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8)-epiafzelechin extracted from Cassia javanica inhibits herpes simplex virus type 2 replication
1 Department of Cosmetic Applications & Management, Tung Fang Institute of Technology, 829 Kaohsiung County, Taiwan
2 Graduate Institute of Pharmaceutical Sciences, College of Pharmacy, Kaohsiung Medical University, 807 Kaohsiung, Taiwan
3 General Education Center, Chungtai Institute of Health Sciences and Technology, Taichung, Taiwan
4 Department of Food Science and Technology, Tajen Institute of Technology, 907 Ping-Tung, Taiwan
Correspondence
Chun-Ching Lin
aalin{at}ms24.hinet.net
Received 7 April 2005
Accepted 11 October 2005
8)-epiafzelechin (EEE) extracted from the fresh leaves of Cassia javanica L. agnes de Wit (Leguminosae) was investigated for its in vitro anti-HSV-2 activity using XTT and plaque reduction assays. Results showed that EEE inhibited HSV-2 replication in a dose-dependent manner. The IC50 value was 83·8±10·9 and 166·8±12·9 µM for XTT and plaque reduction assays, respectively. EEE did not affect the viability and the proliferation of cells at antiviral concentrations. Mechanistic studies demonstrated that EEE prevented HSV-2 from penetrating the cell and also interfered with HSV-2 replication at the late stage of its life cycle. It also disturbed virus attachment but the inhibitory effect was minor. In summary, the conclusion of this study was that EEE exhibits various modes of action in suppressing HSV-2 multiplication.
Abbreviations: ACV, acyclovir; 33'DOGPB, 3,3'-di-O-galloyl prodelphinidin B-2; EEE, ent-epiafzelechin-(48)-epiafzelechin; 3OGPB, 3'-O-galloyl prodelphinidin B-2; SI, selectivity index; XTT, sodium 3'-[1-(phenylamino-carbonyl)-3,4-tetrazolium]-bis(4-methoxy-6-nitro) benzene sulfonic acid.
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INTRODUCTION |
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 TOP INTRODUCTION METHODSRESULTSDISCUSSIONREFERENCES |
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Primary HSV-2 infections can be asymptomatic and unrecognized, or present clinically as typical herpes genitalis. In cases of infection during vaginal delivery, HSV-2 can cause life-threatening neonatal herpes. After primary infection, the virus is always transported in a retrograde fashion along peripheral sensory nerves to the ganglia, which subsequently leads to establishment of persistent (latent) lifetime infection. Proper stimuli can reactivate the latent virus to cause recurrent diseases. HSV infection is, therefore, recognized as a life-long infection.
HSV causes illnesses that are varied and frequently characterized by the formation of vesiculo-ulcerative lesions on the skin and mucus membranes at the infection site. The clinical manifestations range from asymptomatic disease to life-threatening illness, with the most common being genital and orolabial disease. Potential fatal sicknesses include neonatal infection, encephalitis and disseminated infection in patients with defects in cellular immunity (Aurelian, 2000).
Antiviral agents for the treatment of HSV infections have been developed for several decades. The reference compound is acyclovir (ACV), which selectively inhibits HSV DNA replication with low host-cell toxicity. Other used antiviral agents include penciclovir, trifluridine, vidarabine, valacyclovir and famciclovir. For ACV-resistant HSV infection, foscarnet and cidofovir are applied (Chilukuri & Rosen, 2003; Morfin & Thouvenot, 2003). But their toxicities have limited the use of both medicines (Brady & Bernstein, 2004).
The widespread use of ACV has lead to the emergence of HSV strains resistant to ACV. Although the prevalence of ACV-resistant HSV infection is rare among immunocompetent patients (below 1 %), it is, however, about 5 % among immunocompromised patients and reaches up to 18·4 % among bone marrow transplant recipients (Englund et al., 1990; Nugier et al., 1992; Christophers et al., 1998; Danve-Szatanek et al., 2004).
The increased number of immunocompromised patients, i.e. transplant, cancer or AIDS patients, has driven the need for improved antiviral agents to treat diseases caused by herpesvirus (Griffiths, 1996; Pillay et al., 2000). Although some non-nucleoside inhibitors of herpesvirus have been developed (Liuzzi et al., 1994; Kleymann et al., 2002; Wathen, 2002), none of them are officially approved for HSV therapy (De Clercq, 2001; Naesens & De Clercq, 2001). Consequently, there is still a need to search for novel and more effective antiviral agents to prevent and/or to treat HSV infection in the future.
Cassia javanica L. agnes de Wit (Leguminosae) is a traditionally used medicinal plant in China and Southeast Asian countries. It is conventionally believed that the medical herb can reduce fever, decrease the virulence of pathogenic organisms, regulate the flow of ‘chi’ and lubricate the intestines. In China, it is applied to treat gastric pain, cold, malaria, measles, chickenpox and constipation (Chinese Herbal Commission, 1999). The property of lessening the virulence of pathogenic organisms, and the traditional usage in the treatment of chickenpox, a varicella-zoster virus (VZV)-caused disease (both VZV and HSV-2 are classified in subfamily alphaherpesvirinae), suggest that this plant might also exhibit anti-HSV-2 activity.
