Research Article Characterization and Quantification of ...downloads.hindawi.com/journals/ecam/2014/676902.pdf · include amendoeira-da-praia, cuca, and chap ´eu de sol. In several

Research ArticleCharacterization and Quantification of Compounds in theHydroalcoholic Extract of the Leaves from Terminalia catappaLinn (Combretaceae) and Their Mutagenic Activity

Francisco Joseacute Mininel1 Carlos Seacutergio Leonardo Junior1 Liacutevia Greghi Espanha2

Flaacutevia Aparecida Resende2 Eliana Aparecida Varanda2 Clarice Queico Fujimura Leite2

Wagner Vilegas3 and Lourdes Campaner dos Santos1

1 Organic Chemistry Department Institute of Chemistry Sao Paulo State University (UNESP) 14800-900 Araraquara SP Brazil2 Department of Biological Sciences Faculty of Pharmaceutical Sciences Sao Paulo State University (UNESP)14801-902 Araraquara SP Brazil

3 Sao Paulo State University (UNESP) Experimental Campus of Sao Vicente 11350-000 Sao Vicente SP Brazil

Correspondence should be addressed to Lourdes Campaner dos Santos loursantgmailcom

Received 21 November 2013 Revised 10 January 2014 Accepted 17 January 2014 Published 9 March 2014

Academic Editor Sandy van Vuuren

Copyright copy 2014 Francisco Jose Mininel et al This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

Terminalia is a genus of Combretaceous plants widely distributed in tropical and subtropical regions Thus the aim of this studywas to quantify the majority compounds of the hydroalcoholic extract (7 3 vv) of the leaves from T catappa by HPLC-PDAchemically characterize by hyphenated techniques (HPLC-ESI-IT-MSn) and NMR and evaluate its mutagenic activity by theSalmonellamicrosome assay on S typhimurium strains TA98 TA97a TA100 and TA102 The quantification of analytes wasperformed using an external calibration standard Punicalagin is themost abundant polyphenol found in the leavesThe presence ofthis compound as a mixture of anomers was confirmed using HPLC-PDA and 1H and 13C NMR Mutagenic activity was observedin strains TA100 and TA97a As the extract is a complex mixture of punicalagin its derivatives and several other compoundsthe observed mutagenicity may be explained in part by possible synergistic interaction between the compounds present in theextract These studies show that mutagenic activity of T catappa in the Ames test can only be observed when measured at highconcentrations However considering the mutagenic effects observed for T catappa this plant should be used cautiously formedicinal purposes

1 Introduction

Aspart of our efforts to identify the substances responsible forthe pharmacological activities of plants utilized in Brazilianpopular medicine (Biota-Fapesp Project number 0952237-9) we have analyzed the hydroalcoholic extract of the leavesfrom Terminalia catappa L which belongs to the familyCombretaceae The trivial names of these plants in Brazilinclude ldquoamendoeira-da-praiardquo ldquocucardquo and ldquochapeu de solrdquo

In several Asian countries physicians have used theleaves bark and fruit of T catappa to treat dermatitis andpyresis [1ndash4] Moreover the antimicrobial and antifungal [5ndash8] antioxidative [9ndash11] anti-inflammatory [12 13] hepato-protective [3 4] antidiabetic [14] carcinogenesis-preventing

effects [15ndash17] antimalaric [18] and antinociceptive [19] of Tcatappa could potentially provide benefits to human health

Natural products have also been isolated from the plantT catappa which contains triterpenoids (ursolic acid asiaticacid) squalene but no caffeine flavonoids (isovitexin vitexinand rutin) gallic acid hydrolysed tannins such as punicalaginanomers as amajor component punicalin terflavins A and Btergallagin tercatain chebulagic acid geranin granato B andcorilagin [13 20 21]

Tannins have been reported to be responsible fordecreases in food intake growth rate feeding efficiencynet metabolizable energy and protein digestibility in exper-imental animals [21] Incidences of certain cancers such

Hindawi Publishing CorporationEvidence-Based Complementary and Alternative MedicineVolume 2014 Article ID 676902 11 pageshttpdxdoiorg1011552014676902

2 Evidence-Based Complementary and Alternative Medicine

as esophageal cancer have been reported to be related toconsumption of tannin-rich foods such as betel nuts andherbal teas suggesting that tannins might be carcinogenic[22] However other reports have indicated that the carcino-genic activity of tannins might be related to componentsthat are associated with tannins rather than the tanninsthemselves [23 24] Tannins have also been reported to exertother physiological effects such as the acceleration of bloodclotting a reduction in blood pressure a decrease in serumlipid levels liver necrosis and modulation of the immuneresponse [21 25]

Ellagitannins have been isolated along with gallotanninsfrom various other plant families and show little chemotaxo-nomic significance The tannins punicalagin and punicalinboth of which contain a gallagyl unit and were first iso-lated from the pericarps of pomegranate (Punica granatumPunicaceae) [26] were a characteristic component in someTerminalia species This supports the hypothesis that Puni-caceae is chemotaxonomically proximate to Combretaceae asclassified by Cronquist [27]

Despite its biological properties the chemical compo-sition may vary according to geographiclocation [28 29]a fact impairing the standardization of raw material andcommercial products for medicinal purposes [30]

Considering that there are no data in the literature onquantification of the majority secondary metabolites of thehydroalcoholic extract of the T catappa by HPLC-PDA andthe popular use of this plant in this study the punicalaginanomers and ellagic acid were isolated and quantified

Moreover the mutagenic effects were evaluated usinga Salmonellamicrosome assay (Ames test) in the presenceand in the absence of in vitro metabolizing systems in theconcentrations 2224 1668 1112 556 and 278mgplate

2 Experimental

21 Chemical and Reagents HPLC-grade methanol (MeOH)and acetonitrile were purchased from JT Baker (Baker-Mallinckrodt Phillipsburg NJ USA) Standards of ellagicacid were purchased from Sigma Chemical Co (St LouisMO USA) and had a purity of 95 The anomers mixtureof punicalagin 120572 and 120573 isolated of the hydroalcoholic extractfrom T catappa (with 98 purity) were used as external stan-dards HPLC-grade water (18 MΩsdotcm) was obtained using adirect Milli-Q purification system (Millipore Co BedfordMA USA) Sep-Pak RP18 cartridges (500mgmL) for solid-phase extraction (SPE) were purchased from PhenomenexCo (Torrance CA USA)

22 Apparatus The HPLC system used was a JASCO 2010HPLC (Jasco Tokyo Japan) equipped with PU-2089S Pluspump a MD-2018 Plus Photodiode Array Detector (PDA)an AS-2055 Plus auto sampler and column oven (CO-2065 plus) The software ChromNav (Workstation JASCO-ChromNav 11803) was used to control the analytical systemand data collection and processing To purify the substancesa semipreparative JASCOHPLC equipped with two PU-2086pumps a high pressure mixer (Model MX-2080-32) a PDA

detector (model MD-2018 Plus) and a Rheodyne 500 120583Lmanual injector loop was used The software used to operatethe equipment and data processing was ChromNAV version1 18 03

The mass spectrometry experiments were performed onLCQ Fleet equipment (Thermo Scientific) equipped witha dispositive of direct insertion of the sample via flowinjection analysis (FIA) The studied matrix was analyzed byelectrospray ionization (ESI) and fragmentation in multiplestages (MS2MS3 andMSn)was performed at an ion trap (IT)interface The negative mode was selected for the generationand analysis of the mass spectra for the first order (MS)and for the remaining multistage experiments under thefollowing conditions capillary voltage minus25V voltage sprayminus5 kV and capillary temperature 275∘C A carrier gas (N

2)

with a flow of 8 arbitrary units (AU) was used and thecollision gas was helium (He)The track acquisition was 100ndash2000119898119911 Xcalibur version 13 software (Thermo Finnigan)was used to acquire and process the data

The 1H- and 13C-NMR experiments were performedusing a 300MHz (117 T) nuclear magnetic resonance spec-trometer (Varian Inova) CD

3OD was used as the solvent

(Sigma Aldrich)

23 Plant Material The leaves of T catappa were collectedfrom the beach-front city of SantosSP with the permissionof the municipality in April 2010 and were identified bybotanist Paulo Salles Penteado ofUniversity Santa Cecilia andregistered (M Tomaz 01) in the Herbarium of the Universityof Santa Cecilia (HUSC) Authorization was obtained fromIBAMA (Instituto Brasileiro do Meio Ambiente e dos Recur-sos Naturais Renovaveis) to the number 33152

24 Extraction The infusion and hydroalcoholic extractwere prepared according to the experimental protocol estab-lished in Biota-Fapesp Project which this work is linked(process number 0952237-9)

241 Preparation of the Infusion of the Leaves from T catappaThe leaves (1 g) of T catappa after drying were prepared byinfusion as their use in folk medicine [31]

242 Preparation of the Hydroalcoholic Extract of the Leavesfrom T catappa The dried and powdered leaves (5246 g) ofT catappawere extracted by percolation at room temperaturewith a mixture of EtOH H

2O (7 3 vv) The solvents were

evaporated to dryness at low pressure to give 331 g ofthe crude hydroalcoholic extract (87) The extract wasanalyzed by FIA-ESI-IT-MSn and HPLC-PDA

