Research Article Garcinia dulcis Fruit Extract Induced ...downloads.hindawi.com/journals/bmri/2015/916902.pdf · Garcinia dulcis Fruit Extract Induced Cytotoxicity and Apoptosis in

Research ArticleGarcinia dulcis Fruit Extract Induced Cytotoxicity andApoptosis in HepG2 Liver Cancer Cell Line

Mohd Fadzelly Abu Bakar12 Nor Ezani Ahmad2 Monica Suleiman2

Asmah Rahmat3 and Azizul Isha4

1Faculty of Science Technology and Human Development Universiti Tun Hussein Onn Malaysia (UTHM) Batu Pahat86400 Parit Raja Johor Malaysia2Institute for Tropical Biology and Conservation Universiti Malaysia Sabah Jalan UMS 88400 Kota Kinabalu Sabah Malaysia3Department of Nutrition and Dietetics Faculty of Medicine and Health Sciences Universiti Putra Malaysia (UPM)43400 Serdang Selangor Malaysia4Laboratory of Natural Products Institute of Bioscience Universiti Putra Malaysia (UPM) 43400 Serdang Selangor Malaysia

Correspondence should be addressed to Mohd Fadzelly Abu Bakar fadzellyuthmedumy

Received 16 March 2015 Revised 31 May 2015 Accepted 1 June 2015

Academic Editor Kodappully S Siveen

Copyright copy 2015 Mohd Fadzelly Abu Bakar et al This is an open access article distributed under the Creative CommonsAttribution License which permits unrestricted use distribution and reproduction in any medium provided the original work isproperly cited

Garcinia dulcis or locally known inMalaysia as ldquomundurdquo belongs to the family of ClusiaceaeThe studywas conducted to investigatethe anticancer potential of different parts of G dulcis fruit extracts and their possible mechanism of action in HepG2 liver cancercell line MTT assay showed that the peel flesh and seed extracts of G dulcis induced cytotoxicity in HepG2 cell line with IC

50

values of 4633 plusmn 451 3833 plusmn 351 and 75 plusmn 252 120583gmL respectively The flesh extract of G dulcis induced cell cycle arrest at sub-G1(apoptosis) phase in a time-dependent manner Staining with Annexin V-FITC and propidium iodide showed that 412 of the

cell population underwent apoptosis after 72 hours of exposure of the HepG2 cell line toG dulcis flesh extract Caspase-3 has beenshown to be activatedwhich finally leads to the death ofHepG2 cell (apoptosis) GC-MS analysis showed that the highest percentageof compound identified in the extract of G dulcis flesh was hydroxymethylfurfural and 3-methyl-25-furandione together withxanthones and flavonoids (based on literature) could synergistically contribute to the observed effects This finding suggested thatthe flesh extract of G dulcis has its own potential as cancer chemotherapeutic agent against liver cancer cell

1 Introduction

In recent years studies on the exploitation of natural com-pounds from fruits for medicinal purposes have received agreat attention due to their health-promoting effects Plantsparticularly fruits and vegetables contain a diverse sourceof phytochemicals such as phenolics Daily consumptionof fruits and vegetables has been shown to lower the riskof some chronic noncommunicable diseases such as cancerand cardiovascular diseases [1 2] Phenolic compoundshave drawn increasing attention since they possess potentantioxidant potential and are abundantly present in fruits andvegetables and have been shown to exhibit anticancer effectsHowever it is believed that synergistic or additive biologicaleffects of multiple phytochemicals were involved rather than

a single compound or a group of compounds that contributeto cancer treatment and prevention It is estimated that morethan 5000 phytochemicals have been identified and differentantiproliferation mechanisms have been reported [3]

The latest world cancer report from International Agencyfor Research on Cancer (AIRC) a specialized cancer agencyof the World Health Organization (WHO) has revealed thatthe liver cancer is now the second leading cause of deathworldwide [4] Liver cancer is caused by Hepatitis B virus(HBV) which passes from person to person through bloodsemen or other body fluids and can be marked by liverinflammation The Sun Daily [5] reported that liver cancerin Malaysia (developing country) is on the rise and the fifthmost common cancer affecting men where rates are morethan twice as high inmales as in femalesGlobally it is also the

Hindawi Publishing CorporationBioMed Research InternationalVolume 2015 Article ID 916902 10 pageshttpdxdoiorg1011552015916902

2 BioMed Research International

seventh most commonly diagnosed cancer in adult womenwhere up to a million people die each year from liver cancerTherefore early detection is desperately needed to preventthis cancer as this can be prevented by taking Hepatitis Bvaccines Worldrsquos First Anticancer Vaccines In 2006 only27 of infants worldwide received the first dose of vaccineswithin 24 hours of birth As of 2008 a total of 177 countries(91) had introduced the HBV vaccine into their nationalinfant immunization schedule as recommended by theWHO[6]

Garcinia dulcis belongs to the family of Clusiaceae (Gut-tiferae) and is a less common fruit locally known as ldquomundurdquoamong Sabahan orMa-phut inThailandThe fruit is endemicto Borneo Java the Philippines andThailand It is amedium-sized tree 5 to 20m high often with yellow latex in the fruitsand white latex (turns to light brown on exposure to the air)in entire parts of the tree The leaf is large and leathery withdark green colour The fruit is spheroid slightly pointed andturns to yellow-orange when ripe There are usually 1 to 5brown seeds embedded in the flesh The skin of the fruit isthin and soft when ripe while the flesh is pulpy and yellowin colour The flesh is slightly acidic but pleasant to eat [7 8]However local people inThailand used it to enhance the tastein cooking dishes or make pickles because the fruits are toosour to be eaten rawfresh [9]

Previous literature reported that the leaf and seed ofG dulcis have been used traditionally for the treatment oflymphatitis parotitis and goitre whose effects may havebeen caused by endophytic fungi known to be present inGarcinia spp [10] In Thailand the stem bark has beenused traditionally as anti-inflammatory agent while the fruitjuice was used as expectorant In addition to that ripe fruitof G dulcis contained at least 22 known compounds andtwo new compounds (Dulcisflavan and Dulcisxanthone B)[11] These bioactive compounds played an important role asthey might be a great contributor to treat various chronicdiseases as reported in previous literature [12] Therefore themain objectives of this present study were to determine theanticancer potential of the 80 aqueous methanol extractof G dulcis towards the proliferation of liver carcinoma(HepG2) cancer cell line in vitro and investigate their possiblemechanism of action

2 Materials and Methods

21 Plant Materials and Sample Preparation The fruit of Gdulcis was collected from Tenom Sabah Malaysian Borneoin November to December 2012 The plant sample was col-lected identified and deposited at BORNEENSISHerbarium(BORH) Universiti Malaysia Sabah The samples were thenseparated into peel flesh and seedThe small-cut pieces werestored at minus80∘C before being freeze-dried by using freezedryer The lyophilized samples were grounded to obtain finepowderThese ground samples were stored in air-tight plasticbag at minus20∘C for further analysis

22 Sample Extraction The extraction procedure was con-ducted according to Ikram et al [13] with slight modifi-cations The samples were extracted using 80 methanol

by mixing 005 g of lyophilized fruit powder with 15mLsolvent The mixture was placed in a 100mL beaker wrappedwith aluminium foil and agitated at 200 rpm with the aidof orbital shaker for 2 hours The mixture was then filteredthrough filter paper (Whatman number 4) in order to obtaina clear solution The filtrate was then subjected to vacuumrotary evaporator at 40∘C to remove methanol residue Theconcentrated methanolic extract was freeze-dried to ensurethe excess water was removed and crude extracts can beobtained

