research article triterpenoid saponins isolated from ...triterpenoid saponins isolated from...

Hindawi Publishing CorporationEvidence-Based Complementary and Alternative MedicineVolume 2013 Article ID 560417 11 pageshttpdxdoiorg1011552013560417

Research ArticleTriterpenoid Saponins Isolated from Platycodon grandiflorumInhibit Hepatitis C Virus Replication

Jong-Woo Kim12 Sang Jin Park1 Jong Hwan Lim1 Jae Won Yang1 Jung Cheul Shin1

Sang Wook Lee1 Joo Won Suh2 and Soon B Hwang3

1 BampC Biopharm Advanced Institutes of Convergence Technology Suwon 443-270 Republic of Korea2Division of Bioscience and Bioinformatics Myongji University Cheoin-gu Yongin 449-728 Republic of Korea3 National Research Laboratory of Hepatitis C Virus Ilsong Institute of Life Science Hallym University 1605-4 Gwanyang-dongDongan-gu Anyang 431-060 Republic of Korea

Correspondence should be addressed to Joo Won Suh jwsuhmjuackr and Soon B Hwang sbhwanghallymackr

Received 22 May 2013 Accepted 9 September 2013

Academic Editor Roman Huber

Copyright copy 2013 Jong-Woo Kim et alThis is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Hepatitis C virus (HCV) infection is a major cause of liver disease including cirrhosis and hepatocellular carcinoma Due tosignificant adverse effects and emergence of resistant strains of currently developed anti-HCV agents plant extracts have beenconsidered to be potential sources of new bioactive compounds against HCV The aim of this study was to evaluate the functionaleffects of triterpenoid saponins contained in the root extract of Platycodon grandiflorum (PG) on viral enzyme activities andreplication in both HCV replicon cells and cell culture grown HCV- (HCVcc-) infected cells Inhibitory activities of triterpenoidsaponins fromPGwere verified byNS5BRNA-dependent RNApolymerase assay andwere further confirmed in the context ofHCVreplication Six triterpenoid saponins (platycodin D platycodin D

2 platycodin D

3 deapioplatycodin D deapioplatycodin D

2 and

platyconic acid A) PG saponin mixture (PGSM) were identified as active components exerting anti-HCV activity ImportantlyPGSM exerted synergistic anti-HCV activity in combination with either interferon-120572 or NS5A inhibitors We demonstrated thatcombinatorial treatment of PGSM and IFN-120572 efficiently suppressed colony formation with significant reduction in drug resistantvariant of HCVThese data suggest that triterpenoid saponin may represent a novel anti-HCV therapeutic agent

1 Introduction

Hepatitis C virus (HCV) currently affects nearly 170 millionpeople worldwide and 3-4 million people are newly infectedeach yearThemajority of these individualswill be chronicallyinfected andmay lead to fibrosis cirrhosis and hepatocellularcarcinoma [1] HCV is a positive-sense single-stranded RNAgenome of sim96 kb The HCV genome encodes a 3010-amino-acid protein from a single open reading frame Thispolyprotein is processed into structural (core E1 and E2)and nonstructural proteins (p7 NS2-NS5B) Nonstructuralproteins have been attractive to be targets for developinganti-HCV therapy [2ndash4] There is no vaccine available forHCV yet The current standard therapy for chronic HCVa combination of pegylated interferon- (PEG-IFN-) 120572 andribavirin (RBV) is effective in approximately 70ndash80 ofpatients with HCV genotype 2 or 3 infection but effective

in less than 50 of those with HCV genotype 1 [5] Fur-thermore therapy with PEG-IFN and ribavirin has seriousside effects including flu-like symptoms hemolytic anemiaand depression which often lead to the discontinuance oftherapy [6] Recently two HCV NS34A protease inhibitorsboceprevir (Victrelis) and telaprevir (Incivek) are approvedby the US Food and Drug Administration (FDA) Howevertherapeutic strategy using viral proteins is not fully successfuldue to error prone nature of HCV RNA replication Forthese reasons there is an urgent need to develop additionaltherapies that are less toxic and inexpensive and resultin higher sustained virological response (SVR) either ascombination or replacement therapies Discovery of a potentdrug candidate from natural products would be useful inovercoming side effects and in generating more synergisticactivity

2 Evidence-Based Complementary and Alternative Medicine

Platycodi Radix is the root of Platycodon grandiflorum(PG) This plant grows wild in east Asian countries andis reported to contain significant amounts of carbohydrateprotein lipid and ash Additionally PG contains variouskinds of triterpenoid saponins Much attention from phar-maceutical industry has been paid to PG due to its variousefficacies on diseases PG extract has been used as a foodadditive and oriental herb medicine to cure various diseasesincluding bronchitis asthma and pulmonary tuberculosisand inflammatory diseases It has been also reported thatPlatycodi Radix stimulates antioxidants immunostimula-tion and antitumor activity and prevents hyperlipidaemiaand obesity [7ndash9] Moreover it has been shown to improvethe immunogenicity of virus vaccines in mice by enhancingboth humoral and cellular immune responses [10 11] Inter-estingly the extracts from PG prevented chemical-inducedhepatotoxicity [12ndash14] Nevertheless the inhibitory effect oftriterpenoid saponin extract fromPGonHCV replication hasnever been explored thus far

The present study investigated the suppressive activity ofroot extract from PG against HCV replication using Huh7cells harboring HCV genotype 1b subgenomic replicon Theactive fraction pooled from Platycodon grandiflorum saponinmixture (PGSM)was found to have potent anti-HCV activityWe identified 6 triterpenoid saponins (PD PD

2 PD3 DPD

DPD2 and PA) as active components exerting inhibitory

activity againstHCVreplication in subgenomic replicon cellsWe further verified antiviral activity of these compoundsusing RNA-dependent RNA polymerase (RdRp) assay Fur-thermore antiviral properties and synergistic effects oftriterpenoid saponins on interferon and other direct actingantiviral (DAA) drugs were verified in HCV replicon cells

2 Materials and Methods

21 Preparation of Crude Extract and Various FractionsPlatycodon grandiflorum was cultivated for three years inGyeongbuk Province South Korea One kg of dried rootsof Platycodon grandiflorum was cut into slices and rootslices were extracted using 5 L of distilled water at 90∘Cfor 6 h filtered and concentrated under reduced pressureto yield a PG extract (85 g) The powder of PG extract wasdissolved in distilled water and then subjected to a reversephase C

18 The sample was serially eluted with water 3ndash5

acetonitrile 10 methanol 30 methanol 50 methanol70 methanol and 100 methanol The sample isolated in50 methanol fraction contained triterpenoid saponins andexerted the highest anti-HCV activity and was designated asPGSM PGSMwas further purified by using preparative high-performance liquid chromatography (HPLC) as reportedpreviously [15]

22 Analysis of PGSM by HPLCELSD and LCMSD HPLCanalysis of PGSM was performed on an Agilent 1100 seriesHPLC (USA) equipped with a Sedex 55 evaporative lightscattering detector (ELSD SEDERE Alfortville France) Asample was separated in a Gemini C

18column (100mm times

46mm 3120583m particle size Phenomenex Torrance CA

USA) with a precolumn (C18 35 120583m 2 times 20mm) at room

temperature The mobile phase consisted of 01 formicacidmethanolacetonitrile (75 5 20 vvv A) and 01formic acidmethanolacetonitrile (70 5 25 vvv B) andgradient runs were programed as follows 0ndash10min in theabsence of B 10ndash17min (0ndash50 B) 25ndash34min (50ndash80 B)42ndash52min (100 B) and then equilibration in the absence ofB for 10min at a flow rate of 1 mLmin The injection volumewas 20120583LThe ELSD was set to a probe temperature of 70∘Ca gain of 7 and the nebulizer gas nitrogen adjusted to 25 bar

The electrospray ionization-mass spectrometric (ESI-MS) analysis was performed on an Agilent 5989 massspectrometer with an ESI interface fitted with a hexapoleion guide Chromatographic separation of the PGSM wasperformed as described above The optimal condition forthe analysis of triterpenoids employed pneumatic nebuliza-tion with nitrogen (45 psi) and a counterflow of nitrogen(9 Lmin) heated to 350∘C for the nebulization and des-olvation of the introduced liquid Mass spectrometry wasperformed using the negative ion mode and the scan modethis process detected from 100 to 2000mz with a dwell timeof 300ms

