CN114957500A - Selenylation modification method for improving immunocompetence of Chinese yam polysaccharide and application thereof - Google Patents
Selenylation modification method for improving immunocompetence of Chinese yam polysaccharide and application thereof Download PDFInfo
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Abstract
The invention relates to a selenylation modification method for improving immunocompetence of Chinese yam polysaccharide and application thereof, comprising the following steps: step 1, extracting yam polysaccharide; and 2, selenizing the yam polysaccharide. Researches find that the selenized polysaccharide prepared by structurally modifying the yam polysaccharide can regulate intestinal flora and enhance body immunity, has obvious immunoregulation effect, and has better effect than the yam polysaccharide and sodium selenite, so that the selenized polysaccharide can be used for preparing medicaments or health care products for resisting oxidation or improving immunity.
Description
Technical Field
The invention belongs to the field of biological medicines, and relates to a selenylation modification method for improving immunocompetence of Chinese yam polysaccharide and application of selenylation Chinese yam polysaccharide in preparation of medicines or health-care products.
Background
In recent years, a large number of polysaccharides of natural origin have been extracted and developed, and they show good therapeutic effects on metabolic diseases induced by meals. Polysaccharides can regulate intestinal microflora structure, reduce the ratio of Firmicutes and bacteroidetes, and reduce the relative abundance of Proteobacteria (Proteobacteria). After the polysaccharide is utilized by the intestinal flora, the diversity, the composition and the distribution of the intestinal flora are dynamically regulated, the intestinal microenvironment is regulated by regulating and controlling the secretion of IFN-gamma, IL-4, IL-17 and TGF-beta of epithelial tissues, and the immunity of the organism is enhanced.
Selenium (Se) is a mineral trace with biological functions and has a very important impact on human health. Selenium affects important physiological functions of the human body, such as glutathione peroxidase, thioredoxin reductase and iodothymine deiodinase, by altering the expression of at least 30 selenoproteins. The selenase has antioxidant effect, and can regulate thyroid hormone metabolism and immune system function, improve sperm production and quality, and prevent cancer. In addition, selenium can also enhance immune function by maintaining the integrity of the cell membrane structure. Therefore, selenium has strong nutritional value and medicinal value.
The selenium polysaccharide is a substance with higher content and stronger activity in organic selenium, not only integrates the advantages of selenium and polysaccharide, but also embodies the biological function of selenium and polysaccharide combined into selenium polysaccharide. A large number of researches show that the selenium polysaccharide plays more and more roles in the aspects of immunoregulation, oxidation resistance, virus resistance, tumor resistance, blood pressure reduction, blood fat reduction and the like, and the research on the biological activity of the selenium polysaccharide is concerned.
The yam polysaccharide shows good pharmacological activity in the aspects of reducing blood sugar and blood fat, protecting liver, resisting tumor, enhancing immunity, resisting oxidation, resisting aging, protecting nerves, regulating intestinal flora and the like. However, no report on the research of selenizing the yam polysaccharide in the aspects of regulating intestinal flora and enhancing immunity is found.
Disclosure of Invention
The invention designs a selenylation modification method for improving immunocompetence of Chinese yam polysaccharide and application thereof, and solves the technical problems of how to modify the Chinese yam polysaccharide by selenium, optimize physiological and pharmacological functions and how to improve the body immunologic function by regulating intestinal flora.
In order to solve the technical problems, the invention adopts the following scheme:
a selenylation modification method for improving immunocompetence of yam polysaccharide comprises the following steps: step 1, extracting yam polysaccharide; and 2, selenizing the yam polysaccharide.
Preferably, the step 1 and the extraction of the yam polysaccharide comprise the following steps: step 11, crushing rhizome parts of the dioscorea opposita thumb; step 12, extracting a yam polysaccharide extracting solution; step 13, concentrating the yam polysaccharide extracting solution and then carrying out alcohol precipitation; and step 14, removing protein from the yam polysaccharide.
Preferably, the rhizome part of the dioscorea opposita thumb is firstly crushed, sieved by a 20-mesh sieve, mixed according to the material-liquid ratio of 1:25, and subjected to microwave extraction at the temperature of 80 ℃ for 20 minutes; the polysaccharide water solution is decompressed and concentrated to 1/5 volumes, ethanol with 4 times of volume is added, and the mixture is kept stand overnight; centrifuging, collecting the precipitate, adding water for redissolution according to the material-liquid ratio of 1:20, and mixing the polysaccharide water solution, the ethanol and the ammonium sulfate according to the mass ratio of 5.159: 2.811: 2.03 preparing into an ethanol/ammonium sulfate aqueous two-phase system, standing overnight, collecting the lower phase solution, dialyzing, and freeze-drying to obtain rhizoma Dioscoreae polysaccharide.
