CN113956341A - Preparation method of red deer abomasum glycoprotein with hyaluronidase inhibition activity - Google Patents
Preparation method of red deer abomasum glycoprotein with hyaluronidase inhibition activity Download PDFInfo
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- CN113956341A CN113956341A CN202111360384.4A CN202111360384A CN113956341A CN 113956341 A CN113956341 A CN 113956341A CN 202111360384 A CN202111360384 A CN 202111360384A CN 113956341 A CN113956341 A CN 113956341A
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- abomasum
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- red deer
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
- C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
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- Life Sciences & Earth Sciences (AREA)
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Abstract
The invention relates to a preparation method of red deer abomasum glycoprotein with hyaluronidase inhibition activity, which comprises the steps of taking red deer abomasum, washing with tap water, cutting, freezing, carrying out low-temperature vacuum freeze drying, and crushing into powder; adding petroleum ether, placing in a Soxhlet extractor, heating, refluxing, defatting, extracting the defatted residue at room temperature under stirring, filtering with a vibrating screen, and vacuum filtering; precipitating to obtain crude glycoprotein; then carrying out primary separation by using anion exchange resin column chromatography, and screening the hyaluronidase inhibition activity; separating and purifying the active components by using a gel column HW-55F molecular sieve column chromatography system to obtain the red-spotted deer abomasum glycoprotein, and evaluating the hyaluronidase inhibition function of the red-spotted deer abomasum glycoprotein by using an in-vitro activity screening method. The red-spotted deer abomasum glycoprotein obtained by the invention has high purity, the protein content is 91.63%, the sugar content is 3.09%, the method adopted by the invention has simple process, strong operability and easy realization, fully utilizes the raw materials, increases the yield and the variety of the glycoprotein, and has wide industrial application prospect.
Description
Technical Field
The invention relates to the technical field of animal food processing and biological activity, in particular to a preparation method of red deer abomasum glycoprotein with hyaluronidase inhibition activity.
Background
Glycoproteins are formed by covalent linkage of oligosaccharides to polypeptide chains. Glycoproteins are widely present in eukaryotes, most of proteins have glycosylation modification, and glycoproteins play an important role in organisms and are closely related to the occurrence of some diseases. The sugar chain in the glycoprotein has the characteristics of micro heterogeneity and non-template synthesis, determines the structural complexity and composition diversity of the glycoprotein, so that the analysis of the sugar chain cannot make comprehensive and complete structural identification. The extraction of glycoproteins is generally performed by dilute salt solution extraction, acid-base solution extraction, enzyme extraction, and the like, and the extraction method can be determined according to the characteristics of the extraction target. With the rapid development of glycobiology research, glycoproteins have become the hot spots of biochemical research, attracting many disciplines, including workers in chemistry, biology, biochemistry, immunology, cell biology, medicine and food science to conduct research in this field. The glycoprotein has the characteristics of enhancing the immune regulation, inhibiting tumors, reducing blood sugar and blood fat, resisting oxidation, preventing aging and other biological activities and low toxicity, so the glycoprotein has wider and wider research and application in the fields of medicine and food science, and has wide application prospect in the aspects of novel characteristic medicines and functional food development.
China is a main breeding country of world red deer, and the number of artificial breeding stock pens is more than 110 and accounts for about 97 percent of the total world breeding amount. Xinjiang has rich red deer resources. At present, 31 breeding enterprises in the Xinjiang singular general farm pasture, the Arietai, Changji, Yili, Ba Zhou and other places in Xinjiang province are owned by Xinjiang, the Arietu breeding farm is subjected to the chess cloth, more than 10 thousands of Arieten are artificially domesticated, and the Arieten is a wild animal with the largest domestication and breeding scale in Xinjiang. The abomasum is the fourth stomach of deer, also called the fourth stomach of deer, and is a specific stomach structure of ruminant. The abomasum of deer has gastric glands, similar to those of ordinary monogastric animals, and can secrete digestive juice, so it is also called abomasum. The abomasum of deer is rich in protein and protease. The deer stomach mainly has the effects of warming cold and nourishing stomach, and has good effects on gastritis, stomachache, gastric ulcer, gastrectasia, gastroparesis, gastroptosis, prolapse of gastric mucosa and hyperacidity. The deer stomach also has the effects of strengthening the middle-jiao and replenishing qi, invigorating stomach and strengthening body, promoting digestion, promoting absorption, treating dyspepsia, tonifying deficiency and enriching blood, has high medicinal value, and is also a good health food.
The hyaluronidase inhibitor is a substance having an inhibitory effect on the activation of hyaluronidase. Hyaluronidase is a specific lyase of hyaluronic acid, and hyaluronic acid plays an important role in many development and regulation processes of human body, and inhibition of the activity of hyaluronidase can prevent hyaluronic acid from being decomposed and maintain normal physiological functions.
