Antiviral effect of catechins in green tea on influenza virus
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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