Inclusion Complexes of Litsea cubeba (Lour.) Pers Essential Oil into ß-Cyclodextrin: Preparation, Physicochemical Characterization, Cytotoxicity and Antifungal Activity.

The uses of natural compounds, such as essential oils (EOs), are limited due to their instability to light, oxygen and temperature, factors that affect their application. Therefore, improving stability becomes necessary. The objective of this study was to prepare inclusion complexes of Litsea cubeba essential oil (LCEO) with ß-cyclodextrin (ß-CD) using physical mixing (PM), kneading (KN) and co-precipitation (CP) methods and to evaluate the efficiency of the complexes and their physicochemical properties using ATR-FTIR, FT-Raman, DSC and TG. The study also assessed cytotoxicity against human colorectal and cervical cancer cells and antifungal activity against Aspergillus flavus and Fusarium verticillioides. The complexation efficiency results presented significant evidence of LCEO:ß-CD inclusion complex formation, with KN (83%) and CP (73%) being the best methods used in this study. All tested LCEO:ß-CD inclusion complexes exhibited toxicity to HT-29 cells. Although the cytotoxic effect was less pronounced in HeLa tumor cells, LCEO-KN was more active against Hela than non-tumor cells. LCEO-KN and LCEO-CP inclusion complexes were efficient against both toxigenic fungi, A. flavus and F. verticillioides. Therefore, the molecular inclusion of LCEO into ß-CD was successful, as well as the preliminary biological results, evidencing that the ß-CD inclusion process may be a viable alternative to facilitate and increase future applications of this EO as therapeutic medication, food additive and natural antifungal agent.

Risk evaluation of occupational exposure of southern Brazilian flower farmers to pesticides potentially leading to cholinesterase inhibition and metals exposure.

This work presents a frequency matched observational study comparing flower farmers exposed to pesticides and unexposed individuals as controls. All subjects were interviewed before plasma and urine collection. Manganese and Zinc were measured in theses samples by using dynamic reaction cell inductively coupled mass spectrometry. Cholinesterase activity was analyzed through spectrophotometry by using a modified version of the Ellman method. Seventy-eight percent of subjects reported occupational contact with pesticides, from which 37% reported exposure for over 9 years. Flower farms farmers had increased odds of having headache and irritability, respectively, by factors of 6.2 and 2.4 than the control subjects. While the odds of exposed subjects to have insomnia was smaller than control subjects by a factor of 0.34. Exposure to pesticides had a significant effect regarding the plasmatic plasma and urinary manganese levels and whole blood cholinesterase activity (p < 0.05). High levels of plasma and urinary manganese, as well as cholinesterase inhibition in whole blood, were evident in the flower farmers who participated in the study.

Anti-mycotoxigenic and antifungal activity of ginger, turmeric, thyme and rosemary essential oils in deoxynivalenol (DON) and zearalenone (ZEA) producing Fusarium graminearum.

This study aimed to evaluate the antimycotoxigenic effect of essential oils (EOs) obtained from four different aromatic plants on the production of deoxynivalenol (DON) and zearalenone (ZEA) by Fusarium graminearum. The EOs from ginger (GEO), turmeric (TEO), thyme (ThEO) and rosemary (REO) were obtained by hydrodistillation and identified by gas chromatography/mass spectrometry (GC/MS). The major compounds found were mostly monoterpenes and sesquiterpenes. The minimum inhibitory concentration (MIC) and minimum fungicide concentration (MFC) were 11.25, 364, 366 and 11,580 µg mL-1 for ThEO, GEO, REO and TEO, respectively. The results evidenced that the assessed EOs inhibited DON and partially ZEA production by F. graminearum. ThEO and GEO were the EOs with most potent antimycotoxigenic action for DON and ZEA, respectively. These EOs have shown promising results in vitro regarding inhibition of mycotoxin production and might be used in the future as substitutes for synthetic fungicides.

