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1.  Application of immobilized tannase from Aspergillus niger for the removal of tannin from myrobalan juice 
Indian Journal of Microbiology  2010;50(Suppl 1):46-51.
Tannase produced optimally on an agroresidue by an Aspergillus niger isolate under submerged fermentation immobilized on sodium alginate beads with 93.6% efficiency was applied for tannin removal from myrobalan/aonla (Phyllanthus emblica) juice. The pH and temperature optima of the immobilized enzyme were found to be 5.4 and 40°C while the corresponding values of the soluble enzyme were 5.8 and 35°C. Maximum tannin removal of 73.6% was obtained at 40°C and 150 rpm in 180 min with 36.6 U/ml of immobilized enzyme while the same amount of the soluble enzyme removed 45.2% of tannin at 37°C and 150 rpm in the same time period. The immobilized beads could be used repeatedly till 7th cycle with 77% efficiency. When preserved at 6°C the beads retained 71.7% of enzyme activity after 60 days. Reduction in vitamin C content, which is responsible for antioxidant property of the fruit, was minimum at only 2% during the treatment.
doi:10.1007/s12088-010-0029-6
PMCID: PMC3396397  PMID: 22815571
Aspergillus niger; Immobilized tannase; Myrobalan; Submerged fermentation; Sodium alginate
2.  Characterization And Application Of Tannase Produced By Aspergillus Niger ITCC 6514.07 On Pomegranate Rind 
Brazilian Journal of Microbiology  2009;40(4):782-789.
Extracellular tannase and gallic acid were produced optimally under submerged fermentation at 37 0C, 72 h, pH 5.0, 10 %(v/v) inoculum and 4 %(w/v) of the agroresidue pomegranate rind (PR) powder by an Aspergillus niger isolate. Tannic acid (1 %) stimulated the enzyme production by 245.9 % while with 0.5 % glucose, increase was marginal. Tannase production was inhibited by gallic acid and nitrogen sources such as NH4NO3, NH4Cl, KNO3, asparatic acid, urea and EDTA. The partially purified enzyme showed temperature and pH optima of 35 0C and 6.2 respectively which shifted to 40 0C and 5.8 on immobilization in alginate beads. Activity of the enzyme was inhibited by Zn+2, Ca+, Mn+2, Mg+2, Ba+2and Ag+. The immobilized enzyme removed 68.8 % tannin from juice of aonla/myrobalan (Phyllanthus emblica), a tropical fruit, rich in vitamin C and other essential nutrients. The enzymatic treatment of the juice with minimum reduction in vitamin C is encouraging as non enzymatic treatments of myrobalan juice results in vitamin C removal.
doi:10.1590/S1517-83822009000400008
PMCID: PMC3768578  PMID: 24031425
tannase; pomegranate rind; myrobalan; Aspergillus niger
3.  Implications of fungal infections and mycotoxins in camel diseases in Saudi Arabia 
Natural feed ingredients (corn, barley and wheat bran) and compound feed (manufactured pellet) are two types of fodder used for animal feeding, especially camel in Saudi Arabia. Twenty samples of each type of fodder were collected from seven different regions and screened for the presence of fungi, aflatoxins, ochratoxin and zearalenone. Fungal isolation of natural feed ingredients yielded 10 genera and 38 species of different fungi. Compound fodder samples were contaminated with 16 genera and 32 species of fungi. Total counts of Aspergillus, Penicillium and Fusarium in the animal feed samples were ranged from 54 to 223 × 103, 31.9 to 60 × 103 and 18 to 29 × 103 CFU/g, respectively. These isolates when tested for aflatoxin, ochratoxin and zearalenone producing ability, revealed this property in only four isolate, identified as Aspergillus flavus, A. parasiticus, A. ochraceus and Fusarium graminaerum. The percentage of toxigenic fungi was ranged from 5.5% to 30% for natural feed ingredients and from 4.5% to 20% for compound feed. The incidence of aflatoxins (AFT) in samples of natural feed ingredients was found to be ranged from 1 to 24.8 ppb, ochratoxin A (OTA) ranged from 1 to 44 ppb and zearalenone (ZON) ranged from 1 to 23 ppb. Contamination of compound feed with aflatoxin and ochratoxin A was ranged from 1 to 6.4 ppb and 1 to 4.7 ppb, respectively. All samples collected were found contaminated with fungi or their toxins and natural feed samples were more contaminated compared to compound feed samples. The concentrations detected were in the allowed limit (<20 ppb) except four samples of natural feed ingredients which were above the allowed limit of the tested mycotoxins. In conclusion, feed samples were contaminated with fungi and some toxigenic isolates which were responsible about mycotoxin production. Some samples had exceeded amount of AFT, OTA and ZON and may be contaminated with other mycotoxins which mean implication of fungi in camel health problems and death in Saudi Arabia.
