Hindawi Publishing CorporationBioMed Research InternationalVolume 2014 Article ID 843263 10 pageshttpdxdoiorg1011552014843263

2 BioMed Research InternationalRe

lativ

e abu

ndan

ce

100

95

90

85

80

75

70

65

60

55

50

45

40

35

30

25

20

15

10

5

0

6560555045403530252015105

Time (min)

Sb1

Sb2

Sb3

Sb4 Sb5Sb6

Sb7

Sb8

Sb9

Sb10

Sb11

Sb12

Sb13 Sb14

Sb15

Figure 1 LC-MS of aqueous alcohol Schotia brachypetala stalkextract

[8] and monoamine oxidase inhibitors are useful in treatingAD [9] Phenolic glycosides from Markhamia stipulata wereisolated mainly being verbascosides [10] Verbascosides arephenylpropanoids that have cytoprotective action connectedto their antioxidant and chelating capacity [11] This datasuggests genus Markhamia can be used to treat oxidativestress related neurodegenerative disease as AD Thus thecurrent study was performed with the main aim of phar-macological screening of piceatannol and the phenolic richextracts of leaves of Camellia sinensis Markhamia platycalyxand Schotia brachypetala and stalks of Schotia brachypetalausing behaviouralmousemodels (ymaze object recognition)as promisingAlzheimerrsquos disease drug leads andusingCamel-lia sinensis as reference standard Further pharmacologicalscreening of extracts was performed by mouse amyloid beta42 ELISA assay Characterization of phenolicsrsquo profiling instalks of Schotia brachypetala aqueous alcohol extract wasexecuted by LC-MS and X calibur software (Figure 1)

2 Materials and Methods

21 Plant Material Aerial parts of Markhamia platycalyxCamellia sinensis and Schotia brachypetala and stalks ofSchotia brachypetalawere collected from the Orman GardenGiza Egypt in May 2012 The authenticity of species wasconfirmed by Professor Dr Mohamed El Gebaly Professor ofTaxonomy at the National Research Center Egypt Voucherspecimen was deposited at the herbarium of the Faculty ofPharmacy and Biotechnology Department of Pharmaceuti-cal Biology German University in Cairo Egypt

22 Extraction The leaves of Markhamia platycalyx Camel-lia sinensis and Schotia brachypetala (500 g) and stalk ofSchotia brachypetala (500 g) [12] were exhaustively extractedwith distilled water (5 L) individually The extract was evap-orated in vacuo at low temperature till dryness to yield (12 g)for each of Markhamia platycalyx Camellia sinensis andSchotia brachypetala leaves and (15 g) of Schotia brachypetala

stalksThen the dry solid residues obtained from evaporationof aqueous extracts were extracted with ethanol The driedaqueous extract of each plant was dissolved in ethanolindividually This step was followed by evaporation of thefiltrate to obtain crude phenolic content 7 g from leaves ofMarkhamia platycalyx Camellia sinensis and Schotia brachy-petala and 5 g from Schotia brachypetala stalks The dryresidual powder of aqueous ethanol extract was kept in tightlyclosed sample tubes at room temperature

23 LC-HRESI-MS-MS LC-HRESI-MS-MS was performedon a Bruker micro-TOF-Q Daltonics (API) Time-of-Flightmass spectrometer (Bremen Germany) coupled to 1200series HPLC system (Agilent Technologies Waldbronn Ger-many) equipped with a high performance autosamplerbinary pump and PDA detector G 1314 C (SL) Chromato-graphic separation was performed on a Superspher 100 RP-18 (75 times 4mm id 4 120583m) column (Merck DarmstadtGermany)

24 Identification of Phenolic Compounds of Aqueous EthanolExtract of Schotia brachypetala Stalk by LC-HRESI-MS-MSThe solvents were (A) 2 acetic acid (pH 26) and (B) 80methanol 2 acetic acid and pH 26 The gradient elutionwas from 5 to 50 B at 30∘C at a flow rate of 100 120583LminThe ionization technique was an ion spray (pneumaticallyassisted electrospray) The mass spectrometer was operatedin the negativemode Negativemode conditions were appliedto the instrument as follows capillary voltage 4000V endplate offset minus500V heated dry nitrogen gas temperature200∘C at flow rate 10 Lmin the gas flow to the nebulizerwas set at pressure 16 bar For collision-induced dissociation(CID) MS-MS measurements the voltage over the collisioncell varied from 20 to 70 eV Argon was used as collisiongas Data analysis software was used for data interpretationSodium formate was used for calibration at the end of LC-MSrun Interpretation for ESI-MSwas performed by Xcalibur 21software fromThermo Scientific (Berlin Germany)

25 Animals Adult male Swiss albino mice weighing from20 to 30 g each were used They were brought from theNational Institute of Research in Egypt All animals werehoused in alternating 12 h darknight cycles at suitable roomtemperature one week before experimental interventionThey had unrestricted access to food and water throughoutthe period of investigation All experiments were performedunder the anesthesia with 2 isoflurane to minimize thesuffering Animal procedures were approved by the EthicsCommittee at the German University in Cairo in agreementwith recommendations of the National Institute of HealthGuide for Care and Use of Laboratory Animals

26 Experimental Protocol Alzheimerrsquos disease was inducedby intraperitoneal injections of Lipopolysaccharide (08mgkg) once [19] Green tea extract from leaves ofCamellia sinen-sis was used as the reference standard Doses and vehiclesused to dissolve drugs were chosen based on the literatureThe extracts and piceatannol were dissolved in DMSO andthen completed to final volume with saline [20] The mice

BioMed Research International 3

were divided into groups of 8ndash10 mice each receiving ipinjections after being weighed LPS group (Group I) received01mL of 08mgkg LPS ip injections once [19] The normalgroup (Group II) received 01mL DMSO or saline ip injec-tions once The green tea treated group (Group III) received08mgkg LPS ip injection once followed by 100mgkgdaygreen tea extract from Camellia sinensis [21] for 6 days TheSchotia brachypetala leaves treated group (Group IV) received08mgkg LPS ip injection once followed by 100mgkgday[22] Schotia brachypetala leaves extract for 6 days TheSchotia brachypetala stalk treated group (Group V) received08mgkg LPS ip injection once followed by 100mgkgday[22] Schotia brachypetala stalk extract for 6 daysMarkhamiaplatycalyx leaves treated group (GroupVI) received 08mgkgLPS ip injection once followed by 100mgkgday [23]Markhamia platycalyx extract for 6 days Piceatannol treatedgroup (Group VII) received 08 mgkg LPS ip injection oncefollowed by 25mgkgday [6] piceatannol for 6 days Y mazeand object recognition were performed as in vivo tests onthe 7th day to evaluate the extracts and piceatannolrsquos anti-ADpotential and confirmedby ex vivo tests After the in vivo teststhe mice brains were harvested Each brain was divided into2 hemispheres and frozen at minus80 degrees Celsius to be laterhomogenized and used for ex vivo ELISA analysis

27 Object Recognition Test Object recognition test wasperformed in accordance with the study done by [24] Thenatural propensity of animals to explore novel objects morerelative to familiar ones is used as an index of stimulusrecognition The test involves a ldquosamplerdquo phase in which ato-be-remembered object is encountered followed by a delayand then a test phase in which the sample object is presentedtogether with a novel object Discrimination ratio is theratio of attempts towards the new object against the old oneExploratory attempt is defined as the animal directing its nosetowards the object at a distance of 2 cm or less Climbingon object was not considered an exploration Increase indiscrimination ratio is indicative that the mouse is exploringthe new object more

It was calculated by the following equation

Discrimination ratio

=Number of attempts of animal to novel object

Total number of attempts of the animal to both objects(1)

see [25]

