Antiviral agent containing crude drug

FIELD OF THE INVENTION
The present invention relates to an antiviral agent containing a crude drug.
BACKGROUND OF THE INVENTION
In recent years, opportunistic infectious diseases caused by viruses, such as cytomegalovirus (hereinafter abbreviated as CMV), in patients maintained on immunosuppressants, such as recipients of organ transplantation, have given rise to a problem. For instance, such infectious diseases have arisen the problem in the first living liver transplantation conducted in Shimane Medical Collage, Japan.
It has been clinically observed that the incidence of CMV infectious diseases varies depending on the combination of immunosuppressants administered to post-transplantation patients. Based on this observation, the present inventors studied the influences of individual immunosuppressants and combinations thereof on proliferation of CMV through in vitro testing systems. As a result, of various known immunosuppressants, cyclosporine and predonine accelerated CMV proliferation, while mizoribine and azathioprine inhibited CMV proliferation. These results are in good agreement with case reports as described below. Therefore, it is believed that substances exhibiting antiviral activity in vitro also possess antiviral activity in vivo.
More specifically, the cases have been reported in which CMV infection developed in 100% of patients on cyclosporine+predonine therapy whereas the incidence off CMV infection was as low as 51.5% or 63.6% in patients on the therapy with cyclosporine+predonine in combination with mizoribine or azathioprine, respectively, suggesting the contribution of mizoribine or azathioprine to the anti-CMV effect (see Shiraki K., et al., rinsho to virus, Vol. 18, pp. 25-29, "men-ekifuzenjotai ni okeru virus no saikasseika (Reactivation of virus in immunodeficiency)" (1990); Shiraki K., et al., Biomedica., vol. 5, pp. 65-69, "men-eki yokuseizai to CMV (Immunosuppressants and CMV)" (1990); Shiraki K., et al., Transplant. Proc., Vol. 22, pp. 1682-1685, "Effect of cyclosporine, azathioprine, mizoribine and predonine on replication of human cytomegalovirus" (1990); and Shiraki K., et al., Arch. Virol., Vol. 117, pp. 165-171, "Immunosuppressive dose of azathioprine inhibits replication of human cytomegalovirus" (1991).
The like observation applies to FK 506 and cyclosporine as immunosuppressants. It has been reported that FK 506 has no or slight inhibitory influence on CMV growth (see Shiraki K., et al., J. Antibiotics, Vol. 44, pp. 909-911, "Effect of FK 506 replication of human cytomegalovirus in vitro" (1991)), while cyclosporine reveals the above-mentioned results.
According to the clinical report of Alessiani, et al., no difference was recognized in the incidence of bacterial and fungal infectious diseases between liver transplantation recipients on FK 506+predonine therapy and those on cyclosporine+predonine therapy, while symptomatic CMV infectious disease developed in 0 out of 20 post-transplantation patients on the former therapy and 5 out of 20 post-transplantation patients on the latter therapy (see Alessiani M., et al., Transplant. Proc., Vol. 22, pp. 44-46, "Infection with FK 506 Immunosuppression; Preliminary results with primary therapy" (1990)). These clinical reports back up the CMV proliferation accelerating effect of cyclosporine observed in vitro.
Further, Bia, et al. made a case report that the incidence of CMV infection in post-transplantation patients on azathioprine+steroid therapy was about half that in those on cyclosporine+steroid therapy, and no severe case was observed in the former, both groups of patients having received no anti-T cell globulin (see Bia M. J., et al., Transplantation, Vol. 40, pp. 610-614, "Effect of treatment with cyclosporine versus azathioprine on incidence and severity of cytomegalovirus infection post-transplantation" (1985)).
Development of CMV infection is largely influenced by the immune condition of the host, the degree of immune suppression, and the like and does not seem to be decided simply by the combination of immunosuppressants. Nevertheless, it is understood that the clinical observations of post-transplantation patients account for the influences of immunosuppressants on CMV proliferation in vitro more duly than expected.
The above situation implies possibility to alleviate CMV infectious disease by use of some of drugs currently employed as immunosuppressants which exhibit mild, while not potent, antiviral activity.
The reason why immunosuppressants essentially having weaker anti-CMV activity than general antiviral agents eventually exhibit effective anti-CMV activity will be explained below. Taking CMV-caused pneumonia for instance, it takes about 2 weeks for the very early stage (in which the X-ray picture of the chest demonstrates changes which retrospectively appear abnormal) to develop into the stage which is clinically recognized as CMV penumonia. Tentatively setting the doubling time of CMV in the body at 72 hours (3 days), CMV increases 4 to 5 times within 2 weeks. Assuming that mizoribine or azathioprine controls CMV proliferation to half as described above, the amount of the virus will be controlled by administering mizoribine or azathioprine to one-thirty second through the 5-fold doubling period, i.e., about 3% of the amount of the virus in the case of using no mizoribine or azathioprine. It is considered natural that no CMV-caused disease occurs with such a small amount of CMV in patients on mizoribine or azathioprine therapy.
On the other hand, FK 506 hardly affects CMV proliferation, while cyclosporine accelerates CMV proliferation about twice. Accordingly, the CMV amount in the case of cyclosporine therapy will be 32 times that in the case of FK 506 therapy as calculated in the same manner as above. In addition, FK 506 is concentrated in the lung, the target organ of CMV infection, and the CMV proliferation is suppressed at that concentration by FK 506. This fact appears to contribute to the difference between FK 506 therapy and cyclosporine therapy.
Thus, it has now been revealed that use of an immunosuppressant having weak but effective anti-CMV activity possibly alleviates CMV infectious disease. Conventional studies have never been directed to the relationship between CMV and immunosuppressants from this point of view.
On the other hand, traditional medicines (such as traditional Chinese medicines and traditional Japanese medicines) (it is called crude drugs hereinafter) have been used for therapy for a long number of years, and ample knowledge of their usage, dosage, etc. has been accumulated. Many of crude drugs have minor or substantially no side effects. However, studies on antiviral activity of crude drugs are rare. The literature on this subject now available includes Ito M., et al., Antiviral Research, Vol. 7, pp. 127-137, "Inhibitory effect of glycyrrhizin on the in vitro infectivity and cytopathic activity of the human immunodeficiency virus [HIV (HTLV-III/LAV)]", (1987); Takechi M. and Tanaka Y., Planta Medica, Vol. 42, pp. 69-74, "Purification and characterization of antiviral substance from the bud of Syzygium aromaticum", (1981); Kane C. J. M., et al., Bioscience Reports, Vol. 8, pp. 85-94, "Methyl gallate, methyl-3,4,5-trihydroxybenzoate, is a potent and highly specific inhibitor of herpes simplex virus in vitro: I. Purification and characterization of methyl gallate from Sapium sebiferum", (1988); Nagai T., et al., Chem. Pharm. Bull., Vol. 38, pp. 1329-1332, "Inhibition of influenza virus sialidase and anti-influenza virus activity by plant flavonoids", (1990); and Arai Y., et al., J. of Medical and Pharmaceutical Society for WAKAN-YAKU, Vol. 4, pp. 402-403, "Effect of Bezoar bovis against Chikungunya virus", (1987).
SUMMARY OF THE INVENTION
An object of the present invention is to provide an antiviral agent containing a crude drug having effectiveness on viral diseases, taking the above-mentioned relationship between CMV and immunosuppressants as persuasive and theoretical grounds for usefulness of some crude drugs which possess even non-potent but mild antiviral activity in the treatment of virus-caused diseases.
Another object of the present invention is to provide an antiviral agent containing a crude drug effective on a broad range of viruses.
The present inventors have conducted extensive investigations on a variety of crude drugs, including traditional Chinese medicines, Japanese medicines, and Indonesian medicines, which have conventionally been used as medicines, usage or dosage of which is known, and which involve no or a little side effect. As a result, they have found among them crude drugs having antiviral activity and thus reached the present invention.
The present invention relates to an antiviral agent containing at least one crude drug selected from the group consisting of (A1) the whole plant of Ainsliaea fragrans Champ., (A2)the rhizome of Alpinia officinarum Hance, (A3)the bark of Alyxia stellata Roem., (A4)the bark of Andrographis paniculate Nees, (A5) the root of Andropogon zizaniodes (L.) Urban, (A6)the rhizone of Anemarrhena asphodeloides Bunge, (A7)the leaf of Arctostaphylos uva-ursi (L.) Sprengel, (A8)the seed of Areca catechu L., (A9)the leaf of Artemisia princeps Pamp., (A10)the whole plant of Asiasarum heterotropoides F. Maekawa var. mandshuricum F. Maekawa, (A11)the rhizone of Belamcanda chinensis (L.) DC., (A12)the rhizome of Brainia insignis (Hook.) J. Sm., (A13)the seed of Brucea javanica (L.) Merr., (A14)the root of Bupleurum fakatum L., (A15)the bark of Caesalpinia sappan L., (A16)the leaf of Camellia japonica L., (A17)the bark of Cassia fistula L., (A18)the whole plant of Chamaesyce hyssopifolia, (A19)the bark and branch of Cinnamomum cassia Blume, (A20)the bark of Cinnamomum sintok Blume, (A21)the rhizome of Cnidium officinale Makino, (A22)the rhizome of Coptis chinensis Franch., (A23)the leaf of Cordia spinescens, (A24)the fruit of Cornus officinalis Sieb. et Zucc., (A25)the tuber of Corvdalis hurtscharinorii Besser forma yanhusuo Y. H. Chou et C. C. Hsu, (A26)the fruit of Curculigo orchioides Gaertn., (A27)the rhizome of Curcuma aeroginosa Roxb., (A28)the rhizome of Curcuma xanthorrhiza Roxb., (A29)the rhizome of Cyrtomium fortunei J. Sm., (A30)the rhizome of Drynaria fortunei (Kunze) J. Smith, (A31)the rhizome of Dryopteris crassirhizoma Nakai, (A32)the fruit of Elaeocarpus grandiflorus Smith, (A33)the leaf of Elephantopus scaber L., (A34)the leaf of Epimedium koreanum Nakai, (A35)the leaf of Erythroxylum lucidum, (A35')the trunk of Erythroxylum citrifolium, (A36)the fruit of Evodia rutaecarpa Hook. f. et Thoms., (A37)the fruit of Foeniculum vulgare Mill, (A38)the fruit of Forsythia suspensa Vahl., (A39)the whole plant of Geranium thunbergii Sieb. et Zucc., (A40)the whole plant of Geum japonicum Thung., (A41)the root and stolon of Glycyrrhiza uralensis Fisher (A42)the leaf of Hamelia axillaris Swartz, (A43)the branch and leaf of Jatropha curcas L., (A44)the bark of Juglans mandshurica Maxim., (A45)the root of Lithospermum erythrorhizon Sieb. et Zucc., (A46)the aerial part of Loranthus parasiticus (L.) Merr., (A47)the bark of Machilus thunbergii Sieb. et Zucc., (A48)the bark of Magnolia officinalis Rehd. et Wils., (A49)the rhizome of Matteuccia struthiopteris (L.) Todaro, (A50)the whole insect of Mylabris sidae Fabr., (A51)the root bark of Paeonia suffruticosa Andrews, (A52)the root of Panax ginseng C. A. Meyer, (A53)the bark of Parameria laevigata Moldenke, (A54)the leaf of Perilla frutescens Britton var. acuta Kudo, (A55)the bark of Phellodendron amurense Ruprecht, (A56)the aerial part of Physalis anqulata L., (A57)the rhizome of Plagiogyria matsumureana Makino, (A58)the root of Platycodon grandiflorum (Jacquin) A. DC., (A59)the root of Polygala tenuifolia Willd., (A60)the root and rhizome of Polygonum cuspidatum Sieb. et Zucc., (A61)the hoelen of Poria cocos Wolf, (A62)the spike of Prunella vulgaris L. subsp. asiatica Hara, (A63)the bark of Prunus jamasakura Siebold, (A64)the fruit of Prunus mume Sieb. et Zucc., (A65)the root bark and fruit peel of Punica granatum L., (A66)the bark of Quercus acutissima Carruthers, (A67)the leaf of Quercus salicina Blume, (A68)the fruit of Quisqualis indica L., (A69)the rhizome of Rheum palmatum L., (A70)the gall of Rhus javanica L., (A71)the root of Salvia miltiorrhiza Bunge, (A72)the leaf of Sarcandra glabra (Thunb.) Nakai, (A73)the flores of Schizonepeta tenuifolia Briquet, (A74)the root of Scutellaria baicalensis Georgi, (A75)the whole plant of Serjania mexicana, (A76)the flower bud of Sophora japonica L., (A77) the root of Sophora subprostrata Chun et T. Chen, (A78) the stem of Spatholobus suberectus Dunn, (A79) the rhizome of Struthiopteris niponica (Kunze) Nakai, (A80) the seed of Strychnos nux-vomica L., (A81) the flower bud of SyzygiUm aromaticum (L.) Merr. et Perry, (A82) the bark of Terminalia arjuna Wight et Arn., (A83) the fruit peel of Terminalia belerica Roxb., (A84) the fruit of Terminalia chebula Retzus, (A85) the leaf and branch of Uncaria gambir Roxb., (A86) the whole plant of Usnea misaminensis Vain., (A87) the branch and leaf of Waltheria indica L., (A88) the flower and leaf of Woodfordia floribunda Salisb., (A89) the rhizome of Woodwardia orientalis Sw., (A90) the fruit peel of Zanthoxylum bungeanum Maxim., and (A91) the fruit of Zizyphus jujuba Miller var. inermis Rehder (hereinafter referred to as group A).
