Effects of flavonoids on CYP1 expression in RL95-2 endometrial carcinoma cells

RL95-2 endometrial cancer cells were used to study cytochrome P450-mediated chemopreventative mechanisms of four flavonoids found in foods. To investigate enzymatic CYP1 inhibition, intact cells were induced with benzo(a)pyrene or 2,3,7,8-tetrachlorodibenzo-p-dioxin. Quercetin, kaempferol and myricetin inhibited CYP1 activity dose-dependently with IC₅₀s ranging from 2.2 to 4 μM; while amentoflavone was inactive. Further experiments were designed to determine if flavonoids also interacted with the AhR or caused a decrease in CYP1 protein or mRNA expression. CYP1A1 protein expression was inhibited in cells co-treated with TCDD and quercetin, kaempferol or myricetin compared with TCDD alone, but amentoflavone was ineffective. Relatively higher (∼7-fold) basal levels of CYP1B1 protein were not significantly affected by flavonoid treatments. In general, at the message level significant inhibition of induced CYP1A1 or CYP1B1 was not detected following flavonoid cotreatment. Despite the common inhibitory effects of quercetin, kaempferol, and myricetin on induced CYP1A1-dependent activity and protein expression, the mechanisms of CYP1 inhibition in this cell line are complex and dependent on the CYP gene, AhR inducer and the flavonoid.     

不同生理状态下人参总皂苷对HepG2细胞CYP2B6 mRNA和蛋白表达的影响

本文研究人参总皂苷在正常和炎症状态下对HepG2细胞CYP2B6 mRNA和蛋白表达的影响。采用索式提取法提取人参总皂苷,Real-Time PCR及Western Blot技术检测细胞中CYP2B6 mRNA和蛋白表达。结果显示,正常状态下人参总皂苷低(15 g/mL)、中(60 g/mL)与高(240 g/mL)剂量均显著诱导CYP2B6 mRNA表达(p0.05),中、高剂量均显著诱导CYP2B6蛋白的表达(p0.05)。使用LPS孵育细胞制造炎症模型,LPS对CYP2B6 mRNA表达无影响,但显著诱导CYP2B6蛋白的表达(p0.05)。使用人参总皂苷和LPS共同孵育细胞后,炎症状态下人参总皂苷中、高剂量对CYP2B6 mRNA的表达仍有显著诱导效果(p0.05),低和中剂量则均能显著诱导CYP2B6蛋白的表达,但高剂量则对CYP2B6的蛋白表达则呈现抑制作用(p0.05)。提示炎症状态能够改变人参总皂苷对CYP2B6 mRNA和蛋白的表达,因此联合用药时,应考虑不同生理状态下人参对代谢性药物相互作用的影响。     

Dehydroepiandrosterone (DHEA) metabolism in Saccharomyces cerevisiae expressing mammalian steroid hydroxylase CYP7B: Ayr1p and Fox2p display 17beta-hydroxysteroid dehydrogenase activity

We have engineered recombinant yeast to perform stereospecific hydroxylation of dehydroepiandrosterone (DHEA). This mammalian pro-hormone promotes brain and immune function; hydroxylation at the 7alpha position by P450 CYP7B is the major pathway of metabolic activation. We have sought to activate DHEA via yeast expression of rat CYP7B enzyme. Saccharomyces cerevisiae was found to metabolize DHEA by 3beta-acetylation; this was abolished by mutation at atf2. DHEA was also toxic, blocking tryptophan (trp) uptake: prototrophic strains were DHEA-resistant. In TRP(+) atf2 strains DHEA was then converted to androstene-3beta,17beta-diol (A/enediol) by an endogenous 17beta-hydroxysteroid dehydrogenase (17betaHSD). Seven yeast polypeptides similar to human 17betaHSDs were identified: when expressed in yeast, only AYR1 (1-acyl dihydroxyacetone phosphate reductase) increased A/enediol accumulation, while the hydroxyacyl-CoA dehydrogenase Fox2p, highly homologous to human 17betaHSD4, oxidized A/enediol to DHEA. The presence of endogenous yeast enzymes metabolizing steroids may relate to fungal pathogenesis. Disruption of AYR1 eliminated reductive 17betaHSD activity, and expression of CYP7B on the combination background (atf2, ayr1, TRP(+)) permitted efficient (>98%) bioconversion of DHEA to 7alpha-hydroxyDHEA, a product of potential medical utility.     