23 Antiproliferative Assay The MTT (3-(45-dimethylthia-zol-2-yl)-25-diphenyltetrazolium bromide) assay was con-ducted based on the method described by Mosmann [14]The HepG2 cancer cell line was first cultured in RPMI 1640medium with L-glutamine and supplemented with 10 offetal calf serumand 1penicillin streptomycin in 75 cm2 flaskand incubated at 37∘C with 5 CO

2in a humidified atmo-

sphere The viability of cells was determined by staining withtrypan blue Exponentially growing cells were harvested andcounted using haemocytometer A specific culture mediumwas used to dilute the cells to a density of 1 times 106 cellsmLFrom this cell suspension 100 120583L was pipetted into eachwell of a 96-well plate and incubated for 24 h in a 5 CO

2

incubator at 37∘C The old media were discarded before thesample extracts were added with the highest concentrationof 100 120583gmL and make up the final volume of 100 120583L ineach well The plate was then incubated in 5 CO

2incubator

at 37∘C for 72 h Then 20120583L of MTT reagent (3-(45-dimethylthiazol-2-yl)-25-diphenyltetrazolium bromide) wasadded to each well and left in incubator for 4 h Subsequently100 120583L of solubilization solution (DMSO) was added to eachwell and reading was taken at wavelength of 570 nm usingELISA reader The cytotoxicity percentage was determinedusing the following formula

cytotoxicity =optical density of sampleoptical density of control

times 100 (1)

The final results were expressed as inhibition concentration(IC50) the concentration of sample able to inhibit cell

proliferation by 50 that was calculated graphically for eachcell proliferation curve

24 Cell Cycle Analysis Cell cycle analysis was conductedaccording to Abu-Bakar et al [15] Cells at density of 1 times 106cellsmLwere incubated with the extracts at IC

50value for 24

48 and 72 h All adhering and floating cells were harvestedand transferred into sterile centrifuge tube and centrifugedat 1200 rpm for 10min (4∘C) The pellets were then washedwith cold phosphate buffered saline (PBS) (10mM sodiumphosphate pH72 150mMsodiumchloride) and resuspendedin 05mL of cold PBS Then 5mL of ice-cold 70 ethanolwas added to the cell suspension and incubated at minus20∘Cfor 2 hours Then the cells were washed twice with coldPBS and centrifuged again at 1200 rpm for 10min (4∘C)After that 500120583L of propidium iodideRNAse (10 120583gmLpropidium iodide containing 1mgmL RNAse) solution wasadded to the cells and incubated in the dark for 30min at

BioMed Research International 3

room temperature before being analysed by flow cytometerwithin 3 hours of staining

25 Discrimination of Early and Late Apoptosis The proce-dure was conducted according to methodology provided inAnnexin V-FITC Apoptosis Detection Kit (catalog numberAPOAF SigmaUSA) Cells at density of 1times 106 cellsmLwereincubated with the extracts at IC

50value for 24 48 and 72 h

All adhering and floating cells were harvested and washedtwice with PBS before being transferred into sterile centrifugetube The cell pellet was then suspended in binding buffer(100mM HEPESNaOH pH 75 containing 14M NaCl and25mM CaCl

2) at a concentration of sim1 times 106 cells per mL

A sample (500 120583L) of this cell suspension was transferred to a5mL falcon tube towhich 5120583L ofAnnexinV-FITC conjugateand 10 120583L of propidium iodide were added The cells wereincubated in the dark for 10minutes at room temperatureThefluorescence of the cells was determined by flow cytometry

26 Assay of Caspase Activity Caspase-3 Colorimetric Assay(catalog number BF3100 RampD Systems) was used to deter-mine the activity of caspase-3 Cells (sim3 times 106 cells) (treatedand untreated) were collected and transferred into sterilecentrifuge tube The cells were centrifuged at 500 rpm for5min (25∘C) Supernatant was discarded and 25 120583L of coldlysis buffer was added per 1 times 106 cells The cell lysates wereincubated on ice for 10min Samples (50120583L) of the lysate(supernatant) were aliquoted into 96-well microplate and50120583L of 2X reaction buffer 3 followed by 05 120583L of 10mMDTT was added to sample wells Then 5 120583L of caspase-3 colorimetric substrate (DEVD-pNA) was added to eachreaction well and incubated at 37∘C for 2 h Absorbance valuewas read at 405 nm using ELISA reader

27 Gas Chromatography-Mass Spectroscopy (GC-MS) Themethanolic crude extract of flesh of G dulcis was analysedby gas chromatography equipped with mass spectrometry(GC-MS-2010 Plus-Shimadzu)The column temperature wasset to 50∘C for 4min then increased to 320∘C at the rate of7∘Cmin and then held for 20min The injector temperaturewas set at 280∘C (split mode with the ratio being adjustedto 20 1 injection volume = 01 120583L) The flow rate of thehelium carrier gas was set to 1mLmin with total run timeof 60min Mass spectra were obtained from the rangemz 40to 700 and the electron ionization was obtained at 70 eV Thechromatograms of the sample were identified by comparingtheir mass spectra with the library data and the GC retentiontime against known standards (NIST 11 Library and WileyLibrary)

28 Statistical Analysis Data were analysed using SPSSsoftware version 170 and reported as mean plusmn standarddeviation Analysis of variance (ANOVA) with Tukey testand independent sample 119905-test were conducted to identify thesignificant difference (119875 lt 005) All the analysis was carriedout in triplicate in three independent experiments

Table 1 IC50 value of samples tested against HepG2 cancer line byusing MTT

Parts IC50 value (mean plusmn SD)Peel 4633 plusmn 451Flesh 3833 plusmn 351Seed 75 plusmn 252Doxorubicin 20 plusmn 087SD standard deviation 119899 = 3

3 Results and Discussion

31 Anticancer Potential of G dulcis Extract on HepG2 CancerCell Line MTT assay is a commonly used method to inves-tigate the cytotoxicity of natural products This assay is basedon the ability of mitochondrial dehydrogenase enzymes fromviable cells to cleave the tetrazolium rings of the pale yellowMTT and form dark blue formazan crystals which is largelyimpermeable to cellmembranes resulting in its accumulationwithin healthy cells Thus the absorbance of the solubilizedformazan crystal is proportional to the number of living cellsin the system [14]

The incubation of G dulcis peel flesh and seed extractwith HepG2 cancer cell line induced cytotoxicity with IC

50

values of 4633 plusmn 451 3833 plusmn 351 and 75 plusmn 252 120583gmLrespectively (Table 1) Doxorubicin was used as positivecontrol The seed extract showed the lowest IC

50value

followed by flesh and peelThe lower the IC50value the more

effective the anticancer potential of the pure compound orcrude extract Some edible fruit extracts have been previouslyreported to possess anticancer properties such as cranberrylemon apple strawberry red grape banana and grapefruitthat showed potent antiproliferative activity towards HepG2cell line [16] rowanberry raspberry lingonberry cloudberryarctic bramble and strawberry towards HeLa (human cervi-cal cancer) [17]

