Culture System and Nutrient Restriction Shape Antioxidant Activity in In Vitro Spearmint (Mentha spicata L.) Shoots

Spearmint phytochemicals exhibit remarkable antidiabetic, antioxidant, and broad pharmacological activities. <i>In vitro</i> organ cultures offer an efficient and sustainable platform for enhancing the production of these bioactive metabolites, although optimized media and cultivation strategies are essential to maximize yields. Here, four Murashige and Skoog (MS) medium variants and three cultivation systems-agar-solidified, static-liquid (L), and agitated-liquid (LA)-were evaluated to assess phenolics, antioxidant capacity, antidiabetic potential, and metabolic biochemical markers in <i>in vitro</i>-grown spearmint shoots. Half-strength MS (MS/2) consistently produced the highest antioxidant activity and accumulation of phenolics and sugars across all systems. The MS/2-L combination markedly boosted antioxidant responses, increasing 2,2-diphenyl-1-picrylhydrazyl (DPPH) values up to 27-fold and ferric reducing antioxidant power (FRAP) values tenfold relative to full-strength MS. Antioxidant capacity strongly correlated with total phenolics, flavonoids, rosmarinic acid, antidiabetic activity, and carbohydrate levels. Lipid peroxidation analysis further revealed that shoots cultured under LA conditions showed the highest level of malondialdehyde (MDA) accumulation, whereas MSN/2 (half-strength nitrogen) consistently yielded the lowest MDA levels across all cultivation systems. Collectively, these results highlight the strong influence of nutrient availability and culture system on the metabolic performance of <i>in vitro</i>-grown spearmint. In conclusion, nutrient limitation combined with liquid cultivation effectively enhances antioxidant metabolite accumulation, providing valuable criteria for the future design and optimization of scalable bioreactor systems.