【作者】 聂乾忠；

【导师】 夏延斌；

【作者基本信息】 湖南农业大学 ， 园艺产品采后科学与技术， 2009， 博士

【摘要】 本研究首先对辣椒素类物质（辣椒碱）的急性和亚急性毒性进行了相关研究。通过辣椒素类物质的急性毒性试验,测定LD₅₀,了解受试物的毒性强度、性质和可能的靶器官,为进一步毒性试验剂量和毒性观测指标提供依据,并根据LD₅₀进行毒性分级。接着进行了30天喂养试验,进一步了解其毒性作用,观察其对生长发育的影响,并估计最大未观察到有害作用剂量（No Observed Adverse Effect Level, NOAEL）。最后对辣椒素类物质可能的降血脂和胆固醇及抗氧化的效果进行了相关的动物试验；以期为辣椒素类物质的进一步开发利用提供一些基础数据。辣椒素类物质急性毒性试验试验方法按GB 15193.3—1994中的相关规定进行；30天喂养试验则按GB 15193.13—2003的相关规定进行；辣椒素类物质可能的降血脂和胆固醇及抗氧化效果及机理研究则采用单一雄性6周龄SD大鼠60只（普通级,体重206.3±5.4g）,按体重随机分为6组；分别按下列分组进行灌胃。1)基础日粮对照组（BDC）；基础饲料+lml蒸馏水；2)高胆固醇日粮对照组（HCC）；高脂高胆固醇饲料+1ml金龙鱼油；3)阳性对照组（JPC）：高脂高胆固醇饲料+1 ml绞股兰总苷片溶液（7.000 mg／ml）；4)辣椒碱低剂量组（LD）：高脂高胆固醇饲料+1 m1溶有辣椒碱的金龙鱼油,其中辣椒素浓度为3.0171 mg.g^-1。5)辣椒碱中剂量组（MD）：高脂高胆固醇饲料+1 ml溶有辣椒碱的金龙鱼油,其中辣椒素浓度为4.0281 mg.g^-16)辣椒碱高剂量组（HD）：高脂高胆固醇饲料+1 ml溶有辣椒碱的金龙鱼油,其中辣椒素浓度为5.0145 mg·g^-1。每天灌胃一次,连续8周。试验结束时,将已断食一晚的试验动物用乙醚麻醉,用心脏穿刺的方法收集血液,离心得到血清,用于测定各项血清指标（TG、TC、HDL-C、SOD、GSHPx、MDA）。同时迅速摘除肝脏,秤肝重并留取部分肝脏组织用于制备匀浆测定肝脂指标（TG、TC、SOD、GSH-Px、MDA）。辣椒素类物质急性毒性试验结果显示：高剂量的辣椒素类物质对小鼠具有致死作用,根据对各组死亡数的相关统计和Horn法剂量递增法测定LD₅₀计算用表查得辣椒素类物质的LD₅₀为190 mg／kg,表明辣椒碱具有一定的毒性或刺激性。接着对死亡小鼠胃肠进行解剖后发现：高剂量组小鼠的胃内表面均有一定程度的充血现象,进一步说明了辣椒素类物质具有一定的毒性或较强的刺激性。30天喂养试验的结果表明：1)亚急性剂量的辣椒素类物质也会影响试验小鼠的行为表现,各试验组小鼠会表现出明显不适的现象如嘴角上翘,上窜下跳,兴奋异常,有的个体甚至将头埋入垫料中,也出现颤栗现象,但一般在2-3 hr后个体的行为恢复正常。各剂量组均没有出现个体死亡的现象,表明亚急性剂量的辣椒素类物质没有致死效应。2)亚急性剂量的辣椒素类物质对小鼠的血液学指标有一定的影响。如各不同剂量的辣椒素类物质会使小鼠血液中的红细胞数（RBC）,血红蛋白（Hb）含量,白细胞（WBC）及血小板数目均有一定程度地增加,有些和对照组相比较还会显著增加（P<0.05）。这种差异在雌性小鼠中表现更为明显。说明亚急性毒性辣椒碱可能引起小鼠体内一定的炎症反应,而且早个体较（?）个体对辣椒素类物质更为敏感。3)辣椒素类物质对小鼠血液生化指标的影响主要表现在对丙氨酸氨基转移酶（ALT）,尿素氮（BUN）和血液中总胆固醇（TC）含量的影响。辣椒素类物质会使小鼠血液中丙氨酸氨基转移酶（ALT）的活性显著低于溶剂对照组（P<0.05）；尿素氮（BUN）的含量均低于对照组（P<0.05）；血液中总胆固醇（TC）的含量低于对照组（P<0.05）。提示亚急性剂量的辣椒素类物质可能对肝功能有一定的影响：但不会对小鼠的肾功能产生损害,同时具有促进代谢和明显降低小鼠血清胆固醇的作用（P<0.05）。4)各亚急性剂量的辣椒素类物质没有对小鼠的胃或胃黏膜产生明显不利的影响；也不会对小鼠的肝重／体重比、肾重／体重比产生影响（P>0.05）。小鼠肝脏和肾脏外观和蒸馏水对照组比较也没有显著差异。5)通过30天喂养试验发现辣椒素类物质对试验小鼠未观察到有害作用剂量（NOAEL）为16.2 mg／kg。辣椒素类物质（辣椒碱）在高胆固醇血症大鼠中降血脂和抗氧化效果的研究结果为：1)利用不同剂量的辣椒碱灌胃SD雄性大鼠,灌胃后20-30min有不适感觉,但一般1-2 h后基本恢复正常。8周试验期间各试验组大鼠的外观体征、毛色、粪便性状等均没有产生显著性差异。2)在高脂高胆固醇饲料中添加不同剂量的辣椒碱对SD雄性大鼠体重增长无显著性影响（P>O.05）；8周试验期间各组大鼠的日平均摄食量也没有显著性差异。3)本试验条件下不同剂量的辣椒碱能显著降低SD雄性大鼠血液中胆固醇的水平（P<0.05）,但对血清中甘油三酯水平则没有显著性影响（P>0.05）。4)辣椒碱能显著降低血清中低密度脂蛋白胆固醇（LDL—C）的含量（P<0.05）,虽然其效果没有绞股兰含片作用显著,而且其降LDL——C含量的作用似乎与辣椒碱的剂量还存在一定的量效关系；同时,各试验组中高密度脂蛋白胆固醇（HDL-C）的含量则相对稳定,均保持御.52-0.56mmol/L的水平。5)辣椒碱各剂量组对大鼠血清抗氧化酶SOD和GSH-Px活性均有不同程度的提高作用。辣椒碱各剂量组（LD、MD、HD） SOD的活性较高脂高胆固醇日粮对照组（HCC）显著增加（P<0.05）; GSH-Px的活性也较HCC组显著增加（P<0.05）；与之相对应的是辣椒碱各剂量组可以显著降低MDA的含量（P<0.05）。提示辣椒碱各剂量组对大鼠血清抗氧化酶活性有显著地提高作用。6)与高脂高胆固醇日粮对照组（HCC）相比较,辣椒碱各试验组对肝中总胆固醇（TC）的含量影响不显著（P>0.05）；但却使肝中甘油三酯（TG）水平显著降低（P<0.05）。7)辣椒碱各剂量组对大鼠肝脏中抗氧化酶SOD和GSH-Px、舌性影响不显著（P>0.05）。8)与高脂高胆固醇日粮对照组（HCC）相比较,辣椒碱各剂量组可显著促进大鼠体内胆固醇类物质通过粪便排泄（P<0.05）；同时也可以显著促进大鼠体内胆固醇通过代谢转变成胆汁酸,随粪便排出体外。结论：急性毒性试验表明辣椒类物质对昆明种小白鼠经口灌胃的LD5。为190mg／kg,具有一定程度的毒性。30天喂养试验表明：辣椒素类物质（辣椒碱）对昆明种小白鼠未观察到有害作用剂量（NOAEL）为16.2 mg／kg,这相当于食辣人群人体摄入量的3-5倍。表明低剂量摄入辣椒素类物质（辣椒碱）并不会对人体产生不利的影响。辣椒素类物质（辣椒碱）在高胆固醇血症大鼠中降血脂和抗氧化效果研究表明：经8周低剂量摄取辣椒碱能显著降低SD大鼠血清中低密度脂蛋白胆固醇（LDL—C）含量；辣椒碱各剂量组没有显著减少肝脏中胆固醇含量的作用,但却使肝脏中甘油三酯水平显著降低。同时辣椒碱可明显促进SD大鼠体内胆固醇类物质通过粪便排泄或通过代谢转变成胆汁酸,随粪便排出体外。提示长期低剂量摄取辣椒碱对于预防心血管疾病及防止脂肪（甘油三酯）在肝脏中的聚集均具有良好的作用。辣椒碱对SD大鼠血清抗氧化酶SOD、GSH-Px活性均有显著地提高作用,同时显著降低血清中MDA的含量,提示中长期低剂量摄取辣椒碱具有良好的抗脂质氧化的作用。更多还原

