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].

Environmental occurrence of Aspergillus and other fungal spores are hazardous to humans and animals. They cause a broad spectrum of clinical complications. Contamination of aflatoxins in agri-food and feed due to A. flavus and A. parasiticus result in toxicity in humans and animals. Recent advances in aspergillus genomics and aflatoxin management practices are encouraging to tackle the challenges posed by important aspergillus species [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
Most aflatoxins administered to sheep appear to be destroyed in the body, with only 8% of the dose recovered in milk, urine, and feces. Analysis of umbilical cord blood samples from 64 pregnant women in Sierra Leone revealed the presence of ochratoxin A (OTA) and aflatoxin in 25% and 58% of the samples, respectively. Of eight maternal blood samples collected during farrowing, one contained OTA, and six tested positive for aflatoxin. There was no correlation between mycotoxins in maternal blood and umbilical cord blood. This article discusses the potential impact of these toxins on infant birth weight. Approximately 90% of the total amount of aflatoxin administered orally to sheep is excreted within 48 hours after treatment. Six days later, aflatoxin was undetectable in sheep milk; eight days later, it was undetectable in urine; and nine days later, it was undetectable in feces. In this species, after a single oral administration of 1 mg/kg body weight of aflatoxin mixture, only 8.1% was recovered in an identifiable form, with 0.1% in milk, 6.4% in urine, and 1.6% in feces. Aflatoxin is excreted in the milk of lactating animals as its metabolite aflatoxin M1. In cattle given a single oral dose of aflatoxin, 85% of the total amount detected in milk and urine was detected within the first 48 hours after administration. No aflatoxin was detected in milk after 4 days, and also in urine and feces after 6 days. The total aflatoxin detected in milk was 0.39% of the ingested amount. …Less than 0.6% of ingested aflatoxin B1 was excreted in milk. The aflatoxin content in milk is not related to milk production and is cleared from milk within three to four days after cessation of feeding toxic feed. For more complete data on the absorption, distribution, and excretion of aflatoxins (a total of 8), please visit the HSDB records page. Aflatoxin B1 and G1, and their metabolites, exist in the bloodstream as protein conjugates. This binding specifically binds to plasma albumin and is enzymatically catalyzed by liver and kidney cells. The albumin-aflatoxin conjugate is permanent and irreversible. Four Large White sows in each of three groups were fed diets containing 800 ppb purified aflatoxin B1 (Group 1), 800 ppb purified aflatoxin G1 (Group 2), or 400 ppb aflatoxin B1 and 400 ppb aflatoxin G1 (Group 3) during gestation and lactation, respectively. A separate control group of four sows was fed a diet free of aflatoxins. Aflatoxin B1 and M1 were detected in milk samples collected from sows in Group 1 at 5 and 25 days postpartum, aflatoxin G1 was detected in milk samples from sows in Group 2, and all three aflatoxins were detected in milk samples from sows in Group 3. The concentration of aflatoxin in milk was approximately 1000 times lower than in feed, but gradually increased within 25 days postpartum. Metabolism/Metabolites…Aflatoxin is metabolized in the liver into epoxides, which have short half-lives and primarily act on the liver. Current in vivo and in vitro studies indicate that differences in aflatoxin responses among different animals can be attributed to differences in their metabolism. The metabolic rate and the formation of intermediate products are important factors determining the type of toxicity of aflatoxin B1. Based on these criteria, monkeys and humans are more susceptible to acute aflatoxin poisoning but relatively resistant to carcinogenic effects. On the other hand, animals such as sheep and mice are more susceptible to carcinogenic effects. Aflatoxin B1 requires metabolic activation by cytochrome P450-dependent mixed-function oxidases to be converted into active 2,3-epoxides, the ultimate carcinogen. Aflatoxins, such as aflatoxin B1, are genotoxic carcinogens whose active metabolites react with DNA. In intracellular reactions, the main adduct formed with DNA is formed at position 2 of aflatoxin B1 and position N-7 of guanine in DNA. CYP1A2, 2B6, 3A4, 3A5, 3A7, and GSTM1 are enzymes that mediate aflatoxin metabolism in the human body. The overall contribution of these enzymes to aflatoxin B1 metabolism in the body depends not only on their affinity but also on their expression levels in the human liver, with CYP3A4 being the dominant enzyme. This enzyme mediates the formation of exo-epoxides and aflatoxin Q1, while CYP1A2 can generate some exo-epoxides but also a large amount of endo-epoxides and aflatoxin M1. In vitro experimental evidence shows that both enzymes are involved in the metabolism of aflatoxin in the human body, a conclusion confirmed by biomarker studies. Aflatoxins M1 and Q1, produced by CYP1A2 and 3A4 respectively, are present in the urine of individuals exposed to aflatoxin. Aflatoxins are secondary metabolites produced by a group of fungal strains (mainly Aspergillus and Penicillium). These fungal toxins are difuranocoumarin derivatives and, based on their blue or green fluorescence under ultraviolet light and chromatographic separation results, can be divided into four main products: B1, B2, G1, and G2. Aflatoxins B1 and B2, after being metabolized in mammals, produce two metabolites, M1 and M2, which are hydroxylated derivatives of the parent compound… Aflatoxin B3 is produced in Rhizopus… Aflatoxin gm1 is produced in rats.
Intravenous injection of aflatoxin B1, aflatoxin B2, and aflatoxin G1 in rats resulted in their rapid metabolism into seven metabolites, six of which were excreted via bile. All three toxins were hydroxylated at positions 2 and 4. The bile of rats treated with aflatoxin G1 contained glucuronide.
...Incubation of human liver microsomes with aflatoxin B1 or aflatoxin G1...produced genotoxic metabolites that induced the expression of the umuC gene in Salmonella typhimurium (TA-1535/psK1002). The genotoxicity was ranked as...aflatoxin B1>aflatoxin G1. Incubation with an anti-p450NF polyclonal antibody...completely inhibited aflatoxin microsomal activation, and immunochemical assays of p450NF (nifedipine oxidase) in the liver microsomal formulation were correlated with...microsomal activation of aflatoxin G1 and aflatoxin B1. P450NF converts aflatoxin into genotoxic metabolites in a recombinant monooxygenase system containing a purifying enzyme and an NADPH-generating system. ...
Aflatoxin is metabolized in the liver to less toxic metabolites via a cytochrome P-450-dependent multi-substrate monooxygenase system. The main metabolic reactions of aflatoxin are hydroxylation, oxidation, and demethylation. (A2973)

