Study On Isolation And Fermentation Condition Of A Huperzine A–producing Endophytic Fungus, Shiraia Sp. Slf14, From Huperzia Serrata

Huperzine A (HupA), a Lycopodium alkaloid, was first isolated from a traditional Chinese medicine Huperzia serrata (Thunb. ex Murray) Trev. classified to Huperziaceae Pteridophyta. HupA has been proved to be medically effective to Aezheimer's disease (AD). So far, most of the HupA used in herbal supplements and medicines is extracted from H. serrata and some other species in the Huperziaceae. However, H. serrata actually possesses a very low content of HupA, has very limited distribution, and grows very slowly.. Meanwhile, no successful commercial cultivation has been reported for H. serrata or other species in the Huperziaceae. Because of this, increasing efforts have been made to develop alternative means of HupA production. Otherwise, wild populations of these plants will continue be threatened and may soon become extinct if current harvest practices are not curtailed. Recently, endophytic fungi from plants have been widely accepted as an important source of drugs. A large number of compounds with new structures and various bioactivities are continuously isolated. It is known that many bioactive agents produced by plants could also be produced by endophytic fungi. Thus, if a microbial source of HupA would be available, then fermentation processes using HupA-producing endophytic fungi may be an alternative way to produce HupA, and it would eliminate the need to harvest and extract the slow growing and relatively rare Huperiaceae plant for this drug. In present study, the endophytic fungus of H. serrata was isolated and the HupA-producing endophytic fungus were screened, and fermentation conditions that endophytic fungus produced HupA were optimized.The 56 endophytic fungi were isolated from the roots, stems and leaves of H. serrata, 14 isolates of 56 isolates showed the high inhibitory activity to AChE. The inhibition rate of the extract from strain Slf14 was 96.47%, so the strain and product identification and fermentation conditions of strain Slf14 were investigated. According to the morphology, ITS and 18SrDNA phylogenetic analysis, strain Slf14 was classified to the genus Shiraia and designated as Shiraia sp. Slf14. The HupA produced by this endophytic fungus was shown to be identical to authentic HupA analyzed by TLC, HPLC, LC-MS and ¹HNMR. In PDA medium with yeast extract 5g/L, KH₂PO₄ 1.0g/L, MgSO₄ 0.3g/L, KNO₃ 0.5g/L, KCl 0.5g/L, PH 5.5,quantity of inoculation 10%, cultural temperature 28℃, rotational speed of rocking bed 140r/min, fermentation 14 days. Under these conditions,the production of huperzine A from the strain Slf14 amounted to 0.66 mg/L,with a maximal value of 0.75 mg/L.