In an effort to search for new anti-HSV-2 compounds from natural products, we conducted a series of experiments to investigate the in vitro antiviral activity and the mechanism of action of ent-epiafzelechin-(48)-epiafzelechin (EEE) extracted from the fresh leaves of C. javanica. EEE has not been reported formerly as having anti-HSV activity. This is believed to be the first account of the anti-HSV activity of EEE.
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METHODS |
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TOPINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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) was determined by comparing its physical and spectral data with published data (Kashiwada et al., 1990). The purity of EEE was >95 %.
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Cell and virus. All reagents and media for cell culture were purchased from Gibco-BRL. African green monkey kidney cells (Vero) (ATCC CCR-81) were obtained from the hospital of Kaohsiung Medical University.
HSV-2 strain 196 was kindly provided by Dr Lien-Chai Chiang (Department of Microbiology, College of Medicine, Kaohsiung Medical University). Its titre was determined by plaque assay and was expressed as p.f.u. ml–1. Virus stocks were stored at –80 °C until use.
Antiviral assays: XTT assay. The antiviral activity of EEE was assayed using XTT, sodium 3'-[1-(phenylamino-carbonyl)-3,4-tetrazolium]-bis(4-methoxy-6-nitro) benzene sulfonic acid, (Sigma) (Cheng et al., 2002a; Weislow et al., 1989). Briefly, 104 cells per well were seeded into 96-well culture plates (Falcon; BD Biosciences) and incubated for 4 h. Cells were then infected with HSV-2 at m.o.i. 0·5, and various concentrations of EEE were added. After another 72 h of incubation, the minimal concentration of EEE required to inhibit 50 % HSV-2 multiplication (IC50) was evaluated according to Cheng et al. (2002a).
Antiviral assays: plaque reduction assay. The assay was performed as described previously (Cheng et al., 2002a). Briefly, 105 cells per well were seeded into 24-well culture plates (Falcon; BD Biosciences) and then incubated until reaching at least 95 % confluency. The cell monolayer was then infected with 100 p.f.u. HSV-2 in the absence or presence of EEE. After 2 days incubation, the minimal concentration of EEE required to reduce the 50 % plaque number (IC50), was calculated by regression analysis of the dose-response curves generated from the data (Cheng et al., 2002a).
Cytotoxicity assay and selectivity index (SI). The cytotoxic effect of EEE on growing Vero cells was assayed with an XTT-based method (Lin et al., 2002). The initial seeding cell number was 5·0x103 cells per well, and after 72 h incubation, the cell number had increased to around 3·0x104 cells per well. The 50 % cytotoxic concentration (CC50) of EEE was calculated as described by Lin et al. (2002). The SI was evaluated as the ratio of CC50 to IC50.
Virucidal assay. Virucidal activity of EEE was evaluated as described by Carlucci et al. (1999) with minor modifications. Briefly, a virus suspension containing 2x107 p.f.u. HSV-2 was mixed with or without various concentrations of EEE for 6 h at room temperature (about 26 °C). The sample was then diluted and its residual infectivity was determined by plaque assay. Putranjivain A was selected as the positive control because of its virucidal activity (Cheng et al., 2004a).
Time-of-addition studies. The study was performed as described by Boulware et al. (2001) and Cheng et al. (2004a). Cells, 2x105 per well, were seeded into 12-well culture plates (Nunc; Nagle Nunc International), and then infected with 1x105 p.f.u. HSV-2 per well. EEE (250·0 µM) was added into wells either concurrent with HSV-2 infection (0 h) or at intervals of 2, 4, 7 and 12 h post-infection. After 24 h of infection, cells were scraped and viruses were harvested. Virus titre was determined by plaque assay, and the percentage of inhibition was calculated.
Attachment assay. The attachment assay was performed as described by De Logu et al. (2000) and Cheng et al. (2004a). Briefly, a Vero cell monolayer, which was grown in a 24-well culture plate, was pre-chilled at 4 °C for 1 h, and then infected with 200 p.f.u. HSV-2 in the absence or presence of serial dilution of EEE. The plaque assay was performed and the percentage inhibition was calculated.
Penetration assay. The penetration assay of HSV-2 into Vero cells was performed according to reported procedures with minor modifications (Albin et al., 1997; Cheng et al., 2004b). A Vero cell monolayer, which was grown in a 24-well culture plate, was pre-chilled at 4 °C for 1 h, and then infected with 200 p.f.u. HSV-2. After a further 3 h incubation at 4 °C, 250·0 µM EEE was added. Infected cells were then incubated at 37 °C for 60 min. At 10 min intervals, the infected-cell monolayer was treated with PBS at pH 3 for 1 min, and then immediately neutralized by PBS at pH 11. The percentage inhibition of penetration was calculated based on the reduction of the plaque number.
Statistical analysis. Data are presented are the mean±SD for triplicates of each experiment from three independent determinations. Student's unpaired t-test was used to test the significance between the test sample and solvent control. A P value of less than 0·05 was considered to be statistically significant.
