DESIGNER fraction concept unmasks minor bioactive constituents in red clover (Trifolium pratense L.).

In botanical extracts, highly abundant constituents can mask or dilute the effects of other, and often, more relevant biologically active compounds. To facilitate the rational chemical and biological assessment of these natural products with wide usage in human health, we introduced the DESIGNER approach of Depleting and Enriching Selective Ingredients to Generate Normalized Extract Resources. The present study applied this concept to clinical Red Clover Extract (RCE) and combined phytochemical and biological methodology to help rationalize the utility of RCE supplements for symptom management in postmenopausal women. Previous work has demonstrated that RCE reduces estrogen detoxification pathways in breast cancer cells (MCF-7) and, thus, may serve to negatively affect estrogen metabolism-induced chemical carcinogenesis. Clinical RCE contains ca. 30% of biochanin A and formononetin, which potentially mask activities of less abundant compounds. These two isoflavonoids are aryl hydrocarbon receptor (AhR) agonists that activate P450 1A1, responsible for estrogen detoxification, and P450 1B1, producing genotoxic estrogen metabolites in female breast cells. Clinical RCE also contains the potent phytoestrogen, genistein, that downregulates P450 1A1, thereby reducing estrogen detoxification. To identify less abundant bioactive constituents, countercurrent separation (CCS) of a clinical RCE yielded selective lipophilic to hydrophilic metabolites in six enriched DESIGNER fractions (DFs 01-06). Unlike solid-phase chromatography, CCS prevented any potential loss of minor constituents or residual complexity (RC) and enabled the polarity-based enrichment of certain constituents. Systematic analysis of estrogen detoxification pathways (ERα-degradation, AhR activation, CYP1A1/CYP1B1 induction and activity) of the DFs uncovered masked bioactivity of minor/less abundant constituents including irilone. These data will allow the optimization of RCE with respect to estrogen detoxification properties. The DFs revealed distinct biological activities between less abundant bioactives. The present results can inspire future carefully designed extracts with phytochemical profiles that are optimized to increase in estrogen detoxification pathways and, thereby, promote resilience in women with high-risk for breast cancer. The DESIGNER approach helps to establish links between complex chemical makeup, botanical safety and possible efficacy parameters, yields candidate DFs for (pre)clinical studies, and reveals the contribution of minor phytoconstituents to the overall safety and bioactivity of botanicals, such as resilience promoting activities relevant to women's health.

Breast cancer prevention with liquiritigenin from licorice through the inhibition of aromatase and protein biosynthesis in high-risk women's breast tissue.

Breast cancer risk continues to increase post menopause. Anti-estrogen therapies are available to prevent postmenopausal breast cancer in high-risk women. However, their adverse effects have reduced acceptability and overall success in cancer prevention. Natural products such as hops (Humulus lupulus) and three pharmacopeial licorice (Glycyrrhiza) species have demonstrated estrogenic and chemopreventive properties, but little is known regarding their effects on aromatase expression and activity as well as pro-proliferation pathways in human breast tissue. We show that Gycyrrhiza inflata (GI) has the highest aromatase inhibition potency among these plant extracts. Moreover, phytoestrogens such as liquiritigenin which is common in all licorice species have potent aromatase inhibitory activity, which is further supported by computational docking of their structures in the binding pocket of aromatase. In addition, GI extract and liquiritigenin suppress aromatase expression in the breast tissue of high-risk postmenopausal women. Although liquiritigenin has estrogenic effects in vitro, with preferential activity through estrogen receptor (ER)-ß, it reduces estradiol-induced uterine growth in vivo. It downregulates RNA translation, protein biosynthesis, and metabolism in high-risk women's breast tissue. Finally, it reduces the rate of MCF-7 cell proliferation, with repeated dosing. Collectively, these data suggest that liquiritigenin has breast cancer prevention potential for high-risk postmenopausal women.

Cannabidiol inhibits SARS-CoV-2 replication through induction of the host ER stress and innate immune responses.

