A Lectin from Spatholobus parviflorus Inhibits Aspergillus flavus α‐Amylase: Enzyme Kinetics and Thermodynamic Studies

Aspergillus flavus is a commonly found fungal pathogen which produces structurally related and highly toxic secondary metabolites, aflatoxins. It has been proposed that α‐amylase inhibitors may limit the ability of the fungus to produce aflatoxins. Hence, this enzyme is a potent target for the development of antifungal agents. In this study, it was found that Spatholobus parviflorus seed lectin (SPL) can inhibit the growth of A. flavus with a MIC value of 1.5 mg/mL. The enzyme kinetics, molecular modeling and isothermal titration calorimetric studies suggest that SPL can inhibit α‐amylase with K_i value of 0.0042 mm . Hence, it is suggested that the antifungal activity of SPL might be partly due to its ability to inhibit the enzyme α‐amylase.     

Probing the Active Site of Candida glabrata Dihydrofolate Reductase with High Resolution Crystal Structures and the Synthesis of New Inhibitors

Candida glabrata, a fungal strain resistant to many commonly administered antifungal agents, has become an emerging threat to human health. In previous work, we validated that the essential enzyme, dihydrofolate reductase, is a drug target in C. glabrata. Using a crystal structure of dihydrofolate reductase from C. glabrata bound to an initial lead compound, we designed a class of biphenyl antifolates that potently and selectively inhibit both the enzyme and the growth of the fungal culture. In this work, we explore the structure–activity relationships of this class of antifolates with four new high resolution crystal structures of enzyme:inhibitor complexes and the synthesis of four new inhibitors. The designed inhibitors are intended to probe key hydrophobic pockets visible in the crystal structure. The crystal structures and an evaluation of the new compounds reveal that methyl groups at the meta and para positions of the distal phenyl ring achieve the greatest number of interactions with the pathogenic enzyme and the greatest degree of selectivity over the human enzyme. Additionally, antifungal activity can be tuned with substitution patterns at the propargyl and para‐phenyl positions.     

New N-phenylacetamide-linked 1,2,3-triazole-tethered coumarin conjugates: Synthesis,bioevaluation, and molecular docking study

A series of new 1,2,3-triazole-tethered coumarin conjugates linked by N-phenylacetamide was efficiently synthesized via the click chemistry approach in excellent yields. The synthesized conjugates were evaluated for their in vitro antifungal and antioxidant activities. Antifungal activity determination was carried out against fungal strains such as Candida albicans, Fusarium oxysporum, Aspergillus flavus, Aspergillus niger and Cryptococcus neoformans. Compounds 7b , 7d , 7e , 8b and 8e displayed higher potency than the standard drug miconazole, with lower minimum inhibitory concentration values. Also, compound 7a exhibited potential radical scavenging activity as compared with the standard antioxidant butylated hydroxytoluene. In addition, a molecular docking study of the newly synthesized compounds was carried out, and the results showed a good binding mode at the active site of the fungal (C. albicans) P450 cytochrome lanosterol 14α-demethylase enzyme. Furthermore, the synthesized compounds were also tested for ADME properties, and they demonstrated potential as good candidates for oral drugs.     

Chemoprevention by essential oil of turmeric leaves (Curcuma longa L.) on the growth of Aspergillus flavus and aflatoxin production

Turmeric is well known for a wide range of medicinal properties. Essential oil of turmeric leaves (Curcuma longa L.) were evaluated at varying concentrations of 0.01, 0.05, 0.1, 0.5, 0.75, 1.0 and 1.5% (v/v) in Yeast Extract Sucrose (YES) broth inoculated with spore suspension of Aspergillus flavus of 10⁶ conidia/ml. These were evaluated for their potential in the control of aflatoxigenic fungus A. flavus and aflatoxin production. Turmeric leaf oil exhibited 95.3% and 100% inhibition of toxin production respectively at 1.0% and 1.5%. The extent of inhibition of fungal growth and aflatoxin production was dependent on the concentration of essential oil used. The oil exhibited significant inhibition of fungal growth as well as aflatoxins B₁ and G₁ production. The LD₅₀ and LD₉₀ were also determined. GC-MS analysis of the oil showed α-phellandrene, p-cymene and terpinolene as the major components in turmeric leaf oil. The possibility of using these phytochemical components as bio-preservatives for storage of spices is discussed.     

