Study On Tetrandrine As A Synergist To Bifonazole In Trichophyton

Objective:To investigate the synergistic effect of tetrandrine to the anti-dermatophyte activity of bifonazole in vivo and in vitro for the sake of offering the experimental basis for the therapeutic strategies of refractory mycosis.Materials and Methods:The most non-cytotoxic dose of tetrandrine which was chosen as combined dose in the 15 Trichophyton rubrum was determined by the microdilution test.The minimum inhibitory concentration(MICs) of bifonazole alone and combined with tetrandrine are determined in 15 Trichophpyton rubrum by the CLSI proposed M38-A microdilution method.Animal model of dermatophytosis in guinea pig was made by inoculating Trichophyton mentagrophytes conidia on razed skin.The infected animals were scparated into 4 groups randomly and tested with the following formulation: bifonazole/tetrandrine compound cream,bifonazole cream,,tetrandrine cream and matrix cream once daily for 7days.The alterations in the skin lesions,microscopy and culture as well as the pathological changes were observed to investigate the synergistic effect of tetrandrine to the anti-Dermatophyte activity of bifonazole in vivo.Results:The most non-cytotoxin dose of tetrandrine to the Trichophyton rubrum is 40μg/mL.and the reservation rate of Trichophyton rubrum under this concentration is 95%.The MICs of bifonazole alone to the Trichophyton rubrum are 0.25～1μg/mL and are decreased to 0.0313～0.0625μg/mL when combined with tetrandrine (40μg/mL).They have statistical difference(paired -sample t-test,t = 8.614,P = 0.000). After 2 days treatment of bifonazole/tetrandrine compound cream,the skin lesionse recover quickly.There are few erythematous patch and scale.It can be seen from the repeated measurement design that the decrease amplitude of bifonazole/tetrandrine compound cream is more than bifonazole cream.The score of bifonazole/tetrandrine compound cream is lower than bifonazole cream after 2 days' treatment.After 5 days' treatment,the microscopy and culture are negative.But in bifonazole,the microscopy and culture are negative after 7 day's treatment.Meanwhile, there are no improvement in tetrandrine cream and matrix cream,skin lesion are thickening and increasing,the microscopy and culture are positive continuously in both treatment period and observation period.The efficacy of bifonazole/tetrandrine compound cream is better than bifonazole alone after tested by repeated measurement design.(P =0.022 ).There are no difference between tetrandrine cream and matrix cream(P = 0.552).Skin biopsy samples were obtained from one animal per group on the 5^th day and 10^th day.The samples use HE stain and PAS stain.The skin biopsy of bifonazole/tetrandrine compound cream is observed the permeating of neutrophils in 5^th. The skin biopsy is normal in 10^th therapy.There is no hyphae observed in PAS stain.Inflammation was severe in matrix cream in both 5^th and 10^th with more hyperkaratosis and acanthosis, There are a lot of hyphae in PAS stain.The hyperkeratosis and acanthosis are observed in the tetrandrine therapy with more inflammatory cells.There is no improvement in 10^th.The skin biopsy of the Bifonazole therapy is hyperkeratosis lightly and more inflammatory cells,mostly neutrophils and lymphocytes.There is a little hyphae in PAS stain.The skin biopsy is normal in 10^th without any hyphae.Conclusion1.The highest non-cytotoxin dose of tetrandrine to the Trichophyton rubrum is 40μg/mL.2.The highest non-cytotoxin dose of tetrandrine can improve the effect of the anti-dermatophyte activity of bifonazole in vitro.3.There is synergistic effect of tetrandrine to the anti- Trichophyton mentagrophytes of bifonazole in vivo.