丹参、山楂调脂作用有效部位提取、分离及其分子机理研究

【作者】 王文祥；

【导师】 顾振纶；

【作者基本信息】 苏州大学 ， 心内科， 2002， 博士

【摘要】 脂质代谢紊乱是目前临床常见疾病之一，对冠心病动脉粥样硬化发生发展具有重要作用。丹参(Saliva miltiorrhiza Bye)与山楂(Crmaegus pinatigide Bge)是治疗高脂血症及动脉粥样硬化的传统药物，本研究运用现代方法分别提取分离丹参与山楂调脂作用的有效部位，通过高脂小鼠模型，优选丹参山楂提取物的最佳配比。观察了该配比对高脂血症大鼠和兔的防治作用，同时也观察了在高脂血症状态下对血液流变学的影响。在此基础上，从细胞、分子水平研究了丹参与山楂调脂有效部位最佳配比混合物对高脂血症防治作用的机制。 1．丹参、山楂调脂作用有效部位提取、分离、质量控制及结构鉴定研究 (1)丹参调脂有效部位提取工艺研究 根据丹参中丹参总酚酸的性质，选择水为提取溶剂，运用正交的方法对提取丹参总酚酸的提取时间、提取次数和溶媒用量三个因素进行了考察，优选提取工艺。运用明胶乙醇沉淀提取液中鞣质和其他大分子物质，在调节适合pH基础上，运用乙酸乙酯萃取丹参总酚酸有效部位。 结果表明，该提取工艺简便稳定，适合工业生产。 (2)山楂调脂有效部位提取工艺研究 山楂中主要调脂成分为三萜酸，根据三萜酸的性质，选择乙醇为提取溶剂，运用正交的方法对提取山楂三萜酸的提取时间、提取次数、溶媒用量及醇浓度进行考察，优选合理提取工艺。醇提浸膏用水洗除水溶性杂质，再用乙酸乙酯二步提取的方法，提取山楂三萜酸。 结果表明，该提取工艺简便稳定，适合工业生产。 (3)丹参、山楂调脂有效部位及药材的含量测定 丹参的调脂有效部位为丹参总酚酸，选用该部位中有效成分丹参素、原儿茶酸为对照品，分别用HPLC法和比色法对提取物以及药材进行含量测定。结果HPLC法测定得河南洛阳产丹参(Saliva miltiorrhiza Bye)药材丹参素含量最高，为0.477％，上述工艺提取的丹参调脂有效部位其丹参素的含量大于10％。药材的平均回收率为98.75％，RSD为0.31％。提取物的回收率为99.81％，RSD为0.47％。比色法测得提取物中总酚酸含量大于80％，回收率为99.77％，RSD为0.31％，且对相关比色条件进行了优选。 丹参、山檀调脂作用有效部位提取、分离、及其分了机理研穴 中义摘要 山植的调脂有效部位为山植三荫酸，选用该部位中的有效成分熊果酸为对照 U 品，分别用HPLC tL和比色法对山檀三砧酸提取物以及药材中熊果酸的含量和总 三砧酸的含量进行测定，并对相关影响条件进行考察。结果HPLC法测定山东青 州产山楷（O成＊＊g＊sd＊扣d尔B2e）熊果酸含量最高，为0．灼＊％，其回收率为 98石3％，RSD为0．48％。山檀提取物中熊果酸含量大于50％，其回收率为99．29％， RSD为0．48％。比色法测得提取物中总三菇酸的含量大于60％，回收率为97．69％，－RSD为1刀7％。 以上结果表明，该法测定山碴。丹参及其提取物，其结果稳定可靠。 （4）有效部位化学成分分离与鉴定＞山植三砧酸提取物，用石油醚热提得石油醚部位和非石油醚部位。石油醚部 位经气质联用分析得 5个化合物分别为正h十六烷（I）、正十六烷酸（11）、2－ 0 甲基十六烷酸甲酯（Ill）、正十八烷酸（IV）、1． 2、3、三甲基氧苑4－酚（V）。 非石油醚部位经硅胶多次柱层析，石油醚：氯仿、石油醚：乙酸乙酯洗脱，硅胶 薄层纯化，物理常数测定、波谱分析等共分离与鉴定9个化合物，分别为：十六 烷酸三十烷醇酯（VI）、十六烷酸二十八烷醇酯（Vll）、三十九烷醇．10（Vlll）。 烨皮醇（IX）、烨皮酸（X）、熊果醇（XI）、熊果酸（Xll）、齐墩果酸（Xlll）、 p－谷镶醇（XIV）。其中化合物I、11、Ill、IV、V。VI、X为首次从植物中获得。 丹参总酚酸提取物拌硅胶后，分别用乙酸乙酯、丙酮和甲醇回流提取得乙酸 乙酯、丙酮和甲醇三个部位，乙酸乙酯部位经硅胶防复柱层析得五个化合物，经 物理常数测定、彼谱分析等鉴定为，4、5、4”、5”－四羟基卜2双苯醚［11，原 二茶醛［111，原二茶酸rllll，异阿魏酸［IV，咖啡酸［V。丙酮部位经硅胶柱和 ODS 柱层析得二个化合物，经物理常数测定、波谱分析和已知品对照分析等鉴定为， 丹参甲素［VI］，丹酚酸DfVlll，其中化合物1为首次从丹参中分得新的化合物。 2．丹参、山碴调脂有效部位及其不同配比对高脂小鼠调脂作用的比较研究。雄性昆明种小鼠80只，随机分为8组，给药组分别给予，丹参水溶性总酚 提取物、山碴总三砧酸提取物，以及其二者提取物不同配比混合物，剂量为 160mg＆g。给药一周后，除正常对照组外一次lP75％蛋黄乳液后20h测定血脂， 结果各给药组血清TC、TG、LDL（、pOB与模型组相比均有不同程度降低，更多还原