The spread of SARS-CoV-2 and ongoing COVID-19 pandemic underscores the need for new treatments. Here we report that cannabidiol (CBD) inhibits infection of SARS-CoV-2 in cells and mice. CBD and its metabolite 7-OH-CBD, but not THC or other congeneric cannabinoids tested, potently block SARS-CoV-2 replication in lung epithelial cells. CBD acts after viral entry, inhibiting viral gene expression and reversing many effects of SARS-CoV-2 on host gene transcription. CBD inhibits SARS-CoV-2 replication in part by up-regulating the host IRE1α RNase endoplasmic reticulum (ER) stress response and interferon signaling pathways. In matched groups of human patients from the National COVID Cohort Collaborative, CBD (100 mg/ml oral solution per medical records) had a significant negative association with positive SARS-CoV-2 tests. This study highlights CBD as a potential preventative agent for early-stage SARS-CoV-2 infection and merits future clinical trials. We caution against use of non-medical formulations including edibles, inhalants or topicals as a preventative or treatment therapy at the present time.

Selective Preparation and High Dynamic-Range Analysis of Cannabinoids in "CBD Oil" and Other Cannabis sativa Preparations.

Much confusion exists about the chemical composition of widely sold Cannabis sativa products that utilize the cannabidiol (CBD) acronym and related terms such as "CBD oil", "CBD plus hemp oil", "full spectrum CBD", "broad spectrum CBD", and "cannabinoids". Their rational chemical and subsequent biological assessment requires both knowledge of the chemical complexity and the characterization of significant individual constituents. Applicable to hemp preparations in general, this study demonstrates how the combination of liquid-liquid-based separation techniques, NMR analysis, and quantum mechanical-based NMR interpretation facilitates the process of natural product composition analysis by allowing specific structural characterization and absolute quantitation of cannabinoids present in such products with a large dynamic range. Countercurrent separation of a commercial "CBD oil" yielded high-purity CBD plus a more polar cannabinoid fraction containing cannabigerol and cannabidivarin, as well as a less polar cannabinoid fraction containing cannabichromene, trans-Δ9-tetrahydrocannabinol, cis-Δ9-tetrahydrocannabinol, and cannabinol. Representatives of six cannabinoid classes were identified within a narrow range of polarity, which underscores the relevance of residual complexity in biomedical research on cannabinoids. Characterization of the individual components and their quantitation in mixed fractions were undertaken by TLC, HPLC, 1H (q)NMR spectroscopy, 1H iterative full spin analysis (HiFSA), 13C NMR, and 2D NMR. The developed workflow and resulting analytical data enhance the reproducible evaluation of "CBD et al." products, which inevitably represent complex mixtures of varying molecular populations, structures, abundances, and polarity features.

Prenylated Coumaric Acids from Artemisia scoparia Beneficially Modulate Adipogenesis.

Two new diprenylated coumaric acid isomers (1a and 1b) and two known congeners, capillartemisin A (2) and B (3), were isolated from Artemisia scoparia as bioactive markers using bioactivity-guided HPLC fractionation. Their structures were determined by spectroscopic means, including 1D and 2D NMR methods and LC-MS, with their purity assessed by 1D 1H pure shift qNMR spectroscopic analysis. The bioactivity of compounds was evaluated by enhanced accumulation of lipids, as measured using Oil Red O staining, and by increased expression of several adipocyte marker genes, including adiponectin in 3T3-L1 adipocytes relative to untreated negative controls. Compared to the plant's 80% EtOH extract, these purified compounds showed significant but still weaker inhibition of TNFα-induced lipolysis in 3T3-L1 adipocytes. This suggests that additional bioactive substances are responsible for the multiple metabolically favorable effects on adipocytes observed with Artemisia scoparia extract.

Auto-hydrolysis of red clover as "green" approach to (iso)flavonoid enriched products.

