细梗香草皂苷A制备工艺及药代动力学研究
发布时间：2019-04-17 09:10
【摘要】：细梗香草(Lysimachia capillipes Hemsl.)为报春花科(Primulaceae)珍珠菜属(Lysimachia)植物,具有清热解毒、祛风、止咳、调经、宁神等功效,民间用于治疗感冒咳嗽、风湿痹痛、月经不调、神经衰弱和恶性肿瘤,其化学成分以黄酮类和皂苷类为主。课题组前期研究发现,细梗香草总皂苷是其抗肿瘤活性的有效成分,其中LC-A、LC-B、LC-C三个单体皂苷均具有显著的抗肿瘤活性,但LC-B和LC-C溶血性较强,制备成单体药物具有一定的困难,LC-A溶血性低,抗肿瘤作用也较强,具有相对较大的研究利用价值。因此,本文以LC-A为研究对象,对其制备工艺和药代动力学进行考察。首先,本文采用水解的方法对LC-A标准品进行制备。利用氢氧化钠对LC-B单体进行水解,再依次经过大孔树脂、反相硅胶柱纯化得到单体化合物,经质谱和核磁鉴定,确定水解产物为LC-A,采用面积归一化法经高效液相-蒸发光散射检测器(HPLC-ELSD)进行标定后,确定含量大于98%。其次,本文以LC-A为指标性成分,建立了总皂苷水解液的HPLC-ELSD含量测定方法;通过单因素筛选和正交设计,对细梗香草总皂苷的水解时间、水解温度、催化剂种类、浓度和投料比进行优选,确定最佳水解工艺为:总皂苷投料比为1g:40mL,催化剂为0.08mol·L-1的NaOH水溶液,水解温度为50℃,水解时间为144h。按照最佳工艺进行水解后,LC-A得率为39.77%;以LC-A转移率和纯度为综合评价指标,通过静态吸附实验筛选出HP-20为最佳树脂,通过动态吸附实验对上样浓度、上样量等因素进行考察,通过正交设计优选上样和洗脱参数,得到最佳工艺为:上样浓度20g·L-1,上样量为80%饱和上样量,重复上样3次,吸附0.5 h,上样流速为每小时2倍柱体积,径高比1:8,水洗脱3倍柱体积,40%乙醇洗脱3倍柱体积,70%乙醇洗脱4倍柱体积,流速每小时2倍柱体积。按照最佳工艺进行纯化后,LC-A含量为63.25%,转移率为71.36%;以LC-A纯度和含量为指标,采用单因素和正交试验,对洗脱体积、上样量和上样浓度、洗脱流速进行考察,筛选出最佳的中压反相色谱柱纯化工艺为:上样量为0.006倍柱体积、上样浓度为0.1 g·mL-1、洗脱流速为每小时8倍柱体积、洗脱体积为14～17倍柱体积。按照最佳工艺进行纯化,得到LC-A单体纯度达到98%以上,得率为86.54%。最后,本文建立了 LC-MS/MS技术测定大鼠血浆、尿液和粪便中LC-A浓度的方法。大鼠体内的药代动力学结果表明,LC-A经口给药后大鼠体内暴露量很低,5～15 mg·kg-1的静脉注射剂量范围内LC-A在大鼠体内呈剂量依赖型药动学特征;LC-A在尿液和粪便中排泄量均不高,总排泄比例为15.64%。综上所述,本文主要研究了以总皂苷为底物水解得到LC-A的制备工艺以及纯化工艺,为之后的大规模生产提供依据,同时,进行LC-A的部分药代动力学研究,为其剂型的选择和进一步的开发利用奠定理论基础。
[Abstract]:(Lysimachia capillipes Hemsl.) It is a (Lysimachia) plant of the genus (Primulaceae) of the family Primulaceae, which has the functions of clearing heat and detoxification, dispelling wind, relieving cough, regulating menstruation and tranquilizing the mind, and is used in the treatment of colds and cough, rheumatic arthralgia, irregular menstruation, neurasthenia and malignant tumors, etc. Its chemical constituents are mainly flavonoids and saponins. Previous studies in our research group found that total saponins of Herba strobilis were the effective components of its antitumor activity, among which the three saponins of LC-A,LC-B,LC-C had significant antitumor activity, but LC-B and LC-C had strong hemolytic activity. The preparation of monomeric drugs has some difficulties, low hemolytic activity of LC-A and strong antitumor effect, so it has a relatively great value in research and utilization. Therefore, the preparation technology and pharmacokinetics of LC-A were investigated in this paper. Firstly, the LC-A standard was prepared by hydrolysis. LC-B monomer was hydrolyzed by sodium hydroxide, then purified by macroporous resin and reversed-phase silica gel column. The monomer compound was identified by mass spectrometry and NMR. The hydrolysis product was identified as LC-A,. The area normalization method was used to calibrate it by high performance liquid phase evaporative light scattering detector (HPLC-ELSD), and the content was determined to be more than 98%. Secondly, the HPLC-ELSD content of total saponins hydrolysate was determined by using LC-A as the index component. Through single factor selection and orthogonal design, the optimum hydrolysis time, hydrolysis temperature, catalyst type, concentration and feed ratio of total saponins were determined as follows: the ratio of total saponins to total saponins was 1 g / 40 mL, and the optimum hydrolysis conditions were as follows: 1 g / L of total saponins, 40 mL / L of total saponins, The catalyst is NaOH aqueous solution of 0.08mol 路L-1, the hydrolysis temperature is 50 鈩，
本文编号：2459296
[Abstract]:(Lysimachia capillipes Hemsl.) It is a (Lysimachia) plant of the genus (Primulaceae) of the family Primulaceae, which has the functions of clearing heat and detoxification, dispelling wind, relieving cough, regulating menstruation and tranquilizing the mind, and is used in the treatment of colds and cough, rheumatic arthralgia, irregular menstruation, neurasthenia and malignant tumors, etc. Its chemical constituents are mainly flavonoids and saponins. Previous studies in our research group found that total saponins of Herba strobilis were the effective components of its antitumor activity, among which the three saponins of LC-A,LC-B,LC-C had significant antitumor activity, but LC-B and LC-C had strong hemolytic activity. The preparation of monomeric drugs has some difficulties, low hemolytic activity of LC-A and strong antitumor effect, so it has a relatively great value in research and utilization. Therefore, the preparation technology and pharmacokinetics of LC-A were investigated in this paper. Firstly, the LC-A standard was prepared by hydrolysis. LC-B monomer was hydrolyzed by sodium hydroxide, then purified by macroporous resin and reversed-phase silica gel column. The monomer compound was identified by mass spectrometry and NMR. The hydrolysis product was identified as LC-A,. The area normalization method was used to calibrate it by high performance liquid phase evaporative light scattering detector (HPLC-ELSD), and the content was determined to be more than 98%. Secondly, the HPLC-ELSD content of total saponins hydrolysate was determined by using LC-A as the index component. Through single factor selection and orthogonal design, the optimum hydrolysis time, hydrolysis temperature, catalyst type, concentration and feed ratio of total saponins were determined as follows: the ratio of total saponins to total saponins was 1 g / 40 mL, and the optimum hydrolysis conditions were as follows: 1 g / L of total saponins, 40 mL / L of total saponins, The catalyst is NaOH aqueous solution of 0.08mol 路L-1, the hydrolysis temperature is 50 鈩，
本文编号：2459296
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