Birgit M. Dietz

In vitro Serotonergic Activity of Black Cohosh and Identification of Nω-Methylserotonin as a Potential Active Constituent

Cimicifuga racemosa (L.) Nutt. (syn. Actaea racemosa L., black cohosh) is used to relieve menopausal hot flashes, although clinical studies have provided conflicting data, and the active constituent(s) and mechanism(s) of action remain unknown. Because serotonergic receptors and transporters are involved with thermoregulation, black cohosh and its phytoconstituents were evaluated for serotonergic activity using 5-HT7 receptor binding, cAMP induction, and serotonin selective re-uptake inhibitor (SSRI) assays. Crude extracts displayed 5-HT7 receptor binding activity and induced cAMP production. Fractionation of the methanol extract led to isolation of phenolic acids and identification of N(omega)-methylserotonin by LC-MS/MS. Cimicifuga triterpenoids and phenolic acids bound weakly to the 5-HT7 receptor with no cAMP or SSRI activity. In contrast, N(omega)-methylserotonin showed 5-HT7 receptor binding (IC50 = 23 pM), induced cAMP (EC50 = 22 nM), and blocked serotonin re-uptake (IC50 = 490 nM). These data suggest N(omega)-methylserotonin may be responsible for the serotonergic activity of black cohosh.

Evaluation of Estrogenic Activity of Licorice Species in Comparison with Hops Used in Botanicals for Menopausal Symptoms

The increased cancer risk associated with hormone therapies has encouraged many women to seek non-hormonal alternatives including botanical supplements such as hops (Humulus lupulus) and licorice (Glycyrrhiza spec.) to manage menopausal symptoms. Previous studies have shown estrogenic properties for hops, likely due to the presence of 8-prenylnarigenin, and chemopreventive effects mainly attributed to xanthohumol. Similarly, a combination of estrogenic and chemopreventive properties has been reported for various Glycyrrhiza species. The major goal of the current study was to evaluate the potential estrogenic effects of three licorice species (Glycyrrhiza glabra, G. uralensis, and G. inflata) in comparison with hops. Extracts of Glycyrrhiza species and spent hops induced estrogen responsive alkaline phosphatase activity in endometrial cancer cells, estrogen responsive element (ERE)-luciferase in MCF-7 cells, and Tff1 mRNA in T47D cells. The estrogenic activity decreased in the order H. lupulus > G. uralensis > G. inflata > G. glabra. Liquiritigenin was found to be the principle phytoestrogen of the licorice extracts; however, it exhibited lower estrogenic effects compared to 8-prenylnaringenin in functional assays. Isoliquiritigenin, the precursor chalcone of liquiritigenin, demonstrated significant estrogenic activities while xanthohumol, a metabolic precursor of 8-prenylnaringenin, was not estrogenic. Liquiritigenin showed ERβ selectivity in competitive binding assay and isoliquiritigenin was equipotent for ER subtypes. The estrogenic activity of isoliquiritigenin could be the result of its cyclization to liquiritigenin under physiological conditions. 8-Prenylnaringenin had nanomolar estrogenic potency without ER selectivity while xanthohumol did not bind ERs. These data demonstrated that Glycyrrhiza species with different contents of liquiritigenin have various levels of estrogenic activities, suggesting the importance of precise labeling of botanical supplements. Although hops shows strong estrogenic properties via ERα, licorice might have different estrogenic activities due to its ERβ selectivity, partial estrogen agonist activity, and non-enzymatic conversion of isoliquiritigenin to liquiritigenin.

