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

## TL;DR

This study shows that certain plant growth hormones boost antioxidant compound production in Clerodendrum species grown in labs, offering a sustainable way to make valuable phytochemicals.

## Contribution

The study demonstrates that M-T and BPA outperform BAP in promoting biomass and antioxidant phenolic production in Clerodendrum spp. in vitro.

## Key findings

- M-T and BPA treatments increased shoot proliferation and biomass more effectively than BAP in both Clerodendrum species.
- M-T significantly enhanced acteoside levels, with a 3.3-fold increase in C. colebrookianum and C. trichotomum.
- Optimized cultures showed strong antioxidant activity, especially in C. colebrookianum, likely due to higher acteoside content.

## Abstract

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.