ABSTRACT
In this study, methasterone urinary metabolic profiles were investigated by liquid chromatography quadrupole time of flight mass spectrometry (LC-QTOF-MS) in full scan and targeted MS/MS modes with accurate mass measurement. A healthy male volunteer was asked to take the drug and liquid-liquid extraction was employed to process urine samples. Chromatographic peaks for potential metabolites were hunted out with the theoretical [M - H](-) as a target ion in a full scan experiment and actual deprotonated ions were studied in targeted MS/MS experiment. Fifteen metabolites including two new sulfates (S1 and S2), three glucuronide conjugates (G2, G6 and G7), and three free metabolites (M2, M4 and M6) were detected for methasterone. Three metabolites involving G4, G5 and M5 were obtained for the first time in human urine samples. Owing to the absence of helpful fragments to elucidate the steroid ring structure of methasterone phase II metabolites, gas chromatography mass spectrometry (GC-MS) was employed to obtain structural information of the trimethylsilylated phase I metabolite released after enzymatic hydrolysis and the potential structure was inferred using a combined MS method. Metabolite detection times were also analyzed and G2 (18-nor-17ß-hydroxymethyl-2α, 17α-dimethyl-androst-13-en-3α-ol-ξ-O-glucuronide) was thought to be new potential biomarker for methasterone misuse which can be detected up to 10 days.
Subject(s)
Androstanols/urine , Biomarkers/urine , Chromatography, High Pressure Liquid , Tandem Mass Spectrometry , Androstanols/isolation & purification , Androstanols/metabolism , Doping in Sports , Gas Chromatography-Mass Spectrometry , Glucuronides/chemistry , Humans , Liquid-Liquid Extraction , Sulfates/chemistryABSTRACT
AIM OF THE STUDY: Pregnane glycosides are potent cytotoxic agents which may represent new leads in the development of anti-tumour drugs, particularly in the treatment of breast cancer, because of the structural similarity to estrogenic agonists. Caralluma species are natural sources of a wide variety of pregnane glycosides. The aim of the study was to isolate, using an activity-guided fractionation approach, novel pregnane glycosides for testing on breast cancer and other tumour lines. MATERIALS AND METHODS: The effect of crude extracts, specific organic fractions and isolated compounds from Caralluma tuberculata was tested on the growth and viability of MCF-7 estrogen-dependent, and MDA-MB-468 estrogen-independent breast cancer cells, Caco-2 human colonic cells, HUVECs and U937 cells. Neutral red uptake and MTT assays were used. Apoptosis was detected by Western blot of poly-(ADP ribose) polymerase (PARP) as were other markers of nuclear fragmentation (DNA ladder assay, staining of cells with nuclear dye DAPI). The involvement of caspases was investigated using the pan-caspase inhibitor Z-VAD-FMK. RESULTS: The ethyl acetate fraction of Caralluma tuberculata was found to be the most potent anti-proliferative fraction against all three cancer cell lines. Two novel steroidal glycosides were isolated from the active fraction after a series of chromatographic experiments. The structure of the isolated compounds was elucidated solely based on 2D-NMR (HMBC, HETCOR, DQF-COSY) and MS spectral analysis as compound 1: 12-O-benzoyl-20-O-acetyl-3ß,12ß,14ß,20ß-tetrahydroxy-pregnan-3-ylO-ß-D-glucopyranosyl-(1â4)-ß-D-glucopyranosyl-(1 â 4)-3-methoxy-ß-D-ribopyranoside, and as compound 2: 7-O-acetyl-12-O-benzoyl-3ß,7ß,12ß,14ß-tetrahydroxy-17ß-(3-methylbutyl-O-acetyl-1-yl)-androstan-3-ylO-ß-D-glucopyranosyl-(1 â 4)-6-deoxy-ß-D-allopyranosyl-(1 â 4)-ß-D-cymaropyranosyl-(1 â 4)-ß-D-cymapyranosyl-(1â 4)-ß-D-cymaropyranoside. Compound 1 (pregnane glycoside) and compound 2 (androstan glycoside) induced apoptosis at <25 µM after 48 h as assessed by cell shrinkage, PARP cleavage, DNA fragmentation, and reversal with the caspase inhibitor. CONCLUSIONS: Two novel steroid glycosides isolated from Caralluma tuberculata possess moderate, micromolar cytotoxic activity on breast cancer and other cells in vitro, which may indicate a source of activity in vivo of interest to future drug design.
