Elsevier

Antiviral Research

Volume 68, Issue 2, November 2005, Pages 66-74
Antiviral Research

Antiviral effect of catechins in green tea on influenza virus

https://doi.org/10.1016/j.antiviral.2005.06.010Get rights and content

Abstract

Polyphenolic compound catechins ((−)-epigallocatechin gallate (EGCG), (−)-epicatechin gallate (ECG) and (−)-epigallocatechin (EGC)) from green tea were evaluated for their ability to inhibit influenza virus replication in cell culture and for potentially direct virucidal effect. Among the test compounds, the EGCG and ECG were found to be potent inhibitors of influenza virus replication in MDCK cell culture and this effect was observed in all influenza virus subtypes tested, including A/H1N1, A/H3N2 and B virus. The 50% effective inhibition concentration (EC50) of EGCG, ECG, and EGC for influenza A virus were 22–28, 22–40 and 309–318 μM, respectively. EGCG and ECG exhibited hemagglutination inhibition activity, EGCG being more effective. However, the sensitivity in hemagglutination inhibition was widely different among three different subtypes of influenza viruses tested. Quantitative RT-PCR analysis revealed that, at high concentration, EGCG and ECG also suppressed viral RNA synthesis in MDCK cells whereas EGC failed to show similar effect. Similarly, EGCG and ECG inhibited the neuraminidase activity more effectively than the EGC. The results show that the 3-galloyl group of catechin skeleton plays an important role on the observed antiviral activity, whereas the 5′-OH at the trihydroxy benzyl moiety at 2-position plays a minor role. The results, along with the HA type-specific effect, suggest that the antiviral effect of catechins on influenza virus is mediated not only by specific interaction with HA, but altering the physical properties of viral membrane.

Introduction

Green tea is produced from the leaves of the evergreen plant Camellia sinensis. The major active ingredients of green tea are polyphenolic compounds, known as catechins. Catechins of green tea leaves are account for about 10% of dry weight, including (−)-epigallocatechin gallate (EGCG), (−)-epigallocatechin (EGC), (−)-epicatechin gallate (ECG) (Fig. 1) and (−)-epicatechin (EC), where EGCG accounts for approximately 50% of the total amounts of catechins in green tea (Balentine et al., 1997).

Various biological and pharmacological activities have been reported for EGCG, including antioxidative (Valcic et al., 1999), antibacterial (Toda et al., 1992), antitumor and antiviral activity. The antitumor effects of EGCG have been investigated in detail and the compound demonstrated inhibitory action against carcinogenesis on several different organs in animal models (Gensler et al., 1996, Katiyar and Mukhtar, 1996, Mimoto et al., 2000, Yamane et al., 1995, Yang et al., 2002). Studies have shown that antitumor effect correlates with inactivation of tumor-related proteases (Jankun et al., 1997), nitric oxide synthase (NOS) (Lin and Lin, 1997), and is mediated by PI3-to Akt kinase in the NF-kB pathway (Pianetti et al., 2002). Recently, laminin receptor has been identified as a receptor for EGCG mediating the anti cancer activity (Tachibana et al., 2004).

With regard to the antiviral activities, EGCG acts as a strong inhibitor of HIV replication in cultured peripheral blood cells (Fassina et al., 2002), and EGCG and ECG were found more effective than EGC or EC in the inhibition of the HIV-1 reverse transcriptase in vitro (Nakane and Ono, 1990). EGCG also binds directly to CD4 molecule with consequent inhibition of gp120 binding (Kawai et al., 2003). EGCG also induced inactivation of virus in vitro by deformation of phospholipids (Yamaguchi et al., 2002). Distinct antiviral activities of EGCG were reported for Epstein–Barr virus inhibition of expression of viral proteins (Chang et al., 2003) and inhibition of host factors (Weber et al., 2003). Antiviral effects of EGCG have also been reported for influenza virus. EGCG affected the infectivity of influenza virus in cell culture, and it was shown to agglutinate the viruses, preventing the viruses from absorbing to MDCK cells (Nakayama et al., 1993). It was also shown that green tea extract exerted an inhibitory effect on the acidification of intracellular compartments such as endosomes and lysosomes, resulting in inhibition of growth of influenza virus in cell culture (Imanishi et al., 2002). So far, however, biochemical studies on the anti-influenza effects of green tea polyphenols have focused on EGCG. With a view to investigate the structure-activity relationships of the green tea polyphenolic compounds, we investigated in this report the antiviral effects of various catechin compounds, EGCG, ECG and EGC, on influenza virus. The studies were further extended to all three currently circulating virus subtypes, including two different H3N2 and H1N1 influenza A types and one influenza B type. Here, we found that ECG and EGCG were much more effective than EGC, and besides the known inhibition of hemagglutination, the compounds also exerted inhibitory effect on neuraminidase and affects viral RNA synthesis at high concentration.

Section snippets

Preparation of catechins

Green tea extract was prepared by infusing the leaves (Sulloc Cha, Amore-Pacific Co. Ltd., Korea) with 75 °C distilled water in the ratio of 1:7 (w/w). After 20 min of infusion, the tea extract was quickly separated from the tea leaves by filtration and the tea extract was freeze-dried for further tests. To isolate catechins, green tea leaves were extracted with five times of 90 °C distilled water for 5 h, and the tea extract was washed with chloroform and extracted with ethyl acetate three times.

Inhibitory effects of catechins on plaque formation by influenza A and B viruses in MDCK cells

For initial screening of the antiviral activity, each compound was tested by plaque inhibition assay in MDCK cells at a fixed concentration. As shown in Fig. 2, EGCG and ECG at 50 μM concentration inhibited more than 50% of the plaque forming activity of influenza A and B viruses whereas EGC exhibited little inhibition. Notably, polyphenols mixture is more efficient than any other single compounds in plaque inhibition. For a given compound, similar inhibitory effect was observed for all

Discussion

Green tea contains various useful chemical compounds such as catechins, caffeine and vitamins, most notable components being catechins including EGCG, ECG, ECG and EC. As the most abundant component, EGCG has been most extensively studied for various biological activities. Previous reports have demonstrated that EGCG inhibits influenza virus infection when they contact with influenza virus directly (Nakayama et al., 1993), but indirect effect on host cell that might interfere with the

Acknowledgements

This work was supported, in part, by the Strategic Research Fund for Emerging/Re-emerging Viruses from the Korean National Institute of Health (KNIH), the Chemical and Biological Terrorism Research Fund from the Ministry of Commerce, Industry and Energy (MOCIE) and Nano-biotechnology Research Initiatives from the Ministry of Science and Technology (MOST) of the Korean Government.

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