Reduction of aflatoxin in fermented milks during production and storage

Aflatoxins are a group of secondary fungal metabolites produced by toxigenic strains of Aspergillus flavus, A. parasiticus and A. nomius. They are one of the most common food contamination problems. Aflatoxins M₁ and M₂ are the hydroxylated metabolites of aflatoxins B₁ and B₂ and emerge in raw milk when lactating animals ingest feed contaminated with these mycotoxins. They may contaminate other dairy products including fermented milks. Aflatoxins are fairly stable in many foods and are rather resistant to degradation. Its stability can be affected by different factors. This article mainly reviews the impact of process, formulation and storage factors on the amount of aflatoxins in fermented milks while consider related mechanisms.     

In Vitro Biological Control of Aspergillus flavus by Hanseniaspora opuntiae L479 and Hanseniaspora uvarum L793, Producers of Antifungal Volatile Organic Compounds

Aspergillus flavus is a toxigenic fungal colonizer of fruits and cereals and may produce one of the most important mycotoxins from a food safety perspective, aflatoxins. Therefore, its growth and mycotoxin production should be effectively avoided to protect consumers’ health. Among the safe and green antifungal strategies that can be applied in the field, biocontrol is a recent and emerging strategy that needs to be explored. Yeasts are normally good biocontrol candidates to minimize mold-related hazards and their modes of action are numerous, one of them being the production of volatile organic compounds (VOCs). To this end, the influence of VOCs produced by Hanseniaspora opuntiae L479 and Hanseniaspora uvarum L793 on growth, expression of the regulatory gene of the aflatoxin pathway (aflR) and mycotoxin production by A. flavus for 21 days was assessed. The results showed that both yeasts, despite producing different kinds of VOCs, had a similar effect on inhibiting growth, mycotoxin biosynthetic gene expression and phenotypic toxin production overall at the mid-incubation period when their synthesis was the greatest. Based on the results, both yeast strains, H. opuntiae L479 and H. uvarum L793, are potentially suitable as a biopreservative agents for inhibiting the growth of A. flavus and reducing aflatoxin accumulation.     

Loss of msnA, a Putative Stress Regulatory Gene, in Aspergillus parasiticus and Aspergillus flavus Increased Production of Conidia, Aflatoxins and Kojic Acid

Production of the harmful carcinogenic aflatoxins by Aspergillus parasiticus and Aspergillus flavus has been postulated to be a mechanism to relieve oxidative stress. The msnA gene of A. parasiticus and A. flavus is the ortholog of Saccharomyces cerevisiae MSN2 that is associated with multi-stress response. Compared to wild type strains, the msnA deletion (∆msnA) strains of A. parasiticus and A. flavus exhibited retarded colony growth with increased conidiation. The ∆msnA strains also produced slightly higher amounts of aflatoxins and elevated amounts of kojic acid on mixed cereal medium. Microarray assays showed that expression of genes encoding oxidative stress defense enzymes, i.e., superoxide dismutase, catalase, and cytochrome c peroxidase in A. parasiticus ∆msnA, and the catalase A gene in A. flavus ∆msnA, was up-regulated. Both A. parasiticus and A. flavus ∆msnA strains produced higher levels of reactive oxygen species (ROS), and ROS production of A. flavus msnA addback strains was decreased to levels comparable to that of the wild type A. flavus. The msnA gene appears to be required for the maintenance of the normal oxidative state. The impairment of msnA resulted in the aforementioned changes, which might be used to combat the increased oxidative stress in the cells.     