Optimal parameters for the auto-hydrolysis of (iso)flavone glycosides to aglycones in ground Trifolium pratense L. plant material were established as a "green" method for the production of a reproducible red clover extract (RCE). The process utilized 72-h fermentation in DI water at 25 and 37 °C. The aglycones obtained at 25 °C, as determined by UHPLC-UV and quantitative 1H NMR (qHNMR), increased significantly in the auto-hydrolyzed (ARCE) (6.2-6.7% w/w biochanin A 1, 6.1-9.9% formononetin 2) vs a control ethanol (ERCE) extract (0.24% 1, 0.26% 2). After macerating ARCE with 1:1 (v/v) diethyl ether/hexanes (ARCE-d/h), 1 and 2 increased to 13.1-16.7% and 14.9-18.4% w, respectively, through depletion of fatty components. The final extracts showed chemical profiles similar to that of a previous clinical RCE. Biological standardization revealed that the enriched ARCE-d/h extracts produced the strongest estrogenic activity in ERα positive endometrial cells (Ishikawa cells), followed by the precursor ARCE. The glycoside-rich ERCE showed no estrogenic activity. The estrogenicity of ARCE-d/h was similar to that of the clinical RCE. The lower potency of the ARCE compared to the prior clinical RCE indicated that substantial amounts of fatty acids/matter likely reduce the estrogenicity of crude hydrolyzed preparations. The in vitro dynamic residual complexity of the conversion of biochanin A to genistein was evaluated by LC-MS-MS. The outcomes help advance translational research with red clover and other (iso)flavone-rich botanicals by inspiring the preparation of (iso)flavone aglycone-enriched extracts for the exploration of new in vitro and ex vivo bioactivities that are unachievable with genuine, glycoside-containing extracts.

Proanthocyanidin Block Arrays (PACBAR) for Comprehensive Capture and Delineation of Proanthocyanidin Structures.

Proanthocyanidins (PACs) are near-ubiquitous and chemically complex metabolites, prototypical of higher plants. Their roles in food/feed/nutrition and ethnomedicine are widely recognized but poorly understood. With the analysis of evidence that underlies this challenge, this perspective identifies shortcomings in capturing and delineating PAC structures as key factors. While several groups have forwarded new representations, a consensus method that captures PAC structures concisely and offers high integrity for electronic storage is required to reduce confusion in this expansive field. The PAC block arrays (PACBAR) system fills this gap by providing precise and human- and machine-readable structural descriptors that capture PAC metabolomic structural diversity. PACBAR enables communication of PAC structures for the development of precise structure-activity relationships and will assist in advancing PAC research to the next level.

6-Prenylnaringenin from Hops Disrupts ERα-Mediated Downregulation of CYP1A1 to Facilitate Estrogen Detoxification.

Botanical dietary supplements (BDS) containing hops are sold as women's health supplements due to the potent hop phytoestrogen, 8-prenylnaringenin (8-PN), and the cytoprotective chalcone, xanthohumol. Previous studies have shown a standardized hop extract to beneficially influence chemical estrogen carcinogenesis in vitro by fostering detoxified 2-hydroxylation over genotoxic 4-hydroxylation estrogen metabolism. In this study, hop extract and its bioactive compounds were investigated for its mechanism of action within the chemical estrogen carcinogenesis pathway, which is mainly mediated through the 4-hydroxylation pathway catalyzed by CYP1B1 that can form gentoxic quinones. Aryl hydrocarbon receptor (AhR) agonists induce CYP1A1 and CYP1B1, while estrogen receptor alpha (ERα) inhibits transcription of CYP1A1, the enzyme responsible for 2-hydroxylated estrogens and the estrogen detoxification pathway. An In-Cell Western MCF-7 cell assay revealed hop extract and 6-prenylnaringenin (6-PN) degraded ERα via an AhR-dependent mechanism. Reverse transcription PCR and xenobiotic response element luciferase assays showed hop extract and 6-PN-mediated activation of AhR and induction of CYP1A1. A reduction in estrogen-mediated DNA (cytosine-5)-methyltransferase 1 (DNMT1) downregulation of CYP1A1 accompanied this activity in a chromatin immunoprecipitation assay. Ultimately, hop extract and 6-PN induced preferential metabolism of estrogens to their detoxified form in vitro. These results suggest that the standardized hop extract and 6-PN activate AhR to attenuate epigenetic inhibition of CYP1A1 through degradation of ERα, ultimately increasing 2-hydroxylated estrogens. A new mechanism of action rationalizes the positive influence of hop BDS and 6-PN on oxidative estrogen metabolism in vitro and, thus, potentially on chemical estrogen carcinogenesis. The findings underscore the importance of elucidating various biological mechanisms of action and standardizing BDS to multiple phytoconstituents for optimal resilience promoting properties.

