纯种发酵淡豆豉制备工艺的优化研究

目的:优化纯种发酵淡豆豉的制备工艺,明确工艺参数,为纯种发酵淡豆豉的质量控制提供依据。方法:以样本中大豆黄素和染料木素的含量为指标,分别考察不同原料和辅料的使用及发酵时间对其质量的影响,从而规范纯种发酵淡豆豉的制备工艺。结果:纯种发酵淡豆豉最佳制备方法为以黑大豆为原料,青蒿、桑叶各20g,用水浸泡辅料20～30min后煎煮3次,每次1h,合并煎煮液并定容至2L,拌入200g黑大豆中,105℃灭菌15min。接菌比例为大豆30g∶菌液10mL,发酵温度为25℃,发酵时间为9d。结论:优化工艺适合纯种发酵淡豆豉的制备。     

HPLC法测定淡豆豉中异黄酮的含量

目的 :探讨以 HPL C法对淡豆豉中异黄酮的定量分析。 方法 :采用 HPL C法 ,YWGC1 8柱 ,以甲醇∶水∶乙酸( 10∶ 10∶ 1)为流动相 ,检测波长 2 6 0 nm ,测定淡豆豉中染料木素、大豆黄素含量。 结果 :淡豆豉中游离染料木素含量为( 2 30 .6 4± 9.14)μg/ g、大豆黄素含量为 ( 2 6 4.2 6± 4.2 2 )μg/ g;用盐酸水解处理后样品中染料木素总含量为 ( 2 76 .0 0± 7.81)μg/ g,大豆黄素总含量为 ( 2 87.6 5± 5 .70 ) μg/ g。 结论 :用 HPL C法测定淡豆豉中异黄酮类成分含量 ,建立的方法准确、简便 ,可用于控制和评价淡豆豉的品质。     

淡豆豉异黄酮的提取分离及对DPPH自由基清除能力的研究

目的:考察纯种发酵淡豆豉中异黄酮的最佳提取和纯化工艺,并考察异黄酮提取物的体外抗氧化能力。方法:以异黄酮中的主要有效成分大豆黄素和染料木素含量为指标,采用正交试验优化淡豆豉异黄酮的超声辅助提取工艺和大孔树脂纯化工艺,并考察异黄酮体外清除DPPH自由基的能力。结果:淡豆豉异黄酮超声辅助提取最佳工艺为超声时间90 min,酶量10 U,料液比1∶10,在此条件下淡豆豉中异黄酮的提取率为0.598%。使用HPD-100型大孔树脂进行分离纯化效果最好。在DPPH自由基清除实验中,淡豆豉异黄酮提取物IC_(50)(半数有效抑制浓度)为8.18μg/mL,BHA为15.79μg/mL。结论:按本实验结果提取的淡豆豉异黄酮提取率较高,且提取物具有显著的DPPH自由基清除效果。     

染料木素和大豆黄素对海马神经细胞H19—7增殖和神经营养因子表达的影响

目的 探讨染料木素和大豆黄素对海马神经细胞的活性和增殖作用及其可能机制.方法 H19-7/IGF-IR神经细胞在无酚红无血清DMEM培养液中培养72h后,分别加入一定浓度的染料木素、大豆黄素和雌二醇,用MTT和BrdU法分别检测细胞的活性和增殖,流式细胞术检测神经细胞周期,ELISA和RT-PCR法检测脑源性神经营养因子（BDNF）的表达.结果 处理细胞72h后,与对照组相比,20nM、200 Nm雌激素组H19-7细胞增殖分别提高了33%、36%;20 Nm、200 Nm染料木素组H19-7细胞增殖分别提高了15%、13%;200nM大豆黄素组H19-7细胞增殖分别提高了11%,差异有显著性（P＜0.05）.200nM染料木素和大豆黄素的S期细胞比例[分别为（17.64±0.43）%,（19.48±1.01）%]显著高于对照组（14.21 ± 1.75）%,差异具有显著性（P＜0.05）.染料木素和大豆黄素显著增加海马神经细胞成熟型BDNF水平及其Mrna的表达（P＜0.05）.酪氨酸激酶受体阻断剂K252a能阻断染料木素和大豆黄素的促海马神经细胞增殖作用.结论 染料木素和大豆黄素改善海马神经细胞的增殖和活性能力,其作用与促进海马细胞BDNF的表达有关.     

