Use of crude plant extract for the preparation of a medicament for treating HIV infection

Abstract: In order to resolve problems that occur when single administration of a dideoxynucleotide such as AZT, ddI or the like is carried out, this invention provides a number of anti-HIV agents which are effective in treating the onset of HIV and have fewer side effects. Particularly, it provides an anti-HIV composition which includes an active agent in a therapeutically effective amount and a pharmaceutically acceptable carrier, wherein the active agent is at least one crude drug selected from the group consisting of Alpinia officinarum Hance, Geum japonicum Thunb., Paeonia suffruticosa Andrews, Phellodendron amurence Ruprecht, Punica granatum L., Rhus javanica L., Syzygium aromaticum (L.) Merr. et Perry, Terminalia arjuna Wight et Arn., and Terminalia chebula Retzus. It also provides a method for treating HIV infection, including orally administering to a subject in need thereof a therapeutically effective amount of an anti-HIV agent containing as an active agent at least one crude drug is selected from the group consisting of Alpinia officinarum Hance, Geum japonicum Thunb., Paeonia suffruticosa Andrews, Phellodendron amurence Ruprecht, Punica granatum L., Rhus javanica L., Syzygium aromaticum (L.) Merr. et Perry, Terminalia arjuna Wight et Arn., and Terminalia chebula Retzus.

Description: This invention relates to the use of a crude extract for preparing a medicament for treating HIV infection. HIV (human immunodeficiency virus) is the pathogen of acquired immunodeficiency syndrome (AIDS) whose disease concept was established in the 1980's. Soon after HIV infection, the antibody reaction changes to positive via influenza-like initial symptoms (3 to 15 days). After a latent period of one to several years, T-cell insufficiency is developed which subsequently causes AIDS-related syndrome (lymph node enlargement, body weight loss, pyrexia, sweating and chronic diarrhea), AIDS (worsening of the above symptoms, pneumocystis carinii pneumonia, opportunistic infection of candidasis and Kaposi sarcoma) and sometimes neurosis. Only AZT and ddI, which belong to the dideoxynucleotide family, are now used in the clinical field for the treatment of AIDS other than the HIV symptomatic therapy. In many cases, treatment with these drugs is forced to be suspended due to their side effects. In addition to this, the generation of resistant viruses is causing a serious problem. Since these problems mean that a prolonged period of continuous administration cannot be made by a single drug administration, development of a variety of drugs is a pressing need. Starting around 1989, development of HIV protease inhibitors has been reported in succession as new generation drugs instead of dideoxynucleotides. It has been confirmed that these HIV protease inhibitors are effective on various viral strains including HIV-2 type virus and show sufficient in vitro activities. However, these HIV protease inhibitors have a disadvantage in that their activities are reduced due to their nonspecific reaction with blood protein. On the other hand, development of anti-HIV agents making use of crude drugs is also in progress (a crude drug is a drug which is obtained from a natural source such as a plant). It is said that licopyranocoumarin extracted from Glycyrrhiza Root shows anti-HIV activity at a concentration of 20 µg/ml (Miyamoto et al., 1st Meeting Jap.. Soc. AIDS Res. Abstract, 1987). Also, it has been reported that water or alcohol extracts of 6 Indonesian crude drugs Supratul ( Sindora sumatrana ) , Klules ( Helicteres isora ) , Mahoni ( Swietenia mahagoni ), Sambiloto ( Andrographis paniculate ), Temu ireng ( Curcuma aeruginosa ) and Benaluteh ( Loranthus parasiticus ) (scientific names are shown in parentheses) show anti-HIV activity at respective concentrations of 134 µg/ml (water extract), 65 µg/ml (methanol extract), 28.4 µg/ml (methanol extract), 