Preventive and curative medicament against infection with influenza virus, containing tea or tea polyphenols

Abstract: The effective ingredient in the inventive medicament against infection with influenza virus is tea, e.g., black tea, or a tea polyphenol as a constituent of tea including epigallocatechin gallate, epicatechin gallate, epigallocatechin, epicatechin, (+)catechin and the isomer thereof, free theaflavin, theaflavin monogallates A and B and theaflavin digallate.

Description: BACKGROUND OF THE INVENTION The present invention relates to the use of tea polyphenol extracted from tea against infection with influenze virus or, more particularly, to a preventive and curative medicament against infection with influenza viruses of the type A and type B. As is well known, influenza is an important disease of mankind which has attacked human beings periodically in the past up to now sometimes killing more than ten million people in one epidemic. By virtue of the great improvement in recent years in nutrition and hygienic conditions along with the rapid progress of the medical technology in overcoming other infectious diseases, the menace of influenza is no longer so great as in the past. Nevertheless, many and unspecified people are infected by airborne infection with the influenza virus to cause one or more epidemics in every year. Many medicaments to prevent infection by influenza virus have been hitherto proposed but none of them exhibit fully satisfactory preventive effects. The method considered to be most effective for prevention against infection by influenza today is inoculation with vaccine. A problem in the vaccination against influenza, however, is that great difficulties are encountered, despite the relatively low probability of success, in the development of a new influenza vaccine in order to comply with the incessant mutation of the antigen of the influenza virus. Accordingly, it is eagerly desired to develop a novel preventive and curative medicament against infection by influenza virus capable of effectively exhibiting the desired preventive effect regardless of the antigenicity of the virus and usable without problems of safety because of absence of any harmful side effects against human body. In Proc.Soc.Exp. Biol. Med., 71, 1949, 84 extracts of tea are described , which yield an unidentified fraction inhibiting the multiplication of influenza virus in embryonated eggs. However, the effective compounds are not specified, and the fractions used are mixtures of uncertain composition, alcohol-insoluble fractions being more inhibiting than alcohol-soluble fractions. In letters in Applied Microbiology 1990, 11 , 38 - 40, it is described that extracts of black tea inhibit the infectivity of influenza virus A and influenza virus B for Madin-Darby canine kidney cells. Tea extracts inhibited virus adsorption to the cells but did not inhibit virus replication in the cells. SUMMARY OF THE INVENTION An object of the present invention is to provide a use of specific compounds against infection with influenza virus as mentioned above. The inventor has continued extensive investigations of natural products to discover a substance capable of exhibiting the desired effect without the problems which usually ensue in chemically synthesized compounds. Thus present invention relates to the use of at least one tea polyphenol extracted from tea, said tea polyphenol being selected from the group consisting of epigallocatechin gallate, epicatechin gallate, epigallocatechin, epicatechin, free theaflavin, theaflavin monogallate A, theaflavin monogallate B and theaflavin digallate, for the manufacture of a medicament for treating or inhibiting infection caused by influenza virus. BRIEF DESCRIPTION OF THE DRAWING Figure 1 is a graph showing the infection preventing effect in % as a function of the concentration of the prepared medicament. Figure 2 shows the effect on the decrease of the plaques by the antiviral activity exhibited by black tea and several commercial products of gargle as a function of ratio of dilution. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS Influenza viruses can be classified into three types of A, B and C depending on the serum types of nucleoproteins and membranous proteins. Not only human beings but also domestic fowls, pigs, horses and the like are infected with each of these three types of influenza viruses, of which the type A is the most malignant. While influenza viruses proliferate by infecting animal cells, the hemaglutinin (HA) as a glycoprotein found on the surface of the viral particles greatly participate in the initiation of infection. The hemaglutinin effects fusion of the viral membrane and cell membrane so that intrusion of the viral nucleic acid into cells is facilitated with resultant infection by influenza. When red blood cells are used as the target cells, agglutination of the red blood cells by the action of the hemaglutinin takes place so that cell fusion and hemolysis are observed. On the other hand, release of the viruses through the cell membrane is controlled by the neuraminidase (NA) so that inhibition of the activity thereof also leads to prevention of viral infection. The inventor unexpectedly has arrived at a discovery that a medicament, of which the effective ingredient is a tea polyphenol compound, has a power to deactivate the activity of both of the hemaglutinin and neuraminidase so that infection