1. Introduction. Correspondence should be addressed to Maznah Ismail; Received 2 January 2013; Accepted 7 March 2013

Transcription

1 Evidence-Bsed Complementry nd Alterntive Medicine Volume 213, Article ID 54975, 8 pges Reserch Article Cytotoxic Activity of Kenf Seed Oils from Supercriticl Crbon Dioxide Fluid Extrction towrds Humn Colorectl Cncer (HT29) Cell Lines Siti Aisyh Abd Ghfr, 1 Mznh Ismil, 1,2 Ltifh Siful Yzn, 2 Shrid Fkurzi, 2,3 Norshrin Ismil, 1 Kim Wei Chn, 1 nd Pridh Md Thir 4 1 Nutricosmeceuticl nd Nutrigenomic Progrmme, Lbortory of Moleculr Biomedicine, Institute of Bioscience, Universiti Putr Mlysi, 434 Serdng, Selngor Drul Ehsn, Mlysi 2 Fculty of Medicine nd Helth Sciences, Universiti Putr Mlysi, 434 UPM Serdng, Selngor Drul Ehsn, Mlysi 3 Lbortory of Vccines nd Immunotherpeutics, Institute of Bioscience, Universiti Putr Mlysi, 434 Serdng, Selngor Drul Ehsn, Mlysi 4 Institute of Tropicl Forestry nd Forest Products (INTROP), Universiti Putr Mlysi, 434 UPM Serdng, Selngor, Mlysi Correspondence should be ddressed to Mznh Ismil; mznh@medic.upm.edu.my Received 2 Jnury 213; Accepted 7 Mrch 213 Acdemic Editor: Ki-Wn Oh Copyright 213 Siti Aisyh Abd Ghfr et l. This is n open ccess rticle distributed under the Cretive Commons Attribution License, which permits unrestricted use, distribution, nd reproduction in ny medium, provided the originl work is properly cited. Kenf (Hibiscus cnnbinus) from the fmily Mlvcee, is vluble fiber plnt ntive to Indi nd Afric nd is currently plnted s the fourth commercil crop in Mlysi. Kenf seed oil contins lph-linolenic cid, phytosterol such s β-sitosterol,vitmin E, nd other ntioxidnts with chemopreventive properties. Kenf seeds oil (KSO) ws from supercriticl crbon dioxide extrction fluid (SFE) t 9 different permuttions of prmeters bsed on rnge of pressures from 2 to 6 brs nd temperture from 4 to 8 C. They were 2/4, 2/6, 2/8, 4/4, 4/6, 4/8, 6/4, 6/6, nd 6/8. Extrction from 9 prmeters of KSO-SFE ws screened for cytotoxicity towrds humn colorectl cncer cell lines (HT29) nd mouse embryonic fibroblst (NIH/3T3) cell lines using MTS ssy. KSO-SFE t 6/4 showed the strongest cytotoxicity towrds HT29 with IC 5 of 2 μg/ml. The IC 5 for NIH/3T3 ws not detected even t highest concentrtion employed. Cell cycle nlysis showed significnt increse in the ccumultion of KSO-SFE-treted cells t sub-g1 phse, indicting the induction of poptosis by KSO-SFE. Further poptosis induction ws confirmed by Annexin V/PI nd AO/PI stining. 1. Introduction Kenf (Hibiscus cnnbinus L, fmily Mlvcee) is vluble fiberplntntivetoindindafric[1]. The plnt tht is composed of vrious ctive components including tnnins, sponins, polyphenolics, lkloids, essentil oils, nd steroids hs long been prescribed in trditionl folk medicine in Afric nd Indi [2]. Kenf seeds yield vegetble oil tht is edible for humn consumption [1]. The oil contins vitmin E with high ntioxidnt, β-sitosterol with nticncer effects, nd lph-linolenic cid (ALA), s the essentil omeg-3 ftty cid with nti-inflmmtory nd ntithrombotic ctivity. The 3 min bioctive compounds (vitmin E, β-sitosterol, nd ALA) in kenf seed oil re known to contribute gretly s chemopreventive gents [3 5]. From the conventionl method, kenf seed oil cn be extrcted out using orgnic solvents such s n-hexne or petroleum ether. However, the oils extrcted from solventextrction method re usully doubted for their sfety due to the presence of solvent residue. Therefore, supercriticl fluid extrction (SFE) seems to be the sfest wy to extrct theoil.asupercriticlfluidisnysubstncettemperture nd pressure bove its thermodynmic criticl point. It hs unique bility to diffuse through solids like gs nd dissolve