doi:10.1016/j.sjbs.2009.12.011
PMCID: PMC3730929  PMID: 23961061
Mycotoxin; Aflatoxins; Ochratoxin A; Zearalenone; Aspergillus; Camel feed; Fodder; Fungal infection
4.  Direct visual detection of aflatoxin synthesis by minicolonies of Aspergillus species. 
Single-spore colonies of Aspergillus flavus and Aspergillus parasiticus, grown for 4 to 5 days at 25 degrees C on a coconut extract agar containing sodium desoxycholate as a growth inhibitor, produced aflatoxin, readily detectable as blue fluorescent zones under long-wave (365 nm) UV light. Over 100 colonies per standard petri dish were scored for aflatoxin production by this procedure. Progeny from some strains remained consistently stable for toxin production after repeated subculture, whereas instability for toxin synthesis was revealed among progeny from other strains. Spore color markers were used to rule out cross-contamination in monitoring strains. A yellow-spored and nontoxigenic strain of A. flavus, reported previously to produce aflatoxin in response to cycloheximide treatment, proved to be toxin negative even after repeated exposure to cycloheximide. Extended series of progeny from another strain of A. flavus and from a strain of A. parasiticus were each compared by this plating procedure and by fluorometric analysis for aflatoxin when grown in a coconut extract broth. Both of these strains showed variation for toxin synthesis among their respective progeny, and specific progeny showed a good correlation for aflatoxin synthesis when examined by the two procedures.
Images
PMCID: PMC202954  PMID: 2504116
5.  Effect of zero energy cool chamber and post-harvest treatments on shelf-life of fruits under semi-arid environment of Western India. Part 2. Indian gooseberry fruits 
Effect of zero energy cool chamber (ZECC) along with post-harvest treatments including CaCl2, mustard oil and K2SO4 separately on shelf-life and fruit quality attributes of Indian gooseberry or aonla (Emblica officinalis Gaertn) during storage under semi-arid ecosystem of Gujarat was studied. Increase in physiological loss in weight (PLW), spoilage loss, total soluble solids, total sugar and reducing sugars, reduction in titratable acidity and ascorbic acid were observed during storage period in all the treatments. Fruits treated with 1.5% CaCl2 and stored in ZECC recorded least PLW (16%), spoilage loss (16.5%), respiratory activity (83 mg CO2 /kg/h) and exhibited 11 days of shelf-life, while untreated control had 6 days economic life. It was closely followed by 1% CaCl2 + ZECC treatment. Fruits stored in ZECC recorded 9 days shelf-life. Highest respiration rate was in control (88.1 mg CO2 /kg/h) on 13th day of storage. It may be concluded that 1.5% CaCl2 and storage in ZECC treatment was found most efficient to retain the fruit quality attributes till 13th day of storage under semi-arid environment of western India.
doi:10.1007/s13197-010-0075-4
PMCID: PMC3551009  PMID: 23572669
Aonla; Emblica officinalis; Zero energy cool chamber; Calcium chloride; Shelf-life; Spoilage
6.  Mycotoxin-Producing Ability and Chemotype Diversity of Aspergillus Section Flavi from Soils of Peanut-Growing Regions in Iran 
Indian Journal of Microbiology  2012;52(4):551-556.
Invasion of crops with Aspergillus flavus may result in contamination of food and feed with carcinogenic mycotoxins such as aflatoxins (AF) and cyclopiazonic acid (CPA). In the present study, distribution and toxigenicity of Aspergillus flavus and A. parasiticus in soils of five peanut fields located in Guilan province, Northern Iran was investigated. From a total of 30 soil samples, 53 strains were isolated which all of them were finally identified as A. flavus by a combination of colony morphology, microscopic criteria and mycotoxin profiles. Chromatographic analysis of fungal cultures on yeast extract sucrose broth by tip culture method showed that 45 of the 53 A. flavus isolates (84.9 %) were able to produce either CPA or AFB1, while eight of the isolates (15.1 %) were non-toxigenic. The amounts of CPA and AFB1 produced by the isolates were reported in the range of 18.2–403.8 μg/g and 53.3–7446.3 μg/g fungal dry weights, respectively. Chemotype classification of A. flavus isolates based on the ability for producing mycotoxins and sclerotia showed that 43.4 % were producers of CPA, AFB1 and sclerotia (group I), 13.2 % of CPA and AFB1 (group II), 9.4 % of AFB1 and sclerotia (group III), 13.2 % of AFB1 (group IV), 5.7 % of CPA and sclerotia (group V) and 15.1 % were non-toxigenic with no sclerotia (group VI). No strain was found as producer of only CPA or sclerotia. These results indicate different populations of mycotoxigenic A. flavus strains enable to produce hazardous amounts of AFB1 and CPA are present in peanuts field soils which can be quite important regard to their potential to contaminate peanuts as a main crop consumed in human and animal nutrition.
doi:10.1007/s12088-012-0275-x
PMCID: PMC3516660  PMID: 24293709
Aspergillus flavus; Cyclopiazonic acid; Aflatoxin; Chemotype diversity; Soil; Peanuts
7.  Aflatoxins, hepatocellular carcinoma and public health 
Hepatocellular carcinoma (HCC) is one of the leading causes of cancer deaths worldwide, primarily affecting populations in the developing countries. Aflatoxin, a food contaminant produced by the fungi Aspergillus flavus and Aspergillus parasiticus, is a known human carcinogen that has been shown to be a causative agent in the pathogenesis of HCC. Aflatoxin can affect a wide range of food commodities including corns, oilseeds, spices, and tree nuts as well as milk, meat, and dried fruit. Many factors affect the growth of Aspergillus fungi and the level of aflatoxin contamination in food. Drought stress is one of the factors that increase susceptibility of plants to Aspergillus and thus aflatoxin contamination. A recent drought is thought to be responsible for finding of trace amounts of aflatoxin in some of the corn harvested in the United States. Although it’s too soon to know whether aflatoxin will be a significant problem, since United States is the world’s largest corn producer and exporter, this has raised alarm bells. Strict regulations and testing of finished foods and feeds in the United States should prevent a major health scare, and prevent human exposure to deleterious levels of aflatoxin. Unfortunately, such regulations and testing are not in place in many countries. The purpose of this editorial is to summarize the current knowledge on association of aflatoxin and HCC, encourage future research and draw attention to this global public health issue.