Disclosure of Invention
The invention aims to provide a preparation method of red-spotted deer abomasum glycoprotein with hyaluronidase inhibition activity, which comprises the steps of firstly carrying out reflux degreasing on pretreated fresh red-spotted deer abomasum by using petroleum ether, extracting, centrifuging to obtain supernatant, carrying out reduced pressure concentration, carrying out alcohol precipitation, and carrying out freeze drying to obtain crude glycoprotein; and separating and purifying the crude glycoprotein by anion exchange chromatography and gel chromatography to obtain the high-purity red deer abomasum glycoprotein. The yield of the obtained red deer abomasum glycoprotein is 4.7%, the protein content is 91.63%, the sugar content is 3.09%, the molecular weight is 15.11Da, and the hyaluronidase inhibition rate is 79.23%. The method has the advantages of simple process, convenient operation, high extraction rate, high purity, stable property, no need of using a large amount of toxic solvents, low cost, easy popularization and application and the like.
The preparation method of the red-spotted deer abomasum glycoprotein with hyaluronidase inhibition activity, disclosed by the invention, comprises the following steps of:
a. taking abomasum of Tianshan red deer, washing with running water to remove the content in abomasum, and storing in a refrigerator at-20 deg.C for use;
b. b, removing fat and connective tissues from the red deer abomasums cleaned by using pre-cooled distilled water flowing water in the step a, shearing the red deer abomasums to 5-8mm in width, freezing the red deer abomasums at the temperature of-80 ℃ for 12 hours, and then carrying out freeze drying to obtain red deer abomasum dry powder;
c. b, crushing the red deer abomasum dry powder obtained in the step b at low temperature by using liquid nitrogen, crushing by using a crusher at the rotating speed of 5000r/min, pausing for 0.5min every 1min, sieving by using a 80-mesh sieve after accumulative crushing for 3-5min to obtain red deer abomasum powder, and storing the red deer abomasum powder in a refrigerator at the temperature of-20 ℃ for later use;
d. c, adding precooled petroleum ether into the red deer abomasum powder obtained in the step c at the temperature of 4 ℃ according to the material-liquid ratio of 1:10-15, mixing, placing in a Soxhlet extractor, heating at the temperature of 60-75 ℃ for 3-5h, carrying out reflux degreasing treatment, then carrying out suction filtration to remove the petroleum ether, and drying at room temperature to obtain degreased red deer abomasum powder;
e. mixing the degreased abomasum powder obtained in the step d with a NaCl solution with the concentration of 2-5% according to the mass ratio of 1:10-15, stirring and extracting for 3-5h at room temperature, filtering the extracting solution by using an oscillating screen, centrifuging the filtering solution at the temperature of 4 ℃ for 10-20min at 6000-8000r/min to obtain a supernatant, concentrating the supernatant by using a rotary evaporator under reduced pressure of 35-60mbar at the temperature of 50 ℃ to 1/10-1/15 times of the original volume, desalting the concentrated solution by using a 3500Da dialysis bag for 48 hours, and centrifuging for 10-20min at the temperature of 4 ℃ at 6000-8000r/min after desalting to obtain a total extracting solution;
f. adding absolute ethanol into the extracting solution obtained in the step e at the temperature of 0-4 ℃ to ensure that the volume concentration of the ethanol is 70-80%, standing for 24h for complete precipitation, dissolving the precipitate with distilled water, centrifuging at 8000r/min at the temperature of 4 ℃ for 15min, taking supernatant, and freeze-drying at the pressure of 10Pa, -80 ℃, and 24h to obtain crude glycoprotein of the red-spotted deer abomasum;
g. f, carrying out first purification on the crude glycoprotein obtained in the step f, completely dissolving the crude glycoprotein into a phosphate buffer solution with the concentration of 0.02M and the pH value of 7.8 to the concentration of 15mg/mL, centrifuging, taking supernatant, carrying out chromatography by using DEAE-Sephadex A-25 anion exchange resin column, respectively eluting by using NaCl solutions with the concentrations of 0.1mol/mL, 0.2mol/mL, 0.3mol/mL and 0.4mol/mL, detecting the absorption peak of sugar at 492nm by using a phenol-sulfuric acid method, detecting the absorption peak of protein at 280nm by using an ultraviolet spectrophotometer, collecting the eluates with the absorption peaks overlapping parts of the sugar and the protein, concentrating the eluent with the overlapping parts under reduced pressure of 35-60mbar at the temperature of 50 ℃, desalting concentrated solution by using a 3500Da dialysis bag for 48h, freezing and drying at the temperature of-80 ℃ and the pressure of 10Pa for 8h after desalting, obtaining a first purified glycoprotein;
h. g, detecting the hyaluronidase inhibition activity of the first purified glycoprotein obtained in the step g, and collecting components with the hyaluronidase inhibition activity;
i. and (4) carrying out secondary purification on the components with the hyaluronidase inhibition activity collected in the step h, dissolving the components with the hyaluronidase inhibition activity by using distilled water, dissolving the components by using distilled water, separating and purifying the components by using a gel column HW-55F to obtain a pure red deer abomasum glycoprotein solution, and carrying out concentration under the pressure of 35-60mbar and the temperature of 50 ℃, and freeze-drying under the temperature of-80 ℃ and the pressure of 10Pa to obtain the Tianshan red deer abomasum glycoprotein.