28 Y Maze Test Ymaze was carried out in accordance withthe study done by [26] An animalmust rememberwhich armit had entered on a previous occasion to enable it to alternateits choice on a following trial In recent years spontaneousalternation behaviour has been enthusiastically embracedby behavioral pharmacologists and others as a quick andrelatively simple test ofmemory [27]The test is carried out inYmaze shaped apparatuswith three arms Each arm is labeledeither A B or C The test consists of two phases with thefirst being the training phase The mouse is allowed to move

freely through the maze for 8 minutes 24 hours later themouse is allowed to move for 8 minutes and its movementsare recorded Every time the mouse enters an arm with allof its limbs inside its letter is written down The number ofalternations means the successive entries into three differentarms in overlapping triplet sets [28] (eg ABCBACA =3) Total arm entries are simply the total number of armsentered (eg ABCBACA= 7)The percentage alternationwascalculated according to [28] using the following formula

number of alternations(Total arm Entries minus 2)

times 100 (2)

29 Tissue Sampling After performing the behavioural testseach brain was split into 2 hemispheres and placed in aneppendorf tube and weighed and then stored at minus80∘C Thebrain was used later for homogenization using appropriatevolume of PBSprotease inhibitor and guanidinetris HClsolution to bring final concentration of guanidine to 5MThis solution was added in aliquots of 100 120583L while grindingthe hemisphere The homogenates were mixed for 3-4 hoursat room temperature At this stage the samples were stableand can be freeze-thawedmany timesThey were frozen untilassay of amyloid beta 42 levels with ELISA (Invitrogen USA)[29]

210 ELISA and Measurement of Amyloid Beta 42 Sampleswere centrifuged at 16000 g for 20min at 4∘C Supernatantwas diluted to bring the final guanidine concentration to01M using Dulbeccorsquos phosphate buffered saline with 5BSA and 003 Tween-20 The supernatant was stored onice until assay of amyloid beta 42 levels The Mouse A12057342levels were assayed with solid phase sandwich enzyme linkedimmunosorbent assay (ELISA) according to manufacturerrsquosinstructions [29]

211 Statistical Analysis Statistical analysis was performedusing instant automated software (Graph Pad Prism softwareversion 501 Inc San Diego California USA) Results wereexpressed as mean plusmn standard error of mean (SEM) Resultsfrom behavioural tests were analyzed using unpaired 119905-testto compare the two selected groups of Y maze or objectrecognition test The level of significance was set at 119875 = 005with 119875 lt 005 indicating significant change The confidenceinterval was fixed at 95 Graphical representation of resultswas conducted using Graph Pad Prism 5 software

3 Results

31 In Vivo Results

311 Effects of Lipopolysaccharide Camellia sinensis Picea-tannol Markhamia platycalyx and Schotia brachypetalaLeaves and Schotia brachypetala Stalk on the Mean Alterna-tion Percentage Using Y Maze Test Administration of LPS(08mgkg ip) then on the 7th day subjecting mice to Ymaze showed a significant decrease in the mean percentagealternations by 4225 compared to normal group Themean percentage alternations were significantly increased in

4 BioMed Research International

Table 1 Peak assignment mass spectrometry detection parameters and tentative identification of compounds in aqueous alcohol stalksextract of Schotia brachypetala by LC-ESI(-ve)-MS

Peaknumber Identified compounds 119905

119877

(min) [M-H]minus (119898119911) Fragment ions (119898119911) Peak area References

Sb1 Daidzein 176 253 253 13 [13]Sb2 Unidentified 122 495 253 25Sb3 Procyanidin isomer B1B2 1579 577 425 407 65 [14]Sb4 Naringin 1876 579 459 271 52 [14]Sb5 Procyanidin isomer B1B2 2377 577 425 407 48 [14]Sb6 Procyanidin dimer gallate 2979 729 577 62 [3]Sb7 Unidentified 3884 497 441 253 32Sb8 Quercetin 3-O-glucoronide 4207 477 301 151 56 [15]Sb9 Quercetin hexose gallic acid 4682 615 463 301 155 [16]Sb10 Quercetin hexose gallic acid 4763 615 463 301 10 [16]Sb11 Unidentified 4882 463 298 253 83Sb12 Unidentified 508 495 239 220 66Sb13 Quercetin hexose-protocatechuic acid 52 599 463 300 72 [17]Sb14 Quercetin 3-O-rhamnoside 5704 447 301 92 [18]Sb15 Ellagic acid 6464 301 257 229 41 [18]

animals that were treated with piceatannol (25mgkgday)Camellia sinensis leaves (100mgkgday) Markhamia plat-ycalyx leaves (100mgkgday) Schotia brachypetala leaves(100mgkgday) and Schotia brachypetala stalk (100mgkgday) for 6 days by 6878 3974 7214 409 and5785 respectively compared to LPS group (Figure 2)

312 Effects of Lipopolysaccharide Camellia sinensis Picea-tannol Markhamia platycalyx and Schotia brachypetalaLeaves and Schotia brachypetala Stalks on the Mean Discrimi-nation Ratio Using Object Recognition Test Administrationof 08mgkg LPS and then subjecting animals to objectrecognition test on 7th day showed significant decrease inmean discrimination ratio by 4616 when compared to thenormal group

Discrimination ratio was significantly increased in ani-mals that were treated with piceatannol (25mgkgday)Camellia sinensis (100mgkgday) Markhamia platycalyx(100mgkgday) and Schotia brachypetala leaves (100mgkgday) and Schotia brachypetala stalk (100mgkgday) for 6days of treatment by 645 6875 83 6825 and 65respectively when compared with LPS group (Figure 3)

32 Ex Vivo Results

321 Effects of Lipopolysaccharide Camellia sinensis Picea-tannol Markhamia platycalyx and Schotia brachypetalaLeaves and Schotia brachypetala Stalk on the Mean MouseAmyloid Beta 42 Concentration Using ELISA Assay Meanamyloid beta 42 present in mouse brains was evaluated bymouse A12057342 ELISA kit LPS group showed significantlyhigher concentration of amyloid beta when compared tonormal group by almost 2-fold

Themean amyloid beta 42 concentrationwas significantlyreduced in animals that were treated with Camellia sinensis

(100mgkgday) Piceatannol (25mgkgday) Markhamiaplatycalyx (100mgkgday) and Schotia brachypetala leaves(100mgkgday) and Schotia brachypetala stalk (100mgkgday) for 6 days by 726 737 751 762 and 728respectively when compared to LPS group (Figure 4)

4 Discussion

Hyphenated HPLC-MS technique is an important methodused for identifying complex mixtures especially the pheno-lics in the crude extracts or its fraction found in the planteither by using standard compounds (cochromatography) orby comparing mass spectrum obtained with the literature(tentative identification) [16] In this part of the studyaqueous alcohol extract of Schotia brachypetalawas subjectedto HPLC-ESI-MS analysis aiming at developing a robust LC-ESI-MSn method in the first instance for the identification ofmajor compounds within this extract (Figure 1)

The obtained data (Table 1) was interpreted as followsThe negative ion mode LC-ESI-MSn showed major peaks

(Sb9 and Sb10) which were previously identified in [16] asquercetin hexose gallic acid and its sugar isomer (Sb9 andSb10) fragments showed deprotonated molecule [M-H]minus mz615 the fragment peak ion corresponding to the deprotonatedquercetin hexoside (mz 463) and deprotonated quercetinmolecule at (mz 301) [16] the appearance of two peaks hav-ing the samemolecular ion and fragmentation pattern assuresthe presence of the same compound having two different(hexose) isomers the identification of the sugar moiety couldbe further confirmed using NMR spectroscopical analysis[30]

Compound (Sb8) showed the product ion spectrumof quercetin-3-O-glucuronide known as miquelianin withdeprotonated molecule (mz 477) and MS2 at mz 301 a dif-ference of 176 indicates glucuronic acid moiety besides the


