Eight botanical preparations that are commonly used for the treatment of menopausal symptoms were tested for estrogenic activity. Methanol extracts of red clover (Trifolium pratense L.), chasteberry (Vitex agnus-castus L.), and hops (Humulus lupulus L.) showed significant competitive binding to estrogen receptors alpha (ER alpha) and beta (ER beta). With cultured Ishikawa (endometrial) cells, red clover and hops exhibited estrogenic activity as indicated by induction of alkaline phosphatase (AP) activity and up-regulation of progesterone receptor (PR) mRNA. Chasteberry also stimulated PR expression, but no induction of AP activity was observed. In S30 breast cancer cells, pS2 (presenelin-2), another estrogen-inducible gene, was up-regulated in the presence of red clover, hops, and chasteberry. Interestingly, extracts of Asian ginseng (Panax ginseng C.A. Meyer) and North American ginseng (Panax quinquefolius L.) induced pS2 mRNA expression in S30 cells, but no significant ER binding affinity, AP induction, or PR expression was noted in Ishikawa cells. Dong quai [Angelica sinensis (Oliv.) Diels] and licorice (Glycyrrhiza glabra L.) showed only weak ER binding and PR and pS2 mRNA induction. Black cohosh [Cimicifuga racemosa (L.) Nutt.] showed no activity in any of the above in vitro assays. Bioassay-guided isolation utilizing ER competitive binding as a monitor and screening using ultrafiltration LC-MS revealed that genistein was the most active component of red clover. Consistent with this observation, genistein was found to be the most effective of four red clover isoflavones tested in the above in vitro assays. Therefore, estrogenic components of plant extracts can be identified using assays for estrogenic activity along with screening and identification of the active components using ultrafiltration LC-MS. These data suggest a potential use for some dietary supplements, ingested by human beings, in the treatment of menopausal symptoms.

We have developed a high-throughput screen (HTS) to search for novel molecules that can synergize with TRAIL, thus promoting apoptosis of ACHN renal tumor cells in a combinatorial fashion. The HTS detects synthetic compounds and pure natural products that can pre-sensitize the cancer cells to TRAIL-mediated apoptosis, yet have limited toxicity on their own. We have taken into account the individual effects of the single agents, versus the combination, and have identified hits that are synergistic, synergistic-toxic, or additive when combined with TRAIL in promoting tumor cell death. Preliminary mechanistic studies indicate that a subset of the synergistic TRAIL sensitizers act very rapidly to promote cleavage and activation of caspase-8 following TRAIL binding. Caspase-8 is an apical enzyme that initiates programmed cell death via the extrinsic apoptotic pathway. Thus, these TRAIL sensitizers may potentially reduce resistance of tumor cells to TRAIL-mediated apoptosis. Two representative sensitizers were found to increase levels of p53 but did not inhibit the proteasome, suggesting that early DNA damage-sensing pathways may be involved in their mechanisms of action.

The major and minor chemical and active estrogenic components of a preformulated phase II red clover clinical extract were identified, quantitatively measured, and the final capsule doses were calculated. The extract is currently under evaluation in a year-long clinical study for the alleviation of menopausal hot flashes. This is the first report to thoroughly summarize the chemistry and biology of all major peaks observed in the HPLC-UV chromatogram of a clinical red clover dietary supplement.

Red clover (Trifolium pratense L., Fabaceae) dietary supplements are currently used to treat menopausal symptoms because of their high content of the mildly estrogenic isoflavones daidzein, genistein, formononetin and biochanin A. These compounds are estrogenic in vitro and in vivo, but little information exists on the best time to harvest red clover fields to maximize content of the isoflavones and thus make an optimal product. Samples of cultivated red clover aboveground parts and flower heads were collected in parallel over one growing season in northeastern Illinois. Generally, autohydrolytic extracts of aboveground parts contained more isoflavones and had more estrogenic activity in Ishikawa endometrial cells, compared with extracts of flower heads. Daidzein and genistein content peaked around June to July, while formononetin and biochanin A content peaked in early September. Flower head and total aboveground parts extracts exhibited differential estrogenic activity in an Ishikawa (endometrial) cell-based alkaline phosphatase (AP) induction assay, whereas nondifferential activity was observed for most extracts tested in an MCF-7 (breast) cell proliferation assay when tested at the same final concentrations. Ishikawa assay results could be mapped onto the extracts' content of individual isoflavones, but MCF-7 results did not show such a pattern. These results suggest that significant metabolism of isoflavones may occur in MCF-7 cells, but not in Ishikawa cells, and therefore caution is advised in the choice of bioassay used for the biological standardization of botanical dietary supplements.