170 µg/ml (water extract), 323 µg/ml (water extract) and 79 µg/ml (water extract) (JP-A-6-40930, the term "JP-A" as used herein means an "unexamined published Japanese patent application"). In Chem. Pharm. Bull. 43(4) 641-648 (1995) it is reported that extracts inter alia of fruits of Terminalia chebula showed a significant inhibitory activity on human immuno-deficiency virus-1 reverse transcriptase. It is an object of the present invention to provide a number of agents which are effective in treating the onset of HIV and have fewer side effects, in order to resolve the prior art problems that occur when a single administration of a dideoxynucleotide such as AZT or is carried out. The inventors of the present invention have examined a broad range of crude drugs conventionally used as therapeutic drugs whose dose, administration route, and the like are well known and which show no, or slight, side effects. The present invention relates to the use of a one crude extract selected from the group consisting of Alpinia officinarum Hance, Geum japonicum Thunb., Paeonia suffruticosa Andrews, Phellodendron amurence Ruprecht, Punica granatum L., Rhus javanica L., Syzygium aromaticum (L.) Merr. et Perry and Terminalia arjuna Wight et Arn., for the preparation of a medicament for the prevention and treatment of HIV. A pharmaceutically acceptable carrier used in the medicament can be, e.g., water. The therapeutically effective amount varies depending on the crude drug being used and is determined based on the crude drug being used. For example, water is added to 2 to 10 g of at least one crude drug selected from the crude extracts in an amount of 50 ml per g of the crude extract. Then, the water containing the crude extract is boiled down to about a half of the original volume at a heating power to such an extent that the water does not boil over. The thus obtained extract may be orally administered three times a day at a dose of one-third of the extract. The methods for using the extracts are not particularly limited to the above method. The extract may be concentrated and dried to use as powders. Furthermore, the dried extract obtained may be formulated into any dose form such as granules, tablets, and powders. In these formulations, excipients which are usually pharmaceutically used, such as starch, lactose, and magnesium stearate, may be mixed with the extract. An example of the present invention is given below by way of illustration. Unless otherwise indicated, all parts percents, ratios, and the like are by weight. EXAMPLE Crude extracts from the following parts are used in the example. 1. Alpinia officinarum Hance (rhizome) 2. Geum japonicum Thunb. (whole plant) 3. Paeonia suffruticosa Andrews (root bark) 4. Phellodendron amurence Ruprecht (bark) 5. Punica granatum L. (root bark and fruit peel) 6. Rhus javanica L. (insect gall) 7. Syzygium aromaticum (L.) Merr. et Perry (flower bud) 8. Terminalia arjuna Wight et Arn. (bark) A 100 mg portion of powder obtained by pulverizing each of the above crude extracts was mixed with 5 ml of high purity water, the resulting suspension was boiled for 5 minutes and centrifuged for 10 minutes at every 400 g and the thus obtained supernatant fluid was adjusted to 20 mg/ml to be used as an extract in the following experiments. Next, the experimental method and judging method used in the following anti-HIV activity test are described. The test was carried out in accordance with the method developed by Mitsuya et al. ( Proc. Natl. Acad. Sci. USA, 82: 7096 - 7100, 1985). MT-4 cells established from an HIV-infected patient were infected with HIV-I LAI for 1 hour at an amount of 3,000 TCID 50 /5×10 5 cells and mixed with a predetermined concentration of each drug, and a 1 ml portion of the resulting cell suspension was cultured for 4 days after the infection using a 24 well plate. Thereafter, viable cells and dead cells were distinguished by trypan blue staining to count the number of