Y maze

0

20

40

60

80

100

Groups

Mea

n pe

rcen

tage

of a

ltera

tions

+ +

++

++

+++

lowastlowastlowast

Nor

mal

LPS

LPS+

GT

LPS+

PCT

LPS+

MP

LPS+

SBS

LPS+

SBL

Figure 2 Effects of Lipopolysaccharide green tea piceatannolMarkhamia platycalyx and Schotia brachypetala leaves and Schotiabrachypetala stalks on mean alternation percentage using Y mazetest Normal group animals were injected with DMSO or saline09 ip once with nonsignificant results between themThe 6 othergroups had Alzheimerrsquos disease that was induced by ip injectionof LPS once Five of these groups were treated with either PCT(25mgkg) or 100mgkg of GT MP SBS or SBL daily for 6 daysThe animals of each group (119899 = 8ndash10) were subjected to Y mazetesting on the 7th day and the sequence of arm entries was recordedfor 8 minutes for every mouse Then the percentage alternationswere calculated for each mouse Statistical analysis was performedusing unpaired 119905-test to compare every two groups Each valuerepresents mean plusmn standard error of mean lowastSignificantly differentfrom normal at 119875 lt 005lowastlowastSignificantly different from normal at119875 lt 001 lowastlowastlowastSignificantly different from normal at 119875 lt 0001+Significantly different from LPS at 119875 lt 005 ++Significantlydifferent from LPS at 119875 lt 001 +++Significantly different fromLPS at 119875 lt 0001 MPA mean alternation percentage DMSOdimethyl sulfoxide LPS Lipopolysaccharide GT green tea extractof Camellia sinensis PCT piceatannol SBS Schotia brachypetalaleaves SBL Schotia brachypetala stalk MP Markhamia platycalyxip intraperitoneal injection

MS3 at mz 151 of ring A in the quercetin aglycone provingthat it is quercetin aglycone and not ellagic acid that isassociated with these compounds [15]

Furthermore the deprotonated ion of (Sb14) showedfragment at (mz) 447 identified as quercetin 3-O-rhamnoside similar to what was reported previously [31]The result of hemolytic cleavage of the O-glycosidic bondrenders a radical aglycone (mz 301) the ion referring toquercetin ion The fragment ion at mz 301 is formed by lossof the glucose or galactose moiety from the glycosides Noions characteristics of the sugar part were observed in thenegative ion mode [32]

Compound (Sb13) showedmolecular ion peak atmz 599with fragmentsmz 463 andmz 300The difference betweenions at mz 599 of 463 and 300 revealed the loss of twoconsecutive ions with 162 and 136 mass units respectively

00

02

04

06

08

10Object recognition

Mea

n di

scrim

inat

ion

ratio

Groups

++++++

+++

++++++

lowastlowastlowast

Nor

mal

LPS

LPS+

GT

LPS+

PCT

LPS+

MP

LPS+

SBS

LPS+

SBL

Figure 3 Effects of Lipopolysaccharide green tea piceatannolMarkhamia platycalyx and Schotia brachypetala leaves and Schotiabrachypetala stalk on the mean discrimination ratio using objectrecognition test Normal group animals were injected with DMSOor saline 09 ip once with nonsignificant results between themThe 6 other groups had Alzheimerrsquos disease that was induced byip injection of LPS once Five of these groups were treated witheither PCT (25mgkg) or 100mgkg of GT MP SBS or SBL dailyfor 6 days The animals of each group (119899 = 8ndash10) were subjectedto object recognition testing on the 7th day testing and explorationattempts towards each object within 4 minutes were recordedfor each mouse Then the discrimination ratio was calculated foreach mouse Statistical analysis was performed using unpaired 119905-test to compare every two groups Each value represents mean plusmnstandard error of mean lowastSignificantly different from normal at119875 lt 005 lowastlowastSignificantly different from normal at 119875 lt 001lowastlowastlowastSignificantly different from normal at 119875 lt 0001 +Significantlydifferent from LPS at 119875 lt 005 ++Significantly different fromLPS at 119875 lt 001 +++Significantly different from LPS at 119875 lt0001 MDRmean discrimination ratio DMSO dimethyl sulfoxideLPS Lipopolysaccharide GT green tea extract of Camellia sinensisPCT piceatannol SBS Schotia brachypetala leaves SBL Schotiabrachypetala stalk MP Markhamia platycalyx ip intraperitonealinjection

which is characteristic to hexose sugar and protocatechuicacid Then this compound could be identified as quercetinhexose-protocatechuic acid isomer [17] In addition the twomajor peaks (Sb3 and Sb5) having the same deprotonatedions ([M-H]minus) atmz 577 identified tentatively as procyanidinisomer gave [M-H-152]minus fragments ions at mz 425 fromRetro-Diels-Alder (RDA) rearrangement of the heterocyclicring and atmz 407 ([M-H-170]minus) from the rearrangement ofthe heterocyclic ring and loss of H

2O [14]

Consequently the identification of these two peaks (Sb3amp Sb5) gave further evidence to presence of the majorcompound (Sb6) recognized as procyanidin dimer gallatehaving itsmajormolecular ion atmz 577 [3 14] Additionallythe ESI-MS-MS spectra of compound (Sb6) with [M-H]minus atmz 729 showed the fragment ion at mz 577 this could beattributed to the ion arising from the loss of galloyl moiety


0

2

4

6

Mea

n am

yloi

d be

ta42

(pg

mL)

ELISA assay of amyloid beta 42

Groups

++++ +++ +++ +++

lowastlowastlowastN

orm

al

LPS

LPS+

GT

LPS+

PCT

LPS+

MP

LPS+

SBS

LPS+

SBL

Figure 4 Effects of Lipopolysaccharide green tea piceatannolMarkhamia platycalyx and Schotia brachypetala leaves and Schotiabrachypetala stalk on mean mouse amyloid beta 42 concentrationusing ELISA assay The normal group was injected with saline 09or DMSO ip once with nonsignificant results between them Allthe other groups were injected with LPS 08mgkg once to induceAlzheimerrsquos disease Five of them were treated starting 1st day afterLPS intraperitoneal injection with PCT 25mgkg or 100mgkgdayGT MP SBS or SBL for 6 days The brains of all the animalsin each group were harvested on the 7th day and preserved untilamyloid beta 42 concentration was assayed Statistical analysis wascarried out using unpaired 119905-test to compare every two groupsEach value represents mean plusmn standard error of mean lowastSignificantlydifferent from normal at 119875 lt 005 lowastlowastSignificantly different fromnormal at 119875 lt 001 lowastlowastlowastSignificantly different from normal at119875 lt 0001 +Significantly different from LPS at 119875 lt 005++Significantly different from LPS at 119875 lt 001 +++Significantlydifferent from LPS at 119875 lt 0001 LPS Lipopolysaccharide GTgreen tea extract ofCamellia sinensis PCT piceatannol SBS Schotiabrachypetala leaves SBL Schotia brachypetala stalkMPMarkhamiaplatycalyx DMSO dimethyl sulfoxide PgmL picogrammilliliterA120573 amyloid beta ELISA enzyme linked immunosorbent assay ipintraperitoneal injection

from the procyanidin dimer having a mass unit as [galloyl-OH]minus Therefore it is identified as procyanidin dimer gallate[3]

Moreover LC-MS-MS method was also used to dis-tinguish between conjugates of quercetin and ellagic acidsince their aglycones produce identical molecular ions onfragmentation both giving the same base peak mz 301[18] On MSMS analyses the quercetin mz 301 ion furtherfragments to form characteristicmz 179 and 151 ions whereasthe equivalent ellagic acid mz 301 ion yields ions at mz 257and 229 The use of LC-MS-MS methods is therefore usefulto differentiate between ellagic acid and quercetin aglyconesthus the mz 257 and 229 ions in the MS-MS analysisshowed that an ellagic acid moiety and not quercetin wasassociated with compound shown in peak (Sb15) Compound

