Estrogenic Activity of Derris scandens Stem Extract and its Major Compounds Using MCF-7 Cell Proliferation Assay and Estrogen-Related Gene Expression.

Derris scandens, which contains isoflavones and prenylated derivatives, has analgesic and anti-inflammatory properties and is an ingredient in traditional Thai medicine for perimenopause and menopause. However, the estrogenic activity of D. scandens has not yet been explored. Therefore, this study aimed to examine the estrogenic activity of the stem extract of D. scandens and its isoflavone derivatives. In this study, we conducted a proliferation assay in MCF-7 cells, and used quantitative reverse transcription polymerase chain reaction to assess gene expression. We found that the relative cell proliferation of the compounds (1 µM) was ranked in the following order as compared to 0.1 nM 17ß-estradiol (100%): genistein (97.84%) > derrisisoflavone A (83.17%) > genistein-7-O-[α-rhamnopyranosyl-(1 → 6)-glucopyranoside] (69.55%) > 6,8-diprenylgenistein (51.91%) > lupalbigenin (18.72%). Furthermore, cotreatment with 1 µM lupalbigenin and 0.1 nM 17ß-estradiol was performed, which decreased cell proliferation to 80.38%. In vitro results suggest that lupalbigenin has an estrogen-antagonistic effect. At a dose of 1 µM, genistein had the strongest efficacy in increasing the expression of human estrogen receptor ß by 4.0-fold compared to the control. Furthermore, genistein-7-O-[α-rhamnopyranosyl-(1 → 6)]-ß-glucopyranoside augmented the gene expression of human estrogen receptor α and human estrogen receptor ß by 1.5- and 3.4-fold, respectively. Prenylated derivatives of genistein (derrisisoflavone A, 6,8-diprenylgenistein, and lupalbigenin) significantly suppressed the gene expression of the human androgen receptor. The administration of the crude extract at 10 µg/mL significantly suppressed human androgen receptor (0.6-fold) and transmembrane protease serine 2 (0.1-fold) expression but did not significantly affect human estrogen receptor α and human estrogen receptor ß gene expression. This herbal medicine may be safe for estrogen-exposed breast cancer patients.

Estrogenic Effects of Extracts and Isolated Compounds from Belowground and Aerial Parts of Spartina anglica.

Menopause, caused by decreases in estrogen production, results in symptoms such as facial flushing, vaginal atrophy, and osteoporosis. Although hormone replacement therapy is utilized to treat menopausal symptoms, it is associated with a risk of breast cancer development. We aimed to evaluate the estrogenic activities of Spartina anglica (SA) and its compounds and identify potential candidates for the treatment of estrogen reduction without the risk of breast cancer. We evaluated the estrogenic and anti-proliferative effects of extracts of SA and its compounds in MCF-7 breast cancer cells. We performed an uterotrophic assay using an immature female rat model. Among extracts of SA, belowground part (SA-bg-E50) had potent estrogenic activity. In the immature female rat model, the administration of SA-bg-E50 increased uterine weight compared with that in the normal group. Among the compounds isolated from SA, 1,3-di-O-trans-feruloyl-(-)-quinic acid (1) had significant estrogenic activity and induced phosphorylation at serine residues of estrogen receptor (ER)α. All extracts and compounds from SA did not increase MCF-7 cell proliferation. Compound 1 is expected to act as an ERα ligand and have estrogenic effects, without side effects, such as breast cancer development.

Utilization of Isoflavones in Soybeans for Women with Menopausal Syndrome: An Overview.

