Studies On The Meroterpenoids Constituents And Bioactivities Of Ganoderma Petchii (Lloyd) Steyaert

As a rare traditional Chinese medicine,Ganoderma lucidum has a long history of use.In the Chinese Pharmacopoeia,Ganoderma medicines were recorded as dry fruiting bodies of Ganoderma lucidum or Ganoderma sinense which belong to Ganodermataceae Ganoderma fungi.The G.lucidum has effects of Qi soothe the nerves,relieving cough and asthma,also for neurasthenia,pyelonephritis and other diseases.Because of its good biological activities with less toxic and side effects,G.lucidum has been one of the hot species of medicinal fungi to be researched for a long time.G.petchii is a kind of fungus of Ganodermataceae fungi,and a wild medical species of G.lucidum.It has the same way as G.lucidum to be used by the people in China.Although G.petchii could be used as G.lucidum to treatment of neurasthenia,pyelonephritis,chronic hepatitis,bronchitis,antitumor and other diseases,the clinical material basis of G.petchii is not still clear,and no documents studied on chemical constituents of G.petchii have been reported ether.Some meroterpenoids have been reported with potent reno-and neuro-protective activities,these findings raised the idea of meroterpenoids’ important role to the material base of Ganoderma.In order to find out more new compounds with better activies and enrich the knowledge of the material fondation of G.petchii,the meroterpenoids constituents of the ethyl acetate extract of the dry fruiting bodies of G.petchii were studied systemally.In our investigation of G.petchii,via different chromatographic separation and purification technologies,including MCI,RP,Sephadex LH-20,preparation TLC,HSCCC,HPLC and semi-preparation HPLC,simultaneously,by the aid of 1D NMR,2D NMR,X-ray diffraction,and UV spectroscopic methods,53 compounds,including 28 meroterpenoids,were isolated,purified and elucidated from G.petchii.Among these compounds,24 compounds(1-10,18-21,24,28-35,37),including 16 meroterpenoids,are new compounds.Particularly,compounds 1-4,and 24 are aromatic meroterpenoids with novel polycyclic skeletons.Meanwhile,the rest of other known compounds,other type small molecules,are isolated from this species for the first time.The compounds are characterised as:petchiol A(1),petchiol B(2),petchiol C(3),petchiol D(4),petchioic C(5),petchioic D(6),spiropetchine A(7),spiropetchine B(8),spiropetchine C(9),spiropetchine D(10),(2R,2’S,5’S)-5-hydroxy-3-oxo-5’-(3-oxoprop-1-en-2-yl)-3Hspiro[ben-zofuran-2,1’-cyclopentane]-2’-carboxylic acid(11),spirolingzhine A(12),spirolingzhine B(13),spirolingzhine D(14),methyl(2R,2’R,5’S)-5-hydroxy-5’-(3hydroxyprop-l-en-2-yl)-3-oxo-3H-spiro[benzofuran-2,1 ’-cyclopentane]-2’-carboxylate(15),spirolingzhine C(16),(2R,2’R,5’R)-5-hydroxy-5’-(3-hydroxyprop-1-en-2-yl)-3-oxo3H-spiro-[benzofuran-2,1’-cyclopentane]-2’-carboxylic acid(17),spiropetchine E(18),petchine B(19),petchine C(20),petchine D(21),(2aR,2a1S,4aS,5S,7aS)-7a-(2,5dihydroxy-phenyl)-5-(hydroxymethyl)-octahydro-2H-1,7-dioxacyclopenta[cd]inden-2-one(22),lingzhiol(23),petchine E(24),lingzhilactone B(25),ingzhilactone D(26),lingzhilactone C(27),(E)-3-(7,8-dihydroxy-4,8-dimethylnon-3-en-l-yl)-5-(2,5-dihydroxyphenyl)-5-hydroxyfuran-2(5H)-one(28),3-(2-(2,5-dihydroxy-phenyl)-2-methoxyethyl)benzoic acid(29),6,7-dihydroxyisochroman-l-one(30),petchioic A(31),petchioic B(32),petchiate