2 2 Evidence-Bsed Complementry nd Alterntive Medicine mterils like liquid [6]. Additionlly, it cn redily chnge in density upon minor chnges in temperture or pressure. Compred with liquid solvents, SFE hs severl more dvntges: (1) the dissolving power of supercriticl fluid solvent depends on its density which is highly djustble by chnging the pressure or/nd temperture; (2) supercriticl fluid hs higher diffusion coefficient nd lower viscosity nd surfce tension thn liquid solvent, leding to more fvorble mss trnsfer. These properties mke it suitble s substitute for orgnic solvents in process clled supercriticl fluid extrction (SFE) [6]. Previous study hs shown tht kenf seed oil extrcted by SFE (KSO-SFE) is cytotoxic towrds humncerviclcncercellline[7]. However, there hs been lck of study tht ws conducted on oil extrcted from kenf seed using SFE technology towrds colon cncer. Therefore, this study determined the cytotoxicity of KSO-SFE on colon cncer cells. 2. Mterils nd Methods 2.1. Mterils nd Chemicls. Kenf seed ws purchsed from the Ntionl Kenf nd Tobcco Bord (NKTB), Psir Putih, Kelntn. Kenf seed ws clened nd dried t constnt temperture (5 C) overnight in n oven (FD 115, Fisher Scientific). The finl moisture content of the dried seed ws less thn 5%. The dried seeds were stored t 4 Cuntilfurther use. Humn colorectl cncer (HT29) nd mouse embryonic fibroblst (NIH/3T3) cell lines were purchsed from the Americn Type Culture Collection (ATCC), USA. Dulbecco s modified egle medium (DMEM), fetl bovine serum (FBS), penicillin, streptomycin, trypsin, sodium bicrbonte, propidium iodide, RNse A, Acridine ornge, trypn blue, nd phosphte buffer sline (PBS) were purchsed from Sigm- Aldrich Co. (Sigm-Aldrich Co., St. Louis, MO, USA). Methyl thizolyl tetrzolium (MTS) nd Annexin V-FITC poptosis detection kit were purchsed from Promeg (Southmpton, UK) Extrction of Kenf Seed Oil. Kenf seed oils were prepred using supercriticl crbon dioxide fluid extrctor (Thr 1 F) t 9 different extrction prmeters (pressure (brs)/temperture ( C)), which were 2/4, 2/6, 2/8, 4/4, 4/6, 4/8, 6/4, 6/6, nd 6/8. This ws done following method developed in our lb s described by Chn nd Ismil (29) with slight modifictions [8]. In brief, 1 g of seed ws ground using Wring blender for one min nd plced into 1 L extrction vessel. The desired temperture nd pressure were then set. The flow rte of crbon dioxide (CO 2 )wssett25g/min nd regulted by n utomted bckpressure regultor. The extrction strted fter the desired temperture nd pressure were obtined. The whole extrction process lsted for 2.5 h, ndtheyieldobtinedwsclculted. For Soxhlet extrction, 5 g of kenf seeds were ground using Wring blender for 1 min nd divided eqully into 2 extrction thimbles. Ech thimble ws then trnsferred into Soxhlet extrctor (Witeg, Germny). Next, 3 ml of hexne ws dded to ech flsk. After