David G. Popovich, Shi Yun Yeo, and Wei Zhang. 1. Introduction

Transcription

1 Hindwi Pulishing Corportion Evidence-Bsed Complementry nd Alterntive Medicine Volume 211, Article ID 48273, 9 pges doi:1.193/ecm/nep74 Originl Article Ginseng (Pnx quinquefolius) nd Licorice (Glycyrrhiz urlensis) Root Extrct Comintions Increse Heptocrcinom Cell (Hep-G2) Viility Dvid G. Popovich, Shi Yun Yeo, nd Wei Zhng Deprtment of Chemistry, Ntionl University of Singpore, Science Drive 4, Singpore Correspondence should e ddressed to Dvid G. Popovich, chmpdg@nus.edu.sg Received 29 Decemer 28; Accepted 26 My 29 Copyright 211 Dvid G. Popovich et l. This is n open ccess rticle distriuted under the Cretive Commons Attriution License, which permits unrestricted use, distriution, nd reproduction in ny medium, provided the originl work is properly cited. The comined cytoctive effects of Americn ginseng (Pnx quinquefolius) nd licorice (Glycyrrhiz urlensis) root extrcts were investigted in heptocrcinom cell line (Hep-G2). An isoologrphic nlysis ws utilized to express the possiility of synergistic, dditive or ntgonistic interction etween the two extrcts. Both ginseng nd licorice roots re widely utilized in trditionl Chinese medicine preprtions to tret vriety of ilments. However, the effect of the hers in comintion is currently unknown in cultured Hep-G2 cells. Ginseng (GE) nd licorice (LE) extrcts were oth le to reduce cell viility. The LC5 vlues, fter 72 h, were found to e.64 ±.2 mg/ml (GE) nd.53 ±.2 mg/ml (LE). An isoologrm ws plotted, which included five theoreticl LC5s clculted, sed on the fixed frction method of comintion ginseng to licorice extrcts to estlish line of dditivity. All comintions of GE to LE (1/5, 1/3, 1/2, 2/3, 4/5) produced n effect on Hep-G2 cell viility ut they were ll found to e ntgonistic. The LC5 of frctions 1/3, 1/2, 2/3 were 23%, 21% nd 18% ove the theoreticl LC5. Lctte dehydrogense relese indicted tht s the proportion of GE to LE incresed eyond 5%, the influence on memrne permeility incresed. Cell-cycle nlysis showed slight ut significnt rrest t the G1 phse of cell cycle for LE. Both GE nd LE reduced Hep-G2 viility independently; however, the comintions of oth extrcts were found to hve n ntgonistic effect on cell viility nd incresed cultured Hep-G2 survivl. 1. Introduction Americn ginseng (Pnx quinquefolius) nd Asin licorice (Glycyrrhiz urlensis) roots hve long history of use in trditionl Chinese medicine (TCM). These roots re often used in comintion with other herl ingredients with the gol of improving the effectiveness of the TCM preprtions. Ginseng nd licorice roots re likely the most utilized TCM ingredients [1], nd oth roots re lso now utilized y the nutrceuticl nd functionl food industries for their purported ioctive nd functionl properties [2 4]. The min ioctive ingredients found in ginseng re thought to e group of dmmrne triterpene sponins, which re lso known s ginsenosides [5], nd one of the min ctive ingredient in licorice is glycyrrhizic cid, which is n olenne triterpene sponin. Both roots lso contin vriety of other phytochemicl compounds such s polyscchrides nd polyphenols [4, 6] tht my dd dditionl enefits to tinctures or decoctions. Licorice root extrcts nd glycyrrhizic