antioxidants, phytochemicals, and cytotoxicity studies on phaleria

14
Research Article Antioxidants, Phytochemicals, and Cytotoxicity Studies on Phaleria macrocarpa (Scheff.) Boerl Seeds Ma Ma Lay, 1 Saiful Anuar Karsani, 1,2 Behrooz Banisalam, 1 Sadegh Mohajer, 1 and Sri Nurestri Abd Malek 1 1 Institute of Biological Sciences, Faculty of Science, University of Malaya, 50603 Kuala Lumpur, Malaysia 2 University of Malaya Centre for Proteomics Research (UMCPR), University of Malaya, 50603 Kuala Lumpur, Malaysia Correspondence should be addressed to Ma Ma Lay; [email protected] Received 30 November 2013; Revised 6 February 2014; Accepted 18 February 2014; Published 27 March 2014 Academic Editor: Fabio Ferreira Perazzo Copyright © 2014 Ma Ma Lay et al. is is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. In recent years, the utilization of certain medicinal plants as therapeutic agents has drastically increased. Phaleria macrocarpa (Scheff.) Boerl is frequently used in traditional medicine. e present investigation was undertaken with the purpose of developing pharmacopoeial standards for this species. Nutritional values such as ash, fiber, protein, fat, and carbohydrate contents were investigated, and phytochemical screenings with different reagents showed the presence of flavonoids, glycosides, saponin glycosides, phenolic compounds, steroids, tannins, and terpenoids. Our results also revealed that the water fraction had the highest antioxidant activity compared to the methanol extract and other fractions. e methanol and the fractionated extracts (hexane, chloroform, ethyl acetate, and water) of P. macrocarpa seeds were also investigated for their cytotoxic effects on selected human cancer cells lines (MCF-7, HT-29, MDA-MB231, Ca Ski, and SKOV-3) and a normal human fibroblast lung cell line (MRC-5). Information from this study can be applied for future pharmacological and therapeutic evaluations of the species, and may assist in the standardization for quality, purity, and sample identification. To the best of our knowledge, this is the first report on the phytochemical screening and cytotoxic effect of the crude and fractionated extracts of P. macrocarpa seeds on selected cells lines. 1. Introduction Herbal medicine plays a key role in the development of pharmaceuticals and thus there is a high demand in natural medicine for the global market. Although there are thousands of species listed as medicinal plants, only a small number are commercially used in traditional treatments. In this respect, there are very few in-depth scientific studies on the medicinal properties of plants. However, traditional herbal medicine is still prominent and is considered an important alternative to conventional medicine particularly in devel- oping countries. Despite its well-known benefits, Phaleria macrocarpa (Scheff.) Boerl is still relatively unknown in terms of its biochemical constituents and biological activity. P. macrocarpa is a plant commonly used in East Asian herbal medicines. P. macrocarpa is used as a remedy for a variety of ailments such as cancer, diabetes mellitus, allergies, liver and heart diseases, kidney failure, blood diseases, high blood pressure, and stroke. It is also used to treat various skin diseases including acne [1, 2]. P. macrocarpa plantshave round, oval shaped seeds that have a diameter of approximately 1 cm. e seed is the most poisonous part of the plant, having higher toxicity levels than the stem, roots, and leaves. P. macrocarpa fruits and leaves are used in traditional medicine as a concoction. e seeds which have an unpleasant odor are usually used for the treatment of skin diseases. e compounds quercetin and naringin have been found in the seeds [3]. e essential oils of the seedsconsist of heptadecane, octadecane, diclosan, triclosan, vinyl laurate, and diocthyl ester [4]. Another study reported the presence of Mahkoside A and kaempferol 3-O- -D-glucoside in the seeds [5]. Two novel compounds, 29- norcucurbitacin and desacetylfevicordin A, and three known 29-norcucurbitacin derivatives have also been isolated from the ethyl acetate fraction of P. macrocarpa seeds [6]. Hindawi Publishing Corporation BioMed Research International Volume 2014, Article ID 410184, 13 pages http://dx.doi.org/10.1155/2014/410184

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Research ArticleAntioxidants Phytochemicals and Cytotoxicity Studies onPhaleria macrocarpa (Scheff) Boerl Seeds

Ma Ma Lay1 Saiful Anuar Karsani12 Behrooz Banisalam1

Sadegh Mohajer1 and Sri Nurestri Abd Malek1

1 Institute of Biological Sciences Faculty of Science University of Malaya 50603 Kuala Lumpur Malaysia2 University of Malaya Centre for Proteomics Research (UMCPR) University of Malaya 50603 Kuala Lumpur Malaysia

Correspondence should be addressed to Ma Ma Lay mamalaystergmailcom

Received 30 November 2013 Revised 6 February 2014 Accepted 18 February 2014 Published 27 March 2014

Academic Editor Fabio Ferreira Perazzo

Copyright copy 2014 Ma Ma Lay et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

In recent years the utilization of certain medicinal plants as therapeutic agents has drastically increased Phaleria macrocarpa(Scheff) Boerl is frequently used in traditional medicineThe present investigation was undertaken with the purpose of developingpharmacopoeial standards for this species Nutritional values such as ash fiber protein fat and carbohydrate contents wereinvestigated and phytochemical screenings with different reagents showed the presence of flavonoids glycosides saponinglycosides phenolic compounds steroids tannins and terpenoids Our results also revealed that the water fraction had the highestantioxidant activity compared to the methanol extract and other fractions The methanol and the fractionated extracts (hexanechloroform ethyl acetate and water) of P macrocarpa seeds were also investigated for their cytotoxic effects on selected humancancer cells lines (MCF-7 HT-29 MDA-MB231 Ca Ski and SKOV-3) and a normal human fibroblast lung cell line (MRC-5)Information from this study can be applied for future pharmacological and therapeutic evaluations of the species and may assistin the standardization for quality purity and sample identification To the best of our knowledge this is the first report on thephytochemical screening and cytotoxic effect of the crude and fractionated extracts of P macrocarpa seeds on selected cells lines

1 Introduction

Herbal medicine plays a key role in the development ofpharmaceuticals and thus there is a high demand in naturalmedicine for the globalmarket Although there are thousandsof species listed as medicinal plants only a small numberare commercially used in traditional treatments In thisrespect there are very few in-depth scientific studies on themedicinal properties of plants However traditional herbalmedicine is still prominent and is considered an importantalternative to conventional medicine particularly in devel-oping countries Despite its well-known benefits Phaleriamacrocarpa (Scheff) Boerl is still relatively unknown in termsof its biochemical constituents and biological activity Pmacrocarpa is a plant commonly used in East Asian herbalmedicines P macrocarpa is used as a remedy for a varietyof ailments such as cancer diabetes mellitus allergies liverand heart diseases kidney failure blood diseases high blood

pressure and stroke It is also used to treat various skindiseases including acne [1 2]

P macrocarpa plantshave round oval shaped seeds thathave a diameter of approximately 1 cm The seed is the mostpoisonous part of the plant having higher toxicity levelsthan the stem roots and leaves P macrocarpa fruits andleaves are used in traditional medicine as a concoction Theseeds which have an unpleasant odor are usually used forthe treatment of skin diseases The compounds quercetinand naringin have been found in the seeds [3] The essentialoils of the seedsconsist of heptadecane octadecane diclosantriclosan vinyl laurate and diocthyl ester [4] Another studyreported the presence of Mahkoside A and kaempferol 3-O-120573-D-glucoside in the seeds [5] Two novel compounds 29-norcucurbitacin and desacetylfevicordin A and three known29-norcucurbitacin derivatives have also been isolated fromthe ethyl acetate fraction of P macrocarpa seeds [6]

Hindawi Publishing CorporationBioMed Research InternationalVolume 2014 Article ID 410184 13 pageshttpdxdoiorg1011552014410184

2 BioMed Research International

Dried and ground plant material

Extraction with 80 aqueous methanol (three times)

Concentration under reduced pressure

Fractionation with hexane

Methanol extract

Hexane-insoluble fraction

Chloroform fraction Chloroform-insoluble fraction

Ethyl acetate-soluble fraction Water fraction

Phenolic flavonoid flavonol antioxidant and cytotoxicity assays

Fractionation with chloroform

Washed dried and ground

Phaleria macrocarpa (Scheff) Boerl seeds

Partition (vv) ethyl acetate-water (ratio 1 1)

Hexane-soluble fraction

Figure 1 Extraction procedure and its fractionations of P macrocarpa (Scheff) Boerl seeds

The ethanol extract of P macrocarpa seeds exhibitedtoxicity towards T47D breast cancer cell lines (LC

501512 plusmn

321 120583gmL) through COX-2 inhibition [7] Additionally theethanol extract of the seeds and the fruitsrsquo flesh have beenshown to increase p53 gene expression but had no effect onBcl-2 gene expression Moreover the n-hexane extract of theseeds had a greater effect in increasing p53 gene expressionthan that of the flesh of the fruit but had no effect on Bcl-2 gene expression [8] The ethanol extracts of P macrocarpaseeds and fruits have been found to be nontoxic to humanmononuclear peripheral normal cells but were slightly toxicto Vero cell lines [9]

Although P macrocarpa has been used extensively inIndonesia there is limited scientific research available onthe biological properties of this plant in relation to itsmedicinal benefitsThe present research investigated the totalphenolic and flavonoid content and the antioxidative andcytotoxic activities of the crude and fractionated extracts ofP macrocarpa seeds

2 Material and Methods

21 Plant Materials Seeds of Phaleria macrocarpa (Scheff)Boerl were collected from Yogyakarta Indonesia in Decem-ber 2011 A voucher specimen (ID no KLU 47923) wasdeposited into a repository at the Institute of BiologicalSciences Faculty of Science University of Malaya MalaysiaSamples were washed and dried in an oven at approximately50∘CThe seeds were then ground into powder and stored inairtight containers

22 Preparation for Extraction The dried powder was mac-erated with 80 aqueous methanol and extracted for 72 h

before filtration (three times) The filtrate obtained wasconcentrated under reduced pressure (60 rpm at 37∘C) Thiscrude methanol extract was then fractionated initially withhexane followed by chloroform The chloroform insolublefraction was subjected to partition with ethyl acetate andwater (Figure 1) The methanol extract and its fractions(hexane chloroform ethyl acetate and water) were refilteredand evaporated at low pressure (60 rpm at 37∘C) to removeexcess solvent

23 Phytochemical Screening Analysis In order to classify thetypes of organic constituents present in the plant samplespreliminary phytochemical screening tests were carried outon the plant samples according to the qualitative and quanti-tative methods of Trease and Evans [10] and Sofowora [11]The organic constituents that were investigated were thoselisted in Table 2 120572-Amino acids carbohydrates cyanogenicglycosides organic acids reducing sugars saponin glyco-sides and starch content determination were carried outon water extracts Alkaloids tests were used with 1 HClextract Determination of flavonoids glycosides phenoliccompounds and tannins were performed on the ethanolextract Test for steroids was performed on the petroleum -ether extract

24 Physiochemical Determination The nutritional valuesof the P macrocarpa seeds including moisture ash fiberprotein fat carbohydrate contents and energy value weredetermined by using the methods of AOAC [12] Trease andEvans [10] and Harbone [13]

25 Determination of Moisture Content The moisture con-tent of the dried powder samples was determined by using

BioMed Research International 3

the oven drying method [14 15] A clean dried crucible wasweighed and 10 g of the driedPmacrocarpa seeds powderwasplaced in a beaker The sample was dried in an electric ovenat 105∘C until all the moisture was removed from the sampleand constant weight was achieved The crucible containingthe dried sample was weighed again and the loss of weightwas recorded as the moisture content of the dried powderedP macrocarpa seeds The experiment was repeated threetimes The moisture content () was calculated by using thefollowing equation

Mositure percentage () =1198822minus1198821

1198820

times 100 (1)

where1198821= weight of sample after drying (g)119882

2= weight of

sample before drying (g) and1198820= weight of the sample (g)

26 Determination of Ash Content Ash content was deter-mined by using the method of AOCS [12] Briefly theash value of the samples represented the inorganic residuewhen the organic matter has been burnt away An accuratelyweighted amount (10 g) of the sample was placed in a pre-heated cooled and weighed porcelain crucible The cruciblewas heated carefully on a hot plate until the organic matterwas dried and burnt off without flaming and finally heated ina furnace at 550 plusmn 50∘C

The percentage of ash content was calculated using thefollowing formula

Percentage of Ash () =Weight of Ash

Weight of sampletimes 100 (2)

27 Determination of Crude Fiber Content The crude fibercontent was determined by using themethod ofAOAC [16]Dried powdered sample was weighed (10 g) and extractedwith petroleum ether (100mL) three times The extractedsample was air-dried and transferred to a round-bottomedflask In the flask 30mL of sulphuric acid (01275M) wasadded followed by 170mL of hot sulphuric acidThe solutionwas then refluxed for approximately 30 minutes and filteredthrough a Buchner funnel The insoluble matter was washedwith boiling water until the final filtrate was free from acid

The residue was placed back into the flask with 30mLof sodium hydroxide (0313M) and 170mL of hot sodiumhydroxide (0313M) was then added The mixture was againrefluxed for about 30minutes and filtered using sintered glassThe residue was washed with 1 HCl and then washed againwith boilingwater until therewas no acid presentThe residuewas finally washed with ethanol and ether and dried in anoven at 100∘C and thenweighedThis procedure was repeateduntil the fiber content was constant The fiber content wascalculated using the following equation

Percentage of Crude fiber () =Loss in weight

Weight of sampletimes 100

(3)

28 Determination of Carbohydrate Content Carbohydratecontent was calculated by multiplying the reducing sugar

content The reducing sugar content was determined usingthe Fehlingrsquos reducing method of Lane and Eynonrsquos [16]Weighted sample (10 g) was placed into a 250mL round-bottomed flask and 20mL of sulphuric acid (05M) wasadded Reflux was then performed in a sand bath for 25hoursThe residue was washed after filtration with warm dis-tilled water The solution was then neutralized with sodiumcarbonate power and the mixturersquos volume was made up to100mL with distilled water This was followed by titrationwith Fehlingrsquos solution equal amounts of solution A (coppersulphate solution) and solution B (sodium potassium tartrateand sodium hydroxide solution) using methylene blue asindicator The mixed Fehlingrsquos solution (5mL) was pipettedinto a conical flask and distilled water (5mL) was addedThesolution was then boiled for 15 seconds Methylene blue indi-cator (a few drops) was then titrated with the solution untilthe colour changed from blue to green The carbohydratecontent was then calculated according to following equation

Percentage of carbohydrate content ()

=5 times 0005 times 100 times 100 times 100

119881 times 10 times119882times 09

(4)

where 119881 = volume of sample solution (titration volume) and119882 = weight of powdered sample

29 Determination of Fat Content Fat content of P macro-carpa seeds was determined using the soxhlet extractionmethod [12] A seed sample (10 g) was placed in a soxhletextractor Petroleum ether was then poured into the extractorand extraction was performed for 12 hoursThe volume of thepetroleum ether extract was reduced to 15mL by evaporationThis was then dried at 105∘C in an oven until constant weightwas achievedThe fat content was then calculated by using thefollowing equation

of Fat content

=Weight of fat obtained from sample times 100

Weight of sample

(5)

210 Determination of Protein Content Protein content wasdetermined using the AOAC method [12] Briefly pow-dered samples (1 g) 50mL of distilled water 5 g of coppersulphate and 15mL of concentrated sulphuric acid wereadded to a Kjeldahl flask The flask was partially closed bymeans of a funnel and the content was digested by heatingthe flask at an inclined position in the digester The mixturewas heated for 30minutes until there was 40mL of clear 01Mstandard sulphuric A few drops of methyl red indicator weremixed into the clear solutionThe flask was then placed belowthe condenser and the end of the adapter tube was dippedin the acid The flasks were set up for Kjeldahl distillationand 70mL of 40 sodium hydroxide was added through thefunnel The funnel was washed twice with 50mL of distilledwater Distillation was then performed for one hour Thedistilled ammonia was then nitrated with 01M standardsolution until the color changed from yellow to colorlessTheexperiment was repeated three times The nitrogen content

4 BioMed Research International

and protein content in the sample were calculated using thefollowing relation

Percentage of Nitrogen ()

=(119881119904minus 119881119887) times 119872

119860times 00140 times 100

Weight of sample (119882)

Protein content = percentage of Nitrogen times 625

(6)

where 119881119904= volume in cm3 of standard acid used in the

titration of sample119881119887= volume in cm3 of standard acid used

in the blank titration119872119860=morality of standard acid solution

in mol dmminus3 and119882 = weight of the sample in grams

211 Determination of Phenolic Content The total phenoliccontent of the crude methanol and the fractionated extracts(hexane chloroform ethyl acetate and water) was deter-mined using the Folin-Ciocalteu method [17ndash20] Briefly200120583L of each extract solution of different concentrationswas mixed with 1mL of Folin-Ciocalteu reagent (1 10 dilutedwith H

2O) and 800 120583L of Na

2CO3(7505 gL) The mixture

was thoroughly shaken for 15 minutes and then held ina water bath at a temperature of 37∘C The solution wasallowed to stand for 1 hour at room temperature in a darkplace and the absorption was measured at 750 nm using aspectrophotometer Distilled water was used as blank andgallic acid (0ndash250mgL) was used to construct a standardcalibration curve Gallic acid concentration was establishedfrom the calibration curve 119910 = 00221119909 + 02189 1198772 =09914

212 Determination of Flavonoid Content Total flavonoidcontent of P macrocarpa seeds was measured using themethods of Ebrahimzadeh Nabavi and Ordonez [20ndash22]with minor modifications To determine the flavonoid con-tent 1mL of each sample was added to 01mL of 10Al (NO

3)3solution 01mL of 1M potassium acetate and

38mLofmethanolThe solutionwas thoroughlymixed usinga vortex mixer for two to three minutes and then stooduntouched for 10 minutes at room temperature Absorbancewas determined at 415 nm using a spectrophotometer Thetotal content of flavonoids was measured and expressed asquercetin equivalent on a dry weight basis (119910 = 00855119909+02004 1198772 = 09813)

213 Determination of Flavonol Content The method ofKumaran [23] and Mbaebie [24] with slight modificationswas used to measure the total flavonol content of themethanol extract Briefly 1mL of extract was added to a cen-trifuge tube with 2mL of prepared AlCl

3in ethanol and 3mL

of sodium acetate (50 gL) solution The mixture was stirredthoroughly with a vortex and was then incubated for 1 hourThe absorbance was measured with a spectrophotometer at440 nm A calibration curve was constructed using quercetin(1 5 10 15 and 20mgmL) The total flavonol content wascalculated using the calibration curve with the followingequation119910 = 00353119909 + 01456 1198772 = 09807

214 Antioxidant DPPH Assay Screenings of antioxidantactivity of the crude methanol extract chloroform hexaneethyl acetate and water fractions of P macrocarpa seeds werecarried out by determination ofDPPH free radical scavengingproperty using UV spectrophotometric methods [21 25]Based on this protocol 50120583L of test solutions from differentdry extracts and concentrations (1 5 10 15 and 20mgmL)was dissolved in water This solution was then combinedwith 195mL of DPPHmethanol solution After being mixedsolutions were kept at room temperature in the dark for 30minutes After the reaction the increase in absorbance wasrecorded at 517 nm Methanol was used as a blank DPPHsolution was used as negative control (119860

0) and gallic acid

was used as positive control The antioxidant activity wasexpressed as an IC

50value All experiments were carried out

in triplicate The scavenging effect was obtained from thefollowing relation

Scavenging effect () =(1198600minus 1198601) times 100

1198600

(7)

where 1198600was the absorbance of the control reaction and

1198601was the absorbance of the sample of the tested extracts

Gallic acid was used as standard Percentage of inhibitionwas calculated using the following formula inhibition =[(119860negative minus 119860 test)119860negative] times 100 (119860 is absorbance)

215 Cytotoxicity Screening

2151 Cell Culture andCultureMedium Human cervical car-cinoma cells (Ca Ski) hormone-dependent breast carcinomacells (MCF-7) human breast adenocarcinoma cells (MDA-MB231) human ovarian carcinoma cells (SKOV-3) humancolon carcinoma cells (HT-29) and noncancer humanfibrob-last cells (MRC-5) were purchased from the American TissueCulture Collection (ATCC USA)

HT 29 Ca Ski andMCF-7 cells weremaintained in RPMI1640 medium (Sigma) MDA-MB231 and SKOV-3 cells inDulbeccorsquos Modified Eaglersquos medium (DMEM Sigma) andMRC-5 cells in Eaglersquos Minimum Essential medium (EMEMSigma) supplemented with 10 fetal bovine serum (FBSPAA Lab Austria) 100 120583gmL penicillin or streptomycin(PAA Lab Austria) and 50 120583gmL kanamycinamphotericinB (PAA Lab Austria) The cells were cultured in a CO

2

incubator (5) and kept at 37∘C in a humidified atmosphereThe cultures were subcultured every 2-3 days and checkedfrequently under an inverted microscope (Leica Germany)for any contamination

2152 MTT Cell Proliferation Assay MTT [3-(4 5-dimeth-ylthiazol2yl)-2 5-diphenyl tetrazolium bromide)] assay wasperformed on the cultured cells in 96-well plates according tothe method of Mosmann [26 27] Briefly cells were culturedto confluence They were then centrifuged at 1000 rpm for 5minutes and resuspendedwith 10mLof growthmediumThedensity of the viable cells was counted using 04 trypan blueexclusion dye in a haemocytometer with a microscope Thecells were then seeded into microtiter plates and incubated ina CO2incubator at 37∘C for 24 hrs After reaching 70ndash80

BioMed Research International 5

Table 1 Yield of methanol extract and its fractions from P macro-carpa of seedsExtract and fractions Yield (g) Percentage of yield ()Methanolic extract (ME) 1987 1199Hexane fraction (HF) 1587 947Chloroform fraction (CF) 72 043Ethyl acetate fraction (EAF) 62 037Water fraction (WF) Freeze dry

confluence each extract at concentrations of 1 10 25 50and 100 120583gmL (in 200120583L of 10 media) was added to therespective wells containing the cells Wells with untreatedcells were used as the negative control and cells exposedto doxorubicin were used as positive control After 24 48and 72 hours 10 120583L MTT stock solution was added to eachwell OD was then determined by measuring absorbance at540 nm using an ELISA microplate reader The percentageof inhibition () was calculated according to the followingformula

Percentage of inhibition ()

=OD control minusOD sample times 100

OD control

(8)

Cytotoxicity of each sample is expressed as plusmnIC50value The

extract that gave IC50

of 30 120583gmL or less was consideredactive [26]

216 Gas Chromatography-Mass Spectrometry (GC-MS) GC-MS was used to determine the molecular weight of thecomponents collected and the purity of the collected extractsRunning conditions were as follows oven temperature wasprogrammed with an initial temperature of 100∘C andincreased at a ramp rate of 5∘Cmin and reached a finaltemperature of 300∘C The carrier gas or mobile phase usedwas helium and a flow rate of 1mLmin was programmedThe mass spectrometry mode used was electron ionization(EI) mode with a current of 70 eV The injection mode wasprogrammed with a sample injection volume of 1120583L with asplit mode at a ratio of 1 20 The injection port temperaturewas set to 230∘C and the detectorinterface temperature wasset to 250∘C The results were collected for 40 minutes Thetotal ion chromatogram obtained was autointegrated usingChemStation and the chemical compounds or componentswere analysed by comparison with the suppliedmass spectraldatabase (NIST 05 Mass Spectral Library USA)

217 Statistical Analysis All data for each test are the averageof triplicate experiments for comparison of values and wererecorded as the mean plusmn standard deviation using MicrosoftExcel software and statistical data analyses were performedusing SPSS software

3 Results

31 Extraction Theextracts were concentrated using a rotaryevaporator (Buchi USA) under reduced pressure at 35∘CThe

yield of extracts from P macrocarpa seeds is shown in Table 1The highest yield from P macrocarpa seeds was the hexanefraction (947)

32 Preliminary Phytochemical Studies In order to determinethe types of phytoorganic constituents present in P macro-carpa seeds a preliminary phytochemical investigation wascarried out according to conventional methods The resultsobtained from these experiments are summarized in Table 2

The phytochemical tests showed that there were sec-ondary metabolites including carbohydrate flavonoids gly-cosides saponin glycosides phenolic compounds steroidstannins and terpenoids present in different extracts ofP macrocarpa seeds Small amounts of alkaloids 120572-aminoacids cyanogenic glycosides organic acids reducing sugarsand starches were also found to be present

The main constituents such as flavonoids glycosidessaponin glycosides phenolic compounds steroids tanninsand terpenoids present in P macrocarpa seeds may con-tribute to the presence of bioactivities such as antibacterialan analgesic an antifungal an anti-inflammatory agentand cytotoxicity Moreover the toxic chemical constituentscyanogenic glycosides were also present in the seeds

33 Physicochemical Studies The determination of nutri-tional values such as moisture ash fiber protein fat andcarbohydrate contents was carried out using the AOACmethod as well as the Lane and Eynon titration method andthe results obtained are shown in Figure 2(a) and Table 3Different physiochemical parameters for the purpose ofstandardization such as moisture (631 plusmn 143) ash (296 plusmn186) protein (2073 plusmn 244) crude fiber (2276 plusmn 279)crude fat (184 plusmn 311) and carbohydrate (2934 plusmn 198)were determined

34 Flavonoid Flavonol and Phenolic Determinations It wasfound that the methanol extract had the highest flavonoidcontent (933 plusmn 08mgmL) followed by ethyl acetate waterchloroform and hexane fractions which were 838 plusmn 10808 plusmn 03 678 plusmn 11 and 418 plusmn 15mgmL respectively

Themethanol extract (893plusmn11mgmL) of Pmacrocarpaseeds exhibited the highest amount of total flavonol contentfollowed by water ethyl acetate chloroform and hexanefractions which were 893 plusmn 10 613 plusmn 05 429 plusmn 09 and429 plusmn 07mgmL respectively

The total phenolic content of the P macrocarpa seedsextracts and fractions was expressed as gallic acid equiva-lents The methanol extract of P macrocarpa seeds exhibitedthe highest amount (720 plusmn 07mgmL) of total phenolicsfollowed by water ethyl acetate chloroform and hexanefractions which were 512 plusmn 14 358 plusmn 11 326 plusmn 10 and263 plusmn 01mgmL respectively

These data are shown in Figure 2(b) and Table 4

35 DPPH (2 2-Diphenyl-1-picryl-hydazyl) AntioxidantAssay DPPH assay was used to determine the free radicalscavenging ability of extracts and fractions of P macrocarpaseeds and to determine the antioxidant activity of its

6 BioMed Research International

Table 2 Results of chemical constituents of P macrocarpa seeds

No Tests Reagents Observation

1 AlkaloidsWagnerrsquos reagentMayerrsquos reagentDragendorff rsquos reagentSodium picrate solution

++++

2 120572-Amino acids Ninhydrin reagent +3 Carbohydrates 10 120572-naphthol and conc H2SO4 ++4 Cyanogenic glycosides Sodium picrate solution +5 Flavonoids Mg and conc HCl ++6 Glycosides 10 lead acetate ++7 Organic acids Bromothymol blue +8 Phenolic compounds 1 FeCl3 ++

9 Reducing sugars Fehlingrsquos solutionsA and B +

10 Saponin glycosides Distilled water ++11 Starch Iodine solution +12 Steroids Acetic anhydride and conc H2SO4 ++13 Tannins 1 Gelatin ++14 Terpenoids Acetic anhydride and conc H2SO4 +++++ large amount ++ medium amount + small amount minus absent + present

0

5

10

15

20

25

30

35

Con

tent

()

Moi

sture

Ash

Prot

ein

Crud

e fibe

r

Carb

ohyd

rate

Crud

e fat

(a)

123456789

1011

ME HF CF EAF WF

FlavonoidsFlavonolPhenolic

Con

cent

ratio

n (m

gm

L)

(b)

Figure 2 (a)The percentage ofmoisture ash protein crude fiber crude fat and carbohydrate contents on PmacrocarpaBoerl seeds (b) Totalamount of concentration of flavonoid flavonol and phenolic contents of P macrocarpa seeds ME methanol extract HF hexane fractionEAF ethyl acetate fraction CF chloroform fractionWF water fraction

phytoconstituents It is important to note that a lower IC50

value equals a higher scavenging activity The scavengingactivity was presented as the percentage of inhibitionof DPPH free radicals (Figure 3 and Table 4) Based onIC50

values the samples can be ranked in the followingdescending order water fraction gt methanol extract gt ethylacetate fraction gt hexane fraction gt chloroform fractionTheresults revealed that the water fraction was most active forantioxidant activity compared to other fractions The radical

scavenging effect was found to increase with increasingconcentrations

36 Cytotoxicity Screening of MTT Cell Proliferation AssayThe methanol and fractionated extracts (hexane chloro-form ethyl acetate and water) were investigated for cyto-toxic effects in human cervical adenocarcinoma cells (CaSki) humanhormone-dependent breast carcinoma (MCF-7)

BioMed Research International 7

Table 3The percentage of moisture ash protein crude fiber crudefat and carbohydrate contents and the amount of nutrition valueson P macrocarpa seeds

Tests parameter Percentage of contents ()Moisture 631 plusmn 143Ash 296 plusmn 186Protein 2073 plusmn 244Crude fiber 2276 plusmn 279Crude fat 1840 plusmn 311Carbohydrate 2934 plusmn 198

0102030405060708090

100

1 5 10 15 20

ME

HF

CF

EAF

WF

Concentration (mgmL)

Inhi

bitio

n (

)

Figure 3 Percentage of inhibition of DPPH free radical scavengingof P macrocarpa seeds ME methanol extract HF hexane fractionEAF ethyl acetate fraction CF chloroform fractionWF waterfraction

human colon adenocarcinoma cells (HT-29) human ovar-ian carcinoma cells (SKOV-3) human hormone-dependentbreast carcinoma (MDA-MB231) and normal cells (MRC-5) using MTT cells proliferation assay at 24 hrs 48 hrs and72 hrs respectively

The methanol extract of P macrocarpa seeds showedexcellent cytotoxic effects with IC

50value of 82plusmn 466 120583gmL

in Ca Ski cells at 24 hrs and 120 plusmn 22 85 plusmn 168 and30 plusmn 250 120583gmL in MCF-7 cells at 24 hrs 48 hrs and72 hrs respectively The methanol extract also showed goodcytotoxic activity in HT-29 cells with IC

50values of 293 plusmn

226 250 plusmn 135 and 215 plusmn 330 120583gmL at 24 hrs 48 hrsand 72 hrs respectively in Ca Ski cells with IC

50values of

197 plusmn 092 120583gmL and in SKOV-3 cells with IC50

values of165 plusmn 252 221 plusmn 247 and 360 plusmn 355 120583gmL at 24 hrs48 hrs and 72 hrs respectively However this extract had nocytotoxic effect in MDA-MB231 cells with IC

50gt100120583gmL

and the extract also had low cytotoxic effect in the MRC-5cells with IC

50gt 50 120583gmL

The hexane fraction of P macrocarpa seeds showedmoderate cytotoxic effects with IC

50values of 452 plusmn 149

and 555 plusmn 197 120583gmL at 24 hrs and 48 hrs on MCF-7 400 plusmn315 120583gmL inHT-29 cells at 24 hrs and 405plusmn352 and 500plusmn302 120583gmL in SKOV-3 at 24 hrs and 48 hrs In addition the

hexane fraction also displayed low cytotoxic effect in MCF-7at 72 hrs HT-29 at 48 hrs and 72 hrs and in SKOV-3 at 72 hrsThe IC

50value of treatment with hexane fraction in MCF-7

was 725plusmn152 120583gmL for 72 hrs exposure IC50values in HT-

29 were 640plusmn203 and 750plusmn314 120583gmL at 48 hrs and 72 hrswhile IC

50value in SKOV-3 was 703 plusmn 353 120583gmL at 72 hrs

In contrast the hexane fraction exhibited no cytotoxic effectsin MDA-MB231 cells or MRC-5 cells

The chloroform fraction of P macrocarpa seeds exhibitedthe highest cytotoxic effect with IC

50value of 100plusmn131 82plusmn

104 and 220 plusmn 186 120583gmL at 24 hrs 48 hrs and 72 hrs in CaSki cells 95plusmn295 87plusmn159 and 210plusmn198 120583gmL at 24 hrs48 hrs and 72 hrs on HT29 and 248 plusmn 206 165 plusmn 321 and900 plusmn 26 120583gmL in SKOV-3 cells The chloroform fractionexhibitedmoderate cytotoxic effect inMCF-7 with IC

50value

of 575 plusmn 264 400 plusmn 148 and 465 plusmn 345 120583gmL for 24 hrs48 hrs and 72 hrs respectively In contrast the chloroformfraction had no cytotoxic effect against theMDA-MB231 cellsand normal MRC-5 cells with IC

50gt100 120583gmL

The ethyl acetate fraction ofPmacrocarpa seeds exhibitedthe highest cytotoxic effect with IC

50lt 25 120583gmL in SKOV-3

cellsMDA-MB231 cellsMCF-7 cells andCa Ski cells On theother hand the ethyl acetate fraction exhibited low cytotoxiceffect in MRC-5 normal cells with IC

50value of 35 120583gmL

The ethyl acetate fraction exhibited the highest cytotoxiceffect in all selected cells (Ca Ski MCF-7 HT-29 and MDA-MB231) The water extract showed no cytotoxic effect againstall selected cancer cell lines with IC

50gt 100 120583gmL and

exhibited a mild cytotoxic effects against MRC-5 cells Thecorresponding data is shown in Figure 4 and Table 5

37 Characterization and Identification of Hexane and Chlo-roform Fractions Six compounds were identified from thehexane fraction of P macrocarpa seeds using GC-MS Theywere methyl stearate oleic acid methyl oleate linoleic acidmethyl linolenate and palmitic acid Moreover a GCMSanalysis of the chloroform fraction of P macrocarpa seedsshowed the presence of methyl myristate palmitic acidmethyl oleate methyl linoleate oleic acid and (119911)- and917-octadecadienal (119911) A comparison with the NIST massspectral library (NIST 05MS library 2002) andAdams (2001)confirmed the identity of the compounds The chemicalstructures of the identified compounds in the hexane andchloroform fractions of P macrocarpa seeds are as shown inFigure 5

4 Discussion

A longer shelf life can be achieved by reducing moisturecontent Thus moisture content is a critical factor for thestability of an extractMoisture enhances fungal and bacterialgrowth therefore decreasing the longevity of the extract Thetime it takes for plant material to deteriorate depends on howmuch water the plant material contains

Carbohydrates play a vital role in the immune sys-tem fertilization pathogenesis blood clotting and humandevelopment Foods that contain carbohydrates can raiseblood glucose and the three main types of carbohydrate are

8 BioMed Research International

0102030405060708090

100

ME

HF

CF

EAF

24h 48h 72h

IC50

valu

e of c

once

ntra

tion

(120583g

mL)

Ca Ski

0102030405060708090

100

ME

HF

CF

EAF

24h 48h 72h

IC50

valu

e of c

once

ntra

tion

(120583g

mL)

MCF 7

0102030405060708090

100

ME

HF

CF

EAF

24h 48h 72h

IC50

valu

e of c

once

ntra

tion

(120583g

mL)

HT-29

minus10

0

10

20

30

40

50

60

70

80

90

100

ME

HF

CF

EAF

24h 48h 72h

IC50

valu

e of c

once

ntra

tion

(120583g

mL)

SKOV-3

0102030405060708090

100

ME

HF

CF

EAF

24h 48h 72h

IC50

valu

e of c

once

ntra

tion

(120583g

mL)

MDA-MB231

Figure 4 In vitro cytotoxic effects of P macrocarpa seedson Ca Ski MCF-7 HT-29 SKOV-3 and MDA-MB231 cell lines ME methanolextract HF hexane fraction EAF ethyl acetate fraction CF chloroform fractionWF water fraction Each value is expressed as mean plusmnstandard deviation of three measurements

BioMed Research International 9

Methyl linoleate

Methyl stearate

Palmitic acid

Oleic acid

Methyl myristate

Linoleic acid

Methyl oleate

H3C

H3C

H3C

H3C

H3C

H3C

H3C

O

O

O

O

O

O

O

O

O

O

O

CH3

CH3

CH3

CH3

OH

OH

OH

(294mz)

(298mz)

(282mz)

(242mz)

(280mz)

(298mz)

(256mz)

Figure 5

10 BioMed Research International

Table 4 Total flavonoid total flavonol total phenolic contents and result of DPPH free radical scavenging property of P macrocarpa seeds

Tests ME (mgmL) HF (mgmL) CF (mgmL) EAF (mgmL) WF (mgmL)Flavonoid 933 plusmn 08 418 plusmn 15 678 plusmn 11 838 plusmn 03 808 plusmn 10Flavonol 893 plusmn 11 425 plusmn 07 429 plusmn 09 613 plusmn 05 818 plusmn 10Phenolic 720 plusmn 07 263 plusmn 01 326 plusmn 1 358 plusmn 11 512 plusmn 14DPPH 1150 plusmn 003 1500 plusmn 004 1575 plusmn 004 1400 plusmn 005 975 plusmn 003ME methanol extract HF hexane fraction EAF ethyl acetate fraction CF chloroform fraction WF water fraction

Table 5 In vitro cytotoxic effects of methanol extract and its fractions of P macrocarpa cancer cells lines and normal human fibroblast breastcells line

Cell line Incubation periods (hours) ME HF CF EAF Doxorubicin24 82 plusmn 466 ge100 100 plusmn 131 56 plusmn 117 092 plusmn 064

Ca Ski 48 197 plusmn 092 ge100 82 plusmn 104 77 plusmn 185 069 plusmn 10572 407 plusmn 226 ge100 220 plusmn 186 60 plusmn 322 045 plusmn 09924 120 plusmn 22 452 plusmn 149 575 plusmn 264 223 plusmn 158 105 plusmn 108

MCF7 48 85 plusmn 168 555 plusmn 197 400 plusmn 148 168 plusmn 170 012 plusmn 06972 63 plusmn 250 725 plusmn 152 465 plusmn 345 84 plusmn 171 092 plusmn 17024 293 plusmn 226 400 plusmn 315 95 plusmn 295 11 plusmn 120 092 plusmn 072

HT29 48 250 plusmn 135 640 plusmn 203 87 plusmn 159 35 plusmn 200 032 plusmn 21672 215 plusmn 330 750 plusmn 314 210 plusmn 198 12 plusmn 228 088 plusmn 09424 165 plusmn 252 405 plusmn 352 248 plusmn 206 68 plusmn 18 102 plusmn 179

SKOV-3 48 221 plusmn 247 500 plusmn 302 165 plusmn 321 65 plusmn 23 032 plusmn 09772 360 plusmn 355 703 plusmn 353 90 plusmn 26 32 plusmn 281 062 plusmn 09824 ge100 ge100 ge100 65 plusmn 23 043 plusmn 050

MDA-MB231 48 ge100 ge100 ge100 15 plusmn 343 059 plusmn 10172 ge100 ge100 ge100 186 plusmn 126 095 plusmn 132

ME methanol extract HF hexane fraction EAF ethyl acetate fraction CF chloroform fractionWF water fraction Doxorubicin was used as positive controlEach value is expressed as mean plusmn standard deviation of three measurements

starches sugars and fiber A macronutrient protein consistsof amino acids which is required for proper growth andhuman body function The seeds were found to containa high level of crude fiber which is a potential source ofphenolic antioxidants The major components of fibers arecellulose hemicelluloses lignin 120573-glucans gums and pectinand hydrocolloids These components can behave as proan-tioxidants [28 29] In our present physicochemical study Pmacrocarpa seeds were mainly made up of carbohydratesfollowed by crude fiber proteins fatmoisture and ashHigh-protein and low-carbohydrate diets are often effective forweight loss Dietary fiber can aid in digestion is helpfulfor weight management and reduces constipation Howevermany high-protein and low-carbohydrate foods are low infiberWomen aged 50 and older need at least 21 grams of fiberdaily whilst those aged 19 to 50 require 25 grams men aged50 and older need 30 grams and those between the age of 19to 50 should consume at least 38 grams of fiber

Steroids possess biological activities which are insec-ticidal cardiotonic and antimicrobial activities and theseare potentially useful for development into therapeuticdrugs Tannins are known to be important for their antivi-ral antibacterial antiparasitic effects anti-inflammatoryantiulcer and antioxidant properties [30ndash33] Saponinshave been found to have antimicrobial anti-inflammatoryantifeedant and hemolytic effects [34 35] Some alkaloids

have been reported to have anticancer and antiviral activityand saponins have been reported to be cardiotonic whileflavonoids have anticancer and anti-inflammatory activity[10 36] The presence of tannins may be responsible for theability of P macrocarpa seeds to be used in the treatmentof diseases such as diabetes diarrhea and dysentery Ourpreliminary phytochemical studies revealed that the chemicalcomponents of P macrocarpa seeds also include flavonoidsphenolics steroids tannins terpenoids glucosides saponinsand carbohydrates

Previous researchers reported that kaempferol myrice-tin naringin quercetin rutin [3] 29-norcucurbitacinderivatives fevicordin A fevicordin A glucoside fevicordinD glucoside [6] mahkoside A dodecanoic acid palmiticacid des-acetyl flavicordin-A flavicordin-A flavicordin-Dflavicordin-A glucoside ethyl stearate lignans and sucrose[37 38] mangiferin (a C-glucosylxantone) kaempferol-3-o-120573-D-glucoside dodecanoic acid palmitic acid ethyl stearatesucrose [5] pinoresinol lariciresinol matairesinol alkaloidsand saponins [37 39] saponins alkaloids polyphenolicsphenols flavanoids lignans tannins [40ndash42] icarisideC3 mangiferin gallic acid phalerin glycoside (345trihydroxy-4-methoxy-benzophenone-3-O-120573-D-glucoside)[43] and 2410158406 trihydroxy-4-methoxy-benzophenone-3-O-120573-D-glucoside [44] were isolated from different partsof P macrocarpa The hexane extract of the seeds was

BioMed Research International 11

found to contain methyl myristate methyl stearate oleicacid methyl oleate linoleic acid methyl linoleate palmiticacid methyl palmitate 6-octadecenoic acid and 917-octadecadial 120573-sitosterol as major components A further30 to 40 of components were not identified as thesewere polar compounds that require HPLC analysis Othercompounds reported in the literature for the seeds leavesand fruits were not identified in this investigation It isprobable that the components reported in the literaturemay be present in other fractions (methyl acetate CHCl

3

and water fractions)Phenolics are able to transfer protons to radicals [45]

DPPH which provides free radicals is normally a blue-violet color However the DPPH turned yellow after it wasconverted to 1 1-diphenyl-2-picrylhydrazine which has lessfree-radical activity Hendra et al reported that the IC

50

values of free radical and reducing power in methanolwith HCl of P macrocarpa seeds were 2450 plusmn 194 and1502 plusmn 128 [42] In our investigation using DPPH assay thewater fraction displayed the highest IC

50value followed by

methanol ethyl acetate hexane and chloroform fractionThe presence of flavonoids in P macrocarpa seeds may be

responsible for the traditional use of the plants in treatingcancer inflammations and allergies Recently it has beenshown that phenolic compounds in crude methanol andethyl acetate extract of P macrocarpa leaves displayed goodantioxidant and antimicrobial activites [46] Hendra et al(2011) examined the amount of total phenolic (477plusmn 104mggallic acid equivalentg DW) and the amount of flavonoid(359 plusmn 247mg rutin equivalentg DW) contents in themethanol extract with HCl of P macrocarpa seeds [42] Inthis study the total flavonoids content was higher than that inthe results previously reported by Rohyami [47] and phenolflavonol and flavonoid contents were the highest in theaqueous methanol extract followed by ethyl acetate waterchloroform and hexane extracts

MTT assay is a sensitive and reliable colorimetric assaythat uses quantitative measurements to calculate the via-bility proliferation and activation of cells This method iscommonly applied to screen anticancer agents [48] Themost well-known methods used to calculate cytotoxicityare the neutral red (NR) uptake and dimethylthiazole-diphenyl tetrazolium bromide (MTT) metabolism [27 49]Earlier investigations showed that the ethanol extract of Pmacrocarpa seeds and fruit meat were not toxic to normalhuman cells but slightly toxic to a Vero cell line [9] Theethanol extract of P macrocarpa seed fruit also showedtoxicity towards T47D breast cancer cell line through COX-2 expression inhibition [7] Desacetylfevicordin A has beenisolated from the ethyl acetate extract of P macrocarpaseeds and this compound displayed excellent cytotoxicityin brine shrimp [43] The ethyl acetate of P macrocarpaseeds exhibited mild cytotoxic effect against HepG2 cells(IC50values between 30 and 60 120583gmL) [46] Previous studies

showed that the cytotoxic activity of the seeds on HT-29MCF-7 Hela and Chang liver cells lines were 384 plusmn 037255 plusmn 137 295 plusmn 10 and 678 plusmn 027 receptively [42] Inour study the methanolic extract of P macrocarpa seeds wasfound to pose cytotoxic effect against HT-29 MCF-7 Cas Ki

and SKOV-3 cell lines (IC50

values giving from 11 plusmn 120to 360 plusmn 355) and mild toxicity on normal cell lines Therewere significant cytotoxic effects on selected cancer cells linesthat were both time- and dose-dependent manner due to thepresence of many secondary metabolites

The phytochemical screening also indicated the presenceof a small amount of cyanogenic glycosides Cyanogenicglucosides which can cause acute cyanide poisoning causerapid respiration and pulse decrease the blood pressure andinduce vomiting diarrhea headache dizziness and so onThus these components have to be removed (usually byboiling) prior to consumption for therapeutic purposes

5 Conclusion

P macrocarpa seeds exhibited antioxidant and cytotoxicactivities It is highly probable that these activities are dueto the presence of phenolic and flavonoid compounds inappreciable amounts in the plant Furthermore the cytotox-icity activity suggested that the seed may contain a potentialanticancer agent The outcome of this study is encouragingdemonstrating the potential for the P macrocarpa as a sourceof multiple therapeutic agents

Abbreviations

EAF Ethyl acetate fractionME Methanol extractHF Hexane fractionCF Chloroform fractionWF Water fractiong Grammg MilligrammL Milliliter

Conflict of Interests

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

Authorsrsquo Contribution

Ma Ma Lay was involved in all experiments and BehroozBanisalamwas involved in antioxidant assay All authors readand approved the submitted version of the paper

Acknowledgments

This work was supported by grants from the MalaysianMinistry of Higher Education (nos PS2862009C andPS4702010B) Ma Ma Lay is a scholarship recipient fromthe ldquoIslamic Development Bank Saudi Arabiardquo The authorswould like to thank Professor Dr Amru BinNasrulhaq Boycefor generously allowing the use of his laboratory for thisresearch

12 BioMed Research International

References

[1] N HarmantoMahkota DewaObat Pusaka ParaDewa Cetakankeempat Agromedia Pustaka Jakarta Indonesia 2002

[2] A Jemal R Siegel E Ward T Murray J Xu and M J ThunldquoCancer statistics 2007rdquoCa ACancer Journal for Clinicians vol57 no 1 pp 43ndash66 2007

[3] R Hendra S Ahmad A Sukari M Y Shukor and EOskoueian ldquoFlavonoid analyses and antimicrobial activity ofvarious parts of Phaleria macrocarpa (Scheff) Boerl fruitrdquoInternational Journal of Molecular Sciences vol 12 no 6 pp3422ndash3431 2011

[4] W Wijayani Sitotoksisitas Minyak Atsiri Biji Dan Daging BuahMahkota Dewa (Phaleria Macrocarpa [Scheff] Boerl) TerhadapMyeloma Cell Line Dan HeLa Cell Line Serta IdentifikasiSenyawa Minyak Atsiri Dengan Kromatografi Gas-SpektrometriMassa (GC-MS) Skripsi FMIPA UGM Yogyakarta Indonesia2005

[5] Y-B Zhang X-J Xu and H-M Liu ldquoChemical con-stituents fromMahkotadewardquo Journal of Asian Natural ProductsResearch vol 8 no 1-2 pp 119ndash123 2006

[6] D Kurnia K Akiyama and H Hayashi ldquo29-Norcucurbitacinderivatives isolated from the Indonesian medicinal plant Pha-leria macrocarpa (Scheff) Boerlrdquo Bioscience Biotechnology andBiochemistry vol 72 no 2 pp 618ndash620 2008

[7] M Bakhriansyah Pengaruh Ekstrak Etanol Biji Mahkota Dewa(Phaleria Macrocarpa [Scheff] Boerl) Pada Sel Kanker Payu-dara T47D Kajian Aktivitas Sitotoksik Dan PenghambatanEkspresi Siklooksigenase-2 Program Pascasarjana UGM 2004

[8] L Wardani The Effect of Phaleria Macrocarpa [Scheff] Boerl)Seed and Flesh Fruit Extract to P53 and Bcl-2 Gene Expression inHela Cell Line FMIPA UGM 2005

[9] A Endang R Tri Joko and E Dewi ldquoCyototoxicity of Phaleriamacrocarpa (Scheff) boerl fruits flesh and seed extract ofethanol and its effect against p53 and Bcl-2 genes expressionof normalrdquo in Proceedings of the International Conference onComputational Science (ICCS rsquo07) 2007

[10] C Evans Trease and Evans Pharmacognosy Saunder 2002[11] A Sofowora Medicinal Plants and Traditional Medicine in

Africa Spectrum Books Ltd Ibadan Nigeria 2nd edition 1993[12] AOAC Official Methods of Analysis Association of Official