SAR Study on Estrogen Receptor α/ß Activity of (Iso)flavonoids: Importance of Prenylation, C-Ring (Un)Saturation, and Hydroxyl Substituents.

Many botanicals used for women's health contain estrogenic (iso)flavonoids. The literature suggests that estrogen receptor beta (ERß) activity can counterbalance estrogen receptor alpha (ERα)-mediated proliferation, thus providing a better safety profile. A structure-activity relationship study of (iso)flavonoids was conducted to identify ERß-preferential structures, overall estrogenic activity, and ER subtype estrogenic activity of botanicals containing these (iso)flavonoids. Results showed that flavonoids with prenylation on C8 position increased estrogenic activity. C8-prenylated flavonoids with C2-C3 unsaturation resulted in increased ERß potency and selectivity [e.g., 8-prenylapigenin (8-PA), EC50 (ERß): 0.0035 ± 0.00040 µM], whereas 4'-methoxy or C3 hydroxy groups reduced activity [e.g., icaritin, EC50 (ERß): 1.7 ± 0.70 µM]. However, nonprenylated and C2-C3 unsaturated isoflavonoids showed increased ERß estrogenic activity [e.g., genistein, EC50 (ERß): 0.0022 ± 0.0004 µM]. Licorice (Glycyrrhiza inflata, [EC50 (ERα): 1.1 ± 0.20; (ERß): 0.60 ± 0.20 µg/mL], containing 8-PA, and red clover [EC50 (ERα): 1.8 ± 0.20; (ERß): 0.45 ± 0.10 µg/mL], with genistein, showed ERß-preferential activity as opposed to hops [EC50 (ERα): 0.030 ± 0.010; (ERß): 0.50 ± 0.050 µg/mL] and Epimedium sagittatum [EC50 (ERα): 3.2 ± 0.20; (ERß): 2.5 ± 0.090 µg/mL], containing 8-prenylnaringenin and icaritin, respectively. Botanicals with ERß-preferential flavonoids could plausibly contribute to ERß-protective benefits in menopausal women.

The Multiple Biological Targets of Hops and Bioactive Compounds.

Botanical dietary supplements for women's health are increasingly popular. Older women tend to take botanical supplements such as hops as natural alternatives to traditional hormone therapy to relieve menopausal symptoms. Especially extracts from spent hops, the plant material remaining after beer brewing, are enriched in bioactive prenylated flavonoids that correlate with the health benefits of the plant. The chalcone xanthohumol (XH) is the major prenylated flavonoid in spent hops. Other less abundant but important bioactive prenylated flavonoids are isoxanthohumol (IX), 8-prenylnaringenin (8-PN), and 6-prenylnaringenin (6-PN). Pharmacokinetic studies revealed that these flavonoids are conjugated rapidly with glucuronic acid. XH also undergoes phase I metabolism in vivo to form IX, 8-PN, and 6-PN. Several hop constituents are responsible for distinct effects linked to multiple biological targets, including hormonal, metabolic, inflammatory, and epigenetic pathways. 8-PN is one of the most potent phytoestrogens and is responsible for hops' estrogenic activities. Hops also inhibit aromatase activity, which is linked to 8-PN. The weak electrophile, XH, can activate the Keap1-Nrf2 pathway and turn on the synthesis of detoxification enzymes such as NAD(P)H-quinone oxidoreductase 1 and glutathione S-transferase. XH also alkylates IKK and NF-κB, resulting in anti-inflammatory activity. Antiobesity activities have been described for XH and XH-rich hop extracts likely through activation of AMP-activated protein kinase signaling pathways. Hop extracts modulate the estrogen chemical carcinogenesis pathway by enhancing P450 1A1 detoxification. The mechanism appears to involve activation of the aryl hydrocarbon receptor (AhR) by the AhR agonist, 6-PN, leading to degradation of the estrogen receptor. Finally, prenylated phenols from hops are known inhibitors of P450 1A1/2; P450 1B1; and P450 2C8, 2C9, and 2C19. Understanding the biological targets of hop dietary supplements and their phytoconstituents will ultimately lead to standardized botanical products with higher efficacy, safety, and chemopreventive properties.