Toxicity Summary
Aflatoxins produce singlet oxygen under ultraviolet (365 nm) irradiation. Singlet oxygen, in turn, activates aflatoxins, making them mutagens and DNA binders. Aflatoxin metabolites can intercalate into DNA and alkylate bases through their epoxy groups, particularly binding to N7-guanine bases. In addition to randomly mutagenic DNA, this binding is also thought to cause mutations in the p53 gene. Mutations in the p53 gene can halt cell cycle progression or signal apoptosis. (L1877, A2859, A2972) Interactions Low concentrations (100 ppb [AN] of each aflatoxin) of aflatoxins B1, B2, G1, and G2 were added to the diet of Baladi rabbits. Another group of rabbits was fed the same contaminated diet with an additional 0.25% activated charcoal (CC). Both groups of rabbits were compared to a control group fed a diet without AN. Adding AN to the diet reduced feed intake, water consumption, body weight, and survival rate. Compared to the AN group, the activated charcoal group slightly increased feed intake, water consumption, and growth rate. However, the activated charcoal group had a greater effect on the digestibility of organic matter than the AN group. The relative weights of the liver, kidneys, heart, and adrenal glands in both the AN and activated charcoal groups were significantly higher than those in the control group. The hemoglobin content, hematocrit percentage, and erythrocyte sedimentation rate were lower in the AN group than in the control group. Despite increases in the AN group, serum calcium, inorganic phosphorus, cholesterol, phospholipids, and alanine aminotransferase (ALT) levels were elevated, while serum nitrogen and aspartate aminotransferase (AST) levels were decreased. Activated charcoal mitigated the effects of AN on serum nitrogen, GPT, and phospholipids. Chemical analysis showed that the AN group animals had increased water, ash, and silica content in the liver and increased water content in the muscle. On the other hand, the liver fat content, AST and vitamin A content, and muscle ash content were decreased. Adding activated charcoal (CC) to the diet mitigated the effects of aflatoxin (AN) on liver fat, ash, and silica, but led to increased water content in the liver and muscle, as well as increased GPT activity in the liver. Activated charcoal also significantly reduced vitamin A content in the liver. Aflatoxin treatment (AN and CC groups) reduced ash, silica, and magnesium content in bones, as well as bone volume. Activated charcoal administration increased calcium content in bones. Aflatoxin feeding (AN group) resulted in a high AN residue rate. The aflatoxin content ratio in muscle, serum, liver, heart, and kidney was 51:24:3:2:1, respectively. Only 1.42% of the feed was excreted in feces. Activated charcoal was effective because it prevented aflatoxin accumulation in organs. Aflatoxin-contaminated feed (AN and CC groups) caused paralysis, disordered fat deposition, discoloration, and hemorrhage in some organs. Scanning electron microscopy showed that neither AN nor AN + CC had adverse effects on the surface structure of the small intestine. Pathological examination revealed that the primary affected organs were the liver, heart, and spleen… Lesions included round cell infiltration of the liver, irregular lobular structure, focal necrosis, and periportal fibrosis. A more subtle effect of aflatoxin intake is its synergistic or antagonistic effect with various vitamins. In a mink experiment, mink were fed diets containing 0, 34, or 102 ppb (μg/kg) of aflatoxin, with or without the addition of 0.5% hydrated