Pharmacokinetics of Prenylated Hop Phenols in Women Following Oral Administration of a Standardized Extract of Hops

SCOPE Women seeking alternatives to hormone-replacement therapy for menopausal symptoms often try botanical dietary supplements containing extracts of hops (Humulus lupulus L.). Hops contain 8-prenylnaringenin (8-PN), a potent phytoestrogen, the related flavanones 6-prenylnaringenin and isoxanthohumol (IX), and the prenylated chalcone xanthohumol (XN). METHODS AND RESULTS After chemically and biologically standardizing an extract of spent hops to these marker compounds, an escalating dose study was carried out in menopausal women to evaluate safety and pharmacokinetics. 8-PN, 6-prenylnaringenin, IX, and XN, sex hormones, and prothrombin time were determined in blood samples and/or 24 h urine samples. There was no effect on sex hormones or blood clotting. The maximum serum concentrations of the prenylated phenols were dose-dependent and were reached from 2 to 7 h, indicating slow absorption. The marker compounds formed glucuronides that were found in serum and urine. Secondary peaks at 5 h in the serum concentration-time curves indicated enterohepatic recirculation. The serum concentration-time curves indicated demethylation of IX to form 8-PN and cyclization of XN to IX. Slow absorption and enterohepatic recirculation contributed to half-lives exceeding 20 h. CONCLUSION This human study indicated long half-lives of the estrogenic and proestrogenic prenylated phenols in hops but no acute toxicity.

Hops (Humulus lupulus) Inhibits Oxidative Estrogen Metabolism and Estrogen-Induced Malignant Transformation in Human Mammary Epithelial cells (MCF-10A)

Long-term exposure to estrogens including those in traditional hormone replacement therapy (HRT) increases the risk of developing hormone-dependent cancers. As a result, women are turning to over-the-counter (OTC) botanical dietary supplements, such as black cohosh (Cimicifuga racemosa) and hops (Humulus lupulus), as natural alternatives to HRT. The two major mechanisms which likely contribute to estrogen and/or HRT cancer risk are: the estrogen receptor–mediated hormonal pathway; and the chemical carcinogenesis pathway involving formation of estrogen quinones that damage DNA and proteins, hence initiating and promoting carcinogenesis. Because, OTC botanical HRT alternatives are in widespread use, they may have the potential for chemopreventive effects on estrogen carcinogenic pathways in vivo. Therefore, the effect of OTC botanicals on estrogen-induced malignant transformation of MCF-10A cells was studied. Cytochrome P450 catalyzed hydroxylation of estradiol at the 4-position leads to an o-quinone believed to act as the proximal carcinogen. Liquid chromatography/tandem mass spectrometry analysis of estradiol metabolites showed that 4-hydroxylation was inhibited by hops, whereas black cohosh was without effect. Estrogen-induced expression of CYP450 1B1 and CYP450 1A1 was attenuated by the hops extract. Two phenolic constituents of hops (xanthohumol, XH; 8-prenylnaringenin, 8-PN) were tested: 8-PN was a potent inhibitor, whereas XH had no effect. Finally, estrogen-induced malignant transformation of MCF-10A cells was observed to be significantly inhibited by hops (5 μg/mL) and 8-PN (50 nmol/L). These data suggest that hops extracts possess cancer chemopreventive activity through attenuation of estrogen metabolism mediated by 8-PN. Cancer Prev Res; 5(1); 73–81. ©2011 AACR.

Integrated standardization concept for Angelica botanicals using quantitative NMR.

Despite numerous in vitro/vivo and phytochemical studies, the active constituents of Angelica sinensis (AS) have not been conclusively identified for the standardization to bioactive markers. Phytochemical analyses of AS extracts and fractions that demonstrate activity in a panel of in vitro bioassays, have repeatedly pointed to ligustilide as being (associated with) the active principle(s). Due to the chemical instability of ligustilide and related issues in GC/LC analyses, new methods capable of quantifying ligustilide in mixtures that do not rely on an identical reference standard are in high demand. This study demonstrates how NMR can satisfy the requirement for simultaneous, multi-target quantification and qualitative identification. First, the AS activity was concentrated into a single fraction by RP-solid-phase extraction, as confirmed by an alkaline phosphatase, (anti-)estrogenicity and cytotoxicity assay. Next, a quantitative (1)H NMR (qHNMR) method was established and validated using standard compounds and comparing processing methods. Subsequent 1D/2D NMR and qHNMR analysis led to the identification and quantification of ligustilide and other minor components in the active fraction, and to the development of quality criteria for authentic AS preparations. The absolute and relative quantities of ligustilide, six minor alkyl phthalides, and groups of phenylpropanoids, polyynes, and poly-unsaturated fatty acids were measured by a combination of qHNMR and 2D COSY. The qNMR approach enables multi-target quality control of the bioactive fraction, and enables the integrated biological and chemical standardization of AS botanicals. This methodology can potentially be transferred to other botanicals with active principles that act synergistically, or that contain closely related and/or constituents, which have not been conclusively identified as the active principles.

