陆英提取物保肝作用研究

【作者】 杨威；

【导师】 王敏伟；

【作者基本信息】 沈阳药科大学 ， 药理学， 2005， 硕士

【摘要】 本研究以民间常用抗肝炎中药陆英为研究对象，以AST、ALT和病理检查为指标，观察了不同溶剂制备的陆英提取物对CCl₄诱导的小鼠急性肝损伤的影响，确定75％乙醇为最佳提取溶剂，经系统溶剂萃取和大孔吸附树脂纯化，确立了陆英75％乙醇提取物经大孔吸附树脂纯化后30％乙醇洗脱物（LY）为主要有效部位，并进行了初步药效学研究，结果表明：LY对四氯化碳（CCl₄）、D-半乳糖胺（D-Gal）、刀豆蛋白（ConA）诱导的急性肝损伤显示出良好的抗肝损伤作用，有良好的剂量依赖性；并能明显提高正常小鼠单核-巨嗜细胞吞噬功能，增加血清中异物清除速率，提高肝脾网状内皮系统吞噬指数，提示LY有增强正常小鼠非特异性免疫功能的作用。由于文献报道乌索酸（熊果酸）为陆英保肝降酶的主要有效成分，故此我们进行LY药效学初探时，研究了乌索酸对CCl₄、D-GalN诱导的急性肝损伤的保肝作用，结果表明乌索酸标准品经口给药没有抗肝损伤作用。为此，我们进一步建立了LC-MS分析方法测定了大鼠血中乌索酸的方法，比较研究了大鼠灌胃给予陆英提取物和相当于等量的乌索酸对照品溶液后的药物动力学，测定了乌索酸的血药浓度-时间曲线，计算其相应药物动力学参数。大鼠灌胃给予乌索酸标准品后，大鼠血浆中几乎检测不到乌索酸，而给予陆英提取液后乌索酸在1h左右达峰，达峰浓度C_max为294.8ng·mL^-1，消除半衰期t_1/2为4.3h。可以推测陆英中的其他化学成分与乌索酸相互作用，促进乌索酸的吸收，能显著影响乌索酸在大鼠体内的药动学行为。经过药效学初探，在确定了“陆英75％乙醇提取物的乙酸乙酯层和经大孔吸附树脂纯化后30％乙醇洗脱”这条工艺后，为进一步考察LY的保肝作用，我们用陆英颗粒（LYKL）作对照，对LY与陆英颗粒进行了较为系统的保肝作用研究。实验结果表明：LY与陆英颗粒对CCl₄、D-Gal、ConA、硫代乙酰胺（TAA）致小鼠急性肝损伤有保护作用，可明显降低上述各种化学物质造成的小鼠急性肝损伤血清ALT，AST升高，其中LY量效关系明显；另外，LY与陆英颗粒均能减轻TG在CCl₄中毒小鼠肝组织内的蓄积和降低MDA的含量，提高CCl₄中毒小鼠肝组织T-SOD的活力及总抗氧化能力（T-AOC），亦能提高CCl₄中毒小鼠肝细胞膜和微粒体膜的Ca²⁺-ATPase的活性，减少GSH代偿性增高的程度；病理结果也表明LY与陆英颗粒对肝细胞损伤具有明显的保护作用；并且LY的作用要明显强于陆英颗粒。同时，LY与陆英颗粒亦能对抗D—半乳糖胺盐酸盐致大鼠急性肝损伤导致的血清中ALP、TBIL、TG、NEFA的升高以及Glu含量急剧降低，亦能显著对抗D—半乳糖胺盐酸盐致大鼠急性肝损伤肝组织肝糖原含量、Na⁺-K⁺—ATPase、Mg²⁺—ATPase、Ca²⁺—ATPase、ca²⁺-Mg²⁺—ATPase活性降低，LY的作用要明显强于陆英颗粒，并且LY还能抑制本模型大鼠SDH活性降低。在慢性肝损伤实验中，LY能较好的改善肝功能和降低动物病死率，陆英颗粒也能一定程度改善肝功能，却不能明显降低动物病死率，并且高剂量组病死率大于中剂量组；LY与陆英颗粒可一定程度降低复合因素致肝硬化大鼠血清LN，PⅢP，HA及肝组织匀浆HYP含量的升高，在病理切片中可明显看到LY与陆英颗粒各剂量组与模型组比较肝脏病理有不同程度的改善，显示了其保肝作用，并有一定的抑制肝纤维化形成的作用，LY的作用强于陆英颗粒；抗氧化能力检测表明LY与陆英颗粒均能提高肝组织抗氧化的能力，LY还能增强本模型肝组织Ca²⁺—ATPase的活性。LY与陆英颗粒能较好的对抗豚鼠同种免疫性肝炎模型引起的肝脏病变，二者功效相似。退黄利胆试验结果表明LY与陆英颗粒无明显利胆作用，退黄功效亦较差。对免疫系统作用试验研究结果表明LY与陆英颗粒有提高LY致免疫低下小鼠网状内皮功能的作用；有提高HY致免疫低下小鼠体液免疫的作用；在胎儿脾淋巴细胞转化实验中，试验结果显示CTX与陆英颗粒能促进正常胎儿脾淋巴细胞的增值，而在有ConA及LPS诱导胎儿脾淋巴细胞转化的实验中，LY显示了一定的抑制作用，陆英颗粒却无明显影响。综上所述，可以认为中药陆英75％乙醇提取物经大孔吸附树脂纯化后30％乙醇洗脱物（LY）具有较好的保肝作用，有一定的抑制肝纤维化形成的作用，并且明显优于已上市制剂陆英颗粒，其机制可能与LY中有效成分稳定肝细胞膜，抗脂质过氧化，增强肝组织抗氧化的能力，提高肝细胞膜和微粒体膜ATPase的活性，提高肝细胞线粒体SDH的活性，以及对免疫的双向调节作用有关。更多还原