(Sb1) showed molecular ion peak at (mz) 253 identified asdaidzein [13]

In this study LPS injected at a dose of 08mgkg resultedin significant changes in mice working memory This wasproven by Y maze where the percentage spontaneous alter-nations significantly decreased compared to normal miceThese results are in accordance with a study which showedthat the injection of LPS had a disruptive effect on theability of mice to learn the Y maze [33] Using the Morriswater maze task LPS-treated mice took a longer time toreach the hidden platform than normal mice In additioninjection of LPS decreased the percent of correct choicesin the Y maze test [34] LPS also successfully inducedmemory impairment in the Y maze test neuroinflammatoryresponses and oxidative stress such as increases in mRNAlevels of interleukin (IL)-1120573 and IL-6 heme oxygenase-1microglial activation and iNOS activity in hippocampus[28] Neuroinflammation and oxidative stress are significantcomponents of the pathogeneses of AD Therefore thesechanges are successful in inducing learning and memoryimpairment in rats In this study intraperitoneal injectionof LPS at dose of 08 mgkg significantly reduced discrimi-nation ratio in object recognition test when compared withnormal mice These results are similar to a study wherespatial learning and object recognition memory deficits wereobserved in animals dosed with the increasing LPS doseregime [35] In another study there was significant decreaseof cognition function 4 days after systemic injection of LPSanalyzed by object recognition test [36] It has been foundthat amyloid beta 42 is more toxic than amyloid beta 40[37] In this study LPS injected mice showed significantlyhigher concentrations of amyloid beta 42 compared withnormal mice These results are in agreement with anotherstudy inwhich transgenicAPPswemicersquos Abeta1-4042was 3-fold higher when compared to normal as detected by ELISAwestern blots and immunoprecipitation-mass spectrometry(IP-MS) ProteinChip analysis [38] In a study done by [39]LPS was able to induce long lastingmodifications in behaviorand brain protein levels of TNF-120572 TNF-120572 levels after sevendays of LPS exposure were the same as those after 10 monthsthese results suggest that LPS reaches its peak effect after 7days of its injection after which it reaches a plateau

Another study in harmony with this one showed adecrease in preference of the novel object after LPS injection[28] in which intraperitoneal injection of green tea extractresulted in significantly higher discrimination ratio in objectrecognition test compared with nontreated LPS mice In thisstudy treatment of LPS injected mice with green tea phenolicrich extract significantly increased their spontaneous alterna-tions percentage in the Y maze test compared to nontreatedmice This is in accordance with a study in which long-termadministration of green tea catechins to mice also suppressedcognitive dysfunction by assessment of working memoryin the Y maze [40] Administration of green tea catechinsimproved spatial cognition learning ability in rats assessedby partially baited 8-arm radial maze and was thought tobe due to green tea catechins involved in protecting againstneuronal degenerative stress and in the accumulation of lipidperoxides (LPO) and reactive oxygen species (ROS) Tea is


rich in polyphenols contained in the leaves and stems of thetea plant EGCG the major and most active component ofgreen tea catechins acts as an antioxidant in the biologicalsystem and is rapidly absorbed and distributed mainly intothe mucous membranes of the small intestine and the livermore interestingly it can cross the blood brain barrier Itwas found that oxidative stress-induced neuronal apoptosisis prevented by EGCG treatment of neuronal cells [41] Theworkingmemory tested using Ymaze was improved inmicefed a high-fat diet containing green tea catechin [42] EGCGtreatment significantly improved results of the Y maze instreptozotocin diabetic rats [43] In this study intraperitonealinjections of green tea extract caused significantly decreasedlevels of amyloid beta 42 compared with LPS mice This maybe due to the ability of EGCG to increase alpha secretaseactivity (the nonamyloidogenic pathway of APP processing)as discovered in a transgenic Alzheimerrsquos disease mousemodel [44]

In this study piceatannol administered to mice producedsignificantly higher percentage spontaneous alternations inthe Y maze compared to nontreated mice Piceatannol(trans-343101584051015840-tetrahydroxystilbene) is a naturally occurringhydroxylated analogue of resveratrol Tetrahydroxy stilbeneglucoside treatment in aged rats showed a remarkableimprovement in their learning andmemory function as notedby a marked decrease in the escape latency time an increasein the time spent in the target quadrant and an increase inthe times of crossing the quadrant where the platform waspreviously placed in the Morris water maze test [45] A studyinvestigating the possible protective effects of piceatannolon amyloid beta-induced PC12 neuronal cell death foundthat piceatannol exerted much stronger protective effectsthan resveratrol did Piceatannol treatment attenuated theintracellular accumulation of ROS induced by treatmentof PC12 cells with amyloid beta inhibited amyloid beta-induced apoptotic features including internucleosomal DNAfragmentation nucleus condensation cleavage of poly(ADP-ribose) polymerase (PARP) and activation of caspase-3These results suggest that piceatannol blocks amyloid beta-induced accumulation of ROS thereby protecting PC12 cellsfrom oxidative stress [46] In this study intraperitonealinjection of piceatannol resulted in significantly higher dis-crimination ratio in the object recognition test compared toLPS nontreated mice In another study in which the effectof piceatannol to stop apoptosis of cells was investigated (asapoptosis plays an important role in Alzheimerrsquos disease)piceatannol inhibited 4 hydroxynonenal induced apoptosisof PC12 cells It inhibited the phosphorylation of c-Jun N-terminal kinase a key regulator of HNE-induced PC12 celldeath [47] This can explain why piceatannol treated micehad greater discrimination ratio In this study intraperitonealinjections of piceatannol were able to significantly reduceamyloid beta 42 compared with normal mice Piceatannoldecreased amyloid beta 1ndash42 induced fluorescence in a TH-tfluorescence assay [7] It is able to inhibit the toxic effect ofamyloid beta on cells which causes neuronal cell death [46]

Two phenolic rich extracts of Schotia brachypetala andMarkhamia platycalyx were investigated for the first time inthis studyTheir administration to LPS injected mice resulted

in higher percentage spontaneous alternations comparedto nontreated mice in the Y maze Polyphenols influencemental health and cognition namely via energy metabolismand modification of signaling pathways and gene expressioninvolved in the ability of a neuron to strengthen and changesynaptic connections In addition to their antioxidant andanti-inflammatory activity polyphenols have been coupledwith the increased expression of BDNF (a neurotrophinknown for its influence on themaintenance survival growthand differentiation of neurons) assisting in the reversalof neuronal atrophy and behavior deficits [48] Polyphenolrich foods spinach strawberry and blueberry extracts wereeffective in reversing cognitive deficits in Morris water mazeperformance function among aged rats Effects of blueberrieson both motor and cognitive behavior might involve actionsother than antioxidant or antiinflammatory activities Severalstudies showed that at least some of these actionsmay includealterations in signaling [49] Pomegranate containing highlevels of polyphenols including ellagic acids administeredto transgenic mice with a mutated APP gene resulted inimproved performance in cued and spatial learning taskscompared to sugar water controls Grape juice containinghigh levels of flavonoids when administered to rats improvedcognitive performance inMorris water maze [50] Silibinin apolyphenol belonging to the flavonoid class was able to over-come the impairment caused by injected amyloid beta A12057325-35-injected mice showed significantly reduced spontaneousalternation behaviour compared with vehicle-injected miceTreatment with silibinin dose-dependently attenuated theimpairment of spontaneous alternation behaviour in A12057325-35-injected mice [51] In this study mice receiving phenolicrich injections of Markhamia platycalyx and Schotia brachy-petala extracts produced significantly higher discriminationratio in the object recognition tests compared to nontreatedmice In a model of amyloid beta deposition the polyphenololeuropein aglycone improved cognition assayed by objectrecognition test [52] In this study intraperitoneal injectionsof phenolic extracts of Markhamia platycalyx and Schotiabrachypetalawere able to significantly reduce amyloid beta 42compared to LPS mice From the compounds that were iden-tified in Schotia brachypetala in this study extracts of flavan-3-ols procyanidin B isomers (Sb3 Sb5) have been shown tobe potentially useful in the treatment of degenerative diseasesassociated with oxidative stress as Alzheimerrsquos disease Thiswas proven in a study in which they were able to preventlipid and protein oxidative damage in the cerebral cortexcerebellum and hippocampus tissues of rats [53] Moreoverprocyanidins significantly suppressed A12057342 aggregation andwere able to break apart A12057342 aggregates in a dose-dependentmanner [54] From compounds identified in Schotia brachy-petala in this study daidzein (Sb1) is an isoflavone which hasbeen associatedwith a decreased risk of hormones dependentneurodegenerative diseases [13] One of the hypotheses ofAlzheimerrsquos disease involves oxidative stress The antioxi-dant activity of procyanidin dimers (Sb6) (identified fromSchotia brachypetala) containing the interflavan bond C4ndashC8and C4ndashC6 was very significant suggesting that interflavanlinkage contributes importantly to the antioxidant activity ofprocyanidin dimers [55] Ellagic acid (Sb15) (also identified


