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Clinical and Experimental Pharmacology and Physiology 2006 33 612 616 Blackwell Publishing AsiaOriginal Article Antiviral effects of saikosaponinsP W Cheng et al ANTIVIRAL EFFECTS OF SAIKOSAPONINS ON HUMAN CORONAVIRUS 229E IN VITRO Pei Win Cheng Lean Teik Ng Lien Chai Chiang and Chun Ching Lin Department of Microbiology and Graduate Institute of Natural Products Kaohsiung Medical University Kaohsiung and Department of Biotechnology Tajen University Pingtung Taiwan SUMMARY 1 Saikosaponins represent a group of oleanane derivatives usually as glucosides that are found in a number of plant families Saikosaponins isolated from medicinal plants such as Bupleurum spp Heteromorpha spp and Scrophularia scorodonia have been reported to possess various biological activities specifi cally antihepatitis antinephritis antihepatoma anti inflammation immunomodulation and antibacterial effects 2 The aim of the present study was to examine the anticoro naviral activity of saikosaponins A B 2 C and D and their mode of action Using the 2 3 bis 2 methoxy 4 nitro 5 sulfophenyl 5 phenylamino carbonyl 2H tetrazolium hydroxide XTT assay results showed that all saikosaponins tested demonstrated antiviral activity at concentrations of 0 25 25 mmol L with the strongest activity being noted for saikosaponin B 2 IC 50 1 7 0 1 mmol L Interestingly both saikosaponins A 50 cellular cytotoxicity CC 50 concentration 228 1 3 8 mmol L selectivity index SI 26 6 and B 2 CC 50 383 3 0 2 mmol L SI 221 9 exhibited no cytotoxic effects on target cells at concentrations that achieved antiviral activity In the time of addition studies saikosaponin B 2 at 6 mmol L significantly inhibited human coronavirus 229E infection following its addition at various time pre infection 4 to 1 h coinfection 0 h and post infection 1 4 h Furthermore saikosaponin B 2 also showed an inhibitory effect on viral attachment and penetration 3 The present results indicate that saikosaponin B 2 has potent anticoronaviral activity and that its mode of action possibly involves interference in the early stage of viral replication such as absorption and penetration of the virus Key words antiviral activity saikosaponins viral replication INTRODUCTION Nosocomial respiratory viral infections NRVI are increasingly documented in paediatric units The most common aetiological viral agents that infect the respiratory system are respiratory syncytial virus influenza virus and rhinovirus Recently a novel human coronavirus HCoV was identified as a causative agent of the severe acute respiratory syndrome SARS 1 Both HCoV 229E and HCoV OC43 are HCoV that have been recognized for the past 30 years 2 of the two HCoV 229E has been recently recognized as an important cause of NRVI in high risk infants 3 4 Over the past decades a number of phytochemicals have been reported to possess potent antiviral activity 5 7 Saikosaponins represent a group of oleanane derivatives usually as glucosides that are found in a number of plant families Saikosaponins isolated from medicinal plants such as Bupleurum spp Heteromorpha spp and Scrophularia scorodonia have been reported to possess various biological activities namely antihepatitis antinephritis antihepatoma anti inflammation and antibacterial effects 8 10 as well as being able to modulate immune function 11 12 Furthermore saikosaponins A B 2 C and D have been shown to be active against human immunodeficiency virus HIV 13 measles 14 influenza virus 15 16 herpes simplex virus 14 and varicella zoster virus 17 In an attempt to find new anti HCoV compounds we conducted a series of experiments to investigate the antiviral activity and mode of action of saikosaponins against HCoV 229E in vitro METHODS Chemicals Actinomycin D dimethylsulphoxide DMSO and saikosaponins A B 2 C and D Fig 1 were purchased from Sigma Chemical St Louis MO USA 2 3 Bis 2 methoxy 4 nitro 5 sulfophenyl 5 phenylamino carbonyl 2H tetrazolium hydroxide XTT kits were