Metals in Brazilian family farming grapes and estimated daily intake.

The aim of this study was to determine concentrations of metals in peel, pulp, and seeds of grapes obtained from family farms in Brazil, compare them to the maximum threshold levels and to evaluate the risk by estimating the daily intake (EDI). Grape samples were collected from farms and levels of Cd, Cr, Cu, Mn, Ni, Pb and Zn were assessed via ICP-MS. The highest metal levels were found in grape peels, Cu at the highest concentration (107.6 mg kg-1). Cr, Cu, and Pb were found at concentrations which exceeded maximum threshold levels. The EDI of Cd, Cu and Pb through consumption of grapes for the assessed Brazilian population was 0.29, 1822 and 3.02 µg/kg bw/day, respectively. The EDI of Cu was above the Provisionary Tolerable Daily Intake (PTDI). Thus, there are possible health risks due to the occurrence of Cu in Brazilian grapes.

Litsea cubeba essential oil: chemical profile, antioxidant activity, cytotoxicity, effect against Fusarium verticillioides and fumonisins production.

The purpose of this study was to determine the chemical profile of Litsea cubeba essential oil, carry out an in vitro evaluation of its antioxidant potential and its cytotoxicity, as well as its antifungal and antimicotoxigenic activities against Fusarium verticillioides. Most of the compounds observed in the EO were neral (32.75%) and geranial (37.67%). The radical scavenging capacity of 2,2-diphenyl-1-picrylhydrazyl and 2,2'-azino-bis-3-ethylbenzothiazoline-6-sulfonic acid was 104.4 and 56.4 mmol Trolox mg-1, respectively, indicating good antioxidant activity. The EO studied by us revealed cytotoxic effect against HT-29 and HeLa cancer cells. The Minimum Inhibitory and Minimum Fungicidal Concentrations against F. verticillioides were both 125 µg mL-1. Morphological investigation, performed by fluorescence microscopy and scanning electron microscopy, showed that hyphae and microconidia structures underwent changes after treatment with the EO. Analyses performed with the EO strongly reduced the mycelial development of F. verticillioides and the synthesis of fumonisins B1 and B2 in dose-dependence effect compared (P < 0.01) with the fungal control (105 conidia mL-1) and positive control (fludioxonil + metalaxyl-M). Thus, the results obtained in vitro suggest that L. cubeba EO has excellent antioxidant, fungicidal, and antimycotoxigenic effects.

Efeitos antifúngico e antiaflatoxigênico de timol e carvacrol em Aspergillus flavus / Antifungal and antiaflatoxigenic activities of thymol and carvacrol against Aspergillus flavus

O objetivo deste estudo foi avaliar os efeitos do timol e carvacrol contra Aspergillus flavus e a produção de aflatoxinas. Foram determinadas a concentração inibitória mínima (CIM), o espectro fungitóxico e a atividade inibidora de micotoxinas pelo timol e carvacrol. Os resultados mostraram que timol e carvacrol exibiram ação fungicida de acordo com a CIM de 2500 e 30 µg mL-1, respectivamente. Tanto o timol quanto o carvacrol inibiram significativamente o crescimento de A. flavus (p<0,05) a partir de 600 e 15 µg mL-1, respectivamente. A biomassa fúngica, estimada pela determinação da concentração de ergosterol, foi significativamente reduzida (p <0,05) em 2500 µg mL-1 de timol e 250 µg mL-1 de carvacrol. O timol e o carvacrol exibiram efeitos antiaflatoxigênicos em 600 e 125 µg mL-1, respectivamente. O timol e o carvacrol exibiram atividade antifúngica, mas não exibiram alta atividade antiaflatoxigênica. Carvacrol e timol podem ser considerados como potentes compostos naturais antifúngicos contra A. flavus.