cid hve een reported to hve oth cytotoxic nd hepto-protective properties. Glycyrrhizic cid extrcted from licorice root ws reported to protect ginst fltoxin B 1 injury in Hep- G2 cells [7] nd reduced free ftty cid-induced heptic lipotoxicity in oth cultured Hep-G2 cells nd high-ftdiet-induced lipotoxicity in rts [8]. Furthermore, licorice nd glycyrrhizic cid protected primry heptocytes ginst zthioprine (1 μm) injury, n immune suppressnt drug tht hs heptic side effects [9]. Glycyrrhizic cid hs lso een cliniclly used in Jpn to tret chronic heptitis [1]. Licorice extrcts hve exhiited nti-inflmmtory ctivity nd inhiited cultured heptic crcinom cell (Hep-3B) prolifertion [3]. A licorice extrct lso reduced prostte specific ntigen relese, nd reduced cultured prostte cncer cell line (LNCP) prolifertion [11] nd is one of seven ingredients in the herl supplement PC-SPES [12]. Ginseng root nd ginsenosides hve een ssocited with wide rnge of iologicl ctivities such s nti-dietic

2 2 Evidence-Bsed Complementry nd Alterntive Medicine [13, 14], immune stimultion [15, 16] nd n ility to inhiit the growth of vriety of cultured cncer cells [5, 17, 18]. The precise mechnism ttriuted to ginseng nd ginsenoside cellulr cytotoxicity is not entirely cler ut n interction with the cell memrnes leding to memrne permetion hs een previously reported [18]. The ojective of this study ws to determine if the comintion of Americn ginseng (Pnx quinquefolius)nd licorice extrcts (Glycyrrhiz urlensis) would synergisticlly or ntgonisticlly ffect the viility of cultured heptocrcinom cell line (Hep-G2). An isoologrphic nlysis ws utilized to efficiently depict synergistic, ntgonistic nd dditive responses in Hep-G2 cells. Isoologrphic nlysis provides cler grphicl view of the interctions etween the two components nd hs een utilized to depict interctions etween two drug comintions [19]. 2. Methods 2.1. Plnt Mterils. Dried ginseng (Pnx quinquefolius) nd licorice (Glycyrrhiz urlensis) roots were purchsed loclly (Singpore). Ginsenoside HPLC stndrds (Rg1, Re, R1, Rc, Rd) were purchsed from Chromdex Inc. (Snt An, CA, USA) nd glycyrrhizic cid stndrd ws purchsed from Sigm (Steinheim, Germny), trifluorocetic cid (TFA) ws otined from Merck (Whitehouse Sttion, NJ, USA). Acetonitrile nd methnol were of HPLC grde (Tedi Inc., Firfield, OH, USA), ethnol ws of nlyticl grde (Fisher Scientific, UK). Dried ginseng root (Pnx quinquefolius) ws ground into powder nd refluxed in methnol (5 ml) for 3 h, filtered twice (Whtmn no 4) nd the extrction ws repeted three times. The solution ws concentrted under vcuum, nd the extrct ws pplied to preconditioned polymeric sorent Amerlite XAD-4 (Sigm, St. Louis, MO,USA)column(poredimeterof4Å, ed volume of 6 cm 3, flow rte of 1 ml/min) nd wshed with distilled wter (1 L) to remove polr compounds s previously descried [2]. Ginsenosides were eluted from the column using solute ethnol (5 ml) nd concentrted under vcuum, lyophilized nd is herein referred to s the ginseng extrct (GE). Powdered dried licorice (Glycyrrhiz urlensis) root ws refluxed for 2.5 h in methnol (7%, 4 ml), filtered twice, extrcted three times nd concentrted under vcuum nd is herein referred to s the licorice extrct (LE) HPLC