purchased from Roche Diagnostics Mannheim Germany Actinomycin D as a positive control and saikosaponins of purity greater than 95 were dissolved in DMSO They were further diluted with RPMI 1640 medium Sigma Chemical to the required concentrations with the final concentration of DMSO no greater than 0 1 Virus and cells The HCoV 229E was obtained from American Type Culture Collection ATCC VR 740 Rockville MD USA Human fetal lung fibroblasts MRC 5 ATCC CCL 171 were used as target cells for viral infection Cells was grown in RPMI 1640 medium supplemented with 3 fetal calf serum FCS 100 units mL penicillin G 100 mg mL streptomycin and 0 25 mg mL amphotericin B at 37 C in a humidified atmosphere of 5 CO 2 Virus titres were determined by cytopathic effects and were expressed as 50 tissue culture infective dose TCID 50 per mL All viruses were stored at 70 C until use Cytotoxicity assay The MRC 5 cells were seeded onto a 96 well plate at a concentration of 9 10 3 cells A volume of 10 mL well with different concentrations of saikosaponins A B 2 C and D was applied to culture wells in triplicate Dimethylsulphoxide 0 1 and actinomycin D 0 2 0 02 and 0 002 mol L Correspondence Professor CC Lin Graduate Institute of Natural Products Kaohsiung Medical University 100 Shih Chuan 1st Road Kaohsiung 807 Taiwan ROC Email aalin Received 1 August 2005 revision 8 December 2005 accepted 26 December 2005 2006 Blackwell Publishing Asia Pty Ltd Antiviral effects of saikosaponins 613 2006 Blackwell Publishing Asia Pty Ltd were used as negative and positive controls respectively After incubation at 37 C with 5 CO 2 for 4 days a mixture of 0 1 mL phenazine methosulphate PMS electron coupling reagent and 50 mL XTT was added to each well The trays were further incubated for 3 h to allow the production of XTT formazan Absorbance was determined with an ELISA reader Multiskan EX Labsystems Helsinki Finland at a test wavelength of 492 nm and a reference wavelength of 690 nm Data were calculated as percentage inhibition using the following formula Inhibition 100 At As 100 where At and As refer to the absorbance of the test substances and the solvent control respectively The 50 cellular cytotoxicity CC 50 concentration of test substances was calculated according to Chiang et al 18 The selectivity index SI was calculated as the ratio of the CC 50 to the EC 50 Antiviral assay using the XTT method The antiviral activity of pure compounds against HCoV 229E was evaluated using the XTT method 18 The MRC 5 cells treated with trypsin were seeded onto 96 well plates at a concentration of 7 10 3 cells mL and a volume of 70 mL well After incubation at 34 C with 5 CO 2 overnight 20 mL test virus 100 TCID 50 was added and cells were incubated for a further 2 h The XTT test was performed as described above The percentage protection was calculated as Protection Atv Acv Acd Acv 100 where Atv is the absorbance of the viral infected cells in the presence of test compounds and Acv and Acd are the absorbance of the virus control and cell control respectively The antiviral concentration of 50 effectiveness EC 50 was defined as the concentration that achieved 50 inhibition of virus induced cytopathic effects The number of viruses used in each experiment was based on infection target cells by 100 TCID 50 of virus to produce 50 XTT formazan products as in uninfected control cells Time course and dose response effects of saikosaponin B 2 on HCoV 229E Twenty microlitres of 100 TCID 50 HCoV 229E per well was absorbed onto confluent monolayers of MRC 5 cells for 2 h followed by the addition of different concentrations of saikosaponin B 2 0 0 2 1 and 6 mmol L to culture cells at various times pre infection 4 to 1 h coinfection 0 h or post infection 1 4 h of the virus to MRC 5 cells at 34 C After 4 days incubation the XTT test was performed and antiviral activity was determined as described above Attachment assay The attachment assay was conducted according to the procedures described by Albin et al 19 and