Study On The Relativity Of Paeoniflorin And Paeonifolia Decoction By Analgesic Based On Monoclonal Antibody Specific Knocking Technique

ObjectiveThis study aimed to explore the correlation between paeoniflorin (PF) and Shaoyao Gancao decoction (SGD) on the analgesic effect, which provided a new idea and a novel technology for determination the effective components of Chinese herbs in Chinese traditional medicine efficacy material basis research and pharmacokinetic research or finding targets in similar prescriptions.1. Preparation of specific and sensitive monoclonal antibody against PF (anti-PF MAb) and establishment of PF immunoassay method, which provides a reliable tool for next study.2. Application of the established enzyme-linked immunoassay method for PF in mice serum to research the interaction of the PF and glycyrrhizic acid (GA) in vivo.3. Preparation of PF immune affinity chromatography column, optimization of the knockout and elution conditions, establishment of the PF knockout technology in SGD, and the preparation the sample of SGD knockout of PF.4. Exploration the different doseages of PF in SGD on the analgesic effect and the influence of the biological indicators to indicate the PF contribution in SGD analgesic action.Methods1. The PF artificial antigen were synthesized by a periodate oxidation procedure. Then the syntheses of artificial antigens were identified by ultraviolet spectrophotometry and thin layer chromatography (TLC). BALB/c female mice were immunized by subcutaneous multipoint for many times, followed by ELISA method to detect the immune mice serum antibody titer and specificity. An anti-PF MAb was produced from a hybridoma created through fusion of splenocytes immunized with PF-bovine serum albumin (PF-BSA) and conjugated with the HAT-sensitive mouse myeloma cell line SP2/0. The ascites obtained from the abdominal cavity of BALB/c mice implanted with the established hybridoma were purified by the caprylic acid-ammonium sulfate method. SDS-PAGE was applied for identification the antibodies purity, ELISA method was used for determination the antibody titer and specificity. The resultant antibody was used to develop a rapid, specific and sensitive indirect competitive enzyme-linked immunosorbent assay (icELISA) and fluorescence immunosorbent assay (FLISA) for the measurement of PF. Then icELISA was applied to determine PF content in different prescriptions.2. The established enzyme-linked immunoassay method for the measurement of PF in mice serum was used to study the interaction between PF and GA in vivo.3. The anti-PF MAb as ligands were coupled on solid phase carrier, hydrogen bromide activation on agarose gel 4 b. Then we detected the coupling ratio, prepared of PF immune affinity chromatography column, optimized the knockout and elution conditions, and got the sample of SGD knockout of PF.4. Preparation of glacial acetic acid induced pain model in mice, compare the different analgesic action between the different doses of SGD and with different content of PF, and the change of the biological indicators associated with pain in the body.Results1. By ultraviolet spectrophotometry and thin layer chromatography (TLC) identification, PF artificial antigen was synthesized successfully. The immunized mouse antiserum titer was betweenl:8000 and 1:32000. Via cell fusion, cloning culture, several monoclonal cell lines with the ability of stable secretion specific and sensitive anti-PF MAb were chose. SDS-PAGE showed that the purified anti-PF MAb obtained by ascites were pure with a little of impurities. Ascites titer both before and after purification at around 1:60000, which implied that there was no influence on the activity of anti-PF MAb in purification process. The sensitivity (IC50) of enzyme-linked immunoassay against PF is 42.72 ng/mL, the lowest detection limit was 4.75 ng/mL with little cross reaction with its isomers Albiflorin(<0.27%) and others(<0.01%).This method has a good correlation with high performance liquid chromatography (HPLC) in determination the samples, the correlation coefficient R2= 0.9929.The results of the content of PF in different prescriptions showed that compatibility of different ingredients lead to different effects on PF dissolution. In some prescriptions, the dissolution of PF was promoted, such as Chaihu Guizhi decoction, Da Chaihu decoction, Zhenwu decoction and Shaoyao Gancao Fuzi decoction, which comparing with radix paeoniae alba, the PF content increased. However, the PF content decreased in Fuzi decoction. In some other prescriptions such as Maziren pill, Guizhi decoction, Huangqin decoction, Sini powder, SGD, the content of PF was the same with in