THE ROLE OF ACETYLCHOLINE IN THE BIOSYNTHESIS OF ALKALOIDS IN IN VITRO CULTURES OF HUPERZIA SELAGO (L.) GAMETOPHYTES
DOI:
https://doi.org/10.31435/ijitss.3(51).2026.5882Keywords:
Huperzia Selago, Acetylcholine, Huperzine A, Huperzine B, Lycopodine, Alkaloid Biosynthesis, In Vitro CulturesAbstract
Introduction: Huperzia selago is a natural source of huperzine A (HupA), a potent and reversible acetylcholinesterase (AChE) inhibitor with therapeutic potential in Alzheimer’s disease. Due to limited natural resources, alternative methods for alkaloid production—such as in vitro cultures—are being explored. Acetylcholine (ACh) functions as a signaling molecule in plants, but its influence on alkaloid biosynthesis in clubmosses has not yet been investigated [1-5].
Aim: To evaluate the effect of exogenous ACh on the biosynthesis of HupA, huperzine B (HupB), and lycopodine in H. selago gametophyte cultures.
Methods: Gametophytes were grown in vitro on Whittier and Storchova media supplemented with acetylcholine chloride at concentrations of 0 (control), 1, 10, 100, and 1000 µM. Biomass was collected after 2, 4, 6, 8, and 10 weeks, lyophilized, and extracted with methanol. Quantitative analysis of HupA, HupB, and lycopodine was performed using HPLC-DAD [2, 4-12].
Results: Control cultures showed high HupA content (mean 3.34 mg/g DW), ranging from 3.01 to 4.22 mg/g DW, with a maximum at week 8. HupB was detected only at week 8 (0.44 mg/g DW). In contrast, none of the ACh-treated cultures contained detectable HupA or HupB. A significant increase (p<0.05) in lycopodine content was observed in cultures treated with 10 µM ACh, reaching up to 16.72 mg/g DW at week 8. Other ACh concentrations did not significantly affect lycopodine levels compared to the control.
Conclusions:
- selago gametophytes in vitro are an efficient source of HupA and HupB.
- Acetylcholine inhibits the biosynthesis of HupA and HupB.
- Acetylcholine at 10 µM significantly enhances lycopodine biosynthesis.
- Culture duration influences alkaloid content. The results indicate a significant and selective role of ACh in regulating alkaloid biosynthetic pathways in H. selago.
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Copyright (c) 2026 Mateusz Michalak, Piotr Kucharczyk, Tomasz Szwarc, Karolina Parzęcka, Paulina Jaruga, Vanessa Gąsiorowska, Ewa Jachimczak, Karolina Grodkowska, Aleksandra Czapla
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