243 Isolation of the Fraction Containing the PunicalaginAnomers by SemipreparativeHPLC A total of 14 g of hydroal-coholic extract (7 3 vv) was dissolved inH

2O MeOH (8 2

vv) and partitioned successively with hexane (500mL) andethyl acetate (500mL) giving 068 g (48) (hexane fraction)193 g (1378) (ethyl acetate fraction) and the 1015 g (725)(hydromethanol fraction 8 2 vv)

Evidence-Based Complementary and Alternative Medicine 3

200 400 600 800 1000 1200 1400 1600 1800 20000

10

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40

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90

100

Rela

tive a

bund

ance

108319

7814654165311756353232577

9327969203 11148030130 3397111322 114403128348 14393046776 157117 172447185395

mz

Figure 1 First-order mass spectrum of the hydroalcoholic extract from the leaves of T catappa in the negative mode Range of ions with119898119911100ndash2000Da

Table 1 Identification of substances in T catappa by FIA-ESI-IT-MSn

[MndashH]minus MSn ions Identification

1083 781 [M-152-152-H]minus601 [M-152-152-180-H]minus Punicalagin (1)

781 601 [M-180-H]minus Punicalin (2)601 409 [M-191-H]minus Gallagic acid (3)

301 257 [M-44-H]minus229 [M-44-28-H]minus Ellagic acid (4)

25 Identification of the Punicalagin Anomers and EllagicAcid in the Hydroalcoholic Extract Identification of thepunicalagin anomers was performed using FIA-ESI-IT-MSnand compared with the literature data [32] 1H and 13CNMR[33] and HPLC-PDA

For the FIA-ESI-IT-MSn assay 10mg of the crudehydroalcoholic extract was dissolved in 1mL of MeOH H

2O

(1 1 vv) after using an ultrasonic bath for 5 minutes Thesamples were then filtered through a 022120583mPTFE filter andaliquots of 20120583L were directly injected into the FIA-ESI-IT-MSn system

For the HPLC-PDA a clean-up step was performed toremove any contaminants the solution was purified bysolid phase extraction (SPE) using Phenomenex Strata C

18

cartridges (500mg of stationary phase) that were previouslyactivated with 5mL of MeOH and equilibrated with 5mL ofMeOH H

2O (1 1 vv) The compounds were eluted from

cartridges using 1mL of MeOH H2O (1 1 vv) with a final

volume of 5mL The samples were then filtered through a022120583m PTFE filter and dried The dry extract was diluted to10mgmL in HPLC solvent Aliquots of 20 120583L were injecteddirectly into the HPLC-PDA with detection at 270 nm The

identification of the different compounds in the chromato-graphic profile of the hydroalcoholic extract was done bycomparing their retention times (119905

119903) and UV spectra with

those obtained for the pure standards (ellagic acid Sigma)and the compounds isolated from the hydroalcoholic extractof T catappa (punicalagin anomers)

For the confirmation of the presence of ellagic acidexperiments were done by coinjection The hydroalcoholicextract (15mg) was dissolved in 1mL of MeOH H

2O (1 1

vv) and subjected to a clean-up procedure using SPE car-tridge RP18 (50mg) The sample was then filtered througha PTFE filter (022 120583m) and dried at room temperatureThe resulting solid was redissolved in MeOH H

2O to a

concentration of 10mgmL Two milligrams of ellagic acidstandard (Sigma) was then added to this solution and thesolution was centrifuged for 2min and filtered through a022120583m filter A hydro column with a flow rate of 1mLminwas usedwith a gradient of 5ndash60MeOH for 60minutes andthe absorbance at 270 nm was detected The chromatogramsof the hydroalcoholic extract were compared before and afterthe addition of the ellagic acid standard From the coinjectionof ellagic acid and the hydroalcoholic extract we confirmedthe presence of ellagic acid from the significant increase inthe peak eluting at 119905

1199034730min (peak 3)

26 Conditions for Quantification of Punicalagin (Anomers)and Ellagic Acid The quantification of analytes was per-formed using an external calibration standard [34 35] Thecurves were constructed using standards of the commercialellagic acid (Sigma) and the isolated punicalagin anomersWe evaluated the content of each punicalagin anomer andthe total content of punicalagins in leaves of T catappa Astock solution of 1000 120583gmL ellagic acid was prepared and


HOOH

OHOHHO

OO

O

O

OHOH

O

O

OHOH

OO

O

O

OH

O

O

HO

O

O

HOHO

HO

OO

O

O

OH

OHHO

OH

O

O

OHOHO

HO

OHHO

HO

OHOH

HOOH

OHOHHO

OO

O

O

OHOH

O

HO

HO

OH

O

O RO

HO

HO OH HO OH

O

OO

O

OH

O

H

H

CH2

CH2

Punicalagin (1) (mz 1083)

Punicalin (2) (mz 781)

Ellagic acid (4) (mz 301)

Gallagic acid (3) (mz 601)

17

1615

1319

1418

12

89

11

10

7

3 2 1

54

6

20

484745

4443 42

46

39

38 37

36354041

32

31

30 2928

3334

27 2223

242526

21

R998400

Adapted from Seeram et al [32]




Figure 2 Punicalagin (1) punicalin (2) gallagic acid (3) and ellagic acid (4) Adapted from Seeram et al [32]

Table 2 Chemical shifts (1H and 13C) of the 120572- and 120573-punicalagins in CD3OD isolated from T catappa

Position 120572-punicalagin 120573-punicalagin120575 (1H) Multiplicity (Hz) 120575 (13C) 120575 (1H) Multiplicity (Hz) 120575 (13C)

1 508 d(35) 8987 466 d(80) 93772 487 dd(30 80) 7353 462 dd(30 81) 75483 520 t(93) 7608 492 t(100) 78784 478 t(90) 7023 485 t(100) 74885 323 t(90) 6633 257 dd(99 110) 71586a 210 d(99) 6363 405 d(99) 63036b 409 dd(70 70) 6363 410 d(78) 63039 652 s 10546 654 s 1062118 666 s 10741 668 s 1062123 673 s 10876 677 s 1095146 694 s 11011 697 s 11071


Table 3 Parameters for quantification of the isolated compounds

Linear coefficient (119886) Slope (119887) Correlation coefficient LOD120583gmL

LOQ120583gmL

Ellagic acid 21753 71514 0998 100 304120572-punicalagin 37326 11861 0997 1038 3146120573-punicalagin 44760 16830 0999 877 2659Total punicalagins 82086 28691 0998 944 2861

Table 4 Determining the accuracy of the method for the determination of each of the substances quantified

Substances investigated Repetition () Recovery mean DP RSD ()1 2 3

Ellagic acid 904 8775 8452 8756 294 003Total punicalagins 869 8626 8586 8634 052 001RSD () = RSD relative standard deviation (100 times SDmean)

dilutions to 500 250 125 625 156 78 and 39 120583gmL weremade For punicalagin a stock solution at concentrationsof 4000 120583gmL of the 120572-anomer (peak 1) and 4000120583gmLof the 120573-anomer (peak 2) was prepared From the stocksolution serial dilutions were made to reduce the concen-tration in 2 1 increments (2000 1000 500 250 125 6250and 3125 120583gmL) for both the 120572- and 120573-anomers Eachconcentration level was analyzed in triplicateThemean areasof the chromatographic peaks obtained were interpolated asa function of concentration using linear regression and wereused to generate the calibration curvesThe linear coefficients(a) and angle (b) were obtained from the calibration curvesThe correlation coefficient (1199032) and limits of detection (LOD)and quantitation (LOQ) for peaks 1 and 2 corresponding tothe 120572- and 120573-anomers of punicalagin [36] were obtainedThe accuracy of the HPLC method was estimated fromthe recovery tests The recovery tests were performed byadding known concentrations (lowmedium and high) of thereference ellagic acid (15 60 and 250 120583gmL) and isolatedpunicalagin anomer (250 500 and 2000120583gmL) standardsThe average recovery value was calculated using the followingformula recovery = [(119862f119862nf + 119862pd)] times 100 where 119862fcorresponds to fortified concentration 119862nf corresponds tothe unfortified concentration and 119862pd corresponds to theconcentration of the added standard

Repeatability of intra- and interday was done to deter-mine the accuracy of the developed method The relativestandard deviation (RSD)was taken as ameasure of accuracyRepeatability of intra- and interday was determined in sixreplicates within the range found in the extract in a day andon three consecutive days respectively

27 SalmonellaMicrosome Assay Mutagenic activity wasevaluated using the Salmonellamicrosome assay with theSalmonella typhimurium tester strains TA98 TA100 TA97aand TA102 which were kindly provided by Dr B N Ames(Berkeley CA USA) with (+S9) and without (minusS9) metab-olization using the preincubation method [37] The strainswere grown overnight from frozen cultures for 12ndash14 h inOxoid Nutrient Broth number 2 The metabolic activation

mixture (S9 fraction) prepared from the livers of Sprague-Dawley rats treated with the polychlorinated biphenyl mix-ture Aroclor 1254 (500mgkg) was purchased fromMolecu-lar Toxicology Inc (Boone NC USA) and freshly preparedbefore each test The metabolic activation system consistedof 4 S9 fraction 1 04M MgCl

