Cytokinin-Regulated Enhancement of Antioxidant Phenolic Compound Accumulation in Clerodendrum spp. In Vitro Cultures

This study examined shoot proliferation and phenolic compounds accumulation in Clerodendrum colebrookianum and Clerodendrum trichotomum in vitro culture. The cultures were treated with 6-benzylaminopurine (BAP), meta-topolin (M-T), or N-benzyl-9-(2-tetrahydropyranyl)adenine (BPA) (0.5, 1.0 and 2.0 mg/L), and their biomass accumulation, shoot proliferation, and phenolic profiles were quantitatively assessed. In C. colebrookianum, BPA and M-T at 0.5 and 1.0 mg/L yielded higher proliferation rates (11.0-12.0 shoots per explant) and biomass production than BAP. In C. trichotomum, maximal shoot multiplication was achieved with 2.0 mg/L M-T (24.47 shoots per explant), and peak biomass accumulation was achieved with 1.0 mg/L BPA. The two species demonstrated polyphenolic fingerprints, with C. colebrookianum extract containing seven polyphenols and C. trichotomum ten, predominantly represented by acteoside and related compounds. M-T treatments markedly enhanced phenolic biosynthesis, yielding a 3.3-fold increase in acteoside in C. colebrookianum (82.73 mg/g DW) at 2.0 mg/L and in C. trichotomum (41.3 mg/g DW) at 1.0 mg/L relative to controls. TOPSIS multi-criteria decision analysis, integrating growth parameters, acteoside, and total phenolic content, found the optimal supplementation to be 1.0 mg/L M-T in the presence of 0.1 mg/L IAA for both species (closeness coefficients: 0.821 and 0.792, respectively). The extracts derived from optimized cultures exhibited significant radical-scavenging and metal reduction capacity in DPPH, ABTS, FRAP, and CUPRAC assays; a stronger effect was observed for C. colebrookianum, which may be associated with acteoside enrichment. Overall, M-T and BPA were found to be superior to BAP in promoting biomass accumulation and high-value bioactive phenolic production in Clerodendrum spp. Our findings underscore the potential of in vitro culture systems as a sustainable source of antioxidant phytochemicals with prospective nutraceutical and pharmaceutical relevance.