da Silva Anderson Ramos, de Andrade Neto João Batista, da Silva Cecília Rocha, Campos Rosana de Sousa, Costa Silva Rose Anny, Freitas Daniel Domingues, do Nascimento Francisca Bruna Stefany Aires, de Andrade Larissa Nara Dantas, Sampaio Letícia Serpa, Grangeiro Thalles Barbosa, Magalhães Hemerson Iury Ferreira, Cavalcanti Bruno Coêlho, de Moraes Manoel Odorico, Nobre Júnior Hélio Vitoriano
School of Pharmacy, Laboratory of Bioprospection and Experiments in Yeast (LABEL), Federal University of Ceara, Fortaleza, CE, Brazil.
Department of Biology, Science Center, Molecular Genetics Laboratory, Federal University of Ceara, Fortaleza, CE, Brazil.
Antimicrob Agents Chemother. 2016 May 23;60(6):3551-7. doi: 10.1128/AAC.01846-15. Print 2016 Jun.
The incidence of fungal infections and, in particular, the incidence of fungal antibiotic resistance, which is associated with biofilm formation, have significantly increased, contributing to morbidity and mortality. Thus, new therapeutic strategies need to be developed. In this context, natural products have emerged as a major source of possible antifungal agents. Berberine is a protoberberine-type isoquinoline alkaloid isolated from the roots, rhizomes, and stem bark of natural herbs, such as Berberis aquifolium, Berberis vulgaris, Berberis aristata, and Hydrastis canadensis, and of Phellodendron amurense Berberine has been proven to have broad antibacterial and antifungal activity. In the present study, the potential antifungal effect of berberine against fluconazole-resistant Candida and Cryptococcus neoformans strains, as well as against the biofilm form of Candida spp., was assessed. The antifungal effect of berberine was determined by a broth microdilution method (the M27-A3 method of the Clinical and Laboratory Standards Institute) and flow cytometry techniques, in which the probable mechanism of action of the compound was also assessed. For biofilm assessment, a colorimetric 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay was used to determine the susceptibility of sessile cells. The isolates used in the study belonged to the Laboratory of Bioprospection and Experiments in Yeast (LABEL) of the Federal University of Ceará. After 24 and 72 h, fluconazole-resistant Candida and Cryptococcus neoformans strains showed berberine MICs equal to 8 μg/ml and 16 μg/ml, respectively. Cytometric analysis showed that treatment with berberine caused alterations to the integrity of the plasma and mitochondrial membranes and DNA damage, which led to cell death, probably by apoptosis. Assessment of biofilm-forming isolates after treatment showed statistically significant reductions in biofilm cell activity (P < 0.001).
真菌感染的发生率，尤其是与生物膜形成相关的真菌抗生素耐药性的发生率显著增加，这导致了发病率和死亡率的上升。因此，需要开发新的治疗策略。在这种背景下，天然产物已成为可能的抗真菌剂的主要来源。黄连素是一种原小檗碱型异喹啉生物碱，从天然草药如小叶小檗、欧洲小檗、刺檗和加拿大黄连的根、根茎和茎皮中分离得到，以及从黄柏中分离得到。黄连素已被证明具有广泛的抗菌和抗真菌活性。在本研究中，评估了黄连素对氟康唑耐药的念珠菌和新型隐球菌菌株以及念珠菌生物膜形式的潜在抗真菌作用。黄连素的抗真菌作用通过肉汤微量稀释法（临床和实验室标准研究所的M27 - A3方法）和流式细胞术技术来确定，其中还评估了该化合物可能的作用机制。对于生物膜评估，使用比色法3 -（4,5 - 二甲基 - 2 - 噻唑基）- 2,5 - 二苯基 - 2H - 四氮唑溴盐（MTT）测定法来确定固着细胞的敏感性。该研究中使用的分离株属于塞阿拉联邦大学酵母生物勘探与实验实验室（LABEL）。24小时和72小时后，氟康唑耐药的念珠菌和新型隐球菌菌株的黄连素最低抑菌浓度分别等于8μg/ml和16μg/ml。细胞分析表明，黄连素处理导致质膜和线粒体膜完整性改变以及DNA损伤，这可能通过凋亡导致细胞死亡。处理后对生物膜形成分离株的评估显示生物膜细胞活性有统计学意义的降低（P < 0.001）。