from Schotia brachypetala) can have therapeutic potential inAlzheimerrsquos disease as it was able to significantly reduceAbeta42 induced neurotoxicity towards SH-SY5Y cells (humanderived cell line) [56] Quercetin 3-O-glucuronide (identifiedfrom Schotia brachypetala) greatly reduced the generationof 120573-amyloid (A120573) peptides by primary neuron culturesgenerated from the Tg2576 AD mouse model Quercetin 3-O-glucuronide (Sb8) is also capable of interfering with theinitial protein-protein interaction of A120573(1ndash40) and A120573(1ndash42)that is necessary for the formation of neurotoxic oligomericA120573 species Moreover quercetin 3-O-glucuronide treat-ment compared to vehicle-control treatment significantlyimproved AD-type deficits in hippocampal formation basalsynaptic transmission and long-term potentiation possiblythrough mechanisms involving the activation of the c-Jun N-terminal kinases and the mitogen-activated protein kinasesignaling pathways Therefore quercetin 3-O-glucuronideis an effective intervention for AD [57] Pretreatment ofprimary hippocampal cultures with quercetin significantlyattenuated Abeta (1ndash42) induced cytotoxicity protein oxida-tion lipid peroxidation and apoptosis Therefore quercetingreatly protects neuronal cells from oxidative stress-inducedneurotoxicity [58] In another study phenolic glycosides fromMarkhamia stipulata were isolated mainly being verbasco-sides [10] Verbascosides are phenylpropanoids that havecytoprotective action connected to their antioxidant andchelating capacity They were found protective against 1-methyl 4 phenylpyridine ion induced neurotoxicity in cul-tured neurons They attenuated neuronal apoptosis caspase3 activation and the collapse of mitochondrial membranepotential This data is proof that Markhamia genus can havea therapeutic potential for treating oxidative stress relatedneurodegenerative disease [11] and supports the positiveresults in this study

Finally binding and autoradiographic studies revealedthe existence of specific polyphenols binding sites in the ratbrain in particular in the choroid plexus Structure-activitydata support the hypothesis that these specific bindingsites may be responsible for the neuroprotective actions ofpolyphenols [7]

5 Conclusion

The results of this study provide evidence that the pheno-lic rich extracts of Schotia brachypetala Camellia sinensisMarkhamia platycalyx and piceatannol have high potential tobe anti-Alzheimerrsquos disease drug leads The pharmacologicalscreening indicated in the Y maze test that all the drugs hadsignificantly higher spontaneous alternation percentage thanthe nontreated LPS injected mice In the object recognitiontest all the drugs had significantly higher discrimination ratiothan the nontreated LPS injected mice as well The drugswere also able to significantly decrease the amyloid beta 42burden in mice that were treated compared to nontreatedLPS injected mice as detected by ELISA The identificationof compounds procyanidin isomers daidzein naringin pro-cyanidin dimer gallate quercetin 3-O-glucoronide quercetinhexose gallic acid quercetin hexose-protocatechuic acidquercetin 3-O-rhamnoside and ellagic acid seems to be

limited within the complexity of the extract but this studyis directing towards the major compounds in the extract andto be correlated with the biological activity the informationprovided by our study will aid in the evaluation of thebiological importance of Schotia brachypetala consumptionon human health

Further work would involve the identification and iso-lation of the identified compounds to further enrich theliterature concerning such a plant by other tools such as LC-UV and nuclear magnetic resonance (NMR) Further workwould also involve the investigation of the mechanisms ofactions of these compounds and physiochemical identifica-tion of polyphenolic compounds in Markhamia platycalyxPharmacological screening using other methods such aswestern blot can further confirm the relative pharmacologicalactivity of the extracts

Abbreviations

AD Alzheimerrsquos diseaseCNS Central nervous systemAch AcetylcholineAchE AcetylcholinesteraseDMSO DimethylsulfoxideLPS LipopolysaccharideLC-HRESI-MSn Liquid chromatography high

resolution electrospray ionizationmass spectrometry

Conflict of Interests

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

References

[1] M T Ferretti and A C Cuello ldquoDoes a pro-inflammatoryprocess precede Alzheimerrsquos disease andmild cognitive impair-mentrdquo Current Alzheimer Research vol 8 no 2 pp 164ndash1742011

[2] J Kim H J Lee and K W Lee ldquoNaturally occurring phyto-chemicals for the prevention of Alzheimerrsquos diseaserdquo Journal ofNeurochemistry vol 112 no 6 pp 1415ndash1430 2010

[3] R Flamini ldquoRecent applications of mass spectrometry in thestudy of grape and wine polyphenolsrdquo ISRN Spectroscopy vol2013 Article ID 813563 45 pages 2013

[4] F Guedj C Sebrie I Rivals et al ldquoGreen tea polyphenols rescueof brain defects induced by overexpression of DYRK1Ardquo PloSONE vol 4 no 2 Article ID e4606 2009

[5] C Riviere T Richard L Quentin S Krisa J Merillon andJ Monti ldquoInhibitory activity of stilbenes on Alzheimerrsquos 120573-amyloid fibrils in vitrordquo Bioorganic and Medicinal Chemistryvol 15 no 2 pp 1160ndash1167 2007

[6] J W Lee Y K Lee J O Ban et al ldquoGreen tea (-)-epigallocat-echin-3-gallate inhibits 120573-amyloid-induced cognitive dysfunc-tion through modification of secretase activity via inhibition ofERKandNF-120581Bpathways inmicerdquo Journal of Nutrition vol 139no 10 pp 1987ndash1993 2009


[7] S Bastianetto Y Dumont and R Quirion ldquoCatechins andresveratrol as protective polyphenols against beta-amyloid-induced toxicity possible significance to Alzheimerrsquos diseaserdquoRecent Advances on Nutrition and the Prevention of AlzheimerrsquosDisease vol 661 no 2 pp 145ndash154 2010

[8] G I Stafford P D Pedersen A K Jager and J Van StadenldquoMonoamine oxidase inhibition by southernAfrican traditionalmedicinal plantsrdquo South African Journal of Botany vol 73 no3 pp 384ndash390 2007

[9] P Riederer L Lachenmayer and G Laux ldquoClinical applicationsofMAO-inhibitorsrdquoCurrentMedicinal Chemistry vol 11 no 15pp 2033ndash2043 2004

[10] T Kanchanapoom R Kasai and K Yamasaki ldquoPhenolic glyco-sides fromMarkhamia stipulatardquo Phytochemistry vol 59 no 5pp 557ndash563 2002

[11] L G Korkina ldquoPhenylpropanoids as naturally occurringantioxidants from plant defense to human healthrdquo Cellular andMolecular Biology vol 53 no 1 pp 15ndash25 2007