Based on their nutrient composition, soybeans and related foods have been considered to be nutritious and healthy for humans. Particularly, the biological activity and subsequent benefits of soy products may be associated with the presence of isoflavone in soybeans. As an alternative treatment for menopause-related symptoms, isoflavone has gained much popularity for postmenopausal women who have concerns related to undergoing hormone replacement therapy. However, current research has still not reached a consensus on the effects of isoflavone on humans. This overview is a summary of the current literature about the processing of soybeans and isoflavone types (daidzein, genistein, and S-equol) and supplements and their extraction and analysis as well as information about the utilization of isoflavones in soybeans. The processes of preparation (cleaning, drying, crushing and dehulling) and extraction of soybeans are implemented to produce refined soy oil, soy lecithin, free fatty acids, glycerol and soybean meal. The remaining components consist of inorganic constituents (minerals) and the minor components of biologically interesting small molecules. Regarding the preventive effects on diseases or cancers, a higher intake of isoflavones is associated with a moderately lower risk of developing coronary heart disease. It may also reduce the risks of breast and colorectal cancer as well as the incidence of breast cancer recurrence. Consumption of isoflavones or soy foods is associated with reduced risks of endometrial and bladder cancer. Regarding the therapeutic effects on menopausal syndrome or other diseases, isoflavones have been found to alleviate vasomotor syndromes even after considering placebo effects, reduce bone loss in the spine and ameliorate hypertension and in vitro glycemic control. They may also alleviate depressive symptoms during pregnancy. On the other hand, isoflavones have not shown definitive effects regarding improving cognition and urogenital symptoms. Because of lacking standardization in the study designs, such as the ingredients and doses of isoflavones and the durations and outcomes of trials, it currently remains difficult to draw overall conclusions for all aspects of isoflavones. These limitations warrant further investigations of isoflavone use for women's health.

Isolation and Characterization of Phenylpropanoid and Lignan Compounds from Peperomia pellucida [L.] Kunth with Estrogenic Activities.

Extracts of Peperomia pellucida [L.] Kunth have previously been demonstrated to have in vivo estrogenic-like effects, thereby functioning as an anti-osteoporotic agent. However, the compounds responsible for these effects have not yet been determined. Therefore, the aim of this study is to isolate and elucidate potential compounds with estrogenic activity. The structures of the isolated compounds were identified using 1D 1H and 13C-NMR and confirmed by 2D FT-NMR. The estrogenic activity was evaluated using the E-SCREEN assay, and a molecular docking study was performed to predict the binding affinity of the isolated compounds to estrogen receptors. In this experiment, we successfully isolated three phenylpropanoids and two lignan derivatives, namely, 6-allyl-5-methoxy-1,3-benzodioxol-4-ol (1), pachypostaudin B (2), pellucidin A (3), dillapiole (4), and apiol (5). Among these compounds, the isolation of 1 and 2 from P. pellucida is reported for the first time in this study. Activity assays clearly showed that the ethyl acetate extract and its fractions, subfractions, and isolated compounds exerted estrogenic activity. Methanol fraction of the ethyl acetate extract produced the highest estrogenic activity, while 1 and 2 had partial agonist activity. Some compounds (derivates of dillapiole and pellucidin A) also had, in addition, anti-estrogenic activity. In the docking study, the estrogenic activities of 1-5 appeared to be mediated by a classical ligand-dependent mechanism as suggested by the binding interaction between the compounds and estrogen receptors; binding occurred on Arg 394 and His 524 of the alpha receptor and Arg 346 and His 475 of the beta receptor. In summary, we reveal that P. pellucida is a promising anti-osteoporotic agent due to its estrogenic activity, and the compounds responsible for this activity were found to be lignan and phenylpropanoid derivatives. The presence of other compounds in either the extract or fraction may contribute to a synergistic effect, as suggested by the higher estrogenic activity of the methanol fraction. Hence, we suggest further research on the osteoporotic activity and safety of the identified compounds, especially regarding their effects on estrogen-responsive organs.

Estrogenic activity of constituents from the rhizomes of Rheum undulatum Linné.

Stilbenes have been reported to be phytoestrogen compounds owing to its structural similarity to the estrogenic agent diethylstilbestrol. To find new stilbene-derivative phytoestrogens, isolation of stilbene-rich R. undulatum was performed and led to identify six new compounds (1-5 and 28), one newly determined absolute configurations compound (27) together with 21 previously reported compounds (6-26). The structures of compounds were determined on the basis of extensive spectroscopic methods including 1D and 2D NMR and CD spectra data. All the isolated compounds were tested for their estrogenic activities in HepG2 cells transiently transfected with ERα, ERß and ERE-reporter plasmid. Among them, stilbene-derivatives, piceatannol 3'-O-ß-d-xylopyranoside (12), cis-rhaponticin (16) and rhapontigenin 3'-O-ß-d-glucopyranoside (17), showed the more potent binding affinity for estrogen receptors than 17ß-estrodiol.

