CN117017970A - Pharmaceutical composition for relieving oxidative stress and application thereof - Google Patents
Pharmaceutical composition for relieving oxidative stress and application thereof Download PDFInfo
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- CN117017970A CN117017970A CN202310929376.XA CN202310929376A CN117017970A CN 117017970 A CN117017970 A CN 117017970A CN 202310929376 A CN202310929376 A CN 202310929376A CN 117017970 A CN117017970 A CN 117017970A
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- allicin
- selenomethionine
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- 230000036542 oxidative stress Effects 0.000 title claims abstract description 27
- 239000008194 pharmaceutical composition Substances 0.000 title abstract description 19
- JDLKFOPOAOFWQN-UHFFFAOYSA-N allicin Chemical compound C=CCSS(=O)CC=C JDLKFOPOAOFWQN-UHFFFAOYSA-N 0.000 claims abstract description 83
- JDLKFOPOAOFWQN-VIFPVBQESA-N Allicin Natural products C=CCS[S@](=O)CC=C JDLKFOPOAOFWQN-VIFPVBQESA-N 0.000 claims abstract description 82
- 235000010081 allicin Nutrition 0.000 claims abstract description 82
- RJFAYQIBOAGBLC-BYPYZUCNSA-N Selenium-L-methionine Chemical compound C[Se]CC[C@H](N)C(O)=O RJFAYQIBOAGBLC-BYPYZUCNSA-N 0.000 claims abstract description 80
- RJFAYQIBOAGBLC-UHFFFAOYSA-N Selenomethionine Natural products C[Se]CCC(N)C(O)=O RJFAYQIBOAGBLC-UHFFFAOYSA-N 0.000 claims abstract description 78
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- 230000000694 effects Effects 0.000 claims abstract description 32
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Classifications
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/21—Esters, e.g. nitroglycerine, selenocyanates
- A61K31/255—Esters, e.g. nitroglycerine, selenocyanates of sulfoxy acids or sulfur analogues thereof
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K20/00—Accessory food factors for animal feeding-stuffs
- A23K20/10—Organic substances
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K20/00—Accessory food factors for animal feeding-stuffs
- A23K20/10—Organic substances
- A23K20/142—Amino acids; Derivatives thereof
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
- A61K31/19—Carboxylic acids, e.g. valproic acid
- A61K31/195—Carboxylic acids, e.g. valproic acid having an amino group
- A61K31/197—Carboxylic acids, e.g. valproic acid having an amino group the amino and the carboxyl groups being attached to the same acyclic carbon chain, e.g. gamma-aminobutyric acid [GABA], beta-alanine, epsilon-aminocaproic acid or pantothenic acid
- A61K31/198—Alpha-amino acids, e.g. alanine or edetic acid [EDTA]
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P39/00—General protective or antinoxious agents
- A61P39/06—Free radical scavengers or antioxidants
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- Veterinary Medicine (AREA)
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- Pharmacology & Pharmacy (AREA)
- Animal Behavior & Ethology (AREA)
- General Health & Medical Sciences (AREA)
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- Proteomics, Peptides & Aminoacids (AREA)
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Abstract
The invention provides a pharmaceutical composition for relieving oxidative stress and application thereof, and relates to the field of pharmaceutical application. The pharmaceutical composition comprises allicin and selenomethionine. According to the invention, in an in-vitro experiment, selenomethionine and allicin are singly used, so that the selenomethionine and allicin have a certain antioxidation function and have the effect of improving the IPEC-J2 cell activity of the pig small intestine epithelial cells, but compared with the combined use of the selenomethionine and allicin, the selenomethionine and allicin can be singly used for improving the jejunum morphology, improving the ratio of the villus length to the recess depth and improving the jejunum antioxidation function, and compared with the combined use of the selenomethionine and allicin, the selenomethionine and allicin can be singly used for improving the intestinal index and jejunum antioxidation capability. I.e., selenomethionine and allicin both form a synergistic antioxidant effect.
Description
Technical Field
The invention relates to the field of pharmaceutical application, in particular to a pharmaceutical composition for relieving oxidative stress and application thereof.
