Isolation And Identification Of Pathogens From Dairy Cow Endometritis And Study On The Antibacterialactive Components Of Salvia Miltiorrhiza

Dairy cow endometritis is a common disease of dairy cows,which is mainly caused by pathogenic microbial infection.Application of antibiotics and chemosynthesis drugs are efficient way for treatment of dairy cow endometritis.However,attention should be payed owing to the increasing of severe pathogen resistance,drug residues and environmental disasters.Most Traditional Chinese Medicines are natural,with a diverse of chemical compositions and verified clinical efficacies.As a result,they become important source for new drug development.Salvia Miltiorrhizae Radix et Rhizoma(SMRR)is the dried root and rhizome of Salvia miltiorrhiza Bge,which have effects of promoting blood circulation and removing blood stasis,promoting menstruation and relieving pain,cooling blood and eliminating carbuncle.Modern pharmacological studies have shown that SMRR has obvious function on anti-oxidation,anti-bacteria,anti-inflammation and improving microcirculation and so on.In this study,dairy cow endometritis pathogens were isolated and identified,SMRR antimicrobial active fractions were screened,SMRR antimicrobial active constituents were isolated and identified,and the antibacterial activity of purified compounds were studied.The following results were obtained:(1)Isolation and identification of pathogens of dairy cow endometritis The pathogens were isolated from uterine secretions of endometritis infection dairy cows by plate culture method,and identified according to the morphological and molecular biological features.The results showed that four pathogens were isolated from the uterine secretions of infected cows.The four pathogens were identified to be Staphylococcus aureus,Streptococcus,Bacillus subtilis and Escherichia coli serotype O111:K58(B4).(2)Screening of antimicrobial active fractions from SMRR The chemical constituents of SMRR were pretested by the test tube method.The extracts of SMRR were prepared by petroleum ether,ethyl acetate,n-butanol and water,and antibacterial activitieswere tested by agar diffusion and broth micro dilution method.The results showed that SMRR contains flavonoids and their glycosides,phenols,alkaloids,amino acids,terpenes and lactones and so on.Petroleum ether and ethyl acetate extracts have different degrees of antibacterial activities on Staphylococcus aureus,Streptococcus,Salmonella and Bacillus subtilis.Whereas,n-butanol and water extracts had no obvious antibacterial activity.The above results demonstrate that petroleum ether and ethyl acetate extracts are main antibacterial active fractions of SMRR.(3)Isolation and identification of antimicrobial active constituents from SMRR The extract of petroleum ether and ethyl acetate were extracted and purified by silica gel column chromatography,eluted and separated with different proportions of petroleum ether and ethyl acetate sequentially,performed secondary silica gel column chromatography and recrystallized.Six compounds were identified as Tanshinone IIA,Cryptotanshinone,Betasitosterol-D-glucoside,Tanshinone I,Tihydrotanshinone I and Beta-sitosterol according to their physicochemical properties,13C-NMR and 1H-NMR spectrograms.Among them,the contents of cryptotanshinone and tanshinone ?A in petroleum ether extract,tanshinone ? and dihydrotanshinone I in ethyl acetate extract were high,respectively.(4)Determination of antibacterial activity of monomeric compound Take agar diffusion and broth micro dilution method,in vitro antibacterial experiments were performed by Cryptotanshinone,Tanshinone I,Tanshinone IIA and Tihydrotantanone I.The results showed that Cryptotanshinone,Tanshinone I,tanshinone IIA and Dihydrotantanone I can inhibit Staphylococcus aureus and Bacillus subtilis from cow endometritis potently;and can inhibit ATCC12386,ATCC25922 and clinical isolated Escherichia coli serotype O111:K58(B4)and Streptococcus with different potencies.