Analytical Chemists Arlington Tex USA 2002[13] J Harborne Phytochemical Methods Chapman and Hall Lon-

don UK 1973[14] AOAC Official Methods of Analysis Association of Official

Analytical Chemists Gaithersburg Md USA 17th edition2000

[15] J Lako V C Trenerry M Wahlqvist N WattanapenpaiboonS Sotheeswaran and R Premier ldquoPhytochemical flavonolscarotenoids and the antioxidant properties of a wide selection ofFijian fruit vegetables and other readily available foodsrdquo FoodChemistry vol 101 no 4 pp 1727ndash1741 2007

[16] W Horwitz Official Method of Analysis AOAC InterantionalGaithersburg Md USA 17th edition 2000

[17] A A Adedapo F O Jimoh S Koduru P J Masika and AJ Afolayan ldquoAssessment of the medicinal potentials of themethanol extracts of the leaves and stems of Buddleja salignardquoBMCComplementary andAlternativeMedicine vol 9 article 212009

[18] M Zovko Koncic D Kremer J Gruz et al ldquoAntioxidantand antimicrobial properties ofMoltkia petraea (Tratt) Griseb

flower leaf and stem infusionsrdquo Food and Chemical Toxicologyvol 48 no 6 pp 1537ndash1542 2010

[19] S McDonald P D Prenzler M Antolovich and K RobardsldquoPhenolic content and antioxidant activity of olive extractsrdquoFood Chemistry vol 73 no 1 pp 73ndash84 2001

[20] S M Nabavi M A Ebrahimzadeh S F Nabavi A Hamidiniaand A R Bekhradnia ldquoDetermination of antioxidant activityphenol and flavonoid content of Parrotia persicaMeyrdquo Pharma-cologyonline vol 2 pp 560ndash567 2008

[21] M A Ebrahimzadeh S F Nabavi and S M Nabavi ldquoAntiox-idant activities of methanol extract of Sambucus ebulus Lflowerrdquo Pakistan Journal of Biological Sciences vol 12 no 5 pp447ndash450 2009

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

[23] A Kumaran and R Joel Karunakaran ldquoIn vitro antioxidantactivities of methanol extracts of five Phyllanthus species fromIndiardquo Food Science and Technology vol 40 no 2 pp 344ndash3522007

[24] B O Mbaebie H O Edeoga and A J Afolayan ldquoPhy tochemical analysis and antioxidants activities of aqueous stembark extract of Schotia latifolia Jacqrdquo Asian Pacific Journal ofTropical Biomedicine vol 2 no 2 pp 118ndash124 2012

[25] L I Mensor F S Menezes G G Leitao et al ldquoScreening ofBrazilian plants extracts for antioxidants activity by the use ofDPPH free radical methodrdquo Phytotherapy Research vol 15 pp127ndash130 2001

[26] F Denizot and R Lang ldquoRapid colorimetric assay for cellgrowth and survival modifications to the tetrazolium dyeprocedure giving improved sensitivity and reliabilityrdquo Journalof Immunological Methods vol 89 no 2 pp 271ndash277 1986

[27] T Mosmann ldquoRapid colorimetric assay for cellular growth andsurvival application to proliferation and cytotoxicity assaysrdquoJournal of Immunological Methods vol 65 no 1-2 pp 55ndash631983

[28] P Maisuthisakul R Pongsawatmanit and M H GordonldquoAntioxidant properties of Teaw (Cratoxylum formosum Dyer)extract in soybean oil and emulsionsrdquo Journal of Agriculturaland Food Chemistry vol 54 no 7 pp 2719ndash2725 2006

[29] P Maisuthisakul S Pasuk and P Ritthiruangdej ldquoRelationshipbetween antioxidant properties and chemical composition ofsome Thai plantsrdquo Journal of Food Composition and Analysisvol 21 no 3 pp 229ndash240 2008

[30] H Kolodziej and A F Kiderlen ldquoAntileishmanial activity andimmune modulatory effects of tannins and related compoundson Leishmania parasitised RAW 2647 cellsrdquo Phytochemistryvol 66 no 17 pp 2056ndash2071 2005

[31] T Nakagawa and T Yokozawa ldquoDirect scavenging of nitricoxide and superoxide by green teardquo Food and Chemical Toxi-cology vol 40 no 12 pp 1745ndash1750 2002

[32] V Schulz R Hansel and V Tyler Fitoterapia Racional UmGuiade Fitoterapia Para as ciencias da saude Manole Barueri Brazil2002

[33] S D Kapu Y B Ngwai O Kayode P A Akah C Wambebeand K Gamaniel ldquoAnti-inflammatory analgesic and anti-lymphocytic activities of the aqueous extract of Crinum gigan-teumrdquo Journal of Ethnopharmacology vol 78 no 1 pp 7ndash132001

[34] G Francis Z Kerem H P S Makkar and K Becker ldquoThebiological action of saponins in animal systems a reviewrdquoBritish Journal of Nutrition vol 88 no 6 pp 587ndash605 2002

BioMed Research International 13

[35] R Xu W Zhao J Xu B Shao and G Qin ldquoStudies onbioactive saponins fromChinesemedicinal plantsrdquoAdvances inExperimental Medicine and Biology vol 404 pp 371ndash382 1996

[36] J A Manthey N Guthrie and K Grohmann ldquoBiologicalproperties of citrus flavonoids pertaining to cancer and inflam-mationrdquo Current Medicinal Chemistry vol 8 no 2 pp 135ndash1532001

[37] P Hendra Y Fukushi and Y Hashidoko ldquoSynthesis of ben-zophenone glucopyranosides from Phaleria macrocarpa andrelated benzophenone glucopyranosidesrdquo Bioscience Biotech-nology and Biochemistry vol 73 no 10 pp 2172ndash2182 2009

[38] A Rabia Z B A Mohammad D Aidiahmad S Amirin and IU Muhammad ldquoPhytochemistry and medicinal properties ofPhaleria macrocarpa (Scheff) Boerl extractsrdquo PharmacognosyReview vol 7 no 13 pp 73ndash80 2013

[39] A Saufi C B I VonHeimendahl AWAlfermann andE FussldquoStereochemistry of lignans in Phaleria macrocarpa (Scheff)Boerlrdquo Zeitschrift fur Naturforschung C vol 63 no 1-2 pp 13ndash16 2008

[40] S C Chong M A Dollah P P Chong and A Maha ldquoPhaleriamacrocarpa (Scheff) Boerl fruit aqueous extract enhances LDLreceptor and PCSK9 expression in vivo and in vitrordquo Journal ofEthnopharmacology vol 137 no 1 pp 817ndash827 2011

[41] R R Tjandrawinata D Nofiarny L W Susanto P Hendra andA Clarissa ldquoSymptomatic treatment of premenstrual syndromeandor primary dysmenorrhea with DLBS1442 a bioacitveextract ofPhaleriamacrocaraprdquo International Journal of GeneralMedicine vol 4 pp 465ndash476 2011

[42] R Hendra S Ahmad E Oskoueian A Sukari and M YShukor ldquoAntioxidant anti-inflammatory and cytotoxicity ofPhaleriamacrocarap (Boerl) Scheff fruitrdquoBMCComplementaryand Alternative Medicine vol 11 article 110 2011

[43] D Kurnia K Akiyama and H Hayashi ldquo29-Norcucurbitacinderivatives isolated from the Indonesian medicinal plant Pha-leria macrocarpa (Scheff) Boerlrdquo Bioscience Biotechnology andBiochemistry vol 72 no 2 pp 618ndash620 2008

[44] S Oshimi K Zaima Y Matsuno et al ldquoStudies on theconstituents from the fruits of Phaleria macrocaraprdquo Journal ofNatural Medicines vol 62 no 2 pp 207ndash210 2008

[45] C Sanchez-Moreno J A Larrauri and F Saura-Calixto ldquoAprocedure tomeasure the antiradical efficiency of polyphenolsrdquoJournal of the Science of Food and Agriculture vol 76 pp 270ndash276 1998

[46] Y Andriani M A W Effendy T M T Sifzizul and HMohamad ldquoAntibacterial radical-scavenging activities andcytotoxicit properties of Phaleria macrocarpa (Scheff) BoerlLeaves in HepG2 cell linesrdquo International Journal of Pharma-ceutical Sciences and Research vol 2 no 7 pp 1700ndash1706 2011

[47] Y Rohyami ldquoPenentuan Kandungan Flavonoid dari EkstrakMetanol Daging Buah Mahkota Dewa (Phaleria macrocarpaScheff Boerl)rdquo Jurnal Logika vol 5 2009

[48] R D Petty L A Sutherland E M Hunter and I A CreeldquoComparison of MTT and ATP-based assays for the measure-ment of viable cell numberrdquo Journal of Bioluminescence andChemiluminescence vol 10 no 1 pp 29ndash34 1995

[49] J R Sellers S Cook andV S Goldmacher ldquoA cytotoxicity assayutilizing a fluorescent dye that determines accurate survivingfractions of cellsrdquo Journal of Immunological Methods vol 172no 2 pp 255ndash264 1994

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

2 BioMed Research International

Dried and ground plant material

Extraction with 80 aqueous methanol (three times)

Concentration under reduced pressure

Fractionation with hexane

Methanol extract

Hexane-insoluble fraction

Chloroform fraction Chloroform-insoluble fraction

Ethyl acetate-soluble fraction Water fraction

Phenolic flavonoid flavonol antioxidant and cytotoxicity assays

Fractionation with chloroform

Washed dried and ground

Phaleria macrocarpa (Scheff) Boerl seeds

Partition (vv) ethyl acetate-water (ratio 1 1)

Hexane-soluble fraction

Figure 1 Extraction procedure and its fractionations of P macrocarpa (Scheff) Boerl seeds

The ethanol extract of P macrocarpa seeds exhibitedtoxicity towards T47D breast cancer cell lines (LC

501512 plusmn

321 120583gmL) through COX-2 inhibition [7] Additionally theethanol extract of the seeds and the fruitsrsquo flesh have beenshown to increase p53 gene expression but had no effect onBcl-2 gene expression Moreover the n-hexane extract of theseeds had a greater effect in increasing p53 gene expressionthan that of the flesh of the fruit but had no effect on Bcl-2 gene expression [8] The ethanol extracts of P macrocarpaseeds and fruits have been found to be nontoxic to humanmononuclear peripheral normal cells but were slightly toxicto Vero cell lines [9]

Although P macrocarpa has been used extensively inIndonesia there is limited scientific research available onthe biological properties of this plant in relation to itsmedicinal benefitsThe present research investigated the totalphenolic and flavonoid content and the antioxidative andcytotoxic activities of the crude and fractionated extracts ofP macrocarpa seeds

2 Material and Methods

21 Plant Materials Seeds of Phaleria macrocarpa (Scheff)Boerl were collected from Yogyakarta Indonesia in Decem-ber 2011 A voucher specimen (ID no KLU 47923) wasdeposited into a repository at the Institute of BiologicalSciences Faculty of Science University of Malaya MalaysiaSamples were washed and dried in an oven at approximately50∘CThe seeds were then ground into powder and stored inairtight containers

22 Preparation for Extraction The dried powder was mac-erated with 80 aqueous methanol and extracted for 72 h

before filtration (three times) The filtrate obtained wasconcentrated under reduced pressure (60 rpm at 37∘C) Thiscrude methanol extract was then fractionated initially withhexane followed by chloroform The chloroform insolublefraction was subjected to partition with ethyl acetate andwater (Figure 1) The methanol extract and its fractions(hexane chloroform ethyl acetate and water) were refilteredand evaporated at low pressure (60 rpm at 37∘C) to removeexcess solvent

23 Phytochemical Screening Analysis In order to classify thetypes of organic constituents present in the plant samplespreliminary phytochemical screening tests were carried outon the plant samples according to the qualitative and quanti-tative methods of Trease and Evans [10] and Sofowora [11]The organic constituents that were investigated were thoselisted in Table 2 120572-Amino acids carbohydrates cyanogenicglycosides organic acids reducing sugars saponin glyco-sides and starch content determination were carried outon water extracts Alkaloids tests were used with 1 HClextract Determination of flavonoids glycosides phenoliccompounds and tannins were performed on the ethanolextract Test for steroids was performed on the petroleum -ether extract

24 Physiochemical Determination The nutritional valuesof the P macrocarpa seeds including moisture ash fiberprotein fat carbohydrate contents and energy value weredetermined by using the methods of AOAC [12] Trease andEvans [10] and Harbone [13]

25 Determination of Moisture Content The moisture con-tent of the dried powder samples was determined by using

BioMed Research International 3

the oven drying method [14 15] A clean dried crucible wasweighed and 10 g of the driedPmacrocarpa seeds powderwasplaced in a beaker The sample was dried in an electric ovenat 105∘C until all the moisture was removed from the sampleand constant weight was achieved The crucible containingthe dried sample was weighed again and the loss of weightwas recorded as the moisture content of the dried powderedP macrocarpa seeds The experiment was repeated threetimes The moisture content () was calculated by using thefollowing equation

Mositure percentage () =1198822minus1198821

1198820

times 100 (1)

where1198821= weight of sample after drying (g)119882

2= weight of

sample before drying (g) and1198820= weight of the sample (g)

26 Determination of Ash Content Ash content was deter-mined by using the method of AOCS [12] Briefly theash value of the samples represented the inorganic residuewhen the organic matter has been burnt away An accuratelyweighted amount (10 g) of the sample was placed in a pre-heated cooled and weighed porcelain crucible The cruciblewas heated carefully on a hot plate until the organic matterwas dried and burnt off without flaming and finally heated ina furnace at 550 plusmn 50∘C

The percentage of ash content was calculated using thefollowing formula

Percentage of Ash () =Weight of Ash

Weight of sampletimes 100 (2)

27 Determination of Crude Fiber Content The crude fibercontent was determined by using themethod ofAOAC [16]Dried powdered sample was weighed (10 g) and extractedwith petroleum ether (100mL) three times The extractedsample was air-dried and transferred to a round-bottomedflask In the flask 30mL of sulphuric acid (01275M) wasadded followed by 170mL of hot sulphuric acidThe solutionwas then refluxed for approximately 30 minutes and filteredthrough a Buchner funnel The insoluble matter was washedwith boiling water until the final filtrate was free from acid

The residue was placed back into the flask with 30mLof sodium hydroxide (0313M) and 170mL of hot sodiumhydroxide (0313M) was then added The mixture was againrefluxed for about 30minutes and filtered using sintered glassThe residue was washed with 1 HCl and then washed againwith boilingwater until therewas no acid presentThe residuewas finally washed with ethanol and ether and dried in anoven at 100∘C and thenweighedThis procedure was repeateduntil the fiber content was constant The fiber content wascalculated using the following equation

Percentage of Crude fiber () =Loss in weight

Weight of sampletimes 100

(3)

28 Determination of Carbohydrate Content Carbohydratecontent was calculated by multiplying the reducing sugar

content The reducing sugar content was determined usingthe Fehlingrsquos reducing method of Lane and Eynonrsquos [16]Weighted sample (10 g) was placed into a 250mL round-bottomed flask and 20mL of sulphuric acid (05M) wasadded Reflux was then performed in a sand bath for 25hoursThe residue was washed after filtration with warm dis-tilled water The solution was then neutralized with sodiumcarbonate power and the mixturersquos volume was made up to100mL with distilled water This was followed by titrationwith Fehlingrsquos solution equal amounts of solution A (coppersulphate solution) and solution B (sodium potassium tartrateand sodium hydroxide solution) using methylene blue asindicator The mixed Fehlingrsquos solution (5mL) was pipettedinto a conical flask and distilled water (5mL) was addedThesolution was then boiled for 15 seconds Methylene blue indi-cator (a few drops) was then titrated with the solution untilthe colour changed from blue to green The carbohydratecontent was then calculated according to following equation

Percentage of carbohydrate content ()

=5 times 0005 times 100 times 100 times 100

119881 times 10 times119882times 09

(4)

where 119881 = volume of sample solution (titration volume) and119882 = weight of powdered sample

29 Determination of Fat Content Fat content of P macro-carpa seeds was determined using the soxhlet extractionmethod [12] A seed sample (10 g) was placed in a soxhletextractor Petroleum ether was then poured into the extractorand extraction was performed for 12 hoursThe volume of thepetroleum ether extract was reduced to 15mL by evaporationThis was then dried at 105∘C in an oven until constant weightwas achievedThe fat content was then calculated by using thefollowing equation

of Fat content

=Weight of fat obtained from sample times 100

Weight of sample

(5)

210 Determination of Protein Content Protein content wasdetermined using the AOAC method [12] Briefly pow-dered samples (1 g) 50mL of distilled water 5 g of coppersulphate and 15mL of concentrated sulphuric acid wereadded to a Kjeldahl flask The flask was partially closed bymeans of a funnel and the content was digested by heatingthe flask at an inclined position in the digester The mixturewas heated for 30minutes until there was 40mL of clear 01Mstandard sulphuric A few drops of methyl red indicator weremixed into the clear solutionThe flask was then placed belowthe condenser and the end of the adapter tube was dippedin the acid The flasks were set up for Kjeldahl distillationand 70mL of 40 sodium hydroxide was added through thefunnel The funnel was washed twice with 50mL of distilledwater Distillation was then performed for one hour Thedistilled ammonia was then nitrated with 01M standardsolution until the color changed from yellow to colorlessTheexperiment was repeated three times The nitrogen content

4 BioMed Research International

and protein content in the sample were calculated using thefollowing relation

Percentage of Nitrogen ()

=(119881119904minus 119881119887) times 119872

119860times 00140 times 100

Weight of sample (119882)

Protein content = percentage of Nitrogen times 625

(6)

where 119881119904= volume in cm3 of standard acid used in the

titration of sample119881119887= volume in cm3 of standard acid used

in the blank titration119872119860=morality of standard acid solution

in mol dmminus3 and119882 = weight of the sample in grams

211 Determination of Phenolic Content The total phenoliccontent of the crude methanol and the fractionated extracts(hexane chloroform ethyl acetate and water) was deter-mined using the Folin-Ciocalteu method [17ndash20] Briefly200120583L of each extract solution of different concentrationswas mixed with 1mL of Folin-Ciocalteu reagent (1 10 dilutedwith H

2O) and 800 120583L of Na

2CO3(7505 gL) The mixture

was thoroughly shaken for 15 minutes and then held ina water bath at a temperature of 37∘C The solution wasallowed to stand for 1 hour at room temperature in a darkplace and the absorption was measured at 750 nm using aspectrophotometer Distilled water was used as blank andgallic acid (0ndash250mgL) was used to construct a standardcalibration curve Gallic acid concentration was establishedfrom the calibration curve 119910 = 00221119909 + 02189 1198772 =09914

212 Determination of Flavonoid Content Total flavonoidcontent of P macrocarpa seeds was measured using themethods of Ebrahimzadeh Nabavi and Ordonez [20ndash22]with minor modifications To determine the flavonoid con-tent 1mL of each sample was added to 01mL of 10Al (NO

3)3solution 01mL of 1M potassium acetate and

38mLofmethanolThe solutionwas thoroughlymixed usinga vortex mixer for two to three minutes and then stooduntouched for 10 minutes at room temperature Absorbancewas determined at 415 nm using a spectrophotometer Thetotal content of flavonoids was measured and expressed asquercetin equivalent on a dry weight basis (119910 = 00855119909+02004 1198772 = 09813)

213 Determination of Flavonol Content The method ofKumaran [23] and Mbaebie [24] with slight modificationswas used to measure the total flavonol content of themethanol extract Briefly 1mL of extract was added to a cen-trifuge tube with 2mL of prepared AlCl

3in ethanol and 3mL

of sodium acetate (50 gL) solution The mixture was stirredthoroughly with a vortex and was then incubated for 1 hourThe absorbance was measured with a spectrophotometer at440 nm A calibration curve was constructed using quercetin(1 5 10 15 and 20mgmL) The total flavonol content wascalculated using the calibration curve with the followingequation119910 = 00353119909 + 01456 1198772 = 09807

214 Antioxidant DPPH Assay Screenings of antioxidantactivity of the crude methanol extract chloroform hexaneethyl acetate and water fractions of P macrocarpa seeds werecarried out by determination ofDPPH free radical scavengingproperty using UV spectrophotometric methods [21 25]Based on this protocol 50120583L of test solutions from differentdry extracts and concentrations (1 5 10 15 and 20mgmL)was dissolved in water This solution was then combinedwith 195mL of DPPHmethanol solution After being mixedsolutions were kept at room temperature in the dark for 30minutes After the reaction the increase in absorbance wasrecorded at 517 nm Methanol was used as a blank DPPHsolution was used as negative control (119860

0) and gallic acid

was used as positive control The antioxidant activity wasexpressed as an IC

50value All experiments were carried out

in triplicate The scavenging effect was obtained from thefollowing relation

Scavenging effect () =(1198600minus 1198601) times 100

1198600

(7)

where 1198600was the absorbance of the control reaction and

1198601was the absorbance of the sample of the tested extracts

Gallic acid was used as standard Percentage of inhibitionwas calculated using the following formula inhibition =[(119860negative minus 119860 test)119860negative] times 100 (119860 is absorbance)

215 Cytotoxicity Screening

2151 Cell Culture andCultureMedium Human cervical car-cinoma cells (Ca Ski) hormone-dependent breast carcinomacells (MCF-7) human breast adenocarcinoma cells (MDA-MB231) human ovarian carcinoma cells (SKOV-3) humancolon carcinoma cells (HT-29) and noncancer humanfibrob-last cells (MRC-5) were purchased from the American TissueCulture Collection (ATCC USA)

HT 29 Ca Ski andMCF-7 cells weremaintained in RPMI1640 medium (Sigma) MDA-MB231 and SKOV-3 cells inDulbeccorsquos Modified Eaglersquos medium (DMEM Sigma) andMRC-5 cells in Eaglersquos Minimum Essential medium (EMEMSigma) supplemented with 10 fetal bovine serum (FBSPAA Lab Austria) 100 120583gmL penicillin or streptomycin(PAA Lab Austria) and 50 120583gmL kanamycinamphotericinB (PAA Lab Austria) The cells were cultured in a CO

2

incubator (5) and kept at 37∘C in a humidified atmosphereThe cultures were subcultured every 2-3 days and checkedfrequently under an inverted microscope (Leica Germany)for any contamination

2152 MTT Cell Proliferation Assay MTT [3-(4 5-dimeth-ylthiazol2yl)-2 5-diphenyl tetrazolium bromide)] assay wasperformed on the cultured cells in 96-well plates according tothe method of Mosmann [26 27] Briefly cells were culturedto confluence They were then centrifuged at 1000 rpm for 5minutes and resuspendedwith 10mLof growthmediumThedensity of the viable cells was counted using 04 trypan blueexclusion dye in a haemocytometer with a microscope Thecells were then seeded into microtiter plates and incubated ina CO2incubator at 37∘C for 24 hrs After reaching 70ndash80

BioMed Research International 5

Table 1 Yield of methanol extract and its fractions from P macro-carpa of seedsExtract and fractions Yield (g) Percentage of yield ()Methanolic extract (ME) 1987 1199Hexane fraction (HF) 1587 947Chloroform fraction (CF) 72 043Ethyl acetate fraction (EAF) 62 037Water fraction (WF) Freeze dry

confluence each extract at concentrations of 1 10 25 50and 100 120583gmL (in 200120583L of 10 media) was added to therespective wells containing the cells Wells with untreatedcells were used as the negative control and cells exposedto doxorubicin were used as positive control After 24 48and 72 hours 10 120583L MTT stock solution was added to eachwell OD was then determined by measuring absorbance at540 nm using an ELISA microplate reader The percentageof inhibition () was calculated according to the followingformula

Percentage of inhibition ()

=OD control minusOD sample times 100

OD control

(8)

Cytotoxicity of each sample is expressed as plusmnIC50value The

extract that gave IC50

of 30 120583gmL or less was consideredactive [26]

216 Gas Chromatography-Mass Spectrometry (GC-MS) GC-MS was used to determine the molecular weight of thecomponents collected and the purity of the collected extractsRunning conditions were as follows oven temperature wasprogrammed with an initial temperature of 100∘C andincreased at a ramp rate of 5∘Cmin and reached a finaltemperature of 300∘C The carrier gas or mobile phase usedwas helium and a flow rate of 1mLmin was programmedThe mass spectrometry mode used was electron ionization(EI) mode with a current of 70 eV The injection mode wasprogrammed with a sample injection volume of 1120583L with asplit mode at a ratio of 1 20 The injection port temperaturewas set to 230∘C and the detectorinterface temperature wasset to 250∘C The results were collected for 40 minutes Thetotal ion chromatogram obtained was autointegrated usingChemStation and the chemical compounds or componentswere analysed by comparison with the suppliedmass spectraldatabase (NIST 05 Mass Spectral Library USA)

217 Statistical Analysis All data for each test are the averageof triplicate experiments for comparison of values and wererecorded as the mean plusmn standard deviation using MicrosoftExcel software and statistical data analyses were performedusing SPSS software

3 Results

31 Extraction Theextracts were concentrated using a rotaryevaporator (Buchi USA) under reduced pressure at 35∘CThe

yield of extracts from P macrocarpa seeds is shown in Table 1The highest yield from P macrocarpa seeds was the hexanefraction (947)

32 Preliminary Phytochemical Studies In order to determinethe types of phytoorganic constituents present in P macro-carpa seeds a preliminary phytochemical investigation wascarried out according to conventional methods The resultsobtained from these experiments are summarized in Table 2

The phytochemical tests showed that there were sec-ondary metabolites including carbohydrate flavonoids gly-cosides saponin glycosides phenolic compounds steroidstannins and terpenoids present in different extracts ofP macrocarpa seeds Small amounts of alkaloids 120572-aminoacids cyanogenic glycosides organic acids reducing sugarsand starches were also found to be present

The main constituents such as flavonoids glycosidessaponin glycosides phenolic compounds steroids tanninsand terpenoids present in P macrocarpa seeds may con-tribute to the presence of bioactivities such as antibacterialan analgesic an antifungal an anti-inflammatory agentand cytotoxicity Moreover the toxic chemical constituentscyanogenic glycosides were also present in the seeds

33 Physicochemical Studies The determination of nutri-tional values such as moisture ash fiber protein fat andcarbohydrate contents was carried out using the AOACmethod as well as the Lane and Eynon titration method andthe results obtained are shown in Figure 2(a) and Table 3Different physiochemical parameters for the purpose ofstandardization such as moisture (631 plusmn 143) ash (296 plusmn186) protein (2073 plusmn 244) crude fiber (2276 plusmn 279)crude fat (184 plusmn 311) and carbohydrate (2934 plusmn 198)were determined

34 Flavonoid Flavonol and Phenolic Determinations It wasfound that the methanol extract had the highest flavonoidcontent (933 plusmn 08mgmL) followed by ethyl acetate waterchloroform and hexane fractions which were 838 plusmn 10808 plusmn 03 678 plusmn 11 and 418 plusmn 15mgmL respectively

Themethanol extract (893plusmn11mgmL) of Pmacrocarpaseeds exhibited the highest amount of total flavonol contentfollowed by water ethyl acetate chloroform and hexanefractions which were 893 plusmn 10 613 plusmn 05 429 plusmn 09 and429 plusmn 07mgmL respectively

The total phenolic content of the P macrocarpa seedsextracts and fractions was expressed as gallic acid equiva-lents The methanol extract of P macrocarpa seeds exhibitedthe highest amount (720 plusmn 07mgmL) of total phenolicsfollowed by water ethyl acetate chloroform and hexanefractions which were 512 plusmn 14 358 plusmn 11 326 plusmn 10 and263 plusmn 01mgmL respectively

These data are shown in Figure 2(b) and Table 4

35 DPPH (2 2-Diphenyl-1-picryl-hydazyl) AntioxidantAssay DPPH assay was used to determine the free radicalscavenging ability of extracts and fractions of P macrocarpaseeds and to determine the antioxidant activity of its

6 BioMed Research International

Table 2 Results of chemical constituents of P macrocarpa seeds

No Tests Reagents Observation

1 AlkaloidsWagnerrsquos reagentMayerrsquos reagentDragendorff rsquos reagentSodium picrate solution

++++

2 120572-Amino acids Ninhydrin reagent +3 Carbohydrates 10 120572-naphthol and conc H2SO4 ++4 Cyanogenic glycosides Sodium picrate solution +5 Flavonoids Mg and conc HCl ++6 Glycosides 10 lead acetate ++7 Organic acids Bromothymol blue +8 Phenolic compounds 1 FeCl3 ++

9 Reducing sugars Fehlingrsquos solutionsA and B +

10 Saponin glycosides Distilled water ++11 Starch Iodine solution +12 Steroids Acetic anhydride and conc H2SO4 ++13 Tannins 1 Gelatin ++14 Terpenoids Acetic anhydride and conc H2SO4 +++++ large amount ++ medium amount + small amount minus absent + present

0

5

10

15

20

25

30

35

Con

tent

()

Moi

sture

Ash

Prot

ein

Crud

e fibe

r

Carb

ohyd

rate

Crud

e fat

(a)

123456789

1011

ME HF CF EAF WF

FlavonoidsFlavonolPhenolic

Con

cent

ratio

n (m

gm

L)

(b)

Figure 2 (a)The percentage ofmoisture ash protein crude fiber crude fat and carbohydrate contents on PmacrocarpaBoerl seeds (b) Totalamount of concentration of flavonoid flavonol and phenolic contents of P macrocarpa seeds ME methanol extract HF hexane fractionEAF ethyl acetate fraction CF chloroform fractionWF water fraction

phytoconstituents It is important to note that a lower IC50

value equals a higher scavenging activity The scavengingactivity was presented as the percentage of inhibitionof DPPH free radicals (Figure 3 and Table 4) Based onIC50

values the samples can be ranked in the followingdescending order water fraction gt methanol extract gt ethylacetate fraction gt hexane fraction gt chloroform fractionTheresults revealed that the water fraction was most active forantioxidant activity compared to other fractions The radical

scavenging effect was found to increase with increasingconcentrations

36 Cytotoxicity Screening of MTT Cell Proliferation AssayThe methanol and fractionated extracts (hexane chloro-form ethyl acetate and water) were investigated for cyto-toxic effects in human cervical adenocarcinoma cells (CaSki) humanhormone-dependent breast carcinoma (MCF-7)

BioMed Research International 7

Table 3The percentage of moisture ash protein crude fiber crudefat and carbohydrate contents and the amount of nutrition valueson P macrocarpa seeds

Tests parameter Percentage of contents ()Moisture 631 plusmn 143Ash 296 plusmn 186Protein 2073 plusmn 244Crude fiber 2276 plusmn 279Crude fat 1840 plusmn 311Carbohydrate 2934 plusmn 198

0102030405060708090

100

1 5 10 15 20

ME

HF

CF

EAF

WF

Concentration (mgmL)

Inhi

bitio

n (

)

Figure 3 Percentage of inhibition of DPPH free radical scavengingof P macrocarpa seeds ME methanol extract HF hexane fractionEAF ethyl acetate fraction CF chloroform fractionWF waterfraction

human colon adenocarcinoma cells (HT-29) human ovar-ian carcinoma cells (SKOV-3) human hormone-dependentbreast carcinoma (MDA-MB231) and normal cells (MRC-5) using MTT cells proliferation assay at 24 hrs 48 hrs and72 hrs respectively

The methanol extract of P macrocarpa seeds showedexcellent cytotoxic effects with IC

50value of 82plusmn 466 120583gmL

in Ca Ski cells at 24 hrs and 120 plusmn 22 85 plusmn 168 and30 plusmn 250 120583gmL in MCF-7 cells at 24 hrs 48 hrs and72 hrs respectively The methanol extract also showed goodcytotoxic activity in HT-29 cells with IC

50values of 293 plusmn

226 250 plusmn 135 and 215 plusmn 330 120583gmL at 24 hrs 48 hrsand 72 hrs respectively in Ca Ski cells with IC

50values of

197 plusmn 092 120583gmL and in SKOV-3 cells with IC50

values of165 plusmn 252 221 plusmn 247 and 360 plusmn 355 120583gmL at 24 hrs48 hrs and 72 hrs respectively However this extract had nocytotoxic effect in MDA-MB231 cells with IC

50gt100120583gmL

and the extract also had low cytotoxic effect in the MRC-5cells with IC

50gt 50 120583gmL

The hexane fraction of P macrocarpa seeds showedmoderate cytotoxic effects with IC

50values of 452 plusmn 149

and 555 plusmn 197 120583gmL at 24 hrs and 48 hrs on MCF-7 400 plusmn315 120583gmL inHT-29 cells at 24 hrs and 405plusmn352 and 500plusmn302 120583gmL in SKOV-3 at 24 hrs and 48 hrs In addition the

hexane fraction also displayed low cytotoxic effect in MCF-7at 72 hrs HT-29 at 48 hrs and 72 hrs and in SKOV-3 at 72 hrsThe IC

50value of treatment with hexane fraction in MCF-7

was 725plusmn152 120583gmL for 72 hrs exposure IC50values in HT-

29 were 640plusmn203 and 750plusmn314 120583gmL at 48 hrs and 72 hrswhile IC

50value in SKOV-3 was 703 plusmn 353 120583gmL at 72 hrs

In contrast the hexane fraction exhibited no cytotoxic effectsin MDA-MB231 cells or MRC-5 cells

The chloroform fraction of P macrocarpa seeds exhibitedthe highest cytotoxic effect with IC

50value of 100plusmn131 82plusmn

104 and 220 plusmn 186 120583gmL at 24 hrs 48 hrs and 72 hrs in CaSki cells 95plusmn295 87plusmn159 and 210plusmn198 120583gmL at 24 hrs48 hrs and 72 hrs on HT29 and 248 plusmn 206 165 plusmn 321 and900 plusmn 26 120583gmL in SKOV-3 cells The chloroform fractionexhibitedmoderate cytotoxic effect inMCF-7 with IC

50value

of 575 plusmn 264 400 plusmn 148 and 465 plusmn 345 120583gmL for 24 hrs48 hrs and 72 hrs respectively In contrast the chloroformfraction had no cytotoxic effect against theMDA-MB231 cellsand normal MRC-5 cells with IC

50gt100 120583gmL

The ethyl acetate fraction ofPmacrocarpa seeds exhibitedthe highest cytotoxic effect with IC

50lt 25 120583gmL in SKOV-3

cellsMDA-MB231 cellsMCF-7 cells andCa Ski cells On theother hand the ethyl acetate fraction exhibited low cytotoxiceffect in MRC-5 normal cells with IC

50value of 35 120583gmL

The ethyl acetate fraction exhibited the highest cytotoxiceffect in all selected cells (Ca Ski MCF-7 HT-29 and MDA-MB231) The water extract showed no cytotoxic effect againstall selected cancer cell lines with IC

50gt 100 120583gmL and

exhibited a mild cytotoxic effects against MRC-5 cells Thecorresponding data is shown in Figure 4 and Table 5

37 Characterization and Identification of Hexane and Chlo-roform Fractions Six compounds were identified from thehexane fraction of P macrocarpa seeds using GC-MS Theywere methyl stearate oleic acid methyl oleate linoleic acidmethyl linolenate and palmitic acid Moreover a GCMSanalysis of the chloroform fraction of P macrocarpa seedsshowed the presence of methyl myristate palmitic acidmethyl oleate methyl linoleate oleic acid and (119911)- and917-octadecadienal (119911) A comparison with the NIST massspectral library (NIST 05MS library 2002) andAdams (2001)confirmed the identity of the compounds The chemicalstructures of the identified compounds in the hexane andchloroform fractions of P macrocarpa seeds are as shown inFigure 5

4 Discussion

A longer shelf life can be achieved by reducing moisturecontent Thus moisture content is a critical factor for thestability of an extractMoisture enhances fungal and bacterialgrowth therefore decreasing the longevity of the extract Thetime it takes for plant material to deteriorate depends on howmuch water the plant material contains

Carbohydrates play a vital role in the immune sys-tem fertilization pathogenesis blood clotting and humandevelopment Foods that contain carbohydrates can raiseblood glucose and the three main types of carbohydrate are

8 BioMed Research International

0102030405060708090

100

ME

HF

CF

EAF

24h 48h 72h

IC50

valu

e of c

once

ntra

tion

(120583g

mL)

Ca Ski

0102030405060708090

100

ME

HF

CF

EAF

24h 48h 72h

IC50

valu

e of c

once

ntra

tion

(120583g

mL)

MCF 7

0102030405060708090

100

ME

HF

CF

EAF

24h 48h 72h

IC50

valu

e of c

once

ntra

tion

(120583g

mL)

HT-29

minus10

0

10

20

30

40

50

60

70

80

90

100

ME

HF

CF

EAF

24h 48h 72h

IC50

valu

e of c

once

ntra

tion

(120583g

mL)

SKOV-3

0102030405060708090

100

ME

HF

CF

EAF

24h 48h 72h

IC50

valu

e of c

once

ntra

tion

(120583g

mL)

MDA-MB231

Figure 4 In vitro cytotoxic effects of P macrocarpa seedson Ca Ski MCF-7 HT-29 SKOV-3 and MDA-MB231 cell lines ME methanolextract HF hexane fraction EAF ethyl acetate fraction CF chloroform fractionWF water fraction Each value is expressed as mean plusmnstandard deviation of three measurements

BioMed Research International 9

Methyl linoleate

Methyl stearate

Palmitic acid

Oleic acid

Methyl myristate

Linoleic acid

Methyl oleate

H3C

H3C

H3C

H3C

H3C

H3C

H3C

O

O

O

O

O

O

O

O

O

O

O

CH3

CH3

CH3

CH3

OH

OH

OH

(294mz)

(298mz)

(282mz)

(242mz)

(280mz)

(298mz)

(256mz)

Figure 5

10 BioMed Research International

Table 4 Total flavonoid total flavonol total phenolic contents and result of DPPH free radical scavenging property of P macrocarpa seeds

Tests ME (mgmL) HF (mgmL) CF (mgmL) EAF (mgmL) WF (mgmL)Flavonoid 933 plusmn 08 418 plusmn 15 678 plusmn 11 838 plusmn 03 808 plusmn 10Flavonol 893 plusmn 11 425 plusmn 07 429 plusmn 09 613 plusmn 05 818 plusmn 10Phenolic 720 plusmn 07 263 plusmn 01 326 plusmn 1 358 plusmn 11 512 plusmn 14DPPH 1150 plusmn 003 1500 plusmn 004 1575 plusmn 004 1400 plusmn 005 975 plusmn 003ME methanol extract HF hexane fraction EAF ethyl acetate fraction CF chloroform fraction WF water fraction

Table 5 In vitro cytotoxic effects of methanol extract and its fractions of P macrocarpa cancer cells lines and normal human fibroblast breastcells line

Cell line Incubation periods (hours) ME HF CF EAF Doxorubicin24 82 plusmn 466 ge100 100 plusmn 131 56 plusmn 117 092 plusmn 064

Ca Ski 48 197 plusmn 092 ge100 82 plusmn 104 77 plusmn 185 069 plusmn 10572 407 plusmn 226 ge100 220 plusmn 186 60 plusmn 322 045 plusmn 09924 120 plusmn 22 452 plusmn 149 575 plusmn 264 223 plusmn 158 105 plusmn 108

MCF7 48 85 plusmn 168 555 plusmn 197 400 plusmn 148 168 plusmn 170 012 plusmn 06972 63 plusmn 250 725 plusmn 152 465 plusmn 345 84 plusmn 171 092 plusmn 17024 293 plusmn 226 400 plusmn 315 95 plusmn 295 11 plusmn 120 092 plusmn 072

HT29 48 250 plusmn 135 640 plusmn 203 87 plusmn 159 35 plusmn 200 032 plusmn 21672 215 plusmn 330 750 plusmn 314 210 plusmn 198 12 plusmn 228 088 plusmn 09424 165 plusmn 252 405 plusmn 352 248 plusmn 206 68 plusmn 18 102 plusmn 179

SKOV-3 48 221 plusmn 247 500 plusmn 302 165 plusmn 321 65 plusmn 23 032 plusmn 09772 360 plusmn 355 703 plusmn 353 90 plusmn 26 32 plusmn 281 062 plusmn 09824 ge100 ge100 ge100 65 plusmn 23 043 plusmn 050

MDA-MB231 48 ge100 ge100 ge100 15 plusmn 343 059 plusmn 10172 ge100 ge100 ge100 186 plusmn 126 095 plusmn 132

ME methanol extract HF hexane fraction EAF ethyl acetate fraction CF chloroform fractionWF water fraction Doxorubicin was used as positive controlEach value is expressed as mean plusmn standard deviation of three measurements

starches sugars and fiber A macronutrient protein consistsof amino acids which is required for proper growth andhuman body function The seeds were found to containa high level of crude fiber which is a potential source ofphenolic antioxidants The major components of fibers arecellulose hemicelluloses lignin 120573-glucans gums and pectinand hydrocolloids These components can behave as proan-tioxidants [28 29] In our present physicochemical study Pmacrocarpa seeds were mainly made up of carbohydratesfollowed by crude fiber proteins fatmoisture and ashHigh-protein and low-carbohydrate diets are often effective forweight loss Dietary fiber can aid in digestion is helpfulfor weight management and reduces constipation Howevermany high-protein and low-carbohydrate foods are low infiberWomen aged 50 and older need at least 21 grams of fiberdaily whilst those aged 19 to 50 require 25 grams men aged50 and older need 30 grams and those between the age of 19to 50 should consume at least 38 grams of fiber

Steroids possess biological activities which are insec-ticidal cardiotonic and antimicrobial activities and theseare potentially useful for development into therapeuticdrugs Tannins are known to be important for their antivi-ral antibacterial antiparasitic effects anti-inflammatoryantiulcer and antioxidant properties [30ndash33] Saponinshave been found to have antimicrobial anti-inflammatoryantifeedant and hemolytic effects [34 35] Some alkaloids

have been reported to have anticancer and antiviral activityand saponins have been reported to be cardiotonic whileflavonoids have anticancer and anti-inflammatory activity[10 36] The presence of tannins may be responsible for theability of P macrocarpa seeds to be used in the treatmentof diseases such as diabetes diarrhea and dysentery Ourpreliminary phytochemical studies revealed that the chemicalcomponents of P macrocarpa seeds also include flavonoidsphenolics steroids tannins terpenoids glucosides saponinsand carbohydrates

Previous researchers reported that kaempferol myrice-tin naringin quercetin rutin [3] 29-norcucurbitacinderivatives fevicordin A fevicordin A glucoside fevicordinD glucoside [6] mahkoside A dodecanoic acid palmiticacid des-acetyl flavicordin-A flavicordin-A flavicordin-Dflavicordin-A glucoside ethyl stearate lignans and sucrose[37 38] mangiferin (a C-glucosylxantone) kaempferol-3-o-120573-D-glucoside dodecanoic acid palmitic acid ethyl stearatesucrose [5] pinoresinol lariciresinol matairesinol alkaloidsand saponins [37 39] saponins alkaloids polyphenolicsphenols flavanoids lignans tannins [40ndash42] icarisideC3 mangiferin gallic acid phalerin glycoside (345trihydroxy-4-methoxy-benzophenone-3-O-120573-D-glucoside)[43] and 2410158406 trihydroxy-4-methoxy-benzophenone-3-O-120573-D-glucoside [44] were isolated from different partsof P macrocarpa The hexane extract of the seeds was

BioMed Research International 11

found to contain methyl myristate methyl stearate oleicacid methyl oleate linoleic acid methyl linoleate palmiticacid methyl palmitate 6-octadecenoic acid and 917-octadecadial 120573-sitosterol as major components A further30 to 40 of components were not identified as thesewere polar compounds that require HPLC analysis Othercompounds reported in the literature for the seeds leavesand fruits were not identified in this investigation It isprobable that the components reported in the literaturemay be present in other fractions (methyl acetate CHCl

3

and water fractions)Phenolics are able to transfer protons to radicals [45]

DPPH which provides free radicals is normally a blue-violet color However the DPPH turned yellow after it wasconverted to 1 1-diphenyl-2-picrylhydrazine which has lessfree-radical activity Hendra et al reported that the IC

50

values of free radical and reducing power in methanolwith HCl of P macrocarpa seeds were 2450 plusmn 194 and1502 plusmn 128 [42] In our investigation using DPPH assay thewater fraction displayed the highest IC

50value followed by

methanol ethyl acetate hexane and chloroform fractionThe presence of flavonoids in P macrocarpa seeds may be

responsible for the traditional use of the plants in treatingcancer inflammations and allergies Recently it has beenshown that phenolic compounds in crude methanol andethyl acetate extract of P macrocarpa leaves displayed goodantioxidant and antimicrobial activites [46] Hendra et al(2011) examined the amount of total phenolic (477plusmn 104mggallic acid equivalentg DW) and the amount of flavonoid(359 plusmn 247mg rutin equivalentg DW) contents in themethanol extract with HCl of P macrocarpa seeds [42] Inthis study the total flavonoids content was higher than that inthe results previously reported by Rohyami [47] and phenolflavonol and flavonoid contents were the highest in theaqueous methanol extract followed by ethyl acetate waterchloroform and hexane extracts

MTT assay is a sensitive and reliable colorimetric assaythat uses quantitative measurements to calculate the via-bility proliferation and activation of cells This method iscommonly applied to screen anticancer agents [48] Themost well-known methods used to calculate cytotoxicityare the neutral red (NR) uptake and dimethylthiazole-diphenyl tetrazolium bromide (MTT) metabolism [27 49]Earlier investigations showed that the ethanol extract of Pmacrocarpa seeds and fruit meat were not toxic to normalhuman cells but slightly toxic to a Vero cell line [9] Theethanol extract of P macrocarpa seed fruit also showedtoxicity towards T47D breast cancer cell line through COX-2 expression inhibition [7] Desacetylfevicordin A has beenisolated from the ethyl acetate extract of P macrocarpaseeds and this compound displayed excellent cytotoxicityin brine shrimp [43] The ethyl acetate of P macrocarpaseeds exhibited mild cytotoxic effect against HepG2 cells(IC50values between 30 and 60 120583gmL) [46] Previous studies

showed that the cytotoxic activity of the seeds on HT-29MCF-7 Hela and Chang liver cells lines were 384 plusmn 037255 plusmn 137 295 plusmn 10 and 678 plusmn 027 receptively [42] Inour study the methanolic extract of P macrocarpa seeds wasfound to pose cytotoxic effect against HT-29 MCF-7 Cas Ki

and SKOV-3 cell lines (IC50

values giving from 11 plusmn 120to 360 plusmn 355) and mild toxicity on normal cell lines Therewere significant cytotoxic effects on selected cancer cells linesthat were both time- and dose-dependent manner due to thepresence of many secondary metabolites

The phytochemical screening also indicated the presenceof a small amount of cyanogenic glycosides Cyanogenicglucosides which can cause acute cyanide poisoning causerapid respiration and pulse decrease the blood pressure andinduce vomiting diarrhea headache dizziness and so onThus these components have to be removed (usually byboiling) prior to consumption for therapeutic purposes

5 Conclusion

P macrocarpa seeds exhibited antioxidant and cytotoxicactivities It is highly probable that these activities are dueto the presence of phenolic and flavonoid compounds inappreciable amounts in the plant Furthermore the cytotox-icity activity suggested that the seed may contain a potentialanticancer agent The outcome of this study is encouragingdemonstrating the potential for the P macrocarpa as a sourceof multiple therapeutic agents

Abbreviations

EAF Ethyl acetate fractionME Methanol extractHF Hexane fractionCF Chloroform fractionWF Water fractiong Grammg MilligrammL Milliliter

Conflict of Interests

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

Authorsrsquo Contribution

Ma Ma Lay was involved in all experiments and BehroozBanisalamwas involved in antioxidant assay All authors readand approved the submitted version of the paper

Acknowledgments

This work was supported by grants from the MalaysianMinistry of Higher Education (nos PS2862009C andPS4702010B) Ma Ma Lay is a scholarship recipient fromthe ldquoIslamic Development Bank Saudi Arabiardquo The authorswould like to thank Professor Dr Amru BinNasrulhaq Boycefor generously allowing the use of his laboratory for thisresearch

12 BioMed Research International

References

[1] N HarmantoMahkota DewaObat Pusaka ParaDewa Cetakankeempat Agromedia Pustaka Jakarta Indonesia 2002

[2] A Jemal R Siegel E Ward T Murray J Xu and M J ThunldquoCancer statistics 2007rdquoCa ACancer Journal for Clinicians vol57 no 1 pp 43ndash66 2007

[3] R Hendra S Ahmad A Sukari M Y Shukor and EOskoueian ldquoFlavonoid analyses and antimicrobial activity ofvarious parts of Phaleria macrocarpa (Scheff) Boerl fruitrdquoInternational Journal of Molecular Sciences vol 12 no 6 pp3422ndash3431 2011

[4] W Wijayani Sitotoksisitas Minyak Atsiri Biji Dan Daging BuahMahkota Dewa (Phaleria Macrocarpa [Scheff] Boerl) TerhadapMyeloma Cell Line Dan HeLa Cell Line Serta IdentifikasiSenyawa Minyak Atsiri Dengan Kromatografi Gas-SpektrometriMassa (GC-MS) Skripsi FMIPA UGM Yogyakarta Indonesia2005

[5] Y-B Zhang X-J Xu and H-M Liu ldquoChemical con-stituents fromMahkotadewardquo Journal of Asian Natural ProductsResearch vol 8 no 1-2 pp 119ndash123 2006

[6] D Kurnia K Akiyama and H Hayashi ldquo29-Norcucurbitacinderivatives isolated from the Indonesian medicinal plant Pha-leria macrocarpa (Scheff) Boerlrdquo Bioscience Biotechnology andBiochemistry vol 72 no 2 pp 618ndash620 2008

[7] M Bakhriansyah Pengaruh Ekstrak Etanol Biji Mahkota Dewa(Phaleria Macrocarpa [Scheff] Boerl) Pada Sel Kanker Payu-dara T47D Kajian Aktivitas Sitotoksik Dan PenghambatanEkspresi Siklooksigenase-2 Program Pascasarjana UGM 2004