[12] H Handoussa N Osmanova N Ayoub and L Mahran ldquoSpi-catic acid a 4-carboxygentisic acid from Gentiana spicataextract with potential hepatoprotective activityrdquoDrugDiscoveryTherapy vol 3 no 6 pp 278ndash286 2009

[13] D Hanganu L Vlase and N Olah ldquoLCMS analysis ofisoflavones from fabaceae species extractsrdquo Farmacia vol 58no 2 pp 177ndash183 2010

[14] J Sun F Liang Y Bin P Li and C Duan ldquoScreening non-colored phenolics in red wines using liquid chromatog-raphyultraviolet and mass spectrometrymass spectrometrylibrariesrdquoMolecules vol 12 no 3 pp 679ndash693 2007

[15] N P Seeram R Lee H S Scheuller and D Heber ldquoIden-tification of phenolic compounds in strawberries by liquidchromatography electrospray ionization mass spectroscopyrdquoFood Chemistry vol 97 no 1 pp 1ndash11 2006

[16] L S Chua N A Latiff S Y Lee C T Lee M R Sarmidi andR A Aziz ldquoFlavonoids and phenolic acids from Labisia pumila(Kacip Fatimah)rdquo Food Chemistry vol 127 no 3 pp 1186ndash11922011

[17] E S S Abdel-Hameed S A Bazaid and M S SalmanldquoCharacterization of the phytochemical constituents of Taif roseand its antioxidant and anticancer activitiesrdquo BioMed ResearchInternational vol 2013 Article ID 345465 13 pages 2013

[18] W Mullen T Yokota M E J Lean and A Crozier ldquoAnalysisof ellagitannins and conjugates of ellagic acid and quercetin inraspberry fruits by LC-MSnrdquo Phytochemistry vol 64 no 2 pp617ndash624 2003

[19] N S El Sayed L A Kassem and O A Heikal ldquoPromisingtherapy forAlzheimerrsquos disease targeting angiotensinconvertingenzyme and the cyclooxygense-2 isoformrdquo Drug Discoveriesand Therapeutics vol 3 no 6 pp 307ndash315 2009

[20] A E Khalifa ldquoHypericum perforatum as a nootropic drugEnhancement of retrieval memory of a passive avoidanceconditioning paradigm inmicerdquo Journal of Ethnopharmacologyvol 76 no 1 pp 49ndash57 2001

[21] Z Zahedifar and J Baharara ldquoEffect of green tea extract inreducing genotoxic injuries of cell phone microwaves on bonemarrowrdquo Zahedan Journal of Research in Medical Sciences vol15 no 11 pp 39ndash44 2013

[22] A Pandey S Sharma and P Chaturvedi ldquoAntipyretic activity ofPongamia pinnata in ratsrdquo IJPIrsquos Journal of Pharmacognosy andHerbal Formulations vol 2 article 10 2012

[23] J M Makinde O O G Amusan and E K AdesoganldquoThe antimalarial activity of Spathodea campanulata stem barkextract onPlasmodiumberghei berghei inmicerdquoPlantaMedicavol 54 no 2 pp 122ndash125 1988

[24] S N Burke J L Wallace S Nematollahi A R Uprety and CA Barnes ldquoPattern separation deficits may contribute to age-associated recognition impairmentsrdquo Behavioral Neurosciencevol 124 no 5 pp 559ndash573 2010

[25] S N Burke L Ryan and C A Barnes ldquoCharacterizing cog-nitive aging of recognition memory and related processes inanimal models and in humansrdquo Frontiers in Aging Neurosciencevol 4 article 15 2012

[26] N Hidaka K Suemaru K Takechi B Li and H ArakildquoInhibitory effects of valproate on impairment of Y-maze alter-nati onbehavior induced by repeated electroconvulsive seizuresand c-Fos protein levels in rat brainsrdquo Acta Medica Okayamavol 65 no 4 pp 269ndash277 2011

[27] R N Hughes ldquoThe value of spontaneous alternation behavior(SAB) as a test of retention in pharmacological investigations ofmemoryrdquo Neuroscience and Biobehavioral Reviews vol 28 no5 pp 497ndash505 2004

[28] MMiwaM Tsuboi Y Noguchi A Enokishima T Nabeshimaand M Hiramatsu ldquoEffects of betaine on lipopolysaccharide-induced memory impairment in mice and the involvement ofGABA transporter 2rdquo Journal of Neuroinflammation vol 8article 153 2011

[29] E Masliah E Rockenstein I Veinbergs et al ldquo120573-amyloid pep-tides enhance 120572-synuclein accumulation and neuronal deficitsin a transgenic mouse model linking Alzheimerrsquos disease andParkinsonrsquos diseaserdquo Proceedings of the National Academy ofSciences of theUnited States of America vol 98 no 21 pp 12245ndash12250 2001

[30] K S Chandrashekar B J Arun D Satyanarayana and E V SSubramanyam ldquoFlavonoid glycoside from Leucas lavandulae-folia (Rees) aerial partsrdquo Indian Journal of Chemistry B Organicand Medicinal Chemistry vol 45 no 8 pp 1968ndash1969 2006

[31] F Sanchez-Rabaneda O Jauregui I Casals C Andres-Lacueva M Izquierdo-Pulido and R M Lamuela-RaventosldquoLiquid chromatographicelectrospray ionization tandemmassspectrometric study of the phenolic composition of cocoa(Theobroma cacao)rdquo Journal of Mass Spectrometry vol 38 no1 pp 35ndash42 2003

[32] M N Clifford S Stoupi and N Kuhnert ldquoProfiling andcharacterization by LC-MSn of the galloylquinic acids of greentea tara tannin and tannic acidrdquo Journal of Agricultural andFood Chemistry vol 55 no 8 pp 2797ndash2807 2007

[33] J M Holden J B Overmier E T Cowan and L MatthewsldquoEffects of lipopolysaccharide on consolidation of partial learn-ing in the Y-mazerdquo Integrative Physiological and BehavioralScience vol 39 no 4 pp 334ndash340 2004

[34] K Arai N Matsuki Y Ikegaya and N Nishiyama ldquoDeterio-ration of spatial learning performances in lipopolysaccharide-treated micerdquo Japanese Journal of Pharmacology vol 87 no 3pp 195ndash201 2001

[35] W F Kuan Cognition and Potential Mechanisms InvolvedNational University of Singapore 2009

[36] M Jacewicz G A Czapski I Katkowska and R P Strosz-najder ldquoSystemic administration of lipopolysaccharide impairsglutathione redox state and object recognition inmalemiceTheeffect of PARP-1 inhibitorrdquo Folia Neuropathologica vol 47 no 4pp 321ndash328 2009


[37] I Kuperstein K Broersen I Benilova et al ldquoNeurotoxicity ofAlzheimerrsquos disease A120573 peptides is induced by small changes inthe A12057342 to A12057340 ratiordquoThe EMBO Journal vol 29 no 19 pp3408ndash3420 2010

[38] J G Sheng S H Bora G Xu D R Borchelt D L Price and VE Koliatsos ldquoLipopolysaccharide-induced-neuroinflammationincreases intracellular accumulation of amyloid precursor pro-tein and amyloid 120573 peptide in APPswe transgenic micerdquoNeurobiology of Disease vol 14 no 1 pp 133ndash145 2003

[39] P Bossu D Cutuli I Palladino et al ldquoA single intraperitonealinjection of endotoxin in rats induces long-lasting modifica-tions in behavior and brain protein levels of TNF-120572 and IL-18rdquoJournal of Neuroinflammation vol 9 article 101 2012

[40] K Unno F Takabayashi T Kishido and N Oku ldquoSuppressiveeffect of green tea catechins on morphologic and functionalregression of the brain in agedmice with accelerated senescence(SAMP10)rdquo Experimental Gerontology vol 39 no 7 pp 1027ndash1034 2004