【Abstract】 This study focused first of all on the acute toxicity test of capsaicinoids to determine the possible LD₅₀ and to have some knowledge about their toxicity effects and affected organs, and at the same time to provide an orientation for further sub-chronic test concerning the dose design and indicative items to be analyzed. Then 30 days feeding experiment of capsaicinoids was undertaken so to have a further understanding about the sub-chronic toxicities of capsaicinoids and their influence on the growth of mice and to determine the no-observed adverse effect level （NOAEL） of capsaicinoids to mice. Finally hypolipidemic and anti-oxidation effects of capsaicinoids were systematically studied so to provide some basic data for further exploitation of capsaicinoids.The acute toxicity test of capsaicinoids was undertaken as stressed in the relevant National Standard of Acute Toxicity Test GB 15193.3-1994 and 30 days feeding experiment according to the National Standard of Sub-Chronic Toxicity Test 15193.13-2003. Study on hypolipidemic and anti-oxidation effects of capsaicinoids in induced hypercholesterolemic rats was as following:60 SD male rats were randomly divided into 6 groups with an average body weight of 206.3±5.4 g, then all 6 groups were orally administered by I.g daily for 8 weeks as followings:1) Basal diet control group （BDC）; Basal diet+lml distilled water; 2) High Cholesterol control group （HCC）; High Cholesterol diet+1ml salad oil brand "Jinlongyu"; 3) Jiaogulan pill positive control group （JPC）:High Cholesterol diet+1 ml Jiaogulan solution with concentration of 7.000 mg/ml; 4) Low dosage of capsaicinoids（LD）:High Cholesterol diet+1 ml Salad oil brand "Jinlongyu"in which the concentration of capsaicinoids is 3.0171 mg·g^-1.5) Medium dosage of capsaicinoids（MD）:High Cholesterol diet+1 ml Salad oil brand "Jinlongyu"in which the concentration of capsaicinoids is 4.0281 mg·g^-16) High dosage of capsaicinoids（HD）:High Cholesterol diet+1ml Salad oil brand "Jinlongyu"in which the concentration of capsaicinoids is 5.0145 mg·g^-1. At the end of experiment, overnight fasted rats were sacrificed under light ether anesthesia. Serum and related hepatic Triglyceride（TG）, Total cholesterol（TC）, High density lipoprotein cholesterol （HDL-C）,Low density lipoprotein cholesterol （LDL-C）, Superoxide dismutase （SOD）,Glutathione peroxidase （GSH-Px） and Malondialdehyde（MDA） were analyzed and determined.The acute toxicity test of capsaicinoids showed that the acute oral administration of capsaicinoids at high dosage by I.g was lethal to mice and the LD₅₀ of capsaicinoids was 190 mg/kg. The histopathological observation of stomach of dead mice also showed congestive phenomena in the stomach, indicating that capsaicinoids at acute