doi:10.3748/wjg.v19.i10.1508
PMCID: PMC3602466  PMID: 23539499
Aflatoxins; Hepatocellular carcinoma; Environmental health; Food safety; Public health
8.  Multilocus variable-number tandem-repeat analysis of clinical isolates of Aspergillus flavus from Iran reveals the first cases of Aspergillus minisclerotigenes associated with human infection 
BMC Infectious Diseases  2014;14:358.
Background
Aspergillus flavus is intensively studied for its role in infecting crop plants and contaminating produce with aflatoxin, but its role as a human pathogen is less well understood. In parts of the Middle East and India, A. flavus surpasses A. fumigatus as a cause of invasive aspergillosis and is a significant cause of cutaneous, sinus, nasal and nail infections.
Methods
A collection of 45 clinical and 10 environmental A. flavus isolates from Iran were analysed using Variable-Number Tandem-Repeat (VNTR) markers with MICROSAT and goeBURST to determine their genetic diversity and their relatedness to clinical and environmental A. flavus isolates from Australia. Phylogeny was assessed using partial β-tubulin and calmodulin gene sequencing, and mating type was determined by PCR. Antifungal susceptibility testing was performed on selected isolates using a reference microbroth dilution method.
Results
There was considerable diversity in the A. flavus collection, with no segregation on goeBURST networks according to source or geographic location. Three Iranian isolates, two from sinus infections and one from a paranasal infection grouped with Aspergillus minisclerotigenes, and all produced B and G aflatoxin. Phylogenic analysis using partial β-tubulin and calmodulin sequencing confirmed two of these as A. minisclerotigenes, while the third could not be differentiated from A. flavus and related species within Aspergillus section flavi. Based on epidemiological cut-off values, the A. minisclerotigens and A. flavus isolates tested were susceptible to commonly used antifungal drugs.
Conclusions
This is the first report of human infection due to A. minisclerotigenes, and it raises the possiblity that other species within Aspergillus section flavi may also cause clinical disease. Clinical isolates of A. flavus from Iran are not distinct from Australian isolates, indicating local environmental, climatic or host features, rather than fungal features, govern the high incidence of A. flavus infection in this region. The results of this study have important implications for biological control strategies that aim to reduce aflatoxin by the introduction of non-toxigenic strains, as potentially any strain of A. flavus, and closely related species like A. minisclerotigenes, might be capable of human infection.
doi:10.1186/1471-2334-14-358
PMCID: PMC4099206  PMID: 24986045
9.  Influence of Fungicides and Irrigation Practice on Aflatoxin in Peanuts Before Digging 1 
Applied Microbiology  1971;22(4):629-634.
Peanuts grown under dryland conditions where drought stress occurred accumulated more aflatoxin before digging than peanuts grown under irrigation. Kernels became more susceptible to Aspergillus flavus and A. parasiticus invasion when the soil moisture in the pod zone approached levels at which moisture moved from the pod into the soil and the kernel moisture dropped below 31%. Isolation frequencies of these aspergilli from fresh-dug kernels were lowest in 1968 (maximum of 3%). In 1967 and 1969, maximum percentages of 100 and 74, respectively, were noted. Kernel infestation was correlated with degree of aflatoxin contamination. Dryland fresh-dug kernels contained a maximum of 35,800 parts per billion aflatoxin while a maximum of 50 parts per billion was detected in kernels from irrigated plots. In 1969 A. flavus infestation was as high as 59% in peanuts from irrigated plots; however, no aflatoxin was detected. Absence of aflatoxin in these samples is attributed to the higher kernel moisture content which reduced the aflatoxin-producing potential of A. flavus. Statistical analysis of the data revealed no significant differences in degree of fungal infestation, production levels, and grade factors between any fungicide treatments.
PMCID: PMC376377  PMID: 5130434
10.  Quantification of Gallic Acidin Fruits of Three Medicinal Plants 
Triphala is a traditional herbal formulation consisting of dried fruits originating from three medicinal plants, namely Terminalia chebula, Terminalia bellerica and Phyllanthus emblica. It is used in folk medicine for the treatment of headaches, dyspepsia and leucorrhoea. There are some reports regarding Triphala’s pharmacological effects including its anti-cancer, radioprotective, hypocholesterolaemic, hepatoprotective and anti-oxidant activities. The most important components of these plants are the tannins and gallic acid which they contain. Gallic acid being a compound with tannin structure existing in the Triphala fruit. In this research, the gallic acid content contained in the three plants constituting Triphala was determined. Plant fruits were purchased from available Iranian markets. Milled and powdered fruits from each plant were extracted with 70% acetone and subjected to a reaction with rhodanine reagent in the process forming a colored complex. The complex’s absorbance was measured at 520 nm and the amount of gallic acid was determined using its calibration curve. According to the results, the highest amount of gallic acid was observed in Phyllanthus embelica (1.79-2.18%) and the lowest amount was found in Terminalia chebula (0.28-0.80%). Moreover, differences between plant samples from different markets places were found to be statistically significant (p < 0.05). These differences can possibly be due to the source of plant preparation, storage condition and period of Triphala storage. In general, the rhodanine assay is a simple, rapid and reproducible method for the standardization of Triphala as gallic acid.
