Use of a plant essential oil composition in the preparation of a mycotoxin-resistant medicament

Abstract

The application relates to application of a plant essential oil composition in preparation of an anti-mycotoxin medicine and relates to the technical field of medicines; the plant essential oil composition in the application can inhibit mycotoxin growth and toxicity of the mycotoxin, wherein limonene can effectively reduce DON concentration and toxin level, and carvacrol can inhibit aspergillus flavus growth and present dose dependence; the plant essential oil composition in the application can treat oxidative damage caused by mycotoxin to a body; the plant essential oil composition is a safe and green antioxidant, can inhibit growth of toxin-producing fungi and toxicity of mycotoxin, reduce oxidative stress caused by mycotoxin, and further prevent and control mycotoxin pollution.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology, and in particular to a plant essential oil composition and its application in the preparation of antimycotoxin drugs. Background Technology

[0002] Currently, mycotoxin contamination is widespread in feed, and feed sources typically include various grains susceptible to mold contamination. Furthermore, most fungi can produce several mycotoxins simultaneously, and the coexistence of multiple mycotoxins in feed and feed ingredients is quite common. Related technologies detect four or more mycotoxins in feed and feed ingredients, with detection rates of 99.1% for DON (deoxynivalenol), 92.1% for AFB1 (aflatoxin B1), and 95.1% for OTA (ochratoxin A).

[0003] Mycotoxin contamination of feed not only reduces its utilization and nutritional value but also induces poisoning in animals, causing varying degrees of damage to different systems and seriously endangering animal and human health. AFB1, primarily produced by the metabolism of Aspergillus flavus and Aspergillus parasiticus, is recognized worldwide as one of the most widely distributed, highly toxic, and harmful toxins, primarily affecting the liver and also severely damaging the gallbladder, kidneys, spleen, and other organs. DON, mainly produced by Fusarium graminearum and Fusarium oxysporum, primarily causes vomiting as a symptom of poisoning, with pigs being the most susceptible. OTA, a secondary metabolite produced during the metabolism of Aspergillus ochraceus and Penicillium spp., is a type 2B potential carcinogen, with pigs and poultry being the most susceptible, primarily affecting the kidneys and liver. DON, AFB1, and OTA can all induce oxidative stress and cause oxidative damage, and combined exposure to all three has a synergistic effect on cellular oxidative damage.

[0004] Plant essential oils (PEOs) are important secondary metabolites of plants, consisting of volatile aromatic substances extracted from plants using specific processes. In recent years, plant essential oils have been widely used as feed additives to replace antibiotics in livestock and poultry feed production, attracting attention from livestock farmers both domestically and internationally. Plant essential oils can improve the production performance of pigs and poultry, exhibiting antibacterial, antiviral, antioxidant, anti-inflammatory, immune-enhancing, nutrient absorption and development-promoting, and residue-free and drug-resistant properties. Their significant antioxidant capacity has led to their widespread use in livestock and poultry feed production. Adding 100g / t of plant essential oil extract containing 2% thymol, 1% geraniol, and 11% bergamot oil to the diets of weaned piglets significantly improved their growth performance and serum GSH and SOD activities.

[0005] Currently, my country mainly uses feed mycotoxin binders to control mycotoxin contamination, and no specific antidote has yet been developed. Therefore, this invention addresses the issue of plant essential oils inhibiting oxidative damage caused by mycotoxins by combining two common plant essential oil monomers with porcine alveolar macrophages exposed to combined mycotoxins. This aims to solve the problem of the lack of a specific antidote for mycotoxin infection and to provide an effective drug component. Summary of the Invention

[0006] The purpose of this invention is to address the shortcomings of the prior art by providing an application of a plant essential oil composition in the preparation of antifungal drugs, thereby solving the problems in the prior art.

[0007] To achieve the above objectives, the technical solution adopted by the present invention is: the application of a plant essential oil composition in the preparation of antimycotoxin drugs, wherein the plant essential oil composition comprises the following raw materials: carvacrol and limonene.

[0008] According to one application of the technical solution of the present invention, at least the following beneficial effects are achieved:

[0009] The plant essential oil composition of this invention can inhibit the growth of mold and the toxicity of its toxins. Limonene can effectively reduce the concentration and level of DON, while carvacrol can inhibit the growth of Aspergillus flavus in a dose-dependent manner. The plant essential oil composition of this invention can treat oxidative damage to the body caused by mycotoxins. It is a safe and green antioxidant that can inhibit the growth of toxin-producing fungi and the toxic effects of mycotoxins, reduce oxidative stress caused by mycotoxins, and thus prevent mycotoxin pollution.

[0010] According to some embodiments of the present invention, the mass ratio of carvacrol to limonene is 1:0.01 to 99.

[0011] According to some embodiments of the present invention, the mass ratio of carvacrol to limonene in the plant essential oil composition is 9 to 6:4.

