Dose-dependent effects of melatonin on berberine production, antioxidant activity, and metabolite profiles in Berberis vulgaris L. suspension cultures

Melatonin is a regulatory molecule that increases secondary metabolite biosynthesis in plants. Berberis vulgaris L. is a well-known medicinal plant, valued for its alkaloids and phenolic compounds. This study aimed to determine whether and how melatonin affects biosynthesis of alkaloids (particularly berberine) and phenolics in B. vulgaris. For this, cell cultures were treated with 0, 25, 50, and 100 µM melatonin, and the contents of berberine, phenolics, and amino acids, activity of phenylalanine ammonia-lyase (PAL) and tyrosine ammonia-lyase (TAL), and oxidative status were assessed. Melatonin altered total alkaloid and berberine contents, with the highest level observed at 50 µM (1.63 mg g^-1 FW and 60.63 µg g^-1 DW, respectively). Melatonin also significantly increased tyrosine (Tyr) level, a precursor to berberine. Besides, phenylalanine (Phe) content and PAL activity enhanced in response to exogenous melatonin, leading to a dose-dependent cinnamic acid and coumaric acid accumulation. The HPLC results revealed that melatonin also influences the individual flavonoids profile. The highest increase was detected in catechin, kaempferol, and myricetin contents, which were 19.59, 14.73, and 9.26 times higher than the control levels, respectively. Oxidative balance was also disturbed in melatonin-treated B. vulgaris cells, indicated by increased generation of hydrogen peroxide (H₂O₂) and nitric oxide (NO). The resulting oxidative stress was controlled by activation of antioxidant enzymes, including superoxide dismutase (SOD), peroxidase (POD), and ascorbate peroxidase (APX), along with increased ascorbic acid (AsA) levels. These findings suggest that melatonin can allocate Tyr and Phe towards berberine and phenolics accumulation, respectively, possibly by modulating the oxidative status.