Chapter 11
Inhibition of Influenza Virus Infection by Tea Polyphenols Tadakatsu Shimamura
Downloaded by UNIV OF TEXAS AT DALLAS on July 13, 2016 | http://pubs.acs.org Publication Date: May 5, 1994 | doi: 10.1021/bk-1994-0547.ch011
Department of Microbiology and Immunology, Showa University School of Medicine, Tokyo 142, Japan
Influenza is a disease with a high mortality rate throughout the world. Despite efforts to develop effective vaccines and therapeutic agents against influenza virus infection, it is still virtually uncon trolled. The use of vaccines against influenza is presently limited because of the frequent conversion of viral antigens. Symptomatic therapy is the only treatment possible for influenza virus infection except in a few countries where the antiviral compound amantadine is in current use. Amantadine, however, is effective as prophylaxis and therapy only against influenza A virus. Furthermore, it has adverse side effects, and resistant virus mutants arise. Therefore, new vaccines and antiviral strategies are being explored. Tea, one of the most popular beverages in the world, is well known to possess many medicinal properties — numerous laboratories have reported many therapeutic effects of tea In this chapter, the effects of tea and its constituents against influenza virus infection are discussed.
We have previously reported that tea possesses antibacterial activity against various bacteria that cause diarrheal (1-3), respiratory (4,5) and skin (6) diseases. In ancient China tea was first used as an antidote. There were, however, no scientific reports of antitoxin activity by tea until our laboratory found that tea inhibited the activity of bacterial exotoxins (7). We have further demonstrated that polyphenols found in tea are among the components responsible for these activities (4-6,8-11). More recently, we found that tea markedly inhibited the infectivity of both influenza A and Β viruses in cultured cells by blocking adsorption of virus to the cells (12). This led us to study the inhibitory activity of polyphenols against influenza viruses. In Vitro Studies Inhibition of Plaque Formation by Tea Polyphenols. (-)-Epigallocatechin gallate (EGCg) and theaflavin digallate (TF3) were purified from green tea (13) and
0097-6156/94/0547-0101$06.00/0 © 1994 American Chemical Society
Ho et al.; Food Phytochemicals for Cancer Prevention II ACS Symposium Series; American Chemical Society: Washington, DC, 1994.
FOOD PHYTOCHEMICALS II: TEAS, SPICES, AND HERBS
Downloaded by UNIV OF TEXAS AT DALLAS on July 13, 2016 | http://pubs.acs.org Publication Date: May 5, 1994 | doi: 10.1021/bk-1994-0547.ch011
102
black tea (14), respectively. The plaque assay was performed using Madin-Darby canine kidney (MDCK) cells as the influenza virus target. We first studied the capacity of EGCg and TF3 to inhibit infection of influenza A/Yamagata/120/86(HINI) or influenza B/USSR/100/83 viruses in M D C K cells. The virus was mixed with EGCg or TF3 for either 5 min or 60 min before attempting adsorption to the cells. Contact for a short time of EGCg and TF3 with either virus effectively inhibited infectivity of the virus in vitro. Even concentrations of polyphenols as low as about 1 μΜ inhibited the plaque formation by 100%. A control experiment using amantadine mixed with influenza A virus for 10 min before addition to M D C K cells showed that the concentration of amantadine required to inhibit plaque formation was approximately 100 times that of polyphenols. We next determined if polyphenols are effective when added during adsorption of virus to cells. Influenza A virus was adsorbed to cells at 4°C for 30 min, then polyphenol was added to virus-adsorbed cells for 15 min, and the cells were cultured. Although the effective concentration of polyphenols was higher than when added before adsorption, inhibition of plaque formation was observed. When polyphenol was added 30 min after adsorption of the virus to cells at 37°C, however, plaque forming activity of the virus was not inhibited at any concen tration. These findings suggest that polyphenol prevents the virus from entering into cells, but not virus multiplication in the cells. These results also suggest that polyphenols may bind to surface proteins of influenza virus. Observation by Electron Microscopy We used electron microscopy to study the possibility that polyphenols may bind to surface proteins of influenza virus. When the capacities of polyphenols and anti-influenza A virus antibody to bind to influenza A virus was compared, both EGCg and TF3 agglutinated virus particles after a short time, but anti-influenza A virus antibody agglutinated the viruses only weakly. We further examined the capacity of virus pretreated with polyphenol or antibody to bind to M D C K cells by scanning electron microscopy. Untreated virus bound to M D C K cells, whereas virus pretreated with either EGCg or antibody could not. These observations by electron microscopy suggest that polyphenols bind to hemagglutinin of influenza virus and prevent the virus from adsorbing to cells. Inhibition of Hemagglutination by Tea Polyphenols Influenza virus can bind to chicken red blood cells by their hemagglutinin and agglutinate the cells. Examination of the capacity of polyphenols to inhibit hemagglutination by the virus revealed that, like anti-influenza A virus antibody, EGCg and TF3 inhibited the hemagglutination dose-dependently. In Vivo Studies Inhibition of Influenza Virus Infection in Mice. The capacity of tea extracts containing polyphenols to inhibit influenza virus infection was examined in C3H/He mice. Two groups of 10 mice were intranasally administered 4 x l 0 influenza A viruses or a mixture of virus and black tea extract at the beverage concentration. The body weight of mice administered virus alone decreased 5
Ho et al.; Food Phytochemicals for Cancer Prevention II ACS Symposium Series; American Chemical Society: Washington, DC, 1994.