Evidence to the role of interflavan linkages and galloylation of proanthocyanidins at sustaining long-term dentin biomodification.

OBJECTIVES: The interactivity of proanthocyanidins (PACs) with collagen modulates dentin matrix biomechanics and biostability. Herein, PAC extracts selected based on structural diversity were investigated to determine key PAC features driving sustained effects on dentin matrices over a period of 18months. METHODS: The chemical profiles of PAC-rich plant sources, Pinus massoniana (PM), Cinnamomum verum (CV) and Hamamelis virginiana (HV) barks, as well as Vitis vinifera (VV) seeds, were obtained by diol HPLC analysis after partitioning of the extracts between methyl acetate and water. Dentin matrices (n=15) were prepared from human molars to determine the apparent modulus of elasticity over 18months of aging. Susceptibility of the dentin matrix to degradation by endogenous and exogenous proteases was determined by presence of solubilized collagen in supernatant, and resistance to degradation by bacterial collagenase, respectively. Data were analyzed using ANOVA and Games-Howell post hoc tests (α=0.05). RESULTS: After 18months, dentin matrices modified by PM and CV extracts, containing only non-galloylated PACs, were highly stable mechanically (p<0.05). Dentin matrices treated with CV exhibited the lowest degradation by bacterial collagenase after 1h and 18months of aging (p<0.05), while dentin matrices treated with PM showed the least mass loss and collagen solubilization by endogenous enzymes over time (p<0.05). SIGNIFICANCE: Resistance against long-term degradation was observed for all experimental groups; however, the most potent and long-lasting dentin biomodification resulted from non-galloylated PACs.

Evidence for Chemopreventive and Resilience Activity of Licorice: Glycyrrhiza Glabra and G. Inflata Extracts Modulate Estrogen Metabolism in ACI Rats.

Women are increasingly using botanical dietary supplements (BDS) to reduce menopausal hot flashes. Although licorice (Glycyrrhiza sp.) is one of the frequently used ingredients in BDS, the exact plant species is often not identified. We previously showed that in breast epithelial cells (MCF-10A), Glycyrrhiza glabra (GG) and G. inflata (GI), and their compounds differentially modulated P450 1A1 and P450 1B1 gene expression, which are responsible for estrogen detoxification and genotoxicity, respectively. GG and isoliquiritigenin (LigC) increased CYP1A1, whereas GI and its marker compound, licochalcone A (LicA), decreased CYP1A1 and CYP1B1 The objective of this study was to determine the distribution of the bioactive licorice compounds, the metabolism of LicA, and whether GG, GI, and/or pure LicA modulate NAD(P)H quinone oxidoreductase (NQO1) in an ACI rat model. In addition, the effect of licorice extracts and compounds on biomarkers of estrogen chemoprevention (CYP1A1) as well as carcinogenesis (CYP1B1) was studied. LicA was extensively glucuronidated and formed GSH adducts; however, free LicA as well as LigC were bioavailable in target tissues after oral intake of licorice extracts. GG, GI, and LicA caused induction of NQO1 activity in the liver. In mammary tissue, GI increased CYP1A1 and decreased CYP1B1, whereas GG only increased CYP1A1 LigC may have contributed to the upregulation of CYP1A1 after GG and GI administration. In contrast, LicA was responsible for GI-mediated downregulation of CYP1B1 These studies highlight the polypharmacologic nature of botanicals and the importance of standardization of licorice BDS to specific Glycyrrhiza species and to multiple constituents.

Estrogen Receptor (ER) Subtype Selectivity Identifies 8-Prenylapigenin as an ERß Agonist from Glycyrrhiza inflata and Highlights the Importance of Chemical and Biological Authentication.