23 RdRp Assay Recombinant HCV NS5B polymerasefrom HCV genotype 1b carrying an N-terminal GST-tagand C-terminal 21-amino acid truncation (NS5BCΔ21) wasexpressed in Escherichia coli and purified as previouslydescribed with some modifications [16] The colorimetricreverse transcriptase assay kit (Roche Applied Science) wasapplied to perform RdRp assayThe inhibition of HCV RdRpactivity was determined by the amount of double-strandedRNA synthesized by the recombinant NS5BΔC21 in thepresence of various test compounds using HCV 31015840-UTR asa template

24 HCV NS34A Protease Assay The NS34A serine pro-tease assay was performed using a HCV protease assaykit (SensoLyte AnaSpec Fremont CA) according to themanufacturerrsquos instructions Briefly HCV-NS34A proteasepurified from E coli was mixed with the test compound inthe assay buffer After 15min incubation at room tempera-ture EDANSDABCYL-based fluorescence resonance energytransfer (FRET) peptide substrate solution was added andmixed The FRET substrate was cleaved specifically by HCVNS34A protease thereby liberating the C-terminal peptide-fluorophore fragment from the proximity quenching effectof the dark quencher resulting in increase of fluorescenceThe fluorescence intensity was measured immediately andcontinuously at excitationemission at 340 nm and 490 nm

25 In Vitro Anti-HCV Assay in HCV Replicon Cells Huh7cells harboring HCV subgenomic replicon (genotype 1b)[17] were maintained in the presence of 025mgmL G418(Invitrogen Carlsbad CA) HCV replicon cells were seededat a density of 1 times 104 cellswell in a 96-well plate andincubated at 37∘C and 5 CO

2 Following 24 h incubation

the culture medium was replaced with a medium containingserially diluted test compounds in the presence of 2FBS and

Evidence-Based Complementary and Alternative Medicine 3

1 DMSO After cells were treated for 72 h with PGMS andtriterpenoid saponins total RNAswere extracted using a Cel-lAmpDirect RNAPrepKit (Korea Biomedical Seoul Korea)The HCV RNA levels were quantified by a quantitative real-time polymerase chain reaction (qRT-PCR) assay using IQ5real-time PCR detection system (Bio-Rad Hercules CAUSA) with HCV-specific primers (51015840-GAC ACT CCA CCATAG ATC ACT C-31015840 and 51015840-CCC AAC ACT ACT CGGCTA G-31015840) and probe (51015840-FAM-CCC AAA TCT CCA GGCATT GAG CGG-31015840 BHQ-1) Results were normalized toglyceraldehyde-3-phosphate dehydrogenase gene (GAPDH)Anti-HCV activity was determined by HCV RNA levels incompound-treated cells as compared to mock-treated cells

26 In Vitro Anti-HCV Assay in HCV-Infected Cells Infec-tious JFH1 viruses (HCV genotype 2a) were generated asdescribed previously [18] Huh7 cells were seeded in 24-wellplates at a density of 5times 104 cells per well At 24 h after platingcells were infectedwith JFH1 viruses for 2 h After incubationthe supernatants were replaced with fresh medium Increas-ing concentrations of PGMS and its triterpenoid saponinswere added in a medium containing 2 DMSO and cellswere incubated at 37∘C At 48 h after infection total RNAswere extracted and the HCV RNA levels were quantified byqRT-PCR assay Anti-HCV activity was determined by HCVRNA levels in compound-treated cells as compared to mock-treated cells

27 Western Blotting Total cell lysates were separated bySDS-PAGE and electrotransferred to a PVDF membrane(Bio-Rad)Themembrane was blocked with 5 skimmilk inTris-buffered saline (TBS) and incubated with an anti-HCVNS5A monoclonal antibody (Santa Cruz Biotechnology)Proteins were detected using an enhanced chemilumines-cence detection system (Intron Biotechnology)

28 Cell Viability Assay Cell viability was determined bythe colorimetric 3-(45-dimethylthiazol-2-yl)-25-diphenyl-tetrazolium bromide (MTT) reagent (Promega Corporation)according to themanufacturerrsquos instructionsThe absorbancewas detected at 570 nm using a Bio-Tek plate reader (Bio-Tekinstrument Weymouth MA)

29 In Vitro Combination Studies HCV Replicon cells weretreated with various concentrations of inhibitors in twofoldserial dilutions at a final dimethyl sulfoxide concentrationof 1 including titrations of each of the compounds aloneas reference controls The concentration used for PGSMranged from 5 ugmL to 016 ugmL for interferon-120572 theconcentration used ranged from 80UmL to 25UmL theconcentration used for R7227 ranged from 25 ngmL to008 ngmL and for BMS 790052 the concentration usedranged from 20 pgmL to 063 pgmL At three days aftertreatments intracellular HCV RNA levels were determinedby qRT-PCR For the Loewe additivemodel the experimentaldata were analyzed by using CalcuSyn (Biosoft FergusonMo) a computer program based on the method of Chouand Talalay [19 20] A combination index (CI) value for each

experimental combination was calculated by a quantitativemeasure of the degree of drug interaction in terms of additiveeffect (CI = 1) synergism (CI lt 1) or antagonism (CI gt 1) fora given endpoint of the effect measurement

210 Anti-HCV Assay for 14 Days HCV replicon cells wereplated at 2 times 105 cells per well in a 6-well plate Eachcompound was serially diluted in DMEM containing 10FBS and 02 DMSO as reported previously [21 22] Culturemedia were replaced with fresh medium and compoundsevery 3 days Cells were harvested at 3 6 9 and 14 daysand intracellular HCV RNA levels were analyzed by qRT-PCR The relative copy number of HCV RNA per cell wascalculated by comparing RNAs in cells treated with eachcompoundwith thoseRNAs in cells treatedwith 02DMSO

211 Colony Forming Assays Huh7 cells harboring HCVsubgenomic replicon (genotype 1b) were treated with eithervarious concentrations of PGSM alone or in combinationwith IFN-120572 BMS790052 (NS5Ai) and VX-950 (PI) respec-tively G418 was included in the media to provide selectivepressure on HCV replicon cells After three weeks of treat-ment surviving cells were stained with crystal violet [23]HCV RNAs isolated from colonies were also subjected tosequence analysis

3 Results

31 Identification of Active Components Bearing Anti-HCVActivity in the Root of PG PG-extract exerted anti-HCVactivity in a dose-dependent manner in HCV subgenomicreplicon cells The half maximal effective concentration(EC50) of PGSM to inhibit HCV replication was 35 120583gmL

PG-extract was further fractioned by reverse phase C18open

column chromatography and eluted with water (341 g) 3ndash5 acetonitrile (052 g) 10methanol (128 g) 30methanol(212 g) 50 methanol (022 g) 70 methanol (007 g)and 100 methanol (004 g) respectively (data not shown)Based on bioactivity-guided screening HCV subgenomicreplicon (genotype 1b) cells were used to assess the anti-HCV activities of various fractions isolated from PG-extractHCV replicon cells were treated with various fractions usingfixed concentration of 10120583gmL for three days The fractioneluted with 50 methanol (PGSM) showed a potent anti-HCV activity (EC

50= 078120583gmL) as compared to other

fractions To further verify the anti-HCV activity HCVreplicon cells were incubated with various concentrationsof PGSM Cell lysates harvested at two days after PGSMtreatment were immunoblotted with anti-NS5A antibody Asshown in Figure 1(a)HCVprotein expressionwas completelyinhibited by 5120583g of PGSMWe demonstrated that intracellu-lar HCV RNA levels were significantly reduced by treatmentof 2120583g of PGSM (Figure 1(b))We also showed that treatmentof HCV replicon cells with PGSM at concentrations as highas 5 120583gmL induced no cell toxicity as measured by anMTT assay (Figure 1(b)) HPLCELSD and LCMS analysisdata showed that PGSM was found in triterpenoid saponin-rich fraction (Figure 2) Triterpenoid saponin-rich fraction


