Studies On Litsea Cubeba Oil Against Sporothrix Schenckii

Objective: Via the experiments of Litsea Cubeba oil against Sporothrix schenckii to investigate the anti- Sporothrix schenckii activity of Litsea Cubeba oil, and to evaluate the effect of Litsea Cubeba oil against Sporothrix schenckii in mice by the animal laboratory. This can provide initial evidence for clinical use of Litsea Cubeba oil. At last, we explored the anti-fungi mechanism of Litsea Cubeba oil through electron microscope.Methods:1 Drug sensitivity test: The anti- Sporothrix schenckii activity of Litsea Cubeba oil was initially evaluated by disc diffusion method, then the minimum inhibitory concentration (MIC) of Litsea Cubeba oil was determined in fourteen clinically isolated strains by microdilution method, as well as fluconazole was used as quality control.2 Animal experiment:2.1 Establishing murine model of Sporothrix: The fungi preserved in our department were removed from the freezer and let warm, then inoculated into the Sabouraud's Agar, culturing in 27℃. The fungal suspension solution was made up after ten days. Then mice in immunosuppressed states were inoculated through intradermal pathway. 2.2 Evaluating murine model: The murine model was proved by tissue culture and histopathological examination.2.3 Drug treatment: We treated mice infected Sporothrix respectively with Litsea Cubeba oil (83.53mg/kg), potassium iodide (18ml/kg) and isotonic Na chloride(0.2ml/d), and observed the result and the untoward reaction during four weeks.3 Electron Microscope: Sporothrix schenckii was soaked in Litsea Cubeba oil for 24 hours, which concentration was in MIC. As well as the normal Sporothrix schenckii was used as control strain. Both of them were fixed by glutaric dialdehyde before sent the electron microscope department.Results:1 Disc diffusion method: The clear inhibitory ring could be seen after one week. The 10% Litsea Cubeba oil'inhibitory ring diameter was 16mm, and 5% Litsea Cubeba oil'inhibitory ring diameter was 9mm. The inhibitory ring diameter had no manifest change after two weeks.2 Microdilution method: Fluconazole's MIC ranged from 1ug/ml to 2ug/ml. The fixed strains and lymph-vascular strains were all susceptible to Litsea Cubeba oil, and their MIC all ranged from 156.25ug/ml to 1250ug/ml. Subcultivation experiment showed that Litsea Cubeba oil was a fungicide.3 Murine model: Nodules could be seen in back of mice injected fungal cells after about two weeks. At about the fourth week, the lesions got to the peak. Via tissue culture and histopathological examination, the murine model was proved successfully established.4 Drug treatment: The lesions of mice with Litsea Cubeba oil and potassium iodide disappeared or became small after four weeks treatment. We have got satisfying results, and not found adverse effects. In negative control group the lesions transferred or became small. There was significant difference in the effects of treating Sporothrix between the treated group and the negative control group (P<0.05). There was no statistically significant difference between Litsea Cubeba oil group and potassium iodide group. The fixed strains group and lymph-vascular strains group also had no statistically significant difference in the effects of Litsea Cubeba oil or potassium iodide.5 Electron Microscope results: The photos of scanning electron microscope (SEM) showed that hyphae withered, surface became deformed, fade and shrunken. Some hyphae and conidia wall were broken after Litsea Cubeba oil having affected for 24 hours. The photos of transmission electron microscope (TEM) showed that hyphae were deformed. Their cell wall became roughly and loosely. Cytoplasm texture was obscure and mitochondrion was swelling after Litsea Cubeba oil having affected for 24 hours.Conclusion:1 Disc diffusion method shows Sporothrix schenckii is susceptible to Litsea Cubeba oil, and Litsea Cubeba oil may be a fungicide.2 Microdilution method determines quantitatively the MIC of Litsea Cubeba oil about Sporothrix schenckii. Subcultivation experiment shows that Litsea Cubeba oil is a fungicide.3 There is no statistically significant difference between fixed group and lymph-vascular group in getting disease and lesion's appearance. That shows fixed strain and lymph-vascular strain has the same virulence.4 There is statistically significant difference between Litsea Cubeba oil group and negative control group. There is no statistically significant difference between Litsea Cubeba oil group and potassium iodide group. That all show Litsea Cubeba oil has better therapeutic effect for the mice infected by Sporothrix schenckii.5 Litsea Cubeba oil firstly deforms the integrated texture of cell wall, and then cytoplasm gets out. That can affect cell-organ's normal works.