[8] L Wardani The Effect of Phaleria Macrocarpa [Scheff] Boerl)Seed and Flesh Fruit Extract to P53 and Bcl-2 Gene Expression inHela Cell Line FMIPA UGM 2005

[9] A Endang R Tri Joko and E Dewi ldquoCyototoxicity of Phaleriamacrocarpa (Scheff) boerl fruits flesh and seed extract ofethanol and its effect against p53 and Bcl-2 genes expressionof normalrdquo in Proceedings of the International Conference onComputational Science (ICCS rsquo07) 2007

[10] C Evans Trease and Evans Pharmacognosy Saunder 2002[11] A Sofowora Medicinal Plants and Traditional Medicine in

Africa Spectrum Books Ltd Ibadan Nigeria 2nd edition 1993[12] AOAC Official Methods of Analysis Association of Official

Analytical Chemists Arlington Tex USA 2002[13] J Harborne Phytochemical Methods Chapman and Hall Lon-

don UK 1973[14] AOAC Official Methods of Analysis Association of Official

Analytical Chemists Gaithersburg Md USA 17th edition2000

[15] J Lako V C Trenerry M Wahlqvist N WattanapenpaiboonS Sotheeswaran and R Premier ldquoPhytochemical flavonolscarotenoids and the antioxidant properties of a wide selection ofFijian fruit vegetables and other readily available foodsrdquo FoodChemistry vol 101 no 4 pp 1727ndash1741 2007

[16] W Horwitz Official Method of Analysis AOAC InterantionalGaithersburg Md USA 17th edition 2000

[17] A A Adedapo F O Jimoh S Koduru P J Masika and AJ Afolayan ldquoAssessment of the medicinal potentials of themethanol extracts of the leaves and stems of Buddleja salignardquoBMCComplementary andAlternativeMedicine vol 9 article 212009

[18] M Zovko Koncic D Kremer J Gruz et al ldquoAntioxidantand antimicrobial properties ofMoltkia petraea (Tratt) Griseb

flower leaf and stem infusionsrdquo Food and Chemical Toxicologyvol 48 no 6 pp 1537ndash1542 2010

[19] S McDonald P D Prenzler M Antolovich and K RobardsldquoPhenolic content and antioxidant activity of olive extractsrdquoFood Chemistry vol 73 no 1 pp 73ndash84 2001

[20] S M Nabavi M A Ebrahimzadeh S F Nabavi A Hamidiniaand A R Bekhradnia ldquoDetermination of antioxidant activityphenol and flavonoid content of Parrotia persicaMeyrdquo Pharma-cologyonline vol 2 pp 560ndash567 2008

[21] M A Ebrahimzadeh S F Nabavi and S M Nabavi ldquoAntiox-idant activities of methanol extract of Sambucus ebulus Lflowerrdquo Pakistan Journal of Biological Sciences vol 12 no 5 pp447ndash450 2009

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

[23] A Kumaran and R Joel Karunakaran ldquoIn vitro antioxidantactivities of methanol extracts of five Phyllanthus species fromIndiardquo Food Science and Technology vol 40 no 2 pp 344ndash3522007

[24] B O Mbaebie H O Edeoga and A J Afolayan ldquoPhy tochemical analysis and antioxidants activities of aqueous stembark extract of Schotia latifolia Jacqrdquo Asian Pacific Journal ofTropical Biomedicine vol 2 no 2 pp 118ndash124 2012

[25] L I Mensor F S Menezes G G Leitao et al ldquoScreening ofBrazilian plants extracts for antioxidants activity by the use ofDPPH free radical methodrdquo Phytotherapy Research vol 15 pp127ndash130 2001

[26] F Denizot and R Lang ldquoRapid colorimetric assay for cellgrowth and survival modifications to the tetrazolium dyeprocedure giving improved sensitivity and reliabilityrdquo Journalof Immunological Methods vol 89 no 2 pp 271ndash277 1986

[27] T Mosmann ldquoRapid colorimetric assay for cellular growth andsurvival application to proliferation and cytotoxicity assaysrdquoJournal of Immunological Methods vol 65 no 1-2 pp 55ndash631983

[28] P Maisuthisakul R Pongsawatmanit and M H GordonldquoAntioxidant properties of Teaw (Cratoxylum formosum Dyer)extract in soybean oil and emulsionsrdquo Journal of Agriculturaland Food Chemistry vol 54 no 7 pp 2719ndash2725 2006

[29] P Maisuthisakul S Pasuk and P Ritthiruangdej ldquoRelationshipbetween antioxidant properties and chemical composition ofsome Thai plantsrdquo Journal of Food Composition and Analysisvol 21 no 3 pp 229ndash240 2008

[30] H Kolodziej and A F Kiderlen ldquoAntileishmanial activity andimmune modulatory effects of tannins and related compoundson Leishmania parasitised RAW 2647 cellsrdquo Phytochemistryvol 66 no 17 pp 2056ndash2071 2005

[31] T Nakagawa and T Yokozawa ldquoDirect scavenging of nitricoxide and superoxide by green teardquo Food and Chemical Toxi-cology vol 40 no 12 pp 1745ndash1750 2002

[32] V Schulz R Hansel and V Tyler Fitoterapia Racional UmGuiade Fitoterapia Para as ciencias da saude Manole Barueri Brazil2002

[33] S D Kapu Y B Ngwai O Kayode P A Akah C Wambebeand K Gamaniel ldquoAnti-inflammatory analgesic and anti-lymphocytic activities of the aqueous extract of Crinum gigan-teumrdquo Journal of Ethnopharmacology vol 78 no 1 pp 7ndash132001

[34] G Francis Z Kerem H P S Makkar and K Becker ldquoThebiological action of saponins in animal systems a reviewrdquoBritish Journal of Nutrition vol 88 no 6 pp 587ndash605 2002

BioMed Research International 13

[35] R Xu W Zhao J Xu B Shao and G Qin ldquoStudies onbioactive saponins fromChinesemedicinal plantsrdquoAdvances inExperimental Medicine and Biology vol 404 pp 371ndash382 1996

[36] J A Manthey N Guthrie and K Grohmann ldquoBiologicalproperties of citrus flavonoids pertaining to cancer and inflam-mationrdquo Current Medicinal Chemistry vol 8 no 2 pp 135ndash1532001

[37] P Hendra Y Fukushi and Y Hashidoko ldquoSynthesis of ben-zophenone glucopyranosides from Phaleria macrocarpa andrelated benzophenone glucopyranosidesrdquo Bioscience Biotech-nology and Biochemistry vol 73 no 10 pp 2172ndash2182 2009

[38] A Rabia Z B A Mohammad D Aidiahmad S Amirin and IU Muhammad ldquoPhytochemistry and medicinal properties ofPhaleria macrocarpa (Scheff) Boerl extractsrdquo PharmacognosyReview vol 7 no 13 pp 73ndash80 2013

[39] A Saufi C B I VonHeimendahl AWAlfermann andE FussldquoStereochemistry of lignans in Phaleria macrocarpa (Scheff)Boerlrdquo Zeitschrift fur Naturforschung C vol 63 no 1-2 pp 13ndash16 2008

[40] S C Chong M A Dollah P P Chong and A Maha ldquoPhaleriamacrocarpa (Scheff) Boerl fruit aqueous extract enhances LDLreceptor and PCSK9 expression in vivo and in vitrordquo Journal ofEthnopharmacology vol 137 no 1 pp 817ndash827 2011

[41] R R Tjandrawinata D Nofiarny L W Susanto P Hendra andA Clarissa ldquoSymptomatic treatment of premenstrual syndromeandor primary dysmenorrhea with DLBS1442 a bioacitveextract ofPhaleriamacrocaraprdquo International Journal of GeneralMedicine vol 4 pp 465ndash476 2011

[42] R Hendra S Ahmad E Oskoueian A Sukari and M YShukor ldquoAntioxidant anti-inflammatory and cytotoxicity ofPhaleriamacrocarap (Boerl) Scheff fruitrdquoBMCComplementaryand Alternative Medicine vol 11 article 110 2011

[43] D Kurnia K Akiyama and H Hayashi ldquo29-Norcucurbitacinderivatives isolated from the Indonesian medicinal plant Pha-leria macrocarpa (Scheff) Boerlrdquo Bioscience Biotechnology andBiochemistry vol 72 no 2 pp 618ndash620 2008

[44] S Oshimi K Zaima Y Matsuno et al ldquoStudies on theconstituents from the fruits of Phaleria macrocaraprdquo Journal ofNatural Medicines vol 62 no 2 pp 207ndash210 2008

[45] C Sanchez-Moreno J A Larrauri and F Saura-Calixto ldquoAprocedure tomeasure the antiradical efficiency of polyphenolsrdquoJournal of the Science of Food and Agriculture vol 76 pp 270ndash276 1998

[46] Y Andriani M A W Effendy T M T Sifzizul and HMohamad ldquoAntibacterial radical-scavenging activities andcytotoxicit properties of Phaleria macrocarpa (Scheff) BoerlLeaves in HepG2 cell linesrdquo International Journal of Pharma-ceutical Sciences and Research vol 2 no 7 pp 1700ndash1706 2011

[47] Y Rohyami ldquoPenentuan Kandungan Flavonoid dari EkstrakMetanol Daging Buah Mahkota Dewa (Phaleria macrocarpaScheff Boerl)rdquo Jurnal Logika vol 5 2009

[48] R D Petty L A Sutherland E M Hunter and I A CreeldquoComparison of MTT and ATP-based assays for the measure-ment of viable cell numberrdquo Journal of Bioluminescence andChemiluminescence vol 10 no 1 pp 29ndash34 1995

[49] J R Sellers S Cook andV S Goldmacher ldquoA cytotoxicity assayutilizing a fluorescent dye that determines accurate survivingfractions of cellsrdquo Journal of Immunological Methods vol 172no 2 pp 255ndash264 1994

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

BioMed Research International 3

the oven drying method [14 15] A clean dried crucible wasweighed and 10 g of the driedPmacrocarpa seeds powderwasplaced in a beaker The sample was dried in an electric ovenat 105∘C until all the moisture was removed from the sampleand constant weight was achieved The crucible containingthe dried sample was weighed again and the loss of weightwas recorded as the moisture content of the dried powderedP macrocarpa seeds The experiment was repeated threetimes The moisture content () was calculated by using thefollowing equation

Mositure percentage () =1198822minus1198821

1198820

times 100 (1)

where1198821= weight of sample after drying (g)119882

2= weight of

sample before drying (g) and1198820= weight of the sample (g)

26 Determination of Ash Content Ash content was deter-mined by using the method of AOCS [12] Briefly theash value of the samples represented the inorganic residuewhen the organic matter has been burnt away An accuratelyweighted amount (10 g) of the sample was placed in a pre-heated cooled and weighed porcelain crucible The cruciblewas heated carefully on a hot plate until the organic matterwas dried and burnt off without flaming and finally heated ina furnace at 550 plusmn 50∘C

The percentage of ash content was calculated using thefollowing formula

Percentage of Ash () =Weight of Ash

Weight of sampletimes 100 (2)

27 Determination of Crude Fiber Content The crude fibercontent was determined by using themethod ofAOAC [16]Dried powdered sample was weighed (10 g) and extractedwith petroleum ether (100mL) three times The extractedsample was air-dried and transferred to a round-bottomedflask In the flask 30mL of sulphuric acid (01275M) wasadded followed by 170mL of hot sulphuric acidThe solutionwas then refluxed for approximately 30 minutes and filteredthrough a Buchner funnel The insoluble matter was washedwith boiling water until the final filtrate was free from acid

The residue was placed back into the flask with 30mLof sodium hydroxide (0313M) and 170mL of hot sodiumhydroxide (0313M) was then added The mixture was againrefluxed for about 30minutes and filtered using sintered glassThe residue was washed with 1 HCl and then washed againwith boilingwater until therewas no acid presentThe residuewas finally washed with ethanol and ether and dried in anoven at 100∘C and thenweighedThis procedure was repeateduntil the fiber content was constant The fiber content wascalculated using the following equation

Percentage of Crude fiber () =Loss in weight

Weight of sampletimes 100

(3)

28 Determination of Carbohydrate Content Carbohydratecontent was calculated by multiplying the reducing sugar

content The reducing sugar content was determined usingthe Fehlingrsquos reducing method of Lane and Eynonrsquos [16]Weighted sample (10 g) was placed into a 250mL round-bottomed flask and 20mL of sulphuric acid (05M) wasadded Reflux was then performed in a sand bath for 25hoursThe residue was washed after filtration with warm dis-tilled water The solution was then neutralized with sodiumcarbonate power and the mixturersquos volume was made up to100mL with distilled water This was followed by titrationwith Fehlingrsquos solution equal amounts of solution A (coppersulphate solution) and solution B (sodium potassium tartrateand sodium hydroxide solution) using methylene blue asindicator The mixed Fehlingrsquos solution (5mL) was pipettedinto a conical flask and distilled water (5mL) was addedThesolution was then boiled for 15 seconds Methylene blue indi-cator (a few drops) was then titrated with the solution untilthe colour changed from blue to green The carbohydratecontent was then calculated according to following equation

Percentage of carbohydrate content ()

=5 times 0005 times 100 times 100 times 100

119881 times 10 times119882times 09

(4)

where 119881 = volume of sample solution (titration volume) and119882 = weight of powdered sample

29 Determination of Fat Content Fat content of P macro-carpa seeds was determined using the soxhlet extractionmethod [12] A seed sample (10 g) was placed in a soxhletextractor Petroleum ether was then poured into the extractorand extraction was performed for 12 hoursThe volume of thepetroleum ether extract was reduced to 15mL by evaporationThis was then dried at 105∘C in an oven until constant weightwas achievedThe fat content was then calculated by using thefollowing equation

of Fat content

=Weight of fat obtained from sample times 100

Weight of sample

(5)

210 Determination of Protein Content Protein content wasdetermined using the AOAC method [12] Briefly pow-dered samples (1 g) 50mL of distilled water 5 g of coppersulphate and 15mL of concentrated sulphuric acid wereadded to a Kjeldahl flask The flask was partially closed bymeans of a funnel and the content was digested by heatingthe flask at an inclined position in the digester The mixturewas heated for 30minutes until there was 40mL of clear 01Mstandard sulphuric A few drops of methyl red indicator weremixed into the clear solutionThe flask was then placed belowthe condenser and the end of the adapter tube was dippedin the acid The flasks were set up for Kjeldahl distillationand 70mL of 40 sodium hydroxide was added through thefunnel The funnel was washed twice with 50mL of distilledwater Distillation was then performed for one hour Thedistilled ammonia was then nitrated with 01M standardsolution until the color changed from yellow to colorlessTheexperiment was repeated three times The nitrogen content

4 BioMed Research International

and protein content in the sample were calculated using thefollowing relation

Percentage of Nitrogen ()

=(119881119904minus 119881119887) times 119872

119860times 00140 times 100

Weight of sample (119882)

Protein content = percentage of Nitrogen times 625

(6)

where 119881119904= volume in cm3 of standard acid used in the

titration of sample119881119887= volume in cm3 of standard acid used

in the blank titration119872119860=morality of standard acid solution

in mol dmminus3 and119882 = weight of the sample in grams

211 Determination of Phenolic Content The total phenoliccontent of the crude methanol and the fractionated extracts(hexane chloroform ethyl acetate and water) was deter-mined using the Folin-Ciocalteu method [17ndash20] Briefly200120583L of each extract solution of different concentrationswas mixed with 1mL of Folin-Ciocalteu reagent (1 10 dilutedwith H

2O) and 800 120583L of Na

2CO3(7505 gL) The mixture

was thoroughly shaken for 15 minutes and then held ina water bath at a temperature of 37∘C The solution wasallowed to stand for 1 hour at room temperature in a darkplace and the absorption was measured at 750 nm using aspectrophotometer Distilled water was used as blank andgallic acid (0ndash250mgL) was used to construct a standardcalibration curve Gallic acid concentration was establishedfrom the calibration curve 119910 = 00221119909 + 02189 1198772 =09914

212 Determination of Flavonoid Content Total flavonoidcontent of P macrocarpa seeds was measured using themethods of Ebrahimzadeh Nabavi and Ordonez [20ndash22]with minor modifications To determine the flavonoid con-tent 1mL of each sample was added to 01mL of 10Al (NO

3)3solution 01mL of 1M potassium acetate and

38mLofmethanolThe solutionwas thoroughlymixed usinga vortex mixer for two to three minutes and then stooduntouched for 10 minutes at room temperature Absorbancewas determined at 415 nm using a spectrophotometer Thetotal content of flavonoids was measured and expressed asquercetin equivalent on a dry weight basis (119910 = 00855119909+02004 1198772 = 09813)

213 Determination of Flavonol Content The method ofKumaran [23] and Mbaebie [24] with slight modificationswas used to measure the total flavonol content of themethanol extract Briefly 1mL of extract was added to a cen-trifuge tube with 2mL of prepared AlCl

3in ethanol and 3mL

of sodium acetate (50 gL) solution The mixture was stirredthoroughly with a vortex and was then incubated for 1 hourThe absorbance was measured with a spectrophotometer at440 nm A calibration curve was constructed using quercetin(1 5 10 15 and 20mgmL) The total flavonol content wascalculated using the calibration curve with the followingequation119910 = 00353119909 + 01456 1198772 = 09807

214 Antioxidant DPPH Assay Screenings of antioxidantactivity of the crude methanol extract chloroform hexaneethyl acetate and water fractions of P macrocarpa seeds werecarried out by determination ofDPPH free radical scavengingproperty using UV spectrophotometric methods [21 25]Based on this protocol 50120583L of test solutions from differentdry extracts and concentrations (1 5 10 15 and 20mgmL)was dissolved in water This solution was then combinedwith 195mL of DPPHmethanol solution After being mixedsolutions were kept at room temperature in the dark for 30minutes After the reaction the increase in absorbance wasrecorded at 517 nm Methanol was used as a blank DPPHsolution was used as negative control (119860

0) and gallic acid

was used as positive control The antioxidant activity wasexpressed as an IC

50value All experiments were carried out

in triplicate The scavenging effect was obtained from thefollowing relation

Scavenging effect () =(1198600minus 1198601) times 100

1198600

(7)

where 1198600was the absorbance of the control reaction and

1198601was the absorbance of the sample of the tested extracts

Gallic acid was used as standard Percentage of inhibitionwas calculated using the following formula inhibition =[(119860negative minus 119860 test)119860negative] times 100 (119860 is absorbance)

215 Cytotoxicity Screening

2151 Cell Culture andCultureMedium Human cervical car-cinoma cells (Ca Ski) hormone-dependent breast carcinomacells (MCF-7) human breast adenocarcinoma cells (MDA-MB231) human ovarian carcinoma cells (SKOV-3) humancolon carcinoma cells (HT-29) and noncancer humanfibrob-last cells (MRC-5) were purchased from the American TissueCulture Collection (ATCC USA)

HT 29 Ca Ski andMCF-7 cells weremaintained in RPMI1640 medium (Sigma) MDA-MB231 and SKOV-3 cells inDulbeccorsquos Modified Eaglersquos medium (DMEM Sigma) andMRC-5 cells in Eaglersquos Minimum Essential medium (EMEMSigma) supplemented with 10 fetal bovine serum (FBSPAA Lab Austria) 100 120583gmL penicillin or streptomycin(PAA Lab Austria) and 50 120583gmL kanamycinamphotericinB (PAA Lab Austria) The cells were cultured in a CO

2

incubator (5) and kept at 37∘C in a humidified atmosphereThe cultures were subcultured every 2-3 days and checkedfrequently under an inverted microscope (Leica Germany)for any contamination

2152 MTT Cell Proliferation Assay MTT [3-(4 5-dimeth-ylthiazol2yl)-2 5-diphenyl tetrazolium bromide)] assay wasperformed on the cultured cells in 96-well plates according tothe method of Mosmann [26 27] Briefly cells were culturedto confluence They were then centrifuged at 1000 rpm for 5minutes and resuspendedwith 10mLof growthmediumThedensity of the viable cells was counted using 04 trypan blueexclusion dye in a haemocytometer with a microscope Thecells were then seeded into microtiter plates and incubated ina CO2incubator at 37∘C for 24 hrs After reaching 70ndash80

BioMed Research International 5

Table 1 Yield of methanol extract and its fractions from P macro-carpa of seedsExtract and fractions Yield (g) Percentage of yield ()Methanolic extract (ME) 1987 1199Hexane fraction (HF) 1587 947Chloroform fraction (CF) 72 043Ethyl acetate fraction (EAF) 62 037Water fraction (WF) Freeze dry

confluence each extract at concentrations of 1 10 25 50and 100 120583gmL (in 200120583L of 10 media) was added to therespective wells containing the cells Wells with untreatedcells were used as the negative control and cells exposedto doxorubicin were used as positive control After 24 48and 72 hours 10 120583L MTT stock solution was added to eachwell OD was then determined by measuring absorbance at540 nm using an ELISA microplate reader The percentageof inhibition () was calculated according to the followingformula

Percentage of inhibition ()

=OD control minusOD sample times 100

OD control

(8)

Cytotoxicity of each sample is expressed as plusmnIC50value The

extract that gave IC50

of 30 120583gmL or less was consideredactive [26]

216 Gas Chromatography-Mass Spectrometry (GC-MS) GC-MS was used to determine the molecular weight of thecomponents collected and the purity of the collected extractsRunning conditions were as follows oven temperature wasprogrammed with an initial temperature of 100∘C andincreased at a ramp rate of 5∘Cmin and reached a finaltemperature of 300∘C The carrier gas or mobile phase usedwas helium and a flow rate of 1mLmin was programmedThe mass spectrometry mode used was electron ionization(EI) mode with a current of 70 eV The injection mode wasprogrammed with a sample injection volume of 1120583L with asplit mode at a ratio of 1 20 The injection port temperaturewas set to 230∘C and the detectorinterface temperature wasset to 250∘C The results were collected for 40 minutes Thetotal ion chromatogram obtained was autointegrated usingChemStation and the chemical compounds or componentswere analysed by comparison with the suppliedmass spectraldatabase (NIST 05 Mass Spectral Library USA)

217 Statistical Analysis All data for each test are the averageof triplicate experiments for comparison of values and wererecorded as the mean plusmn standard deviation using MicrosoftExcel software and statistical data analyses were performedusing SPSS software

3 Results

31 Extraction Theextracts were concentrated using a rotaryevaporator (Buchi USA) under reduced pressure at 35∘CThe

yield of extracts from P macrocarpa seeds is shown in Table 1The highest yield from P macrocarpa seeds was the hexanefraction (947)

32 Preliminary Phytochemical Studies In order to determinethe types of phytoorganic constituents present in P macro-carpa seeds a preliminary phytochemical investigation wascarried out according to conventional methods The resultsobtained from these experiments are summarized in Table 2

The phytochemical tests showed that there were sec-ondary metabolites including carbohydrate flavonoids gly-cosides saponin glycosides phenolic compounds steroidstannins and terpenoids present in different extracts ofP macrocarpa seeds Small amounts of alkaloids 120572-aminoacids cyanogenic glycosides organic acids reducing sugarsand starches were also found to be present

The main constituents such as flavonoids glycosidessaponin glycosides phenolic compounds steroids tanninsand terpenoids present in P macrocarpa seeds may con-tribute to the presence of bioactivities such as antibacterialan analgesic an antifungal an anti-inflammatory agentand cytotoxicity Moreover the toxic chemical constituentscyanogenic glycosides were also present in the seeds

33 Physicochemical Studies The determination of nutri-tional values such as moisture ash fiber protein fat andcarbohydrate contents was carried out using the AOACmethod as well as the Lane and Eynon titration method andthe results obtained are shown in Figure 2(a) and Table 3Different physiochemical parameters for the purpose ofstandardization such as moisture (631 plusmn 143) ash (296 plusmn186) protein (2073 plusmn 244) crude fiber (2276 plusmn 279)crude fat (184 plusmn 311) and carbohydrate (2934 plusmn 198)were determined

34 Flavonoid Flavonol and Phenolic Determinations It wasfound that the methanol extract had the highest flavonoidcontent (933 plusmn 08mgmL) followed by ethyl acetate waterchloroform and hexane fractions which were 838 plusmn 10808 plusmn 03 678 plusmn 11 and 418 plusmn 15mgmL respectively

Themethanol extract (893plusmn11mgmL) of Pmacrocarpaseeds exhibited the highest amount of total flavonol contentfollowed by water ethyl acetate chloroform and hexanefractions which were 893 plusmn 10 613 plusmn 05 429 plusmn 09 and429 plusmn 07mgmL respectively

The total phenolic content of the P macrocarpa seedsextracts and fractions was expressed as gallic acid equiva-lents The methanol extract of P macrocarpa seeds exhibitedthe highest amount (720 plusmn 07mgmL) of total phenolicsfollowed by water ethyl acetate chloroform and hexanefractions which were 512 plusmn 14 358 plusmn 11 326 plusmn 10 and263 plusmn 01mgmL respectively

These data are shown in Figure 2(b) and Table 4

35 DPPH (2 2-Diphenyl-1-picryl-hydazyl) AntioxidantAssay DPPH assay was used to determine the free radicalscavenging ability of extracts and fractions of P macrocarpaseeds and to determine the antioxidant activity of its

6 BioMed Research International

Table 2 Results of chemical constituents of P macrocarpa seeds

No Tests Reagents Observation

1 AlkaloidsWagnerrsquos reagentMayerrsquos reagentDragendorff rsquos reagentSodium picrate solution

++++

2 120572-Amino acids Ninhydrin reagent +3 Carbohydrates 10 120572-naphthol and conc H2SO4 ++4 Cyanogenic glycosides Sodium picrate solution +5 Flavonoids Mg and conc HCl ++6 Glycosides 10 lead acetate ++7 Organic acids Bromothymol blue +8 Phenolic compounds 1 FeCl3 ++

9 Reducing sugars Fehlingrsquos solutionsA and B +

10 Saponin glycosides Distilled water ++11 Starch Iodine solution +12 Steroids Acetic anhydride and conc H2SO4 ++13 Tannins 1 Gelatin ++14 Terpenoids Acetic anhydride and conc H2SO4 +++++ large amount ++ medium amount + small amount minus absent + present

0

5

10

15

20

25

30

35

Con

tent

()

Moi

sture

Ash

Prot

ein

Crud

e fibe

r

Carb

ohyd

rate

Crud

e fat

(a)

123456789

1011

ME HF CF EAF WF

FlavonoidsFlavonolPhenolic

Con

cent

ratio

n (m

gm

L)

(b)

Figure 2 (a)The percentage ofmoisture ash protein crude fiber crude fat and carbohydrate contents on PmacrocarpaBoerl seeds (b) Totalamount of concentration of flavonoid flavonol and phenolic contents of P macrocarpa seeds ME methanol extract HF hexane fractionEAF ethyl acetate fraction CF chloroform fractionWF water fraction

phytoconstituents It is important to note that a lower IC50

value equals a higher scavenging activity The scavengingactivity was presented as the percentage of inhibitionof DPPH free radicals (Figure 3 and Table 4) Based onIC50

values the samples can be ranked in the followingdescending order water fraction gt methanol extract gt ethylacetate fraction gt hexane fraction gt chloroform fractionTheresults revealed that the water fraction was most active forantioxidant activity compared to other fractions The radical

scavenging effect was found to increase with increasingconcentrations

36 Cytotoxicity Screening of MTT Cell Proliferation AssayThe methanol and fractionated extracts (hexane chloro-form ethyl acetate and water) were investigated for cyto-toxic effects in human cervical adenocarcinoma cells (CaSki) humanhormone-dependent breast carcinoma (MCF-7)

BioMed Research International 7

Table 3The percentage of moisture ash protein crude fiber crudefat and carbohydrate contents and the amount of nutrition valueson P macrocarpa seeds

Tests parameter Percentage of contents ()Moisture 631 plusmn 143Ash 296 plusmn 186Protein 2073 plusmn 244Crude fiber 2276 plusmn 279Crude fat 1840 plusmn 311Carbohydrate 2934 plusmn 198

0102030405060708090

100

1 5 10 15 20

ME

HF

CF

EAF

WF

Concentration (mgmL)

Inhi

bitio

n (

)

Figure 3 Percentage of inhibition of DPPH free radical scavengingof P macrocarpa seeds ME methanol extract HF hexane fractionEAF ethyl acetate fraction CF chloroform fractionWF waterfraction

human colon adenocarcinoma cells (HT-29) human ovar-ian carcinoma cells (SKOV-3) human hormone-dependentbreast carcinoma (MDA-MB231) and normal cells (MRC-5) using MTT cells proliferation assay at 24 hrs 48 hrs and72 hrs respectively

The methanol extract of P macrocarpa seeds showedexcellent cytotoxic effects with IC

50value of 82plusmn 466 120583gmL

in Ca Ski cells at 24 hrs and 120 plusmn 22 85 plusmn 168 and30 plusmn 250 120583gmL in MCF-7 cells at 24 hrs 48 hrs and72 hrs respectively The methanol extract also showed goodcytotoxic activity in HT-29 cells with IC

50values of 293 plusmn

226 250 plusmn 135 and 215 plusmn 330 120583gmL at 24 hrs 48 hrsand 72 hrs respectively in Ca Ski cells with IC

50values of

197 plusmn 092 120583gmL and in SKOV-3 cells with IC50

values of165 plusmn 252 221 plusmn 247 and 360 plusmn 355 120583gmL at 24 hrs48 hrs and 72 hrs respectively However this extract had nocytotoxic effect in MDA-MB231 cells with IC

50gt100120583gmL

and the extract also had low cytotoxic effect in the MRC-5cells with IC

50gt 50 120583gmL

The hexane fraction of P macrocarpa seeds showedmoderate cytotoxic effects with IC

50values of 452 plusmn 149

and 555 plusmn 197 120583gmL at 24 hrs and 48 hrs on MCF-7 400 plusmn315 120583gmL inHT-29 cells at 24 hrs and 405plusmn352 and 500plusmn302 120583gmL in SKOV-3 at 24 hrs and 48 hrs In addition the

hexane fraction also displayed low cytotoxic effect in MCF-7at 72 hrs HT-29 at 48 hrs and 72 hrs and in SKOV-3 at 72 hrsThe IC

50value of treatment with hexane fraction in MCF-7

was 725plusmn152 120583gmL for 72 hrs exposure IC50values in HT-

29 were 640plusmn203 and 750plusmn314 120583gmL at 48 hrs and 72 hrswhile IC

50value in SKOV-3 was 703 plusmn 353 120583gmL at 72 hrs

In contrast the hexane fraction exhibited no cytotoxic effectsin MDA-MB231 cells or MRC-5 cells

The chloroform fraction of P macrocarpa seeds exhibitedthe highest cytotoxic effect with IC

50value of 100plusmn131 82plusmn

104 and 220 plusmn 186 120583gmL at 24 hrs 48 hrs and 72 hrs in CaSki cells 95plusmn295 87plusmn159 and 210plusmn198 120583gmL at 24 hrs48 hrs and 72 hrs on HT29 and 248 plusmn 206 165 plusmn 321 and900 plusmn 26 120583gmL in SKOV-3 cells The chloroform fractionexhibitedmoderate cytotoxic effect inMCF-7 with IC

50value

of 575 plusmn 264 400 plusmn 148 and 465 plusmn 345 120583gmL for 24 hrs48 hrs and 72 hrs respectively In contrast the chloroformfraction had no cytotoxic effect against theMDA-MB231 cellsand normal MRC-5 cells with IC

50gt100 120583gmL

The ethyl acetate fraction ofPmacrocarpa seeds exhibitedthe highest cytotoxic effect with IC

50lt 25 120583gmL in SKOV-3

cellsMDA-MB231 cellsMCF-7 cells andCa Ski cells On theother hand the ethyl acetate fraction exhibited low cytotoxiceffect in MRC-5 normal cells with IC

50value of 35 120583gmL

The ethyl acetate fraction exhibited the highest cytotoxiceffect in all selected cells (Ca Ski MCF-7 HT-29 and MDA-MB231) The water extract showed no cytotoxic effect againstall selected cancer cell lines with IC

50gt 100 120583gmL and

exhibited a mild cytotoxic effects against MRC-5 cells Thecorresponding data is shown in Figure 4 and Table 5

37 Characterization and Identification of Hexane and Chlo-roform Fractions Six compounds were identified from thehexane fraction of P macrocarpa seeds using GC-MS Theywere methyl stearate oleic acid methyl oleate linoleic acidmethyl linolenate and palmitic acid Moreover a GCMSanalysis of the chloroform fraction of P macrocarpa seedsshowed the presence of methyl myristate palmitic acidmethyl oleate methyl linoleate oleic acid and (119911)- and917-octadecadienal (119911) A comparison with the NIST massspectral library (NIST 05MS library 2002) andAdams (2001)confirmed the identity of the compounds The chemicalstructures of the identified compounds in the hexane andchloroform fractions of P macrocarpa seeds are as shown inFigure 5

4 Discussion

A longer shelf life can be achieved by reducing moisturecontent Thus moisture content is a critical factor for thestability of an extractMoisture enhances fungal and bacterialgrowth therefore decreasing the longevity of the extract Thetime it takes for plant material to deteriorate depends on howmuch water the plant material contains

Carbohydrates play a vital role in the immune sys-tem fertilization pathogenesis blood clotting and humandevelopment Foods that contain carbohydrates can raiseblood glucose and the three main types of carbohydrate are

8 BioMed Research International

0102030405060708090

100

ME

HF

CF

EAF

24h 48h 72h

IC50

valu

e of c

once

ntra

tion

(120583g

mL)

Ca Ski

0102030405060708090

100

ME

HF

CF

EAF

24h 48h 72h

IC50

valu

e of c

once

ntra

tion

(120583g

mL)

MCF 7

0102030405060708090

100

ME

HF

CF

EAF

24h 48h 72h

IC50

valu

e of c

once

ntra

tion

(120583g

mL)

HT-29

minus10

0

10

20

30

40

50

60

70

80

90

100

ME

HF

CF

EAF

24h 48h 72h

IC50

valu

e of c

once

ntra

tion

(120583g

mL)

SKOV-3

0102030405060708090

100

ME

HF

CF

EAF

24h 48h 72h

IC50

valu

e of c

once

ntra

tion

(120583g

mL)

MDA-MB231

Figure 4 In vitro cytotoxic effects of P macrocarpa seedson Ca Ski MCF-7 HT-29 SKOV-3 and MDA-MB231 cell lines ME methanolextract HF hexane fraction EAF ethyl acetate fraction CF chloroform fractionWF water fraction Each value is expressed as mean plusmnstandard deviation of three measurements

BioMed Research International 9

Methyl linoleate

Methyl stearate

Palmitic acid

Oleic acid

Methyl myristate

Linoleic acid

Methyl oleate

H3C

H3C

H3C

H3C

H3C

H3C

H3C

O

O

O

O

O

O

O

O

O

O

O

CH3

CH3

CH3

CH3

OH

OH

OH

(294mz)

(298mz)

(282mz)

(242mz)

(280mz)

(298mz)

(256mz)

Figure 5

10 BioMed Research International

Table 4 Total flavonoid total flavonol total phenolic contents and result of DPPH free radical scavenging property of P macrocarpa seeds

Tests ME (mgmL) HF (mgmL) CF (mgmL) EAF (mgmL) WF (mgmL)Flavonoid 933 plusmn 08 418 plusmn 15 678 plusmn 11 838 plusmn 03 808 plusmn 10Flavonol 893 plusmn 11 425 plusmn 07 429 plusmn 09 613 plusmn 05 818 plusmn 10Phenolic 720 plusmn 07 263 plusmn 01 326 plusmn 1 358 plusmn 11 512 plusmn 14DPPH 1150 plusmn 003 1500 plusmn 004 1575 plusmn 004 1400 plusmn 005 975 plusmn 003ME methanol extract HF hexane fraction EAF ethyl acetate fraction CF chloroform fraction WF water fraction

Table 5 In vitro cytotoxic effects of methanol extract and its fractions of P macrocarpa cancer cells lines and normal human fibroblast breastcells line

Cell line Incubation periods (hours) ME HF CF EAF Doxorubicin24 82 plusmn 466 ge100 100 plusmn 131 56 plusmn 117 092 plusmn 064

Ca Ski 48 197 plusmn 092 ge100 82 plusmn 104 77 plusmn 185 069 plusmn 10572 407 plusmn 226 ge100 220 plusmn 186 60 plusmn 322 045 plusmn 09924 120 plusmn 22 452 plusmn 149 575 plusmn 264 223 plusmn 158 105 plusmn 108

MCF7 48 85 plusmn 168 555 plusmn 197 400 plusmn 148 168 plusmn 170 012 plusmn 06972 63 plusmn 250 725 plusmn 152 465 plusmn 345 84 plusmn 171 092 plusmn 17024 293 plusmn 226 400 plusmn 315 95 plusmn 295 11 plusmn 120 092 plusmn 072

HT29 48 250 plusmn 135 640 plusmn 203 87 plusmn 159 35 plusmn 200 032 plusmn 21672 215 plusmn 330 750 plusmn 314 210 plusmn 198 12 plusmn 228 088 plusmn 09424 165 plusmn 252 405 plusmn 352 248 plusmn 206 68 plusmn 18 102 plusmn 179

SKOV-3 48 221 plusmn 247 500 plusmn 302 165 plusmn 321 65 plusmn 23 032 plusmn 09772 360 plusmn 355 703 plusmn 353 90 plusmn 26 32 plusmn 281 062 plusmn 09824 ge100 ge100 ge100 65 plusmn 23 043 plusmn 050

MDA-MB231 48 ge100 ge100 ge100 15 plusmn 343 059 plusmn 10172 ge100 ge100 ge100 186 plusmn 126 095 plusmn 132

ME methanol extract HF hexane fraction EAF ethyl acetate fraction CF chloroform fractionWF water fraction Doxorubicin was used as positive controlEach value is expressed as mean plusmn standard deviation of three measurements

starches sugars and fiber A macronutrient protein consistsof amino acids which is required for proper growth andhuman body function The seeds were found to containa high level of crude fiber which is a potential source ofphenolic antioxidants The major components of fibers arecellulose hemicelluloses lignin 120573-glucans gums and pectinand hydrocolloids These components can behave as proan-tioxidants [28 29] In our present physicochemical study Pmacrocarpa seeds were mainly made up of carbohydratesfollowed by crude fiber proteins fatmoisture and ashHigh-protein and low-carbohydrate diets are often effective forweight loss Dietary fiber can aid in digestion is helpfulfor weight management and reduces constipation Howevermany high-protein and low-carbohydrate foods are low infiberWomen aged 50 and older need at least 21 grams of fiberdaily whilst those aged 19 to 50 require 25 grams men aged50 and older need 30 grams and those between the age of 19to 50 should consume at least 38 grams of fiber

Steroids possess biological activities which are insec-ticidal cardiotonic and antimicrobial activities and theseare potentially useful for development into therapeuticdrugs Tannins are known to be important for their antivi-ral antibacterial antiparasitic effects anti-inflammatoryantiulcer and antioxidant properties [30ndash33] Saponinshave been found to have antimicrobial anti-inflammatoryantifeedant and hemolytic effects [34 35] Some alkaloids

have been reported to have anticancer and antiviral activityand saponins have been reported to be cardiotonic whileflavonoids have anticancer and anti-inflammatory activity[10 36] The presence of tannins may be responsible for theability of P macrocarpa seeds to be used in the treatmentof diseases such as diabetes diarrhea and dysentery Ourpreliminary phytochemical studies revealed that the chemicalcomponents of P macrocarpa seeds also include flavonoidsphenolics steroids tannins terpenoids glucosides saponinsand carbohydrates

Previous researchers reported that kaempferol myrice-tin naringin quercetin rutin [3] 29-norcucurbitacinderivatives fevicordin A fevicordin A glucoside fevicordinD glucoside [6] mahkoside A dodecanoic acid palmiticacid des-acetyl flavicordin-A flavicordin-A flavicordin-Dflavicordin-A glucoside ethyl stearate lignans and sucrose[37 38] mangiferin (a C-glucosylxantone) kaempferol-3-o-120573-D-glucoside dodecanoic acid palmitic acid ethyl stearatesucrose [5] pinoresinol lariciresinol matairesinol alkaloidsand saponins [37 39] saponins alkaloids polyphenolicsphenols flavanoids lignans tannins [40ndash42] icarisideC3 mangiferin gallic acid phalerin glycoside (345trihydroxy-4-methoxy-benzophenone-3-O-120573-D-glucoside)[43] and 2410158406 trihydroxy-4-methoxy-benzophenone-3-O-120573-D-glucoside [44] were isolated from different partsof P macrocarpa The hexane extract of the seeds was

BioMed Research International 11

found to contain methyl myristate methyl stearate oleicacid methyl oleate linoleic acid methyl linoleate palmiticacid methyl palmitate 6-octadecenoic acid and 917-octadecadial 120573-sitosterol as major components A further30 to 40 of components were not identified as thesewere polar compounds that require HPLC analysis Othercompounds reported in the literature for the seeds leavesand fruits were not identified in this investigation It isprobable that the components reported in the literaturemay be present in other fractions (methyl acetate CHCl

3

and water fractions)Phenolics are able to transfer protons to radicals [45]

DPPH which provides free radicals is normally a blue-violet color However the DPPH turned yellow after it wasconverted to 1 1-diphenyl-2-picrylhydrazine which has lessfree-radical activity Hendra et al reported that the IC

50

values of free radical and reducing power in methanolwith HCl of P macrocarpa seeds were 2450 plusmn 194 and1502 plusmn 128 [42] In our investigation using DPPH assay thewater fraction displayed the highest IC

50value followed by

methanol ethyl acetate hexane and chloroform fractionThe presence of flavonoids in P macrocarpa seeds may be

responsible for the traditional use of the plants in treatingcancer inflammations and allergies Recently it has beenshown that phenolic compounds in crude methanol andethyl acetate extract of P macrocarpa leaves displayed goodantioxidant and antimicrobial activites [46] Hendra et al(2011) examined the amount of total phenolic (477plusmn 104mggallic acid equivalentg DW) and the amount of flavonoid(359 plusmn 247mg rutin equivalentg DW) contents in themethanol extract with HCl of P macrocarpa seeds [42] Inthis study the total flavonoids content was higher than that inthe results previously reported by Rohyami [47] and phenolflavonol and flavonoid contents were the highest in theaqueous methanol extract followed by ethyl acetate waterchloroform and hexane extracts

MTT assay is a sensitive and reliable colorimetric assaythat uses quantitative measurements to calculate the via-bility proliferation and activation of cells This method iscommonly applied to screen anticancer agents [48] Themost well-known methods used to calculate cytotoxicityare the neutral red (NR) uptake and dimethylthiazole-diphenyl tetrazolium bromide (MTT) metabolism [27 49]Earlier investigations showed that the ethanol extract of Pmacrocarpa seeds and fruit meat were not toxic to normalhuman cells but slightly toxic to a Vero cell line [9] Theethanol extract of P macrocarpa seed fruit also showedtoxicity towards T47D breast cancer cell line through COX-2 expression inhibition [7] Desacetylfevicordin A has beenisolated from the ethyl acetate extract of P macrocarpaseeds and this compound displayed excellent cytotoxicityin brine shrimp [43] The ethyl acetate of P macrocarpaseeds exhibited mild cytotoxic effect against HepG2 cells(IC50values between 30 and 60 120583gmL) [46] Previous studies

showed that the cytotoxic activity of the seeds on HT-29MCF-7 Hela and Chang liver cells lines were 384 plusmn 037255 plusmn 137 295 plusmn 10 and 678 plusmn 027 receptively [42] Inour study the methanolic extract of P macrocarpa seeds wasfound to pose cytotoxic effect against HT-29 MCF-7 Cas Ki

and SKOV-3 cell lines (IC50

values giving from 11 plusmn 120to 360 plusmn 355) and mild toxicity on normal cell lines Therewere significant cytotoxic effects on selected cancer cells linesthat were both time- and dose-dependent manner due to thepresence of many secondary metabolites

The phytochemical screening also indicated the presenceof a small amount of cyanogenic glycosides Cyanogenicglucosides which can cause acute cyanide poisoning causerapid respiration and pulse decrease the blood pressure andinduce vomiting diarrhea headache dizziness and so onThus these components have to be removed (usually byboiling) prior to consumption for therapeutic purposes

5 Conclusion

P macrocarpa seeds exhibited antioxidant and cytotoxicactivities It is highly probable that these activities are dueto the presence of phenolic and flavonoid compounds inappreciable amounts in the plant Furthermore the cytotox-icity activity suggested that the seed may contain a potentialanticancer agent The outcome of this study is encouragingdemonstrating the potential for the P macrocarpa as a sourceof multiple therapeutic agents

Abbreviations

EAF Ethyl acetate fractionME Methanol extractHF Hexane fractionCF Chloroform fractionWF Water fractiong Grammg MilligrammL Milliliter

Conflict of Interests

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

Authorsrsquo Contribution

Ma Ma Lay was involved in all experiments and BehroozBanisalamwas involved in antioxidant assay All authors readand approved the submitted version of the paper

Acknowledgments

This work was supported by grants from the MalaysianMinistry of Higher Education (nos PS2862009C andPS4702010B) Ma Ma Lay is a scholarship recipient fromthe ldquoIslamic Development Bank Saudi Arabiardquo The authorswould like to thank Professor Dr Amru BinNasrulhaq Boycefor generously allowing the use of his laboratory for thisresearch

12 BioMed Research International

References

[1] N HarmantoMahkota DewaObat Pusaka ParaDewa Cetakankeempat Agromedia Pustaka Jakarta Indonesia 2002

[2] A Jemal R Siegel E Ward T Murray J Xu and M J ThunldquoCancer statistics 2007rdquoCa ACancer Journal for Clinicians vol57 no 1 pp 43ndash66 2007

[3] R Hendra S Ahmad A Sukari M Y Shukor and EOskoueian ldquoFlavonoid analyses and antimicrobial activity ofvarious parts of Phaleria macrocarpa (Scheff) Boerl fruitrdquoInternational Journal of Molecular Sciences vol 12 no 6 pp3422ndash3431 2011

[4] W Wijayani Sitotoksisitas Minyak Atsiri Biji Dan Daging BuahMahkota Dewa (Phaleria Macrocarpa [Scheff] Boerl) TerhadapMyeloma Cell Line Dan HeLa Cell Line Serta IdentifikasiSenyawa Minyak Atsiri Dengan Kromatografi Gas-SpektrometriMassa (GC-MS) Skripsi FMIPA UGM Yogyakarta Indonesia2005

[5] Y-B Zhang X-J Xu and H-M Liu ldquoChemical con-stituents fromMahkotadewardquo Journal of Asian Natural ProductsResearch vol 8 no 1-2 pp 119ndash123 2006

[6] D Kurnia K Akiyama and H Hayashi ldquo29-Norcucurbitacinderivatives isolated from the Indonesian medicinal plant Pha-leria macrocarpa (Scheff) Boerlrdquo Bioscience Biotechnology andBiochemistry vol 72 no 2 pp 618ndash620 2008

[7] M Bakhriansyah Pengaruh Ekstrak Etanol Biji Mahkota Dewa(Phaleria Macrocarpa [Scheff] Boerl) Pada Sel Kanker Payu-dara T47D Kajian Aktivitas Sitotoksik Dan PenghambatanEkspresi Siklooksigenase-2 Program Pascasarjana UGM 2004

[8] L Wardani The Effect of Phaleria Macrocarpa [Scheff] Boerl)Seed and Flesh Fruit Extract to P53 and Bcl-2 Gene Expression inHela Cell Line FMIPA UGM 2005

[9] A Endang R Tri Joko and E Dewi ldquoCyototoxicity of Phaleriamacrocarpa (Scheff) boerl fruits flesh and seed extract ofethanol and its effect against p53 and Bcl-2 genes expressionof normalrdquo in Proceedings of the International Conference onComputational Science (ICCS rsquo07) 2007

[10] C Evans Trease and Evans Pharmacognosy Saunder 2002[11] A Sofowora Medicinal Plants and Traditional Medicine in

Africa Spectrum Books Ltd Ibadan Nigeria 2nd edition 1993[12] AOAC Official Methods of Analysis Association of Official

Analytical Chemists Arlington Tex USA 2002[13] J Harborne Phytochemical Methods Chapman and Hall Lon-

don UK 1973[14] AOAC Official Methods of Analysis Association of Official

Analytical Chemists Gaithersburg Md USA 17th edition2000

[15] J Lako V C Trenerry M Wahlqvist N WattanapenpaiboonS Sotheeswaran and R Premier ldquoPhytochemical flavonolscarotenoids and the antioxidant properties of a wide selection ofFijian fruit vegetables and other readily available foodsrdquo FoodChemistry vol 101 no 4 pp 1727ndash1741 2007

[16] W Horwitz Official Method of Analysis AOAC InterantionalGaithersburg Md USA 17th edition 2000

[17] A A Adedapo F O Jimoh S Koduru P J Masika and AJ Afolayan ldquoAssessment of the medicinal potentials of themethanol extracts of the leaves and stems of Buddleja salignardquoBMCComplementary andAlternativeMedicine vol 9 article 212009

[18] M Zovko Koncic D Kremer J Gruz et al ldquoAntioxidantand antimicrobial properties ofMoltkia petraea (Tratt) Griseb

flower leaf and stem infusionsrdquo Food and Chemical Toxicologyvol 48 no 6 pp 1537ndash1542 2010

[19] S McDonald P D Prenzler M Antolovich and K RobardsldquoPhenolic content and antioxidant activity of olive extractsrdquoFood Chemistry vol 73 no 1 pp 73ndash84 2001

