research article antioxidant and antiproliferative...

Hindawi Publishing CorporationEvidence-Based Complementary and Alternative MedicineVolume 2013 Article ID 950272 10 pageshttpdxdoiorg1011552013950272

Research ArticleAntioxidant and Antiproliferative Activities of Leaf Extractsfrom Plukenetia volubilis Linneo (Euphorbiaceae)

Ana Karina Lima Nascimento12 Raniere Fagundes Melo-Silveira2

Nednaldo Dantas-Santos2 Juacutelia Morais Fernandes3 Silvana Maria Zucolotto3

Hugo Alexandre Oliveira Rocha2 and Katia Castanho Scortecci1

1 Departamento de Biologia Celular e Genetica Centro de Biociencias Universidade Federal do Rio Grande do Norte59072-970 Natal RN Brazil

2 Laboratorio de Biotecnologia de Polımeros Naturais-Biopol Departamento de Bioquımica Centro de BiocienciasUniversidade Federal do Rio Grande do Norte 59072-970 Natal Brazil

3 Departamento de Farmacia Centro de Ciencias da Saude Universidade Federal do Rio Grande do Norte 59010-180 Natal Brazil

Correspondence should be addressed to Katia Castanho Scortecci kcscortecciufrnetbr

Received 18 March 2013 Revised 6 July 2013 Accepted 30 July 2013

Academic Editor Jose Luis Rıos

Copyright copy 2013 Ana Karina Lima Nascimento et al This is an open access article distributed under the Creative CommonsAttribution License which permits unrestricted use distribution and reproduction in any medium provided the original work isproperly cited

Plukenetia volubilisLinneo or Sacha inca is an oleaginous plant from the Euphorbiaceae familyThe aimof this workwas to performa chemical and biological analysis of different leaf extracts from P volubilis such as aqueous extract (AEL)methanol (MEL) ethanol(EEL) chloroform (CEL) and hexane (HEL) Thin layer chromatography analysis revealed the presence of phenolic compoundssteroids andor terpenoıdes Furthermore the antioxidant activities were analyzed by in vitro assays and their effects on cell lineagesby in vivo assaysThe Total Antioxidant Capacity (TCA) was expressed as equivalent ascorbic acid (EEAg) and it was observed thatthe extracts showed values ranging from 5931 to 9776 EAAg Furthermore the DPPH assay values ranged from 628 to 883The cell viability assay showed that the extracts were able to reduce viability from cancer cells such as HeLa and A549 cells Theextracts MEL and HEL (250 120583gmL) were able to reduce the proliferation of HeLa cells up to 543 and 485 respectively Theflow cytometer results showed that these extracts induce cell death via the apoptosis pathway On the other hand the extracts HELand AEL were able to induce cell proliferation of normal fibroblast 3T3 cells

1 Introduction

Natural products have long been investigated for their poten-tial benefits In the 1900s most medicines were obtained fromthe cooking infusion ormaceration of roots barks leaves orflowers Today natural products still have a huge importanceas a source of new drugs and leads Approximately 60of anticancer compounds and 75 of drugs for infectiousdisease come from natural products or their derivatives[1ndash4] In order to develop new drugs different plants arebeing selected randomly or based on ethnobotanic andethnopharmacologic knowledge The extracts obtained fromthese plants are then evaluated using different in vitro assaysas well as their effects on normal or tumor cells Then

the potential extracts are purified in order to obtain theirpotential fractions or molecules to be tested by in vitro andin vivo assays again followed by analysis of their potential fornew drugs [5]

Recently many diseases such as cancer diabetes arte-riosclerosis inflammatory disease autoimmunity cardiovas-cular disease and Alzheimerrsquos have been associated with theincrease of reactive oxygen species (ROS) or the inabilityof the organism to reduce these ROS that were normallyproduced by the organism cells a process known as oxidativestress [6] Antioxidants are important substances that havethe ability to protect the organism from the damage causedby the oxidative stress Due to this ability there is a specialinterest in the presence of natural antioxidants in medicinal

2 Evidence-Based Complementary and Alternative Medicine

plants that may help an organism to keep the normal balanceof ROS In the case of cancer these antioxidant substancesare being used in the treatment to prevent the normal cellfrom becoming a cancer cell as well as to kill cancer cellsfurthermore these compounds had no negative effects onnormal cells [7 8]

The Euphorbiaceae is formed by more than 6000 specieswith extreme diversity of secondary compounds produced [910] This variability of compounds may explain the differentuses of the plants from this family such as antiprolifera-tive antimicrobial cytotoxic activity and anti-inflammatoryamong others [9] Moreover many times the latex producedby these plants has been used as medicine for skin diseaseas well as skin healing for example Euphorbia thymifolia Eneriifolia andE nivulia aswell as theCroton bonplandianumhave been used to treat scabies and other skin diseases [11]

Plukenetia volubilis Linneo or sacha inchi the target ofthis work is a climbing shrub plant from the Euphorbiaceaethat grows mostly in the Amazon region [12] Some studieshave shown that seeds from this plant are an excellent sourceof protein (27ndash33) and oil (35ndash60) [13] The oil compo-sition is mostly omega 3 and omega 6 (4861 and 368resp) [13 14] Some studies have been done using seedsand leaves in order to reduce the lipid profile from patientswith postprandial lipemia [15] and hypercholesterolemia [16]However there is no scientific data about the use of leavesfor skin disease as it has been used in folk medicine inthe Amazon region The aim of the work presented herewas to evaluate the effects from five extracts obtained fromfresh leaves methanol (MEL) ethanol (EEL) chloroformCEL hexane (HEL) and aqueous (AEL) These extractswere analyzed using antioxidant assays and antiproliferativeassays using normal and tumor cells The results showed thatsome of these extracts had antioxidant and antiproliferativeactivities against HeLa cells Furthermore it was observedthat the extracts were able to stimulate the cell proliferationin fibroblast cells-3T3

2 Materials and Methods

21 Plant Material Plukenetia volubilis plants were grownin soil (soil and sand 1 1) in Parnamirim RN Brazil(minus05∘ 471015840 4210158401015840 +35∘ 121015840 3410158401015840) After six months leaves werecollected to obtain the different extracts Two specimenswere deposited in a herbarium at Centro de Biociencias daUniversidade Federal do RioGrande doNorte (UFRN) underthe number 10854

22 Preparation of Leaf Extracts Fresh leaves were collectedand 15 gwas divided into small pieces by hand and then trans-ferred to a flask in a proportion of 1 10 (drug solvent wv)using the following solvents methanol ethanol chloroformhexane andwaterThematerial was protected from light withaluminum foil and these flasks were shaken at 50 rpm for24 h Then the extracts were filtered on Whatman paper n∘1 and dried in Rotavapor at 40∘C resulting in five extractsmethanol ethanol chloroform hexane and aqueous Afterthat the extracts were weighed and then resuspended into

DMSO (CRQnuclear) to final concentration of 10mgmL(extract stock solution)

23 Phytochemistry Screening by Thin Layer Chromatography(TLC) This assay was conducted by TLC using aluminumsheets of silica gel F