[0012] According to some embodiments of the present invention, the mold includes at least one of Aspergillus flavus, Aspergillus parasiticus, Fusarium graminearum, Fusarium pink, Aspergillus ochreus, and Penicillium verticillatum.

[0013] According to some embodiments of the present invention, the mycotoxin includes at least one of DON, AFB1 and OTA.

[0014] According to some embodiments of the present invention, the plant essential oil composition has a mass fraction of 0.1% to 99.9% in the antimycotoxin drug.

[0015] According to some embodiments of the present invention, the plant essential oil composition is an inhibitor of oxidative stress caused by mycotoxins.

[0016] According to some embodiments of the present invention, the plant essential oil composition is a therapeutic agent for oxidative damage caused by mycotoxins.

[0017] According to some embodiments of the present invention, the antimycotoxin drug is used in feed preparation.

[0018] According to some embodiments of the present invention, the antifungal drug is administered to mammals.

[0019] According to some embodiments of the present invention, the mammal includes at least one of human, rat, rabbit, sheep, pig, cow, cat, dog and monkey.

[0020] According to some embodiments of the present invention, the raw materials for preparing the antimycotoxin drug also include a pharmaceutical carrier.

[0021] According to some embodiments of the present invention, the pharmaceutical carrier is a conventional drug carrier in the pharmaceutical field.

[0022] According to some embodiments of the present invention, the pharmaceutical carrier includes at least one of a diluent, excipient, filler, binder, disintegrant, absorption enhancer, surfactant, adsorbent, lubricant, sweetener, and flavoring agent.

[0023] According to some embodiments of the present invention, the excipient includes at least one selected from water, lactose, corn starch, glucose, sorbitol, crystalline cellulose, and silicon dioxide.

[0024] According to some embodiments of the present invention, the filler includes at least one of starch and sucrose.

[0025] According to some embodiments of the present invention, the adhesive includes at least one selected from polyvinyl alcohol, cellulose derivatives, alginate, gum arabic, gelatin, and polyvinylpyrrolidone.

[0026] According to some embodiments of the present invention, the cellulose derivative includes at least one of ethyl cellulose, methyl cellulose, hydroxypropyl cellulose, and hydroxypropyl methyl cellulose.

[0027] According to some embodiments of the present invention, the wetting agent includes glycerin.

[0028] According to some embodiments of the present invention, the disintegrant includes at least one of agar, calcium carbonate, and sodium bicarbonate.

[0029] According to some embodiments of the present invention, the absorption enhancer comprises a quaternary ammonium compound.

[0030] According to some embodiments of the present invention, the surfactant comprises hexadecyl alcohol.

[0031] According to some embodiments of the present invention, the adsorbent carrier includes at least one of kaolin and soap clay.

[0032] According to some embodiments of the present invention, the lubricant includes at least one selected from talc, calcium stearate, magnesium stearate, and polyethylene glycol.

[0033] According to some embodiments of the present invention, the pharmacologically permissible salts of the present invention include salts formed with inorganic acids, organic acids, alkali metal ions, alkaline earth metal ions, and basic amino acids.

[0034] According to some embodiments of the present invention, the inorganic acid includes at least one of hydrochloric acid, nitric acid, sulfuric acid, phosphoric acid, and hydrobromic acid.

[0035] According to some embodiments of the present invention, the organic acid includes at least one selected from maleic acid, fumaric acid, tartaric acid, lactic acid, citric acid, acetic acid, methanesulfonic acid, p-toluenesulfonic acid, adipic acid, palmitic acid, and tannic acid.

[0036] According to some embodiments of the present invention, the alkali metal ion includes at least one of lithium ion, sodium ion and potassium ion.

[0037] According to some embodiments of the present invention, the alkaline earth metal ions include at least one of calcium ions and magnesium ions.

[0038] According to some embodiments of the present invention, the basic amino acid includes lysine.

[0039] According to some embodiments of the present invention, the dosage form of the drug is any of the various dosage forms conventional in the art.

[0040] According to some embodiments of the present invention, the antimycotoxin drug may achieve its function via other chemical and biological forms in the field.

[0041] According to some embodiments of the present invention, the dosage form of the drug is a solid, semi-solid, or liquid.

[0042] According to some embodiments of the present invention, the dosage form of the drug is an aqueous solution, a non-aqueous solution, or a suspension.

[0043] According to some embodiments of the present invention, the dosage form of the drug is tablet, capsule, soft capsule, granule, pill, oral liquid, dry suspension, drop pill, dry extract, injection or infusion.

[0044] According to some embodiments of the present invention, the tablets or granules are coated with sugar coating, gelatin coating, and other necessary coatings.

[0045] According to some embodiments of the present invention, when preparing the injection, at least one of the following is added to the main drug as needed: pH adjuster, buffer, suspending agent, solubilizer, stabilizer, isotonic agent, and preservative.