Anlysis. AWtersHPLC(Milford,MA,USA) equipped with quternry grdient pump nd photodioderry (PDA) detector ws used to ssess the mount of either ginsenosides or glycyrrhizic cid in the respective extrcts. The HPLC nlysis ws conducted using reverse-phse C18 column (C18, mm, 5 μm prticle size, Wters) with column temperture of 25 C, injection volume of 2 μl nd detection wvelength of 23 nm for ginsenosides nd 254 nm for GA. GE, ginsenosides stndrds, LE nd GA were seprtely dissolved in methnol, filtered through.45 μm syringe filter (Minisrt, Germny), prior to nlysis. HPLC nlysis for GE consisted of two seprte grdient progrms (referred to s grdients 1 nd 2), due to the similr retention times of ginsenosides Rg1 nd Re. Both solvent progrms consisted of the comintion of wter (A) nd cetonitrile (B). Grdient progrm 1 (percentge cetonitrile nd flow rte re enclosed in prenthesis) ws s listed: Time (2% B, 1 ml/min), 2 min (25% B,.7 ml/min), 4 min (49% B,.7 ml/min), 5 min (1% B,.7 ml/min), nd 6 min (8% B,.7 ml/min). Grdient progrm 2 consisted of: Time (2% B, 1 ml/min), 45 min 22% B,.7 ml/min), 5 min, (6% B,.7 ml/min), 6 min (2% B,.7 ml/min). HPLC seprtion grdient progrm for LE nd GA utilized.5% TFA wter (A) nd cetonitrile (B) nd ws s follows: Time (2% B), 5 min (4% B), 1 2 min (5% B), nd the flow rte ws constnt t.8 ml/min. Ginsenoside Rg1, Re, R1, Rc, Rd in the GE nd GA in the LE were identified ccording to the respective stndrd curves nd were ssessed in triplicte Cell Culture. Humn heptocrcinom (Hep-G2) cells were purchsed from Americn Type Culture Collection (Mnsss, VA, USA). The cells were mintined in Dulecco s modified Egle s medium (DMEM) supplemented with 1% fetl ovine serum (Sigm), 1 U penicillin, nd 1 μg/ml streptomycin (Gico, Invitrogen, Cnd) in humidified tmosphere of 5% CO 2 t 37 C. Cells were mintined t concentrtion etween nd cells/ml. Cells were sucultured every 2-3 dys y totl medium replcement using.25% (w/v) trypsin.53 mm EDTA solution (GIBCO). Vile cells were ssessed y.4% trypn lue exclusion dye (MP Biomedicls, Solon, OH, USA) using hemocytometer nd ssessed in qudruplicte Cell Viility MTT Assy. Cell viility ws mesured y 3-(4,5-dimethylthizol-2-yl)-2,5-diphenyl tetrzolium romide, MTT (Sigm) ssy, in order to estlish n LC5 vlue (concentrtion to inhiit 5% of cells). Cells were seeded t concentrtion of cells/well in 96- microwellpltendwerellowedtodherefor24h.stock solutions of GE nd LE (1. mg/ml) were dissolved nd filtered through sterile.2 μm syringe filter (Millex GP, Irelnd) efore dding to the cells. Extrcts were dded t vrious concentrtions (.2.9 mg/ml) nd incuted for 72 h. Controls contined Hep-G2 cells, culture medium, ut no extrcts. At the end of 72 h, the extrct-contining medium ws removed nd 1 μlof.5mg/mlofmttws dded nd incuted for 4 h s previously descried [21], crystls were soluilized in 1 μl of 1% SDS (Ntionl University of Medicl Institution, Singpore) in.1 N HCl nd incuted overnight. The sornce ws mesured t 55 nm using microplte reder (Multiskn Spectrum, Thermo Electron Corportion, Wlthm, MA, USA) nd the results were expressed s the percentge of vile cells with respect to the untreted control cells. Cell viility (%) ws clculted s (men sornce of the smple/men sornce of the control) 1.