254(Merck) All chromatograms

were developed in a saturated chamber Two systemswere used as mobile phases (1) ethyl acetate formicacid water methanol (10 05 06 02) and (2)toluene ethyl acetate methanol (5 5 05) The eluentsystems employed in this study were selected or developedaccording to data from the literature [17] After thechromatograms were developed the plates were driedand the spots were visualized sequentially under UV lightat 254 and 365 nm The plates were then sprayed withspecific chromogenic agents according to the chemicalsubstances analyzed The following solutions were used asvisualizing agents vanillin sulfuric +105∘C natural reagentA 05 ferric chloride 1 in methanol and Dragendorffreagent Kaempferol luteolin and apigenin all obtainedfrom Sigma-Aldrich were used as standard sample

24 Total Content of Sugar Protein and Phenolic CompoundsTotal sugar was estimated by the phenol-H

2SO4reaction

using D-glucose (Sigma-Aldrich) as a standard [18] Proteincontent was measured using Spectorrsquos method with bovinealbumin as a standard [19] Total phenolic compounds inthe extract solution were determined using Folin-Ciocalteursquoscoloimetric method and gallic acid as a standard [20]

25 Antioxidant Activity The antioxidant activity was exam-ined by conducting in vitro tests total antioxidant capacityhydroxyl radical scavenging superoxide radical scavengingferric chelating and reducing power as previously describedin [21 22]

251 Determination of Total Antioxidant Capacity (TAC)The assay for total antioxidant capacity is based on the reduc-tion of Mo+6 to Mo+5 by the plant extracts and subsequentformation of green phosphateMo+5 complexes at acid pH[23] The tubes containing the plant extracts at 250 120583gmLconcentration and the reagent solution (06M sulfuric acid28mM sodium phosphate and 4mM ammonium molyb-date) were incubated at 95∘C for 90min After this time themixture was cooled at room temperature and the absorbanceof each tube was measured at 695 nm against a blank Theantioxidant capacity was expressed as mg of ascorbic acidgdescribed as ascorbic acid equivalent (EEAg)

252 Hydroxyl Radical Scavenging Activity Assay The scav-enging activity of plant extracts against the hydroxyl radicalwas measured based on Fentonrsquos reaction (Fe2+ + H

2O2rarr

Fe3+ + OHminus + OH∙) The results were expressed as an inhibi-tion rate Hydroxyl radicals were generated using a modifiedmethod [24] where 3mLof 150mMsodiumphosphate buffer(pH 74) contained 10mM FeSO

4times 7H2O 10mM EDTA

2mM sodium salicylate 30 H2O2(200mL) and the plant

extracts at 250120583gmL concentration The positive control

Evidence-Based Complementary and Alternative Medicine 3

the sodium phosphate buffer was replaced by H2O2 The

solutions were incubated at 37∘C for 1 h and the presence ofthe hydroxyl radical was detected by monitoring absorbanceat 510 nm

253 Ferric Chelating The ferrous ion chelating ability ofplant extracts was measured according to [25] The plantextract samples (250 120583gmL concentration) were added to themixture of 50 120583L of FeCl

2(2mM) and 200 120583L of ferrozine

(5mM) in a final volume of 1mL This mixture was mixedwell and then incubated for 10min at room temperature Theabsorbance was measured at 562 nm against a blank

254 Superoxide Radical Scavenging Activity Assay Theassay was based on the capacity of the leaf extracts to inhibitthe photochemical reduction of nitroblue tetrazolium (NBT)in the riboflavin-light-NBT system [26] Then 3mL reactionmixture (50mM phosphate buffer (pH 78) 13mM methio-nine 2mM riboflavin 100mM EDTA and 75mM NBT)was transferred to a tube and then 1mL from plant extractsolution was added The production of blue formazan wasmonitored as absorbance increased at 560 nm after a 10minillumination from a fluorescent lamp The entire reactionassembly was enclosed in a box covered by aluminum foil

255 Reducing Power The reducing power of the sampleswas quantified as described previously [20 27] The plantextract at 250 120583gmL concentration was added to the reactionmixture (02M phosphate buffer (pH 66) and potassiumferricyanide (1 wv)) with a final volume of 4mL Thismixture was incubated at 50∘C for 20min The reactionwas terminated by adding the TCA solution (10 wv)The solution was then mixed with distilled water and ferricchloride (01 wv) and the absorbance was measured at700 nm The result was expressed as a percentage of theactivity shown by 02mgmL of vitamin C

256 DPPH Free-Radical Scavenging Activity The scaveng-ing activity of the DPPH radical was assayed according to themethod of Shimada et al [28] with some modification In atest tube 15mLofmethanolwas added then the plant extractat 250120583gmL concentration and finally 1mL of a methanolsolution of 01mM DPPH (Fluka) Then the mixture wasshaken vigorously and allowed to stand at room temperaturefor 30min The absorbance was then measured at 517 nmLower absorbance of the reaction mixture indicates higherfree-radical scavenging activity The DPPH free-radical scav-enging activity was calculated as follows scavenging activity() = (1 minus 119860

11198600) times 100 where 119860

0is the absorbance from

the control and 1198601is the absorbance from samples

26 Cell Proliferation Analysis HeLa A549 3T3 and CHOcells were grown in 75 cm2 culture flasks in DulbeccorsquosModified Eagle Medium (DMEM) For the assay cells weretransferred into 96-well plates at a density of 5 times 103 cellwelland were allowed to attach overnight in 200120583L mediumat 37∘C and 5 CO

2 After this period plant extracts were

added at a final concentration of 100 250 and 500120583gmL

and for a period of 24 h 48 h or 72 h at 37∘C and 5CO2 After incubation the plant extracts were removed by

washing the cells twice with 200120583L PBS and 100 120583L of freshmedium was added Then 100120583L of 5mgmL MTT (3-(45-dimethylthiazol-2-yl)-25-diphenyltetrazoliumbromide) dis-solved in DMEM was added to determine the effects of thesamples on cell proliferation The cells were then incubatedfor 4 h at 37∘C and 5 CO

2 To solubilize the product of

MTT reduced 100 120583L of ethanol was added to each welland thoroughly mixed using a multichannel pipette Within20min of ethanol addition the absorbance at 570 nm wasread using a Multiskan Ascent Microplate Reader (ThermoLabsystems Franklin MA USA) The percentage of cellproliferation was calculated as follows

cell proliferation = Abs 570 nmof sampleAbs 570 nmof control

times 100 (1)

27 DAPI Cells were seeded at a density of 2 times 106 cellsper well in 6-well plates After 24 h of incubation cells werecultured with leaf extract at 250120583gmL for 24 h at 37∘CTreated cells were washed with cold PBS following fixation in4 paraformaldehyde at room temperature for 30min Thenthe cells were washed twice with PBS and maintained in PBSsolution containing 01 Triton X-100 at room temperaturefor another 30min Samples were subsequently incubated inDAPI (1 120583gmL) solution at room temperature for 30minwashed with PBS and examined under a fluorescence micro-scope within 24 h