[0046] According to some embodiments of the present invention, the injectable is prepared as an intravenous, subcutaneous, or intramuscular injection using conventional methods.

[0047] According to some embodiments of the present invention, the injection is prepared as a freeze-dried product using conventional methods.

[0048] According to some embodiments of the present invention, the suspending agent includes at least one of methylcellulose, Tween 80, hydroxyethylcellulose, gum arabic, sodium carboxymethylcellulose, and polyoxyethylene sorbitol monolaurate.

[0049] According to some embodiments of the present invention, the solubilizer includes at least one selected from polyethylene oxide hydrogenated castor oil, Tween 80, nicotinamide, polyoxyethylene sorbitol monolaurate, polyethylene glycol, and castor oil fatty acid ethyl ester.

[0050] According to some embodiments of the present invention, the stabilizer includes at least one of sodium sulfite and sodium metasulfite.

[0051] According to some embodiments of the present invention, the preservative includes at least one selected from methylparaben, ethylparaben, sorbic acid, phenol, cresol, and chlorocresol.

[0052] According to some embodiments of the present invention, the preparation methods of the pharmaceutical formulation can all be obtained by conventional preparation methods used by those skilled in the art to prepare this dosage form.

[0053] According to some embodiments of the present invention, each unit of the pharmaceutical preparation contains 0.001 mg to 50 mg of the plant essential oil composition.

[0054] According to some embodiments of the present invention, the administration method of the drug can be a conventional administration method in the art, including but not limited to injection or oral administration.

[0055] According to some embodiments of the present invention, the antimycotoxin drug includes a variety of acceptable dosage forms.

[0056] According to some embodiments of the present invention, the injection administration can be carried out via intravenous injection, intramuscular injection, intraperitoneal injection, intradermal injection, or subcutaneous injection.

[0057] According to some embodiments of the present invention, the mass fraction of the plant essential oil composition in the antimycotoxin drug is 0.1% to 99.9%.

[0058] According to some embodiments of the present invention, the mass fraction of the plant essential oil composition in the antimycotoxin drug is 0.5% to 95%.

[0059] According to some embodiments of the present invention, the mass fraction of the plant essential oil composition in the antimycotoxin drug is 10% to 20%.

[0060] According to some embodiments of the present invention, the standard dosage of the antimycotoxin drug is 0.1 mg / day to 1000 mg / day of plant essential oil composition.

[0061] The term "dosage" as used herein refers to an amount that can alleviate or delay the progression of a disease, degenerative or damaging condition. It can vary depending on the specific disease being treated and other factors, including age, weight, health status, severity of symptoms, route of administration, frequency of treatment, and whether other medications are being used concurrently during treatment.

[0062] The term "antimycotoxin drug" as used in this article refers to reducing the severity of mycotoxins and their complications, or curing mycotoxins and their complications to normalize them, or slowing the progression of mycotoxins and their complications. Attached Figure Description

[0063] Figure 1 The intracellular MDA content of PAM cells was determined by combining DON, AFB1, and OTA at different IC50 values ​​for 24 hours (n=4).

[0064] Figure 2 The intracellular ROS levels of PAM cells were determined by combining different IC50 values ​​of DON, AFB1, and OTA for 24 hours (n=4).

[0065] Figure 3 The effect of different concentrations of carvacrol and combined mycotoxins on the survival rate of PAM cells after 24 hours (n=4).

[0066] Figure 4 The effect of different concentrations of limonene and combined mycotoxins on the survival rate of PAM cells after 24 hours (n=4).

[0067] Figure 5 The effect of the combined action of compound plant essential oils and combined mycotoxins on the survival rate of PAM cells after 24 hours (n=4).

[0068] Figure 6 The effect of the combined action of compound plant essential oils and combined mycotoxins on the intracellular MDA content of PAM cells for 24 hours (n=4).

[0069] Figure 7The effect of the combined action of compound plant essential oils and combined mycotoxins on the intracellular SOD activity of PAM cells for 24 hours (n=4).

[0070] Figure 8 The effect of the combined action of compound plant essential oils and combined mycotoxins on the intracellular CAT activity of PAM cells for 24 hours (n=4).

[0071] Figure 9 The effect of the combined action of compound plant essential oils and combined mycotoxins on the intracellular T-AOC level of PAM cells for 24 hours (n=4).

[0072] Figure 10 The effect of the combined action of compound plant essential oils and combined mycotoxins on the intracellular GSH-Px activity of PAM cells for 24 hours was investigated (n=4).

[0073] Figure 11 The effect of the combined action of compound plant essential oils and combined mycotoxins on the intracellular GR activity of PAM cells for 24 hours (n=4).

[0074] Figure 12 The effect of the combined action of compound plant essential oils and combined mycotoxins on the intracellular GST activity of PAM cells for 24 hours (n=4).

