β-Ecdysterone accumulation and regulation in Ajuga multiflora Bunge suspension culture

Ajuga multiflora Bunge cells contain beta-ecdysterone (beta-EC) that regulates the molting process of insect larvae. In this study, different conditions of culture have been studied to optimize the production of beta-EC. A. multiflora Bunge growth fitted the curve of logistic equation with one growth cycle of 17 days. The electric conductivity of medium had a negative correlation with not only the weight of dry cell but also the beta-EC accumulation, and thus, could be used for monitoring the peak of both cell growth and beta-EC accumulation. The pH value of the culture medium varied from 4.67 to 5.84 and reached the maximum at the end of the culture (on the 17th day). The relation of cell growth and nutrient consumption in A. multiflora Bunge cell suspension culture was distinctly correlated. Continuous subculture caused a reduction in beta-EC synthesis; passages 7-15, the beta-EC content declined (p < 0.05). At passage 11, the beta-EC content was only 42.72% of that at passage 5. Additives such as mevalonic acid (MVA), L-phenylalanine (L-Phe), alpha-pinene, terpineol, and nitric oxide (NO) in the suspension culture medium, could significantly promote the cell growth and stimulate beta-EC accumulation. The optimal concentrations of L-Phe, MVA, terpineol, and alpha-pinene were 0.2 mmol/l, 10 mg/l, 1 mmol/l and 6 mmol/l, respectively, with the beta-EC concentrations as 1.914 +/- 0.1948 mg/g (p < 0.01), 6.012 +/- 0.4252 mg/g (p < 0.01), 5.147 +/- 0.4819 mg/g (p < 0.01), 2.801 +/- 0.1253 mg/g (p < 0.01), respectively. The optimal concentration of sodium nitroprusside, the provider of NO, was 3 mmol/l with the beta-EC concentration 2.87 +/- 0.2493 mg/g (p < 0.01). The results offer a strategy for massive production of beta-EC.