【作者】 胡楠；

【导师】 熊兴耀；

【作者基本信息】 湖南农业大学 ， 药用植物资源工程， 2013， 博士

【摘要】 甜叶悬钩子(Rubus suavissimus S. Lee)是蔷薇科悬钩子属的一个变种,主要生长于我国广西省的山区,故又称广西甜茶。长期以来,民间一直将其当茶饮用或作为代糖加工,亦或作为药材使用,具有清热润肺,止咳祛痰,消肿生肌等功能。从上世纪80年代发现该物种以来,我国逐步展开了对甜叶悬钩子的研究,在其食用和药用等方面获得一定的进展。为对甜叶悬钩子进行比较系统的化学成分研究以及功能性的分析,从而更好的开发该资源,本文通过一系列研究主要取得了以下结果：(1)本课题首先对甜叶悬钩子叶片中有效成分进行了提取,并研究其功能。甜叶悬钩子叶片经过体积分数为50%的乙醇溶液回流提取,通过离子交换树脂,用不同体积分数的乙醇溶液(按照体积分数10%为梯度变化从100%-10%)分别洗脱,得到4个洗脱部分(通过TLC薄层色谱分析将移值相近的部分合并,分别标记为提取物I,提取物II,提取物Ⅲ和提取物Ⅳ)。对各部分进行抗氧化,促进胰岛素分泌以及抑菌活性的检测,结果表明：其中提取物II与提取物Ⅳ表现出强抑菌活性,提取物I表现出抗氧化活性,提取物Ⅲ和提取物Ⅳ拥有最强的促进胰岛素分泌能力。(2)本课题随后针对提取物进行了系统的化学成分研究,从中分离并鉴定了24个化合物,通过ID,2D-NMR, IR, UPLC-MS, HPLC等手段分析了它们的结构。其中包括11个二萜类物质：斯替维醇；对映16β,17—二羟基—贝壳杉—3—酮；对映16β,17—羟基—贝壳杉—19—羧酸；16α,17,19—三羟基贝壳杉；3β,16α,17—三羟基贝壳杉烷,7β—羟基斯替维醇；甜叶悬钩子苷；甜叶悬钩子苷—A；11,15—二羟基—贝壳杉—16烯—18—羧酸；16α—17—二羟基—贝壳杉—19—羧酸；13,17—二羟基—贝壳杉—15—烯—19—羧酸；对映贝壳杉—3α,16β,17—三醇—17—O—β—D—葡萄糖苷；5个三萜类物质：2α,3β—二羟基—乌苏—12—烯—28—羧酸；2α,3β,23—二羟基—乌苏—12—烯—28—羧酸；2α,3卢,19α,23—四羟基—乌苏—12—烯—28—羧酸；2α,3β,19α,23,24—五羟基—乌苏—12—烯—28—羧酸；4个黄酮类物质：槲皮素；槲皮素—3—O—β—D—吡喃葡萄糖苷；槲皮素—3—O—α—D—核糖苷；山奈酚—3—O—β—D—6—O—对羟基桂皮酰基—吡喃葡萄糖苷；以及其他类物质：7,8—二氢—醉鱼草醇B(苏型)；7,8—二氢—醉鱼草醇B(赤型)；7S,8R—二氢去氢二愈创木基醇；蔗糖。(3)经过检测,甜叶悬钩子苷在致龋方面有较强的功效。在200μgmL的时候对变形链球菌生长的抑制率达到了99%以上,因此本课题针对甜叶悬钩子苷对于变形链球菌生长的抑制机理进行研究。结果表明：甜叶悬钩子苷对于变形链球菌产酸能力,表面疏水性,粘附能力,葡萄糖基转移酶GTF的活力,水不溶性胞外多糖的合成有明显的抑制作用。(4)研究表明,甜叶悬钩子对于糖尿病有辅助治疗的作用。设计将小鼠腹腔注射四氧嘧啶制造高血糖糖尿病小鼠模型。成模后与正常小鼠—起分别按血糖随机分为6组：糖尿病对照组,低、高剂量治疗组,正常对照组以及低、高剂量组。连续灌胃直至实验结束时测小鼠血糖水平,并探讨其作用机理。结果表明,甜叶悬钩子苷能够促进胰岛素分泌,且具有显著的降糖作用。其中200,300mg/kg.d的甜叶悬钩子苷降糖幅度均超过20%,而300mg/kg.d的甜叶悬钩子苷对于高血糖小鼠降糖幅度达到27%。(5)最后,课题针对甜叶悬钩子苷的安全性和毒性进行了评价,实验确定了甜叶悬钩子苷经口LDso为3.709g/kg (小鼠)4.735g/kg(大鼠),为低毒物质。经过长期和急性的毒性检测,甜叶悬钩子苷各实验组动物体重,增重,血液学,血液生化学指标均处于正常范围,主要器官经组织学检测未见明显表征变化,表明甜叶悬钩子苷对于小鼠生长发育,血液,肝肾器官等无明显毒性。更多还原