28 Annexin V-FITC Apoptotic Activity To detect earlyapoptotic activity an Annexin V-FITC apoptosis detectionkit (Invitrogen Catalog no V13242) was used according tothe manufacturerrsquos instructions with slight modificationsThe cells were seeded at 8 times 104 cells per well in 6-wellplates After treatment with 250120583gmL of leaf extract for24 h cells were harvested and washed twice with ice-coldphosphate-buffered saline (PBS) and resuspended in 100120583Lof binding buffer A total of 5 120583L of Annexin V-FITC and 5 120583Lof propidium iodide (PI) were added and the mixture wasincubated for 30min in the dark Finally 400 120583L of bindingbuffer was added to the cells and the mixture was analyzed byflow cytometer (Becton Dickinson Co San Jose CA USA)using an FITC signal detector (FL1) and by PI staining with aphycoerythrin emission signal detector (FL2) The apoptoticpercentage of 10000 cells was determined after which allthe experiments reported in this study were performed 3times The data were analyzed using FlowJo software [29] forcalculation of percentage cells with apoptosis per group

29 Statistical Analysis All data were expressed as meansplusmn standard of triplicates Each experiment was performedat least 3 times Statistical analysis was performed by one-way ANOVA using the Graphpad Prism [30] Tukey posttestwas performed to compare different groups Differences wereconsidered significant when 119875 value was less than 005


Table 1 Chemical composition

Plantextract

Total Phenolic()

Sugar()

Protein()

Total()

AEL 802 9149 049 100MEL 608 9288 104 100EEL 534 9344 123 100CEL 1085 8417 498 100HEL 946 8694 360 100

3 Results and Discussion

31 Chemical Analysis In the literature only one reportwas found about the phytochemical screening of P volubilisperformed by chemical reactions to identify the secondarymetabolite present in hydroethanolic leaf extract [31] Thenin this work 5 leaf extracts obtained from P volubilis wereanalyzed by TLC which is an important tool not only for thequality control of medicinal plants but also for the analysisof herbal drugs [32] Some bands with characteristic colorof phenolic compounds steroids andor terpenoids wereobserved in all extracts after the use of eluent systems andchromogenic agents described in item 23 Then it wereobserved bands with color characteristics of steroids andorterpenes in all extracts yellow bands in MEL and EEL andcolor bands characteristic of sugar in MEL EEL and AELWhen the plates were sprayed with natural reagent A it waspossible to observe the presence of yellowbands inMEL EELand AEL suggesting the presence of flavonoids In addition3 flavonoids were used as standard The color developed andthe retention factor suggested the presence of kaempferolin MEL After the use of ferric chloride blue bands wereobserved especially in MEL EEL and AEL Finally afterusing the Dragendorff reagent the development of orangebands characteristic of the presence of alkaloids was notobserved in the plates However the absence of alkaloidsneeds to be confirmed because the five extracts analyzed inthis work were not prepared by classic acid-basic extractionmethod to obtain alkaloids The MEL and EEL were theextracts that showed a richer chemical profile Our resultsare different from those published by Saavedra et al [31] inthat they described the presence of tannins and alkaloids inthe P volubilis leaf extract This difference may be explainedby different soil and environmental conditions during theplant growth because their sample was collected in the cityof Trujillo Peru as well as by the techniques used in theirphytochemical analysis [33] They used the classic chemicalreactions while our study analyzed the chemical compositionof the extracts using TLC

Another analysis identified the chemical composition asshown in Table 1 A high concentration of sugar was observedin all five plant extracts The total polyphenol content of theextracts was different depending on the type of extract Thehighest concentration was found for CEL (108) and thenHEL (946) followed by AEL (802) The EEL and MELhad the lowest concentration compared to the other threeat 534 and 609 respectively (Table 1) Some studies

have shown that the antioxidant anti-inflammatory andantiproliferative activities may be related to the presence offlavonoids terpenes or phenolic compounds In Euphorbiafischeriana it was shown that antiproliferative activitieswere associated with terpenoids [34] Duarte et al [35]tested different phenolic compounds that were isolated fromEuphorbia lagascae Euphorbia tuckeyana and Pycnanthusfor antiproliferative activity and multidrug-resistant humancancer cells (MDR) Newman and Cragg [2] showed theimportance of isolating and characterizing the activity fromplant compounds as at least 60 of chemical therapeuticagents come from plants

32 AntioxidantAssay Antioxidants are compounds that canprevent biological and chemical substances from radical-induced oxidation damage Because radical oxidation ofsubstrates occurs through a chain reaction involving threestages (ie initiation propagation and termination) antiox-idants show their effects through various mechanisms [36]Thus we used different methods to evaluate the effect ofextracts on initiation (total antioxidant capacity DPPH andreducing power) propagation (iron chelating) and termina-tion (superoxide and hydroxyl radical scavenging activities)stages

Thus in order to evaluate the antioxidant activity the fiveextracts from P volubilis were analyzed by in vitro assaystotal antioxidant capacity (TAC) hydroxyl radical scavengingactivity ferric chelating superoxide radical scavenging andreducing power These different antioxidant assays werechosen as the bioactive molecules present in these extractsmay have different antioxidant activities according to theassay Three different concentrations of plant extract (100250 and 500 120583gmL) were analyzed however the best resultsfor these assays were obtained with the concentration of250120583gmL

The ferric chelating superoxide radical scavenging andreducing power assays did not show any significant activityHowever the TAC assay showed interesting results It wasverified with this assay that all five plant extracts hadantioxidant activity (TAC) with values ranging from 5931to 9776 EAAg Moreover the better results for antioxidantactivity were observed for MEL HEL and CEL 97768342 and 8921 EAAg respectively (Figure 1) The otherantioxidant assay evaluated was the DPPH Figure 2 showsthe results obtained for the P volubilis extracts at 250 120583gmLThe values ranged from 628 to 883 This assay measuresthe ability of the plant extract to donate an electron orH+ ionand the results obtained were higher than 50 of what can beconsidered excellent as the plant extract concentration was250120583gmL Guo et al [37] worked with an ethanol extractsfrom Tuber indicum in order to have 80 activity in theDPPH assay They used a concentration of 5mgmL fromthe ethanol extract this concentration was 25-fold that ofthe P volubilis extract concentration that was shown hereFurthermore Basma et al [38] working with extracts fromE hirta leaves observed a DPPH scavenging activity of 729using 1mgmL of extractThese DPPH values were similar toours but the extract concentration used was 4 times higher


AEL MEL EEL CEL HEL0

50

100

150

aabb

c

b

Extracts

Xm

g eq

uiva

lent

s of A

C as

corb

ic

Figure 1 Total capacity antioxidant activity from plant extractsThe119910-axis corresponds to mg of ascorbate acid equivalents (EEAg) andthe 119909-axis the plant extract EML corresponds to methanol extractEHL to to hexane extract ECL to chloroform extract EEL to ethanolextract and EAL to aqueous extract The plant extract was used at250 120583gmL concentration abcThe different letters correspond to thesignificant dataThe results were analyzed using ANOVA-Tukey test(119875 le 005)

AEL MEL EEL CEL HELExtracts

0

20

40

60

80

100a

b

c c c

DPP

H sc

aven

ging

()