超高效液相色谱-串联质谱法测定食品中黄曲霉毒素B1和M1的方法研究

应用超高效液相色谱-串联质谱联用技术（UPLC-MS/MS）,在多反应离子检测（MRM）方式下对食品中的黄曲霉毒素B1（AFB1）和M1（AFM1）污染量进行检测.样品经乙腈-水混合试剂提取,中性氧化铝柱净化,经Agilem ZORBAX Eclipse plus C18色谱柱（100 mm×4.6 mm,1.8 μm）,以甲醇和10 mmol/L NH4Ac溶液（含0.1％的甲酸）为流动相洗脱分离,以MRM方式进行定量分析.AFB1和AFM1分别在0.12～6.12 μg/L和0.11～2.28 μg/L质量浓度范围内线性关系良好,相对标准偏差（RSD）＜5％,加标回收率为81.3％～97.2％.该方法具有低成本、准确、快速、简便、灵敏度高等优点,可满足我国对AFB1和AFM1检测限要求.     

Co-occurrence of aflatoxins B 1 , B 2 , G 1 , G 2 , zearalenone and fumonisin B 1 in Brazilian corn

Two hundred and fourteen unprocessed corn samples (1997-98 harvest), collected at wholesale markets in different regions in Brazil, were surveyed for the occurrence of mycotoxins. The samples were analysed for aflatoxins B 1 , B 2 , G 1 , G 2 , zearalenone and fumoni1sin B 1 using in-house validated methods. The occurrence of aflatoxin B 1 , zearalenone and fumonisin B 1 was found in 38.3, 30.4 and 99.1% of the samples, respectively. Aflatoxin B 1 , zearalenone and fumonisin B 1 contamination levels varied from 0.2 to 129, 36.8 to 719, and 200 to 6100 μg/kg, respectively. The cooccurrence of the two carcinogenic mycotoxins aflatoxin B 1 and fumonisin B 1 was observed in 100% of the aflatoxin-contaminated samples (82 samples). Cooccurrences of aflatoxin B 1 : zearalenone: fumonisin B 1 and aflatoxin B 1 : aflatoxin B 2 : fumonisin B 1 were found in 18 and 43 samples, respectively.     

比对实验中黄曲霉毒素B1的检验与分析

本文综合国标法,色谱法及试剂盒法三种方法进行修正,把1：1的甲醇水改为7：3的甲醇水直接加进样品中,删除国标法及试剂盒自带方法中采用石油醚转移步骤,把手动振摇、蒸发等步骤改为漩涡振荡及离心方法,改良了样品的提取方法,操作方便,减少交叉污染,使结果更稳定。     

二乙烯三胺脱除花生粕中黄曲霉毒素B1的工艺优化及机制研究

旨在为霉变花生粕中黄曲霉毒素B₁(AFB₁)的脱除提供技术支持,以自然霉变花生粕为实验材料,用二乙烯三胺脱除花生粕中的AFB₁。采用单因素实验优化脱毒条件,并对二乙烯三胺脱除AFB₁的机制进行研究。结果表明：二乙烯三胺脱除花生粕中AFB₁的最佳工艺条件为液料比5∶1、处理温度50℃、处理时间45 min、二乙烯三胺溶液质量浓度10 mg/mL,在此条件下AFB₁脱除率为95.5%;高效液相色谱和高分辨质谱分析表明,AFB₁可能的降解机制是其酮羰基与二乙烯三胺发生脱水反应,并且反应产物通过离心随着上清液被脱除。二乙烯三胺具有应用于花生粕中AFB₁脱除的潜力。     