Postmenopausal women are increasingly using botanicals for menopausal symptom relief due to the increased breast cancer risk associated with traditional estrogen therapy. The deleterious effects of estrogens are associated with estrogen receptor (ER)α-dependent proliferation, while ERß activation could enhance safety by opposing ERα effects. Three medicinal licorice species, Glycyrrhiza glabra ( G. glabra), G. uralensis, and G. inflata, were studied for their differential estrogenic efficacy. The data showed higher estrogenic potency for G. inflata in an alkaline phosphatase induction assay in Ishikawa cells (ERα) and an estrogen responsive element (ERE)-luciferase assay in MDA-MB-231/ß41 breast cancer cells (ERß). Bioassay-guided fractionation of G. inflata led to the isolation of 8-prenylapigenin (3). Surprisingly, a commercial batch of 3 was devoid of estrogenic activity. Quality control by MS and qNMR revealed an incorrect compound, 4'- O-methylbroussochalcone B (10), illustrating the importance of both structural and purity verification prior to any biological investigations. Authentic and pure 3 displayed 14-fold preferential ERß agonist activity. Quantitative analyses revealed that 3 was 33 times more concentrated in G. inflata compared to the other medicinal licorice extracts. These data suggest that standardization of G. inflata to 3 might enhance the safety and efficacy of G. inflata supplements used for postmenopausal women's health.

Centrifugal partition chromatography enables selective enrichment of trimeric and tetrameric proanthocyanidins for biomaterial development.

Proanthocyanidins (PACs) find wide applications for human use including food, cosmetics, dietary supplements, and pharmaceuticals. The chemical complexity associated with PACs has triggered the development of various chromatographic techniques, with countercurrent separation (CCS) gaining in popularity. This study applied the recently developed DESIGNER (Depletion and Enrichment of Select Ingredients Generating Normalized Extract Resources) approach for the selective enrichment of trimeric and tetrameric PACs using centrifugal partition chromatography (CPC). This CPC method aims at developing PAC based biomaterials, particularly for their application in restoring and repairing dental hard tissue. A general separation scheme beginning with the depletion of polymeric PACs, followed by the removal of monomeric flavan-3-ols and a final enrichment step produced PAC trimer and tetramer enriched fractions. A successful application of this separation scheme is demonstrated for four polyphenol rich plant sources: grape seeds, pine bark, cinnamon bark, and cocoa seeds. Minor modifications to the generic DESIGNER CCS method were sufficient to accommodate the varying chemical complexities of the individual source materials. The step-wise enrichment of PAC trimers and tetramers was monitored using normal phase TLC and Diol-HPLC-UV analyses. CPC proved to be a reliable tool for the selective enrichment of medium size oligomeric PACs (OPACs). This method plays a key role in the development of dental biomaterials considering its reliability and reproducibility, as well as its scale-up capabilities for possible larger-scale manufacturing.

Red Clover Aryl Hydrocarbon Receptor (AhR) and Estrogen Receptor (ER) Agonists Enhance Genotoxic Estrogen Metabolism.

Many women consider botanical dietary supplements (BDSs) as safe alternatives to hormone therapy for menopausal symptoms. However, the effect of BDSs on breast cancer risk is largely unknown. In the estrogen chemical carcinogenesis pathway, P450 1B1 metabolizes estrogens to 4-hydroxylated catechols, which are oxidized to genotoxic quinones that initiate and promote breast cancer. In contrast, P450 1A1 catalyzed 2-hydroxylation represents a detoxification pathway. The current study evaluated the effects of red clover, a popular BDS used for women's health, and its isoflavones, biochanin A (BA), formononetin (FN), genistein (GN), and daidzein (DZ), on estrogen metabolism. The methoxy estrogen metabolites (2-MeOE1, 4-MeOE1) were measured by LC-MS/MS, and CYP1A1 and CYP1B1 gene expression was analyzed by qPCR. Nonmalignant ER-negative breast epithelial cells (MCF-10A) and ER-positive breast cancer cells (MCF-7) were derived from normal breast epithelial tissue and ER+ breast cancer tissue. Red clover extract (RCE, 10 µg/mL) and isoflavones had no effect on estrogen metabolism in MCF-10A cells. However, in MCF-7 cells, RCE treatments downregulated CYP1A1 expression and enhanced genotoxic metabolism (4-MeOE1/CYP1B1 > 2-MeOE1/CYP1A1). Experiments with the isoflavones showed that the AhR agonists (BA, FN) preferentially induced CYP1B1 expression as well as 4-MeOE1. In contrast, the ER agonists (GN, DZ) downregulated CYP1A1 expression likely through an epigenetic mechanism. Finally, the ER antagonist ICI 182,780 potentiated isoflavone-induced XRE-luciferase reporter activity and reversed GN and DZ induced downregulation of CYP1A1 expression. Overall, these studies show that red clover and its isoflavones have differential effects on estrogen metabolism in "normal" vs breast cancer cells. In breast cancer cells, the AhR agonists stimulate genotoxic metabolism, and the ER agonists downregulate the detoxification pathway. These data may suggest that especially breast cancer patients should avoid red clover and isoflavone based BDSs when making choices for menopausal symptom relief.

