【作者】 李芳；

【导师】 吴向阳；

【作者基本信息】 江苏大学 ， 食品科学， 2012， 博士

【摘要】 糖尿病是一种与遗传因素有关又与多种环境因素相关的慢性全身性疾病,主要表现为胰岛素抵抗、高血糖和糖尿,已被世界卫生组织列为三大疑难病之一。因此,辅助降血糖功能食品的研究开发是当前研究的热点课题之一。铬(Ⅲ)是人体重要的微量元素之一,并具有降血糖功能。当体内缺铬时,胰岛素水平下降,诱发糖代谢、脂代谢紊乱。研究发现补充铬可增强胰岛素敏感性,调节糖代谢和脂代谢。相对无机铬,有机铬的吸收率高且安全,作为辅助降血糖功能食品的功效成分和营养素补充剂已得到广泛应用。目前,市场上应用最广的有机铬为吡啶甲酸铬,但有研究报道,吡啶甲酸铬会产生DNA毒性、细胞毒性和生殖毒性,且毒性与配体吡啶甲酸有关,其安全性已受到广泛的关注,因此,降血糖活性好、安全性高的新型有机铬配合物的研究仍是当前重要课题之一。本文以天然活性有机化合物苹果酸、水苏糖和牛磺酸为配体,与Cr3+合成有机铬配合物,表征其结构；通过药效学实验考察5种有机铬(苹果酸铬、芦丁铬、叶酸铬、水苏糖铬和牛磺酸铬,其中芦丁铬和叶酸铬为前期合成)对四氧嘧啶所致糖尿病小鼠的降血糖活性,筛选出降血糖活性较好的有机铬进行急性毒性和亚急性毒性评价,进一步优选出安全性高的有机铬进行转化和转运研究,探讨其转运机理；除铬化合物外,蜂胶作为辅助降血糖功能食品的功效成分也应用广泛,仅次于铬化合物,本文对有机铬和蜂胶组合物的降血糖活性进行研究,探讨其降血糖的增效作用。本论文研究可为安全性高的新型有机铬辅助降血糖功能食品和营养素补充剂的研究开发提供科学依据,对人类健康和社会经济发展具有重要意义。主要研究内容如下：(1)以天然活性有机化合物苹果酸、水苏糖和牛磺酸与Cr3+反应合成苹果酸铬、水苏糖铬和牛磺酸铬配合物。采用元素分析、红外光谱、紫外-可见光谱、原子吸收光谱等方法表征配合物的结构。结果表明,苹果酸与Cr3+的配位点为羧基中的羟基,水苏糖与Cr3+的配位点为羟基,牛磺酸与Cr3+的配位点为氨基。3种有机铬的分子式分别推测为Cr2C12H22O20、CrC24H48O27和CrN6C12S6H54O24。(2)采用四氧嘧啶所致糖尿病小鼠模型,考察苹果酸铬、芦丁铬、叶酸铬、水苏糖铬和牛磺酸铬(其中芦丁铬和叶酸铬为前期合成)的降血糖活性。结果表明,以剂量为3.0 mg Cr/kg b.w.苹果酸铬、芦丁铬和叶酸铬给药治疗糖尿病小鼠14d后,对糖尿病小鼠的血糖下降率分别为45.91%、29.1%和34.7%,而水苏糖铬和牛磺酸铬的降血糖活性不明显。苹果酸铬、芦丁铬和叶酸铬可显著促进肝糖原的合成,抑制肝糖原分解从而发挥降血糖作用。因此,不同配体的有机铬,其降血糖活性存在明显差异,苹果酸铬、芦丁铬和叶酸铬具有较好的降血糖活性。(3)苹果酸铬、芦丁铬和叶酸铬的急性毒性和亚急性毒性评价采用一次最大限量给药法评价该3种有机铬(苹果酸铬、芦丁铬和叶酸铬)的小鼠经口急性毒性,并设无机铬(CrCl3·6H2O)对照组。结果表明,CrCl3·6H2O的雌、雄性小鼠经口LD50(半数致死剂量)为2.39 g/kg b.w.(95%置信区间为1.776-3.218 g/kgb.w.),属于低毒级；苹果酸铬、芦丁铬和叶酸铬的雌、雄性小鼠经口LDso均大于5.0 g/kg b.w.,属于实际无毒级,可见CrCl3·6H2O与苹果酸、芦丁和叶酸合成有机铬后其毒性明显降低。叶酸铬和苹果酸铬的LDmin(最小致死剂量)大于5.0 g/kgb.w.,远大于芦丁铬的LDmin (2.0 g/kg b.w.)采用连续28 d灌胃给药法评价该3种有机铬的小鼠经口亚急性毒性。结果表明,芦丁铬、叶酸铬和苹果酸铬对雌、雄受试小鼠的体重、脏器指数、血液常规指标和生化指标均无显著影响,且不会造成肝组织和肾组织细胞的脂质过氧化反应和DNA的氧化损伤。然而,补充叶酸铬或叶酸,肝脏中的锌浓度显著降低,而肾脏中的锌浓度显著升高。综上所述,不同配体的有机铬的安全性也存在明显差异,该3种有机铬的小鼠经口急性毒性均达到实际无毒级。苹果酸铬和叶酸铬对小鼠的LDmin远大于芦丁铬的LDmin。在亚急性毒性试验范围内,苹果酸铬未见明显毒性,而叶酸铬导致了人体必需微量元素锌的代谢紊乱,且与其配体叶酸有关。因此,苹果酸铬不仅降血糖活性好,而且安全性好,在辅助降血糖功能食品和营养素补充剂方面具有很好的应用前景。(4)采用静脉注射给药,ICP法考察苹果酸铬在大鼠体内的药代动力学行为,获得基本药代动力学参数,初步阐明了苹果酸铬在大鼠体内分布、排泄规律。经单次静脉注射苹果酸铬在30 min后,铬已从大鼠血液中消除85.0%,肝脏和肾脏中的铬含量在1h后均达到最大值,说明苹果酸铬从血液中消除后分布到各组织中,36 h后几乎所有铬都从各组织中排出。大鼠体内的苹果酸铬主要通过尿液和粪便排泄,36 h内已排出82.4%。(5)采用紫外光谱、荧光光谱和等温微量热法(ITC)研究苹果酸铬与脱铁-转铁蛋白和人血清白蛋白的相互作用形式。结果表明,苹果酸铬可与脱铁-转铁蛋白和人血清白蛋白形成了复合物,导致该两种蛋白与铬离子结合部位的色氨酸残基和酪氨酸残基的苯环π→π*跃迁受到干扰,从而使225 nm处的吸光度增加,280 nm处吸光度逐渐减小,并引起该两种蛋白内源荧光猝灭；苹果酸铬与该两种蛋白之间的相互作用力以氢键和范德华力为主。(6)采用四氧嘧啶所致糖尿病小鼠模型考察苹果酸铬和蜂胶组合物的降血糖增效作用和一次最大限量给药法评价该组合物的小鼠经口急性毒性。降血糖活性试验结果表明,该组合物可降低血清谷草转氨酶(AST)、谷丙转氨酶(ALT)和碱性磷酸酶(ALP)的含量,说明该组合物可显著减轻糖尿病小鼠的肝损伤,同时该组合物对糖尿病小鼠具有显著的降血糖活性,并达到增效作用。小鼠经口急性毒性结果表明,在14 d的试验期内,雌、雄受试小鼠未出现异常情况及死亡,尸解未见异常。该组合物对主要脏器指数、血液常规指标、血生化学指标均无明显影响。雌、雄性小鼠经口LD50均大于5.0 g/kg b.w.,属于实际无毒级。因此,苹果酸铬和蜂胶的组合物减轻了四氧嘧啶所致小鼠肝损伤的作用,对糖尿病小鼠具有显著的降血糖活性,并达到增效作用,且该组合物在经口急性毒性范围内未见明显毒性,达到实际无毒级。综上所述,针对营养素补充剂和含铬化合物功能食品中广泛使用的吡啶甲酸铬存在的安全性问题,本文以天然活性有机化合物为配体合成出了几种新型有机铬配合物,通过降血糖活性研究及急性毒性和亚急性毒性评价筛选出了降血糖活性好、安全性高的苹果酸铬,初步阐明了其在体内的分布、排泄规律及转运机理；苹果酸铬和蜂胶的组合物可减轻四氧嘧啶所致糖尿病小鼠的肝损伤,并达到降血糖增效作用。该成果为新型有机铬辅助降血糖功能食品和营养素补充剂的研究开发提供了科学依据。更多还原

【Abstract】 Diabetes mellitus is a disorder with high abnormal blood glucose levels caused by the failure of the body to produce enough insulin, or the inability of the insulin to make glucose accessible to blood cells. Diabetes mellitus has been identified as one of the three diseases (cardiovascular diseases, caners and diabetes mellitus) difficult to cure. This has therefore triggered the research into secondary anti-diabetic functional foods or nutrient supplement as a measure to help cure the disease. Chromium(Ⅲ) as one of the important trace elements in the body has shown significantly anti-diabetic activity. The lack of chromium in the body system