ABSTRACT
This study aims at evaluating the effects of Zataria multiflora (Z. multiflora) essential oil (EO) on growth, aflatoxin production and transcription of aflatoxin biosynthesis pathway genes. Total RNAs of Aspergillus parasiticus (A.parasiticus) ATCC56775 grown in yeast extract sucrose (YES) broth medium treated with Z. multiflora EO were subjected to reverse transcription-polymerase chain reaction (RT-PCR). Specific primers of nor-1, ver-1, omt-A and aflR genes were used. In parallel mycelial dry weight of samples were measured and all the media were assayed by high-pressure liquid chromatography (HPLC) for aflatoxinB1 (AFB1), aflatoxinB2 (AFB2), aflatoxinG1 (AFG1), aflatoxinG2 (AFG2) and aflatoxin total (AFTotal) production. The results showed that mycelial dry weight and aflatoxin production reduce in the presence of Z. multiflora EO (100 ppm) on day 5 of growth. It was found that the expression of nor-1, ver-1, omt-A and aflR genes was correlated with the ability of fungus to produce aflatoxins on day 5 in YES medium. RT-PCR showed that in the presence of Z.multiflora EO (100 ppm) nor-1, ver-1 and omtA genes expression was reduced. It seems that toxin production inhibitory effects of Z. multiflora EO on day 5 may be at the transcription level and this herb may cause reduction in aflatoxin biosynthesis pathway genes activity.
Subject(s)
Aflatoxins/biosynthesis , Antifungal Agents/metabolism , Aspergillus/drug effects , Biosynthetic Pathways/drug effects , Lamiaceae/chemistry , Oils, Volatile/metabolism , Transcription, Genetic/drug effects , Antifungal Agents/isolation & purification , Aspergillus/genetics , Aspergillus/growth & development , Aspergillus/metabolism , Biosynthetic Pathways/genetics , Chromatography, High Pressure Liquid , Gene Expression Profiling , Oils, Volatile/isolation & purification , Reverse Transcriptase Polymerase Chain ReactionABSTRACT
The goals of this study were to evaluate the effectiveness of Cuminum cyminum, Ziziphora clinopodioides and Nigella sativa essential oils to inhibit the growth of Aspergillus fumigatus and A. flavus and to evoke ultrastructural changes. The fungi were cultured into RPMI 1640 media in the presence of oils at concentrations of 8, 6, 5, 4, 3, 2, 1.5, 1.25, 1, 0.75 and 0.5 mg/ml in broth microdilution and 2, 1.5, 1 and 0.5 mg/ml in broth macrodilution methods with shaking for 48 h at 28ºC. Conidial and mycelial samples exposed to 0.25, 0.5, 1, 1.5 and 2 mg essential oils/ml for 5 days in 2 percent yeast extract granulated plus 15 percent Saccharose media were processed for transmission electron microscopy (TEM). Based on broth dilution methods, C. cyminum and to a lesser extent Z. clinopodioides oils exhibited the strongest activity against A. fumigatus and A. flavus with MIC90 ranging from 0.25 to 1.5 mg/ml, while the oil from N. sativa exhibited relatively moderate activity against two above fungi with MIC90 ranging from 1.5 to 2 mg/ml. The main changes observed by TEM were in the cell wall, plasma membrane and membranous organelles; in particular, in the nuclei and mitochondria. These modifications in fungal structure were associated with the interference of the essential oils with the enzymes responsible for cell wall synthesis, which disturbed normal growth. Moreover, the essential oils caused high vacuolation of the cytoplasm, detachment of fibrillar layer of cell wall, plasma membrane disruption and disorganization of the nuclear and mitochondrial structures. Aspergillus fumigatus and A. flavus growth inhibition induced by these oils were found to be well-correlated with subsequent morphological changes of the fungi exposed to different fungistatic concentrations of the oils. Our results show the anti-Aspergillus activities of C. cyminum, Z. clinopodioides and N. sativa essential oils, which strengthens the potential use of these substances as anti-mould in the future.