[0075] Figure 13 The effect of the same concentration (40 μg / mL) of carvacrol, limonene, compound plant essential oil and combined mycotoxins on the intracellular MDA content of PAM cells for 24 h (n=4).

[0076] Figure 14 The study investigated the effects of the same concentration (40 μg / mL) of carvacrol, limonene, compound plant essential oils, and combined mycotoxins on the intracellular SOD activity of PAM cells after 24 h (n=4).

[0077] Figure 15 The effect of the same concentration (40 μg / mL) of carvacrol, limonene, compound plant essential oil and combined mycotoxin on the intracellular CAT activity of PAM cells for 24 h (n=4).

[0078] Figure 16 The study investigated the effects of the same concentration (40 μg / mL) of carvacrol, limonene, compound plant essential oils, and combined mycotoxins on the intracellular T-AOC level of PAM cells after 24 h (n=4).

[0079] Figure 17 The study investigated the effects of the same concentration (40 μg / mL) of carvacrol, limonene, compound plant essential oils, and combined mycotoxins on the intracellular GSH-Px activity of PAM cells after 24 h (n=4).

[0080] Figure 18 The effect of the same concentration (40 μg / mL) of carvacrol, limonene, compound plant essential oil and combined mycotoxin on the intracellular GR activity of PAM cells for 24 h (n=4).

[0081] Figure 19 The effect of the same concentration (40 μg / mL) of carvacrol, limonene, compound plant essential oil and combined mycotoxin on the intracellular GR activity of PAM cells for 24 h (n=4). Detailed Implementation

[0082] To make the objectives, technical solutions, and advantages of this application clearer, the application is described and illustrated below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the scope of this application. All other embodiments obtained by those skilled in the art based on the embodiments provided in this application without inventive effort are within the scope of protection of this application.

[0083] Obviously, the accompanying drawings described below are merely some examples or embodiments of this application. Those skilled in the art can apply this application to other similar scenarios based on these drawings without any inventive effort. Furthermore, it is understood that although the efforts made in this development process may be complex and lengthy, for those skilled in the art related to the content disclosed in this application, any changes to design, manufacturing, or production based on the technical content disclosed in this application are merely conventional technical means and should not be construed as insufficient disclosure of the content of this application.

[0084] In this application, the reference to "embodiment" means that a specific feature, structure, or characteristic described in connection with an embodiment may be included in at least one embodiment of this application. The appearance of this phrase in various places in the specification does not necessarily refer to the same embodiment, nor is it a separate or alternative embodiment that is mutually exclusive with other embodiments. It will be explicitly and implicitly understood by those skilled in the art that the embodiments described in this application may be combined with other embodiments without conflict.

[0085] Unless otherwise defined, the technical or scientific terms used in this application shall have the ordinary meaning understood by one of ordinary skill in the art to which this application pertains. The terms “a,” “an,” “an,” “the,” and similar words used in this application do not indicate quantity limitation and may indicate singular or plural. The terms “comprising,” “including,” “having,” and any variations thereof used in this application are intended to cover non-exclusive inclusion; for example, a process, method, system, product, or apparatus that includes a series of steps or units (components) is not limited to the listed steps or units but may also include steps or units not listed, or may include other steps or units inherent to these processes, methods, products, or apparatus. The terms “multiple” / “several” used in this application refer to two or more. “And / or” describes the relationship between related objects, indicating that three relationships may exist; for example, “A and / or B” can represent: A alone, A and B simultaneously, and B alone. The character “ / ” generally indicates that the preceding and following related objects are in an “or” relationship. The terms “first,” “second,” “third,” etc., used in this application are merely to distinguish similar objects and do not represent a specific ordering of the objects.

[0086] This invention involves culturing porcine alveolar macrophages (PAM) in vitro, exposing them to a combination of mycotoxins (DON, AFB1, OTA), and treating them with different concentrations of carvacrol and limonene. The optimal concentration for each treatment was determined, and the two plant essential oil monomers at the optimal concentration were combined for use. Finally, the effects of the three treatments were compared, and the best application method was selected.

[0087] Determination of the combined concentration of DON, AFB1, and OTA toxins:

[0088] The cytotoxic effects of DON, AFB1, and OTA exposure alone on PAM cell proliferation were previously determined, and the half-maximal inhibitory concentrations (IC50) of DON, AFB1, and OTA were established. 50 The concentrations were 0.371 μg / mL, 1.877 μg / mL, and 2.508 μg / mL, respectively.