Con 1 5 10

NS5A

120573-actin

PGSM (120583gmL)

(a)

120

100

80

60

40

20

0

120

140

100

80

60

40

20

0

Relat

ive H

CV R

NA

( o

f con

trol)

Cel

l via

bilit

y (

of c

ontro

l)0 1 2 3 4 5 6

PGSM concentration (120583gmL)

(b)

Figure 1 Effect of PGSM on HCV protein expression and RNAreplication in HCV replicon cells Huh7 cells harboring HCVreplicon were treated with different concentrations of PGSM for3 days 1 DMSO was used as a control (vehicle) (a) Total celllysates were immunoblotted with an anti-HCV NS5A antibody andanti-actin antibody respectively (b) Total RNAs were extractedfrom cells at 72 h after PGSM treatment and intracellular HCVRNAs were quantified by qPCR Relative HCV RNA levels werenormalized by cellular GAPDH mRNA Cell viability was assessedby the MTT assay

0 10 20 30 40 50(min)

(mV

)

350030002500200015001000

5000

1

2

3

45 6 7

8

910

1112 13

Figure 2 Representative HPLCELSD chromatograms of triter-penoid saponins in PGSM The numbers indicate each triterpenoidsaponin 1 deapi-platycoside E 2 platycoside E 3 deapi-platycodinD3 4 platycodin D

3 5 platyconic acid A 6 deapi-platycodin

D 7 platycodin D2 8 platycodin D 9 polygalacin D 10 310158401015840-O-

acetylpolygalacin D 11 platycodin A 12 deapio-310158401015840-acetyl poly-galacin D 13 210158401015840-O-acetyl platycodin D

was further fractionated into six groups of triterpenoidsaponin including platycodin D (PD) platycodin D

2(PD2)

platycodin D3(PD3) deapioplatycodin D (DPD) deapio-

platycodin D2(DPD

2) and platyconic acid (PA) (Figure 3)

The purities were ge95 for the different saponin compoundsThe structure of each compound was analyzed by IR NMRand MS

32 Triterpenoid Saponin Extracts Exerted Anti-RdRpActivity To investigate whether triterpenoid saponins

had anti-HCV activity RdRp assay was performed usingrecombinant HCV NS5B protein and HCV 31015840 UTR asa RNA template As shown in Figure 4(a) N-terminallytruncated (21 aa deletion) NS5B protein was expressed as aGST-tagged protein and further purified as approximately87 kDa This protein was verified by immunoblot analysisusing anti-HCV NS5B antibody (Figure 4(b)) We showedthat all triterpenoid saponins including PGSM PD PD

2

PD3 DPD DPD

2 and PA inhibited RdRp activity and

inhibition occurred in a dose-dependent manner with IC50

value of 5120583gmL 5120583gmL 6120583gmL 8 120583gmL 7120583gmL10 120583gmL and 15120583gmL respectively (Table 1) Howeverthese triterpenoid a sponins showed no inhibitory activitieson HCV NS34A protease in FRET assays (data not shown)

33 Triterpenoid Saponins Suppress RNA Replication in HCVReplicon Cells and in HCV-Infected Cells To further verifythe antiviral activity of triterpenoid saponins in the contextof HCV replication the effect of triterpenoid saponins onHCV RNA replication was assessed in both Huh7 cellsharboring HCV subgenomic replicon and HCVcc-infectedHuh7 cells As shown in Table 2 PGSM and its subfractionsof triterpenoid saponins inhibited HCV RNA replicationin HCV replicon cells and in HCVcc-infected cells Thisinhibition occurred in a dose-dependent manner (data notshown) The EC

50values of triterpenoid saponins for RNA

replication in HCV subgenomic replicon cells ranged from035 to 245 120583gmL It was noteworthy that the EC

50values

of HCVcc-infected cells were approximately ten times higherthan those of HCV replicon cells (Table 2) We furthershowed that CC

50values of the triterpenoid saponins in

Huh7 cells were very high as compared to EC50

valueswhich indicate that these compounds were not cytotoxic atinhibitory concentrations To further investigate the effectof triterpenoid saponins on HCV protein expression levelHCV replicon cells were treated with the indicated amountsof triterpenoid saponins and HCV protein expression wasdetected by immunoblotting with anti-NS5A antibody Asdemonstrated in Figure 5 triterpenoid saponins prominentlyinhibited the HCV protein expression level Of note PD

2

and PD3appear to be more potent than other triterpenoid

saponins in anti-HCV activity

34 Synergistic Effect of PGSM on IFN-120572 and HCV Inhibitor-Mediated Antiviral Activity in HCV Replicon Cells The cur-rent standard of care for the treatment of chronic HCV is thecombination of pegylated IFN-120572 and ribavirin Combinationtherapy with two or three drugs which have different modesof action is regarded as a promising way to enhance SVRmore than 90 and to suppress mutant strains The antiviralactivities of PGSM in combination with either IFN-120572 theHCVNS5A inhibitor Daclatasvir (BMS 790052) [24] and theNS34A protease inhibitor Danoprevir (ITMN-191(R7227))[25]were examined inHCVsubgenomic replicon cells Repli-con cells were incubated with PGSM in combination withIFN-120572 BMS-790052 and R7227 at various concentrationsand the anti-HCV activity and cytotoxicity were examinedas described in Materials and Methods The combination


H

HO

OHOH

OH

O

C

HOOC CH2OH

OR2

R1O

Platycogenic acid A

H

HO

O

C

CH2OH

OR2

R1O

Platycodigenin

H

HO

O

C

CH2OH

OR2

R1

R1 R2

O

Polygalacic acid

H3C

HOH2C

2930

2021

2218

19

1716

15141326

12

11251

2

34

5

109

8

76

24

28

Saponins Aglycone

Platycodin D

Platycodigenin

Glc-Lam-Gen-

Deapioplatycodin D Glc-Lam-

Platyconic acid A Platycogenic acid A Glc-

Polygalacin D Polygalacic acid Glc-

Deapioplatycodin D2

Platycodin D2

Platycodin D3

Api-(1rarr3)-Xyl-(1rarr4)-Rha-(1rarr2)-Ara-Api-(1rarr3)-Xyl-(1rarr4)-Rha-(1rarr2)-Ara-Api-(1rarr3)-Xyl-(1rarr4)-Rha-(1rarr2)-Ara-Xyl-(1rarr4)-Rha-(1rarr2)-Ara-Xyl-(1rarr4)-Rha-(1rarr2)-Ara-

Api-(1rarr3)-Xyl-(1rarr4)-Rha-(1rarr2)-Ara-


Figure 3 Chemical structures of triterpenoid saponins from the root of PG Glc 120573-D-glucopyranosyl Lam laminaribosyl Gen gentiobiosylApi 120573-D-apiofuranosyl Xyl 120573-D-xylopyranosyl Rha 120572-L-rhamnopyranosyl Ara 120572-L-arabinofuranosyl

index (CI) values were analyzed using CalcuSyn software toexamine whether the effect of the combination was additiveor synergistic A CI value of 1 indicates an additive effect aCI value of less than 1 indicates a synergistic effect and aCI value of greater than 1 indicates antagonism As shown inTable 3 most CI values were significantly less than 1 (034ndash072) when cells were treatedwith IFN-120572 in combinationwithvarious inhibitorsThese results indicate that combinations ofPGSM with other DAAs have synergistic effect on inhibitionof HCV RNA replication in the replicon cells There was nosignificant increase in cytotoxicity when PGSM was treatedin combination with other DAAs (data not shown)

35 PGSM Potentiates IFN-120572-Mediated Anti-HCV ActivityTo investigate whether PGSM was able to induce a multilogreduction of RNA replication in HCV replicon cells weassessed PGSM activity for 14 days As shown in Figure 6PGSM continuously reduced HCV RNA levels in a time-and concentration-dependent manner At 14 days after treat-ments of PGSM with 02 ugmL 05 ugmL 10 ugmL and20 ugmL reduced HCV RNA levels by 09 log