calcium aluminum silicate and/or 1.0% activated charcoal, for 77 days. Results showed that the mortality rate of mink fed a diet containing 34 ppb of aflatoxin was 20%, while all mink fed a diet containing 102 ppb of aflatoxin died within 53 days. Adding activated charcoal to a diet containing 102 ppb of aflatoxin reduced mortality and prolonged survival in mink, while adding hydrated calcium aluminum silicate alone or in combination with activated charcoal prevented death. Histological examination of the liver and kidneys of mink showed that mink fed a diet containing 102 ppb of aflatoxin had extremely severe liver lesions, while those fed a diet containing 34 ppb of aflatoxin had mild to moderate liver lesions. Adding sodium calcium aluminum silicate hydrate and/or activated charcoal to the diet containing 102 ppb of aflatoxin reduced or essentially eliminated the histopathological damage to the liver. No histopathological changes related to the diet treatment were observed in the kidneys. A long-term feeding experiment with rats for 18 months determined the effectiveness of ammoniation in detoxifying aflatoxin-contaminated peanut oil cake. Pressurized ammonia gas significantly reduced the aflatoxin content in the oil cake, decreasing it from 1000 ppb to 140 ppb at 2 bar and to 60 ppb at 3 bar. No rebound of aflatoxin levels was observed during the experiment. The incidence of liver tumors in untreated oil cake was very high, but decreased rapidly after treatment with moderate pressure, reaching zero after treatment with 3 bar pressure. A satisfactory dose-response relationship was found between the residual aflatoxin content in the oil cake and the observed incidence of liver tumors. These results indicate that ammonia treatment is a practical method for addressing the carcinogenicity of contaminated oil cake. For more complete data on interactions of aflatoxins (11 in total), please visit the HSDB record page. Non-human toxicity values: Oral LD50 in monkeys: 1750 μg/kg; Intramuscular LD50 in monkeys: 2020 mg/kg; Intraperitoneal LD50 in rats: 14,900 μg/kg. The Committee reaffirmed the conclusions of the 49th JECFA meeting that, based on experimental animal studies and human epidemiological studies, aflatoxins are among the most potent known mutagens and carcinogens, and that hepatitis B virus (HBV) infection is a key factor in aflatoxin-induced liver cancer. At its eighty-third session, the Committee also assessed co-exposure to aflatoxin and fumonisin. Both fumonisin and aflatoxin are common contaminants in cereals and cereal products. Aflatoxin is a common contaminant in peanuts and nuts. In areas where these foods are frequently consumed, people are likely to be simultaneously exposed to both mycotoxins. Although previous and laboratory animal evidence in this assessment suggests an additive or synergistic effect of co-exposure to fumonisin and aflatoxin in the development of precancerous lesions or hepatocellular carcinoma, there are currently no data on such effects in humans. The Committee concluded that the available data offer little support that co-exposure is a contributing factor to human disease. However, the interaction between aflatoxin B1 (AFB1), a compound known to be genotoxic, and fumonisin, which has the potential to induce regenerative cell proliferation (especially at exposure levels above PMTDI), remains a concern. This is because of the high incidence of chronic liver disease and the high rates of stunting in areas of the world with high mycotoxin exposure, and biomarkers have confirmed co-exposure to mycotoxins.

