白藜芦醇对亚硝酸盐的清除及亚硝胺阻断作用研究

首页设计目录摘要·································································································1关键词······························································································1前言·································································································11试剂和仪器·····················································································21.1试剂···························································································21.2仪器···························································································22原理······························································································22.1亚硝酸钠的清除率的测定································································22.2阻断亚硝胺合成的原理···································································23实验方法························································································33.1溶液的配制··················································································33.2NaNO2标准液的制备及吸光度的测定·················································33.3不同浓度白藜芦醇溶液在pH=3时对亚硝酸钠的清除率的测定·················43.4不同浓度白藜芦醇溶液在pH=3时对亚硝胺合成的阻断率测定·················44结果及分析····················································································54.1NaNO2溶液的标准曲线的绘制···························································54.2不同浓度的白藜芦醇在pH=3时对亚硝酸钠的清除率及对亚硝胺的阻断率··55讨论及结论·····················································································7致谢·································································································7参考文献···························································································8英文摘要···························································································9加入页眉本科毕业论文1白藜芦醇对亚硝酸盐的清除及亚硝胺阻断作用研究药物制剂：刘玉锋指导老师：张孝林摘要：目的：以亚硝酸盐的清除率和亚硝胺合成的阻断率为指标，研究不同浓度的白藜芦醇在酸性下对亚硝酸盐的清除和对亚硝胺合成的阻断作用。方法：分别配制0.1mg/mL、0.2mg/mL、0.4mg/mL、0.8mg/mL、0.16mg/mL、0.32mg/mL和0.64mg/mL浓度的白藜芦醇溶液和5ug/mL亚硝酸钠溶液,在pH=3模拟胃酸条件下,采用分光光度法，分别测定不同浓度的白藜芦醇对亚硝酸钠的清除率及亚硝胺生成的阻断率[1]。结果：在酸性条件下，不同浓度的白藜芦醇对亚硝酸钠均有较强清除作用，在浓度小于0.32mg/mL时随着浓度增大而增大，最大达到63.48%。同时对亚硝胺的生成有较强的阻断率，最大达到40.94%。结论：白藜芦醇对亚硝酸盐有很强的清除能力，对亚硝胺的生成有较强的阻断能力。关键词：亚硝酸盐，白藜芦醇，分光光度法，亚硝胺，清除，阻断前言癌症是当前威胁人类生活的最大杀手,根据世界卫生组织统计,全世界每年约有500万左右的人被癌症夺去生命。在致癌物中,亚硝胺是最令人关注的一类化学致癌物质。亚硝酸盐作为一种发色剂和防腐剂[2]，在肉制品加工中得到了普遍的应用[3]。许多蔬菜中天然含有的硝酸盐，在人体内通过细菌的作用也可被还原为亚硝酸盐。亚硝酸盐是合成亚硝胺的前体之一[4]，人体内的这些亚硝酸盐在一定条件下可以与食物中的仲胺、叔胺等形成亚硝胺。N-亚硝基化合物是一种致癌性很强的化合物[5]，已研究的近300种N-亚硝基化合物中，约90%以上具有动物致癌性，并证明N-亚硝基化合物可在体内外由前体物胺类与硝酸盐或亚硝酸盐形成[6]。