Screening Of Chinese Herbal Medicines Against Candida Albicans And The Study For Antimicrobial Efficacy And Mechanism Of MZ1711B

Bacterial/fungal infections and their drug resistance have been a major medical chalenge.In recent decades,due to the irrational use or abuse of antibiotics,new drug resistance mechanisms have emerged and spread around the world,threatening not only our ability to treat microbial infections,but also the success rate of major surgery and cancer chemotherapy,leading to long-term ilness,disability or even death.The dialectical treatment of Chinese medicine together with the theory of the formula compatibility provide new research ideas for the treatment of antimicrobial infection.There are many medicinal plant resources in China,among which there are many plants with antimicrobial effect,which is one of the important raw material sources of Traditional Chinese Medicine Formulas.When curing bacterial/fungal infections,the Traditional Chinese Medicine Formula emphasizes the integrity,and has the advantages of small toxic and side effects and difficulty in producing drug resistance.In this paper,the antimicrobial Traditional Chinese Medicine Formula MZ1711 B was developed by combining the screening results of medicinal plants and the compatibility theory of traditional Chinese medicine,and then the effect and the mechanism were studied by in vitro and in vivo experiments.In this paper,the ethanol extracts of 83 medicinal plants were firstly tested for their efficacy.Plants like Rheum palmatum L.and Dendrobium officinale Kimura et Migo showed strong antimicrobial effects.Combined with the results of pharmacodynamic test and Chinese medicine compatibility theory,the antimicrobial formula was developed and named as MZ1711 B.Then the Candida albicans standard strain was used as the research object to evaluate the antimicrobial activity of MZ1711 B.It was found that the growth of C.albicans could be completely inhibited at 2 min.The minimum inhibition of MZ1711 B against C.albicans standard strain was determined by the dilution method.The results of Minimum Inhibitory Concentration(MIC)and Minimum Fungicidal Concentration(MFC)were respectively 10% and 12.5%,showing strong inhibition.In addition,the increased alkaline phosphatase content and conductivity of the treated liquid,together with the morphology of the depressed and damaged cels under Scanning Electron Microscopy(SEM),indicate that MZ1711 B may exert its fungicidal effect by destroying the permeability of cell membrane and the integrity of cell wal.Subsequently,we used 11 clinically isolated strains of C.albicans as the research objects,and found that the MIC and MFC of MZ1711 B against the 10 sensitive strains were respectively between 5%-10% and 7.5%-12.5%,lower than that of the standard strain.MIC and MFC of drug-resistant C.albicans were respectively 10% and 12.5%,demonstrating the same inhibition effect with the standard strain,which indicates that the MZ1711 B wouldn't be affected by its antibiotic resistance when acting on drug-resistant strains.Furthermore,we investigated the fungicidal mechanism of MZ1711 B against the 5-fluorocytosine-resistant C.albicans: the cells treated under SEM were destroyed to different degree;the results of mycelial induction experiments showed that the blank control group and the 5-fluorocytosine group cels grew in mycelial state while the cels treating with the 5% concentration of MZ1711 B demonstrated obvious yeast state.This indicates that 5% MZ1711 B was highly effective in inhibition of the transformation into the mycelial state and thus reducing its virulence and drug resistance.In the phospholipase precipitation circle experiment,the activity of phospholipase was negative after MZ1711 B treatment,which greatly reduced the toxicity of the resistant strain.Finally,in order to explore the broadspectrum antimicrobial effect of MZ1711 B,we determined the Minimum Inhibitory Concentration(MIC)and the Minimum Bactericidal Concentration(MBC)of three types of clinically isolated strains,and found that the MIC and MBC against the Escherichia coli were 10% and 12.5%,respectively;and the MIC and MBC of the three clinically isolated Staphylococcus aureus were 5% and 7.5%,respectively;and the MIC and MBC against Pseudomonas aeruginosa were 5% and 7.5%,indicating the high-efficiency and broadspectrum antibacterial effect of MZ1711 B.We also explored the in vivo toxicity of MZ1711 B and its effect on skin infections.In the oral acute toxicity test of mice,no death occurred in the mice within 7 days of intragastric administration,and there was no abnormality in the diet and behavior of the mice during the observation period either.In addition,there were no significant differences in other indicators such as liver,body weight and blood when the low-and high-concentration groups were compared with the blank control group,except for the spleen index.This indicates that the MZ1711 B is not toxic in the acute toxicity test of mice,and it is preliminarily judged that the drug is of safety and reliability.In the skin infection experiment,we established a model of mouse skin abscess infected by S.aureus,and found that MZ1711 B can cure the skin abscess and significantly promote the healing and recovery of the abscess.HE staining results also showed that MZ1711 B can significantly improve the skin damage caused by S.aureus infection.Immunohistochemical analysis showed that MZ1711 B can significantly downregulate the expression of inflammatory factors TNF-? and IL-6,which is consistent with the result of ELISA on these two kinds of proteins.In summary,we have developed the antimicrobial formula MZ1711 B by screening of 83 kinds of anti-microbial Chinese herbal medicines.The antimicrobial efficacy and preliminary mechanism of the formula against various microorganisms such as Candida albicans were explored by in vitro and in vivo experiments.This study provides important preliminary work and foundation for the research and development of new antimicrobial drugs from traditional Chinese medicine.