[1]. Aflatoxins: Implications on Health. Indian J Clin Biochem. 2017 Jun;32(2):124-133.

[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 G belongs to the coumarin class of compounds. Aflatoxin G1 has been reported to be present in corn, Aspergillus flavus, and Aspergillus parasiticus, and relevant data are available for reference. Aflatoxins are a class of fungal toxins, primarily produced by Aspergillus flavus and Aspergillus parasiticus. Their toxic metabolites consist of a fused coumarin-bis(dihydrofuran) ring structure, exhibiting potential acute and chronic toxicity. Acute exposure to high concentrations of aflatoxin can lead to liver damage. Long-term exposure to aflatoxin increases the risk of liver cancer, possibly due to increased DNA damage caused by aflatoxin metabolites through DNA intercalation and epoxide-mediated nucleoalkylation, potentially leading to mutations. Aflatoxin G is a fungal toxin produced by Aspergillus flavus and Aspergillus parasiticus. Aflatoxin G belongs to the difuranocoumarin lactone class of compounds. These compounds are polycyclic aromatic compounds whose structure includes a δ-pentanolactone ring fused to the coumarin portion of the difuranocoumarin backbone. Difuranocoumarolide is a subclass of aflatoxins and related compounds.
See also: Aflatoxin G1 (note moved to).