Extracts from Pelargonium sidoides. Inhibit the Growth of Bacteria and Fungi

Abstract

Pelargonium sidoides. DC (Geraniaceae) is a medicinal plant used by the people of the Eastern Cape, South Africa, for the treatment of various diseases in man and livestock. Acetone and methanol extracts of the shoot and root of the herb were investigated for antimicrobial activity against 10 bacterial and 5 fungal species by the dilution method on solid agar medium. With the exception of Staphylococcus epidermidis., extracts obtained from both solvents demonstrated significant activity against all the Gram-positive and two of the Gram-negative bacteria tested in this study. The extracts also showed appreciable inhibitory activity against all the fungal species tested with inhibition ranging from 52.5% on Aspergillus flavus. to 82.5% on Mucor hiemalis. at 5 mg ml^?1, the highest concentration tested in the study. The ability of the extracts of this plant to inhibit the growth of several bacteria and fungi is an indication of the broad-spectrum antimicrobial potential of P. sidoides. that further validates the use of this plant for the treatment of various ailments by the people of the Eastern Cape.     

Design,synthesis, and biological evaluation of pyrimidine derivatives as potential inhibitors of human calcium/calmodulin‐dependent protein kinase IV

Calcium/calmodulin‐dependent protein kinase IV (CAMKIV ) is a multifunctional Ser/Thr kinase, associated with cerebral hypoxia, cancer, and neurodegenerative diseases. Here, we report design, synthesis, and biological evaluation of seven pyrimidine‐substituted novel inhibitors of CAMKIV . We successfully synthesized and extensively characterized (ESI ‐MS , ¹H NMR , and ¹³C NMR studies) seven compounds that are showing appreciable binding affinity to the CAMKIV . Molecular docking and fluorescence binding studies revealed that compound 1 is showing very high binding free energy (ΔG  = ?11.52 kcal/mol) and binding affinity (K  =  9.2 × 10¹⁰ m ^?1) to the CAMKIV . We further performed MTT assay to check the cytotoxicity and anticancer activity of these compounds. An appreciable IC ₅₀ (39 μm ) value of compound 1 was observed on human hepatoma cell line and nontoxic till the 400 μm on human embryonic kidney cells. To ensure anticancer activity of all these compounds, we further performed propidium iodide assay to evaluate cell viability and DNA content during the cell cycle. We found that compound 1 is again showing a better anticancer activity on both human hepatoma and human embryonic kidney cell lines.     

Aflatoxins and Their Detection in Animal Tissues and Fluids

Abstract

The aflatoxins are a group of toxic fungal metabolites that may be produced by the organisms Aspergillus flavus or A. parasiticus growing in animal feeds. When consumed, these metabolites or their metabolic products in the animal may occur as tissue or fluid residues in the animal. This report briefly reviews the effects of aflatoxins on animals, the metabolism of aflatoxins in animals and at least one method is described that is useful for the detection and quantitation of aflatoxins or their metabolites in tissues, milk, rumen contents, blood, bile, urine, and feces. Some problems encountered by the authors in conducting these analyses are also discussed.     

In Vitro Effects of the Neolignan 2,3‐Dihydrobenzofuran Against Leishmania Amazonensis