The inhibition of liver cancer cell lines can be partiallyexplained by the presence of phenolic phytochemicals suchas anthocyanins phenolic acids carotenoids and flavonoidsas well as xanthone compound that is mainly distributedin Garcinia species According to Yang et al [18] specificphytochemicals might act additively synergistically andorantagonistically with other compounds to display antiprolif-erative activity Previous study reported that high antioxidantlevel did not necessarily reflect the high anticancer effect ofthe plant extract and might be due to synergistic effect of thecrude extract itself [19]

In addition to that Mangostin (a type of xanthonecompound) is also found in ripe fruit of G dulcis wherethe exact compound can also be found in the pericarp of Gmangostana especially 120572-mangostin and 120574-mangostin Con-sumption of mangosteen pericarp extract (81 120572-mangostinand 16 of 120574-mangostin) in ratio of 025 and 05 extractto food dosage in daily diet has been reported to inhibittumor growth in HCT116 (human colorectal carcinoma) andreduce blood vessels in tumor towards Athymic NCr nunumice [20] Previous literature reported the occurrence of atleast 24 compounds that have been identified in G dulcis


010203040506070

S

Cel

l pop

ulat

ion

()

Control

lowast

lowast

lowast

lowast

24h

Sub-G1

Apoptosis (sub-G1) and cell cycle distributionG0G1 G2M

(a)

Control 48h

010203040506070

S

Cel

l pop

ulat

ion

()

lowast

lowast

lowast

lowast

Apoptosis (sub-G1) and cell cycle distributionSub-G1 G0G1 G2M

(b)

lowast

lowast

lowast

lowast

Control 72h

0102030405060

S

Cel

l pop

ulat

ion

()


(c)

0 200 400 600 800 1000

FL2-A

0

40

80

120

160

200

Cou

nts

Control

(d)

0 200 400 600 800 1000

0

60

120

180

240

300

FL2-A

Cou

nts

DF 24h

(e)

0

0

60

120

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240

300

200 400 600 800 1000

FL2-A

Cou

nts

DF 48h

(f)

0

60

120

180

240

300

0 200 400 600 800 1000

FL2-A

Cou

nts

DF 72h

(g)

Figure 1 Cell cycle analysis of HepG2 cancer cell treated with the methanolic flesh extract of G dulcis at IC50

value The distribution ofcells undergoing apoptosis in various phases of the cell cycle was determined at 24 hours (a) 48 hours (b) and 72 hours (c) in comparisonto the respective control The values are presented as mean plusmn standard deviation where lowast indicated significant difference relative to control(119875 lt 005) The representative flow cytometric scans of untreated HepG2 cells and those treated with methanolic flesh extract of G dulcis for24 48 and 72 hours are presented in Figures (d) to (g) respectively

such as Dulcisflavan Dulcisxanthone B (-) epicatechin [11]and kaempferol (kaempferol 37-di-O-120572-rhamnopyranoside)[21] Epicatechin and kaempferol were two flavonoid com-pounds that belong to flavanols and flavonols respectivelyKaempferol is known to possess antioxidant and anticanceractivity which inhibit cell proliferation of MDA-MB-453(human breast carcinoma) [22] All these compounds mightact synergistically to inhibit the proliferation of studiedcancer cell lines

32 Cell CycleDistribution on the Flesh of G dulcis Due to thefact that only the flesh part of the fruit can be consumed and

also the seed extract induced cytotoxicity in normal mousefibroblast cell line (data not shown) further experiment oncell cycle distribution and apoptosis-inducing effect of Gdulcis only proceeded with the flesh crude extract ofG dulcisusing the IC

50value of 38 120583gmL Apoptosis and cell cycle

arrest in HepG2 cancer cell were studied after exposure toG dulcis flesh extract at IC

50concentration for 24 48 and

72 hours (Figure 1) Flow cytometric analysis (using RNAseand propidium iodide) was conducted to measure the DNAcontent of HepG2 cancer cell which allows us to identifywhether the growth inhibitory effect of the fruit extract wascaused by specific perturbation of cell cycle-related events


The cell cycle analysis demonstrated that the flesh ofG dulcis caused an accumulation of cells in the G

0G1

phase This causes the inhibition transition of cells towardsS phase and allowed the increase in the proportion of cellsin sub-G

1phase An obvious accumulation of cells in sub-

G1phase can be observed from 2 in control to 34 after

72 hours The increase in sub-G1phase was a reflection

of apoptosis induction Gradual decrease in G0G1phase

S phase and G2M phase can be observed from 24 h to

72 hours as compared to control This condition might bedue to the inhibition of DNA replication affected by theinability of the cells to replicate damaged DNA caused bythe sample extracts condensation of chromatin and nuclearfragmentation [23] The treatment of SK-MEL-28 (humanmelanoma) cells with 120572-mangostin significantly increasedthe sub-G

1peak with a concomitant decrease in G

1phase

indicating induction of apoptosis [24]

33 Early and Late Apoptosis Apoptosis is a physiologicalprocess of killing cells and is an important process to elim-inate tumoursThe apoptosis process can be characterized bymembrane blebbing (without loss of cell integrity) shrinkageof cells and nuclear volume chromatin condensation DNAfragmentation and formation of membrane-bound vesicles(apoptotic bodies) which can be triggered by multiple inde-pendent pathways from within or outside the cell [25]

TheAnnexin V-FITCApoptosis Detection kit is the com-bination of fluorescein isothiocyanate (FITC) and annexin Vwith propidium iodide to distinguish living cells in early andlate apoptosisThe annexin is a group of homologous proteinswhich bind phospholipids in the presence of calcium Duringearly apoptosis phosphatidylserine which is usually locatedin the inner membrane of cells is transported into the outerportion of the membrane which can be detected by its strongaffinity for Annexin V-FITC whereas the dead cells can bedetected by the binding of propidium iodide to the cellularDNA in cell [15]

The result showed that there was significant reductionin the proportion of cells in the viable cell group whentreated with methanolic flesh extract of G dulcis (Figure 2)These were concomitant with an increase in the proportionof cells in early and late apoptosis for 24 48 and 72 hoursof treatment when compared to control The total valuesof apoptosis increased up to 298 340 and 609respectively At 24 hours the values of viable cell started todecline by 698 as compared to control with significantincrease in early and late apoptosis with the values of 167and 131The same trendof reduction in proportion of viablecells and increase of early and late apoptosiswere observed for48 hours At the end of 72 hours the total apoptosis increasedup to 609 which comprise of early apoptosis (197) andlate apoptosis (412) Some of the cells induce cell deaththrough necrosis Necrosis is always considered to be almostldquoaccidental deathrdquo that may cause death of the cell at randomand uncontrollably The morphological changes of the cellundergoing apoptosis were shown in Figure 3 The inductionof apoptosis was confirmed through activation of caspase