Subject(s)
Androstanols/pharmacology , Antineoplastic Agents, Phytogenic/pharmacology , Apocynaceae , Apoptosis/drug effects , Caspases/metabolism , Glycosides/pharmacology , Neoplasms/enzymology , Pregnanes/pharmacology , Acylation , Amino Acid Chloromethyl Ketones/pharmacology , Androstanols/chemistry , Androstanols/isolation & purification , Antineoplastic Agents, Phytogenic/chemistry , Antineoplastic Agents, Phytogenic/isolation & purification , Apocynaceae/chemistry , Blotting, Western , Caco-2 Cells , Caspase Inhibitors , Cell Proliferation/drug effects , Cell Survival/drug effects , Chemical Fractionation/methods , Chromatography , Cysteine Proteinase Inhibitors/pharmacology , Dose-Response Relationship, Drug , Glycosides/chemistry , Glycosides/isolation & purification , Human Umbilical Vein Endothelial Cells/drug effects , Human Umbilical Vein Endothelial Cells/enzymology , Human Umbilical Vein Endothelial Cells/pathology , Humans , Magnetic Resonance Spectroscopy , Mass Spectrometry , Molecular Structure , Neoplasms/pathology , Plants, Medicinal , Poly(ADP-ribose) Polymerases/metabolism , Pregnanes/chemistry , Pregnanes/isolation & purification , Structure-Activity Relationship , U937 CellsABSTRACT
The ability of a genus of cephalosporium-like fungus isolated from soil, Acremonium strictum PTCC 5282, for hydrocortisone biotransformation has been investigated. This potential had not been previously examined. The fermentation yielded 11beta,17beta-dihydroxyandrost-4-en-3-one, 11beta,17alpha,20beta,21-tetrahydroxypregn-4-en-3-one and 21-acetoxy-11beta,17alpha,20-trihydroxypregn-4-en-3-one. Each microbial metabolite was purified and characterized using spectroscopic methods.
Subject(s)
Acremonium/metabolism , Hydrocortisone/metabolism , Androstanols/analysis , Androstanols/chemistry , Androstanols/isolation & purification , Biotransformation , Crystallization , Fermentation , Magnetic Resonance Spectroscopy , Molecular Structure , Spectroscopy, Fourier Transform InfraredABSTRACT
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.
Subject(s)
17-Ketosteroids/isolation & purification , Androstanols/isolation & purification , Magnoliopsida/chemistry , Chromatography, Liquid/methods , Magnetic Resonance Spectroscopy , Mass SpectrometryABSTRACT
The raw material of epitiostanol was examined for preparation of "Epitiostanol Reference Standard". Analytical results for the sample were as follows: melting point 130.7 degrees C (decomposition); UV spectrum indicates absorption maximum at 263 nm in ethanol; IR spectrum indicates specific absorption at 2920, 1383, 1056 and 590 cm-1; optical rotation [alpha]20D+ 24.4 degrees; no impurity was found by TLC, but a small amount of one was found by HPLC analysis and the purity is assumed to be 99.5%; water content 1.52%. Based on the results, the present raw material was authorized to be the Reference Standard of the National Institute of Hygienic Sciences.
Subject(s)
Androstanols/standards , Government Agencies , Androstanols/isolation & purification , Chromatography, High Pressure Liquid , Chromatography, Thin Layer , Hygiene , Japan , Pharmacopoeias as TopicABSTRACT
A new polyhydroxysterol 1 beta,3 beta,5,6 beta-tetrahydroxy-5 alpha-androstn-17-one (1) was isolated from the soft coral Sarcophyton glaucum. The structure of 1 was deduced from comparison of the spectral data with those of known 1 beta,3 beta,5 alpha,6 beta-tetrahydroxysterols and confirmed by the synthesis starting from 1 beta,3 beta-dihydroxy-5,16-pregnadien-20-one (6a).