Background
Oxidative stress is closely related to the occurrence and progression of many animal diseases. Oxidative stress occurs when Reactive Oxygen Species (ROS) are produced beyond the clearance of ROS by cellular antioxidant enzymes. The intestinal tract is not only an important organ for digestion and absorption, but also the largest immune organ in animals. In general, the intestinal barrier is considered to be the first line of defense of animals against invasion of oxidative stress irritants and other foreign substances. Thus, intestinal epithelial cells are extremely prone to generate large amounts of ROS, resulting in destruction of intestinal epithelial cell structure and function. Therefore, how to improve the occurrence of oxidative stress of intestinal epithelial cells and to protect the intestinal barrier is an important problem to be solved in clinical emergency.
Selenium is used as a trace element and plays an important role in various aspects of animal antioxidation, anti-inflammatory, immunoregulation, antiviral defense and the like. Selenium mainly plays a biological role by synthesizing selenoprotein. Recent studies have shown that selenoprotein and selenase play an important role in redox homeostasis. For example, thioredoxin reductase and glutathione peroxidase can significantly inhibit ROS production. Selenomethionine (SeMet) is a safe source of organic selenium and is believed to have better bioavailability than sodium selenite or selenium yeast. Recent studies have shown that selenomethionine can be widely used as an antioxidant and a growth promoter to relieve oxidative stress and improve productivity in livestock and poultry.
Garlic is a plant of homology of medicine and food, and contains a large amount of sulfur compounds. Allicin (Allicin) is the most bioactive compound in garlic. When the garlic tissue is damaged (chewed or mashed), alliinase in the bulb is released into the cytoplasm under the action of the cysteinase, and then Alliin (Alliin) is catalyzed to allicin. Allicin has various biological activities such as antioxidation, anti-inflammatory, etc. Recent researches show that allicin can inhibit not only the activation of NLRP3 inflammatory corpuscles and endoplasmic reticulum stress channels in liver cumulating cells induced by acrylamide, but also NF- κB signal channels to induce colon cancer apoptosis. In addition, allicin can also alleviate cyclophosphamide-induced liver injury by activating the Nrf2 pathway.
However, the combined use of allicin and selenomethionine for the anti-oxidative stress has not been described in the prior art.
Disclosure of Invention
In order to solve the problems, the invention provides a medicine composition for relieving oxidative stress and application thereof, and allicin and selenomethionine are combined to improve the effect of relieving oxidative stress.
In order to achieve the above object, the present invention provides the following technical solutions:
the invention provides a pharmaceutical composition for relieving oxidative stress, which comprises allicin and selenomethionine.
Preferably, when the oxidative stress is induced by hydrogen peroxide, the effective concentration of allicin in the cell assay is 2 μg/mL, the effective concentration of selenomethionine is 0.04 μg/mL, and the ratio of allicin to selenomethionine is 50:1.
Preferably, when the oxidative stress is induced by diquat, the effective concentration of allicin in the animal experiment is 20mg/kg, the effective concentration of selenomethionine is 0.4mg/kg, and the ratio of allicin to selenomethionine is 50:1.
The invention also provides application of the pharmaceutical composition in preparation of drugs or feed additives for relieving oxidative stress.
The invention also provides application of the pharmaceutical composition in preparation of a medicament or a feed additive for improving the activity of the intestinal epithelial cells.
The invention also provides application of the pharmaceutical composition in preparation of a medicine or a feed additive for improving the ratio of the length of villus to the depth of crypt.
The invention also provides application of the pharmaceutical composition in preparation of drugs or feed additives for reducing active oxygen production.
The invention also provides application of the pharmaceutical composition in preparation of the medicine or feed additive for reducing malondialdehyde content.
The invention also provides application of the pharmaceutical composition in preparation of the medicament or feed additive for improving the superoxide dismutase activity.