[20] S M Nabavi M A Ebrahimzadeh S F Nabavi A Hamidiniaand A R Bekhradnia ldquoDetermination of antioxidant activityphenol and flavonoid content of Parrotia persicaMeyrdquo Pharma-cologyonline vol 2 pp 560ndash567 2008

[21] M A Ebrahimzadeh S F Nabavi and S M Nabavi ldquoAntiox-idant activities of methanol extract of Sambucus ebulus Lflowerrdquo Pakistan Journal of Biological Sciences vol 12 no 5 pp447ndash450 2009

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

[23] A Kumaran and R Joel Karunakaran ldquoIn vitro antioxidantactivities of methanol extracts of five Phyllanthus species fromIndiardquo Food Science and Technology vol 40 no 2 pp 344ndash3522007

[24] B O Mbaebie H O Edeoga and A J Afolayan ldquoPhy tochemical analysis and antioxidants activities of aqueous stembark extract of Schotia latifolia Jacqrdquo Asian Pacific Journal ofTropical Biomedicine vol 2 no 2 pp 118ndash124 2012

[25] L I Mensor F S Menezes G G Leitao et al ldquoScreening ofBrazilian plants extracts for antioxidants activity by the use ofDPPH free radical methodrdquo Phytotherapy Research vol 15 pp127ndash130 2001

[26] F Denizot and R Lang ldquoRapid colorimetric assay for cellgrowth and survival modifications to the tetrazolium dyeprocedure giving improved sensitivity and reliabilityrdquo Journalof Immunological Methods vol 89 no 2 pp 271ndash277 1986

[27] T Mosmann ldquoRapid colorimetric assay for cellular growth andsurvival application to proliferation and cytotoxicity assaysrdquoJournal of Immunological Methods vol 65 no 1-2 pp 55ndash631983

[28] P Maisuthisakul R Pongsawatmanit and M H GordonldquoAntioxidant properties of Teaw (Cratoxylum formosum Dyer)extract in soybean oil and emulsionsrdquo Journal of Agriculturaland Food Chemistry vol 54 no 7 pp 2719ndash2725 2006

[29] P Maisuthisakul S Pasuk and P Ritthiruangdej ldquoRelationshipbetween antioxidant properties and chemical composition ofsome Thai plantsrdquo Journal of Food Composition and Analysisvol 21 no 3 pp 229ndash240 2008

[30] H Kolodziej and A F Kiderlen ldquoAntileishmanial activity andimmune modulatory effects of tannins and related compoundson Leishmania parasitised RAW 2647 cellsrdquo Phytochemistryvol 66 no 17 pp 2056ndash2071 2005

[31] T Nakagawa and T Yokozawa ldquoDirect scavenging of nitricoxide and superoxide by green teardquo Food and Chemical Toxi-cology vol 40 no 12 pp 1745ndash1750 2002

[32] V Schulz R Hansel and V Tyler Fitoterapia Racional UmGuiade Fitoterapia Para as ciencias da saude Manole Barueri Brazil2002

[33] S D Kapu Y B Ngwai O Kayode P A Akah C Wambebeand K Gamaniel ldquoAnti-inflammatory analgesic and anti-lymphocytic activities of the aqueous extract of Crinum gigan-teumrdquo Journal of Ethnopharmacology vol 78 no 1 pp 7ndash132001

[34] G Francis Z Kerem H P S Makkar and K Becker ldquoThebiological action of saponins in animal systems a reviewrdquoBritish Journal of Nutrition vol 88 no 6 pp 587ndash605 2002

BioMed Research International 13

[35] R Xu W Zhao J Xu B Shao and G Qin ldquoStudies onbioactive saponins fromChinesemedicinal plantsrdquoAdvances inExperimental Medicine and Biology vol 404 pp 371ndash382 1996

[36] J A Manthey N Guthrie and K Grohmann ldquoBiologicalproperties of citrus flavonoids pertaining to cancer and inflam-mationrdquo Current Medicinal Chemistry vol 8 no 2 pp 135ndash1532001

[37] P Hendra Y Fukushi and Y Hashidoko ldquoSynthesis of ben-zophenone glucopyranosides from Phaleria macrocarpa andrelated benzophenone glucopyranosidesrdquo Bioscience Biotech-nology and Biochemistry vol 73 no 10 pp 2172ndash2182 2009

[38] A Rabia Z B A Mohammad D Aidiahmad S Amirin and IU Muhammad ldquoPhytochemistry and medicinal properties ofPhaleria macrocarpa (Scheff) Boerl extractsrdquo PharmacognosyReview vol 7 no 13 pp 73ndash80 2013

[39] A Saufi C B I VonHeimendahl AWAlfermann andE FussldquoStereochemistry of lignans in Phaleria macrocarpa (Scheff)Boerlrdquo Zeitschrift fur Naturforschung C vol 63 no 1-2 pp 13ndash16 2008

[40] S C Chong M A Dollah P P Chong and A Maha ldquoPhaleriamacrocarpa (Scheff) Boerl fruit aqueous extract enhances LDLreceptor and PCSK9 expression in vivo and in vitrordquo Journal ofEthnopharmacology vol 137 no 1 pp 817ndash827 2011

[41] R R Tjandrawinata D Nofiarny L W Susanto P Hendra andA Clarissa ldquoSymptomatic treatment of premenstrual syndromeandor primary dysmenorrhea with DLBS1442 a bioacitveextract ofPhaleriamacrocaraprdquo International Journal of GeneralMedicine vol 4 pp 465ndash476 2011

[42] R Hendra S Ahmad E Oskoueian A Sukari and M YShukor ldquoAntioxidant anti-inflammatory and cytotoxicity ofPhaleriamacrocarap (Boerl) Scheff fruitrdquoBMCComplementaryand Alternative Medicine vol 11 article 110 2011

[43] D Kurnia K Akiyama and H Hayashi ldquo29-Norcucurbitacinderivatives isolated from the Indonesian medicinal plant Pha-leria macrocarpa (Scheff) Boerlrdquo Bioscience Biotechnology andBiochemistry vol 72 no 2 pp 618ndash620 2008

[44] S Oshimi K Zaima Y Matsuno et al ldquoStudies on theconstituents from the fruits of Phaleria macrocaraprdquo Journal ofNatural Medicines vol 62 no 2 pp 207ndash210 2008

[45] C Sanchez-Moreno J A Larrauri and F Saura-Calixto ldquoAprocedure tomeasure the antiradical efficiency of polyphenolsrdquoJournal of the Science of Food and Agriculture vol 76 pp 270ndash276 1998

[46] Y Andriani M A W Effendy T M T Sifzizul and HMohamad ldquoAntibacterial radical-scavenging activities andcytotoxicit properties of Phaleria macrocarpa (Scheff) BoerlLeaves in HepG2 cell linesrdquo International Journal of Pharma-ceutical Sciences and Research vol 2 no 7 pp 1700ndash1706 2011

[47] Y Rohyami ldquoPenentuan Kandungan Flavonoid dari EkstrakMetanol Daging Buah Mahkota Dewa (Phaleria macrocarpaScheff Boerl)rdquo Jurnal Logika vol 5 2009

[48] R D Petty L A Sutherland E M Hunter and I A CreeldquoComparison of MTT and ATP-based assays for the measure-ment of viable cell numberrdquo Journal of Bioluminescence andChemiluminescence vol 10 no 1 pp 29ndash34 1995

[49] J R Sellers S Cook andV S Goldmacher ldquoA cytotoxicity assayutilizing a fluorescent dye that determines accurate survivingfractions of cellsrdquo Journal of Immunological Methods vol 172no 2 pp 255ndash264 1994

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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4 BioMed Research International

and protein content in the sample were calculated using thefollowing relation

Percentage of Nitrogen ()

=(119881119904minus 119881119887) times 119872

119860times 00140 times 100

Weight of sample (119882)

Protein content = percentage of Nitrogen times 625

(6)

where 119881119904= volume in cm3 of standard acid used in the

titration of sample119881119887= volume in cm3 of standard acid used

in the blank titration119872119860=morality of standard acid solution

in mol dmminus3 and119882 = weight of the sample in grams

211 Determination of Phenolic Content The total phenoliccontent of the crude methanol and the fractionated extracts(hexane chloroform ethyl acetate and water) was deter-mined using the Folin-Ciocalteu method [17ndash20] Briefly200120583L of each extract solution of different concentrationswas mixed with 1mL of Folin-Ciocalteu reagent (1 10 dilutedwith H

2O) and 800 120583L of Na

2CO3(7505 gL) The mixture

was thoroughly shaken for 15 minutes and then held ina water bath at a temperature of 37∘C The solution wasallowed to stand for 1 hour at room temperature in a darkplace and the absorption was measured at 750 nm using aspectrophotometer Distilled water was used as blank andgallic acid (0ndash250mgL) was used to construct a standardcalibration curve Gallic acid concentration was establishedfrom the calibration curve 119910 = 00221119909 + 02189 1198772 =09914

212 Determination of Flavonoid Content Total flavonoidcontent of P macrocarpa seeds was measured using themethods of Ebrahimzadeh Nabavi and Ordonez [20ndash22]with minor modifications To determine the flavonoid con-tent 1mL of each sample was added to 01mL of 10Al (NO

3)3solution 01mL of 1M potassium acetate and

38mLofmethanolThe solutionwas thoroughlymixed usinga vortex mixer for two to three minutes and then stooduntouched for 10 minutes at room temperature Absorbancewas determined at 415 nm using a spectrophotometer Thetotal content of flavonoids was measured and expressed asquercetin equivalent on a dry weight basis (119910 = 00855119909+02004 1198772 = 09813)

213 Determination of Flavonol Content The method ofKumaran [23] and Mbaebie [24] with slight modificationswas used to measure the total flavonol content of themethanol extract Briefly 1mL of extract was added to a cen-trifuge tube with 2mL of prepared AlCl

3in ethanol and 3mL

of sodium acetate (50 gL) solution The mixture was stirredthoroughly with a vortex and was then incubated for 1 hourThe absorbance was measured with a spectrophotometer at440 nm A calibration curve was constructed using quercetin(1 5 10 15 and 20mgmL) The total flavonol content wascalculated using the calibration curve with the followingequation119910 = 00353119909 + 01456 1198772 = 09807

214 Antioxidant DPPH Assay Screenings of antioxidantactivity of the crude methanol extract chloroform hexaneethyl acetate and water fractions of P macrocarpa seeds werecarried out by determination ofDPPH free radical scavengingproperty using UV spectrophotometric methods [21 25]Based on this protocol 50120583L of test solutions from differentdry extracts and concentrations (1 5 10 15 and 20mgmL)was dissolved in water This solution was then combinedwith 195mL of DPPHmethanol solution After being mixedsolutions were kept at room temperature in the dark for 30minutes After the reaction the increase in absorbance wasrecorded at 517 nm Methanol was used as a blank DPPHsolution was used as negative control (119860

0) and gallic acid

was used as positive control The antioxidant activity wasexpressed as an IC

50value All experiments were carried out

in triplicate The scavenging effect was obtained from thefollowing relation

Scavenging effect () =(1198600minus 1198601) times 100

1198600

(7)

where 1198600was the absorbance of the control reaction and

1198601was the absorbance of the sample of the tested extracts

Gallic acid was used as standard Percentage of inhibitionwas calculated using the following formula inhibition =[(119860negative minus 119860 test)119860negative] times 100 (119860 is absorbance)

215 Cytotoxicity Screening

2151 Cell Culture andCultureMedium Human cervical car-cinoma cells (Ca Ski) hormone-dependent breast carcinomacells (MCF-7) human breast adenocarcinoma cells (MDA-MB231) human ovarian carcinoma cells (SKOV-3) humancolon carcinoma cells (HT-29) and noncancer humanfibrob-last cells (MRC-5) were purchased from the American TissueCulture Collection (ATCC USA)

HT 29 Ca Ski andMCF-7 cells weremaintained in RPMI1640 medium (Sigma) MDA-MB231 and SKOV-3 cells inDulbeccorsquos Modified Eaglersquos medium (DMEM Sigma) andMRC-5 cells in Eaglersquos Minimum Essential medium (EMEMSigma) supplemented with 10 fetal bovine serum (FBSPAA Lab Austria) 100 120583gmL penicillin or streptomycin(PAA Lab Austria) and 50 120583gmL kanamycinamphotericinB (PAA Lab Austria) The cells were cultured in a CO

2

incubator (5) and kept at 37∘C in a humidified atmosphereThe cultures were subcultured every 2-3 days and checkedfrequently under an inverted microscope (Leica Germany)for any contamination

2152 MTT Cell Proliferation Assay MTT [3-(4 5-dimeth-ylthiazol2yl)-2 5-diphenyl tetrazolium bromide)] assay wasperformed on the cultured cells in 96-well plates according tothe method of Mosmann [26 27] Briefly cells were culturedto confluence They were then centrifuged at 1000 rpm for 5minutes and resuspendedwith 10mLof growthmediumThedensity of the viable cells was counted using 04 trypan blueexclusion dye in a haemocytometer with a microscope Thecells were then seeded into microtiter plates and incubated ina CO2incubator at 37∘C for 24 hrs After reaching 70ndash80

BioMed Research International 5

Table 1 Yield of methanol extract and its fractions from P macro-carpa of seedsExtract and fractions Yield (g) Percentage of yield ()Methanolic extract (ME) 1987 1199Hexane fraction (HF) 1587 947Chloroform fraction (CF) 72 043Ethyl acetate fraction (EAF) 62 037Water fraction (WF) Freeze dry

confluence each extract at concentrations of 1 10 25 50and 100 120583gmL (in 200120583L of 10 media) was added to therespective wells containing the cells Wells with untreatedcells were used as the negative control and cells exposedto doxorubicin were used as positive control After 24 48and 72 hours 10 120583L MTT stock solution was added to eachwell OD was then determined by measuring absorbance at540 nm using an ELISA microplate reader The percentageof inhibition () was calculated according to the followingformula

Percentage of inhibition ()

=OD control minusOD sample times 100

OD control

(8)

Cytotoxicity of each sample is expressed as plusmnIC50value The

extract that gave IC50

of 30 120583gmL or less was consideredactive [26]

216 Gas Chromatography-Mass Spectrometry (GC-MS) GC-MS was used to determine the molecular weight of thecomponents collected and the purity of the collected extractsRunning conditions were as follows oven temperature wasprogrammed with an initial temperature of 100∘C andincreased at a ramp rate of 5∘Cmin and reached a finaltemperature of 300∘C The carrier gas or mobile phase usedwas helium and a flow rate of 1mLmin was programmedThe mass spectrometry mode used was electron ionization(EI) mode with a current of 70 eV The injection mode wasprogrammed with a sample injection volume of 1120583L with asplit mode at a ratio of 1 20 The injection port temperaturewas set to 230∘C and the detectorinterface temperature wasset to 250∘C The results were collected for 40 minutes Thetotal ion chromatogram obtained was autointegrated usingChemStation and the chemical compounds or componentswere analysed by comparison with the suppliedmass spectraldatabase (NIST 05 Mass Spectral Library USA)

217 Statistical Analysis All data for each test are the averageof triplicate experiments for comparison of values and wererecorded as the mean plusmn standard deviation using MicrosoftExcel software and statistical data analyses were performedusing SPSS software

3 Results

31 Extraction Theextracts were concentrated using a rotaryevaporator (Buchi USA) under reduced pressure at 35∘CThe

yield of extracts from P macrocarpa seeds is shown in Table 1The highest yield from P macrocarpa seeds was the hexanefraction (947)

32 Preliminary Phytochemical Studies In order to determinethe types of phytoorganic constituents present in P macro-carpa seeds a preliminary phytochemical investigation wascarried out according to conventional methods The resultsobtained from these experiments are summarized in Table 2

The phytochemical tests showed that there were sec-ondary metabolites including carbohydrate flavonoids gly-cosides saponin glycosides phenolic compounds steroidstannins and terpenoids present in different extracts ofP macrocarpa seeds Small amounts of alkaloids 120572-aminoacids cyanogenic glycosides organic acids reducing sugarsand starches were also found to be present

The main constituents such as flavonoids glycosidessaponin glycosides phenolic compounds steroids tanninsand terpenoids present in P macrocarpa seeds may con-tribute to the presence of bioactivities such as antibacterialan analgesic an antifungal an anti-inflammatory agentand cytotoxicity Moreover the toxic chemical constituentscyanogenic glycosides were also present in the seeds

33 Physicochemical Studies The determination of nutri-tional values such as moisture ash fiber protein fat andcarbohydrate contents was carried out using the AOACmethod as well as the Lane and Eynon titration method andthe results obtained are shown in Figure 2(a) and Table 3Different physiochemical parameters for the purpose ofstandardization such as moisture (631 plusmn 143) ash (296 plusmn186) protein (2073 plusmn 244) crude fiber (2276 plusmn 279)crude fat (184 plusmn 311) and carbohydrate (2934 plusmn 198)were determined

34 Flavonoid Flavonol and Phenolic Determinations It wasfound that the methanol extract had the highest flavonoidcontent (933 plusmn 08mgmL) followed by ethyl acetate waterchloroform and hexane fractions which were 838 plusmn 10808 plusmn 03 678 plusmn 11 and 418 plusmn 15mgmL respectively

Themethanol extract (893plusmn11mgmL) of Pmacrocarpaseeds exhibited the highest amount of total flavonol contentfollowed by water ethyl acetate chloroform and hexanefractions which were 893 plusmn 10 613 plusmn 05 429 plusmn 09 and429 plusmn 07mgmL respectively

The total phenolic content of the P macrocarpa seedsextracts and fractions was expressed as gallic acid equiva-lents The methanol extract of P macrocarpa seeds exhibitedthe highest amount (720 plusmn 07mgmL) of total phenolicsfollowed by water ethyl acetate chloroform and hexanefractions which were 512 plusmn 14 358 plusmn 11 326 plusmn 10 and263 plusmn 01mgmL respectively

These data are shown in Figure 2(b) and Table 4

35 DPPH (2 2-Diphenyl-1-picryl-hydazyl) AntioxidantAssay DPPH assay was used to determine the free radicalscavenging ability of extracts and fractions of P macrocarpaseeds and to determine the antioxidant activity of its

6 BioMed Research International

Table 2 Results of chemical constituents of P macrocarpa seeds

No Tests Reagents Observation

1 AlkaloidsWagnerrsquos reagentMayerrsquos reagentDragendorff rsquos reagentSodium picrate solution

++++

2 120572-Amino acids Ninhydrin reagent +3 Carbohydrates 10 120572-naphthol and conc H2SO4 ++4 Cyanogenic glycosides Sodium picrate solution +5 Flavonoids Mg and conc HCl ++6 Glycosides 10 lead acetate ++7 Organic acids Bromothymol blue +8 Phenolic compounds 1 FeCl3 ++

9 Reducing sugars Fehlingrsquos solutionsA and B +

10 Saponin glycosides Distilled water ++11 Starch Iodine solution +12 Steroids Acetic anhydride and conc H2SO4 ++13 Tannins 1 Gelatin ++14 Terpenoids Acetic anhydride and conc H2SO4 +++++ large amount ++ medium amount + small amount minus absent + present

0

5

10

15

20

25

30

35

Con

tent

()

Moi

sture

Ash

Prot

ein

Crud

e fibe

r

Carb

ohyd

rate

Crud

e fat

(a)

123456789

1011

ME HF CF EAF WF

FlavonoidsFlavonolPhenolic

Con

cent

ratio

n (m

gm

L)

(b)

Figure 2 (a)The percentage ofmoisture ash protein crude fiber crude fat and carbohydrate contents on PmacrocarpaBoerl seeds (b) Totalamount of concentration of flavonoid flavonol and phenolic contents of P macrocarpa seeds ME methanol extract HF hexane fractionEAF ethyl acetate fraction CF chloroform fractionWF water fraction

phytoconstituents It is important to note that a lower IC50

value equals a higher scavenging activity The scavengingactivity was presented as the percentage of inhibitionof DPPH free radicals (Figure 3 and Table 4) Based onIC50

values the samples can be ranked in the followingdescending order water fraction gt methanol extract gt ethylacetate fraction gt hexane fraction gt chloroform fractionTheresults revealed that the water fraction was most active forantioxidant activity compared to other fractions The radical

scavenging effect was found to increase with increasingconcentrations

36 Cytotoxicity Screening of MTT Cell Proliferation AssayThe methanol and fractionated extracts (hexane chloro-form ethyl acetate and water) were investigated for cyto-toxic effects in human cervical adenocarcinoma cells (CaSki) humanhormone-dependent breast carcinoma (MCF-7)

BioMed Research International 7

Table 3The percentage of moisture ash protein crude fiber crudefat and carbohydrate contents and the amount of nutrition valueson P macrocarpa seeds

Tests parameter Percentage of contents ()Moisture 631 plusmn 143Ash 296 plusmn 186Protein 2073 plusmn 244Crude fiber 2276 plusmn 279Crude fat 1840 plusmn 311Carbohydrate 2934 plusmn 198

0102030405060708090

100

1 5 10 15 20

ME

HF

CF

EAF

WF

Concentration (mgmL)

Inhi

bitio

n (

)

Figure 3 Percentage of inhibition of DPPH free radical scavengingof P macrocarpa seeds ME methanol extract HF hexane fractionEAF ethyl acetate fraction CF chloroform fractionWF waterfraction

human colon adenocarcinoma cells (HT-29) human ovar-ian carcinoma cells (SKOV-3) human hormone-dependentbreast carcinoma (MDA-MB231) and normal cells (MRC-5) using MTT cells proliferation assay at 24 hrs 48 hrs and72 hrs respectively

The methanol extract of P macrocarpa seeds showedexcellent cytotoxic effects with IC

50value of 82plusmn 466 120583gmL

in Ca Ski cells at 24 hrs and 120 plusmn 22 85 plusmn 168 and30 plusmn 250 120583gmL in MCF-7 cells at 24 hrs 48 hrs and72 hrs respectively The methanol extract also showed goodcytotoxic activity in HT-29 cells with IC

50values of 293 plusmn

226 250 plusmn 135 and 215 plusmn 330 120583gmL at 24 hrs 48 hrsand 72 hrs respectively in Ca Ski cells with IC

50values of

197 plusmn 092 120583gmL and in SKOV-3 cells with IC50

values of165 plusmn 252 221 plusmn 247 and 360 plusmn 355 120583gmL at 24 hrs48 hrs and 72 hrs respectively However this extract had nocytotoxic effect in MDA-MB231 cells with IC

50gt100120583gmL

and the extract also had low cytotoxic effect in the MRC-5cells with IC

50gt 50 120583gmL

The hexane fraction of P macrocarpa seeds showedmoderate cytotoxic effects with IC

50values of 452 plusmn 149

and 555 plusmn 197 120583gmL at 24 hrs and 48 hrs on MCF-7 400 plusmn315 120583gmL inHT-29 cells at 24 hrs and 405plusmn352 and 500plusmn302 120583gmL in SKOV-3 at 24 hrs and 48 hrs In addition the

hexane fraction also displayed low cytotoxic effect in MCF-7at 72 hrs HT-29 at 48 hrs and 72 hrs and in SKOV-3 at 72 hrsThe IC

50value of treatment with hexane fraction in MCF-7

was 725plusmn152 120583gmL for 72 hrs exposure IC50values in HT-

29 were 640plusmn203 and 750plusmn314 120583gmL at 48 hrs and 72 hrswhile IC

50value in SKOV-3 was 703 plusmn 353 120583gmL at 72 hrs

In contrast the hexane fraction exhibited no cytotoxic effectsin MDA-MB231 cells or MRC-5 cells

The chloroform fraction of P macrocarpa seeds exhibitedthe highest cytotoxic effect with IC

50value of 100plusmn131 82plusmn

104 and 220 plusmn 186 120583gmL at 24 hrs 48 hrs and 72 hrs in CaSki cells 95plusmn295 87plusmn159 and 210plusmn198 120583gmL at 24 hrs48 hrs and 72 hrs on HT29 and 248 plusmn 206 165 plusmn 321 and900 plusmn 26 120583gmL in SKOV-3 cells The chloroform fractionexhibitedmoderate cytotoxic effect inMCF-7 with IC

50value

of 575 plusmn 264 400 plusmn 148 and 465 plusmn 345 120583gmL for 24 hrs48 hrs and 72 hrs respectively In contrast the chloroformfraction had no cytotoxic effect against theMDA-MB231 cellsand normal MRC-5 cells with IC

50gt100 120583gmL

The ethyl acetate fraction ofPmacrocarpa seeds exhibitedthe highest cytotoxic effect with IC

50lt 25 120583gmL in SKOV-3

cellsMDA-MB231 cellsMCF-7 cells andCa Ski cells On theother hand the ethyl acetate fraction exhibited low cytotoxiceffect in MRC-5 normal cells with IC

50value of 35 120583gmL

The ethyl acetate fraction exhibited the highest cytotoxiceffect in all selected cells (Ca Ski MCF-7 HT-29 and MDA-MB231) The water extract showed no cytotoxic effect againstall selected cancer cell lines with IC

50gt 100 120583gmL and

exhibited a mild cytotoxic effects against MRC-5 cells Thecorresponding data is shown in Figure 4 and Table 5

37 Characterization and Identification of Hexane and Chlo-roform Fractions Six compounds were identified from thehexane fraction of P macrocarpa seeds using GC-MS Theywere methyl stearate oleic acid methyl oleate linoleic acidmethyl linolenate and palmitic acid Moreover a GCMSanalysis of the chloroform fraction of P macrocarpa seedsshowed the presence of methyl myristate palmitic acidmethyl oleate methyl linoleate oleic acid and (119911)- and917-octadecadienal (119911) A comparison with the NIST massspectral library (NIST 05MS library 2002) andAdams (2001)confirmed the identity of the compounds The chemicalstructures of the identified compounds in the hexane andchloroform fractions of P macrocarpa seeds are as shown inFigure 5

4 Discussion

A longer shelf life can be achieved by reducing moisturecontent Thus moisture content is a critical factor for thestability of an extractMoisture enhances fungal and bacterialgrowth therefore decreasing the longevity of the extract Thetime it takes for plant material to deteriorate depends on howmuch water the plant material contains

Carbohydrates play a vital role in the immune sys-tem fertilization pathogenesis blood clotting and humandevelopment Foods that contain carbohydrates can raiseblood glucose and the three main types of carbohydrate are

8 BioMed Research International

0102030405060708090

100

ME

HF

CF

EAF

24h 48h 72h

IC50

valu

e of c

once

ntra

tion

(120583g

mL)

Ca Ski

0102030405060708090

100

ME

HF

CF

EAF

24h 48h 72h

IC50

valu

e of c

once

ntra

tion

(120583g

mL)

MCF 7

0102030405060708090

100

ME

HF

CF

EAF

24h 48h 72h

IC50

valu

e of c

once

ntra

tion

(120583g

mL)

HT-29

minus10

0

10

20

30

40

50

60

70

80

90

100

ME

HF

CF

EAF

24h 48h 72h

IC50

valu

e of c

once

ntra

tion

(120583g

mL)

SKOV-3

0102030405060708090

100

ME

HF

CF

EAF

24h 48h 72h

IC50

valu

e of c

once

ntra

tion

(120583g

mL)

MDA-MB231

Figure 4 In vitro cytotoxic effects of P macrocarpa seedson Ca Ski MCF-7 HT-29 SKOV-3 and MDA-MB231 cell lines ME methanolextract HF hexane fraction EAF ethyl acetate fraction CF chloroform fractionWF water fraction Each value is expressed as mean plusmnstandard deviation of three measurements

BioMed Research International 9

Methyl linoleate

Methyl stearate

Palmitic acid

Oleic acid

Methyl myristate

Linoleic acid

Methyl oleate

H3C

H3C

H3C

H3C

H3C

H3C

H3C

O

O

O

O

O

O

O

O

O

O

O

CH3

CH3

CH3

CH3

OH

OH

OH

(294mz)

(298mz)

(282mz)

(242mz)

(280mz)

(298mz)

(256mz)

Figure 5

10 BioMed Research International

Table 4 Total flavonoid total flavonol total phenolic contents and result of DPPH free radical scavenging property of P macrocarpa seeds

Tests ME (mgmL) HF (mgmL) CF (mgmL) EAF (mgmL) WF (mgmL)Flavonoid 933 plusmn 08 418 plusmn 15 678 plusmn 11 838 plusmn 03 808 plusmn 10Flavonol 893 plusmn 11 425 plusmn 07 429 plusmn 09 613 plusmn 05 818 plusmn 10Phenolic 720 plusmn 07 263 plusmn 01 326 plusmn 1 358 plusmn 11 512 plusmn 14DPPH 1150 plusmn 003 1500 plusmn 004 1575 plusmn 004 1400 plusmn 005 975 plusmn 003ME methanol extract HF hexane fraction EAF ethyl acetate fraction CF chloroform fraction WF water fraction

Table 5 In vitro cytotoxic effects of methanol extract and its fractions of P macrocarpa cancer cells lines and normal human fibroblast breastcells line

Cell line Incubation periods (hours) ME HF CF EAF Doxorubicin24 82 plusmn 466 ge100 100 plusmn 131 56 plusmn 117 092 plusmn 064

Ca Ski 48 197 plusmn 092 ge100 82 plusmn 104 77 plusmn 185 069 plusmn 10572 407 plusmn 226 ge100 220 plusmn 186 60 plusmn 322 045 plusmn 09924 120 plusmn 22 452 plusmn 149 575 plusmn 264 223 plusmn 158 105 plusmn 108

MCF7 48 85 plusmn 168 555 plusmn 197 400 plusmn 148 168 plusmn 170 012 plusmn 06972 63 plusmn 250 725 plusmn 152 465 plusmn 345 84 plusmn 171 092 plusmn 17024 293 plusmn 226 400 plusmn 315 95 plusmn 295 11 plusmn 120 092 plusmn 072

HT29 48 250 plusmn 135 640 plusmn 203 87 plusmn 159 35 plusmn 200 032 plusmn 21672 215 plusmn 330 750 plusmn 314 210 plusmn 198 12 plusmn 228 088 plusmn 09424 165 plusmn 252 405 plusmn 352 248 plusmn 206 68 plusmn 18 102 plusmn 179

SKOV-3 48 221 plusmn 247 500 plusmn 302 165 plusmn 321 65 plusmn 23 032 plusmn 09772 360 plusmn 355 703 plusmn 353 90 plusmn 26 32 plusmn 281 062 plusmn 09824 ge100 ge100 ge100 65 plusmn 23 043 plusmn 050

MDA-MB231 48 ge100 ge100 ge100 15 plusmn 343 059 plusmn 10172 ge100 ge100 ge100 186 plusmn 126 095 plusmn 132

ME methanol extract HF hexane fraction EAF ethyl acetate fraction CF chloroform fractionWF water fraction Doxorubicin was used as positive controlEach value is expressed as mean plusmn standard deviation of three measurements

starches sugars and fiber A macronutrient protein consistsof amino acids which is required for proper growth andhuman body function The seeds were found to containa high level of crude fiber which is a potential source ofphenolic antioxidants The major components of fibers arecellulose hemicelluloses lignin 120573-glucans gums and pectinand hydrocolloids These components can behave as proan-tioxidants [28 29] In our present physicochemical study Pmacrocarpa seeds were mainly made up of carbohydratesfollowed by crude fiber proteins fatmoisture and ashHigh-protein and low-carbohydrate diets are often effective forweight loss Dietary fiber can aid in digestion is helpfulfor weight management and reduces constipation Howevermany high-protein and low-carbohydrate foods are low infiberWomen aged 50 and older need at least 21 grams of fiberdaily whilst those aged 19 to 50 require 25 grams men aged50 and older need 30 grams and those between the age of 19to 50 should consume at least 38 grams of fiber

Steroids possess biological activities which are insec-ticidal cardiotonic and antimicrobial activities and theseare potentially useful for development into therapeuticdrugs Tannins are known to be important for their antivi-ral antibacterial antiparasitic effects anti-inflammatoryantiulcer and antioxidant properties [30ndash33] Saponinshave been found to have antimicrobial anti-inflammatoryantifeedant and hemolytic effects [34 35] Some alkaloids

have been reported to have anticancer and antiviral activityand saponins have been reported to be cardiotonic whileflavonoids have anticancer and anti-inflammatory activity[10 36] The presence of tannins may be responsible for theability of P macrocarpa seeds to be used in the treatmentof diseases such as diabetes diarrhea and dysentery Ourpreliminary phytochemical studies revealed that the chemicalcomponents of P macrocarpa seeds also include flavonoidsphenolics steroids tannins terpenoids glucosides saponinsand carbohydrates

Previous researchers reported that kaempferol myrice-tin naringin quercetin rutin [3] 29-norcucurbitacinderivatives fevicordin A fevicordin A glucoside fevicordinD glucoside [6] mahkoside A dodecanoic acid palmiticacid des-acetyl flavicordin-A flavicordin-A flavicordin-Dflavicordin-A glucoside ethyl stearate lignans and sucrose[37 38] mangiferin (a C-glucosylxantone) kaempferol-3-o-120573-D-glucoside dodecanoic acid palmitic acid ethyl stearatesucrose [5] pinoresinol lariciresinol matairesinol alkaloidsand saponins [37 39] saponins alkaloids polyphenolicsphenols flavanoids lignans tannins [40ndash42] icarisideC3 mangiferin gallic acid phalerin glycoside (345trihydroxy-4-methoxy-benzophenone-3-O-120573-D-glucoside)[43] and 2410158406 trihydroxy-4-methoxy-benzophenone-3-O-120573-D-glucoside [44] were isolated from different partsof P macrocarpa The hexane extract of the seeds was

BioMed Research International 11

found to contain methyl myristate methyl stearate oleicacid methyl oleate linoleic acid methyl linoleate palmiticacid methyl palmitate 6-octadecenoic acid and 917-octadecadial 120573-sitosterol as major components A further30 to 40 of components were not identified as thesewere polar compounds that require HPLC analysis Othercompounds reported in the literature for the seeds leavesand fruits were not identified in this investigation It isprobable that the components reported in the literaturemay be present in other fractions (methyl acetate CHCl

3

and water fractions)Phenolics are able to transfer protons to radicals [45]

DPPH which provides free radicals is normally a blue-violet color However the DPPH turned yellow after it wasconverted to 1 1-diphenyl-2-picrylhydrazine which has lessfree-radical activity Hendra et al reported that the IC

50

values of free radical and reducing power in methanolwith HCl of P macrocarpa seeds were 2450 plusmn 194 and1502 plusmn 128 [42] In our investigation using DPPH assay thewater fraction displayed the highest IC

50value followed by

methanol ethyl acetate hexane and chloroform fractionThe presence of flavonoids in P macrocarpa seeds may be

responsible for the traditional use of the plants in treatingcancer inflammations and allergies Recently it has beenshown that phenolic compounds in crude methanol andethyl acetate extract of P macrocarpa leaves displayed goodantioxidant and antimicrobial activites [46] Hendra et al(2011) examined the amount of total phenolic (477plusmn 104mggallic acid equivalentg DW) and the amount of flavonoid(359 plusmn 247mg rutin equivalentg DW) contents in themethanol extract with HCl of P macrocarpa seeds [42] Inthis study the total flavonoids content was higher than that inthe results previously reported by Rohyami [47] and phenolflavonol and flavonoid contents were the highest in theaqueous methanol extract followed by ethyl acetate waterchloroform and hexane extracts

MTT assay is a sensitive and reliable colorimetric assaythat uses quantitative measurements to calculate the via-bility proliferation and activation of cells This method iscommonly applied to screen anticancer agents [48] Themost well-known methods used to calculate cytotoxicityare the neutral red (NR) uptake and dimethylthiazole-diphenyl tetrazolium bromide (MTT) metabolism [27 49]Earlier investigations showed that the ethanol extract of Pmacrocarpa seeds and fruit meat were not toxic to normalhuman cells but slightly toxic to a Vero cell line [9] Theethanol extract of P macrocarpa seed fruit also showedtoxicity towards T47D breast cancer cell line through COX-2 expression inhibition [7] Desacetylfevicordin A has beenisolated from the ethyl acetate extract of P macrocarpaseeds and this compound displayed excellent cytotoxicityin brine shrimp [43] The ethyl acetate of P macrocarpaseeds exhibited mild cytotoxic effect against HepG2 cells(IC50values between 30 and 60 120583gmL) [46] Previous studies

showed that the cytotoxic activity of the seeds on HT-29MCF-7 Hela and Chang liver cells lines were 384 plusmn 037255 plusmn 137 295 plusmn 10 and 678 plusmn 027 receptively [42] Inour study the methanolic extract of P macrocarpa seeds wasfound to pose cytotoxic effect against HT-29 MCF-7 Cas Ki

and SKOV-3 cell lines (IC50

values giving from 11 plusmn 120to 360 plusmn 355) and mild toxicity on normal cell lines Therewere significant cytotoxic effects on selected cancer cells linesthat were both time- and dose-dependent manner due to thepresence of many secondary metabolites

The phytochemical screening also indicated the presenceof a small amount of cyanogenic glycosides Cyanogenicglucosides which can cause acute cyanide poisoning causerapid respiration and pulse decrease the blood pressure andinduce vomiting diarrhea headache dizziness and so onThus these components have to be removed (usually byboiling) prior to consumption for therapeutic purposes

5 Conclusion

P macrocarpa seeds exhibited antioxidant and cytotoxicactivities It is highly probable that these activities are dueto the presence of phenolic and flavonoid compounds inappreciable amounts in the plant Furthermore the cytotox-icity activity suggested that the seed may contain a potentialanticancer agent The outcome of this study is encouragingdemonstrating the potential for the P macrocarpa as a sourceof multiple therapeutic agents

Abbreviations

EAF Ethyl acetate fractionME Methanol extractHF Hexane fractionCF Chloroform fractionWF Water fractiong Grammg MilligrammL Milliliter

Conflict of Interests

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

Authorsrsquo Contribution

Ma Ma Lay was involved in all experiments and BehroozBanisalamwas involved in antioxidant assay All authors readand approved the submitted version of the paper

Acknowledgments

This work was supported by grants from the MalaysianMinistry of Higher Education (nos PS2862009C andPS4702010B) Ma Ma Lay is a scholarship recipient fromthe ldquoIslamic Development Bank Saudi Arabiardquo The authorswould like to thank Professor Dr Amru BinNasrulhaq Boycefor generously allowing the use of his laboratory for thisresearch

12 BioMed Research International

References

[1] N HarmantoMahkota DewaObat Pusaka ParaDewa Cetakankeempat Agromedia Pustaka Jakarta Indonesia 2002

[2] A Jemal R Siegel E Ward T Murray J Xu and M J ThunldquoCancer statistics 2007rdquoCa ACancer Journal for Clinicians vol57 no 1 pp 43ndash66 2007

[3] R Hendra S Ahmad A Sukari M Y Shukor and EOskoueian ldquoFlavonoid analyses and antimicrobial activity ofvarious parts of Phaleria macrocarpa (Scheff) Boerl fruitrdquoInternational Journal of Molecular Sciences vol 12 no 6 pp3422ndash3431 2011

[4] W Wijayani Sitotoksisitas Minyak Atsiri Biji Dan Daging BuahMahkota Dewa (Phaleria Macrocarpa [Scheff] Boerl) TerhadapMyeloma Cell Line Dan HeLa Cell Line Serta IdentifikasiSenyawa Minyak Atsiri Dengan Kromatografi Gas-SpektrometriMassa (GC-MS) Skripsi FMIPA UGM Yogyakarta Indonesia2005

[5] Y-B Zhang X-J Xu and H-M Liu ldquoChemical con-stituents fromMahkotadewardquo Journal of Asian Natural ProductsResearch vol 8 no 1-2 pp 119ndash123 2006

[6] D Kurnia K Akiyama and H Hayashi ldquo29-Norcucurbitacinderivatives isolated from the Indonesian medicinal plant Pha-leria macrocarpa (Scheff) Boerlrdquo Bioscience Biotechnology andBiochemistry vol 72 no 2 pp 618ndash620 2008

[7] M Bakhriansyah Pengaruh Ekstrak Etanol Biji Mahkota Dewa(Phaleria Macrocarpa [Scheff] Boerl) Pada Sel Kanker Payu-dara T47D Kajian Aktivitas Sitotoksik Dan PenghambatanEkspresi Siklooksigenase-2 Program Pascasarjana UGM 2004

[8] L Wardani The Effect of Phaleria Macrocarpa [Scheff] Boerl)Seed and Flesh Fruit Extract to P53 and Bcl-2 Gene Expression inHela Cell Line FMIPA UGM 2005

[9] A Endang R Tri Joko and E Dewi ldquoCyototoxicity of Phaleriamacrocarpa (Scheff) boerl fruits flesh and seed extract ofethanol and its effect against p53 and Bcl-2 genes expressionof normalrdquo in Proceedings of the International Conference onComputational Science (ICCS rsquo07) 2007

[10] C Evans Trease and Evans Pharmacognosy Saunder 2002[11] A Sofowora Medicinal Plants and Traditional Medicine in

Africa Spectrum Books Ltd Ibadan Nigeria 2nd edition 1993[12] AOAC Official Methods of Analysis Association of Official

Analytical Chemists Arlington Tex USA 2002[13] J Harborne Phytochemical Methods Chapman and Hall Lon-

don UK 1973[14] AOAC Official Methods of Analysis Association of Official

Analytical Chemists Gaithersburg Md USA 17th edition2000

[15] J Lako V C Trenerry M Wahlqvist N WattanapenpaiboonS Sotheeswaran and R Premier ldquoPhytochemical flavonolscarotenoids and the antioxidant properties of a wide selection ofFijian fruit vegetables and other readily available foodsrdquo FoodChemistry vol 101 no 4 pp 1727ndash1741 2007

[16] W Horwitz Official Method of Analysis AOAC InterantionalGaithersburg Md USA 17th edition 2000

[17] A A Adedapo F O Jimoh S Koduru P J Masika and AJ Afolayan ldquoAssessment of the medicinal potentials of themethanol extracts of the leaves and stems of Buddleja salignardquoBMCComplementary andAlternativeMedicine vol 9 article 212009

[18] M Zovko Koncic D Kremer J Gruz et al ldquoAntioxidantand antimicrobial properties ofMoltkia petraea (Tratt) Griseb

flower leaf and stem infusionsrdquo Food and Chemical Toxicologyvol 48 no 6 pp 1537ndash1542 2010

[19] S McDonald P D Prenzler M Antolovich and K RobardsldquoPhenolic content and antioxidant activity of olive extractsrdquoFood Chemistry vol 73 no 1 pp 73ndash84 2001

[20] S M Nabavi M A Ebrahimzadeh S F Nabavi A Hamidiniaand A R Bekhradnia ldquoDetermination of antioxidant activityphenol and flavonoid content of Parrotia persicaMeyrdquo Pharma-cologyonline vol 2 pp 560ndash567 2008

[21] M A Ebrahimzadeh S F Nabavi and S M Nabavi ldquoAntiox-idant activities of methanol extract of Sambucus ebulus Lflowerrdquo Pakistan Journal of Biological Sciences vol 12 no 5 pp447ndash450 2009

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

[23] A Kumaran and R Joel Karunakaran ldquoIn vitro antioxidantactivities of methanol extracts of five Phyllanthus species fromIndiardquo Food Science and Technology vol 40 no 2 pp 344ndash3522007

[24] B O Mbaebie H O Edeoga and A J Afolayan ldquoPhy tochemical analysis and antioxidants activities of aqueous stembark extract of Schotia latifolia Jacqrdquo Asian Pacific Journal ofTropical Biomedicine vol 2 no 2 pp 118ndash124 2012

[25] L I Mensor F S Menezes G G Leitao et al ldquoScreening ofBrazilian plants extracts for antioxidants activity by the use ofDPPH free radical methodrdquo Phytotherapy Research vol 15 pp127ndash130 2001

[26] F Denizot and R Lang ldquoRapid colorimetric assay for cellgrowth and survival modifications to the tetrazolium dyeprocedure giving improved sensitivity and reliabilityrdquo Journalof Immunological Methods vol 89 no 2 pp 271ndash277 1986

[27] T Mosmann ldquoRapid colorimetric assay for cellular growth andsurvival application to proliferation and cytotoxicity assaysrdquoJournal of Immunological Methods vol 65 no 1-2 pp 55ndash631983

[28] P Maisuthisakul R Pongsawatmanit and M H GordonldquoAntioxidant properties of Teaw (Cratoxylum formosum Dyer)extract in soybean oil and emulsionsrdquo Journal of Agriculturaland Food Chemistry vol 54 no 7 pp 2719ndash2725 2006

[29] P Maisuthisakul S Pasuk and P Ritthiruangdej ldquoRelationshipbetween antioxidant properties and chemical composition ofsome Thai plantsrdquo Journal of Food Composition and Analysisvol 21 no 3 pp 229ndash240 2008

[30] H Kolodziej and A F Kiderlen ldquoAntileishmanial activity andimmune modulatory effects of tannins and related compoundson Leishmania parasitised RAW 2647 cellsrdquo Phytochemistryvol 66 no 17 pp 2056ndash2071 2005

[31] T Nakagawa and T Yokozawa ldquoDirect scavenging of nitricoxide and superoxide by green teardquo Food and Chemical Toxi-cology vol 40 no 12 pp 1745ndash1750 2002

[32] V Schulz R Hansel and V Tyler Fitoterapia Racional UmGuiade Fitoterapia Para as ciencias da saude Manole Barueri Brazil2002

[33] S D Kapu Y B Ngwai O Kayode P A Akah C Wambebeand K Gamaniel ldquoAnti-inflammatory analgesic and anti-lymphocytic activities of the aqueous extract of Crinum gigan-teumrdquo Journal of Ethnopharmacology vol 78 no 1 pp 7ndash132001

[34] G Francis Z Kerem H P S Makkar and K Becker ldquoThebiological action of saponins in animal systems a reviewrdquoBritish Journal of Nutrition vol 88 no 6 pp 587ndash605 2002

BioMed Research International 13

[35] R Xu W Zhao J Xu B Shao and G Qin ldquoStudies onbioactive saponins fromChinesemedicinal plantsrdquoAdvances inExperimental Medicine and Biology vol 404 pp 371ndash382 1996

[36] J A Manthey N Guthrie and K Grohmann ldquoBiologicalproperties of citrus flavonoids pertaining to cancer and inflam-mationrdquo Current Medicinal Chemistry vol 8 no 2 pp 135ndash1532001

[37] P Hendra Y Fukushi and Y Hashidoko ldquoSynthesis of ben-zophenone glucopyranosides from Phaleria macrocarpa andrelated benzophenone glucopyranosidesrdquo Bioscience Biotech-nology and Biochemistry vol 73 no 10 pp 2172ndash2182 2009

[38] A Rabia Z B A Mohammad D Aidiahmad S Amirin and IU Muhammad ldquoPhytochemistry and medicinal properties ofPhaleria macrocarpa (Scheff) Boerl extractsrdquo PharmacognosyReview vol 7 no 13 pp 73ndash80 2013

[39] A Saufi C B I VonHeimendahl AWAlfermann andE FussldquoStereochemistry of lignans in Phaleria macrocarpa (Scheff)Boerlrdquo Zeitschrift fur Naturforschung C vol 63 no 1-2 pp 13ndash16 2008

[40] S C Chong M A Dollah P P Chong and A Maha ldquoPhaleriamacrocarpa (Scheff) Boerl fruit aqueous extract enhances LDLreceptor and PCSK9 expression in vivo and in vitrordquo Journal ofEthnopharmacology vol 137 no 1 pp 817ndash827 2011

[41] R R Tjandrawinata D Nofiarny L W Susanto P Hendra andA Clarissa ldquoSymptomatic treatment of premenstrual syndromeandor primary dysmenorrhea with DLBS1442 a bioacitveextract ofPhaleriamacrocaraprdquo International Journal of GeneralMedicine vol 4 pp 465ndash476 2011

[42] R Hendra S Ahmad E Oskoueian A Sukari and M YShukor ldquoAntioxidant anti-inflammatory and cytotoxicity ofPhaleriamacrocarap (Boerl) Scheff fruitrdquoBMCComplementaryand Alternative Medicine vol 11 article 110 2011

[43] D Kurnia K Akiyama and H Hayashi ldquo29-Norcucurbitacinderivatives isolated from the Indonesian medicinal plant Pha-leria macrocarpa (Scheff) Boerlrdquo Bioscience Biotechnology andBiochemistry vol 72 no 2 pp 618ndash620 2008

[44] S Oshimi K Zaima Y Matsuno et al ldquoStudies on theconstituents from the fruits of Phaleria macrocaraprdquo Journal ofNatural Medicines vol 62 no 2 pp 207ndash210 2008

[45] C Sanchez-Moreno J A Larrauri and F Saura-Calixto ldquoAprocedure tomeasure the antiradical efficiency of polyphenolsrdquoJournal of the Science of Food and Agriculture vol 76 pp 270ndash276 1998

[46] Y Andriani M A W Effendy T M T Sifzizul and HMohamad ldquoAntibacterial radical-scavenging activities andcytotoxicit properties of Phaleria macrocarpa (Scheff) BoerlLeaves in HepG2 cell linesrdquo International Journal of Pharma-ceutical Sciences and Research vol 2 no 7 pp 1700ndash1706 2011

[47] Y Rohyami ldquoPenentuan Kandungan Flavonoid dari EkstrakMetanol Daging Buah Mahkota Dewa (Phaleria macrocarpaScheff Boerl)rdquo Jurnal Logika vol 5 2009

[48] R D Petty L A Sutherland E M Hunter and I A CreeldquoComparison of MTT and ATP-based assays for the measure-ment of viable cell numberrdquo Journal of Bioluminescence andChemiluminescence vol 10 no 1 pp 29ndash34 1995

