【作者】 刘卫欣；

【导师】 吴祖泽；

【作者基本信息】 中国人民解放军军事医学科学院 ， 病理学与病理生理学， 2009， 博士

【摘要】 地黄(Radix Rehmanniae)为玄参科植物地黄(Rehmannia glutinosa Libosch.)的块根,根据炮制方法的不同,分鲜地黄、生地黄和熟地黄三种入药。最早收载于《神农本草经》,列为上品,现收载于《中华人民共和国药典》(2005年版,一部),为常用的补益中药,归心、肝、肾经,具有滋阴补血止血等功效。鲜地黄清热生津,凉血止血。用于热病伤阴,舌绛烦渴,发斑发疹,吐血,衄血,咽喉肿痛。生地黄清热凉血,养阴生津。用于热病舌绛烦渴,阴虚内热,骨蒸劳热,内热消渴,吐血,衄血,发斑发疹[1],临床应用极为广泛。主产于河南、河北、山东、山西、内蒙古及东北,现全国大部分地区有栽培。糖尿病在祖国医学上属“消渴”范畴,地黄用于治疗消渴的历史悠久,现代药理学研究亦证明地黄中的低聚糖、环烯醚萜类成分的代表性成分梓醇以及地黄苷D有降糖活性,但是对于各种不同的化学成分之间的药效比较没有研究报道。为了明确地黄中不同化学部位的降糖效果,需要将不同的化学部位进行分离后分别研究。低聚糖类和环烯醚萜类是目前为止发现的地黄中具有降糖活性的主要的两种成分,它们极性相似,在溶剂中溶解性差别不大,都属于小分子物质,分离成两个有效部位难度较大,所以我们首先要解决的问题是这两类成分的分离富集问题,要求低聚糖和环烯醚萜苷的含量分别大于50%,并进一步研究适合于大量生产的提取分离工艺。在整体的药物研发过程中需要测定化学成分的含量,保证半成品和成品的质量。环烯醚萜类化合物的化学性质使得地黄中的环烯醚萜类成分的含量测定方法较难建立,迄今为止,未见地黄中总环烯醚萜的含量测定方法的报道,这在一定程度上影响了地黄有效部位的研究开发。为了解决以上存在的问题,继而开发出较高水平的中药新药,我们探索研究了地黄低聚糖类和总环烯醚萜类成分(主要为环烯醚萜总苷)的提取分离方法、含量测定方法,并对它们降糖的药理活性进行了初步的实验研究。研究主要内容和结果如下:1、低聚糖和总环烯醚萜的提取分离方法研究低聚糖和总环烯醚萜苷虽然化学结构有差别,但是极性相似,虽有文献报道分离了此两类成分,但是经本研究实际实验,几次均不能完全分离。柱层析时,环烯醚萜苷类成分总是与低聚糖同时存在于流出液中。针对此种情况,本研究以高效液相色谱(HPLC)检测梓醇的有无和Molish反应检测糖的存在作为提取分离监测指标的手段,对地黄低聚糖类和总环烯醚萜苷类成分的提取分离方法进行了实验研究,成功使两个部位得到分离。本研究以溶剂提取法提取生药,选择合适的提取溶剂、溶剂用量、提取时间、提取次数使目标组分获得高效提取。提取后,比较了四种型号的大孔吸附树脂对低聚糖类和环烯醚萜苷类成分的分离效果,结果表明H103是分离这两类成分较理想的大孔吸附树脂。地黄的水提物经醇沉后通过H103大孔吸附树脂柱层析,分段收集,得到的低聚糖部位和环烯醚萜苷部位经含量测定知低聚糖和总环烯醚萜类成分含量分别大于50%,符合中药、天然药物注册分类5类新药申报要求。2、含量测定方法的建立及含量测定在分离得到低聚糖和总环烯醚萜成分后,利用紫外分光光度法,进行了总环烯醚萜类成分的含量测定方法研究,首次建立了一种总环烯醚萜含量测定方法,并用此方法首次测定了地黄中总环烯醚萜类成分的含量,同时采用苯酚-硫酸法测定了低聚糖部位中低聚糖的含量。地黄药材、水提醇沉物和环烯醚萜苷部位中总环烯醚萜类成分含量经测定约为9%,16%和87%,低聚糖部位中低聚糖含量为76%。目前还没有适用性比较广的环烯醚萜总苷的含量测定方法,本研究新建立的含量测定方法具有一定的实用性,操作简单易行,结果可靠。可以进一步研究是否适合推广应用。3、地黄中各组分的降糖作用研究在初步确定了地黄全药材、低聚糖代表性成分水苏糖、环烯醚萜类成分梓醇的降糖疗效后,以四氧嘧啶致小鼠糖尿病模型测试地黄中的化学组分——总环烯醚萜部位及其代表成分梓醇,低聚糖部位及其代表成分水苏糖以及总环烯醚萜和低聚糖的加合——的降糖作用,观测了模型小鼠的体重、随机血糖、空腹血糖、餐后2h血糖、高密度脂蛋白胆固醇(HDL-C)含量,血清总胆固醇(TC)、甘油三酯(TG)、天冬氨酸氨基转移酶(AST)和丙氨酸氨基转移酶(ALT)的水平,同时称量了内脏的重量。结果表明各受试药能改善糖尿病小鼠餐后血糖升高现象,减低糖尿病小鼠的随机血糖,降低糖尿病小鼠的血清总胆固醇(TC)和甘油三酯(TG)水平,显示不同程度的治疗效果,提示各受试药物在一定程度上能调节糖尿病小鼠的血脂代谢,对糖尿病并发症具有一定的预防作用。同时使血清生化指标天冬氨酸氨基转移酶(AST)、丙氨酸氨基转移酶(ALT)降低,其中有一些组分能对抗糖尿病引起的脾脏重量的降低,提示对糖尿病小鼠的肝脏和脾脏功能具有一定的保护作用。初期的降糖实验表明地黄可以作为化学药的辅助治疗药,发挥其缓解糖尿病并发症或中医症候方面的特色。同时需要进一步研究各不同部位对2型糖尿病的药理作用。4、天然药物研究的一个重要部分是单体的分离纯化,其在寻找新的有效成分,提供参照,提供先导化合物等方面是不可取代的。我们用比较简单的方法分离了需要的化学单体,通过TLC、熔点测定配以简单的光谱解析鉴定了分得的单体成分,满足实验室的需要,并为工业化生产工艺提供参考。更多还原

