Ochratoxin A Racemate   中文名称: 曲霉素A

Mycotoxin

赭曲霉毒素A（OTA）是一种普遍存在的真菌代谢产物，具有致肾、致癌和致畸作用。流行病学研究表明，OTA可能参与不同形式的人类肾病的发病机制。之前我们已经证明，OTA在C7克隆中激活了细胞外信号调节激酶1和2，它们是丝裂原活化蛋白激酶（MAPK）家族的成员，但在肾上皮Madin-Darby犬肾（MDCK）细胞的C11克隆中没有激活。在这里，我们表明，纳摩尔浓度的OTA导致MAPK家族第二个成员的激活，即MDCK-C7细胞中的c-jun氨基末端激酶（JNK），但在MDCK-C11细胞中几乎没有激活，如激酶测定和蛋白质印迹所确定的。此外，OTA增强了肿瘤坏死因子α对JNK活化的影响。与对JNK的影响并行，纳摩尔OTA在MDCK-C7细胞中诱导了凋亡，但在MDCK-C11细胞中没有诱导凋亡，这是通过DNA断裂、DNA梯状结构和胱天蛋白酶激活来确定的。此外，OTA增强了肿瘤坏死因子α的促凋亡作用。我们的数据提供了额外的证据，表明在没有观察到急性毒性作用的浓度下，OTA以细胞类型特异性的方式与MAPK家族的不同成员相互作用。通过JNK途径诱导细胞凋亡可以解释OTA诱导的肾功能变化及其部分致畸作用[3]。

钙稳态的破坏导致细胞质钙水平的持续升高，这与不同细胞类型对各种试剂的细胞毒性反应有关。我们观察到，对大鼠施用单次高剂量或多次低剂量的致癌肾毒素赭曲霉毒素a/ochratoxin A（OTA）会导致肾皮质内质网ATP依赖性钙泵活性的增加。增加非常迅速，在OTA给药后10分钟内明显，此后至少6小时内保持升高。钙泵活性的增加与之前的观察结果不一致，即OTA会增强体内脂质过氧化（乙烷呼气），这是一种已知会抑制钙泵的情况。然而，由于丙二醛和各种抗氧化酶（包括过氧化氢酶、DT黄递酶、超氧化物歧化酶、谷胱甘肽过氧化物酶、谷胱甘肽还原酶和谷胱甘肽S-转移酶）的水平没有改变或降低，因此肾皮质中没有观察到脂质过氧化增强的证据。在体外研究中，在钙摄取过程中向皮质微粒体中添加OTA会抑制摄取过程，尽管这种作用是可逆的。微粒体与NADPH的预孵育对钙摄取有显著的抑制作用，但OTA的加入能够逆转这种抑制作用。微粒体钙摄取速率的变化与磷酸化Mg2+/Ca（2+）-ATP酶中间体稳态水平的变化相关，表明体内/体外条件正在影响酶磷酸化速率[4]。

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本研究使用免疫亲和柱净化和高效液相色谱荧光检测（IAC-LC-FLD）评估了发酵乳清（FW）和南瓜（P）对大鼠黄曲霉毒素B1（AFB1）和赭曲霉毒素A/ochratoxin A（OTA）排泄的影响。该方法实现了AFB1和OTA的检测限分别为0.1µg/kg和0.3µg/kg，AFB1的回收率为72-92%，OTA的回收率在88-98%之间。对100只大鼠的粪便分析显示，AFB1的峰值浓度为418µg/kg，OTA为1729µg/kg。在毒素暴露组中，OTA水平高于AFB1，仅OTA组的雄性大鼠OTA（1729±712µg/kg）明显高于雌性大鼠（933±512µg/kg）。在仅AFB1组中，雄性粪便水平为52±61µg/kg，雌性为91±77µg/kg。AFB1+FW组显示出显著的AFB1浓度（雄性211±51µg/kg，雌性230±36µg/kg）。FW+P组合进一步影响排泄，AFB1和OTA水平较高。这些发现表明，FW和P调节霉菌毒素排泄，可能在霉菌毒素解毒中发挥作用，为减少霉菌毒素暴露及其有害影响的饮食策略提供了见解[6]。