the extrctions were initited, the solvent flow rte ws djusted mnully to 7 min/cycle. Extrction processes were, respectively, terminted fter 2 cycles of solvent flow for rpid soxhlet extrctions (SOX/S) nd 1 cycles of solvent flow for conventionl soxhlet extrction (SOX/L). For conventionl ultrsonic-ssisted solvent extrction (SONIC), 25 g of kenf seed ws ground nd homogenized with3mlofhexnet135rpmfor3min(ultr-turrx T25 bsic, IKA-Werke). Subsequently, the mixture ws sonicted for 9 min (Power Sonic 55, Microprocess Controlled Benchtop Ultrsonic Clener). After the soniction, the mixture ws filtered through filter pper (Whtmn number 1), nd then hexne ws then removed under reduced pressure (Buchi) Cell Cultures. The humn colonic cncer (HT29) nd mouse embryonic fibroblst (NIH/3T3) cell lines from the Americn Type Culture Collection (ATCC), USA, were grown in Dulbecco s modified Egle s medium (DMEM) supplemented with 1% fetl bovine serum, penicillin (1 units/ml), nd streptomycin (1 μg/ml). Cells were mintined in humidified tmosphere of 5% CO 2 t 37 C. Confluent cells were detched using.25% (w/v) trypsin- EDTA. Cell number nd vibility were determined by using hemocytometer fter stining with trypn blue Cell Vibility. Approximtely cells were seeded into 96-well plte nd incubted for 24 h. Kenf seed oils were then dded into the wells, nd seril dilution ws performed. Negtive control cells were lso included (untreted with KSO-SFE). The cell vibility ws determined by CellTiter 96 Aqueous One Solution Assy (Promeg) tht contins tetrzolium compound [3-(4,5-dimethylthizol-2-yl)-5-(3- crboxymethoxyphenyl)-2-(4-sulfophenyl)-2h-tetrzolium, inner slt] (MTS). After 72 h of incubtion, 2 μl ofmts solutionwsddedintothewell,ndthepltewsincubted for 3.5 h t 37 Cin5%CO 2 tmosphere. Vible cells colonies would produce formzn which could be mesured t 49 nm with 96-well plte ELISA reder (Opsys, USA). Cell vibility ws clculted with the following formul: %Vibility = Absorbnce of smple Absorbnce of blnk Absorbnce of control Absorbnce of blnk 1%. (1) 2.5. Determintion of IC 5. A curve/grph of percentge of vibility cells versus concentrtion ws plotted from three replictes of experiment. Inhibitory concentrtion (IC 5 ), tht is defined s the KSO-SFE concentrtion of the tested mteril tht results in 5% of cell deth, tht ws determined from the cell vibility curve Acridine Ornge(AO)/Propidium Iodide (PI) Double Stining Morphologicl Anlysis. Approximtely cells/ml of HT29 were seeded into ech well of 6-well plte nd incubted for 24 h t 37 C in humidified, 5% CO 2

3 Evidence-Bsed Complementry nd Alterntive Medicine 3 tmosphere. KSO-SFE ws then dded into the well nd incubted for 72 h. After incubtion, the cells were trypsinized nd wshed once with phosphte buffered sline (PBS). Cell suspension (1 μl) ws plced on glss slide nd mixed with 1 μl of AO(5μg/mL) nd PI (5μg/mL). The cells were viewed under fluorescence microscope (Leic, Germny) Cell Cycle Anlysis by Flow Cytometer. Approximtely cells/ml of HT29 were seeded into ech well of 6- well plte nd fter 24 h of incubtion KSO-SFE ws dded. The plte ws further incubted for 24 h, 48 h, nd 72 h. Next, cells were detched nd fixed with 5 ml ice cold 7% ethnol t 2 C for 2 h. Subsequently, the cells were wshed with 