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Phytomedicine 18 (2010) 2–10

Contents lists available at ScienceDirect

Phytomedicine

journa l homepage: www.e lsev ier .de /phymed

reparative chromatography of flavonoids and saponins in Gynostemmaentaphyllum and their antiproliferation effect on hepatoma cell

.C. Tsaia, C.L. Linb, B.H. Chena,c,∗

Department of Food Science, Fu Jen University, Taipei 242, TaiwanDepartment of Endocrinology and Metabolism, Cathay General Hospital, Taipei 106, TaiwanGraduate Institute of Medicine, Fu Jen University, Taipei 242, Taiwan

r t i c l e i n f o

eywords:ynostemma pentaphyllumlavonoidsaponinsPLC–MSepatoma cell

a b s t r a c t

A preparative column chromatographic method was developed to isolate flavonoids and saponins fromGynostemma pentaphyllum, a Chinese Medicinal herb, and evaluate their antiproliferation effect on hep-atoma cell Hep3B, with the standards rutin and ginsenoside Rb3 being used for comparison. Initially thepowdered G. pentaphyllum was extracted with ethanol, followed by eluting flavonoids and saponins withethanol–water (30:70, v/v) and 100% ethanol, respectively, in an open-column containing 5 g of Cosmosil

75C18-OPN, and then subjected to HPLC–MS analysis. The flavonoid fraction was mainly composed ofquercetin- and kaempferol-glycosides, while in saponin fraction, both ginsenoside Rb3 and ginsenosideRd dominated. Both fractions were more effective against Hep3B cells than the standards rutin and gin-senoside Rb3, with the cell cycle being arrested at G0/G1 phase for all the treatments. Additionally, theinhibition effect followed a dose-dependent increase for all the sample treatments. The result of thisstudy may be used as a basis for possible phytopreparations in the future with G. pentaphyllum as raw material.

ntroduction

Gynostemma pentaphyllum (Thunb.) Makino, a traditional Chi-ese herb containing many biologically active phytochemicals, haseen demonstrated to be effective against chronic diseases suchs cancer (Guan et al. 2006; Chen et al. 2009; Schild et al. 2010).he most abundant phytochemicals in G. pentaphyllum includedaponins, flavonoids, carotenoids and chlorophylls (Kao et al. 2008;uang et al. 2008), all of which were believed to be responsible for

he health-enhancing effect. However, the antiproliferation effectf saponins or flavonoids prepared from G. pentaphyllum on cancerell was less investigated.

Saponins, a class of glycosidic compounds widely distributedn plants, are mainly composed of sapogenin, aglycons and hex-ses or galacturonic acid. Sapogenins are divided into three classes:

teroidal saponins, N-containing steroidal-saponins and triter-enoid saponins. It has been well documented that most saponins

n G. pentaphyllum belong to the class of triterpenoid saponins, aammarane-type saponin (Razmovski-Naumovski et al. 2005). Theammarane-type saponins were reported to be efficient in inhibit-

∗ Corresponding author at: Department of Food Science, Fu Jen University, Taipei42, Taiwan. Tel.: +886 2 29053626; fax: +886 2 29093271.

E-mail address: 002622@mail.fju.edu.tw (B.H. Chen).

944-7113/$ – see front matter © 2010 Elsevier GmbH. All rights reserved.oi:10.1016/j.phymed.2010.09.004

© 2010 Elsevier GmbH. All rights reserved.

ing growth of liver, lung and skin cancer cells without affectinggrowth of normal cells (Zhang and Yuan 2008). In a similar studyLu et al. (2008) pointed out that the G. pentaphyllum saponinscould induce G0/G1 arrest and apoptosis of lung cancer cell lineA549 through enhanced expression of Bax, caspase-3 and caspase-9, as well as decreased expression of Bcl-2. Similar outcome wasobserved for the human tongue cancer cell SCC-4, which wasarrested at G0/G1 phase during apoptosis as a result of increasedexpression of Bax and caspase-3 and declined expression of Bcl-2(Chen et al. 2009).

Flavonoids belong to a major class of polyphenolic compoundswidely present in plants. More than 6500 flavonoids have beencharacterized in nature, with the varieties and amounts in plantsdepending on growth season, species, environmental condition andmaturity (Jin et al. 2008). Like saponins, most flavonoids are presentin glycosidic form in plant tissue, and can be hydrolyzed to formaglycone and sugar moiety under acidic, thermal or enzymatic con-dition (Kao et al. 2008). It has been well established that flavonoidsare efficient in inhibiting growth of various types of tumor cellsthrough release of cytochrome C from mitochondria to regulateexpression of Bax and Bcl-2 for caspase activation and result in

apoptosis (Tripoli et al. 2007; Ramos 2008). Nair et al. (2004)reported that the growth of prostate cancer cell lines PC-3 and DU-145 could be retarded by quercetin at a dose from 25 to 50 mmol/l.Similarly, flavonoids were effective in inhibiting growth ofesophageal adenocarcinoma cell line OE33 and followed the order:

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uercetin > luteolin > chrysin > kaempferol > apigenin > myricetinZhang et al. 2008).