Figure 2 DPPH assay The plant extract used for this assay was at250 120583gmL concentrationThegraph to averageplusmn standard deviationfrom DPPH scavenging EML corresponds to methanol extractEHL corresponds to hexane extract ECL to chloroform extractEEL to ethanol extract and EAL to aqueous extract The plantextract was used at 250 120583gmL concentration abcThedifferent letterscorrespond to the significant data The results were analyzed usingANOVA-Tukey test (119875 le 005)

Also the methanol and ethanol extract from Plukenetiaconophora showed 50 activity in the DPPH assay usingan extract concentration of 62120583gmL and 69 120583gmL [39]indicating that the genus Plukenetia may have antioxidantpotential

Peinado et al [40] working with grape extract proposedthat sugars may also have a role as antioxidant moleculesThen in order to analyze whether the sugar presented in theplant extracts may have any effect on the antioxidant activitythe Pearson correlation was done a value was obtained for 119877

Table 2 Proliferative effects from Plukenetia volubilis leaf extractsat 250120583gmL concentration on different cell lines in 24 h 48 h and72 h of incubation

HeLa A549 3T3 CHOAEL

24 h 751 plusmn 23 614 plusmn 39 1350 plusmn 43 1029 plusmn 5248 h 833 plusmn 05 716 plusmn 01 1578 plusmn 37 1056 plusmn 0172 h 766 plusmn 18 906 plusmn 04 1313 plusmn 21 1038 plusmn 32

MEL24 h 666 plusmn 41 788 plusmn 26 1309 plusmn 28 1118 plusmn 5648 h 543 plusmn 23 728 plusmn 29 1204 plusmn 12 868 plusmn 21

72 h 780 plusmn 47 941 plusmn 36 1171 plusmn 33 1018 plusmn 56EEL

24 h 630 plusmn 16 768 plusmn 03 1397 plusmn 07 990 plusmn 57

48 h 633 plusmn 17 774 plusmn 15 1247 plusmn 03 1024 plusmn 12172 h 669 plusmn 30 1033 plusmn 30 1455 plusmn 06 1042 plusmn 22

CEL24 h 580 plusmn 18 683 plusmn 36 1319 plusmn 36 1075 plusmn 6148 h 640 plusmn 06 606 plusmn 09 1340 plusmn 18 1076 plusmn 5172 h 593 plusmn 28 929 plusmn 26 1425 plusmn 39 1000 plusmn 50

HEL24 h 606 plusmn 21 615 plusmn 21 1092 plusmn 10 1002 plusmn 7448 h 485 plusmn 11 624 plusmn 16 1707 plusmn 18 1012 plusmn 2172 h 644 plusmn 52 846 plusmn 60 1300 plusmn 23 996 plusmn 45

of 072when total phenolicwas compared toTAC reinforcingthat the total phenolic content was responsible for the resultsobtained with TAC When analyzed by DPPH assay thevalue obtained was 06 for total phenolic and 057 for sugarsuggesting that for DPPH it was possible to have an additiveeffect for total phenolic and sugar

33 Antiproliferative Activity It has been observed thatextracts from the Euphorbiaceae family plant have potentialantiproliferative activity for different tumor cell lines Fur-thermore the presence of phenolic compounds or terpenoidsmay be related to this antiproliferative activity [35 4142] Based on the antioxidant results the proliferative orantiproliferative effects of P volubilis extracts on differentcell lines (normal cells 3T3 and CHO tumor cells HeLaand A549) were analyzed 3T3 cell line is a fibroblast cellfrom the Swiss mouse CHO is a cell line derived from theChinese hamster ovary HeLa is a cervical cancer cell line andA549 is a tumor cell line from lung tissue For this assay 3extract concentrations (100 250 and 500 120583gmL) were testedat 3 different times (24 h 48 h and 72 h) Figure 3 presentsthe graphs obtained in 48 h of the treatment with 3 extractconcentrations (100 250 and 500120583gmL) In this figuresome dose dependence was observed in the concentrationof 100 120583gmL and 250120583gmL However in the 500120583gmLconcentration little difference was observed Considering thisobservation the data obtained only for the concentrationof 250120583gmL at 3 different times 24 h 48 h and 72 h wasshown in Table 2 For the cell lineage A549 an inhibitoryeffect on the cell proliferation was observed for 24 h and


0255075

100125150175200

0 100 200 300 400 500 600

Prol

ifera

tion

()

Concentration (120583gmL)

HeLaA549

3T3CHO

(a)

0255075

100125150175200

0 100 200 300 400 500 600

Prol

ifera

tion

()


HeLaA549

3T3CHO

(b)

0255075

100125150175200

0 100 200 300 400 500 600

Prol

ifera

tion

()


HeLaA549

3T3CHO

(c)

0255075

100125150175200

0 100 200 300 400 500 600

Prol

ifera

tion

()


HeLaA549

3T3CHO

(d)

0255075

100125150175200

0 100 200 300 400 500 600

Prol

ifera

tion

()


HeLaA549

3T3CHO

(e)

Figure 3 Antiproliferative activity from Plukenetia volubilis extracts The 119909-axis corresponds to different plant concentrations used and the119910-axis corresponds to the cell proliferation percentage In (a) the aqueous extract is represented (AEL) (b) corresponds to MEL extract (c)corresponds to EEL extract (d) corresponds to CEL extract and (e) corresponds to HEL extractThe data correspond to 48 h of cell exposureto the plant extract

48 h A similar effect was also observed for HeLa cell linefor the MEL and HEL extracts where the proliferationwas observed at around 54ndash77 (Table 2) However thisinhibitory effect was not observed for the treatment of 72 hraising the hypothesis that perhaps in 72 h the bioactivemolecules present in the extract were not active anymoreHe et al [43] observed that fractions obtained from Panaxnotoginseng were able to inhibit cervical cancer effectivelyThese authors found an IC

50of 1668 and 2769 120583gmL for

Caco-2 and LoVo cell lines respectively [43] The workpresented here showed that for HeLa cells the better extractswere MEL and HEL with a proliferation only of 543 and485 (Table 2) Furthermore for the A549 cell line thebetter extracts were AEL CEL and HEL with a proliferationof 614 606 and 615 respectively with a reductionof almost 40 (Table 2) Not all the extracts had the sameinhibition pattern (Table 2) These data showed that theextracts from P volubiliswere able to reduce the proliferation


10120583m

(a)

10120583m

(b)

10120583m

(c)

10120583m

(d)

Figure 4 HeLa cell morphology after treatment with P volubilis Extracts HeLa cells were incubated with plant extracts at 250 120583gmLconcentration for 48 h and thenweremarkedwithDAPI to analyze the cell morphology Arrows show regions where the cell hadmodificationin itsmorphology (a) corresponds to the controlHeLa cells without plant extract size 20x (b) corresponds toHeLa cells without plant extractsize 40x (c) corresponds to HeLa cells treated with EML extract size 20x (d) corresponds to HeLa cells treated with EHL size 40x

for the two tumor cell lines tested by 40ndash50 rate On theother hand their effects on normal cells (3T3 and CHO)showed that these extracts had no cytotoxicity for thesecells (Table 2) Furthermore they showed an increase inproliferation on 3T3 cells (fibroblast cells) A significantincrease was observed in the proliferation especially at 24or 48 h (approximately 30ndash40 proliferation rate) (Table 2)However this effect was not observed for CHO cells (Figure 3and Table 2) For CHO these extracts did not modify theproliferation rate even when the extract concentration ortime was changed