Caspase-3活性测定[5]
根据文献17进行测定，将16个细胞接种在24孔板中（2×104个细胞/孔）。毒素孵育后，用冷PBS缓冲液洗涤细胞，并在冰上用100μL细胞裂解缓冲液（10 mM TRIS、100 mM NaCl、1 mM EDTA、0.1%Triton X-100，pH 7.5）孵育15分钟。细胞裂解物在4°C下以7000g离心10分钟。将50微升上清液与含有8μM荧光半胱氨酸天冬氨酸蛋白酶-3底物（Ac-Asp-Glu-Val-Asp-7-氨基-4-三氟甲基香豆素，DEVD-AFC）的50μL反应缓冲液（50 mM PIPES，10 mM EDTA，0.5%CHAPS，10 mM DTT）在37°C下孵育1小时。用酶标仪测量蛋白水解切割释放的7-氨基-4-三氟甲基香豆素（AFC）的荧光（激发：λ=400 nm；发射：λ=505 nm）。使用AFC标准品对释放的AFC浓度进行定量以进行校准，并将其归一化为每个样品中的细胞蛋白含量。使用牛血清白蛋白（BSA）作为校准标准，用双辛可宁酸测定法测定蛋白质浓度。

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乳酸脱氢酶释放试验[5]
根据文献进行测定。18个细胞接种在24孔板中（2×104个细胞/孔）。毒素孵育后，用冷PBS缓冲液洗涤细胞，并在冰上用100μL细胞裂解缓冲液（10 mM TRIS、100 mM NaCl、1 mM EDTA、1%Triton X-100，pH 7.5）孵育15分钟。细胞裂解物在4°C下以7000g离心10分钟。细胞裂解物和细胞培养基的一致样品用反应缓冲液（2μM NADH、10 mM丙酮酸盐、100 mM HEPES，pH 7.0）孵育。每2分钟测量一次λ=340 nm处NADH相关吸收的减少，以使用带温度控制的微孔板读数器在37°C下监测酶反应60分钟的动力学参数。因此，计算每个样本的总细胞乳酸脱氢酶（LDH）酶和释放的LDH的量，并将其归一化为阳性和阴性对照。

细胞毒性试验[5]
根据文献和制造商的说明，使用细胞计数试剂盒-8（CCK-8）比色法评估了ochratoxin A/赭曲霉毒素A衍生物的细胞毒性。简而言之，将细胞接种在96孔微孔板上（4×103个细胞/孔）。毒素暴露后，将染料溶液（WST-8）加入细胞中，然后在37°C下孵育1小时。活细胞的细胞脱氢酶对WST-8染料的还原增加了λ=450nm处的吸光度，并用酶标仪进行了测量。毒素处理细胞的结果被标准化为未处理阴性对照的值。

In Vivo Study Design [6]
The current study builds upon the experimental framework established by Vila-Donat et al, investigating the effects of bioactive dietary ingredients on the urinary excretion of AFB1 and ochratoxin A/OTA in Wistar rats. The animals were housed under standard laboratory conditions (regulated temperature, humidity, and a 12 h light/dark cycle) and were provided ad libitum access to water and the assigned diets. Ethical guidelines for animal welfare were strictly followed throughout the experiment.

The study involved 120 rats (60 males and 60 females), divided into 12 groups, with each assigned a tailored diet. The diets included control feeds and feeds contaminated with AFB1 and ochratoxin A/OTA, which were prepared using grains inoculated with mycotoxin-producing fungi (A. flavus for AFB1 and A steynii for OTA). In addition, bioactive dietary ingredients, such as 1% P and 1% FW, were supplemented in specific diets to evaluate their impact on the fecal excretion of mycotoxins.

Fecal samples were periodically collected over the 28-day study period to measure AFB1 and ochratoxin A/OTA levels, facilitating the assessment of the effects of dietary interventions on mycotoxin elimination. Mycotoxin quantification in the feeds was conducted using LSE and LC-FLD methods. The detailed experimental procedures, including feed preparation recipes and the specific concentrations of mycotoxins in the feeds, have been previously detailed in Vila-Donat et al.