5mLofPBS,centrifugedt3rpmfor1min,ndthe superntnt ws discrded. The pellet ws resuspended with 425 μlpbs,2μl of propidium iodide (4 μg/ml) nd 5 μl of RNse A (1 μg/ml). The mixture ws then incubted in the drk t 4 C for 3 min nd red by flow cytometer (CyAN dp, Denmrk) Annexin V-Propidium Iodide (AnnV-PI) Stining Apoptosis Test. Approximtely cells/ml of HT29 were seeded into ech well of 6-well plte nd fter incubtion for 24 h KSO-SFE ws dded. The cells were then further incubted for 24 h, 48 h, nd 72 h. The subsequent procedures were crried out ccording to the instructions provided by the mnufcturer of APOPTEST-FITC kit. Briefly, cells were wshed with PBS, suspended in binding buffer nd then dded with Annexin-V FITC (AnnV) nd propidium iodide (PI) nd left for 1 min. The smples were then nlyzed by flow cytometer (CyAN dp, Denmrk) Sttisticl Anlysis. Sttisticl nlysis were performed using Sttisticl Pckge for Socil Science (SPSS) version 17, nd significnce ws ccepted t P <.5.Dtpresentedin the study were nlyzed using ANOVA, vlues were given s men ± SD, nd mens were seprted using Duncn multiple rnge test. 3. Results 3.1. Cell Vibility. All extrcts of 9 different prmeters of KSO-SFE (2/4, 2/6, 2/8, 4/4, 4/6, 4/8, 6/4, 6/6, nd 6/8) nd solvent extrcted kenf seed oils (SOX/S, SOX/L, nd SONIC) of kenf seed were tested for cell vibility through MTS ssy. In generl, lower IC 5 vlue wsobservedincellstretedwithksofromsfescompred to the one of solvent extrction (Figure 1). The lowest IC 5 of 2μg/mL ws from KSO-SFE 6/4 nd ws further tested on norml cell line (NIH/3T3) for cytotoxicity. Nevertheless, IC 5 vlue for KSO-SFE (6/4) towrds NIH/3T3 ws not obtined even t the highest concentrtion employed (Figure 2) Acridine Ornge (AO)/Propidium Iodide (PI) Double Stining Morphologicl Anlysis. AO/PI double stining morphologicl nlysis distinguishes vible, poptotic, nd IC 5 vlue SFE 2/4 SFE 2/6 d SFE 2/8 SFE 4/4 SFE 4/6 SFE 4/8 SFE 6/4 SFE 6/6 SFE 6/8 Kenf seeds oil (KSO) Figure 1: IC 5 vlue of kenf seed oil (KSO) from supercriticl fluid (SFE) nd solvent extrction towrds HT29 cell line. The dt shown re mens ± SD. Mens with different letter differ significntly (P <.5) using Duncn multiple rnge test. KSO ws extrcted with 9 different SFE extrction prmeters (pressure (brs)/temperture ( C)). SOX/L = conventionl soxhlet extrction; SOX/S = rpid soxhlet extrction; SONIC = Soniction-ssisted solvent extrction. Vibility cell c c b SOX/S b SOX/L b SONIC Concentrtion (μg/ml) HT29 3T3 Figure 2: Cytotoxic effects of KSO-SFE 6/4 towrds colon cncer (HT29) nd norml (NIH/3T3) cells lines s detected by MTS ssy fter 72 h. Vlues re expressed s men ± SD. An sterisk indictes P <.5 when compred to untreted cells. necrotic cells. The results obtined from AO/PI double stining re shown in Figure 3. Vible cells with intct DNA nd nucleus give round nd green nuclei. Nucleus of the cells undergoing poptosis ws stined green but frgmented. Lte poptotic nd necrotic cells were stined ornge nd red. Fromthefigure,itwsclerthtwiththeincreseofKSO- SFE concentrtion, the number of vible cells decresed. In ddition, poptotic cells showed some other chrcteristics such s like plsm membrne blebbing. This indictes tht most of the cell deth occurred primrily through poptosis nd not necrosis.

