| Prescriptions are the main clinical patterns of traditional Chinese medicine (TCM) and the core of prescription is compatibility, which holds that the therapeutical effect of TCM prescription is due to interactions between multicompounds.Different dosage as well as different compatibility of medicines leads to content variations of compounds in prescription, in which way the solubility and chemical properties of the compounds would be changed in virtue of the interactions between herb medicines. Moreover, studies on compatibility principles of "monarch, minister, assistant, and messenger" are of great importance in revealing the mechanisms of prescription compatibility.Pharmacokinetics provides a way to study the compatibility, since that interactions between compounds of prescriptions can occur in the course of ADME, which may be manifested by affecting bioavailability of ingredients, changing distribution characteristic, regulating compositions of in vivo pharmacodynamic substances, and finally leading to the changes of pharmacological effect. Therefore, studying the pharmacokinetics is an efficient and important way for illustrating the mechanism underlying compatibility.In this paper, a monoclonal antibody (MAb) based incomplete competitive enzyme-linked immunosorbent assay (icELISA) with high sensitivity and selectivity was developed and applied to study the pharmacokinetics of active compounds in Banxia-Xiexin decoction and Gegen-Qinlian decoction with different compatibilities.Objective1 Produce and purify ascites fluids containing MAb against baicalin (BAL).2 Establish an icELISA based on anti-BAL MAb in mice blood and assess this method for further determination of BAL.3 Determinate BAL, puerarin (PUE) and ginsenoside Re (GsRe) in mice blood after administration of Banxia-Xiexin decoction (BXD) and Gegen-Qinlian decoction (GQD) with different compatibilities, following analyze the pharmacokinetics characters.Methods1 Ascites fluids containing anti-BAL MAb was collected from mice and purified by caprylic acid-ammonium sulfate precipitation (CA-AS); Antibodies before and after purification were detected by SDS-PAGE; Protein quantification of antibodies before and after purification were measured by Bradford methods; Titer and competition of the MAb were detected by ELISA.2 The icELISA was developed in mice blood using the anti-BAL MAb. Afterwards, the method was assessed through validation of calibration curve, specificity, precision and accuracy (intra-assay and inter-assay), recovery and stability.3 Studies on compatibility of BXD and GQD were carried out by the developed icELISA and pharmacokinetics. BXD and group K, XK and KG were administrated and the blood samples were collected via mice tail, followed determination of BAL; BXD with high, normal and low doses of Pinellia ternaia was administrated to mice and BAL and GsRe was detected from the obtained mice blood by icELISA. Four groups, GQD and GQD without Pueraha lobate, Scutellaria baicalensis, Coptis chinensis, respectively, were administrated to mice before the blood was collected via tail tip; PUE and BAL were detected by icELISA. The concentration verse time profiles were drawn and pharmacokinetics parameters were calculated.Results1 Result of SDS-PAGE showed reduction of the other protein in anti-BAL contained ascites; Protein quantification of antibodies before and after purification were 6.54 mg/mL and 2.40 mg/mL, respectively; The ELISA results using the MAb before and after purification showed little changes of linearity range.2 A linear correlation was obtained for BAL concentrations ranging from 7.81 to 1000.00 ng/mL. The regression equation was y=-0.1431n(x)+1.2551 with a correlation coefficient of 0.9918. Precision and accuracy were evaluated and these parameters were within the normal range (<15%). The recovery rates ranged from 100.52% to 104.02%, meeting the requirements for biological samples. Results of stability showed that BAL sample solutions were intact for UV detection with enough time. However, long-term storage and especially freeze-thaw procedures were detrimental to BAL.3 After administration of different groups from BXD, the BAL concentrations in blood samples from mice in the K and XK groups were lower than those in the other groups; BAL concentrations peaked at around 1-1.5 h and again at 5-7 h; the Cmax and AUCo-t in the KG and BXD groups were almost 3 times of those in the K and XK groups. Following administration of BXD with three doses, the BAL concentrations peaked at approximately 0.5 h and again at approximately 6-7 h, while GsRe showed more concentration peaks than BAL; the AUCo-t of BAL showed a linear relationship with the increasing dose of Pinellia ternata (R"= 0.9628), and the AUCo-t of GsRe also showed a direct relationship with the dose of Pinellia ternata. After administration of GQD, BAL showed two or more peaks of maximum blood concentration in mice, of which the first main peaks occur at the range of 1-3 h and the second at the range of 7-8 h, the values of Cmax, Tmax and AUCo-t in group B (GQD without Pueraria lobate) were higher than that of the other two groups; As to PUE, the first peak time was at the range of 20-60 min and the second was at the range of 7-9 h, Cmax value of group A (GQD) was less than nearly half of that from the other two groups, while the AUCo-t of group C showed the maximum value among the three groups.ConclusionsThe produced and purified MAb can be employed to develop an icELISA; The established icELISA was validated to be effective and sensitive, it demonstrated a method for the continuous detection of Chinese medicine compounds with low content in mice blood, which was successfully applied to pharmacokinetics of active ingredients in mice blood after administration of various doses or combinations of Chinese medicines in prescriptions.BXD with different taste combined herbs have dissimilar effects on the metabolism of BAL mainly reflected in variations of AUCo.t and Cmax, when combined with sweet herbs, the values of groups exhibited significantly higher than that of other groups, which indirectly verified that the tonic effect of sweet herbs worked significantly in compatibility of BXD; Dose variations of Pinellia ternata in BXD affected the pharmacokinetics of both BAL and GsRe, and the values of AUCo-t showed a positive correlation with the dose of Pinellia ternate, indicating the importance of monarch drug and its dosage in compatibility of BXD; Various herbs in GQD have different effects on metabolism of puerarin and baicalin in mice blood, implying that monarch, minister, assistant herbs played distinct roles in compatibility of GQD. Moreover, these results provide a meaningful basis for evaluation of the interactions between components in a complex prescription on their pharmacokinetics.
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