间接竞争ELISA法检测黄连解毒汤中黄芩苷在大鼠脑脊液中的分布

Distribution of baicalin in cerebrospinal fluid of rats in Huanglian Jiedu decoction by indirect competitive ELISA

目的检测黄连解毒汤灌胃给药后黄芩苷在大鼠脑脊液中的分布。方法通过考察线性关系、精密度、回收率等方法学考察指标,建立基于黄芩苷单克隆抗体的间接竞争ELISA(icELISA)法;通过建立的icELISA法,检测大鼠灌胃给药后1小时、2小时、3小时和4小时脑脊液中黄芩苷的含量。结果在大鼠脑脊液中建立的黄芩苷标准曲线为y=0.1069ln(x)+1.0144(R 2=0.9885),浓度范围为3.2~2000 ng/mL,在400、80、3.2 ng/mL三个浓度的精密度分别为13.14%、6.65%和6.04%(RSD≤15%),回收率分别为91.35%、87.04%和117.67%(SD≤15%)。给药后1小时、2小时、3小时和4小时脑脊液中黄芩苷的平均含量分别为(60.53±18.05)、(223.32±29.72)、(121.35±31.93)、(4.46±2.54)ng/mL。结论在脑脊液中建立的黄芩苷icELISA检测法灵敏度高、精密度和回收率能够满足生物样品的检测要求,可以作为黄芩苷的检测方法。在脑脊液中可以检出黄芩苷,给药后2小时黄芩苷在脑脊液中的浓度最高,然后随时间逐渐降低,说明黄芩苷可以透过血脑屏障从而分布到脑脊液中,为黄芩苷治疗脑部疾病提供了直接的实验依据。

Objective To determine the contents of baicalin in cerebrospinal fluid of rats after intragastric administration.Methods Establishing indirect competitive enzymelinked immunosorbent assay method(icELISA)based on baicalin monoclonal antibody by researching linear relationship,acuracy,recovery to determine the content of baicalin in cerebrospinal fluid of rats after 1h,2h,3h and 4h administration.The linearity,accuracy,the average recovery and the like were detected.Results The baicalin standard curve established in rat cerebrospinal fluid is y=0.1069ln(x)+1.0144(R 2=0.9885),and the concentration range is 3.2~2000 ng/mL;meanwhile,the precision are 13.14%,6.65%and 6.04%(RSD≤15%)respectively in 400,80,and 3.2 ng/ml of concentrations,and the recovery are 91.35%、87.04%and 117.67%(SD≤15%).After 1h,2h,3h and 4h administration,the average content of baicalin in cerebrospinal fluid are(60.53±18.05),(223.32±29.72),(121.35±31.93),(4.46±2.54)ng/mL respectively.Conclusion The baicalin indirect competitive enzyme-linked immunosorbent assay method(icELISA)established in cerebrospinal fluid of rats can be used as the detection method for baicalin,which has high sensitivity and the precision as well as recovery can meet the detection requirements of biological samples.Baicalin can be detected in cerebrospinal fluid.After 2h administration,the baicalin in cerebrospinal fluid of rats holds the highest concentration,and then gradually reduced as the time go on.This indicated that baicalin could be distributed in cerebrospinal fluid through the blood-brain barrier,which provided a direct experimental basis for the treatment of brain diseases by baicalin.