【作者】 姜慧萍；

【导师】 郑群雄；

【作者基本信息】 浙江工商大学 ， 食品科学， 2009， 硕士

【摘要】 NO₂^-在体内外的弱酸性条件下均可能与胺发生亚硝化反应生成强致癌性的N-亚硝胺,从而诱发机理癌变。因此,如何清除NO₂^-或抑制N-亚硝胺的生成,进而减少肿瘤的发病率已成为食品安全领域研究的热点之一。黄酮作为一种天然的植物次级代谢产物广泛分布于植物中,由于其具有抗氧化、抗肿瘤、抗衰老等生理活性,已被开发成多种保健食品。随着对黄酮活性研究的不断深入,发现黄酮还具有清除NO₂^-、抑制N-亚硝胺生成的功效,目前这方面的研究仍停留在黄酮粗提物的水平上,如竹叶黄酮、甘草黄酮、苦荞黄酮等。本研究对黄酮单体抑制N-亚硝基二乙胺（N-nitrosodiethylamine,NDEA）的合成进行了深入的探索,为有效利用黄酮类化合物作为外源性的N-亚硝胺合成抑制剂奠定基础。研究内容包括:1.建立NO₂^-和NDEA的测定方法。2.以表没食子儿茶素没食子酸酯（EGCE）作为参比,选择槲皮素、木犀草素、白杨素三种不同结构的单体黄酮为研究对象,考察黄酮结构、浓度、抗氧化力与NDEA生成量之间的关系。选择邻苯二酚、间苯二酚、对苯二酚、邻苯三酚为对象,探讨酚羟基位置及数量对NDEA生成的影响。3.在模拟胃液条件下（pH3.0,37℃）,分析黄酮的代表性物质槲皮素抑制NDEA生成后所得的产物。利用制备型高效液相（PRE-HPLC）对产物进行分离纯化;用紫外可见光分光光度计（UV/VIS）、高效液相-质谱联用仪（HPLC-MS/MS）及核磁共振氢谱（1^H-NMR）鉴定产物结构;利用混合物的¹H-NMR特征图谱推测产物的含量关系,利用反相高效液相色谱-二极管阵列检测器（RP-HPLC-DAD）验证所推测的结果。4.在此基础上,对菊米、银杏叶、艾草3种植物的黄酬粗提物进行研究,考察了NO₂^-清除率及NDEA生成的抑制率随粗提物浓度、反应时间、pH、温度的变化。研究结果如下:1.槲皮素、木犀草素、白杨素均具有清除NO₂^-的能力,清除率随黄酮浓度的增加而增加,大小依次为:槲皮素>木犀草素>白杨素。三种黄酮对致癌物NDEA的生成具有双重影响——低浓度下起促进作用,高浓度下起抑制作用。清除率及抑制率和黄酮的抗氧化能力成正比。通过比较具有不同酚羟基结构的苯酚类化合物对NDEA生成的影响推测黄酮类化合物的双重作用效果与黄酮摩尔浓度及其酚羟基位置有关:黄酮类化合物各环上的酚羟基活性相差较大,B环酚羟基活性最高,C环酚羟基次之,A环酚羟基活性最弱。当黄酬浓度达到一定量后,NO₂^-完全被黄酬B环上的邻位酚羟基反应生成了稳定的邻苯醌型产物,从而抑制NDEA生成;当黄酮浓度过低时,黄酮B环上的邻位酚羟基不足于和NO₂^-完全反应,过量的NO₂^-进而与A环上的间位酚羟基反应,产生了更强的亚硝化试剂,从而促进NDEA生成。此外,黄酮结构中酚羟基数量的多少与黄酮作用效果的强弱有关,槲皮素、木犀草素、白杨素三者羟基数量依次减少,因此在相同浓度下其作用强度依次减弱,确切的作用机制还有待进一步研究。2.通过分析得出:模拟胃液条件下,槲皮素在抑制NDEA生成的过程中被氧化成化合物C₁[2-（3,4-dihy drxybenzoyl）-2,4,6-trihyd-roxybenzofuran-3（2H）-one],该化合物又会自分解为化合物C₂[3,4-dihydroxybenzoic acid]和C₃[2,4,6-trihydroxyphenylglyoxylicacid],C₁、C₂、C₃的含量比例为1:0.2:0.5。3.对菊米、银杏叶、艾草黄酮粗提物的研究发现,黄酮含量高的两种粗体物（菊米:7.17 mg/mL,艾草:5.20 mg/mL）都具有清除NO₂^-、抑制NDEA生成的能力,清除率及抑制率和反应时间、黄酮用量成正相关性,和pH成负相关性。此外,过低或过高的温度都不利于黄酮抑制NDEA的生成。由于银杏粗提物中含游离酚羟基的黄酮含量过低（0.85mg/mL）,在三种黄酮粗提物中它对NO₂^-的清除率最差,而且始终促进NDEA的生成,。结论:研究结果表明,在弱酸性条件下,当黄酮类化合物的摩尔浓度达到一定量后,黄酮具有与亚硝酸盐发生氧化还原反应竞争性地抑制胺类与NO₂^-结合进而产生致癌性N-亚硝胺生成的能力,抑制效果与黄酮的抗氧化力及浓度呈正相关性;当黄酮浓度过少时,它对N-亚硝胺的生成具有促进作用。黄酮类化合物的这种双重作用可能和黄酮浓度及其结构中游离酚羟基的位置有关,详细的机理还有待进一步研究。通过分析槲皮素抑制NDEA生成后的产物得出,黄酮类化合物在抑制N-亚硝胺生成的过程中,自身被亚硝酸氧化生成相应的氧化型产物。更多还原