In silico estrogen-like activity and in vivo osteoclastogenesis inhibitory effect of Cicer arietinum extract.

Postmenopausal osteoporosis is a common disorder accompanied with estrogen deficiency in women. Plants containing phytoestrogens and amino acids have been used in the osteoporosis treatment. The present study aims to evaluate the estrogen-like activity of the Cicer arietinum extract (CAE) and its ability to inhibit osteoclastogenesis process. These achieved by investigating the binding of its active phytoestrogens (genistein, daidzein, formononetin and biochanin A) to the estrogen receptors (ER) α and ß of rats and human in silico. In addition, in vivo study on ovariectomized (OVX) rats is performed. For in vivo study, twenty four rats were divided into four groups (n= 6). Group I is the sham control rats which administered distilled water. Groups II, III, and IV are OVX groups which administered distilled water, CAE (500 mg/kg), and alendronate; respectively. The docking study revealed that the phytoestrogens docked into the protein active site with binding energies comparable with that of estrogens (estriol and ß-estradiol) which means the similarity between the estrogenic contents of CAE and the ensogenous ones. Additionally, in vivo study revealed that CAE reverse TRAP5b and RANKL levels that drastically increased in the untreated OVX group. But, it trigger upregulation of OPG, enhance the OPG/RANKL ratio and modulate the bone and uterus alterations of OVX group. Phytoestrogens and the bone-protective amino acids contents of CAE could be responsible for their estrogen-like effect and antiosteoporotic activity. These results concluded that CAE is an attractive candidate for developing a potential therapeutic cheap agent used as an alternative to the synthetic estrogen replacement therapy. Further, in vivo validation is required for its clinical application.

Isolation and identification of three new chromones from the leaves of Pimenta dioica with cytotoxic, oestrogenic and anti-oestrogenic effects.

CONTEXT: Pimenta dioica (L.) Merr. (Myrtaceae) is used in Costa Rican traditional medicine for women's health. Our previous work showed that P. dioica extracts were oestrogenic. OBJECTIVES: This work identifies phytochemicals from P. dioica that are responsible for the plant's oestrogen-like activities. MATERIALS AND METHODS: P. dioica leaves were collected in Costa Rica in 2005. Fractions resulting from chromatographic separation of a methanol extract were tested at 50 µg/mL in a competitive oestrogen receptor-binding assay. Active compounds were isolated by HPLC and identified by NMR and MS. Pure compounds were tested at 1 µM in the oestrogen-responsive SEAP reporter gene assay. The effects on cell viability, cytotoxicity and apoptosis were investigated in breast cancer (MCF-7 and SK-BR3) and gastric cancer (AGS and NCI-N87) cell lines using the ApoTox-Glo and Caspase-Glo assays and qPCR. RESULTS: Quercitrin and three new chromones, including a 2-phenoxychromone, 6,8-di-C-methylcapillarisin (1) were isolated and identified. Compound 1 caused a 6.2-fold increase in SEAP expression at 1 µM (p < 0.05). This activity was blocked by the ER antagonist ICI 182,780. Compound 2 caused a 6.0-fold increase in SEAP, inhibited the growth of MCF-7, AGS and NCI-N87 cells (IC50 54.27, 38.13 and 51.22 µg/mL, respectively), and induced apoptosis via caspase 8 and increased the Bax/Bcl-2 mRNA ratio in MCF-7 cells. Compound 3 was anti-oestrogenic in MCF-7 cells. DISCUSSION AND CONCLUSIONS: Compounds from P. dioica have oestrogenic, anti-oestrogenic and cytotoxic effects that may explain the ethnomedical use of this plant.

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.

Soy isoflavones exert beneficial effects on letrozole-induced rat polycystic ovary syndrome (PCOS) model through anti-androgenic mechanism.

