Isolation And Characterization Of Endophytic Huperzine A-producing Fungi From Huperzia Serrata

Huperzine A (HupA), a lycopodium alkaloid isolated originally from medicinal pteridophyta Huperzia serrata (Thunb. ex Murray) Trev., has attracted intense attention since its marked anticholinesterase (AChE) activity and is becoming an important compound in drugs for Alzheimer's disease (AD) treatment. Because of no successful commercial chemical synthesis and H. serrata cultivation and cell culture has been reported in producing HupA, thus far, most of the HupA is sourced from Chinese club moss H. serrata. However, H. serrata actually has very low HupA content, very limited distribution, and extremely slow growth. Wild population of the plant may soon become extinct if current over-collection practices are not curtailed. It is imperative to make great effort on developing alternative means of HupA production.Recently, endophytic fungi from plants have been widely accepted as an important source of drugs. Some endophytic fungi have been found to produce similar chemical compounds to those produced by its host. So far, several endophytic HupA-producing fungi associated with Huperziaceae plants were isolated and identified, which demonstrated that fermentation processes using HupA-producing endophytic fungi may be a promising approach to producing HupA.Thus, the objectives of this study were to isolate the endophytic fungal isolates of H. serrata collected from the natural populations at Lushan Botanical Garden (Jiangxi province, China); and to investigate the diversity of fungi associated with leaves, stems and roots from H. serrata; and to screen the endophytic HupA-producing fungi and acetylcholinesterase inhibitor-producing (AChEI-producing) fungi residing in the tissues of H. serrata.A total of 127 endophytic fungi isolates obtained from the root, stem and leaf segments of H. serrata were grouped into 19 genera, including Penicillium sp., Aspergillus sp., Cladosporium sp., Colletotrichum sp., Chaunopycnis sp., Acremonium sp., Capronia sp., Mortierella sp., Botrytis sp., Coniothyrium sp., Paraphaeosphaeria sp., Leptosphaeria sp., Shiraia sp., Alternaria sp., Podospora sp. and so on. Thereinto, even 10 might represent novel species and 4 might represent new genera.Penicillium sp., Aspergillus sp. and Podospora sp. are dominant populations in H. serrata; with the relative frequency 15.7%, 11.8% and 11.0% respectively. From analysis results we found some endophytic fungi showed a certain degree of tissue preference. The isolation rate, colonization rate and Shannon-Wiener index of stems were both larger than those of leaf and roots.After testing the AChE inhibitory activity of these endophytic fungi, a total of 39 endophytic fungi belonging to 15 genera showed AChE inhibition. 11 endophytic fungi showed potent AChE inhibition, with the inhibition from 60.2% to 96.5%, 7 of which were isolated from leaf.9 endophytic fungi isolates from 7 distinct genera were capable of producing HupA verified by thin layer chromatography (TLC) and reverse-phase high performance liquid chromatography (HPLC) and mass spectrometry (ESI-MS). Among the HupA-producing fungi, the yield of HupA produced by the strains Aspergillus flavus LF40 and Cladosporium cladosporioides LF70 were higher than other strains, with the yield of HupA 80.1μg/gdcw and 56.84μg/gdcw, respectively. Interestingly, in the present study, most of HupA-producing fungi (77.8%, 7 of the 9) were isolated from leaf tissues, only two from stem and root tissues, which showed tissue specificity to some degrees of these endophytic HupA-producing fungi in present study.