【Abstract】 Nitrite might interact with amines under the mildly acidic conditions in vitro and in vivo to generate carcinogenic nitrosamines.Thus how to scavenge nitrite or inhibite the formation of nitrosamines in vivo and in vitro has been becoming one of the hot research topics in food safety field. Flavones as secondary metabolite of plants which were widely distributing in the plant kingdom have been applied in developing health food for many biological acitivities,such as antioxidation,antitumor, antiaging,etc.With the further studying of flavone activities,it has revealed that flavones had the ability of scavenging nitrite and inhibiting the formation of nitrosamines.However,the study of flavones on inhibiting nitrosamine formation still focused on crude flavone’s extractions,such as bambool flavone,liquorice flavone,buckwheat flavone.In the present paper,we systemically studied the ability of different puri flavones in inhibiting the formation of nitrosamine to provide a preliminary theoretical basis for flavones as nitrosation inhibitor.Contents1.The methods for the determination of NO₂ and N-nitrosodiethyllamine were developed.2.Quercetin,luteolin and chrysin were used to evaluate the relationships between structure,concentration,antioxidant activity and the ability of acting in NDEA formation with EGCG as reference compound.To investigate the relationship between the amount and position of the phenolic hydroxyl and the effect action in NDEA formation, 1,2-dihydroxy benzene,1,3-dihydroxy benzene,1,4-dihydroxy benzene and 1,2,3-trihydroxybenzene were studied.3.The products of quercetin which is the typical representative of flavones after it inhibited NDEA formation in simulated gastric condition were analyzed.The products were separated by preparative high performance liquid chromatography.The products were identified using UV/VIS spectrophotometer,high performance liquid chromatography/mass spectrometry and nuclear magnetic resonance spectroscopy.Then the proportional relationship of products was calculated basing on the character of nuclear magnetic resonance spectroscopy.And the result was validated by analyzing with reversed-phase high performance liquid chromatography/diode array detector.4.Furthermore,the three crude flavone’s extractions of Jumi,Ginkgo biloba and Argy wormwood were studied,investigating the influences of extractions’ concentration,reaction time,pH and temperature on the scavenging rate and inhibiting rate.Results1.Quercetin,luteolin and chrysin could scavenge nitrite.The scavenging rate was positively related with flavones concentration and appeared to be quercetin＞luteolin＞chrysin.It was discovered that there were twofold influences of flavone in effecting NDEA formation,catalyzing NDEA forming at a low concentration and inhibiting NDEA formation at a high concentration。The scavenging rate and the inhibiting rate were positively related with the antioxidant activity of flavones.By investigating the effects of phenolic compounds with different phenolic hydroxyls in NDEA formation,it was deduced that the twofold influences of flavones in NDEA formation was depending on the concentration and the phenolic hydroxyl position of flavones.There are greatly differences of phenolic hydroxyl activity in flavones,the hydroxyls of B ring＞the hydroxyls of C ring＞the hydroxyls of A ring.Under the high concentration of flavones,NO₂^- completely interacted with the hydroxyls at B ring of flavones to form stable quinine derivatives inhibiting NDEA forming.Under the low concentration of flavones, the hydroxyls at B ring of flavones was not so enough to interact with NO₂^- that the large excess of NO₂^- would interact with the hydroxyls at A ring to generate a more powerful nitrosating agent catalyzing the NDEA formation.In addition,the amount of hydroxyls influenced the extent of flavones in effecting NDEA formation.The inhibiting rates of quercetin,luteolin and chrysin were decreasing successively as well as the amount of hydroxyls decreasing.The definitive mechanism needs to be further studied.2.The products of quercetin reacting with NO₂ ^- at the simulate gastric condition was identified as C₁[2-（3,4-dihydrxybenzoyl）-2,4,6-trihydroxybenzofuran -3（2H）-one],which could decompose to generate C₂[3,4-dihydroxybenzoic acid]and C₃[2,4,6-trihydroxyphenylglyoxylic acid].The proportional relationship of C₁,C₂ and C₃ was 1:0.2:0.8.3.By studying the effect of the crude flavone’s extractions of Jumi, Ginkgo biloba and Argy wormwood effecting on nitrosation,it was shown that the two crude flavone’s extractions containing flavones at the high concentration（Jumi:7.17 mg/mL,Argy wormwood:5.20 mg/mL） had the effects of scavenging nitrite and inhibiting NDEA formation.The scavenging rate and inhibiting rate were positively related with the reacting time and flavones’ concentration and negatively related with the pH value.That the temperature was too high or low was unfavorable to the inhibiting effect of flavones.The crude flavone’s extraction containing flavones at the low concentration（Ginkgo biloba:0.85 mg/mL） always catalytic NDEA forming and the scavenging rate was the lowest.ConclusionsIt was suggested that flavones could redox react with nitrite under mildly acidic condition and inhibite the formation of nitrosamine if the concentration of flavones was enough.The blocking rate was positively related with its concentration and antioxidant activity.Flavones could accelerate NDEA forming at a low concentration.The twofold influences of flavones in effecting the NDEA formation might be related with the structure of phenolic hydroxyl of flavones,which need to be further studied.By analyzing the products of quercetin reacting with nitrite,it was concluded that flavones were oxidated on the process of inhibiting the nitrosamine formation and produced relevant oxidation products.更多还原