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Abstract Extracts from black cohosh (Cimicifuga racemosa, CR) exert an anti-proliferative action in human breast cancer cell cultures, which has been attributed to an anti-estrogenic effect. However, CR constituents do [...]

Abstract Belamcanda chinensis (BC) belongs to the family of iridaceae and the isoflavone tectorigenin has been isolated from the rhizome of this plant. Whether this isoflavone has estrogenic, possibly selective [...]

Summary Extracts of the fruits of chaste tree (Vitex agnus castus = AC) are widely used to treat premenstrual symptoms. Double-blind placebo-controlled studies indicate that one of the most common [...]

Objectives: This review presents findings with clear statements from the literature as well as own results of effects of soy, red clover and their isoflavones as well as of the Cimicifuga racemosa extract BNO 1055. Experimental and clinical effects on climacteric complaints, osteoprotective effects, activity in the urogenital tract, and risks concerning cardiovascular diseases and mammary and endometrial tissue will be compared, also in comparison to classical hormone preparations. The question whether soy and red clover products and/or Cimicifuga racemosa (CR) preparations are endocrine disrupters or may fulfill the criteria of the so-called phyto-SERMs will be discussed., Methods: Review of selected publications since 1980 and summary of unpublished own results of the authors., Results: Experimental and clinical evidences suggest that soy/red clover and their isoflavones do not fulfill the criteria of an ideal SERM. They appear to have mild osteoprotective effects but do not improve climacteric complaints. Furthermore, they seem to stimulate uterine growth and mammary epithelial proliferation. In ovariectomized rats, the CR extract BNO 1055 showed many of the beneficial effects of 17beta-estradiol, including effects in the brain/hypothalamus to reduce serum LH levels, effects in the bone to prevent osteoporosis and estrogenic effects in the urinary bladder. The CR extract BNO 1055 had no uterotrophic effect., Conclusion: If clinical studies confirm these results, the Cimicifuga racemosa preparation BNO 1055 would appear as an ideal SERM and may therefore be an alternative to hormone replacement therapy.

Isoflavones have been shown to exert antiproliferative effects on cancer cells by steroid receptor signaling. In this study, we demonstrate the potential of plant constituents extracted from Belamcanda chinensis as anticancer drugs, which regulate the aberrant expression of genes relevant in proliferation, invasion, immortalization and apoptosis. LNCaP cells were treated with B.chinensis extract, tectorigenin or other isoflavones and mRNA expression was quantified by using real time RT-PCR. In addition, ELISA, TRAP assays and western blots were used to measure protein expression or activity. Male nude mice (n=18) were injected subcutaneously with LNCaP cells and were fed with extracts from B.chinensis, and tumor development was monitored versus a control animal group (n=18). Tectorigenin and several other phytochemicals downregulated PDEF, PSA and IGF-1 receptor mRNA expression in vitro. Furthermore, PSA secretion and IGF-1 receptor protein expression were diminished, and hTERT mRNA expression and telomerase activity decreased after tectorigenin treatments. However, TIMP-3 mRNA was upregulated on tectorigenin treatment. Growth of subcutaneous tumors in nude mice was delayed and diminished in animals fed with extracts from B.chinensis. The downregulation of PDEF, PSA, hTERT and IGF-1 receptor gene expression by tectorigenin demonstrates the antiproliferative potential of these agents. The upregulation of TIMP-3 gene expression indicates a pro-apoptotic function of the drug and a reduction of the invasiveness of tumors. The animal experiments demonstrate that B.chinensis markedly inhibited the development of tumors in vivo. Thus, these compounds may be useful for the prevention or treatment of human prostate cancer.

