Assessment of the estrogenicity of isoflavonoids,using MCF-7-ERE-Luc cells

In the current study, our research focused on the estrogenic activity of isoflavonoids, mainly genistein, biochanin A and daidzein. Genistein enhanced the reporter gene expression of MCF-7-ERE-Luc cells, at a concentration as low as 10 nM, with a concentration of 100 nM the achieved gene expression effects were similar to those of 10 pM 17beta-estradiol, Based on the estrogenic activities of biochanin A and daidzein, hydroxyl groups at the 4' and 5 positions are needed for the maximal effect of the genistein. The estrogenic effects of these isoflavonoids were inhibited by the concomitant treatment with tamoxifen. The data showed that the estrogenic effects of isoflavonoids were mediated through estrogen receptors. When the isoflavonoids were tested as mixtures, the estrogenic effects were lower than the arithmetic sum of those induced by each individual isoflavonoid. The estrogenic potency of each isoflavonoid was presented at EC50 levels with a 17beta-estradiol equivalent concentration (EEQ) based on the dose response of each chemical. The EC50s and EEQs of genistein, biochanin A and daidzein were 4.15, 0.89 and 0.18 microM, and 15.0, 5.12 and 1.83 microM/M, respectively. Our data clearly demonstrated that the pERE-luciferase reporter gene assay was suited for the sensitive and quantitative measurement, and large scale screening, of the estrogenicity of chemicals in vitro.     

ESR and cell culture studies on free radical-scavenging and antioxidant activities of isoflavonoids

Isoflavonoids are thought to be the biologically active components in soy that play a role in the prevention of coronary heart disease and breast and prostate cancer. Mechanisms to explain how isoflavonoids mediate beneficial effects have not yet been clearly established. This study was undertaken to investigate the free radical-scavenging and antioxidant activities of various structure-related isoflavonoids including genistein, daidzein, biochanin A, and genistin in a cell-free and an endothelial cell model system. Electron spin resonance spectroscopy and spin trapping techniques were applied to evaluate the ability of isoflavonoids to scavenge hydroxyl, superoxide, nitric oxide, diphenylpicrylhydrazyl, galvinoxyl, and lipid-derived radicals. All isoflavonoids tested had no significant scavenging effects on the aforementioned radicals in concentrations up to 1.0 mM. However, at a physiologically achievable concentration of 5 nM, both genistein and daidzein slightly increased intracellular-reduced glutathione levels approximately by 10 and 30%, respectively, in human endothelial cells, whereas cellular alpha-tocopherol and uric acid remained unchanged by the isoflavonoid treatments. Present data indicate that free radical-scavenging activities of the isoflavonoids tested probably do not substantially contribute to their antioxidant properties. The ability of genistein and daidzein to increase cellular GSH (reduced glutathione) might be important for their action in biological system.     

丹酚酸B对大鼠离体心脏缺血再灌注损伤的保护作用

目的：研究丹酚酸B对大鼠离体心脏缺血再灌注损伤的保护作用。方法：大鼠离体心脏采用Langendorff装置,灌注含氧K．H液30min后,夹闭,停止灌流30min,然后再灌注40min。实验分为模型（空白K_H液）组、葛根素（0．24mmol／1）组与丹酚酸B高、中、低浓度（60、30、15gmol／1）组。灌注含氧K．H液20min后加入相应药物灌注10min。实时测定血流动力学指标、左室舒张末期压（LVEDP）、左室发展压（LVDP）、左室收缩压最大变化速率（＋dP/dtmax）、左室舒张压最大下降速率（-dP／dtmax）。收集灌流液,并测定肌酸激酶（CK）的活力;取部分心肌组织测定丙二醛（MDA）含量与超氧化物歧化酶（sou）活性。结果：与模型组比较,丹酚酸B高、中浓度组大鼠离体心脏在复灌10min后L、厂EDP显著降低,LVDP显著升高,＋dP/dtmax。显著升高,-dP／dtmax显著降低（P〈0．01或P〈0．05）;丹酚酸B高浓度组离体大鼠心脏组织CK活性显著减弱（尸〈O．01）,丹酚酸B高、中浓度组大鼠离体心脏MDA含量显著减少,SOD活性显著增强（P〈0．01）;丹酚酸B高、中、低浓度组大鼠离体心脏复灌5min后灌流液显著增加（P〈O．01或P〈0．05）。结论：丹酚酸B对大鼠离体心脏有保护作用,其机制可能是通过清除氧自由基,抵抗氧化应激发挥治疗作用。     