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Extraction of AFB1 and ochratoxin A/OTA from Feces [6]
The extraction of mycotoxins from feces was carried out using a clean-up process with the AflaOchra IAC, as described by Rodrigues et al. (2019) with slight modifications. The feces samples were first thawed and homogenized by grinding, and then, 1 g was mixed with 20 mL of 80% MeOH. The mixture was stirred for 45 min on a digital magnetic plate, followed by centrifugation at 4000 rpm for 10 min. Afterward, 10 mL of PBS were added to 2 mL of the supernatant, and the resulting buffered solution was purified using the AflaOchra IAC. For extraction, a vacuum-based SPE system was used, designed to concentrate, purify, or isolate analytes from complex matrices. Key components include a glass chamber and waste container, a position cover with luer adapters and a seal, luer connectors, stopcocks, guide needles, posts, shelves, a pressure gauge, and a mounting valve. The system accommodates 12 or 24 samples, ensuring efficient and precise sample preparation.

The column setup included a 10 mL syringe on top, an adapter, and a stopcock at the bottom to regulate the flow at 1 drop per second. Buffered samples were loaded into these columns. Then, the columns were washed, and finally, the elution of the compounds of interest was performed. AFB1 and ochratoxin A/OTA were eluted using a 1.5 mL mixture of MeOH and H2O (1:1, v/v) and collected in a glass vial. After the elution, air is passed through the system using the glass vacuum collector to ensure that all residual eluate is completely removed from the IAC columns. The extracted samples were then transferred to vials and directly injected into the LC-FLD system, as described in the following sections (Figure 4).

Absorption, Distribution and Excretion
Since there are pathomorphological similarities between porcine mycotoxic nephropathy caused by ochratoxin A and Balkan endemic nephropathy (BEN), it has been suggested that the same aetiological agent has a role in BEN. Based on the results from several field and experimental studies carried out on pigs, an appropriate analytical method of monitoring possible human exposure to ochratoxin A was developed. The toxicokinetic properties of the toxin were species specific, although in all the animal species studied (with the exception of fish), as well as in humans, two binding proteins were found in the plasma. The monkey had the longest elimination half-life of the toxin, 510 hr, in contrast to the fish whose elimination half-life was only 0.68 hr. The fish kidney displayed a specific pattern of distribution. In the laying quail the most prominent observation was the accumulation of labelled ochratoxin A in egg yolk. Generally, (14C)ochratoxin A was eliminated rapidly from the quail body, but had a long retention time in the circulating blood in the mouse. Although the elimination of ochratoxin A from the body depending on its binding to plasma constituents, the existence of enterohepatic circulation might have been partially responsible for its prolonged retention and elimination from the body of mammals. The toxicokinetic profile of ochratoxin A did not contradict the mycotoxic hypothesis in the aetiology of BEN. PMID:1618443 Fuchs R, Hult K; Food Chem Toxicol 30 (3): 201-4 (1992)

... Ochratoxin A is rapidly absorbed throughout the entire gastrointestinal tract and distributes itself in the body as a two compartment open model and has a particular high affinity for serum albumin. Ochratoxin A is hydrolyzed by the intestinal microflora into nontoxic compounds (7-carboxy-5-chloro-8-hydroxy-3,4-dihydro-3R-methylisocoumarin (Ochratoxin alpha) and phenylalanine). It is excreted as either ochratoxin A, hydroxylated ochratoxin A or Ochratoxin alpha in both the urine and feces. Ochratoxin A appears to exert its toxic effect by promoting an increased level of lipid peroxidation by inhibition of an amino acylation reaction and possibly by conversion into metabolites that are capable of binding DNA. These in turn cause other secondary effects associated with ochratoxin A. It would appear that this compound presents a true potential hazard for humans as its occurrence is wide spread and it is highly carcinogenic. PMID:2200593 Marquardt RR et al; Can J Physiol Pharmacol 68 (7): 991-9 (1990)