[41] A M Haque M Hashimoto M Katakura Y Tanabe Y Haraand O Shido ldquoLong-term administration of green tea catechinsimproves spatial cognition learning ability in ratsrdquo Journal ofNutrition vol 136 no 4 pp 1043ndash1047 2006

[42] K Unno H Yamamoto K Maeda et al ldquoProtection of brainand pancreas from high-fat diet effects of catechin and caf-feinerdquo Physiology and Behavior vol 96 no 2 pp 262ndash269 2009

[43] T Baluchnejadmojarad and M Roghani ldquoChronic epigallo-catechin-3-gallate ameliorates learning and memory deficits indiabetic rats viamodulation of nitric oxide and oxidative stressrdquoBehavioural Brain Research vol 224 no 2 pp 305ndash310 2011

[44] A Mahler S Mandel M Lorenz et al ldquoEpigallocatechin-3-gallate a useful effective and safe clinical approach for targetedprevention and individualised treatment of neurological dis-easesrdquoThe EPMA Journal vol 4 no 1 p 5 2013

[45] Y Hou Q Yang L Zhou et al ldquoTetrahydroxystilbene glucosideimproves learning and (or) memory ability of aged rats andmay be connected to the APP pathwayrdquo Canadian Journal ofPhysiology and Pharmacology vol 89 no 11 pp 801ndash809 2011

[46] H J Kim K W Lee and H J Lee ldquoProtective effects ofpiceatannol against beta-amyloid-induced neuronal cell deathrdquoAnnals of the New York Academy of Sciences vol 1095 pp 473ndash482 2007

[47] Y J Jang J Kim N J Kang K W Lee and H J LeeldquoPiceatannol attenuates 4-hydroxynonenal-induced apoptosisof PC12 cells by blocking activation of c-junN-terminal kinaserdquoAnnals of the New York Academy of Sciences vol 1171 pp 176ndash182 2009

[48] F Gomez-Pinilla and T T J Nguyen ldquoNatural mood foodsthe actions of polyphenols against psychiatric and cognitivedisordersrdquo Nutritional Neuroscience vol 15 no 3 pp 127ndash1332012

[49] J A Joseph B Shukitt-Hale and G Casadesus ldquoReversingthe deleterious effects of aging on neuronal communicationand behavior beneficial properties of fruit polyphenolic com-poundsrdquoThe American Journal of Clinical Nutrition vol 81 no1 pp 313Sndash316S 2005

[50] N Vassallo Ed Polyphenols and Health New and RecentAdvances Nova Science Publishers 2008

[51] P Lu T Mamiya L L Lu et al ldquoSilibinin prevents amyloid bpeptide-induced memory impairment and oxidative stress inmicerdquo British Journal of Pharmacology vol 157 no 7 pp 1270ndash1277 2009

[52] C Grossi S Rigacci S Ambrosini et al ldquoThe polyphenol oleu-ropein aglycone protects TgCRND8 mice against Aszlig plaquepathologyrdquo PloS ONE vol 8 no 8 Article ID e71702 2013

[53] G Scola D Conte P W Spada et al ldquoFlavan-3-ol compoundsfrom wine wastes with in vitro and in vivo antioxidant activityrdquoNutrients vol 2 no 10 pp 1048ndash1059 2010

[54] T Toda T Sunagawa T KandaM Tagashira T Shirasawa andT Shimizu ldquoApple procyanidins suppress amyloid 120573-proteinaggregationrdquo Biochemistry Research International vol 2011Article ID 784698 8 pages 2011

[55] P A da Silva Porto J A Laranjinha and V A de FreitasldquoAntioxidant protection of low density lipoprotein by procyani-dins structureactivity relationshipsrdquo Biochemical Pharmacol-ogy vol 66 no 6 pp 947ndash954 2003

[56] Y Feng S G Yang X T Du et al ldquoEllagic acid promotesAbeta42 fibrillization and inhibits Abeta42-induced neurotox-icityrdquo Biochemical and Biophysical Research Communicationsvol 390 no 4 pp 1250ndash1254 2009

[57] L Ho M G Ferruzzi E M Janle et al ldquoIdentification of brain-targeted bioactive dietary quercetin-3-O-glucuronide as a novelintervention for Alzheimerrsquos diseaserdquo The FASEB Journal vol27 no 2 pp 769ndash781 2013

[58] M A Ansari H M Abdul G Joshi W O Opii and D AButterfield ldquoProtective effect of quercetin in primary neuronsagainst Abeta(1-42) relevance to Alzheimerrsquos diseaserdquo TheJournal of Nutritional Biochemistry vol 20 no 4 pp 269ndash2752009

Submit your manuscripts athttpwwwhindawicom

Neurology Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Alzheimerrsquos DiseaseHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

ScientificaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014


BioMed Research International


Research and TreatmentSchizophrenia

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014


Neural Plasticity


Parkinsonrsquos Disease


Research and TreatmentAutism

Sleep DisordersHindawi Publishing Corporationhttpwwwhindawicom Volume 2014


Neuroscience Journal

Epilepsy Research and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014


Psychiatry Journal


Computational and Mathematical Methods in Medicine

Depression Research and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014


Brain ScienceInternational Journal of

StrokeResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Neurodegenerative Diseases


Journal of

Cardiovascular Psychiatry and NeurologyHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

2 BioMed Research InternationalRe

lativ

e abu

ndan

ce

100

95

90

85

80

75

70

65

60

55

50

45

40

35

30

25

20

15

10

5

0

6560555045403530252015105

Time (min)

Sb1

Sb2

Sb3

Sb4 Sb5Sb6

Sb7

Sb8

Sb9

Sb10

Sb11

Sb12

Sb13 Sb14

Sb15

Figure 1 LC-MS of aqueous alcohol Schotia brachypetala stalkextract

[8] and monoamine oxidase inhibitors are useful in treatingAD [9] Phenolic glycosides from Markhamia stipulata wereisolated mainly being verbascosides [10] Verbascosides arephenylpropanoids that have cytoprotective action connectedto their antioxidant and chelating capacity [11] This datasuggests genus Markhamia can be used to treat oxidativestress related neurodegenerative disease as AD Thus thecurrent study was performed with the main aim of phar-macological screening of piceatannol and the phenolic richextracts of leaves of Camellia sinensis Markhamia platycalyxand Schotia brachypetala and stalks of Schotia brachypetalausing behaviouralmousemodels (ymaze object recognition)as promisingAlzheimerrsquos disease drug leads andusingCamel-lia sinensis as reference standard Further pharmacologicalscreening of extracts was performed by mouse amyloid beta42 ELISA assay Characterization of phenolicsrsquo profiling instalks of Schotia brachypetala aqueous alcohol extract wasexecuted by LC-MS and X calibur software (Figure 1)

2 Materials and Methods

21 Plant Material Aerial parts of Markhamia platycalyxCamellia sinensis and Schotia brachypetala and stalks ofSchotia brachypetalawere collected from the Orman GardenGiza Egypt in May 2012 The authenticity of species wasconfirmed by Professor Dr Mohamed El Gebaly Professor ofTaxonomy at the National Research Center Egypt Voucherspecimen was deposited at the herbarium of the Faculty ofPharmacy and Biotechnology Department of Pharmaceuti-cal Biology German University in Cairo Egypt

22 Extraction The leaves of Markhamia platycalyx Camel-lia sinensis and Schotia brachypetala (500 g) and stalk ofSchotia brachypetala (500 g) [12] were exhaustively extractedwith distilled water (5 L) individually The extract was evap-orated in vacuo at low temperature till dryness to yield (12 g)for each of Markhamia platycalyx Camellia sinensis andSchotia brachypetala leaves and (15 g) of Schotia brachypetala

stalksThen the dry solid residues obtained from evaporationof aqueous extracts were extracted with ethanol The driedaqueous extract of each plant was dissolved in ethanolindividually This step was followed by evaporation of thefiltrate to obtain crude phenolic content 7 g from leaves ofMarkhamia platycalyx Camellia sinensis and Schotia brachy-petala and 5 g from Schotia brachypetala stalks The dryresidual powder of aqueous ethanol extract was kept in tightlyclosed sample tubes at room temperature

