Using Both Chemical and Biological Fingerprints for the Quality Study of Estrogenic Licorice (Glycyrrhiza uralensis)

ABSTRACT: The quality of phytoestrogenic licorice was studied by using both chemical and biological fingerprints. A recombinant yeast strain that consists of an estrogen responsive element linked with a reporter gene ( ADE2 ) and a transformed human estrogen receptor–containing plasmid was used for screening and evaluation of estrogenic activity in licorice. Several estrogen-like components in licorice were screened, and licoisoflavone B and formononetin were identified. Licorice extracted with 70% ethanol showed 5 different patterns of chemical fingerprints (LR-A, LR-E, LR-F, LR-H, LR-K), as identified by chromatographic analysis. Among these, LR-E exhibited the strongest estrogenic activity, whereas LR-A, LR-F, and LR-H were in the middle, and LR-K had the weakest activity.     

Comparison of in vivo and in vitro reporter gene assays for short-term screening of estrogenic activity

Functional in vitro and in vivo reporter gene assays have recently been developed for the rapid determination of exposure to (xeno)estrogens. The in vitro estrogen receptor (ER)-mediated chemically activated luciferase gene expression (ER-CALUX) assay uses T47D human breast cancer cells stably transfected with an ER-mediated luciferase gene construct. In the in vivo assay, transgenic zebrafish are used in which the same luciferase construct has been stably introduced. In both assays, luciferase reporter gene activity can be easily quantified following short-term exposure to chemicals activating endogenous estrogen receptors. The objective of this study was to compare responses by known (xeno)estrogenic compounds in both assays. Exposure to the (xeno)estrogens estradiol (E2), estrone, ethynylestradiol (EE2), o,p'-DDT, nonylphenol (NP), and di(2-ethylhexyl)phthalate (DEHP) revealed that EE2 was the most potent (xeno)estrogen tested and was 100 times more potent than E2 in the transgenic zebrafish assay, whereas in the in vitro ER-CALUX assay, EE2 and E2 were equipotent Although the xenoestrogens o,p'-DDT and NP were full estrogen agonists in the in vitro ER-CALUX assay, only o,p'-DDT demonstrated weak dose-related estrogenic activity in vivo. To determine if differences in reporter gene activity may be explained by differential affinity of (xeno)estrogens to human and zebrafish ERs, full-length sequences of the zebrafish ER subtypes alpha, beta, and gamma were cloned, and transactivation by (xeno)estrogens was compared to human ERalpha and ERbeta. Using transiently transfected recombinant ER and reporter gene constructs, EE2 also showed relatively potent activation of zebrafish ERalpha and ERbeta compared to human ERalpha and ERbeta. Zebrafish ERbeta and ERgamma showed higher transactivation by (xeno)estrogens relative to E2 than human ERbeta.     

Involvement of the oestrogenic receptors in superior mesenteric ganglion on the ovarian steroidogenesis in rat

Oestradiol (E(2)) is a key hormone in the regulation of reproductive processes. The aims of this work were a) to examine the distributions of oestrogen receptor α (ERα) and ERβ in the neurons of the superior mesenteric ganglion (SMG) in the oestrus stage by immunohistochemistry, b) to demonstrate whether E(2) in the SMG modifies progesterone (P(4)), androstenedione (A(2)) and nitrite release in the ovarian compartment on oestrus day and c) to demonstrate whether E(2) in the ganglion modifies the activity and gene expression in the ovary of the steroidogenic enzymes 3β-hydroxysteroid dehydrogenase (3β-HSD) and 20α-hydroxysteroid dehydrogenase (20α-HSD). The ex vivo SMG-ovarian nervous plexus-ovary system was used. E(2), tamoxifen (Txf) and E(2) plus Txf were added in the ganglion to measure ovarian P(4) release, while E(2) alone was added to measure ovarian A(2) and nitrites release. Immunohistochemistry revealed cytoplasmic ERα immunoreactivity only in the neural somas in the SMG. E(2) increased ovarian P(4) and A(2) release at 15, 30 and 60?min but decreased nitrites. The activity and gene expression of 3β-HSD increased, while the activity and gene expression of 20α-HSD did not show changes with respect to the control. Txf in the ganglion diminished P(4) release only at 60?min. E(2) plus Txf in the ganglion reverted the effect of E(2) alone and the inhibitory effect of Txf. The results of this study demonstrate that ERα activation in the SMG has an impact on ovarian steroidogenesis in rats, thus providing evidence for the critical role of peripheral system neurons in the control of ovarian functions under normal and pathological conditions.     

