摘要
茵陈蒿汤出自《伤寒论》，由茵陈蒿，栀子，大黄三味药组成，具有清热利湿，解毒退黄之功，主要用于治疗湿热黄疸。现代研究表明，它具有利胆、保肝、护胰、解热抗炎镇痛、抗菌抗病毒等药理作用。栀子苷、6，7-二甲氧基香豆素目前已被证明为主要活性成分，但是本方的有效成分研究还依旧存在空白。本文建立肝细胞膜固相化生物色谱法对该方保肝作用的效应成分进行了初步研究。
本文第一部分以茵陈蒿汤的水提醇沉液和HL-7702正常人肝细胞株为研究对象，确定了茵陈蒿汤对HL-7702正常人肝细胞株的安全作用范围，试验结果表明：茵陈蒿汤水提醇沉液的浓度范围可以控制在5～0.001mg生药／ml范围内，对HL-7702人肝细胞株无细胞毒性作用。茵陈蒿汤水提醇沉液终浓度大于10mg生药／ml，对HL-7702人肝细胞株有显著的毒性作用(P＜0.001)，作用24小时后导致出现细胞死亡现象；与空白对照组相比较，终浓度为5mg生药／ml的茵陈蒿汤水提醇沉液对HL-7702人肝细胞株生长无抑制作用；终浓度0.1～1mg生药／ml的茵陈蒿汤水提醇沉液能显著促进HL-7702人肝细胞株生长(P＜0.05)。因此，本文的工作浓度确定为0.1～1mg生药／ml。
本文第二部分观察了茵陈蒿汤水提醇沉液在上述浓度范围内对D-GalN损伤的HL-7702正常人肝细胞株的保护作用。试验结果表明：茵陈蒿汤水提醇沉液有降低D-GalN诱导的肝细胞上清液中ALT、AST升高的作用。茵陈蒿汤能够保护D-GalN诱导的肝细胞损伤。
本文第三部分采用二元非线性梯度洗脱方法进行了茵陈蒿汤的反相高效液相谱图研究。研究表明：最佳检测波长为254nm、280nm、310nm，最佳记录时间为55分钟，柱温为25℃。得到的茵陈蒿汤水提醇沉液高效液相谱图作为肝细胞膜固相色谱法研究的基础。
本文应用肝细胞膜固相化色谱法分析茵陈蒿汤水提醇沉液与HL-7702正常人肝细胞膜特异结合的化学成分，初步研究表明主要有四个特征成分与HL-7702肝细胞膜特异性结合，而这四个成分来源于大黄。
Yinchenhao Decoction, a famous recipe from ShangHanLun consisting of three Chinese herbs: Herba Artemisiae Scopariae, Fructus Gardeniae and Radix et Rhizoma Rhei., has an effect on treating jaundice and other hepatitis in clinic. Although Gardenoside and 6, 7-dimethylesculetin of this recipe have been thought to be two active chemicals for its effect, which chemical or chemicals are responsible for its effectiveness is still unknown. To find out these active components, we try to develop a hepatocyte membrane immobilized chromatography, in which the chemicals of Yinchenhao Decoction that would specifically bind to the membrane of hepatocyte HL-7702 were studied.
Firstly, The effect of the water extract of Yinchenhao Decoction, which was precipitated with alcohol, on the hepatocyte HL-7702 was studied. It has been testified that 5~0.001mg crude-herb/ml of this extract was no damage for HL-7702, but the hepatocyte has been killed after 24 hours' incubation with the concentration more than 10mg crude-herb/ml; the extract of 5 mg crude-herb/ml had no inhibition to the growth of HL-7702; and the extract of 0.1~1mg crude-herb/ml could improve the growth of HL-7702 significantly (P<0.05) , so the working concentration is between 0.1-1 mg crude-herb/ml.
Secondly, the effect of this extract on the damaged HL-7702 induced by D-GalN was observed. The results showed that both ALT, AST in the supernate of HL-7702 increased by treated with D-GalN, and decreased by added this extract. It means that the extract had the property of protecting HL-7702 from being hurt by D-GalN.
Thirdly, RP-HPLC was performed to analyze the fingerprints of Yinchenhao Decoction. The results indicated that the optimal wavelengths of detection should be respectively at 254nm, 280nm, 3 10nm, and the best recording time should be retained for 55 minutes, the temperature of column is 25 C.
The Hepatocyte membrane immobilized chromatography was used to analyzed the chemical or chemicals in the water extract of Yinchenhao Decoction specifically binded to targets on hepatocyte membrane. The results showed there were four
characteristic compounds specifically conjugated to HL-7702, which originated from the herb of Radix et Rhizoma Rhei.
引文
1 司马镇强，吴军正．细胞培养．西安：世界图文出版公司，2000，186．