Preferably, the selenizing of yam polysaccharide in step 2 comprises the following steps: step 21, dissolving yam polysaccharide and adding a reaction catalyst; step 22, adding Na 2 SeO 3 Generating selenized polysaccharide; step 23, adjusting pH and then dialyzing; and 24, sequentially concentrating, cooling, freezing and drying to obtain the selenized yam polysaccharide.
Preferably, 500mg of yam polysaccharide is dissolved in 50mL of nitric acid solution (0.6%, v/v), and placed on a magnetic stirrer to completely dissolve the polysaccharide; then Na with the concentration of 0.05g/mL is added 2 SeO 3 8ml, stirring and reacting for 6 hours at the constant temperature of 70 ℃ and 1000 r/min; cooling to room temperature, adjusting pH to 5-6 with sodium bicarbonate, centrifuging at 5000r/min for 5 minTaking the supernatant, and dialyzing for 48 hours by using a 1000Da dialysis bag; concentrating the selenized polysaccharide solution by using a rotary evaporator, cooling to room temperature, pouring into a culture dish, putting into a refrigerator with the temperature of-20 ℃ for freezing, and finally putting the frozen sample into a freeze dryer for drying for 48 hours to obtain the selenized yam polysaccharide.
The polysaccharide is dissolved in the nitric acid solution to provide proper reaction conditions for the selenization reaction, and the nitric acid is a catalyst in the selenization reaction of the polysaccharide.
Because polysaccharide contains a large amount of hydroxyl which is an amphoteric group, protons obtained under acidic conditions are positive, protons lost under alkaline conditions are negative, and the proton loss is relatively stable in a solution with pH of 5-6, the pH needs to be adjusted to be between 5 and 6 after the selenylation reaction is finished.
A selenizing yam polysaccharide is prepared by the selenizing modification method.
The selenized Chinese yam polysaccharide is applied to the preparation of immunopotentiators or health products.
The application of the selenized yam polysaccharide in preparing the medicine or health care product for regulating intestinal flora.
The application of the selenized Chinese yam polysaccharide in preparing the medicine for regulating the content of the cell factors.
The selenized Chinese yam polysaccharide is applied to improving the immunity of mice under the immune deficiency induced by cyclophosphamide.
The selenylation modification method for improving the immunocompetence of the yam polysaccharide and the application thereof have the following beneficial effects:
(1) the selenium and the Chinese yam are combined, and then the synergistic effect of the selenium and the Chinese yam is utilized to improve the oxidation resistance and the immunity of the Chinese yam polysaccharide, reduce the toxicity of inorganic selenium, have better effect than sodium selenite or the Chinese yam polysaccharide and have important significance for improving the anti-stress capability and the immunity of animals.
(2) The research shows that the selenized polysaccharide prepared by structurally modifying the yam polysaccharide can adjust the intestinal flora, so that the selenized yam polysaccharide can be used for preparing the medicine or health care product with oxidation resistance or immunity improvement.
Drawings
FIG. 1 is a graph showing the content of TNF-. alpha.in mouse serum: (
±s，n=12)， *p＜0.05，**p＜0.01vs Model。
FIG. 2 is a graph showing the IL-6 content in mouse serum (
±s，n=12)，*p＜0.05，**p＜0.01vs Model。
FIG. 3 is a graph showing the index of spleen of mouse: (
±s，n=12)，*p＜0.05，**p＜0.01vs Model。
FIG. 4 is a mouse thymus index chart (
±s，n=12)，*p＜0.05，**p＜0.01vs Model。
FIG. 5 shows the secretion of TNF-alpha from mouse macrophages by yam polysaccharides and selenized yam polysaccharides
±s，n=3)，*p＜0.05，**p＜0.01vs Control。
FIG. 6 shows the secretion of IL-6 from mouse macrophage cells by yam polysaccharide and selenized yam polysaccharide: (
±s，n=3)，*p＜0.05，**p＜0.01vs Control。
NC is an abbreviation of Normal Control group Normal Control, and the intraperitoneal injection is physiological saline; LPS is lipopolysaccharide, is a TLR4 receptor agonist, and can elicit an immune response when it acts on RAW264.7 cells.