不同辅料炮制对淡豆豉中大豆异黄酮含量的影响

目的考察用不同辅料炮制淡豆豉对其大豆异黄酮含量的影响。方法采用发酵法制取淡豆豉样品,用HPLC色谱法测定样品中大豆异黄酮的含量。色谱柱为Hanbo（250 mm×4.6 mm,5μm）;流动相为甲醇∶水∶冰醋酸（40∶60∶0.5）;流速：1 m L/min;检测波长：260 nm。结果以大豆苷为考察指标时三者的差别不大,与青蒿、桑叶比较,淫羊藿和人参为辅料发酵的淡豆豉含量略低;以染料木苷为考察指标时,与青蒿、桑叶比较,淫羊藿为辅料发酵的淡豆豉含量略有升高,人参为辅料发酵的淡豆豉含量升高明显。结论炮制辅料不同,对淡豆豉中大豆异黄酮含量有一定影响,且人参为炮制辅料时影响较大。     

大豆异黄酮以雌激素非依赖方式改善自发性高血压大鼠的内皮功能

大豆异黄酮类化合物中染料木素和大豆黄素的结构与17β-雌二醇非常相似，都是雌激素受体α型和β型的配体，且对雌激素受体β型有更强的亲和力。作者比较研究了染料木素、大豆黄素和17β-雌二醇对自发性高血压大鼠（SHR）和Wistar Kyoto大鼠（WKY）离体主动脉环内皮功能的作用和可能机制。     

异黄酮类植物雌激素通过雌激素受体对靶基因的转录调节作用

目的:观察异黄酮类植物雌激素染料木素和大豆苷元通过雌激素受体(estrogen receptor,ER)对靶基因的转录调节作用。方法:采用磷酸钙瞬时转染的方法,利用转染雌激素受体表达质粒的Hela细胞,观察染料木素和大豆苷元对雌激素反应元件(estrogen response element,ERE)报告基因的转录激活作用;此外还观察ER拮抗剂ICI 182780对这两种植物雌激素转录激活作用的影响。结果:染料木素和大豆苷元均可通过ER的两种亚型ERα和ERβ激活ERE报告基因的转录,且这种转录激活作用能被ICI 182780所阻断。结论:染料木素和大豆苷元均能产生类雌激素样作用,通过ERα和ERβ激活ER靶基因的转录。     

高速逆流色谱法分离制备淡豆豉中大豆素和染料木素

淡豆豉是经大豆Glycine maa T（L．）Merr．发酵而来的传统中药，其中所含的大豆异黄酮是该药主要活性成分之一。近年来的研究结果表明，大豆异黄酮是一类重要的生理活性物质，具有预防心血管疾病、防癌抗癌、治疗骨质疏松、降低妇女更年期综合症等功效。经发酵处理后大豆异黄酮中游离型苷元的质量分数明显提高，如大豆素和染料木素，而游离型大豆异黄酮比结合型大豆异黄酮具有更强的生物活性。因此，如何简便、快速从淡豆豉中分离制备大豆异黄酮苷元对进一步进行药效与作用机制研究、     

染料木素和大豆黄素对海马神经细胞H19-7增殖和神经营养因子表达的影响

目的 探讨染料木素和大豆黄素对海马神经细胞的活性和增殖作用及其可能机制.方法 H19-7/IGF-IR神经细胞在无酚红无血清DMEM培养液中培养72h后,分别加入一定浓度的染料木素、大豆黄素和雌二醇,用MTT和BrdU法分别检测细胞的活性和增殖,流式细胞术检测神经细胞周期,ELISA和RT-PCR法检测脑源性神经营养因子(BDNF)的表达.结果 处理细胞72h后,与对照组相比,20nM、200 Nm雌激素组H19-7细胞增殖分别提高了33%、36%;20 Nm、200 Nm染料木素组H19-7细胞增殖分别提高了15%、13%;200nM大豆黄素组H19-7细胞增殖分别提高了11%,差异有显著性(P＜0.05).200nM染料木素和大豆黄素的S期细胞比例[分别为(17.64±0.43)%,(19.48±1.01)%]显著高于对照组(14.21 ± 1.75)%,差异具有显著性(P＜0.05).染料木素和大豆黄素显著增加海马神经细胞成熟型BDNF水平及其Mrna的表达(P＜0.05).酪氨酸激酶受体阻断剂K252a能阻断染料木素和大豆黄素的促海马神经细胞增殖作用.结论 染料木素和大豆黄素改善海马神经细胞的增殖和活性能力,其作用与促进海马细胞BDNF的表达有关.

