Application of glycyrrhiza polysaccharide in preparation of mycotoxin-rescuing antidote product
The antidote products prepared by using licorice polysaccharide were solved, and the problem of intestinal toxicity in pigs was significantly alleviated, cell damage was improved, cell activity and antioxidant capacity were improved, and there was broad application prospect in the treatment of intestinal damage in piglets.
Patent Information
- Application Number
- CN202411368134.9
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-09-29
- Publication Date
- 2025-02-25
AI Technical Summary
The prior art has not yet effectively solved the problem of pig intestinal toxicity induced by deoxyfusarium enol (DON), especially in the lack of effective antidote drugs in the treatment of intestinal damage in piglets.
Licorice polysaccharide is used as the main component to prepare the antidote product of mycotoxin, and use its effect of inhibiting the reduction of jejunum villus height and cell fragmentation, improve the activity of pig small intestine epithelial cells, inhibit apoptosis, reduce intracellular oxidative damage, enhance the antioxidant ability of cells, and regulate mitochondrial dysfunction.
Licorice polysaccharide significantly alleviates DON-induced cell damage, improves cell activity, inhibits apoptosis and oxidative damage, improves antioxidant capacity, and has the effect of rescuing DON-induced intestinal toxic damage.
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Figure CN119499273A_ABST
Abstract
Description
Technical Field
[0001] The invention belongs to the technical field of biomedicine, and specifically relates to the application of licorice polysaccharide in the preparation of mycotoxin detoxification products. Background Art
[0002] Deoxynivalenol (DON), also known as vomitoxin, has the intestinal tract as its main toxic target organ. Pigs are the most sensitive animals to DON. Licorice is a commonly used Chinese medicine. It is clinically known as "Nine Herbs in Ten Formulas". It is used in large quantities in clinical practice. It has the effects of tonifying the spleen and replenishing qi, clearing away heat and detoxifying, removing phlegm and relieving cough, relieving pain, and harmonizing various medicines. It is clinically used for spleen and stomach weakness, fatigue, palpitations and shortness of breath, cough and sputum, abdominal and limb cramps and pain, carbuncle, sore and poison, and can also relieve drug toxicity. The active ingredients of licorice include various types of compounds such as polysaccharides, saponins, and flavonoids. Among them, licorice polysaccharides have immunomodulatory, antioxidant, liver-protecting, antitussive, and antiviral effects. It is one of the most studied chemical components in licorice. Currently, Glycyrrhiza polysaccharides (GCP) have been shown to have antioxidant, antitumor, anticancer, immunomodulatory, antibacterial, anti-inflammatory and antiviral activities, but the effect of GCP on DON-induced porcine enterotoxicity is still unclear. Summary of the invention
[0003] In view of the above problems, the present invention aims to propose the use of licorice polysaccharide in the preparation of mycotoxin detoxification products.
[0004] The technical scheme of the present invention is: the use of the licorice polysaccharide of the present invention in the preparation of a mycotoxin detoxification product, wherein the mycotoxin is deoxynivalenol;
[0005] The mycotoxin is vomitoxin.
[0006] Furthermore, the toxicity of the deoxynivalenol is enterotoxicity.
[0007] Further, enterotoxicity in the preparation of an antidote product: effects on jejunal villus height and cellular debris in piglets.
[0008] Further, enterotoxicity in the preparation of the detoxification product: the effects of deoxynivalenol on the viability, apoptosis, antioxidant and mitochondrial function of porcine small intestinal epithelial cells.
[0009] Furthermore, the detoxification function of the licorice polysaccharide is to inhibit the reduction of jejunal villus height and cell fragmentation.
[0010] Furthermore, the detoxification function of the licorice polysaccharide is to increase the activity of pig small intestinal epithelial cells, inhibit apoptosis, reduce intracellular oxidative damage, enhance the antioxidant capacity of cells, and regulate mitochondrial dysfunction.
[0011] Furthermore, the concentration of the licorice polysaccharide is 0.2-0.8 mg / mL.
[0012] Furthermore, the present invention also provides a product for rescuing intestinal toxicity damage induced by deoxynivalenol, comprising licorice polysaccharide and one or more pharmaceutically acceptable drug carriers.
[0013] Furthermore, the dosage form of the detoxification product includes powder, granules, premix, tablet, capsule, oral solution or ointment.
