aflatoxins ( afs ) are carcinogenic , mutagenic , and teratogenic metabolites mainly produced by aspergillus flavus and the very closely related species a. parasiticus ( 1 ) .
these mycotoxins can grow on a wide range of food commodities , including cereals , peanuts , and crops in the eld , but also during postharvest operations and storage ( 2 ) .
the group of afs includes b1 , b2 , g1 , and g2 , while afbs include afb1 and afb2 , and afgs include afg1 and afg2 ( 3 ) .
afs are potent hepatotoxic , teratogenic , and mutagenic metabolites that are now well - recognized as a public health hazard ( 1 , 4 ) .
although some synthetic fungicides have been improved to protect crops against af production , there are some proven indications that these are themselves hazardous to human health ( 5 ) .
therefore , using natural sources is an affirmative approach to protecting from fungal infections in agriculture ( 6 ) . a medicinal herb , pistacia atlantica subsp .
kurdica , has been traditionally used as a therapeutic agent for infections ( 7 - 9 ) .
the genus pistacia belongs to the family anacardiaceae , and among 15 known species of pistachios , only three grow in iran : p. vera , p. khinjuk , and p. atlantica ( 10 , 11 ) .
p. atlantica has three sub - species : mutica , kurdica , and cabulica ( 11 ) .
this plant is an evergreen bush that grows native in some southern and central american and eastern mediterranean countries ( 10 ) .
kurdica is obtained as an exudate after crushing the trunk and branches , and has numerous qualities .
the gum of this plant is used in medicine , with many researchers reporting that it possesses considerable antimicrobial activity ( 12 , 13 ) .
kurdica , as a natural compound , with regard to antifungal activity and inhibition of af production in a. parasiticus infections .
kurdica was obtained from saghez sazi ( van ) ( kurdistan , iran ) in the summer of 2014 .
the gum was obtained as an exudate of the trunk and branches . the a. parasiticus strain ( american type culture collection 15517 )
was incubated at 28c for 72 hours on sabouraud dextrose agar ( germany , merck ) . in vitro antifungal susceptibility testing for the determination of minimum inhibitory concentration ( mic )
was performed according to recommendations provided in the clinical and laboratory standards institute ( clsi ) m38-a2 document , with some modification in gum concentration ( 14 ) .
rpmi 1640 medium ( sigma chemical co. ) was buffered to ph 7.0 with mops ( sigma ) , with l - glutamine and without bicarbonate .
kurdica was dissolved in water to reach a concentration of 500 mg / ml . it was then diluted to achieve concentrations of 500 to 15.6 mg / ml ( 500 , 250 , 125 , 62.5 , 31.2 , and 15.6 mg / ml ) . the concentration of fungal spores was calculated using the hemacytometry method , and finally adjusted to 2x concentrated suspensions in such a way that each test well contained 5  10 colony - forming units ( cfus)/ml .
negative ( only rpmi 1640 medium ) and positive controls were also run alongside each experiment .
the mic endpoints were determined as the lowest concentrations that prevented any recognizable growth , mostly based on the antifungal capacity of the gum ( 100% inhibition ) .
the amount of afs produced by a. parasiticus alone and in combination with different gum concentrations ( 62.5 mg / ml and 125 mg / ml ) was determined using the high - performance liquid chromatography ( hplc ) technique ( waters 474 scanning fluorescence detector ) ( 6 ) .
pdb plates containing 62.5 mg / ml or 125 mg / ml of gum were inoculated with 5  10 fungal spores .
then , 25 g of each fungal biomass with 2.5 g of sodium chloride was added into 100 ml of hplc - grade methanol and blended for 3 minutes at 18,000 rpm .
cell - free filtrates were obtained using whatman filter paper no.4 and subjected to analysis by hplc .
quantitative changes in expression of the aflr gene were analyzed by use of a quantitative real - time pcr assay .
a. parasiticus was cultured in the presence of 62.5 mg / ml or 125 mg / ml of gum of p. atlantica var .