10 11 log

10

13 log10 and 17 log

10 respectively Likewise treatment of

IFN-120572with 10UmL 50UmL and 100UmL inhibitedHCVRNA levels by 04 log

10 08 log

10 and 15 log

10 respectively

It was noteworthy that combinatorial treatments of PGSM


Table 1 Specification of chemical structure and anti-HCV activities of triterpenoid saponin extracts of Platycodon grandiflorum on NS5BRdRp and NS34A protease functions

Compounds Aglycone Composition of sugar HCV enzyme (IC50 120583gmL)

C-3 C-28 HCVNS5B

HCVNS34A

PG-extract mdash mdash mdash 67 gt100PGSM mdash mdash mdash 5 gt100Platycodin D Platycodigenin Glc- Api-(1rarr 3)-Xyl-(1rarr 4)-Rha-(1rarr 2)-Ara- 5 gt100Platycodin D2 Glc-(1rarr 3)-Glc- Api-(1rarr 3)-Xyl-(1rarr 4)-Rha-(1rarr 2)-Ara- 6 gt100Platycodin D3 Glc-(1rarr 6)-Glc- Api-(1rarr 3)-Xyl-(1rarr 4)-Rha-(1rarr 2)-Ara- 8 gt100Deapioplatycodin D Glc- Xyl-(1rarr 4)-Rha-(1rarr 2)-Ara- 7 gt100Deapioplatycodin D2 Glc-(1rarr 3)-Glc- Xyl-(1rarr 4)-Rha-(1rarr 2)-Ara- 10 gt100Platyconic acid A Platycogenic acid A Glc- Api-(1rarr 3)-Xyl-(1rarr 4)-Rha-(1rarr 2)-Ara- 15 gt100

Table 2 Inhibitory effects of triterpenoid saponins on HCV replication in HCV replicon cells and in HCVcc-infected cells

Compounds Inhibition of HCV RNA replication (EC50 120583gmL) Cytotoxicity (CC50 120583gmL)CON1 (genotype 1b) JHF-1 (genotype 2a)

PG-extract 35 gt100PGSM 078 8 25Platycodin D 035 3 25Platycodin D2 105 12 25Platycodin D3 233 27 100Deapioplatycodin D 12 7 25Deapioplatycodin D2 089 8 100Platyconic acid A 245 22 100

and IFN-120572 resulted in significant reduction of HCV RNAlevels as compared to either PGSMor IFN-120572 alone suggestingthat PGSM may be a potent therapeutic agent for HCV incombination with IFN-120572

36 PGSM Potentiates Colony Suppression in Replicon Cells inCombination with Either IFN-120572 or DAAs To further verifythe antiviral activity of PGSM in HCV replicon cells weperformed HCV colony forming assay using PGSM witheither IFN-120572 or two DAAs BMS790052 (NS5Ai) and VX-950 (PI) As shown in Figure 7 5UmL of IFN-120572 was unableto inhibit colony formation of HCV replicons Howevercotreatment of 1 120583g of PGSM and 5UmL of IFN-120572 efficientlysuppressed the colony formation indicating that PGSMpotentiates IFN-120572-mediated anticolony formation Similaractivities of PGSM were observed in both DAA-treated cells(Figure 7) We further showed that the emergence of drugresistant variant was significantly reduced when IFN-120572 orDAAs was cotreated with PGSM in replicon cells (data notshown)

4 Discussion

HCV is a common infectious agent affecting approximately170 million individuals worldwide [26] Currently there isno protective vaccine available for HCV Although currentstandard therapy combination of PEG-IFN-120572 and RBVoften showed high SVR in certain genotypes this therapyaccompanies significant adverse effects The recent approval

of the first HCV-specific DAAs that was given in a triplecombination with PEG-IFN-120572RBV has increased cure ratesin genotype 1 naıve patients from sim55 to 75 at least underconditions of standardized clinical trials but they still havelimitations in the possible dose-limiting adverse effects andlow genetic barrier to resistance

Natural products could be important sources for anti-HCV agents [27] A variety of medicinal herbs were usedto treat HCV especially for patients who are not eligible forIFNRBV or who fail to respond to IFN [28] In the UnitedStates milk thistle and glycyrrhizin are the most popularherbalmedicines for the treatment ofHCV and they are oftenused as adjuncts to conventional therapies Accumulatingpieces of evidence have shown that natural products derivedfrom plants exerted inhibitory effects on HCV replication[29 30] For example Silibin-related flavoligands exhibitedan inhibitory effect on HCV RdRp activity and multiplehepatoprotective functions [31 32] Epigallocatechin gallate(EGCG) a major component of catechin in tea and certainplants was shown to have anti-HCVNS5B activity [33] It hasbeen previously reported that 2-arylbenzofuran derivativesfrom Mori Cortex Radicis possessed anti-HCV activity [34]Overall natural products could be alternative sources tocontrol HCV propagation

In this study we demonstrated that the root extracts of PGexhibited inhibitory activity against HCV RNA replicationin HCV subgenomic replicon cells Furthermore we showedthat triterpenoid mixture fraction exerted inhibitory activity


Table 3 Synergistic effect of PGSM on IFN-120572- R7227- and BMS790052-mediated anti-HCV activity

Combination with Combination Indexlowast EffectAt EC50 At EC75 At EC90

IFN-120572 045 039 034 SynergisticR7227 (NS3 protease inhibitor ) 072 071 070 SynergisticBMS790052 (NS5A inhibitor) 041 042 044 SynergisticHCV replicon cells (genotype 1b) were treated in combination of PGSM and IFN-120572 R7227 and BMS790052 At 72 h after treatment anti-HCV activity wasdetermined by qRT-PCR (Taqman) CI values at 50 effective concentration (EC50) 75 effective concentration (EC75) and 90 effective concentration(EC90) were calculated using CalcuSyn softwarelowastA CI value of 1 indicates additive effect a CI value of less than 1 indicates synergistic effect a CI value of greater than 1 indicates an antagonistic effect

NS

(a) (b)

5BΔ21

(kD

a)

11697

79

42

30

20

1 2

Figure 4 Purification of HCV NS5B protein (a) HCV NS5Bprotein (1120583g) eluted from glutathione sepharose affinity columnwassubjected to SDS-polyacrylamide gel electrophoresis and visualizedby Coomassie Brilliant Blue R-250 (b) NS5B protein purifiedfrom figure legend to A was immunoblotted with an anti-NS5Bmonoclonal antibody and visualized with the enhanced chemilumi-nescence detection The arrowhead indicates the NS5B protein

against HCV RNA replication We further identified that PDPD2 PD3 DPD DPD

2 and PA were the active components

in triterpenoid mixture The EC50

values of active saponinagainst anti-HCV activity in HCV subgenomic replicon cellsranged from 035 to 245 ugmL In fact all of these activecompounds exerted direct anti-NS5B polymerase activityThe IC

50values of active saponins for RdRp activity were

around 5ndash15 ugmL Of note these triterpenoid saponins didnot show any inhibitory effect on NS3 protease Moreoveranti-HCV activities of triterpenoid saponins were morepotent in the context of HCV replication than in vitro enzymeassay system (Tables 1 and 2) These data suggest that eithercellular factors or cellular immune responsesmay be involvedin triterpenoid saponin-mediated anti-HCV activity Furtherstudies are required to elucidate the mechanisms of triter-penoid saponin-induced anti-HCV activity

We then asked whether IC50

in a 120583gmL range could bereached in patients In our animal studies the half-life ofthese compounds was 657 plusmn 07 h which may be enough toreach the therapeutic concentration in rats Pharmacokinetic

studies showed that the absorption rate was increased 6sim10times higher in intraduodenum (ID) and intraileum (IL) thanin oral (Oral-PO) (data not shown) These results suggestthat therapeutic concentration can be reached in patients ifthese compounds are coated for enteric absorption In factpreliminary coated PGSM study showed thatHCV titers weredecreased ge2 log when was administered to for chronic HCVpatients 8 weeks

Triterpenoid saponins are secondary metabolites of gly-cosidic nature that are widely distributed in higher plantsand are also found in marine invertebrates Saponinsexert a wide range of pharmacological activities includingexpectorant anti-inflammatory vasoprotective hypocholes-terolemic immunomodulatory hypoglycemic molluscici-dal antifungal and antiparasitic functions [35] More than20 triterpenoid saponins have been isolated from PG PDand PD

