Huperzia java']javanica as a Novel Source of Huperzine Alkaloids and Huperzine-producing Fungi

Huperzine A (HupA) and huperzine B (HupB) are potent acetylcholinesterase inhibitor used widely for clinical treatment of Alzheimer's disease, which are mainly extracrted from natural populations of Huperzia species. This study aimed to the evaluate potential of producing Hup of native Huperzia javanica species collected in Vietnam as well as identify endophytic fungi that produce high Hup or simultaneously produce many types of Hup derived from this plant as a material source for natural Hup production. By HPLC-DAD-MS analysis, H. javanica collected from Ha Giang, Vietnam was found as a novel producer of HupA and HupB with a yield of 0.513 mg/g dry weight (wt) and 0.192 mg/g dry wt, respectively. Inspired by this discovery, a total of 63 endophytic fungi were isolated from healthy tissues of the collected H. javanica plants, including 52 fungal strains belonging to nine genera and 11 unidentified strains based on morphological characteristics. Using HPLC, 9 Hup-producing fungi were identified when compared to the standard HupA and HupB. Surprisingly, 7 fungal strains produced both HupA and HupB, among which strain TLC12 produced the highest HupA and HupB contents of 0.271 and 0.163 mg/g mycelium dry cell weight (gdcw), respectively. Fungal strains TLC19 and TLC22 only yielded a respective amount of 0.305 and 0.135 mg/ gdcw of HupA. Combining with Internal Transcribed Spacer sequence analysis, 9 potent fungi were identified as Neurospora calospora (TLC9, TLC10, TLC11), Schizophyllum commune TLC12, Epicoccum sorghinum TLC13, Alternaria tenuissima TLC14, Cephalotrichum sp. TLC20, Daldinia sp. TLC19, and Schizophyllum sp. TLC22. To the best of our knowledge, this is the first report demonstrating H. javanica as a prolific and novel source of endophytic fungi capable of yielding high HupA and HupB contents. The exploitation of 9 Hup-producing fungi is also valuable for both basic research and industrial Hup manufactures. These findings open new perspectives for industrial production of fungal HupA and HupB and conservation of Huperzia species.