The Application Of The Extract Of Salvia Miltiorrhiza Against Drug-Resistantstaphylococcus Aureus Infection

Staphylococcus aureus(S.aureus)was one of the most important pathogens in clinical bacterial infectious diseases and in livestock farming,and the infection of S.aureus usually showed acute symptomssuch as rapid onset and short duration of illness.In recent years,the antibioticresistance of S.aureus has become increasingly serious and the cases of infections of multi-drug resistant S.aureus have emerged in clinical.They have developed varying degrees of resistance to common clinical antibiotics including aminoglycosides,?-lactams,macrolides and quinolones,with deeper and more widespread resistance to penicillins and cephalosporins.The isolationrate of drug-resistant S.aureuswas very high in both animals and their associated environment.For example,methicillin-resistant S.aureus(MRSA)occupied an important position among the pathogenic bacteria associated with mastitis in dairy cows and the infection disease of urinary system.Therefore,it was significantthat research related to the prevention and treatment of S.aureus infections that occur during livestock farming.And it was also important to understand the pathogenicity and drug resistance of bacteria in the prevention and control of bacterial infections.In addition,S.aureus mainly expresses secreted virulence factors such as pore-forming toxins,superantigenic exotoxins and cytotoxic enzymes in the late stages of infection or growth that disrupt the access of host cells to nutrients or interfere with the function of host immune cells.Effective inhibition of the main virulence factor such as haemolysin(Hla)was effective in mitigating the damage caused by S.aureus to the tissues or cells.This anti-virulence strategy has been widely recognised and studied by researchers.In this study,the crude extract of Salvia miltiorrhiza was found to be effective in enhancing the in vitro antibacterial activity of antibiotics such as aminoglycosides and?-lactams,while significantly inhibiting the biological activity of Hla of S.aureus.Therefore,this study was firstly conducted to investigate the extraction process of Salvia miltiorrhiza.The best extraction process was preferred by the method of ultrasonic extraction,single factor investigation and orthogonal test.And we determied the optimum extraction conditions was that extraction with 80%ethanol(ethanol/H₂O=10:1)3 times for 1.5 h each time.Through the exploration and optimization of the extraction process of cryptotanshinone in Salvia miltiorrhiza,the preliminary extract of Salvia miltiorrhiza was obtained,and the contents of its main components were cryptotanshinone,tanshinone I,tanshinone IIA and tannic acid B,and the content were 0.33%,0.05%,0.44%and 9.02%,respectively.The synergistic effects of Salvia miltiorrhiza extract and its active ingredients with aminoglycoside and other antibiotics in vitro were determined by minimum inhibitory concentration test,growth curve test andtime-killingtest in this study.The synergistic indices FIC of Salvia miltiorrhiza extract in combination with gentamicin,cefothiophene sodium and polymyxin B sulfate against typical MRSA strain USA300 were all less than 0.5,indicating that the synergistic effect of Salvia miltiorrhiza extract with antibiotics was broad-spectrum.Further studies determined that the combination of cryptotanshinone or tanshinolate B and gentamicin had a significant synergistic effect(FIC<0.5),while tanshinone Ior tanshinone IIA had only additive or ineffective effects with gentamicin.The in vitro growth of S.aureuswas not significantly affected by the extract of Salvia miltiorrhiza at sub-inhibitory concentrations.The combination of 1/4×MIC of gentamicin and the extract of Salvia miltiorrhizacould kill all the tested bacteria in the treated wells within 10 h compared to drug treatment alone.We determined that the extract of Salvia miltiorrhiza could effectively inhibit the hemolytic activity of Hla and protect against cell damage of Hla by hemolysis assay,RT-PCR assay,westen blot analysis and cytotoxicity assay.The results showed that Salvia miltiorrhiza extract at concentrations of 8?g/m L and above significantly inhibited the haemolytic activity of Hla and prokaryotic expression of recombinant Hla in the supernatant cultures of S.aureus.Cryptotanshinone,the main component of the extract,significantly inhibited the haemolytic activity of Hla at a concentration of 2?g/m L.The results of the live/dead bacterial staining kit assay and LDH assay showed that the extract of Salvia miltiorrhiza was effective in reducing cell damage mediated by the Hla of S.aureusin at a concentration of 128?g/m L.Salvia miltiorrhiza extract injection was prepared with reference to the prescription composition of similar products on the market.And a preliminary pharmacodynamic and toxicological investigation of Salvia miltiorrhiza extract injection was carried out in this study based on the comprehensive analysis of the active ingredients of Salvia miltiorrhiza extract and the screening of the main excipients.The extract of Salvia miltiorrhiza was identified as the main ingredient,sodium sulfite as antioxidant,benzyl alcohol as anti-bacterial agent,polysorbate-80as solubilizer,sodium hydroxide solution and water for injection as solvent to make Salvia miltiorrhiza extract injection.The results showed that Salvia miltiorrhiza extract injection had certain therapeutic effects,and the combination of Salvia miltiorrhiza extract injection and gentamicin sulphate injection had better therapeutic effects,which significantly alleviated the histopathological changes in the lungs of infected mice,reduced the colonization of lung tissues and improved the degree of inflammation in lung tissues.Results of the acute toxicity test in mice showed that there was no mortality in mice given 5400 mg/kg intraperitoneally,and no obvious ocular pathological changes were found when the mice were executed and dissected,indicating that the injection of Salvia miltiorrhiza extract is safe inmice.The therapeutic effect of theinjection of Salvia miltiorrhiza extract in combination with gentamicin sulphate injection was further evaluated by establishing an artificial infection model of S.aureusin fectedegg chicks.The results of the study showed that the combination of theinjection of Salvia miltiorrhiza extractat medium dose(0.4 m L/kg)and high dose(0.8 m L/kg)significantly improved the therapeutic effect of gentamicin sulphate injection on MRSA infected egg chicks.This study will lay the ground work for subsequent extended clinical trials and target animal safety trials.In conclusion,the extract of Salvia miltiorrhiza had an important role in the treatment of S.aureus infections by significantly increasing the antibacterial effect of the main antibiotics while reducing the pathogenicity of S.aureus.The preliminary obtained the injection of Salvia miltiorrhiza extracthas significant therapeutic effects and low toxic effects.