2have shown the most potent biological activities

among platycodin saponins It has been reported that PD andPD2are potentially less hemolytic saponin adjuvant eliciting

Th1 and Th2 immune responses [10 36] PD is also a potentadjuvant of specific cellular and humoral immune responsesagainst recombinant hepatitis B antigen [37] It has beenshown that PD induces apoptosis and decreases telomeraseactivity in human leukemia cells [38] PD and 210158401015840-O-acetyl-polygalacin D2 protect against ischemiareperfusion injuryin the gerbil hippocampus [39] Saponins from PD alsoprotect against carbon tetrachloride induced hepatotoxicityand against acute ethanol-induced hepatotoxicity in mice[40 41]

In the present study we investigated whether combina-tion therapy of PGSM and IFN-120572 Daclatasvir (BMS 790052NS5A inhibitor) and Danoprevir (ITMN-191 R7227) wouldenhance the anti-HCV activity in HCV replicon cells Asshown in Table 3 the highest combination index (CI) valueswere significantly less than 1 (034ndash072) when PGSM wastreated in combination with various inhibitors in HCV repli-con cells We demonstrated that treatment of HCV repliconcells with PGSM for two weeks resulted inmultilog reductionin HCV RNA levels in a time- and dose-dependent mannerWe further showed that PGSM showed a synergistic effecton IFN-120572- BMS 790052- and ITMN-19-induced anti-HCVactivity (Figure 4) These data strongly indicate that PGSMmay be used as a new regimen in combination with IFN-120572 fortreatment of chronic HCV patients Collectively we demon-strated for the first time that the triterpenoid saponins fromPG extracts exerted suppressive activity on HCV replication


Con 05 1 5

NS5A

120573-Actin

NS5A

120573-Actin

PD2 (120583gmL)

Con 05 1 5

PD3 (120583gmL)

Con 1 5 10

PA (120583gmL)

Con 1 5 10Con 1 5 10

DPD (120583gmL) DPD2 (120583gmL)

Figure 5 Inhibition of HCV protein expression by triterpenoid saponins in HCV replicon cells HCV replicon cells were treated with theindicated amounts of triterpenoid saponins Con indicates a vehicle (DMSO) Three days after treatments cell lysates were immunoblottedwith an anti-NS5A antibody Actin protein was used as a loading control

HCV

RN

A (f

old

of co

ntro

l)

0 3 6 9 14Days of treatment

ControlIFN-10UmLIFN-50UmLIFN-100UmLPGSM-02 120583gmL

PGSM-05 120583gmLPGSM-1120583gmLPGSM-2120583gmLIFN-50UmL + PGSM-05 120583gmLIFN-50UmL + PGSM-1120583gmL

100E + 01

100E + 00

100E minus 01

100E minus 02

100E minus 03

Figure 6 Synergistic effect of PGSM on IFN-120572-induced anti-HCV activity in HCV replicon cells HCV subgenomic replicon cells (genotype1b) were treated with the indicated amounts of either PGSM or IFN-120572 alone or both as indicated Culture media containing fresh compoundswere replaced every three days At the indicated time points after treatments intracellular HCV RNA levels were determined by qRT-PCRThe copy number of HCV RNAwas calculated from cells treated with compound as compared to that for control Control indicates that cellswere treated with 02 DMSO (vehicle)

Specifically inhibitory functions of these saponins on HCVRdRp activity could partly explain the antiviral mechanismin HCV replicating cells Further studies are necessary toelucidate the mechanism that how triterpenoid saponinsinhibit RdRp activity

Finally we noticed that therapeutic window for mostof the saponins in genotype 2a was rather small because

all selectivity indeices (SI) were smaller than 20 Althoughtherapeutic index for the saponins in genotype 1b was higherthan genotype 2a we still need to improve therapeutic win-dow with forthcoming studies Nevertheless since the com-bination treatments of saponins and IFN-120572 and other DAAsshowed synergistic effects on anti-HCV activity saponinmaybe used as an effective therapeutic agent for certain HCV


0

5

10

50

0 02 05 1 2 5

PG-extract (120583gmL)

0 02 05 1 2 5


0 02 05 1 2 5PG-extract (120583gmL)

IFN

-120572(U

mL)

0

02

05

1

0

2

10

40

BMS-790052

(pg

mL)

VX-950

(120583g

mL)

Figure 7 PGSM potentiates colony suppression in replicon cells in combination with IFN-120572 and DAAs HCV replicon cells were treatedwith various concentrations of PGSM and IFN-120572 PGSM and BMS-790052 (NS5Ai) and PGSM and VX-950 (PI) as indicated Three weeksafter treatment remaining surviving cells were stained with crystal violet

patients Taken together our data indicate that triterpenoidsaponin may represent a novel anti-HCV therapeutic agentto control HCV replication

Abbreviations

HCV Hepatitis C virusHCVcc Cell culture grown HCVqRT-PCR Quantitative real-time PCRPGSM Platycodon grandiflorum saponin mixtureRdRp RNA-dependent RNA polymeraseDAA Direct acting antiviral

Conflict of Interests

The authors declare that there is no conflict of interests

Acknowledgments

This work was supported by Industrialization Support Pro-gram for Biotechnology of Agriculture and Forestry (810007-03-1-SU000) Ministry for Food Agriculture Forestry andFisheries Republic of Korea by Basic Science ResearchProgram (2012026351) from the Ministry of Science ICT

and Future Planning and by the Next-Generation BioGreen21 Program (PJ009643) Rural Development AdministrationRepublic of Korea

References

[1] G M Lauer and B DWalker ldquoHepatitis C virus infectionrdquoTheNew England Journal of Medicine vol 345 no 1 pp 41ndash52 2001

[2] W P Hofmann and S Zeuzem ldquoA new standard of carefor the treatment of chronic HCV infectionrdquo Nature ReviewsGastroenterology andHepatology vol 8 no 5 pp 257ndash264 2011

[3] K M Marks and I M Jacobson ldquoThe first wave HCVNS3 protease inhibitors telaprevir and boceprevirrdquo AntiviralTherapy vol 17 pp 1191ndash1131 2012

[4] C Welsch A Jesudian S Zeuzem and I Jacobson ldquoNewdirect-acting antiviral agents for the treatment of hepatitis Cvirus infection and perspectivesrdquo Gut vol 61 no 1 pp i36ndashi462012

[5] J Vermehren and C Sarrazin ldquoNew hepatitis C therapies inclinical developmentrdquo European Journal of Medical Researchvol 16 no 7 pp 303ndash314 2011

[6] M Schaefer A Hinzpeter A Mohmand et al ldquoHepatitis Ctreatment in ldquodifficult-to-treatrdquo psychiatric patients with pegy-lated interferon-alpha and ribavirin response and psychiatricside effectsrdquo Hepatology vol 46 no 4 pp 991ndash998 2007


[7] E B Lee ldquoPharmacological studies on Platycodon grandiflo-rumADC IV A comparison of experimental pharmacologicaleffects of crude platycodin with clinical indications of platycodiradixrdquo Yakugaku Zasshi vol 93 no 9 pp 1188ndash1194 1973

[8] K S Kim O Ezaki S Ikemoto and H Itakura ldquoEffects ofPlatycodon grandiflorum feeding on serum and liver lipid con-centrations in rats with diet-induced hyperlipidemiardquo Journal ofNutritional Science andVitaminology vol 41 no 4 pp 485ndash4911995

[9] K J Lee J H Choi H G Kim et al ldquoProtective effect ofsaponins derived from the roots of Platycodon grandiflorumagainst carbon tetrachloride induced hepatotoxicity in micerdquoFood and Chemical Toxicology vol 46 no 5 pp 1778ndash17852008

[10] Y Xie H Pan H Sun and D Li ldquoA promising balanced Th1and Th2 directing immunological adjuvant saponins from theroot of Platycodon grandiflorumrdquo Vaccine vol 26 no 31 pp3937ndash3945 2008

[11] Y D Yoon S B Han J S Kang et al ldquoToll-like receptor 4-dependent activation of macrophages by polysaccharide iso-lated from the radix of Platycodon grandiflorumrdquo InternationalImmunopharmacology vol 3 no 13-14 pp 1873ndash1882 2003