Detection of estrogenic activity in herbal teas by in vitro reporter assays

Herbal teas have become popular as alternatives to caffeinated beverages during past two decades. However, toxicological studies of herbal teas have been limited and the safety of herbal teas thus remains unknown. We focused on the estrogenic activities of herbal teas since some of their ingredients are similar to those used in herbal remedies for menopause relief and therefore contain phytoestrogens. To investigate the potential estrogenic activity of extracts prepared from herbal tea mixtures commercially available and to provide useful information for the safety assessment of those products, we initially screened the estrogenic activity in extracts of 15 different herbal teas by an assay using recombinant yeast cells expressing the human estrogen receptor (YES). A distinct estrogenic activity was thus detected in the ethanolic extracts from four herbal tea mixtures. Licorice root was specified as a ingredient responsible for the estrogenic activity in those extracts. In contrast, the aqueous extracts of all herbal tea mixtures we tested exhibited distinct estrogenic activity in YES, thus suggesting the existence of various ingredients that contain estrogenic constituents extractable with water. Among them, the extract of peppermint tea exhibited the highest estrogenic activity. The estrogenic activity in extracts of herbal tea mixtures and specified ingredients were thereafter confirmed by a reporter assay system using transiently transfected HEK293 cells.     

Screening of Korean edible wild plants for estrogenic activity using MCF-7 cell proliferation assay

This present study was performed to investigate estrogenic activity of Korean edible wild plants for alternative of estrogen replacement therapy in postmenopausal women. When the estrogenic activity was measured by estrogen induced proliferation of MCF-7 cells, of investigated 29 extracts, 3 samples of kudzu vine (Pueraria thunbergiana), Cardamine leucantha, and David Vetchling (Lathyrus davidii) exhibited a significant proliferation of above 20% at concentration of 100 μg/mL. This stimulation of MCF-7 cell proliferation could be completely blocked by addition of estrogen antagonist ICI 182,780 (100 nM), indicating estrogen receptor-dependent mechanism of these estrogenic effects on MCF-7 cell proliferation.     

基于动物模型的大豆球蛋白诱导肠黏膜过敏反应机理研究

目的:探究大豆球蛋白诱导小鼠肠黏膜过敏反应的调控机理。方法:以大豆球蛋白和β-伴球蛋白为试验材料,Balb/c小鼠为受试动物,采用连续灌胃的方式建立肠道过敏动物模型。研究了小鼠小肠绒毛T、B淋巴细胞的数量、肠细胞凋亡率和固有层IgA+浆细胞的数量及小肠液中sIgA的含量。结果:与对照组相比,大豆球蛋白组小肠绒毛固有层淋巴细胞CD3^+、CD4^+数量显著高于对照组(P<0.01);大豆β-伴球蛋白组小肠绒毛固有层及肠上皮细胞中CD3^+、CD8^+、CD4^+及整合素α4β7的数量均显著高于对照。高倍镜下观察显示试验组小鼠小肠固有层中可见大量浆细胞及淋巴细胞等炎性细胞浸润;试验组小鼠小肠固有层IgA+浆细胞呈现明显的阳性染色。结论:大豆球蛋白可以介导细胞免疫为主的过敏反应发生,肠黏膜上皮细胞凋亡亢进是造成肠道通透性加强,进而破坏肠黏膜屏障功能的重要原因。     

