异黄酮对PC-3细胞凋亡的诱导作用

目的 :　通过观察染料木素 (genistein,GS)、大豆甙元 (daidzein,DA)和黄豆黄素(glycitein,GL)对前列腺癌细胞 (PC- 3 )凋亡的影响 ,探讨通过膳食途径预防前列腺癌发生危险性的可行性。方法 :　将 PC- 3细胞在 RPMI1 6 4 0培养液 (含 1 0 %小牛血清 )中采用开放式单层贴壁培养。实验设溶剂对照及三种受试物各三个剂量组 ,采用流式细胞术 ,DNA凋亡片段凝胶电泳和细胞 HE染色法分析三种常见异黄酮类物质对 PC- 3细胞凋亡的影响。结果 :　与对照组相比 ,流式细胞仪分析结果表明 ,5 0μ mol/L GS、75μ mol/L DA和 75μ mol/L GL对 PC- 3细胞处理 72 h,二倍体细胞 (G1期细胞 )相对减少 ,亚二倍体细胞峰 (即细胞凋亡率 )逐渐增多 ,即凋亡细胞比例逐渐增多 ;DNA凋亡片段凝胶电泳和细胞苏木素染色结果也显示 ,三种受试物能够诱导 PC- 3细胞凋亡 ,且这种作用存在剂量 -效应关系。结论 :　异黄酮类化合物染料木素、大豆甙元和黄豆黄素均具有诱导 PC- 3细胞凋亡的作用 ,这一结果提示 ,该类物质是可通过诱导细胞凋亡而发挥其抗肿瘤作用的。     

Identification, quantitation and biological activity of phytoestrogens in a dietary supplement for breast enhancement

A hop-based dietary supplement, marketed for natural breast enhancement, was analysed to determine the identity and biological activity of active constituents and potential biological effects in man. Extracts of the dietary supplement were analysed by LC-MS(n) and phytoestrogens identified and quantitated by reference to appropriate standards. Only hop-associated phytoestrogens were found in the dietary supplement at significant concentrations as follows (mean+/-1 S.D.); 8-prenylnaringenin 10.9+/-0.3, 6-prenylnaringenin 27.4+/-1.2, 6,8-diprenylnaringenin 0.9+/-0.1, xanthohumol 321+/-17 and isoxanthohumol 81.1+/-1.6 microg/g of dietary supplement. The oestrogenic activity of extracts in an ERalpha reporter gene assay was equivalent to 48+/-6.3 ng 17beta-oestradiol/g supplement and consistent with the 8-prenylnaringenin content. The dietary supplement extract also inhibited reductive 17beta-hydroxysteroid oxidoreductase activity, but to a greater extent than a concentration matched reference mixture of hop phytoestrogens. However, the supplement was only weakly active in mouse uterotrophic assays following administration in feed or after subcutaneous injection of extract at doses of 8-PN up to 250 times higher than that recommended for women. These preliminary findings suggest that the dietary supplement is unlikely to produce oestrogenic effects in vivo at the level of the uterus; supporting evidence is still required to demonstrate efficacy.     

Analysis of soy isoflavone plasma levels using HPLC with coulometric detection in postmenopausal women

A reliable chromatographic method for the determination of soy isoflavones (genistein, daidzein and glycitein) using a coulometric detection has been developed and applied to analyse plasma of postmenopausal women. The chromatographic separation was performed on a C18 reversed phase column with a mobile phase composed of acetonitrile–phosphate buffer mixture. Coulometric detection was carried out at +0.500 V. A careful and rapid solid phase extraction procedure on hydrophilic/lipophilic cartridges was chosen for plasma sample purification with and without hydrolysis obtaining good extraction yield values for all the analytes (>90.0%). The enzymatic hydrolysis step was necessary for the determination of the total amount of soy isoflavones. The limit of quantitation was 0.5 ng mL⁻¹ for genistein and 0.25 ng mL⁻¹ for daidzein and glycitein. The method was found to be precise and accurate. Thus, the proposed method is suitable for the analysis of soy isoflavones (free and total amounts) in plasma of postmenopausal women under treatment with the SoymenGN^® dietary supplement.     