[0014] Furthermore, the drug carrier includes a diluent, an excipient, a binder, a wetting agent, an absorption enhancer, a surfactant or a lubricant.
[0015] The beneficial effects of the present invention are as follows: utilizing licorice polysaccharide has a rescue effect on DON-induced intestinal toxicity damage, and has broad application prospects in solving the problem of no specific antidote for vomitoxin infection and treating intestinal damage in piglets. BRIEF DESCRIPTION OF THE DRAWINGS
[0016] Figure 1 This is a comparison diagram of the effects of DON and different concentrations of licorice polysaccharides on the activity of IPEC-J2 cells in the embodiment of the present invention;
[0017] Figure 2 Schematic diagram of the effect of DON and 400 μg / mL liquorice polysaccharide treatment on apoptosis of IPEC-J2 cells in an embodiment of the present invention;
[0018] Figure 3 This is a comparison chart of the effects of DON and 400 μg / mL liquorice polysaccharide on ROS in IPEC-J2 cells after treatment in the embodiment of the present invention;
[0019] Figure 4 It is a schematic diagram of the effect of DON and 400 μg / mL liquorice polysaccharide treatment on the antioxidant enzyme activity in IPEC-J2 cells in the embodiment of the present invention;
[0020] Figure 5 Schematic diagram of the effect of DON and 400 μg / mL liquorice polysaccharide treatment on mitochondrial membrane potential of IPEC-J2 cells in an embodiment of the present invention;
[0021] Figure 6 It is a schematic diagram of HE staining of piglet jejunum tissue after treatment with DON and 400 μg / mL liquorice polysaccharide in an embodiment of the present invention. DETAILED DESCRIPTION
[0022] The specific technical scheme of the present invention is further described in detail below with reference to specific examples.
[0023] Example 1
[0024] Effects of DON poisoning on cell morphology and activity and detoxification effects of different concentrations of licorice polysaccharides
[0025] 1. Detection method:
[0026] (1) IPEC-J2 cells in the logarithmic growth phase were selected and cultured at 1.0×10 5 The cells were evenly inoculated at a density of 100 cells / mL in a 96-well cell culture plate, and the culture plate was placed in a cell culture incubator at 37°C and 5% CO2 to promote cell attachment and growth, and incubated for 24 hours;
[0027] (2): After the cells adhered to the wall, the nutrient solution was discarded, and different concentrations (200, 400, 800 μg / mL) of glycyrrhizic polysaccharides were added and incubated for 6 h, and then 0 or 0.5 μg / mL DON was added for testing;
[0028] (3): After 24 h of DON treatment, the culture medium was discarded, the cells were washed twice with PBS, and the pre-prepared CCK-8 working solution was added and incubated for 2 h. The absorbance (OD) value at 450 nm was detected by a microplate reader;
[0029] 2. Experimental results:
[0030] Test results such as Figure 1 As shown in the figure, the cell activity of the 0.5 μg / mL DON treatment group was significantly reduced, while the cell activity of the glycyrrhizic polysaccharide pretreatment group was significantly higher than that of the DON group, and the cell activity of 400 μg / mL and 800 μg / mL was close to that of the control group, indicating that glycyrrhizic polysaccharides can alleviate DON-induced cell damage. Combined with the cell activity test results, 400 μg / mL glycyrrhizic polysaccharide was selected for subsequent experiments.
[0031] Example 2
[0032] Effects of DON poisoning on cell apoptosis and detoxification effects of different concentrations of licorice polysaccharides
[0033] 1. Detection method:
[0034] (1) IPEC-J2 cells in the logarithmic growth phase were selected and cultured at 1.0×10 5 The cells were evenly inoculated at a density of 100 cells / mL in a 6-well cell culture plate, and the culture plate was placed in a cell culture incubator at 37°C and 5% CO2 to promote cell attachment and growth, and incubated for 24 hours;
[0035] (2): After the cells adhered to the wall, the nutrient solution was discarded, 400 μg / mL of glycyrrhizic polysaccharide was added, and the cells were incubated for 6 h. Then, 0 or 0.5 μg / mL of DON was added for related experiments, which were divided into a blank control group, a DON group, and a glycyrrhizic polysaccharide + DON group. To ensure uniform cell adhesion, a slide was pre-laid. After the glycyrrhizic polysaccharide and DON treatment was completed, the cells were fixed with 4% paraformaldehyde fixative at room temperature for 15 min.
