An innovative approach to the safety evaluation of natural products: cranberry (Vaccinium macrocarpon Aiton) leaf aqueous extract as a case study.

Assessment of safety for a food or dietary ingredient requires determination of a safe level of ingestion compared to the estimated daily intake from its proposed uses. The nature of the assessment may require the use of different approaches, determined on a case-by-case basis. Natural products are chemically complex and challenging to characterize for the purpose of carrying out a safety evaluation. For example, a botanical extract contains numerous compounds, many of which vary across batches due to changes in environmental conditions and handling. Key components integral to the safety evaluation must be identified and their variability established to assure that specifications are representative of a commercial product over time and protective of the consumer; one can then extrapolate the results of safety studies on a single batch of product to other batches that are produced under similar conditions. Safety of a well-characterized extract may be established based on the safety of its various components. When sufficient information is available from the public literature, additional toxicology testing is not necessary for a safety determination on the food or dietary ingredient. This approach is demonstrated in a case study of an aqueous extract of cranberry (Vaccinium macrocarpon Aiton) leaves.

Effects of cucurbitacins on cell morphology are associated with sensitization of renal carcinoma cells to TRAIL-induced apoptosis.

Cucurbitacins B and D were among the compounds identified as sensitizers of cancer cells to TRAIL-mediated apoptosis in a high-throughput screen. Therefore a series of cucurbitacins was further investigated for TRAIL sensitization and possible mechanisms of action. A total of six cucurbitacins promoted TRAIL-induced apoptosis (B, I, E, C, D, and K) and one (P) was inactive. Sensitization of renal adenocarcinoma cells to TRAIL was apparent after as little as 1-4 h pretreatment and did not require continued presence of cucurbitacin. Active cucurbitacins induced caspase-8 activation only after subsequent TRAIL addition and caspase activation was required for apoptosis suggesting amplified proximal signaling from TRAIL death receptors. Cucurbitacin-sensitized TRAIL-induced cytotoxicity was inhibited by N-acetyl cysteine. Structure-activity relationship analysis in comparison to published studies suggests that TRAIL-sensitizing and general cytotoxic activities of cucurbitacins may be decoupled. Cucurbitacins are reported to be inhibitors of STAT3 activation. However, their TRAIL-sensitizing activity is STAT3-independent. Treatment of renal carcinoma cells with active cucurbitacins produced rapid and dramatic changes in cell morphology and cytoskeletal organization (also prevented by NAC). Therefore, cucurbitacins may be useful as tools for investigating the molecular mechanism(s) of action of TRAIL sensitizers, particularly with regard to temporal aspects of sensitization and modulation of TRAIL signaling by cell morphology, and could form the basis for future therapeutic development in combination with TRAIL death receptor agonists.

A cell-based high-throughput screen to identify synergistic TRAIL sensitizers.

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.

A red clover (Trifolium pratense) phase II clinical extract possesses opiate activity.

Trifolium pratense (TP) is one of the most common herbs for the relief of menopausal symptoms. Little is known about its mechanisms of action. In this study, we investigated the affinity of TP at the mu- and delta-opiate receptors. We found that a clinically used TP extract bound to the mu-opiate receptor with a high affinity (K(i)=9.7+/-1.6microg/ml). The same extract was also found to have affinity at the delta-opiate receptor with K(i) of 15.9+/-2.4microg/ml. These results for the first time suggest a potential new mechanism of action of TP at the opiate receptors. Given the essential role of the opioid system in regulating temperature, mood, and hormonal levels and actions, this may explain in part the beneficial effect of TP in alleviating menopausal symptoms.

Clinical studies of red clover (Trifolium pratense) dietary supplements in menopause: a literature review.