CONTEXT: Soy is the main source of phytoestrogens, which has long been used as traditional food. One major subtype of phytoestrogens includes isoflavones and they are scientifically validated for their beneficial actions on many hormone-dependent conditions. OBJECTIVE: The present study examines the effect of soy isoflavones on letrozole-induced polycystic ovary syndrome (PCOS) rat model. MATERIALS AND METHODS: PCOS was induced in Sprague-Dawley rats with of 1 mg/kg letrozole, p.o. once daily for 21 consecutive days. Soy isoflavones (50 and 100 mg/kg) was administered for 14 days after PCOS induction. Physical parameters (body weight, oestrous cycle determination, ovary and uterus weight) metabolic parameters (oral glucose tolerance test, total cholesterol), steroidal hormone profile (testosterone and 17ß-oestradiol), steroidogenic enzymes (3ß-hydroxy steroid dehydrogenase (HSD) and 17ß-HSD), oxidative stress and histopathology of ovary were studied. RESULTS: Soy isoflavones (100 mg/kg) treatment significantly altered the letrozole-induced PCOS symptoms as observed by decreased body weight gain (p < 0.05), percentage diestrous phase (p < 0.001), testosterone (p < 0.001), 3ß-HSD (p < 0.01) and 17ß-HSD (p < 0.001) enzyme activity and oxidative stress. Histological results reveal that soy isoflavones treatment in PCOS rats resulted in well-developed antral follicles and normal granulosa cell layer in rat ovary. DISCUSSION: Treatment with soy isoflavones exerts beneficial effects in PCOS rats (with decreased aromatase activity) which might be due to their ability to decrease testosterone concentration in the peripheral blood. CONCLUSION: Analysis of physical, biochemical and histological evidences shows that soy isoflavones may be beneficial in PCOS.

Humudifucol and Bioactive Prenylated Polyphenols from Hops (Humulus lupulus cv. "Cascade").

Humulus lupulus (hop plant) has long been used in traditional medicine as a sedative and antimicrobial agent. More recently, attention has been devoted to the phytoestrogenic activity of the plant extracts as well as to the anti-inflammatory and chemopreventive properties of the prenylated chalcones present. In this study, an Italian sample of H. lupulus cv. "Cascade" has been investigated and three new compounds [4-hydroxycolupulone (6), humudifucol (7) and cascadone (8)] have been purified and identified by means of NMR spectroscopy along with four known metabolites. Notably, humudifucol (7) is the first prenylated dimeric phlorotannin discovered in nature. Because structurally related phloroglucinols from natural sources were found previously to inhibit microsomal prostaglandin E2 synthase (mPGES)-1 and 5-lipoxygenase (5-LO), the isolated compounds were evaluated for their bioactivity against these pro-inflammatory target proteins. The prenylated chalcone xanthohumol inhibited both enzymes at low µM concentrations.

Proteome analysis of Pueraria mirifica tubers collected in different seasons.

Pueraria mirifica-derived tuberous powder has been long-term consumed in Thailand as female hormone-replacement traditional remedies. The protein profiles of tubers collected in different seasons were evaluated. Phenol extraction, 2D-PAGE, and mass spectrometry were employed for tuberous proteome analysis. Out of the 322 proteins detected, over 59% were functionally classified as being involved in metabolism. The rest proteins were involved in defense, protein synthesis, cell structure, transportation, stress, storage, and also unidentified function. The proteins were found to be differentially expressed with respect to harvest season. Importantly, chalcone isomerase, isoflavone synthase, cytochrome p450, UDP-glycosyltransferase, and isoflavone reductase, which are all involved in the biosynthesis pathway of bioactive isoflavonoids, were most abundantly expressed in the summer-collected tubers. This is the first report on the proteomic patterns in P. mirifica tubers in relevant with seasonal variation. The study enlights the understanding of variance isoflavonoid production in P. mirifica tubers.

The Stimulatory Effect of Strontium Ions on Phytoestrogens Content in Glycine max (L.) Merr.