The beneficial effects of the invention are as follows:
according to the invention, in an in-vitro experiment, selenomethionine and allicin are independently used, so that the effects of resisting oxidization and improving the activity of IPEC-J2 cells of pig small intestine epithelial cells are achieved, but compared with the combined application of the selenomethionine and allicin, the combined application of the selenomethionine and allicin can be used independently, the antioxidation capability and the cell activity can be remarkably improved, and in an in-vivo experiment, the jejunum morphology can be improved, and the ratio of the villus length to the recess depth can be improved. And improves jejunum antioxidation function, and compared with the combination of the two, the single administration can obviously improve the intestinal index and jejunum antioxidation capability. I.e., selenomethionine and allicin both form a synergistic antioxidant effect.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are required to be used in the embodiments will be briefly described below.
FIG. 1 shows the effect of selenomethionine and allicin alone or in combination on cell viability and morphology; wherein, a in fig. 1 is a cell viability map of IPEC-J2 cells treated with selenomethionine at various concentrations (0.01, 0.04, 0.16, 0.64, 2.56, 10.24, and 40.96 μg/mL) for 12 h; FIG. 1B is a cell viability map of IPEC-J2 cells treated with different concentrations (0.5, 1, 2, 4, 6, and 8 μg/mL) of allicin for 12 h; FIG. 1C is a graph showing cell viability following 12h treatment of IPEC-J2 cells with various concentrations (0.01, 0.04, 0.16 and 0.64. Mu.g/mL) of selenomethionine followed by 100. Mu.M hydrogen peroxide treatment; FIG. 1D is a graph showing the morphology of IPEC-J2 cells treated with selenomethionine at a specified concentration (0.04. Mu.g/mL) for 12h followed by 100. Mu.M hydrogen peroxide treatment; FIG. 1E is a graph of cell viability under 100. Mu.M hydrogen peroxide after 12h treatment of IPEC-J2 cells with different concentrations (1, 2, 4 and 8. Mu.g/mL) of allicin; FIG. 1F is a graph showing the morphology of IPEC-J2 cells treated with allicin at a prescribed concentration (2. Mu.g/mL) for 12h and then with 100. Mu.M hydrogen peroxide; FIG. 1G is a graph of cell viability under 100. Mu.M hydrogen peroxide treatment after 12h of IPEC-J2 cells treated with a specified concentration (0.04. Mu.g/mL) of selenomethionine in combination with different concentrations (1, 2, 4, and 8. Mu.g/mL) of allicin, or with different concentrations (1, 2, 4, and 8. Mu.g/mL) of allicin alone; FIG. 1H is a cell morphology of IPEC-J2 cells treated with a combination of selenomethionine at a specified concentration (0.04. Mu.g/mL) and allicin at a specified concentration (2. Mu.g/mL) for 12H, followed by 100. Mu.M hydrogen peroxide treatment; ns P > 0.05; * P <0.05; * P <0.01; * P <0.001; * P <0.0001. The following is the same.
FIG. 2 is a graph showing the effect of selenomethionine and allicin alone or in combination on intracellular reactive oxygen species;
FIG. 3 is the effect of selenomethionine and allicin alone or in combination on intracellular redox homeostasis;
FIG. 4 is the effect of selenomethionine and allicin alone or in combination on jejunal morphology and jejunal redox homeostasis in mice;
Detailed Description
The invention provides a pharmaceutical composition for relieving oxidative stress, which comprises allicin and selenomethionine. In the present invention, when the oxidative stress is induced by hydrogen peroxide, the effective concentration of allicin in the cell test is preferably 2. Mu.g/mL, the effective concentration of selenomethionine is preferably 0.04. Mu.g/mL, and the ratio of allicin to selenomethionine is preferably 50:1. In the present invention, when the oxidative stress is induced by diquat, the effective concentration of allicin in the animal experiment is preferably 20mg/kg, the effective concentration of selenomethionine is 0.4mg/kg, and the ratio of allicin to selenomethionine is 50:1.
The invention also provides application of the pharmaceutical composition in preparation of drugs or feed additives for relieving oxidative stress. The preparation method of the medicine or the feed additive is not particularly limited, and the medicine or the feed additive can be prepared by a person skilled in the art according to a conventional method.