Rats intubated daily with 500 ug ochratoxin A or fed 250 ug daily in barley. There was little accumulation of cmpd in liver or kidneys. Avg total amount excreted daily in urine & feces was just over 10% of administered dose. Small amount of hydrolysis product also excreted. VAN WALBEEK W ET AL; TOXICOL APPL PHARMACOL 20 (3): 439 (1971)

Rats given single ip injection of 1 mg ochratoxin A labelled with (14)C. Reached highest levels in serum (90%), liver (4.5%), & kidney (4.4%) 30 min later. Ochratoxin A was excreted primarily in urine as unchanged toxin or metabolites. Excretion in feces less significant. PMID:892675 CHANG FC, CHU FS; FOOD COSMET TOXICOL 15 (3): 199 (1977)

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Metabolism / Metabolites ... Ochratoxin A is hydrolyzed by the intestinal microflora into nontoxic compounds (7-carboxy-5-chloro-8-hydroxy-3,4-dihydro-3R-methylisocoumarin (Ochratoxin alpha) and phenylalanine). It is excreted as either ochratoxin A, hydroxylated ochratoxin A or Ochratoxin alpha in both the urine and feces. ... PMID:2200593 Marquardt RR et al; Can J Physiol Pharmacol 68 (7): 991-9 (1990)

Hydroxyochratoxin A was isolated & identified from urine of rats after injection with ochratoxin A. By incubating ochratoxin A with rat liver microsomes & NADPH, 1 major (90%) & 2 minor metabolites, more polar than ochratoxin A, were formed. PMID:7396488 Full text: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC291461 STORMER FC, PEDERSEN JI; APPL ENVIRON MICROBIOL 39 (5): 971 (1980)

Single oral or iv dose (2.5 mg/kg) ochratoxin A admin to healthy adult rats. Ochratoxin alpha only metabolite recovered from cecum & large intestine. Ochratoxin A excreted via urine & feces, both as free drug & ochratoxin alpha. Unidentified metabolites in urine. PMID:43233 GALTIER P ET AL; DRUG METAB DISPOS 7 (6): 429 (1979)

Ochratoxin A is cleaved into phenylalanine and a less toxic iso-coumarin derivative (ochratoxin alpha) by the microbial flora of the colon ... and by carboxypeptidase A and alpha-chymotrypsin ... . IARC. Monographs on the Evaluation of the Carcinogenic Risk of Chemicals to Humans. Geneva: World Health Organization, International Agency for Research on Cancer, 1972-PRESENT. (Multivolume work). Available at:

Ochratoxin A is cleaved into phenylalanine and a less toxic iso-coumarin derivative (ochratoxin alpha) by the microbial flora of the colon, and by carboxypeptidase A and alpha-chymotrypsim. This is is the major metabolic pathway. 4-Hydroxyochratoxin A is the main hepatic metabolite and its formation appears to be via a polymorphic-like debrisoquine 4-hydroxylation. Some cytochrome P-450 enzymes, such as CYP2C9, and known to metabolize ochratoxin A into more cytotoxic compounds. (T35, A2870, A3099)

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Biological Half-Life Pregnant ICR mice were administered a single ip injection of 5 mg/kg ochratoxin A (OA) on day 11 or 13 of gestation. The half-life of OA in serum was calculated to be 28.7 hr on day 11 and 23.6 hr on day 13 of gestation. Fukui Y et al; Fd Chem Toxic 25: 17-24 (1987)

In rats ... the plasma half-life of ochratoxin A is about 60 hr ... . IARC. Monographs on the Evaluation of the Carcinogenic Risk of Chemicals to Humans. Geneva: World Health Organization, International Agency for Research on Cancer, 1972-PRESENT. (Multivolume work). Available at: https://monographs.iarc.fr/ENG/Classification/index.php, p. V31 198 (1983)

The apparent plasma elimination half-time of ochratoxin A after oral administration at 50 ug/kg bw varied from 0.68 hr in fish to 120 hr in rats and 510 hr in monkeys ... . IARC. Monographs on the Evaluation of the Carcinogenic Risk of Chemicals to Humans. Geneva: World Health Organization, International Agency for Research on Cancer, 1972-PRESENT. (Multivolume work). Available at: https://monographs.iarc.fr/ENG/Classification/index.php, p. V56 499 (1993)

