【作者】 尹忠平；

【导师】 上官新晨；

【作者基本信息】 南昌大学 ， 食品科学与工程， 2011， 博士

【摘要】 青钱柳(Cyclocarya paliurus (Batal.) Iljinsk)为我国特有的胡桃科青钱柳属珍稀濒危植物,零星分布于我国南方各省,可用于保健食品、医药、园林绿化、工业用材等多个领域。研究表明,青钱柳叶含三萜、黄酮、多糖、皂苷及各种微量元素等多种功能活性成分,是具有很高利用价值的天然保健食品资源,但其繁育十分困难,资源稀缺严重制约了其开发利用。本文研究了青钱柳细胞悬浮培养及其三萜酸积累规律,从悬浮培养细胞中提取分离出了主要的三萜酸并进行了结构鉴定,同时对其降糖作用效果及机制进行了研究,旨在探索以青钱柳细胞悬浮培养方式生产三萜酸类降糖功能因子的可行性,为深度开发和利用青钱柳资源、生产三萜酸类降糖功能食品提供理论依据。以青钱柳无菌幼叶诱导愈伤组织,通过长期的筛选和继代培养,获得了疏松易碎、生长旺盛、适合于悬浮培养的优良愈伤组织,转入悬浮培养后,再经反复的筛选、继代和优化培养条件,建立了良好的细胞悬浮培养体系。该培养体系最佳继代时间为接种后第8天,最大细胞干重产量出现在第10天,最高总三萜酸产量出现在第14天。在保持MS培养基中总氮源浓度及铵态氮和硝态氮比例的同时,将磷酸盐、钙离子和镁离子浓度分别调整至1.875 mmol·L-1、2 mmol·L-1和2 mmol·L-1,可获得较高的总三萜酸产量。以三氯化铈和水杨酸对悬浮细胞进行诱导,总三萜酸产量分别可提升35.70%和22.31%。悬浮培养细胞总三萜酸、熊果酸和齐墩果酸产量分别可达1179.85 mg·L-1、443.64 mg·L-1和159.00 mg·L-1。本文首次对青钱柳悬浮培养细胞中的主要三萜酸进行了提取、分离和鉴定。以乙酸乙酯为提取溶剂超声辅助提取悬浮细胞总三萜酸,得率高且杂质少,提取物易于纯化,优化后的主要工艺参数为提取时间40 min、提取温度50℃、料液比1：10,优化条件下总三萜酸提取得率达5.56%。经大孔树脂吸附、硅胶柱层析、Sephadex LH-20凝胶层析、半制备高效液相色谱和重结晶等分离纯化,从总三萜酸粗提物中获得了5个单体三萜酸,经HPLC、TLC、RRLC-TOF/MS、1H NMR、13C NMR和DEPT等检测分析,化合物Ⅰ-V分别被鉴定为熊果酸、齐墩果酸、白桦脂酸、2αl-羟基熊果酸、2a-羟基齐墩果酸,其中白桦脂酸和2α-羟基齐墩果酸是首次在青钱柳中得到分离、鉴定。本文以3T3-L1脂肪细胞为模型,在前脂肪细胞、成熟脂肪细胞、胰岛素抵抗脂肪细胞多个层次上进行了青钱柳悬浮培养细胞三萜酸降糖作用效果研究。试验结果表明：(1)TTA(总三萜酸)、UA(熊果酸)、OA(齐墩果酸)、BA(白桦脂酸)、CA(2α-羟基熊果酸)对3T3-L1前脂肪细胞增殖没有促进作用,2.5μg·mL-1 MA(2α-羟基齐墩果酸)有明显的促进作用；(2)TTA、UA、OA、BA对前脂肪细胞葡萄糖消耗没有促进作用,而CA、MA在浓度为2.5至5μg·mL-1时有促进作用,并且在没有胰岛素刺激的情况下也能显著提高葡萄糖消耗；(3)5μg·mL-1的TTA、UA、OA能极显著地促进前脂肪细胞分化,而5μg·mL-1的CA明显地抑制分化,BA和MA则基本无影响；(4)TTA、UA、OA、CA和MA均对成熟脂肪细胞的葡萄糖消耗具有较好的促进作用,且在无胰岛素存在的条件下也能提升葡萄糖消耗,表明三萜酸促进成熟脂肪细胞葡萄糖消耗可能存在与Ros(罗格列酮)不同的作用机制；(5)TTA、UA、MA能防止Dex(地塞米松)所诱导的胰岛素抵抗的形成,OA和CA虽然不能防止胰岛素抵抗形成但能减轻抵抗的程度,而BA对胰岛素抵抗的形成没有影响；(6)TTA和UA能改善已经形成胰岛素抵抗细胞的抵抗状态,具有较好的胰岛素增敏作用,MA、OA、CA也有一定的增敏效果,但BA对抵抗状态没有改善作用；(7)各三萜酸在合适的浓度下,均能显著地减少胰岛素抵抗脂肪细胞培养上清液中的FFA(游离脂肪酸)的含量,这也可能是三萜酸改善胰岛素抵抗、促进葡萄糖消耗的作用机制之一。在全面研究三萜酸降糖作用效果的基础上,选择了效果较好的TTA 7.5μg·mL-1组、UA 5μg·mL-1组、OA 5μg·mL-1组和MA 5μg·mL-1组进行进一步的降糖机制研究,通过检测三萜酸对胰岛素抵抗3T3-L1脂肪细胞中PPARγ、CAP、Glut4、Resistin和IRS-1五个胰岛素信号传导相关关键靶点的mRNA表达水平,初步探索其作用的分子机理。试验结果表明：(1)UA可能通过两条途径增强胰岛素抵抗3T3-L1脂肪细胞的胰岛素敏感性、促进葡萄糖消耗：一是上调PPARγ及其下游基因Glut4 mRNA的表达,二是提高IRS-1 mRNA的表达；(2)OA可能是通过下调Resistin mRNA的表达来发挥降糖作用；(3)MA可能通过提高胰岛素抵抗3T3-L1脂肪细胞CAP和IRS-1 mRNA的表达来增强胰岛素敏感性、促进葡萄糖消耗,虽然MA没有表现出促进PPARγmRNA表达的活性,但还是明显促进了CAP mRNA的表达,提示CAP的表达除受PPARγ调控之外还可能存在其它调控途径；(4)TTA为各三萜酸的混合物,其降糖作用的效果和机制基本上是各三萜酸作用的综合体现,一是通过促进PPARγmRNA的表达来提高其下游CAP、Glut4 mRNA的表达；二是上调IRS-1 mRNA的表达；(5)青钱柳三萜酸在改善胰岛素抵抗、促进葡萄糖消耗上存在与Ros不一样的作用机制。综上所述可得出如下结论：(1)青钱柳悬浮培养细胞中含熊果酸、齐墩果酸、白桦脂酸、2α-羟基熊果酸、2α-羟基齐墩果酸五种主要的三萜酸；(2)青钱柳细胞悬浮培养可用于熊果酸、齐墩果酸等三萜酸的生产；(3)青钱柳悬浮培养细胞三萜酸具有较好的降糖作用效果；(4)青钱柳悬浮培养细胞三萜酸可通过提升PPARy、CAP、Glut4、IRS-1 mRNA的表达、下调Resistin mRNA的表达水平来实现降糖作用。更多还原

【Abstract】 Cyclocarya paliurus (Batal.) Iljinskaja (C. paliurus) is a rare and endangered plant growing in southern provinces of China and now only a few populations remained due to the great difficulty in propagation and cultivation. This plant is often used for functional foods, drugs, landscape and timber. Researches indicated that C. paliurus leaves contained many functional components such as triterpenoids, flavonoids, polysaccharides, saponins, and microelements, and therefore were valuable natural resource for functional food. Development and utilization of this plant are seriously hampered for scarce resources due to the