can result in the decline in insulin secretion and increases blood glucose levels. As a result of the low absorption rate of chromium salts, it has become necessary to find possible mechanisms such as the designing and development of new organic chromium complexes to help address the menace. Chromium picolinate is said to the most commonly applied dietary supplement. However, it has been reported that chromium picolinate exhibits genotoxicity and cytotoxicity which is likely caused by the ligand (picolinate). Hence the safety aspect of chromium picolinate in the body has been a matter of concern to the majority.In order to reduce the toxicity and improve the bioactivity of chromium, the organic ligand with less or no toxicity has drawn the attention of researchers.In this study, organic ligands including L-malic acid, stachyose, and taurine were selected. The aim of the study was to determine the effects of chromium malate complex, chromium stachyose complex and chromium taurine complex on alloxan-induced diabetic rats. The specific objectives of the study include; (a) to investigate the anti-hyperglycemic activity of five organic chromium complexes (chromium malate complex, chromium rutin complex, chromium folate complex, chromium stachyose complex and chromium taurine complex) administered to alloxan-induced diabetic rats, (b) to evaluate the acute and sub-acute oral toxicity of chromium malate complex, chromium rutin complex and chromium folate complex on the rabbits, and (c) to study the transportation and conversion of the chromium malate complex on alloxan-induced diabetic rats.Propolis, as the functional component of secondary anti-diabetic functional foods has been used over the world. In the domestic market for example, propolis accounts for 29.6% of the domestic functional foods; second to chromium compounds (45.3%). In order to investigate further the additional benefits of anti-diabetic activity and protective effects of liver injury on diabetes, this aspect of the study focused on evaluating the anti-diabetic effect of the combination of chromium malate complex and propolis in alloxan-induced diabetic mice and to test the acute oral toxicity of the combination.The stepwise experimental procedures and the results of the study is given below:(1) Chromium malate complex, chromium stachyose complex and chromium taurine complex were synthesized in a single step reaction in aqueous solution by chelating chromium (Ⅲ) with L-malic acid, stachyose, and taurine respectively. The structures of the chromium complexes were determined using infrared,UV-visible and atomic absorption spectroscopy and elemental analysis. The results of this study (1) indicated that the coordination sites of chromium malate complex, chromium stachyose complex and chromium taurine complex were hydroxyls in the carboxyl groups, hydroxyls and amino respectively. The molecular formulas of the three chromium complexes were inferred as Cr2C12H22O20, Crt24H48O27 and CrN6C12S6H54O24, respectively.In the case of second experiment (2), anti-hyperglycemic activities of chromium malate complex, chromium rutin complex, chromium folate complex, chromium stachyose complex and chromium taurine complex were examined in alloxan-induced diabetic mice with daily oral gavage for a period of two weeks at the dose of 0.5-3.0 mg Cr/kg. b.w. The results indicated that chromium malate complex, chromium rutin complex, chromium folate complex and anti-diabetic activity resulted in percentage reduction of blood