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| Author(s): |
Luciani Gaspar de Toledo
Total Authors: 1
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| Document type: | Doctoral Thesis |
| Press: | Araraquara. 2020-04-20. |
| Institution: | Universidade Estadual Paulista (Unesp). Faculdade de Ciências Farmacêuticas. Araraquara |
| Defense date: | 2020-03-26 |
| Advisor: | Taís Maria Bauab; Margarete Teresa Gottardo de Almeida |
| Abstract | |
Vulvovaginal candidiasis (VVC) is an opportunistic fungal infection that causes relevant problem to woman’s health, due to unpleasant symptoms, difficulties of the therapy and the emergence of resistant strains. In this context, the medicinal plants associated to alternatives that improve the antifungal potential, such as the nanotechnology, is an important source in the eradication and control of fungal infections. Thus, the objective of this study was to evaluate the antifungal activity of the essential oil (EO) of Cymbopogon nardus (L.) Rendle, free form and loaded into a lipid microemulsion (ME+EO) against ATCC and clinical strains of C. albicans, C. glabrata and C. krusei. The EO was obtained by hydrodistillation. The chemical analysis of the EO was performed by gas chromatography-mass spectrometry (GC-MS). The lipid microemulsions (ME) were developed and a phase diagram was constructed. The ME and ME+EO were characterized by scattering, zeta potential, polarized light microscopy, rheological assays, bioadhesiveness and transmission electronic microscopy (TEM). The antifungal activity of the EO and ME+EO were evaluated by determination of minimal inhibitory concentration (MIC) and minimal fungicidal concentration (MFC) by microdilution, sorbitol and ergosterol assay. The inhibition of C. albicans hyphae formation in presence of probiotic and vaginal pathogen was carried out by transwells® system. Moreover, the coated and clean surfaces were characterized by contact angle (captive bubble). The effect of EO and ME+EO was evaluated on biofilm adhesion process by flow chamber in parallel plates and adhesion force between coating surfaces and C. albicans by atomic force microscopy (AFM). Additionally, the action of EO and ME+EO was performed against monotypic and heterotypic mature antibiofilm (C. albicans + Lactobacillus acidophilus). The toxicity of EO and ME+EO was analyzed on human cell line HaCat and using alternative model assay with A. salina. The experimental in vivo VVC was carried out in female mice (C57BL/6). The chemical analysis of the EO showed as main compounds the oxygen-containing monoterpenes: citronellal, geranial, geraniol, citronellol, and neral. The characterization of ME and ME+EO exhibited suitable size and homogeneity, negative charge and stability. By polarized light microscopy, ME and ME + OE demonstrated an isotropic behavior, suggesting the formation of a microemulsified system. ME and ME+EO showed non-Newtonian characteristics, with pseudoplastic behavior. The bioadhesiveness was not altered after the incorporation of the EO in the ME. TEM images showed spherical structures for ME, however, the ME+EO photomicrographs showed possible droplets of OE within the spherical structures, indicating the incorporation of OE in the ME. The EO showed effective inhibitory action for all tested strains. The EO when loaded in the ME (ME+EO) exhibited expressive improvement of the antifungal action with reduction of the MIC (from 500 to 31, 2 µg/mL). For all Candida species (C. krusei e C. glabrata) the ME+EO did not cause fungal inhibition. Thus, the subsequent antifungal activity tests were carried out with C. albicans. The EO and ME+EO have the action in the fungal cell membrane, since the MIC was increased in presence of ergosterol. EO and ME+EO prevented the C. albicans hyphae formation, however, the EO was more active. The presence of probiotic and vaginal pathogen decreased C. albicans hyphae formation. ME+EO showed a significant reduction on C. albicans adhesion to the glass surface in flow chamber. Above all, after the passage of the air-liquid interface (air bubble) effective detachment was observed detachment on the ME+EO coated glass surface. EO and ME+EO coated surfaces were less hydrophilic than the control by contact angle measurement The EO and ME+EO inhibited the biofilm of standard and clinical strain of C. albicans, however, the EO was able to promote the absence of fungal growth on agar (CFU/mL). The L. acidophilus monotypic biofilm exhibited high bacterial metabolic activity in presence of EO and ME+EO. The EO showed acceptable standard of toxicity on cell line HaCat. The EO and ME+EO demonstrated low toxicity in alternative model assay with A. salina. In vivo VVC assay the ME+EO was able to promote the cure of the 100% of the animals in the third day of the treatment and the EO caused the cure in 50% of the animals in the second day of treatment. The use of nanotechnology as a lipid microemulsion, for the incorporation of EO, improves the antifungal activity of OE from C. nardus on planktonic cells, inhibiting the formation of hyphae in the presence of probiotic and vaginal pathogen. Moreover, ME+EO promotes the prevention of adherence of C. albicans in a parallel chamber flow chamber and increased the detachment of adhered yeasts to a ME+EO coated glass surface. Additionally, OE and ME + OE inhibit monotypic and heterotypic biofilms. Above all, the EO incorporated in the ME shows a relevant effect in in vivo CVV assay with mice, being a promising alternative to control of infections caused by C. albicans. (AU) | |
| FAPESP's process: | 15/23959-7 - Cymbopogon nardus (L.) Rendle (citronella): chemical-biological prospecting of essential oil highlighted the biofilm study and control of vulvovaginal candidiasis |
| Grantee: | Luciani Gaspar de Toledo |
| Support Opportunities: | Scholarships in Brazil - Doctorate |