The invention also provides application of the pharmaceutical composition in preparation of a medicament or a feed additive for improving the activity of the intestinal epithelial cells. The preparation method of the medicine or the feed additive is not particularly limited, and the medicine or the feed additive can be prepared by a person skilled in the art according to a conventional method.
The invention also provides application of the pharmaceutical composition in preparation of a medicine or a feed additive for improving the ratio of the length of villus to the depth of crypt. The preparation method of the medicine or the feed additive is not particularly limited, and the medicine or the feed additive can be prepared by a person skilled in the art according to a conventional method.
The invention also provides application of the pharmaceutical composition in preparation of drugs or feed additives for reducing active oxygen production. The preparation method of the medicine or the feed additive is not particularly limited, and the medicine or the feed additive can be prepared by a person skilled in the art according to a conventional method.
The invention also provides application of the pharmaceutical composition in preparation of the medicine or feed additive for reducing malondialdehyde content. The preparation method of the medicine or the feed additive is not particularly limited, and the medicine or the feed additive can be prepared by a person skilled in the art according to a conventional method.
The invention also provides application of the pharmaceutical composition in preparation of the medicament or feed additive for improving the superoxide dismutase activity. The preparation method of the medicine or the feed additive is not particularly limited, and the medicine or the feed additive can be prepared by a person skilled in the art according to a conventional method.
The present invention will be described in detail with reference to examples for further illustration of the invention, but they should not be construed as limiting the scope of the invention.
Example 1
Research on IPEC-J2 cell viability by combined action of selenomethionine and allicin
The experiment researches the protection effect of the combined or independent effect of selenomethionine and allicin with different concentrations on IPEC-J2 cells, and determines the optimal treatment concentration.
(1) IPEC-J2 cells in the logarithmic growth phase were inoculated at a density of 5000 cells/well into 96-well plates (100. Mu.L/well) and allowed to grow on the walls for 6h, and IPEC-J2 cells were treated with different concentrations (0.01, 0.04, 0.16, 0.64, 2.56, 10.24 and 40.96. Mu.g/mL) of selenomethionine or different concentrations (0.5, 1, 2, 4, 6 and 8. Mu.g/mL) of allicin for 12h, with 100. Mu.L of fresh complete medium and 10. Mu.L of CCK-8 reagent added to each well, 5 replicates per group. After further culturing at 37 ℃ for 1 hour, absorbance of each well was detected at a wavelength of 450nm using an enzyme-labeled instrument according to the formula: cell viability (%) = [ a (dosing) -a (blank) ]/[ a (0 dosing) -a (blank) ]x100 the results of cell viability are shown in fig. 1 a, 1B. As can be seen from FIG. 1A, selenomethionine treatment of cells at a concentration of 0.01-40.96 μg/mL for 12h had no significant effect on cell viability (P > 0.05). As can be seen from FIG. 1B, allicin treatment of cells at a concentration of 0.5-8 μg/mL for 12h had no significant effect on cell viability (P > 0.05).
(2) The pre-experiment shows that when the hydrogen peroxide concentration is 100 mu mol/L and IPEC-J2 cells are treated for 6 hours, the cell activity is reduced to 50%, and the intracellular ROS level is obviously increased, so that the method can be used as an oxidative stress model of the experiment.
IPEC-J2 cells in logarithmic growth phase were inoculated at a density of 5000 cells/well into 96-well plates (100. Mu.L/well) and allowed to grow on the walls for 6h, after IPEC-J2 cells were treated with selenomethionine at different concentrations (0.01, 0.04, 0.16 and 0.64. Mu.g/mL) for 12h, cells were treated with 100. Mu.M hydrogen peroxide for 6h, and 100. Mu.L fresh complete medium and 10. Mu.L CCK-8 reagent were added to each well, 5 replicates were performed. After further culturing at 37℃for 1 hour, the absorbance of each well was measured at a wavelength of 450nm using a microplate reader, and the cell viability was calculated. And observing the cell morphology with an inverted microscope (Leica). The results are shown as C in FIG. 1 and D in FIG. 1. As can be seen from FIG. 1C, each concentration (0.04, 0.16 and 0.64. Mu.g/mL) of selenomethionine significantly increased the cell viability of hydrogen peroxide treated IPEC-J2 cells (P <0.05, P <0.01, P < 0.001) and as selenomethionine concentration increased. As can be seen from FIG. 1D, 0.04. Mu.g/mL selenomethionine can improve cell morphology, so the following experiment was performed with this concentration.