The fate of ochratoxin A has been studied in laboratory rodents and in breeding animals. In rats, orally administered ochratoxin A is readily absorbed, and considerable amounts of the toxin are detected in plasma, where maximal concentrations occur 2-4 hr after administration. Pharmacokinetic analysis of curves of plasma level versus time suggests its distribution in two distinct body compartments. The half-time of the toxin depends on both the dose and the animal species, varying from 0.7 hr in fish to 840 h in monkeys. In plasma, the toxin is bound to albumin, like many acidic compounds. This interaction is competitively inhibited by phenylbutazone, ethylbiscoumacetate and sulfamethoxy-pyridazine and is decreased in albumin-deficient rats. Galtier P; IARC Sci Publ (115): 187-200 (1991)

The toxicokinetic properties of the toxin were species specific, although in all the animal species studied (with the exception of fish), as well as in humans, two binding proteins were found in the plasma. The monkey had the longest elimination half-life of the toxin, 510 hr, in contrast to the fish whose elimination half-life was only 0.68 hr. The fish kidney displayed a specific pattern of distribution. In the laying quail, the most prominent observation was the accumulation of labelled ochratoxin A in egg yolk. Generally, (14)C-ochratoxin A was eliminated rapidly from the quail body, but had a long retention time in the circulating blood in the mouse. Although the elimination of ochratoxin A from the body depending on its binding to plasma constituents, the existence of enterohepatic circulation might have been partially responsible for its prolonged retention and elimination from the body of mammals. The toxicokinetic profile of ochratoxin A did not contradict the mycotoxic hypothesis in the etiology of BEN. PMID:1618443 Fuchs R, Hult K; Food Chem Toxicol 30 (3): 201-4 (1992)

Metabolism / Metabolites
3s14s-ochratoxin a has known human metabolites that include 2-[(5-Chloro-4,8-dihydroxy-3-methyl-1-oxo-3,4-dihydroisochromene-7-carbonyl)amino]-3-phenylpropanoic acid.

Toxicity Summary
Ochratoxin A has been shown to be weakly mutagenic, possibly by induction of oxidative DNA damage. The nephrotoxin ochratoxin A (OTA) causes a reduction of glomerular filtration rate (GFR) and of para-aminohippuric acid (PAH) clearance. It is a nephrotoxin which blocks plasma membrane anion conductance in Madin-Darby canine kidney (MDCK) cells. Some cytochrome P-450 enzymes, such as CYP2C9, are known to metabolize ochratoxin A into more cytotoxic compounds capable of forming DNA adducts. (A2869, A3099)

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Evidence for Carcinogenicity
Evaluation: There is inadequate evidence in humans for the carcinogenicity of ochratoxin A. There is sufficient evidence in experimental animals for the carcinogenicity of ochratoxin A. Overall evaluation: Ochratoxin A is possibly carcinogenic to humans (Group 2B).

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Toxicity Data
LD50: 20 mg/kg (Oral, Rat) (A716) LD50: 12,600 ug/kg (Intraperitoneal, Rat) (A716) LD50: 12,750 ug/kg (Intravenous, Rat ) (A716) LD50: 46 mg/kg (Oral, Mouse) (A716)

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Treatment
Care is symptomatic and supportive. (A704)

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Interactions
... Ochratoxin A (OA) toxicity and the effect of supplemental ascorbic acid (AA) /was examined/ in laying hens housed under two environmental temperatures. /Two groups of/ ... 24 hens were randomly assigned to four dietary treatments in six replications. Treatments consisted of a control and three diets containing either 300 ppm AA, 3 ppm OA, or 300 ppm AA plus 3 ppm OA. Treatment diets were fed for 14 days following the feeding of the basal diet for 14 days. The test period temperature was 25 °C ... /for the first group/ and 33 °C in ... /the other group/. ... When laying hens were fed 3 ppm OA compared with those fed the control diet/, there were significant reductions in feed intake, body weight change, and egg production, and increased shell elasticity/. An analysis of plasma constituents showed that OA also increased Cl- concn and aspartate transaminase activity and decreased plasma calcium concentrations. Exposing hens to 33 °C (compared with 25 °C) appeared to aggravate the negative effects of OA. All the negative effects of OA, apart from body-weight changes, reductions in feed intake, and increases in egg shell elasticity at 33 °C were either moderated or significantly ... reversed by dietary AA supplementation. ... The results /indicate/ that the detrimental effects of OA in the diet of the laying hen can be counteracted by dietary /administration/ of AA. Haazele FM et al; Can J Anim Sci 73 (1): 149-57 (1993)