纳洛酮对离体大鼠心脏缺血再灌流损伤的保护作用

本实验采用离体大鼠心脏Langendorff灌流装置.使心脏停灌(旷置)40 min后再恢复灌流30min.夏制心肌缺血再灌流损伤.观察了纳洛酮对再灌流后冠脉流出液中乳酸脱氢酶(LOH)活性,心肌细胞内钙含量.心肌脂质过氧化产物——丙二醛(MDA)含量和超氧化物歧化酶(SOD)活力变化的影响。结果表明.再灌流后应用纳洛酮能显著减少心肌LDH的释放量.心肌细胞内钙聚集、心肌MDA的生成量及SOD活力降低的程度.提示纳洛酮对缺血再灌流心肌具有保护作用。     

Interaction of isoflavonoids with human liver microsomal cytochromes P450: inhibition of CYP enzyme activities

1.?The possibility of interaction of isoflavonoids with concomitantly taken drugs to determined isoflavonoids safety was studied. Inhibition of nine forms of cytochrome P450 (CYP3A4, CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C19, CYP2C9, CYP2D6 and CYP2E1) by 12 isoflavonoids (daidzein, genistein, biochanin A, formononetin, glycitein, equol and six glucosides, daidzin, puerarin, genistin, sissotrin, ononin and glycitin) was studied systematically.

2.?The most potent inhibitors were genistein and daidzein inhibiting noncompetitively the CYP2C9 with K_i of 35.95?±?6.96 and 60.56?±?3.53?μmol/l and CYP3A4 (inhibited by genistein with K_i of 23.25?±?5.85?μmol/l also by a noncompetitive mechanism). Potent inhibition of CYP3A4 was observed also with biochanin A (K_i of 57.69?±?2.36?μmol/l) and equol (K_i of 38.47?±?2.32?μmol/l).

3.?Genistein and daidzein inhibit noncompetitively CYP3A4 and CYP2C9. With plasma levels in micromolar range, a clinically important interaction with concomitantly taken drugs does not seem to be probable.     

Antiinflammatory activity of isoflavonoids fromPueraria radix and biochanin A derivatives

For comparing with flavones/flavonols, isoflavonoids isolated fromPueraria radix and chemically synthesized from biochanin A were evaluated for the antiinflammatory activity using mouse ear edema test. Isoflavonoids such as daidzein and puerarin showed the significant antiinflammatory activity at a dose of 2 mg/mouse, although their activity was generally less than that of flavones/flavonols. 7-O-Substitution of biochanin A was not favorable for the antiinflammatory activity.     

Reduction of genistein clastogenicity in Chinese hamster V79 cells by daidzein and other flavonoids.

A broad spectrum of health benefits has been ascribed to soy products. These products contain soy protein and relatively high levels of polyphenolic compounds known as flavonoids. While they are the most likely candidates for biological activity, flavonoids as a class, and of specific interest, genistein, are well known to be genotoxic due to their ability to "poison" cellular DNA topoisomerase II (topo II) resulting in stable chromosome breakage and mutation and raising questions about the long term health effects associated with chronic flavonoid exposure. Interestingly, some flavonoids, such as biochanin, galangin and daidzein, are catalytic topo II inhibitors (not poisons) and actually antagonize the clastogenicity of topo II poisons. It is shown in the present paper that flavonoids possessing catalytic topo II inhibitory activity, strongly antagonize the clastogenicity of genistein in Chinese hamster V79 cells. Importantly, one of these, daidzein, is a major constituent of marketed soy products. It is conjectured that the potential human clastogenic risk of soy products containing genistein might be mitigated or abolished due to the presence of daidzein or other flavonoids in those products.     

Delay of occurrence of reperfusion-induced ventricular fibrillation in the isolated rat heart with superoxide dismutase.