De Logu et al 20 with minor modification Briefly the MRC 5 cell monolayer was grown in a 24 well culture plate and then prechilled at 4 C for 1 h After the medium had been aspirated the cell monolayer was infected with 100 TCID 50 HCoV 229E in the absence or presence of various concentrations of saikosaponin B 2 0 75 25 mmol L After further incubation of the infected cell monolayer at 4 C for 3 h the medium was aspirated to remove unabsorbed virus The cell monolayer was then washed with phosphate buffered saline PBS three times and overlaid with 2 FCS medium After 4 days of incubation the XTT test was performed as described above Absorbance was measured at a test wavelength of 450 nm and a reference wavelength of 690 nm using an ELISA reader The percentage inhibition of attachment was calculated as Inhibition 100 ODt ODs 100 where ODt and ODs are the optical density of the test substances and the solvent control respectively Penetration assay The penetration assay of HCoV 229E into MRC 5 cells was performed according to published procedures with minor modifications 19 20 The MRC 5 cell monolayer was grown in a 24 well culture plate and prechilled at 4 C for 1 h After the cell monolayer had been infected with 100 TCID 50 HCoV 229E and incubated at 4 C for 3 h to allow the attachment of HCoV 229E 6 mmol L saikosaponin B 2 or solvent control 0 1 DMSO was added to the culture medium The infected cell monolayer was incubated at 37 C to maximize the penetration of the virus At 10 min intervals the infected cell monolayer was treated with acidic PBS pH 3 for 1 min to inactivate non penetrating virus Immediately after the addition of PBS at pH 7 to neutralize acidic PBS pH 3 the neutral PBS was removed and the cell monolayer was overlaid with 2 FCS medium After 4 days of incubation the XTT test was performed as described above Absorbance was measured at a test wavelength of 450 nm and a reference wavelength of 690 nm using an ELISA reader The percentage inhibition of penetration was calculated as Inhibition 100 ODt ODs 100 where ODt and ODs indicate the optical density of the test substances and the solvent control respectively Statistical analysis Results are expressed as the mean SD from three independent experiments Statistical analysis was performed with one way anova followed by the least significant difference LSD multiple range test Differences between test compounds and control were evaluated using Student s t test P 0 05 was considered statistically different RESULTS Cytotoxic effects of saikosaponins A B 2 C D and actinomycin D on MRC 5 cells We first evaluated the cytotoxicity of saikosaponins A B 2 C D and actinomycin D on MRC 5 cells using the XTT assay The results Fig 1 Structure of saikosaponins 614 P W Cheng et al 2006 Blackwell Publishing Asia Pty Ltd show that saikosaponins A B 2 C and D have no cytotoxic effect on the cells at concentrations of 2 5 mmol L Fig 2 Interestingly more than 60 cell viability was noted when saikosaponins A B 2 C and D were used at a concentration of 25 mmol L Furthermore no visible changes in cell morphology or cell density were observed at the concentrations tested The concentrations estimated to reduce cell viability by 50 or the CC 50 of the various saikosaponins were in the range 121 5 383 3 mmol L which was better than for actinomycin D positive control CC 50 2 8 mol L Inhibitory effect of saikosaponins A B 2 C and D on HCoV 229E Figure 3 shows the antiviral activity of saikosaponins A B 2 C and D The results show that saikosaponins A B 2 C and D at concentra tions of 25 mmol L or less significantly inhibited HCoV 229E viral infection P 0 05 with saikosaponin B 2 exhibiting the strongest potency The percentage viral inhibition for 0 25 2 5 and 25 mmol L saikosaponin B 2 was 35 7 0 7 63 0 0 8 and 100 0 0 2 respectively Table 1 gives the EC 50 and SI values of saikosaponins A B 2 C and D in inhibiting infection by HCoV 229E Because saikosaponin B 2 showed the strongest anticoronaviral activity and