glucose levels of 45.91%,29.1% and 34.7%, respectively. However, after a two-week period of administrating the mice with the chromium stachyose complex and chromium taurine complex, the effect on anti-diabetic activity of the rabbits were not significant. The significant increase in the glycogen levels of diabetic rabbits which were treated by chromium malate complex, chromium rutin complex, chromium folate complex were determined. This however, indicates that the ligand of the trivalent chromium compounds markedly affected its efficacy. Not all the trivalent chromium compounds possessed equal efficacy. Chromium malate complex, chromium rutin complex and chromium folate complex had hypoglycemic effect on alloxan-induced diabetic mice, while chromium stachyose complex and chromium taurine complex did not.In further investigation (3), ie the safety toxicological evaluation (the first step and the second step) of 3 chromium complexes,Acute toxicities of chromium malate complex, chromium rutin complex and chromium folate complex were tested using ICR mice with a single oral gavage and observed for a period of two weeks. CrCl3·6H2O was used as the positive control in this test. For the acute toxicities of chromium malate complex, chromium rutin complex and chromium folate complex tested using ICR mice with a single oral gavage (3), LD50 (median lethal dose) for CrCl3·6H2O obtained was 2.39 g/kg body weight (95% confidence intervals:1.776-3.218 g/kg), that of chromium rutin complex and chromium folate complex were greater than 5.0 g/kg each. The minimum lethal dose for both chromium malate complex and chromium folate complex were over 5.0 g/kg b.w., while that of chromium rutin complex was 2.0 g/kg b.w.Three chromium (Ⅲ) complexes with different ligands (rutin, folic acid or L-malic acid) were compared to examine whether they have similar effect on sub-acute toxicity status. ICR mice were orally administered with chromium rutin complex, chromium folate complex and/or chromium malate complex at doses of 6 and 36 mg Cr/kg bodyweight/day for a period of 28 days. The results showed that oral administration of all these chromium complexes do not cause adverse effects on body weight, organ weights, hematology and serum biochemical parameters, hepatic and renal lipid peroxidation and cellular 8-OHdG levels. However, supplemental chromium folate complex or folic acid, not chromium malate complex or chromium rutin complex could induce abnormalities in metabolism of Zn. This can be inferred from the increase in renal Zn concentration and a decrease in hepatic Zn concentration.Collectively, the ligands affected the metal-toxicity of chromium in their complexes. In the acute toxicity study, the three chromium complexes were "non toxic". The minimum lethal dose for chromium malate complex and chromium folate complex was above 5.0 g/kg while that of chromium rutin complex was 2.0 g/kg b.w. Chromium malate complex and chromium rutin complex were safe in the sub-acute toxicity status while chromium folate complexes were not since chromium folate complex induced abnormalities in metabolism of Zn in the body.Following the investigation in (3), additional study (4) where pharmacokinetic study was performed using non-diabetic rats after intravenous administration at a single dose of 500 Crμg/kg b.w. Chromium levels in the blood stream, tissue samples, excreta (urine and faeces) were determined using ICP method. The pharmacokinetic parameters were obtained from this study. The results in this