Salvia officinalis for hot flushes: towards determination of mechanism of activity and active principles.

Herbal medicinal products are commonly used in alternative treatment of menopausal hot flushes. In a recent clinical study, Salvia officinalis tincture was found to reduce hot flush frequency and intensity. The aim of the current study was the investigation of the mechanism(s) responsible for the anti-hot flush activity of S. officinalis and determination of its active principle(s). The 66% ethanolic tincture, as well as the n-hexane, CHCl3, and aqueous ethanolic subextracts obtained from the tincture were studied in vitro for two of the most relevant activities, estrogenicity and selective serotonin reuptake inhibition. Because of an increased risk of menopausal women to suffer from Alzheimer's disease, an in vitro acetylcholinesterase inhibition assay was also employed. No activity was observed in the selective serotonin reuptake inhibition or the acetylcholinesterase inhibition assays at the highest test concentrations. The tincture showed no estrogenic effects whereas the aqueous ethanolic subextract exhibited estrogenicity in the ERLUX assay with an EC50 value of 64 µg/mL. Estrogenic activity-guided fractionation of the aqueous ethanolic subextract by a combination of reverse-phase vacuum liquid chromatography and gel chromatography identified luteolin-7-O-glucuronide (EC50 129 µg/mL) as the active component of the vacuum liquid chromatography fraction 4 (EC50 69 µg/mL). Luteolin-7-O-glucoside was identified as the putative estrogenic principle of the most potent minor fraction (7.6.7.6, EC50 0.7 µg/mL) obtained from the initial vacuum liquid chromatography fraction 7 (EC50 3 µg/mL). This study suggests the involvement of common and ubiquitous estrogenic flavonoids in the anti-hot flush effect of Salvia officinalis, a safe and commonly used herbal medicinal product during the menopause.

Inability to confirm estrogenicity of the heterocyclic amine PhIP in two in vitro assays.

2-Amino-1-methyl-6-phenylimidazo-[4,5-b]pyridine (PhIP) is a heterocyclic amine which is found in food after cooking and which is a known mutagen. Reports from several laboratories have proposed that PhIP has estrogenic activity, which would classify PhIP as a xenoestrogen with human exposure via food. We tested PhIP in two cell-based assays for estrogenicity, both based on human cell lines but utilising different outcome measures: ERLUX (reporter-gene activation) and ESCREEN (cell proliferation). PhIP was inactive in both assays at concentrations spanning the picomolar to micromolar range. To eliminate supplier differences as an explanation for the disparity between these results and positive findings in the literature, we purchased PhIP from three suppliers and found no detectable estrogenic activity in any batch. (1)H NMR spectroscopy confirmed the chemical identity of the tested stock solutions. Correct assay performance was confirmed by including positive and vehicle controls on every assay plate, and by demonstrating the expected responses to a panel of known estrogens (estradiol, bisphenol A, and genistein). Our results differ from those in the literature and, whilst the exact reason for this is unknown, we discuss possible explanations of the disparity. Our results provide no in vitro evidence for the classification of PhIP as an estrogen.