In view of the impact of saponins and flavonoids to humanealth, this study was undertaken to develop a preparative columnhromatographic method for isolation of saponins and flavonoidsrom G. pentaphyllum, determine their composition by high per-ormance liquid chromatography–mass spectrometry (HPLC–MS)nd evaluate their antiproliferation effect on hepatoma cell, withhe standards rutin and ginsenoside Rb3 being used for comparison.

aterials and methods

aterials

A total of 15 kg of G. pentaphyllum grown in Puli, Taiwan, wasainly composed of leaves and procured from a local drug store in

aipei, Taiwan. All the sample leaves were divided and placed intoeveral bags and sealed under vacuum and stored at −30 ◦C prioro use.

Saponin standards including ginsenoside Rb3 and internal stan-ard protopanaxatriol were purchased from LKT Laboratories (St.aul, MN, USA), while ginsenoside Rd was from ExtrasyntheseGenay, France). Flavonoids standards, including rutin and internaltandard kaempferol were from Sigma (St. Louis, MO, USA), whileaempferol-3-O-rutinoside was from Chromadex (Santa Ana, CA,SA).

The HPLC-grade solvents such as methanol, ethanol, ethylcetate, isopropanol, acetone, hexane, acetonitrile, methylene chlo-ide and toluene were from Lab-Scan (Gliwice, Poland). Both formiccid and hydrochloric acid were from Riedel-de Haën (Seelze, Ger-any). Deionized water was made using a Millipore Milli-Q water

urification system (Bedford, MA, USA).Ascorbic acid, butylated hydroxyanisole (BHA),

thylenediamine-tetraacetic acid (EDTA), sodium nitrite, alu-inum chloride and sodium carbonate were from Nacalai Tesque

Kyoto, Japan). Trichloroacetic acid (TCA), �-tocopherol and 1-etane sulfuric acid sodium salt (PICB) were from Sigma. Iron(III)hloride and potassium ferricyanide were from Showa ChemicalTokyo, Japan). Folin-Ciocalteu reagent and glacial acetic acid wererom Merck (Darmstadt, Germany). Meta-phosphoric acid andodium hydroxide were from Riedel-de Haën.

Adsorbent Cosmosil 75C18-OPN (particle size 75 �m, pore size20 A, pH range 2–7) was from Nacalai Tesque, and ion-exchangeesin Diaion HP-20 (particle size 200–600 �m) was from Mitsubishihemical (Tokyo, Japan).

Hepatoma cell Hep3B was obtained from Food Industryevelopment Institute/National Health Research Institute of Tai-an (Hsinchu, Taiwan). Sodium bicarbonate, dimethyl sulfoxide

DMSO), phosphate buffered saline (PBS), amphotericin B solu-ion (fungizone), thymidine, hypoxanthine, minimum essential

edium alpha modification (�-MEM), propidium iodide (PI), RNase, and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bro-ide (MTT) were from Sigma. Hank’s balanced salt solution (HBSS),

.5% trypsin–EDTA, trypan blue stain and penicillin–streptomycinere from Gibco (CA, USA). Fetal bovine serum (FBS) was fromyclone (Logan, UT, USA).

nstrumentation

The HPLC instrument (Agilent Technologies 1100 series) is com-

osed of a G1311A quaternary pump, a G1312A binary pump, a1315B photodiode-array detector, a G1379A degasser, a G1316Aolumn controller and an Alltech ELSD 800 detector (Deerfield, IL,SA). An Agilent Technologies 6130 quadrupole mass spectrom-ter with multi-mode ion source (ESI and APCI) and a 1200 Cap

cine 18 (2010) 2–10 3

Series 6510 Q-TOF LC/MS/MS were also used. The spectrophotome-ter (DU 640) was from Beckman (Fullerton, CA, USA). The rotaryevaporator (N1) was from Eyela (Tokyo, Japan). The Sorvall RC5Chigh-speed centrifuge was from Du Pont (Wilmington, Delaware,USA). The ultra high-speed centrifuge (5810R) was from Eppen-dorf (Hamburg, Germany). The laminar flow (FD24) was from Baker(Sanford, Maine, USA). The inverted microscope (CK2-VI/CK2-TR)was from Olympus (Tokyo, Japan). The hemacytometer was fromReichert-Jung (New York, USA). The CO2 incubator (TC2323) wasfrom Shel Lab (OR, USA). The ELISA reader (MRS 2CXB-2523) wasfrom Dynatech Laboratories (VA, USA). The flow cytometer (Coul-ter EPICS XL-MCL) was from Beckman. The temperature-controlledwater bath (BU-410) was from Yihder (Taipei, Taiwan).

Extraction of saponins and flavonoids

A method similar to that described by Kao et al. (2008) wasused to extract saponins and flavonoids from G. pentaphyllum. A10 g powder sample of G. pentaphyllum was mixed with 50 ml ofethanol, after which the mixture was shaken at 140 rpm at 60 ◦Cfor 3 h, followed by centrifuging at 6000 rpm (5470 × g) at 25 ◦Cfor 30 min. The supernatant was collected and filtered through aglass filter paper (diameter 110 mm, pore size 6 �m) to obtain crudeextract of saponins and flavonoids, which were stored at −20 ◦C foruse.