在体内代谢过程中，特别是在胃液中容易形成亚硝胺[7]。因此，降低胃内的亚硝酸盐含量及阻断亚硝胺前提的合成[7]从而达到防癌抗癌的目的是人类同癌症斗争的有效途径之一，而白藜芦醇在这方面的研究鲜见报道。白藜芦醇(resveratrol，简称Res)是非黄酮类的多酚化合物，日本人Tokaoka1940年首次从毛叶藜芦的根部分离得到。1963年Nonomura等提出白藜芦醇是某些草药治疗炎症、脂类代谢紊乱和心脏疾病等的有效成分，是存在于植物中的天然抗氧化剂，如葡萄、虎杖等[8]被列为抗心血管、抗癌最有前景的药物之一。白藜芦醇具有优良的药理活性和保健功能，其市场需求很大且与日剧增。目前，已有大部分国家和地区都开发了白藜芦醇的相关保健品和其他产品。中国已将含白藜芦醇的植物提取物制成降脂美容的天然保健食品，美国也已把白藜芦醇作为膳食补充剂，日本已将从植物提取的白藜芦醇作为食品添加剂[9]。白藜芦醇的药理功能引起了人们的广泛兴趣，因为它涉及到与人体健康密切相关的许多生理疾病，例如，动脉粥样化、老年痴呆症、病毒性肝炎、胃溃疡、炎症与过敏反应加入页眉本科毕业论文2等。随着人们对白藜芦醇的深入研究，已经显示白藜芦醇具有抗癌、抗氧化、抗菌、抗炎以及预防食物过敏等作用，可广泛地应用于医药、保健品、食品、化妆品等领域。所以，对白藜芦醇的研究及开发，特别是白藜芦醇对亚硝酸盐的清除及阻断作用具有良好的市场前景及重要的意义。1试剂和仪器1.1试剂白藜芦醇（纯度99%，购于陕西西慈生物技术有限公司）、蒸馏水、二乙胺盐酸盐、二水合柠檬酸三钠、十二水合磷酸氢二钠、二甲胺盐酸盐、磷酸二氢钠（均为AR，购于国药集团化学试剂有限公司）、亚硝酸钠（AR，淮化试剂厂）、无水对氨基苯磺酸（AR，上海山浦化工有限公司）、95%乙醇、氢氧化钠、盐酸（均为AR，合肥工业大学化学试剂厂）1.2仪器紫外可见分光光度计UV-5500PC型（上海元析仪器有限公司）、电子分析天平（上海精密科学仪器有限公司）、恒温水浴锅（江苏省金坛市荣华仪器制造有限公司）、超净工作台（苏州苏净有限公司）2原理2.1亚硝酸钠的清除率的测定[10]亚硝酸钠在弱酸性的条件下,能与氨基苯磺酸重氮化后,再与N-1-萘乙二胺盐酸盐偶合生成红色的化合物，本实验通过测定相同条件下亚硝酸钠含量的变化来反映白藜芦醇对亚硝酸根离子的清除能力，并计算清除率：清除率=(A0-Ax)/A0×100%A0—没有加入白藜芦醇的亚硝酸钠溶液空白实验的吸光度值；Ax—加入不同浓度白藜芦醇溶液时的反应液的平均吸光度值。温度为37℃在pH=3的条件下，当用相同量的亚硝酸钠溶液同具有一定浓度梯度的白藜芦醇溶液反应后，再测量吸光度。吸光度越小，溶液中残存的亚硝酸钠含量就越小，表明白藜芦醇对亚硝酸盐的清除率越高；反之，表明白藜芦醇对亚硝酸盐的清除率越低。2.2阻断亚硝胺合成的原理[11]二甲胺与亚硝酸钠在37℃条件下,可适宜地生成二甲基亚硝胺,反应式如下:(CH3)2NH+NaNO2HCl(CH3)2NH·NO+NaCl+H2O其实质为:加入页眉本科毕业论文3(CH3)2NH+HONO(CH3)2NH·NO+H2O当往白藜芦醇溶液中依次加入二甲胺与亚硝酸钠时,白藜芦醇优先同亚硝酸钠作用,使得二甲胺不能与亚硝酸钠反应,达到阻止二甲基亚硝胺生成的目的。据此可以比较相同条件下生成二甲基亚硝胺量的多少来反映白藜芦醇阻断能力的强弱,生成二甲基亚硝胺量少,白藜芦醇的阻断能力就强,反之则弱。在紫外光照射下,二甲基亚硝胺可分解成二甲基仲胺和亚硝酸根,反应式如下:H2O+(CH3)2N-N=O紫外光(CH3)2NH2++NO2-亚硝酸根与对氨基苯磺酸重氮化后,再与1-萘胺偶合生成红色化合物。用紫外分光光度计测出该红色化合物的吸光度值可反映出溶液中亚硝酸根离子的含量，亚硝酸根离子的含量可反映出原来溶液中二甲基亚硝胺含量的多少。3方法3.1溶液的配制3.1.10.4%对氨基苯磺酸溶液的配制：用分析天平精确称取对氨基苯磺酸0.4g充分溶解后，定容至100mL。3.1.20.2%盐酸萘乙二胺溶液的配制：精确称取盐酸萘乙二胺0.2g充分溶解后，定容至100mL。3.1.30.1%1-萘胺溶液的配制：精确称取1-萘胺0.1g充分溶解后，定容至100mL。3.1.4浓度为1mmol/L的二甲胺盐酸盐溶液的配制：精确称取二甲胺盐酸盐0.08541g充分溶解后，定容至100mL。3.1.5浓度为1mmol/L的亚硝酸钠溶液的配制：精确称取亚硝酸钠0.069g充分溶解后，定容至1000mL。3.1.6不同浓度的白藜芦醇溶液的配制：精确称取64mg白藜芦醇，置于烧杯中，加适量95%乙醇溶解，用蒸馏水定容于100mL容量瓶中，配制成0.64mg/mL的白藜芦醇溶液并标记为7号容量瓶，再分别稀释配制成0.01mg/mL、0.02mg/mL、0.04mg/mL、0.08mg/mL、0.16mg/mL和0.32mg/mL白藜芦醇溶液，分别标记为1,2,3,4,5,6号容量瓶，避光保存，备用。3.2NaNO2标准液的制备及吸光度的测定[12]NaNO2标准液制备:用天平精确称取亚硝酸钠20mg，充分溶解后，定容至1L即得20mg/L的NaNO2溶液。然后准确取20mg/L的NaNO2溶液250mL于一1L的容量瓶中并用蒸加入页眉本科毕业论文4馏水稀释、定容至刻度，摇匀后得到浓度为5ug/mL的NaNO2标准液。NaNO2标准曲线的绘制:分别准确吸取浓度为5ug/mL的NaNO2标准液0mL,0.2mL,0.4mL,0.6mL,0.8mL,1.0mL,1.5mL,2.0mL置于10mL具塞试管中，并分别编号为0，1，2，3，4，5，6，7号试管。然后分别向各试管中加入0.4%对氮基苯磺酸1mL，摇匀，静置3—5min，再加入0.2%的盐酸萘乙二胺溶液0.5mL,用水稀释至刻度，摇匀得到浓度分别为0ug/mL，0.1ug/mL，0.2ug/mL