Leishmaniasis is an infectious disease complex caused by protozoa from the Leishmania genus, which presents a broad spectrum of clinical manifestations: cutaneous, mucocutaneous and visceral forms. The current treatments are unsatisfactory considering that few drugs are available and present some level of toxicity. Many lignans and neolignans have been used for the development of new antileishmania drugs. The capability in vitro of the neolignan 2,3‐dihydrobenzofuran (2,3‐DBF), a commonly found constituent of propolis and other plants, to inhibit the growth of promastigote and macrophage‐internalized amastigote forms of Leishmania amazonensis was investigated. The cytotoxicity of this compound was assessed by MTT (3‐[4,5‐dimethylthiazol‐2‐yl]‐2,5‐diphenyltetrazolium bromide) test in BALB/c murine macrophages and human erythrocyte lysis assay. The 2,3‐DBF was active against promastigote (IC₅₀=1.042 μM) and amastigote (IC₅₀=1.43 μM) forms, indicating a potent antileishmanial effect. There was no evidence of cytotoxicity to macrophages or erythrocytes at concentrations ranging from 13 to 0.5 μM, after 48 hr of exposure. The antileishmanial activity is probably mediated by the activation of macrophages, because treatment with 2,3‐DBF increases both phagocytic and lysosomal activities, as well as the nitrite (NO₂^?) levels. These results suggest that 2,3‐DBF may be a potential candidate for the development of a new promising antileishmanial drug. Further studies are needed to determine its potential in vivo effect as well as additional mechanisms underlying the antileishmanial and immunomodulatory activities.     

Influence of Temperature and Humidity on Biodeterioration and Aflatoxin Production in Groundnut Fodder by Aspergillus Flavus

Abstract

The Influence of temperature and relative humidity (RH) on biodeterioration of groundnut fodder and aflatoxin elaboration by Aspergillus flavus was investigated. Though aflatoxin production was observed at all incubation temperature tried, its production was maximum at 25°C. The amount of aflatoxin production decreased significantly with increase in incubation temperature. When a marginal increase in total proteins and ash content was recorded, cellulose, lignin, starch and total nitrogen content decreased significantly due to A. flavus infestation. This was maximum at 25°C and decreased both with an increase or decrease of incubation temperature. Groundnut fodder supported significant weight loss due to A. flavus infestation and it was maximum at 25°C. RH of 90 and above was more conducive for aflatoxin elaboration. A. flavus could elaborate aflatoxins even at RH 30 which, however, was only in trace amounts.     

Bio-detoxification of aflatoxin B1 in artificially contaminated maize grains using lactic acid bacteria

Aflatoxins are a group of carcinogenic mycotoxins produced by Aspergillus flavus, A. parasiticus, and A. nomius. Due to the ubiquitous occurrence of aflatoxins, preventive and remediative measures are necessary including detoxification techniques. Physical and chemical decontamination strategies are inconvenient. In this study a biological detoxification strategy was tested using bacteria of the Lactobacillus species collected from the biotechnology laboratory at University of Ibadan, Nigeria. Maize grains with moisture content of 17% were artificially inoculated with toxigenic A. flavus (LA 32G_28) and atoxigenic A. flavus (LA32_79) at ambient temperature and four samples of bulk maize grains were prepared at aflatoxin B1 concentrations of 50, 100, 200, and 500?ng/g. To evaluate the detoxifying potential of lactic acid bacteria five different cultures consisting of Lactobacillus acidophilus, L. brevis, L. casei, L. delbruekii, and L. plantarum were used to inoculate the aflatoxin B1–contaminated maize samples at 37°C. After 5 days, the residual aflatoxin B1 on maize was determined. All treatments showed significant reductions (P<?0.05) in aflatoxin B1. Lactic acid bacteria decreased the pH of the medium from 5.0 to 4.0. Pronounced aflatoxin B1 reduction was observed in maize contaminated at 50?ng/g (44.5%), while maize contaminated at 500ng/g was the least reduced (29.9%). L. plantarum was the most efficient organism in degrading aflatoxin B1. Use of lactic acid bacteria, which already has Generally Regarded As Safe (GRAS) status, should be encouraged for use as a bio-detoxification agent for aflatoxins.     

Selection of Aspergillus flavus isolates for biological control of aflatoxins in corn

The fungus Aspergillus flavus is responsible for producing carcinogenic mycotoxins, the aflatoxins, on corn (maize) and other crops. An additional harmful toxin, cyclopiazonic acid, is produced by some isolates of A. flavus. Several A. flavus strains that do not produce one or both of these mycotoxins are being used in biological control to competitively exclude the toxin-producing strains from the agroecosystem, particularly from seeds, grain and other marketable commodities. Three well-studied non-aflatoxigenic strains, including two that are commercially available, have been compared in side-by-side field trials. The results of that study, together with a growing understanding of A. flavus ecology and new genetic insights, are guiding the selection of biocontrol strains and influencing crop management decisions for safe and sustainable production.     