The effects of superoxide dismutase (SOD) on reperfusion-induced ventricular fibrillation (R-VF) were determined in isolated, perfused rat hearts with reperfusion after durations of regional myocardial ischemia ranging from 5 to 37.5 min. SOD (100 U/ml) was perfused during both ischemia and reperfusion periods. Regional myocardial ischemia was produced by acute occlusion of the left anterior descending coronary artery (LAD). Reperfusion after a brief period of ischemia (8 min) resulted in R-VF in 33% of SOD-perfused hearts as compared with 100% of control hearts that exhibited this arrhythmia (p less than 0.05). The incidence of R-VF was not affected by SOD with intermediate duration of ischemia of 10, 15, and 22.5 min. Reperfusion after a relatively long 30-min period of ischemia did not result in R-VF in control hearts, but 87% of SOD-treated hearts still exhibited this arrhythmia (p less than 0.05). No hearts exhibited R-VF with reperfusion after 37.5 min of ischemia. Thus, SOD shifted the occurrence of R-VF to longer durations of ischemia without affecting the peak incidence of this arrhythmia. In contrast to effects of SOD on incidence of R-VF, SOD had no effect on onset times of this arrhythmia. Nor did SOD affect reperfusion-induced ventricular tachycardia (VT), heart rate (HR), or coronary flow. These results suggest that SOD may have delayed onset of electrophysiologic derangements that were specifically responsible for R-VF. SOD may be classified as a modulator of R-VF.     

Free Radical Scavenging and Hepatoprotective Constituents from the Leaves of Juglans sinensis

In the course of searching for hepatoprotective agents from natural products, six compounds were isolated from the MeOH extract of the leaves of Juglans sinensis, as guided by their DPPH free radical scavenging activity. The structures were determined as juglanoside B (1), quercetin 3-O-alpha-L-arabinofuranoside (avicularin, 2), quercetin 3-O-alpha-L-arabinopyranoside (guaijaverin, 3), quercetin 3-O-alpha-L-rhamnopyranoside (quercitrin, 4), (+)-catechin (5) and quercetin 3-O-beta-D-galactopyranoside (hyperin, 6). Compounds 2-6 showed significant DPPH free radical scavenging effects. An evaluation for the hepatoprotective activity of the isolated compounds on drug-induced cytotoxicity was conducted, and compounds 1, 2, and 5 showed protective effects against nitrofurantoin-induced cytotoxicity, and compound 5 also exhibited a moderate protective effect on amiodarone-induced cytotoxicity in Hep G2 cells.     

The effects of phytoestrogenic isoflavones on the formation and disposition of paracetamol sulfate in the isolated perfused rat liver

This study examines the potential for the phytoestrogenic isoflavones, a type of complementary medicine, to be involved in pharmacokinetic interactions in the liver. Rat livers were isolated and perfused to steady state, in single-pass mode, with either 5 microM paracetamol (n = 6), or 5 microM paracetamol with a 50:50 molar mixture of genistein and biochanin A or daidzein and formononetin, at a total isoflavone concentration of 1 and 10 microM (n = 6 for each mixture at each concentration). At 1 microM, neither isoflavone mixture had any effect, while at 10 microM both mixtures decreased the clearance of paracetamol and the formation clearance to paracetamol sulfate. Genistein and biochanin A (10 microM) also increased the biliary extraction of hepatically-generated paracetamol sulfate. Additional livers were perfused with an infusion of 5 microM (14)C-paracetamol in the absence (n = 4), or presence, of a 10 microM genistein and biochanin A mixture (n = 4). Analysis of washout perfusate and bile samples (up to 30 min after stopping the infusion) revealed that the isoflavones reduced the first-order rate constant for paracetamol sulfate transport into perfusate, but not for transport into bile. The results indicate that isoflavones can reduce the formation of paracetamol sulfate and that its enhanced excretion into bile arises from the inhibition of sinusoidal efflux transport.     

beta-Galactosidase-inhibiting new isoflavonoids produced by actinomycetes.

