Specific Knockout Of Glycyrrhizic Acid Using An Immunoaffinity Column

Objective:The immunoaffinity chromatography column(GA-IAC)of Glycyrrhizic acid(GA)was prepared for the specifically knocking out of GA from Licorice extraction(LE),and the anti-inflammatory effect of LE and the solution after knocked out of GA were compared.Also,the anti-inflammatory effect of three different species of Licorice(Glycyrrhiza uralensis Fisch,Glycyrrhiza glabra Licoric,Glycyrrhiza inflat Bat)were compared.1.Methodology test of GA-IAC.The parameters including maximum coupling capacity,stability,precision and accuracy of the IAC column for GA were examined.2.The LE and the solution after specific knockout of GA from LE were prepared by the GA-IAC,and the knockout compounds were identified by LC-MS.3.The anti-inflammatory effect of LE and the solution after specific knockout of GA from LE were compared.4.The anti-inflammatory effects of three different kinds of liquorice before and after the knock out extract of GA were evaluatedMethods:1.We prepared the anti-G A-MAb by the mice ascites induced method,and the titer,competition and cross reaction rate were determined.Then purified MAbs by the caprylic acid-ammonium sulfate method.The purity of GA-antibodies by SDS-PAGE method was examined.2.The GA-IAC was prepared by coupling the purified GA-Mabs with agarose gel.The coupling process includes:the activation and swelling of the gelatin,cleaning of gelatin,antibody coupling,sealing,packing,and balance of the column.And the maximum coupling capacity,stability,precision and accuracy of the IAC column were evaluated.3.1.0 mL LE(2.0 mg/mL)was used to knock out the target compounds from the LE after the steps of loading,adsorbing,eluting,balancing by the GA-IAC.The spectrums of Licorice extraction and knockout sample were obtained by HPLC,and the knockout compounds were identified by LC-MS.4.The anti-inflammatory effects of LE and knock out sample solutions were analyzed by measuring the IL-6 and TNF-a secreted by LPS-induced raw264.7 macrophages.The material basis for the anti-inflammatory effects of the three different species of liquorice in inhibiting the secrete of IL-6 and TNF-a efficacy of raw264.7 cell were analyzed.Results:1.The titer of anti-GA-MAb in the ascites were above 64 000:1 and showed a good competition.The detection range was ranging from 78.12 to 2500.0ng/mL and the regression equation was:y=-0.1891n(x)+1.7113,(R2=0.9973).The MAb was highly specific for GA for the cross-reactivity with structurally related chemicals was less than 0.01%,except for glycyrrhetinic acid(31.57%).2.The GA-IAC was prepared.The antibody-coupling rate of the MAb and the gel was 74.9%,and the antibody gel coupling ratio is 5.99:1.The distilled water-glycine hydrochloride elution buffer(pH=2.7)was used as the buffer solution system of the chromatographic column.The maximum capacity of the IAC column for GA was 1326.11 ?g.RSD of the GA knock out rate of the affinity chromatography column stayed 1.37%during 32 d,which showed a pretty well stability.The precision(RSD)of the GA was 0.65%,and the accuracy(RE)was 0.37%,which was suitable for the requirement of methodology.3.The results of MS showed that the IAC using anti-GA-MAb can specifically knock out 18?-Glycyrrhizic acid,Liquorice saponin A3,liquorice saponin G2,22-Acetoxyglycyrrhizin,Mono-Ammonium-Ural saponin A,Yunganoside G1 and 18?-Glycyrrhizic acid.4.The inhibition efficacy of three different species of glycyrrhizin acid knockout solution by GA-IAC from liquorice root extract on LPS-induced raw264.7 macrophages to secrete IL-6 and TNF-? was compared.As the result shown,the Glycyrrhiza uralensis Fisch extraction group content IL-6:1.27±0.009 pg/mL,TNF-a:7.63±0.100pg/mL,which was lower than in LPS group(IL-6:1.31 ±0.006pg/mL,TNF-a:7.95±0.107 pg/mL),and there was significant difference between two groups(P<0.05);The concentration of IL-6(7.89±0.013 pg/mL)and TNF-?(1.33±0.055 pg/mL)in the knockout solution of Glycyrrhiza uralensis Fisch group was no significant differences in LPS group(P>0.05);The Glycyrrhiza uralensis Fisch before and after GA knockout solutions was significant difference in inhibiting inflammatory cytokines IL-6 and TNF-a secretion(P<0.05);The Glycyrrhiza glabra and Glycyrrhiza inflat Bat before and after GA knockout solutions was no significant difference in anti-inflammatory effects(P>0.05).Conclusion:For the first time the GA-IAC was prepared by the anti-GA MAbs and the GA-IAC was first applied into the knock out studies of GA in Licorice alcohol extraction;The negative samples of GA solutions knocked out of GA by the GA-IAC was first prepared by immunoaffinity chromatography,results showed that GA was the main pharmacological effects of the anti-inflammatory effect of liquorice;with the before and after comparison study of knock out GA solutions,the true pharmacological effects material basis was proved and the contribution of GA was studied.This study also provided the evidence for material base research of different kinds of licorice.