[0089] 1 / 6 to 1 IC of DON 50 1 / 6 to 1 IC of AFB1 50 Five combined concentration gradients were prepared by combining 1 / 6 to 1 IC50 of OTA: 1 / 6 IC50, etc. 50 (DON+AFB1+OTA), 1 / 3IC 50 (DON+AFB1+OTA), 1 / 2IC 50 (DON+AFB1+OTA), 2 / 3IC 50 (DON+AFB1+OTA), 1IC 50(DON+AFB1+OTA);

[0090] Taking a 1 / 6 IC50 (DON+AFB1+OTA) mycotoxin solution as an example: the mass concentration of DON in the combined toxin is 0.371 / 6 μg / mL, the mass concentration of AFB1 is 1.877 / 6 μg / mL, and the mass concentration of OTA is 2.508 / 6 μg / mL.

[0091] (1) MDA level detection

[0092] The cell suspension was seeded into 96-well plates (5 × 10⁶ cells / well). 5 Cells were cultured in a constant temperature incubator at 37°C for 24 hours (2 cells / well, 5% CO2, volume fraction) for 24 hours. Different combined concentrations of mycotoxins were added to the experimental groups; the control group received an equal volume of maintenance medium. Each group was repeated four times, and cultured for another 24 hours. After culture, 0.25% trypsin was added to each well for 4 minutes of digestion, and digestion was stopped with a stop solution. All liquids were transferred to EP tubes and centrifuged at 1000 rpm for 4 minutes. The supernatant was discarded, and the cell pellet was collected. Residual trypsin was washed away with PBS. Extraction solution was added according to the cell count, followed by sonication at 200W or 20% power on ice for 3 seconds, with 10-second intervals, repeated 30 times. After sonication, the cells were centrifuged at 8000g for 10 minutes at 4°C. The supernatant was collected and placed on ice to determine protein concentration and MDA content.

[0093] The results of combined exposure of PAM cells to different IC50 values ​​of DON, AFB1, and OTA for 24 hours to intracellular MDA content (n=4) are shown in the figure. Figure 1 (* indicates all experimental groups compared with the control; # indicates the 1 / 3 IC50 combination group compared with the 1 / 2 IC50 group, 2 / 3 IC50 group, and 1 IC50 group, respectively. * and # indicate significant differences (P<0.05), and ** and ## indicate extremely significant differences (P<0.01)). 50 The (DON+AFB1+OTA) combination group significantly increased MDA content without causing excessive damage to cell viability.

[0094] (2) ROS experiment

[0095] The cell suspension was seeded into 96-well plates (5 × 10⁶ cells / well). 5Cells were cultured in a constant temperature incubator at 37℃ for 24 h using 2 mL / well of 5% CO2. Different combined concentrations of mycotoxins were added to the experimental groups, while the control group received an equal volume of maintenance medium. Each group was repeated four times, and cultured for another 24 h. After culture, 0.25% trypsin was added to each well for 4 min of digestion, followed by a stop solution to terminate the digestion. All liquids were transferred to EP tubes and centrifuged at 1000 rpm for 4 min. The supernatant was discarded, and the cell pellet was collected. Residual trypsin was washed away with PBS. Fluorescence intensity was measured within 10 min using a multifunctional fluorescence chemiluminescence immunoassay analyzer at 37℃. The excitation wavelength was 499 nm, and the emission wavelength was 521 nm. Fluorescence value changes within 10 min were recorded. Linear regression fitting was performed, and the regression coefficient, i.e., the slope of the line (k), was calculated. Intracellular ROS production rate = k(measured) - k(blank). Results are shown below. Figure 2 1 / 2 IC 50 The combined group showed a significant increase in ROS levels, with the most pronounced effect.

[0096] Preliminary experiment: In vitro effects of two plant essential oil monomers against combined mycotoxin oxidative stress.

[0097] Based on the preliminary experimental results, carvacrol at concentrations of 40 μg / mL, 50 μg / mL, and 60 μg / mL, and limonene at concentrations of 20 μg / mL, 30 μg / mL, and 40 μg / mL were selected for testing. A control group (C) with no additives was set up, along with a mycotoxin-only treatment group (MD), carvacrol and limonene-only treatment groups (A1 (carvacrol 40 μg / mL), A2 (carvacrol 50 μg / mL), A3 (carvacrol 60 μg / mL), L1 (limonene 20 μg / mL), L2 (limonene 30 μg / mL), L3 (limonene 40 μg / mL)), and carvacrol and limonene-only treatment groups (AI (carvacrol 40 μg / mL + 1 / 2 IC)). 50 Combined mycotoxins), AII (carvacrol 50 μg / mL + 1 / 2 IC 50 Combined mycotoxins), AIII (carvacrol 60 μg / mL + 1 / 2 IC50) 50 Combined mycotoxins), LI (limonene 20 μg / mL + 1 / 2 IC 50 Combined mycotoxins), LII (limonene 30 μg / mL + 1 / 2 IC) 50 Combined mycotoxins), LIII (limonene 40 μg / mL + 1 / 2 IC50) 50 Combined mycotoxins), with 4 replicates per group.