cells which were saved by the influence of the drug from cell death caused by the viral infection. In order to examine the cytotoxicity of the drug, the same test was carried out using a group which was not infected with the HIV, and the results were used in the evaluation of the anti-HIV activity. In this connection, when the anti-viral activity is examined a plurality of times using the same concentration of extract solution, the test results sometimes differ from one another slightly, which, however, seems to be a judging error formed by a slight difference in the sensitivity due to culture conditions (serum lot, culture plate, cell density and the like). The drug effect of each crude extract was evaluated in the following manner. First, the number of viable mock-infected cells to which the crude extract was not added and the number of viable infected cells to which the crude drug extract was not added were counted to estimate the number of survived cells. Next, the number of viable infected cells to which the crude extract was added was counted to estimate the number of survived cells in the presence of the crude drug extract. Evaluation of each crude extract was made based on the increasing ratio of viable count effected by the administration of the crude extract (viable cell percentage). The results are shown in Table 1. In the table, a higher viable cell percentage means a stronger anti-HIV effect. Also, a lower cytotoxicity percentage means a weaker toxicity for cells. In this test, the anti-HIV effect was judged positive when a viable cell percentage of 10% or more was obtained by the administration of each crude extract. Each of the crude extracts showed an anti-HIV effect at a concentration of 2 µg/ml or less in the case of Alpinia officinarum, Phellodendron amurence, Punica granatum, Rhus javanica, Syzygium aromaticum and Geum japonicum and at a concentration of 20 µg/ml or less in the case of Paeonia suffruticosa and Terminalia arjuna. None of them showed strong cytotoxicity at such concentrations. Furthermore, in Table 1, some crude drugs showed a lower viable cell percentage at a higher concentration. These results were because many cultured cells were died by the cytotoxicity which was originally possessed by the crude extracts and as a result, the number of viable cells was reduced. However, all tested crude extracts showed anti-HIV activity at a concentration which was lower than that showing the cytotoxicity. Results of anti-HIV activity test Extracts Conc. Viable count (× 10 5 ) (cell percentage or cytotoxicity percentage ) HIV infection HIV mock infection (no infection) none - 1.8 8.4 Alpinia officinarum 2 µg/ml 3.3 (23) 8.4 (0) 20 µg/ml 3.3 (23) 7.9 (6) 200 µg/ml 4.0 (33) 5.8 (31) Paeonia suffruticosa 2 µg/ml 0.9 (0) 8.1 (4) 20 µg/ml 2.7 (14) 8.8 (0) 200 µg/ml 3.7 (29) 4.9 (42) Phellodendron amurence 2 µg/ml 3.8 (30) 6.5 (23) 20 µg/ml 2.1 (5) 2.3 (73) 200 µg/ml 0 (0) 0.2 (98) Punica granatum 2 µg/ml 2.8 (15) 9.0 (0) 20 µg/ml 3.8 (30) 3.5 (58) 200 µg/ml 0 (0) 0 (100) Terminalia arjuna 2 µg/ml 2.4 (9) 8.5 (0) 20 µg/ml 4.8 (45) 7.7 (8) 200 µg/ml 0 (0) 0.1 (99) Rhus javanica 2 µg/ml 2.7 (14) 5.9 (30) 20 µg/ml 1.3 (0) 1.9 (77) 200 µg/ml 0 (0) 0 (100) Syzygium aromaticum 2 µg/ml 2.9 (17) 8.5 (0) 20 µg/ml 4.2 (36) 5.9 (30) 200 µg/ml 0 (0) 0 (100) Geum japonicum 2 µg/ml 3.1 (20) 8.2 (2) 20 µg/ml 3.7 (29) 6.7 (20) 200 µg/ml 0.6 (0) 0.6 (93) Dideoxyinosine 20 µM 8.6 (103) 8.7 (0) (ddI) 50 µM 8.8 (106) 8.4 (0) In the above test, an examination was made to find the degree of the effect of the crude extracts in decreasing the number of dead cells caused by HIV. As the action points which generate the anti-HIV activity, inhibition of adsorption of HIV to cells, inhibition of an HIV-originated reverse transcriptase, inhibition of an HIV-originated protease and the like can be exemplified. In the following test, inhibition of HIV-originated reverse transcriptase was examined. The reverse transcriptase inhibition