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RESULTS |
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TOPINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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) showed that EEE suppressed HSV-2 multiplication in a dose-dependent fashion. The IC50 of EEE on virus replication was 83·8±10·9 and 166·8±12·9 µM for XTT and plaque reduction assays, respectively. ACV, which was used as a positive control, had an IC50 of 0·8±0·1 and 0·3±0·1 µM for XTT and plaque reduction assays, respectively. However, 500·0 µM EEE inhibited less than 30 % of HSV-1infection, which suggested that it was less active against HSV-1 than HSV-2 (data not shown).
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Effect of EEE on virus infectivity
The effect of EEE on HSV-2 infectivity was investigated to determine whether the inhibitory effect of EEE on virus replication was derived from its ability to inactivate the virus or not. Results demonstrated that EEE up to the concentration of 500·0 µM did not affect virus infectivity (data not shown).
Effect of the EEE treatment time on the antiviral activity
Table 1 shows that EEE inhibited virus replication when it was added at 0–12 h. Delaying the time of addition of EEE did not affect its antiviral activity. However, adding of EEE at 20 h post-infection did not inhibit HSV-2 infection. A similar result was observed when the time of addition of ACV, a known inhibitor of viral DNA replication, was performed.
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). EEE prevented less than 70 % virus from penetrating into the cell membrane during the first 30 min period. The inhibition rate was, however, more than 90 % at 50–60 min.
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DISCUSSION |
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TOPINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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The initial step of HSV-2 infection is the binding of virus envelope glycoprotein C to the heparan sulfate residues present on the proteoglycans on the surface of the target cells (Tal-Singer et al., 1995). The presence of viral glycoprotein D enhances the binding (Rajcani & Vojvodova, 1998). After binding, membrane fusion between the virus envelope and the plasma membrane of the target cell occurs. This requires the action of a number of viral glycoproteins including glycoprotein B, D, H and L (Spear, 1993; Roizman & Sears, 1996). EEE was found to inhibit virus penetration. It, however, had only slight activity in blocking virus binding. The inhibitory effect on virus penetration gradually amplified as incubation time increased. We, therefore, propose that EEE may affect the penetration of virus into cells through the disturbance of viral glycoproteins.
In addition to virus penetration, EEE was also found to interfere with the late stage of virus infection. This result was anticipated as similar outcomes have been observed in our previous studies of related compounds, 3'-O-galloyl prodelphinidin B-2 (3OGPB) (Cheng et al., 2002b) and 3,3'-di-O-galloyl prodelphinidin B-2 (33'DOGPB) (Cheng et al., 2003). However, EEE showed a discrepant effect in inhibiting viral attachment and also in reducing viral infectivity as compared to 3OGPB and 33'DOGPB. Both 3OGPB and 33'DOGPB can extensively block HSV-2 attachment to Vero cells and diminish the virus infectivity (Cheng et al., 2002b; Cheng et al., 2003), but EEE had only slight activity.
The diversity of the activity in inhibiting viral attachment is suggested to account for the differentiation of the structure between EEE, and 3OGPB and 33'DOGPB. EEE contains two units of epiafzelechin that are different in configuration [one is (+)-epiafzelechin and the other one is (–)-epiafzelechin], while both 3OGPB and 33'DOGPB consist of two units of (–)-epigallocatechin. Both epigallocatechin and epiafzelechin have the same skeleton structure except that the C-11 and the C-13 of epiafzelechin are substituted by OH groups thus becoming epigallocatechin. The other difference in structure is the presence of the galloyl group at C-3 for 3OGPB, and at C-3 and C-3' for 33'DOGPB. EEE lacks the galloyl group at either C-3 or C-3'. From the above, it is supposed that the lower potency of EEE in inhibiting viral attachment may be due to the differences in structural configuration, such as the absence of the OH group at C-11 and C-13, and/or the lack of the galloyl group at C-3 and/or C-3'. Nonetheless, further investigations are required to elucidate the critical factor among those above that affects viral attachment inhibition.
A different magnitude for the antiviral potency of EEE was detected by XTT and plaque reduction assays, due to the two assays having diverse principles for detecting antiviral activity. The activity of the cellular enzyme mitochondrial dehydrogenase, which converts XTT to formazan, was monitored during the XTT assay. On the other hand, the number of plaques, formed as the result of lysis of virus-infected cells, was calculated by the plaque reduction assay. Similar differences were also observed between the two assays for the antiviral activity of ACV.
In summary, the anti-HSV-2 activity of EEE from C. javanica was mediated through inhibition of viral penetration and disturbance of the late stage (the stage after 12 h but before 20 h) of virus infection. Although EEE is less potent than ACV in suppressing the multiplication of HSV-2, its differences and various mechanisms of action, encourage its future study.
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), putranjivain A (
) and ACV (
) on the penetration of HSV-2. Each point represents the mean±SD for triplicates of each experiment from three independent determinations. The asterisk (*) indicates a significant difference between test sample and solvent control (P <0·05).