Melatonin (MT) plays a number of key roles in regulating plant growth and secondary metabolite accumulation. Prunella vulgaris is an important traditional Chinese herbal medicinal plant which is used for the treatment of lymph, goiter, and mastitis. However, the effect of MT on the yield and medicinal component content of P. vulgaris remains still unclear. In this research, we have examined the influence of different concentrations of MT (0, 50, 100, 200, 400 μM) on the physiological characteristics, secondary metabolite contents, and yield of P. vulgaris biomass. The results showed that 50–200 μM MT treatment had a positive effect on P. vulgaris. MT treatment at 100 μM greatly increased the activities of superoxide dismutase and peroxidase, the contents of soluble sugar and proline, and obviously decreased the relative electrical conductivity, the contents of malondialdehyde and hydrogen peroxide of leaves. Furthermore, it markedly promoted the growth and development of the root system, increased the content of photosynthetic pigments, improved the performance of photosystems I and II and the coordination of both photosystems, and enhanced the photosynthetic capacity of P. vulgaris. In addition, it significantly increased the dry mass of whole plant and spica and promoted the accumulation of total flavonoids, total phenolics, caffeic acid, ferulic acid, rosmarinic acid, and hyperoside in the spica of P. vulgaris. These findings demonstrated that the application of MT could effectively activate the antioxidant defense system of P. vulgaris, protect the photosynthetic apparatus from photooxidation damage, and improve the photosynthetic capacity and the root absorption capacity, thereby promoting the yield and accumulation of secondary metabolites in P. vulgaris.

As a species of significant traditional medicinal importance, Prunella vulgaris is severely limited by drought stress, given its high sensitivity to this environmental constraint. 24-epibrassinolide (EBR) has shown promise in enhancing plant stress resilience and secondary metabolite production, yet its efficacy in mitigating drought effects on P. vulgaris requires further elucidation. In this study, foliar application of EBR (0, 0.01, 0.05, 0.1, 0.2 μmol·L⁻¹) was applied to drought-stressed P. vulgaris seedlings (maintained at 60% ± 5% field capacity, FC, for 20 days during the flowering stage; control at 75% ± 5% FC). The results showed that drought inhibited the growth and development of P. vulgaris. Compared with the control group, malondialdehyde, hydrogen peroxide, and superoxide anion increased by 77.82%, 27.47%, and 44.95%, respectively. The total chlorophyll content and the coordination between photosystem I and photosystem II decreased by 42.33% and 46.62%, respectively. Additionally, the net photosynthetic rate and biomass of P. vulgaris significantly decreased by 45.12% and 34.66%, respectively. In contrast, the 0.1 μmol·L⁻¹ EBR significantly enhanced the antioxidant and osmoregulation systems. Compared with drought stress treatment, the activities of SOD, POD, CAT, APX and GPX increased by 10.78%, 45.86%, 48.44%, 40.58% and 63.37%, respectively; soluble sugar, soluble protein and proline contents increased by 53.38%, 29.09% and 45.95%, respectively; and malondialdehyde, hydrogen peroxide and superoxide anion levels decreased by 28.37%, 15.77% and 25.73%, respectively. Total chlorophyll content, photosystem coordination and net photosynthetic rate increased by 55.68%, 43.08% and 45.88%, respectively, along with a significant 42.23% increase in total biomass. Furthermore, EBR upregulated the transcription levels of key phenylpropanoid pathway genes and elevated secondary metabolite contents. The expression of Pv4CL, PvC4H, PvPAL and PvTAT increased by 26.97%, 90.42%, 35.52% and 84.35%, respectively. Accordingly, total phenolic content, caffeic acid, ferulic acid, rosmarinic acid and hyperoside increased by 36.44%, 121.01%, 100.27%, 72.38% and 80.77%, respectively. Lower EBR concentrations (0.01 μmol·L⁻¹) had no significant effect on most indices, while 0.2 μmol L⁻¹ EBR showed weakened effects. In summary, under 60% ± 5% field capacity (FC) drought, 2,4-epibrassinolide (EBR) enhances drought adaptation, medicinal yield, and quality of P. vulgaris, with 0.1 μmol L⁻¹ EBR as the optimal concentration. This improvement is driven by enhanced antioxidant capacity, optimized photosynthesis, promoted root–shoot growth, and activated biosynthesis of medicinal compounds.