Research Institute for Wakan-Yaku
(Traditional Sino-Japanese Medicines)
Toyama Medical and Pharmaceutical University
1) Sahar El-Mekkawy, Meselhy and R. Meselhy
Abstract : A new highly oxygenated triterpene has been isolated from the 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 active as anti-HIV with an inhibitory concentration of 7.8 g/ml for both, and ganoderic acid B, ganoderiol B, ganoderic acid Cl, 3 -5 -dihydroxy-6 -methoxyergosta-7,22-diene, compound 1, ganoderic acid H and ganoderiol A were moderately active inhibitors against HIV-1 PR with a 50% inhibitory concentration of 0.17 mM.
Over the past decade, substantial progress has been made in defining strategies for the treatment of human immunodeficiency virus (HIV) disease, the cause of acquired immunodeficiency syndrome (AIDS) , where natural products can serve as a source of structurally novel chemicals that are worth investigating as specific inhibitors of HIV as well as its essential enzymes, protease (PR) and reverse transcriptase (RT).
Ganoderma lucidum (Japanese name: Reishi) is one of the valuable crude drugs, which has long been used in China and Japan as a traditional Chinese medicine or a folk medicine for the treatment of various kinds of diseases1). Several biologically active triterpenes and sterols have been isolated from this mushroom and proved effective as cytotoxic2,3), antiviral4) and anti-inflammatory agents5,6). Besides, polysaccharides and glycoproteins possessing hypoglycemic7,8) and immunostimulant9-13) activities have also been isolated from its water extract. In the course of our continuing search for natural products as anti-HIV agents, the MEOH extract of the fruiting bodies was found to be moderately active against HIV-1 as well as its essential enzyme, protease (PR). Therefore this extract was selected for further fractionation. When subjected to bioassay-guided fractionation, the extract yielded several active compounds. This paper describes the isolation of thirteen compounds, and their inhibitory effects against HIV-1 and its enzyme PR.
RESULTS AND DISCUSSION
Isolation and structure determination of compounds isolated from Ganoderma lucidum
Table 2. Inhibitory Activities of Compounds from Ganoderma lucidum against Protease and Proliferation of HIV-1
Item HIV-1 PR
IC50 (mM) IC ( g/ml) HIV-1
CC ( g/ml)
MeOHext 47.7 31.3# 125#
Compound (1) 0.19 NE 1000
Ganododeric acid A (2) NE (1000) 1000
Ganododeric acid B (3) 0.17 NE 1000
Ganoderic acid CI (4) 0.18 NE 1000
Ganoderic acid H (5) 0.20 NE 1000
Ganoderiol A (6) 0.23 NE 1000
Ganoderiol B (7) 0.17 (7.8) 500
Ganoderiol F (8) 0.32 7.8 15.6
Ganodermanontriol (9) NE 7.8 15.6
Ergosterol (10) NE 7NE 1000
Ergosterol peroxide (11) NT NE 15.6
Cerevisterol (12) NE NE 31.3
3 -5 -dihydroxy-6-B-methoxy
ergosta-7,22-dienne (13) 0.18 NE 15.6
IC, the minimum concentration for complete inhibition of HIV-1 induced CEP in MT-4 cells by microscopic observation. CC, the minimum concentration for appearance of MT-4 cell toxicity by microscopic observation. NE, not effective. ( ) , concentration at which weak anti-HIV-1 activity was observed. %Inhibition at 100 g/ml. #As g/ml
Inhibitory effects of isolated compounds on HIV and its enzymes
Investigation of anti-HIV and PR-inhibitory activities of the isolated compounds (1-13) yielded some compounds with moderate activities (Table 2). In the primary screening test for anti-HIV activity, compounds 8 and 9 were found to inhibit HIV1 induced cytopathic effect (CPE) in MT-4 cells with a 100% inhibitory concentration (IC) value of 7.8 g/ml for both compounds, and the IC value for both was a half of the respective cytotoxic concentration (CC) value.
As for HIV-1 PR inhibitory effects, the PR activity was determined by analysing the hydrolysates of a synthetic substrate in the presence or absence of the isolated compounds using high performance liquid chromatography (HPLC) method. Of the tested compounds, 3 and 7 were found to be the most active against HIV-1 PR enzyme with an IC50 of 0.17 mM for both compounds. Other compounds such as ganoderiol B, ganoderic acid Cl, 3 -5 -dihydroxy-6, -methoxyergosta-7,22-diene, compound 1, ganoderic acid H and ganoderiol A inhibited the enzyme activity in a similar extent.
In the present experiment, we found that 7(8), 9(11)-lanostadiene-type triterpenes had relatively strong anti-HIV activity. On the other hand, 8(9) -lanostene-type triterpenes and ergostane-type compounds 10-12 had no inhibition of HIV-induced cytopathic effects. As to HIV-protease, we could not obtain any conclusive findings on the structure-activity relationship. Lanostane-type triterpenes showed IC50 of 0.17-0.32 mM, while ergosterol derivatives had no inhibitory activity. However, it was reported that synthetic cosalane and its derivatives had an anti-HIV effect as well as inhibitory effects on RT and PR27) . Several triterpenes have been described as antiviral compounds. Glycyrrhizin displays some limited activity against a whole range of viruses including HIV-128). Salaspermic acid29) and suberol (a lanostane-type)30) inhibit HIV-1 in H9 cells in the upper micromolar range. Bile acid derivatives were found slightly active (at 10-4 M) against HIV-1 in MT-4 cells31). Betulinic acid derivatives (lupane-type) have been described as potent inhibitors of the cytopathogenicity of HIV-1 in CEM 4 and MT-4 cells without affecting HIV-1 RT or PR activity32).
When compared with other triterpenes reported, compounds 8 and 9 can be used as leads to develop other related compounds with potential anti-HIV activity. This subject will be of particular interest to be investigated in the future
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