---

Mechanism of Action
Studies have shown that the most likely mechanism of action of aflatoxins is their interaction with DNA and inhibition of polymerases responsible for DNA and RNA synthesis.
...Aflatoxin 8,9-oxide covalently binds to the N-7 position of guanine, causing guanine with an adduct to pair with adenine instead of cytosine, resulting in a wrong codon and the insertion of the wrong amino acid into the protein. This type of event is associated with aflatoxin-induced mutations in the ras proto-oncogene and p53 tumor suppressor gene. /Aflatoxin 8,9-oxide/
...In this study, isoproterenol (ISO, 4x10⁻⁹), AFB(1) (3x10⁻⁶ and 6x10⁻⁵ M), and AFG(1) (3x10⁻⁶ and 6x10⁻⁶ M) caused isolated guinea pig atria to contract, making the preparation hypersensitive to ISO. These properties of aflatoxins are noteworthy because they may be the cause of some of the cardiotoxic effects described in the literature.
A G-to-T transversion of codon 249 of the p53 tumor suppressor gene was found in human liver tumors and experimental animals in areas of high-risk exposure to aflatoxins.
The ability of aflatoxin B1, aflatoxin B2, and aflatoxin G1 to inhibit RNA polymerase activity and reduce RNA content in rat hepatocyte nuclei is similar in nature to the carcinogenic effects and acute and subacute toxic effects of these compounds. Aflatoxin G1 induces rapid macroscopic separation of the fibrous and granular portions of the hepatocyte nucleolus. In vitro studies of human liver cells have shown that the primary catalyst for the bioactivation of the hepatotoxic aflatoxin B1 into a genotoxic 2,3-epoxide derivative is cytochrome P-450NF, a previously identified protein that also catalyzes the oxidation of nifedipine and other dihydropyridine drugs, quinidine, macrolide antibiotics, various steroids, and other compounds. Cytochrome P-450NF, or closely related proteins, also appear to be key catalysts for the activation of aflatoxin G1 and ochratoxin, in which ochratoxin exhibits greater genotoxicity than aflatoxin B1. Various drugs and diseases are known to affect the levels and activity of cytochrome P-450NF in the human liver, and the activity of this enzyme can be assessed using non-invasive assays. These findings provide a testing system for the hypothesis that a specific human disease state (liver cancer) is associated with higher levels of oxidative metabolism in individuals with high aflatoxin intake.
Aflatoxin B1, aflatoxin G1, and aflatoxin G2 inhibited the incorporation of (14)-carbon-labeled orotic acid into rat liver slice RNA at a toxin concentration of 100 μmol/3 mL. The corresponding inhibition rates were approximately 90%, 40%, and 20%, respectively. Aflatoxin B1 (20 μmol/3 mL), aflatoxin G1 (150 μmol/3 mL), and aflatoxin G2 (230 μmol/3 mL) inhibited the incorporation of 14-carbon-labeled DL-leucine into rat liver slice proteins by 32%, 35%, and 38%, respectively.
The phagocytic activity of rat peritoneal macrophages exposed to different concentrations and durations of aflatoxin B1, B2, G1, G2, B2a, and M1, the intracellular killing effect on Candida albicans, and the production of superoxide were analyzed to assess the inhibitory intensity of each mycotoxin. All very low concentrations of aflatoxins inhibit macrophage function. In macrophages exposed to aflatoxin B1 and M1, phagocytosis, intracellular killing, and spontaneous superoxide production are significantly impaired. Among various toxic aflatoxins, aflatoxin B1 and G1 have the strongest biological activity, but other derivatives are also carcinogenic. Aflatoxin B1 requires metabolic activation by a cytochrome P450-dependent mixed-function oxidase to be converted into active 2,3-epoxide, the final carcinogen. Aflatoxins, such as aflatoxin B1, are genotoxic carcinogens; their active metabolites can react with DNA. In intracellular reactions, the main adduct formed with DNA is formed by the binding of aflatoxin B1 at position 2 to the N-7 position of guanine in DNA.

C17H12O7

328.27298

328.058

1165-39-5

Aflatoxin G1-13C17;1217444-07-9

14421

White to off-white solid powder

1.6±0.1 g/cm3

612.1±55.0 °C at 760 mmHg

244-246ºC

274.1±31.5 °C

0.0±1.8 mmHg at 25°C

1.680

-0.17

0

7

1

24

666

0

COC1=C2C3=C(C(=O)OCC3)C(=O)OC2=C4C5C=COC5OC4=C1

XWIYFDMXXLINPU-UHFFFAOYSA-N

InChI=1S/C17H12O7/c1-20-9-6-10-12(8-3-5-22-17(8)23-10)14-11(9)7-2-4-21-15(18)13(7)16(19)24-14/h3,5-6,8,17H,2,4H2,1H3

11-methoxy-6,8,16,20-tetraoxapentacyclo[10.8.0.02,9.03,7.013,18]icosa-1,4,9,11,13(18)-pentaene-17,19-dione

AFLATOXIN G1; Aflatoxin; 1165-39-5; 1402-68-2; AFLATOXINS; Aflatoxin G; Aflatoxin, crude; Aflatoxin G1-d3;

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 : ~10 mg/mL (~30.46 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.

---

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).

View More

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)

---

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).

View More

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

---

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.

---

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