[49] J R Sellers S Cook andV S Goldmacher ldquoA cytotoxicity assayutilizing a fluorescent dye that determines accurate survivingfractions of cellsrdquo Journal of Immunological Methods vol 172no 2 pp 255ndash264 1994

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

BioMed Research International 5

Table 1 Yield of methanol extract and its fractions from P macro-carpa of seedsExtract and fractions Yield (g) Percentage of yield ()Methanolic extract (ME) 1987 1199Hexane fraction (HF) 1587 947Chloroform fraction (CF) 72 043Ethyl acetate fraction (EAF) 62 037Water fraction (WF) Freeze dry

confluence each extract at concentrations of 1 10 25 50and 100 120583gmL (in 200120583L of 10 media) was added to therespective wells containing the cells Wells with untreatedcells were used as the negative control and cells exposedto doxorubicin were used as positive control After 24 48and 72 hours 10 120583L MTT stock solution was added to eachwell OD was then determined by measuring absorbance at540 nm using an ELISA microplate reader The percentageof inhibition () was calculated according to the followingformula

Percentage of inhibition ()

=OD control minusOD sample times 100

OD control

(8)

Cytotoxicity of each sample is expressed as plusmnIC50value The

extract that gave IC50

of 30 120583gmL or less was consideredactive [26]

216 Gas Chromatography-Mass Spectrometry (GC-MS) GC-MS was used to determine the molecular weight of thecomponents collected and the purity of the collected extractsRunning conditions were as follows oven temperature wasprogrammed with an initial temperature of 100∘C andincreased at a ramp rate of 5∘Cmin and reached a finaltemperature of 300∘C The carrier gas or mobile phase usedwas helium and a flow rate of 1mLmin was programmedThe mass spectrometry mode used was electron ionization(EI) mode with a current of 70 eV The injection mode wasprogrammed with a sample injection volume of 1120583L with asplit mode at a ratio of 1 20 The injection port temperaturewas set to 230∘C and the detectorinterface temperature wasset to 250∘C The results were collected for 40 minutes Thetotal ion chromatogram obtained was autointegrated usingChemStation and the chemical compounds or componentswere analysed by comparison with the suppliedmass spectraldatabase (NIST 05 Mass Spectral Library USA)

217 Statistical Analysis All data for each test are the averageof triplicate experiments for comparison of values and wererecorded as the mean plusmn standard deviation using MicrosoftExcel software and statistical data analyses were performedusing SPSS software

3 Results

31 Extraction Theextracts were concentrated using a rotaryevaporator (Buchi USA) under reduced pressure at 35∘CThe

yield of extracts from P macrocarpa seeds is shown in Table 1The highest yield from P macrocarpa seeds was the hexanefraction (947)

32 Preliminary Phytochemical Studies In order to determinethe types of phytoorganic constituents present in P macro-carpa seeds a preliminary phytochemical investigation wascarried out according to conventional methods The resultsobtained from these experiments are summarized in Table 2

The phytochemical tests showed that there were sec-ondary metabolites including carbohydrate flavonoids gly-cosides saponin glycosides phenolic compounds steroidstannins and terpenoids present in different extracts ofP macrocarpa seeds Small amounts of alkaloids 120572-aminoacids cyanogenic glycosides organic acids reducing sugarsand starches were also found to be present

The main constituents such as flavonoids glycosidessaponin glycosides phenolic compounds steroids tanninsand terpenoids present in P macrocarpa seeds may con-tribute to the presence of bioactivities such as antibacterialan analgesic an antifungal an anti-inflammatory agentand cytotoxicity Moreover the toxic chemical constituentscyanogenic glycosides were also present in the seeds

33 Physicochemical Studies The determination of nutri-tional values such as moisture ash fiber protein fat andcarbohydrate contents was carried out using the AOACmethod as well as the Lane and Eynon titration method andthe results obtained are shown in Figure 2(a) and Table 3Different physiochemical parameters for the purpose ofstandardization such as moisture (631 plusmn 143) ash (296 plusmn186) protein (2073 plusmn 244) crude fiber (2276 plusmn 279)crude fat (184 plusmn 311) and carbohydrate (2934 plusmn 198)were determined

34 Flavonoid Flavonol and Phenolic Determinations It wasfound that the methanol extract had the highest flavonoidcontent (933 plusmn 08mgmL) followed by ethyl acetate waterchloroform and hexane fractions which were 838 plusmn 10808 plusmn 03 678 plusmn 11 and 418 plusmn 15mgmL respectively

Themethanol extract (893plusmn11mgmL) of Pmacrocarpaseeds exhibited the highest amount of total flavonol contentfollowed by water ethyl acetate chloroform and hexanefractions which were 893 plusmn 10 613 plusmn 05 429 plusmn 09 and429 plusmn 07mgmL respectively

The total phenolic content of the P macrocarpa seedsextracts and fractions was expressed as gallic acid equiva-lents The methanol extract of P macrocarpa seeds exhibitedthe highest amount (720 plusmn 07mgmL) of total phenolicsfollowed by water ethyl acetate chloroform and hexanefractions which were 512 plusmn 14 358 plusmn 11 326 plusmn 10 and263 plusmn 01mgmL respectively

These data are shown in Figure 2(b) and Table 4

35 DPPH (2 2-Diphenyl-1-picryl-hydazyl) AntioxidantAssay DPPH assay was used to determine the free radicalscavenging ability of extracts and fractions of P macrocarpaseeds and to determine the antioxidant activity of its

6 BioMed Research International

Table 2 Results of chemical constituents of P macrocarpa seeds

No Tests Reagents Observation

1 AlkaloidsWagnerrsquos reagentMayerrsquos reagentDragendorff rsquos reagentSodium picrate solution

++++

2 120572-Amino acids Ninhydrin reagent +3 Carbohydrates 10 120572-naphthol and conc H2SO4 ++4 Cyanogenic glycosides Sodium picrate solution +5 Flavonoids Mg and conc HCl ++6 Glycosides 10 lead acetate ++7 Organic acids Bromothymol blue +8 Phenolic compounds 1 FeCl3 ++

9 Reducing sugars Fehlingrsquos solutionsA and B +

10 Saponin glycosides Distilled water ++11 Starch Iodine solution +12 Steroids Acetic anhydride and conc H2SO4 ++13 Tannins 1 Gelatin ++14 Terpenoids Acetic anhydride and conc H2SO4 +++++ large amount ++ medium amount + small amount minus absent + present

0

5

10

15

20

25

30

35

Con

tent

()

Moi

sture

Ash

Prot

ein

Crud

e fibe

r

Carb

ohyd

rate

Crud

e fat

(a)

123456789

1011

ME HF CF EAF WF

FlavonoidsFlavonolPhenolic

Con

cent

ratio

n (m

gm

L)

(b)

Figure 2 (a)The percentage ofmoisture ash protein crude fiber crude fat and carbohydrate contents on PmacrocarpaBoerl seeds (b) Totalamount of concentration of flavonoid flavonol and phenolic contents of P macrocarpa seeds ME methanol extract HF hexane fractionEAF ethyl acetate fraction CF chloroform fractionWF water fraction

phytoconstituents It is important to note that a lower IC50

value equals a higher scavenging activity The scavengingactivity was presented as the percentage of inhibitionof DPPH free radicals (Figure 3 and Table 4) Based onIC50

values the samples can be ranked in the followingdescending order water fraction gt methanol extract gt ethylacetate fraction gt hexane fraction gt chloroform fractionTheresults revealed that the water fraction was most active forantioxidant activity compared to other fractions The radical

scavenging effect was found to increase with increasingconcentrations

36 Cytotoxicity Screening of MTT Cell Proliferation AssayThe methanol and fractionated extracts (hexane chloro-form ethyl acetate and water) were investigated for cyto-toxic effects in human cervical adenocarcinoma cells (CaSki) humanhormone-dependent breast carcinoma (MCF-7)

BioMed Research International 7

Table 3The percentage of moisture ash protein crude fiber crudefat and carbohydrate contents and the amount of nutrition valueson P macrocarpa seeds

Tests parameter Percentage of contents ()Moisture 631 plusmn 143Ash 296 plusmn 186Protein 2073 plusmn 244Crude fiber 2276 plusmn 279Crude fat 1840 plusmn 311Carbohydrate 2934 plusmn 198

0102030405060708090

100

1 5 10 15 20

ME

HF

CF

EAF

WF

Concentration (mgmL)

Inhi

bitio

n (

)

Figure 3 Percentage of inhibition of DPPH free radical scavengingof P macrocarpa seeds ME methanol extract HF hexane fractionEAF ethyl acetate fraction CF chloroform fractionWF waterfraction

human colon adenocarcinoma cells (HT-29) human ovar-ian carcinoma cells (SKOV-3) human hormone-dependentbreast carcinoma (MDA-MB231) and normal cells (MRC-5) using MTT cells proliferation assay at 24 hrs 48 hrs and72 hrs respectively

The methanol extract of P macrocarpa seeds showedexcellent cytotoxic effects with IC

50value of 82plusmn 466 120583gmL

in Ca Ski cells at 24 hrs and 120 plusmn 22 85 plusmn 168 and30 plusmn 250 120583gmL in MCF-7 cells at 24 hrs 48 hrs and72 hrs respectively The methanol extract also showed goodcytotoxic activity in HT-29 cells with IC

50values of 293 plusmn

226 250 plusmn 135 and 215 plusmn 330 120583gmL at 24 hrs 48 hrsand 72 hrs respectively in Ca Ski cells with IC

50values of

197 plusmn 092 120583gmL and in SKOV-3 cells with IC50

values of165 plusmn 252 221 plusmn 247 and 360 plusmn 355 120583gmL at 24 hrs48 hrs and 72 hrs respectively However this extract had nocytotoxic effect in MDA-MB231 cells with IC

50gt100120583gmL

and the extract also had low cytotoxic effect in the MRC-5cells with IC

50gt 50 120583gmL

The hexane fraction of P macrocarpa seeds showedmoderate cytotoxic effects with IC

50values of 452 plusmn 149

and 555 plusmn 197 120583gmL at 24 hrs and 48 hrs on MCF-7 400 plusmn315 120583gmL inHT-29 cells at 24 hrs and 405plusmn352 and 500plusmn302 120583gmL in SKOV-3 at 24 hrs and 48 hrs In addition the

hexane fraction also displayed low cytotoxic effect in MCF-7at 72 hrs HT-29 at 48 hrs and 72 hrs and in SKOV-3 at 72 hrsThe IC

50value of treatment with hexane fraction in MCF-7

was 725plusmn152 120583gmL for 72 hrs exposure IC50values in HT-

29 were 640plusmn203 and 750plusmn314 120583gmL at 48 hrs and 72 hrswhile IC

50value in SKOV-3 was 703 plusmn 353 120583gmL at 72 hrs

In contrast the hexane fraction exhibited no cytotoxic effectsin MDA-MB231 cells or MRC-5 cells

The chloroform fraction of P macrocarpa seeds exhibitedthe highest cytotoxic effect with IC

50value of 100plusmn131 82plusmn

104 and 220 plusmn 186 120583gmL at 24 hrs 48 hrs and 72 hrs in CaSki cells 95plusmn295 87plusmn159 and 210plusmn198 120583gmL at 24 hrs48 hrs and 72 hrs on HT29 and 248 plusmn 206 165 plusmn 321 and900 plusmn 26 120583gmL in SKOV-3 cells The chloroform fractionexhibitedmoderate cytotoxic effect inMCF-7 with IC

50value

of 575 plusmn 264 400 plusmn 148 and 465 plusmn 345 120583gmL for 24 hrs48 hrs and 72 hrs respectively In contrast the chloroformfraction had no cytotoxic effect against theMDA-MB231 cellsand normal MRC-5 cells with IC

50gt100 120583gmL

The ethyl acetate fraction ofPmacrocarpa seeds exhibitedthe highest cytotoxic effect with IC

50lt 25 120583gmL in SKOV-3

cellsMDA-MB231 cellsMCF-7 cells andCa Ski cells On theother hand the ethyl acetate fraction exhibited low cytotoxiceffect in MRC-5 normal cells with IC

50value of 35 120583gmL

The ethyl acetate fraction exhibited the highest cytotoxiceffect in all selected cells (Ca Ski MCF-7 HT-29 and MDA-MB231) The water extract showed no cytotoxic effect againstall selected cancer cell lines with IC

50gt 100 120583gmL and

exhibited a mild cytotoxic effects against MRC-5 cells Thecorresponding data is shown in Figure 4 and Table 5

37 Characterization and Identification of Hexane and Chlo-roform Fractions Six compounds were identified from thehexane fraction of P macrocarpa seeds using GC-MS Theywere methyl stearate oleic acid methyl oleate linoleic acidmethyl linolenate and palmitic acid Moreover a GCMSanalysis of the chloroform fraction of P macrocarpa seedsshowed the presence of methyl myristate palmitic acidmethyl oleate methyl linoleate oleic acid and (119911)- and917-octadecadienal (119911) A comparison with the NIST massspectral library (NIST 05MS library 2002) andAdams (2001)confirmed the identity of the compounds The chemicalstructures of the identified compounds in the hexane andchloroform fractions of P macrocarpa seeds are as shown inFigure 5

4 Discussion

A longer shelf life can be achieved by reducing moisturecontent Thus moisture content is a critical factor for thestability of an extractMoisture enhances fungal and bacterialgrowth therefore decreasing the longevity of the extract Thetime it takes for plant material to deteriorate depends on howmuch water the plant material contains

Carbohydrates play a vital role in the immune sys-tem fertilization pathogenesis blood clotting and humandevelopment Foods that contain carbohydrates can raiseblood glucose and the three main types of carbohydrate are

8 BioMed Research International

0102030405060708090

100

ME

HF

CF

EAF

24h 48h 72h

IC50

valu

e of c

once

ntra

tion

(120583g

mL)

Ca Ski

0102030405060708090

100

ME

HF

CF

EAF

24h 48h 72h

IC50

valu

e of c

once

ntra

tion

(120583g

mL)

MCF 7

0102030405060708090

100

ME

HF

CF

EAF

24h 48h 72h

IC50

valu

e of c

once

ntra

tion

(120583g

mL)

HT-29

minus10

0

10

20

30

40

50

60

70

80

90

100

ME

HF

CF

EAF

24h 48h 72h

IC50

valu

e of c

once

ntra

tion

(120583g

mL)

SKOV-3

0102030405060708090

100

ME

HF

CF

EAF

24h 48h 72h

IC50

valu

e of c

once

ntra

tion

(120583g

mL)

MDA-MB231

Figure 4 In vitro cytotoxic effects of P macrocarpa seedson Ca Ski MCF-7 HT-29 SKOV-3 and MDA-MB231 cell lines ME methanolextract HF hexane fraction EAF ethyl acetate fraction CF chloroform fractionWF water fraction Each value is expressed as mean plusmnstandard deviation of three measurements

BioMed Research International 9

Methyl linoleate

Methyl stearate

Palmitic acid

Oleic acid

Methyl myristate

Linoleic acid

Methyl oleate

H3C

H3C

H3C

H3C

H3C

H3C

H3C

O

O

O

O

O

O

O

O

O

O

O

CH3

CH3

CH3

CH3

OH

OH

OH

(294mz)

(298mz)

(282mz)

(242mz)

(280mz)

(298mz)

(256mz)

Figure 5

10 BioMed Research International

Table 4 Total flavonoid total flavonol total phenolic contents and result of DPPH free radical scavenging property of P macrocarpa seeds

Tests ME (mgmL) HF (mgmL) CF (mgmL) EAF (mgmL) WF (mgmL)Flavonoid 933 plusmn 08 418 plusmn 15 678 plusmn 11 838 plusmn 03 808 plusmn 10Flavonol 893 plusmn 11 425 plusmn 07 429 plusmn 09 613 plusmn 05 818 plusmn 10Phenolic 720 plusmn 07 263 plusmn 01 326 plusmn 1 358 plusmn 11 512 plusmn 14DPPH 1150 plusmn 003 1500 plusmn 004 1575 plusmn 004 1400 plusmn 005 975 plusmn 003ME methanol extract HF hexane fraction EAF ethyl acetate fraction CF chloroform fraction WF water fraction

Table 5 In vitro cytotoxic effects of methanol extract and its fractions of P macrocarpa cancer cells lines and normal human fibroblast breastcells line

Cell line Incubation periods (hours) ME HF CF EAF Doxorubicin24 82 plusmn 466 ge100 100 plusmn 131 56 plusmn 117 092 plusmn 064

Ca Ski 48 197 plusmn 092 ge100 82 plusmn 104 77 plusmn 185 069 plusmn 10572 407 plusmn 226 ge100 220 plusmn 186 60 plusmn 322 045 plusmn 09924 120 plusmn 22 452 plusmn 149 575 plusmn 264 223 plusmn 158 105 plusmn 108

MCF7 48 85 plusmn 168 555 plusmn 197 400 plusmn 148 168 plusmn 170 012 plusmn 06972 63 plusmn 250 725 plusmn 152 465 plusmn 345 84 plusmn 171 092 plusmn 17024 293 plusmn 226 400 plusmn 315 95 plusmn 295 11 plusmn 120 092 plusmn 072

HT29 48 250 plusmn 135 640 plusmn 203 87 plusmn 159 35 plusmn 200 032 plusmn 21672 215 plusmn 330 750 plusmn 314 210 plusmn 198 12 plusmn 228 088 plusmn 09424 165 plusmn 252 405 plusmn 352 248 plusmn 206 68 plusmn 18 102 plusmn 179

SKOV-3 48 221 plusmn 247 500 plusmn 302 165 plusmn 321 65 plusmn 23 032 plusmn 09772 360 plusmn 355 703 plusmn 353 90 plusmn 26 32 plusmn 281 062 plusmn 09824 ge100 ge100 ge100 65 plusmn 23 043 plusmn 050

MDA-MB231 48 ge100 ge100 ge100 15 plusmn 343 059 plusmn 10172 ge100 ge100 ge100 186 plusmn 126 095 plusmn 132

ME methanol extract HF hexane fraction EAF ethyl acetate fraction CF chloroform fractionWF water fraction Doxorubicin was used as positive controlEach value is expressed as mean plusmn standard deviation of three measurements

starches sugars and fiber A macronutrient protein consistsof amino acids which is required for proper growth andhuman body function The seeds were found to containa high level of crude fiber which is a potential source ofphenolic antioxidants The major components of fibers arecellulose hemicelluloses lignin 120573-glucans gums and pectinand hydrocolloids These components can behave as proan-tioxidants [28 29] In our present physicochemical study Pmacrocarpa seeds were mainly made up of carbohydratesfollowed by crude fiber proteins fatmoisture and ashHigh-protein and low-carbohydrate diets are often effective forweight loss Dietary fiber can aid in digestion is helpfulfor weight management and reduces constipation Howevermany high-protein and low-carbohydrate foods are low infiberWomen aged 50 and older need at least 21 grams of fiberdaily whilst those aged 19 to 50 require 25 grams men aged50 and older need 30 grams and those between the age of 19to 50 should consume at least 38 grams of fiber

Steroids possess biological activities which are insec-ticidal cardiotonic and antimicrobial activities and theseare potentially useful for development into therapeuticdrugs Tannins are known to be important for their antivi-ral antibacterial antiparasitic effects anti-inflammatoryantiulcer and antioxidant properties [30ndash33] Saponinshave been found to have antimicrobial anti-inflammatoryantifeedant and hemolytic effects [34 35] Some alkaloids

have been reported to have anticancer and antiviral activityand saponins have been reported to be cardiotonic whileflavonoids have anticancer and anti-inflammatory activity[10 36] The presence of tannins may be responsible for theability of P macrocarpa seeds to be used in the treatmentof diseases such as diabetes diarrhea and dysentery Ourpreliminary phytochemical studies revealed that the chemicalcomponents of P macrocarpa seeds also include flavonoidsphenolics steroids tannins terpenoids glucosides saponinsand carbohydrates

Previous researchers reported that kaempferol myrice-tin naringin quercetin rutin [3] 29-norcucurbitacinderivatives fevicordin A fevicordin A glucoside fevicordinD glucoside [6] mahkoside A dodecanoic acid palmiticacid des-acetyl flavicordin-A flavicordin-A flavicordin-Dflavicordin-A glucoside ethyl stearate lignans and sucrose[37 38] mangiferin (a C-glucosylxantone) kaempferol-3-o-120573-D-glucoside dodecanoic acid palmitic acid ethyl stearatesucrose [5] pinoresinol lariciresinol matairesinol alkaloidsand saponins [37 39] saponins alkaloids polyphenolicsphenols flavanoids lignans tannins [40ndash42] icarisideC3 mangiferin gallic acid phalerin glycoside (345trihydroxy-4-methoxy-benzophenone-3-O-120573-D-glucoside)[43] and 2410158406 trihydroxy-4-methoxy-benzophenone-3-O-120573-D-glucoside [44] were isolated from different partsof P macrocarpa The hexane extract of the seeds was

BioMed Research International 11

found to contain methyl myristate methyl stearate oleicacid methyl oleate linoleic acid methyl linoleate palmiticacid methyl palmitate 6-octadecenoic acid and 917-octadecadial 120573-sitosterol as major components A further30 to 40 of components were not identified as thesewere polar compounds that require HPLC analysis Othercompounds reported in the literature for the seeds leavesand fruits were not identified in this investigation It isprobable that the components reported in the literaturemay be present in other fractions (methyl acetate CHCl

3

and water fractions)Phenolics are able to transfer protons to radicals [45]

DPPH which provides free radicals is normally a blue-violet color However the DPPH turned yellow after it wasconverted to 1 1-diphenyl-2-picrylhydrazine which has lessfree-radical activity Hendra et al reported that the IC

50

values of free radical and reducing power in methanolwith HCl of P macrocarpa seeds were 2450 plusmn 194 and1502 plusmn 128 [42] In our investigation using DPPH assay thewater fraction displayed the highest IC

50value followed by

methanol ethyl acetate hexane and chloroform fractionThe presence of flavonoids in P macrocarpa seeds may be

responsible for the traditional use of the plants in treatingcancer inflammations and allergies Recently it has beenshown that phenolic compounds in crude methanol andethyl acetate extract of P macrocarpa leaves displayed goodantioxidant and antimicrobial activites [46] Hendra et al(2011) examined the amount of total phenolic (477plusmn 104mggallic acid equivalentg DW) and the amount of flavonoid(359 plusmn 247mg rutin equivalentg DW) contents in themethanol extract with HCl of P macrocarpa seeds [42] Inthis study the total flavonoids content was higher than that inthe results previously reported by Rohyami [47] and phenolflavonol and flavonoid contents were the highest in theaqueous methanol extract followed by ethyl acetate waterchloroform and hexane extracts

MTT assay is a sensitive and reliable colorimetric assaythat uses quantitative measurements to calculate the via-bility proliferation and activation of cells This method iscommonly applied to screen anticancer agents [48] Themost well-known methods used to calculate cytotoxicityare the neutral red (NR) uptake and dimethylthiazole-diphenyl tetrazolium bromide (MTT) metabolism [27 49]Earlier investigations showed that the ethanol extract of Pmacrocarpa seeds and fruit meat were not toxic to normalhuman cells but slightly toxic to a Vero cell line [9] Theethanol extract of P macrocarpa seed fruit also showedtoxicity towards T47D breast cancer cell line through COX-2 expression inhibition [7] Desacetylfevicordin A has beenisolated from the ethyl acetate extract of P macrocarpaseeds and this compound displayed excellent cytotoxicityin brine shrimp [43] The ethyl acetate of P macrocarpaseeds exhibited mild cytotoxic effect against HepG2 cells(IC50values between 30 and 60 120583gmL) [46] Previous studies

showed that the cytotoxic activity of the seeds on HT-29MCF-7 Hela and Chang liver cells lines were 384 plusmn 037255 plusmn 137 295 plusmn 10 and 678 plusmn 027 receptively [42] Inour study the methanolic extract of P macrocarpa seeds wasfound to pose cytotoxic effect against HT-29 MCF-7 Cas Ki

and SKOV-3 cell lines (IC50

values giving from 11 plusmn 120to 360 plusmn 355) and mild toxicity on normal cell lines Therewere significant cytotoxic effects on selected cancer cells linesthat were both time- and dose-dependent manner due to thepresence of many secondary metabolites

The phytochemical screening also indicated the presenceof a small amount of cyanogenic glycosides Cyanogenicglucosides which can cause acute cyanide poisoning causerapid respiration and pulse decrease the blood pressure andinduce vomiting diarrhea headache dizziness and so onThus these components have to be removed (usually byboiling) prior to consumption for therapeutic purposes

5 Conclusion

P macrocarpa seeds exhibited antioxidant and cytotoxicactivities It is highly probable that these activities are dueto the presence of phenolic and flavonoid compounds inappreciable amounts in the plant Furthermore the cytotox-icity activity suggested that the seed may contain a potentialanticancer agent The outcome of this study is encouragingdemonstrating the potential for the P macrocarpa as a sourceof multiple therapeutic agents

Abbreviations

EAF Ethyl acetate fractionME Methanol extractHF Hexane fractionCF Chloroform fractionWF Water fractiong Grammg MilligrammL Milliliter

Conflict of Interests

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

Authorsrsquo Contribution

Ma Ma Lay was involved in all experiments and BehroozBanisalamwas involved in antioxidant assay All authors readand approved the submitted version of the paper

Acknowledgments

This work was supported by grants from the MalaysianMinistry of Higher Education (nos PS2862009C andPS4702010B) Ma Ma Lay is a scholarship recipient fromthe ldquoIslamic Development Bank Saudi Arabiardquo The authorswould like to thank Professor Dr Amru BinNasrulhaq Boycefor generously allowing the use of his laboratory for thisresearch

12 BioMed Research International

References

[1] N HarmantoMahkota DewaObat Pusaka ParaDewa Cetakankeempat Agromedia Pustaka Jakarta Indonesia 2002

[2] A Jemal R Siegel E Ward T Murray J Xu and M J ThunldquoCancer statistics 2007rdquoCa ACancer Journal for Clinicians vol57 no 1 pp 43ndash66 2007

[3] R Hendra S Ahmad A Sukari M Y Shukor and EOskoueian ldquoFlavonoid analyses and antimicrobial activity ofvarious parts of Phaleria macrocarpa (Scheff) Boerl fruitrdquoInternational Journal of Molecular Sciences vol 12 no 6 pp3422ndash3431 2011

[4] W Wijayani Sitotoksisitas Minyak Atsiri Biji Dan Daging BuahMahkota Dewa (Phaleria Macrocarpa [Scheff] Boerl) TerhadapMyeloma Cell Line Dan HeLa Cell Line Serta IdentifikasiSenyawa Minyak Atsiri Dengan Kromatografi Gas-SpektrometriMassa (GC-MS) Skripsi FMIPA UGM Yogyakarta Indonesia2005

[5] Y-B Zhang X-J Xu and H-M Liu ldquoChemical con-stituents fromMahkotadewardquo Journal of Asian Natural ProductsResearch vol 8 no 1-2 pp 119ndash123 2006

[6] D Kurnia K Akiyama and H Hayashi ldquo29-Norcucurbitacinderivatives isolated from the Indonesian medicinal plant Pha-leria macrocarpa (Scheff) Boerlrdquo Bioscience Biotechnology andBiochemistry vol 72 no 2 pp 618ndash620 2008

[7] M Bakhriansyah Pengaruh Ekstrak Etanol Biji Mahkota Dewa(Phaleria Macrocarpa [Scheff] Boerl) Pada Sel Kanker Payu-dara T47D Kajian Aktivitas Sitotoksik Dan PenghambatanEkspresi Siklooksigenase-2 Program Pascasarjana UGM 2004

[8] L Wardani The Effect of Phaleria Macrocarpa [Scheff] Boerl)Seed and Flesh Fruit Extract to P53 and Bcl-2 Gene Expression inHela Cell Line FMIPA UGM 2005

[9] A Endang R Tri Joko and E Dewi ldquoCyototoxicity of Phaleriamacrocarpa (Scheff) boerl fruits flesh and seed extract ofethanol and its effect against p53 and Bcl-2 genes expressionof normalrdquo in Proceedings of the International Conference onComputational Science (ICCS rsquo07) 2007

[10] C Evans Trease and Evans Pharmacognosy Saunder 2002[11] A Sofowora Medicinal Plants and Traditional Medicine in

Africa Spectrum Books Ltd Ibadan Nigeria 2nd edition 1993[12] AOAC Official Methods of Analysis Association of Official

Analytical Chemists Arlington Tex USA 2002[13] J Harborne Phytochemical Methods Chapman and Hall Lon-

don UK 1973[14] AOAC Official Methods of Analysis Association of Official

Analytical Chemists Gaithersburg Md USA 17th edition2000

[15] J Lako V C Trenerry M Wahlqvist N WattanapenpaiboonS Sotheeswaran and R Premier ldquoPhytochemical flavonolscarotenoids and the antioxidant properties of a wide selection ofFijian fruit vegetables and other readily available foodsrdquo FoodChemistry vol 101 no 4 pp 1727ndash1741 2007

[16] W Horwitz Official Method of Analysis AOAC InterantionalGaithersburg Md USA 17th edition 2000

[17] A A Adedapo F O Jimoh S Koduru P J Masika and AJ Afolayan ldquoAssessment of the medicinal potentials of themethanol extracts of the leaves and stems of Buddleja salignardquoBMCComplementary andAlternativeMedicine vol 9 article 212009

[18] M Zovko Koncic D Kremer J Gruz et al ldquoAntioxidantand antimicrobial properties ofMoltkia petraea (Tratt) Griseb

flower leaf and stem infusionsrdquo Food and Chemical Toxicologyvol 48 no 6 pp 1537ndash1542 2010

[19] S McDonald P D Prenzler M Antolovich and K RobardsldquoPhenolic content and antioxidant activity of olive extractsrdquoFood Chemistry vol 73 no 1 pp 73ndash84 2001

[20] S M Nabavi M A Ebrahimzadeh S F Nabavi A Hamidiniaand A R Bekhradnia ldquoDetermination of antioxidant activityphenol and flavonoid content of Parrotia persicaMeyrdquo Pharma-cologyonline vol 2 pp 560ndash567 2008

[21] M A Ebrahimzadeh S F Nabavi and S M Nabavi ldquoAntiox-idant activities of methanol extract of Sambucus ebulus Lflowerrdquo Pakistan Journal of Biological Sciences vol 12 no 5 pp447ndash450 2009

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

[23] A Kumaran and R Joel Karunakaran ldquoIn vitro antioxidantactivities of methanol extracts of five Phyllanthus species fromIndiardquo Food Science and Technology vol 40 no 2 pp 344ndash3522007

[24] B O Mbaebie H O Edeoga and A J Afolayan ldquoPhy tochemical analysis and antioxidants activities of aqueous stembark extract of Schotia latifolia Jacqrdquo Asian Pacific Journal ofTropical Biomedicine vol 2 no 2 pp 118ndash124 2012

[25] L I Mensor F S Menezes G G Leitao et al ldquoScreening ofBrazilian plants extracts for antioxidants activity by the use ofDPPH free radical methodrdquo Phytotherapy Research vol 15 pp127ndash130 2001

[26] F Denizot and R Lang ldquoRapid colorimetric assay for cellgrowth and survival modifications to the tetrazolium dyeprocedure giving improved sensitivity and reliabilityrdquo Journalof Immunological Methods vol 89 no 2 pp 271ndash277 1986

[27] T Mosmann ldquoRapid colorimetric assay for cellular growth andsurvival application to proliferation and cytotoxicity assaysrdquoJournal of Immunological Methods vol 65 no 1-2 pp 55ndash631983

[28] P Maisuthisakul R Pongsawatmanit and M H GordonldquoAntioxidant properties of Teaw (Cratoxylum formosum Dyer)extract in soybean oil and emulsionsrdquo Journal of Agriculturaland Food Chemistry vol 54 no 7 pp 2719ndash2725 2006

[29] P Maisuthisakul S Pasuk and P Ritthiruangdej ldquoRelationshipbetween antioxidant properties and chemical composition ofsome Thai plantsrdquo Journal of Food Composition and Analysisvol 21 no 3 pp 229ndash240 2008

[30] H Kolodziej and A F Kiderlen ldquoAntileishmanial activity andimmune modulatory effects of tannins and related compoundson Leishmania parasitised RAW 2647 cellsrdquo Phytochemistryvol 66 no 17 pp 2056ndash2071 2005

[31] T Nakagawa and T Yokozawa ldquoDirect scavenging of nitricoxide and superoxide by green teardquo Food and Chemical Toxi-cology vol 40 no 12 pp 1745ndash1750 2002

[32] V Schulz R Hansel and V Tyler Fitoterapia Racional UmGuiade Fitoterapia Para as ciencias da saude Manole Barueri Brazil2002

[33] S D Kapu Y B Ngwai O Kayode P A Akah C Wambebeand K Gamaniel ldquoAnti-inflammatory analgesic and anti-lymphocytic activities of the aqueous extract of Crinum gigan-teumrdquo Journal of Ethnopharmacology vol 78 no 1 pp 7ndash132001

[34] G Francis Z Kerem H P S Makkar and K Becker ldquoThebiological action of saponins in animal systems a reviewrdquoBritish Journal of Nutrition vol 88 no 6 pp 587ndash605 2002

BioMed Research International 13

[35] R Xu W Zhao J Xu B Shao and G Qin ldquoStudies onbioactive saponins fromChinesemedicinal plantsrdquoAdvances inExperimental Medicine and Biology vol 404 pp 371ndash382 1996

[36] J A Manthey N Guthrie and K Grohmann ldquoBiologicalproperties of citrus flavonoids pertaining to cancer and inflam-mationrdquo Current Medicinal Chemistry vol 8 no 2 pp 135ndash1532001

[37] P Hendra Y Fukushi and Y Hashidoko ldquoSynthesis of ben-zophenone glucopyranosides from Phaleria macrocarpa andrelated benzophenone glucopyranosidesrdquo Bioscience Biotech-nology and Biochemistry vol 73 no 10 pp 2172ndash2182 2009

[38] A Rabia Z B A Mohammad D Aidiahmad S Amirin and IU Muhammad ldquoPhytochemistry and medicinal properties ofPhaleria macrocarpa (Scheff) Boerl extractsrdquo PharmacognosyReview vol 7 no 13 pp 73ndash80 2013

[39] A Saufi C B I VonHeimendahl AWAlfermann andE FussldquoStereochemistry of lignans in Phaleria macrocarpa (Scheff)Boerlrdquo Zeitschrift fur Naturforschung C vol 63 no 1-2 pp 13ndash16 2008

[40] S C Chong M A Dollah P P Chong and A Maha ldquoPhaleriamacrocarpa (Scheff) Boerl fruit aqueous extract enhances LDLreceptor and PCSK9 expression in vivo and in vitrordquo Journal ofEthnopharmacology vol 137 no 1 pp 817ndash827 2011

[41] R R Tjandrawinata D Nofiarny L W Susanto P Hendra andA Clarissa ldquoSymptomatic treatment of premenstrual syndromeandor primary dysmenorrhea with DLBS1442 a bioacitveextract ofPhaleriamacrocaraprdquo International Journal of GeneralMedicine vol 4 pp 465ndash476 2011

[42] R Hendra S Ahmad E Oskoueian A Sukari and M YShukor ldquoAntioxidant anti-inflammatory and cytotoxicity ofPhaleriamacrocarap (Boerl) Scheff fruitrdquoBMCComplementaryand Alternative Medicine vol 11 article 110 2011

[43] D Kurnia K Akiyama and H Hayashi ldquo29-Norcucurbitacinderivatives isolated from the Indonesian medicinal plant Pha-leria macrocarpa (Scheff) Boerlrdquo Bioscience Biotechnology andBiochemistry vol 72 no 2 pp 618ndash620 2008

[44] S Oshimi K Zaima Y Matsuno et al ldquoStudies on theconstituents from the fruits of Phaleria macrocaraprdquo Journal ofNatural Medicines vol 62 no 2 pp 207ndash210 2008

[45] C Sanchez-Moreno J A Larrauri and F Saura-Calixto ldquoAprocedure tomeasure the antiradical efficiency of polyphenolsrdquoJournal of the Science of Food and Agriculture vol 76 pp 270ndash276 1998

[46] Y Andriani M A W Effendy T M T Sifzizul and HMohamad ldquoAntibacterial radical-scavenging activities andcytotoxicit properties of Phaleria macrocarpa (Scheff) BoerlLeaves in HepG2 cell linesrdquo International Journal of Pharma-ceutical Sciences and Research vol 2 no 7 pp 1700ndash1706 2011

[47] Y Rohyami ldquoPenentuan Kandungan Flavonoid dari EkstrakMetanol Daging Buah Mahkota Dewa (Phaleria macrocarpaScheff Boerl)rdquo Jurnal Logika vol 5 2009

[48] R D Petty L A Sutherland E M Hunter and I A CreeldquoComparison of MTT and ATP-based assays for the measure-ment of viable cell numberrdquo Journal of Bioluminescence andChemiluminescence vol 10 no 1 pp 29ndash34 1995

[49] J R Sellers S Cook andV S Goldmacher ldquoA cytotoxicity assayutilizing a fluorescent dye that determines accurate survivingfractions of cellsrdquo Journal of Immunological Methods vol 172no 2 pp 255ndash264 1994

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

6 BioMed Research International

Table 2 Results of chemical constituents of P macrocarpa seeds

No Tests Reagents Observation

1 AlkaloidsWagnerrsquos reagentMayerrsquos reagentDragendorff rsquos reagentSodium picrate solution

++++

2 120572-Amino acids Ninhydrin reagent +3 Carbohydrates 10 120572-naphthol and conc H2SO4 ++4 Cyanogenic glycosides Sodium picrate solution +5 Flavonoids Mg and conc HCl ++6 Glycosides 10 lead acetate ++7 Organic acids Bromothymol blue +8 Phenolic compounds 1 FeCl3 ++

9 Reducing sugars Fehlingrsquos solutionsA and B +

10 Saponin glycosides Distilled water ++11 Starch Iodine solution +12 Steroids Acetic anhydride and conc H2SO4 ++13 Tannins 1 Gelatin ++14 Terpenoids Acetic anhydride and conc H2SO4 +++++ large amount ++ medium amount + small amount minus absent + present

0

5

10

15

20

25

30

35

Con

tent

()

Moi

sture

Ash

Prot

ein

Crud

e fibe

r

Carb

ohyd

rate

Crud

e fat

(a)

123456789

1011

ME HF CF EAF WF

FlavonoidsFlavonolPhenolic

Con

cent

ratio

n (m

gm

L)

(b)

Figure 2 (a)The percentage ofmoisture ash protein crude fiber crude fat and carbohydrate contents on PmacrocarpaBoerl seeds (b) Totalamount of concentration of flavonoid flavonol and phenolic contents of P macrocarpa seeds ME methanol extract HF hexane fractionEAF ethyl acetate fraction CF chloroform fractionWF water fraction

phytoconstituents It is important to note that a lower IC50

value equals a higher scavenging activity The scavengingactivity was presented as the percentage of inhibitionof DPPH free radicals (Figure 3 and Table 4) Based onIC50

values the samples can be ranked in the followingdescending order water fraction gt methanol extract gt ethylacetate fraction gt hexane fraction gt chloroform fractionTheresults revealed that the water fraction was most active forantioxidant activity compared to other fractions The radical

scavenging effect was found to increase with increasingconcentrations

36 Cytotoxicity Screening of MTT Cell Proliferation AssayThe methanol and fractionated extracts (hexane chloro-form ethyl acetate and water) were investigated for cyto-toxic effects in human cervical adenocarcinoma cells (CaSki) humanhormone-dependent breast carcinoma (MCF-7)

BioMed Research International 7

Table 3The percentage of moisture ash protein crude fiber crudefat and carbohydrate contents and the amount of nutrition valueson P macrocarpa seeds

Tests parameter Percentage of contents ()Moisture 631 plusmn 143Ash 296 plusmn 186Protein 2073 plusmn 244Crude fiber 2276 plusmn 279Crude fat 1840 plusmn 311Carbohydrate 2934 plusmn 198

0102030405060708090

100

1 5 10 15 20

ME

HF

CF

EAF

WF

Concentration (mgmL)

Inhi

bitio

n (

)

Figure 3 Percentage of inhibition of DPPH free radical scavengingof P macrocarpa seeds ME methanol extract HF hexane fractionEAF ethyl acetate fraction CF chloroform fractionWF waterfraction

human colon adenocarcinoma cells (HT-29) human ovar-ian carcinoma cells (SKOV-3) human hormone-dependentbreast carcinoma (MDA-MB231) and normal cells (MRC-5) using MTT cells proliferation assay at 24 hrs 48 hrs and72 hrs respectively

The methanol extract of P macrocarpa seeds showedexcellent cytotoxic effects with IC

50value of 82plusmn 466 120583gmL

in Ca Ski cells at 24 hrs and 120 plusmn 22 85 plusmn 168 and30 plusmn 250 120583gmL in MCF-7 cells at 24 hrs 48 hrs and72 hrs respectively The methanol extract also showed goodcytotoxic activity in HT-29 cells with IC

50values of 293 plusmn

226 250 plusmn 135 and 215 plusmn 330 120583gmL at 24 hrs 48 hrsand 72 hrs respectively in Ca Ski cells with IC

50values of

197 plusmn 092 120583gmL and in SKOV-3 cells with IC50

values of165 plusmn 252 221 plusmn 247 and 360 plusmn 355 120583gmL at 24 hrs48 hrs and 72 hrs respectively However this extract had nocytotoxic effect in MDA-MB231 cells with IC

50gt100120583gmL

and the extract also had low cytotoxic effect in the MRC-5cells with IC

50gt 50 120583gmL

The hexane fraction of P macrocarpa seeds showedmoderate cytotoxic effects with IC

50values of 452 plusmn 149

and 555 plusmn 197 120583gmL at 24 hrs and 48 hrs on MCF-7 400 plusmn315 120583gmL inHT-29 cells at 24 hrs and 405plusmn352 and 500plusmn302 120583gmL in SKOV-3 at 24 hrs and 48 hrs In addition the

hexane fraction also displayed low cytotoxic effect in MCF-7at 72 hrs HT-29 at 48 hrs and 72 hrs and in SKOV-3 at 72 hrsThe IC

50value of treatment with hexane fraction in MCF-7

was 725plusmn152 120583gmL for 72 hrs exposure IC50values in HT-

29 were 640plusmn203 and 750plusmn314 120583gmL at 48 hrs and 72 hrswhile IC

50value in SKOV-3 was 703 plusmn 353 120583gmL at 72 hrs

In contrast the hexane fraction exhibited no cytotoxic effectsin MDA-MB231 cells or MRC-5 cells

The chloroform fraction of P macrocarpa seeds exhibitedthe highest cytotoxic effect with IC

50value of 100plusmn131 82plusmn

104 and 220 plusmn 186 120583gmL at 24 hrs 48 hrs and 72 hrs in CaSki cells 95plusmn295 87plusmn159 and 210plusmn198 120583gmL at 24 hrs48 hrs and 72 hrs on HT29 and 248 plusmn 206 165 plusmn 321 and900 plusmn 26 120583gmL in SKOV-3 cells The chloroform fractionexhibitedmoderate cytotoxic effect inMCF-7 with IC

50value

of 575 plusmn 264 400 plusmn 148 and 465 plusmn 345 120583gmL for 24 hrs48 hrs and 72 hrs respectively In contrast the chloroformfraction had no cytotoxic effect against theMDA-MB231 cellsand normal MRC-5 cells with IC

50gt100 120583gmL

The ethyl acetate fraction ofPmacrocarpa seeds exhibitedthe highest cytotoxic effect with IC

50lt 25 120583gmL in SKOV-3

cellsMDA-MB231 cellsMCF-7 cells andCa Ski cells On theother hand the ethyl acetate fraction exhibited low cytotoxiceffect in MRC-5 normal cells with IC

50value of 35 120583gmL

The ethyl acetate fraction exhibited the highest cytotoxiceffect in all selected cells (Ca Ski MCF-7 HT-29 and MDA-MB231) The water extract showed no cytotoxic effect againstall selected cancer cell lines with IC

50gt 100 120583gmL and

exhibited a mild cytotoxic effects against MRC-5 cells Thecorresponding data is shown in Figure 4 and Table 5

37 Characterization and Identification of Hexane and Chlo-roform Fractions Six compounds were identified from thehexane fraction of P macrocarpa seeds using GC-MS Theywere methyl stearate oleic acid methyl oleate linoleic acidmethyl linolenate and palmitic acid Moreover a GCMSanalysis of the chloroform fraction of P macrocarpa seedsshowed the presence of methyl myristate palmitic acidmethyl oleate methyl linoleate oleic acid and (119911)- and917-octadecadienal (119911) A comparison with the NIST massspectral library (NIST 05MS library 2002) andAdams (2001)confirmed the identity of the compounds The chemicalstructures of the identified compounds in the hexane andchloroform fractions of P macrocarpa seeds are as shown inFigure 5

4 Discussion

A longer shelf life can be achieved by reducing moisturecontent Thus moisture content is a critical factor for thestability of an extractMoisture enhances fungal and bacterialgrowth therefore decreasing the longevity of the extract Thetime it takes for plant material to deteriorate depends on howmuch water the plant material contains

Carbohydrates play a vital role in the immune sys-tem fertilization pathogenesis blood clotting and humandevelopment Foods that contain carbohydrates can raiseblood glucose and the three main types of carbohydrate are

8 BioMed Research International

0102030405060708090

100

ME

HF

CF

EAF

24h 48h 72h

IC50

valu

e of c

once

ntra

tion

(120583g

mL)

Ca Ski

0102030405060708090

100

ME

HF

CF

EAF

24h 48h 72h

IC50

valu

e of c

once

ntra

tion

(120583g

mL)

MCF 7

0102030405060708090

100

ME

HF

CF

EAF

24h 48h 72h

IC50

valu

e of c

once

ntra

tion

(120583g

mL)

HT-29

minus10

0

10

20

30

40

50

60

70

80

90

100

ME

HF

CF

EAF

24h 48h 72h

IC50

valu

e of c

once

ntra

tion

(120583g

mL)

SKOV-3

0102030405060708090

100

ME

HF

CF

EAF

24h 48h 72h

IC50

valu

e of c

once

ntra

tion

(120583g

mL)

MDA-MB231

Figure 4 In vitro cytotoxic effects of P macrocarpa seedson Ca Ski MCF-7 HT-29 SKOV-3 and MDA-MB231 cell lines ME methanolextract HF hexane fraction EAF ethyl acetate fraction CF chloroform fractionWF water fraction Each value is expressed as mean plusmnstandard deviation of three measurements

BioMed Research International 9

Methyl linoleate

Methyl stearate

Palmitic acid

Oleic acid

Methyl myristate

Linoleic acid

Methyl oleate

H3C

H3C

H3C

H3C

H3C

H3C

H3C

O

O

O

O

O

O

O

O

O

O

O

CH3

CH3

CH3

CH3

OH

OH

OH

(294mz)

(298mz)

(282mz)

(242mz)

(280mz)

(298mz)

(256mz)

Figure 5

10 BioMed Research International

Table 4 Total flavonoid total flavonol total phenolic contents and result of DPPH free radical scavenging property of P macrocarpa seeds

Tests ME (mgmL) HF (mgmL) CF (mgmL) EAF (mgmL) WF (mgmL)Flavonoid 933 plusmn 08 418 plusmn 15 678 plusmn 11 838 plusmn 03 808 plusmn 10Flavonol 893 plusmn 11 425 plusmn 07 429 plusmn 09 613 plusmn 05 818 plusmn 10Phenolic 720 plusmn 07 263 plusmn 01 326 plusmn 1 358 plusmn 11 512 plusmn 14DPPH 1150 plusmn 003 1500 plusmn 004 1575 plusmn 004 1400 plusmn 005 975 plusmn 003ME methanol extract HF hexane fraction EAF ethyl acetate fraction CF chloroform fraction WF water fraction

Table 5 In vitro cytotoxic effects of methanol extract and its fractions of P macrocarpa cancer cells lines and normal human fibroblast breastcells line

Cell line Incubation periods (hours) ME HF CF EAF Doxorubicin24 82 plusmn 466 ge100 100 plusmn 131 56 plusmn 117 092 plusmn 064

Ca Ski 48 197 plusmn 092 ge100 82 plusmn 104 77 plusmn 185 069 plusmn 10572 407 plusmn 226 ge100 220 plusmn 186 60 plusmn 322 045 plusmn 09924 120 plusmn 22 452 plusmn 149 575 plusmn 264 223 plusmn 158 105 plusmn 108

MCF7 48 85 plusmn 168 555 plusmn 197 400 plusmn 148 168 plusmn 170 012 plusmn 06972 63 plusmn 250 725 plusmn 152 465 plusmn 345 84 plusmn 171 092 plusmn 17024 293 plusmn 226 400 plusmn 315 95 plusmn 295 11 plusmn 120 092 plusmn 072

HT29 48 250 plusmn 135 640 plusmn 203 87 plusmn 159 35 plusmn 200 032 plusmn 21672 215 plusmn 330 750 plusmn 314 210 plusmn 198 12 plusmn 228 088 plusmn 09424 165 plusmn 252 405 plusmn 352 248 plusmn 206 68 plusmn 18 102 plusmn 179

SKOV-3 48 221 plusmn 247 500 plusmn 302 165 plusmn 321 65 plusmn 23 032 plusmn 09772 360 plusmn 355 703 plusmn 353 90 plusmn 26 32 plusmn 281 062 plusmn 09824 ge100 ge100 ge100 65 plusmn 23 043 plusmn 050

MDA-MB231 48 ge100 ge100 ge100 15 plusmn 343 059 plusmn 10172 ge100 ge100 ge100 186 plusmn 126 095 plusmn 132

