Study On The Screening And Formulation Of Substituted Antimicrobial TCMs Based On The Theory Of "Strengthening Vital Qi And Removing Pathogens"

OBJECTIVE:By establishing a library of pathogenic bacteria in poultry and livestock,potential effective traditional Chinese medicines that inhibit the main pathogenic bacteria in livestock and poultry were screened.Based on the theory of " Strengthening Vital Qi and Removing Pathogens",and the principle of differentiation and treatment of bacterial diseases using traditional Chinese veterinary medicine,the optimal combination of traditional Chinese medicine is established,providing new ways and methods for healthy breeding of poultry and livestock.METHODS:PCR method was used to identify bacteria collected and isolated from aquaculture farms in Qingdao and its surrounding areas.After gene sequencing using the whole genome shotgun strategy and the second generation sequencing technology,BLAST software was used to analyze antibiotic resistance related genes and virulence factor related genes.The Oxford Cup method was used to determine the antibacterial activity of 38 traditional Chinese medicines against pathogenic bacteria in poultry and livestock.Under the guidance of the theory of " Strengthening Vital Qi and Removing Pathogens",as well as the theory of" compatibility between Principals,associates,adjuvants and messengers",the formulation of traditional Chinese veterinary medicine was established.Analyze its antibacterial synergistic effect using the chessboard dilution method.Optimize the extraction process of the formula through orthogonal experiments.Analyze the chemical components contained in it using ultra-high performance liquid chromatography highresolution mass spectrometry technology.Using acute toxicity experiments to test the tolerance of mice to formula milk powder.Evaluate the safety of the formula by recording the weight of mice and observing the pathological changes in their viscera.The in vitro antibacterial activity of this formula was studied using agar drilling method,double dilution method,and intracellular ATP content determination method.Study the efficacy of the formula by preparing a bacterial infection model.After the experiment,blood and colon were collected for testing.Observe the pathological changes of the colon using HE staining and detect the content of inflammatory factors in serum using ELISA method.RESULTS:After collection and isolation,14 types of bacteria were identified,with Shigella being the most abundant.81.58%of the strains have multiple antibiotic resistance,with a total of 213 antibiotic resistance genes;The virulence factor mainly belongs to Salmonella and Escherichia coli,and the bacterial virulence of virulence factor is related to intestinal diseases.33 traditional Chinese medicines with different antibacterial effects were selected through in vitro antibacterial experiments,among which Viola yedoensis,Silymarin,Geranium sibiricum,and Fructus viticis have the most prominent effects.According to the guidance of the theory of "Strengthening Vital Qi and Removing Pathogens" and the clinical drug experience,Viola yedoensis,Geranium sibiricum,and Fructus viticis were selected,and the compatibility of Atractylodes macrocephala and Glycyrrhiza uralensis was added to establish the formula.After verification,its compatibility is reasonable and can exert certain antibacterial synergistic effects.The optimal extraction process is to use 20 times the amount of water to reflux and extract twice,each time for 2 hours.The combined extract is then concentrated under reduced pressure and freeze-dried.Under this process condition,the yield is high and has good antibacterial effect.The main components of the formula are flavonoids,terpenoids,phenylpropanoids,phenols,and alkaloids.The acute toxicity test did not detect LD50,and the MTD test showed that the formula was non-toxic.In vitro antibacterial activity is good,which can have a bactericidal effect and is less affected by the dosage form.The establishment of a stable and reliable model of intestinal inflammation caused by bacterial attack(P<0.01).The optimized extraction process can effectively improve spleen swelling caused by pathogenic bacteria infection and reduce IL-1 in mouse serum β And IL-6 levels.The HE staining results showed that the formula HY-3 can effectively promote the recovery of colitis.CONCLUSION:This study established a library of pathogenic bacteria in poultry and livestock,and identified and analyzed various strains.The strains widely exhibit multiple drug resistance,and the main bacterial virulence is related to causing intestinal inflammation.A screening of potential antibacterial Chinese herbal medicines was conducted for current resistant strains,and several effective Chinese herbal medicines were selected.Based on the guidance of the theory of " Strengthening Vital Qi and Removing Pathogens",as well as the theory of " compatibility between Prineipals,assoeiates,adjuvants and messengers",the formula HY-3 for Chinese veterinary medicine was established.After verification,the formula is scientifically and reasonably compatible,has good antibacterial and anti-inflammatory effects,and has low toxicity,which can be safely used for animal husbandry.Research has shown that the combination of formulas can reduce the levels of serum inflammatory factors in mice,improve the pathological condition of the colon,and have good therapeutic effects on intestinal inflammation caused by pathogenic bacteria.