Detection of multiple hormonal activities in wastewater effluents and surface water, using a panel of steroid receptor CALUX bioassays

It is generally known that there are compounds present in the aquatic environment that can disturb endocrine processes, for example via interaction with the endogenous hormone receptors. Most research so far has focused on compounds that bind to the estrogen and/or androgen receptor, but ligands for other hormone receptors might also be present. In this study, a newly completed panel of human cell derived CALUX reporter gene bioassays was utilized to test water extracts for estrogen (ER), as well as androgen (AR), progesterone (PR), and glucocorticoid (GR) receptor mediated transactivation activity. Effluents from industry, hospital, and municipal sewage treatment plants, as well as tap water and different sources of surface water were tested. The CALUX reporter gene panel showed high sensitivity and specificity to known agonists, enabling discrimination between different receptor based endocrine responses present in the aquatic environment. Our results clearly showed the presence of agonistic activity on the ER, as well as on the AR, PR, and GR in the raw and wastewater and surface water extracts. However, no hormone receptor-mediated transactivation was detected in the drinking water or in the blank water. The levels of estrogenic activity were 0.2-0.5 ng E2-equiv/L for surface water and 0.4-1.0 ng E2-equiv/L for municipal effluents, which was consistent with previous studies. Surprisingly, the other hormonal activities were found to be present in similar or much higher levels. Most notably, glucocorticoid-like activity was detected in all samples, at surprisingly high levels ranging from 0.39-1.3 ng Dex-equiv/L in surface water and 11-243 ng Dex-equiv/L in effluents. When regarding the fact that dexamethasone in the GR CALUX bioassay is a factor 12 more potent than the natural hormone cortisol, results expressed as cortisol equivalents would range up to 2900 ng cortisol equiv/L. Further studies are needed to establish the identity of the active compounds and to understand the significance of the level of activities with regard to human and ecotoxicological risks.     

Oxidation products of stigmasterol interfere with the action of the female sex hormone 17beta-estradiol in cultured human breast and endometrium cell lines

Phytosterols are constituents of plant membranes and are thus contained in low concentrations in vegetable products as well as at high concentrations in functional food designed to reduce serum cholesterol levels. Similar to ChOL, phytosterols are oxidized chemically in food and by biotransformation in vivo. Although oxyphytosterols have been detected in the serum of healthy human subjects, little is known of their biological activity. Therefore, the estrogenic and antiestrogenic activities of a mixture of six oxidation products of stigmasterol (oxy-StOL) were determined at the following endpoints: (i) the affinity to isolated human estrogen receptors (ER), (ii) the basal and 17beta-estradiol (E2)-induced expression of the alkaline phosphatase (AlP) in human endometrial adenocarcinoma (Ishikawa) cells, and (iii) the basal and E2-induced proliferation of human breast adenocarcinoma (MCF-7) cells. Oxy-StOL was able to replace E2 from human ERalpha and ERbeta and induced a weak estrogenic response in MCF-7 cells. Moreover, the E2-induced activity of the AlP in Ishikawa cells as well as the E2-induced proliferation of MCF-7 cells were decreased at noncytotoxic concentrations (up to 10 microM), indicating that at least one component of oxy-StOL represents an estrogen-active compound which might interfere with endogenous estrogens.     

Indolylfuran,a potent aryl hydrocarbon receptor agonist from sauerkraut,interacts with the oestrogen pathway

Numerous ligands of the aryl hydrocarbon receptor (AhR) have been found in plants, especially edible plants, such as cruciferous vegetables, which exert beneficent health effects. A potent activator of the AhR was found in sauerkraut juice. The isolated active ingredient was identified as the novel AhR ligand, (1-(2-furanyl)2-(3-indolyl)ethanone, common name indolylfuran. The isolated and the synthesised compound exerted similar potencies; their EC₅₀-values in an AhR transactivation assay were 160 and 123 nM, respectively. Our in vivo studies confirm and enlighten basic interactions between the AhR and oestrogen receptors (ERs). Further anti-oestrogenic effects of sauerkraut extract were shown. Indolylfuran regulates ER α and β expression, most likely via the AhR pathway, since indolylfuran had no effect on uterus weight and did not agonise ERα. Sauerkraut and indolylfuran may have potential for the prevention or treatment of diseases through modulation of AhR regulation and, indirectly, the ER pathway.     