The first embodiment of the present invention relates to an antiherpesviral agent containing at least one crude drug selected from the group consisting of (B1) the whole plant of Ainsliaea fragrans Champ., (B2) the rhizome of Alpinia officinarum Hance, (B3) the bark of Alyxia stellata Roem., (B4) the root of Andropogon zizaniodes (L.) Urban, (B5) the seed of Areca catechu L., (B6) the leaf of Artemisia princeps Pamp., (B7) the rhizome of Brainia insignis (Hook.) J. Sm., (B8) the seed of Brucea javanica (L.) Merr., (B9) the bark of Caesalpinia sappan L., (B10) the leaf of Camellia japonica L., (B11) the bark of Cassia fistula L., (B12) the whole plant of Chamaesyce hyssopifolia, (B13) the bark and branch of Cinnamomum cassia Blume, (B14) the bark of Cinnamomum sintok Blume, (B15) the rhizome of Coptis chinensis Franch., (B16) the leaf of cordia spinescens, (B17) the rhizome of Cyrtomium fortunei J. Sm., (B18) the rhizome of Drynaria fortunei (Kunze) J. Smith, (B19) the rhizone of Dryopteris crassirhizoma Nakai, (B20) the fruit of Elaeocarpus grandiflorus Smith, (B21) the leaf of Epimedium koreanum Nakai, (B22) the leaf of Erythroxylum lucidum, (B22') the trunk of Erythroxylum citrifolium, (B23) the fruit of Foeniculum vulgare Mill., (B24) the whole plant of Geranium thunbergii Sieb. et Zucc., (B25) the whole plant of Geum japonicum Thunb., (B26) the leaf of Hamelia axillaris Swartz, (B27) the branch and leaf of Jatropha curcas L., (B28) the bark of Juglans mandshurica Maxim., (B29) the bark of Machilus thunbergii Sieb. et Zucc., (B30) the root bark of Paeonia suffruticosa Andrews, (B31) the leaf of Perilla frutescens Britton var. acuta Kudo, (B32) the bark of Phellodendron amurense Ruprecht, (B33) the rhizome of Plagiogyria matsumureana Makino, (B34) the root of Polygala tenuifolia Willd., (B35) the root and rhizome of Polygonum cuspidatum Sieb. et Zucc., (B36) the spike of Prunella vulgaris L. subsp. asiatica Hara, (B37) the root bark and fruit peel of Punica granatum L., (B38) the bark of Quercus acutissima Carruthers, (B39) the rhizome of Rheum palmatum L., (B40) the gall of Rhus javanica L., (B41) the root of Salvia miltiorrhiza Bunge, (B42) the leaf of Sarcandra glabra (Thunb.) Nakai, (B43) the flores of Schizonepeta tenuifolia Briquet, (B44) the whole plant of Serjania mexicana, (B45) the stem of Spatholobus suberectus Dunn, (B46) the bark of Terminalia arjuna Wight et Arn., (B47) the fruit peel of Terminalia belerica Roxb., (B48) the fruit of Terminalia chebula Retzus, (B49) the branch and leaf of Waltheria indica L., (B50) the flower and leaf of Woodfordia floribunda Salisb., (B51) the rhizome of Woodwardia orientalis Sw., and (B52) the fruit peel of Zanthoxylum bungeanum Maxim. (hereinafter referred to as group B).
The second embodiment of the present invention relates to an antipolioviral agent containing at least one crude drug selected from the group consisting of (C1) the rhizome of Alpinia officinarum Hance, (C2) the bark of Andrographis paniculate Nees, (C3) the root of Andropogon zizaniodes (L.) Urban, (C4) the rhizome of Anemarrhena asphodeloides Bunge, (C5) the leaf of Arctostaphylos uva-ursi (L.) Sprengel, (C6) the seed of Areca catechu L., (C7) the leaf of Artemisia princeps Pamp., (C8) the whole plant of Asiasarum heterotropoides F. Maekawa var. mandshuricum F. Maekawa, (C9) the rhizome of Belamcanda chinensis (L.) DC., (C10) the rhizome of Brainia insignis (Hook.) J. Sm., (C11) the seed of Brucea javanica (L.) Merr, (C12) the bark of Caesalpinia sappan L., (C13) the bark of Cassia fistula L., (C14) the bark of Cinnamomum sintok Blume, (C15) the rhizome of Coptis chinensis Franch., (C16) the fruit of Cornus officinalis Sieb. et Zucc., (C17) the tuber of Corydalis hurtscharinorii Besser forma yanhusuo Y. H. Chou et C. C. Hsu, (C18) the fruit of Curculigo orchioides Gaertn., (C19) the rhizome of Curcuma aeroginosa Roxb., (C20) the rhizome of Curcuma xanthorrhiza Roxb., (C21) the rhizome of Cyrtomium fortunei J. Sm., (C22) the rhizome of Dryopteris crassirhizoma Nakai, (C23) the fruit of Elaeocarpus grandiflorus Smith, (C24) the leaf of Elephantopus scaber L., (C25) the fruit of Eyodia rutaecarpa Hook. f. et Thoms., (C26) the fruit of Foeniculum vulgare Mill., (C27) the fruit of Forsythia suspensa Vahl., (C28) the whole plant of Geranium thunbergii Sieb. et Zucc., (C29) the whole plant of Geum japonicum Thunb., (C30) the bark of Juglans mandshurica Maxim., (C31) the root of Lithospermum erythrorhizon Sieb. et Zucc., (C32) the aerial part of Loranthus parasiticus (L.) Merr., (C33) the bark of Machilus thunbergii Sieb. et Zucc., (C34) the rhizome of Matteuccia struthiopteris (L.) Todaro, (C35) the whole insect of Mylabris sidae Fabr., (C36) the root bark of Paeonia suffruticosa Andrews, (C37) the bark of Parameria laevigata Moldenke, (C38) the bark of Phellodendron amurense Ruprecht, (C39) the aerial part of Physalis angulata L., (C40) the rhizome of Plagiogyria matsumureana Makino, (C41) the root of Polygala tenuifolia Willd., (C42) the root and rhizome of Polygonum cuspidatum Sieb. et Zucc., (C43) the bark of Prunus jamasakura Siebold, (C44) the fruit of Prunus mume Sieb. et Zucc., (C45) the root bark and fruit peel of Punica granatum L., (C46) the bark of Quercus acutissima Carruthers, (C47) the leaf of Quercus salicina Blume, (C48) the fruit of Quisgualis indica L., (C49) the rhizome of Rheum palmatum L., (C50) the gall of Rhus javanica L., (C51) the root of Scutellaria baicalensis Georgi, (C52) the flower bud of Sophora japonica L., (C53) the root of Sophora subprostrata Chun et T. Chen, (C54) the stem of Spatholobus suberectus Dunn, (C55) the rhizome of Struthiopteris niponica (Kunze) Nakai, (C56) the seed of Strychnos nux-vomica L., (C57) the flower bud of Syzygium aromaticum (L.) Merr. et Perry, (C58) the bark of Terminalia arjuna Wight et Arn., (C59) the fruit peel of Terminalia belerica Roxb., (C60) the fruit of Terminalia chebula Retzus, (C61) the leaf and branch of Uncaria gambir Roxb., (C62) the whole plant of Usnea misaminensis Vain., (C63) the flower and leaf of Woodfordia floribunda Salisb., (C64) the rhizome of Woodwardia orientalis Sw., and (C65) the fruit peel of Zanthoxylum bungeanum Maxim. (hereinafter referred to as group C).
The third embodiment of the present invention relates to an anti-measles virus agent containing at least one crude drug selected from the group consisting of (D1) the seed of Areca catechu L., (D2) the leaf of Artemisia princeps Pamp., (D3) the rhizome of Belamcanda chinensis (L.) DC., (D4) the rhizome of Brainia insignis (Hook.) J. Sm., (D5) the seed of Brucea javanica (L.) Merr, (D6) the bark of Caesalpinia sappan L., (D7) the bark of Cassia fistula L., (D8) the bark of Cinnamomum sintok Blume, (D9) the rhizome of Cnidium officinale Makino, (D10) the rhizome of Coptis chinensis Franch., (D11) the rhizome of Cyrtomium fortunei J. Sm., (D12) the fruit of Elaeocarpus grandiflorus Smith, (D13) the leaf of Elephantopus scaber L., (D14) the fruit of Foeniculum vulgare Mill., (D15) the fruit of Forsythia suspensa Vahl., (D16) the whole plant of Geranium thunbergii Sieb. et Zucc., (D17) the root and stolon of Glycyrrhiza uralensis Fisher, (D18) the bark of Juglans mandshurica Maxim., (D19) the bark of Magnolia officinalis Rehd. et Wils., (D20) the whole insect of Mylabris sidae Fabr., (D21) the root bark of Paeonia suffruticosa Andrews, (D22) the bark of Phellodendron amurense Ruprecht, (D23) the rhizome of Plagiogyria matsumureana Makino, (D24) the root of Platycodon grandiflorum (Jacquin) A. DC., (D25) the root of Polygala tenuifolia Willd., (D26) the root bark and fruit peel of Punica granatum L., (D27) the bark of Quercus acutissima Carruthers, (D28) the rhizome of Rheum palmatum L., (D29) the gall of Rhus javanica L., (D30) the root of Scutellaria baicalensis Georgi, (D31) the stem of Spatholobus suberectus Dunn, (D32) the flower bud of Syzygium aromaticum (L.) Merr. et Perry, (D33) the bark of Terminalia arjuna Wight et Arn., (D34) the fruit peel of Terminalia belerica Roxb., (D35) the fruit of Terminalia chebula Retzus, (D36) the flower and leaf of Woodfordia floribunda Salisb., (D37) the rhizome of Woodwardia orientalis Sw., and (D38) the fruit of Zizyphus jujuba Miller var. inermis Rehder (hereinafter referred to as group D).