34 Caspase Activity Caspase-3 is themost prevalent caspasewithin cells and responsible for most of apoptotic effectsand upon activation it was able to induce Poly ADP RibosePolymerase (PARP) cleavage and DNA break and finallyleads to apoptosis The exposure to the flesh of G dulcistowards HepG2 cancer cell line led to the activation ofcaspase-3 (Figure 4) This activation confirms that one of theinitiator caspases (caspase-2 caspase-8 and caspase-9) wasfirst activatedwhich eventually could activate the executionercaspase (caspase-3 caspase-6 and caspase-7) which in thiscase was caspase-3 and thus led to the induction of apoptosis[26] Caspase-3 was responsible for DNA fragmentation andmorphological changes associated with apoptosis whereascaspase-2 and caspase-9 were early biomarkers of apoptosiswhich act as downstream targets of cytochrome c release frommitochondria which will then activate caspase-3 [27] Thisfinding was in agreement with previous study [28] where120572-mangostin was able to suppress cell viability and colonyformation which caused cell cycle arrest upon activation ofcaspase-3 towards PC3 and 22Rv1 (human prostate) cancercell line Induction of apoptosis can be observed in COLO205(human colorectal adenocarcinoma) when exposed to 48mgof 120572-mangostin and 640mg of 120574-mangostin per gram extractwhere the apoptosis induction happened at caspase-3 andcaspase-8 pathway [29]

35 Gas Chromatography-Mass Spectroscopy (GC-MS)Methanolic crude extract of flesh of G dulcis was subjectedto GC-MS analysis GC-MS analysis is a widely used methodthat combines the features of gas chromatography andmass spectrometry purposely to identify compounds in asample (Figure 5) The identification and characterization ofchemical compounds of G dulcis extracted in 80 aqueousmethanol are shown in Table 2 The highest percentage ofcompound identified in the crude extract of G dulcis washydroxymethylfurfural or 5-hydroxymethylfurfural (HMF)

HMF is an organic compound absent naturally infood but generated in sugar-containing food during heat-treatments like drying or cooking This compound wascommonly found in bakery products malt fruit juices andcoffee with extremely high concentration in some food itemssuch as dried fruits caramel and vinegar [30] HMFwas usedas marker of quality in processed fruits coffee honey andmilk as well as monitoring the heating processes applied tocereal products such as pasta drying bread baking breadslices toasting and extrusion of baby cereals and breakfastcereals [31] HMF might occur naturally in the fruit or begenerated during high temperature processes such as dryingor during GC-MS analysis

Previous study reported that theHMF compound showedcytoprotective effect by protecting LO2 (normal liver cellline) cell against cytotoxicity induced by hydrogen peroxide(H2O2) Hydrogen peroxide is a major precursor of highly

reactive free radicals and it has been reported to decreasethe viability of cell and induce apoptosis in many cellssuch as cardiomyocytes chondrocytes tendon fibroblasts


0102030405060708090

Earlyapoptosis

Cel

l pop

ulat

ion

()

Control

Viablecells apoptosis

Late

24h

lowastlowast

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(a)

Control

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100

Viable Early Late

Cel

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ulat

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()

cells apoptosis apoptosis

48h

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Viablecells

Earlyapoptosis

Lateapoptosis

Control72h

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(c)

102

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idiu

m io

dide

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A

Annexin FITC-A

HepG2 apoptosis-control

(d)

102

103

104

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103

104

105

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idiu

m io

dide

PI-

A

Annexin FITC-A

HepG2 apoptosis-DF 24h

(e)

102

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104

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102

103

104

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idiu

m io

dide

PI-

A


(f)

102

102

103

103

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104

105

105

Prop

idiu

m io

dide

PI-

A

Annexin FITC-A


(g)

Figure 2 Apoptosis study upon treatment of the methanolic flesh extract of G dulcis at IC50

value The distribution of cells undergoingearly and late apoptosis together with viable cells was determined at 24 hours (a) 48 hours (b) and 72 hours (c) in comparison to respectivecontrol using Annexin V-FITC and propidium iodide flow cytometric analysisThe values are presented asmean plusmn standard deviation wherelowast indicated significant difference relative to control (119875 lt 005) The representative flow cytometric scans of untreated HepG2 cells and thosetreated with methanolic flesh extract of G dulcis for 24 48 and 72 hours are presented in Figures (d) to (g) respectively


Untreated cell at 24h

(a)


(b)


(c)

HepG2 cell treated with flesh of G dulcis at 24h

(d)


(e)


(f)

Figure 3 Morphological changes of HepG2 cancer cell line upon treatment with the flesh of G dulcis at (d) 24 h (e) 48 h and (f) 72 hcompared to untreated cell (a) (b) and (c)

020406080

100120140160180

24 48 72

Relat

ive t

o co

ntro

l (

)

Time (hours)

lowast

lowast

Figure 4 Activation of caspase in HepG2 cancer cell after incu-bation with methanolic flesh extract of G dulcis at IC

50value for

24 48 and 72 hours The values are presented as mean plusmn standarddeviation where lowast indicated significant difference relative to control(119875 lt 005)

hepatocytes and human umbilical vein endothelial cell Thecompound significantly inhibits the effect of cell apoptosis in

a dose-dependent manner after incubation of the cell with1 120583gmL HMF for 24 hours and thus decreased caspase-3activity as well as nitric oxide level [32] HMF which isalso present in roasted coffee residue suppressed cytotoxicityin mouse embryonic fibroblast cell which is induced byhydrogen peroxide [33] Liu et al [34] also reported thatHMFincreases enzyme activities such as superoxide dismutase(SOD) and glutathione peroxidase (GPx) as well as potentialtherapeutic agent for the treatment of Alzheimerrsquos disease

In comparison to previous study by Haroun et al [35]three out of 10 compounds identified in the pomegranatepeels extract were also found in methanolic crude extractof flesh of G dulcis namely HMF n-hexadecanoic acidand octadecanoic acid These compounds were believedto contribute to antimicrobial effects Previous researchrevealed that most of predominate compounds identified incrude extract through GC-MS analysis are biologically activemolecules and considered to be part of plant defence system[36] Hence it is possible that these compounds present inmethanolic crude extract of flesh of G dulcis contributed tothe antioxidant and anticancer properties


Region I

Region II

100 200 300 400 500 600 700 800 90030

Inte

nsity

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2000000

1500000

1000000

500000

0

(min)

68

7 9 1012

15 16 17 18 19 20 21 22 23 24 25

26 27

2829 30

31 321311

531

24

14

(a) Overall GC-MS analysis

Inte

nsity

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2000000

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1000000

500000

0100 200 300

(min)40030

201918

14

1716151312

11109

86

4

2

5

31 7

(b) Enlarged from Region I21

050000

100000150000200000250000300000350000400000450000500000550000600000650000

500(min)

600 700 800 900

22

2324

2526 27

2829

30

31 32

Inte

nsity

(c) Enlarged from Region II

Figure 5 Chemical compounds of methanolic crude extract of G dulcis detected using GC-MS with relative retention time (119905119877) (a) (b)enlarged from Region I and (c) enlarged from Region II

Table 2 Secondary metabolites in the G dulcis flesh extract based on GC-MS analysis

Peak RT Compound name 14 22175 5-Hydroxymethylfurfural 39614 9062 25-Furandione 3-methyl- 26242 5263 Furfural 66513 20732 1-Butanol 2-methyl- propanoate (CAS) 2-methylbutyl propionate 05412 20130 Catechol 4356 13024 25-Furandione dihydro-3-methylene- 35311 17579 4H-Pyran-4-one 23-dihydro-35-dihydroxy-6-methyl- 3338 14697 Furyl hydroxymethyl ketone 30919 34318 D-Allose 2685 10047 24-Dihydroxy-25-dimethyl-3(2H)-furan-3-one 12020 38486 16-Anhydro-alpha-d-galactofuranose 11622 52692 551015840-Oxy-dimethylene-bis(2-furaldehyde) 0941 4556 14-Dioxadiene 0597 14198 135-Triazine-246-triamine 04921 51056 n-Hexadecanoic acid 0369 15298 135-Triazine-246-triamine 03325 57555 Octadecanoic acid 03224 56821 Heptadecene-(8)-carbonic acid-(1) 0273 5713 2-Furanmethanol 014RT retention time