Quercetin‐induced apoptotic cascade in cancer cells: Antioxidant versus estrogen receptor α‐dependent mechanisms

The flavonol quercetin, especially abundant in apple, wine, and onions, is reported to have anti‐proliferative effects in many cancer cell lines. Antioxidant or pro‐oxidant activities and kinase inhibition have been proposed as molecular mechanisms for these effects. In addition, an estrogenic activity has been observed but, at the present, it is poorly understood whether this latter activity plays a role in the quercetin‐induced anti‐proliferative effects. Here, we studied the molecular mechanisms of quercetin committed to the generation of an apoptotic cascade in cancer cells devoid or containing transfected estrogen receptor α (ERα; i.e., human cervix epitheloid carcinoma HeLa cells). Although none of tested quercetin concentrations increase reactive oxygen species (ROS) generation in HeLa cells, quercetin stimulation prevents the H₂O₂‐induced ROS production both in the presence and in the absence of ERα. However, this flavonoid induces the activation of p38/MAPK, leading to the pro‐apoptotic caspase‐3 activation and to the poly(ADP‐ribose) polymerase cleavage only in the presence of ERα. Notably, no down‐regulation of survival kinases (i.e., AKT and ERK) was reported. Taken together, these findings suggest that quercetin results in HeLa cell death through an ERα‐dependent mechanism involving caspase‐ and p38 kinase activation. These findings indicate new potential chemopreventive actions of flavonoids on cancer growth.     

Bioactive compounds and antioxidant activity from harvest to edible ripeness of avocado cv. Hass (Persea americana) throughout the harvest seasons

The influence of regular air cold storage (7 °C and 85 ± 5% RH) followed by ripening at shelf-life conditions (19–21 °C and 65 ± 5% RH), on bioactive compounds of Hass avocados was investigated. Results showed that the content of mannoheptulose and perseitol decreased significantly already during cold storage and ripening period. The fatty acid profile and contents of tocopherols (α- and β-tocopherol) and phytosterols (β-sitosterol, stigmasterol, campesterol) remained unchanged from day 0 to edible ripeness. Total phenolics, hydrophilic and lipophilic antioxidant capacity remained unchanged during cold storage and increased during the ripening period. At edible ripeness, significant amounts of phenolic acids, p-coumaric and caffeic and their derivatives were synthesised. Our results demonstrated that regular air cold storage for up to 37 days followed by ripening at shelf-life conditions enhances the phenolic compounds and mainly the hydrophilic antioxidant capacity without affecting the remaining bioactive compounds in Hass avocado.     

基于电子鼻、HS-GC-IMS和HS-SPME-GC-MS 对5种食用植物油挥发性风味成分分析

采用电子鼻、顶空气相色谱-离子迁移谱(HS-GC-IMS)和顶空固相微萃取-气相色谱-质谱联用(HS-SPME-GC-MS)法对5种食用植物油(花生油、大豆油、玉米油、油茶籽油、棕榈油)中挥发性成分进行分析,并采用相对气味活度法确定关键风味物质。结果表明:电子鼻检测发现花生油与油茶籽油气味轮廓相似,但花生油气味浓度大于油茶籽油,大豆油、玉米油和棕榈油整体气味差异相对较小且浓度较低; HS-GC-IMS共检出5种食用植物油56种化合物(其中定性24种),定性的共有风味化合物为戊醛(单体)、己醛(单体)、庚醛、1-戊醇(单体)、1-丙醇、2-丁酮(单体)、辛醛和丁醛(单体),并得到5种食用植物油差异图谱; HS-SPME-GC-MS鉴定出5种食用植物油86种化合物,共有关键风味化合物为己醛、壬醛和庚醛,花生油、大豆油和棕榈油特有关键风味化合物分别为吡嗪类化合物、(E,E)-2,4-庚二烯醛和6-甲基-5-庚烯-2-酮。     