radix paeoniae alba extract. The FLISA based on anti-PF MAb is more sensitive than icELISA, with the IC50=2.20ng/mL, the lowest detection limit was 0.004ng/mL. The intra-assay and inter-assay precision values of the FLISA method were well within the recommended range (<10%).2. Pharmacokinetic parameters obtained from mouse blood samples at various intervals following oral administration of PF, GA or PF, GA, saikosaponin a (Ssa), naringin (Nar) at three doses (PF:GA=1:0.3, PF:GA=1:1, PF:GA=1:3) or (PF:GA:Ssa:Nar=1:0.3:1:1, PF: GA:Ssa:Nar=1:1:1:1, PF:GA:Ssa:Nar=1:3:1:1), both of them showed that with the content of GA increasing, the Cmax and AUCO-6 values of PF decreased gradually, while the MRT got extension, which implies that the absorption of PF was inhibited when the content of GA increased to a certain amount, but the residence time of PF was prolonged.3. The PF immune affinity chromatography column was prepared successfully, and the knockout, elution conditions were optimized. As a result deionized water was chosen as the knockout buffer,0.1 M glycine-hydrochloric acid buffer as the elution buffer, and the flow rate was 2 mL/min. The coupling rate of the immune affinity chromatography column was 99%, the coupling ratio was 2.3 mg/mL, and column capacity was 1.47μg/mL. The sample of SGD knockout of PF was achieved.4. The glacial acetic acid induced pain model was prepared successfully in mice. The different dose groups of SGD all had analgesic action, and its analgesic effect with the increase of the SGD dose gradually enhanced. PF monomer also had significant analgesic action. When the content of PF in SGD ranged from 50% to 150%, with the increase of concentration of PF, model mice body torsion inhibition rate gradually reduced, that was to say the analgesic action gradually declined, but when the PF concentration reached 200%, the body torsion inhibition rate significantly enhanced. At the same time, the model group mice serum PGE2, cAMP, NO content increased obviously compared with that in the blank group, and positive drug group (aspirin), PF, SGD-50% PF, SGD-100% PF, SGD-150% PF, SGD-200% PF group compared with model group all had different degrees of lower, the difference was statistically significant (P<0.05). In SGD-50% PF, SGD-100% PF and SGD-150% PF the three groups, with the PF content increased, the mice serum PGE2, cAMP, NO content also increased, the difference was statistically significant (P<0.05), but when the content of PF increased to 200%, namely SGD-200% PF group, in the mice serum, PGE2 and cAMP content were lower than SGD-150% PF group (P< 0.05). The content of PGE2 in SGD-200% PF group was similar with it in SGD-100%PF group, while the content of the cAMP were higher than that in the SGD-100%PF group(P< 0.05). The NO content in SGD-200% PF group was no significant difference with SGD-100% PF and SGD-150% PF group (P> 0.05), but higher than that in SGD-50% PF group (P< 0.05). PF group contained the same amount of paeoniflorin with SGD-100% PF group, but in PF group, PGE2 and cAMP levels were lower than that in SGD-100% PF group (P< 0.05), while NO content were the same with that in SGD-100% PF group. Superoxide dismutase (SOD) energy were different in different treatment groups, it was the same in PF group and model group (P> 0.05), but SGD-100% PF had a significant role in promoting the SOD activity. And with the increase of PF content in SGD, namely the SGD-50% PF, SGD-100% PF, SGD-150%PF, SGD-200% PF group, the SOD vigor had a tendency to gradually increase. Compared with SGD-50% PF group, SOD vigor in SGD-150% PF and SGD-200%PF group were significantly enhanced.ConclusionIn this study, the anti-PF MAb with high affinity and specificity is different from the one which was formerly reported. The ELISA and FLIS A established based on the antibody with high sensitivity character, which can be used to detect trace content of PF in biological samples, are more convenient and faster than traditional liquid chromatographic detection technology, In different compatibility environments, the pharmacokinetic studies using the ELISA method showed that high dose of GA inhibits the absorption of PF significantly. The samples of SGD knockout PF were obtained by the immune affinity chromatography column which is based on the anti-PF MAb. Comparing the analgesic actions of different PF content in SGD and PF monomer, the results showed that PF as an important factor on analgesic action in SGD make great contribution, but it is not the only impact factor. PF plays a significant role on regulating PGE2, cAMP and NO to express its analgesic effect. But it does not play a major role on SOD enzyme activity.