4 Conclusion

In conclusion the methanolic flesh extract of G dulcis hasdemonstrated promising anticancer properties against livercancer Increasing awareness promotion and utilization ofthis fruit for public benefits are highly encouraged Pro-teomic genomic in vivo and human clinical studies areneeded to determine the efficacy of the fruit extract to serveas natural cytotoxic agent in cancer patient

Conflict of Interests

The authors declare that there is no conflict of interests

Acknowledgments

The authors would like to acknowledge the Ministry ofHigher Education of Malaysia (MOHE) for financial assis-tance under the Exploratory Research Grant Scheme ERGS(Project no ERGS0028-STWN-12012) the Institute forTropical Biology and Conservation Seaweed Research Lab-oratory Faculty of Science and Natural Resources UniversitiMalaysia Sabah Malaysia Department of Nutrition andDietetic Universiti Putra Malaysia Malaysia and SabahDepartment of Agriculture Kota Kinabalu Sabah for thepermission of using the facilities and technical assistance

References

[1] M S Rabeta S Chan G D Neda K L Lam and M T OngldquoAnticancer effect of underutilized fruitsrdquo International FoodResearch Journal vol 20 no 2 pp 551ndash556 2013

[2] M F Abu Bakar M Mohamed A Rahmat and J FryldquoPhytochemicals and antioxidant activity of different parts ofbambangan (Mangifera pajang) and tarap (Artocarpus odoratis-simus)rdquo Food Chemistry vol 113 no 2 pp 479ndash483 2009

[3] N Jia Y L Xiong B Kong Q Liu and X Xia ldquoRadicalscavenging activity of black currant (Ribes nigrum L) extractand its inhibitory effect on gastric cancer cell proliferation viainduction of apoptosisrdquo Journal of Functional Foods vol 4 no1 pp 382ndash390 2012

[4] World Cancer Report 2014 httplivercancerconnectorg[5] A F Cruez ldquoLiver cancer on the riserdquo The Sun Daily 2014

httpwwwthesundailymynews1150045[6] A Jemal F Bray M M Center J Ferlay E Ward and D

Forman ldquoGlobal cancer statisticsrdquo CAmdashCancer Journal forClinicians vol 61 no 2 pp 69ndash90 2011

[7] M Osman and A R Milan Fruits of the Future 9 MangosteenGarciniamangostana L SouthamptonCentre forUnderutilisedCrops Southampton UK 2006

[8] W W W Wong T C Chong J Tananak H Ramba and NKalitu Fruits of Sabah vol 1 Department of Agriculture SabahSabah Malaysia 2007

[9] C Yapwattanaphun S Subhadrabandhu A Sugiura KYonemori and N Utsunomiya ldquoUtilization of some Garciniaspecies in Thailandrdquo in Proceedings of the International Sympo-sium on Tropical amp Subtropical Fruits pp 563ndash570 Inter-national Society for Horticultural Science Cairns Australia2002

[10] S Phongpaichit N Rungjindamai V Rukachaisirikul and JSakayaroj ldquoAntimicrobial activity in cultures of endophyticfungi isolated from Garcinia speciesrdquo FEMS Immunology andMedical Microbiology vol 48 no 3 pp 367ndash372 2006

[11] S Deachathai W Mahabusarakam S Phongpaichit and W CTaylor ldquoPhenolic compounds from the fruit of Garcinia dulcisrdquoPhytochemistry vol 66 no 19 pp 2368ndash2375 2005

[12] M Hemshekhar K Sunitha M S Santhosh et al ldquoAn overviewon genus Garcinia phytochemical and therapeutical aspectsrdquoPhytochemistry Reviews vol 10 no 3 pp 325ndash351 2011

[13] E H K Ikram K H Eng A M M Jalil et al ldquoAntioxidantcapacity and total phenolic content of Malaysian underutilizedfruitsrdquo Journal of Food Composition and Analysis vol 22 no 5pp 388ndash393 2009

[14] 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

[15] M F Abu-Bakar M Mohamed A Rahmat S A Burr andJ R Fry ldquoCytotoxicity and polyphenol diversity in selectedparts ofMangifera pajang and Artocarpus odoratissimus fruitsrdquoNutrition amp Food Science vol 40 no 1 pp 29ndash38 2010

[16] J Sun Y-F Chu X Wu and R H Liu ldquoAntioxidant andantiproliferative activities of common fruitsrdquo Journal of Agricul-tural and Food Chemistry vol 50 no 25 pp 7449ndash7454 2002

[17] G J McDougall H A Ross M Ikeji and D Stewart ldquoBerryextracts exert different antiproliferative effects against cervicaland colon cancer cells grown in vitrordquo Journal of Agriculturaland Food Chemistry vol 56 no 9 pp 3016ndash3023 2008

[18] J Yang R H Liu and L Halim ldquoAntioxidant and antiprolifera-tive activities of common edible nut seedsrdquo LWTmdashFood Scienceand Technology vol 42 no 1 pp 1ndash8 2009

[19] L J Jing M F Abu Bakar M Mohamed and A RahmatldquoEffects of selected Boesenbergia species on the proliferationof several cancer cell linesrdquo Journal of Pharmacology andToxicology vol 6 no 3 pp 272ndash282 2011

[20] A F A Aisha K M Abu-Salah Z D Nassar M J Siddiqui ZIsmail andAM SAMajid ldquoAntitumorigenicity of xanthones-rich extract from Garcinia mangostana fruit rinds on HCT116 human colorectal carcinoma cellsrdquo Brazilian Journal ofPharmacognosy vol 21 no 6 pp 1025ndash1034 2011

[21] W Mahabusarakam P Wiriyachitra and W C Taylor ldquoChem-ical constituents of Garcinia mangostanardquo Journal of NaturalProducts vol 50 no 3 pp 474ndash478 1987

[22] A E-M M R Afify S A Fayed E A Shalaby and H A El-Shemy ldquoSyzygium cumini (pomposia) active principles exhibitpotent anticancer and antioxidant activitiesrdquo African Journal ofPharmacy and Pharmacology vol 5 no 7 pp 948ndash956 2011

[23] J Li Y ChengWQu et al ldquoFisetin a dietary flavonoid inducescell cycle arrest and apoptosis through activation of p53 andinhibition of NF-Kappa B pathways in bladder cancer cellsrdquoBasic amp Clinical Pharmacology amp Toxicology vol 108 no 2 pp84ndash93 2011

[24] J J Wang B J S Sanderson and W Zhang ldquoCytotoxic effectof xanthones from pericarp of the tropical fruit mangosteen(Garcinia mangostana Linn) on human melanoma cellsrdquo Foodand Chemical Toxicology vol 49 no 9 pp 2385ndash2391 2011

[25] S H Kaufmann and W C Earnshaw ldquoInduction of apoptosisby cancer chemotherapyrdquo Experimental Cell Research vol 256no 1 pp 42ndash49 2000