Abstract：

Objective To determine the effects of genistein and daidzein on the proliferation and survival of the hippocampal neural cells and underlying mechanism. Methods H19-7/IGF-IR neural cell line was cultured in phenol red free DMEM absented of serum for 72h. Genistein, daidzein or 17β-estradiol was added to the culture at various concentrations. Their proliferation and protective effects on the neuronal cells were determined by BrdU and MTT assay respectively. The effect of phytoestrogens on cell cycle regulation was determined using flow cytometry. The effects of the soy isoflavones on brain-derived neurotrophic factor (BDNF) expression were determined by ELISA and RT-PCR respectively. Results It was observed that, with 72h of treatment, 20nM and 200 nM 17B-estradiol significantly promoted the neuronal cell proliferation at 33% and 36% ;20nM and 200nM genistein significantly promoted the neuronal cell proliferation at 15% and 13% ; 200nM daidzein significantly promoted the neuronal cell proliferation at 11% compared to the control (P＜0.05). Genistein and daidzein induced an significantly increase in the S phase arrest at (17.64 ± 0.43) % and (19.48 ± 1.01) % compared to the control (P ＜ 0. 05). Moreover, genistein and daidzein significantly increased the expression of mature BDNF and BDNF mRNA level (P＜0.05). The effect of genistein and daidzein on hippocampal neuronal cell proliferation was blocked by K252a, selective inhibitor of tyrosine kinase receptors. Conclusion Genistein and daidzein improved hippocampal neuronal cell proliferation and viability in vitro. The effects might be mediated by increasing in BDNF expression.     

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。     

Modulation of Isoflavones on Bone—nodule Formation in Rat Calvaria Osteoblasts in vitro

Objective:to observe the effects of two main isoflavones,daidzein and genistein on the bone-nodule formation in rat calvaria osteoblasts in vitro.Methods:Osteoblasts obtained from newborn Sprague-dawley rat calvarias were cultured for several generations.The second generation cells were cultured in Minimum Essential Medium supplemenmted with ascorbic acid and Na-beta-glycerophosphate for several days,in the presence of daidzein and genistein,with or without the estrogen receptor antagonist ICI 182780.Number of nodules was counted at the end of the incubation period(day 20) by staining with Alizarin Red S calcium stain.The release of osteocalcin,as a marker of osteoblast activity,was also determined on day 7 and 12 during the incubation period.Results:compared with the control,the numbers of nodules were both increased by incubation with daidzin and genistein,17β-estradiol was used as a positive control and proved to be a more effective inducer of the increase in bone-nodules formation than daidzein and genisterin.The release of osteocalcin into culture media was also increased in the presence of daidzein and genistein,as well as 17β-estradiol on day 7 and day 12(day 12 were higher).The estrogen receptor antagonist ICI 182780 completely blocked the genistein-and 17β0estradiol-induced increase of nodule numbers and osteocalcin release in osteoblasts.Howerver,the effects induced by daidzein could not be inhibited by ICI 182780.Conclusion:These findings suggest that geinistein can stimulate bone-nodule formation and increase the release of osteocalcin in rat osteoblasts.The effects,like those induced by 17β-estradiol,are mediated by the estrogen receptor dependent pathway,Daidzin also can stimulate bone-nodule formation and increase the release of osteocalcin in rat osteoblasts,but it is not,at least not merely,mediated by the estrogen receptor dependent pathway.     

大豆异黄酮激活PPARγ促进人前列腺癌DU-145细胞凋亡

目的探讨染料异黄酮(genistein,GEN)和大豆苷元(daidzein,DAI)对人前列腺癌DU-145细胞凋亡的影响及其与过氧化物酶体增殖物激活体受体γ(peroxisome proliferators-activated receptorγ,PPARγ)的关系。方法采用过氧化物酶体增殖物反应元件(peroxisome proliferator responsive element,PPRE)驱动的荧光素酶报告基因检测GEN、DAI对DU-145细胞PPARγ的激活作用;GEN、DAI单独或联合PPARγ选择性拮抗剂GW9662处理DU-145细胞,采用免疫荧光化学染色方法观察PPARγ定位分布变化;TUNEL法和AnnexinⅤ/PI双染色流式细胞术检测细胞凋亡的变化。结果 GEN、DAI明显增强转染PPRE-TK-Luc质粒的DU-145细胞中荧光素酶表达活性,且这种作用可被GW9662所逆转。GEN或DAI单独作用于DU-145细胞时,PPARγ发生核移位;细胞凋亡率明显增加(P<0.05)。GW9662分别与GEN或DAI联合作用时,GEN、DAI促进PPARγ核移位和诱导细胞凋亡的作用明显削弱(P<0.05)。结论大豆异黄酮可通过激活PPARγ信号途径,促进人前列腺癌DU-145细胞凋亡。     

金雀黄素和大豆黄酮对人乳腺癌细胞系体外增殖作用的影响

应用体外细胞培养和MTT比色法.研究大豆异黄酮类化合物金雀黄素和大豆黄酮对人乳腺癌细胞系体外增殖作用的影响.结果大豆异黄酮类化合物能明显抑制人乳腺癌细胞系MCF-7 和MDA-MB-231的体外增殖,且呈明显的剂量依赖性和时间依赖性.金雀黄素和大豆黄酮对MDA-MB-231细胞的抑制作用不如MCF-7明显,而金雀黄素的抑制作用优于大豆黄酮.说明金雀黄素和大豆黄酮对乳腺癌细胞系体外增殖具有明显的抑制作用,为进一步阐明其防癌抑癌作用机理奠定了实验基础.     

