Isolation and identification of endophytic fungi from Huperzia serrata and their metabolites' inhibitory activities against acetylcholinesterase and anti-inflammatory activities / 中国中药杂志

A total of 27 endophytic fungal strains were isolated from Huperzia serrata,which were richly distributed in the stems and leaves while less distributed in roots. The 27 strains were identified by Internal Transcribed Spacer( ITS) r DNA molecular method and one of the strains belongs to Basidiomycota phylum,and other 26 stains belong to 26 species,9 general,6 families,5 orders,3 classes of Ascomycota Phylum. The dominant strains were Colletotrichum genus,belonging to Glomerellaceae family,Glomerellales order,Sordariomycetes class,Ascomycota Phylum,with the percentage of 48. 15%. The inhibitory activities of the crude extracts of 27 endophytic fungal strains against acetylcholinesterase( ACh E) and nitric oxide( NO) production were evaluated by Ellman's method and Griess method,respectively. Crude extracts of four fungi exhibited inhibitory activities against ACh E with an IC50 value of 42. 5-62. 4 mg·L~(-1),and some fungi's crude extracts were found to inhibit nitric oxide( NO) production in lipopolysaccharide( LPS)-activated RAW264. 7 macrophage cells with an IC50 value of 2. 2-51. 3 mg·L~(-1),which indicated that these fungi had potential anti-inflammatory activities.The chemical composition of the Et OAc extract of endophytic fungus HS21 was also analyzed by LCMS-IT-TOF. Seventeen compounds including six polyketides,four diphenyl ether derivatives and seven meroterpenoids were putatively identified.

Detection of Huperzine A and Huperzine B in fermentation broth of endophytic fungus Colletotrichum gloesporioides from Huperzia serrate by HPLC / 生物工程学报

In this study, we established a rapid and efficient HPLC method to determine the accumulation of Huperzine A and Huperzine B in the fermentation broth of endophytic fungus Colletotrichum gloesporioides from Huperzia serrate. The chloroform extracts of fermentation broth were dissolved in methanol and filtered before injection for HPLC analysis. The analysis was performed on an Agilent Eclipse plus-C18 column (250 mm×4.6 mm, 5 μm) by isocratic elution. The mobile phase was 0.015 mol/L ammonium acetate-methanol (70:30, V/V), the flow rate was 1 mL/min and the detection wavelength was set at 308 nm. Huperzine A and Huperzine B could be well separated within 25 min. Good linearity of Huperzine A was found in the range of 1.50-48.00 μg/mL (r=0.999 5), and that of huperzine B was in 0.25-7.50 μg/mL (r=0.999 7). The average recoveries of Huperzine A and Huperzine B were 106.83% and 108.06%, respectively (RSD=3.34%, 3.60%). The results demonstrate that this method can detect the content of huperzine A and huperzine B in fermentation broth simply, rapidly, accurately and in good reproducibility. Under the optimized conditions, the accumulated content of huperzine A and huperzine B were measured from the sixth to the fifteenth day. Huperzine A and Huperzine B reached the highest (12.417 0 μg/mL and 4.660 3 μg/mL, respectively) at the fourteenth and eighth days. The analysis methodology could contribute to the future study of huperzine A and huperzine B biosynthesis in C. gloeosporioides, consequently facilitate the development of new drug resources.

Study on lateral buds induction and culture of Huperzia serrata / 中草药

Objective: Due to the low reproductive capacity and scarce resources of Huperzia serrata, and in vitro culture of H. serrata has not been really successful, we wish to make a good progress in rapid propagation in this study. Methods: Living shoot tips were used as the materials, after a variety of surface sterilization, malachite green and AAS were used to eliminate the endophytic fungi of the seedling. Then, the aseptic seedlings were transplanted to the medium with 1/2 MS + 25 g/L sucrose to be cultured. After the new dichotomous branching occurred, and the branches grew to 4-5 cm high, we cut it and subcultured for the new dichotomous branching. Results: After four times of subculture of dichotomous branching, the aseptic seedlings got the different number of proliferating lateral buds, which came from the middle or top of the stem. Comparing the structure of lateral buds with the gemma, we found it was significantly different from the gemma. The number of lateral buds from different individuals was different, the most lateral buds could get to 14 in one seedling, some lateral bud generating point can produce two or more lateral buds. After removing the apical dominance of stem tip, the lateral buds in the middle of the stem grew faster. The surviving rate of different height lateral buds was different when they separated from the stock plant. Lateral buds which beyond 5 mm, the surviving rate could get 90%, while cultured in medium of 1/2 MS + 30 g/L sucrose, after 120 d, it grew into seedling about 2-3 cm high, lateral buds less 5 mm had high mortality with the same cultured medium. Conclusion: Though in vitro culture, H. serrata can break the reproductive way by which the leaf axil could generate spore only but no lateral bud naturally, and can produce the multiple lateral bud proliferation. The lateral buds can easily produce adventitious roots by stripping, which could provide an important view for rapid reproduction of H. serrata.