PMCID: PMC3828909  PMID: 24250348
Triphala; Terminalia chebula; Terminalia bellerica; Phyllanthus emblica; Gallic acid; Rhodanine assay
11.  Improved protocols for functional analysis in the pathogenic fungus Aspergillus flavus 
BMC Microbiology  2007;7:104.
Background
An available whole genome sequence for Aspergillus flavus provides the opportunity to characterize factors involved in pathogenicity and to elucidate the regulatory networks involved in aflatoxin biosynthesis. Functional analysis of genes within the genome is greatly facilitated by the ability to disrupt or mis-express target genes and then evaluate their result on the phenotype of the fungus. Large-scale functional analysis requires an efficient genetic transformation system and the ability to readily select transformants with altered expression, and usually requires generation of double (or multi) gene deletion strains or the use of prototrophic strains. However, dominant selectable markers, an efficient transformation system and an efficient screening system for transformants in A. flavus are absent.
Results
The efficiency of the genetic transformation system for A. flavus based on uracil auxotrophy was improved. In addition, A. flavus was shown to be sensitive to the antibiotic, phleomycin. Transformation of A. flavus with the ble gene for resistance to phleomycin resulted in stable transformants when selected on 100 μg/ml phleomycin. We also compared the phleomycin system with one based on complementation for uracil auxotrophy which was confirmed by uracil and 5-fluoroorotic acid selection and via transformation with the pyr4 gene from Neurospora crassa and pyrG gene from A. nidulans in A. flavus NRRL 3357. A transformation protocol using pyr4 as a selectable marker resulted in site specific disruption of a target gene. A rapid and convenient colony PCR method for screening genetically altered transformants was also developed in this study.
Conclusion
We employed phleomycin resistance as a new positive selectable marker for genetic transformation of A. flavus. The experiments outlined herein constitute the first report of the use of the antibiotic phleomycin for transformation of A. flavus. Further, we demonstrated that this transformation protocol could be used for directed gene disruption in A. flavus. The significance of this is twofold. First, it allows strains to be transformed without having to generate an auxotrophic mutation, which is time consuming and may result in undesirable mutations. Second, this protocol allows for double gene knockouts when used in conjunction with existing strains with auxotrophic mutations.
To further facilitate functional analysis in this strain we developed a colony PCR-based method that is a rapid and convenient method for screening genetically altered transformants. This work will be of interest to those working on molecular biology of aflatoxin metabolism in A. flavus, especially for functional analysis using gene deletion and gene expression.
doi:10.1186/1471-2180-7-104
PMCID: PMC2212646  PMID: 18039373
12.  Hepatoprotective effect of poly herbal formulation against various hepatotoxic agents in rats 
Pharmacognosy Research  2012;4(1):50-56.
Background:
Individually Andrographis paniculata Nees. (Acanthaceae), Phyllanthus niruri Linn.(Euphorbiaceae) and Phyllanthus emblica Linn. single plant extracts have been reported to have hepatoprotective activity. However, literature survey shows that no sufficient scientific data has been publish on pharmacological evaluation of these plants in combined form.
Method:
Hepatoprotective activity of the polyherbal hepatoprotaective formulation (PHF)-containing spray-dried aqueous extracts of Andrographis paniculata Nees. (Acanthaceae), Phyllanthus niruri Linn. (Euphorbiaceae) and Phyllanthus emblica Linn. (Euphorbiaceae), was screened against paracetamol, carbon tetrachloride (CCl4), and ethanol-induced hepatic damage in rats. PHF was evaluated by measuring levels of serum marker enzymes like SGOT, SGPT, ALP, direct bilirubin (DB), and lactate dehydrogenase (LDH). The histological studies were also studied support the biochemical parameters. Silymarin was used as standard drug.
Results:
Administration of PHF (100 and 200 mg/kg p.o.) significantly inhibited paracetamol, CCl4 and ethanol-induced elevation levels of SGPT, SGOT, ALP, DB and LDH. A comparative histopathological study of liver exhibited almost normal architecture as compared to toxicant group.
Conclusion:
Results suggests that the hepatoprotective effects of PHF might be useful for liver protection due to combined action of all plant extracts along with their phytoconstituents.