[0098] (1) CCK-8 assay for cell viability

[0099] The cell suspension was seeded into 96-well plates (1×10⁶ cells / well).4 After culturing PAM cells in a 5% CO2 incubator at 37°C for 24 h (100 μL / well), the supernatant was aspirated and the cells were washed with PBS. Plant essential oil monomers and combined mycotoxins were added according to the above groupings. After 24 h of culture, the liquid in the wells was aspirated, the cells were washed with PBS, and 100 μL of maintenance medium and 10 μL of CCK-8 solution were added. The cells were incubated for 3 h, and the absorbance was measured at 450 nm. Both plant essential oil monomers and combined mycotoxins, when used in combination for 24 h, inhibited cell damage induced by the combined mycotoxins and significantly improved cell viability. Results are shown below. Figures 3-4 Among them, carvacrol and limonene showed the strongest cell-protective ability at concentrations of 60 μg / mL and 40 μg / mL, respectively.

[0100] (2) Detection of MDA, SOD, CAT, GSH-Px, total antioxidant capacity (T-AOC), GR, and GST levels.

[0101] The cell suspension was seeded into 96-well plates (5 × 10⁶ cells / well). 5 After culturing for 24 h in a constant temperature incubator at 37℃ with 5% CO2 (2 mL / well), plant essential oil monomers and combined mycotoxins were added according to the above grouping, and cultured for another 24 h. After the culture, 0.25% trypsin was added to each well for 4 min of digestion, and digestion was terminated with a stop solution. All liquids were transferred to EP tubes, centrifuged at 1000 r / min for 4 min, the supernatant was discarded, and the cell pellet was collected. Residual trypsin was washed off with PBS. Intracellular MDA content, SOD activity, CAT activity, GSH-Px activity, total antioxidant capacity (T-AOC), GR activity, and GST activity were measured. The two plant essential oil monomers antagonized the increase in MDA content and the decrease in SOD activity, CAT activity, T-AOC, GSH-Px activity, GR activity, and GST activity in PAM cells after exposure in a dose-dependent manner. The results are shown in Tables 1-2. Carvacrol and limonene at concentrations of 60 μg / mL and 40 μg / mL, respectively, showed the strongest inhibitory effect on oxidative stress induced by combined mycotoxins.

[0102] Table 1. Changes in indicators of oxidative stress in PAM cells induced by combined exposure to DON, AFB1, and OTA (carvacrol)

[0103]

[0104] Note: Data in the table are presented in the format of mean ± standard deviation. Different uppercase letters in the superscript indicate extremely significant differences (P<0.01), different lowercase letters with the same uppercase letters indicate significant differences (P<0.05), and the same lowercase letters indicate no significant differences (P>0.05).

[0105] Table 2. Changes in indicators of oxidative stress in PAM cells induced by combined exposure to DON, AFB1, and OTA with limonene.

[0106]

[0107]

[0108] Note: Data in the table are presented in the format of mean ± standard deviation. Different uppercase letters in the superscript indicate extremely significant differences (P<0.01), different lowercase letters with the same uppercase letters indicate significant differences (P<0.05), and the same lowercase letters indicate no significant differences (P>0.05).

[0109] Example 1: In vitro anti-oxidative stress effect of compound plant essential oils against combined mycotoxins

[0110] In this embodiment, based on the optimal concentrations of carvacrol and limonene obtained from the above experiments, appropriate doses of the two plant essential oil monomers were dissolved together in 95% ethanol to prepare a compound plant essential oil (composed of 60 μg / mL carvacrol solution and 40 μg / mL limonene solution in a volume ratio of V...). 香芹酚 V 柠檬烯 (Configured in a 3:2 ratio) Set up a control group (C) with no additives, a combined mycotoxin-only group (MD), a compound plant essential oil-only group (B), an optimal concentration of carvacrol-only group (A3, 60 μg / mL), an optimal concentration of limonene-only group (L3, 40 μg / mL), a combined compound plant essential oil and combined mycotoxin-only group (E), an optimal concentration of carvacrol and combined mycotoxin-only group (AIII), and an optimal concentration of limonene and combined mycotoxin-only group (LIII).

[0111] (1) CCK-8 assay for cell viability

[0112] The cell suspension was seeded into 96-well plates (1×10⁶ cells / well). 4 100 μL / well was incubated in a 5% CO2 incubator at 37°C for 24 h. The supernatant was then aspirated and washed with PBS. Plant essential oil monomers, compound plant essential oils, and combined mycotoxins were added according to the above groupings. After 24 h of incubation, the liquid in the wells was aspirated, washed with PBS, and 100 μL of maintenance medium and 10 μL of CCK-8 solution were added. The mixture was incubated for 3 h, and the absorbance was measured at 450 nm. Results are shown below. Figure 5(* indicates comparison with the control group; # indicates comparison between the two plant essential oil monomers and the combined treatment with the combined plant essential oil and the combined mycotoxin group and the combined mycotoxin exposure group. * and # indicate significant differences (P<0.05), ** and ## indicate extremely significant differences (P<0.01), and ### indicate extremely significant differences (P<0.001)). After 24 hours of combined treatment of PAM cells with the combined plant essential oil and the combined mycotoxin, the cell damage caused by the combined mycotoxin was significantly inhibited and the cell survival rate was improved.