The aim of this study was to identify genomic aberrations in endometrial cancer cells treated with the phyto-estrogenic compounds tectorigenin, irigenin and apigenin and to compare with those treated with beta-estradiol using array-based comparative genomic hybridisation (array CGH). The microarray contains 287 targets and includes telomeres, microdeletions, oncogenes and tumour suppressor genes and has increased mapping resolution compared to conventional CGH. An endometrial cancer cell line (Ishikawa) was cultured and treated with the phyto-estrogens. Treated cells were examined using the CGH microarray. Over 20 % of the array genes were aberrated in the cells treated with beta-estradiol, tectorigenin and irigenin compared to 3 % in those treated with the same concentration of apigenin. Protein kinase c zeta form, insulin, insulin receptor and protein-tyrosine phosphatase non-receptor-type 1 which are involved in insulin metabolism were aberrated by tectorigenin and irigenin. Apigenin may play a role in the treatment of endometrial cancer and in the treatment of postmenopausal women. Further studies in normal endometrium and primary endometrial cancer cells are needed to elucidate the role of the phyto-estrogens.

Background: Phytoestrogens may reduce tumorigenesis in prostate cancer. We screened five phytoestrogens for their effect on cell growth and apoptosis in PWR-1E, LNCaP, PC-3, and DU145 prostate epithelial cells in vitro., Methods: We assessed cell number, proliferation, and apoptosis using crystal violet assays, flow cytometric analysis, and TUNEL. Focusing specifically on apigenin we assessed the ability of calpain, serine protease, caspase, estrogen receptor, and ceramide synthase inhibitors to block apigenin induced apoptosis. We also analyzed caspase 3, 7, 8, 9, Bcl-2, Bax, Bid, and cytochrome C by Western analysis, and mitochondrial permeability and reactive oxygen species production by flow cytometry using mitosensor(TM) and DCFH-DA, respectively., Results: Apigenin and silybinin significantly reduced cell number, with apigenin inducing apoptosis in PWR-1E, LNCaP, PC-3, and DU145 cells. The PC-3 and DU145 cells were less susceptible to apigenin induced apoptosis then LNCaP and PWR-1E cells. The induction of apoptosis by apigenin was caspase dependent. Apigenin generated reactive oxygen species, a loss of mitochondrial Bcl-2 expression, mitochondrial permeability, cytochrome C release, and the cleavage of caspase 3, 7, 8, and 9 and the concomitant cleavage of the inhibitor of apoptosis protein, cIAP-2. The overexpression of Bcl-2 in LNCaP B10 cells reduced the apoptotic effects of apigenin., Conclusions: Apigenin induces cell death in prostate epithelial cells using a mitochondrial mediated cell death pathway. Bcl-2 has a role in inhibiting apigenin induced cell death in prostate epithelial cells., ((c) 2004 Wiley-Liss, Inc.)

Extracts from black cohosh (Cimicifuga racemosa, CR) exert an anti-proliferative action in human breast cancer cell cultures, which has been attributed to an anti-estrogenic effect. However, CR constituents do not bind to either of the known estrogen receptors. Thus, the anti-tumor effect of CR me be mediated by mechanisms not involving these receptors. Polycyclic aromatic hydrocarbons are toxic environmental pollutants, which indirectly act as anti-estrogens by activating the aryl hydrocarbon receptor (AhR). The AhR is widely expressed in mammalian tissues and tumors. A recent screening study demonstrated activation of the AhR by a variety of herbal extracts, among others, CR. Since activation of the AhR causes inhibition of growth of prostate cancer cells, we addressed the question, whether CR may not only inhibit growth of breast cancer--but also of prostate cancer cells. In the AhR ligand assay, the CR extract BNO 1055 reduced tracer binding to 71% of the control demonstrating interaction of constituents of this extract with the receptor. Under basal as well as under estradiol- and dihydrotestosterone stimulated conditions, the CR extract dose dependently inhibited proliferation of LNCaP cells. A significant reduction of cell growth was observed at a concentration as low as 50 ng/ml. Thus, it is demonstrated for the first time that CR compounds potently inhibit the growth of human prostate cancer cells in vitro. This anti-proliferative effect may be mediated via the AhR.

