Activities Of Berberine Hydrochloride In Combination With Antifungal Agents Against Microsporum Canis In Vitro

Objective: The distribution of White ringworm is worldwide in children, due to geographical location, climate and environment, the different habits of life, there are also differences in incidence. Recent research shows that most regions in Europe [1 - 3] and our country landlocked [4-- 15] (except Xinjiang) of its major pathogens for Microsporum canis. Treatment of the tinea capitis caused by Microsporum canis, effective drugs, including griseofulvin, itraconazole and terbinafine, etc. All kinds of treatment and efficacy of drug therapy reported mixed.[16-19]. In order to reduce toxicity and improve efficacy of Traditional medicines, domestic and foreign scholars advocate combined.Xuzhen, et al [20] reported that berberine with antifungal agents of fluconazole, ketoconazole, miconazole et al combined, to varying degrees to improve the treatment of fungal infections, and enable the resumption of antifungal drug resistance, it can be used as a synergist of antifungal agents. Other Scholars[32,33] also have reported the application of crude extract Berberine anti-dermatophytes research, but the application of pure berberine hydrochloride with antifungal agents in vitro antifungal activity of the research so far has not been reported .So far, the sensitivity for dermatophytes standard programs and on the results of drug-resistant and sensitive criteria for the definition has no uniform standard [21]. This experiment with reference to the United States National Clinical and Laboratory Standards Institute (CLSI) recommended《Filamentous fungi conidiation rich liquid-based micro-dilution method Antifungal Susceptibility Test program》(M38-A) and reference documentation [21,23-25], berberine hydrochloride alone and in combination of three antifungal agents against clinical isolates from 30 Microsporum canis in vitro with micro-dilution method, and compared the combination method of different concentrations of antifungal agents activity to further explore the optimal combination programs.Methods:1 Strains separation and culture and identification:All of the strains isolated from September 2007 to October 2008 with tinea capitis patients in outpatient clinic in the second hospital of Hebei Medical University. Removal of disease hair and surrounding scale filings with the tweezers, if positive in fungal microscopy, at the same time to do culture. Inoculated with chloramphenicol Sabouraud's dextrose agar, in each case for two specimens at the same time , (25-27)℃cultivate for (2-4) weeks; observation of colony growth and smear microscopy, if it is dermatophytes, then switch species in the rice medium, at the same time to do small culture to identify the strains.2 Antifungal drugs sensitive experiment in vitro2.1 Application of antifungal agents alone against fungal in vitro:Add the 2 times of final concentration of antifungal drug-fold dilution to 96 cell culture plate each line 1st to the 10th row hole, each hole add 100uL, 11th row and the 12th row add RPMI1640 culture medium 100uL. The fungus suspension with RPMI-1640 liquid medium diluted 50 times, to be (0.4～5)×104 CFU / ml of 2 times the final concentration of the fungus suspension. 96 cell culture plate 's each line add the 100uL fungus suspension 1st to the 11th row hole, the 12th row add 100uL culture medium. Cultivate temperature 28℃, 5 days in the first interpretation of the results.2.2 Berberine and antifungal agents in equal combination experiment:The above-mentioned concentration of berberine hydrochloride and antifungal agents each 50uL add 96 cell culture plate in 1st to 10 rows. Other experiment operation are the same as alone,s. Cultivate temperature 28℃, 5 days in the first interpretation of the results.2.3 The hydrochloric Berberine and antifungal agents in Cross combination Experiments :Add the antifungal agents to 96 cell culture plate with micropipettor,the 1st to the 8th line of 1st row (hole) to the 10th row (hole), from the high concentration to the low, followed by add Berberine Series solution each 50μl; the 1st row to the 10th row 1st line (hole) to the 8th line (hole) is the 3 to 10 times series of diluted concentrations of antifungal agents each 50μl. Other experiment operation are the same as alone,s. Cultivate temperature 28℃, 5 days in the first interpretation of the results.3 Statistical