Toru Otake

ANTI-HIV-1 AND ANTI-HIV-1-PROTEASE SUBSTANCES FROM GANODERMA LUCIDUM

A new highly oxygenated triterpene named ganoderic acid alpha has been isolated from a methanol extract of the fruiting bodies of Ganoderma lucidum together with twelve known compounds. The structures of the isolated compounds were determined by spectroscopic means including 2D-NMR. Ganoderiol F and ganodermanontriol were found to be active as anti-HIV-1 agents with an inhibitory concentration of 7.8 micrograms ml-1 for both, and ganoderic acid B, ganoderiol B, ganoderic acid C1, 3 beta-5 alpha-dihydroxy-6 beta-methoxyergosta-7,22-diene, ganoderic acid alpha, ganoderic acid H and ganoderiol A were moderately active inhibitors against HIV-1 PR with a 50% inhibitory concentration of 0.17-0.23 mM.

Anti-HIV-1 phorbol esters from the seeds of Croton tiglium

Five phorbol diesters, together with three known ones, were isolated from a MeOH extract of the seeds of Croton tiglium, and their structures were determined by spectroscopic methods and selective hydrolysis of acyl groups. These compounds were assessed for their abilities to inhibit an HIV-induced cytopathic effect (CPE) on MT-4 cells and to activate protein kinase C (PKC) associated with tumor-promoting action. 12-O-Acetylphorbol-13-decanoate and 12-O-decanoylphorbol-13-(2-methylbutyrate) effectively inhibited the cytopathic effect of HIV-1 [complete inhibitory concentration (IC100) values of 7.6 ng/ml and 7.81 microg/ml, and minimum cytotoxic concentration (CC0) value of 62.5 and 31.3 microg/ml, respectively]; however, 12-O-acetylphorbol-13-decanoate showed no activation of PKC at concentrations of 10 and 100 ng/ml. 12-O-Tetradecanoylphorbol-13-acetate (TPA) was found to be not only the most potent inhibitor of HIV-1-induced CPE (IC100 value of 0.48 ng/ml), but also the most potent activator of PKC (100% activation at 10 ng/ml).

Inhibitory effects of Sudanese plant extracts on HIV-1 replication and HIV-1 protease.

Forty‐eight methanol and aqueous extracts from Sudanese plants were screened for their inhibitory activity on viral replication. Nineteen extracts showed inhibitory effects on HIV‐induced cytopathic effects (CPE) on MT‐4 cells. The extracts were further screened against HIV‐1 protease (PR) using an HPLC assay method. Of the tested extracts, the methanol extracts of Acacia nilotica (bark and pods), Euphorbia granulata (leaves), Maytenus senegalensis (stem‐bark) and aqueous extracts of A. nilotica (pods) and M. senegalensis (stem‐bark) showed considerable inhibitory effects against HIV‐1 PR. Inhibitory principles were isolated from M. senegalensis and their activities were also discussed. Copyright

Inhibitory effect of tachyplesin I on the proliferation of human immunodeficiency virus in vitro

An antimicrobial peptide, tachyplesin I, isolated from hemocytes of the Japanese horseshoe crab (Tachypleus tridentatus) was examined for its inhibitory effects on human immunodeficiency virus (HIV) infection in vitro. At a concentration of 7.5 micrograms/ml, tachyplesin I suppressed the development of cytopathic effects (CPE) by more than 70% in MT-4 cells infected with HIV (lymphadenopathy-associated virus). This inhibitory effect was observed only when the drug was added during the adsorption period of the virus to the cells. In cocultures of MOLT-4 and persistently HIV-infected cells (MOLT-4/HIV), tachyplesin I at the same concentration completely inhibited multinucleated giant cell formation. Infectivity of HIV was reduced by 10(-2.5) in medium free from fetal calf serum containing tachyplesin I at a concentration of 200 micrograms/ml. Tachyplesin I did not show any inhibitory effect on reverse transcriptase activity of HIV at concentrations of 9-80 micrograms/ml at which tachyplesin I inhibited HIV infection. These results suggest that the anti-HIV action of tachyplesin I was due to the inhibition of virus adsorption.