Preparation of saponins and flavonoids

Adsorbents Cosmosil 75C18-OPN and Diaion HP-20 were com-pared with respect to separation efficiency of saponins andflavonoids based on several previous reports by Chiang et al. (2004),Kim et al. (2008) and Kang et al. (2009). However, with DiaionHP-20 as adsorbent, both flavonoids and saponins in G. pentaphyl-lum were inadequately separated by employing a solvent systemcontaining different proportions of ethanol and water. Next, a 5-gadsorbent of Cosmosil 75C18-OPN was poured into a glass col-umn (300 mm × 16 mm, I.D.), which was preactivated with 100 mlof ethanol and 100 ml of deionized water. Then 2-ml of crudeextract was evaporated to dryness and dissolved in 1-ml of 0.1%formic acid, added to the column, followed by elution of high-polarimpurities with 60 ml of deionized water, flavonoids with 60 mlof ethanol–water (30:70, v/v) and saponins with 20 ml of 100%ethanol with flow rate at 4 ml/min. Both fractions were collectedseparately, evaporated to dryness under vacuum, dissolved in 5 mlof methanol, filtered through a 0.2-�m membrane filter and 20 �lwas injected for HPLC analysis.

HPLC analysis of saponins

An HPLC method based on Kao et al. (2008) was used to sep-arate the various saponins in saponin fraction prepared from G.pentaphyllum with a Gemini C18 column (250 mm × 4.6 mm I.D.,5 �m particle size) and a gradient mobile phase of 0.1% formic acidsolution (A) and acetonitrile (B) with flow rate at 1 ml/min, anddetection by ELSD. Additionally, the various saponins were identi-fied by comparing retention times and mass spectra of unknownpeaks with authentic standards and those in the literature, aswell as cochromatography with added standards (Kao et al. 2008).For quantitation, 7 concentrations of 10, 20, 40, 60, 80, 100 and125 �g/ml for ginsenoside Rb3 and ginsenoside Rd were preparedseparately, and mixed with internal standard protopanaxatriol

for a final concentration at 50 �g/ml. Both standard curves wereobtained by plotting concentration ratio against its area ratio, andthe regression equations for ginsenoside Rb3 and ginsenoside Rdwere y = 0.7466x − 0.1343 and y = 1.5549x − 0.3165, respectively,with R2 being 0.9953 and 0.9966. Both ginsenoside Rb3 and gin-

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enoside Rd as well as the other saponins were quantified using aormula as described by Kao et al. (2008).

PLC analysis of flavonoids

An HPLC method based on Kao et al. (2008) was used toeparate various flavonoids in flavonoid fraction prepared from. pentaphyllum with a Gemini C18 column (250 mm × 4.6 mm,

.D., particle size 5 �m) and a gradient solvent system of 0.1%ormic acid (A) and methanol (B). The flow rate was 1 ml/minith column temperature at 35 ◦C and detection wavelength at

80 nm. The criteria used for identification of flavonoids werehe same as described by Kao et al. (2008). For quantitation, 6oncentrations of 2, 5, 10, 40, 60 and 80 �g/ml were preparedor rutin and kaempferol-3-O-rutinoside separately, and 0.5, 1.0,.0, 2.5, 5.0 and 8.0 �g/ml for caffeic acid. Each standard solu-ion was then mixed with internal standard kaempferol with

final concentration at 10 �g/ml. The 3 standard curves werebtained by plotting concentration ratio against its area ratio,nd the regression equations for rutin, kaempferol-3-O-rutinosidend caffeic acid were y = 0.503x + 0.0056, y = 0.6811x + 0.0326 and= 1.037x + 0.0067, respectively, with R2 being all higher than 0.99.ll the three flavonoids as well as the other flavonoids were quan-

ified using a formula as described by Kao et al. (2008).

etermination of various functional components in saponin andavonoid fractions

The various functional components in both saponin andavonoid fractions, including gallic acid, caffeic acid, chlorogeniccid, �-coumaric acid, ascorbic acid, �-tocopherol, total flavonoidsnd total phenolic acids, were determined using a method asescribed by Kao and Chen (2006) and Wang et al. (2009).

educing power test

This test aims to verify that the reduction reaction involved inhe subsequent MTT test is due to high cell activity, but not sam-le components. A 250-�l of saponin fraction, flavonoid fraction,scorbic acid, �-tocopherol, BHA and EDTA with concentration of0 �g/ml each was mixed with 250 �l of 0.2 M phosphate bufferedolution (pH 6.6) and 250 �l of 1% potassium ferricyanide sepa-ately, followed by heating in 50 ◦C water bath for 20 min, coolingmmediately, adding 250 �l of 10% trichloroacetic acid, centrifug-ng at 3000 rpm (402 g) at 25 ◦C for 10 min, collecting 300 �l ofupernatant, adding 300 �l of deionized water and 300 �l of 0.1%erric chloride solution, reacting for 10 min and measuring thebsorbance at 700 nm. Both deionized water and sample solventisopropanol) were used as control treatment.