activity test was carried out in the following manner. The reverse transcriptase inhibition activity was examined using 7 crude extracts which showed high anti-HIV activity. The reverse transcriptase inhibition activity was measured in accordance with the method of B.J. Poiesz et al. ( Proc. Natl. Acad. Sci. USA, vol.77, no.12, pp.7415 - 7419, 1980). To a reverse transcriptase reaction solution [30 U/ml AMV reverse transcriptase (manufactured by Pharmacia), 40 mM Tris-HCl (pH 7.8), 4 mM dithiothreitol, 45 mM KCl, 10 mM MgCl 2 , 50 µg/ml poly(A) dT 12-18 template primer and 50 µg/ml poly(C) dG 12-18 template primer] were added each crude drug extract (final concentration, 2, 20 or 200 µg/ml) and labeled deoxytriphosphate (final concentration, 15 µM), followed by 30 minutes of reaction at 37°C. Total volume of the reaction solution was adjusted to 100 µl, and the reverse transcriptase inhibition activity was measured based on the incorporated amount of the label. AZT and ddI do not show reverse transcriptase inhibition activity since these are not phosphorylated by this test. Therefore, AZT and ddI were not examined at this test. The results are shown in Table 2. Reverse transcriptase inhibition activity of crude extract Reverse transcriptase inhibition activity (%) Extracts Extract conc. (µg/ml) 2 20 200 Alpinia officinarum - 54 92 Geum japonicum 8 29 55 Paeonia suffruticosa - 23 55 Punica granatum 17 38 61 Terminalia arjuna - 6 3 Syzygium aromaticum 11 43 76 Each of the crude extracts excluding Terminalia arjuna showed reverse transcriptase inhibition activity in a concentration-dependent fashion, indicating that the reverse transcriptase inhibition action is included in the action mechanism of these crude extracts. On the other hand, the Terminalia arjuna extract did not show reverse transcriptase inhibition activity, indicating that it has an action mechanism which is different from that of the prior art dideoxynucleotide extracts. Thus, it is apparent that there has been provided an anti-HIV composition by the use of the specified crude extracts which hardly shows side effects.

Claims: The use of a crude extract selected from the group consisting of Alpinia officinarum Hance, Geum japonicum Thunb., Paeonia suffruticosa Andrews, Phellodendron amurence Ruprecht, Punica granatum L., Rhus javanica L., Syzygium aromaticum (L.) Merr. et Perry, and Terminalia arjuna Wight et Arn., for the preparation of a medicament for the prevention and treatment of HIV. The use according to claim 1, wherein the crude extract is selected from the group consisting of Phellodendron amurence Ruprecht, Rhus javanica L., Punica granatum L., Geum japonicum Thunb., Syzygium aromaticum (L.) Merr. et Perry, Paeonia suffruticosa Andrews and Alpinia officinarum Hance. The use according to claim 1, wherein the crude extract comprises Terminalia arjuna Wight et Arn. The use according to claim 1, wherein the crude extract is selected from the group consisting of Phellodendron amurence Ruprecht, Rhus javanica L., Punica granatum L., Geum japonicum Thunb. , Syzygium aromaticum (L.) Merr. et Perry, and Alpinia officinarum Hance and is used in a concentration of at most 2 µg/ml. The use according to claim 1, wherein the crude extract is selected from the group consisting of Terminalia arjuna Wight et Arn. and Paeonia suffruticosa Andrews and is used in a concentration of at most 20 µg/ml.

Inventors: HOZUMI TOYOHARU, KAGEYAMA SEIJI, KURIMURA TAKASHI, KUROKAWA MASAHIKO, NAMBA TSUNEO, OOYAMA HARUO, SATO HITOSHI, SHIRAKI KIMIYASU, TSUCHIE HIDEAKI, HOZUMI, TOYOHARU, KAGEYAMA, SEIJI, KURIMURA, TAKASHI, KUROKAWA, MASAHIKO, NAMBA, TSUNEO, OOYAMA, HARUO, SATO, HITOSHI, SHIRAKI, KIMIYASU, TSUCHIE, HIDEAKI, Hozumi, Toyoharu, c/o Showa Shell Sekiyu K.K., Ooyama, Haruo, c/o Showa Shell Sekiyu K.K.
Applicants: SHOWA SHELL SEKIYU, SHOWA SHELL SEKIYU KABUSHIKI KAISHA
Application year: 1996
Main classification: A61K35
IPC codes: A61K36, C12N9, A61P37, A61P31