[0113] 2) Detection of MDA, SOD, CAT, GSH-Px, total antioxidant capacity (T-AOC), GR, and GST levels.

[0114] The cell suspension was seeded into 96-well plates (5 × 10⁶ cells / well). 5 Cells were cultured in a constant temperature incubator at 37℃ for 24 h using 2 mL / well of 5% CO2. Then, plant essential oil monomers, compound plant essential oils, and combined mycotoxins were added according to the above groupings, and cultured for another 24 h. After culture, 0.25% trypsin was added to each well for 4 min of digestion, and digestion was terminated with a stop solution. All liquids were transferred to EP tubes, centrifuged at 1000 r / min for 4 min, the supernatant was discarded, and the cell pellet was collected. Residual trypsin was washed away with PBS. Intracellular MDA content, SOD activity, CAT activity, GSH-Px activity, total antioxidant capacity (T-AOC), GR activity, and GST activity were measured. The two plant essential oil monomers antagonized the increase in MDA content and decreased SOD, CAT, T-AOC, GSH-Px, GR, and GST activities in PAM cells after exposure in a dose-dependent manner. Results are shown below. Figures 6-12 ("*" indicates comparison with the control group; "#" indicates comparison between the combined treatment with the mycotoxin and the combined mycotoxin exposure group. "*" and "#" indicate significant differences (P<0.05), "**" and "##" indicate extremely significant differences (P<0.01), and "###" indicate extremely significant differences (P<0.001)) and Table 3 show that the combined plant essential oil significantly inhibited the oxidative stress induced by the combined mycotoxin, and its effect was better than that of the two plant essential oil monomers acting alone.

[0115] Table 3. Changes in indicators of oxidative stress in PAM cells induced by combined exposure to DON, AFB1, and OTA induced by compound plant essential oils.

[0116]

[0117] Note: Data in the table are presented in the format of mean ± standard deviation. Different uppercase letters in the superscript indicate extremely significant differences (P<0.01), different lowercase letters with the same uppercase letters indicate significant differences (P<0.05), and the same lowercase letters indicate no significant differences (P>0.05).

[0118] Example 2: Anti-oxidative stress effect of single and compound plant essential oils against combined mycotoxins

[0119] This embodiment is based on the maximum non-toxic concentrations of two plant essential oil monomers to PAM cells obtained from previous experiments (carvacrol: 60 μg / mL, limonene: 40 μg / mL). A 40 μg / mL carvacrol solution and a 40 μg / mL limonene solution were selected, and a 40 μg / mL compound plant essential oil was prepared (composed of 40 μg / mL carvacrol solution and 40 μg / mL limonene solution at a volume ratio of V...). 香芹酚 V 柠檬烯 (Configured in a 3:2 ratio). The following groups were set up: a control group (C) with no additives, a combined mycotoxin treatment group (MD), a 40 μg / mL carvacrol treatment group (A), a 40 μg / mL limonene treatment group (L), a 40 μg / mL compound plant essential oil treatment group (E), a 40 μg / mL carvacrol and combined mycotoxin treatment group (a), a 40 μg / mL limonene and combined mycotoxin treatment group (l), and a 40 μg / mL compound plant essential oil and combined mycotoxin treatment group (e). Each group had four replicates.

[0120] The detection methods for MDA, SOD, CAT, GSH-Px, total antioxidant capacity (T-AOC), GR, and GST levels are as follows:

[0121] The cell suspension was seeded into 96-well plates (5 × 10⁶ cells / well). 5 Cells were cultured in a constant temperature incubator at 37℃ for 24 h using 2 mL / well (5% CO2). Then, plant essential oil monomers, compound plant essential oils, and combined mycotoxins were added according to the above groupings, and cultured for another 24 h. After culture, 0.25% trypsin was added to each well for 4 min of digestion, and digestion was terminated with a stop solution. All liquids were transferred to EP tubes, centrifuged at 1000 r / min for 4 min, the supernatant was discarded, and the cell pellet was collected. Residual trypsin was washed away with PBS. Intracellular MDA content, SOD activity, CAT activity, GSH-Px activity, total antioxidant capacity (T-AOC), GR activity, and GST activity were measured. The results are shown in Table 4. Figure 13-19(* indicates comparison with the control group; # indicates comparison between the treatment with carvacrol (40 μg / mL), limonene (40 μg / mL), and compound plant essential oil (40 μg / mL) and the treatment with combined mycotoxins versus the combined mycotoxins exposure group. * and # indicate significant differences (P<0.05), ** and ## indicate extremely significant differences (P<0.01), and ### indicate extremely significant differences (P<0.001). At the same concentration (40 μg / mL), the compound plant essential oil showed a significantly better effect against oxidative stress caused by combined mycotoxins than the individual effects of the two plant essential oils.)

