Elicitor and LED spectral engineering enhance sustainable production of Cepharanthine in Stephania pierrei Diels callus cultures

Stephania pierrei Diels is a medicinal plant valued for producing cepharanthine, a bioactive bisbenzylisoquinoline alkaloid with diverse pharmaceutical properties. However, natural cepharanthine production is limited by slow plant growth and inconsistent metabolite accumulation. This study aimed to develop an efficient in vitro callus culture system to enhance cepharanthine production through optimization of culture age, elicitor treatment, and light quality. Callus cultures showed rapid biomass accumulation between 15 and 35 days after subculture, while cepharanthine content reached the highest level at 40 days. Among elicitors tested, 100 µM methyl jasmonate (MeJA) applied for 6 days most effectively enhanced cepharanthine accumulation. Under MeJA elicitation, balanced blue: red LED light (B50R50) significantly increased cepharanthine accumulation to 1532.15 ± 52.67 µg g⁻¹ DW and also promoted extracellular release into the culture medium. Exploratory GC-MS profiling provided tentative annotations of lipid-, sterol-, and furan-related features in the analyzed control and treated profiles; these qualitative observations were not used to infer quantitative metabolite changes or pathway relationships. qRT-PCR demonstrated strong upregulation of the BIA biosynthetic genes 6OMT and CNMT under B50R50. Antioxidant assays and MTT-based cell-viability testing further showed treatment-dependent differences in the crude callus extract; however, these in vitro bioactivities cannot be attributed to cepharanthine alone. Sequential optimization of callus developmental stage, MeJA elicitation, and light quality provide an effective in vitro strategy for increasing cepharanthine accumulation in S . pierrei Diels callus cultures. The targeted HPLC and transcriptional results support a MeJA-light-associated response under the tested conditions. The exploratory GC-MS profiles provide only tentative, qualitative chemical annotations and do not establish quantitative metabolite changes, pathway relationships, or causality. The biological activities of the crude extracts require compound-specific validation before attribution to cepharanthine and assessment of its pharmaceutical potential.