In vitro production of metabolism-enhancing phytoecdysteroids from Ajuga turkestanica

In order to develop a sustainable source of metabolism-enhancing phytoecdysteroids, cell suspension and hairy root cultures were established from shoot cultures of wild-harvested Ajuga turkestanica, a medicinal plant indigenous to Uzbekistan. Precursors of phytoecdysteroids (acetate, mevalonic acid cholesterol) or methyl jasmonate (an elicitor) were added to subculture media to increase phytoecdysteroid accumulation. In cell suspension cultures, 20-hydroxyecdysone (20E) content increased 3- or 2-fold with the addition of 125 or 250 μM methyl jasmonate, respectively, compared to unelicited cultures. Precursor addition, however, did not provoke phytoecdysteroid accumulation. In hairy root cultures, addition of sodium acetate, mevalonic acid, and methyl jasmonate, but not cholesterol, increased phytoecdysteroid content compared to unelicited cultures. Hairy root cultures treated with 150 mg l^-1 sodium acetate, or 15 or 150 mg l^-1 mevalonic acid, increased 20E content approximately 2-fold to 19.9, 20.4 or 21.7 μg mg^-1, respectively, compared to control (10.5 μg mg^-1). Older hairy root cultures, extracted after the seventh subculture cycle, also showed increases in 20E content (24.8 μg mg^-1), turkesterone (0.9 μg mg ^-1) and cyasterone (8.1 μg mg^-1) compared to control cultures maintained for a shorter duration of four subculture cycles. Doses of 10 or 20 μg ml^-1 hairy root extract increased protein synthesis by 25.7% or 31.1%, respectively, in a C2C12 mouse skeletal cell line. These results suggest that sustainable production of metabolically active phytoecdysteroid can be achieved through hairy root culture systems.