Botanical Modulation of Menopausal Symptoms: Mechanisms of Action?

Menopausal women suffer from a variety of symptoms, including hot flashes and night sweats, which can affect quality of life. Although it has been the treatment of choice for relieving these symptoms, hormone therapy has been associated with increased breast cancer risk leading many women to search for natural, efficacious, and safe alternatives such as botanical supplements. Data from clinical trials suggesting that botanicals have efficacy for menopausal symptom relief have been controversial, and several mechanisms of action have been proposed including estrogenic, progestogenic, and serotonergic pathways. Plant extracts with potential estrogenic activities include soy, red clover, kudzu, hops, licorice, rhubarb, yam, and chasteberry. Botanicals with reported progestogenic activities are red clover, hops, yam, and chasteberry. Serotonergic mechanisms have also been proposed since women taking antidepressants often report a reduction in hot flashes and night sweats. Black cohosh, kudzu, kava, licorice, and dong quai all either have reported 5-hydroxytryptamine receptor 7 ligands or inhibit serotonin reuptake, therefore have potential serotonergic activities. Understanding the mechanisms of action of these natural remedies used for womens health could lead to more efficacious formulations and to the isolation of active components which have the potential of becoming effective medications in the future.

Structural modulation of reactivity/activity in design of improved benzothiophene selective estrogen receptor modulators: induction of chemopreventive mechanisms

The benzothiophene selective estrogen receptor modulators (SERM) raloxifene and arzoxifene are in clinical use and clinical trials for chemoprevention of breast cancer and other indications. These SERMs are “oxidatively labile” and therefore have potential to activate antioxidant responsive element (ARE) transcription of genes for cytoprotective phase II enzymes such as NAD(P)H-dependent quinone oxidoreductase 1 (NQO1). To study this possible mechanism of cancer chemoprevention, a family of benzothiophene SERMs was developed with modulated redox activity, including arzoxifene and its metabolite desmethylarzoxifene (DMA). The relative antioxidant activity of these SERMs was assayed and correlated with induction of NQO1 in murine and human liver cells. DMA was found to induce NQO1 and to activate ARE more strongly than other SERMs, including raloxifene and 4-hydroxytamoxifen. Livers from female, juvenile rats treated for 3 days with estradiol and/or with the benzothiophene SERMs arzoxifene, DMA, and F-DMA showed substantial induction of NQO1 by the benzothiophene SERMs. No persuasive evidence in this assay or in MCF-7 breast cancer cells was obtained of a major role for the estrogen receptor in induction of NQO1 by the benzothiophene SERMs. These results suggest that arzoxifene might provide chemopreventive benefits over raloxifene and other SERMs via metabolism to DMA and stimulation of ARE-mediated induction of phase II enzymes. The correlation of SERM structure with antioxidant activity and NQO1 induction also suggests that oxidative bioactivation of SERMs may be modulated to enhance chemopreventive activity. [Mol Cancer Ther 2007;6(9):2418–28]

Phytochemistry of Cimicifugic Acids and Associated Bases in Cimicifuga racemosa Root Extracts