the best SI value we investigated the mechanistic effects of this drug on viral infection further Time of addition studies of saikosaponin B 2 on viral replication In order to investigate the mechanism as to how saikosaponin B 2 inhibits infection by the HCoV 229E virus a study was conducted to investigate the time course of effects of various concentrations of saikosaponin B 2 0 2 1 0 and 6 mmol L at 4 h before and 0 4 h after viral infection The results show that saikosaponin B 2 decreases infection by HCoV 229E in a dose and time dependent manner Fig 4 In addition saikosaponin B 2 was noted to inhibit viral infec tion more efficiently when it was added before viral adsorption 45 0 60 0 67 6 74 9 and 91 8 99 6 at 0 2 1 0 and 6 mmol L Fig 2 Efects of saikosaponin A B 2 H17039 C and D and actinomycin D H17040 on the viability of the human fetal lung fibroblast MRC 5 cell line Cells were treated with various concentrations 0 2 5 25 and 250 mmol L of saikosaponins A B 2 C and D for 96 h Cell viability was determined by the XTT assay Data are the mean SD of three independent experiments P 0 05 compared with control Student s t test Fig 3 Antiviral activity of saikosaponin A B 2 C and D against HCoV 229E The virus was treated with various concentrations 0 0 25 2 5 and 25 mmol L of saikosaponin A B 2 C and D for 96 h followed by determination of viral inhibition using the XTT assay Data are the mean SD of three independent experiments P 0 05 compared with control Student s t test Table 1 Assessement of anticoronaviral activity of saikosaponins Test drug CC 50 mmol L EC 50 mmol L SI Actinomycin D 2 8 0 3 0 02 0 0 140 Saikosaponin A 228 1 3 8 8 6 0 3 26 6 Saikosaponin B 2 383 3 0 2 1 7 0 1 221 9 Saikosaponin C 121 5 0 1 19 9 0 1 19 2 Saikosaponin D 176 2 0 2 13 2 0 3 13 3 Data are the mean SD of three independent experiments P 0 05 compared with saikosaponin A P 0 05 compared with saikosaponin B 2 P 0 05 compared with saikosaponin C Student s t test CC 50 the concentration that reduces the growth of target MRC 5 cells by 50 EC 50 the inhibitory concentration of compound that produces 50 inhibition of virus induced cytopathic effects SI selectivity index CC 50 EC 50 Actinomycin D was used as a positive control Fig 4 Time course of effects of saikosaponin B 2 on HCoV 229E Different concentrations of saikosaponin B 2 0 0 2 1 and 6 mmol L were added at various times pre infection 4 to 1 h coinfection 0 h or post infection 1 4 h of HCoV 229E to MRC 5 cells at 34 C After incubation for 4 days the percentage inhibition was evaluated by the XTT method Data are the mean SD of three independent experiments a b c P 0 05 compared with control one way anova followed by the LSD multiple range test Antiviral effects of saikosaponins 615 2006 Blackwell Publishing Asia Pty Ltd saikosaponin B 2 respectively than if it was added after viral adsorp tion 19 3 34 6 37 2 58 7 and 69 8 88 4 at 0 2 1 0 and 6 mmol L saikosaponin B 2 respectively Effect of saikosaponin B 2 on viral attachment and penetration According to the results of time of addition studies saikosaponin B 2 appeared to affect the early stage of HCoV 229E infection This observation suggests that saikosaponin B 2 may disturb the early events of HCV 229E infection including viral attachment and penetration Thus the effect of saikosaponin B 2 on viral attachment and penetration was investigated The results show that HCoV 229E attachment is significantly inhibited by saikosaponin B 2 in a dose dependent manner Fig 5 The percentage attachment inhibition at concentrations of 0 0 75 1 5 3 6 12 and 25 mmol L saikosaponin B 2 was 0 10 4 12 2 20 70 8 81 8 and 89 3 respectively These results suggest that saikosaponin B 2 prevents the attachment of HCoV 229E into host cells Fig 5 Penetration of HCoV 229E into host cells was inhibited by 6 mmol L saikosaponin B 2 in a time dependent manner with an inhibition rate of 0 17 3 19 21 9 24 8 31 35 38 9 53 3 and 81 3 at 0 10 20 30 40 50 60 70 80 and 90 min respectively