Abstract
BackgroundTrifolium pratense (red clover) ethanolic extract (TPEE) has been used as a popular over-the-counter remedy for the management of menopausal symptoms. Prolonged consumption of herbal extract has been shown to regulate the composition of gut microbiota. This study was designed to elucidate the influence of TPEE on the gut microbiota composition in the ovariectomized (OVX) rats.MethodsOVX rats were treated with TPEE at 125, 250, 500 mg/kg/day, or controls (pomegranate extract, 500 mg/kg/day; estradiol, 25 μg/kg/day) for 12 weeks. Gut microbiota analysis was conducted by extracting the microbial DNA from fecal samples and microbiome taxonomic profiling was carried out by using next-generation sequencing. The levels of serum biomarkers were analyzed using enzyme-linked immunosorbent assay (ELISA) kit. The prediction of functional biomarker of microbiota was performed using PICRUSt to investigate the potential pathways associated with gut health and serum lipid profile regulation. To study the correlation between gut microbiota composition and serum lipid levels, Spearman’s correlation coefficients were defined and analyzed. Additionally, gas chromatography–mass spectrometry analysis was conducted to uncover additional physiologically active ingredients.ResultsTPEE-treated OVX rats showed significant reduction in serum triglycerides (TG), total cholesterols (TCHOL), and LDL/VLDL levels but increase in HDL level. The alteration in the pathways involve in metabolism was the most common among the other KEGG categories. Particularly, TPEE also significantly reduced the relative abundance of sequences read associated with inflammatory bowel disease (IBD) and the peroxisome proliferator-activated receptor (PPAR) signalling pathway. TPEE intervention was seen to reduce the Firmicutes to Bacteroidetes (F/B) ratio in the OVX rats, denoting a reduction in microbial dysbiosis in the OVX rats. Correlation analysis at the phylum level revealed that Bacteriodetes and Proteobacteria were strongly correlated with serum TG, TCHOL and HDL levels. At the species level, Bifidobacterium pseudolongum group was seen to positively correlate with serum HDL level and negatively correlated with serum AST, ALT, LDL/VLDL, TCHOL, and TG levels.ConclusionsTPEE treatment showed therapeutic benefits by improving the intestinal microbiota composition which strongly correlated with the serum lipid and cholesterol levels in the OVX rats.
Highlights
Trifolium pratense ethanolic extract (TPEE) has been used as a popular over-the-counter remedy for the management of menopausal symptoms
GC‐MS analysis of Trifolium pratense ethanolic extract Gas chromatography mass spectrometry (GC-MS) analysis of TPEE led to the identification of 18 functional phytocomponents (Table 1)
Effects of Trifolium pratense ethanolic extract on serum biomarker levels Serum biomarker analysis revealed that TPEE treatment significantly reduced serum triglyceride (TG) and lowdensity lipoprotein/very low-density lipoprotein (LDL/ VLDL) levels in OVX rats while significantly increased serum high-density lipoprotein (HDL) levels in OVX rats in a dose-dependent manner (Fig. 2)
Summary
Trifolium pratense (red clover) ethanolic extract (TPEE) has been used as a popular over-the-counter remedy for the management of menopausal symptoms. Research into major physiological changes associated with menopause is receiving increased clinical and commercial interest [1]. The major consequences of menopause such as urogenital atrophy, osteoporosis, cancer, obesity, and cardiovascular diseases are related primarily to estrogen deficiency. Previous clinical findings indicate that the treatment of postmenopausal osteoporosis should consider preventative measures for cardiovascular disease because osteoporotic postmenopausal women are at higher risk of suffering cardiovascular events [2]. This is because estrogens exert a protective role in the cardiovascular system and are produced primarily in the ovaries via a process that utilize low-density lipoprotein cholesterol (LDL) as a substrate. Researchers are attempting to identify alternative therapeutics including nutrients and botanicals which are rich in phytoestrogen to improve the abovementioned postmenopausal symptoms
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