method :The data obtained by statistical treatment SPSS11.5 software. Different antifungal MIC values after conversion by the rank of one-way ANOVA; The different FIC values comparison between Berberine and antifungal agents withχ2 test; further comparison with diremption ofχ2 method. P <0.05 have statistical significance.Results:1 The results of four antifungal agents against Microsporu- m canis in vitro:Griseofulvin, Itraconazole, Terbinafine and Berberine , the minimum inhibitory concentration (MIC) values of geometric mean as follows: 0.9117ug / ml, 0.5236ug/ml, 0.0050ug/ml, 4.5920ug/ml, four antifungal MIC values after conversion by the rank of one-way ANOVA, P> 0.05, the difference was not statistically significant. (Table.1)2 The results of Berberine with griseofulvin, itraconazole and terbinafine in equal combination:Berberine with griseofulvin, itraconazole, terbinafine equivalent united FIC value results: FIC <1, respectively, the proportion of 26.7% (8 / 30), 46.7% (14 / 30), 43.3% (13/30); 1≤FIC <2 separately for the proportion of 33.3% (10/30), 20.0% (6 / 30), 23.3% (7 / 30); FIC≥2 share rates were 40.0% (12/30), 33.3% (10/30), 33.3% (10/30), the overall comparison by theχ2test, P> 0.05, the difference was not statistically significant. (Table.2)3 The result of Berberine and antifungal agents in Cross combination Experiments:3.1 Different concentrations of berberine hydrochloride and griseofulvin combination antifungal results: The concentration of berberine hydrochloride and 8ug/ml, 4ug/ml, 2ug/ml, 1ug/ml, 0.5ug/ml, 0.25ug/ml griseofulvin cross united FIC <1 percentage are as follows: 3.3% (1 / 30), 20.0% (6 / 30), 40.0% (12/30), 50.0% (15/30), 43.3% ( 13/30), 50.0% (15/30); 1≤FIC <2 percentage are as follows: 20.0% (6 / 30), 36.7% (11/30), 33.3% (10/30), 33.3% (10/30), 40.0% (12/30), 33.3% (10/30); FIC≥2 percentage are as follows: 76.7% (23/30), 43.3% (13/30), 26.7% ( 8 / 30), 16.7% (5 / 30), 16.7% (5 / 30), 16.7% (5 / 30); by theχ2 test, P <0.05, the difference has statistical significance. Further comparison of griseofulvin 8ug/ml group and separately 2ug/ml, 1ug/ml, 0.5ug/ml, 0.25 ug/ml group, P <0.0031, differences were statistically significa- nt. Any remaining groups compared were not statistically. (Table.5)3.2 Different concentrations of berberine hydrochloride and itraconazole combination antifungal results: The concentration of berberine hydrochloride and 2ug/ml, 1ug/ml, 0.5ug/ml, 0.25ug/ml, 0.125ug/ml, 0.0625ug / ml Itraconazole cross united FIC <1 percentage are as follows: 33.3% (10/30), 50.0% (15/30), 60.0% (18/30), 60.0% (18/30), 53.3 % (16/30), 56.7% (17/30); 1≤FIC <2 percentage are as follows: 26.7% (8 / 30), 36.7% (11/30), 26.7% (8 / 30), 30.0% (9 / 30), 33.3% (10/30), 36.7% (11/30); FIC≥2 percentage as follows: 40.0% (12/30), 13.3% (4 / 30), 13.3 % (4 / 30), 10.0% (3 / 30), 13.3% (4 / 30), 6.7% (2 / 30); by theχ2 test, P> 0.05, the difference was not statistically significant. (Table.4)3.3 Different concentrations of berberine hydrochloride and terbinafine combination antifungal Results:The concentration of berberine hydrochloride and terbi- nafine 0.04ug/mL, 0.02ug/mL, 0.01ug/mL, 0.005ug/mL, 0.0025 ug/mL, 0.00125ug/mL, 0.000625ug/mL , 0.000313ug / mL concentration of the experimental group from cross-FIC <1 followed by the proportion of 10.0% (3 / 30), 30.0% (9 / 30), 36.7% (11/30), 40.0% (12/30), 50.0% ( 15/30), 40.0% (12/30), 33.3% (10/30), 40.0% (12/30); 1≤FIC <2 followed by the proportion of 26.7% (8 / 30), 20.0% ( 6 / 30), 33.3% (10/30), 30.0% (9 / 30), 33.3% (10/30), 30.0% (9 / 30), 36.7% (11/30), 30.0% (9 / 30); FIC≥2 followed by the proportion was 63.3% (19/30), 50.0% (15/30), 30.0% (9 / 30), 30.0% (9 / 30), 16.7% (5 / 30) , 30.0% (9 / 30), 30.0% (9 / 30), 30.0% (9 / 30). By theχ~2test, P> 0.05, the difference was not statistically significant. (Table.3) Conclusion:1 Berberine , Griseofulvin, Terbinafine and Itraconazole against clinical isolates of Microsporum canis have a very good antifungal activity in vitro.2 Joint Application of Berberine hydrochloride and Griseofulvin, Terbinafine, Itraconazole has in vitro synergistic anti- Microsporum canis activity, synergy and joint with concentrateions. Low concentrations of berberine hydrochloric -de and griseofulvin, terbinafine united was a strongger synergy.3 Berberine and itraconazole united the most synergies, United Griseofulvin with Berberine synergy with the weakest.