The forth embodiment of the present invention relates to an anti-varicella-zoster virus agent containing at least one crude drug selected from the group consisting of (E1) the rhizome of Alpinia officinarum Hance, (E2) the seed of Areca catechu L., (E3) the leaf of Artemisia princeps Pamp., (E4) the root of Bupleurum fakatum L., (E5) the bark of Cassia fistula L., (E6) the rhizome of Coptis chinensis Franch., (E7) the rhizome of Cyrtomium fortunei J. Sm., (E8) the rhizome of Drynaria fortunei (Kunze) J. Smith., (E9) the whole plant of Geranium thunbergii Sieb. et Zucc., (E10) the root and stolon of Glycyrrhiza uralensis Fisher, (E11) the bark of Juglans mandshurica Maxim., (E12) the root bark of Paeonia suffruticosa Andrews, (E13) the root of Panax ginseng C. A. Meyer, (E14) the bark of Phellodendron amurense Ruprecht, (E15) the rhizome of Plagiogyria matsumureana Makino, (E16) the root of Polygala tenuifolia Willd., (E17) the root and rhizome of Polygonum cuspidatum Sieb. et Zucc., (E18) the hoelen of Poria cocos Wolf, (E19) the root bark and fruit peel of Punica granatum L., (E20) the bark of Quercus acutissim Carruthers, (E21) the rhizome of Rheum palmatum L., (E22) the gall of Rhus javanica L., (E23) the flower bud of Syzygium aromaticum (L.) Merr. et Perry, (E24) the bark of Terminalia arjuna Wight et Arn., (E25) the fruit of Terminalia chebula Retzus, (E26) the rhizome of Woodwardia orientalis Sw., and (E27) the rhizome of Dryopteris crassirhizoma Nakai (hereinafter referred to as group E).
The fifth embodiment of the present invention relates to an anti-CMV agent containing at least one crude drug selected from the group consisting of (F1) the seed of Areca catechu L., (F2) theleaf of Artemisia princeps Pamp., (F3) the bark of Cassia fistula L., (F4) the rhizome of Coptis chinensis Franch., (F5) the rhizome of Cyrtomium fortunei J. Sm., (F6) the rhizome of Drynaria fortunei (Kunze) J. Smith, (F7) the whole plant of Geranium thunbergii Sieb. et Zucc., (F8) the whole plant of Geum japonicum Thunb., (F9) the bark of Juglans mandshurica Maxim., (F10) the bark of Machilus thunbergii Sieb. et Zucc., (F11) the root bark of Paeonia suffruticosa Andrews, (F12) the bark of Phellodendron amurense Ruprecht, (F13) the rhizome of Plagiogyria matsumureana Makino, (F14) the root of Polygala tenuifolia Willd., (F15) the root and rhizome of Polygonum cuspidatum Sieb. et Zucc., (F16) the root bark and fruit peel of Punica granatum L., (F17) the bark of Quercus acutissima Carruthers, (F18) the rhizome of Rheum palmatum L., (F19) the gall of Rhus javanica L., (F20) the flower bud of Syzygium aromaticum (L.) Merr. et Perry, (F21) the bark of Terminalia arjuna Wight et Arn., (F22) the fruit of Terminalia chebula Retzus, and (F23) the rhizome of Woodwardia orientalis Sw. (hereinafter referred to as group F).
The sixth embodiment of the present invention relates to an anti-DNA virus and anti-RNA virus agent containing at least one crude drug selected from the group consisting of (G1) the seed of Areca catechu L., (G2) the leaf of Artemisia princeps Pamp., (G3) the rhizome of Brainia insignis (Hook.) J. Sm., (G4) the seed of Brucea javanica (L.) Merr., (G5) the bark of Caesalpinia sappan L., (G6) the bark of Cassia fistula L., (G7) the bark of Cinnamomum sintok Blume, (G8) the rhizome of Coptis chinensis Franch., (G9) the rhizome of Cyrtomium fortunei J. Sm., (G10) the fruit of Elaeocarpus grandiflorus Smith, (G11) the fruit of Foeniculum vulgare Mill., (G12) the whole plant of Geranium thunbergii Sieb. et Zucc., (G13) the bark of Juglans mandshurica Maxim., (G14) the root bark of Paeonia suffruticosa Andrews, (G15) the bark of Phellodendron amurense Ruprecht, (G16) the rhizome of Plagiogyria matsumureana Makino, (G17) the root of Polygala tenuifolia Willd., (G18) the root bark and fruit peel of Punica granatum L., (G19) the bark of Quercus acutissima Carruthers, (G20) the rhizome of Rheum palmatum L., (G21) the gall of Rhus javanica L., (G22) the stem of Spatholobus suberectus Dunn, (G23) the flower bud of Syzygium aromaticum (L.) Merr. et Perry, (G24) the bark of Terminalia arjuna Wight et Arn., (G25) the fruit peel of Terminalia belerica Roxb., (G26) the fruit of Terminalia chebula Retzus, (G27) the flower and leaf of Woodfordia floribunda Salisb., (G28) the rhizome of Woodwardia orientalis Sw. (hereinafter referred to as group G).
The seventh embodiment of the present invention relates to an antiherpesviral agent containing at least one crude drug selected from the group B as mentioned above and the other antiviral agent.

DETAILED DESCRIPTION OF THE INVENTION
All viruses are classified into DNA viruses and RNA viruses. Families included inder DNA viruses include Poxviridae, Herpesviridae, Adenoviridae, Papovaviridae, Hepadnaviridae, and Parvoviridae. Familes included under RNA viruses include Reoviridae, Picornaviridae, Togaviridae, Flaviviridae, Coronaviridae, Rhabdoviridae, Paramyxoviridae, Orthomyxoviridae, Filoviridae, Bunyaviridae, Arenaviridae, Caliciviridae and Retroviridae.
Familes belonging to DNA viruses have many basic traits in common in DNA replication mechanism. RNA viruses also have characteristics in common with each other in the growth step. The common characteristics include a number of cell-dependent DNA replication-related enzymes, complementarily acting cell factors, and RNA synthesis by RNA dependent RNA polymerase (RNA replicase), and so on. Therefore, a drug exhibiting inhibitory activity on proliferation of a typical DNA virus, e.g., the Herepesviridae, or a typical RNA virus, e.g., the Picornaviridae, is expected to be effective as an antiviral agent common to a wide range of DNA or RNA viruses.
Viruses belonging to Picornaviridae include poliovirus, echovirus, coxsackie virus, enterovirus, and rhinovirus. Diseases caused by these viruses include acute anterior poliomyelitis, hand-foot-and-mouth disease, herpangina, myopericarditis, epidermic myalgia, enanthema, acute hemorrhagic conjuctivitis, summer cold, aseptic meningitis, hepatitis type A, coryza, respirator infectious diseases, and the like.
Viruses-zoster belonging to Paramyxoviridae include parainfluenze virus, mumps virus, measles virus, and RS virus. Diseases caused by these viruses include pharyngitis, upper respiratory tract infectious disease, bronchitis, pneumonia, measles, mumps, and acute respiratory tract infectious disease.
Varicella virus belongs to the family Herpesviridae and causes varicella in children aged between 2 and 8 easily by droplet infection or contagen. Herpes zoster mostly occurs in adults having once suffered from varicella and aged over 50.
CMV also belongs to the family Herpesviridae. Besides the above-mentioned problem, if a woman is first infected with CMV during pregnancy, it sometimes happens that the fetus is directly infected to develop congenital cytomegalic inclusion body disease, etc. Further, CMV often plays a main role in opportunistic infection in acquired immunodeficiency syndrome (AIDS).
The crude drugs belonging to group B have antiherpesviral activity; those of group C antipolioviral activity; those of group D anti-measles virus activity; those of group E anti-varicella-zoster virus activity; those of group F anti-CMV activity; and those of group G anti-DNA virus and anti-RNA virus activity. Group A includes all crude drugs of the present invention.
The dosage, administration route, etc. of the crude drugs included under group A, inclusive of groups B to G, are well known, and the known dose levels produce no or slight side effects.
2 to 10 g of the crude drug according to the present invention is added to water at a ratio of 50 ml water per g of the crude drug, and the mixture is boiled and concentrated to half the original volume. The resulting extract can be orally administered three times a day at a dose of one-third of the extract.
In particular, as hereinafter described, when at least one of the crude drugs of group B is used in combination of acyclovir, a known antiviral agent, there is produced synergistic antiviral activity, by which the requisite dose of acyclovir can be reduced, and appearance of acyclovir-resistant viruses will be inhibited.
Accordingly, the crude drug according to the present invention whose fraction having been absorbed through the digestive tracts exhibits antiviral activity, or a combination of such a crude drug and other known antiviral agent is considered effective in prevention and treatment of the above-mentioned diseases caused by herpes simplex virus (e.g., herpes simplex and its complication), diseases caused by varicella-zoster virus (e.g., varicella, shingles and complications thereof), and diseases caused by CMV (e.g., pneumonia, hepatitis, and conjuctivitis).
Test Examples of the present invention are described below. All the plants and insects used in the tests were purchased at the market. The part of the plants or insects used was as described above.
Preparation of Extract
Each plant or insect was extracted at reflux with distilled water under a neutral condition, followed by concentration and drying to obtain an aqueous extract of the crude drug. It is generally spread to use methanol in place of water as an extracting solvent for obtaining hydrophobic substances in higher concentrations. The test of antiviral activity was also conducted on the methanol extract of some of crude drugs.
For example, 100 g of the rhizome of Cyrtomium fortunei J. Sm.# (available on the Hong Kong market) was extracted at reflux with 1.5 l of distilled water, and the extract was concentrated at 40.degree. C. under reduced pressure and lyophilized to obtain 12.3 g of an aqueous extract of Cyrtomium fortunei J. Sm.# (the term "extract" hereinafter used means an aqueous extract unless otherwise specified). Cyrtomium fortunei J. Sm. with a sharp mark means a mixture of Brainia insignis (Hook.) J. Sm., Cyrtonium fortunei J. Sm., Dryopteris crassirhizoma Nakai, Matteuccia struthiopteris (L.) Todaro, Plagiogyria matsumureana Makino, Struthiopteris niponica (Kunze) Nakai and Woodwardia orientalis Sw. The Cyrtomium fortunei J. Sm.# is available on the Hong Kong market.