equal amounts of rna ( 1 g in 20 l ) were subjected to cdna synthesis by using random hexamer primers , according to the manual protocol ( fermentase ) . the primers fafl ( 5'-cggaacagggacttccggcg-3 ' ) and rafl ( 5'-gggtggcgggggactctgat-3 ' ) were designed on the basis of the published sequence of the aflr gene of a. parasiticus ( ncbi , accession no : af441438 ) . the b - actin gene ( act1 ) with fact
( 5'-acggtattgtttccaactgggacg-3 ' ) and ract ( 5'- tggagcttcggtcaacaaaactgg-3 ' ) primers was used as an endogenous reference gene .
quantitative real - time pcr was carried out using the sybr green master mix ( applied biosystems , foster city , ca , usa ) performed with a step one plus real - time pcr system ( applied biosystems , foster city , ca , usa ) .
the aflr gene expression was analyzed with rest software ( 2008 v2.0.7 , corbett research , sydney , australia ) .
kurdica was obtained from saghez sazi ( van ) ( kurdistan , iran ) in the summer of 2014 .
the a. parasiticus strain ( american type culture collection 15517 ) was incubated at 28c for 72 hours on sabouraud dextrose agar ( germany , merck ) . in vitro antifungal susceptibility testing for the determination of minimum inhibitory concentration ( mic )
was performed according to recommendations provided in the clinical and laboratory standards institute ( clsi ) m38-a2 document , with some modification in gum concentration ( 14 ) .
rpmi 1640 medium ( sigma chemical co. ) was buffered to ph 7.0 with mops ( sigma ) , with l - glutamine and without bicarbonate .
kurdica was dissolved in water to reach a concentration of 500 mg / ml . it was then diluted to achieve concentrations of 500 to 15.6 mg / ml ( 500 , 250 , 125 , 62.5 , 31.2 , and 15.6 mg / ml ) . the concentration of fungal spores was calculated using the hemacytometry method , and finally adjusted to 2x concentrated suspensions in such a way that each test well contained 5  10 colony - forming units ( cfus)/ml .
negative ( only rpmi 1640 medium ) and positive controls were also run alongside each experiment .
the mic endpoints were determined as the lowest concentrations that prevented any recognizable growth , mostly based on the antifungal capacity of the gum ( 100% inhibition ) .
the amount of afs produced by a. parasiticus alone and in combination with different gum concentrations ( 62.5 mg / ml and 125 mg / ml ) was determined using the high - performance liquid chromatography ( hplc ) technique ( waters 474 scanning fluorescence detector ) ( 6 ) .
pdb plates containing 62.5 mg / ml or 125 mg / ml of gum were inoculated with 5  10 fungal spores .
then , 25 g of each fungal biomass with 2.5 g of sodium chloride was added into 100 ml of hplc - grade methanol and blended for 3 minutes at 18,000 rpm .
cell - free filtrates were obtained using whatman filter paper no.4 and subjected to analysis by hplc .
quantitative changes in expression of the aflr gene were analyzed by use of a quantitative real - time pcr assay .
a. parasiticus was cultured in the presence of 62.5 mg / ml or 125 mg / ml of gum of p. atlantica var .
equal amounts of rna ( 1 g in 20 l ) were subjected to cdna synthesis by using random hexamer primers , according to the manual protocol ( fermentase ) . the primers fafl ( 5'-cggaacagggacttccggcg-3 ' ) and rafl ( 5'-gggtggcgggggactctgat-3 ' ) were designed on the basis of the published sequence of the aflr gene of a. parasiticus ( ncbi , accession no : af441438 ) . the b - actin gene ( act1 ) with fact
( 5'-acggtattgtttccaactgggacg-3 ' ) and ract ( 5'- tggagcttcggtcaacaaaactgg-3 ' ) primers was used as an endogenous reference gene .
quantitative real - time pcr was carried out using the sybr green master mix ( applied biosystems , foster city , ca , usa ) performed with a step one plus real - time pcr system ( applied biosystems , foster city , ca , usa ) .
the aflr gene expression was analyzed with rest software ( 2008 v2.0.7 , corbett research , sydney , australia ) .