ell culture

Human hepatoma cell line Hep3B was cultured in �-MEMedium (pH 7.4) containing 10% fetal bovine serum (FBS),hich was prepared by mixing 10 g of �-MEM powder, 2.2 g of

odium bicarbonate, 100 ml of FBS, 3.6 ml of 36 �M hypoxan-hine, 3.6 ml of 36 �M thymidine, 5 ml of fungizone and 10 ml ofenicillin–streptomycin, followed by diluting to 1 l with sterilizedater, filtering through a 0.2-�m membrane filter and storing at◦C for use. Cells were incubated under 5% CO2 and humidifiedtmosphere at 37 ◦C, with the medium being replaced every 2 days

o maintain normal cell growth. After growth of cells to a high den-ity at about 80% confluence, the �-MEM medium was removed andells washed with 3 ml of PBS solution, followed by adding 0.5 ml of.125% trypsin-EDTA solution to cover cells uniformly. After incu-ation at 37 ◦C for 1–2 min, cells were observed under microscope

ine 18 (2010) 2–10

and 2-ml of �-MEM medium was added for neutralization to washcells. Then cells were collected and centrifuged at 2000 rpm (805 g)at 4 ◦C for 5 min, followed by removing supernatant, adding 2-mlof �-MEM medium to disperse cell lumps and mixing thoroughly.Next, 0.5 ml of cell suspension was seeded in 10-ml medium forincubation. Likewise, the medium was replaced every 2 days untilcell growth saturation for subsequent incubation.

Cell viability test

This test is intended to verify the cell viability decrease is dueto sample treatment, not overgrowth of cells. Briefly, Hep3B cells(6 × 103) were seeded in a 96-well plate and incubated for 24 h forattachment, replaced with fresh medium and incubated for another72 h. Then cells were collected every 24 h and trypan blue wasadded to count cells with a hemacytometer under microscope.

Sample solvent endurability test

This test aims to elucidate if the solvent, ethanol–water (1:9,v/v), used to dissolve saponin and flavonoid can be harmful toHep3B cell, so that the experimental error can be excluded. Briefly,a 0.2-ml of cell suspension was seeded in a 96-well plate with eachwell containing 6 × 103 cells and subjected to incubation for 24 hfor cell attachment. Then the medium was removed and replacedwith fresh medium containing 1, 2, 3, 4 and 5% of ethanol–water(1:9, v/v), and incubated again for 72 h. The medium was removedagain and 0.2 ml of MTT solution was added and 0.1 ml of DMSO wasadded to each well to dissolve purple crystal, with the absorbancebeing determined at 570 nm with an ELISA reader. Triplicate anal-yses were carried out for each concentration.

Inhibition effect of Hep3B cells by flavonoid and saponin fractionsas well as standards

Both flavonoid and saponin fractions prepared from columnwere collected 5 and 2 times, respectively. After HPLC analy-sis, the contents of flavonoid and saponin were shown to be7412.9 and 13394.1 �g/ml, respectively. By collecting 1350 �l offlavonoid and 746 �l of saponin and mixing with 650 �l and1254 �l of ethanol–water (1:9, v/v) respectively, a concentration of5000 �g/ml was obtained. Similarly, a concentration of 5000 �g/mlwas each prepared for flavonoid standard rutin and saponin stan-dard ginsenoside Rb3 by dissolving 10 mg standard in 2 ml ofethanol–water (1:9, v/v). All the solutions were filtered througha 0.45-�m membrane filter, and 6 doses of 5, 10, 30, 50, 70 and100 �g/ml in �-MEM medium were prepared for each solution. ForMTT test, a 0.2 ml of cell suspension was seeded in a 96-well platewith each well containing 6 × 103 cells. After incubation for 24 h forcell attachment, the medium was removed and replaced with freshones containing 5, 10, 30, 50, 70 and 100 �g/ml for each fractionand standard and incubated for another 72 h. Then the medium wasremoved, washed with PBS 3 times, followed by adding 0.2 ml ofMTT solution to each well, 0.1 ml of DMSO to dissolve crystal andmeasuring the absorbance at 570 nm with an ELISA reader. Bothrelative cell survival rate and IC50 were determined (Wang et al.2009). Triplicate analyses were conducted for each dose.

Cell cycle analysis

Hep3B cells (2 × 106) were seeded in a culture plate (5 ml) and

incubated for 24 h, after which the medium was replaced with freshones containing 10, 50 and 100 �g/ml for each fraction and stan-dard. After 24 h, cells were washed with PBS and trypsin-EDTA wasadded to detach cells for collection. Then cells were centrifugedat 1500 rpm (603 g) at 4 ◦C for 5 min, followed by removing the

Y.C. Tsai et al. / Phytomedicine 18 (2010) 2–10 5

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ig. 1. HPLC chromatograms of saponin and flavonoid fractions isolated by a prepathanol (A, C) and 20 ml of 100% ethanol (B, D), respectively. Peak identification sho

upernatant, washing with PBS 3 times, adding PBS containing0% ethanol slowly and storing at 4 ◦C overnight to fix cells. Next,thanol was removed by centrifuging at 2000 rpm (805 g) at 4 ◦C formin, washed with PBS 3 times, and 0.1 ml of RNase A (100 �g/ml)nd propidium iodide (10 �g/ml) each was added for reaction in a7 ◦C water bath for 30 min. Cells were screened through a nylonesh (40 �m), transferred to a Falcon tube and 0.5 ml of PBS was

dded for cell cycle analysis. Four phases, including sub-G0/G1,0/G1, S and G2/M were automatically calculated by a WinMDI.9 software.

tatistical analysis

All the data were analyzed using SAS software system (SAS009). In addition, the data were subjected to analysis of varianceANOVA) and Duncan’s multiple range test for mean comparisonP < 0.05).