A methanol extract was obtained by, for example, extracting 20 g of a crude drug with 500 ml. of methanol for 3 hours and removing methanol by distillation under reduced pressure.
Each crude drug extract was ground and suspended in water in a prescribed concentration. The suspension was heated in a boiling water bath for 10 minutes and centrifuged (3000 rpm.times.10 min.). The resulting supernatant, an extract solution, was used for the antiviral test.
Test Virus
Herpes simplex virus type I (Seibert strain), poliovirus (vaccine strain and Sabin strain), measles virus (vaccine strain and Tanabe strain), varicella-zoster virus (Kawaguchi strain), and CMV (Town strain) were used.
Vero cells or HEL cells derived from human embryonal lungs were infected with each virus except varicella-zoster virus. After incubation for a prescribed time, the infected cells were subjected to freezing and thawing three times, followed by centrifugation (300 rpm.times.15 mins.). The supernatant was used as a virus suspension.
A varicella-zoster virus suspension was prepared according to the method of Shiraki K and Takahashi M., J. Gen. Virol., vol 61, pp. 271-275, "Virus particles and glycoproteines excreted from cultured cells infected with varicella-zoster virus".
The thus prepared virus liquids were preserved, if necessary, at -80.degree. C.
Cells to be Infected with Virus
1. Vero cells originated from the kidney of African green monkey were cultured in a minimum essential medium (hereinafter abbreviated as MEM) containing 5% bovine fetal serum (BFS).
2. Vero E6 cells were obtained by cloning of the above-mentioned Vero cells and have high susceptibility to infection with various viruses.
3. HEL cells originated from human embryonal lungs which have high susceptibility to infection with varicella-zoster virus and CMV.
Cultivation of all these cells were conducted in CO.sub.2 incubator at 37.degree. C.
Plague Formation Test Method
0.2 ml of a virus suspension diluted to 100 PFU/0.2 ml was inoculated to a monolayer culture of Vero cells, Vero E6 cells or HEL cells in a plastic dish having a diameter of 60 mm. The virus was allowed to adsorb on the monolayer cells at 37.degree. C. for 1 hour.
After adsorption, 5 ml of 2% BFS-added MEM containing each extract solution in a prescribed concentration and 0.8% methyl cellulose was pliled on the monolayer cells, and the system was cultured at 37.degree. C. for 2 to 5 days to observe plaque formation. The cultured monolayer cells were fixed with formalin and stained with a 0.03% Methylene Blue solution for measuring the number of plaques.
Where the above-mentioned test was replicated several times with the same concentration of the extract solution, the results obtained sometimes show slight scatter. This seems attributed to the error in detection and judgement of plaques due to slight variation of susceptibility depending on the cultivation conditions, such as the lot of serum, the culture plate, the cell density, etc.
TEST EXAMPLE 1
Antiherpesviral activity of the crude drugs shown in Table 1 at a concentration shown were examined by using Vero cells or Vero E6 cells by the plaque formation test. The results obtained are shown in Table 1 below. It can be seen from the Table that the extract of the crude drug according to the present invention significantly reduced the efficiency of plaque formation at such a concentration as low as 500 .mu.g/ml or less and thus exhibited herpesvirus growth inhibitory activity.
TABLE 1__________________________________________________________________________Antiherpesviral Activity Plaque Formation Efficiency (%) Species Part 100 .mu.m/ml 300 .mu.m/ml 500 .mu.m/ml__________________________________________________________________________(B1)Ainsliaea fragrans Champ. whole plant 55.7 51.7(B2)Alpinia officinarum Hance rhizome 106.8 0(B3)Alyxia stellata Roem. bark 59.8 57.9(B4)Andropogon zizaniodes (L.) Urban root 73.8 39.3(B5)Areca catechu L. seed 0 0(B6)Artemisia princeps Pamp. leaf 65 0(B7)Brainia insignia (Hook.) J. Sm. rhizome 62.7 0(B8)Brucea jayanica (L.) Merr. seed 6.3 0(B9)Caesalpinia sappan L. bark 0 0(B10)Camellia japonica L. leaf 89.7 54.8(B11)Cassia fistula L. bark 67 0(B12)Chamaesyce hyssopifolia whole plant 35.6(B13)Cinnamomum cassia Blume bark, branch 69.5 21.5(B14)Cinnamomum sintok Blume bark 0 0(B15)Coptis chinensis Franch. rhizome 74.1 0(B16)Cordia spinescens leaf 6(B17)Cyrtomium fortunei J. Sm. rhizome 59.6 0(B18)Drynaria fortunei (Kunze) J. Smith rhizome 87.4 0(B19)Dryopteris crassirhizoma Nakai rhizome 62.1 51.7(B20)Elaeocarpus grandiflorus Smith fruit 0 0(B21)Epimedium koreanum Nakai leaf 65.1 42.3(B22)Erythroxylum citrifolium trunk 33.6(B22')Erythroxylum lucidum leaf 41.8(B23)Foeniculum vulgare Mill. fruit 67.5 47.5(B24)Geranium thunbergii Sieb. et Zucc. whole plant 91.6 23.5(B25)Geum japonicum Thunb. whole plant 81.7 0(B26)Hamelia axillaris Swartz leaf 35.3(B27)Jatropha curcas L. branch, leaf 32(B28)Juglans mandshurica Maxim. bark 73.6 0(B29)Machilus thunbergii Sieb. et Zucc. bark 98.6 17.8(B30)Paeonia suffruticosa Andrews root bark 96.5 0(B31)Perilla frutescens Britton var. leaf 67.2 45.2acuta Kubo(B32)Phellodendron amurense Ruprecht bark 85.3 29.4(B33)Plagiogyria matsumureana Makino rhizome 76.1 50.7(B34)Polygala tenuifolia Willd. root 118.6 0(B35)Polygonum cuspidatum Sieb. et Zucc. root, 93.4 0 rhizome(B36)Prunella vulgaris L. subsp. asiatica Hara spike 66.1 0(B37)Punica granatum L. root bark, 0 0 fruit peel(B38)Quercus acutissima Carruthers bark 38.2 9.8(B39)Rheum palmatum L. rhizome 93.2 0(B40)Rhus javanica L. gall 0 0(B41)Salvia miltiorrhiza Bunge root 89.9 58.6(B42)Sarcandra glabra (Thunb.) Nakai leaf 67.7 51.6(B43)Schizonepeta tenuifolia Briquet flores 73.8 48.4(B44)Serjania mexicana whole plant 37.6(B45)Spatholobus suberectus Dunn stem 92.2 0(B46)Terminalia arjuna Wight et Arn. bark 56.3 0(B47)Terminalia belerica Roxb. fruit peel 70.8 0(B48)Terminalia chebula Retzus fruit 99.6 0(B49)Waltheria indica L. branch, 27.2 leaf(B50)Woodfordia floribunda Salisb. flower, 0 0 leaf(B51)Woodwardia orientalis Sw. rhizome 51.6 0(B52)Zanthoxylum bungeanum Maxim. fruit peel 107.7 20.3__________________________________________________________________________
TEST EXAMPLE 2
Antipolioviral activity of the crude drugs shown in Table 2 (group C) was determined in the same manner as in Test Example 1. The results are shown in the Table.
The crude drug extract solution shown in Table 2 showed cytotoxicity at a concentration of about 300.mu.g/ml or 500 .mu.g/ml. However, from the fact that these crude drugs have been orally administered for years with no or only slight side effects observed, cytotoxicity of these crude drugs appears to give rise to no problem.
Through screening various crude drugs for antiviral activity against poliovirus as a typical species of the viruses belonging to the family Picornaviridae as described above, medicines having antiviral activity against viruses belonging to Picornaviridae and having the similar physicochemical properties and proliferation mechanism in common can be developed.
The crude drugs belonging to group C whose fraction absorbed through the digestive tract exhibits antiviral activity against poliovirus are considered effective for prevention and treatment of the diseases caused by viruses belonging to the family Picornaviridae.
TABLE 2__________________________________________________________________________Antipolioviral Acitivity Plaque Formation Efficiency (%) Species Part 100 .mu.m/ml 300 .mu.m/ml 500 .mu.m/ml__________________________________________________________________________(C1)Alpinia officinarum Hance rhizome 58.9 11.5(C2)Andrographis paniculate Nees bark 88.1 55.1(C3)Andropogon zizaniodes (L.) Urban root 19.5 7.6(C4)Anemarrhena asphodeloides Bunge rhizome 44.2 30.1(C5)Arctostaphylos uva-ursi (L.) Sprengel leaf 63.3 54.6(C6)Areca catechu L. seed 86.5 0.sup.+(C7) Artemisia princeps Pamp. leaf 84.3 40.5(C8)Asiasarum heterotropoides F. Maekawa var. whole plant 73.2 43mandshuricum F. Maekawa(C9)Belamcanda chinensis (L.) DC. rhizome 97.4 55.1(C10)Brainia insignis (Hook.) J. Sm. rhizome 61 54.6(C11)Brucea javanica (L.) Merr. seed 0 0(C12)Caesalpinia sappan L. bark 0 0.sup.+(C13) Cassia fistula L. bark 52.1 2.8(C14)Cinnamomum sintok Blume bark 102.5 0.sup.+(C15) Coptis chinensis Franch. rhizome 89.7 0(C16)Cornus officinalis Sieb. et Zucc. fruit 82.2 57.2(C17)Corydalis hurtscharinorii Besser tuber 77.4 6.4forma yanhusuo Y. H. Chou et C. C. Hsu(C18)Curculigo orchioides Gaertn. fruit 88.1 42.4(C19)Curcuma aeroginosa Roxb. rhizome 34 29.7(C20)Curcuma xanthorrhiza Roxb. rhizome 39 35.6(C21)Cyrtomium fortunei J. Sm. rhizome 66.5 0.sup.+(C22) Dryopteris crassirhizoma Nakai rhizome 93.8 48.4(C23)Elaeocarpus grandifluorus Smith fruit 0 0(C24)Elephantopus scaber L. leaf 20.8 17.7(C25)Evodia rutaecarpa Hook. f. et Thoms. fruit 82.1 41.1(C26)Foeniculum vulgare Mill. fruit 31.3 28.1(C27)Forsythia suspensa Vahl. fruit 72.9 30.7(C28)Geranium thunbergii Sieb. et Zucc. whole plant 30 21.4(C29)Geum japonicum Thunb. whole plant 90.4 0(C30)Juglans nandshurica Maxim. bark 35.5 4.8(C31)Lithospermum erythrorhizon Sieb. et Zucc. root 61.9 50(C32)Loranthus parasiticus (L.) Merr. aerial part 83.3 29.2(C33)Machilus thunbergii Sieb. et Zucc. bark 55.5 0(C34)Matteuccia struthiopteris (L.) Todaro rhizome 65.9 34.6(C35)Mylabris sidae Fabr. whole insect 88.4 0.sup.+(C36) Paeonia suffruticosa Andrews root bark 124.2 35.5(C37)Parameria laevigata Moldenke bark 43.8 32.3(C38)Phellodendron amurense Ruprecht bark 103.4 43.3(C39)Physalis angulata L. aerial part 17.6 0(C40)Plagiogyria matsumureana Makino rhizome 73.9 0(C41)Polygala tenuifolia Willd. root 86.4 0.sup.+(C42) Polygonum cuspidatum Sieb. et Zucc. root, 88 38 rhizome(C43)Prunus jamasakura Siebold bark 99.7 46.9(C44)Prunus mume Sieb. et Zucc. fruit 75.8 38.9(C45)Punica granatum L. root bark, 79.5 0 fruit peel(C46)Quercus acutissima Carruthers bark 98.4 35.6(C47)Quercus salicina Blume leaf 48 30.6(C48)Quisqualis indica L. fruit 28.2 16.9(C49)Rheum palmatum L. rhizome 13.1 0.sup.+(C50) Rhus javanica L. gall 65.1 4.3(C51)Scutellaria baicalensis Georgi root 66 14.7(C52)Sophora japonica L. flower bud 67.1 49.5(C53)Sophora subprostrata Chun et T. Chen root 99.5 57.7(C54)Spatholobus suberectus Dunn stem 80.2 0(C55)Struthiopteris niponica (Kunze) Nakai rhizome 66 33.4(C56)Strychnos nux-vomica L. seed 39.6 47.9(C57)Syzygium aromaticum (L.) Merr. et Perry flower bud 55.1 0.sup.+(C58) Terminalia arjuna Wight et Arn. bark 22.5 0(C59)Terminalia belerica Roxb. fruit peel 7.4 0(C60)Terminalla chebula Retzus fruit 32.6 0.7(C61)Uncaria gambir Roxb. leaf, branch 85.3 24.2(C62)Usnea misaminensis Vain. whole plant 111.1 53.7(C63)Woodfordia floribunda Salisb. flower, 0 0.sup.+leaf(C64) Woodwardia orientalis Sw. rhizome 30.5 0.sup.+(C65) Zanthoxylum bungeanum Maxim. fruit peel 80.4 14.3__________________________________________________________________________ Note: .sup.+ : Cytotoxicity was observed.
