Secondary metabolite from Aspergillus flavus and Aspergillus parasiticus

Aflatoxins (AFs) are hepatogenic, teratogenic, imunosuppressive, and carcinogenic fungal metabolites found in feeds, nuts, wine-grapes, spices, and other grain crops. Humans are exposed to AFs via consumption of mycotoxin-contaminated foods. This study aimed to determine the prevalence of AF contamination in powdered red peppers sold in Sanliurfa. A total of 42 samples were randomly collected from retail shops, supermarkets, open bazaars, and apiaries and examined for the occurrence and levels of AFB1, AFB2, AFG1, and AFG2 toxins. AFs were determined by using an HPLC system after pre-separation utilizing immunoaffinity columns. AFs levels were below 2.5 μg/kg in 16 samples, between 2.5 and 10 μg/kg in 13 samples while 13 samples had AFs higher than the tolerable limit (10 μg/kg) according to the regulations of Turkish Food Codex and European Commission. The occurrence of AF fractions during powdered red pepper processing steps was also evaluated. According to the results obtained in this study, it was found that the highest AF accumulations in powdered red peppers start during perspiration and final drying of the products processed on soil contacted surfaces while there was no limit exceeding aflatoxin contamination in the samples produced on concrete surfaces [2].

The aflatoxin producing fungi, Aspergillus spp., are widely spread in nature and have severely contaminated food supplies of humans and animals, resulting in health hazards and even death. Therefore, there is great demand for aflatoxins research to develop suitable methods for their quantification, precise detection and control to ensure the safety of consumers' health. Here, the chemistry and biosynthesis process of the mycotoxins is discussed in brief along with their occurrence, and the health hazards to humans and livestock. This review focuses on resources, production, detection and control measures of aflatoxins to ensure food and feed safety. The review is informative for health-conscious consumers and research experts in the fields. Furthermore, providing knowledge on aflatoxins toxicity will help in ensure food safety and meet the future demands of the increasing population by decreasing the incidence of outbreaks due to aflatoxins [1].

Aflatoxin analysis by HPLC [2]
Detection and quantification of AFB1, AFB2, AFG1, and AFG2 levels in the samples was carried out by HPLC equipped with an autosampler using a fluorescence detector. The HPLC equipment was a Shimadzu system with Shimadzu LC-20AD pump, Shimadzu SIL-20 ADHT autosampler, CTO-20AC column oven, Shimadzu RF-10AXL fluorescence detector (FLD) set at 360-nm excitation and 460-nm emission. An ODS3 column (ODS3 250 mm × 5 μm × 4.6 mm) was used. The mobile phase was distilled water/acetonitrile (90:10), and the flow rate was 1 ml/min; injection volume was 100 μl (AOAC, 999.07).

Absorption, Distribution and Excretion
Administration of (3)H-aflatoxin b2 to male rats gave levels of hepatic DNA & ribosomal (r)RNA aflatoxin adducts that were about 1% of those for rats given (3)H-aflatoxin b1. Levels of hepatic protein aflatoxin adducts were 35-70% as great for aflatoxin b2-treated rats as for aflatoxin b1-treated rats.
A study was conducted to determine aflatoxin levels in the tissues of broiler chickens that had been fed a diet containing 2057 micrograms aflatoxin B1 and 1323 micrograms aflatoxin B2/kg for 35 days. Results showed that aflatoxins were deposited in all tissues. The highest levels of aflatoxins were present in the gizzards, livers and kidneys. There was evidence that the high levels of aflatoxins B1 and B2 in the gizzards might have been caused by contamination by the gizzard contents during the slaughtering process. After feeding the aflatoxin-contaminated diet for 35 days, mean values for the combined aflatoxins were less than 3 micrograms/kg of tissue. Four days after withdrawal of the aflatoxin-contaminated ration, there were no detectable amounts of aflatoxins in any of the tissues. The results indicate that broiler chickens rapidly clear aflatoxins from their tissues once they are transferred to an aflatoxin-free diet.
To evaluate the rate at which the four main aflatoxins (aflatoxins B1, B2, G1 and G2) are able to cross the luminal membrane of the rat small intestine, a study about intestinal absorption kinetics of these mycotoxins has been made. In situ results obtained showed that the absorption of aflatoxins in rat small intestine is a very fast process that follows first-order kinetics, with an absorption rate constant (ka) of 5.84 +/- 0.05 (aflatoxin B1), 4.06 +/- 0.09 (aflatoxin B2), 2.09 +/- 0.03 (aflatoxin G1) and 1.58 +/- 0.04 (aflatoxin G2) h-1, respectively.