RP-HPLC法测定血脂康胶囊中大豆苷元、黄豆黄素和染料木素

目的建立血脂康胶囊中大豆苷元、黄豆黄素、染料木素的测定方法。方法采用反相高效液相色谱法,YMC-C18分析柱,乙腈-水(0.1%磷酸)梯度洗脱为流动相,检测波长256nm。结果大豆苷元、黄豆黄素、染料木素在测定范围内线性关系良好,平均加样回收率分别为97.8%、99.1%、97.9%,RSD分别为2.02%、1.93%、1.84%(n=5)。结论方法操作简便、准确、重现性好,可用于同时测定血脂康胶囊中大豆苷元、黄豆黄素、染料木素的量。     

异黄酮类化合物对前列腺癌细胞PC-3增殖的抑制

[目的 ]通过观察金雀异黄素 (genistein ,GS)、大豆苷元 (daidzein ,DA)和大豆黄素 (glycitein ,GL)对前列腺癌细胞 (PC 3 )增殖的影响 ,探讨通过膳食干预途径预防前列腺癌发病的可行性。 [方法 ]将PC 3在PRMI 164 0培养液 (含10 %小牛血清 )中采用开放式单层贴壁培养。实验设溶剂对照组、顺铂阳性对照组及三种受试物各三个剂量组 (5× 10 -6mol/L ,2 5× 10 -6mol/L ,75× 10 -6mol/L) ,采用MTT法、3 H TdR掺入法及流式细胞术对PC 3的增殖情况进行分析。 [结果 ]与溶剂对照组相比 ,2 5 μmol/LGS、75 μmol/LDA和 75 μmol/LGL对PC 3处理 72h可抑制PC 3增殖 ,抑制率分别为42 %、5 0 %及 3 9%。三种受试物均可抑制细胞DNA合成并推进G0 /G1期细胞进入S期 ,降低细胞增殖指数。 [结论 ]大豆异黄酮类化合物GS、DA和GL均具有明显抑制前列腺癌细胞PC 3增殖效应 ,并呈现剂量 效应和时间 效应关系 ,提示通过膳食干预方式可预防前列腺癌的发生     

HPLC法同时测定血脂康胶囊中3种异黄酮和洛伐他汀的含量

目的:建立血脂康胶囊中大豆苷元、黄豆黄素、染料木素和洛伐他汀的含量测定方法。方法:采用反相高效液相色谱法,YMC-C_(18)分析柱(4.6 mm×250 mm,5μm),乙腈(A)-0.1%磷酸溶液(B)梯度洗脱[0 min(25%A)→45 min(84% A)→55 min(25% A)],流速1.0 mL·min~(-1),进样量10μL,256 nm 检测。结果:大豆苷元、黄豆黄素、染料木素和洛伐他汀进样浓度分别在13.6×10~(-3)～81.6×10~(-3)μg(r=1.0000),8.0×10~(-3)～48.0×10~(-3)μg(r=1.0000),24.0×10~(-3)～144.0×10~(-3)μg(r=1.0000),4.50～8.25μg(r=0.9996)范围内与峰面积呈良好线性关系;平均加样回收率分别为99.1%,98.5%,98.2%,99.6%,RSD 分别为1.75%,1.37%,1.54%,1.49%(n=5)。结论:方法操作简便,准确,重复性好,可用于同时测定血脂康胶囊中大豆苷元、黄豆黄素、染料木素和洛伐他汀的含量。     