[26] J Cortese ldquoA wealth of regents exists to probe caspases activi-tiesrdquoThe Scientist vol 15 no 13 p 24 2001


[27] S Gupta G E N Kass E Szegezdi and B Joseph ldquoThemitochondrial death pathway a promising therapeutic target indiseasesrdquo Journal of Cellular andMolecularMedicine vol 13 no6 pp 1004ndash1033 2009

[28] J J Johnson S M Petiwala D N Syed et al ldquo120572-Mangostina xanthone from mangosteen fruit promotes cell cycle arrestin prostate cancer and decreases xenograft tumor growthrdquoCarcinogenesis vol 33 no 2 pp 413ndash419 2012

[29] R Watanapokasin F Jarinthanan A Jerusalmi et al ldquoPotentialof xanthones from tropical fruit mangosteen as anti-canceragents caspase-dependent apoptosis induction in vitro and inmicerdquo Applied Biochemistry and Biotechnology vol 162 no 4pp 1080ndash1094 2010

[30] M Murkovic and N Pichler ldquoAnalysis of 5-hydroxymeth-ylfurfual in coffee dried fruits and urinerdquo Molecular Nutritionand Food Research vol 50 no 9 pp 842ndash846 2006

[31] E Capuano and V Fogliano ldquoAcrylamide and 5-hydroxym-ethylfurfural (HMF) a review on metabolism toxicity occur-rence in food and mitigation strategiesrdquo LWTmdashFood Scienceand Technology vol 44 no 4 pp 793ndash810 2011

[32] X DingM-YWang Y-X Yao G-Y Li and B-C Cai ldquoProtec-tive effect of 5-hydroxymethylfurfural derived from processedFructus corni on human hepatocyte LO2 injured by hydrogenperoxide and its mechanismrdquo Journal of Ethnopharmacologyvol 128 no 2 pp 373ndash376 2010

[33] J-S Sung E-B Go and H-S Shin ldquoCytoprotective activityof extract of roasted coffee residue on mouse embryonicfibroblasts cells against apoptosis induced by oxidative stressrdquoFood Science and Biotechnology vol 21 no 1 pp 137ndash143 2012

[34] A Liu X Zhao H Li et al ldquo5-Hydroxymethylfurfural anantioxidant agent from Alpinia oxyphylla Miq improves cog-nitive impairment in A1205731ndash42 mouse model of Alzheimerrsquosdiseaserdquo International Immunopharmacology vol 23 no 2 pp719ndash725 2014

[35] B M Haroun A A Elghamry E H Abdel-Shakour S MElmorsy and Y H Zahem ldquoDetermination of bioactive com-pounds of Punica granatum punicaceae and studying its antimi-crobial activities against bacteria isolated from El-Manzalawater treatment planetrdquo World Rural Observation vol 6 no 1pp 79ndash86 2014

[36] M A Hossain W A S Al-Toubi A M Weli Q A Al-Riyamiand J N Al-Sabahi ldquoIdentification and characterization ofchemical compounds in different crude extracts from leaves ofOmani neemrdquo Journal of TaibahUniversity for Science vol 7 no4 pp 181ndash188 2013

Submit your manuscripts athttpwwwhindawicom

PainResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom

Volume 2014

ToxinsJournal of

VaccinesJournal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

AntibioticsInternational Journal of

ToxicologyJournal of


StrokeResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Drug DeliveryJournal of



Advances in Pharmacological Sciences

Tropical MedicineJournal of


Medicinal ChemistryInternational Journal of


AddictionJournal of



BioMed Research International

Emergency Medicine InternationalHindawi Publishing Corporationhttpwwwhindawicom Volume 2014


Autoimmune Diseases


Anesthesiology Research and Practice

ScientificaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of


Pharmaceutics


MEDIATORSINFLAMMATION

of


seventh most commonly diagnosed cancer in adult womenwhere up to a million people die each year from liver cancerTherefore early detection is desperately needed to preventthis cancer as this can be prevented by taking Hepatitis Bvaccines Worldrsquos First Anticancer Vaccines In 2006 only27 of infants worldwide received the first dose of vaccineswithin 24 hours of birth As of 2008 a total of 177 countries(91) had introduced the HBV vaccine into their nationalinfant immunization schedule as recommended by theWHO[6]

Garcinia dulcis belongs to the family of Clusiaceae (Gut-tiferae) and is a less common fruit locally known as ldquomundurdquoamong Sabahan orMa-phut inThailandThe fruit is endemicto Borneo Java the Philippines andThailand It is amedium-sized tree 5 to 20m high often with yellow latex in the fruitsand white latex (turns to light brown on exposure to the air)in entire parts of the tree The leaf is large and leathery withdark green colour The fruit is spheroid slightly pointed andturns to yellow-orange when ripe There are usually 1 to 5brown seeds embedded in the flesh The skin of the fruit isthin and soft when ripe while the flesh is pulpy and yellowin colour The flesh is slightly acidic but pleasant to eat [7 8]However local people inThailand used it to enhance the tastein cooking dishes or make pickles because the fruits are toosour to be eaten rawfresh [9]

Previous literature reported that the leaf and seed ofG dulcis have been used traditionally for the treatment oflymphatitis parotitis and goitre whose effects may havebeen caused by endophytic fungi known to be present inGarcinia spp [10] In Thailand the stem bark has beenused traditionally as anti-inflammatory agent while the fruitjuice was used as expectorant In addition to that ripe fruitof G dulcis contained at least 22 known compounds andtwo new compounds (Dulcisflavan and Dulcisxanthone B)[11] These bioactive compounds played an important role asthey might be a great contributor to treat various chronicdiseases as reported in previous literature [12] Therefore themain objectives of this present study were to determine theanticancer potential of the 80 aqueous methanol extractof G dulcis towards the proliferation of liver carcinoma(HepG2) cancer cell line in vitro and investigate their possiblemechanism of action

2 Materials and Methods

21 Plant Materials and Sample Preparation The fruit of Gdulcis was collected from Tenom Sabah Malaysian Borneoin November to December 2012 The plant sample was col-lected identified and deposited at BORNEENSISHerbarium(BORH) Universiti Malaysia Sabah The samples were thenseparated into peel flesh and seedThe small-cut pieces werestored at minus80∘C before being freeze-dried by using freezedryer The lyophilized samples were grounded to obtain finepowderThese ground samples were stored in air-tight plasticbag at minus20∘C for further analysis

22 Sample Extraction The extraction procedure was con-ducted according to Ikram et al [13] with slight modifi-cations The samples were extracted using 80 methanol

by mixing 005 g of lyophilized fruit powder with 15mLsolvent The mixture was placed in a 100mL beaker wrappedwith aluminium foil and agitated at 200 rpm with the aidof orbital shaker for 2 hours The mixture was then filteredthrough filter paper (Whatman number 4) in order to obtaina clear solution The filtrate was then subjected to vacuumrotary evaporator at 40∘C to remove methanol residue Theconcentrated methanolic extract was freeze-dried to ensurethe excess water was removed and crude extracts can beobtained

23 Antiproliferative Assay The MTT (3-(45-dimethylthia-zol-2-yl)-25-diphenyltetrazolium bromide) assay was con-ducted based on the method described by Mosmann [14]The HepG2 cancer cell line was first cultured in RPMI 1640medium with L-glutamine and supplemented with 10 offetal calf serumand 1penicillin streptomycin in 75 cm2 flaskand incubated at 37∘C with 5 CO