34 Nuclear Morphology Changes in HeLa Cells To evaluatethe inhibition of the proliferation of the HeLa a DAPIassay was performed HeLa cells were previously treated withMEL and HEL extracts for 48 h as the higher reductionwas observed The control used in this assay was the HeLacells without plant extract No change was observed inHeLa cell morphology these cells were round and stainedhomogeneously with DAPI (Figures 4(a) and 4(b)) Howeverwhen the cells were treated with the P volubilis extractschanges were observed in the cell morphology such aspyknotic bodies membrane blebbing and nuclear condensa-tion (Figures 4(c) and 4(d)) This morphology suggested thatthe inhibition in proliferation previously observed may be

due to an apoptotic mechanism Machana et al [44] workingwith extracts from Cladogynos orientales (Euphorbiaceae)that had phenolic compounds and flavonoids they observeda reduction on the proliferation rate of HepG2 tumor cellsby early and late stages of apoptosis similar to what wasobserved in this work

35 P volubilis Extracts Induce Apoptosis or Necrosis Thenuclear morphology changes previously observed may be aresult of an apoptosis or necrosis process In order to evaluatethis the HeLa cells were treated with P volubilis extracts andthen observed by flow cytometry analysis This techniqueallowed observation of the cell phase and the presence ofDNA fragments that were characteristic from the apoptosisprocess [45] Figure 5 shows the results obtained when threegroups of cells were observed The first group was markedby Annexin V and not by PI This group characterized theearly apoptosis where HEL AEL and MEL extracts had133 172 and 102 cells in early apoptosis respectivelyThe second group of cells was marked by Annexin V andPI suggesting the late stages of the apoptosis process whereAEL and HEL extracts had 101 and 89 respectively(Figure 5) And the third group of cells was marked only byPI which corresponds to the necrosis processThis groupwasobserved for AEL (3) and HEL (5) extracts (Figure 5)


C EA EM EE EC EH0

10

20

30

40

Celu

las (

)

Anexina+PIminusAnexinaminusPI+Anexina+PI+

Figure 5 Distribution of apoptotic cells HeLa cells were treatedwith 250 120583gmL of plant extracts (AELMEL EEL CEL or HEL) for24 h and then stained with Annexine V-FITC and PI C correspondsto HeLa cells not treated with plant extract

Studies done with Croton lechleri (Euphorbiaceae) showedthe induction of the initial apoptosis in HeLa cells [46]Mota et al [47] using the ethanol extract from Synadeniumumbellatum (Euphorbiaceae) on EAT cells also found aproportion of the cells in apoptosis (29) and also in necrosis(1933) Furthermore the euphol (a triterpene) isolatedfrom Euphorbia tirucalli (Euphorbiaceae) showed inductionof apoptosis around 25 for gastric cancer cell lines CS12AGS and MKN45 [48 49] So the results from P volubilisextracts showed the potential of these biomolecules to induceapoptosis (early and late stages)

4 Conclusions

Normally in the cell there is a balance between the freeradicals produced by the organism and antioxidant productsto keep this balance ROS are also an important signal forthe organism but it is important to keep this balance Indisease conditions so many free radicals may be generatedthat the antioxidant products cannot reduce the effect of suchradicals Thus it is important to search for new bioactivemolecules that can be used as a natural antioxidant andas the treatment for cancer and other diseases The datapresented here show that Plukenetia volubilis extracts hadthe presence of terpenoids saponins and phenolic com-pounds (flavonoids) that were known to have antioxidantantiproliferative and other activities The data from CATand DPPH indicated the potential of the AEL MEL andHEL extracts Besides this assays also verified an inhibitoryeffect on proliferation of HeLa and A549 cells consequentlyproducing early apoptosis and late apoptosis However theseextracts were also able to induce proliferation of fibroblastcells 3T3 cells More studies are needed to understand theapoptosis pathway as well as the extract effects on fibroblastproliferation

Conflict of Interests

Noneof the authors of this paper has a direct financial relationwith the commercial identities mentioned in this paper(FlowJo software andBectonDickinsonCo)Moreover thereis no any conflict of interests by any of the authors

Acknowledgments

The authors wish to thank Conselho Nacional de Desen-volvimento Cientıfico e Tecnologico (CNPq) and alsoCoordenacao de Aperfeicoamento Pessoal de Nıvel Superior(CAPES) for the financial support Katia Castanho Scortecciand Hugo Alexandre Oliveira Rocha are CNPq fellowship-honored researchers Ana Karina Lima Nascimento had amaster scholarship from CAPES Raniere Fagundes Melo-Silveira has a PhD scholarship from CAPES and NednaldoDantas-Santos had a PhD scholarship from CNPq

References

[1] S C Sahu N K Dhal and R C Mohanty ldquoPotential medicinalplants used by the tribal of deogarh district Orissa IndiardquoStudies on Ethno-Medicine vol 4 no 1 pp 53ndash61 2010

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

[3] J D McChesney S K Venkataraman and J T Henri ldquoPlantnatural products back to the future or into extinctionrdquo Phyto-chemistry vol 68 no 14 pp 2015ndash2022 2007

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

[5] J GDeMelo T ADe SousaAraujo V TNDeAlmeidaCastroet al ldquoAntiproliferative activity antioxidant capacity and tannincontent in plants of semi-arid northeastern Brazilrdquo Moleculesvol 15 no 12 pp 8534ndash8542 2010

[6] O I Aruoma ldquoMethodological considerations for character-izing potential antioxidant actions of bioactive components inplant foodsrdquoMutation Research vol 523-524 pp 9ndash20 2003

[7] A R M R Amin O Kucuk F R Khuri and D M ShinldquoPerspectives for cancer prevention with natural compoundsrdquoJournal of Clinical Oncology vol 27 no 16 pp 2712ndash2725 2009

[8] K W Lee A M Bode and Z Dong ldquoMolecular targets ofphytochemicals for cancer preventionrdquo Nature Reviews Cancervol 11 no 3 pp 211ndash218 2011

[9] J T Mwine and P van Damme ldquoWhy do euphorbiaceae tickas medicinal plants A review of euphorbiaceae family and itsmedicinal featuresrdquo Journal of Medicinal Plant Research vol 5no 5 pp 652ndash662 2011

[10] R D S Secco I CordeiroII L de Senna-Vale et al ldquoAnoverview of recent taxonomic studies on Euphorbiaceae sl inBrazilrdquo Rodriguesia vol 63 no 1 pp 227ndash242 2012

[11] S C Tripathi and M Srivastava ldquoEthnomedicinal flora ofEuphorbiaceae used in dermatological problemsrdquo Indian Jour-nal of Traditional Knowledge vol 9 no 2 pp 318ndash320 2010