Detailed Description
The invention is further described below with reference to fig. 1 to 6:
example 1: preparing selenized yam polysaccharide:
1. extracting yam polysaccharide;
A. crushing: crushing rhizome parts of the Chinese yam, and sieving the crushed rhizome parts with a 20-mesh sieve;
B. extraction: mixing rhizoma Dioscoreae and water at a ratio of 1:25, and microwave extracting at 80 deg.C for 20 min;
C. alcohol precipitation: b, concentrating the yam polysaccharide extracting solution, adding ethanol with 4 times of volume, and standing overnight;
D. protein removal: and C, centrifugally collecting the yam polysaccharide in the step C, adding water for redissolution according to the material-liquid ratio of 1:20, and mixing the yam polysaccharide with ethanol and ammonium sulfate according to the mass ratio of 5.159: 2.811: 2.03 preparing into an ethanol/ammonium sulfate aqueous two-phase system, standing overnight, collecting the lower phase solution, dialyzing, and freeze-drying to obtain rhizoma Dioscoreae polysaccharide.
Selenization of yam polysaccharide
Dissolving 500mg rhizoma Dioscoreae polysaccharide in 50mL nitric acid solution (0.6%, v/v), placing on magnetic stirrer to dissolve polysaccharide completely, and adding Na with concentration of 0.05g/mL 2 SeO 3 8ml, stirring and reacting at the constant temperature of 70 ℃ for 6h at the constant temperature of 1000r/min, cooling to room temperature, adjusting the pH value to 5-6 by using sodium bicarbonate, centrifuging for 5 min at the speed of 5000r/min, taking the supernatant, and dialyzing for 48h by using a 1000Da dialysis bag. Concentrating the selenized polysaccharide solution by using a rotary evaporator, cooling to room temperature, pouring the solution into a culture dish, freezing the culture dish in a refrigerator at the temperature of-20 ℃, and finally drying the frozen sample in a freeze dryer for 48 hours to obtain the selenized yam polysaccharide.
Test example 1: effect of selenized yam polysaccharide on cyclophosphamide-induced hypoimmunity mice:
the mice were divided into 5 groups (n =12) based on weight balance, namely, a normal control group (normal saline), a cyclophosphamide group (CPA), a yam polysaccharide group (YP), a sodium selenite group (Se), and a selenized yam polysaccharide group (Se-YP). Except for the control group, the mice of other groups are injected with CPA 50 mg/kg in a single abdominal cavity on the next day of the experiment, and the mice of the blank group are injected with normal saline for 5 days continuously to prepare the mice model with low immunity. Normal control and model groups are given physiological saline with the same volume, a selenylation polysaccharide group is given with 0.1mg/kg (calculated by selenium) by intragastric administration, a sodium selenite group is given with 0.1mg/kg (calculated by selenium), a yam polysaccharide group is given with polysaccharide of a corresponding selenylation polysaccharide group by continuous intragastric administration for 14d, after the 14d administration is carried out for 12h, eyeballs are picked for blood taking, and serum is separated to measure the content of TNF-alpha and IL-6 in the serum according to the specification of an ELISA kit.
After blood collection, the mice were sacrificed by supporting the cervical vertebrae, and the thymus and spleen were removed, precisely weighed, and then the thymus index and spleen index of each group of mice were calculated according to the following formulas.
Thymus (spleen) index = thymus (spleen) weight/body weight (mg/10 g)
As shown in fig. 1 and fig. 2, spleen index of the selenized yam polysaccharide group mice is higher than that of the yam polysaccharide group and the sodium selenite group, and is significantly higher than that of the model group; the thymus index of the selenized yam polysaccharide group mice is higher than that of the model group, the yam polysaccharide group and the sodium selenite group, and the difference is not obvious.
Test example 3: influence of selenized yam polysaccharide on cytokine secretion of RAW264.7 macrophage
Taking RAW264.7 macrophage which grows well and grows in logarithmic phase, sucking out the culture solution, adding 2 mL of preheated PBS to wash the residual culture solution, repeating twice, then treating with cold PBS, performing blow beating after 2 min to prepare cell suspension with the concentration of 1 × 106/mL, inoculating the cell suspension into a sterile 96-hole cell culture plate, adding 100 μ L of the cell suspension into each hole, performing adherent culture in an incubator at 5% CO2 and 37 ℃ for 12h, and respectively adding 100 μ L of DMEM (containing 10% fetal bovine serum and 1% double antibody) culture solution and Na with different concentrations 2 SeO 3 And (0.5, 1.5, 3 mug/mL) yam polysaccharide and selenized yam polysaccharide (50, 250, 500 mug/mL) culture medium, and LPS (10 mug/mL) is used as a positive control. Each group was plated with 3 multiple wells, cultured in an incubator at 37 ℃ under 5% CO2 for 24 hours, centrifuged (170 Xg, 10 min), and the culture supernatant was collected. The amount of cytokine secreted from RAW264.7 macrophages was measured by ELISA kit with reference to kit instructions.