2in a humidified atmo-

sphere The viability of cells was determined by staining withtrypan blue Exponentially growing cells were harvested andcounted using haemocytometer A specific culture mediumwas used to dilute the cells to a density of 1 times 106 cellsmLFrom this cell suspension 100 120583L was pipetted into eachwell of a 96-well plate and incubated for 24 h in a 5 CO

2

incubator at 37∘C The old media were discarded before thesample extracts were added with the highest concentrationof 100 120583gmL and make up the final volume of 100 120583L ineach well The plate was then incubated in 5 CO

2incubator

at 37∘C for 72 h Then 20120583L of MTT reagent (3-(45-dimethylthiazol-2-yl)-25-diphenyltetrazolium bromide) wasadded to each well and left in incubator for 4 h Subsequently100 120583L of solubilization solution (DMSO) was added to eachwell and reading was taken at wavelength of 570 nm usingELISA reader The cytotoxicity percentage was determinedusing the following formula

cytotoxicity =optical density of sampleoptical density of control

times 100 (1)

The final results were expressed as inhibition concentration(IC50) the concentration of sample able to inhibit cell

proliferation by 50 that was calculated graphically for eachcell proliferation curve

24 Cell Cycle Analysis Cell cycle analysis was conductedaccording to Abu-Bakar et al [15] Cells at density of 1 times 106cellsmLwere incubated with the extracts at IC

50value for 24

48 and 72 h All adhering and floating cells were harvestedand transferred into sterile centrifuge tube and centrifugedat 1200 rpm for 10min (4∘C) The pellets were then washedwith cold phosphate buffered saline (PBS) (10mM sodiumphosphate pH72 150mMsodiumchloride) and resuspendedin 05mL of cold PBS Then 5mL of ice-cold 70 ethanolwas added to the cell suspension and incubated at minus20∘Cfor 2 hours Then the cells were washed twice with coldPBS and centrifuged again at 1200 rpm for 10min (4∘C)After that 500120583L of propidium iodideRNAse (10 120583gmLpropidium iodide containing 1mgmL RNAse) solution wasadded to the cells and incubated in the dark for 30min at
















50


50value






010203040506070

S

Cel

l pop

ulat

ion

()

Control

lowast

lowast

lowast

lowast

24h

Sub-G1


(a)

Control 48h

010203040506070

S

Cel

l pop

ulat

ion

()

lowast

lowast

lowast

lowast


(b)

lowast

lowast

lowast

lowast

Control 72h

0102030405060

S

Cel

l pop

ulat

ion

()


(c)

0 200 400 600 800 1000

FL2-A

0

40

80

120

160

200

Cou

nts

Control

(d)

0 200 400 600 800 1000

0

60

120

180

240

300

FL2-A

Cou

nts

DF 24h

(e)

0

0

60

120

180

240

300

200 400 600 800 1000

FL2-A

Cou

nts

DF 48h

(f)

0

60

120

180

240

300

0 200 400 600 800 1000

FL2-A

Cou

nts

DF 72h

(g)












0G1









1phase











0102030405060708090

Earlyapoptosis

Cel

l pop

ulat

ion

()

Control


Late

24h

lowastlowast

lowast

(a)

Control

0102030405060708090

100

Viable Early Late

Cel

l pop

ulat

ion

()


48h

lowastlowast

lowast

(b)

0

20

40

60

80

100

Cel

l pop

ulat

ion

()

Viablecells

Earlyapoptosis

Lateapoptosis

Control72h

lowast

lowast

lowast

(c)

102

103

104

105

102

103

104

105

Prop

idiu

m io

dide

PI-

A

Annexin FITC-A


(d)

102

103

104

105

102

103

104

105

Prop

idiu

m io

dide

PI-

A

Annexin FITC-A


(e)

102

103

104

105

102

103

104

105

Annexin FITC-A

Prop

idiu

m io

dide

PI-

A


(f)

102

102

103

103

104

104

105

105

Prop

idiu

m io

dide

PI-

A

Annexin FITC-A


(g)





(a)


(b)


(c)


(d)


(e)


(f)


020406080

100120140160180

24 48 72

Relat

ive t

o co

ntro

l (

)

Time (hours)

lowast

lowast


50value for






Region I

Region II

100 200 300 400 500 600 700 800 90030

Inte

nsity

2500000

2000000

1500000

1000000

500000

0

(min)

68

7 9 1012

15 16 17 18 19 20 21 22 23 24 25

26 27

2829 30

31 321311

531

24

14


Inte

nsity

2500000

2000000

1500000

1000000

500000

0100 200 300

(min)40030

201918

14

1716151312

11109

86

4

2

5

31 7


050000

100000150000200000250000300000350000400000450000500000550000600000650000

500(min)

600 700 800 900

22

2324

2526 27

2829

30

31 32

Inte

nsity






4 Conclusion




Acknowledgments


References










































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of
















50


50value






010203040506070

S

Cel

l pop

ulat

ion

()

Control

lowast

lowast

lowast

lowast

24h

Sub-G1


(a)

Control 48h

010203040506070

S

Cel

l pop

ulat

ion

()

lowast

lowast

lowast

lowast


(b)

lowast

lowast

lowast

lowast

Control 72h

0102030405060

S

Cel

l pop

ulat

ion

()


(c)

0 200 400 600 800 1000

FL2-A

0

40

80

120

160

200

Cou

nts

Control

(d)

0 200 400 600 800 1000

0

60

120

180

240

300

FL2-A

Cou

nts

DF 24h

(e)

0

0

60

120

180

240

300

200 400 600 800 1000

FL2-A

Cou

nts

DF 48h

(f)

0

60

120

180

240

300

0 200 400 600 800 1000

FL2-A

Cou

nts

DF 72h

(g)












0G1









1phase











0102030405060708090

Earlyapoptosis

Cel

l pop

ulat

ion

()

Control


Late

24h

lowastlowast

lowast

(a)

Control

0102030405060708090

100

Viable Early Late

Cel

l pop

ulat

ion

()


48h

lowastlowast

lowast

(b)

0

20

40

60

80

100

Cel

l pop

ulat

ion

()

Viablecells

Earlyapoptosis

Lateapoptosis

Control72h

lowast

lowast

lowast

(c)

102

103

104

105

102

103

104

105

Prop

idiu

m io

dide

PI-

A

Annexin FITC-A


(d)

102

103

104

105

102

103

104

105

Prop

idiu

m io

dide

PI-

A

Annexin FITC-A


(e)

102

103

104

105

102

103

104

105

Annexin FITC-A

Prop

idiu

m io

dide

PI-

A


(f)

102

102

103

103

104

104

105

105

Prop

idiu

m io

dide

PI-

A

Annexin FITC-A


(g)





(a)


(b)


(c)


(d)


(e)


(f)


020406080

100120140160180

24 48 72

Relat

ive t

o co

ntro

l (

)

Time (hours)

lowast

lowast


50value for






Region I

Region II

100 200 300 400 500 600 700 800 90030

Inte

nsity

2500000

2000000

1500000

1000000

500000

0

(min)