4 4 Evidence-Bsed Complementry nd Alterntive Medicine () (b) (c) (d) Figure 3: Morphologicl study of HT29 cell lines treted with vrious concentrtion of KSO-SFE fter 72 h. () μg/ml (control); (b) 2 μg/ml; (c) 1 μg/ml viewed under fluorescence microscope (x mgnifiction). (d) Cell blebbing (1x mgnifiction). Vible cells were round stined green by cridine ornge, while ded cells were stined red by propidium iodide. The rrows in (d) showed blebbed cell tht indicte the poptosis chrcteristics. KSO-SFE: kenf seed oil extrcted by supercriticl fluid extrction Cell Cycle Anlysis by Flow Cytometer. Figure 4 shows the effects of KSO-SFE on the cell cycle of HT29 cells. A significnt (P <.1)increseinthecellpopultiontsub-G1 phse ws observed t 1 μg/ml of KSO-SFE (7.52% ±.62) nd ws more pronounced t the higher concentrtions (2, 5, 1 μg/ml). After 72 h, cell deth ws found to increse significntly up to 77.14% ± 1.13 compred to the control (3.46% ±.8) in cells treted with KSO-SFE t 1 μg/ml Annexin V-Propidium Iodide (AnnV-PI) Stining Apoptosis Test. Bsed on Figure 5, some chnges in the cell popultion were noticed t tretment of 1 μg/ml KSO-SFE nd becoming more pronounced t higher concentrtion (2, 5, nd 1 μg/ml) over time. After 24 h, only 9.73% ±.92 of the untreted cells entered erly poptosis stge, but 14.15% ± 2.17 (1 μg/ml), 21.37% ± 3.26 (2 μg/ml), 19.23% ±.9 (5 μg/ml), nd 25.15% ±.66 (1 μg/ml) of treted cell hve entered erly poptosis stge. After 72 h only 11.% ± 1.38 of untreted cells entering lte poptosis but 22.46% ±.22 (1 μg/ml), 29.74% ±.12 (2μg/mL), 34.34% ± 3.2 (5 μg/ml), nd 56.74% ± 4.72 (1μg/mL) of treted cells entering lte poptosis. Besides, there is no significnt difference (P <.5) whencompredwith1.72%±.12 of untreted cells, 3.46% ±.3 nd 2.18% ±.16 of 5 μg/ml nd 1μg/mL treted cells entering necrosis stge, respectively. The differences between the percentge of untreted cells nd KSO-SFE treted cells in erly poptosis, lte poptosis, nd secondry poptosis stges were ll significnt (P <.5). 4. Discussion There hs been substntil interest in the serch for potentil chemopreventive gents in the pst yers for tretment of cncers. Understnding how dietry components regulte prolifertion nd cell survivl could ply criticl role in the development of new gents tht cn prevent nd tret cncer with low toxicity [9]. Cncer chemoprevention ws described s the use of nturl synthetic chemicls llowing suppression, retrdtion, or inversion of crcinogenesis [1]. Apoptosis induction is one of potent defensive strtegies ginst cncer progression [11 14]. Numerous diet-derived gents re mong promising gents nd gent combintions tht re being evluted cliniclly s chemopreventive gents for mjor cncer trgets including colon, brest, prostte, nd lung cncers [15]. Yield of the oil extrcted is one of the importnt criteri to be considered for commerciliztion of products such s drugs, functionl foods, or dietry supplement. Apprently, rise in the extrction pressure incresed the yield of KSO from SFE ccording to the following sequence: 6/8 6/6 6/4 4/8 4/4 4/6 > 2/4 > 2/6 2/8 (P <.5). Pressure nd temperture re two importnt SFE prmeters tht give huge effects to the yield of kenf seed oil. Elevtion in pressure t given temperture results in n increse in the fluid (CO 2 ) density which mens tht it enhnced solubility of the solutes hence, incresed the yield of KSO-SFE [16]. On the other hnd, t constnt pressure, the density of CO 2 decreses

5 Evidence-Bsed Complementry nd Alterntive Medicine 5 8 μg/ml 7 1 μg/ml Cell deth G/G1 S G2/M Cells phse Cell deth G/G1 S G2/M Cells phse () (b) 7 2 μg/ml μg/ml Cell deth G/G1 S G2/M Cell deth G/G1 S G2/M Cells phse Cells phse (c) (d) μg/ml Cell deth G/G1 S G2/M Cells phse (e) Figure 4: Cell cycle nlysis of HT29 treted with () μg/ml; (b) 1 μg/ml; (c) 2 μg/ml, (d) 5 μg/ml; (e) 1 μg/ml of KSO-SFE for 24 h, 48 h, nd 72 h. P <.5, P <.1 compred to control cells.

