【摘要】：細梗香草(Lysimachia capillipes Hemsl.)為報春花科(Primulaceae)珍珠菜屬(Lysimachia)植物,具有清熱解毒、祛風(fēng)、止咳、調(diào)經(jīng)、寧神等功效,民間用于治療感冒咳嗽、風(fēng)濕痹痛、月經(jīng)不調(diào)、神經(jīng)衰弱和惡性腫瘤,其化學(xué)成分以黃酮類和皂苷類為主。課題組前期研究發(fā)現(xiàn),細梗香草總皂苷是其抗腫瘤活性的有效成分,其中LC-A、LC-B、LC-C三個單體皂苷均具有顯著的抗腫瘤活性,但LC-B和LC-C溶血性較強,制備成單體藥物具有一定的困難,LC-A溶血性低,抗腫瘤作用也較強,具有相對較大的研究利用價值。因此,本文以LC-A為研究對象,對其制備工藝和藥代動力學(xué)進行考察。首先,本文采用水解的方法對LC-A標準品進行制備。利用氫氧化鈉對LC-B單體進行水解,再依次經(jīng)過大孔樹脂、反相硅膠柱純化得到單體化合物,經(jīng)質(zhì)譜和核磁鑒定,確定水解產(chǎn)物為LC-A,采用面積歸一化法經(jīng)高效液相-蒸發(fā)光散射檢測器(HPLC-ELSD)進行標定后,確定含量大于98%。其次,本文以LC-A為指標性成分,建立了總皂苷水解液的HPLC-ELSD含量測定方法;通過單因素篩選和正交設(shè)計,對細梗香草總皂苷的水解時間、水解溫度、催化劑種類、濃度和投料比進行優(yōu)選,確定最佳水解工藝為:總皂苷投料比為1g:40mL,催化劑為0.08mol·L-1的NaOH水溶液,水解溫度為50℃,水解時間為144h。按照最佳工藝進行水解后,LC-A得率為39.77%;以LC-A轉(zhuǎn)移率和純度為綜合評價指標,通過靜態(tài)吸附實驗篩選出HP-20為最佳樹脂,通過動態(tài)吸附實驗對上樣濃度、上樣量等因素進行考察,通過正交設(shè)計優(yōu)選上樣和洗脫參數(shù),得到最佳工藝為:上樣濃度20g·L-1,上樣量為80%飽和上樣量,重復(fù)上樣3次,吸附0.5 h,上樣流速為每小時2倍柱體積,徑高比1:8,水洗脫3倍柱體積,40%乙醇洗脫3倍柱體積,70%乙醇洗脫4倍柱體積,流速每小時2倍柱體積。按照最佳工藝進行純化后,LC-A含量為63.25%,轉(zhuǎn)移率為71.36%;以LC-A純度和含量為指標,采用單因素和正交試驗,對洗脫體積、上樣量和上樣濃度、洗脫流速進行考察,篩選出最佳的中壓反相色譜柱純化工藝為:上樣量為0.006倍柱體積、上樣濃度為0.1 g·mL-1、洗脫流速為每小時8倍柱體積、洗脫體積為14～17倍柱體積。按照最佳工藝進行純化,得到LC-A單體純度達到98%以上,得率為86.54%。最后,本文建立了 LC-MS/MS技術(shù)測定大鼠血漿、尿液和糞便中LC-A濃度的方法。大鼠體內(nèi)的藥代動力學(xué)結(jié)果表明,LC-A經(jīng)口給藥后大鼠體內(nèi)暴露量很低,5～15 mg·kg-1的靜脈注射劑量范圍內(nèi)LC-A在大鼠體內(nèi)呈劑量依賴型藥動學(xué)特征;LC-A在尿液和糞便中排泄量均不高,總排泄比例為15.64%。綜上所述,本文主要研究了以總皂苷為底物水解得到LC-A的制備工藝以及純化工藝,為之后的大規(guī)模生產(chǎn)提供依據(jù),同時,進行LC-A的部分藥代動力學(xué)研究,為其劑型的選擇和進一步的開發(fā)利用奠定理論基礎(chǔ)。
[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，

本文編號：2459296