| Salvia miltiorrhiza is an important Chinese traditional medicine. The water-soluble components of S. miltiorrhiza have many pharmacological activities, especially the antioxidant activity. The water-soluble components of S. miltiorrhiza are phenolic acids,while salvianolic acid B content is significant. In this study, three kinds of cultures in vitro of S. miltiorrhiza were treated with different concentrations of salicylic acid, to improve the contents of salvianolic acid B. The results could provide technological support and reference data of screening the salvianolic acid B-high yield cell lines.First of all, the tissue culture system of wild S. miltiorrhiza was established. The young leaves of S. miltiorrhiza were used as explant to induce callus. Callus induction frequency and growth status were determined and used to evaluate the effects of different hormone concentrations. The best medium to inducing callus from young leaves of S. miltiorrhiza was MS + 2,4-D 2.0 mg / L + 6-BA 1.0 mg / L. Three kinds of cultures in vitro of S.miltiorrhiza, callus, suspension cultured cells, regenerated multiple shoots, were obtained.Phenolic acids were extracted from these three kinds of cultures in vitro by using 70%methanol as solvent and with ultrasonic treatment. The content of salvianolic acid B was determined via HPLC. The results showed that salvianolic acid B was accumulated in all of these three kinds of cultures in vitro, and the content of salvianolic acid B in regenerated multiple shoot was the highest, the value of it was 50.90 mg/g.In this study, effects of different concentrations of salicylic acid on growth and secondary metabolism of three kinds of culture in vitro of S. miltiorrhiza were investigated.The results showed that salicylic acid resulted in decrease in growth of three kinds of cultures in vitro. With respect to the content of salvianolic acid B, its value was obviously affected by adding lower concentrations of salicylic acid into the medium for the undifferentiated cultures in vitro, specifically, 0.05mmol/L of salicylic acid resulted in a significant increase; while higher concentrations of salicylic acid resulted in decreases in the content of salvianolic acid B in cultures in vitro. For regenerated multiple shoot of S.miltiorrhiza, salicylic acid resulting in a content decrease of salvianolic acid B.Finally, the relationship between the antioxidant activity of salvia phenolic acids and the content of salvianolic acid B in cultures in vitro of S. miltiorrhiza were studied. The ability of inhibit the hydroxyl radical, anti-lipid peroxidation capacity and ability of reducing were determined and used to evaluate the antioxidant activity. The results showed that there was a positive correlation between the content of salvianolic acid B in phenolic acids of three kinds of cultures in vitro of S. miltiorrhiza and the antioxidant activity. |
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