68

7 9 1012

15 16 17 18 19 20 21 22 23 24 25

26 27

2829 30

31 321311

531

24

14


Inte

nsity

2500000

2000000

1500000

1000000

500000

0100 200 300

(min)40030

201918

14

1716151312

11109

86

4

2

5

31 7


050000

100000150000200000250000300000350000400000450000500000550000600000650000

500(min)

600 700 800 900

22

2324

2526 27

2829

30

31 32

Inte

nsity






4 Conclusion




Acknowledgments


References










































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of


010203040506070

S

Cel

l pop

ulat

ion

()

Control

lowast

lowast

lowast

lowast

24h

Sub-G1


(a)

Control 48h

010203040506070

S

Cel

l pop

ulat

ion

()

lowast

lowast

lowast

lowast


(b)

lowast

lowast

lowast

lowast

Control 72h

0102030405060

S

Cel

l pop

ulat

ion

()


(c)

0 200 400 600 800 1000

FL2-A

0

40

80

120

160

200

Cou

nts

Control

(d)

0 200 400 600 800 1000

0

60

120

180

240

300

FL2-A

Cou

nts

DF 24h

(e)

0

0

60

120

180

240

300

200 400 600 800 1000

FL2-A

Cou

nts

DF 48h

(f)

0

60

120

180

240

300

0 200 400 600 800 1000

FL2-A

Cou

nts

DF 72h

(g)












0G1









1phase











0102030405060708090

Earlyapoptosis

Cel

l pop

ulat

ion

()

Control


Late

24h

lowastlowast

lowast

(a)

Control

0102030405060708090

100

Viable Early Late

Cel

l pop

ulat

ion

()


48h

lowastlowast

lowast

(b)

0

20

40

60

80

100

Cel

l pop

ulat

ion

()

Viablecells

Earlyapoptosis

Lateapoptosis

Control72h

lowast

lowast

lowast

(c)

102

103

104

105

102

103

104

105

Prop

idiu

m io

dide

PI-

A

Annexin FITC-A


(d)

102

103

104

105

102

103

104

105

Prop

idiu

m io

dide

PI-

A

Annexin FITC-A


(e)

102

103

104

105

102

103

104

105

Annexin FITC-A

Prop

idiu

m io

dide

PI-

A


(f)

102

102

103

103

104

104

105

105

Prop

idiu

m io

dide

PI-

A

Annexin FITC-A


(g)





(a)


(b)


(c)


(d)


(e)


(f)


020406080

100120140160180

24 48 72

Relat

ive t

o co

ntro

l (

)

Time (hours)

lowast

lowast


50value for






Region I

Region II

100 200 300 400 500 600 700 800 90030

Inte

nsity

2500000

2000000

1500000

1000000

500000

0

(min)

68

7 9 1012

15 16 17 18 19 20 21 22 23 24 25

26 27

2829 30

31 321311

531

24

14


Inte

nsity

2500000

2000000

1500000

1000000

500000

0100 200 300

(min)40030

201918

14

1716151312

11109

86

4

2

5

31 7


050000

100000150000200000250000300000350000400000450000500000550000600000650000

500(min)

600 700 800 900

22

2324

2526 27

2829

30

31 32

Inte

nsity






4 Conclusion




Acknowledgments


References










































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of



0G1









1phase











0102030405060708090

Earlyapoptosis

Cel

l pop

ulat

ion

()

Control


Late

24h

lowastlowast

lowast

(a)

Control

0102030405060708090

100

Viable Early Late

Cel

l pop

ulat

ion

()


48h

lowastlowast

lowast

(b)

0

20

40

60

80

100

Cel

l pop

ulat

ion

()

Viablecells

Earlyapoptosis

Lateapoptosis

Control72h

lowast

lowast

lowast

(c)

102

103

104

105

102

103

104

105

Prop

idiu

m io

dide

PI-

A

Annexin FITC-A


(d)

102

103

104

105

102

103

104

105

Prop

idiu

m io

dide

PI-

A

Annexin FITC-A


(e)

102

103

104

105

102

103

104

105

Annexin FITC-A

Prop

idiu

m io

dide

PI-

A


(f)

102

102

103

103

104

104

105

105

Prop

idiu

m io

dide

PI-

A

Annexin FITC-A


(g)





(a)


(b)


(c)


(d)


(e)


(f)


020406080

100120140160180

24 48 72

Relat

ive t

o co

ntro

l (

)

Time (hours)

lowast

lowast


50value for






Region I

Region II

100 200 300 400 500 600 700 800 90030

Inte

nsity

2500000

2000000

1500000

1000000

500000

0

(min)

68

7 9 1012

15 16 17 18 19 20 21 22 23 24 25

26 27

2829 30

31 321311

531

24

14


Inte

nsity

2500000

2000000

1500000

1000000

500000

0100 200 300

(min)40030

201918

14

1716151312

11109

86

4

2

5

31 7


050000

100000150000200000250000300000350000400000450000500000550000600000650000

500(min)

600 700 800 900

22

2324

2526 27

2829

30

31 32

Inte

nsity






4 Conclusion




Acknowledgments


References










































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of


0102030405060708090

Earlyapoptosis

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Earlyapoptosis

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Control72h

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102

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24 48 72

Relat

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)

Time (hours)

lowast

lowast


50value for






Region I

Region II

100 200 300 400 500 600 700 800 90030

Inte

nsity

2500000

2000000

1500000

1000000

500000

0

(min)

68

7 9 1012

15 16 17 18 19 20 21 22 23 24 25

26 27

2829 30

31 321311

531

24

14


Inte

nsity

2500000

2000000

1500000

1000000

500000

0100 200 300

(min)40030

201918

14

1716151312

11109

86

4

2

5

31 7


050000

100000150000200000250000300000350000400000450000500000550000600000650000

500(min)

600 700 800 900

22

2324

2526 27

2829

30

31 32

Inte

nsity






4 Conclusion




Acknowledgments


References










































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of



(a)


(b)


(c)


(d)


(e)


(f)


020406080

100120140160180

24 48 72

Relat

ive t

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l (

)

Time (hours)

lowast

lowast


50value for






Region I

Region II

100 200 300 400 500 600 700 800 90030

Inte

nsity

2500000

2000000

1500000

1000000

500000

0

(min)

68

7 9 1012

15 16 17 18 19 20 21 22 23 24 25

26 27

2829 30

31 321311

531

24

14


Inte

nsity

2500000

2000000

1500000

1000000

500000

0100 200 300

(min)40030

201918

14

1716151312

11109

86

4

2

5

31 7


050000

100000150000200000250000300000350000400000450000500000550000600000650000

500(min)

600 700 800 900

22

2324

2526 27

2829

30

31 32

Inte

nsity






4 Conclusion




Acknowledgments


References










































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of


Region I

Region II

100 200 300 400 500 600 700 800 90030

Inte

nsity

2500000

2000000

1500000

1000000

500000

0

(min)

68

7 9 1012

15 16 17 18 19 20 21 22 23 24 25

26 27

2829 30

31 321311

531

24

14


Inte

nsity

2500000

2000000

1500000

1000000

500000

0100 200 300

(min)40030

201918

14

1716151312

11109

86

4

2

5

31 7


050000

100000150000200000250000300000350000400000450000500000550000600000650000

500(min)

600 700 800 900

22

2324

2526 27

2829

30

31 32

Inte

nsity






4 Conclusion




Acknowledgments


References










































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of


4 Conclusion




Acknowledgments


References










































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of
















Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of





Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of

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