esults and discussion

PLC analysis of saponin and flavonoid fractions

Fig. 1A and B show HPLC chromatograms of flavonoid andaponin fractions with ELSD detection, respectively. Followingdentification criteria described in the method section, no saponineak was detected in Fig. 1A, while 30 saponin peaks detected inig. 1B. Similarly, Fig. 1C and 1D show HPLC chromatograms ofavonoid and saponin fractions with UV detection at 280 nm, withflavonoid peaks being detected in Fig. 1C and no flavonoid peak inig. 1D. It has been reported that a low sensitivity as well as base-

ine noise and drift can occur for saponins with UV detection at 202r 210 nm amid absence of chromophore (Ichikawa et al. 2009).owever, with ELSD detection both baseline noise and drift coulde substantially improved to enhance quantitation accuracy (Kaot al. 2008).

column containing 5 g of Cosmosil 75C18-OPN with a mobile phase of 60 ml of 30%Tables 1 and 2.

Table 1 shows retention time, mass spectra and contents of var-ious saponins in saponin fraction isolated from G. pentaphyllum.A total of 30 saponins were separated within 40 min, in which17 were identified, including gypenoside IV (ginsenoside Rb3) andgypenoside VIII (ginsenoside Rd) as well as their derivatives, withcontents ranging from 0.2 �g/ml (peak 2) to 540.9 �g/ml (peak15). The contents of all the saponins were much lower than thatreported by Kao et al. (2008), which should be caused by the dif-ference in quantitation method, as the former used eluate (ml) asbasis for calculation while the latter used sample weight (g). Table 2shows retention time, UV, MS spectral data and contents of vari-ous flavonoids in flavonoid fraction isolated from G. pentaphyllum.A total of 8 flavonoids were identified, in which kaempferol-rhamno-hexoside was the most abundant (169.5 �g/ml), followedby quercetin-rhamno-hexoside (61.9 �g/ml), rutin (27.8 �g/ml),quercetin-di-(rhamno)-hexoside (20.9 �g/ml), unknown peak(15.4 �g/ml), kaempferol-3-O-rutinoside (9.1 �g/ml) and caffeicacid (2.6 �g/ml). Likewise, the amounts of all the flavonoids weremuch smaller than that reported by Kao et al. (2008) based on thesame reason indicated above.

Functional components in flavonoid and saponin fractions

Both flavonoid and saponin fractions were found to contain totalflavonoids at 37.5 and 4.4 �g/ml, respectively, whereas total phe-nolic compounds were 28.1 and 7.8 �g/ml. For individual phenolicacid, only a level of 1.3 �g/ml of caffeic acid was present in flavonoidfraction, but the other phenolic acids, gallic acid, chlorogenic acidand �-coumaric acid remained undetected in both fractions. Simi-larly, caffeic acid was not detected in saponin fraction.

Reducing power test

The reducing power of both flavonoid and saponin fractions aswell as standards is shown in Fig. 2. As expected, a maximum reduc-

6 Y.C. Tsai et al. / Phytomedicine 18 (2010) 2–10

Table 1Retention time, MS spectral data and contents (�g/mL) of saponin fraction prepared from G. pentaphyllum.

Peak No. Gypenoside tR (min) [M–H]− (On-line) [M–H]− (reported)c Formula (reported)c Content (�g/ml)d

1 Unknown 4.33 995.5 995.4 – 0.4 ± 0.22 Unknown 5.29 995.5 995.4 – 5.5 ± 1.53 Gyp LXI; Gyp LXVIII; Gyp-7; Gyp-3 6.85 1109.5 1109.5 C53H90O24 0.7 ± 0.44 Gyp LXI; Gyp LXVIII; Gyp-7; Gyp-3 7.84 1109.5 1109.5 C53H90O24 0.2 ± 0.15 Unknown 8.24 977.5 977.4 – 0.6 ± 0.16 Gyp-2 8.84 1125.5 1125.5 C53H90O25 1.8 ± 0.37 Gyp-2 9.51 1125.5 1125.5 C53H90O25 1.0 ± 0.38 Unknown 10.65 993.5 993.5 – 1.2 ± 0.69 Unknown 11.48 993.5 993.5 – 2.3 ± 0.510 Gyp-2 - 1125.5 1125.4 C53H90O25 -11 Gyp XLII; Gyp XLVII 17.03 1123.6 1123.5 C54H92O24 2.9 ± 0.112 Gyp LXIII; Gyp-5; Gyp-3; Gyp-4 17.47 1077.5 1077.4 C53H90O22 1.6 ± 1.113 Gyp LXIII; Gyp-5; Gyp-3; Gyp-4 17.81 1077.6 1077.4 C53H90O22 10.4 ± 1.614 Gyp XXII; Gyp LVI; Gyp LXII; Gyp LXVII; Gyp