Biological effect of PCBs, PCQs and PCDFs present in the oil causing yusho and yu-cheng.

Male Sprague-Dawley rats were daily given orally for 22 days a regimen consisting of polychlorinated biphenyls (PCBs), 1 mg/day; polychlorinated quaterphenyls (PCQs), 1 mg/day; polychlorinated dibenzofurans (PCDFs), 10 micrograms/day; or a mixture of PCBs, PCQs and PCDFs (Mix-1, 1 mg + 1 mg + 10 micrograms/day). Female Cynomolgus monkeys were daily administered PCBs (5 mg), PCQs (5 mg) or a mixture (Mix-2) containing 5 mg PCBs + 20 micrograms PCDFs for 20 weeks. The PCBs, and PCDFs had the components of PCBs, PCQs and PCDFs similar to those contained in Japanese yusho oils, respectively. The PCB-treated rats and monkeys showed hepatic hypertrophy, immunosuppression and increased drug-metabolizing enzyme activities in hepatic microsomes, but were devoid of the dermal symptoms characteristic of yusho. PCQs caused an increase in drug-metabolizing enzyme activities in hepatic microsomes and immunosuppression in monkeys, but these effects were much smaller than those found with PCBs treatment. On the other hand, treatment with PCDF or Mix-1 or Mix-2 caused hypertrophy of the liver, immunosuppression, increase in drug-metabolizing enzyme activities of hepatic microsome to much greater extent than observed with PCBs, being more than 100 times as effective as PCBs. In addition PCDFs and the mixtures containing PCDFs caused weight loss and thymic atrophy. PCDFs and Mix-2-treated monkeys showed the dermal symptoms that are characteristic of yusho patients but were not observed in monkeys treated with PCBs and PCQs alone. These results suggest that PCDFs are the primary causative agent of yusho.

Effect of polychlorinated biphenyls and polychlorinated quaterphenyls in cynomolgus monkey (Macaca fascicularis)

Female Cynomolgus monkeys (Macaca fascicularis) with P-KC-400, Y-PCB, PY-PCB or polychlorinated quaterphenyls (PCQ) received a daily dose of 5 mg for 20 weeks, and some monkeys received a daily dose of 10 mg of Y-PCB or 0.5 mg of PCQ. The chemical compositions of the polychlorobiphenyls (PCB) used for the oral administration were as follows: P-KC-400, PCB from which polychlorodibenzofurans (PCDF) have been removed from Kanecklor 400, largely contains tri- and tetrachlorobiphenyls and no PCDF. Whereas, Y-PCB and PY-PCB, PCB with constituents similar to PCB ingested by yusho patients, largely contain penta- and hexachlorobiphenyls, in addition, PCDF of 400 ppm was present only in Y-PCB, but not in PY-PCB. There were immunosuppression, enlargement and histopathological changes of the liver (such as interstitial inflammation, and proliferation of epithelial cells of biliary duct, etc.) in the groups fed P-KC-400 and PY-PCB (free of PCDF). In the group fed Y-PCB (with PCDF), there were more apparent decreases in body weight, immunosuppression, fatty liver and histopathological changes than in the groups P-KC-400 and PY-PCB. In addition, there were hair loss, acneform eruptions, edema of the eyelid, congestion and abscess of the Meibomian gland, and cornifications of the skin, characteristic dermatological findings of yusho disease.