difficulties in reproduction. In order to explore the feasibility of triterpenic acid production with hypoglycemic effect by suspension culture and provide reference for manufactures of triterpenoid functional food and the utilization of C. paliurus, cell suspension culture of C. paliurus was established, and the accumulation, extraction, separation, and identification of triterpenic acids in suspended cultured cells were studied in this paper. At the same time, the hypoglycemic effect and mechanism of triterpenic acids were explored.The crisp vigorous calli which were fit for suspension culture were obtained from the calli induced form germless immature C. paliurus leaves after sub-culturing and screening repeatedly. Good cell suspension cultures have been established by repetitious subculture and optimization of culture conditions when the suitable calli were inoculated into liquid culture medium. The optimum date for subculture was the 8th day after inoculation, and the highest biomass yield appeared at the 10th day. but the maxmum yield of TTA (total triterpenic acid) appeared at the 14th day. Higher yield of TTA could be obtained in MS medium (Murashige and Skoog mediums) when the phosphate, Ca++, and Mg++ concentration was adjusted to 1.875 mM,2 mM, and 2 mM respectively. TTA yield increased 35.70% and 22.31% respectively when the suspended cultured cells were induced by CeCl3 and salicylic acid. The yield of TTA, UA (ursolic acid), and OA (oleanolic acid) were as high as 1179.85 mg·L-1, 443.64 mg·L-1, and 159.00 mg·L-1 respectively in cell suspension culture of C. paliurus. The major triterpenic acids in the suspended cultured cells of C. paliurus were extracted, separated, and identified for the first time. High TTA extraction yield could be obtained from the suspended cultured cells by the ultrasonic extraction with acetoacetate, and the crude extraction was easy to purify for little impurities. The maximum TTA extraction yield reached 5.56% when the extraction was performed under the optimal parameters namely 40 min,50℃, and 1/10 of material/acetoacetate. Five triterpenic acids were separated from TTA by macroporous resin adsorption, silica gel column chromatography, Sephadex LH-20 column chromatography and semipreparative high performance liquid chromatography, and identified as UA, OA, BA (betulinic acid), CA ((2a,3(3)-2,3-Dihydroxy-urs-12-en-28-oic acid), and MA ((2α,3β)-2,3-Dihydroxy- olean-12-en-28-oic acid) respectively by determination of HPLC, TLC, RRLC-TOF/MS,1H NMR 13C NMR, and DEPT. This is the first report that BA and MA was separated and identified from C. paliurus.The hypoglycemic effects of triterpenic acids from suspended cultured cells of C. paliurus were investigated with in vitro model of 3T3-L1 preadipocytes, mature adipocytes, and IR mature adipocytes. The main results showed that:(1) Improvement on the proliferation of 3T3-L1 preadipocytes wasn’t observed when TTA, UA, OA, and BA were added into the culture medium, but 2.5μg·mL-1 MA did improve the proliferation. (2) TTA, UA, OA, and BA didn’t stimulate the glucose consumption of preadipocytes, but 2.5-5μg·mL-1 CA and MA increased glucose consumption with or without the stimulation of insulin. (3) 5μg·mL-1 TTA, UA, and OA significantly improved the differentiation of preadipocytes, but 5μg·mL-1 CA inhibited differentiation, and BA and MA didn’t show effects on differentiation. (4) TTA, UA, OA, CA, and MA all increased the glucose consumption of mature adipocytes with or without the stimulation of insulin, which indicated that the mechanism of triterpenic acids on the enhancement of glucose consumption differed from Ros (rosiglitazone). (5) TTA, UA, and MA could prevent mature adipocytes from formation of insilin resistance (IR). Although OA and CA couldn’t prevent IR formation, but both could alleviate IR symptom. BA had no effect on the IR formation. (6) TTA and UA could significantly alleviate IR symptom and enhance the sensitivity to insulin for IR adipocytes, MA, OA, CA showed weaker effect than that of TTA and UA, but BA had no effect on IR adipocytes. (7) All triterpenic acids significantly decreased FFA (free fatty acids) content of culture medium at suitable concentration, which might interpret the mechanism of alleviation of IR symptom and improvement of glucose consumption.IR adipocytes treated by 7.5μg·mL-1 TTA,5μg·mL-1 UA,5μg·mL-1 OA,5μg·mL-1 MA,3.6μg·mL-1 Ros and adipocytes of control, IR model were collected for experiments on hypoglycemic mechanism. This paper investigated the moleculer hypoglycemic mechanism by detecting on mRNA expression of PPARy, CAP, Glut4, Resistin, and IRS-1 which played important role in the signal transduction of insulin. The main results showed that:(1) UA might improve the sensitivity to insulin and the consumption of glucose by two pathways:firstly up-regulated PPARy and Glut4 (downstream responsive gene of PPARy) mRNA expression, secondly enhanced IRS-1 mRNA expression. (2) OA stimulated glucose consumption by down-regulation of Resistin mRNA expression. (3) MA might improve the sensitivity to insulin by increasing the expression of CAP and IRS-1 mRNA. It should be noted that MA improved CAP mRNA expression although it didn’t enhance the expression of PPARγmRNA, which suggested that there were still other pathways to regulate the expression of CAP mRNA except PPARy pathway. (4) As mixture of triterpenic acids, TTA showed comprehensive effects as a whole by two pathways:firstly increasing the expression of downstream responsive genes such as CAP and Glut4 by up-regulation of PPARγmRNA, secondly increasing the expression of IRS-1 mRNA. (5) Triterpenic acids from suspended cultured cells of C. paliurus improved the sensitivity to insulin and the consumption of glucose by the pathways different from Ros.The main results can be concluded as:(1) There were five triterpenic acids namely UA, OA, BA, CA, and MA in the suspended cultured cells of C. paliurus. (2) Cell suspension culture of C. paliurus could be used to produce triterpenic acids such as UA and OA. (3) Triterpenic acids from suspended cultured cells of C. paliurus showed good hypoglycemic effect. (4) Triterpenic acids from suspended cultured cells of C. paliurus improved glucose consumption by increasing the expression of PPARy, CAP, Glut4. and IRS-1 mRNA and decreasing the expression of Resistin mRNA.更多还原