LXX; Gyp LXXI19.80 1093.6 1093.5 C53H90O23 1.8 ± 0.5

15 Gyp XXII; Gyp LVI; Gyp LXII; Gyp LXVII; GypLXX; Gyp LXXI

20.55 1093.6 1093.5 C53H90O23 540.9 ± 6.0

16 Unknown 22.41 1135.6 1135.5 – 51.3 ± 23.217 Gyp XXIII; Gyp XLIV; Gyp XLVI 24.20 961.5 961.4 C48H82O19 338.4 ± 9.918 Gyp IV (Ginsenoside Rb3)b 24.98 1077.6 1077.5 C53H90O22 68.1 ± 23.619 Unknown 26.83 1003.5 1003.5 – 108.3 ± 33.420 Unknown 28.14 1135.6 1135.5 – 71.9 ± 2.021 Gyp VIII (Ginsenoside Rd)b 29.58 945.4 945.4 C48H82O18 18.9 ± 2.422 Unknown 31.51 1003.5 1003.5 – 95.6 ± 25.323 Unknown 32.20 1135.4 1135.4 – 8.3 ± 2.424 Unknown 32.65 1003.4 1003.4 – 5.7 ± 1.425 Gyp LVII; Gyp LXIV 33.43 931.5 931.5 C47H80O18 12.2 ± 13.026 Unknown 34.31 987.4 987.4 – 13.5 ± 2.927 Gyp XXX; Gyp XXXI; Gyp XXXII; Gyp XXXVIII;

Gyp XXIX(20R); Gyp L; Gyp LI; Gyp LXXIV;Gynos U; Ginsenoside Rf; Gyp-1; Gynoside B

35.20 799.2 799.2 C42H72O14 13.1 ± 8.3

28 Gyp XXX; Gyp XXXI; Gyp XXXII; Gyp XXXVIII;Gyp XXIX(20R); Gyp L; Gyp LI; Gyp LXXIV;Gynos U; Ginsenoside Rf; Gyp-1; Gynoside B

35.97 799.4 799.4 C42H72O14 10.6 ± 9.0

29 Gyp XXI; Gyp LIV; Gyp LXV; Gyp LXXVII;Gynoside A; Gynoside C; Gynoside E

36.87 769.4 769.4 C41H70O13 9.5 ± 4.6

30 Unknown 38.58 673.3 673.3 – 6.6 ± 3.4ISa Protopanaxatriol 39.26 – – –

Gyp, Gypenoside; Gynos, Gynosaponin.a IS: internal standard.b Compound identified by comparison with authentic standards.c Based on a reference by Kao et al. (2008).d Average of triplicate analyses ± standard deviation.

Table 2Retention time, UV, MS spectral data and contents (�g/mL) of flavonoid fraction from G. pentaphyllum.

Peak No. Flavonoid tR (min) �max

(on-line)c�max

(reported)m/z (on-line) m/z (reported) Content (�g/ml)h

1 Caffeic acidb 17.14 296, 322 296, 324d 179 [M–H]− , 135 [M–H–CO2]− 179, 135f 2.6 ± 0.152 Unknown 22.31 278 – 325 [M–H]− – 15.4 ± 2.793 Quercetin-di-(rhamno)-hexoside 25.15 256, 354 256, 354e 755 [M–H]− , 609 [M–H–Rham]− 755, 609g 20.9 ± 0.364 Quercetin-rhamno-hexoside 25.93 256, 354 256, 354e 609 [M–H]− 609g 61.9 ± 5.405 Kaempferol-rhamno-hexoside 28.74 264, 348 264, 348e 593 [M–H]− 593g 62.5 ± 0.426 Kaempferol-rhamno-hexoside 29.24 264, 348 264, 348e 593 [M–H]− 593g 102.9 ± 3.217 Rutinb 30.42 256, 354 256, 354e 609 [M–H]− , 463 [M–H–Rham]− ,

301 [M–H–Rham–Glu]−609, 463, 301g 27.8 ± 5.22

8 Kaempferol-rhamno-hexoside 33.04 264, 348 264, 348e 593 [M–H]− , 447 [M–H–Rham]− ,284 [M–2H–Rham–Hexose]−

593, 447, 284g 4.1 ± 0.28

9 Kaempferol-3-O-rutinosideb 34.02 264, 348 264, 348e 593 [M–H]− , 447 [M–H–Rham]− ,284 [M–2H–Rham–Hexose]−

593, 447, 284g 9.1 ± 2.18

ISa Kaempferol 44.56 264, 366 – – –

a IS: internal standard.b Compound conclusively identified by comparison with authentic standards.c A gradient mobile phase of 0.1% formic acid in water and acetonitrile (from 91:9, v/v to 32:68, v/v) was used.d A gradient mobile phase of 1% acetic acid in water and 1% acetic acid in methanol (from 90:10, v/v to 5:95, v/v) was used by Sun et al. (2007).e A gradient mobile phase of 0.1% formic acid in water and acetonitrile (from 91:9, v/v to 32:68, v/v) was used by Kao et al. (2008).f Based on a reference by Hollecker et al. (2009).g Based on a reference by Kao et al. (2008).h Average of triplicate analyses ± standard deviation.

omedicine 18 (2010) 2–10 7

iVrtnM

C

hntc4flH

AH

aA

Fg

Y.C. Tsai et al. / Phyt

ng power was observed for Vit C (1.10), followed by BHA (0.68) andit E (0.34). Both ETDA and ginsenoside Rb3 standards showed noeducing power, and a low reducing power was found for the otherreatments. This outcome revealed that flavonoids and saponins didot interfere with the reduction reaction involved in subsequentTT test.

