Confirmation Of Components And Establishment Of Quality Inspection Method Of "Huangren Tang" Against Actinobacillus Pleuropneumoniae Infection

Porcine infectious pleuropneumonia(PCP)is a bacterial respiratory infection caused by Actinobacillus pleuropneumoniae(APP).Pigs of all ages are susceptible and have a high mortality rate.The emergence of this disease has caused economic losses to the pig industry worldwide.At present,there are up to 18 APP serotypes,and there are differences in the predominant epidemic strains in various countries and regions.In addition,the cross-protection between the serotypes is not strong,which makes it difficult to prevent and treat this disease.Commercial vaccines currently used in clinical is inactivated vaccines,which still cannot reduce the incidence of animals.And in recent years,the emergence of drug-resistant genes of strains and the slow development of new antibiotic drugs have brought huge threats to the health of humans and animals,and the development of new treatments is urgently needed to solve the current problems.The traditional Chinese medicine "Huangren Tang" is a clinical veterinary medicine that has been changed through traditional prescriptions.After years of clinical trials,it has a relatively good therapeutic effect on respiratory infections.But its active ingredients are not clear.In order to facilitate the establishment of quality control,we established the mice infection model by observing the clinical symptoms and weight changes of each group of animals,the changes in lung histopathology and detection of differences in lung bacterial load,analysis of changes in immune cells and detection of related chemokine expression differences,finally,the main immunological indicators of "Huangren Tang" were determined.Then,the animal experiment was performed to detect the main components of "Huangren Tang".The clinical symptoms,the degree of lung injury,the bacterial load,immunological indicators(cytokines,and the number and function of immune cells),and the survival rate were analyzed to confirm its effective therapeutic ingredients and content.On the basis of determining the effective substance of "Huangren Tang",we establish the high performance liquid chromatography tandem mass spectrometry to determine the content of effective monomers,and formulate the quality inspection standard of "Huangren Tang",to ensure the credibility,effectiveness and scientific in application of clinical treatment.The results showed that the scores of clinical symptoms of the mice that treated with the "Huangren Tang" were significantly lower than those of the infected group(P <0.05);the weight change trend was stable and the loss was less than that of the infected group;the apparent pathological lesion of the lung was mild,with only small areas of congestion and edema.The lung bacterial load of mice in the treatment group was lower than that in the infection group,and the difference was significant at 6 hours after infection(P <0.05);the gene levels of neutrophil-related chemokines in mouse lung tissue: CXCL1,CXCL2,CXCL12,and CXCL15 were significantly increased;and mainly by mobilizing PMN-I neutrophils to play its role in eliminating bacteria.After treating the infected mice with the 8 monomer components contained in “Huangren Tang”: baicalin,baicalein,quercetin,geniposide,emodin,chlorogenic acid,magnolol,and β-sitosterol,examined the clinical symptoms,lung index,lung bacteria load,blood circulating immune cells,and survival rate of mice from different groups,evaluate the therapeutic effect of drugs.We found that baicalin,baicalein,quercetin,geniposide,and chrysophanol exerted anti-infection effects to reduce the clinical symptoms in infected mice,change the body weight of mice in treatment group from a decrease to an upward trend,and significantly inhibiting the lung fungal load.The amount of circulating immune cells(neutrophils,monocytes)was down-regulated;and the quercetin was found to have a protective effect on infected mice.Further research found that these five components screen effective monomer components,significantly inhibit the expression of IL-6,IL-1β,TNF-α in the blood and lungs of infected mice,especially,baicalin,baicalein and quercetin can promote the formation of extracellular trapping nets(NETs)of neutrophils,thereby assisting the bactericidal effect.Finally,a liquid chromatography-mass spectrometry method was established for the identified five effective components.In this study,the effective monomer components(baicalin,baicalein,quercetin,geniposide,emodin)in "Huangren Decoction" were determined through in vivo infection experiments.A high performance liquid chromatography-tandem mass spectrometry detection method was established for the active components.Finally,it was determined that baicalin,baicalein,and chrysophanol can be used as detection items for the quality control of semi-finished products and finished products,and can be used to formulate production quality control standards.