6 6 Evidence-Bsed Complementry nd Alterntive Medicine Vible Erly poptosis Concentrtion (μg/ml) Lte pop Necrosis Figure 5: Annexin V-PI flow cytometry nlysis of HT29 cells treted with μg/ml, 1 μg/ml, 2μg/mL, 5 μg/ml, nd 1 μg/ml of KSO-SFE. The cells were clssified into four stges: vible, erly poptosis, lte poptosis, nd necrosis/secondry necrosis. Vlues re expressed s men ± SD. P <.5 compred to untreted cells. when the temperture is incresed. In ddition, temperture lso ffects the voltility of the solute. Hence the effect of temperture elevtion is difficult to predict becuse of its dependence on the nture of the smple. For instnce, nonvoltile solute would result in lower yield, wheres for voltile solute it will increse in yield [16]. The results of cell vibility nlysis showed tht ll 9 prmeters of KSO-SFE were cytotoxic towrds HT29 cells with the IC 5 vlues rnging from 2 μg/ml to 375 μg/ml (Figure1). Apprently, there ws rise in the cytotoxic ctivity of KSO-SFE ccording to the following sequence: 6/4 4/6 4/4 2/4 6/6 2/6 > 6/8 > 4/8 2/8 (P <.5). Wheres the IC 5 towrds NIH/3T3 cells line could not be determined (Figure 2). This indictes tht KSO-SFE especilly the 6/4 ws cytotoxic towrds HT29 cell line. The pressure of 6 brs nd temperture t 4 C were probbly the most suitble condition where most of the bioctive components nd nutritive vlues of kenf seed hve been successfully extrcted. Kenf seed oil ws extrcted t 8 C t ny pressure, nd the IC 5 towrds HT29 cell lines ws t 375 μg/ml. The temperture might be high enough to degrde some of the bioctive components, or tht some biologicl ctive substnces my not be extrcted t tht temperture compred to others (4 Cnd6 C) [17]. For soxhlet extrcts [(SOX/L) nd (SOX/S)], higher IC 5 ws noted when compred to the SFE extrcts. The results proved the dvntge using SFE thn conventionl methods (solvents extrction) for extrction of oil from kenf seed. The results obtined cn be used s bsis for development of disese-oriented drug discovery. From Mnosroi (26), (1) extrct tht hd IC 5 vlueoflessthn125μg/ml cn be possible cndidte for further development to cncer therpeutic gent; (2) extrct tht hd the IC 5 vlue between 125 nd 5μg/mL hs moderte possibility to be developed into cncer therpeutic gent but high possibility to be developed s chemopreventive gents nd; (3) extrct tht hd the IC 5 more thn 5 μg/ml hs low possibility to be developed s cncer therpeutic gent. Bsed on criteri nd Figure 3(b), KSO-SFE flls into the group tht hs the potentil to be developed s chemopreventive gent [18]. This result hs been supported by previous study showing tht kenf seed oil extrcted by SFE reduced berrnt crypt foci (ACF) number on zoxymethne (AOM) induced rts [19].Forthepurposeofthisstudy,KSO-SFEof6/4ws chosen s the most potentil extrct, since it hs the lowest IC 5 (2 μg/ml) mong others. It is importnt to determine mode of cell deth induced by KSO-SFE. Since cell deth could be divided into poptosis or necrosis. Apoptosis is more fvourble thn necrosis becuse it is progrmmed cell deth tht does not trigger inflmmtory responses [13]. In AO/PI stining, DNA-binding dye cridine ornge (AO) nd propidium iodide (PI) were used for morphologicl detection of poptotic nd necrotic cells. AO intercltes into DNA giving it green ppernce. Thus, vible cells hve bright green nucleus. PI is only tken up by nonvible cells. This dye lso intercltes into DNA, mking it ppers ornge. The live cells with intct membrne hve uniform green color in their nuclei. Erly poptotic cells hve chromtin condenstion with bright colored nuclei. Lte poptotic cells hve bright ornge res of condensed chromtin in the nucleus tht distinguished them from necrotic cells, which hve uniform ornge color [2, 21]. Bsed on the AO/PI dul