3 Evidence-Bsed Complementry nd Alterntive Medicine Isoologrm Anlysis. The interctions etween GE nd LE were nlyzed with the use of n isoologrm nd test of significnce ccording to the method of Tllrid (2) [22]. The LC5 vlues for ginseng nd licorice extrcts descried ove were used to plot the isoologrm. The LC5 vlue for GE ws plotted on the x-xis, while the LC5 vlue for LE on the y-xis. The line connecting the two points is referred to s the line of dditivity nd represents the predicted LC5 (refer to (1)) of different comintions of extrcts. Antgonistic comintions re points tht lie ove the line, while synergistic comintions re points elow the line [22]. The fixed rtio design ws used. Briefly, five frctions (1/5, 1/3, 1/2, 2/3, 4/5) of GE to LE were used to test the interctions of the two extrcts (refer to (2)). LC5 vlues were first otined for GE nd LE lone nd then the LC5 vlues were otined for ech of the five frctions Cell LDH Activity. Hep-G2 cells were seeded t concentrtion of cells/ml in 24-well pltes nd llowed to dhere for 24 h. The medium ws removed nd extrcts (ginseng, licorice or the five frctions) t their respective LC5 concentrtion were dded to the wells. The concentrtions used were.64 mg/ml (GE),.53 mg/ml (LE),.64 mg/ml (f1/5),.7 mg/ml (f1/3),.74 mg/ml (f1/2),.74 mg/ml (f2/3) nd.69 mg/ml (f4/5). Cells were incutedfor24,48nd72h,nduntretedcellscteds control. At the end of ech incution time, the medium ws removed nd tested for LDH ctivity s previously descried [5] Cell-Cycle Anlysis. GE, LE nd five frctions (1/5, 1/3, 1/2, 2/3, 4/5) were dded to Hep-G2 cells t their respective LC5 concentrtions. Cells were incuted for 24, 48 nd 72 h with untreted cells cting s controls. Cellcycle nlysis ws determined s previously descried [21]. Briefly, cells were trypsinized, wshed with PBS nd the cellulr pellet ws fixed in ice-cold 7% ethnol overnight t 4 C. The superntnt ws removed y centrifugtion, 1 ml of PBS contining 5 μg/ml propidium iodide (Sigm) nd 1 U/mL RNAse A (Applichem Inc, Cheshire, CT, USA) were dded nd incuted t room temperture for 1 h efore dt cquisition using Dko Cytomtion Cyn LX Flow Cytometry (Beckmn Coulter, Fullerton, CA, USA) nd nlyzed with Dko Summit v4.3 softwre pckge Sttisticl nd Dt Anlysis. TheLC5vluesofGE, LE nd the five frctions were determined from five seprte experiments with five replictes for ech experiment. LDH nd cell-cycle nlysis were repeted on three seprte occsions with three replictes. Dt re expressed s men ± stndrd devition (SD). A one-wy ANOVA with Tukey post hoc comprison of mens ws used to test for significnce (P <.5) of LDH nd cell-cycle nlysis using the SPSS sttisticl softwre (v12., Chicgo, IL, USA). Determintion of Theoreticl LC5 (CAL5). CAL5 = f GE + ( 1 f ) LE (1) Concentrtion (mg/ml) GE LE Figure 1: The effect of GE nd LE on Hep-G2 viility. The results re expressed s men ± SD of five seprte experiments with five replictes. GE nd LE vriles represent the experimentlly determined LC5 vlues of ginseng nd licorice extrcts, nd vrile f represents the fix rtio frctions (1/5, 1/3, 1/2, 2/3, 4/5) of GE to LE comintions, CAL5 is the theoreticl or clculted LC5. Determintion of Concentrtions of Individul Extrct in Ech Frction. GE f = f GE ( CAL5 ), 1 f LE LE f = CAL5 Vriles f, GE, LE nd CAL5 re descried ove, GE f nd LE f represent the mount ech root extrct to dd to ech frction. Additive, Synergy nd Antgonism. Additive: CAL5 EXPLC5 = Synergy: EXPLC5 < CAL5 Antgonism: EXPLC5 > CAL5 Vrile EXPLC5 is the experimentlly derived LC5 of the frctions nd vrile CAL5 is descried ove. The test of significnce nd the ove equtions re sed on the reported literture [22, 23]. (2) (3)