The application of the red-spotted deer abomasum glycoprotein obtained by the method in preparing anti-inflammatory drugs.
According to the preparation method of the red-spotted deer abomasum hyaluronidase inhibition activity glycoprotein, in the method, the hyaluronidase inhibition activity is detected by adopting Elson-Morgan, wherein 0.5mL and 0.5mL of hyaluronidase is 500unit/mL in sample liquid, and the sample liquid is cultured for 20min at 37 ℃; adding 2.5mol/L CaCl20.1mL, and preserving the heat at 37 ℃ for 20 min; adding 0.5mg/mL potassium hyaluronate at 37 deg.CThe temperature is 40min, the mixture is placed for 5min at the normal temperature, 2 drops of 5mol/L NaOH are added, 0.5mL of acetylacetone solution is added, accurate boiling water bath is carried out for 15min, ice water is immediately cooled for 5min, the mixture is placed for 10min at the normal temperature, 0.5mL of deionized water and 0.5mL of Ehrlich reagent are added, absolute ethyl alcohol is added to 10mL, the mixture is shaken and mixed evenly, the mixture is placed for 30min, color development is carried out, and the ABS value is measured at 530 nm.
Drawings
FIG. 1 is a schematic view of the process of the present invention;
FIG. 2 is a chromatogram of the present invention for separating and purifying red deer abomasum glycoprotein using anion exchange resin;
FIG. 3 is a chromatogram of the present invention using gel chromatography to purify the red deer abomasum glycoprotein;
FIG. 4 is a chart showing HPLC molecular weight determination and purity identification of red deer abomasum glycoprotein of the present invention;
FIG. 5 is an infrared spectrum of red deer abomasum glycoprotein of the present invention;
FIG. 6 is a GC chart of the analytical analysis of monosaccharides from red deer abomasum glycoprotein, wherein 1 is rhamnose, 2 is arabinose, 3 is xylose, 4 is mannose, 5 is glucose, and 6 is galactose;
FIG. 7 is a graph of the ultraviolet full wavelength of red deer abomasum glycoprotein in accordance with the present invention;
FIG. 8 is a 15% SDS-polyacrylamide gel electrophoresis of red deer abomasum glycoprotein of the present invention;
FIG. 9 shows hyaluronidase inhibitory activity of the red-spotted deer abomasum glycoprotein of the present invention.
Detailed Description
The present invention will be described in further detail with reference to the following examples and drawings, but the present invention is not limited thereto in any way.
Example 1
a. Taking abomasum of Tianshan red deer, washing with running water to remove the content in abomasum, and storing in a refrigerator at-20 deg.C for use;
b. b, removing fat and connective tissues from the red deer abomasums cleaned by using pre-cooled distilled water flowing water in the step a, shearing the red deer abomasums to 5mm, freezing the red deer abomasums at the temperature of-80 ℃ for 12 hours, and then carrying out freeze drying to obtain red deer abomasum dry powder;
c. b, crushing the red deer abomasum dry powder obtained in the step b at low temperature by using liquid nitrogen, crushing by using a crusher at the rotating speed of 5000r/min, pausing for 0.5min every 1min, sieving by using a 80-mesh sieve after accumulative crushing for 3-5min to obtain red deer abomasum powder, and storing the red deer abomasum powder in a refrigerator at the temperature of-20 ℃ for later use;
d. c, adding pre-cooled petroleum ether into the red deer abomasum powder obtained in the step c at the temperature of 4 ℃ according to the material-liquid ratio of 1:10, mixing, placing in a Soxhlet extractor, heating at the temperature of 60 ℃ for 5 hours, carrying out reflux degreasing treatment, then carrying out suction filtration to remove the petroleum ether, and drying at room temperature to obtain degreased red deer abomasum powder;
e. mixing the degreased abomasum powder obtained in the step d with a NaCl solution with the concentration of 2% according to the mass ratio of 1:10, stirring and extracting for 4 hours at room temperature, filtering the extracting solution by using a vibrating screen, centrifuging the filtered solution at the temperature of 4 ℃ and 6000r/min for 10 minutes to obtain a supernatant, concentrating the supernatant by using a rotary evaporator under reduced pressure at the pressure of 35mbar and at the temperature of 50 ℃ to 1/10 times of the original volume, desalting the concentrated solution by using a 3500Da dialysis bag for 48 hours, and centrifuging for 10 minutes at the temperature of 4 ℃ and 6000r/min after desalting to obtain a total extracting solution;
f. adding absolute ethanol into the extracting solution obtained in the step e at the temperature of 4 ℃ according to the volume ratio of 1:4, mixing until the volume concentration of the ethanol is 70%, standing for 24h for complete precipitation, dissolving the precipitate with distilled water, centrifuging for 15min at the temperature of 4 ℃ at 8000r/min, taking supernatant, and freeze-drying for 10Pa, -80 ℃, and obtaining crude glycoprotein of red-rooted red deer abomasum after 24h, wherein the extraction rate is 10.29%, the protein content is 41.73%, and the sugar content is 8.06%;