2 1 165M KCl 05

1M D-glucose-6-phosphate disodium 4 01M NADP 5002M phosphate buffer and 395 sterile distilled water Forthe determination of the mutagenic activity five differentconcentrations of extract (156 to 2224mgplate) diluted inDMSO were assayed The concentrations of the sample wereselected on the basis of a preliminary toxicity test In allsubsequent assays the upper limit of the dose range testedwas either the highest nontoxic dose or the lowest toxic dosedetermined in the preliminary assay Toxicity was detectedeither as a reduction in the number of histidine revertants(His+) or as a thinning of the auxotrophic background (iebackground lawn)

The various concentrations of extract to be tested wereadded to either 05mL of 02M phosphate buffer or 05mLof 4 S9 mixture with 01mL of bacterial culture and thenincubated at 37∘C for 20ndash30min Next 2mL of top agar wasadded and the mixture was poured onto a plate containingminimal agar

The plates were incubated at 37∘C for 48 h and the His+revertant colonies were counted manually All experimentswere analyzed in triplicate The results were analyzed withthe statistical software package Salanal 10 (US Environ-mental Protection Agency Monitoring Systems LaboratoryLas Vegas NV from the Research Triangle Institute RTPNC USA) adopting the Bernstein et al [38] model Thedata (revertantsplate) were assessed by analysis of variance(ANOVA) followed by linear regression The mutagenicindex (MI) which is the average number of revertantsper plate with the test compound divided by the averagenumber of revertants per plate with the negative (solvent)control was also calculated for each concentration testedA sample was considered mutagenic when a dose-responserelationship was detected and a twofold increase in thenumber of mutants (MI ge 2) was observed for at least


00 100 200 300 400 500 600 Retention time (min)

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(120583V

)

Figure 3 Chromatogram of hydroalcoholic extract of the leaves of T catappa dissolved in MeOH H2O (1 1 vv) at 10mgmL The method

utilized a hydro column at a flow rate of 1mLmin with a gradient of 5ndash60MeOH for 60min HPLC-PDA (Jasco) 270 nmThe UV spectrafrom 119905

1199031475 (peak 1) min and at 119905

1199031886min (peak 2) characteristic of the 120572- and 120573-anomers of punicalagin (1 2) and ellagic acid (3)

one concentration [39] The standard mutagens used aspositive controls in experiments without the S9 mix were4-nitro-o-phenylenediamine (100 120583gplate TA98) sodiumazide (125 120583gplate TA100) mitomycin (05 120583gplate TA102)and 2-anthramine (125120583gplate TA98 TA100) and 2-aminofluorene (100 120583gplate TA102) in the presence of S9DMSO served as the negative (solvent) control

3 Results and Discussion

The analysis of mass spectra of the hydroalcoholic extract(Figure 1) showed the precursor ions [M-H]minus 119898119911 1083(punicalagin) 119898119911 781 (punicalin) 119898119911 601 (gallagic acid)and 119898119911 301 (ellagic acid) [32] Second-order fragmentation(MS2) confirmed the presence of the metabolites shown inthe scheme of Figure 2 for substances 1ndash4 (Table 1) Seeramet al [32] studying the commercial juice industry of thepomegranate husk identified the same fragmentation of thesesame compounds

The chromatographic profile by HPLC-PDA of thehydroalcoholic extract and the infusion are shown in Figures3 and 4 In the chromatogram threemajor peaks (1ndash3) elutingat 1199051199031475min 119905

1199031880min and 119905

1199034730min are observed

Figures 3 and 4 show the UV region of the spectrumobtained using a PDA detector for the peaks eluting at

1199051199031475min (peak 1) and 119905

1199031880min (peak 2) The data

confirm the presence of both punicalagin anomers withabsorption maxima at 120582max 218 260 and 379 nm The com-pound eluting at 119905

1199034730min (peak 3) corresponds to ellagic

acid with 120582max values of 250 306 and 368 nm as confirmedby coinjection experiments with a standard analyte (Figures3 and 4) The data obtained for UV anomers punicalagin andellagic acid are consistent with those found in the literature[40] and are characteristic of gallagyl chromophore [41]

Peaks 1 and 2 (Figure 3) were partitioned (item 243)collected separately and analyzed by HPLC-PDA (Figure 5)The analysis of the peaks by HPLC-PDA with 119905

1199031475min

and 1880min respectively display an interesting behavioras they resolve into two peaks with distinct retention timeswhen analyzed under the same conditions to obtain peaksat similar retention times with the same UV spectra Thisindicates that the compounds are the mixture of anomers(120572- and 120573-punicalagin) These compounds have absorptionmaxim at 120582max 218 260 and 379 nm and are characteristicof the chromophore gallagyl [41] For peak 1 the HPLC-PDA analysis shows the presence of two peaks (1a and 1b)with retention times of 1529min and 2107min respectively(Figure 5)

In solution punicalagin rapidly interconverts betweenthe 120572- and 120573-anomers (Figure 2 compound 1)The 13CNMR(Table 2) spectrum reveals the presence of two anomeric


1

2

3

200 220 240 260 280 300 320 340 360 380 400Wavelength (nm)

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0

1000000

2000000

Inte

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(120583V

)

Figure 4 Chromatogram of infusion of the leaves of T catappa Gradient 5ndash60MeOH 60min 270 nm Peaks 1 and 2 = punicalagins 3 =ellagic Acid The UV spectra of peak 1 eluting at 119905

1199031470min and peak 2 at 119905

1199031870min characteristic of the 120572- and 120573-punicalagin anomers

Peak 3 at 1199051199034730min

carbons at 120575 8987 and 120575 9377 corresponding to punicalagins120572 (present in lesser proportion in peak 1) and 120573 (presentin a greater proportion in peak 2) respectively The stereo-chemistry at the anomeric position can be easily determinedby measuring the coupling constant between the anomericproton H-1 and the adjacent proton H-2 in the 1H NMRspectrum A large coupling constant (119869H-1H-2 gt 5Hz) gen-erally indicates a 120573-glycosidic configuration (substituent inthe equatorial orientation) whereas a small coupling constant(119869H-1H-2 = 0ndash5 Hz) indicates an 120572-glycosidic configuration(substituent in the axial orientation) The chemical shift andthe coupling constant corresponding to the 120572-anomer is 120575508 (119869 = 35Hz) and for the 120573-anomer is 120575 466 (119869 = 80Hz)NMRdata of the isolated anomers (120572- and 120573-punicalagin) areshown in the Table 2 (enumeration of carbons and hydrogensin Figure 2) compared with data found in the literature [33]The deprotonated molecule generated in the FIA-ESI-IT-MS [M-H]minus at 119898119911 1083 (1084 MW) (Table 1) and the UVspectra exhibited by 120572- and 120573-punicalagin confirmed thisidentification (Figure 5)

The linear regression analysis of the peak area for 120572-and 120573-punicalagin the total punicalagins and ellagic acidare showed in the Table 3 Thus it becomes necessary to usesufficient number of standard solutions to adequately definethe relationship between concentration and response [42]These results demonstrate the linearity of the method overthe analyzed concentration range

The recovery percentages for ellagic acid and totalpunicalagins were found to be 87 and 86 respectively(Table 4) The results were presented as mean and standarddeviation of the percentages recovered The results showedthat the accuracy of the method is very good for all analytes(RSDlt 2)The relative standard deviations also showed thatthe process of sample preparation is reproducible [43]

The relative standard deviations (RSDs) for repeatabilitytesting intra- and interday analyses are shown in Table 5 Theresult is presented as relative standard deviation from theaverage The method showed excellent repeatability with allRSD values lower than 1 [44]

Table 6 shows the mean number of revertantsplate (M)the standard deviation (SD) and the mutagenic index (MI)after the treatments with the T catappa hydroalcoholicextract observed in S typhimurium strains TA98 TA100TA102 and TA97a in the presence (+S9) and absence (minusS9)of metabolic activation

In the TA98 strain the T catappa hydroalcoholic extractdid not induce any increase in the number of revertantcolonies under the conditions used in this study The otherstrains were more sensitive to the toxic effects of extract andit was thus necessary to decrease the doses

Only in the absence of the external metabolizing systemS9 mix in the S typhimurium strains TA100 and TA97athe T catappa hydroalcoholic extract induced an increasein the number of revertant colonies relative to the negative


00 50 100 150 200 250 300 350 400

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)

Retention time (min)

Figure 5 Chromatogram peak 1a (120572-punicalagin) and peak 1b (120573-punicalagin) collected from H2O MeOH (8 2 vv) at 10mgmL of the

leaves of T catappa eluting at 1199051199031529min (1a) and 119905

1199032107min (1b)The flow rate was 1mLmin with a gradient 5ndash30MeOH for 30minutes

using a hydro columnHPLC-PDA (Jasco) 270 nmTheUV spectra of peak 1 eluting at 1199051199031529min (1a) and at 119905

1199032107 (1b)min characteristic

of the 120572- and 120573-punicalagin anomers

Table 5 Repeatability test results for each substance quantified

Substances investigated Intraday (119899 = 6) Interday (119899 = 6)Mean () DP RSD () Mean () DP RSD ()

Ellagic acid 8744 012 0001 8753 019 0002Total Punicalagins 8634 019 0002 8619 017 0002RSD () = RSD relative standard deviation (100 times SDmean)

control with a MI higher than 20 at the concentration of312mgplate in strain TA100 and 938mgplate in strainTA97a indicating the direct mutagenic activity for thesestrains