doi:10.4103/0974-8490.91040
PMCID: PMC3250040  PMID: 22224062
Andrographis paniculata Nees; carbon tetrachloride; hepatoprotective activity; Marker enzymes; paracetamol; Phyllanthus niruri Linn
13.  Non-aflatoxigenic Aspergillus flavus to prevent aflatoxin contamination in crops: advantages and limitations 
Aspergillus flavus is a diverse assemblage of strains that include aflatoxin-producing and non-toxigenic strains with cosmopolitan distribution. The most promising strategy currently being used to reduce preharvest contamination of crops with aflatoxin is to introduce non-aflatoxin (biocontrol) A. flavus into the crop environment. Whether or not introduction of biocontrol strains into agricultural fields is enough to reduce aflatoxin contamination to levels required for acceptance of the contaminated food as fit for consumption is still unknown. There is no question that biocontrol strains are able to reduce the size of the populations of aflatoxin-producing strains but the available data suggests that at most only a four- to five-fold reduction in aflatoxin contamination is achieved. There are many challenges facing this strategy that are both short term and long term. First, the population biology of A. flavus is not well understood due in part to A. flavus’s diversity, its ability to form heterokaryotic reproductive forms, and its unknown ability to survive for prolonged periods after application. Second, biocontrol strains must be selected that are suitable for the environment, the type of crop, and the soil into which they will be introduced. Third, there is a need to guard against inadvertent introduction of A. flavus strains that could impose an additional burden on food safety and food quality, and fourth, with global warming and resultant changes in the soil nutrients and concomitant microbiome populations, the biocontrol strategy must be sufficiently flexible to adapt to such changes. Understanding genetic variation within strains of A. flavus is important for developing a robust biocontrol strategy and it is unlikely that a “one size fits all” strategy will work for preharvest aflatoxin reduction.
doi:10.3389/fmicb.2014.00050
PMCID: PMC3918586  PMID: 24575088
aflatoxin; Aspergillus flavus; biocontrol; food safety; recombination; maize; cottonseed; population diversity
14.  Mycoflora study in a wheat flour mill of Argentina 
Brazilian Journal of Microbiology  2012;43(4):1444-1451.
The mycoflora of the environment: wheat conditioning, milling and screening, and filling zone, as well as, raw material -wheat-, intermediate product -grits- and end product -flour- on day 1, and after cleaning improvements -days 45 and 90- were studied in an Argentine wheat mill. Samples were incubated at 28°C for 5–7 days on Malt Extract Agar with chloramphenicol (100 mg L-1) and the results were expressed in colony forming units per cubic meter of air (CFU m-3) or per gram of sample (CFU g-1), respectively. Fungal genera and species were isolated and identified and the potential toxicogenic capacity of the Aspergillus flavus and Fusarium graminearum isolated was studied. Time-Place and Time-Product multifactorial ANOVA were carried out. After cleaning improvements, CFU m-3 of air decreased as a function of time. Cladosporium and Alternaria were abundant in every zone, Aspergillus predominated in the wheat conditioning zone and Penicillium and Eurotium decreased with time. Wheat was more contaminated than grits and flour; Aspergillus, Eurotium and Mucoraceae family were the most abundant. Deoxynivalenol was above the levels allowed in wheat, being acceptable in grits and flour. Aflatoxin and Zearalenone showed acceptable levels. When studied in vitro, 53% of Aspergillus flavus and 100% of Fusarium graminearum isolates, produced Total Aflatoxins, and Deoxynivalenol and Zearalenone, respectively.
doi:10.1590/S1517-838220120004000028
PMCID: PMC3769011  PMID: 24031975
mycoflora; environment; wheat mill
15.  Effects of Phyllanthus emblica extract on endothelial dysfunction and biomarkers of oxidative stress in patients with type 2 diabetes mellitus: a randomized, double-blind, controlled study 
Background
It has been reported that hyperglycemia can induce endothelial dysfunction via increased oxidative stress and that it plays a central role in the development of atherosclerosis and coronary heart disease. Phyllanthus emblica (Emblica officinalis, amla) is known for its antioxidant and antihyperlipidemic activity. The present study compared the effects of an aqueous extract of P. emblica (highly standardized by high-performance liquid chromatography to contain low molecular weight hydrolyzable tannins, ie, emblicanin A, emblicanin B, pedunculagin, and punigluconin) versus those of atorvastatin and placebo on endothelial dysfunction and biomarkers of oxidative stress in patients with type 2 diabetes.
Methods
Eligible patients were randomized to receive either P. emblica 250 mg twice daily, P. emblica 500 mg twice daily, atorvastatin 10 mg in the evening and matching placebo in the morning, or placebo twice daily for 12 weeks. The primary efficacy parameter was the change in endothelial function identified on salbutamol challenge at baseline and after 12 weeks of treatment. Secondary efficacy parameters were changes in biomarkers of oxidative stress (malondialdehyde, nitric oxide, and glutathione), high sensitivity C-reactive protein levels, the lipid profile, and glycosylated hemoglobin (HbA1c) levels. Laboratory safety parameters were measured at baseline and after 12 weeks of treatment.
Results
Eighty patients completed the study. Treatment with P. emblica 250 mg, P. emblica 500 mg, or atorvastatin 10 mg produced significant reductions in the reflection index (−2.25%±1.37% to −9.13%±2.56% versus −2.11%±0.98% to −10.04%±0.92% versus −2.68%±1.13% to −11.03%±3.93%, respectively), suggesting improvement in endothelial function after 12 weeks of treatment compared with baseline. There was a significant improvement in biomarkers of oxidative stress and systemic inflammation compared with baseline and placebo. Further, the treatments significantly improved the lipid profile and HbA1c levels compared with baseline and placebo. All treatments were well tolerated.