Aflatoxins in Maize: A Mexican Perspective

Aflatoxins are carcinogenic metabolites produced by Aspergillus flavus, a fungal pathogen that infects maize both in the field and during storage. Mexico is the center of origin of maize and its production in most parts in the country is characterized by the employment of a wide diversity of open‐pollinated genotypes adapted to certain environments. In most regions, maize is produced under rain fed conditions with low fertilizer and pesticide input and consequent low yields, probably fostering A. flavus infection in drought‐stressed plants. In addition, poor pest control increases insect damage, facilitating fungal infection and aflatoxin contamination. Ideally, management of aflatoxin contamination should begin with the employment of resistant genotypes as has been demonstrated by several U.S. breeding programs. However, in Mexico the wide genetic diversity of maize has not been fully exploited to identify resistance to aflatoxin contamination in breeding programs, thus impeding the reduction of aflatoxin levels in the field. Additional complications come from the fact that transgenic maize expressing insecticidal protein or any other trait to reduce aflatoxin is not viable in Mexico due to a government prohibition on the use of genetically modified maize. Maize is a staple crop in Mexico with high consumption in forms such as tortillas; thus, aflatoxin contamination is a significant threat to human health. Although aflatoxins are partially destroyed during the alkaline cooking procedure (called nixtamalization) to prepare tortillas, residual levels of aflatoxins might be considerable. Although important research has been conducted in several aspects of aflatoxin contamination of maize by universities, agricultural centers, and some government agencies, a full mycotoxin research program is needed in Mexico to ascertain the extents of aflatoxin contamination in different parts of the country and to develop economically viable technology to reduce aflatoxin exposure.     

New perspectives for the application of bioplastic materials in the biocontrol of Aspergillus flavus in corn

Mycotoxins are secondary metabolites produced by certain filamentous fungi that can contaminate a large variety of agricultural commodities before and after harvest. Among different mycotoxins, aflatoxins and especially aflatoxin B1 are of particular concern because they are potent natural carcinogens. Aflatoxin-producing fungi, mainly Aspergillus flavus and A. parasiticus, are ubiquitous, being commonly isolated from agricultural soil and crop debris. Although many aspects of the ecology of aflatoxin-producing fungi have been elucidated, control of aflatoxin contamination of agricultural crops remains a difficult task. Agronomical practices promoting general plant health have shown variable and more frequently limited success in preharvest control of aflatoxin contamination. Competitive replacement of indigenous toxigenic soil isolates is considered a more promising and effective approach. This biocontrol strategy is based on field application of a large number of propagules of nontoxigenic strains of A. flavus. Biocontrol strains are typically formulated as inoculated or spore-coated grain seeds. More recently, efforts to explore new approaches and technologies have resulted in the development of other practical solutions, including a bioplastic-based formulation. This formulation originally developed in 2008, consists of bioplastic granules entrapping spores of the nontoxigenic biocontrol strain, A. flavus NRRL 30797. Laboratory and field studies that have been conducted until now have clearly shown that granules of the starch-based bioplastic Mater-Bi^® are effective in delivering this biocontrol strain. In addition to having a satisfactory shelf life, the granules are easy to prepare, handle, and apply to agricultural fields. More importantly, this novel bioplastic formulation is capable of efficiently reducing aflatoxin contamination of corn. The bioplastic Mater-Bi^® can also have other applications. For instance, rods or granules prepared using a slightly modified Mater-Bi^® bioplastic matrix can be used to selectively isolate A. flavus from soil and corn kernels.     

