Skullcap Of Endophytic Fungi Preliminary Study

Endophytic fungi are fungi that colonize the inside of healthy plant tissues, without causing visible disease symptoms, for all or part of their life cycle. It was found that some endophytic strains could produce the bioactive compounds, similar to their host, in research for the past few years, and therefore becoming a novel source of potentially useful medicinal compounds. As a new sources of microorganisms, these secondary metabolites have gradually been a research focus on biological pharmaceutical production, biological control of pest and industrial fermentation. As a well-known traditional Chinese herbal medicine, Scutellaria baicalensis Georgi has been commonly used for sedation, hypotension, antimicrobic, anti-virus, anti-tumor etc., treated tepid disease, upper respiratory tract infection, and pulmonary hot cough, hot and humid HuangDan, pneumonia and diarrhea disorders. In this paper, endophytic fungi from medicinal plant Scutellaria baicalensis Georgi were studied, and the results are as follows.(1)A total of 17 strains of endophytic fungi were isolated from healthy, and symptomless tissues of Scutellaria baicalensis Georgi using traditionary method of microbiology, among which 3 strains were gained from the root,7 strains from the stem, 1 strains from the leaf,6 strains from the flower.(2) In the present study,10 kinds of indicator bacteria were used as test antimicrobial actions targets to assess antibacterial activity, including Escherichia coli(A70), Bacillus subtilis(A99), Staphylococcus aureus(26003) etc., which was measured by Oxford plate agar diffusion bioassay. The result shows that most strains take on antibacterial activity on at least one indicator bacteria to some extent, and 4 strains (J1, J4, J5 and H3)have antibacterial activity against multiple kinds of indicator bacteria, manifesting a relatively wider antibacterial spectrum.(3) Besides, thin layer chromatography (TLC) and high-performance liquid chromatography (HPLC) were carried out to select the strains, which could produce bacailin like its host plant Scutellaria baicalensis Georgi. The result shows that extracts from Endophytic fungal strains, labled as J1 and H3 (inside cell), had a component with the same Rf value in TLC as that of authentic bacailin and one ingredient of the J1and H3 extract had a retention time identical with that of authentic bacailin in HPLC. Therefore, it was concluded that the fungal strains J1 and H3 appears to produce the bioactive ingredient bacailin like its host plant.(4) According to the Colony morphology of the fungal culture and the microscopic observation with staining, more combined with ITS nucleotide sequences analysis, it was concluded Endophytic fungi J1 and H3 should be Penicillium sp. of Deuteromycota.(5) As strain J1 produce more bacailin, the optimization tests are done to gain the most optimized culture medium and conditions. Through the test among different basic culture medium in common use, we found that bean sprouts liquid medium was propitious to J1 both in mycelium growth and bacailin accumulation. By the ANOVA analysis, the best carbon resource was approved to be sucrose, and the best nitrogen resource was approved to be ammonium nitrate. Further ferment conditions were optimized by orthogonal experiment. Among three factor seclected, the results showed that carbon resource concentration was most influencing factor to the growth of the mycelia and the yield of bacailin. The results of optimization were that:the concentration of sucrose was 4.0%, the concentration of ammonium nitrate was 0.15% and the pH was 7.0 could gain the most mycelium yield, while the concentration of sucrose was 4.0%, the concentration of ammonium nitrate was 0.25% and the pH was 6.0 would gain the maximum bacailin yield. Therefore in order to gain the maximum yield of bacailin, the ferment conditions should be:the concentration of sucrose was 4.0%, the concentration of ammonium nitrate was 0.15% and the pH was 7.0, and when the strain was reach to the stable period, a little improvement in the concentration of ammonium nitrate and reducing of pH should be done to improve the yield of bacailin.