Studies On The Chemical Constituents And Pharmacological Activities Of Inonotus Hispidus And Fomitiporia Ellipsoidea

Based on the theories of natural product chemistry and pharmacology, the chemical constituents and bioactivities of Inonotus hispidus and Fomitiporia ellipsoidea were studied. Twenty eight compounds, including four new ones, have been isolated from the fruiting bodies of two medicinal fungi by variety of chromatographic methods (silica gel, reversed-phase (ODS), sephadex LH-20and pre-HPLC) and structures of twenty seven compounds were elucidated on the basis of extensive spectroscopic analysis including1D and2D-NMR(1H-NMR,13C-NMR,1H-1H COSY, HMQC and HMBC) as well as mass spectrometry (EI-MS, ESI-MS and HR-ESI-MS). The new compounds involved pyrone and furfuran ester, and named inonotusin A, inonotusin B, fomitiporiaester A and phelligridin K. Some of the isolates and extracts were evaluated for their vitro and vivo antitumor, antioxidant and antibacterial activities.Ⅰ. hispidus is a parasitic fungus preferably living on mulberry of more than40years in the southern region of Xinjiang province, and has been used as traditional medicines for the treatment of cancer, diabetes, arthritides and other stomach problems. Fifteen compounds were isolated from the fruiting body of Ⅰ. hispidus, including7(8),22(23)-diene-3-one-ergostane, ergosterol,5α,8α-epidioxy-(22E,24R)-ergosta-6,22-dien-3β-ol,4,6,8(14),22(23)-tetraen-3-one-ergostane, eburicoic acid, hispidin, hispolon, inonotusin A, inonotusin B,(E)-4-(3,4-dihydroxyphenyl) but-3-en-2-one, inoscavin C, inoscavin D, protocatechuic acid, protocatechualdehyde and inosine. Compounds inonotusin A and B,(E)-4-(3,4-dihydroxyphenyl) but-3-en-2-one, hispidin and protocatechualdehyde were tested for cytotoxic activity against two cell lines, human hepatoblastoma(HepG-2) and human breast carcinoma(MCF-7) by SRB method. The result showed only inonotusin A had moderate cytotoxicity against MCF-7with IC50values of19.6μM, and other four compounds were noncytotoxic to the two cell lines. In vivo anti-tumor activity of petroleum ether and water extracts from fruiting bodies in different stage also carried out by H22tumor-bearing mice transplanting model. The result showed that petroleum ether and water extracts in maturation stage had significant antitumor activities, and the inhibitory value were44.40%(petroleum ether low dosage group,50mg/Kg),55.24%(petroleum ether high dosage group,100mg/Kg),54.01%(water low dosage group,500mg/Kg),52.19%(water high dosage group,1000mg/Kg), and closed to the inhibition of CTX group (61.36%). The water extracts could regulate the immune function of tumor-bearing mice, but petroleum ether extract had effection to immune organ negatively. In darkening fruiting body stage, the inhibitory effects of petroleum ether and water extracts were more lower than the maturation stage, and the value were34.73%(50mg/Kg) and29.99%(500mg/Kg), but the high dosage group had no activity to H22. The inhibition value of methanol extracts was29.77%(500mg/Kg). The extracts had the similar effects to immune organ of tumor-bearing mice, the water extracts could regulate the immune function, but petroleum ether extracts had effection to immune organ negatively. The leucocyte, spleen index and thymus index of water extracts increased higher than CTX group. Moreover, the concentrations of phenolic compounds in the70%ethanol extract of Inonotus hispidus were measured with Folin-Ciocalteu reagent, and total flavonoid concentration was determined by the NaNO2-Al(NO3)3method. The result showed the phenolics were64.02±0.73μg/mg(gallic acid) while total flavanoids were5.74±0.13μg/mg (rutin equivalent) in the70%extract. Antioxidant and antimicrobial activities of Ⅰ. hispidus extracts obtained with70%ethanol and compound hispidin were investigated in this study.·DPPH and·OH free radical-scavenging activity were found to90%inhibition at concentration of200μg/mL. Compound inonotusin B potently scavenged the ABTS radical cation and reached to the level of59.53±9.70μmol.70%ethanol extract and hispidin showed narrow antibacterial activities against Staphyloccocus aureus and Bacillus subtilis.70%ethanol extracts inhibited the growth of the Escherichia coli, and inhibition values was40.35%at concentration of40.96mg/mL.Fomitiporia ellipsoidea was first collected in Wanmulin Nature Reserve in2008by Bao-Kai Cui and Yu-Cheng Dai, and local people called "sanghuang". In2010, Cui and Dai were performing field work in tropical woodland on Hainan Island, China, studying wood-rotting fungi. The pair uncovered a very large F. ellipsoidea fruit body on a fallen Quercus asymetrica log, which turned out to be the largest fungal fruit body ever documented. Thirteen compounds were isolated from the fruiting body of F. ellipsoidea, and twelve compounds were elucidated, including fomitiporiaester A,(E)-4-(3,4-dihydroxyphenyl) but-3-en-2-one, hispidin, hispolon, inoscavin A, inoscavin C, methylinoscavin D, inoscavin E, phelligridin K, inonoblins B, protocatechuic acid and protocatechualdehyde. Compounds fomitiporiaester A、inoscavin C and inonoblins B were tested for cytotoxic activity against two cell lines, human hepatoblastoma(HepG-2) and human oophoroma (SKOV3) by SRB method. The result showed that inoscavin C and inonoblins B had moderate cytotoxicity against the two cell lines and fomitiporiaester A was noncytotoxic to the two cell lines. In vivo anti-tumor activity of fomitiporiaester A was carried out by H22tumor-bearing mice transplanting model. The result indicated fomitiporiaester A had manifest antitumor activity, and the inhibition ration were 42.94%,49.17%and58.15%at concentration of5mg/Kg,10mg/Kg and20mg/Kg, respectively. Inhibition value nearly to the CTX group (52.56%) at dosage10mg/Kg and better than the CTX group at dosage10mg/Kg. Inhibition effects had dose-effect relationship, and it had significant difference to compare negative control (p<0.01), In addition,compound can increase the immune organ index. Some of isolated hispidin derivates from fruiting bady of F. ellipsoidea potently scavenged the ABTS radical cation in which new compound phelligridin K showed the highest activity at the level of3.56mM Trolox. But fomitiporiaester A had no activity to scavenge the ABTS radical cation.As a conclusion, the chemical constituents and bioactivities of Ⅰ. hispidus and F. ellipsoidea were studied in this thesis. We isolated some compounds with anti-tumor and antioxidant activitives, including four new compounds.The present results established the basis for discovery of bioactive lead compounds and provided theoretical foundation for further utilization of the two species fungi.