Evaluation of estrogenic potency of a standardized hops extract on mammary gland biology and on MNU-induced mammary tumor growth in rats.

Supplements with estrogenic activities are intensively investigated as potential alternatives for the treatment of menopausal symptoms. These investigations include studies on their safety regarding potential breast cancer risks. Therefore, the aim of this study was to assess whether or not a standardized hops (Humulus lupulus) extract, containing 0.42% of the estrogenic flavanone, 8-prenylnaringenin, would stimulate growth of methyl-nitrosourea (MNU) induced mammary cancer in ovariectomized (OVX) Sprague-Dawley (SD) rats or would impact on the proliferative activity within the normal mammary gland of Wistar rats. To induce tumorigenesis SD-rats received an intraperitoneal injection of 50mg/kg body weight of MNU on postnatal days PND 50 and 52. 28days later animals were OVX or were SHAM operated (positive control) and randomly allocated and maintained for 140days on either a phytoestrogen-free placebo diet (SHAM and negative control) or on the hops fortified diet. For the investigations in the normal mammary gland young adult Wistar rats were bilaterally OVX and randomly allocated to a control group fed to a phytoestrogen-free diet, or to a diet supplemented either with E2-benzoate or the hops extract. As a major result, the tumor incidence was 15% (3 tumors totally) in OVX controls, whereas it was 85% (39 tumors totally) in SHAM operated positive controls. No tumors were detectable in the hops group. In addition, no estrogenic activity of the hops extract was detectable in uterus and liver of these animals. In investigations on the normal mammary gland, no impact of hops extract on the expression of estrogen dependent proliferation markers or of progesterone receptor became apparent. In conclusion, the lack of growth stimulation of MNU-induced breast cancer in OVX SD-rats and the lack of stimulation proliferative events in the normal mammary gland of OVX Wistar rats by standardized hops extracts provides an important piece of evidence regarding the safety of these extracts in the management of menopausal symptoms.

A standardized Humulus lupulus (L.) ethanol extract partially prevents ovariectomy-induced bone loss in the rat without induction of adverse effects in the uterus.

BACKGROUND: Hops (Humulus lupulus (L.)) dietary supplements are of interest as herbal remedies to alleviate menopausal symptoms, such as hot flushes, depression and anxiety. So far, the evidence regarding estrogenic and related properties of hops preparations has been considered insufficient for a market authorization for menopausal indications. PURPOSE: The study aims to investigate a chemically standardized hops extract regarding its safety in the uterus, as wells as its efficacy to prevent bone loss in the ovariectomized rat model. STUDY DESIGN/METHODS: Female Wistar rats were ovariectomized and divided into a control group receiving phytoestrogen-free diet, a group treated with E2benzoate (0.93 mg/kg body weight/d) and a group treated with the standardized hops extract (60 mg/kg body weight/d) for 8 weeks. Micro-computed tomography of the tibiae and vertebrae, as wells as histological changes in the uterus and tibia were analyzed. RESULTS: Neither uterotrophic nor proliferative effects were observed in the endometrium in response to the oral 8-week administration of the hops extract. However, site-dependent skeletal effects were observed. The hops extract significantly decreased the number of osteoclasts in the tibial metaphysis and prevented reduction of the trabecular thickness that resulted from estradiol depletion. In contrast, the hops extract did not prevent the ovariectomy-induced micro-architectural changes in the lumbar vertebra. Certain parameters (e.g. thickness and number of trabeculae) were even found to be below the values determined in the ovariectomized control group. CONCLUSION: Taken together, the results provide evidence for the safety of the standardized hops extract and point to a weak bone type-specific, protective effect on bone loss following estradiol depletion.