Aldrin concentration increased in liver of neonatal rats during 1st 6 hr after oral administration then decreased over 72 hr to less than 0.1% dose. Aldrin and ochratoxin given together; aldrin increased 1st 6 hr then decreased to 0.4% dose over 18 hr. FARB RM ET AL; PESTIC ENVIRON: CONTINUING CONTROVERSY, PAP INTER-AM CONF TOXICOL OCCUP MED, 8TH; 179 (1973)

Dieldrin, detected 2 hr after administration of aldrin to neonatal rats, increased to max 30% of initial aldrin by 18 hr. Aldrin & ochratoxin given together; dieldrin increased from 10% of aldrin dose at 2 hr to max 50% at 24 hr. FARB RM ET AL; PESTIC ENVIRON: CONTINUING CONTROVERSY, PAP INTER-AM CONF TOXICOL OCCUP MED, 8TH; 179 (1973)

Rainbow trout fed a diet containing 20 ug ochratoxin A/kg of diet; together with sterculic acid, developed hepatomas (number unspecified). IARC. Monographs on the Evaluation of the Carcinogenic Risk of Chemicals to Humans. Geneva: World Health Organization, International Agency for Research on Cancer, 1972-PRESENT. (Multivolume work). Available at: https://monographs.iarc.fr/ENG/Classification/index.php, p. V10 193 (1976)

[1]. Role of dopamine and selective dopamine receptor agonists on mouse ductus arteriosus tone and responsiveness. Pediatr Res. 2019 Dec 9.

[2]. First synthesis of a stable isotope of Ochratoxin A metabolite for a reliable detoxification monitoring. Org Lett. 2013 Aug 2;15(15):3888-90.

[3]. Ochratoxin A induces JNK activation and apoptosis in MDCK-C7 cells at nanomolar concentrations. J Pharmacol Exp Ther . 2000 Jun;293(3):837-44.

[4]. Alterations in ATP-dependent calcium uptake by rat renal cortex microsomes following ochratoxin A administration in vivo or addition in vitro. Biochem Pharmacol. 1992 Oct 6;44(7):1401-9.

[5]. Total synthesis and cytotoxicity evaluation of all ochratoxin A stereoisomers. Bioorg Med Chem. 2010 Jan 1;18(1):343-7.

[6]. Impact of Bioactive Ingredients on the Fecal Excretion of Aflatoxin B1 and Ochratoxin A in Wistar Rats. Molecules. 2025 Feb 1;30(3):647.

(R)N-(5-Chloro-3,4-dihydro-8-hydroxy-3-methyl-1-oxo-1H-2-benzopyran-7-yl)phenylalanine has been reported in Aspergillus ochraceus.

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Due to its toxicity and presence in numerous food products, Ochratoxin A (OTA) has drawn attention for decades. This article summarizes the first synthesis of a labeled analogue of Ochratoxin α (OTα), one of the main products generated by the metabolization of OTA by microorganisms. This synthesis also led to a new labeled analogue of OTA with the deuteration located on the dihydroisocoumarin moiety allowing thus both the accurate quantification of OTA and OTα and the establishing of a reliable detoxification rate.[2]

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The mycotoxin ochratoxin A is a potent inhibitor of the protein biosynthesis and known to be cytotoxic in nanomolar concentrations. In order to investigate the relationship between stereochemistry and cytotoxicity of this compound, all four ochratoxin A stereoisomers have been synthesized. Using the liver cell line Hep G2, the compounds were tested for cytotoxic and apoptotic potential. It could be shown, that the l-configuration of the phenylalanine moiety of the molecule is mostly responsible for the high cytotoxicity of ochratoxin A while the stereocenter at the dihydroisocoumarine structure is of less importance.[5]