kurdica inhibited a. parasiticus growth at mic values of 125 mg / ml . according to hplc analysis results , afl - b2 and afl - g2 production was significantly decreased and even entirely inhibited in 125 mg / ml of gum ( p < 0.01 ) .
however , the production of afl - b1 was not fully inhibited at this concentration .
based on quantitative real - time pcr results , the rate of aflr gene expression was significantly decreased after treating the a. parasiticus with 125 mg / ml of p. atlantica subsp .
the results of an analysis of the relative quantification of the aflr gene after treatment with the gum of p. atlantica subsp .
aflatoxins are extremely toxic and carcinogenic metabolites that widely contaminate agricultural commodities and animal feed ( 15 ) . in recent years
, research has been ongoing into the development of new anti - fungal agents or through the search for natural sources to control growth and toxin production of aspergillus species ( 8 , 9 , 16 ) . in several studies ,
ghalem et al . reported that the gum of p. atlantica has antimicrobial activity against gram - positive and -negative bacteria that are resistant to commonly used antimicrobial agents , and this was considerably dependent on concentration ( 17 ) .
hesami et al . suggested the potential substitution of antifungal chemicals by p. atlantica subsp .
kurdica as a natural inhibitor to control the growth of botrytis cinereain ( 19 ) .
aflatoxin inhibition mainly occurs during the primary stages of cell growth , when the genes involved in aflatoxin biosynthesis are at maximum activity within the fungal cells ( 7 ) .
our study revealed that the rate of aflr gene expression was significantly decreased after treating the fungus with 125 mg / ml of p. atlantica subsp .
showed that licorice extract could efficiently inhibit aflr gene expression , and consequently , af production , in a. parasiticus ( 6 ) . in another study ,
. showed potent inhibition of a. parasiticus growth and af production with 1 mg / ml of ephedra major ( 20 ) .
kurdica could efficiently inhibit aflr gene expression and the consequent af production in a. parasiticus .
this gum may therefore be considered a potential anti - mycotoxic agent in medicine or industrial agriculture .
further studies are recommended to determine the effective components of the gum of p. atlantica subsp .
kurdica and to consider it as a potential candidate for controlling af contamination of crops in the field .
 backgroundaflatoxins are highly toxic secondary metabolites mainly produced by aspergillus parasiticus 
 . this species can contaminate a wide range of agricultural commodities , including cereals , peanuts , and crops in the field . in recent years , 
 research on medicinal herbs , such as pistacia atlantica subsp . 
 kurdica , have led to reduced microbial growth , and these herbs also have a particular effect on the production of aflatoxins as carcinogenic compounds.objectivesin this study , we to examine p. atlantica subsp . 
 kurdica as a natural compound used to inhibit the growth of a. parasiticus and to act as an anti-mycotoxin.materials and methodsin vitro antifungal susceptibility testing of p. atlantica subsp . 
 kurdica for a. parasiticus was performed according to clsi document m38-a2 . 
 the rate of aflatoxin production was determined using the hplc technique after exposure to different concentrations ( 62.5 - 125 mg / ml ) of the gum . 
 the changes in expression levels of the aflr gene were analyzed with a quantitative real - time pcr assay.resultsthe results showed that p. atlantica subsp . 
 kurdica can inhibit a. parasiticus growth at a concentration of 125 mg / ml . 
 hplc results revealed a significant decrease in aflatoxin production with 125 mg / ml of p. atlantica subsp . 
 kurdica , and afl - b1 production was entirely inhibited . 
 based on quantitative real - time pcr results , the rate of aflr gene expression was significantly decreased after treatment with p. atlantica subsp . kurdica.conclusionspistacia 
 atlantica subsp . 
 kurdica has anti - toxic properties in addition to an inhibitory effect on a. parasiticus growth , and is able to decrease aflatoxin production effectively in a dose - dependent manner . 
 therefore , this herbal extract maybe considered a potential anti - mycotoxin agent in medicine or industrial agriculture . 
1. Background
2. Objectives
3. Materials and Methods
3.1. Preparation of Gum
3.2. Fungal Strain and Susceptibility Test
3.3. Detection of AFs
3.4. AflR Gene Expression Assay
4. Results
5. Discussion