oral administration dosage is somewhat toxic indeed.30 days feeding experiment showed that first of all, oral administration of capsaicinoids at sub-chronic dosage by I.g would affect the behavior of treated mice such as excessive exciting, trembling or burying its head in the litter etc. But usually all these abnormal behavior of mice would disappear in 2-3 hrs and no lethal effect was observed, indicating that capsaicinoids at sub-chronic dosage was relatively safe. Secondly sub-chronic dosage of capsainoids affected blood parameters of mice to some extent, for instance, the administration of capsaicinoids at tested groups increased the RBC count, Hb content, WBC and platelet count and in some cases even significantly compared with that of control group （P<0.05）, this was especially true for female mice. Thirdly, sub-chronic dosage of capsainoids also influenced some of the hematological index of treated mice such as ALT activity, BUN and TC content in serum. The administration of capsaicinoids significantly reduced the ALT activity （P<0.05）, BUN and TC content in serum compared to that of the control group （P <0.05） indicating that oral administration of capsaicinoids at sub-chronic dosage might also affect liver, but not kidney and actually even promote the metabolism and decrease the TC in serum. Further, no side or harmful effects were observed concerning possible injury to stomach or gastric of treated mouse compared to that of control group, neither did it to the coefficient of liver/bw ratio, kidney/bw ratio or the appearance of liver or kidney. Finally, it was concluded that the no observed adverse effect level （NOAEL） for capsaicinoids to Kunming mice was 16.2 mg/kg. Study on the hypolipidemic and anti-oxidation effects of capsaicinoids in induced male hypercholesterolemic rats showed that:1) Feeding the rats with different concentration of capsaicinoids resulted in anxious behavior of the tested groups but returned to normal in 20-30 min. There were no significant differences regarding to the appearance, color and the fecal characteristics among tested groups compared with that of HCC group during 8 weeks feeding experiment.2) Feeding the rats with high fat, high cholesterol diet could significantly increase the body weight gain for male hypercholesterolemic rats compared with that of BDC group （P<0.05）. However, there was no significant difference of body weight gain among rats of HCC and the other three capsaicinoids tested groups （P> 0.05）, neither did the average daily feed intake.3) Compared with that of HCC group, the serum TC level of JPC, LD, MD and HD was lowered significantly（P<0.05）. But no significant difference was observed between HCC group and the other three capsaicinoids groups （P>0.05） concerning the serum TG level.4) Further analysis showed that the lowering effect of serum TC by capsaicinoids in LD, MD and HD group was mainly due to the decrease of serum LDL-C level （P<0.05）, however, there was hardly any significant difference observed concerning serum HDL-C level among male hypercholesterolemic rats.5) The oral administration of capsaicinoids at different dosage also improved the enzymatic activities of different kinds of anti-oxidation enzymes in serum such as SOD and GSH-Px. The serum enzymatic activities of SOD in LD, MD and HD group were increased significantly compared with that of HCC group （P<0.05）. Similarly, the serum activities of GSH-Px were also increased significantly compared with that of HCC group（P<0.05）. Thanks to the effect of capsaicinoids on these anti-oxidation enzymes in serum lipids profile, the level of MDA in LD, MD and HD group were significantly lower than that of HCC group （P<0.05）.6) The liver cholesterol level was not significantly influenced in male hypercholesterolemic rats （P>0.05） However, the hepatic triglyceride level of LD, MD and HD rats were significantly lowered than that of HCC group（P<0.05）.7) There was no significant difference between HCC group and the other three capsaicinoids groups （P>0.05） regarding to the hepatic enzymatic activities of SOD and neither did it to the hepatic GSH-Px activity indicating that capsaicinoids did not influence the hepatic enzymatic activities of SOD and GSH-Px significantly （P>0.05）.8) Compared with the tested rats of HCC group, oral administration of capsaicinoids at set dosages significantly increased the excretion of fecal cholesterol and also the bile acid from fecal （P<0.05）.From the acute toxicity test it is concluded the LD₅₀ of capsaicinoids by I.g is 190 mg/kg and therefore can be categorized as a kind of somewhat toxic substances. 30 days feeding experiment reveals that the no observed adverse effect level （NOAEL） for capsaicinoids to mice by I.g is 16.2 mg/kg. This amount is roughly 3-5 times that of average daily intake of ordinary chili consumers indicating that sub-chronic intake of capsaicinoids is actually safe. From the last experiment, it can be concluded that capsaicinoids at different dosage can significantly reduce the serum TC level but not necessarily the hepatic TC of tested rats and the main reducing fraction is actually due to the decrease of serum LDL-C. Similarly, oral administration of capsaicinoids also promotes the fecal excretion of cholesterol either directly or in form of bile acid. Hence the consumption of proper amount of capsaicinoids may be helpful in prevention of blood and heart diseases and possible accumulation of lipids in hepatic tissue. Simultaneously, Capsaicinoids may also enhance the activities of certain anti-oxidation enzymes in serum lipids profile such as SOD and GSH-Px so that the related serum MDA content is correspondingly decreased. However, such phenomenon is not observed in liver tissue of tested rats. All these indicated that capsaicinoids could function as a kind of potential anti-oxidative agent in lipids profile metabolism of rats.更多还原