of cells with the influenza virus can be inhibited thereby. The inhibitive effect on infection could be confirmed not only relative to the phenomenon of agglutination of red blood cells but also by means of the plaque formation as a consequence of proliferation of the viruses. The infection-inhibiting effect can be exhibited by the treatment of either the viruses or the cells with a medicament comprising the above mentioned tea polyphenol compounds to give a possibility of influenza prevention. Furthermore, unlike vaccines, the effectiveness of the medicament is not limited by the antigenicity which does not affect the effectiveness of a medicament comprising the above mentioned tea polyphenol compounds. Namely, the present invention provides a novel preventive and curative medicament against infection with influenza viruses, of which tea polyphenols are the medically effective ingredient. The tea polyphenol compounds as the principal effective ingredients in medicament against infection with influenza virus include the tea catechin compounds represented by the general formula (I) given below and the theaflavin compounds represented by the general formula (II) given below: in which R₁ is a hydrogen atom or a hydroxy group and R₂ is a hydrogen atom or a 3,4,5-trihydroxy benzoyl group; and in which R₃ and R₄ are, each independently from the other, a hydrogen atom or a 3,4,5-trihydroxy benzoyl group. Particular examples of the tea catechin compounds represented by the general formula (I) include: (-)epicatechin, which is a compound of the formula (I) with R₁ = H and R₂ = H; (-)epigallocatechin, which is a compound of the formula (I) with R₁ = OH and R₂ = H; (-)epicatechin gallate, which is a compound of the formula (I) with R₁ = H and R₂ = 3,4,5-trihydroxy benzoyl group; and (-)epigallocatechin gallate, which is a compound of the formula (I) with R₁ = OH and R₂ = 3,4,5-trihydroxy benzoyl group. Particular examples of the theaflavin compounds include: free theaflavin, which is a compound of the formula (II) with R₃ = H and R₄ = H; theaflavin monogallate A, which is a compound of the formula (II) with R₃ = 3,4,5-trihydroxy benzoyl group and R₄ = H; theaflavin monogallate B, which is a compound of the formula (II) with R₃ = H and R₄ = 3,4,5-trihydroxy benzoyl group; and theaflavin digallate, which is a compound of the formula (II) with R₃ = 3,4,5-trihydroxy benzoyl group and R₄ = 3,4,5-trihydroxy benzoyl group. The above described tea polyphenol compounds can be prepared from tea leaves as the starting material and a method for the preparation thereof and a typical example of the product composition are described,for example, in Japanese Patent Kokai 59-219384, 60-13780 and 61-130285 and elsewhere. When the medicament against infection with influenza virus is prepared in a medicament form such as food, e.g., candies, collutoria, troches and the like, the tea polyphenol compounds as the effective ingredients of tea can be admixed with the medicament base either as such or as dissolved in water, alcohol and the like. An effective concentration of tea is obtained by diluting a conventional drinking tea of about 2 g/100 ml concentration by up to 260 times while the effective concentration of the tea polyphenol compounds is in the range from 1 to 100 µg/ml. In the following, the present invention is described in more detail by way of examples conducted against type A influenza virus of the A/Sichuan/2/87(H3N2) strain and type B influenza virus of the B/USSA/100/83E strain. The medicinal agents tested include a 20 wt./vol. % extract of black tea buffered with a phosphate buffer solution as well as epigallocatechin gallate and theaflavin digallate isolated and purified from green tea and black tea, each of which was dissolved in a phosphate buffer solution in a concentration of 1 mg/ml. The tests were conducted by further diluting the above obtained solutions. Assay of the viral activity for agglutination of red blood cells Measurements were undertaken on 0.5% red blood cells of domestic fowls after a length of time of 1 hour. Plaque measurement Subcultured canine kidney cells (MDCK) were used as the test cells. The culture medium for the virus proliferation was a 0.9% agar culture medium containing 2 µg/ml of trypsin, 0.4% of bovine serum albumin and the basal medium Eagle's vitamin. After infection of the cells with the virus, the culture medium was poured thereon and culturing was undertaken at 33.5°C for 3 to 5 days. After fixation thereof with formalin, the agar was removed and the plaques were counted by dyeing with methylene blue. Observation with electron microscope The phosphotransacetylase (PTA) dyeing method was utilized. Example 1 The type A influenza virus was treated for 30 minutes to 1 hour with an extract solution of black tea or epigallocatechin gallate (EGCg) and the blood coagulation (hemaglutinin activity) and infectiousness (number of plaques) were examined. The results were as shown below. Serum coagulation: serum coagulation after a treatment for 30 minutes (hemaglutinin activity) with a 10-fold diluted black tea was completely deactivated. Infectiousness (plaque counting): the number of plaques after a treatment for 1 hour was zero with each of a 2560-fold diluted black tea and 0.5 µg/ml of EGCg indicating 100% inhibition of infection. As is shown above, infection with the virus could be inhibited by a direct treatment of the virus. Figure 