(3) IPEC-J2 cells in logarithmic growth phase were inoculated at a density of 5000 cells/well into 96-well plates (100. Mu.L/well) and allowed to grow on the walls for 6h, after 12h treatment of IPEC-J2 cells with allicin at different concentrations (1, 2, 4 and 8. Mu.g/mL), cells were treated with 100. Mu.M hydrogen peroxide for 6h, and 100. Mu.L fresh complete medium and 10. Mu.L CCK-8 reagent were added per well, 5 replicates per group. After further culturing at 37℃for 1 hour, the absorbance of each well was measured at a wavelength of 450nm using a microplate reader, and the cell viability was calculated. And observing the cell morphology. The results are shown as E in FIG. 1 and F in FIG. 1. As can be seen from FIG. 1E, allicin at each concentration (1, 2, 4 and 8. Mu.g/mL) significantly increased the cell viability of hydrogen peroxide treated IPEC-J2 cells (P <0.001, P < 0.0001). As can be seen from FIG. 1F, 2. Mu.g/mL allicin significantly improved cell morphology.
(4) IPEC-J2 cells in logarithmic growth phase were inoculated at a density of 5 000cells/well into 96-well plates (100. Mu.L/well) and allowed to grow on the walls for 6h, IPEC-J2 cells were treated with selenomethionine at the indicated concentration (0.04. Mu.g/mL) in combination with allicin at different concentrations (1, 2, 4 and 8. Mu.g/mL) for 12h, or IPEC-J2 cells were treated with allicin at different concentrations (1, 2, 4 and 8. Mu.g/mL) alone for 12h, and then treated with 100. Mu.M hydrogen peroxide for 6h, with 100. Mu.L fresh complete medium and 10. Mu.LCCK-8 reagent added to each well, 5 replicates per group. After further culturing at 37℃for 1 hour, the absorbance of each well was measured at a wavelength of 450nm using a microplate reader, and the cell viability was calculated. And observing the cell morphology. The results are shown as G in fig. 1 and H in fig. 1. As can be seen from FIG. 1G, the combined effect of allicin at a concentration of 2, 4. Mu.g/mL and selenomethionine at the indicated concentration (0.04. Mu.g/mL) significantly improved the cell viability of IPEC-J2 cells (P <0.01 and P < 0.05) in hydrogen peroxide treatment compared to allicin (2, 4. Mu.g/mL) alone. Moreover, the combined effect significantly improved the cell viability of IPEC-J2 cells under hydrogen peroxide treatment (P <0.0001 and P < 0.0001) compared to selenomethionine alone. As can be seen from FIG. 1H, 0.04. Mu.g/mL selenomethionine combined with 2. Mu.g/mL allicin significantly improved cell morphology under hydrogen peroxide treatment. Thus, the combined use of allicin and selenomethionine has stronger antioxidation protection effect on the cells of the hydrogen peroxide treatment group.
