title: The influence of Aspalathus linearis (Rooibos) and dihydrochalcones on adrenal steroidogenesis: quantification of steroid intermediates and end products in H295R cells.
abstract: The steroid hormone output of the adrenal gland is crucial in the maintenance of hormonal homeostasis, with hormonal imbalances being associated with numerous clinical conditions which include, amongst others, hypertension, metabolic syndrome, cardiovascular disease, insulin resistance and type 2 diabetes. Aspalathus linearis (Rooibos), which has been reported to aid stress-related symptoms linked to metabolic diseases, contains a wide spectrum of bioactive phenolic compounds of which aspalathin is unique. In this study the inhibitory effects of Rooibos and the dihydrochalcones, aspalathin and nothofagin, were investigated on adrenal steroidogenesis. The activities of both cytochrome P450 17α-hydroxylase/17,20 lyase and cytochrome P450 21-hydroxylase were significantly inhibited in COS-1 cells. In order to study the effect of these compounds in H295R cells, a human adrenal carcinoma cell line, a novel UPLC-MS/MS method was developed for the detection and quantification of twenty-one steroid metabolites using a single chromatographic separation. Under both basal and forskolin-stimulated conditions, the total amount of steroids produced in H295R cells significantly decreased in the presence of Rooibos, aspalathin and nothofagin. Under stimulated conditions, Rooibos decreased the total steroid output 4-fold and resulted in a significant reduction of aldosterone and cortisol precursors. Dehydroepiandrosterone-sulfate levels were unchanged, while the levels of androstenedione (A4) and 11β-hydroxyandrostenedione (11βOH-A4) were inhibited 5.5 and 2.3-fold, respectively. Quantification of 11βOH-A4 showed this metabolite to be a major product of steroidogenesis in H295R cells and we confirm, for the first time, that this steroid metabolite is the product of the hydroxylation of A4 by human cytochrome P450 11β-hydroxylase. Taken together our results demonstrate that Rooibos, aspalathin and nothofagin influence steroid hormone biosynthesis and the flux through the mineralocorticoid, glucocorticoid and androgen pathways, thus possibly contributing to the alleviation of negative effects arising from elevated glucocorticoid levels.
authors.affiliation: Department of Biochemistry, University of Stellenbosch, Stellenbosch 7600, South Africa.
authors.forename: Lindie
authors.initials: L
authors.lastname: Schloms
authors.forename: Karl-Heinz
authors.initials: KH
authors.lastname: Storbeck
authors.forename: Pieter
authors.initials: P
authors.lastname: Swart
authors.forename: Wentzel C A
authors.initials: WC
authors.lastname: Gelderblom
authors.forename: Amanda C
authors.initials: AC
authors.lastname: Swart
mesh_terms: D000067956:Adenylyl Cyclase Inhibitors; D000311:Adrenal Glands / Q000187:drug effects* / Q000201:enzymology / Q000378:metabolism*; D000818:Animals; D036903:Aspalathus / Q000737:chemistry*; D019556:COS Cells; D002460:Cell Line; D047188:Chalcones / Q000494:pharmacology*; D002522:Chlorocebus aethiops; D005576:Colforsin / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006801:Humans; D006900:Hydroxylation / Q000187:drug effects; D015394:Molecular Structure; D010215:Papio; D010936:Plant Extracts / Q000494:pharmacology*; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D013254:Steroid 17-alpha-Hydroxylase / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D013255:Steroid 21-Hydroxylase / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D013256:Steroids / Q000737:chemistry / Q000378:metabolism*
chemical_list: D000067956:Adenylyl Cyclase Inhibitors; D047188:Chalcones; D004791:Enzyme Inhibitors; D010936:Plant Extracts; D011994:Recombinant Proteins; D013256:Steroids; C517016:aspalathin; C528728:nothofagin; D005576:Colforsin; D013255:Steroid 21-Hydroxylase; D013254:Steroid 17-alpha-Hydroxylase