23 LC-HRESI-MS-MS LC-HRESI-MS-MS was performedon a Bruker micro-TOF-Q Daltonics (API) Time-of-Flightmass spectrometer (Bremen Germany) coupled to 1200series HPLC system (Agilent Technologies Waldbronn Ger-many) equipped with a high performance autosamplerbinary pump and PDA detector G 1314 C (SL) Chromato-graphic separation was performed on a Superspher 100 RP-18 (75 times 4mm id 4 120583m) column (Merck DarmstadtGermany)

24 Identification of Phenolic Compounds of Aqueous EthanolExtract of Schotia brachypetala Stalk by LC-HRESI-MS-MSThe solvents were (A) 2 acetic acid (pH 26) and (B) 80methanol 2 acetic acid and pH 26 The gradient elutionwas from 5 to 50 B at 30∘C at a flow rate of 100 120583LminThe ionization technique was an ion spray (pneumaticallyassisted electrospray) The mass spectrometer was operatedin the negativemode Negativemode conditions were appliedto the instrument as follows capillary voltage 4000V endplate offset minus500V heated dry nitrogen gas temperature200∘C at flow rate 10 Lmin the gas flow to the nebulizerwas set at pressure 16 bar For collision-induced dissociation(CID) MS-MS measurements the voltage over the collisioncell varied from 20 to 70 eV Argon was used as collisiongas Data analysis software was used for data interpretationSodium formate was used for calibration at the end of LC-MSrun Interpretation for ESI-MSwas performed by Xcalibur 21software fromThermo Scientific (Berlin Germany)

25 Animals Adult male Swiss albino mice weighing from20 to 30 g each were used They were brought from theNational Institute of Research in Egypt All animals werehoused in alternating 12 h darknight cycles at suitable roomtemperature one week before experimental interventionThey had unrestricted access to food and water throughoutthe period of investigation All experiments were performedunder the anesthesia with 2 isoflurane to minimize thesuffering Animal procedures were approved by the EthicsCommittee at the German University in Cairo in agreementwith recommendations of the National Institute of HealthGuide for Care and Use of Laboratory Animals

26 Experimental Protocol Alzheimerrsquos disease was inducedby intraperitoneal injections of Lipopolysaccharide (08mgkg) once [19] Green tea extract from leaves ofCamellia sinen-sis was used as the reference standard Doses and vehiclesused to dissolve drugs were chosen based on the literatureThe extracts and piceatannol were dissolved in DMSO andthen completed to final volume with saline [20] The mice








see [25]




times 100 (2)




3 Results

31 In Vivo Results





119877






32 Ex Vivo Results




4 Discussion






Y maze

0

20

40

60

80

100

Groups

Mea

n pe

rcen

tage

of a

ltera

tions

+ +

++

++

+++

lowastlowastlowast

Nor

mal

LPS

LPS+

GT

LPS+

PCT

LPS+

MP

LPS+

SBS

LPS+

SBL





00

02

04

06

08


Mea

n di

scrim

inat

ion

ratio

Groups

++++++

+++

++++++

lowastlowastlowast

Nor

mal

LPS

LPS+

GT

LPS+

PCT

LPS+

MP

LPS+

SBS

LPS+

SBL



2O [14]



0

2

4

6

Mea

n am

yloi

d be

ta42

(pg

mL)


Groups

++++ +++ +++ +++

lowastlowastlowastN

orm

al

LPS

LPS+

GT

LPS+

PCT

LPS+

MP

LPS+

SBS

LPS+

SBL















5 Conclusion




Abbreviations





References









































































Neural Plasticity










Psychiatry Journal









Journal of









see [25]




times 100 (2)




3 Results

31 In Vivo Results





119877






32 Ex Vivo Results




4 Discussion






Y maze

0

20

40

60

80

100

Groups

Mea

n pe

rcen

tage

of a

ltera

tions

+ +

++

++

+++

lowastlowastlowast

Nor

mal

LPS

LPS+

GT

LPS+

PCT

LPS+

MP

LPS+

SBS

LPS+

SBL





00

02

04

06

08


Mea

n di

scrim

inat

ion

ratio

Groups

++++++

+++

++++++

lowastlowastlowast

Nor

mal

LPS

LPS+

GT

LPS+

PCT

LPS+

MP

LPS+

SBS

LPS+

SBL



2O [14]



0

2

4

6

Mea

n am

yloi

d be

ta42

(pg

mL)


Groups

++++ +++ +++ +++

lowastlowastlowastN

orm

al

LPS

LPS+

GT

LPS+

PCT

LPS+

MP

LPS+

SBS

LPS+

SBL















5 Conclusion




Abbreviations





References









































































Neural Plasticity










Psychiatry Journal









Journal of





119877






32 Ex Vivo Results




4 Discussion






Y maze

0

20

40

60

80

100

Groups

Mea

n pe

rcen

tage

of a

ltera

tions

+ +

++

++

+++

lowastlowastlowast

Nor

mal

LPS

LPS+

GT

LPS+

PCT

LPS+

MP

LPS+

SBS

LPS+

SBL





00

02

04

06

08


Mea

n di

scrim

inat

ion

ratio

Groups

++++++

+++

++++++

lowastlowastlowast

Nor

mal

LPS

LPS+

GT

LPS+

PCT

LPS+

MP

LPS+

SBS

LPS+

SBL



2O [14]



0

2

4

6

Mea

n am

yloi

d be

ta42

(pg

mL)


Groups

++++ +++ +++ +++

lowastlowastlowastN

orm

al

LPS

LPS+

GT

LPS+

PCT

LPS+

MP

LPS+

SBS

LPS+

SBL















5 Conclusion




Abbreviations





References









































































Neural Plasticity










Psychiatry Journal









Journal of



Y maze

0

20

40

60

80

100

Groups

Mea

n pe

rcen

tage

of a

ltera

tions

+ +

++

++

+++

lowastlowastlowast

Nor

mal

LPS

LPS+

GT

LPS+

PCT

LPS+

MP

LPS+

SBS

LPS+

SBL





00

02

04

06

08


Mea

n di

scrim

inat

ion

ratio

Groups

++++++

+++

++++++

lowastlowastlowast

Nor

mal

LPS

LPS+

GT

LPS+

PCT

LPS+

MP

LPS+

SBS

LPS+

SBL



2O [14]



0

2

4

6

Mea

n am

yloi

d be

ta42

(pg

mL)


Groups

++++ +++ +++ +++

lowastlowastlowastN

orm

al

LPS

LPS+

GT

LPS+

PCT

LPS+

MP

LPS+

SBS

LPS+

SBL















5 Conclusion




Abbreviations





References









































































Neural Plasticity










Psychiatry Journal









Journal of



0

2

4

6

Mea

n am

yloi

d be

ta42

(pg

mL)


Groups

++++ +++ +++ +++

lowastlowastlowastN

orm

al

LPS

LPS+

GT

LPS+

PCT

LPS+

MP

LPS+

SBS

LPS+

SBL















5 Conclusion




Abbreviations





References









































































Neural Plasticity










Psychiatry Journal









Journal of










5 Conclusion




Abbreviations





References









































































Neural Plasticity










Psychiatry Journal









Journal of




































































Neural Plasticity










Psychiatry Journal









Journal of


Research Article Evaluation of Plant Phenolic Metabolites as...

Documents

Transcript of Research Article Evaluation of Plant Phenolic Metabolites as...