Effects of sex, weight, diet and hCG administration on levels of skatole and indole in the liver and hepatic activities of cytochromes P4502E1 and P4502A6 in pigs

Cytochromes P4502E1 (CYP2E1) and P4502A6 (CYP2A6) catalyse metabolic reactions of skatole and indole metabolism. The objectives of this study were as follows: to evaluate whether activities of CYP2E1 and CYP2A6 in pigs of two live weights (LW) differ between males and females; to investigate whether activities of CYP2E1 and CYP2A6 are affected by hCG stimulation; and to investigate whether the levels of skatole and indole in the liver and the activities of CYP2E1 and CYP2A6 are affected by raw potato starch (RPS). Female pigs expressed higher CYP2A6 activity at 90kg LW, and higher CYP2E1 activity at 115kg LW compared to male pigs. Skatole levels in the liver were higher in male pigs than in female pigs at both LW, whereas indole levels were higher in males only at 115 kg LW. Neither levels of indolic compounds in the liver nor enzyme activities were affected by hCG stimulation. The inclusion of RPS in the diet reduced skatole levels in the liver in both sexes and increased CYP2A6 activity in female pigs. It was concluded that the incidence of boar taint may depend on both skatole amount, which reach the liver, and the activities of enzymes involved in skatole metabolism, which may vary depending on sex, live weight, and diet.     

间接竞争ELISA检测试剂盒测定粮油食品中的 黄曲霉毒素B1

目的建立一套适用于食品中黄曲霉毒素B1的快速、简便、准确的检测方法,同时,进一步验证本公司研制的黄曲霉毒素ELISA检测试剂盒的检测效果。方法用酶联免疫法和高效液相色谱法分别对市售的食用油、花生、谷物样品中AFB1的污染程度进行检测分析。结果用ELISA法和HPLC法测定食用油样品的回收率分别为87.1%~92.7%和85.8%~100.8%;花生样品的回收率分别为76.0%~92.8%和76.3%~92.9%;谷物样品的回收率分别为82.6%~92.7%和82.7%~106.4%,花生加标样品6次平行测定的RSD分别为1.29%~2.46%和5.15%~8.70%,11份阳性样品测定结果表明2种方法具有很好的一致性(r2=0.995)。结论 ELISA法和HPLC法均有很好的线性关系,且测定结果相近。本公司研制的黄曲霉毒素ELISA试剂盒可以快速地检测食品中黄曲霉毒素B1,分析周期短,分析效率高。     

生物技术对黄曲霉毒素B1的脱除及机制研究进展

黄曲霉毒素是危害最大的真菌毒素之一,而黄曲霉毒素B₁(AFB₁)是黄曲霉毒素中毒性最大的一种,具有高毒性、致癌、致畸和致突变的作用,对动物和人类健康造成危害。为了有效脱除AFB₁,综述了近年来研究的具有解毒作用的微生物,介绍了采用生物技术脱除AFB₁的方法,重点探讨了生物脱除AFB₁机制。用于脱除AFB₁的菌株有芽孢杆菌、假单胞菌、乳酸菌、非产毒曲霉等,可采用单菌种发酵、多菌种协同发酵和菌-酶协同作用脱除AFB₁。生物脱除AFB₁机制主要为降解脱除和吸附脱除。可进一步筛选具有高优良能力的菌株以及更深层次地研究微生物脱毒的机制,探索微生物制剂对AFB₁的降解作用。     

2011-2012年食用植物油中黄曲霉毒素B1的调查

为调查我国市场食用植物油油中黄曲礞毒素B1是螽符合国家标准规定,本论义膈两年的时间,对988个传统烹调油样品、342个新兴功放类油样品中的黄曲霉毒素B1进行了检测。结果显示：2011年传统烹调油合格率为99．22％,新必功效类油样,铺及凋味汕合格宰为95．03％;2012年传统烹调油合格率为99．37％,新必功效类油样品及调味油合格半为96．90％。由此可见,食用油产品的合格牢征逐年提高。     

