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

Abstract

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 Huperzia selago 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 H. selago 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 H. serrata, its current source for the pharmaceutical industry. Moreover, the biosynthesis of HupB was several-fold more efficient than in H. selago sporophytes growing in the wild. HPLC-HR-MS analyses of the extracts identified eight new alkaloids previously unreported in H. selago: deacetylfawcettine, fawcettimine, 16-hydroxyhuperzine B, deacetyllycoclavine, annopodine, lycopecurine, des-N-methylfastigiatine and flabelline.

Fractionation of Lycopodiaceae Alkaloids and Evaluation of Their Anticholinesterase and Cytotoxic Activities

“…When tentatively analyzing the TLC bioautography results, by far the strongest zones of AChE inhibition are seen on TLC plates made for fractions obtained from H. selago. These data are also confirmed by the literature, where it is repeatedly mentioned that this plant species of the Lycopodiaceae family possesses strong AChE inhibitors, and it is the main source of HupA [25]. The results are shown in Figure 2.…”

“…Currently, HupA is isolated from Huperzia serrata, occurring commonly in Asia. However, the uncontrolled harvesting of this species has reduced its original range and depleted its natural resources [10,25]. Therefore, the aim of our work was to search for alternative sources of HupA and new potent AChE inhibitors from three species commonly encountered in the Euro-Asia region, belonging to the Lycopodiaceae family: Lycopodium clavatum L., Lycopodium annotinum L. and Huperzia selago (L.) Bernh.…”

Polish Achievements in Bioactive Compound Production From In Vitro Plant Cultures

“…With the growing interest in the therapeutic applications of HupA, the demand for club mosses as a source of HupA and other alkaloids is increasing rapidly, and studies on in vitro cultures of Lycopod are gaining importance (Szypuła & Pietrosiuk, 2021). Till date, procedures for establishing and maintaining in vitro cultures of Huperzia selago sporophytes (Figure 2A) and gametophytes (Figure 2B) have been developed using various starting materials, including shoot fragments, vegetative propagules, somatic embryos, and spores (Szypuła & Pietrosiuk, 2021;Szypuła et al, 2005Szypuła et al, , 2013Szypuła et al, , 2020. e first studies on HupA biosynthesis were conducted by Szypuła et al (2005Szypuła et al ( , 2011Szypuła et al ( , 2013.…”

“…Using spores from sporophytes procured from natural populations is an effective method for establishing in vitro axenic cultures of H. selago gametophytes (Szypuła et al, 2020). Prothalli were obtained aer 7-18 months, and optimal results were achieved on the medium originally developed by Whittier and Storchova (2007) and on Moore medium as modified by Freeberg and Wetmore (1957).…”

Biological Activities of Natural Products

“…Szypuła et al [21] proposed the in vitro cultures of gametophytes of the European club-moss species Huperzia selago as a rich source of two potent reversible acetylcholinesterase inhibitors: huperzine A (HA) and huperzine B (HB). They documented that the contents of (HA) were higher than in the H. serrata Asian species grown in open air, which is a current source of (HA) for pharmaceutical industry.…”