Fig 6 This observation suggests that saikosaponin B 2 also prevents the penetration of HCoV 229E into host cells DISCUSSION In the present study we demonstrated that saikosaponins A B 2 C and D possess anti HCoV 229E activity with saikosaponin B 2 exhibiting the strongest potency Furthermore saikosaponins A and B 2 showed no cytotoxic effect on cell viability The time course study revealed that the anti HCoV 229E activity of saikosaponin B 2 was higher when it was added before the virus entered the host cells than if it was added after the virus infected host cells These results suggest that saikosaponin B 2 may interfere with the early stage of viral replication such as the adsorption and penetration of the virus Natural products from plants and micro organisms have tradition ally provided the pharmaceutical industry with many important leads in the search for new drugs Over the past decades many compounds isolated from medicinal plants have been found to possess inhibitory activity against viruses 5 7 Viral infection remains a serious problem in many countries coupled with the recent outbreak of SARS for which no effective drug is available and scientists worldwide are searching for new cures for this disease The present study indicates that saikosaponin B 2 has potent activity against HCoV 229E With further investigation and a better understanding of its molecular mode of action saikosaponin B 2 could be a novel lead for the develoment of a potential chemopreventive agent for this viral infection In previous studies triterpenoid saponins have been shown to possess marked antiviral activity at con centrations at which the saponin showed no cytotoxic effect on Vero cells 13 15 The mechanism of the antiviral activity was suggested to be associated with an early event in the viral infectious cycle through the inhibition of virus host cell attachment based on a direct interaction of saikosaponin D with the surface membrane of host cells 16 17 In the present study we postulated that the antiviral activity of saikosaponin B 2 on anti HCoV 229E could be through a direct inactivation of the virus or by direct interaction with the host cell membrane resulting in morphological changes in the cell membrane These events could consequently lead to an arrest in viral adsorption and penetration In previous studies although saikosaponins have been shown to have inhibitory effect against herpes simplex type I HSV 1 in vitro the mechanism of action was not elucidated 21 In another study saikosaponin C showed activity in inhibiting hepatitis B virus DNA replication 12 whereas saikosaponin D was able to inactivate the measles Fig 5 Effect of saikosaponin B 2 on HCoV 229E attachment Cells were prechilled at 4 C for 1 h followed by inoculation with HCoV 229E 100 TCID 50 of the cell monolayer in the absence or presence of test compound After 3 h incubation percentage attachment of the test compound treated group was evaluated using the XTT assay Data are the mean SD of three independent experiments P 0 05 between different doses of saikosaponin B 2 one way anova Fig 6 Effect of saikosaponin B 2 on the penetration of HCoV 229E to MRC 5 cells The cell monolayer was prechilled at 4 C for 1 h and then infected with 100 TCID 50 HCoV 229E at 4 C for 3 h After 3 h incubation 6 mmol L saikosaponin B 2 was added to the culture At 10 min intervals extracellular virus was inactivated by acidic phosphate buffered saline PBS pH 3 for 1 min Then PBS pH 7 was added to neutralize the acidic PBS The neutral PBS was removed and 3 fetal calf serum was added The percentage penetration of virus was evaluated using the XTT assay Data are the mean SD of three independent experiments P 0 05 between different time points one way anova 616 P W Cheng et al 2006 Blackwell Publishing Asia Pty Ltd virus and herpes simplex virus in vitro 14 In other phytochemical studies 22 baicalein has been reported to functionally block the epidermal growth factor receptor tyrosine kinase activity and h