Mild Salinity Stimulates Biochemical Activities and Metabolites Associated with Anticancer Activities in Black Horehound (Ballota nigra L.)

Black horehound (Ballota nigra L.) is one of the most important medicinal plants, as a rich source of health-promoting essential oils and metabolites. Salinity stress affects plant development and alters antioxidant activity and plant metabolite composition. The present research aimed to study the effect of salinity on physiological and biochemical changes and metabolites of B. nigra under greenhouse and in vitro culture conditions. The plants were treated with different concentrations of NaCl (25, 50, 75, 100 mM), and morphological characteristics of the plant were measured. The growth-related traits and soil plant analysis development (SPAD) were decreased both in vivo and in vitro. Additionally, increased salt concentration negatively affected the cell membrane integrity. The total phenolic content and flavonoids of plants growing in the greenhouse increased by 21% at 50 mM of NaCl, but the amounts decreased significantly at higher stress levels (100 mM of NaCl). Antioxidant activity was also measured. Among the NaCl treatments, the most increased DPPH scavenging activities (IC50) under greenhouse and in vitro conditions were detected at mild salinity stress, but the activity significantly decreased in higher salinity treatments (i.e., 75 and 100 mM). In general, with increasing the salinity level to 75 mM, the activities of CAT and APX were significantly upregulated in both greenhouse and in vitro culture conditions. A correlation between total phenolics and flavonoids contents as well as antioxidant activity was obtained. Salinity level caused a shift in the metabolite expression. Mild salinity stress elevated the metabolites involved in anticancer and anti-inflammatory activities, such as &beta:-ionone and caryophyllene oxide. However, the higher salt stress resulted in a significant reduction in their expression. Differential expression of metabolites to various levels of salt stress can be further exploited for the in vitro biosynthesis of metabolites.