Red clover (Trifolium pratense L., Fabaceae) botanical dietary supplements have received much attention recently for their potential use in the treatment of menopause symptoms, maintenance/improvement of bone and cardiovascular health, and reported benign effects on the breast and endometrium. Literature searches of four computerized databases were run to identify clinical studies of red clover botanical dietary supplements. The manufacturer of the red clover products used in the majority of the studies was contacted for unpublished information and/or clarification regarding the chemical content of their products. Red clover studies were reviewed that pertained to women's health or menopause. Clinical evidence is presently lacking to support the efficacy of semipurified red clover isoflavone extracts for alleviation of climacteric vasomotor symptoms or reduction of low-density lipoprotein levels in the blood. Furthermore, the safety of use of red clover isoflavone supplements in patients with breast or endometrial cancer has not been established. Limited evidence suggests possible efficacy in maintenance of bone health and improvement of arterial compliance, a risk factor for atherosclerosis. This literature review covers red clover botanical dietary supplement clinical studies having a possible impact on the health care of mature and menopausal women, and provides historical perspective regarding the traditional uses of red clover.

The chemical and biologic profile of a red clover (Trifolium pratense L.) phase II clinical extract.

OBJECTIVES: To document the chemical and biologic profile of a clinical phase II red clover (Trifolium pratense L.) extract by identifying and measuring the major and minor components visible in the high-performance liquid chromatography-ultraviolet (HPLC-UV) chromatogram and evaluating each compound for estrogenic and antioxidant activity. DESIGN: Individual compounds in the preformulated (i.e., no excipients present) extract were identified by either chemical isolation followed by structure elucidation or matching to retention time and molecular mass of chemical standards via liquid chromatography-mass spectrometry (LC-MS) analysis. Quantitation of the amounts of compounds found in the preformulated extract was done using HPLC-UV or LC-MS. Isolated compounds or standards were evaluated for their ability to: (1) induce alkaline phosphatase (AP) in an endometrial carcinoma cell line, (2) competitively bind to recombinant human estrogen receptors (ERs) alpha (alpha) and beta (beta), and (3) act as antioxidants by scavenging 2,2-di(4-tert-octylphenyl)-1-picrylhydrazyl (DPPH) free radicals. RESULTS: The preformulated red clover extract had 50% effective concentration (EC 50) of 2.0 to 2.2 microg/mL in the AP estrogenicity assay, and 50% inhibitory concentrations (IC(50)s) of 18.4 to 32.6 microg/mL and 1.9 to 3.4 microg/mL in the ERalpha and ERbeta binding assays, respectively. The preformulated extract was composed of 35.54% isoflavones, 1.11% flavonoids, 0.06% pterocarpans, < or =0.03% coumarins, and < or =0.03% tyramine. Daidzein, genistein, formononetin, biochanin A, coumestrol, and naringenin were estrogenic in the AP assay, and all of these, except formononetin, bound to one or both ERs. CONCLUSIONS: 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.

Seasonal variation of red clover (Trifolium pratense L., Fabaceae) isoflavones and estrogenic activity.

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 above-ground parts and flower heads were collected in parallel over one growing season in northeastern Illinois. Generally, autohydrolytic extracts of above-ground parts contained more isoflavones and had more estrogenic activity in Ishikawa endometrial cells as compared with extracts of flower heads. Daidzein and genistein contents peaked around June to July, while formononetin and biochanin A contents peaked in early September. Flower head and total above-ground parts extracts exhibited differential estrogenic activity in an Ishikawa (endometrial) cell-based alkaline phosphatase 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; therefore, caution is advised in the choice of bioassay used for the biological standardization of botanical dietary supplements.

Trifolium pratense (red clover) exhibits estrogenic effects in vivo in ovariectomized Sprague-Dawley rats.

Studies were conducted using an ovariectomized rat model to determine the estrogenic and antiestrogenic activity of Trifolium pratense L. (red clover) extracts. A red clover extract, standardized to contain 15% isoflavones was administered by gavage [250, 500 and 750 mg/(kg x d)] to virgin, ovariectomized 50-d-old Sprague-Dawley rats, for 21 d in the presence and absence of 17beta-estradiol [50 microg/(kg x d)]. Estrogenic effects included an increase in uterine weight, vaginal cell cornification and mammary gland duct branching. Red clover produced a dose-dependent increase in uterine weight and differentiated vaginal cells at the two higher doses, but it did not stimulate cell proliferation in the mammary glands. Neither antiestrogenic nor additive estrogenic properties were observed in any of the tissues studied. These data suggest that red clover extract is weakly estrogenic in the ovariectomized rat model.