4 4 Evidence-Bsed Complementry nd Alterntive Medicine LC5 =.65 ±.1 mg/ml Frction(f1/5) (mg/ml) () LC5 =.7 ±.2 mg/ml Frction(1/3) (mg/ml) (c) LC5 =.75 ±.2 mg/ml Frction(1/2) (mg/ml) (e) LC5 =.74 ±.2 mg/ml Frction(2/3) (mg/ml) () LC5 =.69 ±.2 mg/ml LC5 (mg/ml) Frction(4/5) (mg/ml) (d) cd GE LE f1/5 f1/3 f1/2 f2/3 f4/5 Figure 2: The effect of five distinct frctions (1/5, 1/3, 1/2, 2/3, 4/5) on Hep-G2 viility () (e). (f) corresponds to the LC5s vlues of GE, LE nd five frctions. Brs with different letters re significntly different from ech other (P <.5). Results re expressed s men ± SD of five seprte experiments with five replictes. (f) cd d c 3. Results 3.1. HPLC Anlysis. Individul ginsenoside nlysis showed tht the percentge of ginsenosides in the GE ws determined to e Re (2.2 ± 7.8), R1 (8.8 ±.2), Rg1 (1.5 ±.4) nd Rd (.71 ±.1), nd the totl ginsenosides content wsdetermined to e3.9 ± 7.8% (dry weight). Ginsenoside Rc ws detected ut the mount ws elow the limit of detection. For LE, the totl percentge of glycyrrhizic cid ws determined to e 7.1 ±.2% (dry weight) Dose-Response LC5 Determintion of Ginseng nd Licorice Extrcts. Figure 1 shows the dose-response reltionship of GE nd LE. The LC5 ws clculted y plotting cell viility (%) versus log concentrtion (grph not shown) which yielded liner eqution of y = x (r 2 =.982) for GE nd y = x (r 2 =.981) for the LE. The LC5s were determined to e.64 ±.2 mg/ml for GE nd.53 ±.2 mg/ml for LE. Both GE nd LE showed dose dependent effect on Hep-G2 viility, nd the LE LC5 vlue ws found to e significntly (P <.5) lower LC5 compred to GE Dose-Response LC5 Determintion of Frctions. The LC5 vlues of five frctions corresponding to rtio of ginseng to licorice extrcts of 1/5, 1/3, 1/2, 2/3 nd 4/5 nd re shown in Figures 2() 2(e)) nd clculted s descried ove. Compred to the LC5 of GE, frctions (1/3, 1/2, 2/3, 4/5) were ll significntly (P <.5) greter thn GE with the exception of f1/5 which ws similr to GE. Furthermore,

5 Evidence-Bsed Complementry nd Alterntive Medicine 5 G. urlensis (mg/ml) f(1/5) f(1/3) f(1/2) f(2/3) f(4/5) P. quinquefolius (mg/ml) Figure 3: Isoologrphic representtion of the interction etween experimentlly mesured GE, LE nd frctions (grphicl points) nd the theoreticl line of dditively (solid line) on Hep-G2 viility. The dotted line represents the theoreticl stndrd devition of plus/minus 2.5%. Experimentl points nd error rs re expressed s men ± SD of five seprte experiments with five replictes. the LC5 for ll frctions were significntly (P <.5) greter when compred to the LC5 of LE (Figure 2(f)) Isoologrphic Anlysis. Figure 3 represents grphicl view of the effects of the comintions of GE nd LE on Hep-G2 cells viility. An isoologrm ws creted using the LC5 vlue for GE, which intersects the x-xis, nd the LC5 vlue for LE, which intersects the y-xis. The two points re joined together to form the line of dditivity. This line of dditivity refers to the concentrtions of GE nd LE extrcts needed to produce the sme effect if they were to ct lone. Points lying on the line re termed dditive, ove the line re ntgonistic nd elow the line re synergistic [22, 23]. All comintions (1/5, 1/3, 1/2, 2/3 nd 4/5) of