Conclusion
Both atorvastatin and P. emblica significantly improved endothelial function and reduced biomarkers of oxidative stress and systemic inflammation in patients with type 2 diabetes mellitus, without any significant changes in laboratory safety parameters.
doi:10.2147/DMSO.S46341
PMCID: PMC3735284  PMID: 23935377
Phyllanthus emblica; atorvastatin; endothelial dysfunction; type 2 diabetes
16.  Cloning of the afl-2 gene involved in aflatoxin biosynthesis from Aspergillus flavus. 
Aflatoxins are extremely potent carcinogens produced by Aspergillus flavus and Aspergillus parasiticus. Cloning of genes in the aflatoxin pathway provides a specific approach to understanding the regulation of aflatoxin biosynthesis and, subsequently, to the control of aflatoxin contamination of food and feed. This paper reports the isolation of a gene involved in aflatoxin biosynthesis by complementation of an aflatoxin-nonproducing mutant with a wild-type genomic cosmid library of A. flavus. Strain 650-33, blocked in aflatoxin biosynthesis at the afl-2 allele, was complemented by a 32-kb cosmid clone (B9), resulting in the production of aflatoxin. The onset and profile of aflatoxin accumulation was similar for the transformed strain and the wild-type strain (NRRL 3357) of the fungus, indicating that the integrated gene is under the same control as in wild-type strains. Complementation analyses with DNA fragments from B9 indicated that the gene resides within a 2.2-kb fragment. Because this gene complements the mutated afl-2 allele, it was designated afl-2. Genetic evidence obtained from a double mutant showed that afl-2 is involved in aflatoxin biosynthesis before the formation of norsolorinic acid, the first stable intermediate identified in the pathway. Further, metabolite feeding studies with the mutant, transformed, and wild-type cultures and enzymatic activity measurements in cell extracts of these cultures suggest that afl-2 regulates gene expression or the activity of other aflatoxin pathway enzymes. This is the first reported isolation of a gene for aflatoxin biosynthesis in A. flavus.
Images
PMCID: PMC202071  PMID: 8439147
17.  Preparation and Physico-Chemical Evaluation of Kshiramandura 
Ancient Science of Life  2010;29(4):7-12.
Mandura (Iron rust) is known by names lohkitta, malayas, ayomala, meaning waste of iron. Among different formulations prescribed for Parinamashoola (Peptic ulcer), in Ayurvedic classics Kshiramandura (Preparation of Iron rust in Milk) is one. Ancient authorities have given similar recipes of Mandura and these medicines are being successfully administered in the management of Peptic ulcer. In Parinamashoola (Peptic ulcer), Acharya Chakradatta mentioned Kshiramandura, as a formulation prepared by taking 384gms of Mandura Bhasma (Incinerated Iron rust), 3 Kg 73 gm of cow's urine and 768gms of cow's milk, boiled and administered in a dose of 500mg. To establish Physical and Chemical factors present in Mandura before and after purification and incineration, the preparation ofKshira mandura was attempted by adopting Quantitative and Qualitative methods. The drugwas identified by the qualities as described in the classics, viz., unctuous, heavy, hard and black in color and absence of hollow space. Mandura was heated in burning charcoal (600-800° c) and dipped in 5 liters of Cow's urine. This process was repeated 7 times, till the Mandura broke. This purified Mandura was then powdered and triturated with decoction ofTerminalia chebula, Terminalia belerica and Emblica officinalis (Triphala kashaya). Thereafter pellets were prepared and dried. The pellets were then sealed in crucibles and heated 30 times in a special type of furnace with temperature of 1000°c (Gajaputa method) to incinerate Mandura and prepare its ash (Bhasma). Cow's urine and milk were added to this Mandura Bhasma and Kshiramandura was prepared. When analyzed it showed 68.3 5% Ferric oxide, 0.66%MgCO3 and 1.32% CaCO3.
PMCID: PMC3336292  PMID: 22557361
Mandura; Parinama shoola; Triphala Kvatha; Gaja puta; Kshira Mandura; Tridosa
18.  Occurrence of aflatoxin B1 in natural products 
Brazilian Journal of Microbiology  2012;43(4):1428-1436.
The media claims for the consumption of natural resource-based food have gradually increased in both developing and developed countries. The interest in the safety of these products is partially due to the possible presence of toxigenic fungi acting as mycotoxin producers, such as aflatoxins produced during the secondary metabolism of Aspergillus flavus, A. parasiticus and A. nomius. Aflatoxins, mainly aflatoxin B1, are directly associated with liver cancer in human beings. This paper is aimed at evaluating the presence of aflatoxin B1 in a few vegetable drugs, dried plant extracts and industrialized products traded in 2010 in the city of Belo Horizonte, State of Minas Gerais, Brazil. The method used for the quantification of aflatoxin B1 was based on extraction through acetone:water (85:15), immunoaffinity column purification followed by separation and detection in high efficiency liquid chromatography. Under the conditions of analysis, the Limits of Detection and Quantification were 0.6 µg kg-1 and 1.0 µg kg-1 respectively. The complete sets of analyses were carried out in duplicate. Aflatoxin B1 was noticed in a single sample (< 1.0 µg kg-1). The results revealed low aflatoxin B1 contamination in the products under analysis. However, it is required to establish a broad monitoring program in order to obtain additional data and check up on the actual extension of contamination.
doi:10.1590/S1517-838220120004000026
PMCID: PMC3769045  PMID: 24031973
medicinal plants; fungi; mycotoxin; high-performance liquid chromatography (HPLC)
19.  Identification of seed proteins associated with resistance to pre-harvested aflatoxin contamination in peanut (Arachis hypogaea L) 
BMC Plant Biology  2010;10:267.