[0036] (3): Remove the fixative solution, wash twice with PBS, add 0.5 mL Hoechst 33258 staining solution, and stain for 10 min; aspirate the staining solution, add PBS to wash three times, drop anti-fade mounting medium on the slide, cover with a coverslip, and observe under a fluorescence microscope, λex = 350 nM, λem = 460 nM;
[0037] 2. Experimental results:
[0038] Results from cell apoptosis staining Figure 2 From the above, it can be seen that the cells in the DON group were densely and intensely stained with a white and shiny color, while most of the cells in the DON group after pretreatment with glycyrrhizic polysaccharide were normal blue like the control group, with only a few showing whitening and the cell nuclei were in good condition; the control group showed that the cells rescued by glycyrrhizic polysaccharide tended to be normal, indicating that glycyrrhizic polysaccharide had a good rescue effect on DON-induced cell apoptosis.
[0039] Example 3
[0040] Effects of DON poisoning on intracellular ROS and the detoxification effect of licorice polysaccharide
[0041] 1. Detection method:
[0042] (1): IPEC-J2 cells were treated with the poisoning treatment and grouping method according to Example 2;
[0043] (2): In a light-proof environment, 10 μM DCFH-DA probe prepared in serum-free medium was added to the cells and incubated in a 37°C constant temperature incubator in the dark for 20 min;
[0044] (3): Remove the staining solution and wash with serum-free DMEM three times. Use a fluorescence microplate reader to measure the fluorescence at λex = 488 nM and λem = 525 nm;
[0045] (4): After the test, 10 μL of CCK-8 was added to each well of cells and cultured for 2 h at 37°C and 5% CO2. The OD value at 450 nm was measured using a multifunctional microplate reader, and the fluorescence intensity / cell viability was used to represent the cellular ROS level.
[0046] 2. Experimental results:
[0047] like Figure 3 As shown, compared with the blank control group, the ROS production of cells treated with DON alone was significantly increased (P<0.05); however, compared with treatment with DON alone, the combined treatment of glycyrrhizic polysaccharide and DON significantly reduced the production of ROS (P<0.05); pretreatment with glycyrrhizic polysaccharide at a concentration of 400 μg / mL significantly attenuated the level of DON-induced ROS in a dose-dependent manner (P<0.05).
[0048] Example 4
[0049] Effects of DON poisoning on intracellular redox levels (CAT, SOD and GPx) and the detoxification effect of licorice polysaccharides
[0050] 1. Detection method:
[0051] (1): IPEC-J2 cells were treated with the poisoning treatment and grouping method according to Example 2;
[0052] (2): After the stimulation, place the plate on ice and wash once with pre-cooled PBS; add an appropriate amount of lysis buffer (the special sample collection buffer provided in the kit is used for SOD sample collection) and lyse on ice for 10 min. Use a cell scraper to collect the cells into a centrifuge tube, centrifuge at 12000g for 5 min at 4°C, take the supernatant, and store it in a freezer at -80°C;
[0053] (3): Use GSH-Px, SOD, and CAT kits according to the instructions to process the collected samples, detect the OD values at wavelengths of 340, 450, and 520 nm on a microplate reader, and calculate the results;
[0054] 2. Experimental results:
[0055] like Figure 4 As shown in the results, compared with the control group, DON significantly reduced the activities of catalase (CAT), superoxide dismutase (SOD), and glutathione peroxidase (GPX) in the cells. Compared with the DON group, licorice polysaccharide significantly increased the activities of antioxidant enzymes CAT, SOD, and GPx, thereby enhancing the antioxidant capacity of IPEC-J2.
[0056] Example 5
[0057] Effects of DON poisoning on intracellular mitochondrial membrane potential and the detoxification effect of licorice polysaccharide
[0058] 1. Detection method:
[0059] (1): IPEC-J2 cells were treated with the poisoning treatment and grouping method of Example 2.