ell viability and solvent endurability tests

The cell survival rates during incubation of Hep3B were alligher than 98%, demonstrating even after 72 h incubation, theormal cell growth was maintained. For solvent endurability test,here was no significant difference in Hep3B growth betweenontrol treatment and the other 5 treatments. But, a level of% ethanol–water (1:9, v/v) was selected in practice to dissolveavonoid and saponin fractions to maintain normal growth ofep3B cells.

ntiproliferation effect of saponoin and flavonoid fractions on

ep3B cells

Fig. 3 shows antiproliferation effect of hepatoma cell Hep3B asffected by flavonoid and saponin fractions as well as standards.poor inhibition effect with a relative cell survival rate ranging

ig. 3. Inhibition effect of saponin and flavonoid fractions as well as standards on Hep3Binsenoside Rb3 standard; Ru, rutin standard. Data with different letters are significantly

Fig. 2. Reducing power of saponin and flavonoid fractions from G. pentaphyllum aswell as standards and control. S, saponin fraction; F, flavonoid fraction; Rb3, ginseno-side Rb3 standard; Ru, rutin standard. Data with different letters are significantlydifferent at P < 0.05.

from 62.2 to 93.3% was shown for Hep3B cells for all the treat-ments with a dose from 5 to 30 �g/ml. However, following a risein dose, the inhibition effect showed an increased trend, with aplateau (85.4%) being attained for flavonoid fraction at 100 �g/ml,

followed by saponin fraction (77.6%), rutin (39.3%) and ginseno-side Rb3 (22.1%). Compared to standards, a more pronounced effectwas observed for both flavonoid and saponin fractions in inhibitinggrowth of Hep3B cells, being due to the presence of many func-

cell growth as determined by MTT. S, saponin fraction; F, flavonoid fraction; Rb3,different at P < 0.05.

8 Y.C. Tsai et al. / Phytomedicine 18 (2010) 2–10

F fracts ndard

tHfrCafttraofliaegcM

C

fcmahnsftor

dat

ig. 4. Flow cytometric analysis of Hep3B cells treated with saponin and flavonoidaponin fraction; F, flavonoid fraction; Rb3, ginsenoside Rb3 standard; Ru, rutin sta

ional components in each fraction (Tables 1 and 2). The IC50 ofep3B cells were 47.6, 57.8, 166.4 and 137.4 �g/ml for saponin

raction, flavonoid fraction, ginsenoside Rb3 and rutin, respectively,evealing both fractions to be more effective in antiproliferation.omparatively, flavonoid fraction was less efficient in antiprolifer-tion effect of Hep3B cells than saponin fraction at a dose rangingrom 5 to 70 �g/ml, probably caused by the glycosidic-type struc-ure of the former as shown in Table 2. It has been well documentedhat flavonoids containing sugar moiety were inferior to their cor-esponding aglycones in inhibiting cancer cell growth (Mantheynd Guthrie 2002; Shen et al. 2003; Campbell et al. 2006). Inur experiment the sugar-containing flavonoids dominated in theavonoid fraction should be responsible for this effect. At this stage,

t is difficult to assess the possible synergistic effect of flavonoidsnd saponins in combination, as the verification of real synergisticffects can be achieved through detailed pharmacological investi-ations and by means of controlled clinical studies performed inomparison with synthetic reference drugs (Wagner and Ulrich-erzenich 2009).

ell cycle analysis

Fig. 4 and Table 3 show the effect of saponin and flavonoidractions as well as standards on cell cycle distribution of Hep3Bells after 24 h incubation. With the exception of control treat-ent, the sub-G0/G1 ratio showed an increased trend followingrise in concentration for the other 4 treatments, implying the

igher the concentration, the larger proportion the cells undergoecrosis or apoptosis. At a low dose of 10 �g/ml, all the treatmentshowed a slight difference in sub-G0/G1 ratio. But, the flavonoidraction exhibited a much higher sub-G0/G1 ratio than the otherreatments at a high dose of 100 �g/ml, indicating the proportionf cells to undergo necrosis or apoptosis could be substantially

aised.

A similar tendency was followed at G0/G1 phase, with a slightifference being shown in G0/G1 ratio between control treatmentnd the other four treatments at a low dose (10 �g/ml). However,he difference became more pronounced at a high dose (100 �g/ml),

ions as well as standards. CtlE/H, control of ethanol–water (1:9, v/v) in medium; S,. a, sub-G0/G1; b, G0/G1; c, S; and d, G2/M.

especially for the saponin fraction with a G0/G1 ratio at 69.2%. Asthe G0/G1 ratio followed a dose-dependent increase for all the sam-ple treatments, it may be postulated that the cell cycle of Hep3B tobe more readily arrested at G0/G1 phase under the condition ofelevated concentration.

In contrast to both sub-G0/G1 and G0/G1 ratios, the S ratiodeclined following a raise in concentration, revealing cells to beprevented from entering into DNA synthesis stage for all thesample treatments. Compared to control treatment, most treat-ments showed a lower S ratio, especially for saponin and flavonoidfractions at high dose (100 �g/ml), which equaled 4.7 and 4.8%,respectively. Like S ratio, the G2/M ratio also displayed a dimin-ished trend. However, there was no significant difference in G2/Mratio between 10 and 50 �g/ml for most treatments. At a highdose of 100 �g/ml, a lower G2/M ratio occurred for all the sam-ple treatments, which amounted to 14.3, 15.8, 16.2 and 15.3% forsaponin fraction, flavonoid fraction, ginsenoside Rb3 and rutin,respectively, all of which were significantly lower than the controltreatment.