In the TA102 strain (+S9 and ndashS9) the T catappahydroalcoholic extract shows signs of mutagenicity with MIvalues approximately 2

The S typhimurium test strain TA97a detects frameshiftmutations in C-C-C-C-C-C +1 cytosine and TA98 frameshiftin DNA target-C-G-C-G-C-G-C-G the S typhimuriumtester strain TA100 is capable of revealing base-pair substi-tution point mutations and the S typhimurium tester strainTA102 is normally used to detect cross-linking agents andbase-pair substitution mutations [39] Thus according to the

strains involved theT catappa hydroalcoholic extract mainlyinduces substitution of base pairs (TA100) and frameshiftmutations (TA97a)

Ko et al [17] evaluated the toxicity and mutagenicity ofsupercritical carbon dioxide (SC-CO

2) extracts of T catappa

leaves at a dose of 05mgplate in the strain TA100 (detectbase-pair substitution) and observed absence of mutageniceffect The absence of mutagenic effect was also observed forTA102 and TA98 when the ethanolic extract was evaluated

These studies show that mutagenic activity of T catappain the Ames test can only be observed whenmeasured at highconcentrations The results of this study are consistent witha review of the literature indicating that high concentrationsof tannins (15 and 60mM) can cause DNA damage These


Table6Mutagenicactiv

ityexpressedas

them

eanandsta

ndarddeviationof

then

umbero

frevertantsplatea

ndmutagenicindex(in

parenthesis

)inSalm

onellatyphim

urium

TA98TA100

TA97aandTA

102strainstreated

with

hydroalcoh

olicextracto

fTcatappa

atvario

usdo

seswith

(+S9)o

rwith

out(minusS9)m

etabolicactiv

ation

Treatm

ents

Num

bero

frevertants(MplusmnSD

)platea

ndMI

TA98

TA100

TA102

TA97a

mgplate

minusS9

+S9

mgplate

minusS9

+S9

minusS9

+S9

minusS9

+S9

Term

inaliacatapp

a

00a

16plusmn3

68plusmn1

00a

93plusmn6

87plusmn4

352plusmn30

435plusmn43

172plusmn2

99plusmn1

278

17plusmn6(11)75plusmn7(11)

156

91plusmn2(10)88plusmn9(10)461plusmn11lowast(13)481plusmn23(11)

223plusmn3lowast(13)127plusmn3(13)

556


312

188plusmn19lowastlowast(20)90plusmn6(10)514plusmn16lowast(15)668plusmn75lowast(15)252plusmn12lowast(15)102plusmn7(10)

1112


625


1668


938

40plusmn4(04)95plusmn1(11)563plusmn32lowast(16)728plusmn28lowast(17)337plusmn19lowastlowast(20)93plusmn10(09)

2224


125

Nd

88plusmn4(10)648plusmn32lowast(18)621plusmn23lowast(14)397plusmn19lowastlowast(23)83plusmn2(08)

Ctrol+

720plusmn63

500plusmn57

Ctrol+

1225plusmn75

700plusmn80

1143plusmn53

1309plusmn38

1426plusmn67

700plusmn45

lowast119875lt005(A

NOVA

)lowastlowast119875lt001(A

NOVA

)0aN

egativecontroldimethylsulfoxide

(DMSO

mdash75120583Lplate)P

ositive

Con

trol4-nitro

-o-phenylenediam

ine(100120583

gplatemdash

TA98

andTA

97a)sod

ium

azide

(125120583

gplatemdash

TA100)m

itomycin

(05120583gplatemdash

TA102)intheabsenceof

S9and

2-anthramine(125120583

gplatemdash

TA98T

A100

TA97a)2-aminofl

uorene

(100120583

gplatemdash

TA102)inthepresence

ofS9N

dno

tdeterm

ined


data corroborate the observations of Gupta et al [45] whichindicated that all tannins result in polyphenol toxicity at highconcentrations [46]

4 Conclusions

In the hydroalcoholic extract of T catappa tannins were themost abundant compounds observed and the anomers 120572-and 120573-punicalagin were the major compounds Moreoverconsidering that the extract is a complex mixture of severalunknown organic compounds the mutagenicity observedmay be explained in part by a synergy between compoundspresent in the extract

The dosage and type of tannin involved are critical forthese effects Thus the results obtained in this study are use-ful for better understanding the pharmacological activitiesof T catappa However considering the mutagenic effectsobserved in this study this plant should be used cautiouslyfor medicinal purposes

Based on the obtained results it can be concludedthat the developed method (HPLC-PDA) is suitable for itspurpose namely the determination of anomers punicalaginand ellagic acid in extract of T catappa

Conflict of Interests

The authors declare that they have no conflict of interests

Acknowledgments

The authors are grateful for Grant 0952237-9 Sao PauloResearch Foundation (FAPESP) for providing financial aidto W Vilegas and L C Santos The authors also thank Con-selho Nacional de Desenvolvimento Cientıfico e Tecnologico(CNPq) for grants to L C Santos E A Varanda and WVilegas

References

[1] C B Yeh M J Hsieh M H Chien P Y Lin H L Chiouand S F Yang ldquoTerminalia catappa exerts antimetastatic effectson hepatocellular carcinoma through transcriptional inhibitionof matrix metalloproteinase-9 by modulating NF-120581B and AP-1 activityrdquo Evidence Based and Complementary AlternativeMedicine vol 2012 Article ID 595292 11 pages 2012

[2] T Morioka M Suzui V Nabandith et al ldquoModifying effectsof Terminalia catappa on azoxymethane-induced colon car-cinogenesis in male F344 ratsrdquo European Journal of CancerPrevention vol 14 no 2 pp 101ndash105 2005

[3] X Tang J Gao Y Wang et al ldquoEffective protection of Ter-minalia catappa L leaves from damage induced by carbontetrachloride in liver mitochondriardquo Journal of NutritionalBiochemistry vol 17 no 3 pp 177ndash182 2006

[4] X-H Tang L Gao J Gao et al ldquoMechanisms of hepatopro-tection of Terminalia catappa L extract on D-galactosamine-induced liver damagerdquo American Journal of Chinese Medicinevol 32 no 4 pp 509ndash519 2004

[5] P Fyhrquist L Mwasumbi C-A Haeligggstrom H Vuorela RHiltunen and P Vuorela ldquoEthnobotanical and antimicrobial

investigation on some species of Terminalia and Combretum(Combretaceae) growing in Tanzaniardquo Journal of Ethnopharma-cology vol 79 no 2 pp 169ndash177 2002

[6] R Nair and S Chanda ldquoAntimicrobial activity of Terminaliacatappa Manilkara zapota and Piper betel leaf extractrdquo IndianJournal of Pharmaceutical Sciences vol 70 no 3 pp 390ndash3932008

[7] A Manzur A Raju and S Rahman ldquoAntimicrobial activity ofTerminalia catappa extracts against some pathogenic microbialstrainsrdquo Pharmacology amp Pharmacy vol 2 pp 299ndash305 2011

[8] S Chanda K Rakholiya and R Nair ldquoAntimicrobial activityof Terminalia catappa L leaf extracts against some clinicallyimportant pathogenic microbial strainsrdquo Chinese Medicine vol2 pp 171ndash177 2011

[9] P-S Chen and J-H Li ldquoChemopreventive effect of punicalagina novel tannin component isolated from Terminalia catappa onH-ras-transformed NIH3T3 cellsrdquo Toxicology Letters vol 163no 1 pp 44ndash53 2006

[10] C-C Chyau P-T Ko and J-L Mau ldquoAntioxidant properties ofaqueous extracts from Terminalia catappa leavesrdquo LWT vol 39no 10 pp 1099ndash1108 2006

[11] T Masuda S Yonemori Y Oyama et al ldquoEvaluation of theantioxidant activity of environmental plants activity of the leafextracts from seashore plantsrdquo Journal of Agricultural and FoodChemistry vol 47 no 4 pp 1749ndash1754 1999

[12] C-C Lin Y-F Hsu and T-C Lin ldquoEffects of punicalaginand punicalin on carrageenan-induced inflammation in ratsrdquoAmerican Journal of Chinese Medicine vol 27 no 3-4 pp 371ndash376 1999

[13] YM Fan L Z Xu J Gao et al ldquoPhytochemical and antiinflam-matory studies on Terminalia catappardquo Fitoterapia vol 75 no3-4 pp 253ndash260 2004

[14] A N Nagappa P A Thakurdesai N V Rao and J SinghldquoAntidiabetic activity ofTerminalia catappa Linn fruitsrdquo Journalof Ethnopharmacology vol 88 no 1 pp 45ndash50 2003

[15] S-C Chu S-F Yang S-J Liu W-H Kuo Y-Z Chang andY-S Hsieh ldquoIn vitro and in vivo antimetastatic effects ofTerminalia catappa L leaves on lung cancer cellsrdquo Food andChemical Toxicology vol 45 no 7 pp 1194ndash1201 2007

[16] N B Pandya P Tigari K Dupadahalli H Kamurthy and RR Nadendla ldquoAntitumor and antioxidant status of Terminaliacatappa against Ehrlich ascites carcinoma in Swiss albinomicerdquoIndian Journal of Pharmacology vol 45 no 5 pp 464ndash469

[17] T-F Ko Y-M Weng S-B Lin and R Y-Y Chiou ldquoAntimu-tagenicity of supercritical CO