g. f, carrying out first purification on the crude glycoprotein obtained in the step f, completely dissolving the crude glycoprotein into a phosphate buffer solution with the concentration of 0.02M and the pH value of 7.8 to the concentration of 15mg/mL, centrifuging, taking supernatant, carrying out DEAE-Sephadex A-25 anion exchange resin column chromatography, respectively eluting with NaCl solutions with the concentrations of 0.1mol/mL, 0.2mol/mL, 0.3mol/mL and 0.4mol/mL, detecting the sugar absorption peak at 492nm by using a phenol-sulfuric acid method, detecting the protein absorption peak at 280nm by using an ultraviolet spectrophotometer, collecting the eluates with the mutually overlapped absorption peaks of the sugar and the protein, carrying out reduced pressure concentration on the mutually overlapped eluates at the pressure of 35mbar and at the temperature of 50 ℃, desalting concentrated solution by using a 3500Da dialysis bag for 48h, freezing and drying at the temperature of-80 ℃ and the pressure of 10Pa for 8h after desalting, obtaining a first purified glycoprotein (figure 2);
h. g, detecting the hyaluronidase inhibition activity of the first purified glycoprotein obtained in the step g, and collecting components with the hyaluronidase inhibition activity;
i. performing secondary purification on the components with the hyaluronidase inhibition activity collected in the step h, dissolving the components with the hyaluronidase inhibition activity by distilled water, separating and purifying the components by adopting a gel column HW-55F to obtain a pure red deer abomasum glycoprotein solution, and performing freeze drying at the temperature of 50 ℃ and the temperature of-80 ℃ under the pressure of 35mbar to obtain the red deer abomasum glycoprotein; named RDA4-1 (FIG. 3).
Example 2
a. Taking abomasum of Tianshan red deer, washing with running water to remove the content in abomasum, and storing in a refrigerator at-20 deg.C for use;
b. b, removing fat and connective tissues from the red deer abomasums cleaned by using pre-cooled distilled water flowing water in the step a, shearing into pieces with the width of 6mm, freezing the red deer abomasums at the temperature of-80 ℃ for 12 hours, and then carrying out freeze drying to obtain red deer abomasum dry powder;
c. b, crushing the red deer abomasum dry powder obtained in the step b at low temperature by using liquid nitrogen, crushing by using a crusher at the rotating speed of 5000r/min, pausing for 0.5min every 1min, sieving by using a 80-mesh sieve after cumulative crushing for 4min to obtain red deer abomasum powder, and storing the red deer abomasum powder in a refrigerator at the temperature of-20 ℃ for later use;
d. c, adding pre-cooled petroleum ether into the red deer abomasum powder obtained in the step c at the temperature of 4 ℃ according to the material-liquid ratio of 1:13, mixing, placing in a Soxhlet extractor, heating at the temperature of 70 ℃ for 3 hours, carrying out reflux degreasing treatment, then carrying out suction filtration to remove the petroleum ether, and drying at room temperature to obtain degreased red deer abomasum powder;
e. mixing the degreased abomasum powder obtained in the step d with a NaCl solution with the concentration of 3% according to the mass ratio of 1:13, stirring and extracting for 3 hours at room temperature, filtering the extracting solution by using a vibrating screen, centrifuging the filtered solution at the temperature of 4 ℃ and 7000r/min to obtain a supernatant, concentrating the supernatant by using a rotary evaporator under reduced pressure at the pressure of 50mbar and at the temperature of 50 ℃ to 1/13 times of the original volume, desalting the concentrated solution by using a 3500Da dialysis bag for 48 hours, and centrifuging for 15 minutes at the temperature of 4 ℃ and 7000r/min to obtain a total extracting solution;
f. adding absolute ethanol into the extracting solution obtained in the step e at the temperature of 0 ℃ according to the volume ratio of 1:4 to enable the volume concentration of the ethanol to be 75%, standing for 24h to completely precipitate, dissolving the precipitate with distilled water, centrifuging for 15min at the temperature of 4 ℃ at 8000r/min, taking supernatant, and freeze-drying for 10Pa, 80 ℃ below zero, 24h to obtain crude glycoprotein of red-rooted red deer abomasum, wherein the extraction rate is 8.54%, the protein content is 35.63%, and the sugar content is 5.91%;
g. f, carrying out first purification on the crude glycoprotein obtained in the step f, completely dissolving the crude glycoprotein into a phosphate buffer solution with the concentration of 0.02M and the pH value of 7.8 to the concentration of 15mg/mL, centrifuging, taking supernatant, carrying out DEAE-Sephadex A-25 anion exchange resin column chromatography, respectively eluting with NaCl solutions with the concentrations of 0.1mol/mL, 0.2mol/mL, 0.3mol/mL and 0.4mol/mL, detecting the sugar absorption peak at 492nm by using a phenol-sulfuric acid method, detecting the protein absorption peak at 280nm by using an ultraviolet spectrophotometer, collecting the eluates with the mutually overlapped absorption peaks of the sugar and the protein, carrying out reduced pressure concentration on the mutually overlapped eluates at the pressure of 50mbar, concentrating at the temperature of 50 ℃, desalting concentrated solution by using a 3500Da dialysis bag for 48h, carrying out freeze drying at the temperature of-80 ℃ and the pressure of 10Pa for 8h after desalting, obtaining a first purified glycoprotein (figure 2);