GE nd LE showed ntgonism. They were ll greter thn the line of dditivity. Frctions 1/2 nd 2/3 showed the gretest ntgonistic effect. Tle 1 shows the theoreticl LC5 nd the experimentlly oserved LC5. Frctions 1/3 nd 1/2 showed the gretest difference from the theoreticl LC5 t 23 nd 21%, respectively Cell-Cycle Anlysis. Representtive DNA histogrms of the vrious tretments re shown in Figure 4 nd corresponding nlysis is listed in Tle 2. Apoptotic cells (sug1) were generlly not oserved during the cell-cycle nlysis. A mjority of frctions tested did not show ny significnt increse of poptotic cells t 24 h of tretment s compred to the untreted control. GE significntly (P <.5) incresed su G1 cells ut the increse ws only mrginl t 48 nd 72 h. Overll, Hep-G2 cells generlly showed significnt (P <.5) increse in the proportion of cells in the G1 phse t 24 nd 48 h time periods with the exception of GE t 24 h. A decrese in the proportion of G2/M cells ws oserved for ll extrcts nd comintions compred to the control. At 48 nd 72 h of tretment, significnt reductions (P <.5) in the Tle 1: Theoreticl nd oserved LC5 vlues for ginseng nd licorice extrcts. Frctions, Theoreticl LC5 CAL5 (mg/ml) Oserved LC5 c EXPLC5 (mg/ml) Difference (%) f1/ ±.1 d 16. f1/ ±.2,c 22.9 f1/ ±.2, 21.3 f2/ ± f4/ ±.2 c 11.2 For exmple, f1/5 refers to the mount of ginseng (1/5) to licorice (4/5) in the mixture. Refer to the Methods section for description of the clcultions nd vriles CAL5 nd EXPLC5. c Vlues with different letters re significntly different (P <.5). proportion of G2/M cells were oserved in frctions 1/3, 1/2, 2/3 nd 4/5. Cells oserved in the S phse were reduced ut not significnt with the exception of frctions 1/3, 1/2, 2/3 nd 4/5 fter 24 h of tretment LDH Anlysis. The effect of different tretments nd exposure times on LDH relese, mrker of memrne integrity nd dmge, is shown in Figure 5. LEtretment from 24 to 72 h did not significntly ffect the relese of LDH compred to the corresponding control cells. GE tretment produced the gretest significnt increse in LDH relese, while frctions tht contined proportion of t lest one hlf GE (e.g., f1/2, f2/3, f4/5) hd significnt (P <.5) increse in LDH relese compred to untreted control cells fter 72 h of incution. Frctions 1/2, 2/3 nd 4/5 were found to hve percentge increse of 57% (f1/2), 192% (f2/3) nd 263% (f4/5). GE showed gretest significnt LDH increse t ll time periods followed y f4/5 t 48 h nd frctions f2/3 nd f4/5. 4. Discussion By utilizing n isoologrphic nlysis, we hve effectively shown tht the comintions of ginseng (Pnx quinquefolius) nd licorice (Glycyrrhiz urlensis) extrcts do not produce synergistic effect on reducing Hep-G2 cell viility. On the contrry, the effect ws ntgonistic. Frctions 1/2 nd 2/3 were found to hve the highest LC5 vlues (.75 ±.2 nd.74 ±.2 mg/ml, resp.), compred to the other frctions nd frctions 1/3 nd 1/2 showed the gretest percentge difference when compred to the theoreticl or clculted LC5 (CAL5). Adms et l. [19] lso reported ntgonism with the comintion G. urlensis with either Rdosi ruescens or Scuteellri iclensis in equl prts (1 : 1 frction) in cultured prostte cncer cells. Glycyrrhiz urlensis, R. ruescens nd S. iclensis re lso found in TCM prescriptions [24] nd PC-SPES formultions [12]. Rdosi ruescens nd S. iclensis hve een reported to reduce cultured cncer cell viility in numer of cell lines (Hep-G2, MCF-7, MCF-1A) [25, 26].