The amount of secondary metabolites in plants can be enhanced or reduced by various external factors. In this study, the effect of strontium ions on the production of phytoestrogens in soybeans was investigated. The plants were treated with Hoagland's solution, modified with Sr(2+) with concentrations ranging from 0.5 to 3.0 mM, and were grown for 14 days in hydroponic cultivation. After harvest, soybean plants were separated into roots and shoots, dried, and pulverized. The plant material was extracted with methanol and hydrolyzed. Phytoestrogens were quantified by HPLC. The significant increase in the concentration of the compounds of interest was observed for all tested concentrations of strontium ions when compared to control. Sr(2+) at a concentration of 2 mM was the strongest elicitor, and the amount of phytoestrogens in plant increased ca. 2.70, 1.92, 3.77 and 2.88-fold, for daidzein, coumestrol, genistein and formononetin, respectively. Moreover, no cytotoxic effects were observed in HepG2 liver cell models after treatment with extracts from 2 mM Sr(2+)-stressed soybean plants when compared to extracts from non-stressed plants. Our results indicate that the addition of strontium ions to the culture media may be used to functionalize soybean plants with enhanced phytoestrogen content.

Erythroidine alkaloids: a novel class of phytoestrogens.

Erythrina poeppigiana is a medicinal plant which is widely used in Asia, Latin America, and Africa in traditional remedies for gynecological complications and maladies. In continuation of studies for the discovery of novel phytoestrogens, four erythroidine alkaloids, namely α-erythroidine, ß-erythroidine, and their oxo-derivatives 8-oxo-α-erythroidine and 8-oxo-ß-erythroidine, were isolated and structurally characterized from the methanolic extract of the stem bark of E. poeppigiana. Due to the high amounts of erythroidines in the extract and considering the widespread utilization of Erythrina preparations in traditional medicine, the exploration of their estrogenic properties was performed. The estrogenicity of the isolated erythroidines was assayed in various estrogen receptor-(ER)-dependent test systems, including receptor binding affinity, cell culture based ER-dependent reporter gene assays, and gene expression studies in cultured cells using reverse transcription polymerase chain reaction techniques. α-Erythroidine and ß-erythroidine showed binding affinity values for ERα of 0.015 ± 0.010% and 0.005 ± 0.010%, respectively, whereas only ß-erythroidine bound to ERß (0.006 ± 0.010%). In reporter gene assays, both erythroidines exhibited a significant dose-dependent estrogenic stimulation of ER-dependent reporter gene activity in osteosarcoma cells detectable already at 10 nM. Results were confirmed in the MVLN cells, a bioluminescent variant of MCF-7 breast cancer cells. Further, α-erythroidine and ß-erythroidine both induced the enhanced expression of the specific ERα-dependent genes trefoil factor-1 and serum/glucocorticoid regulated kinase 3 in MCF-7 cells, confirming estrogenicity. Additionally, using molecular docking simulations, a potential mode of binding on ERα, is proposed, supporting the experimental evidences. This is the first time that an estrogenic profile is reported for erythroidine alkaloids, potentially a new class of phytoestrogens.

Analysis of compounds with phytoestrogenic activity in dietary supplements with use of HPTLC-densitometry method.

Soy (Glycine max L., Fabaceae) and soy products are becoming more popular because of their low toxicity and therapeutic effects. Soy possesses antioxidant, anti-inflammatory and anti-allergic properties, however, the most important is its estrogenic activity associated with occurrence of phytoestrogens. Isoflavones with phytoestrogenic effects were determined in four commercially available soya formulations. Analyses were performed with the use of high performance thin-layer chromatography (HPTLC) combined with densitometry. The compounds were extracted, hydrolyzed in order to obtain aglycone forms and separated on HPTLC silica gel 60 F254 plates with the use of mobile phase consisting of chloroform--ethyl acetate--formic acid 4:6:0.1 (v/v/v). After drying, the spots on the plates were determined in absorbance/reflectance mode at a wavelength of 260 nm using a computer-controlled densitometer Desaga CD 60.

Drynaria fortunei-derived total flavonoid fraction and isolated compounds exert oestrogen-like protective effects in bone.