11. SHIMAMURA
Inhibition of Influenza Virus Infection by Tea Polyphenols 103
Downloaded by UNIV OF TEXAS AT DALLAS on July 13, 2016 | http://pubs.acs.org Publication Date: May 5, 1994 | doi: 10.1021/bk-1994-0547.ch011
gradually from four days after infection but increased in mice administered the mixture of virus and black tea extract. A l l mice administered virus alone died within 10 days after infection while all other mice survived. Anti-influenza virus antibodies in surviving mice were examined by the hemagglutination inhibition test 3 weeks after virus administration. Nine of ten mice administered the mixture of virus and black tea extract did not develop specific antibody, even though in this mouse model specific antibody appears in mice administered only 100 virus particles. These findings suggest that tea extracts inhibit the infectivity of influenza virus in nasal epithelial cells. Inhibition of Natural Influenza Virus Infection in Swine It is well known that swine are as susceptible to influenza virus as human. Field trials to test the inhibitory activity of polyphenols against natural influenza virus infection in swine were performed in the northern Japanese prefecture of Miyagi from October 1990 to March 1991 using a polyphenol mixture prepared from Japanese green tea (75). Two thousand one-month-old shoats were exposed to 0.1% polyphenols sprayed from a sprinkler for 20 seconds every 30 minutes for six months. Specific anti-influenza virus antibodies in the sera of 40 shoats were examined once a month using swine influenza A virus isolated at that time. In the case of the control group, specific antibodies that were naturally transferred from sows decreased gradually for two months after birth. Then antibodies increased in the third month, meaning that natural infection had occurred. On the other hand, specific antibodies of shoats exposed to polyphenols continued to decrease—by the end of the third month, no antibody was detected in any polyphenol-treated shoat. Unfortunately, the sprinkler system broke down at the end of the third month. After that, specific antibodies appeared at higher levels. These results suggest that polyphenols can prevent natural infection in swine. Use of Tea Polyphenols as a New Strategy Against Influenza Tea polyphenols may be useful as a strategy against influenza virus infection, for example, in a gargle. We compared black tea to four kinds of gargles now available in Japan in their capacities to inhibit the infectivity of influenza virus in M D C K cells. Unlike tea, most gargles could not inhibit the infectivity of the virus. A l l pupils in one of the primary schools in the Shizuoka district of Japan used Japanese green tea as a gargle several times per day during one winter season. It was reported that tea as a gargle was effective at preventing influenza infection and that no class in the school was closed down by influenza outbreak. The Japanese Buddhist priest Eisai wrote a book entitled Tea Drinking For Health about eight hundred years ago. In the first paragraph of the book, he described tea as "a wonder drug for health." Echoing Eisai, I would say that tea is a wonder drug against influenza. Literature Cited
1. Toda, M.; Okubo, S.; Hiyoshi, R.; Shimamura, T. Letters in Appl. Microbiol. 1989, 8, 123-125. 2. Toda, M.; Okubo, S.; Ohnishi, R.; Shimamura, T. Jpn. J. Bacteriol. 1989, 44, 669-672.
Ho et al.; Food Phytochemicals for Cancer Prevention II ACS Symposium Series; American Chemical Society: Washington, DC, 1994.
Downloaded by UNIV OF TEXAS AT DALLAS on July 13, 2016 | http://pubs.acs.org Publication Date: May 5, 1994 | doi: 10.1021/bk-1994-0547.ch011
104
FOOD PHYTOCHEMICALS II: TEAS, SPICES, AND HERBS
3. Toda, M.; Okubo, S.; Ikigai, H.; Suzuki, T.; Suzuki, Y.; Shimamura, T. J. Appl. Bacteriol. 1991, 70, 109-112. 4. Horiuchi, Y.; Toda, M.; Okubo, S.; Hara, Y.; Shimamura, T. J. J. A. Inf. D. 1992, 66, 599-605. 5. Chosa, H.; Toda, M.; Okubo, S.; Hara, Y.; Shimamura, T. J. J. A. Inf. D. 1992, 66, 606-611. 6. Okubo, S.; Toda, M.; Hara, Y.; Shimamura T. Jpn. J. Bacteriol. 1991, 46, 509514. 7. Okubo, S.; Ikigai, H.; Toda, M.; Shimamura, T. Letters in Appl. Microbiol. 1989, 9, 65-66. 8. Toda, M.; Okubo, S.; Ikigai, H.; Shimamura, T. Jpn. J. Bacteriol. 1990, 45, 561-566. 9. Ikigai, H.; Toda, M.; Okubo, S.; Hara, Y.; Shimamura, T. Jpn. J. Bacteriol. 1990, 45, 913-919. 10. Toda, M.; Okubo, S.; Hara, Y.; Shimamura, T. Jpn. J. Bacteriol. 1991, 46, 839845. 11. Toda, M.; Okubo, S.; Ikigai, H.; Suzuki, T.; Suzuki, Y.; Hara, Y.; Shimamura, T. Microbiol. Immunol. 1992, 36, 999-1001. 12. Nakayama, M . ; Toda, M . ; Okubo, S.; Shimamura, T. Letters in Appl. Microbiol. 1990, 11, 38-40. 13. Matsuzaki, T.; Hara, Y. Nippon Nogeikagaku Kaishi 1985, 59, 129-134. 14. Hara, Y.; Matsuzaki, T.; Suzuki, T. Nippon Nogeikagaku Kaishi 1987, 61, 803808. 15. Hara, Y.; Watanabe, M. Nippon Shokuhin Kogyo Gakkaishi 1989, 36, 951-955. RECEIVED
June 7, 1993
Ho et al.; Food Phytochemicals for Cancer Prevention II ACS Symposium Series; American Chemical Society: Washington, DC, 1994.