INTRODUCTION Earlier studies reported serotonergic activity for cimicifugic acids (CA) isolated from Cimicifuga racemosa. The discovery of strongly basic alkaloids, cimipronidines, from the active extract partition and evaluation of previously employed work-up procedures has led to the hypothesis of strong acid/base association in the extract. OBJECTIVE Re-isolation of the CAs was desired to permit further detailed studies. Based on the acid/base association hypothesis, a new separation scheme of the active partition was required, which separates acids from associated bases. METHODOLOGY A new 5-HT(7) bioassay guided work-up procedure was developed that concentrates activity into one partition. The latter was subjected to a new two-step centrifugal partitioning chromatography (CPC) method, which applies pH zone refinement gradient (pHZR CPC) to dissociate the acid/base complexes. The resulting CA fraction was subjected to a second CPC step. Fractions and compounds were monitored by (1)H NMR using a structure-based spin-pattern analysis facilitating dereplication of the known acids. Bioassay results were obtained for the pHZR CPC fractions and for purified CAs. RESULTS A new CA was characterised. While none of the pure CAs was active, the serotonergic activity was concentrated in a single pHZR CPC fraction, which was subsequently shown to contain low levels of the potent 5-HT(7) ligand, N(omega)-methylserotonin. CONCLUSION This study shows that CAs are not responsible for serotonergic activity in black cohosh. New phytochemical methodology (pHZR CPC) and a sensitive dereplication method (LC-MS) led to the identification of N(omega)-methylserotonin as serotonergic active principle.

Estrogen receptor α enhances the rate of oxidative DNA damage by targeting an equine estrogen catechol metabolite to the nucleus

Exposure to estrogens increases the risk of breast and endometrial cancer. It is proposed that the estrogen receptor (ER) may contribute to estrogen carcinogenesis by transduction of the hormonal signal and as a “Trojan horse” concentrating genotoxic estrogen metabolites in the nucleus to complex with DNA, enhancing DNA damage. 4-Hydroxyequilenin (4-OHEN), the major catechol metabolite of equine estrogens present in estrogen replacement formulations, autoxidizes to a redox-cycling quinone that has been shown to cause DNA damage. 4-OHEN was found to be an estrogen of nanomolar potency in cell culture using a luciferase reporter assay and, using a chromatin immunoprecipitation assay, was found to activate ERα binding to estrogen-responsive genes in MCF-7 cells. DNA damage was measured in cells by comparing ERα(+) versus ERα(-) cells and 4-OHEN versus menadione, a reactive oxygen species (ROS)-generating, but non-estrogenic, quinone. 4-OHEN selectively induced DNA damage in ERα(+) cells, whereas menadione-induced damage was not dependent on cellular ER status. The rate of 4-OHEN-induced DNA damage was significantly enhanced in ERα(+) cells, whereas ER status had no effect on the rate of menadione-induced damage. Imaging of ROS induced by 4-OHEN showed accumulation selective for the nucleus of ERα(+) cells within 5 min, whereas in ERα(-) or menadione-treated cells, no selectivity was observed. These data support ERα acting as a Trojan horse concentrating 4-OHEN in the nucleus to accelerate the rate of ROS generation and thereby amplify DNA damage. The Trojan horse mechanism may be of general importance beyond estrogen genotoxins.

Biological Reactive Intermediates (BRIs) Formed from Botanical Dietary Supplements

The use of botanical dietary supplements is increasingly popular, due to their natural origin and the perceived assumption that they are safer than prescription drugs. While most botanical dietary supplements can be considered safe, a few contain compounds, which can be converted to biological reactive intermediates (BRIs) causing toxicity. For example, sassafras oil contains safrole, which can be converted to a reactive carbocation forming genotoxic DNA adducts. Alternatively, some botanical dietary supplements contain stable BRIs such as simple Michael acceptors that react with chemosensor proteins such as Keap1 resulting in induction of protective detoxification enzymes. Examples include curcumin from turmeric, xanthohumol from hops, and Z-ligustilide from dang gui. Quinones (sassafras, kava, black cohosh), quinone methides (sassafras), and epoxides (pennyroyal oil) represent BRIs of intermediate reactivity, which could generate both genotoxic and/or chemopreventive effects. The biological targets of BRIs formed from botanical dietary supplements and their resulting toxic and/or chemopreventive effects are closely linked to the reactivity of BRIs as well as dose and time of exposure.