2extracts of Terminalia catappa

leaves and cytotoxicity of the extracts to human hepatoma cellsrdquoJournal of Agricultural and Food Chemistry vol 51 no 12 pp3564ndash3567 2003

[18] S Y Mudi and A Muhammad ldquoAntimalaria activity of ethano-lic extracts of leaves of Terminalia catappa L Combretaceae[indian almond]rdquo Bayero Journal of Pure and Applied Sciencesvol 2 pp 14ndash18 2010

[19] W D Ratnasooriya M G Dharmasiri R A S Rajapakse etal ldquoTender leaf extract of Terminalia catappa antinociceptiveactivity in ratsrdquo Pharmaceutical Biology vol 40 no 1 pp 60ndash66 2002

[20] Y-L Lin Y-H Kuo M-S Shiao C-C Chen and J-C OuldquoFlavonoid glycosides fromTerminalia catappa Lrdquo Journal of theChinese Chemical Society vol 47 no 1 pp 253ndash256 2000

[21] K-T Chung T Y Wong C-I Wei Y-W Huang and Y LinldquoTannins and human health a reviewrdquo Critical Reviews in FoodScience and Nutrition vol 38 no 6 pp 421ndash464 1998


[22] I Orr ldquoOral cancer in betel nut chewersin travancore itsetiology pathology and treatmentrdquo The Lancet vol 222 no5741 pp 575ndash580 1933

[23] K J Ranadive S N Ranadive N M Shivapurkar and S VGothoskar ldquoBetel quid chewing and oral cancer experimentalstudies on hamstersrdquo International Journal of Cancer vol 24 no6 pp 835ndash843 1979

[24] L G Butler and J C Rogler ldquoBiochemicalmechanisms of theantinutritional effectsof tanninsrdquo in Phenolic Compounds inFood andTheir Effects on Health I C-T Ho C Y Lee and M-T Huang Eds vol 506 ofACS Symposium Series chapter 23 p298 American Chemical Society Washington DC USA 1992

[25] F Marzo A Tosar and S Santidrian ldquoEffect of tannic acid onthe immune response of growing chickensrdquo Journal of AnimalScience vol 68 no 10 pp 3306ndash3312 1990

[26] T Tanaka G-I Nonaka and I Nishioka ldquoTannins andrelated compounds XL Revision of the structures of punicalinand punicalagin and isolation and characterization of 2-O-galloylpunicalin from the bark of Punica granatum LrdquoChemicaland Pharmaceutical Bulletin vol 34 no 2 pp 650ndash655 1986

[27] A CronquistAn Integrated System of Classification of FloweringPlants Columbia University Press New York NY USA 1988

[28] F A Resende J M Alves C C Munari et al ldquoInhibition ofdoxorubicin-induced mutagenicity by Baccharis dracunculifo-liardquoMutation Research vol 634 no 1-2 pp 112ndash118 2007

[29] J M Sforcin R O Orsi and V Bankova ldquoEffect of propolissome isolated compounds and its source plant on antibodyproductionrdquo Journal of Ethnopharmacology vol 98 no 3 pp301ndash305 2005

[30] D P D S Leitao A A Da Silva Filho A C M PolizelloJ K Bastos and A C C Spadaro ldquoComparative evaluationof in-vitro effects of Brazilian green propolis and Baccharisdracunculifolia extracts on cariogenic factors of Streptococcusmutansrdquo Biological amp Pharmaceutical Bulletin vol 27 no 11 pp1834ndash1839 2004

[31] P-S Chen J-H Li T-Y Liu and T-C Lin ldquoFolk medicine Ter-minalia catappa and its major tannin component punicalaginare effective against bleomycin-induced genotoxicity inChinesehamster ovary cellsrdquo Cancer Letters vol 152 no 2 pp 115ndash1222000

[32] N Seeram R Lee M Hardy and D Heber ldquoRapid largescale purification of ellagitannins from pomegranate husk aby-product of the commercial juice industryrdquo Separation andPurification Technology vol 41 no 1 pp 49ndash55 2005

[33] MKraszni AMarosi andCK Larive ldquoNMRassignments andthe acid-base characterization of the pomegranate ellagitanninpunicalagin in the acidic pH-rangerdquo Analytical and Bioanalyti-cal Chemistry vol 405 no 17 pp 5807ndash5816 2013

[34] K Robards ldquoStrategies for the determination of bioactive phe-nols in plants fruit and vegetablesrdquo Journal of ChromatographyA vol 1000 no 1-2 pp 657ndash691 2003

[35] M Ribani C B G Bottoli C H Collins I C S Jardimand L C F Melo ldquoValidacao em metodos cromatograficos eeletroforeticosrdquoQuımica Nova vol 27 no 5 pp 771ndash780 2004

[36] W Qu A P Breksa III Z Pan and H Ma ldquoQuantitative deter-mination of major polyphenol constituents in pomegranateproductsrdquo Food Chemistry vol 132 no 3 pp 1585ndash1591 2012

[37] D M Maron and B N Ames ldquoRevised methods for theSalmonella mutagenicity testrdquo Mutation Research vol 113 no3-4 pp 173ndash215 1983

[38] L Bernstein J Kaldor JMcCann andMC Pike ldquoAn empiricalapproach to the statistical analysis of mutagenesis data from theSalmonella testrdquo Mutation Research vol 97 no 4 pp 267ndash2811982

[39] K Mortelmans and E Zeiger ldquoThe Ames Salmonellamicro-some mutagenicity assayrdquo Mutation Research vol 455 no 1-2pp 29ndash60 2000

[40] T D B Machado I C R Leal A C F Amaral K R NDos Santos M G Da Silva and R M Kuster ldquoAntimicrobialellagitannin of Punica granatum fruitsrdquo Journal of the BrazilianChemical Society vol 13 no 5 pp 606ndash610 2002

[41] A J Doig D H Williams P B Oelrichs and L BaczynskyjldquoIsolation and structure elucidation of punicalagin a toxichydrolysable tannin from Terminalia oblongatardquo Journal of theChemical Society Perkin Transactions 1 no 8 pp 2317ndash23211990

[42] F Bressolle M Bromet-Petit and M Audran ldquoValidation ofliquid chromatographic and gas chromatographic methodsApplications to pharmacokineticsrdquo Journal of ChromatographyB vol 686 no 1 pp 3ndash10 1996

[43] R E Ardrey Liquid Chromatography-Mass Spectrometry AnIntroduction John Wiley amp Sons San Francisco Calif USA2003

[44] L R Snyder J J Kirkland and J L Glajch Practical HPLCMethod Development John Wiley amp Sons New York NY USA2nd edition 1997

[45] S Gupta B Saha and A K Giri ldquoComparative antimutagenicand anticlastogenic effects of green tea and black tea a reviewrdquoMutation Research vol 512 no 1 pp 37ndash65 2002

[46] M Labieniec and T Gabryelak ldquoResponse of DNA proteinsand membrane bilayer in the digestive gland cells of freshwatermussel Unio tumidus to tannins exposurerdquo Toxicology in Vitrovol 18 no 6 pp 773ndash781 2004

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


as esophageal cancer have been reported to be related toconsumption of tannin-rich foods such as betel nuts andherbal teas suggesting that tannins might be carcinogenic[22] However other reports have indicated that the carcino-genic activity of tannins might be related to componentsthat are associated with tannins rather than the tanninsthemselves [23 24] Tannins have also been reported to exertother physiological effects such as the acceleration of bloodclotting a reduction in blood pressure a decrease in serumlipid levels liver necrosis and modulation of the immuneresponse [21 25]

Ellagitannins have been isolated along with gallotanninsfrom various other plant families and show little chemotaxo-nomic significance The tannins punicalagin and punicalinboth of which contain a gallagyl unit and were first iso-lated from the pericarps of pomegranate (Punica granatumPunicaceae) [26] were a characteristic component in someTerminalia species This supports the hypothesis that Puni-caceae is chemotaxonomically proximate to Combretaceae asclassified by Cronquist [27]

Despite its biological properties the chemical compo-sition may vary according to geographiclocation [28 29]a fact impairing the standardization of raw material andcommercial products for medicinal purposes [30]

Considering that there are no data in the literature onquantification of the majority secondary metabolites of thehydroalcoholic extract of the T catappa by HPLC-PDA andthe popular use of this plant in this study the punicalaginanomers and ellagic acid were isolated and quantified

Moreover the mutagenic effects were evaluated usinga Salmonellamicrosome assay (Ames test) in the presenceand in the absence of in vitro metabolizing systems in theconcentrations 2224 1668 1112 556 and 278mgplate

2 Experimental

21 Chemical and Reagents HPLC-grade methanol (MeOH)and acetonitrile were purchased from JT Baker (Baker-Mallinckrodt Phillipsburg NJ USA) Standards of ellagicacid were purchased from Sigma Chemical Co (St LouisMO USA) and had a purity of 95 The anomers mixtureof punicalagin 120572 and 120573 isolated of the hydroalcoholic extractfrom T catappa (with 98 purity) were used as external stan-dards HPLC-grade water (18 MΩsdotcm) was obtained using adirect Milli-Q purification system (Millipore Co BedfordMA USA) Sep-Pak RP18 cartridges (500mgmL) for solid-phase extraction (SPE) were purchased from PhenomenexCo (Torrance CA USA)

