Studies On Antibacterial Mechanism Of Persimmon Tannin Water Extract On Candida Albicans

Candida Albicans is a clinically common conditioned pathogenic bacteria,which is easy to exist in the normal population cavity,upper respiratory tract,intestinal tract and vagina,and is the main pathogenic bacteria of common candidiasis.Candida albicans are diploid and do not have a sexual cycle,and are capable of transforming from single-celled blastomyces to filament-like forms.Extensive filament-like growth leads to the formation of mycelia,or budding reproduction.The energy needed for Candida Albicans to grow and reproduce is provided by Candida Albicans mitochondria.Succinate dehydrogenase?SDH?,an enzyme in the mitochondria,is involved in the tricarboxylic acid cycle of energy supply in the mitochondria;Ca²⁺-Mg²⁺ATPase is an enzyme that maintains the normal physiological function of cells and fungi and plays an important role in maintaining the stability of Ca²⁺ and Mg²⁺ concentrations in its internal environment.Therefore,succinic acid dehydrogenase and Ca²⁺-Mg²⁺ATPase play an indispensable role in maintaining the growth and reproduction of Candida Albicans.With the wide application of various immunosuppressants and antifungal drugs,candidiasis infection gradually increases and candidiasis resistance becomes increasingly serious.Therefore,it is urgent to research and develop new antifungal targets instead of antifungal targets.Tannin,also known as tannin,is a water-soluble multiphenol compound with complex structure,while persimmon fruit is rich in condensed tannins.Tannin can solidify the protoplasm of microorganism,so it has strong bacteriostasis effect.In this study,the influence mechanism of persimmon tannin water extract on the activity of succinic acid dehydrogenase and Ca²⁺-Mg²⁺ATPase enzyme in the mitochondria of candida albicans were investigated to understand the bacteriostatic effect of persimmon tannin water extract and provide theoretical basis for the clinical application of tannin compounds in oral cavity.Part ? Extraction and Determination of Mitochondrial Protein from Candida AlbicansObjective: The mitochondrial protein content was determined by Lowry method under different conditions by repeated freezing-thawing ultrasonic wall breaking method and repeated freezing-thawing rapid grinding instrument wall breaking method.The optimum conditions of repeated freezing-thawing-ultrasonic wall breaking method were screened by orthogonal test.Methods: The mitochondrial protein content was determined by Lowry method under different conditions by repeated freezing-thawing ultrasonic crushing and repeated freezing-thawing rapid grinding.The optimum conditions of repeated freezing-thawing and ultrasonic crushing were screened by orthogonal test.Results: 1.The standard protein solution showed a good linear relationship in the range of 0-100 ?g/ml.The standard curve regression equation was y =0.0018x-0.0056 and the correlation coefficient was R2=0.9994.2.Repeated freezing-thawing-ultrasonic wall breaking method: The influence of total working time?A?,ultrasonic power?B?and working time?C?on protein content was: C>A>B.The best determination conditions were as follows: A1B2C2,that is,repeated freezing-thawing at-20 ?combined with ultrasonic crushing to break the wall,that is,the total working time was 5 min,the power was 50%,and the working time was 5 s.From the analysis of variance,the effect of working time on mitochondrial protein content of Candida albicans was the most significant?P<0.01?.3.Repeated freezing-thawing-rapid grinding instrument wall breaking method: rapid grinding instrument wall breaking method under different action time,different rotational speed to determine the content of candida albicans protein significant difference?P<0.05?,the optimal condition is that the working time is 2.0 min,the rotational speed is 1600 rpm.There was no significant difference in mitochondrial protein content between the 3.0 min group and 4.0 min group in each time group?P>0.05?.There was no significant difference in mitochondrial protein content among rotational speed groups?P>0.05?.4.The optimal choice of two different wall breaking methods: repeated freezing-thawing ultrasonic wall breaking method and repeated freezing-thawing rapid grinding instrument wall breaking method showed significant difference in the determination of candida albicans protein content?P<0.05?.Part ? Studies on Antibacterial Mechanism of Persimmon Tanned Water Extract on Candida albicansObjective: The activity changes of succinic dehydrogenase?SDH?and Ca²⁺-Mg²⁺ATPase in candida albicans with different concentration of persimmon tannin water extract were detected to explore the antifungal mechanism of persimmon tannin water extract.Methods: Determination of minimum inhibitory concentration?MIC?of persimmon tannin water extract against candida albicans by microdilution method;the SDH enzyme activity and Ca²⁺-Mg²⁺ATPase activity of candida albicans were detected after the activity of persimmon tannin water extract.The concentration of persimmon tannin water extract was 0 ?g/m L,61 ?g/m L,122 ?g/m L,244 ?g/m L,after the treatment of candida albicans for 48 h,candida albicans were collected,the bacterial suspension was prepared,the wall was broken,and the mitochondrial suspension of candida albicans was extracted as the sample solution by differential centrifugation;the protein content of mitochondria was measured by Lowry method.According to the different enzymes in the sample solution,corresponding reagents were added,and the absorbance?OD?value was recorded by spectrophotometer at 650 nm and 636 nm respectively,so as to calculate the activity of SDH enzyme and Ca²⁺-Mg²⁺ATPase.Result:1.The MIC value of persimmon tanning water extract for candida albicans was 77 ?g/ml.2.The activity of succinic dehydrogenase in the 0 ?g/m L group and the 61 ?g/m L group was not significantly different?P>0.05?,but it was significantly higher than 122 ?g/ml,244 ?g/ml in the concentration group?P<0.05?,and the higher the concentration of persimmon tannin,the lower the activity of SDH was.3.The change of Ca²⁺-Mg²⁺ATPase in Candida albicans after the effect of persimmon tannin concentration: the difference between persimmon tanning concentration 61 ?g/ml and blank group 0 ?g/ml was not statistically significant?P>0.05?,the activity of Ca²⁺-Mg²⁺ATPase in the 122 ?g/m L and 244 ?g/m L groups was significantly lower than that in the blank group at 0 ?g/m L?P<0.05?,the higher the tannin concentration,the lower the activity of Ca²⁺-Mg²⁺ATPase;there were statistically significant differences in Ca²⁺-Mg²⁺ATPase activity between groups?P< 0.05?.Conclusion: 1.Repeated freezing-thawing and ultrasonic wall breaking method was used for candida albicans wall breaking to obtain mitochondrial protein.2.The tannin water extract of persimmon can reduce the activity of SDH enzyme and Ca²⁺-Mg²⁺ATPase enzyme of candida albicans.