h. g, detecting the hyaluronidase inhibition activity of the first purified glycoprotein obtained in the step g, and collecting components with the hyaluronidase inhibition activity;
i. and (4) performing secondary purification on the components with the hyaluronidase inhibition activity collected in the step h, dissolving the components with the hyaluronidase inhibition activity by using distilled water, separating and purifying the components by using a gel column HW-55F to obtain a pure red deer abomasum glycoprotein solution, and performing freeze drying at the pressure of 50mbar and the temperature of 50 ℃, at the temperature of-80 ℃ and under the pressure of 10Pa to obtain the red deer abomasum glycoprotein named as RDA4-1 (figure 3).
Example 3
a. Taking abomasum of Tianshan red deer, washing with running water to remove the content in abomasum, and storing in a refrigerator at-20 deg.C for use;
b. b, removing fat and connective tissues from the red deer abomasums cleaned by using pre-cooled distilled water flowing water in the step a, shearing into pieces with the width of 8mm, freezing the red deer abomasums at the temperature of-80 ℃ for 12 hours, and then carrying out freeze drying to obtain red deer abomasum dry powder;
c. b, crushing the red deer abomasum dry powder obtained in the step b by a crusher at the rotating speed of 5000r/min, pausing for 0.5min every 1min, sieving by a 80-mesh sieve after cumulative crushing for 5min to obtain red deer abomasum powder, and storing the red deer abomasum powder in a refrigerator at the temperature of-20 ℃ for later use;
d. c, adding pre-cooled petroleum ether into the red deer abomasum powder obtained in the step c at the temperature of 4 ℃ according to the material-liquid ratio of 1:15, mixing, placing in a Soxhlet extractor, heating at the temperature of 75 ℃ for 5 hours, carrying out reflux degreasing treatment, then carrying out suction filtration to remove the petroleum ether, and drying at room temperature to obtain degreased red deer abomasum powder;
e. mixing the degreased abomasum powder obtained in the step d with a NaCl solution with the concentration of 5% according to the mass ratio of 1:15, stirring and extracting for 3-5h at room temperature, filtering the extracting solution by using a vibrating screen, centrifuging the filtrate at the temperature of 4 ℃ for 20min at 8000r/min to obtain a supernatant, concentrating the supernatant by using a rotary evaporator under reduced pressure at the pressure of 60mbar and at the temperature of 50 ℃ to 1/15 times of the original volume, desalting the concentrated solution by using a 3500Da dialysis bag for 48 hours, and centrifuging for 20min at the temperature of 4 ℃ at 8000r/min after desalting to obtain a total extracting solution;
f. d, adding absolute ethyl alcohol into the extracting solution obtained in the step e at the temperature of 2 ℃ to enable the volume concentration of the ethyl alcohol to be 80%, standing for 24 hours to completely precipitate, dissolving the precipitate with distilled water, and freeze-drying at the temperature of-80 ℃, the pressure of 10Pa and the time of 48 hours to obtain crude glycoprotein of the red deer abomasum;
g. f, carrying out first purification on the crude glycoprotein obtained in the step f, completely dissolving the crude glycoprotein into a phosphate buffer solution with the concentration of 0.02M and the pH value of 7.8 to the concentration of 15mg/mL, centrifuging, taking supernatant, carrying out DEAE-Sephadex A-25 anion exchange resin column chromatography, respectively eluting with NaCl solutions with the concentrations of 0.1mol/mL, 0.2mol/mL, 0.3mol/mL and 0.4mol/mL, detecting the sugar absorption peak at 492nm by using a phenol-sulfuric acid method, detecting the protein absorption peak at 280nm by using an ultraviolet spectrophotometer, collecting the eluates with the mutually overlapped absorption peaks of the sugar and the protein, carrying out reduced pressure concentration on the mutually overlapped eluates at the pressure of 60mbar and at the temperature of 50 ℃, desalting concentrated solution by using a 3500Da dialysis bag for 48h, freezing and drying at the temperature of-80 ℃ and the pressure of 10Pa for 8h after desalting, obtaining a first purified glycoprotein (figure 2);
h. g, detecting the hyaluronidase inhibition activity of the first purified glycoprotein obtained in the step g, and collecting components with the hyaluronidase inhibition activity;
i. and (4) performing secondary purification on the components with the hyaluronidase inhibition activity collected in the step h, dissolving the components with the hyaluronidase inhibition activity by using distilled water, separating and purifying the components by using a gel column HW-55F to obtain a pure red deer abomasum glycoprotein solution, and performing freeze drying at the pressure of 60mbar and the temperature of 50 ℃ and the temperature of-80 ℃ and the pressure of 10Pa to obtain the red deer abomasum glycoprotein named as RDA4-1 (figure 3).