study indicated that chromium malate complex leaves the blood stream after intravenous administration for 30 min, and the elimination rate of chromium was around 85% in the blood stream. The complex leaves of chromium malate in the blood stream ends up appearing in the urine and entering tissue cells. For liver and kidney examined, the chromium content (livers and kidneys) is maximal 1 h after injection. After 36 h, however, no chromium was left in the livers and kidneys. Urine and fecal samples collected over time intervals of 30 min to 36 h after injection of the complex revealed that chromium loss of urine and fecal occurs rapidly for the first 2 h after injection and continues at a substantially lower rate over the next 34 h. The excretion rate of chromium in the urine and fecal was 82.4% after 36 injections.In order to determine the interaction between chromium (Ⅲ) malate complex with the high molecular mass components of the human serum albumin and apo-transferrin, a fifth (5) study was embarked upon. In this study, ultraviolet and fluorescence spectra as well as isothermal titration calorimetry was used. Cr3+ was binded with human serum albumin and apo-transferrin. The results showed that the interaction of Cr3+ with the human serum albumin and apo-transferrin obstructed atπ→π* transition of benzene rings (the bind sites of the tryptophan and tyrosine residues) cause the increase of the absorbance at 225 nm and decrease at 280 nm. The interaction also quenched the fluorescence of the two proteins. The interactions between Cr3+with the human serum albumin and apo-transferrin were mainly hydrogen bonds and van der Waals.To obtain the additional benefits of anti-diabetic activity and protective effects of liver injury on diabetes, anti-diabetic effect and acute oral toxicity of a combination of chromium (Ⅲ) malate complex and propolis were assessed (6). The anti-diabetic effect of the combination of chromium (Ⅲ) malate complex and propolis was determined and compared with chromium (III) malate complex and propolis only in alloxan-induced diabetic mice through daily oral gavage for 2 weeks. Acute oral toxicity of the combination of chromium (Ⅲ) malate complex and propolis was tested in ICR mice at the dose of 1.0-5.0 g/kg body weight with a single oral gavage and also observed for two weeks. The results therefore indicated that the increase in anti-diabetic efficacy and the protective effects of liver injury for diabetes of chromium malate complex combined with propolis was significant. In acute toxicity study, LD50 (median lethal dose) values for the combination was greater than 5.0 g/kg body weight. The combination might represent the nutritional supplement with potential therapeutic value to control blood glucose and exhibit protective effects of liver injury for diabetes and non-toxicity in acute toxicity.Taken together; three chromium complexes with natural active organic ligands have been synthesized. Chromium malate complex represent an optimal chromium supplement among those chromium complexes with potential therapeutic value to control blood glucose in diabetes and non-toxicity in acute and sub-acute toxicity. The fates of distribution and excretion, and transportation mechanism have been elaborated. The combination of chromium (Ⅲ) malate complex and propolis represent the nutritional supplement with potential therapeutic value to control blood glucose and exhibit protective effects of liver injury for diabetes and non-toxicity in acute toxicity.更多还原