Purpose: Phytoestrogens are nonsteroidal plant derived compounds with estrogenic activity that have been implicated in protecting against prostate cancer progression. We hypothesized that these compounds would alter cell number and increase the ability of antiandrogens to induce cell death in prostate cancer cells., Materials and Methods: RWPE-1, LNCaP and PC-3 cells were treated with or without an extract of Belamcanda chinensis, 2 purified phytoestrogens derived from this extract (irigenin and tectorigenin) and the antiandrogen bicalutamide. We assessed the effect on cell number, proliferation and apoptosis., Results: Phytoestrogens (50 to 100 microM) and bicalutamide (10 to 50 microM) alone decreased the cell number in all 3 cell lines. Phytoestrogens (50 microM) combined with bicalutamide (10 microM) further decreased the number of RWPE-1 and PC-3 cells compared to these agents alone. Tectorigenin and irigenin inhibited the proliferation of RWPE-1, LNCaP and PC-3 cells, causing G1 arrest and the induction of p21WAF1 or p27 protein expression, whereas bicalutamide induced apoptosis in a dose dependent manner in all 3 cell lines. Phytoestrogens did not have antiandrogenic activity., Conclusions: These in vitro studies demonstrate a role for tectorigenin and irigenin in regulating prostate cancer cell number by inhibiting proliferation through cell cycle regulation.

Belamcanda chinensis (BC) belongs to the family of iridaceae and the isoflavone tectorigenin has been isolated from the rhizome of this plant. Whether this isoflavone has estrogenic, possibly selective estrogen receptor modulator activities and if so, whether they are mediated via the estrogen receptor alpha or beta is unknown at present. Therefore, we performed binding studies with recombinant human ERalpha and ERbeta to show that tectorigenin binds to both receptor subtypes. In ERalpha-expressing MCF7 and ERbeta-expressing MDA-MB231 reporter gene transfected cells tectorigenin causes transactivation. When given intravenously to ovariectomized (ovx) rats, it inhibits pulsatile pituitary LH secretion. In postmenopausal women estrogen-unopposed LH pulses correlate with hot flushes. Therefore, suppression of pulsatile LH secretion may be beneficial in women suffering from hot flushes. Upon chronic application to ovx rats a BC extract containing 5% Belamcanda at a daily dose of 33 mg or 130 mg of the extract had no effect on uterine weight or on estrogen-regulated uterine gene expression while estrogenic effects in the bone, on bone mineral density of the metaphysis of the tibia could be established. Hence, tectorigenin may have antiosteoporotic effects also in postmenopausal women. Immunohistochemical staining of proliferating cell nuclear antigen--a proliferation marker--in the mammary gland did not indicate a mammotrophic effect of the tectorigenin-containing BC extract at both tested doses. In summary, tectorigenin or the B. chinensis extract containing tectorigenin had a strong hypothalamotropic and osteotropic effect but no effect in the uterus or the mammary gland. Therefore, tectorigenin may be in the future a clinically useful selective estrogen receptor modulator.

Recent cell culture experiments indicated that extracts of Vitex agnus-castus (VAC) may contain yet unidentified phytoestrogens. Estrogenic actions are mediated via estrogen receptors (ER). To investigate whether VAC compounds bind to the currently known isoforms ERalpha or ERss, ligand binding assays (LBA) were performed. Subtype specific ER-LBA revealed a binding of VAC to ERss only. To isolate the ERss-selective compounds, the extract was fractionated by bio-guidance. The flavonoid apigenin was isolated and identified as the most active ERss-selective phytoestrogen in VAC. Other isolated compounds were vitexin and penduletin. These data demonstrate that the phytoestrogens in VAC are ERss-selective.

