Studies On The Fungal Metabolites Affecting The Growth And Active Components Of Three Traditional Chinese Medicine

Two species of fungi (Armillaria mellea and Epulorhiza sp.) affecting the growths, developments and active components of their hosts, were studied by the methods of chemistry, physiology, biochemistry and analytical chemistry. The three portions of research thesis included studies on chemical constituents of Armillaria mellea and their effects on the mycelium growth and the effective components of Polyporus umbellatus, the effects of A. mellea metabolites on Gastrodia elata seed germination and development, and studies on chemical constituents of Epulorhiza sp. and their effects on the growth and effective components of Anoectochilus roxburghii.Chemical constituents of natural A. mellea rhizomorphs were systematically studied for the first time. Thirteen compounds were isolated and identified on the basis of modern chromatography and spectroscopic analysis and they are friedelin, 3α-hydroxyfriedel-2-one, 3-hydroxyfriedel-3-en-2-one, ergosterol, ergosterol peroxide, 6, 9-epoxy-ergosta-7, 22-dien-3β-ol, 3β-hydroxyglutin-5-ene, 2- acetyl-1,3,8-trihydroxyxanthone, octadecylic acid, isoselachoceric acid, octadecylic methyl ester, D-mannitol and glucose. To the best of our knowledge, the occurrence of triterpenes whose skeletons are friedelin derivatives was reported for the first time in the fungal kingdom. 6, 9-epoxy-ergosta-7, 22-dien-3β-ol and. all of the triterpenes were reported in the genus Armillaria for the first time. 2-Acetyl-1,3, 8-trihydroxyxanthone was isolated as a natural product for the first time. It was a supplement for the information of 3β-hydroxyfriedel-2-one, 3-hydroxyfriedel-3-en-2-one and 6, 9-epoxy-ergosta-7, 22-dien-3β-ol that ~1H NMR spectral data for them was described completely for the first time.The effects of A. mellea metabolites on P. umbellatus mycelium growth and active components were studied. The results showed that water extract (W) of A. mellea could supply carbon source and nitrogen source for P. umbellatus mycelium growth. Methanol extract (ME) of A. mellea could significantly accelerate P. umbellatus mycelium growth and its petroleum ether extract was the most effective part which increased coenobium diameter by 1.26-flod bigger than that of control. Ergosterol and friedelin isolated from the petroleum ether extract could promote P. umbellatus mycelium growth, especially in its early stage of growth that showed they were the active components to accelerate P. umbellatus mycelium growth. All the extracts and compounds mentioned above could induce the production of polyporusterones like compounds from P. umbellatus mycelium, and the more complex the extract is, the more effective it does. Petroleum ether extract of A. mellea was the best treatment which could induce 14 polyporusterones in the mycelium of P. unbellatus.The effects of A. mellea metabolites on seed germination and development of G. elata were studied.The results indicated that the components inhibiting seed germination of G. elata existed manily in water extract. Both supematant fluid and polysaccharides from water extract of A. mellea could significantly inhibit seed germination of G. elata, which could delay the germination and decrease germination rate in a dose dependent way. Mannitol isolated from supernatant fluid also could inhibit G. elata seed germination, but the inhibition was not as effective as that of supernanate fluid and polysaccharides. Thereafter, we proposed that the inhibition of supernatant fluid against G. elata may be a result of synergistic effects by multiple components. Methanol extract of A. mellea inhibited G. elata seed germination, but accelerated vegetative propagation of G. elata protocorms in suit concentrations. Ergosterol isolated from methanol extract of A. mellea could promote G. elata seeds germination rate and accelerate vegetative propagation of G. elata protocorms.Fermentation culture of Epulorhiza sp. was successfully accomplished, and both fermentation broth and mycelium were obtained. Chemical constituents of the fungal fermentation broth and mycelium were systematically investigated for the first time. Thirty five compounds were isolated on the basis of modern chromatography methods, among which 33 compounds were identified by spectroscopic analysis. We found that steroids are the main constituents (8 steroids obtained), in addition, there were 2 flavanoids, 1 anthraquinone, 6 nucleosides or their analogues, 2 cyclopeptides, 8 micromolecule compounds, 4 aliphatic series, and 2 monosaccharides. All the compounds were isolated from the genus Epulorhiza for the first time.The effects of AR 18 metabolites on the growth of A. roxburghii and its flavonoids constituentswere explored. The results indicated that ethanol extracts of both fermentation broth and mycelium of Epulorhiza sp. in suit concentration could extensively increase the growth of A. roxburghii. The active components mainly distributed in acetic ether extract from ethanol extract of fermentation broth and n-butanol extract from ethanol extract of mycelium. Compounds AR7 and AR10 could improve the growth of A. roxburghii. The fact that AR7 existed in both active extract suggested that AR7 might be the active ingredient of Epulorhiza sp..Every elicitors from the fungus Epulorhiza sp. affectecd the content but not the category of flavonoids in A. roxburghii. Ethanol extracts of both fermentation broth and mycelium promoted the total count of known flavonoids and mainly promoted the content of fla-3, the maximum increased 18-fold higher than the control in A. roxburghii. Acetic ether extract of fermentation broth could promote the content of every known flavone in a dose dependent way. Petroleum ether, n-butanol and water extract of fermentation broth could promote the content of fla-5, water extract had the most induction and could increase 5.15-fold higher than the control. Petroleum ether extracts of mycelium decreased the total content of known flavonoids. The total content of known flavonoids of acetic ether and water extract were close to control, n-Butanol extracts promoted the total content of known flavonoids in a dose dependent way. Compounds AR7 and AR10 decreased the total content of known flavonoids in a dose dependent way. To sum up, AR7(m) as elicitor could not only promote the growth of A. roxburghii but also have little effect on the accumulation of flavonoids. The further research showed that AR7 could promote the photosynthetic rate and induce the production of IAA to promote the growth of A. roxburghii.