| The compatibility of traditional Chinese medicine(TCM)is one of the essence part of TCM.The unique rules and utility advantages of it have been proved by long-term clinical practice.However,the scientific significance of the compatibility which was summarized through the theory of traditional Chinese medicine and treatment experience,still cannot be explained by modern scientific method.So it became the main threshold of the TCM development.In recent years,with the progress of scientific technology,the researches of compatibility have made a lot of achievements.But due to the complex system of compatibility,the traditional resurch ideas of reductionism can not effectively evaluate the compatibility of TCM.The lack of comprehensive study make it difficult to reach the real scientific connotation of compatibility.The lack of suitable technical methods for the study of compatibility with strong operability and reliability makes the progresses of many key questions be slow.The monoclonal antibody technology,with strong specificity,high sensitivity,convenient and efficient,is more suitable for the resurches of compatibility of TCM.In this paper,we carried on the comprehensive research on various aspects of the compatibility of paeonia and glycyrrhiza to resolve the mechanism using the monoclonal antibody technology.ObjectiveThis study aimed to research on the chemical composition,the pharmacokinetics,pharmacological efficacy,etc.in order to comprehensive analysis the scientific connotation of compatibility using the monoclonal antibody technology.Methods1.The icELISA was developed in mice blood using the anti-PF MAb.Afterwards,the method was assessed through validation of calibration curve,precision and accuracy(intra-assay and inter-assay)and recovery.2.The HPLC was established for detection of glycyrrhizic acid and paeoniflorin of peony liquorice decoction(SGD).3.Studies on compatibility of SGD were carried out by the developed icELISA and HPLC.The decoctions with different proportion of peony and liquorice were tested with HPLC.The decoctions were given to the kunming mice by gastric.The blood samples were collected via mice tail,followed determination of PF using the established paeoniflorin enzyme-linked immunoassay method.Using hot plate model in mice,compare the different analgesic action of different groups of SGD over time.Using the xylene induced ear swelling model in mice,compare the anti-inflammatory effect of different groups of SGD.4.The SGD with different medicines were tested with HPLC.The decoctions were given to the kunming mice by gastric.The blood samples were collected via mice tail,followed determination of PF using the established paeoniflorin enzyme-linked immunoassay method.Using hot plate model in mice,compare the different analgesic action of different groups of decoctions over time.Using the xylene induced ear swelling model in mice,compare the anti-inflammatory effect of different groups of decoctions.5.The anti-GA Mab as ligands were coupled on solid phase carrier,hydrogen bromide activation on agarose gel 4 b.Then we got the sample of SGD knockout of GA.Preparation of human umbilical vein endothelial cells(HUVEC)treated with lipopolysaccharide(LPS),compare the secretion of interleukin 6(IL-6)and tumor necrosis factor ?(TNF-?)among different doses of SGD with different content of GA.Results1.A linear correlation was obtained for PF concentrations ranging from 6.25 to 800.00 ng/mL.The regression equation was y =-0.1651n(x)+1.1141 with a correlation coefficient of 0.9929.Precision and accuracy were evaluated and these parameters were within the normal range(<15%).The average recovery rates was 105.13%,meeting the requirements for biological samples.2.HPLC method for testing Glycyrrhizic acid(GA)was established with linear ranging from 125 to 2000 ?g/mL,with the R2 of 0.9992.Established HPLC method of paeoniflorin(PF),linear ranging from 5 to 300 ?g/mL,with the R2 of 1.Established the HPLC fmethod for testing GA and PF of peony liquorice decoction at the same time,which can be used for the ingredients detection of peony liquorice decoction.3.Detection of different peony liquorice decoction showed that a linear relationship of GA and licorice content.Although peony content is constant,but the content of PF reduced with the increase of licorice content.The suppression was more obvious when the contents of licorice were more than the contents of peony.The results of pharmacokinetic studies showed that the Tmax of PF in SGDs were all at 60 mins after the treatment,while the Tmax of PF in peony group was at 40 mins.When the contents of licorice were less than the content of peony,the Cmax of PF increased with the contents of licorice.When the contents of licorice were more than the content of peony,the Cmax of PF were decreased.The highest Cmax of PF was 1.74±1.57 ?g/mL when the ratio of peony and licorice was 1:1.Analgesic experiments showed that the strongest analgesic action of peony group appared at 30 mins after the treatment,while the strongest analgesic action of SGD groups were in 60-90 mins.The analgesic action were increased with the proportion of licorice.The response latency of SG7 in 60 min after the treatment,SG4,5,6,7 groups in 90 min after the treatment,SG5,6,7 groups in 120 min after the treatment were significantly different(P<0.001,P<0.01,P<0.05)from the control group.The auricle swelling degrees of SGD groups were significantly inhibit(P<0.05,P<0.01,P<0.001)compared with the control group.The anti-inflammatory effects of SG3(3:2)and SG2(2:1)were stronger(P<0.001)than other groups.4.The results of HPLC showed that the contents of GA in SGD with different medicines were significantly decreased compared with SG group,the content of PF in SGH was significantly higher than that of SG group,while decreased also in other groups.Pharmacokinetic studies showed that the Tmax of PF in SGH and SGF were about 20 mins,in SGG was about 40 mins,and inSGC was about 80 mins.Compared with the SG group,the Cmax and AUCtot were decreased.The Cmax of SGH was the highest,1.22 ±0.24?g/mL.The AUCtot of SGG was the highest,459.89±92.70 ?g/mL.The average retention times(MRT)of SGC group was the longest,985.90±441.34 min.The analgesic test showed that the response latency of SGG was the longest at 90 min(P<0.01)after treatment,SGC at 120 min(P<0.05),SGH and SGF at 60 min(P<0.01).The response latencies were stronger than that of the SG group at each time point.The analgesic effect was the strongest in SGF group.The anti-inflammatory effect of SG,SGG and SGH groups were significantly increased compared with the control group(P<0.05,P<0.01).5.The GA immune affinity chromatography column was prepared successfully.The sample of SGD knockout of GA was achieved.Compared with the model group,the content of IL-6 and TNF-a of 100%GA and the GA group were significantly lower(P<0.05).While the other groups had no significant effect on IL-6 and TNF-?(P>0.05).Conclusions1.In this study,the method of icELISA for the content of PF in mouse blood was established.The wide linear range,the high sensitivity and the great reproducibility were showed that this method was suitable for the determination of PF in large number of biological samples.2.Through the comprehensive research with different proportions of SGD:?The effects of in vitro dissolution and in vivo absorption of paeoniflorin were both different when the concentration of licorice is lower than that of peony and the amount of licorice is higher than that of peony.?The analgesic effect of peony had been delayed by licorice.? The suitable proportions of peony and licorice were different when used for different efficacy.3.The study of the effect of different medicines with peony licorice compatibility showed that:?The in vitro dissolution and in vivo absorption of paeoniflorin were inhibited to varying degrees by the addition of other herbs.?The Tmax of paeoniflorin and the onset time of the compatibility was significantly correlated,indicating that paeoniflorin may had its unique role in the mechanism of analgesic efficacy.4.The anti-inflammatory effect of SDG is similar to that of GA.But both the increase and decrease of glycyrrhizic acid had inhibited the anti-inflammatory effect of SGD.Indicating that there is a best ratio of ingredients,rather than into a simple linear relationship. |
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