1 of the accompanying drawing shows that the hemaglutinin activity inhibiting power and the infection inhibiting power of black tea and EGCg depend on the concentration thereof. Example 2 Inhibition of proliferation of the type A influenza virus was examined after infection of cells. Animal cells (MDCK) infected with the virus were added to a culture medium with addition of black tea having a concentration of 80-fold dilution or to a control culture medium without addition of black tea and culturing was performed for 48 to 96 hours. After culturing, the supernatant was taken from each culture medium and subjected to the assay of the hemaglutinin (HA) activity. No difference could be detected by the plaque assay between the two groups in the activity of the virus at the starting moment of culturing. The result was that the control culture medium exhibited an HA activity of 512 after 48 hours from infection while no HA activity could be detected in the culture medium to which black tea at an 80-fold dilution had been added. Example 3 A study was performed to examine the inhibitive power against infection by type A influenza virus of a pretreatment of uninfected cells. Thus, MDCK cells were treated beforehand for 1 hour with black tea or EGCg and then infected with the virus. Thereafter a plaque assay was conducted. The results showed that the percentage infection inhibition was 85% with black tea at a 20-fold dilution and 45% with EGCg in a concentration of 100 µg/ml. These results support the conclusion that the pretreatment of the uninfected cells is effective in inhibiting infection with the influenza virus. Example 4 Similar experiments to Example 1 were undertaken to examine inhibition of infection with the type B influenza virus by treatment with EGCg or theaflavin digallate (TF3) each for 1 hour at a concentration of 1 µg/ml and for 5 minutes at a concentration of 4 µg/ml. The results obtained in these four experiments were that the percentage of inhibition against infection was 100% in each. These results support the conclusion that the pretreatment of the cells is also effective in inhibiting infection by type B influenza virus. Example 5 A comparative study was conducted for the percentage of infection inhibition of the type B influenza virus in terms of the neuraminidase (NA) activity by the determination of the isolated sialic acid by the TBA method with Fetuin as the substrate. As is summarized in the table shown below, the results were that, while the NA activity in the control test without treatment was 11, the NA activity was decreased to 8 to 2 by the treatment with black tea, epigallocatechin gallate (EGCg) or theaflavin digallate (TF3) supporting the conclusion that the treatment therewith was effective for inhibiting infection with the virus. Sample NA activity Control 11.0 Black tea, 10-fold dilution 1.25 Black tea, 20-fold dilution 2.90 Black tea, 40-fold dilution 5.00 EGCg, 50 µg/ml 8.00 EGCg, 100 µg/ml 5.10 TF3, 25 µg/ml 3.70 TF3, 50 µg/ml 2.70 TF3, 100 µg/ml 1.70 Example 6 EGCg was added to a suspension of the virus and the behavior of the virus was inspected with an electron microscope to find instantaneous formation of agglutinated lumps of the viruses indicating that EGCg had an activity as an anti-viral agent. Example 7 Influenza virus was treated for 1 hour with black tea or one of the gargle (or collutoria) A, B, C and D purchased on the market in varied dilutions to examine the effect for decreasing the number of plaques for each concentration. The effective ingredients in the gargle were as follows. A: decalinium chloride, thymol B: popidon iodine C: water-soluble azulene, benzetonium chloride D: cetyl pyridinium chloride The results of the assay are shown graphically in Figure 2 from which it is understood that, although the gargle B and D have effectiveness for inhibiting viral infection when used in a normal concentration, the effectiveness thereof is not high as compared with black tea. The above described preventive and curative medicament against infection with influenza virus comprises, as the effective ingredient, a natural product which is a drinkable taken in daily life in a considerably large volume so that it is absolutely free from the problem of undesirable side effects against human body not only when it is used as a medicine but also when it is used as an additive of food. Moreover, the effectiveness thereof is so high that infection with influenza virus can be effectively inhibited by the addition thereof even in a very low concentration to provide a means for preventing infection with influenza virus.

Claims: Use of at least one tea polyphenol extracted from tea, said tea polyphenol being selected from the group consisting of epigallocatechin gallate, epicatechin gallate, epigallocatechin, epicatechin, free theaflavin, theaflavin monogallate A, theaflavin monogallate B and theaflavin digallate, for the manufacture of a medicament for treating or inhibiting infection caused by influenza virus. The use of Claim 1, wherein the influenza virus is influenza virus A. The use of Claim 1, wherein the influenza virus is influenza virus B.

Inventors: HARA YUKIHIKO, SHIMAMURA TADAKATSU, HARA, YUKIHIKO, SHIMAMURA, TADAKATSU
Applicants: MITSUI NORIN KK, SHIMAMURA TADAKATSU, MITSUI NORIN CO., LTD., SHIMAMURA, TADAKATSU
Application year: 1990
Main classification: A61K35
IPC codes: A61K31, A61P31, A61K36, C07D311, A23F3