Stereochemistry of a Second Riolozane and Other Diterpenoids from Jatropha dioica.

The dichloromethane extract of the roots of Jatropha dioica afforded riolozatrione (1) and a C-6 epimer of riolozatrione, 6-epi-riolozatrione (2), as a new structure and only the second reported riolozane diterpenoid. The two known diterpenoids jatrophatrione (3) and citlalitrione (4) were also isolated and characterized. Both epimers 1 and 2 are genuine plant constituents, with 2 likely being the biosynthesis precursor of 1 due to the tendency for the quantitative transformation of 2 into 1 under base catalysis. The structural characterization and distinction of the stereoisomers utilized 1H iterative full-spin analysis, yielding complete J-correlation maps that were represented as quantum interaction and linkage tables. The absolute configuration of compounds 1-4 was established by means of vibrational circular dichroism and via X-ray diffraction analysis for 1, 2, and 4. Additionally, the cytotoxic and antiherpetic in vitro activities of the isolates were evaluated.

Cytochrome P450 inhibition by three licorice species and fourteen licorice constituents.

The potential of licorice dietary supplements to interact with drug metabolism was evaluated by testing extracts of three botanically identified licorice species (Glycyrrhiza glabra L., Glycyrrhiza uralensis Fish. ex DC. and Glycyrrhiza inflata Batalin) and 14 isolated licorice compounds for inhibition of 9 cytochrome P450 enzymes using a UHPLC-MS/MS cocktail assay. G. glabra showed moderate inhibitory effects against CYP2B6, CYP2C8, CYP2C9, and CYP2C19, and weak inhibition against CYP3A4 (testosterone). In contrast, G. uralensis strongly inhibited CYP2B6 and moderately inhibited CYP2C8, CYP2C9 and CYP2C19, and G. inflata strongly inhibited CYP2C enzymes and moderately inhibited CYP1A2, CYP2B6, CYP2D6, and CYP3A4 (midazolam). The licorice compounds isoliquiritigenin, licoricidin, licochalcone A, 18ß-glycyrrhetinic acid, and glycycoumarin inhibited one or more members of the CYP2C family of enzymes. Glycycoumarin and licochalcone A inhibited CYP1A2, but only glycycoumarin inhibited CYP2B6. Isoliquiritigenin, glabridin and licoricidin competitively inhibited CYP3A4, while licochalcone A (specific to G. inflata roots) was a mechanism-based inhibitor. The three licorice species commonly used in botanical dietary supplements have varying potential for drug-botanical interactions as inhibitors of cytochrome P450 isoforms. Each species of licorice displays a unique profile of constituents with potential for drug interactions.

DESIGNER Extracts as Tools to Balance Estrogenic and Chemopreventive Activities of Botanicals for Women's Health.

Botanical dietary supplements contain multiple bioactive compounds that target numerous biological pathways. The lack of uniform standardization requirements is one reason that inconsistent clinical effects are reported frequently. The multifaceted biological interactions of active principles can be disentangled by a coupled pharmacological/phytochemical approach using specialized ("knock-out") extracts. This is demonstrated for hops, a botanical for menopausal symptom management. Employing targeted, adsorbent-free countercurrent separation, Humulus lupulus extracts were designed for pre- and postmenopausal women by containing various amounts of the phytoestrogen 8-prenylnaringenin (8-PN) and the chemopreventive constituent xanthohumol (XH). Analysis of their estrogenic (alkaline phosphatase), chemopreventive (NAD(P)H-quinone oxidoreductase 1 [NQO1]), and cytotoxic bioactivities revealed that the estrogenicity of hops is a function of 8-PN, whereas their NQO1 induction and cytotoxic properties depend on XH levels. Antagonization of the estrogenicity of 8-PN by elevated XH concentrations provided evidence for the interdependence of the biological effects. A designed postmenopausal hop extract was prepared to balance 8-PN and XH levels for both estrogenic and chemopreventive properties. An extract designed for premenopausal women contains reduced 8-PN levels and high XH concentrations to minimize estrogenic while retaining chemopreventive properties. This study demonstrates the feasibility of modulating the concentrations of bioactive compounds in botanical extracts for potentially improved efficacy and safety.