【Abstract】 Radix Rehmanniae is the tuberous root of Rehmannia glutinosa Libosch. of the family Scrophulariaceae, which can be processed to fresh rehmannia root, rehmanniae praeparatum and Radix Rehmanniae preparata. It backs to Shennong bencao jing and is recorded by Chinese Pharmacopoeia (2005 Edition) now and it is mainly distributed in Henan, Hebei, Shandong, Shanxi, Inner mongolia, and now cultivated in most parts of China. It is widely used in clinic and diabetes with a long history. Documents reported that the hypoglycemic activity is saccharide, catalpol and rehmannioside D, which of the last two are the relative higher contents in iridoid glycosides.Research of the pharmacological effect of Radix Rehmanniae reported that the active component of hypoglycemic is more than one content, but did not report the difference of them. In order to identify the difference of them, need to get the departed components. Up to now, the main components of lowing blood sugar in Radix rehmanniae is oligosaccharide and iridoid glycosides, which are similar in polarity and solubility, so it is difficult to be separated. First of all, we should separate and enrich them with meeting the requirements of the total content of effective components is more than 50% respectively.In order to guarantee the quality of finished products and semi-manufactured goods and provide reasons of dosage, the content of the effective compositions needed to know in the whole process of discovery and development. The determination method of iridoid glycosides is not easy to establish because its chemical characters, which obstructs the development of Radix rehmanniae.To solve the above problems and develop Chinese new drug, we investigated the extraction and isolation technology, the determination method and studied the pharmacological effect of Rehmannia glutinosa oligosaccharide (ROS) and iridoid glycosides.1. Studies on the extraction and separation technology of ROS and iridoid glycosidesDetection of indexes: catalpol and saccharide which was monitored by the means of HPLC and Molish teast respectively. We studied the technology of extraction and separation of saccharide and iridod glycosides, then separated them successfully.The objective products were extracted effectively by solvent distillation witn using suitable solvent, quantity of solvent, extraction times and extraction process. Then, the efficiency of 4 types of macroreticular resins were compared and draw the conclusion that H103 was the most suitable resin to separate ROS and iridoid glycosides. And the content of ROS and iridoid glycosides was more than 50% respectively, which meets the requirements of the V class of new Chinese drugs.2. Studies on the determination method and content determinationAfter getting ROS and iridoid glycosides, studied the determination method by UV spectrophotometry and established a method to determine the content of iridoid glycosides of Radix Rehmanniae finally, by which we determined the total content of iridoid glycosdies , at one time, we determined the content of ROS in effective compositions by phenol-sulfuric acid method. The iridoid glycosides content of medical material, water extract-alcohol precipitated and effective parts is 2.7%, 16.2% and 87.4% respectively, the ROS content in the effective compositions of polysaccharides is 76%.3. Studies on hypoglycemic effect of components in Radix RehmanniaeIn alloxan-induced diabetic mice, we compared the hypoglycemic effect of chemical compositions which including iridoid glycosides and its chief constituent catalpol, ROS and its chief constituent stachyose and the mixtrue of iridoid glycosides and ROS that comed from Radix Rehmanniae. We observed these indexes:body weight, random blood sugar, fasting plasma glucose, two-hour postprandial plasma glucose, high density lipoprotein cholesterol(HDL-C), total cholesterol(TC), triglycerides(TG), alanine aminotransferase(ALT), aspartate aminotransferase(AST) and weight of viscera, which indicated that those components could lower two-hour postprandial plasma glucose, decrease total cholesterol (TC) and triglycerides (TG) level , but the effect is difference from each other. It was observed that the drugs could increase high density lipoprotein cholesterol (HDL-C) content in alloxan-induced diabetic mice, suggesting that the drugs had some preventive effects on diabetic complications. Hyperglycemia-resulted injury of liver, spleen was also ameliorated. In conclusion, the mechanisms of hypoglycemic activity of objective drugs might relate to modulating carbohydrate and lipid metabolism, protecting liver and spleen against impairment by hyperglycemia.The preliminary research indicated that Radix Rehmanniae could be used as assistant drugs of chemical medicine, which would take effect in complication in diabetes.4. One of the important part of natural products is the extraction–separation- purification of monomers, which is could not be replaced by anything else. We obtained several monomers in simple way and identified the structure by TLC, determination of melting point, analysis of spectrum. These monomers could meet the requirement of the lab, and it is also available for industrial production.更多还原