高效液相色谱法测定食品中大豆异黄酮

目的：建立高效液相色谱法测定食品中大豆异黄酮（包括染料木黄酮和大豆苷元）的含量，探讨样品的提取方法及测定影响因素。方法：样品经0.50mol/L盐酸回液和乙醇提取大豆异黄酮后，采用甲醇－0.01mol/L乙酸铵溶液（pH4.5)(60 40)作为流动相分离待测物质，等度洗脱，采用紫外检测器于254nm波长下测定。结果：染料木黄酮的检出限为0．04μg/ml，大豆苷元为0．05μg/ml，样品的加标回收率为91．4％－113．1％，日内和日间相对标准偏差分别为1．1％－2．8％和2．9％－5．1％。结论：本法简便、快速，易于推广普及，为大豆及其制品和保健食品中大豆异黄酮的检测和膳食摄取植物性雌激素提供了分析方法。     

Influence of Isoflavones on Cadmium-induced Adverse Effects in Vascular Endothelial Cells （ECV 304）

To study the possible intervention of isoflavones in cytotoxicity induced by cadmium in vascular endothelial cells. Methods An ECV 304 cell line derived from human umbilical vein endothelial cells was adopted. Genistein / daidzein was added prior to or simultaneously with CdCl2, cell viability was determined by MTT assay, and metallothionein mRNA expression was monitored by RT-PCR method. Results Cell viability was higher in isoflavone and CdCl2. co-treated groups than that in CdCl2 treated group, with CdCl2 concentration at 10, 20, 40, and 80 μmol/L, respectively. However this increase was not observed in the group treated with CdCl2 at a concentration of 60 μmol/L, lsoflavones (10^-1mol/L to 10^-5 mol/L) were added 24 h before cells were challenged with 80 μmol/L CdCl2 for 24 h or simultaneously with 80 ^-10mol/L CdCl2. Genisteinincreased cell viability only at 10^-5 mol/L, while daidzein caused a dose-dependent increase from 10^-10 mol/L to 10^-5 mol/L in co-treatment with CdCl2. In pre-treatment, genistein (10^-7 to 10^-5 mol/L) increased cell viability whereas only 10^-5 mol/L of daidzein exerted protection. Apparent protection could be found when the cells were pre-treated with 10^-5 mol/L isoflavones for over 12 h, whereas 24 h incubation was required in such a co-treatment, with the exception of daidzein that had a significant protection in only 3 h. Isoflavones (10^-6 mol/L) incubated for 3 h to 24 h, increased MT ⅡA and MT IF mRNA expression, but the induction could not last for more than 24 h. Co-treatment with isoflavones could induce an additional induction of MT ⅡA mRNA expression in cells exposed to cadmium. However, the additional induction of MT ⅡA and MT IF mRNA was not seenwhen pre-treatment was carried out with isoflavones, with the exception of an increase in MT ⅡA mRNA expression in the daidzein pre-treated group. Conclusion Genistein/daidzein could reverse the cytotoxicity of cadmium either in pre-treatment or in co-treatment. The protection is the strongest in 10^-5 mol/L of isoflavones with a dose-dependent pattern. There are differences between genistein and daidzein in their protective effects. Whether the protection of isoflavones is related to their capacity of inducing MT mRNA expression remains to be elucidated.     

C5位上羟基对染料木黄酮抑制重组人蛋白激酶CK2活性的影响

目的观察染料木黄酮C5位上的羟基对其抑制重组人蛋白激酶CK2全酶活性的影响。方法将利用基因工程技术获得的重组人CK2α’及β亚基在体外等摩尔混合构成CK2全酶。通过测定药物作用后转移到CK2底物上的[γ-32P]ATP的32P的放射性活度,探讨染料木黄酮和大豆甙元对重组人CK2全酶活性的抑制作用,并采用半数效量概率单位法计算其IC50值。结果染料木黄酮和大豆甙元能明显抑制重组人CK2全酶活性,其IC50值分别是27.95μmol/L和2.38μmol/L,作用效果接近或者强于目前已知的CK2抑制剂N-(2-氨乙基)-5-氯萘-1-硫胺(A3)。结构效应分析表明:C5位上的羟基可减弱染料木黄酮对重组人CK2全酶的抑制作用。结论染料木黄酮和大豆甙元是两种有效的重组人CK2全酶的抑制剂。