Insights into total phenolic, flavonoid, and antioxidant activity of callus subculture frequency in rare endemic Ajuga xylorrhiza Kit Tan

This study assessed the callus morphology, growth index, callus diameter, total antioxidant activity, and total phenolic and flavonoid contents (TPC and TFC) of callus extracts from Ajuga xylorrhiza, a severely endangered plant found in small populations in Türkiye. Calluses obtained from a total of 18 applications formed in culture media supplemented with Kin and 2,4-D at six ratios (A, B, C, D, E, and F) and their subcultures (A1, A2, B1, B2, C1, C2, D1, D2, E1, E2, F1, and F2) were used for examination. Extracts A and D had a higher TFC than the other groups (27.26 and 18.08 µg QEs/mg extract, respectively). Cultures D, A, and B had high TPCs of 66.29, 58.85, and 51.41 GAEs/mg extract, respectively, while F, F2, and E1 had the lowest. Total antioxidant activity varied by the method (DPPH, ABTS, and CUPRAC). Extracts A, D, and B (IC₅₀ = 4.52, 9.51, and 16.18 µg/mL, respectively) demonstrated higher radical scavenging activity with the ABTS technique than the positive controls, BHT and BHA. The findings showed that an increase in plant growth regulators negatively impacted TPC, TFC, and total antioxidant activity. A. xylorrhiza extracts displayed higher antioxidant activity than their respective subcultures, indicating that the plant has a significant phytochemical content (total phenol and flavonoid contents). Moreover, the data suggest a possible relationship between TPC and TFC in extracts A, B, and D and radical scavenging activity (total antioxidant), which is worth investigating in further research.