[0122] Table 4. Changes in indicators of oxidative stress in PAM cells induced by combined exposure to DON, AFB1, and OTA at the same concentration (40 μg / mL) of carvacrol, limonene, and compound plant essential oils.

[0123]

[0124]

[0125] Note: Data in the table are presented as mean ± standard deviation. Different uppercase letters in the superscript indicate highly significant differences (P < 0.01), different lowercase letters among identical uppercase letters indicate significant differences (P < 0.05), and identical lowercase letters indicate no significant differences (P > 0.05).

[0126] Example 3: Application of carvacrol as an antifungal drug

[0127] PAM cells were treated with 40 μg / mL carvacrol and a combination of mycotoxins in vitro for 24 h. The cell culture medium was then centrifuged at 1000 rpm for 4 min, the supernatant was discarded, and the cell pellet was collected to assess the cells' antioxidant capacity. The results showed that 40 μg / mL carvacrol significantly inhibited oxidative stress induced by the combination of mycotoxins.

[0128] Example 4: Application of limonene as an antifungal drug

[0129] PAM cells were treated with 40 μg / mL limonene and a combination of mycotoxins in vitro for 24 h. The cell culture medium was then centrifuged at 1000 rpm for 4 min, the supernatant was discarded, and the cell pellet was collected to assess the cells' antioxidant capacity. The results showed that 40 μg / mL limonene significantly inhibited oxidative stress induced by the combination of mycotoxins.

[0130] Example 5: Application of compound plant essential oils as antifungal drugs

[0131] Prepare a compound plant essential oil (V) with a concentration of 40 μg / mL. 香芹酚 V 柠檬烯A 3:2 ratio of compound plant essential oils to combined mycotoxins was used to treat PAM cells in vitro for 24 h. The cell culture medium was then centrifuged at 1000 rpm for 4 min, the supernatant was discarded, and the cell pellet was collected to assess the cells' antioxidant capacity. The results showed that 40 μg / mL of the compound plant essential oils significantly inhibited oxidative stress induced by the combined mycotoxins, and the effect was better than that of either of the individual plant essential oils acting alone.

[0132] The comparative results of Examples 3-5 above show that carvacrol and limonene have the best anti-combined mycotoxin oxidative stress effect.

[0133] In summary, the plant essential oil composition of this invention can inhibit the growth of molds and the toxicity of their toxins. Limonene effectively reduces DON concentration and toxin levels, while carvacrol inhibits Aspergillus flavus growth in a dose-dependent manner. The plant essential oil composition of this invention can treat oxidative damage caused by mycotoxins. It is a safe and green antioxidant that can inhibit the growth of toxin-producing fungi and suppress the toxic effects of mycotoxins, reducing oxidative stress caused by mycotoxins and thus controlling mycotoxin pollution. This invention utilizes two plant essential oil monomers (carvacrol and limonene) to antagonize the oxidative stress induced by DON, AFB1, and OTA in porcine alveolar macrophages (PAM) through in vitro culture, demonstrating broad application prospects.

[0134] The above description is merely a preferred embodiment of the present invention and does not limit the implementation and protection scope of the present invention. Those skilled in the art should realize that any equivalent substitutions and obvious changes made based on the description and illustrations of the present invention should be included within the protection scope of the present invention.

Claims

1. The use of a plant essential oil composition in the preparation of an antifungal drug, characterized in that, The plant essential oil composition comprises the following raw materials: carvacrol and limonene; the mycotoxin includes at least one of DON, AFB1 and OTA; the mass fraction of the plant essential oil composition in the antimycotoxin drug is 0.1% to 99.9%; the mass ratio of carvacrol to limonene in the plant essential oil composition is 9 to 6:

4.

2. The application according to claim 1, characterized in that, The mold includes at least one of Aspergillus flavus, Aspergillus parasiticus, Fusarium graminearum, Fusarium pink, Aspergillus ochreus, and Penicillium verticillatum.

3. The application according to claim 1, characterized in that, The plant essential oil composition is an inhibitor of oxidative stress caused by mycotoxins.

4. The application according to claim 1, characterized in that, The plant essential oil composition is a therapeutic agent for oxidative damage caused by mycotoxins.

5. The application according to claim 1, characterized in that, The antimycotoxin drug is used in feed preparation.

6. The application according to claim 1, characterized in that, The antifungal drugs are administered to mammals.

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