[12] M D Guillen A Ruiz N Cabo R Chirinos and G PascualldquoCharacterization of sacha inchi (Plukenetia volubilis L) Oil byFTIR spectroscopy and 1HNMR Comparison with linseed oilrdquoJournal of the American Oil Chemistsrsquo Society vol 80 no 8 pp755ndash762 2003


[13] B R Hamaker C Valles R Gilman et al ldquoAmino acid fattyacid profiles of the Inca peanut (Plukenetia volubilis)rdquo CerealChemistry vol 69 no 4 pp 461ndash463 1992

[14] L A Follegatti-Romero C R Piantino R Grimaldi and FA Cabral ldquoSupercritical CO

2

extraction of omega-3 rich oilfrom Sacha inchi (Plukenetia volubilis L) seedsrdquo Journal ofSupercritical Fluids vol 49 no 3 pp 323ndash329 2009

[15] J Huaman K Chavez E Castaneda et al ldquoEffect of Plukenetiavolubilis Linneo (sacha inchi) on postprandial triglyceridesrdquoAnales de la Facultad de Medicina vol 69 no 4 pp 263ndash2662008

[16] F Garmendia R R Pando and G Ronceros ldquoEffect of sachainchi oil (Plukenetia volubilis L) on the lipid profile of patientswith Hyperlipoproteinemiardquo Revista Peruana de Medicina deExperimental y Salud Publica vol 28 no 4 pp 628ndash632 2011

[17] S M Zucolotto J A Palermo and E P Schenkel ldquoEstudofitoquımico das raızes de Passiflora edulis forma flavicarpaDegenerrdquo Acta Farmaceutica vol 25 no 1 pp 5ndash9 2006

[18] M Dubois K A Gilles J K Hamilton P A Rebers and FSmith ldquoColorimetric method for determination of sugars andrelated substancesrdquoAnalytical Chemistry vol 28 no 3 pp 350ndash356 1956

[19] M M Bradford ldquoA rapid and sensitive method for the quanti-tation of microgram quantities of protein utilizing the principleof protein dye bindingrdquoAnalytical Biochemistry vol 72 no 1-2pp 248ndash254 1976

[20] Y Athukorala K-N Kim and Y-J Jeon ldquoAntiproliferative andantioxidant properties of an enzymatic hydrolysate from brownalga Ecklonia cavardquo Food and Chemical Toxicology vol 44 no7 pp 1065ndash1074 2006

[21] R F Melo-Silveira G P Fidelis M S S Pereira Costa et al ldquoInvitro antioxidant anticoagulant and antimicrobial activity andin inhibition of cancer cell proliferation by xylan extracted fromcorn cobsrdquo International Journal of Molecular Sciences vol 13no 1 pp 409ndash426 2012

[22] L S Costa G P Fidelis S L Cordeiro et al ldquoBiologicalactivities of sulfated polysaccharides from tropical seaweedsrdquoBiomedicine amp Pharmacotherapy vol 64 no 1 pp 21ndash28 2010

[23] P Prieto M Pineda and M Aguilar ldquoSpectrophotometricquantitation of antioxidant capacity through the formation ofa phosphomolybdenum complex specific application to thedetermination of vitamin Erdquo Analytical Biochemistry vol 269no 2 pp 337ndash341 1999

[24] N Smirnoff and Q J Cumbes ldquoHydroxyl radical scavengingactivity of compatible solutesrdquo Phytochemistry vol 28 no 4 pp1057ndash1060 1989

[25] E A Decker and B Welch ldquoRole of ferritin as a lipid oxidationcatalyst in muscle foodrdquo Journal of Agricultural and FoodChemistry vol 38 no 3 pp 674ndash677 1990

[26] N Dasgupta and B De ldquoAntioxidant activity of Piper betle Lleaf extract in vitrordquo Food Chemistry vol 88 no 2 pp 219ndash2242004

[27] J Wang Q Zhang Z Zhang and Z Li ldquoAntioxidant activityof sulfated polysaccharide fractions extracted from Laminariajaponicardquo International Journal of Biological Macromoleculesvol 42 no 2 pp 127ndash132 2008

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

[29] FlowJo Version 763 Tree Star Ashland OR USA 1997

[30] GraphPad Software ldquoPrism (data analysis software system)version 5rdquo 2013 httpwwwgraphpadcomwelcomehtml

[31] C Saavedra E Felix C Viera S Felix and R Alfaro ldquoPhyto-chemical screening of Plukenetia volubilis L and its antioxidanteffects of the Fe3+ascorbate stimulated lipid peroxidation inhepatic of Rattus rattus var Albinusrdquo Revista Cientıfica de laUniversidad Cesar Vallejo vol 2 no 1 pp 11ndash21 2010

[32] R R Duron L C Almaguer A de J Garza-Juarez Ma LuzS Cavazos and N Waksman-De-Torres ldquoDevelopment andvalidation of thin-layer chromatographic methods for qualitycontrol of herbal productsrdquo Acta Chromatography vol 21 pp203ndash215 2009

[33] E Bagdonaite P Martonfi M Repcak and J Labokas ldquoVari-ation in concentrations of major bioactive compounds inHypericum perforatum L from Lithuaniardquo Industrial Crops andProducts vol 35 no 1 pp 302ndash308 2012

[34] Y-X Sun and J-C Liu ldquoChemical constituents and biologicalactivities of Euphorbia fischeriana SteudrdquoChemistryampBiodiver-sity vol 8 no 7 pp 1205ndash1214 2011

[35] N Duarte H Lage M Abrantes and M-J U FerreiraldquoPhenolic compounds as selective antineoplasic agents againstmultidrug-resistant human cancer cellsrdquo PlantaMedica vol 76no 10 pp 975ndash980 2010

[36] A Somogy K Rosta P Pusztai Z Tulassay and G NagyldquoAntioxidant measurementsrdquo Physiological Measurement vol28 pp 41ndash55 2007

[37] T Guo L Wei J Sun C-L Hou and L Fan ldquoAntioxidantactivities of extract and fractions from Tuber indicum Cooke ampMasseerdquo Food Chemistry vol 127 no 4 pp 1634ndash1640 2011

[38] A A Basma Z Zakaria L Y Latha and S Sasidharan ldquoAntiox-idant activity and phytochemical screening of the methanolextracts of Euphorbia hirta Lrdquo Asian Pacific Journal of TropicalMedicine vol 4 no 5 pp 386ndash390 2011

[39] O U Amaeze G A Ayoola M O Sofidiya A A Adepoju-Bello A O Adegoke andH A B Coker ldquoEvaluation of antiox-idant activity of Tetracarpidium conophorum (Mull Arg) Hutchamp Dalziel leavesrdquo Oxidative Medicine and Cellular Longevityvol 2011 Article ID 976701 7 pages 2011

[40] J Peinado N L de Lerma and R A Peinado ldquoSynergisticantioxidant interaction between sugars and phenolics from asweet winerdquo European Food Research and Technology vol 231no 3 pp 363ndash370 2010

[41] J Xu D Jin P Guo C Xie L Fang and Y Guo ldquoThreenew myrsinol diterpenes from Euphorbia prolifera and theirneuroprotective activitiesrdquo Molecules vol 17 no 8 pp 9520ndash9528 2012