Drynaria fortunei (Kunze) J. Sm. (DF), a Chinese herb commonly used for the treatment of bone fracture, was previously shown to exert anabolic effects on bone. However, its active ingredients as well as the mechanisms of action are far from clear. The present study aimed to characterise the bone anabolic effects of DF flavonoid fraction (DFTF) in ovariectomised (OVX) mice and to determine if DFTF and its isolated compounds exert oestrogen-like effects in rat osteoblast-like UMR-106 cells. Young OVX C57/BL6J mice were treated orally with DFTF (0·087, 0·173 or 0·346 mg/g per d), 17b-oestradiol (2 mg/g per d) or its vehicle for 6 weeks. Serum and urine samples were collected for biochemical marker analysis. Bones were collected for computed tomography analysis. UMR-106 cells were treated with DFTF and isolated compounds naringin, (2S)-5,7,30,50-tetrahydroxy-flavonone 7-O-neohesperidoside (compound 1) and 5,7-dihydroxychromone 7-O-neohesperidoside (compound 2). DFTF exerted dose-dependent effects in improving bone mineral densities as well as bone strength at the femur, tibia and lumbar spine L1 in OVX mice. DFTF and the three isolated compounds stimulated osteoblastic cell proliferation and alkaline phosphatase activities in a dose-dependent manner. In addition, they stimulated the ratio of osteoprotegrin and receptor-activator NF-kB ligand mRNA expression, suggesting their involvement in inhibiting osteoclastogenesis. These stimulatory effects on osteoblastic functions were abolished in the presence of oestrogen receptor (ER) antagonist, ICI 182780. The present results suggested that DFTF is effective in protecting against OVX-induced bone loss in mice, and its actions in regulating osteoblastic activities appear to be mediated by ER.

Assessment of the estrogenic activities of chickpea (Cicer arietinum L) sprout isoflavone extract in ovariectomized rats.

AIM: Chickpea (Cicer arietinum L) is a traditional Uighur herb. In this study we investigated the estrogenic activities of the isoflavones extracted from chickpea sprouts (ICS) in ovariectomized rats. METHODS: Ten-week-old virgin Sprague-Dawley female rats were ovariectomized (OVX). The rats were administered via intragastric gavage 3 different doses of ICS (20, 50, or 100 mg·kg(-1)·d(-1)) for 5 weeks. Their uterine weight and serum levels of 17ß-estradiol (E2), follicle stimulating hormone (FSH) and luteinizing hormone (LH) were measured. The epithelial height, number of glands in the uterus, and number of osteoclasts in the femur were histologically quantified, and the expression of proliferating cell nuclear antigen (PCNA) was assessed immunohistochemically. Bone structural parameters, including bone mineral density (BMD), bone volume/tissue volume (BV/TV), trabecular thickness (Tb.Th) and trabecular separation (Tb.Sp) were measured using Micro-CT scanning. RESULTS: Treatments of OVX rats with ICS (50 or 100 mg·kg(-1)·d(-1)) produced significant estrogenic effects on the uteruses, including the increases in uterine weight, epithelial height and gland number, as well as in the expression of the cell proliferation marker PCNA. The treatments changed the secretory profile of ovarian hormones and pituitary gonadotropins: serum E2 level was significantly increased, while serum LH and FSH levels were decreased compared with the vehicle-treated OVX rats. Furthermore, the treatments significantly attenuated the bone loss, increased BMD, BV/TV and Tb.Th and decreased Tb.Sp and the number of osteoclasts. Treatment of OVX rats with the positive control drug E2 (0.25 mg·kg(-1)·d(-1)) produced similar, but more prominent effects. CONCLUSION: ICS exhibits moderate estrogenic activities as compared to E2 in ovariectomized rats, suggesting the potential use of ICS for the treatment of menopausal symptoms and osteoporosis caused by estrogen deficiency.

Nitric oxide signalling is involved in diarylheptanoid-induced increases in femoral arterial blood flow in ovariectomized rats.

