Phytoecdysteroids, which are structurally similar or identical to insect molting hormones, produce a range of effects in mammals, including increasing growth and physical performance. To study the mechanism of action of phytoecdysteroids in mammalian tissue, an in vitro cellular assay of protein synthesis was developed. In C2C12 murine myotubes and human primary myotubes, phytoecdysteroids increased protein synthesis by up to 20%. In vivo, ecdysteroids increased rat grip strength. Ecdysteroid-containing plant extracts produced similar results. The effect was inhibited by a phosphoinositide kinase-3 inhibitor, which suggests a PI3K-mediated mechanism.

Phytoecdysteroids, structurally similar to insect molting hormones, produce a range of effects in mammals, including increasing growth and physical performance. In skeletal muscle cells, phytoecdysteroids increase protein synthesis. In this study we show that in a mouse skeletal muscle cell line, C2C12, 20-hydroxyecdysone (20HE), a common phytoecdysteroid in both insects and plants, elicited a rapid elevation in intracellular calcium, followed by sustained Akt activation and increased protein synthesis. The effect was inhibited by a G-protein Coupled Receptor (GPCR) inhibitor, a phospholipase C (PLC) inhibitor, and a phosphoinositide kinase-3 (PI3K) inhibitor.

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 lM 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 lg mg -1 , respectively, compared to control (10.5 lg mg -1 ). Older hairy root cultures, extracted after the seventh subculture cycle, also showed increases in 20E content (24.8 lg mg -1 ), turkesterone (0.9 lg mg -1 ) and cyasterone (8.1 lg mg -1 ) compared to control cultures maintained for a shorter duration of four subculture cycles. Doses of 10 or 20 lg 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. Keywords 20-Hydroxyecdysone Á Cell suspension culture Á Cyasterone Á Hairy root Á Methyl jasmonate Á Mevalonic acid Á Turkesterone Abbreviations 20E 20-Hydroxyecdysone CC Column chromatography CD 3 OD Deuterated methanol DAD Diode array detector DMEM Dulbecco's modified eagle's media DPM Decays per minute DW Dry weight ESI-MS Electrospray ionization-mass spectroscopy FW Fresh weight HPLC High performance liquid chromatography Electronic supplementary material The online version of this article (

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