ME methanol extract HF hexane fraction EAF ethyl acetate fraction CF chloroform fractionWF water fraction Doxorubicin was used as positive controlEach value is expressed as mean plusmn standard deviation of three measurements

starches sugars and fiber A macronutrient protein consistsof amino acids which is required for proper growth andhuman body function The seeds were found to containa high level of crude fiber which is a potential source ofphenolic antioxidants The major components of fibers arecellulose hemicelluloses lignin 120573-glucans gums and pectinand hydrocolloids These components can behave as proan-tioxidants [28 29] In our present physicochemical study Pmacrocarpa seeds were mainly made up of carbohydratesfollowed by crude fiber proteins fatmoisture and ashHigh-protein and low-carbohydrate diets are often effective forweight loss Dietary fiber can aid in digestion is helpfulfor weight management and reduces constipation Howevermany high-protein and low-carbohydrate foods are low infiberWomen aged 50 and older need at least 21 grams of fiberdaily whilst those aged 19 to 50 require 25 grams men aged50 and older need 30 grams and those between the age of 19to 50 should consume at least 38 grams of fiber

Steroids possess biological activities which are insec-ticidal cardiotonic and antimicrobial activities and theseare potentially useful for development into therapeuticdrugs Tannins are known to be important for their antivi-ral antibacterial antiparasitic effects anti-inflammatoryantiulcer and antioxidant properties [30ndash33] Saponinshave been found to have antimicrobial anti-inflammatoryantifeedant and hemolytic effects [34 35] Some alkaloids

have been reported to have anticancer and antiviral activityand saponins have been reported to be cardiotonic whileflavonoids have anticancer and anti-inflammatory activity[10 36] The presence of tannins may be responsible for theability of P macrocarpa seeds to be used in the treatmentof diseases such as diabetes diarrhea and dysentery Ourpreliminary phytochemical studies revealed that the chemicalcomponents of P macrocarpa seeds also include flavonoidsphenolics steroids tannins terpenoids glucosides saponinsand carbohydrates

Previous researchers reported that kaempferol myrice-tin naringin quercetin rutin [3] 29-norcucurbitacinderivatives fevicordin A fevicordin A glucoside fevicordinD glucoside [6] mahkoside A dodecanoic acid palmiticacid des-acetyl flavicordin-A flavicordin-A flavicordin-Dflavicordin-A glucoside ethyl stearate lignans and sucrose[37 38] mangiferin (a C-glucosylxantone) kaempferol-3-o-120573-D-glucoside dodecanoic acid palmitic acid ethyl stearatesucrose [5] pinoresinol lariciresinol matairesinol alkaloidsand saponins [37 39] saponins alkaloids polyphenolicsphenols flavanoids lignans tannins [40ndash42] icarisideC3 mangiferin gallic acid phalerin glycoside (345trihydroxy-4-methoxy-benzophenone-3-O-120573-D-glucoside)[43] and 2410158406 trihydroxy-4-methoxy-benzophenone-3-O-120573-D-glucoside [44] were isolated from different partsof P macrocarpa The hexane extract of the seeds was

BioMed Research International 11

found to contain methyl myristate methyl stearate oleicacid methyl oleate linoleic acid methyl linoleate palmiticacid methyl palmitate 6-octadecenoic acid and 917-octadecadial 120573-sitosterol as major components A further30 to 40 of components were not identified as thesewere polar compounds that require HPLC analysis Othercompounds reported in the literature for the seeds leavesand fruits were not identified in this investigation It isprobable that the components reported in the literaturemay be present in other fractions (methyl acetate CHCl

3

and water fractions)Phenolics are able to transfer protons to radicals [45]

DPPH which provides free radicals is normally a blue-violet color However the DPPH turned yellow after it wasconverted to 1 1-diphenyl-2-picrylhydrazine which has lessfree-radical activity Hendra et al reported that the IC

50

values of free radical and reducing power in methanolwith HCl of P macrocarpa seeds were 2450 plusmn 194 and1502 plusmn 128 [42] In our investigation using DPPH assay thewater fraction displayed the highest IC

50value followed by

methanol ethyl acetate hexane and chloroform fractionThe presence of flavonoids in P macrocarpa seeds may be

responsible for the traditional use of the plants in treatingcancer inflammations and allergies Recently it has beenshown that phenolic compounds in crude methanol andethyl acetate extract of P macrocarpa leaves displayed goodantioxidant and antimicrobial activites [46] Hendra et al(2011) examined the amount of total phenolic (477plusmn 104mggallic acid equivalentg DW) and the amount of flavonoid(359 plusmn 247mg rutin equivalentg DW) contents in themethanol extract with HCl of P macrocarpa seeds [42] Inthis study the total flavonoids content was higher than that inthe results previously reported by Rohyami [47] and phenolflavonol and flavonoid contents were the highest in theaqueous methanol extract followed by ethyl acetate waterchloroform and hexane extracts

MTT assay is a sensitive and reliable colorimetric assaythat uses quantitative measurements to calculate the via-bility proliferation and activation of cells This method iscommonly applied to screen anticancer agents [48] Themost well-known methods used to calculate cytotoxicityare the neutral red (NR) uptake and dimethylthiazole-diphenyl tetrazolium bromide (MTT) metabolism [27 49]Earlier investigations showed that the ethanol extract of Pmacrocarpa seeds and fruit meat were not toxic to normalhuman cells but slightly toxic to a Vero cell line [9] Theethanol extract of P macrocarpa seed fruit also showedtoxicity towards T47D breast cancer cell line through COX-2 expression inhibition [7] Desacetylfevicordin A has beenisolated from the ethyl acetate extract of P macrocarpaseeds and this compound displayed excellent cytotoxicityin brine shrimp [43] The ethyl acetate of P macrocarpaseeds exhibited mild cytotoxic effect against HepG2 cells(IC50values between 30 and 60 120583gmL) [46] Previous studies

showed that the cytotoxic activity of the seeds on HT-29MCF-7 Hela and Chang liver cells lines were 384 plusmn 037255 plusmn 137 295 plusmn 10 and 678 plusmn 027 receptively [42] Inour study the methanolic extract of P macrocarpa seeds wasfound to pose cytotoxic effect against HT-29 MCF-7 Cas Ki

and SKOV-3 cell lines (IC50

values giving from 11 plusmn 120to 360 plusmn 355) and mild toxicity on normal cell lines Therewere significant cytotoxic effects on selected cancer cells linesthat were both time- and dose-dependent manner due to thepresence of many secondary metabolites

The phytochemical screening also indicated the presenceof a small amount of cyanogenic glycosides Cyanogenicglucosides which can cause acute cyanide poisoning causerapid respiration and pulse decrease the blood pressure andinduce vomiting diarrhea headache dizziness and so onThus these components have to be removed (usually byboiling) prior to consumption for therapeutic purposes

5 Conclusion

P macrocarpa seeds exhibited antioxidant and cytotoxicactivities It is highly probable that these activities are dueto the presence of phenolic and flavonoid compounds inappreciable amounts in the plant Furthermore the cytotox-icity activity suggested that the seed may contain a potentialanticancer agent The outcome of this study is encouragingdemonstrating the potential for the P macrocarpa as a sourceof multiple therapeutic agents

Abbreviations

EAF Ethyl acetate fractionME Methanol extractHF Hexane fractionCF Chloroform fractionWF Water fractiong Grammg MilligrammL Milliliter

Conflict of Interests

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

Authorsrsquo Contribution

Ma Ma Lay was involved in all experiments and BehroozBanisalamwas involved in antioxidant assay All authors readand approved the submitted version of the paper

Acknowledgments

This work was supported by grants from the MalaysianMinistry of Higher Education (nos PS2862009C andPS4702010B) Ma Ma Lay is a scholarship recipient fromthe ldquoIslamic Development Bank Saudi Arabiardquo The authorswould like to thank Professor Dr Amru BinNasrulhaq Boycefor generously allowing the use of his laboratory for thisresearch

12 BioMed Research International

References

[1] N HarmantoMahkota DewaObat Pusaka ParaDewa Cetakankeempat Agromedia Pustaka Jakarta Indonesia 2002

[2] A Jemal R Siegel E Ward T Murray J Xu and M J ThunldquoCancer statistics 2007rdquoCa ACancer Journal for Clinicians vol57 no 1 pp 43ndash66 2007

[3] R Hendra S Ahmad A Sukari M Y Shukor and EOskoueian ldquoFlavonoid analyses and antimicrobial activity ofvarious parts of Phaleria macrocarpa (Scheff) Boerl fruitrdquoInternational Journal of Molecular Sciences vol 12 no 6 pp3422ndash3431 2011

[4] W Wijayani Sitotoksisitas Minyak Atsiri Biji Dan Daging BuahMahkota Dewa (Phaleria Macrocarpa [Scheff] Boerl) TerhadapMyeloma Cell Line Dan HeLa Cell Line Serta IdentifikasiSenyawa Minyak Atsiri Dengan Kromatografi Gas-SpektrometriMassa (GC-MS) Skripsi FMIPA UGM Yogyakarta Indonesia2005

[5] Y-B Zhang X-J Xu and H-M Liu ldquoChemical con-stituents fromMahkotadewardquo Journal of Asian Natural ProductsResearch vol 8 no 1-2 pp 119ndash123 2006

[6] D Kurnia K Akiyama and H Hayashi ldquo29-Norcucurbitacinderivatives isolated from the Indonesian medicinal plant Pha-leria macrocarpa (Scheff) Boerlrdquo Bioscience Biotechnology andBiochemistry vol 72 no 2 pp 618ndash620 2008

[7] M Bakhriansyah Pengaruh Ekstrak Etanol Biji Mahkota Dewa(Phaleria Macrocarpa [Scheff] Boerl) Pada Sel Kanker Payu-dara T47D Kajian Aktivitas Sitotoksik Dan PenghambatanEkspresi Siklooksigenase-2 Program Pascasarjana UGM 2004

[8] L Wardani The Effect of Phaleria Macrocarpa [Scheff] Boerl)Seed and Flesh Fruit Extract to P53 and Bcl-2 Gene Expression inHela Cell Line FMIPA UGM 2005

[9] A Endang R Tri Joko and E Dewi ldquoCyototoxicity of Phaleriamacrocarpa (Scheff) boerl fruits flesh and seed extract ofethanol and its effect against p53 and Bcl-2 genes expressionof normalrdquo in Proceedings of the International Conference onComputational Science (ICCS rsquo07) 2007

[10] C Evans Trease and Evans Pharmacognosy Saunder 2002[11] A Sofowora Medicinal Plants and Traditional Medicine in

Africa Spectrum Books Ltd Ibadan Nigeria 2nd edition 1993[12] AOAC Official Methods of Analysis Association of Official

Analytical Chemists Arlington Tex USA 2002[13] J Harborne Phytochemical Methods Chapman and Hall Lon-

don UK 1973[14] AOAC Official Methods of Analysis Association of Official

Analytical Chemists Gaithersburg Md USA 17th edition2000

[15] J Lako V C Trenerry M Wahlqvist N WattanapenpaiboonS Sotheeswaran and R Premier ldquoPhytochemical flavonolscarotenoids and the antioxidant properties of a wide selection ofFijian fruit vegetables and other readily available foodsrdquo FoodChemistry vol 101 no 4 pp 1727ndash1741 2007

[16] W Horwitz Official Method of Analysis AOAC InterantionalGaithersburg Md USA 17th edition 2000

[17] A A Adedapo F O Jimoh S Koduru P J Masika and AJ Afolayan ldquoAssessment of the medicinal potentials of themethanol extracts of the leaves and stems of Buddleja salignardquoBMCComplementary andAlternativeMedicine vol 9 article 212009

[18] M Zovko Koncic D Kremer J Gruz et al ldquoAntioxidantand antimicrobial properties ofMoltkia petraea (Tratt) Griseb

flower leaf and stem infusionsrdquo Food and Chemical Toxicologyvol 48 no 6 pp 1537ndash1542 2010

[19] S McDonald P D Prenzler M Antolovich and K RobardsldquoPhenolic content and antioxidant activity of olive extractsrdquoFood Chemistry vol 73 no 1 pp 73ndash84 2001

[20] S M Nabavi M A Ebrahimzadeh S F Nabavi A Hamidiniaand A R Bekhradnia ldquoDetermination of antioxidant activityphenol and flavonoid content of Parrotia persicaMeyrdquo Pharma-cologyonline vol 2 pp 560ndash567 2008

[21] M A Ebrahimzadeh S F Nabavi and S M Nabavi ldquoAntiox-idant activities of methanol extract of Sambucus ebulus Lflowerrdquo Pakistan Journal of Biological Sciences vol 12 no 5 pp447ndash450 2009

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

[23] A Kumaran and R Joel Karunakaran ldquoIn vitro antioxidantactivities of methanol extracts of five Phyllanthus species fromIndiardquo Food Science and Technology vol 40 no 2 pp 344ndash3522007

[24] B O Mbaebie H O Edeoga and A J Afolayan ldquoPhy tochemical analysis and antioxidants activities of aqueous stembark extract of Schotia latifolia Jacqrdquo Asian Pacific Journal ofTropical Biomedicine vol 2 no 2 pp 118ndash124 2012

[25] L I Mensor F S Menezes G G Leitao et al ldquoScreening ofBrazilian plants extracts for antioxidants activity by the use ofDPPH free radical methodrdquo Phytotherapy Research vol 15 pp127ndash130 2001

[26] F Denizot and R Lang ldquoRapid colorimetric assay for cellgrowth and survival modifications to the tetrazolium dyeprocedure giving improved sensitivity and reliabilityrdquo Journalof Immunological Methods vol 89 no 2 pp 271ndash277 1986

[27] T Mosmann ldquoRapid colorimetric assay for cellular growth andsurvival application to proliferation and cytotoxicity assaysrdquoJournal of Immunological Methods vol 65 no 1-2 pp 55ndash631983

[28] P Maisuthisakul R Pongsawatmanit and M H GordonldquoAntioxidant properties of Teaw (Cratoxylum formosum Dyer)extract in soybean oil and emulsionsrdquo Journal of Agriculturaland Food Chemistry vol 54 no 7 pp 2719ndash2725 2006

[29] P Maisuthisakul S Pasuk and P Ritthiruangdej ldquoRelationshipbetween antioxidant properties and chemical composition ofsome Thai plantsrdquo Journal of Food Composition and Analysisvol 21 no 3 pp 229ndash240 2008

[30] H Kolodziej and A F Kiderlen ldquoAntileishmanial activity andimmune modulatory effects of tannins and related compoundson Leishmania parasitised RAW 2647 cellsrdquo Phytochemistryvol 66 no 17 pp 2056ndash2071 2005

[31] T Nakagawa and T Yokozawa ldquoDirect scavenging of nitricoxide and superoxide by green teardquo Food and Chemical Toxi-cology vol 40 no 12 pp 1745ndash1750 2002

[32] V Schulz R Hansel and V Tyler Fitoterapia Racional UmGuiade Fitoterapia Para as ciencias da saude Manole Barueri Brazil2002

[33] S D Kapu Y B Ngwai O Kayode P A Akah C Wambebeand K Gamaniel ldquoAnti-inflammatory analgesic and anti-lymphocytic activities of the aqueous extract of Crinum gigan-teumrdquo Journal of Ethnopharmacology vol 78 no 1 pp 7ndash132001

[34] G Francis Z Kerem H P S Makkar and K Becker ldquoThebiological action of saponins in animal systems a reviewrdquoBritish Journal of Nutrition vol 88 no 6 pp 587ndash605 2002

BioMed Research International 13

[35] R Xu W Zhao J Xu B Shao and G Qin ldquoStudies onbioactive saponins fromChinesemedicinal plantsrdquoAdvances inExperimental Medicine and Biology vol 404 pp 371ndash382 1996

[36] J A Manthey N Guthrie and K Grohmann ldquoBiologicalproperties of citrus flavonoids pertaining to cancer and inflam-mationrdquo Current Medicinal Chemistry vol 8 no 2 pp 135ndash1532001

[37] P Hendra Y Fukushi and Y Hashidoko ldquoSynthesis of ben-zophenone glucopyranosides from Phaleria macrocarpa andrelated benzophenone glucopyranosidesrdquo Bioscience Biotech-nology and Biochemistry vol 73 no 10 pp 2172ndash2182 2009

[38] A Rabia Z B A Mohammad D Aidiahmad S Amirin and IU Muhammad ldquoPhytochemistry and medicinal properties ofPhaleria macrocarpa (Scheff) Boerl extractsrdquo PharmacognosyReview vol 7 no 13 pp 73ndash80 2013

[39] A Saufi C B I VonHeimendahl AWAlfermann andE FussldquoStereochemistry of lignans in Phaleria macrocarpa (Scheff)Boerlrdquo Zeitschrift fur Naturforschung C vol 63 no 1-2 pp 13ndash16 2008

[40] S C Chong M A Dollah P P Chong and A Maha ldquoPhaleriamacrocarpa (Scheff) Boerl fruit aqueous extract enhances LDLreceptor and PCSK9 expression in vivo and in vitrordquo Journal ofEthnopharmacology vol 137 no 1 pp 817ndash827 2011

[41] R R Tjandrawinata D Nofiarny L W Susanto P Hendra andA Clarissa ldquoSymptomatic treatment of premenstrual syndromeandor primary dysmenorrhea with DLBS1442 a bioacitveextract ofPhaleriamacrocaraprdquo International Journal of GeneralMedicine vol 4 pp 465ndash476 2011

[42] R Hendra S Ahmad E Oskoueian A Sukari and M YShukor ldquoAntioxidant anti-inflammatory and cytotoxicity ofPhaleriamacrocarap (Boerl) Scheff fruitrdquoBMCComplementaryand Alternative Medicine vol 11 article 110 2011

[43] D Kurnia K Akiyama and H Hayashi ldquo29-Norcucurbitacinderivatives isolated from the Indonesian medicinal plant Pha-leria macrocarpa (Scheff) Boerlrdquo Bioscience Biotechnology andBiochemistry vol 72 no 2 pp 618ndash620 2008

[44] S Oshimi K Zaima Y Matsuno et al ldquoStudies on theconstituents from the fruits of Phaleria macrocaraprdquo Journal ofNatural Medicines vol 62 no 2 pp 207ndash210 2008

[45] C Sanchez-Moreno J A Larrauri and F Saura-Calixto ldquoAprocedure tomeasure the antiradical efficiency of polyphenolsrdquoJournal of the Science of Food and Agriculture vol 76 pp 270ndash276 1998

[46] Y Andriani M A W Effendy T M T Sifzizul and HMohamad ldquoAntibacterial radical-scavenging activities andcytotoxicit properties of Phaleria macrocarpa (Scheff) BoerlLeaves in HepG2 cell linesrdquo International Journal of Pharma-ceutical Sciences and Research vol 2 no 7 pp 1700ndash1706 2011

[47] Y Rohyami ldquoPenentuan Kandungan Flavonoid dari EkstrakMetanol Daging Buah Mahkota Dewa (Phaleria macrocarpaScheff Boerl)rdquo Jurnal Logika vol 5 2009

[48] R D Petty L A Sutherland E M Hunter and I A CreeldquoComparison of MTT and ATP-based assays for the measure-ment of viable cell numberrdquo Journal of Bioluminescence andChemiluminescence vol 10 no 1 pp 29ndash34 1995

[49] J R Sellers S Cook andV S Goldmacher ldquoA cytotoxicity assayutilizing a fluorescent dye that determines accurate survivingfractions of cellsrdquo Journal of Immunological Methods vol 172no 2 pp 255ndash264 1994

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

BioMed Research International 7

Table 3The percentage of moisture ash protein crude fiber crudefat and carbohydrate contents and the amount of nutrition valueson P macrocarpa seeds

Tests parameter Percentage of contents ()Moisture 631 plusmn 143Ash 296 plusmn 186Protein 2073 plusmn 244Crude fiber 2276 plusmn 279Crude fat 1840 plusmn 311Carbohydrate 2934 plusmn 198

0102030405060708090

100

1 5 10 15 20

ME

HF

CF

EAF

WF

Concentration (mgmL)

Inhi

bitio

n (

)

Figure 3 Percentage of inhibition of DPPH free radical scavengingof P macrocarpa seeds ME methanol extract HF hexane fractionEAF ethyl acetate fraction CF chloroform fractionWF waterfraction

human colon adenocarcinoma cells (HT-29) human ovar-ian carcinoma cells (SKOV-3) human hormone-dependentbreast carcinoma (MDA-MB231) and normal cells (MRC-5) using MTT cells proliferation assay at 24 hrs 48 hrs and72 hrs respectively

The methanol extract of P macrocarpa seeds showedexcellent cytotoxic effects with IC

50value of 82plusmn 466 120583gmL

in Ca Ski cells at 24 hrs and 120 plusmn 22 85 plusmn 168 and30 plusmn 250 120583gmL in MCF-7 cells at 24 hrs 48 hrs and72 hrs respectively The methanol extract also showed goodcytotoxic activity in HT-29 cells with IC

50values of 293 plusmn

226 250 plusmn 135 and 215 plusmn 330 120583gmL at 24 hrs 48 hrsand 72 hrs respectively in Ca Ski cells with IC

50values of

197 plusmn 092 120583gmL and in SKOV-3 cells with IC50

values of165 plusmn 252 221 plusmn 247 and 360 plusmn 355 120583gmL at 24 hrs48 hrs and 72 hrs respectively However this extract had nocytotoxic effect in MDA-MB231 cells with IC

50gt100120583gmL

and the extract also had low cytotoxic effect in the MRC-5cells with IC

50gt 50 120583gmL

The hexane fraction of P macrocarpa seeds showedmoderate cytotoxic effects with IC

50values of 452 plusmn 149

and 555 plusmn 197 120583gmL at 24 hrs and 48 hrs on MCF-7 400 plusmn315 120583gmL inHT-29 cells at 24 hrs and 405plusmn352 and 500plusmn302 120583gmL in SKOV-3 at 24 hrs and 48 hrs In addition the

hexane fraction also displayed low cytotoxic effect in MCF-7at 72 hrs HT-29 at 48 hrs and 72 hrs and in SKOV-3 at 72 hrsThe IC

50value of treatment with hexane fraction in MCF-7

was 725plusmn152 120583gmL for 72 hrs exposure IC50values in HT-

29 were 640plusmn203 and 750plusmn314 120583gmL at 48 hrs and 72 hrswhile IC

50value in SKOV-3 was 703 plusmn 353 120583gmL at 72 hrs

In contrast the hexane fraction exhibited no cytotoxic effectsin MDA-MB231 cells or MRC-5 cells

The chloroform fraction of P macrocarpa seeds exhibitedthe highest cytotoxic effect with IC

50value of 100plusmn131 82plusmn

104 and 220 plusmn 186 120583gmL at 24 hrs 48 hrs and 72 hrs in CaSki cells 95plusmn295 87plusmn159 and 210plusmn198 120583gmL at 24 hrs48 hrs and 72 hrs on HT29 and 248 plusmn 206 165 plusmn 321 and900 plusmn 26 120583gmL in SKOV-3 cells The chloroform fractionexhibitedmoderate cytotoxic effect inMCF-7 with IC

50value

of 575 plusmn 264 400 plusmn 148 and 465 plusmn 345 120583gmL for 24 hrs48 hrs and 72 hrs respectively In contrast the chloroformfraction had no cytotoxic effect against theMDA-MB231 cellsand normal MRC-5 cells with IC

50gt100 120583gmL

The ethyl acetate fraction ofPmacrocarpa seeds exhibitedthe highest cytotoxic effect with IC

50lt 25 120583gmL in SKOV-3

cellsMDA-MB231 cellsMCF-7 cells andCa Ski cells On theother hand the ethyl acetate fraction exhibited low cytotoxiceffect in MRC-5 normal cells with IC

50value of 35 120583gmL

The ethyl acetate fraction exhibited the highest cytotoxiceffect in all selected cells (Ca Ski MCF-7 HT-29 and MDA-MB231) The water extract showed no cytotoxic effect againstall selected cancer cell lines with IC

50gt 100 120583gmL and

exhibited a mild cytotoxic effects against MRC-5 cells Thecorresponding data is shown in Figure 4 and Table 5

37 Characterization and Identification of Hexane and Chlo-roform Fractions Six compounds were identified from thehexane fraction of P macrocarpa seeds using GC-MS Theywere methyl stearate oleic acid methyl oleate linoleic acidmethyl linolenate and palmitic acid Moreover a GCMSanalysis of the chloroform fraction of P macrocarpa seedsshowed the presence of methyl myristate palmitic acidmethyl oleate methyl linoleate oleic acid and (119911)- and917-octadecadienal (119911) A comparison with the NIST massspectral library (NIST 05MS library 2002) andAdams (2001)confirmed the identity of the compounds The chemicalstructures of the identified compounds in the hexane andchloroform fractions of P macrocarpa seeds are as shown inFigure 5

4 Discussion

A longer shelf life can be achieved by reducing moisturecontent Thus moisture content is a critical factor for thestability of an extractMoisture enhances fungal and bacterialgrowth therefore decreasing the longevity of the extract Thetime it takes for plant material to deteriorate depends on howmuch water the plant material contains

Carbohydrates play a vital role in the immune sys-tem fertilization pathogenesis blood clotting and humandevelopment Foods that contain carbohydrates can raiseblood glucose and the three main types of carbohydrate are

8 BioMed Research International

0102030405060708090

100

ME

HF

CF

EAF

24h 48h 72h

IC50

valu

e of c

once

ntra

tion

(120583g

mL)

Ca Ski

0102030405060708090

100

ME

HF

CF

EAF

24h 48h 72h

IC50

valu

e of c

once

ntra

tion

(120583g

mL)

MCF 7

0102030405060708090

100

ME

HF

CF

EAF

24h 48h 72h

IC50

valu

e of c

once

ntra

tion

(120583g

mL)

HT-29

minus10

0

10

20

30

40

50

60

70

80

90

100

ME

HF

CF

EAF

24h 48h 72h

IC50

valu

e of c

once

ntra

tion

(120583g

mL)

SKOV-3

0102030405060708090

100

ME

HF

CF

EAF

24h 48h 72h

IC50

valu

e of c

once

ntra

tion

(120583g

mL)

MDA-MB231

Figure 4 In vitro cytotoxic effects of P macrocarpa seedson Ca Ski MCF-7 HT-29 SKOV-3 and MDA-MB231 cell lines ME methanolextract HF hexane fraction EAF ethyl acetate fraction CF chloroform fractionWF water fraction Each value is expressed as mean plusmnstandard deviation of three measurements

BioMed Research International 9

Methyl linoleate

Methyl stearate

Palmitic acid

Oleic acid

Methyl myristate

Linoleic acid

Methyl oleate

H3C

H3C

H3C

H3C

H3C

H3C

H3C

O

O

O

O

O

O

O

O

O

O

O

CH3

CH3

CH3

CH3

OH

OH

OH

(294mz)

(298mz)

(282mz)

(242mz)

(280mz)

(298mz)

(256mz)

Figure 5

10 BioMed Research International

Table 4 Total flavonoid total flavonol total phenolic contents and result of DPPH free radical scavenging property of P macrocarpa seeds

Tests ME (mgmL) HF (mgmL) CF (mgmL) EAF (mgmL) WF (mgmL)Flavonoid 933 plusmn 08 418 plusmn 15 678 plusmn 11 838 plusmn 03 808 plusmn 10Flavonol 893 plusmn 11 425 plusmn 07 429 plusmn 09 613 plusmn 05 818 plusmn 10Phenolic 720 plusmn 07 263 plusmn 01 326 plusmn 1 358 plusmn 11 512 plusmn 14DPPH 1150 plusmn 003 1500 plusmn 004 1575 plusmn 004 1400 plusmn 005 975 plusmn 003ME methanol extract HF hexane fraction EAF ethyl acetate fraction CF chloroform fraction WF water fraction

Table 5 In vitro cytotoxic effects of methanol extract and its fractions of P macrocarpa cancer cells lines and normal human fibroblast breastcells line

Cell line Incubation periods (hours) ME HF CF EAF Doxorubicin24 82 plusmn 466 ge100 100 plusmn 131 56 plusmn 117 092 plusmn 064

Ca Ski 48 197 plusmn 092 ge100 82 plusmn 104 77 plusmn 185 069 plusmn 10572 407 plusmn 226 ge100 220 plusmn 186 60 plusmn 322 045 plusmn 09924 120 plusmn 22 452 plusmn 149 575 plusmn 264 223 plusmn 158 105 plusmn 108

MCF7 48 85 plusmn 168 555 plusmn 197 400 plusmn 148 168 plusmn 170 012 plusmn 06972 63 plusmn 250 725 plusmn 152 465 plusmn 345 84 plusmn 171 092 plusmn 17024 293 plusmn 226 400 plusmn 315 95 plusmn 295 11 plusmn 120 092 plusmn 072

HT29 48 250 plusmn 135 640 plusmn 203 87 plusmn 159 35 plusmn 200 032 plusmn 21672 215 plusmn 330 750 plusmn 314 210 plusmn 198 12 plusmn 228 088 plusmn 09424 165 plusmn 252 405 plusmn 352 248 plusmn 206 68 plusmn 18 102 plusmn 179

SKOV-3 48 221 plusmn 247 500 plusmn 302 165 plusmn 321 65 plusmn 23 032 plusmn 09772 360 plusmn 355 703 plusmn 353 90 plusmn 26 32 plusmn 281 062 plusmn 09824 ge100 ge100 ge100 65 plusmn 23 043 plusmn 050

MDA-MB231 48 ge100 ge100 ge100 15 plusmn 343 059 plusmn 10172 ge100 ge100 ge100 186 plusmn 126 095 plusmn 132

ME methanol extract HF hexane fraction EAF ethyl acetate fraction CF chloroform fractionWF water fraction Doxorubicin was used as positive controlEach value is expressed as mean plusmn standard deviation of three measurements

starches sugars and fiber A macronutrient protein consistsof amino acids which is required for proper growth andhuman body function The seeds were found to containa high level of crude fiber which is a potential source ofphenolic antioxidants The major components of fibers arecellulose hemicelluloses lignin 120573-glucans gums and pectinand hydrocolloids These components can behave as proan-tioxidants [28 29] In our present physicochemical study Pmacrocarpa seeds were mainly made up of carbohydratesfollowed by crude fiber proteins fatmoisture and ashHigh-protein and low-carbohydrate diets are often effective forweight loss Dietary fiber can aid in digestion is helpfulfor weight management and reduces constipation Howevermany high-protein and low-carbohydrate foods are low infiberWomen aged 50 and older need at least 21 grams of fiberdaily whilst those aged 19 to 50 require 25 grams men aged50 and older need 30 grams and those between the age of 19to 50 should consume at least 38 grams of fiber

Steroids possess biological activities which are insec-ticidal cardiotonic and antimicrobial activities and theseare potentially useful for development into therapeuticdrugs Tannins are known to be important for their antivi-ral antibacterial antiparasitic effects anti-inflammatoryantiulcer and antioxidant properties [30ndash33] Saponinshave been found to have antimicrobial anti-inflammatoryantifeedant and hemolytic effects [34 35] Some alkaloids

have been reported to have anticancer and antiviral activityand saponins have been reported to be cardiotonic whileflavonoids have anticancer and anti-inflammatory activity[10 36] The presence of tannins may be responsible for theability of P macrocarpa seeds to be used in the treatmentof diseases such as diabetes diarrhea and dysentery Ourpreliminary phytochemical studies revealed that the chemicalcomponents of P macrocarpa seeds also include flavonoidsphenolics steroids tannins terpenoids glucosides saponinsand carbohydrates

Previous researchers reported that kaempferol myrice-tin naringin quercetin rutin [3] 29-norcucurbitacinderivatives fevicordin A fevicordin A glucoside fevicordinD glucoside [6] mahkoside A dodecanoic acid palmiticacid des-acetyl flavicordin-A flavicordin-A flavicordin-Dflavicordin-A glucoside ethyl stearate lignans and sucrose[37 38] mangiferin (a C-glucosylxantone) kaempferol-3-o-120573-D-glucoside dodecanoic acid palmitic acid ethyl stearatesucrose [5] pinoresinol lariciresinol matairesinol alkaloidsand saponins [37 39] saponins alkaloids polyphenolicsphenols flavanoids lignans tannins [40ndash42] icarisideC3 mangiferin gallic acid phalerin glycoside (345trihydroxy-4-methoxy-benzophenone-3-O-120573-D-glucoside)[43] and 2410158406 trihydroxy-4-methoxy-benzophenone-3-O-120573-D-glucoside [44] were isolated from different partsof P macrocarpa The hexane extract of the seeds was

BioMed Research International 11

found to contain methyl myristate methyl stearate oleicacid methyl oleate linoleic acid methyl linoleate palmiticacid methyl palmitate 6-octadecenoic acid and 917-octadecadial 120573-sitosterol as major components A further30 to 40 of components were not identified as thesewere polar compounds that require HPLC analysis Othercompounds reported in the literature for the seeds leavesand fruits were not identified in this investigation It isprobable that the components reported in the literaturemay be present in other fractions (methyl acetate CHCl

3

and water fractions)Phenolics are able to transfer protons to radicals [45]

DPPH which provides free radicals is normally a blue-violet color However the DPPH turned yellow after it wasconverted to 1 1-diphenyl-2-picrylhydrazine which has lessfree-radical activity Hendra et al reported that the IC

50

values of free radical and reducing power in methanolwith HCl of P macrocarpa seeds were 2450 plusmn 194 and1502 plusmn 128 [42] In our investigation using DPPH assay thewater fraction displayed the highest IC

50value followed by

methanol ethyl acetate hexane and chloroform fractionThe presence of flavonoids in P macrocarpa seeds may be

responsible for the traditional use of the plants in treatingcancer inflammations and allergies Recently it has beenshown that phenolic compounds in crude methanol andethyl acetate extract of P macrocarpa leaves displayed goodantioxidant and antimicrobial activites [46] Hendra et al(2011) examined the amount of total phenolic (477plusmn 104mggallic acid equivalentg DW) and the amount of flavonoid(359 plusmn 247mg rutin equivalentg DW) contents in themethanol extract with HCl of P macrocarpa seeds [42] Inthis study the total flavonoids content was higher than that inthe results previously reported by Rohyami [47] and phenolflavonol and flavonoid contents were the highest in theaqueous methanol extract followed by ethyl acetate waterchloroform and hexane extracts

MTT assay is a sensitive and reliable colorimetric assaythat uses quantitative measurements to calculate the via-bility proliferation and activation of cells This method iscommonly applied to screen anticancer agents [48] Themost well-known methods used to calculate cytotoxicityare the neutral red (NR) uptake and dimethylthiazole-diphenyl tetrazolium bromide (MTT) metabolism [27 49]Earlier investigations showed that the ethanol extract of Pmacrocarpa seeds and fruit meat were not toxic to normalhuman cells but slightly toxic to a Vero cell line [9] Theethanol extract of P macrocarpa seed fruit also showedtoxicity towards T47D breast cancer cell line through COX-2 expression inhibition [7] Desacetylfevicordin A has beenisolated from the ethyl acetate extract of P macrocarpaseeds and this compound displayed excellent cytotoxicityin brine shrimp [43] The ethyl acetate of P macrocarpaseeds exhibited mild cytotoxic effect against HepG2 cells(IC50values between 30 and 60 120583gmL) [46] Previous studies

showed that the cytotoxic activity of the seeds on HT-29MCF-7 Hela and Chang liver cells lines were 384 plusmn 037255 plusmn 137 295 plusmn 10 and 678 plusmn 027 receptively [42] Inour study the methanolic extract of P macrocarpa seeds wasfound to pose cytotoxic effect against HT-29 MCF-7 Cas Ki

and SKOV-3 cell lines (IC50

values giving from 11 plusmn 120to 360 plusmn 355) and mild toxicity on normal cell lines Therewere significant cytotoxic effects on selected cancer cells linesthat were both time- and dose-dependent manner due to thepresence of many secondary metabolites

The phytochemical screening also indicated the presenceof a small amount of cyanogenic glycosides Cyanogenicglucosides which can cause acute cyanide poisoning causerapid respiration and pulse decrease the blood pressure andinduce vomiting diarrhea headache dizziness and so onThus these components have to be removed (usually byboiling) prior to consumption for therapeutic purposes

5 Conclusion

P macrocarpa seeds exhibited antioxidant and cytotoxicactivities It is highly probable that these activities are dueto the presence of phenolic and flavonoid compounds inappreciable amounts in the plant Furthermore the cytotox-icity activity suggested that the seed may contain a potentialanticancer agent The outcome of this study is encouragingdemonstrating the potential for the P macrocarpa as a sourceof multiple therapeutic agents

Abbreviations

EAF Ethyl acetate fractionME Methanol extractHF Hexane fractionCF Chloroform fractionWF Water fractiong Grammg MilligrammL Milliliter

Conflict of Interests

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

Authorsrsquo Contribution

Ma Ma Lay was involved in all experiments and BehroozBanisalamwas involved in antioxidant assay All authors readand approved the submitted version of the paper

Acknowledgments

This work was supported by grants from the MalaysianMinistry of Higher Education (nos PS2862009C andPS4702010B) Ma Ma Lay is a scholarship recipient fromthe ldquoIslamic Development Bank Saudi Arabiardquo The authorswould like to thank Professor Dr Amru BinNasrulhaq Boycefor generously allowing the use of his laboratory for thisresearch

12 BioMed Research International

References

[1] N HarmantoMahkota DewaObat Pusaka ParaDewa Cetakankeempat Agromedia Pustaka Jakarta Indonesia 2002

[2] A Jemal R Siegel E Ward T Murray J Xu and M J ThunldquoCancer statistics 2007rdquoCa ACancer Journal for Clinicians vol57 no 1 pp 43ndash66 2007

[3] R Hendra S Ahmad A Sukari M Y Shukor and EOskoueian ldquoFlavonoid analyses and antimicrobial activity ofvarious parts of Phaleria macrocarpa (Scheff) Boerl fruitrdquoInternational Journal of Molecular Sciences vol 12 no 6 pp3422ndash3431 2011

[4] W Wijayani Sitotoksisitas Minyak Atsiri Biji Dan Daging BuahMahkota Dewa (Phaleria Macrocarpa [Scheff] Boerl) TerhadapMyeloma Cell Line Dan HeLa Cell Line Serta IdentifikasiSenyawa Minyak Atsiri Dengan Kromatografi Gas-SpektrometriMassa (GC-MS) Skripsi FMIPA UGM Yogyakarta Indonesia2005

[5] Y-B Zhang X-J Xu and H-M Liu ldquoChemical con-stituents fromMahkotadewardquo Journal of Asian Natural ProductsResearch vol 8 no 1-2 pp 119ndash123 2006

[6] D Kurnia K Akiyama and H Hayashi ldquo29-Norcucurbitacinderivatives isolated from the Indonesian medicinal plant Pha-leria macrocarpa (Scheff) Boerlrdquo Bioscience Biotechnology andBiochemistry vol 72 no 2 pp 618ndash620 2008

[7] M Bakhriansyah Pengaruh Ekstrak Etanol Biji Mahkota Dewa(Phaleria Macrocarpa [Scheff] Boerl) Pada Sel Kanker Payu-dara T47D Kajian Aktivitas Sitotoksik Dan PenghambatanEkspresi Siklooksigenase-2 Program Pascasarjana UGM 2004

[8] L Wardani The Effect of Phaleria Macrocarpa [Scheff] Boerl)Seed and Flesh Fruit Extract to P53 and Bcl-2 Gene Expression inHela Cell Line FMIPA UGM 2005

[9] A Endang R Tri Joko and E Dewi ldquoCyototoxicity of Phaleriamacrocarpa (Scheff) boerl fruits flesh and seed extract ofethanol and its effect against p53 and Bcl-2 genes expressionof normalrdquo in Proceedings of the International Conference onComputational Science (ICCS rsquo07) 2007

[10] C Evans Trease and Evans Pharmacognosy Saunder 2002[11] A Sofowora Medicinal Plants and Traditional Medicine in

Africa Spectrum Books Ltd Ibadan Nigeria 2nd edition 1993[12] AOAC Official Methods of Analysis Association of Official

Analytical Chemists Arlington Tex USA 2002[13] J Harborne Phytochemical Methods Chapman and Hall Lon-

don UK 1973[14] AOAC Official Methods of Analysis Association of Official

Analytical Chemists Gaithersburg Md USA 17th edition2000

[15] J Lako V C Trenerry M Wahlqvist N WattanapenpaiboonS Sotheeswaran and R Premier ldquoPhytochemical flavonolscarotenoids and the antioxidant properties of a wide selection ofFijian fruit vegetables and other readily available foodsrdquo FoodChemistry vol 101 no 4 pp 1727ndash1741 2007

[16] W Horwitz Official Method of Analysis AOAC InterantionalGaithersburg Md USA 17th edition 2000

[17] A A Adedapo F O Jimoh S Koduru P J Masika and AJ Afolayan ldquoAssessment of the medicinal potentials of themethanol extracts of the leaves and stems of Buddleja salignardquoBMCComplementary andAlternativeMedicine vol 9 article 212009

[18] M Zovko Koncic D Kremer J Gruz et al ldquoAntioxidantand antimicrobial properties ofMoltkia petraea (Tratt) Griseb

flower leaf and stem infusionsrdquo Food and Chemical Toxicologyvol 48 no 6 pp 1537ndash1542 2010

[19] S McDonald P D Prenzler M Antolovich and K RobardsldquoPhenolic content and antioxidant activity of olive extractsrdquoFood Chemistry vol 73 no 1 pp 73ndash84 2001

[20] S M Nabavi M A Ebrahimzadeh S F Nabavi A Hamidiniaand A R Bekhradnia ldquoDetermination of antioxidant activityphenol and flavonoid content of Parrotia persicaMeyrdquo Pharma-cologyonline vol 2 pp 560ndash567 2008

[21] M A Ebrahimzadeh S F Nabavi and S M Nabavi ldquoAntiox-idant activities of methanol extract of Sambucus ebulus Lflowerrdquo Pakistan Journal of Biological Sciences vol 12 no 5 pp447ndash450 2009

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

[23] A Kumaran and R Joel Karunakaran ldquoIn vitro antioxidantactivities of methanol extracts of five Phyllanthus species fromIndiardquo Food Science and Technology vol 40 no 2 pp 344ndash3522007

[24] B O Mbaebie H O Edeoga and A J Afolayan ldquoPhy tochemical analysis and antioxidants activities of aqueous stembark extract of Schotia latifolia Jacqrdquo Asian Pacific Journal ofTropical Biomedicine vol 2 no 2 pp 118ndash124 2012

[25] L I Mensor F S Menezes G G Leitao et al ldquoScreening ofBrazilian plants extracts for antioxidants activity by the use ofDPPH free radical methodrdquo Phytotherapy Research vol 15 pp127ndash130 2001

[26] F Denizot and R Lang ldquoRapid colorimetric assay for cellgrowth and survival modifications to the tetrazolium dyeprocedure giving improved sensitivity and reliabilityrdquo Journalof Immunological Methods vol 89 no 2 pp 271ndash277 1986

[27] T Mosmann ldquoRapid colorimetric assay for cellular growth andsurvival application to proliferation and cytotoxicity assaysrdquoJournal of Immunological Methods vol 65 no 1-2 pp 55ndash631983

[28] P Maisuthisakul R Pongsawatmanit and M H GordonldquoAntioxidant properties of Teaw (Cratoxylum formosum Dyer)extract in soybean oil and emulsionsrdquo Journal of Agriculturaland Food Chemistry vol 54 no 7 pp 2719ndash2725 2006

[29] P Maisuthisakul S Pasuk and P Ritthiruangdej ldquoRelationshipbetween antioxidant properties and chemical composition ofsome Thai plantsrdquo Journal of Food Composition and Analysisvol 21 no 3 pp 229ndash240 2008

[30] H Kolodziej and A F Kiderlen ldquoAntileishmanial activity andimmune modulatory effects of tannins and related compoundson Leishmania parasitised RAW 2647 cellsrdquo Phytochemistryvol 66 no 17 pp 2056ndash2071 2005

[31] T Nakagawa and T Yokozawa ldquoDirect scavenging of nitricoxide and superoxide by green teardquo Food and Chemical Toxi-cology vol 40 no 12 pp 1745ndash1750 2002

[32] V Schulz R Hansel and V Tyler Fitoterapia Racional UmGuiade Fitoterapia Para as ciencias da saude Manole Barueri Brazil2002

[33] S D Kapu Y B Ngwai O Kayode P A Akah C Wambebeand K Gamaniel ldquoAnti-inflammatory analgesic and anti-lymphocytic activities of the aqueous extract of Crinum gigan-teumrdquo Journal of Ethnopharmacology vol 78 no 1 pp 7ndash132001

[34] G Francis Z Kerem H P S Makkar and K Becker ldquoThebiological action of saponins in animal systems a reviewrdquoBritish Journal of Nutrition vol 88 no 6 pp 587ndash605 2002

BioMed Research International 13

[35] R Xu W Zhao J Xu B Shao and G Qin ldquoStudies onbioactive saponins fromChinesemedicinal plantsrdquoAdvances inExperimental Medicine and Biology vol 404 pp 371ndash382 1996

[36] J A Manthey N Guthrie and K Grohmann ldquoBiologicalproperties of citrus flavonoids pertaining to cancer and inflam-mationrdquo Current Medicinal Chemistry vol 8 no 2 pp 135ndash1532001

[37] P Hendra Y Fukushi and Y Hashidoko ldquoSynthesis of ben-zophenone glucopyranosides from Phaleria macrocarpa andrelated benzophenone glucopyranosidesrdquo Bioscience Biotech-nology and Biochemistry vol 73 no 10 pp 2172ndash2182 2009

[38] A Rabia Z B A Mohammad D Aidiahmad S Amirin and IU Muhammad ldquoPhytochemistry and medicinal properties ofPhaleria macrocarpa (Scheff) Boerl extractsrdquo PharmacognosyReview vol 7 no 13 pp 73ndash80 2013

[39] A Saufi C B I VonHeimendahl AWAlfermann andE FussldquoStereochemistry of lignans in Phaleria macrocarpa (Scheff)Boerlrdquo Zeitschrift fur Naturforschung C vol 63 no 1-2 pp 13ndash16 2008

[40] S C Chong M A Dollah P P Chong and A Maha ldquoPhaleriamacrocarpa (Scheff) Boerl fruit aqueous extract enhances LDLreceptor and PCSK9 expression in vivo and in vitrordquo Journal ofEthnopharmacology vol 137 no 1 pp 817ndash827 2011

[41] R R Tjandrawinata D Nofiarny L W Susanto P Hendra andA Clarissa ldquoSymptomatic treatment of premenstrual syndromeandor primary dysmenorrhea with DLBS1442 a bioacitveextract ofPhaleriamacrocaraprdquo International Journal of GeneralMedicine vol 4 pp 465ndash476 2011

[42] R Hendra S Ahmad E Oskoueian A Sukari and M YShukor ldquoAntioxidant anti-inflammatory and cytotoxicity ofPhaleriamacrocarap (Boerl) Scheff fruitrdquoBMCComplementaryand Alternative Medicine vol 11 article 110 2011

[43] D Kurnia K Akiyama and H Hayashi ldquo29-Norcucurbitacinderivatives isolated from the Indonesian medicinal plant Pha-leria macrocarpa (Scheff) Boerlrdquo Bioscience Biotechnology andBiochemistry vol 72 no 2 pp 618ndash620 2008

[44] S Oshimi K Zaima Y Matsuno et al ldquoStudies on theconstituents from the fruits of Phaleria macrocaraprdquo Journal ofNatural Medicines vol 62 no 2 pp 207ndash210 2008

[45] C Sanchez-Moreno J A Larrauri and F Saura-Calixto ldquoAprocedure tomeasure the antiradical efficiency of polyphenolsrdquoJournal of the Science of Food and Agriculture vol 76 pp 270ndash276 1998

[46] Y Andriani M A W Effendy T M T Sifzizul and HMohamad ldquoAntibacterial radical-scavenging activities andcytotoxicit properties of Phaleria macrocarpa (Scheff) BoerlLeaves in HepG2 cell linesrdquo International Journal of Pharma-ceutical Sciences and Research vol 2 no 7 pp 1700ndash1706 2011

[47] Y Rohyami ldquoPenentuan Kandungan Flavonoid dari EkstrakMetanol Daging Buah Mahkota Dewa (Phaleria macrocarpaScheff Boerl)rdquo Jurnal Logika vol 5 2009

[48] R D Petty L A Sutherland E M Hunter and I A CreeldquoComparison of MTT and ATP-based assays for the measure-ment of viable cell numberrdquo Journal of Bioluminescence andChemiluminescence vol 10 no 1 pp 29ndash34 1995

[49] J R Sellers S Cook andV S Goldmacher ldquoA cytotoxicity assayutilizing a fluorescent dye that determines accurate survivingfractions of cellsrdquo Journal of Immunological Methods vol 172no 2 pp 255ndash264 1994

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

8 BioMed Research International

0102030405060708090

100

ME

HF

CF

EAF

24h 48h 72h

IC50

valu

e of c

once

ntra

tion

(120583g

mL)

Ca Ski

0102030405060708090

100

ME

HF

CF

EAF

24h 48h 72h

IC50

valu

e of c

once

ntra

tion

(120583g

mL)

MCF 7

0102030405060708090

100

ME

HF

CF

EAF

24h 48h 72h

IC50

valu

e of c

once

ntra

tion

(120583g

mL)

HT-29

minus10

0

10

20

30

40

50

60

70

80

90

100

ME

HF

CF

EAF

24h 48h 72h

IC50

valu

e of c

once

ntra

tion

(120583g

mL)

SKOV-3

0102030405060708090

100

ME

HF

CF

EAF

24h 48h 72h

IC50

valu

e of c

once

ntra

tion

(120583g

mL)