The aim of this study was to evaluate the effects of thymol and carvacrol upon production of aflatoxins by Aspergillus flavus and upon its growth. Minimal inhibitory concentration (MIC), fungitoxic spectrum and mycotoxin inhibition were assessed. Results showed thymol and carvacrol exhibited fungicidal action, as determined by MIC values of 2500 and 30 µg mL-1, respectively. Both thymol and carvacrol significantly inhibited growth of A. flavus (p<0.05) at concentrations of 600 and 15 µg mL-1, respectively. Fungal biomass, as estimated by determination of ergosterol concentration, was significantly reduced (p<0.05) at thymol concentrations of 2500 µg mL-1 and at carvacrol concentrations of 250 µg mL-1. Thymol and carvacrol exhibited antiaflatoxigenic effects at concentrations of 600 and 125 µg mL-1, respectively. While both thymol and carvacrol showed possessing antifungal activities, neither were highly antiaflatoxigenic. Carvacrol and thymol might be considered for use as potential antifungal natural compounds against A. flavus.

Occurrence, exposure evaluation and risk assessment in child population for aflatoxin M1 in dairy products in Brazil.

This study aimed to evaluate the risk concerning child population's health because of the occurrence of AFM1 in UHT milk, powdered milk (PM) and infant formulae (IF). Determination of AFM1 was performed in 60 samples and evaluation of the mycotoxin exposure was carried out through the determination of the estimated daily intake (EDI), whereas risk characterization was evaluated with the calculation of the risk of Hepatocellular Carcinoma (HCC) and the Margin of Exposure (MOE). AFM1 ranged from 150 to 1020 ng/kg, and all the positive samples exceeded the limits stablished by European Community. The EDI for AFM1 ranged according to the age group of the population studied (0-5 years old) from 0.828 to 2.523, 0-2.113 and 0.029-0.833 ng/kg b. w./day in UHT, PM and IF, respectively. The number of HCC cases associated with AFM1 exposure (0.0015 a 0.0045) was higher than the limit of 0.001 case/100,000. MOE values for AFM1 were 728 to 239, considerably below the security margin of 10,000. These results point to a potential risk to the health of Brazilian child population exposed to AFM1 in dairy products.

Antifungal and antiaflatoxigenic activity of rosemary essential oil (Rosmarinus officinalis L.) against Aspergillus flavus.

The increased risk to health by diverse pathologies, such as cancer, liver diseases, and endocrine alterations, caused by chemical residues in food, has led to the search for sustainable agricultural management alternatives, such as the use of essential oils for the development of natural and eco-friendly fungicides. The aim of this study was to evaluate the antifungal and antiaflatoxigenic activity of Rosmarinus officinalis L. essential oil (REO) against Aspergillus flavus Link. REO was obtained by hydrodistillation and its major components were identified as 1,8-cineole (eucalyptol, 52.2%), camphor (15.2%) and α-pinene (12.4%) by GC/MS and NMR. The minimum inhibitory concentration (MIC) and minimum fungicidal concentration (MFC) were both 500 µg/mL. REO reduced the mycelial growth of A. flavus at a concentration of 250 µg/mL (15.3%). The results obtained from scanning electron microscopy (SEM) demonstrated a reduction in the size of conidiophores and in the thickness of hyphae in A. flavus caused by treatment with REO (250 µg/mL). The production of ergosterol and the biomass of mycelium were both reduced as the REO treatment concentration increased. The production of aflatoxins B1 and B2 was inhibited after treatment with 250 µg/mL REO, a concentration below the MIC/MFC, indicating that the antiaflatoxigenic effect of REO is independent of its antifungal effect and is likely due to its direct action upon toxin biosynthesis. The data demonstrated that REO may be used as an alternative to synthetic fungicides.