Occurrence of fumonisins B1 and B2 in Portuguese maize and maize-based foods intended for human consumption

Fumonisin B₁ (FB₁) and fumonisin B₂ (FB₂) are mycotoxins mainly produced by Fusarium verticillioides and Fusarium proliferatum, which are field pathogens of maize. A survey was conducted on the incidences of FB₁ and FB₂ in both maize and derived products purchased in Portugal. The analytical method involved extraction with methanol-water, clean-up by immunoaffinity column and derivatization with naphthalene-2,3-dicarboxaldehyde. Determination was carried out by high-performance liquid chromatography (HPLC) with spectrofluorimetric detection, with liquid chromatography/mass spectrometry (LC/MS) confirmation. The presence of FB₁ and FB₂ was determined in 67 samples of maize and maize-based foods, such as flour, semolina, starch, sweet maize, cornflakes and other breakfast cereals, and snacks collected in 2005. FBs were found in 15 samples at concentrations ranging from 113 to 2026 µg kg^-1. Two of the samples showed higher contamination levels than the limits established by the European Commission Regulation. None of the samples contained levels of fumonisins that would lead to an exposure exceeding the tolerable daily intake (TDI).     

高效液相色谱法测定婴幼儿配方乳粉中的维生素B1、维生素B2

目的建立高效液相色谱法快速测定婴幼儿配方乳粉中的维生素B1、B2的含量。方法样品进行前处理后,采用Waters Xbridge C18色谱柱(150 mm×4.6 mm, 5μm)进行分离。以0.05 mol/L乙酸钠溶液-甲醇为流动相等度洗脱,柱温为30℃,流速为1 mL/min,荧光检测器进行检测,外标法定量。结果在优化的实验条件下,维生素B1、维生素B2可以有效分离,线性范围为0.05～1.00μg/mL,相关系数均大于0.999。维生素B1的加标回收率为87.4%～99.1%,相对标准偏差为1.2%～3.06%,检出限为0.03 mg/100 g;维生素B2的加标回收率为90.6%～97.8%,相对标准偏差为0.97%～3.44%,检出限为0.02 mg/100 g。结论此方法简单、快捷、高效,适用于婴幼儿配方乳粉中维生素B1、维生素B2的测定。     

Fumonisin B1, fumonisin B2, zearalenone and ochratoxin A contamination of maize in Croatia

Mycotoxins are products of moulds that frequently contaminate maize. In this study the presence of mycotoxins fumonisin B₁ (FB₁), fumonisin B₂ (FB₂), zearalenone (ZEA) and ochratoxin A (OTA) was determined in 49 maize grain samples collected in autumn 2002. The most frequent finding was that of FB₁(100%), followed by ZEA (84%) and OTA (39%), while FB₂ was found only in three samples. The co-occurrence of two and three mycotoxins was found in 55 and 37% of samples, respectively. The concentrations (mean ± SD) of FB₁, ZEA and OTA in positive samples were 459.8 ± 310.7, 3.84 ± 6.68 and 1.47 ± 0.38 µg kg^-1, respectively, and the concentrations of FB₂ in three positive samples were 68.4, 109.2 and 3084.0 µg kg^-1. Although such low concentrations of mycotoxins are not a significant source of exposure in countries with a European diet, a few samples with extreme values indicate that thorough control is needed.     

