Huperzine A and Huperzine B Production by Prothallus Cultures of Huperzia selago (L.) Bernh. ex Schrank et Mart

• Published in: Molecules (Basel, Switzerland) Vol. 25; no. 14; p. 3262
• Main Authors: Szypuła, Wojciech J, Wileńska, Beata, Misicka, Aleksandra, Pietrosiuk, Agnieszka
• Format: Journal Article
• Language: English
• Published: Switzerland MDPI AG 17-07-2020; MDPI
• Subjects: Alkaloids; Alkaloids - biosynthesis; Alkaloids - chemistry; Alkaloids - classification; Alkaloids - isolation & purification; Apoptosis; Biosynthesis; Blood-brain barrier; Cell division; Current sources; Enzymes; gametophyte; Gametophytes; Genes; Growth regulators; Heterocyclic Compounds, 4 or More Rings - chemistry; Heterocyclic Compounds, 4 or More Rings - isolation & purification; High-performance liquid chromatography; Huperzia - chemistry; Huperzia selago; huperzine; Huperzine A; in vitro culture; Liquid chromatography; Metabolites; Pharmaceutical industry; prothallus; secondary metabolites; Sesquiterpenes - chemistry; Spores; Sporophytes; Tissue Culture Techniques; Asia; Huperzia selago; secondary metabolites; Alzheimer’s disease; in vitro culture; gametophyte; huperzine; prothallus
• ISSN: 1420-3049; 1420-3049
• DOI: 10.3390/molecules25143262

This is the first report of an efficient and effective procedure to optimize the biosynthesis of huperzine A (HupA) and huperzine B (HupB) in vitro from
gametophytes. Axenic tissue cultures were established using spores collected from the sporophytes growing in the wild. The prothalia were obtained after 7-18 months. Approximately 90 up to 100% of the gametophytes were viable and grew rapidly after each transfer on to a fresh medium every 3 months. The best biomass growth index for prothallus calculated on a fresh (FW) and dry weight (DW) basis, at 24 weeks of culture, was 2500% (FW) and 2200% (DW), respectively. The huperzine A content in the gametophytes was very high and ranged from 0.74 mg/g to 4.73 mg/g DW. The highest yield HupA biosynthesis at >4 mg/g DW was observed on W/S medium without growth regulators at 8 to 24 weeks of culture. The highest HupB content ranged from 0.10 mg/g to 0.52 mg/g DW and was obtained on the same medium. The results demonstrate the superiority of
gametophyte cultures, with the level of HupA biosynthesis approximately 42% higher compared to sporophyte cultures and 35-fold higher than when the alkaloid was isolated from
its current source for the pharmaceutical industry. Moreover, the biosynthesis of HupB was several-fold more efficient than in
sporophytes growing in the wild. HPLC-HR-MS analyses of the extracts identified eight new alkaloids previously unreported in
: deacetylfawcettine, fawcettimine, 16-hydroxyhuperzine B, deacetyllycoclavine, annopodine, lycopecurine, des-
-methylfastigiatine and flabelline.