基于网络药理学及分子对接探讨淡豆豉抗抑郁的作用机制

目的 采用网络药理学和分子对接技术预测淡豆豉抗抑郁活性成分、作用靶点及通路，探讨其潜在作用机制。方法 利用CNKI、TCMSP、TCMIP检索并筛选淡豆豉抗抑郁潜在活性成分；运用PharmMapper服务器预测活性成分的作用靶点；利用UniProt数据库查询靶点蛋白对应的基因名；运用DrugBank、GeneCards和DisGeNET数据库检索抗抑郁作用靶基因，并将活性成分靶点相互映射获得淡豆豉活性成分抗抑郁靶点；通过String数据库下载靶蛋白相互作用数据，与获取的潜在作用靶点利用Cytoscape 3.7.2.软件构建淡豆豉–活性成分–潜在靶点相互作用网络图。运用DAVID数据库分析潜在靶点的基因本体（GO）分子功能和京都基因与基因组百科全书（KEGG）信号通路，最后利用Autodock Vina和Pymol软件对药物有效活性成分和关键靶点进行分子对接验证。结果 预测获得淡豆豉主要活性成分8个，作用靶点396个，与抑郁症交集潜在靶点334个，GO分子功能和KEGG通路涉及多种生物学过程以及脂质和动脉粥样硬化通路、磷脂酰肌醇-3激酶/蛋白激酶B（PI3K-Akt）信号通路、小分子量G蛋白（Ras）信号通路、叉头框蛋白（FoxO）信号通路等多个信号通路。将关键活性成分和靶点进行对接，其中丝氨酸-苏氨酸蛋白激酶1（AKT1）与大豆苷元结合能力较好；磷酸肌醇3-激酶调节亚基1（PIK3R1）与木犀草素、槲皮素结合能力较好；HSP90-α热休克蛋白（HSP90AA1）与黄豆黄素结合能力较好。结论 淡豆豉以多种活性成分、多个作用靶点和途径发挥其抗抑郁作用，为淡豆豉在临床上用于抑郁症的干预和治疗提供依据。     

GC-MS method for the simultaneous determination of β-blockers, flavonoids, isoflavones and their metabolites in human urine

A sensitive and selective method based on gas chromatography hyphenated to mass spectrometry (GC-MS) for the screening of 23 different compounds including β-blockers, flavonoids, isoflavones and metabolites in human urine sample was developed and validated. The present paper reports, for the first time, the method for the simultaneous determination of β-blockers, isoflavones, flavonoids and metabolites in human urine samples. When flavonoids are ingested in combination with drugs that have a narrow therapeutic range, interactions between flavonoids and drugs should be investigated.Substances of interest were extracted from urine samples by solid-phase extraction (SPE) employing a mixture of tert-butyl methyl ether:methanol:formic acid (4.5:4.5:1; v/v/v) as a mobile phase and Oasis HLB (Waters) as a stationary phase. Before extraction, urine samples were incubated with β-glucuronidase/sulfatase in order to achieve enzymatic hydrolysis. Before GC-MS analysis the analytes had to be derivatized with N-methyl-N-(trimethylsilyl)trifluoroacetamide (MSTFA) into their trimethylsilyl derivatives by incubating for 60 min at 60 °C. Statistical central composite design and response surface analysis were used to optimize the derivatization reagent. These multivariate procedures were efficient in determining the optimal separation condition, using peak areas as responses.The calibration curves were indicative of high linearity (r² ≥ 0.9992) in the range of interest for each analyte. LODs (S/N = 3) ranged between 0.6 and 9.7 ng/ml. Intra-day and inter-day precision (CV, %) was less than 4.96%, accuracy between 0.01 and 4.98% and recovery was found in the range from 70.20 to 99.55%.The developed method can be applied to the routine determination of examined compounds’ concentrations in human urine. Moreover the method is suitable for detecting pharmaceutical compounds containing β-blockers, isoflavones and flavonoids in urine after administration to humans.     