22 Apparatus The HPLC system used was a JASCO 2010HPLC (Jasco Tokyo Japan) equipped with PU-2089S Pluspump a MD-2018 Plus Photodiode Array Detector (PDA)an AS-2055 Plus auto sampler and column oven (CO-2065 plus) The software ChromNav (Workstation JASCO-ChromNav 11803) was used to control the analytical systemand data collection and processing To purify the substancesa semipreparative JASCOHPLC equipped with two PU-2086pumps a high pressure mixer (Model MX-2080-32) a PDA

detector (model MD-2018 Plus) and a Rheodyne 500 120583Lmanual injector loop was used The software used to operatethe equipment and data processing was ChromNAV version1 18 03

The mass spectrometry experiments were performed onLCQ Fleet equipment (Thermo Scientific) equipped witha dispositive of direct insertion of the sample via flowinjection analysis (FIA) The studied matrix was analyzed byelectrospray ionization (ESI) and fragmentation in multiplestages (MS2MS3 andMSn)was performed at an ion trap (IT)interface The negative mode was selected for the generationand analysis of the mass spectra for the first order (MS)and for the remaining multistage experiments under thefollowing conditions capillary voltage minus25V voltage sprayminus5 kV and capillary temperature 275∘C A carrier gas (N

2)

with a flow of 8 arbitrary units (AU) was used and thecollision gas was helium (He)The track acquisition was 100ndash2000119898119911 Xcalibur version 13 software (Thermo Finnigan)was used to acquire and process the data

The 1H- and 13C-NMR experiments were performedusing a 300MHz (117 T) nuclear magnetic resonance spec-trometer (Varian Inova) CD

3OD was used as the solvent

(Sigma Aldrich)

23 Plant Material The leaves of T catappa were collectedfrom the beach-front city of SantosSP with the permissionof the municipality in April 2010 and were identified bybotanist Paulo Salles Penteado ofUniversity Santa Cecilia andregistered (M Tomaz 01) in the Herbarium of the Universityof Santa Cecilia (HUSC) Authorization was obtained fromIBAMA (Instituto Brasileiro do Meio Ambiente e dos Recur-sos Naturais Renovaveis) to the number 33152

24 Extraction The infusion and hydroalcoholic extractwere prepared according to the experimental protocol estab-lished in Biota-Fapesp Project which this work is linked(process number 0952237-9)

241 Preparation of the Infusion of the Leaves from T catappaThe leaves (1 g) of T catappa after drying were prepared byinfusion as their use in folk medicine [31]

242 Preparation of the Hydroalcoholic Extract of the Leavesfrom T catappa The dried and powdered leaves (5246 g) ofT catappawere extracted by percolation at room temperaturewith a mixture of EtOH H

2O (7 3 vv) The solvents were

evaporated to dryness at low pressure to give 331 g ofthe crude hydroalcoholic extract (87) The extract wasanalyzed by FIA-ESI-IT-MSn and HPLC-PDA

243 Isolation of the Fraction Containing the PunicalaginAnomers by SemipreparativeHPLC A total of 14 g of hydroal-coholic extract (7 3 vv) was dissolved inH

2O MeOH (8 2

vv) and partitioned successively with hexane (500mL) andethyl acetate (500mL) giving 068 g (48) (hexane fraction)193 g (1378) (ethyl acetate fraction) and the 1015 g (725)(hydromethanol fraction 8 2 vv)


200 400 600 800 1000 1200 1400 1600 1800 20000

10

20

30

40

50

60

70

80

90

100

Rela

tive a

bund

ance

108319

7814654165311756353232577

9327969203 11148030130 3397111322 114403128348 14393046776 157117 172447185395

mz









2O



18









2O (1 1


2O to a


1199034730min (peak 3)



HOOH

OHOHHO

OO

O

O

OHOH

O

O

OHOH

OO

O

O

OH

O

O

HO

O

O

HOHO

HO

OO

O

O

OH

OHHO

OH

O

O

OHOHO

HO

OHHO

HO

OHOH

HOOH

OHOHHO

OO

O

O

OHOH

O

HO

HO

OH

O

O RO

HO

HO OH HO OH

O

OO

O

OH

O

H

H

CH2

CH2





17

1615

1319

1418

12

89

11

10

7

3 2 1

54

6

20

484745

4443 42

46

39

38 37

36354041

32

31

30 2928

3334

27 2223

242526

21

R998400












LOQ120583gmL









2 1 165M KCl 05






0

1000000

2000000

200 220 240 260 280 300 320 340 360 380 400Wavelength (nm)

0

20

40

60

80

100

Inte

nsity

()

200 220 240 260 280 300 320 340 360 380 400Wavelength (nm)

0

20

40

60

80

100

Inte

nsity

()

200 220 240 260 280 300 320 340 360 380 400Wavelength (nm)

0

20

40

60

80

100

Inte

nsity

()

1

2

3

Inte

nsity

(120583V

)



1199031475 (peak 1) min and at 119905






1199031880min and 119905



1199051199031475min (peak 1) and 119905






1199031475min




1

2

3

200 220 240 260 280 300 320 340 360 380 400Wavelength (nm)

0

20

40

60

80

100

Inte

nsity

()

200 220 240 260 280 300 320 340 360 380 400Wavelength (nm)

0

20

40

60

80

100

Inte

nsity

()

200 220 240 260 280 300 320 340 360 380 400Wavelength (nm)

0

20

40

60

80

100

Inte

nsity

()


0

1000000

2000000

Inte

nsity

(120583V

)


1199031470min and peak 2 at 119905


Peak 3 at 1199051199034730min









00 50 100 150 200 250 300 350 400

0

500000

1000000

1a

1b

200 220 240 260 280 300 320 340 360 380 400 Wavelength (nm)

Wavelength (nm)

0

20

40

60

80

100

Inte

nsity

()

Inte

nsity

()

200 220 240 260 280 300 320 340 360 380 400

0

20

40

60

80

100

Inte

nsity

(120583V

)





















ityexpressedas

them

eanandsta

ndarddeviationof

then

umbero

frevertantsplatea

ndmutagenicindex(in

parenthesis

)inSalm

onellatyphim

urium

TA98TA100

TA97aandTA

102strainstreated

with

hydroalcoh

olicextracto

fTcatappa

atvario

usdo

seswith

(+S9)o

rwith

out(minusS9)m

etabolicactiv

ation

Treatm

ents

Num

bero


)platea

ndMI

TA98

TA100

TA102

TA97a

mgplate

minusS9

+S9

mgplate

minusS9

+S9

minusS9

+S9

minusS9

+S9

Term

inaliacatapp

a

00a

16plusmn3

68plusmn1

00a

93plusmn6

87plusmn4

352plusmn30

435plusmn43

172plusmn2

99plusmn1

278


156



556


312


1112


625


1668


938


2224


125

Nd


Ctrol+

720plusmn63

500plusmn57

Ctrol+

1225plusmn75

700plusmn80

1143plusmn53

1309plusmn38

1426plusmn67

700plusmn45

lowast119875lt005(A

NOVA


NOVA

)0aN


(DMSO


ositive

Con

trol4-nitro

-o-phenylenediam

ine(100120583

gplatemdash

TA98

andTA

97a)sod

ium

azide

(125120583

gplatemdash

TA100)m

itomycin



S9and


gplatemdash

TA98T

A100

TA97a)2-aminofl

uorene

(100120583

gplatemdash

TA102)inthepresence

ofS9N

dno

tdeterm

ined



4 Conclusions






Acknowledgments


References
























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















200 400 600 800 1000 1200 1400 1600 1800 20000

10

20

30

40

50

60

70

80

90

100

Rela

tive a

bund

ance

108319

7814654165311756353232577

9327969203 11148030130 3397111322 114403128348 14393046776 157117 172447185395

mz









2O



18









2O (1 1


2O to a


1199034730min (peak 3)



HOOH

OHOHHO

OO

O

O

OHOH

O

O

OHOH

OO

O

O

OH

O

O

HO

O

O

HOHO

HO

OO

O

O

OH

OHHO

OH

O

O

OHOHO

HO

OHHO

HO

OHOH

HOOH

OHOHHO

OO

O

O

OHOH

O

HO

HO

OH

O

O RO

HO

HO OH HO OH

O

OO

O

OH

O

H

H

CH2

CH2





17

1615

1319

1418

12

89

11

10

7

3 2 1

54

6

20

484745

4443 42

46

39

38 37

36354041

32

31

30 2928

3334

27 2223

242526

21

R998400












LOQ120583gmL









2 1 165M KCl 05






0

1000000

2000000

200 220 240 260 280 300 320 340 360 380 400Wavelength (nm)

0

20

40

60

80

100

Inte

nsity

()

200 220 240 260 280 300 320 340 360 380 400Wavelength (nm)

0

20

40

60

80

100

Inte

nsity

()

200 220 240 260 280 300 320 340 360 380 400Wavelength (nm)

0

20

40

60

80

100

Inte

nsity

()

1

2

3

Inte

nsity

(120583V

)



1199031475 (peak 1) min and at 119905






1199031880min and 119905



1199051199031475min (peak 1) and 119905






1199031475min




1

2

3

200 220 240 260 280 300 320 340 360 380 400Wavelength (nm)

0

20

40

60

80

100

Inte

nsity

()

200 220 240 260 280 300 320 340 360 380 400Wavelength (nm)