stining ssy, round nd helthyviblecellscnbeseeninthecontrolcellsuntreted with KSO-SFE. On the other hnd, reduction in the cncer cell number nd lso feture of poptosis like cell blebbing were noted in the treted cells. This provides qulittive morphologicl proof tht KSO-SFE could induce poptosis nd inhibit cncer cell growth. This is further confirmed with the existence of the sub-g1 re by cell cycle nlysis. It hs been reported tht ppernce of sub-g1 cells is the mrker of cell deth cused by poptosis [22]. Annexin V/PI (AnnV/PI) stining is bsed on bility of the protein Annexin V to bind to phosphtidylserine (PS) exposed on the outer membrne leflet, but upon induction of poptosis it is trnslocted to the outer membrne leflet nd becomes vilble for Annexin V binding. The ddition of PI enbled vible (AnnV /PI ), erly poptotic (AnnV + /PI ), lte poptotic (AnnV + /PI + ), nd necrotic (AnnV /PI + )cellstobedistinguished[23]. Bsed on tht, it shows tht KSO-SFE induced erly poptosis t 24 h nd lte poptosis/secondry necrosis t 72 h necrosis in HT29 cells (Figure 5). As KSO-SFE induced cell deth in sequence from erly poptosis to lte poptosis/secondry necrosis [24], it is concluded tht KSO-SFE ctully induces cell deth vi poptotic rther thn necrotic pthwy. KSO-SFE ws found to contin higher vitmin E by our group compred to the one of solvent extrction, whereby when compred to solvent extrction [1, 5]. Vitmin E is known to induce poptosis in HT29 cell line nd inhibit humn prostte cncer cell growth [25, 26]. KSO-SFE is lso rich in sterol especilly β-sitosterol [5]. Previous studies hve shown tht β-sitosterol reduces crcinogen-induced cncer of the colon in rts [27]. Severl studies hve indicted tht β-sitosterol inhibits the growth of vrious cultured cncer cell lines tht re ssocited with cell cycle rrest

7 Evidence-Bsed Complementry nd Alterntive Medicine 7 nd the stimultion of poptotic cell deth [28 3]. Alphlinolenic cid (ALA), the essentil omeg-3 ftty cid, ws lso found in kenf seed oil tht is known s chemopreventive gents [1, 4]. These bioctive compounds re probbly responsible for the cytotoxicity of KSO-SFE in HT29 cell line. In conclusion, lthough the yield of KSO-SFE is lower thn the one of solvent extrction, this study hs shown tht KSO-SFE possesses better cytotoxic properties towrds HT29 cell line. It induced cell deth vi poptosis nd inhibited prolifertion of the cncer cells. These findings dd to growing body of literture on helth benefit of KSO-SFE. As current studies re focusing on in vitro KSO-SFE effects, further reserch might explore KSO-SFE effects in vivo. Finding of this study will be beneficil for further development of new chemotherpeutic/chemopreventive gents. Acknowledgment This study ws supported by The Ministry of Plnttion Industries nd Commodities, Mlysi (Grnt no ). References [1] A. Mohmed, H. Bhrdwj, A. Hmm, nd C. Webber, Chemicl composition of kenf (Hibiscus cnnbinus L.) seed oil, Industril Crops nd Products,vol.4,no.3,pp ,1995. [2]M.Kobisy,M.R.Tellez,C.L.Webber,F.E.Dyn,K.K. Schrder, nd D. E. Wedge, Phytotoxic nd fungitoxic ctivities of the essentil oil of kenf (Hibiscus cnnbinus L.) leves nd its composition, Journl of Agriculturl nd Food Chemistry, vol. 49, no. 8, pp , 21. [3] M.L.RuizdelCstillo,G.Dobson,R.Brennn,ndS.Gordon, Genotypic vrition in ftty cid content of blckcurrnt seeds, Journl of Agriculturl nd Food Chemistry, vol.5,no. 2, pp , 22. [4]D.Willims,M.Verghese,L.T.Wlker,J.Boteng,L.Shckelford, nd C. B. Chwn, Flx seed oil nd flx seed mel reduce the formtion of berrnt crypt foci (ACF) in zoxymethne-induced colon cncer in Fisher 344 mle rts, Food nd Chemicl Toxicology,vol.45,no.1,pp ,27. [5] A. A. Mriod, S. F. Fthy, nd M. 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