Background
Pre-harvest infection of peanuts by Aspergillus flavus and subsequent aflatoxin contamination is one of the food safety factors that most severely impair peanut productivity and human and animal health, especially in arid and semi-arid tropical areas. Some peanut cultivars with natural pre-harvest resistance to aflatoxin contamination have been identified through field screening. However, little is known about the resistance mechanism, which has slowed the incorporation of resistance into cultivars with commercially acceptable genetic background. Therefore, it is necessary to identify resistance-associated proteins, and then to recognize candidate resistance genes potentially underlying the resistance mechanism.
Results
The objective of this study was to identify resistance-associated proteins in response to A. flavus infection under drought stress using two-dimensional electrophoresis with mass spectrometry. To identify proteins involved in the resistance to pre-harvest aflatoxin contamination, we compared the differential expression profiles of seed proteins between a resistant cultivar (YJ-1) and a susceptible cultivar (Yueyou 7) under well-watered condition, drought stress, and A. flavus infection with drought stress. A total of 29 spots showed differential expression between resistant and susceptible cultivars in response to A. flavus attack under drought stress. Among these spots, 12 protein spots that consistently exhibited an altered expression were screened by Image Master 5.0 software and successfully identified by MALDI-TOF MS. Five protein spots, including Oso7g0179400, PII protein, CDK1, Oxalate oxidase, SAP domain-containing protein, were uniquely expressed in the resistant cultivar. Six protein spots including low molecular weight heat shock protein precursor, RIO kinase, L-ascorbate peroxidase, iso-Ara h3, 50 S ribosomal protein L22 and putative 30 S ribosomal S9 were significantly up-regulated in the resistant cultivar challenged by A. flavus under drought stress. A significant decrease or down regulation of trypsin inhibitor caused by A. flavus in the resistant cultivar was also observed. In addition, variations in protein expression patterns for resistant and susceptible cultivars were further validated by real time RT-PCR analysis.
Conclusion
In summary, this study provides new insights into understanding of the molecular mechanism of resistance to pre-harvest aflatoxin contamination in peanut, and will help to develop peanut varieties with resistance to pre-harvested aflatoxin contamination.
doi:10.1186/1471-2229-10-267
PMCID: PMC3095339  PMID: 21118527
20.  Relationship between Meloidogyne arenaria and Aflatoxin Contamination in Peanut 
Journal of Nematology  2004;36(2):167-170.
Damaged and developing kernels of peanut (Arachis hypogaea) are susceptible to colonization by fungi in the Aspergillus flavus group which, under certain conditions, produces aflatoxins prior to harvest. Our objective was to determine whether infection of peanut roots and pods by Meloidogyne arenaria increases aflatoxin contamination of the kernels when peanut is subjected to drought stress. The experiment was a completely randomized 2-x-2 factorial with 6 replicates/treatment. The treatment factors were nematodes (plus and minus M. arenaria) and fungus (plus and minus A. flavus inoculum). The experiment was conducted in 2001 and 2002 in microplots under an automatic rain-out shelter. In treatments where A. flavus inoculum was added, aflatoxin concentrations were high (> 1,000 ppb) and not affected by nematode infection; in treatments without added fungal inoculum, aflatoxin concentrations were greater (P ≤ 0.05) in kernels from nematode-infected plants (1,190 ppb) than in kernels from uninfected plants (79 ppb). There was also an increase in aflatoxin contamination of kernels with increasing pod galling (r² = 0.83 in 2001, r² = 0.43 in 2002; P ≤ 0.04). Colonization of kernels by A. flavus increased with increasing pod galling (r² = 0.18; P = 0.04) in 2001 but not in 2002. Root-knot nematodes may have a greater role in enhancing aflatoxin contamination of peanut when conditions are not optimal for growth and aflatoxin production by fungi in the A. flavus group.
PMCID: PMC2620756  PMID: 19262803
aflatoxin; Arachis hypogaea; Aspergillus flavus; A. parasiticus; interaction; Meloidogyne arenaria; peanut; root-knot nematode
21.  Biodiversity of Aspergillus section Flavi in Europe in relation to the management of aflatoxin risk 
Aflatoxins and the producing fungi Aspergillus section Flavi are widely known as the most serious and dangerous mycotoxin issue in agricultural products. In Europe, before the outbreak of aflatoxins on maize (2003–2004) due to new climatic conditions, their contamination was confined to imported foods. Little information is available on molecular biodiversity and population structure of Aspergillus section Flavi in Europe. Preliminary reports evidenced the massive presence of Aspergillus flavus L -morphotype as the predominant species in maize field, no evidence of the highly toxigenic S-morphotype and of other aflatoxigenic species are reported. The risk of a shift in traditional occurrence areas for aflatoxins is expected in the world and in particular in South East of Europe due to the increasing average temperatures. Biological control of aflatoxin risk in the field by atoxigenic strains of A. flavus starts to be widely used in Africa and USA. Studies are necessary on the variation of aflatoxin production in populations of A. flavus to characterize stable atoxigenic A. flavus strains. The aim of present article is to give an overview on biodiversity and genetic variation of Aspergillus section Flavi in Europe in relation to the management of aflatoxins risk in the field.