0

20

40

60

80

100

Inte

nsity

()

200 220 240 260 280 300 320 340 360 380 400Wavelength (nm)

0

20

40

60

80

100

Inte

nsity

()


0

1000000

2000000

Inte

nsity

(120583V

)


1199031470min and peak 2 at 119905


Peak 3 at 1199051199034730min









00 50 100 150 200 250 300 350 400

0

500000

1000000

1a

1b

200 220 240 260 280 300 320 340 360 380 400 Wavelength (nm)

Wavelength (nm)

0

20

40

60

80

100

Inte

nsity

()

Inte

nsity

()

200 220 240 260 280 300 320 340 360 380 400

0

20

40

60

80

100

Inte

nsity

(120583V

)





















ityexpressedas

them

eanandsta

ndarddeviationof

then

umbero

frevertantsplatea

ndmutagenicindex(in

parenthesis

)inSalm

onellatyphim

urium

TA98TA100

TA97aandTA

102strainstreated

with

hydroalcoh

olicextracto

fTcatappa

atvario

usdo

seswith

(+S9)o

rwith

out(minusS9)m

etabolicactiv

ation

Treatm

ents

Num

bero


)platea

ndMI

TA98

TA100

TA102

TA97a

mgplate

minusS9

+S9

mgplate

minusS9

+S9

minusS9

+S9

minusS9

+S9

Term

inaliacatapp

a

00a

16plusmn3

68plusmn1

00a

93plusmn6

87plusmn4

352plusmn30

435plusmn43

172plusmn2

99plusmn1

278


156



556


312


1112


625


1668


938


2224


125

Nd


Ctrol+

720plusmn63

500plusmn57

Ctrol+

1225plusmn75

700plusmn80

1143plusmn53

1309plusmn38

1426plusmn67

700plusmn45

lowast119875lt005(A

NOVA


NOVA

)0aN


(DMSO


ositive

Con

trol4-nitro

-o-phenylenediam

ine(100120583

gplatemdash

TA98

andTA

97a)sod

ium

azide

(125120583

gplatemdash

TA100)m

itomycin



S9and


gplatemdash

TA98T

A100

TA97a)2-aminofl

uorene

(100120583

gplatemdash

TA102)inthepresence

ofS9N

dno

tdeterm

ined



4 Conclusions






Acknowledgments


References
























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















HOOH

OHOHHO

OO

O

O

OHOH

O

O

OHOH

OO

O

O

OH

O

O

HO

O

O

HOHO

HO

OO

O

O

OH

OHHO

OH

O

O

OHOHO

HO

OHHO

HO

OHOH

HOOH

OHOHHO

OO

O

O

OHOH

O

HO

HO

OH

O

O RO

HO

HO OH HO OH

O

OO

O

OH

O

H

H

CH2

CH2





17

1615

1319

1418

12

89

11

10

7

3 2 1

54

6

20

484745

4443 42

46

39

38 37

36354041

32

31

30 2928

3334

27 2223

242526

21

R998400












LOQ120583gmL









2 1 165M KCl 05






0

1000000

2000000

200 220 240 260 280 300 320 340 360 380 400Wavelength (nm)

0

20

40

60

80

100

Inte

nsity

()

200 220 240 260 280 300 320 340 360 380 400Wavelength (nm)

0

20

40

60

80

100

Inte

nsity

()

200 220 240 260 280 300 320 340 360 380 400Wavelength (nm)

0

20

40

60

80

100

Inte

nsity

()

1

2

3

Inte

nsity

(120583V

)



1199031475 (peak 1) min and at 119905






1199031880min and 119905



1199051199031475min (peak 1) and 119905






1199031475min




1

2

3

200 220 240 260 280 300 320 340 360 380 400Wavelength (nm)

0

20

40

60

80

100

Inte

nsity

()

200 220 240 260 280 300 320 340 360 380 400Wavelength (nm)

0

20

40

60

80

100

Inte

nsity

()

200 220 240 260 280 300 320 340 360 380 400Wavelength (nm)

0

20

40

60

80

100

Inte

nsity

()


0

1000000

2000000

Inte

nsity

(120583V

)


1199031470min and peak 2 at 119905


Peak 3 at 1199051199034730min









00 50 100 150 200 250 300 350 400

0

500000

1000000

1a

1b

200 220 240 260 280 300 320 340 360 380 400 Wavelength (nm)

Wavelength (nm)

0

20

40

60

80

100

Inte

nsity

()

Inte

nsity

()

200 220 240 260 280 300 320 340 360 380 400

0

20

40

60

80

100

Inte

nsity

(120583V

)





















ityexpressedas

them

eanandsta

ndarddeviationof

then

umbero

frevertantsplatea

ndmutagenicindex(in

parenthesis

)inSalm

onellatyphim

urium

TA98TA100

TA97aandTA

102strainstreated

with

hydroalcoh

olicextracto

fTcatappa

atvario

usdo

seswith

(+S9)o

rwith

out(minusS9)m

etabolicactiv

ation

Treatm

ents

Num

bero


)platea

ndMI

TA98

TA100

TA102

TA97a

mgplate

minusS9

+S9

mgplate

minusS9

+S9

minusS9

+S9

minusS9

+S9

Term

inaliacatapp

a

00a

16plusmn3

68plusmn1

00a

93plusmn6

87plusmn4

352plusmn30

435plusmn43

172plusmn2

99plusmn1

278


156



556


312


1112


625


1668


938


2224


125

Nd


Ctrol+

720plusmn63

500plusmn57

Ctrol+

1225plusmn75

700plusmn80

1143plusmn53

1309plusmn38

1426plusmn67

700plusmn45

lowast119875lt005(A

NOVA


NOVA

)0aN


(DMSO


ositive

Con

trol4-nitro

-o-phenylenediam

ine(100120583

gplatemdash

TA98

andTA

97a)sod

ium

azide

(125120583

gplatemdash

TA100)m

itomycin



S9and


gplatemdash

TA98T

A100

TA97a)2-aminofl

uorene

(100120583

gplatemdash

TA102)inthepresence

ofS9N

dno

tdeterm

ined



4 Conclusions






Acknowledgments


References
























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of



















LOQ120583gmL









2 1 165M KCl 05






0

1000000

2000000

200 220 240 260 280 300 320 340 360 380 400Wavelength (nm)

0

20

40

60

80

100

Inte

nsity

()

200 220 240 260 280 300 320 340 360 380 400Wavelength (nm)

0

20

40

60

80

100

Inte

nsity

()

200 220 240 260 280 300 320 340 360 380 400Wavelength (nm)

0

20

40

60

80

100

Inte

nsity

()

1

2

3

Inte

nsity

(120583V

)



1199031475 (peak 1) min and at 119905






1199031880min and 119905



1199051199031475min (peak 1) and 119905






1199031475min




1

2

3

200 220 240 260 280 300 320 340 360 380 400Wavelength (nm)

0

20

40

60

80

100

Inte

nsity

()

200 220 240 260 280 300 320 340 360 380 400Wavelength (nm)

0

20

40

60

80

100

Inte

nsity

()

200 220 240 260 280 300 320 340 360 380 400Wavelength (nm)

0

20

40

60

80

100

Inte

nsity

()


0

1000000

2000000

Inte

nsity

(120583V

)


1199031470min and peak 2 at 119905


Peak 3 at 1199051199034730min









00 50 100 150 200 250 300 350 400

0

500000

1000000

1a

1b

200 220 240 260 280 300 320 340 360 380 400 Wavelength (nm)

Wavelength (nm)

0

20

40

60

80

100

Inte

nsity

()

Inte

nsity

()

200 220 240 260 280 300 320 340 360 380 400

0

20

40

60

80

100

Inte

nsity

(120583V

)





















ityexpressedas

them

eanandsta

ndarddeviationof

then

umbero

frevertantsplatea

ndmutagenicindex(in

parenthesis

)inSalm

onellatyphim

urium

TA98TA100

TA97aandTA

102strainstreated

with

hydroalcoh

olicextracto

fTcatappa

atvario

usdo

seswith

(+S9)o

rwith

out(minusS9)m

etabolicactiv

ation

Treatm

ents

Num

bero


)platea

ndMI

TA98

TA100

TA102

TA97a

mgplate

minusS9

+S9

mgplate

minusS9

+S9

minusS9

+S9

minusS9

+S9

Term

inaliacatapp

a

00a

16plusmn3

68plusmn1

00a

93plusmn6

87plusmn4

352plusmn30

435plusmn43

172plusmn2

99plusmn1

278


156



556


312


1112


625


1668


938


2224


125

Nd


Ctrol+

720plusmn63

500plusmn57

Ctrol+

1225plusmn75

700plusmn80

1143plusmn53

1309plusmn38

1426plusmn67

700plusmn45

lowast119875lt005(A

NOVA


NOVA

)0aN


(DMSO


ositive

Con

trol4-nitro

-o-phenylenediam

ine(100120583

gplatemdash

TA98

andTA

97a)sod

ium

azide

(125120583

gplatemdash

TA100)m

itomycin



S9and


gplatemdash

TA98T

A100

TA97a)2-aminofl

uorene

(100120583

gplatemdash

TA102)inthepresence

ofS9N

dno

tdeterm

ined



4 Conclusions






Acknowledgments


References
























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


















0

1000000

2000000

200 220 240 260 280 300 320 340 360 380 400Wavelength (nm)