2011~2015年花生和小麦制品中黄曲霉毒素B1的检测

目的 对2011~2015年花生和小麦制品中黄曲霉毒素B1进行检测分析和安全评价。方法 采用酶联免疫法对2011~2015年的1140个花生类和6475个小麦类制品的黄曲霉毒素B1的含量进行检测。结果 2011~2015年检测的花生类制品合格率分别为95.5%、98%、97.4%、98.3%和98.7%; 检测的各种小麦类制品合格率均为100%。结论 花生类制品合格率逐年上升, 但仍存在一定的质量隐患; 小麦类制品质量均符合国家规定限量。     

A method for determining fumonisin B1 in corn using immunoaffinity column clean-up and thin layer chromatography/densitometry

A method for determining fumonisin B₁(FB₁) in corn was developed and the clean-up optimized in order to give an extract suitable for one-dimensional thin layer chromatographic (TLC) analysis. FB₁ was extracted with a solution of methanol:water (80:20,v/v), purified through an immunoaffinity column and separated on a C₁₈ reversed phase TLC plate. The FB₁ was visualized with 0.1mol/l sodium tetraborate, 0.40mg/ml fluorescamine in acetonitrile and 0.01mol/l boric acid:acetonitrile (2:3,v/v) for fluorescence detection, and quantified by densitometric analysis. Water, acetonitrile:water (1:1v/v) and acetonitrile:water (4:1v/v) were evaluated as TLC solvents for running both standards and samples together with derivatization procedures aimed at improving separation, resolution, sensitivity and linearity. The mean recovery for FB₁ for spiked samples was found to be 85% and the linear equation of standard calibration curve by densitometric analysis gave an r2 value higher than 0.99. The maximum coefficient of variation for replicate analysis of spiked samples was 19%. The absolute amount of FB₁ standard detectable on a TLC plate was 2 ng, giving a detection limit for the method of 0.1mg/kg. The method has been shown to be robust in the application of FB₁ monitoring in corn (214 samples) collected in different regions of the country. FB₁ was detected in 99% of these samples in the range of 0.2 to 6 mg/kg.     

高效液相色谱法同时测定保健食品中泛酸、烟酰胺、维生素B1、维生素B2、维生素B6的含量

目的建立高效液相色谱法同时测定保健食品中泛酸、烟酰胺、维生素B_1、维生素B_2、维生素B_6的含量。方法样品经0.2%磷酸溶液超声提取,经Alltima-C18柱分离,以乙腈-5 mmol/L庚烷磺酸钠溶液为流动相进行梯度洗脱,采用二极管阵列检测器(diode array detector,DAD)同时在200 nm、280 nm波长处进行检测。结果泛酸、烟酰胺、维生素B_1、维生素B_2、维生素B_6能实现较好的分离,在各自线性范围内线性关系良好,平均回收率为93.9%~103.2%,相对标准偏差(relative standard deviation,RSD)为0.4%~1.8%。结论该方法操作快速简便、精密度和重复性好,可适用于保健食品中泛酸、烟酰胺、维生素B_1、维生素B_2、维生素B_6含量的同时测定。     

臭氧降解玉米中黄曲霉毒素B1效果及降解动力学研究

玉米是我国重要的食品和饲料原料,当收获、加工和储藏等措施不当时,可能会造成黄曲霉毒素B1（aflatoxin B1,AFB1）污染玉米这一突出问题,AFB1已被国际癌症机构定为1级致癌物。尽管目前已建立了一些物理、化学和生物降解AFB1的方法,但高效、安全、经济的绿色降解方法仍很少。本研究以AFB1污染的玉米为试样,研究臭氧对玉米中AFB1的降解效果。结果表明：AFB1降解率随着臭氧质量浓度的增加和处理时间的延长而显著提高;当水分质量分数为20.37%的玉米经90 mg/L的臭氧处理40 min后,AFB1含量由77.6 μg/kg降低到21.42 μg/kg,降解率达72.4%。臭氧降解AFB1的动力学模拟结果表明,臭氧降解AFB1符合一级动力学模型。玉米中AFB1降解速率常数按以下次序递减：k90 mg/L＞k65 mg/L＞k40 mg/L。实验得到臭氧降解AFB1的动力学方程、反应速率常数、决定系数和半衰期,为最优地控制臭氧降解AFB1的反应条件奠定了理论和实践基础,也为臭氧降解AFB1污染玉米的应用提供了技术保障。