Diversity-oriented synthesis through gamma radiolysis: Preparation of unusual ecdysteroid derivatives activating Akt and AMPK in skeletal muscle cells.

Gamma-ray radiation is a unique way to induce chemical transformations of bioactive compounds. In the present study, we pursued this approach to the diversity-oriented synthesis of analogs of 20-hydroxyecdysone (20E), an abundant ecdysteroid with a range of beneficial, non-hormonal bioactivities in mammals including humans. Gamma irradiations of aqueous solutions of 20E were conducted either in N2- or N2O-saturated solutions. Centrifugal partition chromatography was used to fractionate crude resulting irradiated materials using a biphasic solvent system composed of tert-butyl alcohol - ethyl acetate - water (0.45:0.9:1, v/v/v) in ascending mode. Subsequently, the products were purified by RP-HPLC. Fourteen ecdysteroids, including five new compounds, were isolated, and their structure were elucidated by 1D and 2D NMR and HRMS. Compounds 2-4, 7, 9, 12 and 15 were tested for their capacity to increase the Akt- and AMPK-phosphorylation of C2C12 murine skeletal myotubes in vitro. The compounds were similarly active on Akt as their parent compound. Stachysterone B (7) and a new ring-rearranged compound (12) were more potent than 20E in activating AMPK, indicating a stronger cytoprotective effect. Our results demonstrate the use of gamma irradiation in expanding the chemical diversity of ecdysteroids to obtain new, unusual bioactive metabolites.

Side-chain cleaved phytoecdysteroid metabolites as activators of protein kinase B.

Phytoecdysteroids exert their non-hormonal anabolic and adaptogenic effects in mammals, including humans, through a partially revealed mechanism of action involving the activation of protein kinase B (Akt). We have recently found that poststerone, a side-chain cleaved in vivo metabolite of 20-hydroxyecdysone, exerts potent anabolic activity in rats. Here we report the semi-synthetic preparation of a series of side-chain cleaved ecdysteroids and their activity on the Akt phosphorylation in murine skeletal muscle cells. Twelve C-21 ecdysteroids including 8 new compounds were obtained through the oxidative side-chain cleavage of various phytoecdysteroids, or through the base-catalyzed autoxidation of poststerone. The complete 1H and 13C NMR spectroscopic assignments of the new compounds are presented. Among the tested compounds, 9 could activate Akt stronger than poststerone revealing that side-chain cleaved derivatives of phytoecdysteroids other than 20-hydroxyecdysone are valuable bioactive metabolites. Thus, our results suggest that the expectable in vivo formation of such compounds should contribute to the bioactivity of herbal preparations containing ecdysteroid mixtures.

Capillary electrophoresis study on the base-catalyzed formation of bioactive oxidized metabolites of 20-hydroxyecdysone.

A novel capillary electrophoretic method was developed for the analysis and monitoring of the base-catalyzed autoxidation of 20-hydroxyecdysone, a worldwide used non-hormonal anabolic food supplement. An effective separation of the starting material and its bioactive oxidized derivatives was achieved by using sulfobutyl-ß-cyclodextrin as selector at pH 11 and by fixing the separation voltage at +30kV. Only a dilution step was inserted before injecting the sample, taken from the crude reaction mixture, to the capillary electrophoresis instrument. The same alkaline pH was used for the analysis as for the reaction, unlike the previously reported HPLC study where sample neutralization was required prior to the measurement. Due to the very short analysis time (6min) in capillary electrophoresis, more frequent sampling and more detailed time scale analysis could be carried out. Furthermore, in contrast with the preceding HPLC results, the previously unobserved calonysterone could also be detected by capillary electrophoresis as a minor primary product. Our novel method demonstrated higher resolution than the one before. Baseline separation could be achieved and the resolution values were in the range of 1.9-7.0. The limit of detection was below 71µg/ml, the relative standard deviation values of the migration time and peak area for intra- and inter-day precision were less than 10%. The more precise, direct monitoring of the time dependency of the oxidation process is expected to have a significant impact on yield optimization initiatives to allow related pharmacological studies in the near future.

Centrifugal partition chromatography in the isolation of minor ecdysteroids from Cyanotis arachnoidea.

Phytoecdysteroids are known for their various beneficial bioactivities in mammals including a non-hormonal anabolic and adaptogenic effect. Cyanotis arachnoidea extracts are extensively utilized worldwide as ecdysteroid-rich materials for various purposes, e.g. food supplementation, use in agriculture and aquaculture, etc. Preparative chromatography of ecdysteroids requires extensive use of methods of different selectivity, and only a very limited number of papers are available on related application of modern liquid-liquid chromatographic techniques. In this work, a centrifugal partition chromatography (CPC) method was developed for the isolation of two minor ecdysteroids, dacryhainansterone and calonysterone, from a pre-purified commercial extract of Cyanotis arachnoidea. The biphasic solvent system was optimized by HPLC, and was composed of n-hexane - ethyl acetate - methanol - water (1:5:1:5, v/v/v/v). The isolated dacryhainansterone and calonysterone represented 99.1% and 99.7% purity, respectively. Calonysterone exerts a stronger effect on the protein kinase B (Akt) phosphorylation in mammalian skeletal muscle cells than the abundant 20-hydroxyecdysone, while no related data are available on dacryhainansterone. Despite their presence in food supplements, neither compound has appropriately been assessed for safety and efficacy. The reported method allows the gram scale isolation of these compounds, opening ways to their in-depth pharmacological investigation.