Light spectrum effects on huperzine A and B accumulation in axenic gametophyte cultures of Huperzia selago (L.) Bernh. ex Schrank et Mart.

Abstract

Introduction: Huperzine A (HupA) and huperzine B (HupB) are high-value Lycopodiaceae alkaloids with potent, reversible acetylcholinesterase (AChE) inhibitory activity and NMDA receptor antagonism. Their supply is limited by impractical field cultivation and conservation constraints on wild harvesting. Here, we evaluated axenic gametophyte cultures of Huperzia selago as a controllable and ethically sourced in vitro system and tested LED spectral control as a tool to modulate alkaloid accumulation.

Methods: Cultures were maintained for 24 weeks under three light treatments (WHITE-L, BLUE-L and RED-BLUE-L) or darkness. HupA and HupB were quantified by HPLC-DAD, while LC-MS/IT-TOF and MS/MS-supported profiling were used for target-alkaloid confirmation and characterization of the multi-component extract composition. Extracts standardized to HupA equivalents were evaluated for AChE inhibition.

Results: LED spectral control shaped HupA/HupB accumulation patterns. BLUE-L (≈450 nm) was associated with the strongest increase in HupA content, reaching 4.83 ± 0.21 mg·g^-¹ DW at week 12, whereas RED-BLUE-L (660/450 nm) maintained the highest mean HupA content over 24 weeks (3.61 ± 0.61 mg·g^-¹ DW). Darkness promoted relative biomass accumulation, whereas BLUE-L and RED-BLUE-L favored higher HupA/HupB accumulation during the 8-12-week harvest window. These complementary responses suggest a potential two-stage strategy involving biomass accumulation in darkness followed by induction under blue or red-blue light; however, this sequential approach requires future validation. HupB remained a minor but light-responsive metabolite (≤0.74 ± 0.03 mg·g^-¹ DW). Extracts standardized to HupA equivalents (0.5-2.0 µM) inhibited AChE up to approximately 50%, but HupA content alone did not fully explain the observed activity.

Discussion: The weak-to-moderate relationship between HupA content and AChE inhibition suggests that optimization of H. selago gametophyte cultures should consider not only HupA accumulation, but also the broader alkaloid profile and extract bioactivity. LED spectral control supports the development of reproducible, ethically sourced systems for obtaining Lycopodiaceae alkaloid-rich biomass under controlled conditions.