Experimental example 2
Research on ROS (reactive oxygen species) level of IPEC-J2 cells by combined action of selenomethionine and allicin
Excessive accumulation of reactive oxygen species ROS in cells is an important initiation signal for oxidative stress leading to cell damage. The present experiment examined the alleviation of hydrogen peroxide induced ROS production by IPEC-J2 cells by a specific concentration of the drug combination. The specific method comprises the following steps: cells were seeded in 24-well plates at a density of 50000 cells/well, selenomethionine at a concentration of 0.04. Mu.g/mL and allicin at a concentration of 2. Mu.g/mL were used to treat the `IPEC-J2 cells individually or in combination for 12h after the cells had adhered to the walls, the cells were treated with 100. Mu.M hydrogen peroxide for 6h, each group of cells was washed 3 times with PBS solution, 500. Mu.L DCFH-DA (diluted in serum-free medium at a final concentration of 10. Mu. Mol/L) was added to each well, and incubated in a 37℃cell incubator for 20min. The cells were washed 3 times with serum-free cell culture medium to sufficiently remove DCFH-DA that did not enter the cells. ROS levels were detected using an inverted fluorescence microscope at 488nm excitation wavelength and 525nm emission wavelength, 3 replicates per group. Cells were seeded at 200000 cells/well in 6-well plates, treated as described above, each group of cells was digested with pancreatin and collected in centrifuge tubes, and ROS production levels were quantitatively detected using flow cytometry (LSRFortessa). Each group was repeated 3 times. The results are shown in fig. 2 a and 2B. As can be seen from fig. 2 a, the hydrogen peroxide treated group IPEC-J2 had significantly elevated intracellular ROS levels, whereas the 0.04 μg/mL concentration of selenomethionine and 2 μg/mL concentration of allicin combined treated group had significantly reduced intracellular ROS levels, and were lower than each of the individual treated groups. As can be seen from fig. 2B, the relative ROS content was quantified by flow cytometry, indicating that the intracellular ROS levels were significantly lower in the selenomethionine allicin combination treatment group than in the selenomethionine treatment group (P < 0.05) and allicin alone treatment group (P < 0.001).
Experimental example 3
Effects of selenomethionine and allicin combined action on intracellular redox homeostasis.
The effect of selenomethionine and allicin combination on intracellular redox homeostasis was reflected by detection of SOD (superoxide dismutase) and MDA (malondialdehyde). Cells were inoculated into a petri dish at a density of 200000 cells/dish, after the cells were attached, IPEC-J2 cells were treated with selenomethionine at a concentration of 0.04. Mu.g/mL and allicin at a concentration of 2. Mu.g/mL, either alone or in combination, for 12 hours, then treated with 100. Mu.M hydrogen peroxide for 6 hours, and each group of cells was digested with pancreatin and collected into a centrifuge tube, and the extract was added for sonication. The supernatants were placed on ice and centrifuged at 8000g/min for 10min, after which SOD activity and MDA content were detected according to the kit, 6 replicates per group. The results are shown in fig. 3 a and 3B. As can be seen from fig. 3 a, the hydrogen peroxide treated group IPEC-J2 cells showed significantly lower SOD activity than the blank (P < 0.001). The SOD activity of selenomethionine and allicin alone treatment group is significantly higher than that of hydrogen peroxide treatment group (P <0.001, P < 0.01); the SOD activity of the combined treatment group is significantly higher than that of the selenomethionine and allicin alone treatment group (P <0.001, P < 0.0001). As can be seen from fig. 3B, the hydrogen peroxide treated group showed significantly increased MDA content (P < 0.0001) compared to the blank control group cells, and the selenomethionine and allicin alone treated group showed significantly decreased MDA content (P <0.001, P < 0.01) compared to the hydrogen peroxide treated group; the MDA content of the combined treatment group is lower than that of the selenomethionine and allicin alone treatment group (P <0.05, P < 0.01). Therefore, the selenomethionine and allicin combined can enhance the activity of SOD by inducing cells, reduce the MDA content and play an antioxidant function, and the effect is better than that of selenomethionine and allicin which are independently used.
Experimental example 4
Effects of selenomethionine and allicin combined action on jejunal morphology and redox homeostasis.
Early experiments prove that 25mg/kg diquat is injected into the abdominal cavity to cause acute oxidative stress of the jejunum of a mouse, so that the influence of the combined action of selenomethionine and allicin on jejunum morphology and redox steady state is evaluated by using the acute oxidative stress as a model.