[0060] (2): After the stimulation, IPEC-J2 cells were collected and washed three times with PBS; in addition, IPEC-J2 cells were stained with MitoGreen and TMRE at 37°C and 5% CO2 for 30 min to evaluate mitochondrial membrane potential; then, images were collected under a fluorescence microscope;
[0061] 2. Experimental results:
[0062] TMRE dye can accumulate in intact mitochondria. In inactive mitochondria, the membrane potential is reduced, resulting in less aggregation and lower brightness. Figure 5 It can be seen that the TMRE brightness of the control group and the glycyrrhizic polysaccharide group is almost the same, while the TMRE brightness of the DON group is significantly dimmed. DON treatment significantly reduced the ratio of red fluorescence to green fluorescence, indicating that the membrane potential was reduced (P<0.05). 400 μg / mL glycyrrhizic polysaccharide significantly alleviated the DON-induced membrane potential downregulation (P<0.05). Combined with the changes in ROS in Example 3, it shows that glycyrrhizic polysaccharide can significantly rescue DON-induced cellular mitochondrial dysfunction.
[0063] Example 6
[0064] Effects of DON poisoning on piglet jejunal explants and the detoxification effect of licorice polysaccharide
[0065] 1. Detection method:
[0066] (1) Jejunal explants were prepared from five 4-5 week old weaned piglets in William's Medium supplemented with 100 U / mL penicillin G and 100 ng / mL streptomycin, 50 μg / mL gentamicin, 2.5 g / LD glucose, 30 mM alanine-glutamine and 10% FBS. Collect 5 cm of middle jejunal segment in E (WME) complete medium (the damaged part by forceps cannot be used for tissue morphology); wash with WME 4-6 times; open each jejunal segment longitudinally and use scissors to obtain 6 mm explant segments; add 1 mL of WME complete medium to 24-well plates with the villi facing up, and place 1 explant / well in the cell plate, which are divided into blank control group, DON group, and glycyrrhizin + DON group; the DON concentration is 3 μg / mL, and the glycyrrhizin concentration is 400 μg / mL; all these surgical operations are completed within 1 hour after the piglets are bled to death; the explants are placed in a 37°C CO2 incubator and gently stirred for 4 hours on a horizontal shaker, and then fixed in 10% buffered formalin for histological analysis;
[0067] 2. Experimental results:
[0068] DON can cause obvious tissue autolysis and histological damage. At 3 μg / mL, the intestinal cell nuclei are condensed, the villi are flattened and aggregated, the lamina propria is edematous and necrotic, and a large number of apoptotic cells can be seen. At this concentration, there is obvious diffuse epithelial lining shedding on the surface of the villi, and a consistent and obvious heterogeneous material coating composed of secreted mucus and cell fragments is observed along the surface of the explant. Adding licorice polysaccharide can help alleviate DON-induced intestinal explant damage ( Figure 6 ).
Claims
1. The use of licorice polysaccharide in the preparation of a mycotoxin detoxification product, characterized in that: The mycotoxin is deoxynivalenol; The mycotoxin is vomitoxin.
2. The use according to claim 1, characterized in that: The toxicity of deoxynivalenol is enterotoxicity.
3. The use according to claim 2, characterized in that: The intestinal toxicity is the effect on the height of villi and cell debris in the jejunum of piglets.
4. The use according to claim 2, characterized in that: The intestinal toxicity is the effect of deoxynivalenol on the activity, apoptosis, anti-oxidation and mitochondrial function of porcine small intestinal epithelial cells.
5. The use according to claim 3, characterized in that: The detoxification function of the licorice polysaccharide is to inhibit the reduction of jejunal villus height and cell fragmentation.
6. The use according to claim 4, characterized in that: The detoxification function of the licorice polysaccharide is to increase the activity of pig small intestinal epithelial cells, inhibit apoptosis, reduce intracellular oxidative damage, enhance the antioxidant capacity of cells, and regulate mitochondrial dysfunction.
7. The use according to claim 5 or 6, characterized in that: The concentration of the licorice polysaccharide is 0.2-0.8 mg / mL.
8. A product for rescuing intestinal toxicity damage induced by deoxynivalenol, characterized in that: The invention comprises licorice polysaccharide and one or more pharmaceutically acceptable drug carriers.
9. A product for rescuing intestinal toxicity damage induced by deoxynivalenol according to claim 1, characterized in that: The dosage form of the detoxification product includes powder, granules, premix, tablet, capsule, oral solution or ointment.
10. A product for rescuing intestinal toxicity damage induced by deoxynivalenol according to claim 8, characterized in that: The drug carrier includes a diluent, an excipient, a binder, a wetting agent, an absorption promoter, a surfactant or a lubricant.