In contrast to saponins, most studies focused on the effect offlavonoid standards on cell cycle distribution with the cell cyclebeing arrested at G0/G1 or G2/M phase depending on sample vari-ety or cell type. For instance, a concentration of 200 �M quercetinwas required to raise the G0/G1 ratio to 69% for HepG2 cancercell (Ramos et al. 2005), whereas a dose of 50 �M quercetin wasnecessary in raising G0/G1 ratio to 89.62% (Mu et al. 2007). Also,kaempferol-7-O-�-d-glucoside could induce apoptosis of cervicalcancer cell HeLa to be arrested at G2/M phase (Xu et al. 2008).Likewise, the esophageal adenocarcinoma cell OE33 was arrestedat G2/M phase in the presence of flavonoid standards, includingquercetin, luteolin, chrysin, kaempferol, apigenin and myricetin(Zhang et al. 2008). This phenomenon clearly demonstrated theregulation mechanism in cell cycle arrest can be varied depending

on varieties of flavonoid and cell.

All in all, both saponin and flavonoid fractions were moreeffective in antiproliferation of liver cancer cell Hep3B than theircorresponding standards. Finally, we have to point out here thisis only a preliminary experiment to learn about the composition

Y.C. Tsai et al. / Phytomedi

Tab

le3

Effe

cts

ofsa

pon

inan

dfl

avon

oid

frac

tion

sas

wel

las

stan

dar

ds

once

llcy

cle

dis

trib

uti

onof

Hep

3Bce

lls.

a

Trea

tmen

tsu

b-G

0/G

1(%

)G

0/G

1(%

)S

(%)

G2/

M(%

)

10�

g/m

l50

�g/

ml

100

�g/

ml

10�

g/m

l50

�g/

ml

100

�g/

ml

10�

g/m

l50

�g/

ml

100

�g/

ml

10�

g/m

l50

�g/

ml

100

�g/

ml

Ctl

E/H

0.7

±0.

1bA

0.6

±0.

1cA

0.5

±0.

2cA

58.6

±0.

6cA

58.9

±1.

2cA

59.2

±1.

5bA

9.8

±1.

5aA

8.9

±0.

5aA

8.9

±0.

5aA

18.0

±1.

0bA

18.0

±0.

9aA

18.5

±1.

1aA

S1.

1±

0.2a

B1.

6±

0.1a

B3.

1±

0.9b

A59

.1±

1.1b

cC64

.5±

2.4a

B69

.2±

1.8a

A8.

4±

0.2a

A5.

5±

0.9c

B4.

7±

0.5d

B18

.6±

1.3a

bA17

.0±

1.0a

A14

.3±

0.1b

BF

0.9

±0.

3abB

2.0

±0.

4aB

4.7

±1.

2aA

57.8

±0.

9cB

61.6

±1.

9bcB

66.9

±2.

6aA

10.0

±1.

7aA

7.8

±1.

0abA

4.8

±0.

2dB

19.3

±0.

9abA

17.9

±1.

0aA

15.8

±1.

0bB

Rb3

0.7

±0.

1bB

1.1

±0.

1bB

2.2

±0.

3bA

61.9

±0.

9aC

65.0

±0.

9aB

67.3

±0.

9aA

8.9

±0.

4aA

7.4

±0.

5bB

6.2

±0.

6cC

20.3

±0.

6aA

18.3

±0.

4aB

16.2

±0.

8bC

Ru

0.7

±0.

2bB

1.0

±0.

0bcB

3.0

±0.

8bA

60.4

±1.

1abC

64.0

±0.

3abB

66.0

±0.

5aA

8.6

±1.

2aA

8.6

±0.

1abA

7.9

±0.

5bA

19.4

±0.

3abA

18.4

±0.

9aA

15.3

±1.

9bB

aA

vera

geof

du

pli

cate

anal

yses

±st

and

ard

dev

iati

on.S

ymbo

lsbe

arin

gd

iffe

ren

tle

tter

s(a

–d)

inth

esa

me

colu

mn

are

sign

ifica

ntl

yd

iffe

ren

t(P

<0.

05).

Sym

bols

bear

ing

dif

fere

nt

lett

ers

(A–C

)in

the

sam

ero

ww

ith

inea

chp

erio

dof

cell

cycl

ear

esi

gnifi

can

tly

dif

fere

nt

(P<

0.05

).C

tlE/

H,c

ontr

olof

eth

anol

–wat

er(1

:9,v

/v)

inm

ediu

m;

S,sa

pon

infr

acti

on;

F,fl

avon

oid

frac

tion

;R

b3,g

inse

nos

ide

Rb 3

stan

dar

d;

Ru

,ru

tin

stan

dar

d.

cine 18 (2010) 2–10 9

of functional components in saponin and flavonoid fractions iso-lated from G. pentaphyllum and illustrate their inhibition effect onhepatoma cell growth. Further research is necessary to elucidatethe antiproliferation mechanism of hepatoma cell as affected bysaponin and flavonoid fractions from G. pentaphyllum.

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