摘要
中药委陵菜为蔷薇科(Rosaceae)植物委陵菜(Potentilla chinensis Ser.)的干燥全草,《中国药典》2000,2005年版收载,其性寒,味苦,具有清热解毒、凉血止痢的作用,常用于赤痢腹痛、久痢不止、痔疮出血、痈肿疮毒等。经临床应用调查,天津蓟县中医医院和天津中医药大学附属医院应用委陵菜治疗糖尿病,取得了较好的疗效。本课题组前期研究表明委陵菜降血糖作用的主要成分为委陵菜黄酮(trans-tiliroside),其降血糖作用强于盐酸苯乙双胍,且毒性较低。该化合物与市售双胍类、硫脲类、α-糖苷酶抑制剂、噻唑烷二酮类以及GLP-1R激动剂肽类药物在结构上存在根本差异,则可能存在不同的作用机制,是具有较高开发价值的活性先导化合物。为进一步的委陵菜药材质量控制以及药物动力学研究提供切实可行的分析方法,本文进行委陵菜黄酮对照品的制备及含量分析方法学的研究。
首先,利用高速逆流色谱技术分离制备委陵菜黄酮对照品,结合HPLC选择溶剂体系以及调整各溶剂的比例,将提取制备的委陵菜黄酮粗品用于HSCCC分离纯化。结果表明,二氯甲烷-甲醇-水(体积比为5：4：2)为最佳溶剂体系,以上相为流动相,下相为固定相,转速为800r/min,流速为2.0mL/min，分离得到纯度达98%的委陵菜黄酮。
本文第二部分进行委陵菜黄酮在药材和生物样品中含量分析方法研究。首先,利用高效液相色谱法建立委陵菜药材中委陵菜黄酮的含量方法,通过考察不同比例的甲醇-醋酸液、乙腈-醋酸液色谱条件对委陵菜黄酮与干扰物分离效果和峰形对称性的影响,确定采用甲醇-3%o醋酸(49：51)为流动相。通过测定不同生长期和不同生长部位委陵菜药材中委陵菜黄酮的含量变化,结果表明7月份采集的委陵菜及其叶中委陵菜黄酮的含量最高,分别为1.018%o和1.477%o。
其次,建立了高效液相色谱测定大鼠血浆样品中委陵菜黄酮的含量,流动相乙腈-3‰醋酸液(35：65)对委陵菜黄酮与血浆中内源性物质的分离效果较好,结果表明该方法准确、快速、灵敏、专属性强。在此基础上进一步开展了委陵菜黄酮血浆蛋白结合率的研究,分别考察了平衡透析法和超滤法,由于委陵菜黄酮在磷酸盐缓冲液中溶解度较低,稳定性较差,且平衡透析所需时间较长(1～2天),而超滤法在30min之内能快速分离游离委陵菜黄酮,因此选用超滤法进行委陵菜黄酮与大鼠血浆蛋白结合实验,测得委陵菜黄酮血浆蛋白结合率平均为87.95%。
最后建立了大鼠组织样品中委陵菜黄酮的含量测定方法,流动相乙腈-3‰醋酸液(28：72)能实现委陵菜黄酮与组织中内源性物质的分离度大于1.5,完成方法学考察,该方法快速、灵敏、专属性强,符合药物动力学中生物样品的分析要求。初步进行了组织分布预实验,发现除小肠外,委陵菜黄酮在胰脏中的分布最高,为深入开展委陵菜黄酮的组织分布实验提供了基础数据。
The Chinese folk medicine Potentilla chinensis Ser. Bge (Rosaceae) is a perennial herb. It is also called "Fan-bai-cai", "Bai-tou-weng", "Ha-ma-cao" and "Tian-qing-di-bai", listed in Chinese Pharmacopoeia 2000 and 2005. As a folk medicine, Potentilla chinensis has anti-inflammatory and detoxification properties. In addition, P. Chinensis revealed the therapeutic effects for controlling blood glucose and improving glucose tolerance of body according to clinical researches from the Jixian Chinese Medicine Hospital of Tianjin and hospitals of Tianjin Traditional Chinese Medicine Universty. Our preliminary study showed that the main constituent compound of its hypoglycemic effect is potentilla flavone (trans-tiliroside), and its hypoglycemic effect is better than phenformin hydrochloride. And its structure is specially different from biguanides, sulfonylureas, a-glycosidase inhibitors, thiazolidinedione,GLP-1R peptide agonist drug, which indicates potentilla flavone has a different anti-diabetic mechanism and is an active leading compound worth studying.For furder quality control of herbs P. Chinensis and pharmacokinetic study provide a practical analysis methods, we have done the preparation of reference substance and established analysis methods for potentilla flavone.
Firstly, we have established the separating conditions for reference substance of potentilla flavone with high-speed countercurrent chromatography, combined with HPLC to select solvent systems and adjust the solvents' ratios. The extracting crude of potentilla flavone was for the HSCCC purification. The results showed that the methylene chloride-methanol-water (5:4:2,v/v) was the best solvent system. The separating conditions as follow:stationary phase:upper phase; mobile phase:lower phase; flow late of the mobile phase:2.0mL/min; rotation speed:800r/min. The purity of potentilla flavone was over 98%, which was separated by HSCCC.
And then determination methods in herb P. chinesis and biological samples for potentilla flavone were studied. Firstly, we have estabilshed a content method of potentilla flavone from herb P. chinesis by HPLC, with investigating different proportions of methanol-acetic acid, acetonitrile-acetic acid solutions in order to separate potentilla flavone and disturb chemicals. It was showed that methanol-3%o acetic acid(49:51) was the best mobile phase.By measuring the potentilla flavone from Potentilla chinesis in different growth periods and parts, the determination results showed that the highest content of potentilla flavone are in July and its leaves of P. chinesis, with the contents are 1.018%o and 1.477%o, respectively.
A rapid, precise and sensitive HPLC method for the determination of potentilla flavone in rat plasma has been developed. Acetonitrile-3‰acetic acid solution(35:65) on potentilla flavone and endogenous substances in plasma was better. It could be used for potentilla flavone and plasma protein-binding study. By investigating equilibrium dialysis and ultrafiltration, we have found that potentilla flavone has a low solubility and poor stability in water. Otherwise, its equilibrium dialysis time was longer than one or two days. But free potentilla flavone could be separated fastly within 30 minutes by ultrafiltration method. So we have chosen the ultrafiltration method for the binding rate of potentilla flavone to rat plasma proteins. The plasma protein binding rate of potentilla flavone was meanly 87.95%.
Lastly, a rapid, sesitive and specific method was established for determing potentilla flavone in rat tissue samples by HPLC. Acetonitrile-3‰acetic acid solution(28:72) could be achieved to separate potentilla flavone and endogenous substances in tissue samples, which the resolution was greater than 1.5. It was a good method for the further tissue distribution research of potentilla flavone in rats with oral administration. The initial pretest of tissue distribution was carried out and found that the content in the pancreas was the highest one, except the small intestine. It was useful for providing basic datas of the further tissue distribution study.
引文
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