A(33),petchiate B(34),petchine A(35),3-hydroxy-5-methyl-5,6-dihydro-7Hcyclopenta[b]pyridin-7-one(36),2-(5-hydroxy-4-methyl-6,7-dihydro-5H-cyclopenta[c]pyridin-1-yl)ben-zene-1,4-diol(37),3-benzylhexahydropyrrolo[1,2-α]pyrazine-1,4-dione(38),2-(6,7-dihydro-5H-cyclopenta[c]pyridin-3-yl)benzene-1,4-diol(39),1H-indole-3-carbaldehyde(40),4-(pyridin-4-yl)benzene-1,2-diol(41),6-hydroxy-4H-chromen-4-one(42),2-(4-methoxyphenyl)acetic acid(43),N-phenethylformamide(44),5-hydroxyfuran-2-carbaldehyde(45),5-ethoxyfuran-2-carbaldehyde(46),5-methoxyfuran-2-carbaldehyde(47),6,7-dihydroxy-2H-chromen-2-one(48),1-(4-ethylphenyl)ethane-1,2-diol(49),1-(2-ethylphenyl)ethane-1,2-diol(50),1-(2,5-dihydroxyphenyl)ethan-l-one(51),5-hydroxy-4-(4-hydroxyphenyl)furan-2(5H)-one(52),ganoderic acid C6(53).Most of the new compounds have chiral carbons,the compounds selected to be evalue their bioactivities were chiral separated via chiral separation technology.Their ECD calculations were carried out by the quantum chemistry calculation method,using time-dependent density functional theory(TDFT)and density functional theory(DFT)method,under B3LYP/6-31G(d)//B3LYP/6-31G(d)level to dederminate their absolute configrutions.According to the calculation results,the absoluton configurations of(-)-1,(+)-1,(一)-2,(+)-2,(-)-3 and(+)-3 were determinated as 2’R,3’S,6’S,7’S,10’R;2’S,3’R,6’R,7’R,10’S 2’R,3’S,6’S,7’R;2’S,3’R,6’R,7’S;6’S,7’S,9’S and 6’R,7’R,9’R respectively.In bioassay section,antifibrotic and acetylcholinesterase(AChE)inhibitory activity assays were performed to the now compounds which absolute configuration is clear.The experiments of inhibitor activities to FN secreted by HK-2(human proximal tubular epithelial cell)cells induced by TGF-β1 could illustrate that compounds(-)-1 could inhibit fibronectin(FN)secreted by HK-2 cells to antifibrotic under 5μM level(P<0.05).Noticeably,(+)-2 and(-)-2 shows anti-fibrotic activity under 5μM level(P<0.01).Inhibitory assay of FN protein expression were performed by Western-blot analysis,the results show that compounds(-)-1,(+)-2 and(-)-2 could inhibit FN protein expression in HK-2 cells with TGF-β1 as stimulus.Based on these antifibrotic assays,compounds(+)-2 and(-)-2 were evuled as potent reno-protective active ingredients due to they could inhibit FN secretion to ECM(extracellular matrix)or expression in cell.Acetylcholinesterase(AChE)inhibitory activity assays of the compounds leaded to the results that compound(+)-2 and(-)-2 could inhibit AchE to 57.38%and 56.76%under 50 μM level,respectively,thus,it illustrates that both(+)-2 and(-)-2 have good activities to inhibit AChE.All above tests suggested that meroterpenoids of Ganoderma maybe were the functional constituents of Ganoderma’s pharmaceutical potency.Cytotoxic assay(including 8 different cell lines:K562,MCF-7,A549,Huh-7,Hela,DU145,H1975,A431),cyclooxygenase(COX)inhibitory assay and MDCK cell based anti-influenza assay were futher experimentized on compounds 31-35 to evalue their bioactivities,but unfortunately,none of them was found to be active towards these assays.In a summary,G.petchii is a rare medicinal fungus rarely studied;the results of this thesis would contribute to the researeh on the chemical constituents,it could enhance understanding the diversity of Gnoderma chemical composition,and supplying seientific evidenees for the development and utilization of G.petchii.