Silymarin is a widely used standardized mixture of flavonolignans and its major component Silybinin binds to cytosolic estrogen receptors. Here, we demonstrate that this binding is exclusive to the estrogen receptor beta (ERbeta). Treatment of ovariectomized (ovx) rats with silymarin or estradiol (E2) may allow differentiation of biological effects mediated by the ERalpha or ERbeta. E2 inhibited serum LH, cholesterol, LDL and HDL concentrations in the blood and increased gene expression of IGF1, HbEGF and C3 in the uterus, while silymarin was totally ineffective or antagonistic in altering these parameters. Both, E2 and silymarin inhibited expression of uterine ERbeta gene. Hence, in the pituitary, liver (where the lipoproteins are synthesized) and uterus E2 acts primarily via the ERalpha. Exclusive estrogenic effects of silymarin were observed in the metaphysis of the femur (MF), on osteoblast parameters (gene expression of IGF1, TGFbeta1, osteoprotegerin, collagen-1alpha1, osteocalcin (OC)) and on the osteoclast activity marker tartrate resistant acid phosphatase (TRAP) gene expression of adult ovx rats. Our RT-PCR method detects ERbeta gene expression in all organs including developing bones but not in the MF of adult ovx rats. We conclude therefore, that the effects of silymarin in this part of the bone cannot be exerted via the ERalpha because it does not bind to this receptor subtype. Despite the failure to detect ERbeta mRNA in the MF of our animals the possibility exists that ERbeta protein is present and may mediate the effects of silymarin. Another possibility may be that the effect of silymarin and therefore possibly also of E2 in the MF may be mediated via other possibly not yet identified receptors or via an ERbeta splice variant which is not detected by our PCR-method.

Herbal extracts of Hypericum perforatum L. (St. John's wort, SJW) are now successfully competing for status as a standard antidepressant therapy. Because of this, great effort has been devoted to identifying the antidepressive active compounds. In the present study we used the following strategy to evaluate the relative pharmacological importance of various extract components: 1. preparation of an hydroalcoholic SJW extract containing both hyperforin (3.2%) and hypericin (0.15%) (extract A); 2. step by step removal of hyperforin and hypericin led to the following extracts: Extract B, devoid of hyperforin but still containing hypericin (0.14%) and Extract C, free of hypericin and hyperforin but enriched in flavonoids ( approximately 12%). We characterized the in vivo activity profile of all three preparations using the tail suspension test (TST) in mice and the forced swimming test (FST) in rats as screening models. We further investigated the activity of pure hyperforin. Extract B and C (500 mg/kg each) as well as pure hyperforin (8 mg/kg) significantly shortened immobility time in the TST after acute pre-treatment whereas extract A was inactive. In the FST all three extracts decreased immobility time in a dosage of 500 mg/kg after acute as well as after repeated treatment. The present results clearly show that an SJW extract free of hyperforin and hypericin exerts antidepressant activity in behavioral models, supporting our working hypothesis that flavonoids are part of the constituents responsible for the therapeutic efficacy of SJW extracts. We also could show that hyperforin contributes to the beneficial properties of SJW extract, confirming the hypothesis that the crude SJW extract contains several constituents with antidepressant activity.

Extracts of the fruits of chaste tree (Vitex agnus castus = AC) are widely used to treat premenstrual symptoms. Double-blind placebo-controlled studies indicate that one of the most common premenstrual symptoms, i.e. premenstrual mastodynia (mastalgia) is beneficially influenced by an AC extract. In addition, numerous less rigidly controlled studies indicate that AC extracts have also beneficial effects on other psychic and somatic symptoms of the PMS. Premenstrual mastodynia is most likely due to a latent hyperprolactinemia, i.e. patients release more than physiologic amounts of prolactin in response to stressful situations and during deep sleep phases which appear to stimulate the mammary gland. Premenstrually this unphysiological prolactin release is so high that the serum prolactin levels often approach heights which are misinterpreted as prolactinomas. Since AC extracts were shown to have beneficial effects on premenstrual mastodynia serum prolactin levels in such patients were also studied in one double-blind, placebo-controlled clinical study. Serum prolactin levels were indeed reduced in the patients treated with the extract. The search for the prolactin-suppressive principle(s) yielded a number of compounds with dopaminergic properties: they bound to recombinant DA2-receptor protein and suppressed prolactin release from cultivated lactotrophs as well as in animal experiments. The search for the chemical identity of the dopaminergic compounds resulted in isolation of a number of diterpenes of which some clerodadienols were most important for the prolactin-suppressive effects. They were almost identical in their prolactin-suppressive properties than dopamine itself. Hence, it is concluded that dopaminergic compounds present in Vitex agnus castus are clinically the important compounds which improve premenstrual mastodynia and possibly also other symptoms of the premenstrual syndrome.