TEST EXAMPLE 3
Anti-measles virus activity of the crude drugs shown in Table 3 (group D) was determined in the same manner as in Test Example 1. The results obtained are shown in Table 3. The crude drugs belonging to group D were thus proved to have anti-measles virus activity.
Through screening various crude drugs for antiviral activity against measle virus as a typical species of the viruses belonging to the family Paramyxoviridae as described above, medicines having antiviral activity against viruses belonging to Paramyxoviridae and having the similar physicochemical properties and proliferation mechanism in common can be developed.
The crude drugs belonging to group D whose fraction absorbed through the digestive tract exhibits antiviral activity against measle virus are considered effective for prevention and treatment of the diseases caused by viruses belonging to the family Paramyxoviridae.
TABLE 3__________________________________________________________________________Anti-measle Virus Activity Plaque Formation Efficiency (%) Species Part 100 .mu.m/ml 300 .mu.m/ml 500 .mu.m/ml__________________________________________________________________________(D1)Areca catechu L. seed 93.6 0(D2)Artemisia princeps Pamp. leaf 10.8 13.6(D3)Belamcanda chinensis (L.) DC. rhizome 100 0(D4)Brainia insignis (Hook.) J. Sm. rhizome 109.7 5.1(D5)Brucea javanica (L.) Merr. seed 37.2 0(D6)Caesalpinia sappan L. bark 0 0(D7)Cassia fistula L. bark 100 14.1(D8)Cinnamomum sintok Blume bark 153 0(D9)Cnidium officinale Makino rhizome 64.1 43.5(D10)Coptis chinensis Franch. rhizome 5.6 0(D11)Cyrtomium fortunei J. Sm. rhizome 112.1 0(D12)Elaeocarpus grandiflorus Smith fruit 0 0(D13)Elephantopus scaber L. leaf 85 47.8(D14)Foeniculum vulgare Mill. fruit 99.3 47.5(D15)Forsythia suspensa Vahl. fruit 26.1 6.7(D16)Geranium thunbergii Sieb. et Zucc. whole plant 104.7 0(D17)Glycyrrhiza uralensis Fischer root, stolon 59.4 50(D18)Juglans mandshurica Maxim. bark 96.5 3.5(D19)Magnolia officinalis Rehd. et Wils. bark 71.6 34.1(D20)Mylabris sidae Fabr. whole insect 60.2 0(D21)Paeonia suffruticosa Andrews root bark 119.1 0(D22)Phellodendron amurense Ruprecht bark 17.2 0.6(D23)Plagiogyria matsumureana Makino rhizome 90.8 84.6(D24)Platycodon grandiflorum (Jacquin) A. DC. root 95.1 0(D25)Polygala tenuifolia Willd. root 100 0(D26)Punica granatum L. root bark, 0 0 fruit peel(D27)Quercus acutissima Carruthers bark 83.3 38.5(D28)Rheum palmatum L. rhizome 108.1 0(D29)Rhus javanica L. gall 3.4 0(D30)Scutellaria baicalensis Georgi root 12.3 5(D31)Spatholobus suberectus Dunn stem 75.4 0(D32)Syzygium aromaticum (L.) Merr. et Perry flower bud 98.6 0(D33)Terminalia arjuna Wight et Arn. bark 100 0(D34)Terminalia belerica Roxb. fruit peel 104.4 0(D35)Terminalia chebula Retzus fruit 152 0(D36)Woodfordia floribunda Salisb. flower, 0 0 leaf(D37)Woodwardia orientalis Sw. rhizome 105.4 0(D38)Zizyphus jujuba Miller var. fruit 74.7 51.8inernis Rehder__________________________________________________________________________
TEST EXAMPLE 4
Anti-varicella-zoster virus activity of the crude drugs shown in Table 4 (group E) was determined in the same manner as in the Test Example 1. The results obtained are shown in Table 4. The crude belonging to group E were thus proved to have anti-varicella-zoster virus activity. In the case of using Plagiogyria matsumureana Makino, methanol extract was used in place of water extract.
TABLE 4__________________________________________________________________________Anti-varicella-zoster Virus Activity Plaque Formation Efficiency (%) Species Part 100 .mu.g/ml 200 .mu.g/ml__________________________________________________________________________(E1)Alpinia officinarum Hance rhizome 0(E2)Areca catechu L. seed 0(E3)Artemisia princeps Pamp. leaf 46.7(E4)Bupleurum fakatum L. root 31.2(E5)Cassia fistula L. bark 0(E6)Coptis chinensis Franch. rhizome 0(E7)Cyrtomium fortunei J. Sm. rhizome 0(E8)Drynaria fortunei (Kunze) J. Smith rhizome 0(E9)Geranium thunbergii Sieb. et Zucc. whole plant 14.7(E10)Glycyrrhiza uralensis Fisher root, stolon 17.2(E11)Juglans mandshurica Maxim. bark 0(E12)Panax ginseng C.A. Meyer root 55.9(E13)Paeonia suffruticosa Andrews root bark 39.7(E14)Phellodendron amurense Ruprecht bark 0(E15)Plagiogyria matsumureana Makino rhizome 0(E16)Polygala tenuifolia Willd. root 0(E17)Polygonum cuspidatum Sieb. et Zucc. root, rhizome 0(E18)Poria cocos Wolf hoelen 50.5(E19)Punica granatum L. root bark, 0 fruit peel(E20)Quercus acutissima Carruthers bark 0(E21)Rheum palmatum L. rhizome 0(E22)Rhus javanica L. gall 0(E23)Syzygium aromaticum (L.) Merr. et Perry flower bud 26.6(E24)Terminalia arjuna Wight et Arn. bark 0(E25)Terminalia chebula Retzus fruit 0(E26)Woodwardia orientalis Sw. rhizome 0(E27)Dryopteris crassirhizoma Nakai rhizome 1.6__________________________________________________________________________
TEST EXAMPLE 5
Anti-CMV activity of the crude drugs shown in Table 5 (group F) was determined in the same manner as in Test Example 1. The results obtained are shown in Table 5. From these results, the crude drugs belonging to group F were proved to have anti-CMV activity. In the case of using Plagiogyria matsumureana Makino, methanol extract was used in place of water extract.
TABLE 5__________________________________________________________________________Anti-CMV Activity Plaque Formation Efficiency (%) Species Part 100 .mu.g/ml 200 .mu.g/ml__________________________________________________________________________(F1)Areca catechu L. seed 17.8(F2)Artemisia princeps Pamp. leaf 0(F3)Cassia fistula L. bark 0(F4)Coptis chinensis Franch. rhizome 0(F5)Cyrtomium fortunei J. Sm. rhizome 21.7(F6)Drynaria fortunei (Kunze) J. Smith rhizome 0(F7)Geranium thunbergii Sieb. et Zucc. whole plant 0(F8)Geum japonicum Thunb. whole plant 0(F9)Juglans mandshurica Maxim. bark 0(F10)Machilus thunbergii Sieb. et Zucc. bark 0(F11)Paeonia suffruticosa Andrews root bark 47.4(F12)Phellodendron amurense Ruprecht bark 0(F13)Plagiogyria matsumureana Makino rhizome 33.6(F14)Polygala tenuifolia Willd. root 0(F15)Polygonum cuspidatum Sieb. et Zucc. root, rhizome 0(F16)Punica granatum L. root bark, 0 fruit peel(F17)Quercus acutissima Carruthers bark 0(F18)Rheum palmatum L. rhizome 0(F19)Rhus javanica L. gall 0(F20)Syzygium aromaticum (L.) Merr. et Perry flower bud 0(F21)Terminalia arjuna Wight et Arn. bark 0(F22)Terminalia chebula Retzus fruit 0(F23)Woodwardia orientalis Sw. rhizome 0__________________________________________________________________________
TEST EXAMPLE 6
Antiviral activity of the crude drugs shown in Table 6 below was examined in the same manner as in Test Example 1, except for using Vero E6 cells and changing the concentration of the extract solution. The results obtained are shown in Table 6. It can be seen that the extract of the crude drug according to the present invention significantly reduced the efficiency of plaque formation at such a concentration as low as 300 .mu.g/ml or less and thus exhibited antiviral activity.
Cytotoxicity of each extract solution was also determined. While some of the extract solutions tested showed cytotoxicity at a concentration of 300 .mu.g/ml, they exhibited antiviral activity at low concentrations showing no cytotoxicity.