MDA-MB231

Figure 4 In vitro cytotoxic effects of P macrocarpa seedson Ca Ski MCF-7 HT-29 SKOV-3 and MDA-MB231 cell lines ME methanolextract HF hexane fraction EAF ethyl acetate fraction CF chloroform fractionWF water fraction Each value is expressed as mean plusmnstandard deviation of three measurements

BioMed Research International 9

Methyl linoleate

Methyl stearate

Palmitic acid

Oleic acid

Methyl myristate

Linoleic acid

Methyl oleate

H3C

H3C

H3C

H3C

H3C

H3C

H3C

O

O

O

O

O

O

O

O

O

O

O

CH3

CH3

CH3

CH3

OH

OH

OH

(294mz)

(298mz)

(282mz)

(242mz)

(280mz)

(298mz)

(256mz)

Figure 5

10 BioMed Research International

Table 4 Total flavonoid total flavonol total phenolic contents and result of DPPH free radical scavenging property of P macrocarpa seeds

Tests ME (mgmL) HF (mgmL) CF (mgmL) EAF (mgmL) WF (mgmL)Flavonoid 933 plusmn 08 418 plusmn 15 678 plusmn 11 838 plusmn 03 808 plusmn 10Flavonol 893 plusmn 11 425 plusmn 07 429 plusmn 09 613 plusmn 05 818 plusmn 10Phenolic 720 plusmn 07 263 plusmn 01 326 plusmn 1 358 plusmn 11 512 plusmn 14DPPH 1150 plusmn 003 1500 plusmn 004 1575 plusmn 004 1400 plusmn 005 975 plusmn 003ME methanol extract HF hexane fraction EAF ethyl acetate fraction CF chloroform fraction WF water fraction

Table 5 In vitro cytotoxic effects of methanol extract and its fractions of P macrocarpa cancer cells lines and normal human fibroblast breastcells line

Cell line Incubation periods (hours) ME HF CF EAF Doxorubicin24 82 plusmn 466 ge100 100 plusmn 131 56 plusmn 117 092 plusmn 064

Ca Ski 48 197 plusmn 092 ge100 82 plusmn 104 77 plusmn 185 069 plusmn 10572 407 plusmn 226 ge100 220 plusmn 186 60 plusmn 322 045 plusmn 09924 120 plusmn 22 452 plusmn 149 575 plusmn 264 223 plusmn 158 105 plusmn 108

MCF7 48 85 plusmn 168 555 plusmn 197 400 plusmn 148 168 plusmn 170 012 plusmn 06972 63 plusmn 250 725 plusmn 152 465 plusmn 345 84 plusmn 171 092 plusmn 17024 293 plusmn 226 400 plusmn 315 95 plusmn 295 11 plusmn 120 092 plusmn 072

HT29 48 250 plusmn 135 640 plusmn 203 87 plusmn 159 35 plusmn 200 032 plusmn 21672 215 plusmn 330 750 plusmn 314 210 plusmn 198 12 plusmn 228 088 plusmn 09424 165 plusmn 252 405 plusmn 352 248 plusmn 206 68 plusmn 18 102 plusmn 179

SKOV-3 48 221 plusmn 247 500 plusmn 302 165 plusmn 321 65 plusmn 23 032 plusmn 09772 360 plusmn 355 703 plusmn 353 90 plusmn 26 32 plusmn 281 062 plusmn 09824 ge100 ge100 ge100 65 plusmn 23 043 plusmn 050

MDA-MB231 48 ge100 ge100 ge100 15 plusmn 343 059 plusmn 10172 ge100 ge100 ge100 186 plusmn 126 095 plusmn 132

ME methanol extract HF hexane fraction EAF ethyl acetate fraction CF chloroform fractionWF water fraction Doxorubicin was used as positive controlEach value is expressed as mean plusmn standard deviation of three measurements

starches sugars and fiber A macronutrient protein consistsof amino acids which is required for proper growth andhuman body function The seeds were found to containa high level of crude fiber which is a potential source ofphenolic antioxidants The major components of fibers arecellulose hemicelluloses lignin 120573-glucans gums and pectinand hydrocolloids These components can behave as proan-tioxidants [28 29] In our present physicochemical study Pmacrocarpa seeds were mainly made up of carbohydratesfollowed by crude fiber proteins fatmoisture and ashHigh-protein and low-carbohydrate diets are often effective forweight loss Dietary fiber can aid in digestion is helpfulfor weight management and reduces constipation Howevermany high-protein and low-carbohydrate foods are low infiberWomen aged 50 and older need at least 21 grams of fiberdaily whilst those aged 19 to 50 require 25 grams men aged50 and older need 30 grams and those between the age of 19to 50 should consume at least 38 grams of fiber

Steroids possess biological activities which are insec-ticidal cardiotonic and antimicrobial activities and theseare potentially useful for development into therapeuticdrugs Tannins are known to be important for their antivi-ral antibacterial antiparasitic effects anti-inflammatoryantiulcer and antioxidant properties [30ndash33] Saponinshave been found to have antimicrobial anti-inflammatoryantifeedant and hemolytic effects [34 35] Some alkaloids

have been reported to have anticancer and antiviral activityand saponins have been reported to be cardiotonic whileflavonoids have anticancer and anti-inflammatory activity[10 36] The presence of tannins may be responsible for theability of P macrocarpa seeds to be used in the treatmentof diseases such as diabetes diarrhea and dysentery Ourpreliminary phytochemical studies revealed that the chemicalcomponents of P macrocarpa seeds also include flavonoidsphenolics steroids tannins terpenoids glucosides saponinsand carbohydrates

Previous researchers reported that kaempferol myrice-tin naringin quercetin rutin [3] 29-norcucurbitacinderivatives fevicordin A fevicordin A glucoside fevicordinD glucoside [6] mahkoside A dodecanoic acid palmiticacid des-acetyl flavicordin-A flavicordin-A flavicordin-Dflavicordin-A glucoside ethyl stearate lignans and sucrose[37 38] mangiferin (a C-glucosylxantone) kaempferol-3-o-120573-D-glucoside dodecanoic acid palmitic acid ethyl stearatesucrose [5] pinoresinol lariciresinol matairesinol alkaloidsand saponins [37 39] saponins alkaloids polyphenolicsphenols flavanoids lignans tannins [40ndash42] icarisideC3 mangiferin gallic acid phalerin glycoside (345trihydroxy-4-methoxy-benzophenone-3-O-120573-D-glucoside)[43] and 2410158406 trihydroxy-4-methoxy-benzophenone-3-O-120573-D-glucoside [44] were isolated from different partsof P macrocarpa The hexane extract of the seeds was

BioMed Research International 11

found to contain methyl myristate methyl stearate oleicacid methyl oleate linoleic acid methyl linoleate palmiticacid methyl palmitate 6-octadecenoic acid and 917-octadecadial 120573-sitosterol as major components A further30 to 40 of components were not identified as thesewere polar compounds that require HPLC analysis Othercompounds reported in the literature for the seeds leavesand fruits were not identified in this investigation It isprobable that the components reported in the literaturemay be present in other fractions (methyl acetate CHCl

3

and water fractions)Phenolics are able to transfer protons to radicals [45]

DPPH which provides free radicals is normally a blue-violet color However the DPPH turned yellow after it wasconverted to 1 1-diphenyl-2-picrylhydrazine which has lessfree-radical activity Hendra et al reported that the IC

50

values of free radical and reducing power in methanolwith HCl of P macrocarpa seeds were 2450 plusmn 194 and1502 plusmn 128 [42] In our investigation using DPPH assay thewater fraction displayed the highest IC

50value followed by

methanol ethyl acetate hexane and chloroform fractionThe presence of flavonoids in P macrocarpa seeds may be

responsible for the traditional use of the plants in treatingcancer inflammations and allergies Recently it has beenshown that phenolic compounds in crude methanol andethyl acetate extract of P macrocarpa leaves displayed goodantioxidant and antimicrobial activites [46] Hendra et al(2011) examined the amount of total phenolic (477plusmn 104mggallic acid equivalentg DW) and the amount of flavonoid(359 plusmn 247mg rutin equivalentg DW) contents in themethanol extract with HCl of P macrocarpa seeds [42] Inthis study the total flavonoids content was higher than that inthe results previously reported by Rohyami [47] and phenolflavonol and flavonoid contents were the highest in theaqueous methanol extract followed by ethyl acetate waterchloroform and hexane extracts

MTT assay is a sensitive and reliable colorimetric assaythat uses quantitative measurements to calculate the via-bility proliferation and activation of cells This method iscommonly applied to screen anticancer agents [48] Themost well-known methods used to calculate cytotoxicityare the neutral red (NR) uptake and dimethylthiazole-diphenyl tetrazolium bromide (MTT) metabolism [27 49]Earlier investigations showed that the ethanol extract of Pmacrocarpa seeds and fruit meat were not toxic to normalhuman cells but slightly toxic to a Vero cell line [9] Theethanol extract of P macrocarpa seed fruit also showedtoxicity towards T47D breast cancer cell line through COX-2 expression inhibition [7] Desacetylfevicordin A has beenisolated from the ethyl acetate extract of P macrocarpaseeds and this compound displayed excellent cytotoxicityin brine shrimp [43] The ethyl acetate of P macrocarpaseeds exhibited mild cytotoxic effect against HepG2 cells(IC50values between 30 and 60 120583gmL) [46] Previous studies

showed that the cytotoxic activity of the seeds on HT-29MCF-7 Hela and Chang liver cells lines were 384 plusmn 037255 plusmn 137 295 plusmn 10 and 678 plusmn 027 receptively [42] Inour study the methanolic extract of P macrocarpa seeds wasfound to pose cytotoxic effect against HT-29 MCF-7 Cas Ki

and SKOV-3 cell lines (IC50

values giving from 11 plusmn 120to 360 plusmn 355) and mild toxicity on normal cell lines Therewere significant cytotoxic effects on selected cancer cells linesthat were both time- and dose-dependent manner due to thepresence of many secondary metabolites

The phytochemical screening also indicated the presenceof a small amount of cyanogenic glycosides Cyanogenicglucosides which can cause acute cyanide poisoning causerapid respiration and pulse decrease the blood pressure andinduce vomiting diarrhea headache dizziness and so onThus these components have to be removed (usually byboiling) prior to consumption for therapeutic purposes

5 Conclusion

P macrocarpa seeds exhibited antioxidant and cytotoxicactivities It is highly probable that these activities are dueto the presence of phenolic and flavonoid compounds inappreciable amounts in the plant Furthermore the cytotox-icity activity suggested that the seed may contain a potentialanticancer agent The outcome of this study is encouragingdemonstrating the potential for the P macrocarpa as a sourceof multiple therapeutic agents

Abbreviations

EAF Ethyl acetate fractionME Methanol extractHF Hexane fractionCF Chloroform fractionWF Water fractiong Grammg MilligrammL Milliliter

Conflict of Interests

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

Authorsrsquo Contribution

Ma Ma Lay was involved in all experiments and BehroozBanisalamwas involved in antioxidant assay All authors readand approved the submitted version of the paper

Acknowledgments

This work was supported by grants from the MalaysianMinistry of Higher Education (nos PS2862009C andPS4702010B) Ma Ma Lay is a scholarship recipient fromthe ldquoIslamic Development Bank Saudi Arabiardquo The authorswould like to thank Professor Dr Amru BinNasrulhaq Boycefor generously allowing the use of his laboratory for thisresearch

12 BioMed Research International

References

[1] N HarmantoMahkota DewaObat Pusaka ParaDewa Cetakankeempat Agromedia Pustaka Jakarta Indonesia 2002

[2] A Jemal R Siegel E Ward T Murray J Xu and M J ThunldquoCancer statistics 2007rdquoCa ACancer Journal for Clinicians vol57 no 1 pp 43ndash66 2007

[3] R Hendra S Ahmad A Sukari M Y Shukor and EOskoueian ldquoFlavonoid analyses and antimicrobial activity ofvarious parts of Phaleria macrocarpa (Scheff) Boerl fruitrdquoInternational Journal of Molecular Sciences vol 12 no 6 pp3422ndash3431 2011

[4] W Wijayani Sitotoksisitas Minyak Atsiri Biji Dan Daging BuahMahkota Dewa (Phaleria Macrocarpa [Scheff] Boerl) TerhadapMyeloma Cell Line Dan HeLa Cell Line Serta IdentifikasiSenyawa Minyak Atsiri Dengan Kromatografi Gas-SpektrometriMassa (GC-MS) Skripsi FMIPA UGM Yogyakarta Indonesia2005

[5] Y-B Zhang X-J Xu and H-M Liu ldquoChemical con-stituents fromMahkotadewardquo Journal of Asian Natural ProductsResearch vol 8 no 1-2 pp 119ndash123 2006

[6] D Kurnia K Akiyama and H Hayashi ldquo29-Norcucurbitacinderivatives isolated from the Indonesian medicinal plant Pha-leria macrocarpa (Scheff) Boerlrdquo Bioscience Biotechnology andBiochemistry vol 72 no 2 pp 618ndash620 2008

[7] M Bakhriansyah Pengaruh Ekstrak Etanol Biji Mahkota Dewa(Phaleria Macrocarpa [Scheff] Boerl) Pada Sel Kanker Payu-dara T47D Kajian Aktivitas Sitotoksik Dan PenghambatanEkspresi Siklooksigenase-2 Program Pascasarjana UGM 2004

[8] L Wardani The Effect of Phaleria Macrocarpa [Scheff] Boerl)Seed and Flesh Fruit Extract to P53 and Bcl-2 Gene Expression inHela Cell Line FMIPA UGM 2005

[9] A Endang R Tri Joko and E Dewi ldquoCyototoxicity of Phaleriamacrocarpa (Scheff) boerl fruits flesh and seed extract ofethanol and its effect against p53 and Bcl-2 genes expressionof normalrdquo in Proceedings of the International Conference onComputational Science (ICCS rsquo07) 2007

[10] C Evans Trease and Evans Pharmacognosy Saunder 2002[11] A Sofowora Medicinal Plants and Traditional Medicine in

Africa Spectrum Books Ltd Ibadan Nigeria 2nd edition 1993[12] AOAC Official Methods of Analysis Association of Official

Analytical Chemists Arlington Tex USA 2002[13] J Harborne Phytochemical Methods Chapman and Hall Lon-

don UK 1973[14] AOAC Official Methods of Analysis Association of Official

Analytical Chemists Gaithersburg Md USA 17th edition2000

[15] J Lako V C Trenerry M Wahlqvist N WattanapenpaiboonS Sotheeswaran and R Premier ldquoPhytochemical flavonolscarotenoids and the antioxidant properties of a wide selection ofFijian fruit vegetables and other readily available foodsrdquo FoodChemistry vol 101 no 4 pp 1727ndash1741 2007

[16] W Horwitz Official Method of Analysis AOAC InterantionalGaithersburg Md USA 17th edition 2000

[17] A A Adedapo F O Jimoh S Koduru P J Masika and AJ Afolayan ldquoAssessment of the medicinal potentials of themethanol extracts of the leaves and stems of Buddleja salignardquoBMCComplementary andAlternativeMedicine vol 9 article 212009

[18] M Zovko Koncic D Kremer J Gruz et al ldquoAntioxidantand antimicrobial properties ofMoltkia petraea (Tratt) Griseb

flower leaf and stem infusionsrdquo Food and Chemical Toxicologyvol 48 no 6 pp 1537ndash1542 2010

[19] S McDonald P D Prenzler M Antolovich and K RobardsldquoPhenolic content and antioxidant activity of olive extractsrdquoFood Chemistry vol 73 no 1 pp 73ndash84 2001

[20] S M Nabavi M A Ebrahimzadeh S F Nabavi A Hamidiniaand A R Bekhradnia ldquoDetermination of antioxidant activityphenol and flavonoid content of Parrotia persicaMeyrdquo Pharma-cologyonline vol 2 pp 560ndash567 2008

[21] M A Ebrahimzadeh S F Nabavi and S M Nabavi ldquoAntiox-idant activities of methanol extract of Sambucus ebulus Lflowerrdquo Pakistan Journal of Biological Sciences vol 12 no 5 pp447ndash450 2009

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

[23] A Kumaran and R Joel Karunakaran ldquoIn vitro antioxidantactivities of methanol extracts of five Phyllanthus species fromIndiardquo Food Science and Technology vol 40 no 2 pp 344ndash3522007

[24] B O Mbaebie H O Edeoga and A J Afolayan ldquoPhy tochemical analysis and antioxidants activities of aqueous stembark extract of Schotia latifolia Jacqrdquo Asian Pacific Journal ofTropical Biomedicine vol 2 no 2 pp 118ndash124 2012

[25] L I Mensor F S Menezes G G Leitao et al ldquoScreening ofBrazilian plants extracts for antioxidants activity by the use ofDPPH free radical methodrdquo Phytotherapy Research vol 15 pp127ndash130 2001

[26] F Denizot and R Lang ldquoRapid colorimetric assay for cellgrowth and survival modifications to the tetrazolium dyeprocedure giving improved sensitivity and reliabilityrdquo Journalof Immunological Methods vol 89 no 2 pp 271ndash277 1986

[27] T Mosmann ldquoRapid colorimetric assay for cellular growth andsurvival application to proliferation and cytotoxicity assaysrdquoJournal of Immunological Methods vol 65 no 1-2 pp 55ndash631983

[28] P Maisuthisakul R Pongsawatmanit and M H GordonldquoAntioxidant properties of Teaw (Cratoxylum formosum Dyer)extract in soybean oil and emulsionsrdquo Journal of Agriculturaland Food Chemistry vol 54 no 7 pp 2719ndash2725 2006

[29] P Maisuthisakul S Pasuk and P Ritthiruangdej ldquoRelationshipbetween antioxidant properties and chemical composition ofsome Thai plantsrdquo Journal of Food Composition and Analysisvol 21 no 3 pp 229ndash240 2008

[30] H Kolodziej and A F Kiderlen ldquoAntileishmanial activity andimmune modulatory effects of tannins and related compoundson Leishmania parasitised RAW 2647 cellsrdquo Phytochemistryvol 66 no 17 pp 2056ndash2071 2005

[31] T Nakagawa and T Yokozawa ldquoDirect scavenging of nitricoxide and superoxide by green teardquo Food and Chemical Toxi-cology vol 40 no 12 pp 1745ndash1750 2002

[32] V Schulz R Hansel and V Tyler Fitoterapia Racional UmGuiade Fitoterapia Para as ciencias da saude Manole Barueri Brazil2002

[33] S D Kapu Y B Ngwai O Kayode P A Akah C Wambebeand K Gamaniel ldquoAnti-inflammatory analgesic and anti-lymphocytic activities of the aqueous extract of Crinum gigan-teumrdquo Journal of Ethnopharmacology vol 78 no 1 pp 7ndash132001

[34] G Francis Z Kerem H P S Makkar and K Becker ldquoThebiological action of saponins in animal systems a reviewrdquoBritish Journal of Nutrition vol 88 no 6 pp 587ndash605 2002

BioMed Research International 13

[35] R Xu W Zhao J Xu B Shao and G Qin ldquoStudies onbioactive saponins fromChinesemedicinal plantsrdquoAdvances inExperimental Medicine and Biology vol 404 pp 371ndash382 1996

[36] J A Manthey N Guthrie and K Grohmann ldquoBiologicalproperties of citrus flavonoids pertaining to cancer and inflam-mationrdquo Current Medicinal Chemistry vol 8 no 2 pp 135ndash1532001

[37] P Hendra Y Fukushi and Y Hashidoko ldquoSynthesis of ben-zophenone glucopyranosides from Phaleria macrocarpa andrelated benzophenone glucopyranosidesrdquo Bioscience Biotech-nology and Biochemistry vol 73 no 10 pp 2172ndash2182 2009

[38] A Rabia Z B A Mohammad D Aidiahmad S Amirin and IU Muhammad ldquoPhytochemistry and medicinal properties ofPhaleria macrocarpa (Scheff) Boerl extractsrdquo PharmacognosyReview vol 7 no 13 pp 73ndash80 2013

[39] A Saufi C B I VonHeimendahl AWAlfermann andE FussldquoStereochemistry of lignans in Phaleria macrocarpa (Scheff)Boerlrdquo Zeitschrift fur Naturforschung C vol 63 no 1-2 pp 13ndash16 2008

[40] S C Chong M A Dollah P P Chong and A Maha ldquoPhaleriamacrocarpa (Scheff) Boerl fruit aqueous extract enhances LDLreceptor and PCSK9 expression in vivo and in vitrordquo Journal ofEthnopharmacology vol 137 no 1 pp 817ndash827 2011

[41] R R Tjandrawinata D Nofiarny L W Susanto P Hendra andA Clarissa ldquoSymptomatic treatment of premenstrual syndromeandor primary dysmenorrhea with DLBS1442 a bioacitveextract ofPhaleriamacrocaraprdquo International Journal of GeneralMedicine vol 4 pp 465ndash476 2011

[42] R Hendra S Ahmad E Oskoueian A Sukari and M YShukor ldquoAntioxidant anti-inflammatory and cytotoxicity ofPhaleriamacrocarap (Boerl) Scheff fruitrdquoBMCComplementaryand Alternative Medicine vol 11 article 110 2011

[43] D Kurnia K Akiyama and H Hayashi ldquo29-Norcucurbitacinderivatives isolated from the Indonesian medicinal plant Pha-leria macrocarpa (Scheff) Boerlrdquo Bioscience Biotechnology andBiochemistry vol 72 no 2 pp 618ndash620 2008

[44] S Oshimi K Zaima Y Matsuno et al ldquoStudies on theconstituents from the fruits of Phaleria macrocaraprdquo Journal ofNatural Medicines vol 62 no 2 pp 207ndash210 2008

[45] C Sanchez-Moreno J A Larrauri and F Saura-Calixto ldquoAprocedure tomeasure the antiradical efficiency of polyphenolsrdquoJournal of the Science of Food and Agriculture vol 76 pp 270ndash276 1998

[46] Y Andriani M A W Effendy T M T Sifzizul and HMohamad ldquoAntibacterial radical-scavenging activities andcytotoxicit properties of Phaleria macrocarpa (Scheff) BoerlLeaves in HepG2 cell linesrdquo International Journal of Pharma-ceutical Sciences and Research vol 2 no 7 pp 1700ndash1706 2011

[47] Y Rohyami ldquoPenentuan Kandungan Flavonoid dari EkstrakMetanol Daging Buah Mahkota Dewa (Phaleria macrocarpaScheff Boerl)rdquo Jurnal Logika vol 5 2009

[48] R D Petty L A Sutherland E M Hunter and I A CreeldquoComparison of MTT and ATP-based assays for the measure-ment of viable cell numberrdquo Journal of Bioluminescence andChemiluminescence vol 10 no 1 pp 29ndash34 1995

[49] J R Sellers S Cook andV S Goldmacher ldquoA cytotoxicity assayutilizing a fluorescent dye that determines accurate survivingfractions of cellsrdquo Journal of Immunological Methods vol 172no 2 pp 255ndash264 1994

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

BioMed Research International 9

Methyl linoleate

Methyl stearate

Palmitic acid

Oleic acid

Methyl myristate

Linoleic acid

Methyl oleate

H3C

H3C

H3C

H3C

H3C

H3C

H3C

O

O

O

O

O

O

O

O

O

O

O

CH3

CH3

CH3

CH3

OH

OH

OH

(294mz)

(298mz)

(282mz)

(242mz)

(280mz)

(298mz)

(256mz)

Figure 5

10 BioMed Research International

Table 4 Total flavonoid total flavonol total phenolic contents and result of DPPH free radical scavenging property of P macrocarpa seeds

Tests ME (mgmL) HF (mgmL) CF (mgmL) EAF (mgmL) WF (mgmL)Flavonoid 933 plusmn 08 418 plusmn 15 678 plusmn 11 838 plusmn 03 808 plusmn 10Flavonol 893 plusmn 11 425 plusmn 07 429 plusmn 09 613 plusmn 05 818 plusmn 10Phenolic 720 plusmn 07 263 plusmn 01 326 plusmn 1 358 plusmn 11 512 plusmn 14DPPH 1150 plusmn 003 1500 plusmn 004 1575 plusmn 004 1400 plusmn 005 975 plusmn 003ME methanol extract HF hexane fraction EAF ethyl acetate fraction CF chloroform fraction WF water fraction

Table 5 In vitro cytotoxic effects of methanol extract and its fractions of P macrocarpa cancer cells lines and normal human fibroblast breastcells line

Cell line Incubation periods (hours) ME HF CF EAF Doxorubicin24 82 plusmn 466 ge100 100 plusmn 131 56 plusmn 117 092 plusmn 064

Ca Ski 48 197 plusmn 092 ge100 82 plusmn 104 77 plusmn 185 069 plusmn 10572 407 plusmn 226 ge100 220 plusmn 186 60 plusmn 322 045 plusmn 09924 120 plusmn 22 452 plusmn 149 575 plusmn 264 223 plusmn 158 105 plusmn 108

MCF7 48 85 plusmn 168 555 plusmn 197 400 plusmn 148 168 plusmn 170 012 plusmn 06972 63 plusmn 250 725 plusmn 152 465 plusmn 345 84 plusmn 171 092 plusmn 17024 293 plusmn 226 400 plusmn 315 95 plusmn 295 11 plusmn 120 092 plusmn 072

HT29 48 250 plusmn 135 640 plusmn 203 87 plusmn 159 35 plusmn 200 032 plusmn 21672 215 plusmn 330 750 plusmn 314 210 plusmn 198 12 plusmn 228 088 plusmn 09424 165 plusmn 252 405 plusmn 352 248 plusmn 206 68 plusmn 18 102 plusmn 179

SKOV-3 48 221 plusmn 247 500 plusmn 302 165 plusmn 321 65 plusmn 23 032 plusmn 09772 360 plusmn 355 703 plusmn 353 90 plusmn 26 32 plusmn 281 062 plusmn 09824 ge100 ge100 ge100 65 plusmn 23 043 plusmn 050

MDA-MB231 48 ge100 ge100 ge100 15 plusmn 343 059 plusmn 10172 ge100 ge100 ge100 186 plusmn 126 095 plusmn 132

ME methanol extract HF hexane fraction EAF ethyl acetate fraction CF chloroform fractionWF water fraction Doxorubicin was used as positive controlEach value is expressed as mean plusmn standard deviation of three measurements

starches sugars and fiber A macronutrient protein consistsof amino acids which is required for proper growth andhuman body function The seeds were found to containa high level of crude fiber which is a potential source ofphenolic antioxidants The major components of fibers arecellulose hemicelluloses lignin 120573-glucans gums and pectinand hydrocolloids These components can behave as proan-tioxidants [28 29] In our present physicochemical study Pmacrocarpa seeds were mainly made up of carbohydratesfollowed by crude fiber proteins fatmoisture and ashHigh-protein and low-carbohydrate diets are often effective forweight loss Dietary fiber can aid in digestion is helpfulfor weight management and reduces constipation Howevermany high-protein and low-carbohydrate foods are low infiberWomen aged 50 and older need at least 21 grams of fiberdaily whilst those aged 19 to 50 require 25 grams men aged50 and older need 30 grams and those between the age of 19to 50 should consume at least 38 grams of fiber

Steroids possess biological activities which are insec-ticidal cardiotonic and antimicrobial activities and theseare potentially useful for development into therapeuticdrugs Tannins are known to be important for their antivi-ral antibacterial antiparasitic effects anti-inflammatoryantiulcer and antioxidant properties [30ndash33] Saponinshave been found to have antimicrobial anti-inflammatoryantifeedant and hemolytic effects [34 35] Some alkaloids

have been reported to have anticancer and antiviral activityand saponins have been reported to be cardiotonic whileflavonoids have anticancer and anti-inflammatory activity[10 36] The presence of tannins may be responsible for theability of P macrocarpa seeds to be used in the treatmentof diseases such as diabetes diarrhea and dysentery Ourpreliminary phytochemical studies revealed that the chemicalcomponents of P macrocarpa seeds also include flavonoidsphenolics steroids tannins terpenoids glucosides saponinsand carbohydrates

Previous researchers reported that kaempferol myrice-tin naringin quercetin rutin [3] 29-norcucurbitacinderivatives fevicordin A fevicordin A glucoside fevicordinD glucoside [6] mahkoside A dodecanoic acid palmiticacid des-acetyl flavicordin-A flavicordin-A flavicordin-Dflavicordin-A glucoside ethyl stearate lignans and sucrose[37 38] mangiferin (a C-glucosylxantone) kaempferol-3-o-120573-D-glucoside dodecanoic acid palmitic acid ethyl stearatesucrose [5] pinoresinol lariciresinol matairesinol alkaloidsand saponins [37 39] saponins alkaloids polyphenolicsphenols flavanoids lignans tannins [40ndash42] icarisideC3 mangiferin gallic acid phalerin glycoside (345trihydroxy-4-methoxy-benzophenone-3-O-120573-D-glucoside)[43] and 2410158406 trihydroxy-4-methoxy-benzophenone-3-O-120573-D-glucoside [44] were isolated from different partsof P macrocarpa The hexane extract of the seeds was

BioMed Research International 11

found to contain methyl myristate methyl stearate oleicacid methyl oleate linoleic acid methyl linoleate palmiticacid methyl palmitate 6-octadecenoic acid and 917-octadecadial 120573-sitosterol as major components A further30 to 40 of components were not identified as thesewere polar compounds that require HPLC analysis Othercompounds reported in the literature for the seeds leavesand fruits were not identified in this investigation It isprobable that the components reported in the literaturemay be present in other fractions (methyl acetate CHCl

3

and water fractions)Phenolics are able to transfer protons to radicals [45]

DPPH which provides free radicals is normally a blue-violet color However the DPPH turned yellow after it wasconverted to 1 1-diphenyl-2-picrylhydrazine which has lessfree-radical activity Hendra et al reported that the IC

50

values of free radical and reducing power in methanolwith HCl of P macrocarpa seeds were 2450 plusmn 194 and1502 plusmn 128 [42] In our investigation using DPPH assay thewater fraction displayed the highest IC

50value followed by

methanol ethyl acetate hexane and chloroform fractionThe presence of flavonoids in P macrocarpa seeds may be

responsible for the traditional use of the plants in treatingcancer inflammations and allergies Recently it has beenshown that phenolic compounds in crude methanol andethyl acetate extract of P macrocarpa leaves displayed goodantioxidant and antimicrobial activites [46] Hendra et al(2011) examined the amount of total phenolic (477plusmn 104mggallic acid equivalentg DW) and the amount of flavonoid(359 plusmn 247mg rutin equivalentg DW) contents in themethanol extract with HCl of P macrocarpa seeds [42] Inthis study the total flavonoids content was higher than that inthe results previously reported by Rohyami [47] and phenolflavonol and flavonoid contents were the highest in theaqueous methanol extract followed by ethyl acetate waterchloroform and hexane extracts

MTT assay is a sensitive and reliable colorimetric assaythat uses quantitative measurements to calculate the via-bility proliferation and activation of cells This method iscommonly applied to screen anticancer agents [48] Themost well-known methods used to calculate cytotoxicityare the neutral red (NR) uptake and dimethylthiazole-diphenyl tetrazolium bromide (MTT) metabolism [27 49]Earlier investigations showed that the ethanol extract of Pmacrocarpa seeds and fruit meat were not toxic to normalhuman cells but slightly toxic to a Vero cell line [9] Theethanol extract of P macrocarpa seed fruit also showedtoxicity towards T47D breast cancer cell line through COX-2 expression inhibition [7] Desacetylfevicordin A has beenisolated from the ethyl acetate extract of P macrocarpaseeds and this compound displayed excellent cytotoxicityin brine shrimp [43] The ethyl acetate of P macrocarpaseeds exhibited mild cytotoxic effect against HepG2 cells(IC50values between 30 and 60 120583gmL) [46] Previous studies

showed that the cytotoxic activity of the seeds on HT-29MCF-7 Hela and Chang liver cells lines were 384 plusmn 037255 plusmn 137 295 plusmn 10 and 678 plusmn 027 receptively [42] Inour study the methanolic extract of P macrocarpa seeds wasfound to pose cytotoxic effect against HT-29 MCF-7 Cas Ki

and SKOV-3 cell lines (IC50

values giving from 11 plusmn 120to 360 plusmn 355) and mild toxicity on normal cell lines Therewere significant cytotoxic effects on selected cancer cells linesthat were both time- and dose-dependent manner due to thepresence of many secondary metabolites

The phytochemical screening also indicated the presenceof a small amount of cyanogenic glycosides Cyanogenicglucosides which can cause acute cyanide poisoning causerapid respiration and pulse decrease the blood pressure andinduce vomiting diarrhea headache dizziness and so onThus these components have to be removed (usually byboiling) prior to consumption for therapeutic purposes

5 Conclusion

P macrocarpa seeds exhibited antioxidant and cytotoxicactivities It is highly probable that these activities are dueto the presence of phenolic and flavonoid compounds inappreciable amounts in the plant Furthermore the cytotox-icity activity suggested that the seed may contain a potentialanticancer agent The outcome of this study is encouragingdemonstrating the potential for the P macrocarpa as a sourceof multiple therapeutic agents

Abbreviations

EAF Ethyl acetate fractionME Methanol extractHF Hexane fractionCF Chloroform fractionWF Water fractiong Grammg MilligrammL Milliliter

Conflict of Interests

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

Authorsrsquo Contribution

Ma Ma Lay was involved in all experiments and BehroozBanisalamwas involved in antioxidant assay All authors readand approved the submitted version of the paper

Acknowledgments

This work was supported by grants from the MalaysianMinistry of Higher Education (nos PS2862009C andPS4702010B) Ma Ma Lay is a scholarship recipient fromthe ldquoIslamic Development Bank Saudi Arabiardquo The authorswould like to thank Professor Dr Amru BinNasrulhaq Boycefor generously allowing the use of his laboratory for thisresearch

12 BioMed Research International

References

[1] N HarmantoMahkota DewaObat Pusaka ParaDewa Cetakankeempat Agromedia Pustaka Jakarta Indonesia 2002

[2] A Jemal R Siegel E Ward T Murray J Xu and M J ThunldquoCancer statistics 2007rdquoCa ACancer Journal for Clinicians vol57 no 1 pp 43ndash66 2007

[3] R Hendra S Ahmad A Sukari M Y Shukor and EOskoueian ldquoFlavonoid analyses and antimicrobial activity ofvarious parts of Phaleria macrocarpa (Scheff) Boerl fruitrdquoInternational Journal of Molecular Sciences vol 12 no 6 pp3422ndash3431 2011

[4] W Wijayani Sitotoksisitas Minyak Atsiri Biji Dan Daging BuahMahkota Dewa (Phaleria Macrocarpa [Scheff] Boerl) TerhadapMyeloma Cell Line Dan HeLa Cell Line Serta IdentifikasiSenyawa Minyak Atsiri Dengan Kromatografi Gas-SpektrometriMassa (GC-MS) Skripsi FMIPA UGM Yogyakarta Indonesia2005

[5] Y-B Zhang X-J Xu and H-M Liu ldquoChemical con-stituents fromMahkotadewardquo Journal of Asian Natural ProductsResearch vol 8 no 1-2 pp 119ndash123 2006

[6] D Kurnia K Akiyama and H Hayashi ldquo29-Norcucurbitacinderivatives isolated from the Indonesian medicinal plant Pha-leria macrocarpa (Scheff) Boerlrdquo Bioscience Biotechnology andBiochemistry vol 72 no 2 pp 618ndash620 2008

[7] M Bakhriansyah Pengaruh Ekstrak Etanol Biji Mahkota Dewa(Phaleria Macrocarpa [Scheff] Boerl) Pada Sel Kanker Payu-dara T47D Kajian Aktivitas Sitotoksik Dan PenghambatanEkspresi Siklooksigenase-2 Program Pascasarjana UGM 2004

[8] L Wardani The Effect of Phaleria Macrocarpa [Scheff] Boerl)Seed and Flesh Fruit Extract to P53 and Bcl-2 Gene Expression inHela Cell Line FMIPA UGM 2005

[9] A Endang R Tri Joko and E Dewi ldquoCyototoxicity of Phaleriamacrocarpa (Scheff) boerl fruits flesh and seed extract ofethanol and its effect against p53 and Bcl-2 genes expressionof normalrdquo in Proceedings of the International Conference onComputational Science (ICCS rsquo07) 2007

[10] C Evans Trease and Evans Pharmacognosy Saunder 2002[11] A Sofowora Medicinal Plants and Traditional Medicine in

Africa Spectrum Books Ltd Ibadan Nigeria 2nd edition 1993[12] AOAC Official Methods of Analysis Association of Official

Analytical Chemists Arlington Tex USA 2002[13] J Harborne Phytochemical Methods Chapman and Hall Lon-

don UK 1973[14] AOAC Official Methods of Analysis Association of Official

Analytical Chemists Gaithersburg Md USA 17th edition2000

[15] J Lako V C Trenerry M Wahlqvist N WattanapenpaiboonS Sotheeswaran and R Premier ldquoPhytochemical flavonolscarotenoids and the antioxidant properties of a wide selection ofFijian fruit vegetables and other readily available foodsrdquo FoodChemistry vol 101 no 4 pp 1727ndash1741 2007

[16] W Horwitz Official Method of Analysis AOAC InterantionalGaithersburg Md USA 17th edition 2000

[17] A A Adedapo F O Jimoh S Koduru P J Masika and AJ Afolayan ldquoAssessment of the medicinal potentials of themethanol extracts of the leaves and stems of Buddleja salignardquoBMCComplementary andAlternativeMedicine vol 9 article 212009

[18] M Zovko Koncic D Kremer J Gruz et al ldquoAntioxidantand antimicrobial properties ofMoltkia petraea (Tratt) Griseb

flower leaf and stem infusionsrdquo Food and Chemical Toxicologyvol 48 no 6 pp 1537ndash1542 2010

[19] S McDonald P D Prenzler M Antolovich and K RobardsldquoPhenolic content and antioxidant activity of olive extractsrdquoFood Chemistry vol 73 no 1 pp 73ndash84 2001

[20] S M Nabavi M A Ebrahimzadeh S F Nabavi A Hamidiniaand A R Bekhradnia ldquoDetermination of antioxidant activityphenol and flavonoid content of Parrotia persicaMeyrdquo Pharma-cologyonline vol 2 pp 560ndash567 2008

[21] M A Ebrahimzadeh S F Nabavi and S M Nabavi ldquoAntiox-idant activities of methanol extract of Sambucus ebulus Lflowerrdquo Pakistan Journal of Biological Sciences vol 12 no 5 pp447ndash450 2009

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

[23] A Kumaran and R Joel Karunakaran ldquoIn vitro antioxidantactivities of methanol extracts of five Phyllanthus species fromIndiardquo Food Science and Technology vol 40 no 2 pp 344ndash3522007

[24] B O Mbaebie H O Edeoga and A J Afolayan ldquoPhy tochemical analysis and antioxidants activities of aqueous stembark extract of Schotia latifolia Jacqrdquo Asian Pacific Journal ofTropical Biomedicine vol 2 no 2 pp 118ndash124 2012

[25] L I Mensor F S Menezes G G Leitao et al ldquoScreening ofBrazilian plants extracts for antioxidants activity by the use ofDPPH free radical methodrdquo Phytotherapy Research vol 15 pp127ndash130 2001

[26] F Denizot and R Lang ldquoRapid colorimetric assay for cellgrowth and survival modifications to the tetrazolium dyeprocedure giving improved sensitivity and reliabilityrdquo Journalof Immunological Methods vol 89 no 2 pp 271ndash277 1986

[27] T Mosmann ldquoRapid colorimetric assay for cellular growth andsurvival application to proliferation and cytotoxicity assaysrdquoJournal of Immunological Methods vol 65 no 1-2 pp 55ndash631983

[28] P Maisuthisakul R Pongsawatmanit and M H GordonldquoAntioxidant properties of Teaw (Cratoxylum formosum Dyer)extract in soybean oil and emulsionsrdquo Journal of Agriculturaland Food Chemistry vol 54 no 7 pp 2719ndash2725 2006

[29] P Maisuthisakul S Pasuk and P Ritthiruangdej ldquoRelationshipbetween antioxidant properties and chemical composition ofsome Thai plantsrdquo Journal of Food Composition and Analysisvol 21 no 3 pp 229ndash240 2008

[30] H Kolodziej and A F Kiderlen ldquoAntileishmanial activity andimmune modulatory effects of tannins and related compoundson Leishmania parasitised RAW 2647 cellsrdquo Phytochemistryvol 66 no 17 pp 2056ndash2071 2005

[31] T Nakagawa and T Yokozawa ldquoDirect scavenging of nitricoxide and superoxide by green teardquo Food and Chemical Toxi-cology vol 40 no 12 pp 1745ndash1750 2002

[32] V Schulz R Hansel and V Tyler Fitoterapia Racional UmGuiade Fitoterapia Para as ciencias da saude Manole Barueri Brazil2002

[33] S D Kapu Y B Ngwai O Kayode P A Akah C Wambebeand K Gamaniel ldquoAnti-inflammatory analgesic and anti-lymphocytic activities of the aqueous extract of Crinum gigan-teumrdquo Journal of Ethnopharmacology vol 78 no 1 pp 7ndash132001

[34] G Francis Z Kerem H P S Makkar and K Becker ldquoThebiological action of saponins in animal systems a reviewrdquoBritish Journal of Nutrition vol 88 no 6 pp 587ndash605 2002

BioMed Research International 13

[35] R Xu W Zhao J Xu B Shao and G Qin ldquoStudies onbioactive saponins fromChinesemedicinal plantsrdquoAdvances inExperimental Medicine and Biology vol 404 pp 371ndash382 1996

[36] J A Manthey N Guthrie and K Grohmann ldquoBiologicalproperties of citrus flavonoids pertaining to cancer and inflam-mationrdquo Current Medicinal Chemistry vol 8 no 2 pp 135ndash1532001

[37] P Hendra Y Fukushi and Y Hashidoko ldquoSynthesis of ben-zophenone glucopyranosides from Phaleria macrocarpa andrelated benzophenone glucopyranosidesrdquo Bioscience Biotech-nology and Biochemistry vol 73 no 10 pp 2172ndash2182 2009

[38] A Rabia Z B A Mohammad D Aidiahmad S Amirin and IU Muhammad ldquoPhytochemistry and medicinal properties ofPhaleria macrocarpa (Scheff) Boerl extractsrdquo PharmacognosyReview vol 7 no 13 pp 73ndash80 2013

[39] A Saufi C B I VonHeimendahl AWAlfermann andE FussldquoStereochemistry of lignans in Phaleria macrocarpa (Scheff)Boerlrdquo Zeitschrift fur Naturforschung C vol 63 no 1-2 pp 13ndash16 2008

[40] S C Chong M A Dollah P P Chong and A Maha ldquoPhaleriamacrocarpa (Scheff) Boerl fruit aqueous extract enhances LDLreceptor and PCSK9 expression in vivo and in vitrordquo Journal ofEthnopharmacology vol 137 no 1 pp 817ndash827 2011

[41] R R Tjandrawinata D Nofiarny L W Susanto P Hendra andA Clarissa ldquoSymptomatic treatment of premenstrual syndromeandor primary dysmenorrhea with DLBS1442 a bioacitveextract ofPhaleriamacrocaraprdquo International Journal of GeneralMedicine vol 4 pp 465ndash476 2011

[42] R Hendra S Ahmad E Oskoueian A Sukari and M YShukor ldquoAntioxidant anti-inflammatory and cytotoxicity ofPhaleriamacrocarap (Boerl) Scheff fruitrdquoBMCComplementaryand Alternative Medicine vol 11 article 110 2011

[43] D Kurnia K Akiyama and H Hayashi ldquo29-Norcucurbitacinderivatives isolated from the Indonesian medicinal plant Pha-leria macrocarpa (Scheff) Boerlrdquo Bioscience Biotechnology andBiochemistry vol 72 no 2 pp 618ndash620 2008

[44] S Oshimi K Zaima Y Matsuno et al ldquoStudies on theconstituents from the fruits of Phaleria macrocaraprdquo Journal ofNatural Medicines vol 62 no 2 pp 207ndash210 2008

[45] C Sanchez-Moreno J A Larrauri and F Saura-Calixto ldquoAprocedure tomeasure the antiradical efficiency of polyphenolsrdquoJournal of the Science of Food and Agriculture vol 76 pp 270ndash276 1998

[46] Y Andriani M A W Effendy T M T Sifzizul and HMohamad ldquoAntibacterial radical-scavenging activities andcytotoxicit properties of Phaleria macrocarpa (Scheff) BoerlLeaves in HepG2 cell linesrdquo International Journal of Pharma-ceutical Sciences and Research vol 2 no 7 pp 1700ndash1706 2011

[47] Y Rohyami ldquoPenentuan Kandungan Flavonoid dari EkstrakMetanol Daging Buah Mahkota Dewa (Phaleria macrocarpaScheff Boerl)rdquo Jurnal Logika vol 5 2009

[48] R D Petty L A Sutherland E M Hunter and I A CreeldquoComparison of MTT and ATP-based assays for the measure-ment of viable cell numberrdquo Journal of Bioluminescence andChemiluminescence vol 10 no 1 pp 29ndash34 1995

[49] J R Sellers S Cook andV S Goldmacher ldquoA cytotoxicity assayutilizing a fluorescent dye that determines accurate survivingfractions of cellsrdquo Journal of Immunological Methods vol 172no 2 pp 255ndash264 1994

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

10 BioMed Research International

Table 4 Total flavonoid total flavonol total phenolic contents and result of DPPH free radical scavenging property of P macrocarpa seeds

Tests ME (mgmL) HF (mgmL) CF (mgmL) EAF (mgmL) WF (mgmL)Flavonoid 933 plusmn 08 418 plusmn 15 678 plusmn 11 838 plusmn 03 808 plusmn 10Flavonol 893 plusmn 11 425 plusmn 07 429 plusmn 09 613 plusmn 05 818 plusmn 10Phenolic 720 plusmn 07 263 plusmn 01 326 plusmn 1 358 plusmn 11 512 plusmn 14DPPH 1150 plusmn 003 1500 plusmn 004 1575 plusmn 004 1400 plusmn 005 975 plusmn 003ME methanol extract HF hexane fraction EAF ethyl acetate fraction CF chloroform fraction WF water fraction

Table 5 In vitro cytotoxic effects of methanol extract and its fractions of P macrocarpa cancer cells lines and normal human fibroblast breastcells line

Cell line Incubation periods (hours) ME HF CF EAF Doxorubicin24 82 plusmn 466 ge100 100 plusmn 131 56 plusmn 117 092 plusmn 064

Ca Ski 48 197 plusmn 092 ge100 82 plusmn 104 77 plusmn 185 069 plusmn 10572 407 plusmn 226 ge100 220 plusmn 186 60 plusmn 322 045 plusmn 09924 120 plusmn 22 452 plusmn 149 575 plusmn 264 223 plusmn 158 105 plusmn 108

MCF7 48 85 plusmn 168 555 plusmn 197 400 plusmn 148 168 plusmn 170 012 plusmn 06972 63 plusmn 250 725 plusmn 152 465 plusmn 345 84 plusmn 171 092 plusmn 17024 293 plusmn 226 400 plusmn 315 95 plusmn 295 11 plusmn 120 092 plusmn 072

HT29 48 250 plusmn 135 640 plusmn 203 87 plusmn 159 35 plusmn 200 032 plusmn 21672 215 plusmn 330 750 plusmn 314 210 plusmn 198 12 plusmn 228 088 plusmn 09424 165 plusmn 252 405 plusmn 352 248 plusmn 206 68 plusmn 18 102 plusmn 179

SKOV-3 48 221 plusmn 247 500 plusmn 302 165 plusmn 321 65 plusmn 23 032 plusmn 09772 360 plusmn 355 703 plusmn 353 90 plusmn 26 32 plusmn 281 062 plusmn 09824 ge100 ge100 ge100 65 plusmn 23 043 plusmn 050

MDA-MB231 48 ge100 ge100 ge100 15 plusmn 343 059 plusmn 10172 ge100 ge100 ge100 186 plusmn 126 095 plusmn 132

ME methanol extract HF hexane fraction EAF ethyl acetate fraction CF chloroform fractionWF water fraction Doxorubicin was used as positive controlEach value is expressed as mean plusmn standard deviation of three measurements

starches sugars and fiber A macronutrient protein consistsof amino acids which is required for proper growth andhuman body function The seeds were found to containa high level of crude fiber which is a potential source ofphenolic antioxidants The major components of fibers arecellulose hemicelluloses lignin 120573-glucans gums and pectinand hydrocolloids These components can behave as proan-tioxidants [28 29] In our present physicochemical study Pmacrocarpa seeds were mainly made up of carbohydratesfollowed by crude fiber proteins fatmoisture and ashHigh-protein and low-carbohydrate diets are often effective forweight loss Dietary fiber can aid in digestion is helpfulfor weight management and reduces constipation Howevermany high-protein and low-carbohydrate foods are low infiberWomen aged 50 and older need at least 21 grams of fiberdaily whilst those aged 19 to 50 require 25 grams men aged50 and older need 30 grams and those between the age of 19to 50 should consume at least 38 grams of fiber

Steroids possess biological activities which are insec-ticidal cardiotonic and antimicrobial activities and theseare potentially useful for development into therapeuticdrugs Tannins are known to be important for their antivi-ral antibacterial antiparasitic effects anti-inflammatoryantiulcer and antioxidant properties [30ndash33] Saponinshave been found to have antimicrobial anti-inflammatoryantifeedant and hemolytic effects [34 35] Some alkaloids

have been reported to have anticancer and antiviral activityand saponins have been reported to be cardiotonic whileflavonoids have anticancer and anti-inflammatory activity[10 36] The presence of tannins may be responsible for theability of P macrocarpa seeds to be used in the treatmentof diseases such as diabetes diarrhea and dysentery Ourpreliminary phytochemical studies revealed that the chemicalcomponents of P macrocarpa seeds also include flavonoidsphenolics steroids tannins terpenoids glucosides saponinsand carbohydrates