【Abstract】 Lipometabolism disorder is one of clinic familiar disease. In recent years, It is realized that dyslipidemia plays an important role in the generation and development of coronary heart disease (CHD) and atherosclerosis(AS). Danshen and shanzha are traditional drug to treat lipometabolism disorder. In the studies, the compounds of lipid-regulating function were extracted, isolated and determined in the danshen and shanzha. The best proportion of lipid-regulating extraction of danshen and shanzha were elected with hyperlipidemic mice. Their lipid-regulating effects were observed in hyperlipidemic rats and rabbits. On this basis, cell and molecular mechanisms of lipid-regulating of the best proportion were studied.1.Extraction, isolation and determination of lipid-regulating function part in danshen and shanzha(1). Study on extraction process which effective part of lipid regulating of danshenAccording to properties of total-phenolic acid of danshen selected water was as extractive solvent. The main influence factors including extractive time, extractive times and solvent content were defined by orthogonal design. To find out the optimum extraction process for total-phenolic acid of danshen, in the extractive fluid tannin and other large molecular substance were deposited by gelatin and alcohol. Total phenolic acids of danshen were extracted by ethyl acetate on the basis of regulating propriety pH of fluid.The results showed that the extraction process was simply, good stability and was available for industrial prodution.(2). Study on extraction process which effective part of lipid regulating of shanzhaAccording to properties of total triterpenic acid of shanzha, selected alcohol was as extractive solvent. The main influence factors including extractive time, extractive times, solvent content and alcohol concentration were defined by orthogonal design. To find out the optimum extraction process for total triterpenicacid of shanzha, in alcohol extractive substance, substance which soluble in water was washed by water and extracted by ethyl acetate.The results showed that the extraction process was simply, good stability and was available for industrial production.(3) Determination of danshen,shanzha and their effective part of lipid-regulatingThe effective part of lipid regulating of danshen is total-phenolic acid. Using active compound danshensu,protocatechualdehyde are index of this part. To determine the contents of effective compound and extractive substance HPLC and colorimetric method.were used. The results showed that the content of danshensu was best in danshen of henna louyang. It was 0.477%. The content of danshensu of effective part was 12%. The average recovery of danshen was 98.75%. RSD was 0.3%. The average recovery of extractive substance was 99.81%. RSD was 0.47%. The content of total-phenolic acid in extractive substance was 80%, which detected by colorimetric method. The average recovery was 99.77%. RSD was 0.31%.The effective part of lipid regulating of shanzha is total- triterpenic acid. Using active compound ursolic acid in this part as an index. Adopting HPLC and colorimetric method to determine the contents of effective compound and extractive substance. The results showed that content of ursolic acid in shanzha of shangdong qingzhou, was best. It was 0.454%. The content of ursolic acid of effective part was 55%. The average recovery of shanzha was 98.63%. RSD was 0.48%. The average recovery of extractive substance was 99.29%. RSD was 0.48%. The content of total triterpenic acid of extractive substance was 60% by colorimetric method. The average recovery was 97.69%. RSD was 1.07%.The results showed that the method was simple, reliable, and accurate and could be applied to the quality control of the preparation.(4) Isolation and determination of active constituents in effective parts.15 compounds were isolated from the effective part of lipid regulating of shanzha. On the basis of the properties and spectral analysis, their structures更多还原