Study On The Mechanism Of Anti-inflammation In Vitro Of The Complex Compounds Of Modified Bitongling Prescription

Objective:Based on the herbal prescription of modified Bitongling(MBTL),the main goal of the present study was to construct a new model for rapid evaluation of the anti-inflammatory activity of traditional Chinese medicine in vitro and to assess the application potential for as an index of pharmaceutical performance analysis in the future.Methods:The inhibition assays such as protein denaturation(IAPD)and trypsin activity(IATP),erythrocyte membrane stabilization(EMS)tests including heat-induced hemolysis assay(HIHA)and hypotonic-induced hemolysis assay(HPHA),as well as free radical(1,1-diphenyl-2-picrylhydrazol,DPPH)scavenging assay were conducted as pharmaceutic performance indexes(PPIs)in vitro for rapid evaluation of anti-arthritis activity of BTL complex prescription using Taguchi orthogonal array(TOA),principal component analysis(PCA),and heatmap hierarchical clustering analysis(HMHCA).Results:The detection of total contents of flavonoid and polyphenol compounds in herbal medicines was conducted with various extraction solutions(distilled water,30%ethanol,50%ethanol and 80%ethanol),time(30 and 60 min),as well as methods(heated,ultrasonic frequency 40 KHz and 70 KHz).The results showed that the total compound contents were best under the extracted conditions when 50%ethanol was used as the extraction solution of medicines and treated with ultrasonic frequency of 70 kHz for 60 minutes.The results clearly showed that the inhibition efficiency of protein denaturation was E.sinica,E.divaricata,HMC,C.cassia,S.acutum,which was similar to that of diclofenac sodium compound(P>0.05)in the control.The better performances in the inhibition rate of trypsin activity of herbal medicines included S.divaricata,HMC,C.cassia,E.sinica and A.sinensis,and the inhibition efficiency was also equivalent to that of the control group of aspirin(P>0.05).Except S.divaricata,the free radical(DPPH)scavenging rate of herbal medicines in BTL complex prescription including A.sinensis were almost higher than 80%.In addition,the plant extracts from S.divaricata,C.cassia,HMA(herbal medicine A),HMC(herbal medicine C),S.acutum,and E.sinica had protective-well effects on the test of erythrocyte membrane stabilization under the environment of hypotonic solution.However,only both of S.acutum and E.sinica in the inhibition rate of heat-induced haemolysis assay were better than that of diclofenac sodium(p<0.05),the others of BTL prescription were worse.After variation analysis of Taguchi method,it was found that the top three medicines in BTL prescription for the IAPD assay was E.sinica,HMC and S.acutum in order.The contribution(p value)was 82.70%,12.75%and 1.77%respectively,and the confidence level(CL)was greater than 99%using F test method.For the IATP assay,the CL values were all up to 100%except for the HMA,and the order of contribution(p value)was S.acutum>HMC>E.sinica>S.divaricata>C.cassia>HMA.Four herbal medicines including HMA,E.sinica,S.divaricate and C.cassia,had good protective effects on the test of EMS,whose contributions were 37.26%,27.46%,20.18%and 4.38%,respectively.In terms of DPPH free radical scavenging rate,the contribution order of BTL formula was HMC(35.99%)>HMA(23.06%)>C.cassia(15.90%)S.acutum(11.69%)S.divaricata(4.51%)>E.sinica(0.23%).After principal component analysis(PCA),it was found that the PPIs among IAPD,HAHI and HAHP were closely correlated,whose correlation coefficients were 0.857 and 0.786,respectively(P<0.05).There was a moderate correlation(0.679)between HPHA and HIHA.In addition,HAHP,HAHI,and IAPD contributed respectively 30.7%,34.1%,and 31.7%to the variable of first principal component factor.The contribution percentages of DPPH and IATP in the variable of second principal component factor were 48.4%and 51.1%respectively.Moreover,it was also found that each herbal medicine in BTL complex prescription had its own characteristics using PCA assay.For example,in terms of IAPD,EMS(HAHI and HAHP),and DPPH assay,the better medicines included E.sinica,S.acutum and HMC,HMA,C.cassia and S.divaricata had a weak performance in the IATP assay,while the inhibition efficiency of A.sinensis was the worst.C.cassia,HMC,A.sinensis and E.sinica were mainly good at the DPPH free radical scavenging and IATP assay.After heatmap hierarchical clustering analysis(HMHC),it was found that MBTL prescription including A.sinensis could be rapidly divided into three clusters according to the color patches.E.sinica and HMC were the first cluster S.acutum,HMA and S.divaricata were the second cluster,as well as C.cassia and A.sinensis were the third cluster.In addition,for the determination index items of AIA of herbal medicines,the results of HMHC assay were similar to those of PCA.It was found that it can be also quickly divided into two clusters,DDPH and IATP one cluster,as well as IAPD,HAHI and HAHP another cluster.Conclusions:The pharmaceutical performance indexes(PPIs)of rapid evaluation in vitro of AIA of MBTL prescription are summarized in this paper.These evidences show that five PPIs including IAPD,IATP,HAHI,HAHP and DPPH assays constructed in this study have potential for screening herbal medicines on AIA in vitro.After the analysis of Taguchi method,principal component and heatmap hierarchical clustering,the PPIs are rapidly classified two clusters,that is one cluster of DPPH and IATP,as well as another of IAPD,HIHA and HPHA.The inhibition effects of MBTL prescription on IAPD,IATP,HAHI,HAHP and DPPH is E.sinica,HMC,S acutum,S.divaricate,C.cassia,HMA and A.sinensis in order.Moreover,it can be divided into three clusters according to the PPIs.In short,the determination models of five PPIs such as IAPD,IATP,HAHI,HAHP,and DPPH constructed in this study and combined with the use of methods including Taguchi-method,principal component and heatmap hierarchical clustering analysis,has the potential for rapid screening AIA of herbal medicines in vitro and providing references of pharmaceutic effect and mechanism of herbal prescription in the future.