Ethanolic- and isopropanolic-aqueous extracts of Cimicifuga racemosa are used for the treatment of climacteric complaints. As hot flushes and psychic complaints seem to be special targets for Cimicifuga extracts in clinical studies, these parameters were studied in experimental animals. Hot flush equivalents were measured in castrated rats as a quick increase in peripheral temperature with the aid of a transmitter implanted subcutaneously on the ventral side. The hot flush equivalents proved to respond to estrogen and the antidopaminergic drug veralipride but they were also reduced very effectively by Cimicifuga extract BNO 1055 (which is contained in Klimadynon/Menofem). In addition, an ethanolic-aqueous extract of C. racemosa was studied in the tail suspension test (TST), a behavioural test indicative for antidepressant activity. A significant decrease of the period of immobility was observed after treatment with 30 mg/kg body weight (bw) imipramine or with 50 or 100 mg/kg bw Cimicifuga extract. These findings in pharmacological tests-a reduction of the frequency of hot flush equivalents and hints on antidepressant activity of Cimicifuga extracts-are in good agreement with the therapeutical responses in climacteric women.

Objectives: Extracts of Black cohosh (Cimicifuga racemosa or CR) have been used for the treatment of climacteric complaints since decades. Efficacy, particularly concerning neurovegetative and psychic symptoms, has been proven in clinical trials. As active principle yet unknown substances with selective estrogen receptor modulator (SERM) activity are assumed. Recently, evidence arose that CR may also contain dopaminergic compounds, which may contribute to the therapeutic activity of the extract., Methods: Two subtypes of the estrogen receptor (ERalpha and ERbeta) are known. To examine, whether active substances of CR extract BNO 1055 (which is contained in Klimadynon and Menofem) bind to either of the two estrogen receptors, subtype-specific estrogen receptor ligand-binding assays with recombinant ERalpha or ERbeta were conducted. A ligand-binding assay with recombinant dopamine D(2)-receptor protein was employed to assess possible dopaminergic activity in the CR extract BNO 1055., Results: While a displacement of radiolabeled estradiol from binding sites of a cytosol preparation from procine and human endometrium by CR extract BNO 1055 was shown no such displacement was achieved when either ERalpha or ERbeta protein was used as ligands for tracer. Dopaminergic activity in the CR extract BNO 1055 could be demonstrated with the D(2)-receptor assay. A countercurrent chromatography resulted in a separation of estrogenic and dopaminergic activity in two distinct fractions., Conclusions: It is suggested that not yet identified substances in the CR extract BNO 1055 bind to a yet unknown estrogen-binding site in the endometrium. Also, yet unknown dopaminergic compounds may contribute to the pharmacological profile of CR extract BNO 1055.