Previous researchers reported that kaempferol myrice-tin naringin quercetin rutin [3] 29-norcucurbitacinderivatives fevicordin A fevicordin A glucoside fevicordinD glucoside [6] mahkoside A dodecanoic acid palmiticacid des-acetyl flavicordin-A flavicordin-A flavicordin-Dflavicordin-A glucoside ethyl stearate lignans and sucrose[37 38] mangiferin (a C-glucosylxantone) kaempferol-3-o-120573-D-glucoside dodecanoic acid palmitic acid ethyl stearatesucrose [5] pinoresinol lariciresinol matairesinol alkaloidsand saponins [37 39] saponins alkaloids polyphenolicsphenols flavanoids lignans tannins [40ndash42] icarisideC3 mangiferin gallic acid phalerin glycoside (345trihydroxy-4-methoxy-benzophenone-3-O-120573-D-glucoside)[43] and 2410158406 trihydroxy-4-methoxy-benzophenone-3-O-120573-D-glucoside [44] were isolated from different partsof P macrocarpa The hexane extract of the seeds was

BioMed Research International 11

found to contain methyl myristate methyl stearate oleicacid methyl oleate linoleic acid methyl linoleate palmiticacid methyl palmitate 6-octadecenoic acid and 917-octadecadial 120573-sitosterol as major components A further30 to 40 of components were not identified as thesewere polar compounds that require HPLC analysis Othercompounds reported in the literature for the seeds leavesand fruits were not identified in this investigation It isprobable that the components reported in the literaturemay be present in other fractions (methyl acetate CHCl

3

and water fractions)Phenolics are able to transfer protons to radicals [45]

DPPH which provides free radicals is normally a blue-violet color However the DPPH turned yellow after it wasconverted to 1 1-diphenyl-2-picrylhydrazine which has lessfree-radical activity Hendra et al reported that the IC

50

values of free radical and reducing power in methanolwith HCl of P macrocarpa seeds were 2450 plusmn 194 and1502 plusmn 128 [42] In our investigation using DPPH assay thewater fraction displayed the highest IC

50value followed by

methanol ethyl acetate hexane and chloroform fractionThe presence of flavonoids in P macrocarpa seeds may be

responsible for the traditional use of the plants in treatingcancer inflammations and allergies Recently it has beenshown that phenolic compounds in crude methanol andethyl acetate extract of P macrocarpa leaves displayed goodantioxidant and antimicrobial activites [46] Hendra et al(2011) examined the amount of total phenolic (477plusmn 104mggallic acid equivalentg DW) and the amount of flavonoid(359 plusmn 247mg rutin equivalentg DW) contents in themethanol extract with HCl of P macrocarpa seeds [42] Inthis study the total flavonoids content was higher than that inthe results previously reported by Rohyami [47] and phenolflavonol and flavonoid contents were the highest in theaqueous methanol extract followed by ethyl acetate waterchloroform and hexane extracts

MTT assay is a sensitive and reliable colorimetric assaythat uses quantitative measurements to calculate the via-bility proliferation and activation of cells This method iscommonly applied to screen anticancer agents [48] Themost well-known methods used to calculate cytotoxicityare the neutral red (NR) uptake and dimethylthiazole-diphenyl tetrazolium bromide (MTT) metabolism [27 49]Earlier investigations showed that the ethanol extract of Pmacrocarpa seeds and fruit meat were not toxic to normalhuman cells but slightly toxic to a Vero cell line [9] Theethanol extract of P macrocarpa seed fruit also showedtoxicity towards T47D breast cancer cell line through COX-2 expression inhibition [7] Desacetylfevicordin A has beenisolated from the ethyl acetate extract of P macrocarpaseeds and this compound displayed excellent cytotoxicityin brine shrimp [43] The ethyl acetate of P macrocarpaseeds exhibited mild cytotoxic effect against HepG2 cells(IC50values between 30 and 60 120583gmL) [46] Previous studies

showed that the cytotoxic activity of the seeds on HT-29MCF-7 Hela and Chang liver cells lines were 384 plusmn 037255 plusmn 137 295 plusmn 10 and 678 plusmn 027 receptively [42] Inour study the methanolic extract of P macrocarpa seeds wasfound to pose cytotoxic effect against HT-29 MCF-7 Cas Ki

and SKOV-3 cell lines (IC50

values giving from 11 plusmn 120to 360 plusmn 355) and mild toxicity on normal cell lines Therewere significant cytotoxic effects on selected cancer cells linesthat were both time- and dose-dependent manner due to thepresence of many secondary metabolites

The phytochemical screening also indicated the presenceof a small amount of cyanogenic glycosides Cyanogenicglucosides which can cause acute cyanide poisoning causerapid respiration and pulse decrease the blood pressure andinduce vomiting diarrhea headache dizziness and so onThus these components have to be removed (usually byboiling) prior to consumption for therapeutic purposes

5 Conclusion

P macrocarpa seeds exhibited antioxidant and cytotoxicactivities It is highly probable that these activities are dueto the presence of phenolic and flavonoid compounds inappreciable amounts in the plant Furthermore the cytotox-icity activity suggested that the seed may contain a potentialanticancer agent The outcome of this study is encouragingdemonstrating the potential for the P macrocarpa as a sourceof multiple therapeutic agents

Abbreviations

EAF Ethyl acetate fractionME Methanol extractHF Hexane fractionCF Chloroform fractionWF Water fractiong Grammg MilligrammL Milliliter

Conflict of Interests

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

Authorsrsquo Contribution

Ma Ma Lay was involved in all experiments and BehroozBanisalamwas involved in antioxidant assay All authors readand approved the submitted version of the paper

Acknowledgments

This work was supported by grants from the MalaysianMinistry of Higher Education (nos PS2862009C andPS4702010B) Ma Ma Lay is a scholarship recipient fromthe ldquoIslamic Development Bank Saudi Arabiardquo The authorswould like to thank Professor Dr Amru BinNasrulhaq Boycefor generously allowing the use of his laboratory for thisresearch

12 BioMed Research International

References

[1] N HarmantoMahkota DewaObat Pusaka ParaDewa Cetakankeempat Agromedia Pustaka Jakarta Indonesia 2002

[2] A Jemal R Siegel E Ward T Murray J Xu and M J ThunldquoCancer statistics 2007rdquoCa ACancer Journal for Clinicians vol57 no 1 pp 43ndash66 2007

[3] R Hendra S Ahmad A Sukari M Y Shukor and EOskoueian ldquoFlavonoid analyses and antimicrobial activity ofvarious parts of Phaleria macrocarpa (Scheff) Boerl fruitrdquoInternational Journal of Molecular Sciences vol 12 no 6 pp3422ndash3431 2011

[4] W Wijayani Sitotoksisitas Minyak Atsiri Biji Dan Daging BuahMahkota Dewa (Phaleria Macrocarpa [Scheff] Boerl) TerhadapMyeloma Cell Line Dan HeLa Cell Line Serta IdentifikasiSenyawa Minyak Atsiri Dengan Kromatografi Gas-SpektrometriMassa (GC-MS) Skripsi FMIPA UGM Yogyakarta Indonesia2005

[5] Y-B Zhang X-J Xu and H-M Liu ldquoChemical con-stituents fromMahkotadewardquo Journal of Asian Natural ProductsResearch vol 8 no 1-2 pp 119ndash123 2006

[6] D Kurnia K Akiyama and H Hayashi ldquo29-Norcucurbitacinderivatives isolated from the Indonesian medicinal plant Pha-leria macrocarpa (Scheff) Boerlrdquo Bioscience Biotechnology andBiochemistry vol 72 no 2 pp 618ndash620 2008

[7] M Bakhriansyah Pengaruh Ekstrak Etanol Biji Mahkota Dewa(Phaleria Macrocarpa [Scheff] Boerl) Pada Sel Kanker Payu-dara T47D Kajian Aktivitas Sitotoksik Dan PenghambatanEkspresi Siklooksigenase-2 Program Pascasarjana UGM 2004

[8] L Wardani The Effect of Phaleria Macrocarpa [Scheff] Boerl)Seed and Flesh Fruit Extract to P53 and Bcl-2 Gene Expression inHela Cell Line FMIPA UGM 2005

[9] A Endang R Tri Joko and E Dewi ldquoCyototoxicity of Phaleriamacrocarpa (Scheff) boerl fruits flesh and seed extract ofethanol and its effect against p53 and Bcl-2 genes expressionof normalrdquo in Proceedings of the International Conference onComputational Science (ICCS rsquo07) 2007

[10] C Evans Trease and Evans Pharmacognosy Saunder 2002[11] A Sofowora Medicinal Plants and Traditional Medicine in

Africa Spectrum Books Ltd Ibadan Nigeria 2nd edition 1993[12] AOAC Official Methods of Analysis Association of Official

Analytical Chemists Arlington Tex USA 2002[13] J Harborne Phytochemical Methods Chapman and Hall Lon-

don UK 1973[14] AOAC Official Methods of Analysis Association of Official

Analytical Chemists Gaithersburg Md USA 17th edition2000

[15] J Lako V C Trenerry M Wahlqvist N WattanapenpaiboonS Sotheeswaran and R Premier ldquoPhytochemical flavonolscarotenoids and the antioxidant properties of a wide selection ofFijian fruit vegetables and other readily available foodsrdquo FoodChemistry vol 101 no 4 pp 1727ndash1741 2007

[16] W Horwitz Official Method of Analysis AOAC InterantionalGaithersburg Md USA 17th edition 2000

[17] A A Adedapo F O Jimoh S Koduru P J Masika and AJ Afolayan ldquoAssessment of the medicinal potentials of themethanol extracts of the leaves and stems of Buddleja salignardquoBMCComplementary andAlternativeMedicine vol 9 article 212009

[18] M Zovko Koncic D Kremer J Gruz et al ldquoAntioxidantand antimicrobial properties ofMoltkia petraea (Tratt) Griseb

flower leaf and stem infusionsrdquo Food and Chemical Toxicologyvol 48 no 6 pp 1537ndash1542 2010

[19] S McDonald P D Prenzler M Antolovich and K RobardsldquoPhenolic content and antioxidant activity of olive extractsrdquoFood Chemistry vol 73 no 1 pp 73ndash84 2001

[20] S M Nabavi M A Ebrahimzadeh S F Nabavi A Hamidiniaand A R Bekhradnia ldquoDetermination of antioxidant activityphenol and flavonoid content of Parrotia persicaMeyrdquo Pharma-cologyonline vol 2 pp 560ndash567 2008

[21] M A Ebrahimzadeh S F Nabavi and S M Nabavi ldquoAntiox-idant activities of methanol extract of Sambucus ebulus Lflowerrdquo Pakistan Journal of Biological Sciences vol 12 no 5 pp447ndash450 2009

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

[23] A Kumaran and R Joel Karunakaran ldquoIn vitro antioxidantactivities of methanol extracts of five Phyllanthus species fromIndiardquo Food Science and Technology vol 40 no 2 pp 344ndash3522007

[24] B O Mbaebie H O Edeoga and A J Afolayan ldquoPhy tochemical analysis and antioxidants activities of aqueous stembark extract of Schotia latifolia Jacqrdquo Asian Pacific Journal ofTropical Biomedicine vol 2 no 2 pp 118ndash124 2012

[25] L I Mensor F S Menezes G G Leitao et al ldquoScreening ofBrazilian plants extracts for antioxidants activity by the use ofDPPH free radical methodrdquo Phytotherapy Research vol 15 pp127ndash130 2001

[26] F Denizot and R Lang ldquoRapid colorimetric assay for cellgrowth and survival modifications to the tetrazolium dyeprocedure giving improved sensitivity and reliabilityrdquo Journalof Immunological Methods vol 89 no 2 pp 271ndash277 1986

[27] T Mosmann ldquoRapid colorimetric assay for cellular growth andsurvival application to proliferation and cytotoxicity assaysrdquoJournal of Immunological Methods vol 65 no 1-2 pp 55ndash631983

[28] P Maisuthisakul R Pongsawatmanit and M H GordonldquoAntioxidant properties of Teaw (Cratoxylum formosum Dyer)extract in soybean oil and emulsionsrdquo Journal of Agriculturaland Food Chemistry vol 54 no 7 pp 2719ndash2725 2006

[29] P Maisuthisakul S Pasuk and P Ritthiruangdej ldquoRelationshipbetween antioxidant properties and chemical composition ofsome Thai plantsrdquo Journal of Food Composition and Analysisvol 21 no 3 pp 229ndash240 2008

[30] H Kolodziej and A F Kiderlen ldquoAntileishmanial activity andimmune modulatory effects of tannins and related compoundson Leishmania parasitised RAW 2647 cellsrdquo Phytochemistryvol 66 no 17 pp 2056ndash2071 2005

[31] T Nakagawa and T Yokozawa ldquoDirect scavenging of nitricoxide and superoxide by green teardquo Food and Chemical Toxi-cology vol 40 no 12 pp 1745ndash1750 2002

[32] V Schulz R Hansel and V Tyler Fitoterapia Racional UmGuiade Fitoterapia Para as ciencias da saude Manole Barueri Brazil2002

[33] S D Kapu Y B Ngwai O Kayode P A Akah C Wambebeand K Gamaniel ldquoAnti-inflammatory analgesic and anti-lymphocytic activities of the aqueous extract of Crinum gigan-teumrdquo Journal of Ethnopharmacology vol 78 no 1 pp 7ndash132001

[34] G Francis Z Kerem H P S Makkar and K Becker ldquoThebiological action of saponins in animal systems a reviewrdquoBritish Journal of Nutrition vol 88 no 6 pp 587ndash605 2002

BioMed Research International 13

[35] R Xu W Zhao J Xu B Shao and G Qin ldquoStudies onbioactive saponins fromChinesemedicinal plantsrdquoAdvances inExperimental Medicine and Biology vol 404 pp 371ndash382 1996

[36] J A Manthey N Guthrie and K Grohmann ldquoBiologicalproperties of citrus flavonoids pertaining to cancer and inflam-mationrdquo Current Medicinal Chemistry vol 8 no 2 pp 135ndash1532001

[37] P Hendra Y Fukushi and Y Hashidoko ldquoSynthesis of ben-zophenone glucopyranosides from Phaleria macrocarpa andrelated benzophenone glucopyranosidesrdquo Bioscience Biotech-nology and Biochemistry vol 73 no 10 pp 2172ndash2182 2009

[38] A Rabia Z B A Mohammad D Aidiahmad S Amirin and IU Muhammad ldquoPhytochemistry and medicinal properties ofPhaleria macrocarpa (Scheff) Boerl extractsrdquo PharmacognosyReview vol 7 no 13 pp 73ndash80 2013

[39] A Saufi C B I VonHeimendahl AWAlfermann andE FussldquoStereochemistry of lignans in Phaleria macrocarpa (Scheff)Boerlrdquo Zeitschrift fur Naturforschung C vol 63 no 1-2 pp 13ndash16 2008

[40] S C Chong M A Dollah P P Chong and A Maha ldquoPhaleriamacrocarpa (Scheff) Boerl fruit aqueous extract enhances LDLreceptor and PCSK9 expression in vivo and in vitrordquo Journal ofEthnopharmacology vol 137 no 1 pp 817ndash827 2011

[41] R R Tjandrawinata D Nofiarny L W Susanto P Hendra andA Clarissa ldquoSymptomatic treatment of premenstrual syndromeandor primary dysmenorrhea with DLBS1442 a bioacitveextract ofPhaleriamacrocaraprdquo International Journal of GeneralMedicine vol 4 pp 465ndash476 2011

[42] R Hendra S Ahmad E Oskoueian A Sukari and M YShukor ldquoAntioxidant anti-inflammatory and cytotoxicity ofPhaleriamacrocarap (Boerl) Scheff fruitrdquoBMCComplementaryand Alternative Medicine vol 11 article 110 2011

[43] D Kurnia K Akiyama and H Hayashi ldquo29-Norcucurbitacinderivatives isolated from the Indonesian medicinal plant Pha-leria macrocarpa (Scheff) Boerlrdquo Bioscience Biotechnology andBiochemistry vol 72 no 2 pp 618ndash620 2008

[44] S Oshimi K Zaima Y Matsuno et al ldquoStudies on theconstituents from the fruits of Phaleria macrocaraprdquo Journal ofNatural Medicines vol 62 no 2 pp 207ndash210 2008

[45] C Sanchez-Moreno J A Larrauri and F Saura-Calixto ldquoAprocedure tomeasure the antiradical efficiency of polyphenolsrdquoJournal of the Science of Food and Agriculture vol 76 pp 270ndash276 1998

[46] Y Andriani M A W Effendy T M T Sifzizul and HMohamad ldquoAntibacterial radical-scavenging activities andcytotoxicit properties of Phaleria macrocarpa (Scheff) BoerlLeaves in HepG2 cell linesrdquo International Journal of Pharma-ceutical Sciences and Research vol 2 no 7 pp 1700ndash1706 2011

[47] Y Rohyami ldquoPenentuan Kandungan Flavonoid dari EkstrakMetanol Daging Buah Mahkota Dewa (Phaleria macrocarpaScheff Boerl)rdquo Jurnal Logika vol 5 2009

[48] R D Petty L A Sutherland E M Hunter and I A CreeldquoComparison of MTT and ATP-based assays for the measure-ment of viable cell numberrdquo Journal of Bioluminescence andChemiluminescence vol 10 no 1 pp 29ndash34 1995

[49] J R Sellers S Cook andV S Goldmacher ldquoA cytotoxicity assayutilizing a fluorescent dye that determines accurate survivingfractions of cellsrdquo Journal of Immunological Methods vol 172no 2 pp 255ndash264 1994

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

BioMed Research International 11

found to contain methyl myristate methyl stearate oleicacid methyl oleate linoleic acid methyl linoleate palmiticacid methyl palmitate 6-octadecenoic acid and 917-octadecadial 120573-sitosterol as major components A further30 to 40 of components were not identified as thesewere polar compounds that require HPLC analysis Othercompounds reported in the literature for the seeds leavesand fruits were not identified in this investigation It isprobable that the components reported in the literaturemay be present in other fractions (methyl acetate CHCl

3

and water fractions)Phenolics are able to transfer protons to radicals [45]

DPPH which provides free radicals is normally a blue-violet color However the DPPH turned yellow after it wasconverted to 1 1-diphenyl-2-picrylhydrazine which has lessfree-radical activity Hendra et al reported that the IC

50

values of free radical and reducing power in methanolwith HCl of P macrocarpa seeds were 2450 plusmn 194 and1502 plusmn 128 [42] In our investigation using DPPH assay thewater fraction displayed the highest IC

50value followed by

methanol ethyl acetate hexane and chloroform fractionThe presence of flavonoids in P macrocarpa seeds may be

responsible for the traditional use of the plants in treatingcancer inflammations and allergies Recently it has beenshown that phenolic compounds in crude methanol andethyl acetate extract of P macrocarpa leaves displayed goodantioxidant and antimicrobial activites [46] Hendra et al(2011) examined the amount of total phenolic (477plusmn 104mggallic acid equivalentg DW) and the amount of flavonoid(359 plusmn 247mg rutin equivalentg DW) contents in themethanol extract with HCl of P macrocarpa seeds [42] Inthis study the total flavonoids content was higher than that inthe results previously reported by Rohyami [47] and phenolflavonol and flavonoid contents were the highest in theaqueous methanol extract followed by ethyl acetate waterchloroform and hexane extracts

MTT assay is a sensitive and reliable colorimetric assaythat uses quantitative measurements to calculate the via-bility proliferation and activation of cells This method iscommonly applied to screen anticancer agents [48] Themost well-known methods used to calculate cytotoxicityare the neutral red (NR) uptake and dimethylthiazole-diphenyl tetrazolium bromide (MTT) metabolism [27 49]Earlier investigations showed that the ethanol extract of Pmacrocarpa seeds and fruit meat were not toxic to normalhuman cells but slightly toxic to a Vero cell line [9] Theethanol extract of P macrocarpa seed fruit also showedtoxicity towards T47D breast cancer cell line through COX-2 expression inhibition [7] Desacetylfevicordin A has beenisolated from the ethyl acetate extract of P macrocarpaseeds and this compound displayed excellent cytotoxicityin brine shrimp [43] The ethyl acetate of P macrocarpaseeds exhibited mild cytotoxic effect against HepG2 cells(IC50values between 30 and 60 120583gmL) [46] Previous studies

showed that the cytotoxic activity of the seeds on HT-29MCF-7 Hela and Chang liver cells lines were 384 plusmn 037255 plusmn 137 295 plusmn 10 and 678 plusmn 027 receptively [42] Inour study the methanolic extract of P macrocarpa seeds wasfound to pose cytotoxic effect against HT-29 MCF-7 Cas Ki

and SKOV-3 cell lines (IC50

values giving from 11 plusmn 120to 360 plusmn 355) and mild toxicity on normal cell lines Therewere significant cytotoxic effects on selected cancer cells linesthat were both time- and dose-dependent manner due to thepresence of many secondary metabolites

The phytochemical screening also indicated the presenceof a small amount of cyanogenic glycosides Cyanogenicglucosides which can cause acute cyanide poisoning causerapid respiration and pulse decrease the blood pressure andinduce vomiting diarrhea headache dizziness and so onThus these components have to be removed (usually byboiling) prior to consumption for therapeutic purposes

5 Conclusion

P macrocarpa seeds exhibited antioxidant and cytotoxicactivities It is highly probable that these activities are dueto the presence of phenolic and flavonoid compounds inappreciable amounts in the plant Furthermore the cytotox-icity activity suggested that the seed may contain a potentialanticancer agent The outcome of this study is encouragingdemonstrating the potential for the P macrocarpa as a sourceof multiple therapeutic agents

Abbreviations

EAF Ethyl acetate fractionME Methanol extractHF Hexane fractionCF Chloroform fractionWF Water fractiong Grammg MilligrammL Milliliter

Conflict of Interests

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

Authorsrsquo Contribution

Ma Ma Lay was involved in all experiments and BehroozBanisalamwas involved in antioxidant assay All authors readand approved the submitted version of the paper

Acknowledgments

This work was supported by grants from the MalaysianMinistry of Higher Education (nos PS2862009C andPS4702010B) Ma Ma Lay is a scholarship recipient fromthe ldquoIslamic Development Bank Saudi Arabiardquo The authorswould like to thank Professor Dr Amru BinNasrulhaq Boycefor generously allowing the use of his laboratory for thisresearch

12 BioMed Research International

References

[1] N HarmantoMahkota DewaObat Pusaka ParaDewa Cetakankeempat Agromedia Pustaka Jakarta Indonesia 2002

[2] A Jemal R Siegel E Ward T Murray J Xu and M J ThunldquoCancer statistics 2007rdquoCa ACancer Journal for Clinicians vol57 no 1 pp 43ndash66 2007

[3] R Hendra S Ahmad A Sukari M Y Shukor and EOskoueian ldquoFlavonoid analyses and antimicrobial activity ofvarious parts of Phaleria macrocarpa (Scheff) Boerl fruitrdquoInternational Journal of Molecular Sciences vol 12 no 6 pp3422ndash3431 2011

[4] W Wijayani Sitotoksisitas Minyak Atsiri Biji Dan Daging BuahMahkota Dewa (Phaleria Macrocarpa [Scheff] Boerl) TerhadapMyeloma Cell Line Dan HeLa Cell Line Serta IdentifikasiSenyawa Minyak Atsiri Dengan Kromatografi Gas-SpektrometriMassa (GC-MS) Skripsi FMIPA UGM Yogyakarta Indonesia2005

[5] Y-B Zhang X-J Xu and H-M Liu ldquoChemical con-stituents fromMahkotadewardquo Journal of Asian Natural ProductsResearch vol 8 no 1-2 pp 119ndash123 2006

[6] D Kurnia K Akiyama and H Hayashi ldquo29-Norcucurbitacinderivatives isolated from the Indonesian medicinal plant Pha-leria macrocarpa (Scheff) Boerlrdquo Bioscience Biotechnology andBiochemistry vol 72 no 2 pp 618ndash620 2008

[7] M Bakhriansyah Pengaruh Ekstrak Etanol Biji Mahkota Dewa(Phaleria Macrocarpa [Scheff] Boerl) Pada Sel Kanker Payu-dara T47D Kajian Aktivitas Sitotoksik Dan PenghambatanEkspresi Siklooksigenase-2 Program Pascasarjana UGM 2004

[8] L Wardani The Effect of Phaleria Macrocarpa [Scheff] Boerl)Seed and Flesh Fruit Extract to P53 and Bcl-2 Gene Expression inHela Cell Line FMIPA UGM 2005

[9] A Endang R Tri Joko and E Dewi ldquoCyototoxicity of Phaleriamacrocarpa (Scheff) boerl fruits flesh and seed extract ofethanol and its effect against p53 and Bcl-2 genes expressionof normalrdquo in Proceedings of the International Conference onComputational Science (ICCS rsquo07) 2007

[10] C Evans Trease and Evans Pharmacognosy Saunder 2002[11] A Sofowora Medicinal Plants and Traditional Medicine in

Africa Spectrum Books Ltd Ibadan Nigeria 2nd edition 1993[12] AOAC Official Methods of Analysis Association of Official

Analytical Chemists Arlington Tex USA 2002[13] J Harborne Phytochemical Methods Chapman and Hall Lon-

don UK 1973[14] AOAC Official Methods of Analysis Association of Official

Analytical Chemists Gaithersburg Md USA 17th edition2000

[15] J Lako V C Trenerry M Wahlqvist N WattanapenpaiboonS Sotheeswaran and R Premier ldquoPhytochemical flavonolscarotenoids and the antioxidant properties of a wide selection ofFijian fruit vegetables and other readily available foodsrdquo FoodChemistry vol 101 no 4 pp 1727ndash1741 2007

[16] W Horwitz Official Method of Analysis AOAC InterantionalGaithersburg Md USA 17th edition 2000

[17] A A Adedapo F O Jimoh S Koduru P J Masika and AJ Afolayan ldquoAssessment of the medicinal potentials of themethanol extracts of the leaves and stems of Buddleja salignardquoBMCComplementary andAlternativeMedicine vol 9 article 212009

[18] M Zovko Koncic D Kremer J Gruz et al ldquoAntioxidantand antimicrobial properties ofMoltkia petraea (Tratt) Griseb

flower leaf and stem infusionsrdquo Food and Chemical Toxicologyvol 48 no 6 pp 1537ndash1542 2010

[19] S McDonald P D Prenzler M Antolovich and K RobardsldquoPhenolic content and antioxidant activity of olive extractsrdquoFood Chemistry vol 73 no 1 pp 73ndash84 2001

[20] S M Nabavi M A Ebrahimzadeh S F Nabavi A Hamidiniaand A R Bekhradnia ldquoDetermination of antioxidant activityphenol and flavonoid content of Parrotia persicaMeyrdquo Pharma-cologyonline vol 2 pp 560ndash567 2008

[21] M A Ebrahimzadeh S F Nabavi and S M Nabavi ldquoAntiox-idant activities of methanol extract of Sambucus ebulus Lflowerrdquo Pakistan Journal of Biological Sciences vol 12 no 5 pp447ndash450 2009

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

[23] A Kumaran and R Joel Karunakaran ldquoIn vitro antioxidantactivities of methanol extracts of five Phyllanthus species fromIndiardquo Food Science and Technology vol 40 no 2 pp 344ndash3522007

[24] B O Mbaebie H O Edeoga and A J Afolayan ldquoPhy tochemical analysis and antioxidants activities of aqueous stembark extract of Schotia latifolia Jacqrdquo Asian Pacific Journal ofTropical Biomedicine vol 2 no 2 pp 118ndash124 2012

[25] L I Mensor F S Menezes G G Leitao et al ldquoScreening ofBrazilian plants extracts for antioxidants activity by the use ofDPPH free radical methodrdquo Phytotherapy Research vol 15 pp127ndash130 2001

[26] F Denizot and R Lang ldquoRapid colorimetric assay for cellgrowth and survival modifications to the tetrazolium dyeprocedure giving improved sensitivity and reliabilityrdquo Journalof Immunological Methods vol 89 no 2 pp 271ndash277 1986

[27] T Mosmann ldquoRapid colorimetric assay for cellular growth andsurvival application to proliferation and cytotoxicity assaysrdquoJournal of Immunological Methods vol 65 no 1-2 pp 55ndash631983

[28] P Maisuthisakul R Pongsawatmanit and M H GordonldquoAntioxidant properties of Teaw (Cratoxylum formosum Dyer)extract in soybean oil and emulsionsrdquo Journal of Agriculturaland Food Chemistry vol 54 no 7 pp 2719ndash2725 2006

[29] P Maisuthisakul S Pasuk and P Ritthiruangdej ldquoRelationshipbetween antioxidant properties and chemical composition ofsome Thai plantsrdquo Journal of Food Composition and Analysisvol 21 no 3 pp 229ndash240 2008

[30] H Kolodziej and A F Kiderlen ldquoAntileishmanial activity andimmune modulatory effects of tannins and related compoundson Leishmania parasitised RAW 2647 cellsrdquo Phytochemistryvol 66 no 17 pp 2056ndash2071 2005

[31] T Nakagawa and T Yokozawa ldquoDirect scavenging of nitricoxide and superoxide by green teardquo Food and Chemical Toxi-cology vol 40 no 12 pp 1745ndash1750 2002

[32] V Schulz R Hansel and V Tyler Fitoterapia Racional UmGuiade Fitoterapia Para as ciencias da saude Manole Barueri Brazil2002

[33] S D Kapu Y B Ngwai O Kayode P A Akah C Wambebeand K Gamaniel ldquoAnti-inflammatory analgesic and anti-lymphocytic activities of the aqueous extract of Crinum gigan-teumrdquo Journal of Ethnopharmacology vol 78 no 1 pp 7ndash132001

[34] G Francis Z Kerem H P S Makkar and K Becker ldquoThebiological action of saponins in animal systems a reviewrdquoBritish Journal of Nutrition vol 88 no 6 pp 587ndash605 2002

BioMed Research International 13

[35] R Xu W Zhao J Xu B Shao and G Qin ldquoStudies onbioactive saponins fromChinesemedicinal plantsrdquoAdvances inExperimental Medicine and Biology vol 404 pp 371ndash382 1996

[36] J A Manthey N Guthrie and K Grohmann ldquoBiologicalproperties of citrus flavonoids pertaining to cancer and inflam-mationrdquo Current Medicinal Chemistry vol 8 no 2 pp 135ndash1532001

[37] P Hendra Y Fukushi and Y Hashidoko ldquoSynthesis of ben-zophenone glucopyranosides from Phaleria macrocarpa andrelated benzophenone glucopyranosidesrdquo Bioscience Biotech-nology and Biochemistry vol 73 no 10 pp 2172ndash2182 2009

[38] A Rabia Z B A Mohammad D Aidiahmad S Amirin and IU Muhammad ldquoPhytochemistry and medicinal properties ofPhaleria macrocarpa (Scheff) Boerl extractsrdquo PharmacognosyReview vol 7 no 13 pp 73ndash80 2013

[39] A Saufi C B I VonHeimendahl AWAlfermann andE FussldquoStereochemistry of lignans in Phaleria macrocarpa (Scheff)Boerlrdquo Zeitschrift fur Naturforschung C vol 63 no 1-2 pp 13ndash16 2008

[40] S C Chong M A Dollah P P Chong and A Maha ldquoPhaleriamacrocarpa (Scheff) Boerl fruit aqueous extract enhances LDLreceptor and PCSK9 expression in vivo and in vitrordquo Journal ofEthnopharmacology vol 137 no 1 pp 817ndash827 2011

[41] R R Tjandrawinata D Nofiarny L W Susanto P Hendra andA Clarissa ldquoSymptomatic treatment of premenstrual syndromeandor primary dysmenorrhea with DLBS1442 a bioacitveextract ofPhaleriamacrocaraprdquo International Journal of GeneralMedicine vol 4 pp 465ndash476 2011

[42] R Hendra S Ahmad E Oskoueian A Sukari and M YShukor ldquoAntioxidant anti-inflammatory and cytotoxicity ofPhaleriamacrocarap (Boerl) Scheff fruitrdquoBMCComplementaryand Alternative Medicine vol 11 article 110 2011

[43] D Kurnia K Akiyama and H Hayashi ldquo29-Norcucurbitacinderivatives isolated from the Indonesian medicinal plant Pha-leria macrocarpa (Scheff) Boerlrdquo Bioscience Biotechnology andBiochemistry vol 72 no 2 pp 618ndash620 2008

[44] S Oshimi K Zaima Y Matsuno et al ldquoStudies on theconstituents from the fruits of Phaleria macrocaraprdquo Journal ofNatural Medicines vol 62 no 2 pp 207ndash210 2008

[45] C Sanchez-Moreno J A Larrauri and F Saura-Calixto ldquoAprocedure tomeasure the antiradical efficiency of polyphenolsrdquoJournal of the Science of Food and Agriculture vol 76 pp 270ndash276 1998

[46] Y Andriani M A W Effendy T M T Sifzizul and HMohamad ldquoAntibacterial radical-scavenging activities andcytotoxicit properties of Phaleria macrocarpa (Scheff) BoerlLeaves in HepG2 cell linesrdquo International Journal of Pharma-ceutical Sciences and Research vol 2 no 7 pp 1700ndash1706 2011

[47] Y Rohyami ldquoPenentuan Kandungan Flavonoid dari EkstrakMetanol Daging Buah Mahkota Dewa (Phaleria macrocarpaScheff Boerl)rdquo Jurnal Logika vol 5 2009

[48] R D Petty L A Sutherland E M Hunter and I A CreeldquoComparison of MTT and ATP-based assays for the measure-ment of viable cell numberrdquo Journal of Bioluminescence andChemiluminescence vol 10 no 1 pp 29ndash34 1995

[49] J R Sellers S Cook andV S Goldmacher ldquoA cytotoxicity assayutilizing a fluorescent dye that determines accurate survivingfractions of cellsrdquo Journal of Immunological Methods vol 172no 2 pp 255ndash264 1994

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

12 BioMed Research International

References

[1] N HarmantoMahkota DewaObat Pusaka ParaDewa Cetakankeempat Agromedia Pustaka Jakarta Indonesia 2002

[2] A Jemal R Siegel E Ward T Murray J Xu and M J ThunldquoCancer statistics 2007rdquoCa ACancer Journal for Clinicians vol57 no 1 pp 43ndash66 2007

[3] R Hendra S Ahmad A Sukari M Y Shukor and EOskoueian ldquoFlavonoid analyses and antimicrobial activity ofvarious parts of Phaleria macrocarpa (Scheff) Boerl fruitrdquoInternational Journal of Molecular Sciences vol 12 no 6 pp3422ndash3431 2011

[4] W Wijayani Sitotoksisitas Minyak Atsiri Biji Dan Daging BuahMahkota Dewa (Phaleria Macrocarpa [Scheff] Boerl) TerhadapMyeloma Cell Line Dan HeLa Cell Line Serta IdentifikasiSenyawa Minyak Atsiri Dengan Kromatografi Gas-SpektrometriMassa (GC-MS) Skripsi FMIPA UGM Yogyakarta Indonesia2005

[5] Y-B Zhang X-J Xu and H-M Liu ldquoChemical con-stituents fromMahkotadewardquo Journal of Asian Natural ProductsResearch vol 8 no 1-2 pp 119ndash123 2006

[6] D Kurnia K Akiyama and H Hayashi ldquo29-Norcucurbitacinderivatives isolated from the Indonesian medicinal plant Pha-leria macrocarpa (Scheff) Boerlrdquo Bioscience Biotechnology andBiochemistry vol 72 no 2 pp 618ndash620 2008

[7] M Bakhriansyah Pengaruh Ekstrak Etanol Biji Mahkota Dewa(Phaleria Macrocarpa [Scheff] Boerl) Pada Sel Kanker Payu-dara T47D Kajian Aktivitas Sitotoksik Dan PenghambatanEkspresi Siklooksigenase-2 Program Pascasarjana UGM 2004

[8] L Wardani The Effect of Phaleria Macrocarpa [Scheff] Boerl)Seed and Flesh Fruit Extract to P53 and Bcl-2 Gene Expression inHela Cell Line FMIPA UGM 2005

[9] A Endang R Tri Joko and E Dewi ldquoCyototoxicity of Phaleriamacrocarpa (Scheff) boerl fruits flesh and seed extract ofethanol and its effect against p53 and Bcl-2 genes expressionof normalrdquo in Proceedings of the International Conference onComputational Science (ICCS rsquo07) 2007

[10] C Evans Trease and Evans Pharmacognosy Saunder 2002[11] A Sofowora Medicinal Plants and Traditional Medicine in

Africa Spectrum Books Ltd Ibadan Nigeria 2nd edition 1993[12] AOAC Official Methods of Analysis Association of Official

Analytical Chemists Arlington Tex USA 2002[13] J Harborne Phytochemical Methods Chapman and Hall Lon-

don UK 1973[14] AOAC Official Methods of Analysis Association of Official

Analytical Chemists Gaithersburg Md USA 17th edition2000

[15] J Lako V C Trenerry M Wahlqvist N WattanapenpaiboonS Sotheeswaran and R Premier ldquoPhytochemical flavonolscarotenoids and the antioxidant properties of a wide selection ofFijian fruit vegetables and other readily available foodsrdquo FoodChemistry vol 101 no 4 pp 1727ndash1741 2007

[16] W Horwitz Official Method of Analysis AOAC InterantionalGaithersburg Md USA 17th edition 2000

[17] A A Adedapo F O Jimoh S Koduru P J Masika and AJ Afolayan ldquoAssessment of the medicinal potentials of themethanol extracts of the leaves and stems of Buddleja salignardquoBMCComplementary andAlternativeMedicine vol 9 article 212009

[18] M Zovko Koncic D Kremer J Gruz et al ldquoAntioxidantand antimicrobial properties ofMoltkia petraea (Tratt) Griseb

flower leaf and stem infusionsrdquo Food and Chemical Toxicologyvol 48 no 6 pp 1537ndash1542 2010

[19] S McDonald P D Prenzler M Antolovich and K RobardsldquoPhenolic content and antioxidant activity of olive extractsrdquoFood Chemistry vol 73 no 1 pp 73ndash84 2001

[20] S M Nabavi M A Ebrahimzadeh S F Nabavi A Hamidiniaand A R Bekhradnia ldquoDetermination of antioxidant activityphenol and flavonoid content of Parrotia persicaMeyrdquo Pharma-cologyonline vol 2 pp 560ndash567 2008

[21] M A Ebrahimzadeh S F Nabavi and S M Nabavi ldquoAntiox-idant activities of methanol extract of Sambucus ebulus Lflowerrdquo Pakistan Journal of Biological Sciences vol 12 no 5 pp447ndash450 2009

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

[23] A Kumaran and R Joel Karunakaran ldquoIn vitro antioxidantactivities of methanol extracts of five Phyllanthus species fromIndiardquo Food Science and Technology vol 40 no 2 pp 344ndash3522007

[24] B O Mbaebie H O Edeoga and A J Afolayan ldquoPhy tochemical analysis and antioxidants activities of aqueous stembark extract of Schotia latifolia Jacqrdquo Asian Pacific Journal ofTropical Biomedicine vol 2 no 2 pp 118ndash124 2012

[25] L I Mensor F S Menezes G G Leitao et al ldquoScreening ofBrazilian plants extracts for antioxidants activity by the use ofDPPH free radical methodrdquo Phytotherapy Research vol 15 pp127ndash130 2001

[26] F Denizot and R Lang ldquoRapid colorimetric assay for cellgrowth and survival modifications to the tetrazolium dyeprocedure giving improved sensitivity and reliabilityrdquo Journalof Immunological Methods vol 89 no 2 pp 271ndash277 1986

[27] T Mosmann ldquoRapid colorimetric assay for cellular growth andsurvival application to proliferation and cytotoxicity assaysrdquoJournal of Immunological Methods vol 65 no 1-2 pp 55ndash631983

[28] P Maisuthisakul R Pongsawatmanit and M H GordonldquoAntioxidant properties of Teaw (Cratoxylum formosum Dyer)extract in soybean oil and emulsionsrdquo Journal of Agriculturaland Food Chemistry vol 54 no 7 pp 2719ndash2725 2006

[29] P Maisuthisakul S Pasuk and P Ritthiruangdej ldquoRelationshipbetween antioxidant properties and chemical composition ofsome Thai plantsrdquo Journal of Food Composition and Analysisvol 21 no 3 pp 229ndash240 2008

[30] H Kolodziej and A F Kiderlen ldquoAntileishmanial activity andimmune modulatory effects of tannins and related compoundson Leishmania parasitised RAW 2647 cellsrdquo Phytochemistryvol 66 no 17 pp 2056ndash2071 2005

[31] T Nakagawa and T Yokozawa ldquoDirect scavenging of nitricoxide and superoxide by green teardquo Food and Chemical Toxi-cology vol 40 no 12 pp 1745ndash1750 2002

[32] V Schulz R Hansel and V Tyler Fitoterapia Racional UmGuiade Fitoterapia Para as ciencias da saude Manole Barueri Brazil2002

[33] S D Kapu Y B Ngwai O Kayode P A Akah C Wambebeand K Gamaniel ldquoAnti-inflammatory analgesic and anti-lymphocytic activities of the aqueous extract of Crinum gigan-teumrdquo Journal of Ethnopharmacology vol 78 no 1 pp 7ndash132001

[34] G Francis Z Kerem H P S Makkar and K Becker ldquoThebiological action of saponins in animal systems a reviewrdquoBritish Journal of Nutrition vol 88 no 6 pp 587ndash605 2002

BioMed Research International 13

[35] R Xu W Zhao J Xu B Shao and G Qin ldquoStudies onbioactive saponins fromChinesemedicinal plantsrdquoAdvances inExperimental Medicine and Biology vol 404 pp 371ndash382 1996

[36] J A Manthey N Guthrie and K Grohmann ldquoBiologicalproperties of citrus flavonoids pertaining to cancer and inflam-mationrdquo Current Medicinal Chemistry vol 8 no 2 pp 135ndash1532001

[37] P Hendra Y Fukushi and Y Hashidoko ldquoSynthesis of ben-zophenone glucopyranosides from Phaleria macrocarpa andrelated benzophenone glucopyranosidesrdquo Bioscience Biotech-nology and Biochemistry vol 73 no 10 pp 2172ndash2182 2009

[38] A Rabia Z B A Mohammad D Aidiahmad S Amirin and IU Muhammad ldquoPhytochemistry and medicinal properties ofPhaleria macrocarpa (Scheff) Boerl extractsrdquo PharmacognosyReview vol 7 no 13 pp 73ndash80 2013

[39] A Saufi C B I VonHeimendahl AWAlfermann andE FussldquoStereochemistry of lignans in Phaleria macrocarpa (Scheff)Boerlrdquo Zeitschrift fur Naturforschung C vol 63 no 1-2 pp 13ndash16 2008

[40] S C Chong M A Dollah P P Chong and A Maha ldquoPhaleriamacrocarpa (Scheff) Boerl fruit aqueous extract enhances LDLreceptor and PCSK9 expression in vivo and in vitrordquo Journal ofEthnopharmacology vol 137 no 1 pp 817ndash827 2011

[41] R R Tjandrawinata D Nofiarny L W Susanto P Hendra andA Clarissa ldquoSymptomatic treatment of premenstrual syndromeandor primary dysmenorrhea with DLBS1442 a bioacitveextract ofPhaleriamacrocaraprdquo International Journal of GeneralMedicine vol 4 pp 465ndash476 2011

[42] R Hendra S Ahmad E Oskoueian A Sukari and M YShukor ldquoAntioxidant anti-inflammatory and cytotoxicity ofPhaleriamacrocarap (Boerl) Scheff fruitrdquoBMCComplementaryand Alternative Medicine vol 11 article 110 2011

[43] D Kurnia K Akiyama and H Hayashi ldquo29-Norcucurbitacinderivatives isolated from the Indonesian medicinal plant Pha-leria macrocarpa (Scheff) Boerlrdquo Bioscience Biotechnology andBiochemistry vol 72 no 2 pp 618ndash620 2008

[44] S Oshimi K Zaima Y Matsuno et al ldquoStudies on theconstituents from the fruits of Phaleria macrocaraprdquo Journal ofNatural Medicines vol 62 no 2 pp 207ndash210 2008

[45] C Sanchez-Moreno J A Larrauri and F Saura-Calixto ldquoAprocedure tomeasure the antiradical efficiency of polyphenolsrdquoJournal of the Science of Food and Agriculture vol 76 pp 270ndash276 1998

[46] Y Andriani M A W Effendy T M T Sifzizul and HMohamad ldquoAntibacterial radical-scavenging activities andcytotoxicit properties of Phaleria macrocarpa (Scheff) BoerlLeaves in HepG2 cell linesrdquo International Journal of Pharma-ceutical Sciences and Research vol 2 no 7 pp 1700ndash1706 2011

[47] Y Rohyami ldquoPenentuan Kandungan Flavonoid dari EkstrakMetanol Daging Buah Mahkota Dewa (Phaleria macrocarpaScheff Boerl)rdquo Jurnal Logika vol 5 2009

[48] R D Petty L A Sutherland E M Hunter and I A CreeldquoComparison of MTT and ATP-based assays for the measure-ment of viable cell numberrdquo Journal of Bioluminescence andChemiluminescence vol 10 no 1 pp 29ndash34 1995

[49] J R Sellers S Cook andV S Goldmacher ldquoA cytotoxicity assayutilizing a fluorescent dye that determines accurate survivingfractions of cellsrdquo Journal of Immunological Methods vol 172no 2 pp 255ndash264 1994

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

BioMed Research International 13

[35] R Xu W Zhao J Xu B Shao and G Qin ldquoStudies onbioactive saponins fromChinesemedicinal plantsrdquoAdvances inExperimental Medicine and Biology vol 404 pp 371ndash382 1996

[36] J A Manthey N Guthrie and K Grohmann ldquoBiologicalproperties of citrus flavonoids pertaining to cancer and inflam-mationrdquo Current Medicinal Chemistry vol 8 no 2 pp 135ndash1532001

[37] P Hendra Y Fukushi and Y Hashidoko ldquoSynthesis of ben-zophenone glucopyranosides from Phaleria macrocarpa andrelated benzophenone glucopyranosidesrdquo Bioscience Biotech-nology and Biochemistry vol 73 no 10 pp 2172ndash2182 2009

[38] A Rabia Z B A Mohammad D Aidiahmad S Amirin and IU Muhammad ldquoPhytochemistry and medicinal properties ofPhaleria macrocarpa (Scheff) Boerl extractsrdquo PharmacognosyReview vol 7 no 13 pp 73ndash80 2013

[39] A Saufi C B I VonHeimendahl AWAlfermann andE FussldquoStereochemistry of lignans in Phaleria macrocarpa (Scheff)Boerlrdquo Zeitschrift fur Naturforschung C vol 63 no 1-2 pp 13ndash16 2008

[40] S C Chong M A Dollah P P Chong and A Maha ldquoPhaleriamacrocarpa (Scheff) Boerl fruit aqueous extract enhances LDLreceptor and PCSK9 expression in vivo and in vitrordquo Journal ofEthnopharmacology vol 137 no 1 pp 817ndash827 2011

[41] R R Tjandrawinata D Nofiarny L W Susanto P Hendra andA Clarissa ldquoSymptomatic treatment of premenstrual syndromeandor primary dysmenorrhea with DLBS1442 a bioacitveextract ofPhaleriamacrocaraprdquo International Journal of GeneralMedicine vol 4 pp 465ndash476 2011

[42] R Hendra S Ahmad E Oskoueian A Sukari and M YShukor ldquoAntioxidant anti-inflammatory and cytotoxicity ofPhaleriamacrocarap (Boerl) Scheff fruitrdquoBMCComplementaryand Alternative Medicine vol 11 article 110 2011

[43] D Kurnia K Akiyama and H Hayashi ldquo29-Norcucurbitacinderivatives isolated from the Indonesian medicinal plant Pha-leria macrocarpa (Scheff) Boerlrdquo Bioscience Biotechnology andBiochemistry vol 72 no 2 pp 618ndash620 2008

[44] S Oshimi K Zaima Y Matsuno et al ldquoStudies on theconstituents from the fruits of Phaleria macrocaraprdquo Journal ofNatural Medicines vol 62 no 2 pp 207ndash210 2008

[45] C Sanchez-Moreno J A Larrauri and F Saura-Calixto ldquoAprocedure tomeasure the antiradical efficiency of polyphenolsrdquoJournal of the Science of Food and Agriculture vol 76 pp 270ndash276 1998

[46] Y Andriani M A W Effendy T M T Sifzizul and HMohamad ldquoAntibacterial radical-scavenging activities andcytotoxicit properties of Phaleria macrocarpa (Scheff) BoerlLeaves in HepG2 cell linesrdquo International Journal of Pharma-ceutical Sciences and Research vol 2 no 7 pp 1700ndash1706 2011

[47] Y Rohyami ldquoPenentuan Kandungan Flavonoid dari EkstrakMetanol Daging Buah Mahkota Dewa (Phaleria macrocarpaScheff Boerl)rdquo Jurnal Logika vol 5 2009

[48] R D Petty L A Sutherland E M Hunter and I A CreeldquoComparison of MTT and ATP-based assays for the measure-ment of viable cell numberrdquo Journal of Bioluminescence andChemiluminescence vol 10 no 1 pp 29ndash34 1995

[49] J R Sellers S Cook andV S Goldmacher ldquoA cytotoxicity assayutilizing a fluorescent dye that determines accurate survivingfractions of cellsrdquo Journal of Immunological Methods vol 172no 2 pp 255ndash264 1994

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology