【作者】 陈祥洪；

【导师】 陈家春；

【作者基本信息】 华中科技大学 ， 药理学， 2011， 博士

【摘要】 湖北麦冬(Liriope spicata (Thunb.) Lour. var. prolifera Y. T. Ma)是湖北省的道地药材,是中药麦冬的主流品种之一。在中医临床上湖北麦冬与杭麦冬和川麦冬等同入药,用于治疗热病伤津、心烦口渴等症。在古今治疗糖尿病(消渴病)的中药复方中麦冬常作为主药,近年多位学者就麦冬的“消渴”作用做了一些抗糖尿病研究,表明川、杭麦冬具有抗糖尿病的作用,并初步证明其发挥抗糖尿病作用的活性物质主要是麦冬多糖。对湖北麦冬多糖降血糖的相关研究在国内外还未见公开报道,但湖北麦冬多糖极可能也具有降血糖活性。鉴于此,在国家自然科学基金和国家十一五科技支撑计划的资助下,本论文对湖北麦冬抗Ⅱ型糖尿病活性、活性物质(湖北麦冬多糖)的制备工艺、理化性质及结构特征进行了研究。本论文由四个部分组成。第一部分为湖北麦冬抗Ⅱ型糖尿病的活性及作用机理研究,证实湖北麦冬抗Ⅱ型糖尿病活性,确定其活性物质基础为多糖成分,并推测改善胰岛素抵抗是其抗Ⅱ型糖尿病机理之一。第二部分为湖北麦冬多糖(TLSP)制备工艺研究,确定出一条稳定、天然、高效的湖北麦冬多糖实验室制备路线。第三部分是湖北麦冬多糖(TLSP)的理化性质及结构研究,获得了TLSP及从中分离出的均一多糖(LSP1和LSP2)稳定的理化参数,并解析出LSP1和LSP2的一级结构特征。第四部分是综述,对2005年1月至2010年12月报道的具有潜在抗糖尿病活性的单体化合物进行了概括。第一部分为探究湖北麦冬是否具有抗糖尿病(Ⅱ型糖尿病)活性、活性物质是什么,本研究首先采用高脂高糖饲料+小剂量STZ诱导的Ⅱ型糖尿病小鼠模型,对湖北麦冬抗糖尿病活性成分提取溶剂(95%乙醇或水)进行筛选研究,确定只有水提取物(WE)具有较强的抗糖尿病活性(降低糖尿病小鼠的空腹血糖(P<0.01),改善糖尿病小鼠糖耐量(P<0.01))。接着,本研究对湖北麦冬水提取物(WE)进行分离、纯化,得到湖北麦冬粗多糖(CP)、总多糖(TLSP)及无糖部位(NP),并采用同样的动物模型对各部位进行活性筛选,确定WE、CP、TLSP均具有较强的抗糖尿病活性(降血糖及改善糖耐量,P<0.01),活性强度依次为WE<CP<TLSP,非糖部位(NP)无抗糖尿病活性。多糖纯度最高的TLSP具有最好的活性,说明TLSP是湖北麦冬主要的抗糖尿病活性成分。为进一步探讨湖北麦冬多糖的抗Ⅱ型糖尿病活性物质基础,本研究又对TLSP进行分离纯化,得到均一多糖1(LSP1)和2(LSP2),并采用同样的动物模型对TLSP、LSP1和LSP2进行活性比较。实验结果证实,TLSP、LSP1和LSP2均可显著降低Ⅱ型糖尿病小鼠的血糖(P<0.01),改善糖耐量(P<0.01)和降低胰岛素抵抗指数(P<0.01),各项作用强度与罗格列酮比较没有显著性差异(P>0.05)。而且能降低血总胆固醇(P<0.01)和甘油三酯(P<0.01)、增加高密度脂蛋白相对含量(P<0.01)并降低低密度脂蛋白水平(P<0.01),其效果均较罗格列酮好(P<0.05或P<0.01),体现湖北麦冬多糖能从多方面综合治疗糖尿病、缓解其症状。通过各方面综合比较,确定三者抗Ⅱ型糖尿病活性强度基本相当：TLSP≈LSP1≈LSP2,100mg/kg≈200mg/kg。由于TLSP的制备工艺相对简单,工业生产周期短、成本低。因此本项目组选择TLSP为候选药物进行后续开发,并初步确定其对小鼠的有效剂量为100mg/kg。同时,在对TLSP、LSP1和LSP2的药效学研究中,发现TLSP、LSP1和LSP2均具有改善胰岛素抵抗(HOMA-IR)的作用,因此,本课题顺着胰岛素抵抗这条路线,研究湖北麦冬多糖对改善胰岛素信号转导通路和肝糖代谢的影响,以初步探究抗Ⅱ型糖尿病机理。机理研究结果表明,TLSP、LSP1和LSP2均能明显提高糖尿病小鼠肾脏组织中胰岛素信号因子—胰岛素受体(InsR)、胰岛素受体底物-1(IRS-1)、磷脂酰肌醇3激酶(PI3K)的表达水平(P<0.01),进而改善胰岛素信号传导,增加组织对胰岛素的敏感性,从而改善糖尿病症状,这是湖北麦冬多糖抗糖尿病机理之一。同时,TLSP、LSP1和LSP2还能增加肝糖原含量(P<0.01)及葡萄糖激酶(GcK)活性(P<0.01),降低葡萄糖-6-磷酸酶(G6P)活性(P<0.01),从而改善糖尿病小鼠肝内糖代谢紊乱,增强肝细胞合成糖原及促进葡萄糖利用,进而改善糖尿病症状,这是湖北麦冬多糖抗糖尿病机理之二。第二部分在确定以湖北麦冬多糖TLSP作为抗糖尿病候选药物开发后,本研究分别采用正交和单因素实验对TLSP的沸水提取、酶解除蛋白质、透析除小分子、醇沉及DEAE-纤维素柱层析脱色工艺进行优化。经反复验证,得到一条稳定的TLSP制备路线。确定出的制备路线为：取湖北麦冬干燥块根的粗粉,煎煮3次,每次加入水的体积分别为粗粉重量的4、4和2倍,每次30分钟,合并滤液,60℃减压浓缩得到6倍药材体积粗多糖液；将粗多糖液调节pH 6和水浴温度45℃,加入0.3%药材量的木瓜蛋白酶(12U/mg),酶解2小时,将酶解后的多糖液煮沸5 min,静置放冷,抽滤去沉淀。然后将滤液装入透析袋(Mw1000)(装入的液体量不超过透析袋容积的2/3)用自来水和蒸馏水各透析1天,去除小分子。透析后的袋内多糖液(保留部分)缩到原药材重量的1.5倍体积,加入95%的乙醇,以乙醇终浓度80%在室温下(20±2℃)醇沉24h,收集沉淀；上清液减压浓缩到原药材重量的1.0倍体积,再加入95%的乙醇,以乙醇终浓度80%在室温下(20±2℃)再醇沉24h,收集沉淀；合并沉淀部分,真空／冷冻干燥获得湖北麦冬粗多糖。湖北麦冬粗多糖蒸馏水溶解、调节浓度0.2g/mL,采用DEAE-纤维素52层析柱脱色纯化,上样体积5mL/g填料,上样及洗脱流速2.5柱体积/h(BV/h),蒸馏水／纯水为洗脱剂,水洗量3 BV,收集量6 BV(从上样后第2 BV的下柱液开始收集)。收集到的多糖液减压浓缩、真空／冷冻干燥即得湖北麦冬多糖TLSP。第三部分为了更好的对湖北麦冬多糖(TLSP)进行质量控制,本论文对TLSP的理化性质及其主要的组成成分(LSP1和LSP2)的一级结构特征进行研究。主要结果如下：(1)从TLSP中分离得到2个结构新颖的均一多糖,LSP1和LSP2,两均一多糖分别占TLSP的25.4%和64.2%。(2)运用多种物理、化学方法对TLSP、LSP1和LSP2的常规理化性质进行了研究,获得了TLSP、LSP1和LSP2稳定的理化参数。(3)运用多种光谱、色谱、化学等方法对LSP1和LSP2的一级结构进行研究,获得LSP1和LSP2较精确的结构信息。LSP1为小分子果聚糖(Mw为3199),聚合度为20,由19个β-D-果糖和1个α-D-葡萄糖构成；其主链是以1、2链接的果糖残基构成,有6个重复单元,每个单元包含1个1、2位连接的双取代果糖残基、1个1、2、6位连接的三取代果糖残基和1个2位连接的单取代果糖残基；多糖主链的一个末端以2位羟基与1个α-D-葡萄糖的1位羟基链接成苷,另一个末端以1位羟基与1个β-D-果糖的2位羟基链接成苷。LSP2也为小分子果聚糖(Mw为4287),聚合度为26,由25个β-D-果糖和1个α-D-葡萄糖构成；其主链是以1、2链接的果糖残基构成,有6个重复单元,每个单元包含2个1、2位连接的双取代果糖残基、1个1、2、6位连接的三取代果糖残基和1个2位连接的单取代果糖残基；多糖主链的一个末端以2位羟基与1个α-D-葡萄糖的1位羟基链接成苷,另一个末端以1位羟基与1个β-D-果糖的2位羟基链接成苷。第四部分本部分对2005年1月至2010年12月报道的具有抗糖尿病潜力的单体化合物进行了概括,侧重于介绍从药用植物分离获得的一些具有抗糖尿病活性的化合物(包括已知的和新发现的),包括皂甙、黄酮、生物碱、蒽醌类、萜类、香豆素类、酚类、多糖和其他一些化合物,以说明抗糖尿病天然产物研究的必要性。更多还原

【Abstract】 Liriope spicata (Thunb.) Lour. var. prolifera Y. T. Ma, which belongs to Liliaceae family, is indigenous to Hubei province, China. The tuberous root, recorded as Radix Liriopes in Pharmacopoeia of the People’s Republic of China, is frequently used as "maidong" in prescriptions of traditional Chinese medicine for the treatment of diabetes mellitus (DM). Recently, some scholars have discovered that Radix Ophiopogonis (Ophiopogon japonicus (Thunb.) Ker-Gawl), which is another type of traditional Chinese medicine "maidong", showed considerable hypoglycemic effects and the water extract as well as polysaccharides were the main active components. Thus, the tuberous root of Liriope spicata var. prolifera might also have potential anti-diabetic activities. However, anti-diabetic effects of the tuberous root of Liriope spicata var. prolifera and the polysaccharides from it have not been reported. Therefore, supported by the Key Technologies R & D Program and the National Natural Science Foundation of China, the study on screening of active substance from the tuberous root of Liriope spicata var. prolifera on streptozotocin (STZ)-induced type II diabetic mice was carried out, as well as the preparation, physical-chemical properties and structural characteristics of the active substances (polysaccharides).My work was composed by four parts. The first part was about the anti-diabetic activity of Liriope spicata var. prolifera, as well as the mechanisms of action. The second part was about the preparation of a polysaccharide faction (TLSP) from Liriope spicata var. prolifera. The third part was about physical-chemical properties and structural characteristics of TLSP and two new polysaccharides (LSP1 and LSP2) separated from it. The last part was a review on natural compounds with anti-diabetic potential reported from 2005 to 2010. Part OneTo investigate whether the tuberous root of Liriope spicata var. prolifera possesses anti-diabetic (type 2 diabetic) activities and find out the active substances, this study firstly screened the activities of 95% ethanol extract (EE) and water extract (WE) form the tuberous root on a type 2 diabetic mice model induced by diets high in fat and fructose and intraperitoneal injection of STZ at a low dose (40 mg/kg). The results indicated that only the water extract (WE) showed significant anti-diabetic effects (a marked decrease of fasting blood glucose (FBG) and a significant improvement on glucose tolerance).After that, based on the separation and purification, the crude polysaccharides (CP), a polysaccharide faction (TLSP) and a non-polysaccharide faction (NP, contained no polysaccharides) were obtained from WE. Activity screening of WE, CP, TLSP and NP was also carried out on STZ-induced type 2 diabetic mice. The results proved that WE, CP and TLSP all showed significant anti-diabetic effects (a marked decrease of FBG and a significant improvement on glucose tolerance). Compared the activities, WE<CP<TLSP, but NP did not show any anti-diabetic effect. TLSP with the highest polysaccharide purity showed the highest activity, which indicated that polysaccharides were the main anti-diabetic substances of Liriope spicata var. prolifera.In order to further investigate the anti-diabetic material foundation of polysaccharides from Liriope spicata var. prolifera, separation was performed on TLSP to obtain two polysaccharides both with a single molecular weight, LSP1 and LSP2. Activity screening of TLSP, LSP1 and LSP2 was also carried out on STZ-induced type 2 diabetic mice. The results proved that they all caused a marked decrease of FBG and a significant improvement on glucose tolerance and insulin resistance (HOMA-IR), and all the above effects were the same marked as rosiglitazone. In addition, while lowering total cholesterol (TC), triglyceride (TG) and low-density lipoprotein (LDL) cholesterol levels, TLSP, LSP1 and LSP2 elevated the relative high-density lipoprotein (HDL) cholesterol level (HDL/TC) in serum, and the activities of them were more marked than rosiglitazone. All the above results reflected polysaccharides from Liriope spicata var. prolifera could therapy type 2 diabetes and release symptoms in many ways. Compared with the synthetic activities, there was no difference among TLSP, LSP1 and LSP2 (TLSP≈LSP1≈LSP2,100 mg/kg≈200 mg/kg). Because the preparation of TLSP was easier, shorter (time) and cost-lower than LSP1 and LSP2, our group chose TLSP as a candidate drug for further exploitation.During the pharmacodynamics research of polysaccharides from Liriope spicata var. prolifera, we found that TLSP, LSP1 and LSP2 could improve insulin resistance (HOMA-IR). So, following the idea of insulin resistance, our group studied the effects of TLSP, LSP1 and LSP2 on insulin signaling transduction and glucose metabolism to explain the possible anti-diabetic mechanisms. The results indicated that after oral administration of TLSP, LSP1 and LSP2, the protein expression levels of insulin receptor-α(InsR-α), insulin receptor substrate-1 (IRS-1) and phosphatidylinositol 3-kinase (PI3K) in renal tissues of the diabetic mice were significantly increased. In addition, the glycogen content and glucokinase (GK) activity in liver were significantly increased, yet the hepatic glucose-6-phosphatase (G6Pase) activity was decreased. So, the possible anti-diabetic mechanisms of TLSP, LSP1 and LSP2 may be due to the improvement of insulin signaling transduction and glucose metabolism.Part TwoWhen TLSP was chosen as a candidate drug for further exploitation, orthogonal experiments or/and single-factor designs were investigated to get the best preparation conditions of TLSP from the tuberous root of Liriope spicata var. prolifera, including the extraction by hot water, deproteinization by the Papain enzymolysis, dialysis by a regenerated cellulose membrane tube, repeated precipitation by ethanol, decolorization by a diethylaminoethyl cellulose 52 (DEAE-cellulose 52) column. By repeated verification, we got a stabile preparation of TLSP. The best preparation conditions were as follows:Powdered tuberous root was boiled in distilled water three times,0.5 h each time, and the ratios of liquid to solid were 4∶1 (v/w),4∶1,2∶1, respectively. Each extract was then filtered and combined. The water extract was deproteinated by an enzymolysis method with Papain. Deproteinization conditions were pH 5.91 (phosphate buffer), ratio of Papain (12μ/mg) to raw material was 0.3%, and kept in water-bath (45℃) for 2 h. The deproteinated extract was then boiled for 5 min, stored overnight at 4℃and fitered, the filtrate dialyzed using the regenerated cellulose membrane tube (Mw cut-off 1000) against tap water for 1 days and distilled water for another day. The retentate portion (nondialysate) was concentrated to a volume which was 1.5 times of the powdered material weight, and then precipitated by addition of ethanol to a final concentration of 80% (v/v); after 24 h, the precipitates were collected, the supernatant was concentrated to a volume which was 1.2 times of the powdered material weight, and then precipitated again by addition of ethanol to a final concentration of 80%(v/v); after 24 h, the precipitates were collected. The precipitates obtained by twice ethanol precipitation were combined, vacuum dried to obtain the crude polysaccharides. The crude polysaccharides were dissolved in water in a concentration of 0.2 g/mL, applied to a DEAE-cellulose 52 column. The sample volume was 5 mL/g column filler, the flow was 2.5 column body volumes (BV) per hour, the amount of water used for elution was 3 BV, and the collection amount of elution was 6 BV (from the 2nd BV to 7th BV of the elution). The fraction eluted with water was further vcuum concentrated, vacuum dried or lyophilized to obtain TLSP.Part ThreeIn order to make the quality of TLSP be easy controlled, this study investigated the physical-chemical properties of TLSP, LSP1 and LSP2, as well as the primary structural characteristics of LSP1 and LSP2.The main results as follows:(1) Two new polysaccharides, LSP1 and LSP2, were separated from TLSP. Their yields were 25.4% and 64.2%, respectively.(2) On the basis of several chemical and physical methods, the stable physical-chemical parameters of TLSP, LSP1 and LSP2 were finally determined.(3) The primary structural characteristics of LSP1 and LSP2 were investigated by HPGPC, FT-IR, UV, NMR spectroscopy, periodate oxidation and Smith degradation, methylation analysis and GC-MS. Based on the data obtained, LSP1 and LSP2 were two fructans with the molecular weights 3.20 and 4.29 kDa, respectively. They both have a backbone structure with six repeating units; each repeating unit in LSP1 comprises (1, 2→)-linked, (1,2,6→)-linked, and (2→)-linked fructosyl residues in the ratio of 1∶1∶1 and in LSP2 in the ratio of 2∶1∶1; a glucosyl residue and a fructosyl residue linked with the two terminals of the backbone of both LSP1 and LSP2, respectively.Part FourThe last part was a review on natural compounds with anti-diabetic potential reported in the literature from January 2005 to December 2010, in order to identify the research needs in this area. it focuses on some new and known chemical compounds isolated mainly from medicinal plants possessing anti-diabetic properties, including saponins, flavonoids, alkaloids, anthraquinones, terpenes, coumarins, phenolics, polysaccharides, and some other compounds.更多还原