Male Balb/c mice were divided into 5 groups, namely, blank group, diquat group, selenomethionine+diquat group, allicin+diquat group, selenomethionine+allicin+diquat group. The pre-test result of dosage screening is used for determining that the gastric lavage dosage of selenomethionine is 0.4mg/kg, the gastric lavage dosage of allicin is 20mg/kg, and the dosage is 1 time per day for 7 continuous days. Diquat and placebo mice were gastrically treated with the same volume of PBS. On day 8, 25mg/kg diquat was intraperitoneally injected into each of the remaining groups except the blank control group, and after 24 hours, jejunal samples were collected from each group of mice. The mice jejunal specimens were fixed in 4% paraformaldehyde solution, paraffin embedded and sectioned. Stained with hematoxylin and eosin and scanned with a scanner (3 DHISTECH PANNORAMIC MIDI II) and the intestinal morphology was analyzed. The results are shown in FIG. 4A, 4B, 4C, 4D. Jejunum samples of 0.1g were taken and assayed for SOD activity and MDA content according to the kit, 4 replicates per group. The results are shown as E in FIG. 4 and F in FIG. 4.
From figures 4A, 4B, 4C, 4D, it can be seen that, in morphological observation, the ratio of the small intestine villus height to the crypt depth of the mice was increased by both the gastric lavage of 0.4mg/kg selenomethionine and 20mg/kg allicin (P <0.01, P < 0.05) compared to the diquat group. And the combination of the two significantly increases the ratio of the villus height to the crypt depth (P <0.001 and P < 0.0001) compared with the single administration. As can be seen from fig. 4E, the diquat group showed significantly reduced jejunal SOD activity (P < 0.01) compared to the placebo group. The selenomethionine and allicin alone treatment group significantly improved SOD activity compared to the diquat group (P <0.05 ); the SOD activity of the combined treatment group is higher than that of the selenomethionine and allicin alone treatment group (P <0.05 ). As can be seen from fig. 4F, the diquat group has significantly increased jejunal MDA content compared to the control group (P < 0.001) compared to the control group cells, and the selenomethionine and allicin alone treated group has significantly reduced MDA content (P <0.001 ); the MDA content of the combined treatment group is lower than that of the selenomethionine and allicin alone treatment group (P <0.01 ). Thus, the combined action of selenomethionine and allicin can improve the antioxidant level of the jejunum of the mice more than the independent action of selenomethionine and allicin.
Although the foregoing embodiments have been described in some, but not all, embodiments of the invention, it should be understood that other embodiments may be devised in accordance with the present embodiments without departing from the spirit and scope of the invention.
Claims (9)
1. A pharmaceutical combination for alleviating oxidative stress, comprising allicin and selenomethionine.
2. The pharmaceutical combination of claim 1, wherein the effective concentration of allicin in the cell assay is 2 μg/mL and the effective concentration of selenomethionine is 0.04 μg/mL when the oxidative stress is induced by hydrogen peroxide, the allicin to selenomethionine ratio being 50:1.
3. The pharmaceutical combination according to claim 1, wherein the effective concentration of allicin in the animal experiment is 20mg/kg and the effective concentration of selenomethionine is 0.4mg/kg, when the oxidative stress is induced by diquat, the allicin to selenomethionine ratio is 50:1.
4. Use of a pharmaceutical combination according to any one of claims 1 to 3 for the preparation of a medicament or feed additive for alleviating oxidative stress.
5. Use of a pharmaceutical combination according to any one of claims 1 to 3 for the preparation of a medicament or feed additive for increasing the viability of intestinal epithelial cells.
6. Use of a pharmaceutical combination according to any one of claims 1 to 3 for the preparation of a medicament or feed additive for increasing the ratio of villus length to crypt depth.
7. Use of a pharmaceutical combination according to any one of claims 1 to 3 for the preparation of a medicament or feed additive for reducing the production of active oxygen.
8. Use of a pharmaceutical combination according to any one of claims 1 to 3 for the preparation of a medicament or feed additive for reducing malondialdehyde content.
9. Use of a pharmaceutical combination according to any one of claims 1 to 3 for the preparation of a medicament or feed additive for increasing superoxide dismutase activity.
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