| The purpose of this study was to investigate red clover (Trifolium pratense L.) for use as a standardized estrogenic botanical dietary supplement. A review of the ethnomedical, folkloric, and clinical literature summarizes the past, present and possible future uses of red clover. The seasonal variation of the isoflavones daidzein, genistein, formononetin, and biochanin A in cultivated red clover was examined. Red clover fields cultivated at the UIC Field Station in Downers Grove, IL, had maximum formononetin and biochanin A content in early September, while daidzein and genistein peaked around June--July. Extracts of aboveground parts were found to contain more isoflavones, and had higher estrogenic activity in vitro, compared to extracts of flower heads. Extracts of cultivated red clover flower heads demonstrated weak ability to scavenge diphenylpicrylhydrazyl (DPPH) free radicals, whereas extracts of aboveground parts were not active.; More than one-third of the chemical composition of a Phase II clinical red clover extract was identified and measured. The extract contained 35.54% isoflavones, 1.11 flavones, ≤0.03% coumarins, 0.06% pterocarpans, and 0.03% tyramine. Eight biologically active (estrogenic) compounds comprised 30.02% of the extract. The main estrogenic compounds were daidzein, genistein, formononetin, biochanin A and prunetin. Other isoflavones (pratensein, calycosin, pseudobaptigenin, and irilone) were not active in the estrogenic Ishikawa alkaline phosphatase induction assay and did not bind to estrogen receptors alpha and beta. A new isoflavanone, 3',4'-methylenedioxy-6 '-methoxy-6,7-dihydroxyisoflavone, was isolated from the extract, but was not estrogenic. Coumestrol, xanthotoxol, daphnoretin, fraxidin, and scopoletin were detected in the extract at ≤100 ppm. The latter four are new to Trifolium pratense. Only coumestrol was estrogenic. No dicoumarol was present. Tyramine was found in red clover extracts aged more than one year, but it did not accumulate to levels that might clinically interfere with monoamine oxidase inhibitors.; Results from an accelerated stability study on a Phase I red clover clinical capsule product suggest that daidzein, genistein, formononetin, and biochanin A are stable when formulated with rice flour and silicon dioxide. Genistein declined only moderately when stored at 30°C/65% relative humidity. |