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This study highlights the potential of bioactive compounds like P and FW in modulating the excretion of mycotoxins, specifically AFB1 and OTA, in Wistar rats. The observed effects on toxin bioavailability may result from altered absorption dynamics or enhanced excretion processes, potentially driven by mechanisms like physical adsorption or biochemical interactions. While the exact pathways remain to be clarified, these findings open new avenues for using FW and P as dietary interventions to mitigate mycotoxin exposure. Further research is needed to deepen our understanding of these mechanisms and to assess the practical applications of these compounds in mycotoxin detoxification strategies. Moving forward, research should explore the impact of these dietary interventions on gut microbiota, as shifts in the microbial community may influence the body’s ability to metabolize and eliminate mycotoxins, thereby reducing their toxic effects. Understanding these interactions may provide innovative strategies for improving animal health and public safety. In conclusion, the results suggest that dietary modifications, including the use of bioactive ingredients, could offer promising solutions for managing mycotoxin contamination in agricultural systems and may serve as a crucial strategy to safeguard both food production and public health. [6]

C20H18NO6CL

403.81302

407.107

C, 59.49 H, 4.49 Cl, 8.78 N, 3.47 O, 23.77

303-47-9

12313657

White to off-white solid powder

1.4±0.1 g/cm3

632.4±55.0 °C at 760 mmHg

336.3±31.5 °C

0.0±2.0 mmHg at 25°C

1.628

Aspergillus ochraceus

4.31

113

3

6

5

28

608

2

C[C@@H]1CC2=C(C(O1)=O)C(O)=C(C(N[C@H](C(O)=O)CC3=CC=CC=C3)=O)C=C2Cl

RWQKHEORZBHNRI-GENIYJEYSA-N

InChI=1S/C20H18ClNO6/c1-10-7-12-14(21)9-13(17(23)16(12)20(27)28-10)18(24)22-15(19(25)26)8-11-5-3-2-4-6-11/h2-6,9-10,15,23H,7-8H2,1H3,(H,22,24)(H,25,26)/t10?,15-/m1/s1 SMILES Code

(R)-N-((5-Chloro-3,4-dihydro-8-hydroxy-3-methyl-1-oxo-1H-benzo(c)pyran-7-yl)carbonyl)-3-phenylalanine

Ochratoxin-A; Antibiotic 9663; Alanine, (-)-; 1448049-50-0; ((R)-5-chloro-8-hydroxy-3-(methyl-d3)-1-oxoisochromane-7-carbonyl-3-d)-L-phenylalanine; (R)N-(5-Chloro-3,4-dihydro-8-hydroxy-3-methyl-1-oxo-1H-2-benzopyran-7-yl)phenylalanine; WLN: T66 BVOT & J D1 GG IVMYVQ1R & JQ; NSC-201422; L-Phenylalanine,4-dihydro-8-hydroxy-3-methyl-1-oxo-1H-2-benzopyran-7-yl)carbonyl]-, (R)-; EX-A1468; EX-A-1468; EX-A 1468; EXA1468; EXA-1468; EXA 1468

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)

May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples

注意: 如下所列的是一些常用的体内动物实验溶解配方，主要用于溶解难溶或不溶于水的产品（水溶度<1 mg/mL）。 建议您先取少量样品进行尝试，如该配方可行，再根据实验需求增加样品量。

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注射用配方1: DMSO : Tween 80： Saline = 10 : 5 : 85 (如： 100 μL DMSO → 50 μL Tween 80 → 850 μL Saline)
*生理盐水/Saline的制备：将0.9g氯化钠/NaCl溶解在100 mL ddH ₂ O中，得到澄清溶液。
注射用配方 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (如： 100 μL DMSO → 400 μL PEG300 → 50 μL Tween 80 → 450 μL Saline)
注射用配方 3: DMSO : Corn oil = 10 : 90 (如： 100 μL DMSO → 900 μL Corn oil)
示例: 以注射用配方 3 (DMSO : Corn oil = 10 : 90) 为例说明, 如果要配制 1 mL 2.5 mg/mL的工作液, 您可以取 100 μL 25 mg/mL 澄清的 DMSO 储备液，加到 900 μL Corn oil/玉米油中, 混合均匀。

