Inokosterone

High performance liquid chromatography used for quality control of Achyranthis Radix.[Pubmed:22941488]

Arch Pharm Res. 2012 Aug;35(8):1449-55.

To establish a standard of quality control and to identify reliable Achyranthis Radix, three phytoecdysones including ecdysterone (1), 25R-Inokosterone (2) and 25S-Inokosterone (3) were determined by quantitative HPLC/UV analysis. Three phytoecdysones were separated with an YMC J'sphere ODS C(18) column (250 mm x 4.6 mm, 4 mum) by isocratic elution using 0.1% formic acid in water and acetonitrile (85:15, v/v%) as the mobile phase. The flow rate was 1.0 mL/min and the UV detector wavelength was set at 245 nm. The standards were quantified by HPLC/UV from Achyranthes bidentata Blume and Achyranthes japonica Nakai, as well as Cyathula capitata Moq. and Cyathula officinalis Kuan, which are of a different genus but are comparative herbs. The method was successfully used in the analysis of Achyranthis Radix of different geographical origin or genera with relatively simple conditions and procedures, and the assay results were satisfactory for linearity, recovery, precision, accuracy, stability and robustness. The HPLC analytical method for pattern recognition analysis was validated by repeated analysis of eighteen A. bidentata Blume samples and ten A. japonica Nakai samples. The results indicate that the established HPLC/UV method is suitable for quantitation and pattern recognition analyses for quality evaluation of Achyranthis Radix.

C-26 vs. C-27 hydroxylation of insect steroid hormones: regioselectivity of a microsomal cytochrome P450 from a hormone-resistant cell line.[Pubmed:21763268]

Arch Biochem Biophys. 2011 Sep 1;513(1):27-35.

Hydroxylation of steroids at one of the side chain terminal methyl groups, commonly linked to C-26, represents an important regulatory step established in many phyla. Discrimination between the two sites, C-26 and C-27, requires knowing the stereochemistry of the products. 26-Hydroxylation of the insect steroid hormone 20-hydroxyecdysone by a microsomal cytochrome P450 was previously found to be responsible for hormonal resistance in a Chironomus cell line mainly producing the (25S)-epimer of 20,26-dihydroxyecdysone. Here, we studied the 25-desoxy analog of 20-hydroxyecdysone, ponasterone A, to elucidate the stereochemistry of the expected 26-hydroxy product, Inokosterone, which occurs as C-25 epimers in nature. We identified the predominant metabolite as the C-25 R epimer of Inokosterone on comparison by RP-HPLC with the (25R)- and (25S)-epimers the stereochemistry of which was confirmed by X-ray crystallography. (25R)-Inokosterone was further oxidized to the 26-aldehyde identified by mass spectroscopy, borohydride reduction and metabolic transformation to 26-carboxylic acid. The (25S)-epimers of Inokosterone and its aldehyde were minor products. With 20-hydroxyecdysone as substrate, we newly identified the (25R)-epimer of 20,26-dihydroxyecdysone as a minor product. In conclusion, the present stereochemical studies revealed high regioselectivity of the Chironomus enzyme to hydroxylate both steroids at the same methyl group, denoted C-27.