The mechanisms by which the hexane extract of Curcuma comosa increases femoral blood flow (FBF) in ovariectomized rats are not known. Thus, the aim of the present study was to investigate the acute effects and modes of action of the diarylheptanoid (3R)-1,7-diphenyl-(4E,6E)-4,6-heptadien-3-ol (D3), a phyto-oestrogen isolated from C. comosa, on FBF in ovariectomized rats. On Day 7 after ovariectomy, rats were injected once intra-arterially with D3 (100, 200, 400 and 800 µg/kg), 17ß-oestradiol (E2; 1, 2, 4 and 8 µg/kg) or vehicle. In some experiments, rats were injected with N(G)-nitro-L-arginine methyl ester (L-NAME; 10 mg/kg) 120 min after 800 µg/kg D3 or 4 µg/kg E2. In other experiments, rats were injected with 10 mg/kg L-NAME, 900 µg/kg 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) or 900 µg/kg ICI 182 780 30 min prior to the injection of 800 µg/kg D3 or 4 µg/kg E2. Mean arterial blood pressure (mABP) and FBF were recorded using a pressure transducer and a laser Doppler flow meter, respectively. Both D3 and E2 dose-dependently increased FBF without changing mABP or heart rate. The EC(50) at 120 min for D3 and E2 was 195.8 and 1.8 µg/kg, respectively. In addition, D3 and E2 dose-dependently decreased femoral vascular resistance (FVR). The EC(50) of D3 was about 100-fold greater than that of E2. The effects of D3 and E2 on FBF and FVR were diminished by intravenous injection of 10 mg/kg l-NAME. Furthermore, 30 min pretreatment with L-NAME (10 mg/kg), ODQ (900 µg/kg) or ICI 182 780 (900 µg/kg) blocked the effects of D3 and E2 on FBF and FVR. The results of the present study suggest that the phyto-oestrogen D3 increases FBF in ovariectomized rats via oestrogen receptor and nitric oxide-guanylyl cyclase signalling, which, in turn, relaxes femoral vascular resistance.

Estrogenic and mutagenic activities of Crotalaria pallida measured by recombinant yeast assay and Ames test.

BACKGROUND: Crotalaria pallida Ailton is a plant belonging to the Fabaceae family, popularly known as "rattle or rattlesnake" and used in traditional medicine to treat swelling of the joints and as a vermifuge. Previous pharmacological studies have also reported anti-inflammatory, antimicrobial and antifungal activities. Nevertheless, scientific information regarding this species is scarce, and there are no reports related to its possible estrogenic and mutagenic effects. Thus, the purpose of the present study was to investigate the estrogenic potential of C. pallida leaves by means of the Recombinant Yeast Assay (RYA), seeking an alternative for estrogen replacement therapy during menopause; and to reflect on the safe use of natural products to assess the mutagenic activity of the crude extract from C. pallida leaves, the dichloromethane fraction and stigmasterol by means of the Ames test. METHODS: The recombinant yeast assay with the strain BY4741 of Saccharomyces cerevisiae, was performed with the ethanolic extract, dichloromethane fraction and stigmasterol isolated from the leaves of C. pallida. Mutagenic activity was evaluated by the Salmonella/microsome assay (Ames test), using the Salmonella typhimurium tester strains TA100, TA98, TA97 and TA102, with (+S9) and without (-S9) metabolization, by the preincubation method. RESULTS: All samples showed estrogenic activity, mainly stigmasterol. The ethanolic extract from C. pallida leaves showed mutagenic activity in the TA98 strain (-S9), whereas dichloromethane fraction and stigmasterol were found devoid of activity. CONCLUSION: Considering the excellent estrogenic activity performed by stigmasterol in the RYA associated with the absence of mutagenic activity when evaluated by the Ames test, stigmasterol becomes a strong candidate to be used in hormone replacement therapy during menopause.

2,3-cis-2R,3R-(-)-epiafzelechin-3-O-p-coumarate, a novel flavan-3-ol isolated from Fallopia convolvulus seed, is an estrogen receptor agonist in human cell lines.