[12] T Khanal J H Choi Y P Hwang Y C Chung andHG JeongldquoSaponins isolated from the root of Platycodon grandiflorumprotect against acute ethanol-induced hepatotoxicity in micerdquoFood and Chemical Toxicology vol 47 no 3 pp 530ndash535 2009


[14] K J Lee H J You S J Park et al ldquoHepatoprotective effectsof Platycodon grandiflorum on acetaminophen-induced liverdamage in micerdquo Cancer Letters vol 174 no 1 pp 73ndash81 2001

[15] K K Hyun S C Jae S Y Dae et al ldquoHPLC analysis of saponinsin platycodi radixrdquo Korean Journal of Pharmacognosy vol 38no 2 pp 192ndash196 2007

[16] J-W Oh T Ito and M M C Lai ldquoA recombinant hepatitis Cvirus RNA-dependent RNA polymerase capable of copying thefull-length viral RNArdquo Journal of Virology vol 73 no 9 pp7694ndash7702 1999

[17] V Lohmann F Korner J-O Koch U Herian L Theilmannand R Bartenschlager ldquoReplication of subgenomic hepatitis Cvirus RNAs in a hepatoma cell linerdquo Science vol 285 no 5424pp 110ndash113 1999

[18] T Wakita T Pietschmann T Kato et al ldquoProduction ofinfectious hepatitis C virus in tissue culture from a cloned viralgenomerdquo Nature Medicine vol 11 no 7 pp 791ndash796 2005

[19] T-C Chou and P Talalay ldquoAnalysis of combined drug effectsa new look at a very old problemrdquo Trends in PharmacologicalSciences vol 4 pp 450ndash454 1983

[20] T-C Chou and P Talalay ldquoQuantitative analysis of dose-effectrelationships the combined effects of multiple drugs or enzymeinhibitorsrdquo Advances in Enzyme Regulation vol 22 pp 27ndash551984

[21] K Lin A D Kwong and C Lin ldquoCombination of a hepatitisC virus NS3-NS4A protease inhibitor and alpha interferonsynergistically inhibits viral RNA replication and facilitatesviral RNA clearance in replicon cellsrdquo Antimicrobial Agents andChemotherapy vol 48 no 12 pp 4784ndash4792 2004

[22] K Lin R B Perni A D Kwong and C Lin ldquoVX-950 a novelhepatitis C virus (HCV) NS3-4A protease inhibitor exhibits

potent antiviral activities in HCV replicon cellsrdquo AntimicrobialAgents and Chemotherapy vol 50 no 5 pp 1813ndash1822 2006

[23] E J S Graham R Hunt S M Shaw et al ldquoColony-formingassays reveal enhanced suppression of hepatitis C virus replica-tion using combinations of direct-acting antiviralsrdquo Journal ofVirological Methods vol 174 no 1-2 pp 153ndash157 2011

[24] R A Fridell D Qiu C Wang L Valera and M GaoldquoResistance analysis of the hepatitis C virus NS5A inhibitorBMS-790052 in an in vitro replicon systemrdquo AntimicrobialAgents and Chemotherapy vol 54 no 9 pp 3641ndash3650 2010

[25] Y He M S King D J Kempf et al ldquoRelative replicationcapacity and selective advantage profiles of protease inhibitor-resistant hepatitis C virus (HCV) NS3 protease mutants in theHCV genotype 1b replicon systemrdquo Antimicrobial Agents andChemotherapy vol 52 no 3 pp 1101ndash1110 2008

[26] E Szabo G Lotz C Paska A Kiss and Z Schaff ldquoViralhepatitis new data on hepatitis C infectionrdquo Pathology andOncology Research vol 9 no 4 pp 215ndash221 2003

[27] D Duan Z Li H LuoW Zhang L Chen and X Xu ldquoAntiviralcompounds from traditional Chinese medicines Galla Chineseas inhibitors of HCV NS3 proteaserdquo Bioorganic and MedicinalChemistry Letters vol 14 no 24 pp 6041ndash6044 2004

[28] R E Stauber and V Stadlbauer ldquoNovel approaches for therapyof chronic hepatitis Crdquo Journal of Clinical Virology vol 36 no2 pp 87ndash94 2006

[29] H S Azzam C Goertz M Fritts and W B Jonas ldquoNaturalproducts and chronic hepatitis C virusrdquo Liver International vol27 no 1 pp 17ndash25 2007

[30] L B Seeff K L Lindsay B R Bacon T F Kresina and JH Hoofnagle ldquoComplementary and alternative medicine inchronic liver diseaserdquo Hepatology vol 34 no 3 pp 595ndash6032001

[31] A Ahmed-Belkacem N Ahnou L Barbotte et al ldquoSilibininand related compounds are direct inhibitors of hepatitis C virusRNA-dependent RNA polymeraserdquo Gastroenterology vol 138no 3 pp 1112ndash1122 2010

[32] S J Polyak C Morishima V Lohmann et al ldquoIdentification ofhepatoprotective flavonolignans from silymarinrdquo Proceedings ofthe National Academy of Sciences of the United States of Americavol 107 no 13 pp 5995ndash5999 2010

[33] C Roh and S-K Jo ldquo(-)-Epigallocatechin gallate inhibitshepatitis C virus (HCV) viral protein NS5Brdquo Talanta vol 85no 5 pp 2639ndash2642 2011

[34] Y LHyunH Y Ji K R Yang et al ldquoInhibition ofHCV repliconcell growth by 2-arylbenzofuran derivatives isolated fromMoriCortex Radicisrdquo Planta Medica vol 73 no 14 pp 1481ndash14852007

[35] S G Sparg M E Light and J van Staden ldquoBiological activitiesand distribution of plant saponinsrdquo Journal of Ethnopharmacol-ogy vol 94 no 2-3 pp 219ndash243 2004

[36] Y XieW Deng H Sun and D Li ldquoPlatycodin D2 is a potentialless hemolytic saponin adjuvant eliciting Th1 and Th2 immuneresponsesrdquo International Immunopharmacology vol 8 no 8pp 1143ndash1150 2008

[37] Y Xie H-X Sun and D Li ldquoPlatycodin D is a potent adjuvantof specific cellular and humoral immune responses againstrecombinant hepatitis B antigenrdquoVaccine vol 27 no 5 pp 757ndash764 2009

[38] M-OKimD-OMoon YHChoi et al ldquoPlatycodinD inducesapoptosis and decreases telomerase activity in human leukemiacellsrdquo Cancer Letters vol 261 no 1 pp 98ndash107 2008


[39] J H Choi K-Y Yoo O K Park et al ldquoPlatycodin D and 210158401015840-o-acetyl-polygalacin D2 isolated from Platycodon grandiflorumprotect ischemiareperfusion injury in the gerbil hippocampusrdquoBrain Research vol 1279 pp 197ndash208 2009



Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014


MEDIATORSINFLAMMATION

of


Behavioural Neurology

EndocrinologyInternational Journal of



Disease Markers


BioMed Research International

OncologyJournal of



Oxidative Medicine and Cellular Longevity


PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of



Computational and Mathematical Methods in Medicine

OphthalmologyJournal of


Diabetes ResearchJournal of



Research and TreatmentAIDS


Gastroenterology Research and Practice


Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom


Platycodi Radix is the root of Platycodon grandiflorum(PG) This plant grows wild in east Asian countries andis reported to contain significant amounts of carbohydrateprotein lipid and ash Additionally PG contains variouskinds of triterpenoid saponins Much attention from phar-maceutical industry has been paid to PG due to its variousefficacies on diseases PG extract has been used as a foodadditive and oriental herb medicine to cure various diseasesincluding bronchitis asthma and pulmonary tuberculosisand inflammatory diseases It has been also reported thatPlatycodi Radix stimulates antioxidants immunostimula-tion and antitumor activity and prevents hyperlipidaemiaand obesity [7ndash9] Moreover it has been shown to improvethe immunogenicity of virus vaccines in mice by enhancingboth humoral and cellular immune responses [10 11] Inter-estingly the extracts from PG prevented chemical-inducedhepatotoxicity [12ndash14] Nevertheless the inhibitory effect oftriterpenoid saponin extract fromPGonHCV replication hasnever been explored thus far

