Study Of Ilex Latifolia Thunb On Antifungal Susceptibility And Mouse T Lymphocytes Activation In Vitro

Objective:To study the effects of chemistry extractions(sample I , I , I , I ra, II) from /fee latifolia Thunb on antifungal susceptibility and immunological regulation in vitro. Ilex latifolia Thunb were collected from areas of Yao clansman in the south of Guangdong province. Methods:(1) Antifungal susceptibility testing of 5 fungal strains (C. albicans, S. schenckii, C.glabrata, C.guiliermondii and T.gupseum) for amphotericin B and Itraconazole were performed by reference method of National Committee for Clinical Laboratory Standard (NCCLS). The minimum inhibitory concentration (MIC) were determined by MTT assay and routine direct observing assay separately. These MIC values from two assay were compared with each other.(2) Antifungal susceptibility testing of 5 fungal strains (C. albicans, S. schenckii, C.glabrata, C.guiliermondii and T.gupseum) for 5 chemistry extractions (sample I ,I i , I ii -. I in -. II) from Ilex latifolia Thunb were performed by reference method of National Committee for Clinical Laboratory Standard (NCCLS), The minimum inhibitory concentration (MIC) were determined by MTT assay.(3) The ultrastructure of C. albicans after culturing with sample I [was detected with transmission electron microscope.(4) The effect of chemistry extractions (sample I , I t. In-. I m, II) from llex latifolia Thunb on proliferation of murine spleen cells stimulated by ConA in vitro were examined with MTT assay.(5) The effect of chemistry extractions (sample I -, I i)from Ilex latifolia Thunb on the expression level of CD69 on T cells stimulated by ConA was assessed by flow cytometry.Results:(1) The MTT assay and routine direct observing to determine endpoint are good identical. The A values of MTT assay decreased as increasing of theconcentration of antifungal drugs.(2)Sample I , I iand I , all obviously have inhibitory effects on C. albicans and C.glabrata. Compared with DMSO solvent control, The minimum efficacious antifungal concentrations of C. albicans and C.glabrata for sample I I i I u are 31.25ug/mU 7.81ug/mK 31.25ug/ml and 7.81ug/mK 31.25ug/ml 7.81ug/ml respectively. Both sample I m and II have no inhibitory effects on C. albicans and S. schenckii. Chemistry extractions (sample 1 I , I M I m-. II) from /fee latifolia Thunb all have no inhibitory effects on C.glabrata, C.guiliermondii and T.gupseum.(3) By transmission electron microscope, the wall of C. albicans showed incrassating and dissolving partly, separating from membrane. Mitochondria were swelling and necrosis. Fuscous stains and alveolate structures appeared in cytoplasm.(4) Sample I (25u?mK 50ug/mK l00ug/ml sample I ,(12.5ug/mk 25ug/mU 50ug/mK 100u,g/ml)and sample I ]i(50ug/mK 100ug/ml)can inhibit the proliferation of murine spleen cells stimulated by ConA in vitro(P0.0l). Sample I uj(6.25u.g/mK 12.5ug/mK 25u.g/ml 50u.g/m 100ug/ml)andII(6.25jig/rnK 12.5ug/mK 25 g/mK 50ug/mK 100(4.g/ml)have no inhibitory effect on the proliferation(P>0.05).(5) Sample I (25ug/mK 50ug/mK 100g/ml )and sample I ,(12.5ug/mK 25|iig/mK 50|ig/mk 100p,g/ml) can depress the expression level of CD69 on T cells stimulated by ConA in vitro(P<0.01).Conclusion: (1) MTT assay is more objective, more precise than routine direct observing ,It can replace routine direct observing assay to determine antifungal endpoint .(2) Ilex latifolia Thunb have antifungal susceptibility on C. albicans and C.glabrata.(3) Ilex latifolia Thunb may inhibit fungi through destroying the wall and membrane of cell.(4) Ilex latifolia Thunb can inhibit the proliferation of murine spleen cells stimulated by ConA in vitro.(5) Ilex latifolia Thunb depress the activation of murine T cells stimulated by ConA in vitro.