Kudoa septempunctata was recognised by Toll-like receptor 2 produced by a RAW 264 macrophage-like cell line

Kudoa septempunctata is a myxosporean parasite that infects Paralichthys olivaceus (olive flounder). Previously, we reported that the consumption of raw P. olivaceus meat containing a high concentration of K. septempunctata spores induces transient but severe diarrhoea and emesis. In this study, we investigated the cytokine production of mouse macrophage-like RAW 264 cells stimulated with K. septempunctata. When the RAW 264 cells were incubated with the spores of K. septempunctata for 24 h, they secreted tumour necrosis factor α (TNF-α) and several chemokines, such as IP-10, MIP-1β, and MIP-2. The secretion of TNF-α was induced in a dose-dependent manner in a bioassay using L929 cells and mouse TNF-α-specific enzyme-linked immunosorbent assay (ELISA). To identify the macrophage receptor of K. septempunctata, activation of HEK 293 cells expressing one of the Toll-like receptors (TLR) was measured using an NF-κB-dependent reporter assay. TLR2-expressing HEK 293 cells were strongly activated following stimulation with the spores. These results suggested that K. septempunctata was recognised by TLR2 on the macrophages, which were then activated and produced TNF-α.     

Fermentation by Amylolytic Lactic Acid Bacteria and Consequences for Starch Digestibility of Plantain, Breadfruit, and Sweet Potato Flours

The potential of tropical starchy plants such as plantain (Musa paradisiaca), breadfruit (Artocarpus communis), and sweet potato (Ipomoea batatas) for the development of new fermented foods was investigated by exploiting the capacity of some lactic acid bacteria to hydrolyze starch. The amylolytic lactic acid bacteria (ALAB) Lactobacillus plantarum A6 and Lactobacillus fermentum Ogi E1 were able to change the consistency of thick sticky gelatinized slurries of these starchy fruits and tubers into semiliquid to liquid products. Consequently, a decrease in apparent viscosity and an increase in Bostwick flow were observed. These changes and the production of maltooligosaccharides confirmed starch hydrolysis. Sucrose in sweet potato was not fermented by strain A6 and poorly fermented by strain Ogi E1, suggesting possible inhibition of sucrose fermentation. In all 3 starchy plants, rapidly digestible starch (RDS) was higher than slowly digestible starch (SDS) and resistant starch (RS) represented between 17% and 30% dry matter (DM). The digestibility of plantain was not affected by fermentation, whereas the RDS content of breadfruit and sweet potato decreased and the RS content increased after fermentation. Practical Application: The characteristics resulting from different combinations of gluten free starchy plants (plantain, breadfruit, sweet potato) and amylolytic lactic acid bacteria (ALAB) offer opportunities to develop new functional fermented beverages, mainly for breadfruit and sweet potato, after further investigation of their formulation, sensory attributes, nutritional, and prebiotic characteristics.     

Isolation and identification of ent-kaurane-type diterpenoids from Rabdosia serra (MAXIM.) HARA leaf and their inhibitory activities against HepG-2, MCF-7, and HL-60 cell lines

