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Objective:To develop a method for high frequency plantlets regeneration protocol forEnicostema axillare(Lam.)Raynal ssp.littoralis(Blume)Raynal(E. axillare) without intermediate callus phase and to determine the content and correlation of phenolic compounds and their antioxidant activity of both the plants derived from nature and nodal culture byDPPH assay.Methods:The nodal explants were cultured onMS basal medium fortified with different concentration of various growth regulators such asBAP,KIN,TDZ and2iP(0.5-2 mg/L) individually and in combinations with or withoutGA3 for shoot bud induction and multiplication.Total phenol and flavonoid content was determined in both plants from nature and nodal culture and antioxidant activity was determined byDPPH assay.Results:The highest number of multiple shoot(108.00±3.55 shoots/explants) was obtained onMS medium supplemented withBAP(2 mg/L) in combination withKIN (0.5 mg/L) andGA3(2 mg/L).Rooting was optimized on half-strengthMS medium supplemented withIAA(0.5 mg/L).The rooted plantlets were transferred to paper cups containing vermiculite and hardening was successfully attained with75% survival.Among the four extract of methanol and water extract from both the plantlet from nature and nodal culture, the concentration of flavonoid was found to be higher in methanol extract of the plants from nature(146.57±1.68 mg rutin/g extract) and phenol content was higher in the water extract of plant from nature(52.53±1.67 mgGAE /g extract).The radical scavenging activity of four extracts.Methanol extract of plant grown in nature showed the highest radical scavenging activity(IC50 =87.10 μg/mL) was investigated byDPPH test.Conculsion:The present study not only enables reinforcement of wild plant populations usingex situ growth of individuals, but it also helps for high scale production of plantlets.A high correlation between antioxidant capacities and their total phenolic contents indicated that flavonoid compounds were a major contributor of antioxidant activity of both plants grown from nature and nodal culture.