The Studies On The Constituents Of The Root Of Anemone Tomentosa And Its Secondary Metabolites Bioactivity Of Endophytic Fungi

Anemone tomentosa (Maxim.)Pei is a perennial herbaceous paint which belongs to the Ranunculaceae Anemone L. In China, it is commonly called "Dahuocao"、"Yemianhua"、"Tubaitouwen"、"Baitouwen"(Shanxi). The root and stem of Anemone tomentosa have been used in traditional Chinese medicines for the treatment of various carbuncles, malaria, cough, contusions and strains, ect. Besides, it has injuring effect on some insects. In this paper, Anemone tomentosa was choosed as the experimental material, we mainly taking the power of root and the endophytic fungi which isolated from the root and stem of Anemone tomentosa as the entry point to conduct this studies. This thesis dealt with investigation on the chemical consitituents of the root of Anemone tomentosa, combined the bio-activity of the endophytic fungi of its root and stem, we look forward to laying a solid theoretical foundation for further study and Anemone tomentosa as a new medicine resource.1. Ground air-dried roots of Anemone tomentosa were extracted with95%EtOH, the EtOH extract was suspended in distilled water and then partitioned successively with petroleum ether, EtOAc and n-BuOH. Five compounds were isolated by silica gel column chromatography, macroporous resin colum chromatography, Sephadex LH-20. And the structures of these compounds were identified by modern spectroscopic methods, for example IR, UV, NMR spectrometer and DEPT. and their physical and chemistry nature, combing with their physical and chemistry nature. They were β-sitosterol(1), oleanolic acid(2),23-hydroxy-3-oxo-olean-12-en-28-oic acid(3), daucosterol (4) and2α,3α,23-Tri-hydroxy-olean-12-en-28-oic acid O-β-D-glucopyranosyl ester(5). Compounds3and5were first isolated from Anemone tomentosa. The fllowing is studying the antimicrobial activity and antioxidant activity of the parts of petroleum ether, EtOAc, n-BuOH and five compounds. The results showed that the antioxidant activity of the part of EtOAc was strong.2.23endophytic fungus were isolated from the fresh roots and stems, in which10and13fungus were obtained from the roots and stems respectively. The part of endophytic fungus attribution was made by molecular identification and micromorphologic observation. Then we built a phylogenetic tree to determine the genetic relationship among endophytic fungus. Our results showed that the root-4belongs to Streptococcus; root-3, root-5and root-8belong to Fusarium; root-9, root-10, stem-5and stem-12belong to Alternaria; stem-3belongs to Chaetosphaeronema; stem-4and stem-9belong to Septoria. The endophytic fungus from Anemone tomentosa mainly belongs to Fusarium, Alternaria and Septoria.3. In vitro antimicrobial and antioxidant activity of the endophytic fungi were analyzed by filter paper method and DPPH, reducing power, hydroxyl radical scavenging, respectively. Our results showed that the endophytic fungus of Anemone tomentosa had a certain antimicrobial activity against six test bacteria. Among them, the root-1, root-7and root-8have a strong inhibited effect on six test bacteria, especially the EtOAc and n-BuOH of root-8, which they inhibition zones were more than20mm. In addition, the endophytic fungi isolated from the roots have better antibacterial activity than which from the stems, especially the ethyl acetate extracts. Among13endophytic fungus from stems, except the ethyl acetate of stem-7and stem-10, the mycelia extract of stem-11, the antimicrobial activity of endophytic fungus isolated from stem were not significant. Furthermore, we selected the diameter of inhibition zone≥20mm for testing minimal inhibitory concentration (MIC), the MIC of the mycelia extract of root-1was0.313mg/mL to Streptococcus uberis and Pseudomonas aeruginosa, the rest of MIC was0.156mg/mL to four test bacteria. Both the MIC of the ethyl acetate and the n-BuOH extracts of root-8were0.156mg/mL against to six test bacteria. The study showed that the antioxidant activity of endophytic fungus from Anemone tomentosa was not obvious.