The present study investigated the suppressive activity ofroot extract from PG against HCV replication using Huh7cells harboring HCV genotype 1b subgenomic replicon Theactive fraction pooled from Platycodon grandiflorum saponinmixture (PGSM)was found to have potent anti-HCV activityWe identified 6 triterpenoid saponins (PD PD

2 PD3 DPD

DPD2 and PA) as active components exerting inhibitory

activity againstHCVreplication in subgenomic replicon cellsWe further verified antiviral activity of these compoundsusing RNA-dependent RNA polymerase (RdRp) assay Fur-thermore antiviral properties and synergistic effects oftriterpenoid saponins on interferon and other direct actingantiviral (DAA) drugs were verified in HCV replicon cells

2 Materials and Methods

21 Preparation of Crude Extract and Various FractionsPlatycodon grandiflorum was cultivated for three years inGyeongbuk Province South Korea One kg of dried rootsof Platycodon grandiflorum was cut into slices and rootslices were extracted using 5 L of distilled water at 90∘Cfor 6 h filtered and concentrated under reduced pressureto yield a PG extract (85 g) The powder of PG extract wasdissolved in distilled water and then subjected to a reversephase C

18 The sample was serially eluted with water 3ndash5

acetonitrile 10 methanol 30 methanol 50 methanol70 methanol and 100 methanol The sample isolated in50 methanol fraction contained triterpenoid saponins andexerted the highest anti-HCV activity and was designated asPGSM PGSMwas further purified by using preparative high-performance liquid chromatography (HPLC) as reportedpreviously [15]

22 Analysis of PGSM by HPLCELSD and LCMSD HPLCanalysis of PGSM was performed on an Agilent 1100 seriesHPLC (USA) equipped with a Sedex 55 evaporative lightscattering detector (ELSD SEDERE Alfortville France) Asample was separated in a Gemini C

18column (100mm times

46mm 3120583m particle size Phenomenex Torrance CA

USA) with a precolumn (C18 35 120583m 2 times 20mm) at room

temperature The mobile phase consisted of 01 formicacidmethanolacetonitrile (75 5 20 vvv A) and 01formic acidmethanolacetonitrile (70 5 25 vvv B) andgradient runs were programed as follows 0ndash10min in theabsence of B 10ndash17min (0ndash50 B) 25ndash34min (50ndash80 B)42ndash52min (100 B) and then equilibration in the absence ofB for 10min at a flow rate of 1 mLmin The injection volumewas 20120583LThe ELSD was set to a probe temperature of 70∘Ca gain of 7 and the nebulizer gas nitrogen adjusted to 25 bar

The electrospray ionization-mass spectrometric (ESI-MS) analysis was performed on an Agilent 5989 massspectrometer with an ESI interface fitted with a hexapoleion guide Chromatographic separation of the PGSM wasperformed as described above The optimal condition forthe analysis of triterpenoids employed pneumatic nebuliza-tion with nitrogen (45 psi) and a counterflow of nitrogen(9 Lmin) heated to 350∘C for the nebulization and des-olvation of the introduced liquid Mass spectrometry wasperformed using the negative ion mode and the scan modethis process detected from 100 to 2000mz with a dwell timeof 300ms

23 RdRp Assay Recombinant HCV NS5B polymerasefrom HCV genotype 1b carrying an N-terminal GST-tagand C-terminal 21-amino acid truncation (NS5BCΔ21) wasexpressed in Escherichia coli and purified as previouslydescribed with some modifications [16] The colorimetricreverse transcriptase assay kit (Roche Applied Science) wasapplied to perform RdRp assayThe inhibition of HCV RdRpactivity was determined by the amount of double-strandedRNA synthesized by the recombinant NS5BΔC21 in thepresence of various test compounds using HCV 31015840-UTR asa template

24 HCV NS34A Protease Assay The NS34A serine pro-tease assay was performed using a HCV protease assaykit (SensoLyte AnaSpec Fremont CA) according to themanufacturerrsquos instructions Briefly HCV-NS34A proteasepurified from E coli was mixed with the test compound inthe assay buffer After 15min incubation at room tempera-ture EDANSDABCYL-based fluorescence resonance energytransfer (FRET) peptide substrate solution was added andmixed The FRET substrate was cleaved specifically by HCVNS34A protease thereby liberating the C-terminal peptide-fluorophore fragment from the proximity quenching effectof the dark quencher resulting in increase of fluorescenceThe fluorescence intensity was measured immediately andcontinuously at excitationemission at 340 nm and 490 nm

25 In Vitro Anti-HCV Assay in HCV Replicon Cells Huh7cells harboring HCV subgenomic replicon (genotype 1b)[17] were maintained in the presence of 025mgmL G418(Invitrogen Carlsbad CA) HCV replicon cells were seededat a density of 1 times 104 cellswell in a 96-well plate andincubated at 37∘C and 5 CO

2 Following 24 h incubation

the culture medium was replaced with a medium containingserially diluted test compounds in the presence of 2FBS and










3 Results







Con 1 5 10

NS5A

120573-actin

PGSM (120583gmL)

(a)

120

100

80

60

40

20

0

120

140

100

80

60

40

20

0

Relat

ive H

CV R

NA

( o

f con

trol)

Cel

l via

bilit

y (

of c

ontro

l)0 1 2 3 4 5 6


(b)


0 10 20 30 40 50(min)

(mV

)

350030002500200015001000

5000

1

2

3

45 6 7

8

910

1112 13






2(PD2)


platycodin D2(DPD





2

PD3 DPD DPD







50values





2





H

HO

OHOH

OH

O

C

HOOC CH2OH

OR2

R1O

Platycogenic acid A

H

HO

O

C

CH2OH

OR2

R1O

Platycodigenin

H

HO

O

C

CH2OH

OR2

R1

R1 R2

O

Polygalacic acid

H3C

HOH2C

2930

2021

2218

19

1716

15141326

12

11251

2

34

5

109

8

76

24

28

Saponins Aglycone

Platycodin D

Platycodigenin

Glc-Lam-Gen-




Deapioplatycodin D2

Platycodin D2

Platycodin D3







10 11 log

10

13 log10 and 17 log



10 08 log

10 and 15 log

10 respectively





C-3 C-28 HCVNS5B

HCVNS34A







4 Discussion









NS

(a) (b)

5BΔ21

(kD

a)

11697

79

42

30

20

1 2

















Con 05 1 5

NS5A

120573-Actin

NS5A

120573-Actin

PD2 (120583gmL)

Con 05 1 5

PD3 (120583gmL)

Con 1 5 10

PA (120583gmL)

Con 1 5 10Con 1 5 10

DPD (120583gmL) DPD2 (120583gmL)


HCV

RN

A (f

old

of co

ntro

l)




100E + 01

100E + 00

100E minus 01

100E minus 02

100E minus 03






0

5

10

50

0 02 05 1 2 5


0 02 05 1 2 5


0 02 05 1 2 5PG-extract (120583gmL)

IFN

-120572(U

mL)

0

02

05

1

0

2

10

40

BMS-790052

(pg

mL)

VX-950

(120583g

mL)



Abbreviations




Acknowledgments



References


















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

























3 Results







Con 1 5 10

NS5A

120573-actin

PGSM (120583gmL)

(a)

120

100

80

60

40

20

0

120

140

100

80

60

40

20

0

Relat

ive H

CV R

NA

( o

f con

trol)

Cel

l via

bilit

y (

of c

ontro

l)0 1 2 3 4 5 6


(b)


0 10 20 30 40 50(min)

(mV

)

350030002500200015001000

5000

1

2

3

45 6 7

8

910

1112 13






2(PD2)


platycodin D2(DPD





2

PD3 DPD DPD







50values





2





H

HO

OHOH

OH

O

C

HOOC CH2OH

OR2

R1O

Platycogenic acid A

H

HO

O

C

CH2OH

OR2

R1O

Platycodigenin

H

HO

O

C

CH2OH

OR2

R1

R1 R2

O

Polygalacic acid

H3C

HOH2C

2930

2021

2218

19

1716

15141326

12

11251

2

34

5

109

8

76

24

28

Saponins Aglycone

Platycodin D

Platycodigenin

Glc-Lam-Gen-




Deapioplatycodin D2

Platycodin D2

Platycodin D3







10 11 log

10

13 log10 and 17 log



10 08 log

10 and 15 log

10 respectively





C-3 C-28 HCVNS5B

HCVNS34A







4 Discussion









NS

(a) (b)