基于网络药理学及分子对接的五子衍宗丸治疗肝纤维化作用机制探讨

目的 基于网络药理学和分子对接技术探讨补肾复方五子衍宗丸治疗肝纤维化的作用机制。方法 使用中药系统药理学数据库与分析平台(TCMSP,http://lsp.nwu.edu.cn/tcmspsearch.php)数据库预测五子衍宗丸有效成分的靶点并通过Uniprot数据库进行靶点名称标准化;通过OMIM、Gene Cards、DisGeNET数据库收集肝纤维化靶点,通过韦恩图筛选活性成分靶点和疾病靶点基因的交集,获取五子衍宗丸治疗肝纤维化的潜在靶点;运用Cytoscape 3.9.0构建有效成分-靶点-肝纤维化网络,预测核心靶点,构建基于STRING平台的蛋白质-蛋白质相互作用(PPI)网络;利用微生信平台进行基因本体(GO)功能和京都基因与基因组百科全书(KEGG)通路富集分析;使用AutoDockTools软件,结合PPI网络及GO功能、KEGG通路分析,将核心靶点与有效成分进行对接验证。结果 获得五子衍宗丸活性成分84个,筛选出有效成分作用靶点179个,肝纤维化靶点798个,五子衍宗丸与肝纤维化交集靶点81个,经拓扑属性分析筛选得到五子衍宗丸26个活性成分、37个治疗肝纤维化关键靶点;根据“活性成分-靶点-通路”网络图,预测槲皮素、山柰酚、苦参碱、黄豆黄素为五子衍宗丸作用于肝纤维化的重要活性成分;RAC-α丝氨酸/苏氨酸蛋白激酶(AKT1)、肿瘤坏死因子(TNF)、白细胞介素-6(IL6)、细胞肿瘤抗原p53(TP53)、血管内皮生长因子A(VEGFA)等为核心靶点。GO功能富集分析的生物学过程(BP)主要与RNA聚合酶II启动子转录的正调控、基因表达的正调控等关联;细胞组分(CC)主要与细胞外空间、细胞核等关系密切;分子功能(MF)主要与大分子复合物结合、蛋白激酶活性、RNA聚合酶II核心启动子近端序列特异性DNA结合等相关。五子衍宗丸治疗肝纤维化途径主要包括化学致癌-活性氧信号通路、JAK-STAT信号通路、PI3K-Akt信号通路、MAPK信号通路等。核心成分槲皮素、山柰酚、苦参碱、黄豆黄素与核心作用靶点AKT1、TNF、IL6、VEGFA、CASP3等对接结合稳定,分子对接初步证明关键成分自发地与多个核心蛋白结合,可对多个关键靶点进行调控。结论 通过网络药理学及分子对接初步揭示补肾复方五子衍宗丸影响凋亡过程的正负调控、氧化应激、炎症因子、血管生成等反应过程,具有多成分、多靶点、多通路等作用特点,可为临床肝纤维化治疗中重视补肾法提供参考依据。     

黑豆、半成品和成品淡豆豉中6种异黄酮含量变化比较

目的 比较发酵前的黑豆及半成品和成品淡豆豉中大豆苷、黄豆黄苷、染料木苷、大豆苷元、黄豆黄素和染料木素6种异黄酮的含量变化。方法 采用高效液相色谱法测定异黄酮的含量,色谱条件为DiamonsilC₁₈色谱柱(4.6×250 mm,5μm),柱温30℃,检测波长为260 nm,流动相为0.2%醋酸水(A)-甲醇(B),梯度洗脱,流速为1.0 ml/min。结果 该方法测定6种异黄酮成分在测定范围内线性良好(r≥0.999 3),且回收率符合要求,黑豆经发酵后其中的6种异黄酮含量明显提升,且成品淡豆豉比半成品淡豆豉中含量更多。结论 本研究建立的高效液相色谱法能够准确测定淡豆豉中6种异黄酮的含量。发酵可提升黑豆中的异黄酮的含量,且在发酵过程中结合型异黄酮向游离型异黄酮苷元转化。