Lycopodiaceae herb from Vietnam as a promising medicinal source of natural hupezine and novel huperzine-producing endophytic fungi.

Abstract

Objectives: (1) To evaluate the potential of producing huperzine (Hup) and anticholinesterase (AChE) activities of nine native Lycopodiaceae species collected in Vietnam; (2) Isolation, identification and characterization of a novel fungus producing both HupA and HupB isolated from Lycopodium casuarinoides Spring.

Results: All methanolic extracts of nine plants showed AChE inhibition from 8.55 to 71.81%. Of note, Huperzia serrata (Thunb.) Trevis, L. casuarinoides, Lycopodium clavatum L., Phlegmariurus squarrosus (G. Forst.), and P. phlegmaria (L.) T. Sen & U. Sen were shown to biosynthesize both HupA and HupB by high-performance liquid chromatography (HPLC). Plants H. serrata, L. casuarinoides and L. clavatum showed the most potent AchE IC₅₀ inhibition. HupA and HupB concentrations from six plants were greater than those of previously reported Lycopodiaceae species. Sixty-four endophytic fungi were isolated from tissue of natural L. casuarinoides and then screened for HupA- and HupB-production by HPLC. Out of 64 fungal strains, only TTD2-2.7 extract could produce both HupA and HupB with the yields of 0.034 and 0.028 µg gdcw^-1, respectively. Moreover, TTD2-2.7 extract also had inhibitory effects on AChE with the IC₅₀ of 129.76 ± 4.13 µg ml^-1, which was lower than the extract of host plant L. casuarinoides (94.03 ± 4.13 µg ml^-1). The fungus was identified as Aspergillus sp. TTD2-2.7 by morphological characteristics and Internal Transcribed Spacer sequence analysis.

Conclusions: These are the first reports of (1) two species L. clavatum and L. casuarinoides producing both HupA and HupB, and (2) L. casuarinoides as novel sources of Hup-producing endophytic fungi as well as (3) fungus Aspergillus as a novel HupA- and HupB-producing endophyte isolated from L. casuarinoides.