【Abstract】 Rubus suavissimus S.Lee is a perennial frutex deciduous which widely distributed in Kwangshi China, so it was also called Kwangshi Sweet tea. It has been used as tea drinks and sweetening agents in the folk for a long time. It was also used as traditional medicine for eliminating phlegm by cooling, expelling phlegm to arrest coughing and homeostatic elimination of stagnation.In order to built a foundation for the further exploitation of Rubus suavissimus S Lee, a series pharmacological tests and systematic analyses on the chemical structure of the extraction from the leaves of sweet tea have been carried on, and the results were followed:(1) The active compounds were extracted from the leaves, and the medicinal functions were studied. Four crude fractions (combin ed by thin-layer chromatography test) were separated by the Amberlite column according to the change of the ethanol concentration (from100%to10%, v/v), which marked Fraction Ⅰ～Fraction Ⅳ. According to the result, The Fraction Ⅱ and FractionⅣshows the inhibition on S-mutans, and the Fraction Ⅰ shows the anti-oxidant activities, while the FractionⅢ and FractionⅣ has the strongest insulin-stimulating activities.(2) The further studies on the analyses of chemical structures were carried out, and twenty-four compounds were purified and eluted from these fractions by using the1D and2D-NMR, IR, UPLC-MS and HPLC methods. There were eleven diterpenoid compounds as followed: Steviol; Ent-16β,17-dihydroxy-Kaurane-3-one; Ent-16β,17-dihydroxy-Kaurane-19-oic acid;16α,17,19-trihydroxy-ent-kaurane;3β,16a,17-trihydroxy-ent-kaurane,7β-hydroxyl-steviol; Rubusoside; Rubusoside-A;11,15-dihydroxy-ent-kaurane-16-en-18-oic-acid;16α-17-dihydroxy-ent-kaurane-19-oic-acid;13,17-dihydroxy-ent-kaurane-15-en-19-oic-acid; Ent-kaurane-3α,16β,17-triol-17-O-β-D-glucoside; Five triterpene compounds as followed:2α,3β-dihydroxy-ursane-12-en-28-oic-acid;2α,3β,23-trihydroxy-ursane-12-en-28-oic-acid;2α,3β,19α,23-tetrahydroxy-ursane-12-en-28-oic-acid;2α,3β,19α,23,24-pentacarbonyl-ursane-12-en-28-oic-acid. Four Flavonoid compounds as followed: Quercetin; Quercetin-3-O-β-D-glucopyranoside; Quercetin-3-O-α-D-ribopyra-noside; Kaempferol-3-O-β-D-6-O-trans-P-coumaryol-glucopyranoside; and four other compounds as followed:7,8-dihydro-buddlenol-B (erythro);7,8-dihydro-buddlenol-B (threo);7S,8R-dihydrodyhydroconiferyl alcohol and Sucrose.(3) According to the test, the rubusoside has strong anti-bacteria activity, and the200μg/mL rubusoside exhibited more than99%inhibition against the growth of Streptococcus mutants. In order to explore the mechanism of antibacterial action, series of tests were conducted and the results showed that:the rubusoside has strong anti-effect against the acidogenicity of Streptococcus mutants, cell surface hydrophobicity, adhesive force, the enzyme activity of GTF, and the synthesis of WIG(4) Recent researches showed that the rubusoside was valuable in the treatment of diabetes. The mice were rendered diabetic by a single intra peritoneal injection of alloxan, after confirmation of TIDM, control and diabetic mice were further separated into six groups:control, low dose (C), highdose(C), diabetic, low dose (D), high dose (D).At the end of the experiment, the determinations of serum glucose concentration were carried out, and the Hypoglycemic mechanism was explored. The results showed that the rubusoside could increase the secretion of insulin, and could obviously decrease the serum glucose concentration. The hypoglycemic rates were more than20%(200and300mg/kg.d), while the highest rate reaches27%(300mg/kg.d)(5) At the end, Toxicology tests were carried out in order to study the toxicity and safety of rubusoside. The result showed the LD50of rubusoside were3.709g/kg (Mice)4.735g/kg (Rats) which belongs to the low-toxicity level. After the acute and chronic toxicity experiments, the weights, dynamiting, hematology and blood biochemistry were belongs to the normal range, no obviously representational changes in major organs, which means the rubusoside has no obvious toxicity on the growth and development, blood system or hepatorenal functions.更多还原