0

20

40

60

80

100

Inte

nsity

()

200 220 240 260 280 300 320 340 360 380 400Wavelength (nm)

0

20

40

60

80

100

Inte

nsity

()

200 220 240 260 280 300 320 340 360 380 400Wavelength (nm)

0

20

40

60

80

100

Inte

nsity

()

1

2

3

Inte

nsity

(120583V

)



1199031475 (peak 1) min and at 119905






1199031880min and 119905



1199051199031475min (peak 1) and 119905






1199031475min




1

2

3

200 220 240 260 280 300 320 340 360 380 400Wavelength (nm)

0

20

40

60

80

100

Inte

nsity

()

200 220 240 260 280 300 320 340 360 380 400Wavelength (nm)

0

20

40

60

80

100

Inte

nsity

()

200 220 240 260 280 300 320 340 360 380 400Wavelength (nm)

0

20

40

60

80

100

Inte

nsity

()


0

1000000

2000000

Inte

nsity

(120583V

)


1199031470min and peak 2 at 119905


Peak 3 at 1199051199034730min









00 50 100 150 200 250 300 350 400

0

500000

1000000

1a

1b

200 220 240 260 280 300 320 340 360 380 400 Wavelength (nm)

Wavelength (nm)

0

20

40

60

80

100

Inte

nsity

()

Inte

nsity

()

200 220 240 260 280 300 320 340 360 380 400

0

20

40

60

80

100

Inte

nsity

(120583V

)





















ityexpressedas

them

eanandsta

ndarddeviationof

then

umbero

frevertantsplatea

ndmutagenicindex(in

parenthesis

)inSalm

onellatyphim

urium

TA98TA100

TA97aandTA

102strainstreated

with

hydroalcoh

olicextracto

fTcatappa

atvario

usdo

seswith

(+S9)o

rwith

out(minusS9)m

etabolicactiv

ation

Treatm

ents

Num

bero


)platea

ndMI

TA98

TA100

TA102

TA97a

mgplate

minusS9

+S9

mgplate

minusS9

+S9

minusS9

+S9

minusS9

+S9

Term

inaliacatapp

a

00a

16plusmn3

68plusmn1

00a

93plusmn6

87plusmn4

352plusmn30

435plusmn43

172plusmn2

99plusmn1

278


156



556


312


1112


625


1668


938


2224


125

Nd


Ctrol+

720plusmn63

500plusmn57

Ctrol+

1225plusmn75

700plusmn80

1143plusmn53

1309plusmn38

1426plusmn67

700plusmn45

lowast119875lt005(A

NOVA


NOVA

)0aN


(DMSO


ositive

Con

trol4-nitro

-o-phenylenediam

ine(100120583

gplatemdash

TA98

andTA

97a)sod

ium

azide

(125120583

gplatemdash

TA100)m

itomycin



S9and


gplatemdash

TA98T

A100

TA97a)2-aminofl

uorene

(100120583

gplatemdash

TA102)inthepresence

ofS9N

dno

tdeterm

ined



4 Conclusions






Acknowledgments


References
























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















1

2

3

200 220 240 260 280 300 320 340 360 380 400Wavelength (nm)

0

20

40

60

80

100

Inte

nsity

()

200 220 240 260 280 300 320 340 360 380 400Wavelength (nm)

0

20

40

60

80

100

Inte

nsity

()

200 220 240 260 280 300 320 340 360 380 400Wavelength (nm)

0

20

40

60

80

100

Inte

nsity

()


0

1000000

2000000

Inte

nsity

(120583V

)


1199031470min and peak 2 at 119905


Peak 3 at 1199051199034730min









00 50 100 150 200 250 300 350 400

0

500000

1000000

1a

1b

200 220 240 260 280 300 320 340 360 380 400 Wavelength (nm)

Wavelength (nm)

0

20

40

60

80

100

Inte

nsity

()

Inte

nsity

()

200 220 240 260 280 300 320 340 360 380 400

0

20

40

60

80

100

Inte

nsity

(120583V

)





















ityexpressedas

them

eanandsta

ndarddeviationof

then

umbero

frevertantsplatea

ndmutagenicindex(in

parenthesis

)inSalm

onellatyphim

urium

TA98TA100

TA97aandTA

102strainstreated

with

hydroalcoh

olicextracto

fTcatappa

atvario

usdo

seswith

(+S9)o

rwith

out(minusS9)m

etabolicactiv

ation

Treatm

ents

Num

bero


)platea

ndMI

TA98

TA100

TA102

TA97a

mgplate

minusS9

+S9

mgplate

minusS9

+S9

minusS9

+S9

minusS9

+S9

Term

inaliacatapp

a

00a

16plusmn3

68plusmn1

00a

93plusmn6

87plusmn4

352plusmn30

435plusmn43

172plusmn2

99plusmn1

278


156



556


312


1112


625


1668


938


2224


125

Nd


Ctrol+

720plusmn63

500plusmn57

Ctrol+

1225plusmn75

700plusmn80

1143plusmn53

1309plusmn38

1426plusmn67

700plusmn45

lowast119875lt005(A

NOVA


NOVA

)0aN


(DMSO


ositive

Con

trol4-nitro

-o-phenylenediam

ine(100120583

gplatemdash

TA98

andTA

97a)sod

ium

azide

(125120583

gplatemdash

TA100)m

itomycin



S9and


gplatemdash

TA98T

A100

TA97a)2-aminofl

uorene

(100120583

gplatemdash

TA102)inthepresence

ofS9N

dno

tdeterm

ined



4 Conclusions






Acknowledgments


References
























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















00 50 100 150 200 250 300 350 400

0

500000

1000000

1a

1b

200 220 240 260 280 300 320 340 360 380 400 Wavelength (nm)

Wavelength (nm)

0

20

40

60

80

100

Inte

nsity

()

Inte

nsity

()

200 220 240 260 280 300 320 340 360 380 400

0

20

40

60

80

100

Inte

nsity

(120583V

)





















ityexpressedas

them

eanandsta

ndarddeviationof

then

umbero

frevertantsplatea

ndmutagenicindex(in

parenthesis

)inSalm

onellatyphim

urium

TA98TA100

TA97aandTA

102strainstreated

with

hydroalcoh

olicextracto

fTcatappa

atvario

usdo

seswith

(+S9)o

rwith

out(minusS9)m

etabolicactiv

ation

Treatm

ents

Num

bero


)platea

ndMI

TA98

TA100

TA102

TA97a

mgplate

minusS9

+S9

mgplate

minusS9

+S9

minusS9

+S9

minusS9

+S9

Term

inaliacatapp

a

00a

16plusmn3

68plusmn1

00a

93plusmn6

87plusmn4

352plusmn30

435plusmn43

172plusmn2

99plusmn1

278


156



556


312


1112


625


1668


938


2224


125

Nd


Ctrol+

720plusmn63

500plusmn57

Ctrol+

1225plusmn75

700plusmn80

1143plusmn53

1309plusmn38

1426plusmn67

700plusmn45

lowast119875lt005(A

NOVA


NOVA

)0aN


(DMSO


ositive

Con

trol4-nitro

-o-phenylenediam

ine(100120583

gplatemdash

TA98

andTA

97a)sod

ium

azide

(125120583

gplatemdash

TA100)m

itomycin



S9and


gplatemdash

TA98T

A100

TA97a)2-aminofl

uorene

(100120583

gplatemdash

TA102)inthepresence

ofS9N

dno

tdeterm

ined



4 Conclusions






Acknowledgments


References
























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


















ityexpressedas

them

eanandsta

ndarddeviationof

then

umbero

frevertantsplatea

ndmutagenicindex(in

parenthesis

)inSalm

onellatyphim

urium

TA98TA100

TA97aandTA

102strainstreated

with

hydroalcoh

olicextracto

fTcatappa

atvario

usdo

seswith

(+S9)o

rwith

out(minusS9)m

etabolicactiv

ation

Treatm

ents

Num

bero


)platea

ndMI

TA98

TA100

TA102

TA97a

mgplate

minusS9

+S9

mgplate

minusS9

+S9

minusS9

+S9

minusS9

+S9

Term

inaliacatapp

a

00a

16plusmn3

68plusmn1

00a

93plusmn6

87plusmn4

352plusmn30

435plusmn43

172plusmn2

99plusmn1

278


156



556


312


1112


625


1668


938


2224


125

Nd


Ctrol+

720plusmn63

500plusmn57

Ctrol+

1225plusmn75

700plusmn80

1143plusmn53

1309plusmn38

1426plusmn67

700plusmn45

lowast119875lt005(A

NOVA


NOVA

)0aN


(DMSO


ositive

Con

trol4-nitro

-o-phenylenediam

ine(100120583

gplatemdash

TA98

andTA

97a)sod

ium

azide

(125120583

gplatemdash

TA100)m

itomycin



S9and


gplatemdash

TA98T

A100

TA97a)2-aminofl

uorene

(100120583

gplatemdash

TA102)inthepresence

ofS9N

dno

tdeterm

ined



4 Conclusions






Acknowledgments


References
























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


















4 Conclusions






Acknowledgments


References
























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of





















of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















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Research Article Characterization and Quantification of ...downloads.hindawi.com/journals/ecam/2014/676902.pdf · include amendoeira-da-praia, cuca, and chap ´eu de sol. In several