| Baicalin (BA) is the most abundant flavonoid in Scutellaria baicalensis Georgi, which is available from the dried root of S. baicalensis, one of the most important crude drugs used widly in traditional Chinese medicine. Researchers have found that BA has various pharmacological activities such as sedation, depressurization, free radical scavenging, anti-arrhythmia, anti-inflammatory, antiviral, anticancer, and antineoplastic effects in systemic malignancies.Enzyme-linked immunosorbent assay (EIISA) is increasingly popular in recent years to be used to study effective ingredients of traditional Chinese medicine (TCM). Unlike other traditional analysis methods (such as HPLC or HPLC-MS), ELISA method requires no complicated preparation of test samples, but has high sensitivity and specificity of analyzing. Also, it is economical and environmental friendly.And no large experiment instruments were needed during the ELISA test. At present, the immune analysis method has already been applied to determine the concentration of effective drug compound of TCM.However, few have been reported of immune analysis method of BA. Because the key step of establishing a immune analysis method is to prepare a specific monoclonal antibody (MAb), the study presented in this paper started with the synthesis of immunogen of BA, then followed the preparation of anti-BA MAb through a series of process. After that, ELISA method with anti-BA MAb was established and applied for the determination of BA in the Chinese medicine preparation, the pharmacokinetics study of drug ingredient of Chinese medicine formulae, and the study of percutaneous absorption of BA.In this study, Immunogen BA-BSA (bovine serum albumin) and coating antigen BA-PLL(polylysine) were synthesized successfully by a modification of the procedure of carbodiimide method. Balb/c mice were immunized with BA-BSA7times and its splenocytes were fused with SP2/0myeloma cells by hybridoma technique. Then, a hybridoma cell line secreting a monoclonal antibody (MAb) against BA was selected and the MAb against BA was named BA1H7. According to indirect ELISA and indirect compete ELISA test results, BA1H7showed high titer (>128000) and specific reactivity against BA (IC50=98ng/mL), but not against other natural products like baicalein and rutin (cross activities<0.1%).Next, indirect competitive ELISA for the determination of Baieal in was developed by using anti-BA MAb, and applied for measuring BA in the traditional Chinese medicine injection Jingzhi Qingkai ling. The standard curve of established ELISA was linear between2.669ng/mL and1029ng/mL The average recovery was100.9%, while the relative standard deviations (R. S. D. s) of measurements were <3%in the intra-assay, and <4%in the inter-assay. The test of samples with this ELISA method could be finished in4hs. The analysis result of ELISA method was consistent with that of1IPLC test in determination of BA of Jingzhi Qingkailing.For the first time, the quantitative ElISA with anti-BA MAb was applied and validated for the quantitative analysis of baieal in (BA) in its phannacokinetic study of Ban-xia-xie—xin decoction in consecutive72hs. Besides, the study of percutaneous absorption of BA was conducted with the establ ishded ELISA method. According to the test data, Higuchi equation of skin permeation of BA was Q=5.786X-2.043, and the rate constants was5.786.Therefore, MAb against BA was successfully preparared, and ELISA with this MAb was well developed in this study. Owing to its specialties in the determination of BA, such as low detection limit (peak level), quickness (the test could be finished in4hs), fewer requirements for the test samples (samples could be used for the test directly after dilution), this method showed its great advantage in the field of quality control of TCM, the oral pharmacokinctic study of BA of compound prescription of Chinese medicine, and the study of percutaneous absorption of chinese medicine contains BA. |
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