Example 4
The implementation effect is as follows:
HPLC for purity and molecular weight determination:
preparing chromatographic pure glucan with molecular weights of 5KDa, 25KDa, 50KDa, 80KDa, 150KDa and 410KDa purchased from Sigma and blue glucan respectively into polysaccharide molecular weight standard samples with the concentration of 2mg/mL by using ultrapure water solution, filtering by using a 0.45 mu m filter membrane, injecting samples, and adopting blue glucan with retention time V0Pore volume is normalized by the logarithm of molecular weight (1 g)Mw) Plotting a standard curve with the abscissa and the ordinate as the retention time, and obtaining a regression equation of the standard curve as y-4.2574 x +10.494, R20.9904; simultaneously, sampling the red-spotted deer abomasum glycoprotein obtained in any one of the embodiments 1-3, and calculating the relative molecular mass according to the retention time; chromatographic conditions are as follows: the chromatographic column is TSK-G3000 PWXL (7.8 multiplied by 300mm), the mobile phase is ultrapure water, the flow rate is 0.6mL/min, the temperature of the column incubator is 25 ℃, and the detector shows the differenceA detector; as can be seen from HPLC analysis (FIG. 4), the molecular weight and purity of the red-spotted deer abomasum glycoprotein prepared by the invention are 15.11kDa and 95.41 percent respectively;
infrared (FT-IR) analysis:
the abomasum glycoprotein of any one of the athyris amurensis obtained in examples 1-3 was purified at 4000--1The characteristic absorption peak of the carbohydrate and the protein compound appears in the middle, as shown in figure 5, which is 3304CM-1A distinct broad peak is formed, and is the superposition stretching vibration of O-H bonds and N-H bonds, which indicates that intermolecular or intramolecular hydrogen bonds exist, and the peak is the characteristic absorption peak of polysaccharide and protein molecules. 2927CM-1The weaker bending is C-H vibration; at 1654CM-1The absorption is a deformation vibration of N-H, indicating the presence of protein in the sample; 1541CM-1The absorption strip is C-N telescopic vibration; 1238CM-1And 1077CM-1The absorption peaks at (a) are the stretching vibration of the C ═ O group and the C — N stretching vibration; at 536CM-1Nearby absorption of N-H bending vibration attributable to amide groups;
protein content determination:
adopting a BCA method: accurately measuring a 2mg/mL standard bovine serum albumin solution, and preparing a 25-2000 mu g/mL standard solution; preparing a working solution according to the operation of the BCA protein measurement kit specification, wherein the BCA reagent is divided into a reagent A and a reagent B, and the reagent B with the volume of 1 time is added into the reagent A with the volume of 50 times, and is fully shaken up for standby; measuring 25uL of standard solutions with different concentrations in a 96-well plate, respectively adding 175uL of BCA working solution, slightly oscillating, fully mixing, keeping the temperature in an incubator at 37 ℃ for 30min, and measuring the absorbance of the solution at 562nm by using an enzyme-linked immunosorbent assay; drawing a standard curve by taking the absorbance of the standard substance as an abscissa and taking the mass concentration of bovine serum albumin as an ordinate; accurately weighing 2.0mg of purified and obtained red deer abomasum glycoprotein sample, adding 2mL of distilled water for dissolving, taking 25 mu L of sample solution to a 96-well plate, determining the absorbance value of the sample solution by the same standard curve operation method, and calculating to obtain the glycoprotein RDA4-1 with the protein content of 91.63%;
and (3) total sugar content determination:
the total sugar content is determined by adopting a phenol-sulfuric acid method: accurately weighing 10mg of glucose standard substance, adding distilled water to dissolve, fixing the volume to 50mL, wherein the final concentration of the standard substance is 0.2mg/mL, and respectively and accurately sucking 0, 0.4, 0.8, 1.2, 1.6 and 2.0mL into different test tubes; adding distilled water to make each tube volume reach 2 mL; respectively and accurately sucking 50uL of the concentrated sulfuric acid into a 96-pore plate, quickly adding 150uL of concentrated sulfuric acid into each tested pore, adding 30uL of 5% phenol into each pore, shaking uniformly, standing at room temperature for 20min, standing at room temperature for 10min, and measuring absorbance at 490nm by using an enzyme-linked immunosorbent assay (ELIAS); taking the absorbance as an abscissa, taking the mass concentration of the standard solution as an ordinate, and taking distilled water as a blank control to prepare a standard curve; and (3) sample determination: preparing 200 mug/mL of red deer abomasum glycoprotein sample solution, measuring absorbance by the standard curve operation method, calculating the total sugar content, and calculating to obtain that the total sugar content of the red deer abomasum glycoprotein RDA4-1 is 3.09%;