Subject(s)
Aspergillus flavus/growth & development , Aspergillus fumigatus/growth & development , Cell Wall , Culture Media , Cuminum/growth & development , Enzyme Activation , Oils, Volatile/analysis , Efficacy , Methods , Microscopy, Electron , Plant Preparations , MethodsABSTRACT
Disseminated candidiasis is a serious problem in public health that results from the invasion of Candida species, in particular Candida albicans. The aim of this study was to compare the efficacies of Zataria multiflora essential oil and itraconazole in clearing C. albicans from the visceral organs of BALB/c mice suffered from disseminated candidiasis. Zataria multiflora essential oil was extracted using Clevenger-type apparatus and analyzed by gas chromatography mass spectrometry (GC-MS). For clearance experiment, mice (20-25 g, N=8 per group) received essential oil at doses of 30, 48 and 64 mg/kg and itraconazole at dose of 200 mg/kg intraperitoneally (IP) 2 days before and after intravenous inoculation of 0.5×10(6) C. albicans blastospores. The treated animals were sacrificed on day 20, and 0.1 g of the tissue homogenates was plated onto specific media. In GC-Mass, the main components of the essential oil were carvacrol (61.29 percent) and thymol (25.18 percent). The results demonstrated that IP administration of 64 mg/kg of the essential oil had the highest efficacy in reducing C. albicans and produced 39.5, 21.8, 141.5, 174 and 501-fold reductions in mean CFUs per 0.1 gram in Candida infections of the liver, spleen, lungs, brain and kidneys, respectively, compared to positive control. Itraconazole showed significantly more responsiveness than the essential oil at dose of 30 mg/kg in clearing C. albicans from the kidneys (P<0.02), brain (P<0.02) and spleen (P<0.04), and less responsiveness than that of 64 mg/kg in clearing the organism from the brain (P<0.01), lungs (P<0.0005) and kidneys (P<0.0005), whereas no significant difference was observed between this drug and Z. multiflora at dose of 48 mg/kg. These data explain the increased rate of yeast clearance and reduced dissemination to the viscera of Z. multiflora treated mice.
A candidíase disseminada é um problema sério de saúde publica decorrente da invasão por espécies de Candida, e Candida albicans em particular. O objetivo deste estudo foi comparar a eficiência do óleo essencial de Zataria multiflora e itraconazol na remoção de C. albicans das vísceras de camundongos BALB/c com candidíase disseminada. O óleo essencial de Zataria multiflora foi extraído empregando um aparelho do tipo Clevenger e analisado por cromatografia a gás e espectrometria de massa (GC-MS). Para os experimentos de remoção, camundongos (20-25g, n=8 por grupo) receberam óleo essencial nas doses de 30, 48 e 64 mg/kg e itraconazol na dose de 200 mg/kg via intraperitoneal (IP) por dois dias antes e após a inoculação intravenosa de 0,5 x 10(6) blastósporos de C. albicans. Os animais tratados foram sacrificados no vigésimo dia e 0,1g dos tecidos homogeneizados foram semeados em meios específicos. De acordo com o GC-MS, os principais componentes do óleo essencial foram carvacrol (61,29 por cento) e timol (25,28 por cento). Os resultados mostraram que a administração IP de 64 mg/kg de óleo essencial apresentou a eficiência mais alta na redução de C. albicans e resultou na redução de 39,5, 21,8, 141,5, 174 e 501 vezes na contagem média de C. albicans por 0,1g do fígado, baço, pulmões, cérebro e rins, respectivamente, quando comparado ao controle positivo. O itraconazol apresentou redução de C. albicans maior do que o óleo essencial na dose de 30mg/kg nos rins (P<0,02), cérebro (P<0,02) e baço (P<0,04) e menor no cérebro (P<0,01), pulmões (P<0,0005) e rins (P<0,0005) na dose de 64 mg/kg, enquanto não houve diferença entre esse droga e óleo essencial na dose de 48mg/kg. Estes resultados explicam a remoção aumentada de leveduras e a disseminação reduzida para as vísceras de camundongos tratados com Z. multiflora.