6 6 Evidence-Bsed Complementry nd Alterntive Medicine Cell numer Cell numer Cell numer Cell numer Cell numer Cell numer Cell numer Cell numer 912 Control 151 Control 136 Control h h h DNA content DNA content DNA content LE h f1/ f1/ f1/ h h h f1/ f1/ f1/ h h h f1/ h GE 24 h GE 48 h LE 48 h f1/2 48 h f2/ f2/ h h GE 72 h LE 72 h f1/2 72 h f2/3 72 h f4/ f4/ f4/ h h h Figure 4: DNA cell-cycle histogrms of Hep-G2 cells. Cells were treted with GE, LE nd five frctions (1/5, 1/3, 1/2, 2/3, 4/5) for 24, 48 nd 72 h, respectively, t the respective LC5s. Untreted cells cted s controls. Cells were fixed in 7% ethnol nd stined with PI s descried in the Methods section. DNA histogrms shown re representtives of the ssy repeted in three independent experiments with similr results.

7 Evidence-Bsed Complementry nd Alterntive Medicine 7 Tle 2: Cell-cycle nlysis. Cell cycle Su G1 G1 S G2/M Time (h) 24 Control.46 ± ± ± ±.83 GE 2.5 ± ± ± ± 3.39 LE.79 ± ± 2.5 c 9.54 ± ± 1.76 f1/5.46 ± ± 1.5 c 9.74 ± ± 2.8 f1/3.26 ± ± 2.1 c 8.4 ± ± 2.5 f1/2.25 ± ± 2.59 c 8.18 ± ± 3.1 f2/3.27 ± ± 3.55 c 8.54 ± ± 2.85 f4/5.31 ± ± 4.24 c 7.75 ± ± 3.73 Time (h) 48 Control.4 ± ± ± ± 3.47 GE.97 ± ± ± ± 1.47 LE.67 ± ±.65 c 8.88 ± ± 1.63 f1/5.32 ± ±.73 cd 6.72 ± ± 2.86 f1/3.31 ± ± 1.56 cd 6.27 ± ± 1.63 f1/2.26 ± ± 1.76 d 5.76 ± ± 2.89 f2/3.29 ± ± 1.77 d 5.8 ± ±.96 f4/5.35 ± ± 2.11 cd 5.36 ± ± 1.19 Time (h) 72 Control.54 ± ± ± ± 2.14 c GE 1.14 ±.27 c ±.61 c 6.1 ± ± 2.83 c LE 1.4 ±.11 c ±.62 c 8.71 ± ±.63 f1/5.69 ±.23 c 8.68 ±.24 c 6.8 ± ± 2.55 f1/3.73 ±.16 c ±.62 c 5.95 ± ± 2.36 f1/2.63 ± ± ± ± 1.44 f2/3.45 ± ± ± ± 2.12 f4/5.42 ± ± ± ± 1.39 Columns of the sme ctegory nd time period with different letters re significntly different (P <.5). In this study, ginseng extrct incresed the relese of LDH t the LC5 concentrtion over time with significnt (P <.5) increse t 48 nd 72 h of exposure, wheres licorice extrct did not. Ginseng extrct lone hs een shown to e effective in permeting the cellulr memrne of intestinl cells (Int-47, cco-2 cells) [18] therey relesing LDH from the cytoplsm possily through n interction with memrne cholesterol [27]. Notly, when the proportions of ginseng to licorice contined t lest 5% ginseng (f1/2, f2/3, f4/5) significnt (P <.5) increse in LDH relese ws oserved t 72 h for ginseng nd licorice comintions. Both ginseng nd licorice extrcts did not produce ny sustntil ccumultion of su G1 cells t the LC5 concentrtion tested. With the exception of GE t 24 h, nlysis of the G1 phse of the cell cycle indicted tht there ws significnt (P <.5) cell-cycle rrest for ll treted cells s compred to the control t 24 to 72 h. In ddition, significnt (P <.5) reduction in cell percentges t the G2/M phse ws seen t 72 h. G1 cell-cycle rrest dt re in greement with the reported literture, licorice extrct hve een reported to inhiit cell prolifertion, nd induce G1 phse rrest in n estrogen sensitive rest cncer cell line (MCF-7) [28] nd ffect viility in cultured prostte cncer cells (LNCP) [11]. One possile explntion of these ntgonistic effects on cell viility is tht the ctive compounds in ginseng nd licorice