As shown in figures 4-6, selenized yam polysaccharide can induce RAW264.7 macrophage to secrete TNF-alpha and IL-6, and has obvious dose-effect relationship. The TNF-alpha secretion is close to LPS group, slightly higher than yam polysaccharide group, far higher than sodium selenite group and blank group; the IL-6 secretion is lower than that of LPS group, slightly higher than that of rhizoma Dioscoreae polysaccharide group, and far higher than that of sodium selenite group and blank group.
The invention is described above with reference to the accompanying drawings, it is obvious that the implementation of the invention is not limited in the above manner, and it is within the scope of the invention to adopt various modifications of the inventive method concept and solution, or to apply the inventive concept and solution directly to other applications without modification.
Claims (10)
1. A selenylation modification method for improving immunocompetence of yam polysaccharide comprises the following steps: step 1, extracting yam polysaccharide; and 2, selenizing the yam polysaccharide.
2. The selenization modification method for improving immunocompetence of yam polysaccharide as claimed in claim 1, wherein: step 1, the extraction of the yam polysaccharide comprises the following steps: step 11, crushing rhizome parts of the dioscorea opposita thumb; step 12, extracting a yam polysaccharide extracting solution; step 13, concentrating the yam polysaccharide extracting solution and then carrying out alcohol precipitation; and step 14, removing protein from the yam polysaccharide.
3. The selenization modification method for improving immunocompetence of yam polysaccharide as claimed in claim 2, wherein: crushing rhizome parts of the dioscorea opposita thumb, sieving the crushed rhizome parts by a 20-mesh sieve, mixing the crushed rhizome parts and the sieved rhizome parts according to a material-liquid ratio of 1:25, and performing microwave extraction at the temperature of 80 ℃ for 20 minutes; the polysaccharide water solution is decompressed and concentrated to 1/5 volumes, ethanol with 4 times of volume is added, and the mixture is kept stand overnight; centrifuging, collecting the precipitate, adding water for redissolution according to the material-liquid ratio of 1:20, and mixing the polysaccharide water solution, the ethanol and the ammonium sulfate according to the mass ratio of 5.159: 2.811: 2.03 preparing into an ethanol/ammonium sulfate aqueous two-phase system, standing overnight, collecting the lower phase solution, dialyzing, and freeze-drying to obtain rhizoma Dioscoreae polysaccharide.
4. The selenization modification method for improving immunocompetence of yam polysaccharide as claimed in claim 2, wherein: step 2, selenizing the yam polysaccharide comprises the following steps:
step 21, dissolving yam polysaccharide and adding a reaction catalyst; step 22, adding Na 2 SeO 3 Generating selenized polysaccharide; step 23, adjusting pH and then dialyzing; and 24, sequentially concentrating, cooling, freezing and drying to obtain the selenized yam polysaccharide.
5. The selenylation modification method for improving immunocompetence of yam polysaccharide according to claim 4, wherein the modification method comprises the following steps: dissolving 500mg of Chinese yam polysaccharide in 50mL of nitric acid solution (0.6%, v/v), and putting on a magnetic stirrer to completely dissolve the polysaccharide; then Na with the concentration of 0.05g/mL is added 2 SeO 3 8ml, stirring and reacting for 6 hours at the constant temperature of 70 ℃ and 1000 r/min; cooling to room temperature, adjusting pH to 5-6 with sodium bicarbonate, centrifuging at 5000r/min for 5 min, collecting supernatant, and dialyzing with 1000Da dialysis bag for 48 hr; concentrating the selenized polysaccharide solution by using a rotary evaporator, cooling to room temperature, pouring into a culture dish, putting into a refrigerator with the temperature of-20 ℃ for freezing, and finally putting the frozen sample into a freeze dryer for drying for 48 hours to obtain the selenized yam polysaccharide.
6. A selenized Chinese yam polysaccharide is characterized in that: prepared using the selenization modification process of any of claims 1-5.
7. The use of selenized yam polysaccharide of claim 6 in the preparation of immunopotentiator drugs or health products.
8. The use of selenized yam polysaccharide of claim 6 in the preparation of a medicament or health product for regulating intestinal flora.
9. The use of selenized yam polysaccharide of claim 6 in the preparation of a medicament for regulating cytokine content.
10. The use of selenized yam polysaccharides of claim 6 for enhancing the immunity of cyclophosphamide-induced immunocompromised mice.
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| CN116948049A (en) * | 2023-06-08 | 2023-10-27 | 湖南可诺耶生物科技有限公司 | Extraction method of selenium polysaccharide |
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