A phytochemical investigation was conducted on Rabdosia serra leaf in this work. A new ent-kaurane-type diterpenoid named 6β,14α-dihydroxy-1α,7β-diacetoxy-7α,20-epoxy-ent-kaur-16-en-15-one, together with 6 known compounds were identified, including parvifolin G, effusanin E, lasiodin, nodosin, β-sitosterol, and stigmasterol. It was the first time that parvifolin G and effusanin E were found in R. serra. The assay of inhibition activity against HepG-2, MCF-7, and HL-60 cell lines indicated that 10 compounds (including rosmarinic acid, methyl rosmarinate and pedalitin which were isolated previously), except parvifolin G and stigmasterol, exhibited cytotoxicity against the tested tumour cells. The tumour inhibitory effects of ent-kaurane-type diterpenoids (except parvifolin G) were more effective than those of sterols and phenolics. Both 6β,14α-dihydroxy-1α,7β-diacetoxy-7α,20-epoxy-ent-kaur-16-en-15-one and lasiodin (IC₅₀ < 5 μM) displayed strong cytotoxicity against the tested tumour cells, indicating the potential for new chemotherapeutic drugs.     

Functional associations between two estrogen receptors, environmental estrogens, and sexual disruption in the roach (Rutilus rutilus)

Wild male roach (Rutilus rutilus) living in U.K. rivers contaminated with estrogenic effluents from wastewater treatment works show feminized responses and have a reduced reproductive capability, but the chemical causation of sexual disruption in the roach has not been established. Feminized responses were induced in male roach exposed to environmentally relevant concentrations of the pharmaceutical estrogen 17alpha-ethinylestradiol, EE2 (up to 4 ng/ L), during early life (from fertilization to 84 days posthatch, dph), and these effects were signaled by altered patterns of expression of two cloned roach estrogen receptor (ER) subtypes, ERalpha. and ERbeta, in the brain and gonad/ liver. Transactivation assays were developed for both roach ER subtypes and the estrogenic potencies of steroidal estrogens differed markedly at the different ER subtypes. EE2 was by far the most potent chemical, and estrone (E1, the most prevalent environmental steroid in wastewater discharges) was equipotent with estradiol (E2) in activating the ERs. Comparison of the EC50 values for the compounds tested showed that ERbeta was 3-21-fold more sensitive to natural steroidal estrogens and 54-fold more sensitive to EE2 as compared to ERalpha. These findings add substantial support to the hypothesis that steroidal estrogens play a significant role in the induction of intersex in roach populations in U.K. rivers and that the molecular approach described could be usefully applied to understand interspecies sensitivity to xenoestrogens.     

Construction of a reporter yeast strain to detect estrogen receptor signaling through aryl hydrocarbon receptor activation

The activation mechanism of estrogen receptor (ER) signaling by association with the aryl hydrocarbon receptor (AhR) was elucidated recently (Ohtake, et al., Nature 2003, 423, 545). In the present study, we established a reporter yeast strain to evaluate this ER signaling by association with the activated AhR. This yeast strain expresses human ER and AhR, and has a reporter plasmid with estrogen response elements. With this yeast strain we assayed ER activation by various AhR ligands, i.e., 2,3,7,8-tetrachlorodibenzo-p-dioxin, benzo[a]pyrene, 3-methylcholanthrene, beta-naphthoflavone, and indirubin. All these ligands induced ER activation dose-dependently and their EC50 values were 60, 180, 130, 26, and 0.5 nM, respectively. Then, we measured the activity in water collected at 5 localities in the Ishizu River system in Japan. The activities of water samples ranged from 4.8 pmol/L (1.3 ng/L) to 52 pmol/L (14 ng/ L) (17beta-estradiol (E2) equivalent). These values were higher than those measured with the yeast for ER activation through direct ligand binding to ER. The direct ER ligand binding activities of the water samples ranged from 2.5 to 5.3 pmol/L (E2 equivalent). We also measured AhR activation of the water samples using a reporter yeast for AhR ligand activity. The activities ranged from 102 to 472 pmol/L (beta-naphthoflavone equivalent). These results indicate that the water samples contain substances that bind to AhR, and these substances contribute to ER signaling through AhR activation in the yeast reporter strain. This yeast reporter strain should be a useful tool to evaluate direct and indirect ER activation by environmental samples.