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注射用配方 4: DMSO : 20% SBE-β-CD in Saline = 10 : 90 [如：100 μL DMSO → 900 μL (20% SBE-β-CD in Saline)]
*20% SBE-β-CD in Saline的制备（4°C，储存1周）：将2g SBE-β-CD (磺丁基-β-环糊精) 溶解于10mL生理盐水中，得到澄清溶液。
注射用配方 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (如： 500 μL 2-Hydroxypropyl-β-cyclodextrin (羟丙基环胡精)→ 500 μL Saline)
注射用配方 6: DMSO : PEG300 : Castor oil : Saline = 5 : 10 : 20 : 65 (如： 50 μL DMSO → 100 μL PEG300 → 200 μL Castor oil → 650 μL Saline)
注射用配方 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (如： 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
注射用配方 8: 溶解于Cremophor/Ethanol (50 : 50), 然后用生理盐水稀释。
注射用配方 9: EtOH : Corn oil = 10 : 90 (如： 100 μL EtOH → 900 μL Corn oil)
注射用配方 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (如： 100 μL EtOH → 400 μL PEG300 → 50 μL Tween 80 → 450 μL Saline)

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口服配方 1: 悬浮于0.5% CMC Na (羧甲基纤维素钠)
口服配方 2: 悬浮于0.5% Carboxymethyl cellulose (羧甲基纤维素)
示例: 以口服配方 1 (悬浮于 0.5% CMC Na)为例说明, 如果要配制 100 mL 2.5 mg/mL 的工作液, 您可以先取0.5g CMC Na并将其溶解于100mL ddH2O中，得到0.5%CMC-Na澄清溶液；然后将250 mg待测化合物加到100 mL前述 0.5%CMC Na溶液中，得到悬浮液。

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口服配方 3: 溶解于 PEG400 (聚乙二醇400)
口服配方 4: 悬浮于0.2% Carboxymethyl cellulose (羧甲基纤维素)
口服配方 5: 溶解于0.25% Tween 80 and 0.5% Carboxymethyl cellulose (羧甲基纤维素)
口服配方 6: 做成粉末与食物混合

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注意: 以上为较为常见方法，仅供参考， InvivoChem并未独立验证这些配方的准确性。具体溶剂的选择首先应参照文献已报道溶解方法、配方或剂型，对于某些尚未有文献报道溶解方法的化合物，需通过前期实验来确定（建议先取少量样品进行尝试），包括产品的溶解情况、梯度设置、动物的耐受性等。

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请根据您的实验动物和给药方式选择适当的溶解配方/方案：
1、请先配制澄清的储备液(如：用DMSO配置50 或 100 mg/mL母液(储备液));
2、取适量母液，按从左到右的顺序依次添加助溶剂，澄清后再加入下一助溶剂。以 下列配方为例说明 (注意此配方只用于说明，并不一定代表此产品 的实际溶解配方):
10% DMSO → 40% PEG300 → 5% Tween-80 → 45% ddH2O (或 saline);
假设最终工作液的体积为 1 mL, 浓度为5 mg/mL: 取 100 μL 50 mg/mL 的澄清 DMSO 储备液加到 400 μL PEG300 中，混合均匀/澄清；向上述体系中加入50 μL Tween-80，混合均匀/澄清；然后继续加入450 μL ddH2O (或 saline)定容至 1 mL；
3、溶剂前显示的百分比是指该溶剂在最终溶液/工作液中的体积所占比例;
4、 如产品在配制过程中出现沉淀/析出，可通过加热(≤50℃)或超声的方式助溶;
5、为保证最佳实验结果，工作液请现配现用！
6、如不确定怎么将母液配置成体内动物实验的工作液，请查看说明书或联系我们;
7、 以上所有助溶剂都可在 Invivochem.cn网站购买。