Objectives: This review presents findings with clear statements from the literature as well as own results of effects of soy, red clover and their isoflavones as well as of the Cimicifuga racemosa extract BNO 1055. Experimental and clinical effects on climacteric complaints, osteoprotective effects, activity in the urogenital tract, and risks concerning cardiovascular diseases and mammary and endometrial tissue will be compared, also in comparison to classical hormone preparations. The question whether soy and red clover products and/or Cimicifuga racemosa (CR) preparations are endocrine disrupters or may fulfill the criteria of the so-called phyto-SERMs will be discussed., Methods: Review of selected publications since 1980 and summary of unpublished own results of the authors., Results: Experimental and clinical evidences suggest that soy/red clover and their isoflavones do not fulfill the criteria of an ideal SERM. They appear to have mild osteoprotective effects but do not improve climacteric complaints. Furthermore, they seem to stimulate uterine growth and mammary epithelial proliferation. In ovariectomized rats, the CR extract BNO 1055 showed many of the beneficial effects of 17beta-estradiol, including effects in the brain/hypothalamus to reduce serum LH levels, effects in the bone to prevent osteoporosis and estrogenic effects in the urinary bladder. The CR extract BNO 1055 had no uterotrophic effect., Conclusion: If clinical studies confirm these results, the Cimicifuga racemosa preparation BNO 1055 would appear as an ideal SERM and may therefore be an alternative to hormone replacement therapy.

The effect of an extract of Thymus vulgaris on induced spasms was investigated on guinea-pig trachea preparations. By the experimental setup used, effects of ethanol as the vehicle could be differentiated from the activity of the herbal constituents. The extract reversibly and concentration-dependently antagonized the contraction of the Musculus transversus tracheae, provoked by four different spasmogens (BaCl2, carbachol, histamine, prostaglandin F2 alpha). The degree of the antispasmodic activity was dependent on the individual spasmogen with prostaglandin F2 alpha being most efficiently antagonized.

The relative oral bioavailability of beta-escine (CAS 11072-93-8) from a sugar-coated tablet formulation was compared to a reference preparation available in capsule form in 18 healthy, male volunteers over a 48 h period. The study design was randomized, single-blind and cross-over. Both the test and the reference preparation contained 50 mg standardized horse chestnut seed extract; beta-escine was taken as the reference substance. By means of a newly developed, validated radioimmunosorbent assay (RIA), beta-escine in plasma was determined (blind samples) after oral intake of a single dose of each drug formulation. The confidence limits calculated for the AUC, Cmax and Tmax of the test preparation exceed the upper limit of the specified equivalence range of 80%--125%, but do never fall below the lower limit. Therefore, bioin-equivalence cannot be rejected statistically. All the bioavailability data for the test preparation--measured with the newly developed RIA--exceed the corresponding values for the reference preparation. As the rate of absorption of aesculetinic triterpene glycosides is low, the higher bioavailability of the test preparation is desirable from a therapeutical point of view. Since the reference preparation is classified as being clinically effective, the test preparation must also be estimated as being clinically effective. Adverse drug effects were not observed with either the test preparation or the reference preparation.

Pharmacokinetic studies of lonidamine (1-[2,4-dichlorobenzyl]- 1H- indazole- 3-carboxylic acid, Doridamina: CAS 50264-69-2) in humans showed a wide variation of the plasma concentration-time profiles following a single peroral dose of 300 mg (Cmax between 6.5 and 40.9 micrograms/ml, tmax between 0.75 and 5.5 h). In order to investigate single and multiple dose pharmacokinetics after peroral administration of this chemotherapeutic compound, a study was performed involving 12 patients with non-small-cell malignancies of the lungs. Plasma and urinary concentration profiles were analyzed for determination of the pharmacokinetic parameters for lonidamine after single dose administration and in the steady state. In addition, age dependency and the presence of liver induction or inhibition was evaluated. Results indicate that steady state was reached after 2 dosing intervals of 12 h and no changes in liver metabolism or age dependent pharmacokinetics could be revealed after 4 days of multiple dose treatment.

The sulphated glycoprotein SSG 185 is the monomeric precursor of a highly aggregated structural element in the extracellular matrix of the multicellular green alga Volvox carteri. A phosphodiester of arabinose was isolated from a saccharide fragment of SSG 185. The structure of this phosphodiester was investigated by methylation analysis, 13C-NMR, photometric methods and enzymatic assays and identified as D-Araiota-5-phospho-5-D-Araiota. The function of this phosphodiester bridge as a crosslink of different carbohydrate chains in SSG 185 is discussed.