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
Following injection of (3)H-aflatoxin B2 into male rats, the levels of aflatoxin adducts in their liver DNA and ribosomal (r)RNA were approximately 1% of those in rats injected with (3)H-aflatoxin B1. The levels of aflatoxin adducts in liver proteins of rats injected with aflatoxin B2 were 35-70% of those in rats injected with aflatoxin B1. One study determined aflatoxin levels in broiler tissues after 35 days of feeding them a diet containing 2057 μg/kg aflatoxin B1 and 1323 μg/kg aflatoxin B2. The results showed that aflatoxin was deposited in all tissues. The highest levels of aflatoxin were found in the gizzard, liver, and kidneys. There is evidence that high concentrations of aflatoxin B1 and B2 in chicken gizzards may be due to contamination from gizzard contents during slaughter. After 35 days of feeding with aflatoxin-contaminated feed, the average total aflatoxin level in tissues was less than 3 μg/kg. Four days after stopping the feeding of aflatoxin-contaminated feed, no aflatoxin was detected in any tissues. These results indicate that broilers can rapidly clear aflatoxin from tissues once switched to an aflatoxin-free diet. To assess the rate at which four major aflatoxins (aflatoxins B1, B2, G1, and G2) cross the small intestinal membrane in rats, we conducted a study on the intestinal absorption kinetics of these mycotoxins. In situ experiments showed that the absorption of aflatoxin in the rat small intestine is a rapid process following first-order kinetics, with absorption rate constants (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. Metabolites were produced in rats. See Table. The metabolism of aflatoxin B2 was studied in in vitro systems using mitochondrial postsupernatant fractions from duck, rat, mouse, and human livers. The mitochondrial postsupernatant equivalent to 0.2 g of duck liver metabolized 40–80% of the initial substrate within 30 minutes, while the metabolic rate in other species was less than 6%. Among the various metabolites produced in duck liver, aflatoxin B1 was generated at a rate equivalent to 2-8% of the initial substrate. Small amounts of metabolites with the speculative chromatographic characteristics of aflatoxins 1 and 2, as well as aflatoxins M1 and M2, were also generated. The increased susceptibility of duck liver to aflatoxin B2 toxicity may be attributed to its ability to generate aflatoxin B1, which can be activated through further metabolism. In rats, intravenous injection of aflatoxin B2 rapidly metabolized it into seven metabolites, six of which were excreted via bile. Aflatoxin B2 was hydroxylated at positions 2 and 4. Following injection of aflatoxin B2 into rats, the rat bile contained two glucuronide nucleotides.

Non-Human Toxicity Values
Duck oral LD50: 1700 μg/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 with a hexahydrocyclopentano[c]furano[3',2':4,5]furano[2,3-h]chromene skeleton, containing oxygen functional groups at positions 1, 4, and 11. It has been reported in Aspergillus nomiae, Glycyrrhiza uralensis, and several other organisms with relevant data. Mechanism of Action: Among the four major aflatoxins tested, the order of inhibition against RNA polymerase II was: B1 > G1 > B1 > G2. The ability of aflatoxins B1, B2, and G1 to inhibit RNA polymerase activity and reduce RNA content in rat hepatocyte nuclei is similar in nature to the carcinogenic, acute, and subacute toxic effects of these compounds.
Aflatoxin B2 Interaction: This study investigated the in vivo binding of aflatoxin B2 (AFB2) to rat liver nuclear macromolecules to explore the association between this binding and biological activity. Incorporation of [(3)H]AFB2 residues into rat liver histones and DNA was measured at 2, 24, and 48 hours after intraperitoneal injection of a single dose of 1 mg [(3)H]AFB2/kg body weight in histone H1 and total histone fractions. At each time point, the content of [(3)H]AFB2 (by weight) in histone H1 and total histone fractions was 5–30 times that in DNA. Analytical reversed-phase high-performance liquid chromatography (HPLC) analysis of the acid hydrolysis products generated by AFB2 binding to DNA showed that 85% of the radioactivity was co-chromatographically separated from the major aflatoxin B1-DNA adduct, 2,3-dihydro-2-(N7-guanosyl)-3-hydroxyaflatoxin B1. These studies reveal a clear association between the DNA binding of aflatoxin B2 (AFB2) in rats and its toxic and carcinogenic potency in that species. Aflatoxins generate singlet oxygen upon exposure to ultraviolet light (365 nm). This singlet oxygen, in turn, activates aflatoxins, making them mutagens and DNA binders. Compared to the reaction in water, DNA binding and mutagenicity of aflatoxins are enhanced in heavy water (D2O), while singlet oxygen scavengers inhibit mutagenicity. In the presence of unlabeled aflatoxin B2, the DNA photobinding of 3H-labeled aflatoxin B1 is enhanced, and the addition of aflatoxin B2 synergistically enhances the mutagenicity of aflatoxin B1. These results are consistent with the idea that singlet oxygen generated by one aflatoxin molecule can readily activate another aflatoxin molecule. This could have environmental impacts, as the weakly carcinogenic aflatoxin B2 (which is usually produced in nature along with aflatoxin B1) may play a significant role 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.)