Six isoflavonoids having beta-galactosidase inhibiting activity were isolated from the culture filtrate of Streptomyces xanthophaeus. Their structures were determined by spectral analyses to be daidzein, daidzein 7-alpha-L-rhamnoside, daidzein 4',7-di-alpha-L-rhamnoside, genistein, genistein 7-alpha-L-rhamnoside and genistein 4',7-di-alpha-L-rhamnoside.     

红景天苷对大鼠离体心脏缺血-再灌注损伤的保护作用及机制研究

目的研究红景天的主要成分红景天苷对大鼠离体心脏缺血－再灌注损伤的保护作用及其可能的作用机制。方法将大鼠随机分为对照组和红景天苷组2组,取各大鼠离体心脏,按Langendorff预灌注10min,夹闭30min,再灌注120min,期间实时测定血流动力学指标：左室舒张末期压（LVEDP）、左室发展压（LVDP）、左室收缩压最大变化速率（＋dP／dtmax）、左室舒张压最大下降速率（－dP／dtmin）。复灌末,心脏用1％三苯四氯化磷酸盐缓冲液（TTC）孵育15min,通过TTC染色法测定大鼠心肌梗死面积。实验结束后,测定丙二醛（MDA）含量及心肌组织超氧化物歧化酶（SOD）和一氧化氮合酶（NOS）活力。结果与对照组相比,红景天苷组的LvEDP下降,而LVDP、＋dP／dtmin、－dP／dtmin均上升。给药后,大鼠离体心脏的血流动力学指标有所好转。红景天苷组梗塞面积（14．7±5．5）％明显低于对照组（28．4±4．6）％（P＝0．00012）。生化指标测定结果表明红景天苷组可降低磷酸肌酶（CK）活性和MDA含量,提高SOD和NOS的活性。结论红景天苷对大鼠离体心脏缺血－再灌注损伤的保护作用,这可能是它能抑制氧化应激所致的。     

Anti-inflammatory and antioxidant activities of constituents isolated from Pueraria lobata roots

In order to evaluate the anti-inflammatory and antioxidant activities of Pueraria lobata roots and its active components, in vitro inhibitory activities against lipopolysaccharide (LPS)-induced nitric oxide (NO) production, inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2) protein expression, and tert-butylhydroperoxide (t-BHP)-induced reactive oxygen species (ROS) generation in RAW 264.7 cells, as well as in vitro scavenging activities against 1,1-diphenyl-2-picrylhydrazyl (DPPH), peroxynitrite (ONOO(-)), nitric oxide (NO·), superoxide anion (·O(2)(-)) and total ROS, and inhibitory activities against ONOO(-)-mediated tyrosine nitration, were determined. Repeated column chromatography was performed to isolate four known compounds from the anti-inflammatory and antioxidant EtOAc fraction: daidzein; genistein; puerarin; (+)-puerarol B-2-O-glucopyranoside; four known compounds from the anti-inflammatory n-hexane fraction: lupenone; lupeol; puerarol; coumestrol; seven known compounds from the antioxidant n-BuOH fraction: allantoin; 3'-hydroxypuerarin; daidzein 8-C-apiosyl-(1→6)-glucoside; puerarin; genistin; 3'-methoxypuerarin; daidzin. Among these compounds, lupenone and lupeol reduced NO production, as well as iNOS and COX-2 protein levels in LPS-stimulated RAW 264.7 cells. Furthermore, lupeol showed significant inhibitory activity against intracellular ROS generation by t-BHP. Meanwhile, 3'-hydroxypuerarin showed marked ONOO(-), NO·, total ROS scavenging activities, and weak ·O(2)(-) scavenging activity, while 3'-methoxypuerarin showed ONOO(-) scavenging activity and weak NO· and O(2)(-) scavenging activities, suggesting that existence of the 3'-hydroxyl group in puerarin plays an important role in the scavenging of ONOO(-), NO·, and total ROS, as well as inhibiting the ONOO(-)-mediated tyrosine nitration mechanism. These results indicate that P. lobata roots and its constituents may be a useful therapeutic and preventive approach to various inflammatory diseases and oxidative stress-related disease.     

