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Investigating the mode of action and characterisation of active principles - A Salvia officinalis tincture for the amelioration of hot flushes

Rahte, Sinikka; (2011) Investigating the mode of action and characterisation of active principles - A Salvia officinalis tincture for the amelioration of hot flushes. Doctoral thesis (Ph.D), UCL (University College London). Green open access

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Abstract

The life of most women is at some point affected by side effects of their menopausal transition. The most common menopausal side effects are the hot flushes, which influence the quality of life severely. The physiology behind the hot flushes and the connection between hormones, neurotransmitters and hot flush occurrence is still unclear. However, in vivo studies suggest a correlation between the hormone 17-β-estradiol (E2), the neurotransmitter serotonin (5-HT) and the regulation of the core body temperature. Menosan® (a Salvia officinalis tincture) is an herbal medicinal product (HMP), which has proven to reduce hot flushes, but the mechanism underlying this effect has remained unknown. In order to elucidate the anti-hot flush mechanism of action, the Salvia officinalis tincture as well as three subextracts (n-hexane, chloroform and aqueous-ethanolic (aq-EtOH) subextract (SE)), which were obtained by solvent-solvent partitioning of the tincture, were tested for relevant in vitro activities. In a multi-target approach, the influence of the tincture and SE on the serotonergic system was assessed in a selective serotonin re-uptake inhibition (SSRI) assay and estrogenicity was investigated in an estrogen receptor luciferase reporter gene (ERLUX) assay and an estrogen screen (ESCREEN) assay. None of the extracts showed activity in the SSRI assay. The aq-EtOH SE was found to be estrogenic in the ERLUX assay (EC50 64 μg/ml) and the ESCREEN assay (EC50 130 μg/ml), but none of the other extract exhibited an estrogenic response. In a second approach the Salvia officinalis tincture was investigated for a possible multi-beneficial effect for menopausal women and whether the active principles underlying the anti-hot flush activity and treatment of Alzheimer disease could be linked. The Salvia officinalis tincture and SEs were therefore tested for acetylcholinesterase inhibition (AChEI) activity. The neurotransmitter acetylcholine (ACh) has been linked with Alzheimer diseases (AD) and as it has been suggested that menopausal women may have a higher risk than men to develop AD, hence the effect on ACh levels would be of interest. No activity in the AChEI assay was found for the Salvia officinalis tincture or the SEs. HMPs need to meet high safety standards. Thus, for the safety assessment the inhibitory potential of the Salvia officinalis tincture and its SEs against CYP3A4 was evaluated in an in vitro assay. Cytochrome P450 (CYP) enzyme 3A4 is as key enzyme in human drug metabolism and all four extracts inhibited the CYP3A4 enzyme. While the activity of the aq-EtOH SE was very low, the activity of the Salvia officinalis tincture (EC50 6.8 μg/ml), the chloroform SE (EC50 3.1 μg/ml) and the n-hexane SE (EC50 1.8 μg/ml) were considerably stronger and the inhibitory principle of these extracts warrant further investigation. In the initial screening the aq-EtOH SE was the only SE, which displayed an anti-hot flush activity. Hence, an activity-guided fractionation of the aq-EtOH SE was performed in order to isolate and identify the active constituent(s). Following the estrogenicity in the ERLUX assay resulted in identification of luteolin-7-O-β-glucuronide as the active principle in fraction AQ 4. The estrogenic potential of the isolated luteolin-7-O-β-glucuronide was weak (EC50 129 μg/ml). Luteolin-7-O-glucoside was identified as the major component in the most active fraction (AQ 7.6.7.6, EC50 0.7 μg/ml). The compounds were identified in the estrogenic fractions by nuclear magnetic resonance (NMR) spectroscopy, mass spectroscopy (MS), ultra-high-pressure liquid chromatography at elevated temperature coupled to time-of-flight mass spectrometry (UHPLC-TOF-MS) and capillary (cap) NMR. The estrogenic activity of pure commercially available luteolin-7-O-glucoside (EC50 0.44 μg/ml) was similar to that of fraction AQ 7.6.7.6. Quantitative analysis of luteolin-7-O-glucoside in the aq-EtOH SE and the Salvia officinalis tincture explained the estrogenic activity of the aq-EtOH SE, but failed to explain the absence of estrogenic effects by the Salvia officinalis tincture. In conclusion the investigation revealed noteworthy CYP3A4 inhibitory activity by the Salvia officinalis tincture, luteolin-7-O-β-glucuronide has been identified as the estrogenic principle in fraction AQ 4 and luteolin-7-O-glucoside as the putative estrogenic principle of fraction AQ 7.6.7.6.

Type: Thesis (Doctoral)
Qualification: Ph.D
Title: Investigating the mode of action and characterisation of active principles - A Salvia officinalis tincture for the amelioration of hot flushes
Open access status: An open access version is available from UCL Discovery
Language: English
Additional information: Thesis digitised by ProQuest.
Keywords: Health and environmental sciences; Amelioration; Flushes; Hot; Salvia officinalis; Tincture
URI: https://discovery.ucl.ac.uk/id/eprint/10107029
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