【Abstract】 Sambucus Chinensis L. is a native perennial herb distributed throughout China. All parts of the plant can be used in traditional Chinese medicine （TCM ） as Luying. Luying is one of the important folk medicines in TCM. It has been used to treat hepatitis over a very long period of time and has produced quite a good effect.The effect of therapeutic hepatitis on Luying was studied in this paper. Luying extract by different extracting methods were prepared. Effects of Luying extracts of different processes on ALT and AST in acute hepatic injury mice were studied. As a result, 75% alcohol was selected as the optimal extraction reagent. The fractions of various polarities fractionated and purified with macroporous resin from 75% alcohol extract were further investigated, the fraction eluted from the macroporous resin with 30% alcohol（LY） were found to be primarily active. Then we primarily investigate the pharmacodynamics of against hepatitis of the LY .It showed significantly protective effects on mice or rats acute hepatic injury induced by CCU, D-GalN or Cona. On the other hand, it showed significantly improving the ability of nonspecific immunity of mice. In a word,the LY showed significantly protective effects against many kinds of chemical and immunological liver injuries, what’s more, it has the effects improving the ability of nonspecific immunity of mice.The ursolic acid that was taken orally by mice or rats didn’t show protective effects on mice or rats acute hepatic injury induced by CCl₄ or D-GalN, but it was reported by the literature that the ursolic acid that was taken orally has well effect of therapeutic hepatitis. So, a rapid, sensitive, and accurate liquid chromatography-mass spectrometry （LC-MS） method for the determination of ursolic acid in rat plasma was developed and validated. Plasma samples taken from rats that had received Luying extract orally were acidified with acetic acid and then extracted with a mixture of hexane-dichloromethane-2-propanol （20:10:1, v/v/v）. Atmospheric pressure chemical ionization was operated in negative-ion mode. Using selected ion-monitoring mode, the deprotonated molecules [M-H]^- at m/z 455 and 469 were used to quantify ursolic acid and glycyrrhetic acid （internal standard）, respectively. The assay was shown to be linear over the range of 10～1000 ng·mL^-1 （r≥0.9960） with a lower limit of quantification of 10 ng·mL^-1. The method was shown to be reproducible and reliable with intraday precision below 7.8%, interday precision below 8.1%, accuracy within±4.3%, and mean extraction recovery excess of 83.6%, which were all calculated from the blank plasma sample spiked with ursolic acid at three concentrations of 20, 200, and 800 ng·mL^-1. The LC-MS method has been successfully applied to pharmacokinetic studies of ursolic acid after oral administration of Luying ethanolic extract to rats. The main pharmacokinetic parameters were: t_1／2, 4.3h; C_max, 294.8 ng·mL^-1; and t_max, 1.0 h, respectively.We followed to go deep into studing the pharmacodynamics of against hepatitis of the LY and Luyingkeli （LYKL）, what’s more, there is a contrast between LY and LYKL. From the results, LY and LYKL could prevent liver from acute damage induced by CCl₄,D-Gal,ConA and TAA. They decreased the level of ALT and AST of blood serum in acute hepatic mice, and LY is well displayed the effects in dose-dependent manner. At the same time, LY and LYKL significantly decreased TG、MDA and GSH of liver tissue, and increased the level of T-AOC、T-SOD and Ca²⁺-ATPase of liver tissue of mice acute hepatic injury induced by CCl₄. On the other hand, LY and LYKL significantly decreased ALP、TBIL、TG and NEFA in serum, and increased the content of Glu in serum and glycogen and the activities of Na⁺-K⁺-ATPase、Ca²⁺-Mg²⁺-ATPase、Mg²⁺-ATPase、Ca²⁺-ATPase of liver tissue of mice acute hepatic injury induced by D-Gal. LY could increased the activities of SDH of liver tissue of mice acute hepatic injury induced by D-Gal, too. And the effect of LY is obviously better than LYKL.In chronic hepatic injured experiments, LY and LYKL could checkmate hypohepatia, and the effect of LY is obviously better than LYKL. At the same time, LY could obviously induced death rate, but LYKL not. LY and LYKL also certainly lowered hepatic cirrhosis rat the serum LN,HA,PIIIP contents and the liver homogenate HYP contents. From the pathologic section, compared with model group, LY and LYKL differently certainly improved the rat hepatic phathology. Rats hepatic cell necrosis, lipid degeneration, liver fibrosis were all abated. What’s more, LY and LYKL could increase the ablity against lipoperoxidation of liver tisse. LY could increase the activities of Ca²⁺-ATPase of liver tissue, too. on the side, they showed significantly protective effects on the homogeneous immunological liver injury models of the cavy.In receding jaudice and normalizing gallbladder experiments, LY and LYKL had no obvious effect.In immunology experiments, LY and LYKL enhanced phagocyte function and accelarated carbon clearance in immunodepression mice induced by Hydrocortisone（HY）. At the same time LY and LYKL stimulated antibody forming and increased hymolytic HC₅₀ in hypoimmunity mice induced by Cyclophosphamide（CTX）. In lymphocyte of foetus proliferation experiments, LY and LYKL accelerate lymphocyte immunity. In lymphocyte of foetus proliferation experiments by LPS and ConA stimulated, LY showed certainly restrain effects, but LYKL had no obvious effect.更多还原