doi:10.3389/fmicb.2014.00377
PMCID: PMC4104701  PMID: 25101075
Aspergillus; aflatoxin; genetic variation; climate change; atoxigenic strains; biocontrol
22.  Fungi and Mycotoxins in Feed Intended for Sows at Different Reproductive Stages in Argentina 
The aim of this study was to evaluate fungi and contamination levels of aflatoxin B1, ochratoxin A, fumonisin B1, and zearalenone in raw materials and finished feed intended for sows at different reproductive stages. Total fungi, Aspergillus, Penicillium, and Fusarium species occurrence, were examined. Aspergillus flavus, A. niger aggregate spp., and F. verticillioides were the prevalent species. Fungal counts exceeded the levels proposed as feed hygienic quality limits (1 × 104 colony forming units) at all reproductive stages. Aflatoxin B1, ochratoxin A, fumonisin B1, and zearalenone were detected by high-pressure liquid chromatography. Aflatoxin levels in 80% samples of finished sow feeds were over the permitted levels of 0.02 μg g−1 (mean 228.2 ± 95 μg Kg−1). Fumonisin B1 was detected in all tested raw materials at levels that varied from 50.3 to 1137.64 μg Kg−1 and finished feed samples at levels that ranged from 99.8 to 512.4 μg Kg−1. Aflatoxin B1, zearalenone, and ochratoxin A were not detected in raw materials. All finished feeds were negative for zearalenone contamination whereas all nonpregnant gilt samples were contaminated with low OTA levels (mean 0.259 ± 0.123). This fact requires periodic monitoring to prevent the occurrence of mycotoxicosis in animal production, to reduce the economic losses, and to minimize hazards to human health.
doi:10.4061/2010/569108
PMCID: PMC2896851  PMID: 20613957
23.  Loss of msnA, a Putative Stress Regulatory Gene, in Aspergillus parasiticus and Aspergillus flavus Increased Production of Conidia, Aflatoxins and Kojic Acid 
Toxins  2011;3(1):82-104.
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.
doi:10.3390/toxins3010082
PMCID: PMC3210457  PMID: 22069691
Aspergillus; aflatoxin; kojic acid; oxidative stress; development
24.  Emblica officinalis Garten fruits extract ameliorates reproductive injury and oxidative testicular toxicity induced by chlorpyrifos in male rats 
SpringerPlus  2013;2:541.
Organophosphate pesticides have destroying properties on male reproduction and chlorpyrifos adversely affects the male reproductive system. Emblica offcinalis Garten plays a vital role to challenge many diseases in human body. We investigated the induction of oxidative stress in the male reproductive system of adult rats (Wistar Strain) exposed to widely used organophosphate pesticide, Chlorpyrifos, and tried to establish the ameliorative properties of Emblica officinalis Garten with respect to reproductive reconstruction in them. Rats were divided into 2 groups, control group and experimental group, and the experimental group was divided into 3 groups (G1-G3). All the groups had 5 rats each. Control group received water, experimental group, G1, received 20 mg/kg bw/day Emblica officinalis Garten, G2 received 12 mg/kg bw/day chlorpyrifos and G3 received 12 mg chlorpyrifos with 20 mg Emblica officinalis Garten /kg bw/day. Treatment was done orally from 30 days. Thereafter body weight, male reproductive organs weight, sperm count, sperm morphology, ACP, ALP, total protein, uric acid and testis and serum testosterone level were determined using standard methods. The changes recorded are indicative of infertility in male rats because of chlorpyrifos exposure. When the subjects were treated with Emblica officinalis Garten in conjunction with chlorpyrifos, these parameters exhibited recovery and when treated with Emblica officinalis Garten alone, these parameters were more or less near to the control group. This highlights the debilitating effect of chlorpyrifos and scavenging property of Emblica officinalis Garten.
doi:10.1186/2193-1801-2-541
PMCID: PMC3824715  PMID: 24255841
Organophosphate pesticides; Chlorpyrifos; Emblica officinalis; Testicular toxicity; Reproductive injury; Oxidative stress; Natural medicine
25.  Toward elucidation of genetic and functional genetic mechanisms in corn host resistance to Aspergillus flavus infection and aflatoxin contamination 
Aflatoxins are carcinogenic mycotoxins produced by some species in the Aspergillus genus, such as A. flavus and A. parasiticus. Contamination of aflatoxins in corn profusely happens at pre-harvest stage when heat and drought field conditions favor A. flavus colonization. Commercial corn hybrids are generally susceptible to A. flavus infection. An ideal strategy for preventing aflatoxin contamination is through the enhancement of corn host resistance to Aspergillus infection and aflatoxin production. Constant efforts have been made by corn breeders to develop resistant corn genotypes. Significantly low levels of aflatoxin accumulation have been determined in certain resistant corn inbred lines. A number of reports of quantitative trait loci have provided compelling evidence supporting the quantitative trait genetic basis of corn host resistance to aflatoxin accumulation. Important findings have also been obtained from the investigation on candidate resistance genes through transcriptomics approach. Elucidation of molecular mechanisms will provide in-depth understanding of the host–pathogen interactions and hence facilitate the breeding of corn with resistance to A. flavus infection and aflatoxin accumulation.
doi:10.3389/fmicb.2014.00364
PMCID: PMC4104783  PMID: 25101068
Aspergillus flavus; aflatoxin; corn; host resistance; transcriptomics

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