Biotransformation of a potent anabolic steroid, mibolerone, with Cunninghamella blakesleeana, C. echinulata, and Macrophomina phaseolina, and biological activity evaluation of its metabolites.

Seven metabolites were obtained from the microbial transformation of anabolic-androgenic steroid mibolerone (1) with Cunninghamella blakesleeana, C. echinulata, and Macrophomina phaseolina. Their structures were determined as 10ß,17ß-dihydroxy-7α,17α-dimethylestr-4-en-3-one (2), 6ß,17ß-dihydroxy-7α,17α-dimethylestr-4-en-3-one (3), 6ß,10ß,17ß-trihydroxy-7α,17α-dimethylestr-4-en-3-one (4), 11ß,17ß-dihydroxy-(20-hydroxymethyl)-7α,17α-dimethylestr-4-en-3-one (5), 1α,17ß-dihydroxy-7α,17α-dimethylestr-4-en-3-one (6), 1α,11ß,17ß-trihydroxy-7α,17α-dimethylestr-4-en-3-one (7), and 11ß,17ß-dihydroxy-7α,17α-dimethylestr-4-en-3-one (8), on the basis of spectroscopic studies. All metabolites, except 8, were identified as new compounds. This study indicates that C. blakesleeana, and C. echinulata are able to catalyze hydroxylation at allylic positions, while M. phaseolina can catalyze hydroxylation of CH2 and CH3 groups of substrate 1. Mibolerone (1) was found to be a moderate inhibitor of ß-glucuronidase enzyme (IC50 = 42.98 ± 1.24 µM) during random biological screening, while its metabolites 2-4, and 8 were found to be inactive. Mibolerone (1) was also found to be significantly active against Leishmania major promastigotes (IC50 = 29.64 ± 0.88 µM). Its transformed products 3 (IC50 = 79.09 ± 0.06 µM), and 8 (IC50 = 70.09 ± 0.05 µM) showed a weak leishmanicidal activity, while 2 and 4 were found to be inactive. In addition, substrate 1 (IC50 = 35.7 ± 4.46 µM), and its metabolite 8 (IC50 = 34.16 ± 5.3 µM) exhibited potent cytotoxicity against HeLa cancer cell line (human cervical carcinoma). Metabolite 2 (IC50 = 46.5 ± 5.4 µM) also showed a significant cytotoxicity, while 3 (IC50 = 107.8 ± 4.0 µM) and 4 (IC50 = 152.5 ± 2.15 µM) showed weak cytotoxicity against HeLa cancer cell line. Compound 1 (IC50 = 46.3 ± 11.7 µM), and its transformed products 2 (IC50 = 43.3 ± 7.7 µM), 3 (IC50 = 65.6 ± 2.5 µM), and 4 (IC50 = 89.4 ± 2.7 µM) were also found to be moderately toxic to 3T3 cell line (mouse fibroblast). Interestingly, metabolite 8 showed no cytotoxicity against 3T3 cell line. Compounds 1-4, and 8 were also evaluated for inhibition of tyrosinase, carbonic anhydrase, and α-glucosidase enzymes, and all were found to be inactive.

Isolation of 5 alpha- and 5 beta-dihydrorubrosterone from Silene otites L. (Wib).

5 alpha-Dihydrorubrosterone (2 beta, 3 beta, 14 alpha, 17 beta-tetrahydroxy-5 alpha-androst-7-ene-6-one), a new 19-carbon 5 alpha-ecdysteroid, was isolated together with its 5 beta counterpart from the aerial parts of Silene otites L. (Wib.) (Caryophyllaceae) by a combination of solvent partition, low-pressure column chromatography, thin-layer chromatography (normal-phase and reversed-phase) and finally HPLC. Mass spectrometry and nuclear magnetic resonance spectroscopic procedures were used for compound characterization.

[Analysis of 17-oxosteroids by gas liquid chromatography after purification of the urinary extract by Girard's T reagent (author's transl)]. / Analyse des 17-oxostéroïdes par chromatographie gaz-liquide après purification de l'extrait urinaire par le réactif T de Girard.

The authors describe a method of fractionation of urinary 17-oxosteroids after purification of the urinary extract by Girard's T reagent. After solvolysis, enzyme hydrolysis and purification by Girard's T reagent, the 17-oxosteroids were estimated in the form of trimethyl silyl ethers by gas liquid chromatographye on OV 225 using cholesterol acetate for standardisation. The improvement in the quality of the fraction using this technic, the analytical characteristics of which are studied in this work, is of definite interest, in particular after stimulation by ACTH, HCG, metopirone and in the case of pigmentation of the urine.

[Hydrolytic conditions of the conjugated steroids from human urine (author's transl)]. / Condiciones para la hidrólisis de los esteroides conjugados de la orina humana

The hydrolisis of dehydroepiandrosterone sulfate added to human urine was studied under several hydrolytic procedures. Enzymatic and acid hydrolysis with and without the previous removal of enzymic inhibitors present in the human urine were compared. The simple precipitation of inorganic ions with barium acetate before the enzymatic hydrolysis was considered an efficient procedure giving an 81% recovery of the added dehydroepiandrosterone. Since this procedure does not involve rearrangements in the steroid molecules, and because of its simplicity it is considered adequate for the determination of the urinary steroid excretion patterns in humans allowing the quantitation of some conjugates (16 alpha-hydroxy-dehydroepiandrosterone) otherwise not detectable because of its incomplete hydrolysis and extraction.

[Urinary excretion of 17-ketosteroids studied by gas chromatography in the healthy adult: distribution of values]. / Excrétion urinaire des 17-cétostéroïdes étudiés par chromatographie gazeuse chez l'adulte sain: distribution des valeurs

The level of different urinary 17-ketosteroids were assayed using a method requiring an enzymatic hydrolysis and a solvolysis, a separation by gas-chromatography on OV 225 (cyanopropylsilicone) and a detection by flame ionisation. The method has been discuted. Statistical study of the results obtained in 89 healthy and ambulatory subjects (divided in 4 groups according to sex and age) has shown a log-normal distribution of urinary excretions.

Application of over-run thin-layer and gas-liquid chromatography in the separation of closely related C19O2 steroids.

An improved method for the separation of epimeric C19O2 steroids and their related allylic alcohols is described. In this method, the steroids are first separated by over-run thin-layer chromatography, and the unresolved groups are further analysed as free or as trimethylsilyl ether derivatives by gas-liquid chromatography. The behaviour of twenty-one C19O2 steroids was investigated by thin-layer chromatography in four systems and by gas-liquid chromatography in four liquid phases. All steroid pairs of similar polarity were resolved by the combination of these two fractionation procedures.