Short-term salinity stress enhances phytochemical accumulation and antioxidant activity in Thai Centella asiatica (L.) Urban: A genotype-dependent metabolomic study

Centella asiatica (L.) Urban is a valuable medicinal herb whose therapeutic efficacy depends on bioactive secondary metabolites, including terpenoids, phenolics, flavonoids, iridoids, and alkaloids. Although moderate salinity stress can enhance phytochemical production in medicinal plants, systematic optimization of stress parameters and comprehensive metabolomic characterization across diverse genotypes remain limited. This study investigated short-term salinity stress effects on phytochemical accumulation and antioxidant responses in 39 Thai C. asiatica accessions. Optimization experiments established that 100 mM NaCl applied for 6 days maximized phytochemical enhancement while maintaining cell membrane stability. Population-level screening revealed substantial genotypic variation, with accessions classified into four response groups based on hierarchical clustering. The salt-tolerant accession CMI exhibited remarkable increases in terpenoids (2.2-fold), phenolics (1.5-fold), flavonoids (1.5-fold), iridoids (1.8-fold), and alkaloids (1.4-fold), accompanied by enhanced antioxidant activities (DPPH, FRAP, ABTS). In contrast, the salt-sensitive accession SNK showed severe reductions across all phytochemical classes and antioxidant parameters. Untargeted LC-MS/MS metabolomics revealed that CMI responded through coordinated activation of phenylpropanoid, flavonoid, terpenoid, and alkaloid biosynthetic pathways, with 514 metabolites showing increased accumulation, whereas SNK exhibited metabolic suppression with 409 decreased metabolites. Pathway-level analysis confirmed broad-spectrum biosynthetic activation in tolerant genotypes, encompassing stress hormones (abscisic acid, gibberellins, cytokinin), osmoprotectants (raffinose, proline), defense compounds (phenolics, flavonoids, alkaloids), and membrane-stabilizing triterpenoids. These findings indicate that genotype-dependent metabolic plasticity determines salinity tolerance and establish controlled salt stress as an effective strategy for enhancing C. asiatica medicinal quality, providing a basis for stress-based cultivation protocols aimed at improving phytochemical yield under controlled conditions.