[42] D S Llanes-Coronel L Y Gamez-Dıaz L P Suarez-Quinteroet al ldquoNew promising Euphorbiaceae extracts with activity inhuman lymphocytes from primary cell culturesrdquo Immunophar-macology and Immunotoxicology vol 33 no 2 pp 279ndash2902011

[43] N-W He Y Zhao L Guo J Shang and X-B Yang ldquoAntioxi-dant antiproliferative and pro-apoptotic activities of a saponinextract derived from the roots ofPanax notoginseng (Burk) FHChenrdquo Journal of Medicinal Food vol 15 no 4 pp 350ndash3592012

[44] S Machana N Weerapreeyakul S Barusrux A NonpunyaB Sripanidkulchai and T Thitimetharoch ldquoCytotoxic andapoptotic effects of six herbal plants against the human hepa-tocarcinoma (HepG2) cell linerdquo Chinese Medicine vol 6 article39 2011


[45] R Nunez ldquoDNA measurement and cell cycle analysis by flowcytometryrdquo Current Issues in Molecular Biology vol 3 no 3 pp67ndash70 2001

[46] A J Alonso-Castro E Ortiz-Sanchez F Domınguez et alldquoAntitumor effect of Croton lechleri Mull Arg (Euphor-biaceae)rdquo Journal of Ethnopharmacology vol 140 no 2 pp 438ndash442 2012

[47] M F da Mota P L Benfica A C Batista F S Martins JR De Paula and M C Valadares ldquoInvestigation of Ehrlichascites tumor cell death mechanisms induced by Synadeniumumbellatum Paxrdquo Journal of Ethnopharmacology vol 139 no 2pp 319ndash329 2012

[48] M W Lin A S Lin D C Wu et al ldquoEuphol from Euphorbiatirucalli selectively inhibits human gastric cancer cell growththrough the induction of ERK12-mediated apoptosisrdquo Foodand Chemical Toxicology vol 50 no 12 pp 4333ndash4339 2012

[49] R C Patil S MManohar V I Katchi and A J Rao ldquoEthanolicstem extract of Excoecaria Agallocha induces G1 arrest orapoptosis in human lung cancer cells depending on their P53statusrdquo Taiwania vol 57 no 2 pp 89ndash98 2012

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


plants that may help an organism to keep the normal balanceof ROS In the case of cancer these antioxidant substancesare being used in the treatment to prevent the normal cellfrom becoming a cancer cell as well as to kill cancer cellsfurthermore these compounds had no negative effects onnormal cells [7 8]

The Euphorbiaceae is formed by more than 6000 specieswith extreme diversity of secondary compounds produced [910] This variability of compounds may explain the differentuses of the plants from this family such as antiprolifera-tive antimicrobial cytotoxic activity and anti-inflammatoryamong others [9] Moreover many times the latex producedby these plants has been used as medicine for skin diseaseas well as skin healing for example Euphorbia thymifolia Eneriifolia andE nivulia aswell as theCroton bonplandianumhave been used to treat scabies and other skin diseases [11]

Plukenetia volubilis Linneo or sacha inchi the target ofthis work is a climbing shrub plant from the Euphorbiaceaethat grows mostly in the Amazon region [12] Some studieshave shown that seeds from this plant are an excellent sourceof protein (27ndash33) and oil (35ndash60) [13] The oil compo-sition is mostly omega 3 and omega 6 (4861 and 368resp) [13 14] Some studies have been done using seedsand leaves in order to reduce the lipid profile from patientswith postprandial lipemia [15] and hypercholesterolemia [16]However there is no scientific data about the use of leavesfor skin disease as it has been used in folk medicine inthe Amazon region The aim of the work presented herewas to evaluate the effects from five extracts obtained fromfresh leaves methanol (MEL) ethanol (EEL) chloroformCEL hexane (HEL) and aqueous (AEL) These extractswere analyzed using antioxidant assays and antiproliferativeassays using normal and tumor cells The results showed thatsome of these extracts had antioxidant and antiproliferativeactivities against HeLa cells Furthermore it was observedthat the extracts were able to stimulate the cell proliferationin fibroblast cells-3T3

2 Materials and Methods

21 Plant Material Plukenetia volubilis plants were grownin soil (soil and sand 1 1) in Parnamirim RN Brazil(minus05∘ 471015840 4210158401015840 +35∘ 121015840 3410158401015840) After six months leaves werecollected to obtain the different extracts Two specimenswere deposited in a herbarium at Centro de Biociencias daUniversidade Federal do RioGrande doNorte (UFRN) underthe number 10854

22 Preparation of Leaf Extracts Fresh leaves were collectedand 15 gwas divided into small pieces by hand and then trans-ferred to a flask in a proportion of 1 10 (drug solvent wv)using the following solvents methanol ethanol chloroformhexane andwaterThematerial was protected from light withaluminum foil and these flasks were shaken at 50 rpm for24 h Then the extracts were filtered on Whatman paper n∘1 and dried in Rotavapor at 40∘C resulting in five extractsmethanol ethanol chloroform hexane and aqueous Afterthat the extracts were weighed and then resuspended into

DMSO (CRQnuclear) to final concentration of 10mgmL(extract stock solution)

23 Phytochemistry Screening by Thin Layer Chromatography(TLC) This assay was conducted by TLC using aluminumsheets of silica gel F

254(Merck) All chromatograms

were developed in a saturated chamber Two systemswere used as mobile phases (1) ethyl acetate formicacid water methanol (10 05 06 02) and (2)toluene ethyl acetate methanol (5 5 05) The eluentsystems employed in this study were selected or developedaccording to data from the literature [17] After thechromatograms were developed the plates were driedand the spots were visualized sequentially under UV lightat 254 and 365 nm The plates were then sprayed withspecific chromogenic agents according to the chemicalsubstances analyzed The following solutions were used asvisualizing agents vanillin sulfuric +105∘C natural reagentA 05 ferric chloride 1 in methanol and Dragendorffreagent Kaempferol luteolin and apigenin all obtainedfrom Sigma-Aldrich were used as standard sample

24 Total Content of Sugar Protein and Phenolic CompoundsTotal sugar was estimated by the phenol-H

2SO4reaction

using D-glucose (Sigma-Aldrich) as a standard [18] Proteincontent was measured using Spectorrsquos method with bovinealbumin as a standard [19] Total phenolic compounds inthe extract solution were determined using Folin-Ciocalteursquoscoloimetric method and gallic acid as a standard [20]

25 Antioxidant Activity The antioxidant activity was exam-ined by conducting in vitro tests total antioxidant capacityhydroxyl radical scavenging superoxide radical scavengingferric chelating and reducing power as previously describedin [21 22]

251 Determination of Total Antioxidant Capacity (TAC)The assay for total antioxidant capacity is based on the reduc-tion of Mo+6 to Mo+5 by the plant extracts and subsequentformation of green phosphateMo+5 complexes at acid pH[23] The tubes containing the plant extracts at 250 120583gmLconcentration and the reagent solution (06M sulfuric acid28mM sodium phosphate and 4mM ammonium molyb-date) were incubated at 95∘C for 90min After this time themixture was cooled at room temperature and the absorbanceof each tube was measured at 695 nm against a blank Theantioxidant capacity was expressed as mg of ascorbic acidgdescribed as ascorbic acid equivalent (EEAg)