analysis of monosaccharide composition:
hydrolysis: taking 5mg of a purified red deer abomasum glycoprotein sample, adding 4mL of 2mol/L trifluoroacetic acid into a headspace bottle, sealing, hydrolyzing at 110 ℃ for 6h at constant temperature, adding methanol, decompressing, evaporating, and repeating for 3 times; acetylated derivatives: adding 8mg glycolic acid, 1mL pyridine and 1mL acetic anhydride into the hydrolysate, heating at 90 deg.C for 1h, cooling, drying with nitrogen, diluting the acetylated monosaccharide alcohol with chloroform, and performing gas chromatography-mass spectrometry (GS) analysis (figure 6), wherein GC analysis shows that the rDNA 4-1 of red deer abomasum glycoprotein consists of rhamnose, arabinose, mannose, xylose, glucose and galactose (Table 1).
TABLE 1 composition of glycoprotein monosaccharides (mol%)
Amino acid analysis:
the determination method comprises the following steps: weighing 5mg of purified velvet deerhorn abomasum glycoprotein into a hydrolysis tube, adding 3mL of hydrochloric acid solution with the concentration of 6mol/L, introducing nitrogen for 2min, vacuumizing, sealing the tube, placing the tube in an oven, hydrolyzing at the temperature of 110 ℃ for 24h, evaporating the hydrolyzed hydrolysate under reduced pressure by using a rotary evaporator to fully evaporate hydrochloric acid to dryness, removing acid in the hydrolysate, washing the hydrolysate for 3 times by using methanol, continuously evaporating residual hydrochloric acid to dryness, then diluting the hydrolysate to 1mL by using distilled water to constant volume, performing derivatization, filtering the derivatized hydrolysate by using a 0.45-micrometer microporous filter membrane, and performing amino acid content determination and analysis on the filtrate by using an amino acid analyzer, wherein the determination results are shown in Table 2:
TABLE 2 results of amino acid analysis of abomasum glycoprotein from Cervus elaphus
As can be seen from table 2, the protein chain in the red-spotted deer abomasum glycoprotein obtained by the method of the present invention comprises 17 amino acids, wherein the main amino acids are glycine 25.257%, alanine 20.048%, tyrosine 12.903%, valine 8.115%, and lysine 6.710%;
full wavelength ultraviolet scanning:
full wavelength scan with uv spectrophotometer: switching on the power supply, opening an instrument switch, opening a dark box cover of the sample chamber, and preheating for 10 min. And respectively pouring the blank liquid and the determination liquid into a colorimetric cuvette, wiping the outer wall with a piece of lens wiping paper, and putting the cuvette into a sample chamber to align the blank tube with the light path. After the ultraviolet scanner is corrected by the blank solution, the maximum absorption peak of the sample in the vicinity of 280nm is measured, which also accords with the maximum absorption characteristic of the ultraviolet of the protein substances, and the ultraviolet scanning atlas is shown in figure 7.
SDS-PAGE electrophoresis:
in order to examine the molecular weight range and the separation and purification effect of the red-spotted deer abomasum glycoprotein, 12% SDS-PAGE vertical electrophoresis is carried out by referring to a Laemmli method (figure 8); sample buffer: 0.5mL of 0.5mol/L Tris-HCl solution with pH of 6.8, 2mL of 10% SDS, 1mL of glycerol, 0.5mL of beta-mercaptoethanol, 1mL of ultrapure water and trace bromophenol blue; electrophoresis conditions: the concentration of the sample is 2.5mg/mL, the sample loading amount is 10 muL, the sample is heated for 5min before loading, the voltage of the sample is 75V by taking the voltage as the standard, and the voltage is increased to 150V after the sample is concentrated into a line in the concentrated gel part; electrophoresis buffer solution: 25mM Tris,192mM glycine, 0.1% SDS, pH 8.3; fixing liquid: 20% (v/v) ethanol, 10% (v/v) acetic acid; coomassie brilliant blue staining solution: 0.6g of Coomassie Brilliant blue R250 is dissolved in 200mL of destaining solution; decoloring liquid: 25% (v/v) ethanol, 8% (v/v) acetic acid;
hyaluronidase inhibitory activity:
culturing the separated and purified red deer abomasum glycoprotein at 37 ℃ for 20min by adopting Elson-Morgan and 0.5mL of sample liquid and 500unit/mL of hyaluronidase; adding 2.5mol/L CaCl20.1mL, and preserving the heat at 37 ℃ for 20 min; adding 0.5mg/mL of potassium hyaluronate, keeping the temperature at 37 ℃ for 40min, standing at normal temperature for 5min, adding 2 drops of 5mol/L NaOH, adding 0.5mL of acetylacetone solution, accurately boiling in a water bath for 15min, immediately cooling with ice water for 5min, standing at normal temperature for 10min, adding 0.5mL of deionized water and 0.5mL of Ehrlich reagent, adding absolute ethyl alcohol to 10mL, shaking and uniformly mixing, standing for 30min, developing, and measuring an ABS value at 530nm, wherein as can be seen from fig. 9, the red deer abomasum glycoprotein prepared by the invention has good hyaluronidase inhibition activity, namely 79.23%.