5BΔ21

(kD

a)

11697

79

42

30

20

1 2

















Con 05 1 5

NS5A

120573-Actin

NS5A

120573-Actin

PD2 (120583gmL)

Con 05 1 5

PD3 (120583gmL)

Con 1 5 10

PA (120583gmL)

Con 1 5 10Con 1 5 10

DPD (120583gmL) DPD2 (120583gmL)


HCV

RN

A (f

old

of co

ntro

l)




100E + 01

100E + 00

100E minus 01

100E minus 02

100E minus 03






0

5

10

50

0 02 05 1 2 5


0 02 05 1 2 5


0 02 05 1 2 5PG-extract (120583gmL)

IFN

-120572(U

mL)

0

02

05

1

0

2

10

40

BMS-790052

(pg

mL)

VX-950

(120583g

mL)



Abbreviations




Acknowledgments



References


















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















Con 1 5 10

NS5A

120573-actin

PGSM (120583gmL)

(a)

120

100

80

60

40

20

0

120

140

100

80

60

40

20

0

Relat

ive H

CV R

NA

( o

f con

trol)

Cel

l via

bilit

y (

of c

ontro

l)0 1 2 3 4 5 6


(b)


0 10 20 30 40 50(min)

(mV

)

350030002500200015001000

5000

1

2

3

45 6 7

8

910

1112 13






2(PD2)


platycodin D2(DPD





2

PD3 DPD DPD







50values





2





H

HO

OHOH

OH

O

C

HOOC CH2OH

OR2

R1O

Platycogenic acid A

H

HO

O

C

CH2OH

OR2

R1O

Platycodigenin

H

HO

O

C

CH2OH

OR2

R1

R1 R2

O

Polygalacic acid

H3C

HOH2C

2930

2021

2218

19

1716

15141326

12

11251

2

34

5

109

8

76

24

28

Saponins Aglycone

Platycodin D

Platycodigenin

Glc-Lam-Gen-




Deapioplatycodin D2

Platycodin D2

Platycodin D3







10 11 log

10

13 log10 and 17 log



10 08 log

10 and 15 log

10 respectively





C-3 C-28 HCVNS5B

HCVNS34A







4 Discussion









NS

(a) (b)

5BΔ21

(kD

a)

11697

79

42

30

20

1 2

















Con 05 1 5

NS5A

120573-Actin

NS5A

120573-Actin

PD2 (120583gmL)

Con 05 1 5

PD3 (120583gmL)

Con 1 5 10

PA (120583gmL)

Con 1 5 10Con 1 5 10

DPD (120583gmL) DPD2 (120583gmL)


HCV

RN

A (f

old

of co

ntro

l)




100E + 01

100E + 00

100E minus 01

100E minus 02

100E minus 03






0

5

10

50

0 02 05 1 2 5


0 02 05 1 2 5


0 02 05 1 2 5PG-extract (120583gmL)

IFN

-120572(U

mL)

0

02

05

1

0

2

10

40

BMS-790052

(pg

mL)

VX-950

(120583g

mL)



Abbreviations




Acknowledgments



References


















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















H

HO

OHOH

OH

O

C

HOOC CH2OH

OR2

R1O

Platycogenic acid A

H

HO

O

C

CH2OH

OR2

R1O

Platycodigenin

H

HO

O

C

CH2OH

OR2

R1

R1 R2

O

Polygalacic acid

H3C

HOH2C

2930

2021

2218

19

1716

15141326

12

11251

2

34

5

109

8

76

24

28

Saponins Aglycone

Platycodin D

Platycodigenin

Glc-Lam-Gen-




Deapioplatycodin D2

Platycodin D2

Platycodin D3







10 11 log

10

13 log10 and 17 log



10 08 log

10 and 15 log

10 respectively





C-3 C-28 HCVNS5B

HCVNS34A







4 Discussion









NS

(a) (b)

5BΔ21

(kD

a)

11697

79

42

30

20

1 2

















Con 05 1 5

NS5A

120573-Actin

NS5A

120573-Actin

PD2 (120583gmL)

Con 05 1 5

PD3 (120583gmL)

Con 1 5 10

PA (120583gmL)

Con 1 5 10Con 1 5 10

DPD (120583gmL) DPD2 (120583gmL)


HCV

RN

A (f

old

of co

ntro

l)




100E + 01

100E + 00

100E minus 01

100E minus 02

100E minus 03






0

5

10

50

0 02 05 1 2 5


0 02 05 1 2 5


0 02 05 1 2 5PG-extract (120583gmL)

IFN

-120572(U

mL)

0

02

05

1

0

2

10

40

BMS-790052

(pg

mL)

VX-950

(120583g

mL)



Abbreviations




Acknowledgments



References


















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of



















C-3 C-28 HCVNS5B

HCVNS34A







4 Discussion









NS

(a) (b)

5BΔ21

(kD

a)

11697

79

42

30

20

1 2

















Con 05 1 5

NS5A

120573-Actin

NS5A

120573-Actin

PD2 (120583gmL)

Con 05 1 5

PD3 (120583gmL)

Con 1 5 10

PA (120583gmL)

Con 1 5 10Con 1 5 10

DPD (120583gmL) DPD2 (120583gmL)


HCV

RN

A (f

old

of co

ntro

l)




100E + 01

100E + 00

100E minus 01

100E minus 02

100E minus 03






0

5

10

50

0 02 05 1 2 5


0 02 05 1 2 5


0 02 05 1 2 5PG-extract (120583gmL)

IFN

-120572(U

mL)

0

02

05

1

0

2

10

40

BMS-790052

(pg

mL)

VX-950

(120583g

mL)



Abbreviations




Acknowledgments



References


















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of




















NS

(a) (b)

5BΔ21

(kD

a)

11697

79

42

30

20

1 2

















Con 05 1 5

NS5A

120573-Actin

NS5A

120573-Actin

PD2 (120583gmL)

Con 05 1 5

PD3 (120583gmL)

Con 1 5 10

PA (120583gmL)

Con 1 5 10Con 1 5 10

DPD (120583gmL) DPD2 (120583gmL)


HCV

RN

A (f

old

of co

ntro

l)




100E + 01

100E + 00

100E minus 01

100E minus 02

100E minus 03






0

5

10

50

0 02 05 1 2 5


0 02 05 1 2 5


0 02 05 1 2 5PG-extract (120583gmL)

IFN

-120572(U

mL)

0

02

05

1

0

2

10

40

BMS-790052

(pg

mL)

VX-950

(120583g

mL)



Abbreviations




Acknowledgments



References


















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















Con 05 1 5

NS5A

120573-Actin

NS5A

120573-Actin

PD2 (120583gmL)

Con 05 1 5

PD3 (120583gmL)

Con 1 5 10

PA (120583gmL)

Con 1 5 10Con 1 5 10

DPD (120583gmL) DPD2 (120583gmL)


HCV

RN

A (f

old

of co

ntro

l)




100E + 01

100E + 00

100E minus 01

100E minus 02

100E minus 03






0

5

10

50

0 02 05 1 2 5


0 02 05 1 2 5


0 02 05 1 2 5PG-extract (120583gmL)

IFN

-120572(U

mL)

0

02

05

1

0

2

10

40

BMS-790052

(pg

mL)

VX-950

(120583g

mL)



Abbreviations




Acknowledgments



References


















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















0

5

10

50

0 02 05 1 2 5


0 02 05 1 2 5


0 02 05 1 2 5PG-extract (120583gmL)

IFN

-120572(U

mL)

0

02

05

1

0

2

10

40

BMS-790052

(pg

mL)

VX-950

(120583g

mL)



Abbreviations




Acknowledgments



References


















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of



























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















research article triterpenoid saponins isolated from ...triterpenoid saponins isolated from...

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