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Metabolism / Metabolites
Yields Alfatoxin m2 in rat. From table/
Metabolism of aflatoxin b2 by postmitochondrial supernatant fractions of duck, rat, mouse & human livers was studied in an in vitro system. Postmitochondrial supernatant from duck equivalent to 0.2 g whole liver metabolized 40-80% of the initial substrate in 30 min, compared to less than 6% for other species. Among several metabolites formed by duck liver, aflatoxin b1 was produced in amt equivalent to 2-8% of the initial substrate, & metabolites having chromatographic properties postulated for aflatoxicols 1 & 2 & aflatoxins m1 & m2 were also formed in small amounts. The greater susceptibility of duck liver to the toxicity of aflatoxin b2 may be attributable to its ability to form aflatoxin b1, which could be activated through further metabolism.
Aflatoxin b2 admin iv to rats was rapidly metabolized to 7 groups of metabolites, 6 of which were excreted in the bile. Aflatoxin b2 was hydroxylated at the 2- & 4-positions. Bile from rats given aflatoxin b2 contained 2 glucuronides.

Non-Human Toxicity Values
LD50 Duck oral 1700 ug/kg

[1]. Aflatoxins: A Global Concern for Food Safety, Human Health and Their Management. Front Microbiol. 2017 Jan 17;7:2170.

[2]. Aflatoxins B1, B2, G1, and G2 contamination in ground red peppers commercialized in Sanliurfa, Turkey. Environ Monit Assess. 2015 Apr;187(4):184.

Aflatoxin B2 is an aflatoxin having a hexahydrocyclopenta[c]furo[3',2':4,5]furo[2,3-h]chromene skeleton with oxygen functionality at positions 1, 4 and 11.
Aflatoxin B2 has been reported in Aspergillus nomiae, Glycyrrhiza uralensis, and other organisms with data available.

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Mechanism of Action
With the 4 principal aflatoxins tested, the order of inhibitory effect on RNA polymerase ii was: b1 greater than g1 greater than b1, g2.
Ability of aflatoxin b1, aflatoxin b2, & aflatoxin g1 to inhibit RNA polymerase activity & decr RNA content in rat hepatocyte nuclei was qualitatively similar to the carcinogenic & acute & subacute toxic actions of these compounds.
The interaction of aflatoxin B2 (AFB2) in vivo with rat liver nuclear macromolecules was examined in an attempt to correlate this binding with biological potency. The incorporation of [(3)H]AFB2 residues into rat liver histones and DNA was determined 2, 24 and 48 hr following administration of a single ip dose of 1 mg [(3)H]AFB2/kg bw. At each time point, histone H1 and the total histone fraction contained 5--30-fold more [(3)H]AFB2 moieties than did DNA on a weight basis. Analytical reversed-phase HPLC of the acid hydrolysis products resulting from AFB2 binding to DNA revealed that 85% of the radioactivity co-chromatographed with the major aflatoxin B1-DNA adduct, 2,3-dihydro-2-(N7-guanyl)-3-hydroxyaflatoxin B1. These studies revealed an apparent correlation between AFB2 derived binding to DNA in vivo in rats and its potency as a toxin and carcinogen in this species.
Aflatoxins produce singlet oxygen upon their exposure to UV (365-nm) light. Singlet oxygen in turn activates them to mutagens and DNA binding species. DNA binding and mutagenesis by aflatoxins were enhanced in D2O as compared to reactions in H2O, and a singlet oxygen scavenger inhibited mutagenesis. DNA photobinding of 3H-aflatoxin B1 increased in the presence of unlabeled aflatoxin B2, and the addition of aflatoxin B2 enhanced mutagenesis by aflatoxin B1 in a synergistic manner. These results are compatible with the notion that singlet oxygen, formed by one aflatoxin molecule, can readily activate another aflatoxin molecule. This may bear an environmental implication in that the weakly carcinogenic aflatoxin B2, which is often produced in nature together with aflatoxin B1, may be important in enhancing the activation of aflatoxin B1 by sunlight.

C17H14O6

314.28946

314.079

7220-81-7

Aflatoxin B2-13C17;1217470-98-8

2724360

White to off-white solid powder

1.52±0.1 g/cm3

521.0±50.0 °C at 760 mmHg

305 ºC (分解) (chloroform )

234.0±30.2 °C

0.0±1.4 mmHg at 25°C

1.660

0.37

0

6

1

23

610

2

COC1=C2C3=C(C(=O)CC3)C(=O)OC2=C4[C@@H]5CCO[C@@H]5OC4=C1

WWSYXEZEXMQWHT-WNWIJWBNSA-N

InChI=1S/C17H14O6/c1-20-10-6-11-14(8-4-5-21-17(8)22-11)15-13(10)7-2-3-9(18)12(7)16(19)23-15/h6,8,17H,2-5H2,1H3/t8-,17+/m0/s1

(3S,7R)-11-methoxy-6,8,19-trioxapentacyclo[10.7.0.02,9.03,7.013,17]nonadeca-1,9,11,13(17)-tetraene-16,18-dione

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO : ~2.94 mg/mL (~9.35 mM)

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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 (Please use freshly prepared in vivo formulations for optimal results.)