Novel Potent and Selective Acetylcholinesterase Inhibitors as Potential Drugs for the Treatment of Alzheimer's Disease: Synthesis,Pharmacological Evaluation,and Molecular Modeling of Amino‐Alkyl‐Substituted Fluoro‐Chalcones Derivatives

A new series of‐fluoro chalcones‐substituted amino‐alkyl derivatives ( 3a?3l ) were designed, synthesized, characterized and evaluated for the inhibitory activity against acetylcholinesterase and butyrylcholinesterase. The results showed that the alteration of fluorine atom position and amino‐alkyl groups markedly influenced the activity and the selectivity of chalcone derivates in inhibiting acetylcholinesterase and butyrylcholinesterase. Among them, compound 3l possesses the most potent inhibitory against acetylcholinesterase (IC₅₀ = 0.21 ± 0.03 μmol/L), and the highest selectivity for acetylcholinesterase over butyrylcholinesterase (IC₅₀ (BuChE)/IC₅₀ (AChE) = 65.0). Molecular modeling and enzyme kinetic study on compound 3l supported its dual acetylcholinesterase inhibitory profile, simultaneously binding at the catalytic active and peripheral anionic site of the enzyme.     

Potent antibacterial activity of dihydydropyrimidine‐1,3,5‐triazines via inhibition of DNA gyrase and antifungal activity with favourable metabolic profile

The compounds were tested against panel of three Gram‐positive, viz. Staphylococcus aureus, Bacillus subtilis, Bacillus cereus and three Gram‐negative bacterial strains viz. Pseudomonas aeruginosa, Escherichia coli, and Proteus vulgaris where they showed significant to moderate antibacterial activity. The compound also showed considerable antibiofilm activity against S. aureus and B. subtilis. The most potent compounds 7l and 7m found bacteriostatic in time‐kill assay via inhibition of DNA gyrase enzyme and interacting with Glu58, Val130, Ile175 and Ile186 via numerous H‐bonds as revealed by docking. In S. aureus‐induced murine infection model, compound 7m showed dose‐dependent reduction of viability of bacteria with maximum activity in 25 mg/kg treated group. The antifungal activity against human fungal pathogens was also estimated, where these compounds showed considerable inhibitory activity as compared to standard. The metabolic liability of compound 7m was determined using RS‐Predictor and MetaPrint 2D React. The molecules were proved as effective antibacterial agent via inhibition of DNA gyrase as a mechanism together with significant antifungal activity.     

Aflatoxin biomarkers in hair may facilitate long‐term exposure studies

Aflatoxins are highly toxic fungal metabolites produced by some members of the Aspergillus species. They are low molecular weight lipophilic compounds that are easily absorbed from the gastrointestinal tract. They contaminate most staple foods, including maize, peanuts, peanut butter and sorghum mainly in the tropics where hot and humid conditions promote fungal growth. Absorbed aflatoxins are metabolized by the cytochrome P450 enzyme system in the liver into toxic metabolites. Aflatoxin B (AFB)₁ is the most toxic, carcinogenic and mutagenic naturally occurring toxin. Aflatoxin exposure assessment has been traditionally achieved through food use frequency questionnaires and laboratory analysis of food samples. However, estimation of individual exposure to aflatoxins based on these methods may not be accurate. The use of aflatoxin biomarkers in urine and blood for use in exposure studies has emerged in more recent times. However, the current biomarkers (e.g., AFB‐N⁷‐guanine and AFB₁‐albumin adduct) in use have a short half‐life and are only practically useful to indicate levels over 24 h–3 months post‐exposure. There is therefore an immediate need to study and evaluate alternative biomarkers in non‐conventional matrices such as hair and nails. Hair analysis revealed considerable interest in forensic analysis particularly in the detection of drugs of abuse where it has emerged as a sensitive and specific technique complementary to blood and urinalysis. This article provides an overview of aflatoxins, current aflatoxin biomarkers and proposes the use of hair as a potential matrix for biomarkers of long‐term aflatoxin exposure. Copyright © 2016 John Wiley & Sons, Ltd.