extrcts my compete for the sme cellulr receptor. In the ginseng extrct, ginsenoside Re ws most undnt sponin t 2.2% dry weight, followed y ginsenoside R1 (8.8%), Rg1 (1.5%) nd Rd (.7%). Ginsenosides such s Rg1, protopnxtriol type dmmrne sponin tht includes structurlly relted ginsenoside Re, hs een reported to e functionl lignd of the glucocorticoid receptor [29] nd olenne-type sponins similr to glycyrrhizic cid determined to e 7.1% in the extrct hve een compred to dexmethsone, synthetic glucocorticoid in cell culture experiments [3]. An undnce of ctive compounds would sturte receptor site leding to lower thn expected cellulr response thn predicted. Alterntively, licorice under specific conditions cn exhiit hepto-protection. For exmple, glycyrrhizin, structurl relted to glycyrrhizic cid hs een reported to llevite intrperitonel induced liver injury y cron tetrchloride (CCl 4 ) in mice y down regultion of pro-inflmmtory mrkers [31]. Pre-tretment of cultured heptocytes with licorice or glycyrrhizic cid protected ginst zthioprine injury through incresing the intrcellulr glutthione levels.

8 8 Evidence-Bsed Complementry nd Alterntive Medicine LDH ctivity (μmol/min/l) Control GE LE f1/5 f1/3 f1/2 f2/3 f1/5 Smples 24 hours 48 hours 72 hours Figure 5: The effect of GE, LE nd five frctions (1/5, 1/3, 1/2, 2/3, 4/5) on LDH ctivity. Cells were treted with different extrcts for 24, 48 nd 72 h t their respective LC5 concentrtion determined from 72-h MTT ssy. Results re expressed s men ± SD with three replictes repeted in three seprte experiments. Untreted cells cted s controls. Brs with different letters re significntly different (P <.5). Nkmur et l. reported tht glycyrrhizin cused dosedependent inhiition of LDH lekge cused y CCl 4 in primry rt heptocytes [32]. This protective effect ws evident in this study s frctions tht contin more licorice thn ginseng reduced the cytotoxic effect nd memrne permetion of ginseng. Licorice my protect ginst ginseng cytotoxicity nd thus leding to the oserved ntgonism nd increse in heptocyte survivl. Comintions of ginseng to licorice extrct using fix frction isoologrphic nlysis were found to e ntgonistic rther then synergistic nd reduced the cytotoxicity compred to the individul extrcts. Furthermore, licorice extrct ws found to reduce ginseng extrct memrne permetion properties. Further reserch is needed to determine the precise cellulr trigger nd if these oserved effects re cell specific. Funding Ministry of Eduction, Singpore; Ntionl University of Singpore (NUS) (Grnt R ). References [1] J.ShindF.Z.Chu,The ABC of Trditionl Chinese Medicine, Hi Feng, Hong Kong, 2nd edition, [2] P. M. Brnes, E. Powell-Griner, K. McFnn, nd R. L. Nhin, Complementry nd lterntive medicine use mong dults: United Sttes, 22, Advnce Dt, vol. 343, pp. 1 19, 24. [3] W.T.Chung,S.H.Lee,J.D.Kimetl., Effect of the extrcts from Glycyrrhiz urlensis Fisch on the growth chrcteristics of humn cell lines: nti-tumor nd immune ctivtion ctivities, Cytotechnology, vol. 37, no. 1, pp , 21. [4] Z. Y. Wng nd D. W. 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