TABLE 6__________________________________________________________________________Antiviral Activity (in Vero E6 Cells) Concn. of Extract Solution Plaque Formation Efficiency (%)Species (part) (.mu.g/ml) Herpes I Polio Measles Cytotoxicity__________________________________________________________________________Cassia fistula L. (bark) 0 100 100 100 10 101.4 101.7 97.5 50 92.2 66.9 95.6 100 29.5 37.7 76.5 300 0 2.8 7 .+-.Terminalia arjuna Wight et Arn. 0 100 100 100(bark) 10 108.8 86.8 92.1 50 78.3 88.7 92.7 100 47 0 0 + 300 0 0 0 +Alpinia officinarum Hance (rhizome) 0 100 100 100 10 105.1 98.8 92.4 50 101.8 56 89.5 100 76.5 30.7 106.7 300 0 0 14.9 +Juglans mandshurica Maxim. (bark) 0 100 100 100 10 96.3 75.1 91.4 50 112 57.2 89.2 100 73.6 24.1 86.3 300 0 4.8 3.5 +Punica granatum L. (fruit peel) 0 100 100 100 10 87.9 45.1 84 50 102.1 25.4 121.8 100 83.2 14.6 102.8 300 2.9 5.8 0 +Geranium thunbergii Sieb. et Zucc. 0 100 100 100(whole plant) 10 100.7 66.8 94.7 50 90.7 40.7 122.7 100 91.6 30 104.7 300 23.5 18.3 0 .+-.Drynaria fortunei (Kunze) J. Smith 0 100 100 100(rhizome) 10 110.7 87.8 96.4 50 90.7 68.5 117.8 100 87.4 59 101.8 300 0 25.4 87.1 +Polygonum cuspidatum Sieb. et Zucc. 0 100 100 100(root, rhizome) 10 125 85.4 73.8 50 110 74.6 107.6 100 97.1 36.9 107.1 300 0 0 0 +Punica granatum L. (root bark) 0 100 100 100 10 93.7 52.4 104.8 50 78.7 9.4 96.7 100 79.2 7.4 45.4 .+-. 300 0 0 0 +Terminalia chebula Retzus (fruit) 0 100 100 100 10 89.4 41.8 103.9 50 87 16.8 112.2 100 83.6 18.8 98.8 300 0 4.1 0 +Paeonia suffruticosa Andrews 0 100 100 100(root bark) 10 89.9 103.5 85.1 50 101.4 97.9 86 100 101.4 72.4 97.9 300 76.3 64.4 113.7__________________________________________________________________________
TEST EXAMPLE 7
Antiherpesviral activity (anti-herpesvirus type I activity, anti-varicella-zoster virus activity, and anti-CMV activity) of the crude drugs shown in Table 7 below were examined by the plaque formation test. The anti-herpesvirus type I activity test was carried out using Vero E6 cells and an extract solution diluted to 300 .mu.g/ml except where noted; and the anti-varicella-zoster virus activity test and anti-CMV activity test were carried out by using HEL cells and an extract solution diluted to 200 .mu.g/ml. The results obtained are shown in Table 7. As can be seen from the Table, the extract of the crude drug according to the present invention significantly reduced the efficiency of plaque formation at such a concentration as low as 500 .mu.g/ml or less and thus exhibited antiherpesviral activity. Further, Cyrtomium fortunei J. Sm. exhibited similar effects in its methanol extract and aqueous extract, suggesting that a crude drug whose methanol extract exhibits antiviral activity also exhibits antiviral activity in its aqueous extract as well.
TABLE 7__________________________________________________________________________Antiherpesviral Activity Plaque Formation Efficiency (%) Varicella-zoster Herpesvirus, I Virus CMVSpecies (part) 300 .mu.g/ml 200 .mu.g/ml 200 .mu.g/ml__________________________________________________________________________Cyrtomium fortunei (Kunze) J. Smith.sup.# (rhizome) 0 0 0Cassia fistula L. (bark) 0 0 0Areca catechu L. (seed) 0* 0 17.8Artemisia princeps Pamp. (leaf) 0* 46.7 0Terminalia arjuna Wight et Arn. (bark) 0 0 0Terminalia chebula Retzus (fruit) 0 0 0Punica granatum L. (fruit peel) 0 0 0Punica granatum L. (root bark) 0* 0 0Geum japonicum Thunb. (whole plant) 0* 54.9 0Drynaria fortunei (Kunze) J. Smith (rhizome) 0 0 0Polygonum cuspidatum Sieb. et Zucc. (root, 0* 0 0rhizome)Geranium thunbergii Sieb. et Zucc. (whole 23.5 14.7 0plant)Alpinia officinarum Hance (rhizome) 0 0 73.0**Juglans mandshurica Maxim. (bark) 0 0 0Coptis chinensis Franch. (rhizome) 0* 0 0Polygala tenuifolia Willd. (root) 0* 0 0Machilus thunbergii Sieb. et Zucc. (bark) 17.8 0 0Rheum palmatum L. (rhizome) 0 0 0Paeonia suffruticosa Andrews (root bark) 0* 39.7 47.4Rhus javanica L. (gall) 0* 0 0Phellodendron amurense Ruprecht (bark) 0* 0 0Syzygium aromaticum (L.) Merr. et Perry 0 26.6 0(flower bud)Cyrtonium fortunei J. Sm. (rhizome) 0 0 21.7Woodwardia orientalis Sw. (rhizome) 0 0Dryoperis crassirhizoma Nakai (rhizome) (M) 35.9 1.6 73**Woodwardia orientalis Sw. (rhizome) (M) 0 0 0Cyrtomium fortunei J. Sm. (rhizome) (M) 0 0 0Plagiogyria matsumureana Makino (rhizome) (M) 0 0 33.6__________________________________________________________________________ Note: *: An extract solution having a concentration of 500 .mu.g/ml was used. **: The plaque size was very small. (M): A methanol extract was used in place of an aqueous extract.
TEST EXAMPLE 8
Antiherpesviral activity of a combination of an extract solution of Cyrtomium fortunei (Kunze) J. Smith# (hereinafter abbreviated as Cryt. f.#) and an extract solution of the crude drug shown in Table 8 was determined using herpesvirus type I and Vero E6 cells. Each extract solution was diluted to 100 .mu.g/ml in a single use, and in a combined use, two extract solutions each having a concentration of 100 .mu.g/ml were mixed. The results obtained are shown in Table 8.
Antiherpesviral activity of a combination of Cryt. f.# and Artemisia princeps Pamp. was also determined in the same manner, except for using HEL cells, varicella-zoster virus, and CMV. The results obtained are shown in Table 9.
As is apparent from Tables 8 and 9, a combination of Cryt. f.# and other crude drugs showed appreciable synergism.
TABLE 8__________________________________________________________________________Antiherpesviral Activity (Herpesvirus, Type I) Plaque Formation Efficiency (%)Species (part) Single Use Combined Use__________________________________________________________________________Crytomium fortunei J. Sn..sup.# (rhizome) 82.9 --Cassia fistula L. (bark) 60 0Areca catechu L. (seed) 65.2 0Artemisia princeps Pamp. (leaf) 66.1 37.1Terminalia arjuna Wight et Arn. (bark) 56.7 0Terminalia chebula Retzus (fruit) 83.6 16.1Punica granatum L. (fruit peel) 86.2 0Punica granatum L. (root bark) 63.8 0Geum japonicum Thunb. (whole plant) 98.6 38.7Drynaria fortunei (Kunze) J. Smith (rhizome) 100.5 0Polygonum cuspidatum Sieb. et Zucc. (root, rhizome) 118.6 28.4Geranium thunbergii Sieb. et Zucc. (whole plant) 83.9 0Alpinia officinarum Hance (rhizome) 91 44.1Juglans mandshurica Maxim. (bark) 61.4 25.4Coptis chinensis Franch. (rhizome) 92.9 10.3Polygala tenuifolia Willd. (root) 27.1 0Machilus thunbergii Sieb. et Zucc. (bark) 117.6 0Rheum palmatum L. (rhizome) 76.7 0Paeonia suffruticosa Andrews (root bark) 103.8 67.3Rhus javanica L. (gall) 0 0Phellodendron amurense Ruprecht (bark) 111 64Syzygium aromaticum (L.) Merr. et Perry (flower bud) 102.4 61.7__________________________________________________________________________
TABLE 9__________________________________________________________________________Antiviral Activity Plaque Formation Efficiency (%) Varicella Virus CMVSpecies (part) Single Use Combined Use Single Use Combined Use__________________________________________________________________________Cyrtomium fortunei J. Sm..sup.# (rhizome) 7 -- 49 --Artemisia princeps Pamp. (leaf) 48.4 0 56.9 0__________________________________________________________________________
TEST EXAMPLE 9
Because the crude drugs according to the present invention are usually administered orally, the components which are absorbed through the gastrointestinal tract appear to be effective. Accordingly, the antiherpesviral activity of the serum prepared from the blood of a guinea pig having orally or intragastrointestinally received an extract solution of Cryt. f.# was determined according to the following animal test method.
For comparison, the antiherpesviral activity of the serum prepared in the same manner, except for replacing the Cryt. f.# extract with acyclovir ("Zovirax 200" sold by Wellcome Co.) (hereinafter abbreviated as Acv) known as an antiviral agent, was also determined. Acv tablets were ground to powder in a mortar and suspended in water or a crude drug extract solution in a prescribed concentration.
Animal Test Method:
Laparotomy was performed on a Hartley female guinea pig (body weight: 300-350 g) under etherization. Into each of the stomach, the small intestine, and the large intestine was infused 10 ml of water, an Acv suspension, a Cryt. f.# extract solution, or a mixed solution of Acv and the extract solution, and the abdomen was immediately closed. Two hours from the operation, blood was taken from the heart. The serum separated from the blood was inactivated by heating at 56.degree. for 30 minutes.
Vero cells was monolayer-cultured in a 25 cm.sup.2 -volume plastic flask and infected with 0.01 PFU/ml of herpesvirus. After adsorption for 1 hour, the infected cells were cultured in MEM containing 30 to 40% of the above-prepared inactivated serum. After cultivation for a given time (1 to 4 days), the cells were destroyed by freezing and thawing three times, followed by centrifugation. The amount of the virus in the supernatant liquor was measured by the plaque formation test using Vero cells. The results obtained are shown in Tables 10 and 11.
As is apparent from the results in Tables 10 and 11, antiherpesviral activity was displayed in both the Cryt. f.# group and the Acv group, and the combined use of Cryt. f.# and Acv showed significant synergism.
TABLE 10__________________________________________________________________________ Amount of Virus (PFU/ml)Run Sample Normal Serum of Serum of Serum of Cryt.No. No. Serum Cryt. f..sup.# Group Acv Group f..sup.# + Acv Group__________________________________________________________________________1 a 2.05 .times. 10.sup.8 2.15 .times. 10.sup.8 2.58 .times. 10.sup.7 1.17 .times. 10.sup.7 b 2.33 .times. 10.sup.8 2.18 .times. 10.sup.8 5.83 .times. 10.sup.7 1.64 .times. 10.sup.7 c 2.35 .times. 10.sup.8 1.98 .times. 10.sup.8 6.85 .times. 10.sup.7 1.97 .times. 10.sup.7 mean .+-. .sigma.* (2.24 .+-. 0.17) .times. 10.sup.8 (2.10 .+-. 0.11) .times. 10.sup.8 (5.09 .+-. 2.20) .times. 10.sup.7 (1.59 .+-. 0.40) .times. 10.sup.7 (p = 0.29)** (p = 0.056)***2 a 3.25 .times. 10.sup.8 1.80 .times. 10.sup.8 1.68 .times. 10.sup.7 8.30 .times. 10.sup.5 b 2.30 .times. 10.sup.8 1.83 .times. 10.sup. 8 1.54 .times. 10.sup.7 4.88 .times. 10.sup.5 c -- 1.58 .times. 10.sup.8 -- 2.17 .times. 10.sup.6 mean .+-. .sigma. 2.78 .times. 10.sup.8 (1.74 .+-. 0.14) .times. 10.sup.8 1.61 .times. 10.sup.7 (1.33 .+-. 1.30) .times. 10.sup.6 (p = 0.067)** (p < 0.01)***3 a 1.55 .times. 10.sup.8 1.10 .times. 10.sup.8 7.67 .times. 10.sup.5 1.75 .times. 10.sup.5 b 1.20 .times. 10.sup.8 1.93 .times. 10.sup.8 1.25 .times. 10.sup.6 5.00 .times. 10.sup.4 c 1.63 .times. 10.sup.8 2.75 .times. 10.sup.7 1.02 .times. 10.sup.6 2.50 .times. 10.sup.4 mean .+-. .sigma. (1.46 .+-. 0.23) .times. 10.sup.8 (1.10 .+-. 0.83) .times. 10.sup.8 (5.09 .+-. 2.20) .times. 10.sup.6 (8.33 .+-. 8.04) .times. 10.sup.4 (p = 0.51)** (p < 0.01)***4 a 1.73 .times. 10.sup.8 3.50 .times. 10.sup.7 4.67 .times. 10.sup.6 3.67 .times. 10.sup.5 b 1.80 .times. 10.sup.8 1.05 .times. 10.sup.8 2.65 .times. 10.sup.6 1.37 .times. 10.sup.6 c 2.03 .times. 10.sup.8 1.50 .times. 10.sup.8 6.00 .times. 10.sup.6 9.00 .times. 10.sup.5 mean .+-. .sigma. (1.85 .+-. 0.16) .times. 10.sup.8 (9.67 .+-. 5.80) .times. 10.sup.7 (4.44 .+-. 1.68) .times. 10.sup.6 (8.79 .+-. 5.00) .times. 10.sup.5 (p = 0.063)** (p = 0.025)***__________________________________________________________________________ Note: *: Standard deviation (hereinafter the same) **: tTest value with respect to the normal serum. ***: ttest value with respect to the serum of the Acv group.