BACKGROUND: The plant genus Fallopia is well-known in Chinese traditional medicine and includes many species that contain bioactive compounds, namely phytoestrogens. Consumption of phytoestrogens may be linked to decreased incidence of breast and prostate cancers therefore discovery of novel phytoestrogens and novel sources of phytoestrogens is of interest. Although phytoestrogen content has been analyzed in the rhizomes of various Fallopia sp., seeds of a Fallopia sp. have never been examined for phytoestrogen presence. METHODS: Analytical chemistry techniques were used with guidance from an in vitro estrogen receptor bioassay (a stably transfected human ovarian carcinoma cell line) to isolate and identify estrogenic components from seeds of Fallopia convolvulus. A transiently transfected human breast carcinoma cell line was used to characterize the biological activity of the isolated compounds on estrogen receptors (ER) α and ß. RESULTS: Two compounds, emodin and the novel flavan-3-ol, (-)-epiafzelechin-3-O-p-coumarate (rhodoeosein), were identified to be responsible for estrogenic activity of F. convolvulus seed extract. Absolute stereochemistry of rhodoeosein was determined by 1 and 2D NMR, optical rotation and circular dichroism. Emodin was identified by HPLC/DAD, LC/MS/MS, and FT/ICR-MS. When characterizing the ER specificity in biological activity of rhodoeosein and emodin, rhodoeosein was able to exhibit a four-fold greater relative estrogenic potency (REP) in breast cells transiently-transfected with ERß as compared to those transfected with ERα, and emodin exhibited a six-fold greater REP in ERß-transfected breast cells. Cell type-specific differences were observed with rhodoeosein but not emodin; rhodoeosein produced superinduction of reporter gene activity in the human ovarian cell line (> 400% of maximum estradiol [E2] induction) but not in the breast cell line. CONCLUSION: This study is the first to characterize the novel flavan-3-ol compound, rhodoeosein, and its ability to induce estrogenic activity in human cell lines. Rhodoeosein and emodin may have potential therapeutic applications as natural products activating ERß, and further characterization of rhodoeosein is necessary to evaluate its selectivity as a cell type-specific ER agonist.

Improvements of insulin resistance in ovariectomized rats by a novel phytoestrogen from Curcuma comosa Roxb.

BACKGROUND: Curcuma comosa Roxb. (C. comosa) is an indigenous medicinal herb that has been used in Thailand as a dietary supplement to relieve postmenopausal symptoms. Recently, a novel phytoestrogen, (3R)-1,7-diphenyl-(4E,6E)-4,6-heptadien-3-ol or compound 049, has been isolated and no study thus far has investigated the role of C. comosa in preventing metabolic alterations occurring in estrogen-deprived state. The present study investigated the long-term effects (12 weeks) of C. comosa hexane extract and compound 049 on insulin resistance in prolonged estrogen-deprived rats. METHODS: Female Sprague-Dawley rats were ovariectomized (OVX) and treated with C. comosa hexane extract (125 mg, 250 mg, or 500 mg/kg body weight (BW)) and compound 049 (50 mg/kg BW) intraperitoneally three times per week for 12 weeks. Body weight, food intake, visceral fat weight, uterine weight, serum lipid profile, glucose tolerance, insulin action on skeletal muscle glucose transport activity, and GLUT-4 protein expression were determined. RESULTS: Prolonged ovariectomy resulted in dyslipidemia, impaired glucose tolerance and insulin-stimulated skeletal muscle glucose transport, as compared to SHAM. Treatment with C. comosa hexane extract and compound 049, three times per week for 12 weeks, markedly reduced serum total cholesterol and low-density lipoprotein levels, improved insulin sensitivity and partially restored uterine weights in ovariectomized rats. In addition, compound 049 or high doses of C. comosa hexane extract enhanced insulin-mediated glucose uptake in skeletal muscle and increased muscle GLUT-4 protein levels. CONCLUSIONS: Treatment with C. comosa and its diarylheptanoid derivative improved glucose and lipid metabolism in estrogen-deprived rats, supporting the traditional use of this natural phytoestrogen as a strategy for relieving insulin resistance and its related metabolic defects in postmenopausal women.