Anti-human immunodeficiency virus-type 1 activity of constituents fromJuglans mandshurica

Three naphthalene glycosides (1-3), four flavonoids (4-7), and two galloyl glycosides (8-9) were isolated from the stem-bark ofJuglans mandshurica (Juglandaceae). Their structures were determined by chemical and spectral means, including to 2D-NMR (COSY, HMQC, and HMBC) experiments. Amongst the isolated compounds, taxifolin (4) showed the most potent HIV-induced cytopethic activity against MT-4 cells with complete inhibitory concentration (IC100) value of 25 μg/ml and maximum cytotoxic concentration (CC100) value of above 100 μg/ml. However, naphthalene glycosides (1-3), flavonoids (5-7)), and galloyl tannins (8-9) were inactive against anti-HIV-1 activity.

Lignan, sesquilignans and dilignans, novel HIV-1 protease and cytopathic effect inhibitors purified from the rhizomes of Saururus chinensis.

Five lignans were isolated from the ethyl acetate extracts of Saururus chinensis rhizomes and evaluated for anti-HIV-1 activity. Their structures were elucidated as two dilignans, manassantin A (1), manassantin B (2), two sesquilignans, saucerneol B (3) and saucerneol C (4), and a new lignan, saururin B (5) by spectroscopic analysis. Of these components, manassantin A (1) and saururin B (5) showed dose-dependent inhibitory activities on HIV-1 protease with IC(50) values of 38.9 and 5.6 microM. In addition, manassantins A (1), B (2) and saucerneol B (3) inhibited HIV-1-induced cytopathic effects in a human T lymphoblastoid cell line with IC(100) values of 1.0, 1.0 and 0.2 microM, respectively. Of these active constituents, saucerneol B (3) showed the most potent and selective anti-HIV-1 activity (IC(100) of 0.2 microM, CC(0) of >125.0 microM, and SI of >520.8).

Synthesis, anti-HIV and anti-oxidant activities of caffeoyl 5,6-anhydroquinic acid derivatives

In our continued research on chlorogenic acid analogues and derivatives with improved bioactivity, we have synthesized some caffeoyl 5,6-anhydroquinic acid derivatives. The 1,7 acetonides of chlorogenic acid (15), and of the mono-caffeoyl 5,6-anhydroquinic acids (7-8) showed appreciable anti-HIV activity. The 3,4-dicaffeoyl 5,6-anhydroquinic acid (12) exhibited an anti-HIV activity twice as that of 3,5-dicaffeoylquinic acid (22). The caffeoyl 5,6-anhydroquinic acid derivatives displayed potent anti-oxidant activities. The mono-caffeoyl 5,6-anhydroquinic acids (10-11) were more than twice stronger than chlorogenic acid (21) on SOD-like activity.

Ability to induce p53 and caspase-mediated apoptosis in primary CD4+ T cells is variable among primary isolates of human immunodeficiency virus type 1.

Infection with human immunodeficiency virus type 1 (HIV-1) is associated with dramatic depletion of CD4(+) T cells, the major HIV-1-induced pathogenesis. Apoptosis has been suggested to play an important role for the T cell depletion and a number of mechanisms have been proposed for the apoptosis in T cells. Here, we compared the levels for apoptosis induction in primary peripheral blood mononuclear cells (PBMCs) among several laboratory strains and primary isolates of the HIV-1 subtypes B and E. The results showed that apoptosis in infected PBMCs, preferentially in CD4+ T cell population, became detectable around the time for virus production by flow cytometric terminal transferase dUTP nick end labeling (TUNEL) technique and staining with the nuclear dye Hoechst 33342. The abilities to induce apoptosis in PBMCs were highly variable in individual isolates. The increase of p53 protein in infected PBMCs, which was initiated before virus production, was observed in infected PBMCs and the levels of p53 protein were almost proportional to the rates of the isolates to induced apoptosis. The cells infected and cultured in the presence of Z-VAD-FMK had significantly decreased cell mortalities, indicating that activated caspases also played a significant role in the apoptosis. Thus, HIV-1-induced apoptosis in primary T cells was accompanied by the p53 protein and caspase activation at varied levels in primary isolates.