硫化氢对离体大鼠心脏缺血/再灌注损伤的影响及机制初探

目的观察内源性CSE/H2S通路的改变以及给予H2S供体对缺血/再灌注心脏的影响,探讨该通路与心脏缺血/再灌注损伤的关系及作用机制。方法采用Langendorff离体灌流装置、通过停灌30min/复灌30min方式造成Wistar大鼠心肌缺血/再灌注损伤模型;采用外源性NaHS(40μmol.L-1)分别在停灌30min前(SIR)与停灌30min后处理(IRS)对缺血/再灌注心脏的影响。记录心脏收缩期左心室内压上升的最大变化速率(+dp/dtmax)、舒张期左心室内压下降的最大变化速率(-dp/dtmax)及左室内压差(LVP=左室收缩压-左室舒张压)。采用比色法检测灌流液中乳酸脱氢酶(LDH)、心肌MDA及SOD;采用比色法检测心肌胱硫醚-γ-裂解酶(CSE)活性;采用RT-PCR方法测定心肌组织CSEmRNA表达。结果与缺血/再灌注组(I/R)30min相比,SIR组及IRS组±dp/dtmax、LVP均增高,LDH降低;I/R组MDA水平高于对照组(CON)、SIR组及IRS组(P<0.05,P<0.01);IR组SOD活性低于SIR组及IRS组(P<0.05),但与CON组差别无显著性;I/R组大鼠心肌CSE活性低于CON组(P<0.05);而大鼠心肌CSEmRNA的表达与CON组差异无显著性。结论在缺血前后给予外源性NaHS均可改善因再灌注损伤引起的心肌收缩及舒张功能障碍;其作用机制可能是通过提高心肌SOD活性,增加氧自由基清除而拮抗缺血/再灌注引起的心功能及细胞膜损伤;心肌缺血/再灌注时内源性CSE活性抑制可能与心功能障碍及细胞损伤有关。     

何首乌炮制前后及其主要成分体外抗氧化活性研究

目的：研究何首乌生品、炮制品及其主要成分的体外抗氧化活性,探讨何首乌的炮制机理及其抗氧化作用物质基础。方法：采用2,2-二苯基-1-苦肼基（DPPH）自由基清除实验及铁离子还原（FRAP）法抗氧化能力检测方法评价何首乌炮制前后及其主要化学成分的体外抗氧化活性。结果：DPPH自由基清除实验中,何首乌生品（S）,随着乙醇浓度的增加,对DPPH自由基的清除率也增加,制品（Q/H/Z）中,以90%乙醇提取所得样品的平均清除率为最高,其次是水、70%乙醇和50%乙醇提取物;FRAP法检测结果显示何首乌生品（S）、黑豆汁制品（H）、清蒸制品（Q）及市售制品（Z）的FRAP值范围分别为2.23~9.27 mmol·L^-1,5.94~9.66 mmol·L^-1,5.74~10.49 mmol·L^-1,3.99~10.40 mmol·L^-1 FeSO₄,不同溶剂提取物的FRAP值由大到小为70%醇 > 50%醇 > 水 > 90%醇;何首乌的组成成分中虎杖苷、白藜芦醇、槲皮苷、槲皮素、芦丁、儿茶素、金丝桃苷、二苯乙烯苷和没食子酸有明显的清除DPPH自由基的作用,没食子酸、槲皮素、槲皮苷、儿茶素、芦丁和白藜芦醇的FRAP值较高,具有较强的抗氧化作用。结论：何首乌炮制前后均有较强的抗氧化作用,虎杖苷、白藜芦醇、槲皮苷、槲皮素、芦丁、儿茶素、金丝桃苷、二苯乙烯苷和没食子酸对何首乌的抗氧化活性有一定的贡献作用。     

Sulforaphane protects ischemic injury of hearts through antioxidant pathway and mitochondrial KATP channels