TABLE 11__________________________________________________________________________ Amount of Virus (PFU/ml) Serum of Cryt. Serum of Serum of Cryt. f..sup.# + Acv GroupRun Sample Normal f..sup.# (20 mg/ml) Acv (200 mg/ 1 mg/ml of 5 mg/ml of 20 mg/ml ofNo. No. Serum Group ml) Group Cryt. f..sup.# Cryt. f..sup.# Cryt. f..sup.#__________________________________________________________________________1 a 3.30 .times. 10.sup.8 1.30 .times. 10.sup.8 4.93 .times. 10.sup.7 8.25 .times. 10.sup.6 2.50 .times. 10.sup.5 2.00 .times. 10.sup.16 b 3.03 .times. 10.sup.8 1.45 .times. 10.sup.8 3.80 .times. 10.sup.7 1.58 .times. 10.sup.7 6.25 .times. 10.sup.6 2.00 .times. 10.sup.6 c 2.75 .times. 10.sup.8 2.08 .times. 10.sup.8 7.83 .times. 10.sup.7 8.25 .times. 10.sup.6 2.50 .times. 10.sup.5 1.00 .times. 10.sup.6 mean .+-. .sigma. (3.03 .+-. 0.28) .times. (1.61 .+-. 0.41) .times. (5.52 .+-. 2.08) .times. (1.59 .+-. 0.40) .times. (2.25 .+-. 3.46) (1.67 .+-. 0.58) .times. 10.sup.8 10.sup.7 10.sup.7 10.sup.7 10.sup.6 10.sup.6 (p < 0.01)* (p < 0.05)** (p < 00.05)** (p < 0.05)**__________________________________________________________________________ Note: *: tTest value with respect to the normal serum. **: tTest value with respect to the serum of the Acv group.
TEST EXAMPLE 10
Since the antiherpesviral activity of Acv is known, whether or not the synergism shown in Test Example 9 had been attributed to the improvement of Acv absorption by the extract solution of Cryt. f.# was examined by measuring Acv concentration in blood when Acv was administered alone or in combination with a Cryt. f.# extract solution.
An Acv suspension or a mixture of Acv and an extract solution of Cryt. f.# was orally or intragastrointestinally administered to a guinea pig. One hour from the administration, blood was taken from the heart, and an inactivated serum was prepared therefrom in the same manner as in Test Example 9.
To a 100 .mu.l aliquot of the serum were added 100 .mu.l of acetonitrile and 20 .mu.l of a 100 mM sodium acetate solution, and the mixture was centrifuged (10000 rpm.times.10 mins.). The supernatant liquor (150 .mu.l) was evaporated to dryness in an evaporator, and the residue was dissolved in 25 .mu.l of a solvent for reverse phase liquid chromatography which contained 16.7 mM of adenine as an internal standard to prepare a sample solution.
The sample solution was separated into Acv and adenine by reverse phase liquid chromatography in a usual manner. Each peak area was measured with an automatic integrator, and the Acv content was obtained from the previously prepared calibration curve. The results obtained are shown in Table 12. From the results in Table 12, it is not recognized that the Acv absorption was accelerated by the combined use of the Cryt. f.# extract solution. It is therefore considered that a Cryt. f.# extract solution per se possesses antiherpesviral activity in its absorbable fraction.
Further, 0.5 ml of an Acv preparation or 0.5 ml of a mixture of Acv and a varied concentration of Cryt. f.# extract solution was orally administered to a 6-week-old DDY male mouse, and the Acv concentration in blood was measured in the same manner as described above. The results obtained are shown in Table 13. As is apparent from Table 13, no increase in Acv concentration in blood was observed in the group having received Acv combined with Cryt. f.#. Accordingly, the Cryt. f.# extract solution is believed to have antiherpesviral activity by itself.
Antiherpesviral activity was examined in combined use of Acv and an extract solution of the crude drug shown in Table 14.
As is apparent from the results in Table 14, antiherpesviral activity was displayed in the combined use of Acv and an extract solution of the crude drug showed significant synergism.
TABLE 12______________________________________Acv Concentration in Guinea Pig BloodAcv Concentration in Blood (.mu.M)Serum Acv Acv (200 mg) + Cryt. f..sup.# GroupSample (200 mg) 1 mg/ml of 5 mg/ml of 20 mg/ml ofNo. Group Cryt. f..sup.# Cryt. f..sup.# Cryt. f..sup.#______________________________________1 21.6 76.4 92.9 152.72 185.1 129.6 103 21.63 59.1 72.7 69.1 42.5Mean .+-. .sigma. 88.6 .+-. 85.6 92.9 .+-. 31.9 88.3 .+-. 17.4 72.2 .+-. 70.4______________________________________
TABLE 13______________________________________Acv Level in Mouse Blood (p.o.)Acv Blood Level (.mu.M) Acv (100 mg/kg) + Cryt. f..sup.# GroupSerum Acv 10 20Sample (10 mg/kg) 2 mg/ml of mg/ml of mg/ml ofNo. Group Cryt. f..sup.# Cryt. f..sup.# Cryt. f..sup.#______________________________________1 3.2 1.7 2.3 0.42 1.7 1.4 1.1 43 0.5 1.6 3.5 3.24 5.1 2.5 1.6 0.55 0.8 4.7 1.9 4.16 1.9 2.9 2.6 27 1.9 2 1.5 3.38 1.7 2.4 5.2 49 2.9 9.5 -- 1.610 4 2.3 -- 4.411 -- -- -- 1Mean .+-. .sigma. 2.28 .+-. 1.49 3.10 .+-. 2.43 2.46 .+-. 1.33 2.59 .+-. 1.53______________________________________
TABLE 14__________________________________________________________________________Antiherpesviral activity incombination use of Acv and hot water extract of crude drug Concn. of Plaque Formation Efficiency (%) Extract solution Acv +Species (.mu.g/ml) Acv Crude drug Crude drug__________________________________________________________________________Acyclovir 0.35 53.6Alpinia officinarum Hance 200 58 30.6Caesalpinia sappan L. 40 53.3 0.2Geum japonicum Thunb. 120 52.8 1.1Paeonia suffruticosa Andrews 400 59.5 26Phellodendron amurense Ruprecht 400 61.5 24.5Polygala tenuifolia Willd. 400 55.2 15.7Polygonum cuspidatum Sieb. 200 59.4 20.6et ZuccPunica granatum L. 50 50.9 1Syzygium aromaticum (L.) 60 61.2 0.2Merr. et PerryTerminalia arjuna Wight et Arn. 50 52.8 24.8__________________________________________________________________________
TEXT EXAMPLE 11
Antiherpesviral activity of various crude drugs shown in Table 15 below in guinea pigs (p.o.) was determined as follows.
A control group of guinea pigs was allowed to take water freely for 5 days. A 5 mg/ml extract solution of Cryt. F.' was given to another group freely by mouth for 5 days. Still another group was allowed to take a 5 mg/ml solution of Areca catechu L. freely for 5 days. Other groups were orally given 10 ml of a 20 mg/ml solution of each of the other crude drug extracts. Blood was taken from each animal, and an inactivated serum was prepared therefrom in the same manner as in Test Example 9. A plaque formation test was conducted using the resulting serum sample. The results obtained are shown in Table 15. It is obvious from the results that each crude drug extract exhibited antiherpesviral activity.
TABLE 15______________________________________ Plaque Formation EfficiencyCrude Drug (%)______________________________________Water (control) 100.0Acyclovir (6.7 mg/ml) 18.2(B2)Alpinia officinarum Hance 6.8(B5)Areca catechu L. 57.3(B17)Crytomium fortunei J. Sm..sup.# 45.5(B8) Brucea javanica (L.) Merr. 20.4(B9)Caesalpinia sappan L. 43.4(B11)Cassia fistula L. 45.0(B14)Cinnamomum sintok Blume 41.9(B18)Drynaria fortunei. (Kunze) J. Smith 34.0(B20)Elaeocarpus grandiflorus Smith 15.4(B24)Geranium thunbergii Sieb. et Zucc. 50.0(B25)Geum japonicum Thunb. 22.5(B28)Juglans mandshurica Maxim. 12.6(B30)Paeonia suffruticosa Andrews 61.4(B32)Phellodendron amurense Ruprecht 30.4(B34)Polygala tenuifolia Willd. 8.3(B35)Polygonum cuspidatum Sieb. at Zucc. 65.6(B36)Prunella vulgaris L. subsp. asiatica Hara 58.5(B37)Punica granatum L. (root bark and fruit 54.3peel)(B40)Rhus javanica L. 87.3(B46)Terminalia arjuna Wight et Arn. 39.0(B47)Terminalia belerica Roxb. 31.2(B48)Terminalia chebula Retzus 87.4(B50)Woodfordia floribunda Salisb. 80.1(B51)Woodwardia orientalis Sw. 82.1Syzygium aromaticum (L.) Merr. et Perry 50.5______________________________________
TEST EXAMPLE 12
Mouse was infected with Herpes simplex virus at the right midflank to examine the antiviral activity of the crude drug by a rate of herpes zoster formation and a rate of death.
In vivo antiviral activity of Geum japonicum Thunb. or Juglans mandshurica Maxim. in mice infected with herpes simplex virus was examined as follows.
The hair on the side abdomen of 6 to 7-week-old male BALB/C mice was removed by a chemical hair remover (produced by Shiseido Co., Ltd.). The hairless skin was scratched at random ten times with an intradermal injection needle 26G, and 10 .mu.l of a herpes simplex virus I (Hayashida strain) (having been proliferated in Vero E6 cells) suspension (10.sup.6 PFU) was applied to the scarified area.