Antifungal activity and inhibition of aflatoxins production by Zingiber officinale Roscoe essential oil against Aspergillus flavus in stored maize grains / Atividade antifúngica e inibição da produção de aflatoxinas pelo óleo essencial de Zingiber officinale Roscoe contra Aspergillus flavus em grãos de milho armazenados

ABSTRACT: Essential oils are possible alternatives to the use of synthetic pesticides for control of fungal contamination. Ginger (Zingiber officinale) essential oil (GEO) is known for having antifungal and antiaflatoxigenic properties, but its use as a fumigant in situ has not been studied yet. The aim of this study was to evaluate GEO's effects upon Aspergillus flavus as a fumigant agent in stored maize grains. The main compounds reported in GEO were α-zingiberene (23.85%) and geranial (14.16%), characterized by gas chromatography-mass spectrometry and nuclear magnetic resonance. The GEO was used as a fumigant in irradiated maize grains in concentrations ranging from 5 to 50 µg/g and the resulting effects were compared to a synthetic antifungal agent (carbendazim and thiram), an antifungal traditionally used for seed treatment. The antifungal efficacy of GEO against A. flavus has been proven in a dose-dependent manner through in situ (maize grains) test. The GEO inhibited aflatoxin production at concentrations 25 and 50 µg/g and controlled fungal growth. Therefore, GEO can be used as an effective and non-toxic alternative to conventional treatments in stored maize grains for the natural control of A. flavus.

RESUMO: Os óleos essenciais são possíveis alternativas ao uso de praguicidas sintéticos no controle da contaminação por fungos. O óleo essencial de gengibre (Zingiber officinale - OEG) é conhecido por possuir propriedades antifúngicas e antiaflatoxigênicas, mas seu uso como fumigante in situ ainda não foi estudado. O objetivo deste estudo foi avaliar os efeitos do OEG sobre Aspergillus flavus como agente fumigante em grãos de milho armazenados. Os principais compostos encontrados no OEG foram α-zingibereno (23,85%) e geranial (14,16%), caracterizados por cromatografia gasosa acoplada a espectrometria de massas e ressonância magnética nuclear. O OEG foi utilizado como fumigante em grãos de milho irradiados em concentrações variando de 5 a 50 µg/g e os efeitos resultantes foram comparados a um agente antifúngico sintético (carbendazim e tiram), antifúngico tradicionalmente usado para o tratamento de sementes. A eficácia antifúngica do OEG contra A. flavus foi comprovada de maneira dependente da dose através do teste in situ (grãos de milho). O OEG inibiu a produção de aflatoxina nas concentrações 25 e 50 µg/g e controlou o crescimento de fungos. Portanto, o OEG pode ser usado como uma alternativa eficaz e não tóxica aos tratamentos convencionais em grãos de milho armazenados para o controle natural de A. flavus.

Occurrence and risk assessment of population exposed to deoxynivalenol in foods derived from wheat flour in Brazil.

Deoxynivalenol (DON) is the most important of the trichothecenes in terms of amounts and occurrence in wheat. This compound was shown to be associated with a glomerulonephropathy involving an increase of immunoglobulin A in humans. This study assessed the occurrence of DON in wheat flour and the exposure of Brazilian teenagers, adults and elderly to this mycotoxin due to intake of wheat flour-based products. DON extraction in wheat flour was carried out by solid phase extraction and the quantification was performed by ultra-high proficiency liquid chromatography with diode-array detection. A total of 77.9% of all samples were positive for DON, with concentrations ranging from 73.50 to 2794.63 µg kg-1. The intake was calculated for the average and 90th percentile of the contamination levels of DON in foods based-wheat for teenagers, adults and elderly in Brazil, and compared with the provisional maximum tolerable daily intakes (PMTDI). Females of all age groups were exposed to DON at higher levels when compared to males in regard of consumption of breads and pastas. Teenagers were the main consumers of foods derived from wheat flour, with maximum probable daily intakes of 1.28 and 1.20 µg kg-1 b.w. day-1 for females and males, respectively. This population is at an increased risk of exposure to DON due to consumption of wheat flour-based foods in Brazil.