252 Hydroxyl Radical Scavenging Activity Assay The scav-enging activity of plant extracts against the hydroxyl radicalwas measured based on Fentonrsquos reaction (Fe2+ + H

2O2rarr

Fe3+ + OHminus + OH∙) The results were expressed as an inhibi-tion rate Hydroxyl radicals were generated using a modifiedmethod [24] where 3mLof 150mMsodiumphosphate buffer(pH 74) contained 10mM FeSO

4times 7H2O 10mM EDTA

2mM sodium salicylate 30 H2O2(200mL) and the plant

extracts at 250120583gmL concentration The positive control










11198600) times 100 where 119860











times 100 (1)






Plantextract

Total Phenolic()

Sugar()

Protein()

Total()











50

100

150

aabb

c

b

Extracts

Xm

g eq

uiva

lent

s of A

C as

corb

ic



0

20

40

60

80

100a

b

c c c

DPP

H sc

aven

ging

()
















0255075

100125150175200

0 100 200 300 400 500 600

Prol

ifera

tion

()


HeLaA549

3T3CHO

(a)

0255075

100125150175200

0 100 200 300 400 500 600

Prol

ifera

tion

()


HeLaA549

3T3CHO

(b)

0255075

100125150175200

0 100 200 300 400 500 600

Prol

ifera

tion

()


HeLaA549

3T3CHO

(c)

0255075

100125150175200

0 100 200 300 400 500 600

Prol

ifera

tion

()


HeLaA549

3T3CHO

(d)

0255075

100125150175200

0 100 200 300 400 500 600

Prol

ifera

tion

()


HeLaA549

3T3CHO

(e)



50of 1668 and 2769 120583gmL for



10120583m

(a)

10120583m

(b)

10120583m

(c)

10120583m

(d)







C EA EM EE EC EH0

10

20

30

40

Celu

las (

)




4 Conclusions




Acknowledgments


References
















2











































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

























11198600) times 100 where 119860











times 100 (1)






Plantextract

Total Phenolic()

Sugar()

Protein()

Total()











50

100

150

aabb

c

b

Extracts

Xm

g eq

uiva

lent

s of A

C as

corb

ic



0

20

40

60

80

100a

b

c c c

DPP

H sc

aven

ging

()
















0255075

100125150175200

0 100 200 300 400 500 600

Prol

ifera

tion

()


HeLaA549

3T3CHO

(a)

0255075

100125150175200

0 100 200 300 400 500 600

Prol

ifera

tion

()


HeLaA549

3T3CHO

(b)

0255075

100125150175200

0 100 200 300 400 500 600

Prol

ifera

tion

()


HeLaA549

3T3CHO

(c)

0255075

100125150175200

0 100 200 300 400 500 600

Prol

ifera

tion

()


HeLaA549

3T3CHO

(d)

0255075

100125150175200

0 100 200 300 400 500 600

Prol

ifera

tion

()


HeLaA549

3T3CHO

(e)



50of 1668 and 2769 120583gmL for



10120583m

(a)

10120583m

(b)

10120583m

(c)

10120583m

(d)







C EA EM EE EC EH0

10

20

30

40

Celu

las (

)




4 Conclusions




Acknowledgments


References
















2











































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


















Plantextract

Total Phenolic()

Sugar()

Protein()

Total()











50

100

150

aabb

c

b

Extracts

Xm

g eq

uiva

lent

s of A

C as

corb

ic



0

20

40

60

80

100a

b

c c c

DPP

H sc

aven

ging

()
















0255075

100125150175200

0 100 200 300 400 500 600

Prol

ifera

tion

()


HeLaA549

3T3CHO

(a)

0255075

100125150175200

0 100 200 300 400 500 600

Prol

ifera

tion

()


HeLaA549

3T3CHO

(b)

0255075

100125150175200

0 100 200 300 400 500 600

Prol

ifera

tion

()


HeLaA549

3T3CHO

(c)

0255075

100125150175200

0 100 200 300 400 500 600

Prol

ifera

tion

()


HeLaA549

3T3CHO

(d)

0255075

100125150175200

0 100 200 300 400 500 600

Prol

ifera

tion

()


HeLaA549

3T3CHO

(e)



50of 1668 and 2769 120583gmL for



10120583m

(a)

10120583m

(b)

10120583m

(c)

10120583m

(d)







C EA EM EE EC EH0

10

20

30

40

Celu

las (

)




4 Conclusions




Acknowledgments


References
















2











































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


















50

100

150

aabb

c

b

Extracts

Xm

g eq

uiva

lent

s of A

C as

corb

ic



0

20

40

60

80

100a

b

c c c

DPP

H sc

aven

ging

()
















0255075

100125150175200

0 100 200 300 400 500 600

Prol

ifera

tion

()


HeLaA549

3T3CHO

(a)

0255075

100125150175200

0 100 200 300 400 500 600

Prol

ifera

tion

()


HeLaA549

3T3CHO

(b)

0255075

100125150175200

0 100 200 300 400 500 600

Prol

ifera

tion

()


HeLaA549

3T3CHO

(c)

0255075

100125150175200

0 100 200 300 400 500 600

Prol

ifera

tion

()


HeLaA549

3T3CHO

(d)

0255075

100125150175200

0 100 200 300 400 500 600

Prol

ifera

tion

()


HeLaA549

3T3CHO

(e)



50of 1668 and 2769 120583gmL for



10120583m

(a)

10120583m

(b)

10120583m

(c)

10120583m

(d)







C EA EM EE EC EH0

10

20

30

40

Celu

las (

)




4 Conclusions




Acknowledgments


References
















2











































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















0255075

100125150175200

0 100 200 300 400 500 600

Prol

ifera

tion

()


HeLaA549

3T3CHO

(a)

0255075

100125150175200

0 100 200 300 400 500 600

Prol

ifera

tion

()


HeLaA549

3T3CHO

(b)

0255075

100125150175200

0 100 200 300 400 500 600

Prol

ifera

tion

()


HeLaA549

3T3CHO

(c)

0255075

100125150175200

0 100 200 300 400 500 600

Prol

ifera

tion

()


HeLaA549

3T3CHO

(d)

0255075

100125150175200

0 100 200 300 400 500 600

Prol

ifera

tion

()


HeLaA549

3T3CHO

(e)



50of 1668 and 2769 120583gmL for



10120583m

(a)

10120583m

(b)

10120583m

(c)

10120583m

(d)







C EA EM EE EC EH0

10

20

30

40

Celu

las (

)




4 Conclusions




Acknowledgments


References
















2











































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















10120583m

(a)

10120583m

(b)

10120583m

(c)

10120583m

(d)







C EA EM EE EC EH0

10

20

30

40

Celu

las (

)




4 Conclusions




Acknowledgments


References
















2











































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















C EA EM EE EC EH0

10

20

30

40

Celu

las (

)




4 Conclusions




Acknowledgments


References
















2











































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of



















2











































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of



























of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















research article antioxidant and antiproliferative...

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