Immediately after the infection, 0.5 ml of the crude drug extract solution having a concentration of from 2 to 20 mg/ml (corresponding to 10 mg-drug/mouse) was orally given to the animal every 8 hours (3 times/day) for at least consecutive 10 days. A control group was orally given the equal volumes of water The development of skin lesions and mortality were monitored three times a day and any change observed on the infected skin was scored on the following basis. The results obtained are shown in Table 16.
______________________________________Score Standard:______________________________________0 . . . No lesion2 . . . Vesicles in local region (bulla and erosion)6 . . . Mild zosteriform lesion8 . . . Moderate zosteriform lesion10 . . . Severe zosteriform lesionDeath______________________________________
As is apparent from Table 16, the group having orally received the extract solution exhibited a reduced rate of death, an increased survival rate and a reduced rate of herpes zoster formation as compared with the control group, proving the antiherpesviral activity of the crude drug in vivo.
TABLE 16______________________________________ Rate of Herpes Average Zoster Death SurvivalCrude Drug Formation Rate Days______________________________________Water (control) 6/6 6/6 8.2 .+-. 0.4 (100%) (100%)Geum japonicum Thunb. 3/4 3/4 10.2 .+-. 1.8* (75%) (75%)Juglans mandshurica Maxim. 2/3 2/3 10.0 .+-. 2.7 (67%) (67%)______________________________________ Note: *significance level p < 0.05
As described in the foregoing test examples, antiherpesviral activity of the crude drugs belonging to group A according to the present invention can be ascertained by the above-described in vitro screening test. Further, the serum prepared from the guinea pig having orally or intragastrointestinally received the extract of the crude drug of group B exhibited antiherpesviral activity. Furthermore, the crude drug extract having antiherpesviral activity obviously inhibited herpesvirus growth in mice (p.o.) (cf. Table 16). The results coincided with the results shown in Table 1 which was conducted in vitro.
From all these test results, it was thus revealed that a crude drug of group B which exhibits antiherpesviral activity in vitro also exhibits the same activity in vivo.
TEST EXAMPLE 13
Antipolioviral activity of Punica granatum L. (fruit peel) was determined by using the guinea pig serum prepared in the same manner as in Text Example 9. The results obtained are shown in Table 17. The results clearly demonstrate the antipolioviral activity of the serum of the Punica granatum group.
TABLE 17______________________________________Amount of Virus (PFU/ml)Sample Normal Serum of Punica granatum GroupNo. Serum 5 mg/ml 20 mg/ml______________________________________a 9.0 .times. 10.sup.6 9.5 .times. 10.sup.6 7.25 .times. 10.sup.6b 9.0 .times. 10.sup.6 8.5 .times. 10.sup.6 4.75 .times. 10.sup.6c 1.48 .times. 10.sup.7 8.25 .times. 10.sup.6 6.25 .times. 10.sup.5d 1.05 .times. 10.sup.7 -- 8.25 .times. 10.sup.6e -- -- 5.75 .times. 10.sup.6Mean .+-. (1.08 .+-. 0.274) .times. (8.75 .+-. 0.661) .times. (6.45 .+-. 1.35) .times..sigma. 10.sup.7 10.sup.6 10.sup.6 (p = 0.265)* (p = 0.016)*______________________________________ Note: *: tTest value with respect to the normal serum.
Only the fruit peel of Punica grantum L. was used in this Example. The fruit peel of Punica grantum L. showed the same effect as the root bark thereof in the present invention. Punia grantum which can be obtained on the market is properly a mixture of the fruit peel and the root bark in this field.
TEST EXAMPLE 14
Antipolioviral activity of the crude drugs shown in Table 18 below in guinea pigs (p.o.) was determined in the same manner as in Text Example 11 as follows.
A group of guinea pigs was allowed to take water or a 5 mg/ml extract solution freely for 5 days. A serum sample was prepared in the same manner as in Test Example 9 and subjected to the plaque formation test. The results obtained are shown in Table 18. It is obvious from the results that each crude drug extract exhibited antipolioviral activity.
TABLE 18__________________________________________________________________________ Amount of Virus (PFU/ml)Run Sample Normal Serum of Serum of Serum ofNo. No. Serum Term. c..sup.1) Group Pun. g..sup.2) Group Are. c..sup.3) Group__________________________________________________________________________1 a 5.53 .times. 10.sup.5 5.42 .times. 10.sup.5 2.70 .times. 10.sup.5 -- b 4.90 .times. 10.sup.5 2.78 .times. 10.sup.5 2.48 .times. 10.sup.5 -- c 5.65 .times. 10.sup.5 5.13 .times. 10.sup.5 4.25 .times. 10.sup.5 -- Mean .+-. .sigma. (5.36 .+-. 0.40) .times. 10.sup.5 (4.44 .+-. 1.45) .times. 10.sup.5 (3.14 .+-. 0.965) .times. 10.sup.5 (p < 0.5)* (p < 0.05)* 100% 85.10%** 60.20%**2 a 1.18 .times. 10.sup.7 6.83 .times. 10.sup.6 7.50 .times. 10.sup.6 7.00 .times. 10.sup.6 b 1.02 .times. 10.sup.7 1.07 .times. 10.sup.7 9.17 .times. 10.sup.6 7.33 .times. 10.sup.6 c 1.22 .times. 10.sup.7 -- 9.12 .times. 10.sup.6 -- Mean .+-. .sigma. (1.14 .+-. 0.11) .times. 10.sup.7 8.77 .times. 10.sup.6 (8.61 .+-. 0.96) .times. 10.sup.6 7.17 .times. 10.sup.6 (p < 0.05)* 100% 76.90%** 75.50%** 62.90%**__________________________________________________________________________ Note: .sup.1) Terminalia chebula Retzus .sup.2) Punica granatum L. (fruit peel) .sup.3) Areca catechu L. *: tTest value with respect to the normal serum **: Plaque formation efficiency (percent virus growth inhibition)
As described above, the antipolioviral activity of various crude drugs belonging to group C can be examined by in vitro screening (Test Example 2). The crude drugs of group C exhibited antipolioviral activity in the serum prepared from the guinea pig having orally or intragastrointestinally received the extract of the drug (Test Examples 13 and 14).
From these test results, it was thus revealed that a crude drug which exhibits antipolioviral activity in vitro also exhibits the same activity in vivo.
The dosage, administration route, etc. of the crude drugs included under group C are well known, and the known dose levels produce no or slight side effects. Accordingly, these crude drugs were proved effective to inhibit poliovirus growth in oral administration or intragastrointestinal administration.
TEST EXAMPLE 15
The crude drugs exhibiting antiviral activity against both DNA viruses and RNA viruses, i.e., the crude drugs of group G, are shown in Table 19 together with their data.
The crude drugs of group G were proved to exhibit antiviral activity against not only any species belonging to the famility Herpesviridae but RNA viruses of different familities (measles virus and poliovirus) and are therefore effective in the prevention and treatment of diseases caused by DNA viruses or RNA viruses.
TABLE 19__________________________________________________________________________ Plaque Formation Efficiency (%) DNA Virus RNA Virus Herpesvirus Measles Virus Poliovirus Concentration of Extract Solution (.mu.g/ml)Crude Drug (part) 100 300 500 100 300 500 100 300 500__________________________________________________________________________(G1)Areca catechu L. (seed) 0 0 93.6 0 86.5 0(G2)Artemisia pinceps Pamp. 65 0 10.8 13.6 84.3 40.5(leaf)(G3)Brainia insignis (Hook.) 62.7 0 109.7 5.1 61 54.6(J. Sm.(rhizome)(G4)Brucea javanica (L.) Merr. 6.3 0 37.2 0 0 0(seed)(G5)Caesalpinia sappan L. 0 0 0 0 0 0(bark)(B6)Cassia fistula L. (bark) 67 0 100 14.1 52.1 2.8(G7)Cinnamomum sintok Blume 0 0 153 0 102.5 0(bark)(G8)Coptis chinensis Franch. 74.1 0 5.6 0 89.7 0(rhizome)(G9)Cyrtomium fortunei J. Sm. 59.6 0 112.1 0 66.5 0(rhizome)(G10)Elaeocarpus grandiflorus 0 0 0 0 0 0Smith (fruit)(G11)Foeniculum vulgare Mill. 67.5 47.5 99.3 47.5 31.3 28.1(fruit)(G12)Geranium thunbergii Sieb. 91.6 23.5 104.7 0 30 21.4et Zucc (whole plant)(G13)Juglans mandshurica Maxim. 73.6 0 96.5 3.5 35.5 4.8(bark)(G14)Paeonia suffruticosa 96.5 0 119.1 0 124.2 35.5Andrews (root bark)(G15)Phellodendron amurense 85.3 29.4 17.2 0.6 103.4 43.3Ruprecht (bark)(G16)Plagiogyria matsumureana 76.1 50.7 90.8 84.6 73.9 0Makino (rhizome)(G17)Polygala tenuifolia Willd. 118.6 0 100 0 86.4 0(root)(G18)Punica granatum L. 0 0 0 0 79.5 0(root bark, fruit peel)(G19)Quercus acutissima 38.2 9.8 83.3 38.5 98.4 35.6Carruthers (bark)(G20)Rheum palmatum L. (rhizome) 93.2 0 108.1 0 13.7 0(G21)Rhus javanica L. (gall) 0 0 3.4 0 65.1 4.3(G22)Spatholobus suberectus 92.2 0 75.4 0 80.2 0Dunn (stem)(G23)Syzygium aromaticum (L.) 79.4 0 98.6 0 55.0Merr. et Perry (flower bud)(G24)Terminalia arjuna Wight et 56.3 0 100 0 22.5 0Arn. (bark)(G25)Terminalia belerica Roxb. 70.8 0 104.4 0 7.4 0(fruit peel)(G26)Terminalia chebula Retzus 99.6 0 152 0 32.6 0.7(fruit)(G27)Woodfordia floribunda 0 0 0 0 0 0Salisb. (flower, leaf)(G28)Woodwardia orientalis Sw. 51.6 0 105.4 0 30.5 0(rhizome)__________________________________________________________________________
The crude drugs in accordance with the present invention exhibit antiviral activity and inhibit virus growth. Therefore, appropriate use of these crude drugs brings about improved effects in the prevention and treatment of virus infectious diseases.
While, in the foregoing test examples, the crude drugs of the present invention were used in the form of an aqueous extract or a methanol extract, extracts with other alcohols, e.g., ethyl alcohol, or a water/alcohol mixed solvent may also be used. A decoction or infusion prepared by decocting the crude drug with boiling water followed by filtration through a strainer, etc. can also be used. Further, a powder preparation prepared by grinding the crude drug is also usable.
While the invention has been described in detail and with reference to specific examples thereof, it will be apparent to one skilled in the art that various changes and modifications can be made therein without departing from the spirit and scope thereof.

Number	Date	Country	Kind
4-107659	Apr 1992	JPX
4-107672	Apr 1992	JPX

Number	Name	Date
4442087	Kojima et al.	Apr 1984
4592910	Wolf et al.	Jun 1986
4595593	Wolf et al.	Jun 1986
4871540	Kojima et al.	Oct 1989

Antiviral agent containing crude drug

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