Reactive oxygen species are important mediators that exert a toxic effect during ischemia–reperfusion (I/R) injury of various organs. Sulforaphane is known to be an indirect antioxidant that acts by inducing Nrf2-dependent phase 2 enzymes. In this study, we investigated whether sulforaphane protects heart against I/R injury. Sprague–Dawley rats received sulforaphane (500 μg/kg/day) or vehicle intraperitoneally for 3 days and global ischemia was performed using isolated perfused Langendorff hearts. Hearts were perfused with Krebs-bicarbonate buffer for 20 min pre-ischemic period followed by a 20 min global ischemia and 50 min reperfusion. Treatment with sulforaphane inhibited an increase in the post-ischemic left ventricular end-diastolic pressure (LVEDP) and improved the post-ischemic left ventricular developed pressure (LVDP), ±dP/dt, and coronary flow as compared with the untreated control hearts. Pretreatment with 5-hydroxydecanoic acid (5-HD), a mitochondrial K_ATP channel blocker, for 10 min before ischemia attenuated the improvement of LVEDP, LVDP, ±dP/dt, and coronary flow induced by sulforaphane. Sulforaphane markedly decreased the infarcted size and attenuated the increased lactate dehydrogenase level in effluent during reperfusion. Pretreatment with 5-HD also blocked these protective effects of sulforaphane. Post-ischemia increased the concentration of atrial natriuretic peptide in coronary effluent, which attenuated by sulforaphane treatment. Decreases on Mn-superoxide dismutase (SOD), catalase, and heme oxygenase-1 levels by I/R were increased by sulforaphane treatment and pretreatment of 5-HD blocked the sulforaphane effects. Increases in Bax and caspase-3 levels, and decrease in Bcl-2 level by I/R were attenuated by sulforaphane treatment. These results suggest that the protective effects of sulforaphane against I/R injury may be partly mediated through mitochondrial K_ATP channels and antioxidant pathway.     

Inhibitory effects of constituents of <Emphasis Type="Italic">Morinda citrifolia</Emphasis> seeds on elastase and tyrosinase

A 50% ethanolic extract (MCS-ext) from seeds of Morinda citrifolia (“noni” seeds) showed more potent in vitro inhibition of elastase and tyrosinase, and 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging activity than extracts of M. citrifolia leaves or flesh. Activity-guided fractionation of MCS-ext using in vitro assays led to the isolation of ursolic acid as an active constituent of elastase inhibitory activity. 3,3′-Bisdemethylpinoresinol, americanin A, and quercetin were isolated as active constituents having both tyrosinase inhibitory and radical scavenging activities. Americanin A and quercetin also showed superoxide dismutase (SOD)-like activity. These active compounds were isolated from noni seeds for the first time.     

Inhibition of mitochondrial proton F0F1-ATPase/ATP synthase by polyphenolic phytochemicals

Mitochondrial proton F0F1-ATPase/ATP synthase synthesizes ATP during oxidative phosphorylation. In this study, we examined the effects of several groups of polyphenolic phytochemicals on the activity of the enzyme. Resveratrol, a stilbene phytoalexin that is present in grapes and red wine, concentration-dependently inhibited the enzymatic activity of both rat brain and liver F0F1-ATPase/ATP synthase (IC(50) of 12 - 28 microM). Screening of other polyphenolic phytochemicals using rat brain F0F1-ATPase activity resulted in the following ranking potency (IC(50) in parenthesis): piceatannol (8 microM)>resveratrol (19 microM)=(-)epigallocatechin gallate (17 microM)>(-)epicatechin gallate, curcumin (45 microM)>genistein=biochanin A=quercetin=kaempferol=morin (55 - 65 microM)>phloretin=apigenin=daidzein (approx. 100 microM). Genistin, quercitrin, phloridzin, (+)catechin, (+)epicatechin, (-)epicatechin and (-)epigallocatechin had little effect at similar concentrations. Tannic acid, theaflavins (tea extract) and grape seed proanthocyanidin extract (GSPE) had IC(50) values of 5, 20 and 30 microg ml(-1), respectively. Several monophenolic antioxidants and non-phenolic compounds were ineffective at concentrations of 210 microM or higher. The inhibition of F0F1-ATPase by resveratrol and genistein was non-competitive in nature. The effects of polyphenolic phytochemicals were additive. Both resveratrol and genistein had little effect on the Na(+)/K(+)-ATPase activity of porcine cerebral cortex, whereas quercetin had similar inhibitory potency as for F0F1-ATPase. In conclusion, the ATP synthase is a target for dietary phytochemicals. This pharmacological property of these phytochemicals should be included in the examination of their health benefits as well as potential cytotoxicity.