The above examples are embodiments of the present invention, but are not limited thereto.
Claims (2)
1. A preparation method of red deer abomasum glycoprotein with hyaluronidase inhibition activity is characterized by comprising the following steps:
a. taking abomasum of Tianshan red deer, washing with running water to remove the content in abomasum, and storing in a refrigerator at-20 deg.C for use;
b. b, removing fat and connective tissues from the red deer abomasums cleaned by using pre-cooled distilled water flowing water in the step a, shearing the red deer abomasums to 5-8mm in width, freezing the red deer abomasums at the temperature of-80 ℃ for 12 hours, and then carrying out freeze drying to obtain red deer abomasum dry powder;
c. b, crushing the red deer abomasum dry powder obtained in the step b at low temperature by using liquid nitrogen, crushing by using a crusher at the rotating speed of 5000r/min, pausing for 0.5min every 1min, sieving by using a 80-mesh sieve after accumulative crushing for 3-5min to obtain red deer abomasum powder, and storing the red deer abomasum powder in a refrigerator at the temperature of-20 ℃ for later use;
d. c, adding precooled petroleum ether into the red deer abomasum powder obtained in the step c at the temperature of 4 ℃ according to the material-liquid ratio of 1:10-15, mixing, placing in a Soxhlet extractor, heating at the temperature of 60-75 ℃ for 3-5h, carrying out reflux degreasing treatment, then carrying out suction filtration to remove the petroleum ether, and drying at room temperature to obtain degreased red deer abomasum powder;
e. mixing the degreased abomasum powder obtained in the step d with a NaCl solution with the concentration of 2-5% according to the mass ratio of 1:10-15, stirring and extracting for 3-5h at room temperature, filtering the extracting solution by using an oscillating screen, centrifuging the filtering solution at the temperature of 4 ℃ for 10-20min at 6000-8000r/min to obtain a supernatant, concentrating the supernatant by using a rotary evaporator under reduced pressure of 35-60mbar at the temperature of 50 ℃ to 1/10-1/15 times of the original volume, desalting the concentrated solution by using a 3500Da dialysis bag for 48 hours, and centrifuging for 10-20min at the temperature of 4 ℃ at 6000-8000r/min after desalting to obtain a total extracting solution;
f. adding absolute ethanol into the extracting solution obtained in the step e at the temperature of 0-4 ℃ to ensure that the volume concentration of the ethanol is 70-80%, standing for 24h for complete precipitation, dissolving the precipitate with distilled water, centrifuging at 8000r/min at the temperature of 4 ℃ for 15min, taking supernatant, and freeze-drying at the pressure of 10Pa, -80 ℃, and 24h to obtain crude glycoprotein of the red-spotted deer abomasum;
g. f, carrying out primary purification on the crude glycoprotein obtained in the step f, completely dissolving the crude glycoprotein into a phosphate buffer solution with the concentration of 0.02M and the pH value of 7.8 to the concentration of 15mg/mL, centrifuging, taking supernatant, carrying out DEAE-Sephadex A-25 anion exchange resin column chromatography, respectively eluting with NaCl solutions with the concentrations of 0.1mol/mL, 0.2mol/mL, 0.3mol/mL and 0.4mol/mL, detecting the sugar absorption peak at 492nm by using a phenol-sulfuric acid method, detecting the protein absorption peak at 280nm by using an ultraviolet spectrophotometer, collecting the eluates with the mutually overlapped absorption peaks of the sugar and the protein, concentrating the mutually overlapped eluates under reduced pressure at the temperature of 35-60mbar, concentrating at the temperature of 50 ℃, desalting the concentrated solution by using a 3500Da dialysis bag for 48h, desalting at the temperature of-80 ℃, freeze-drying for 8h under the pressure of 10Pa to obtain the first purified glycoprotein;
h. g, detecting the hyaluronidase inhibition activity of the first purified glycoprotein obtained in the step g, and collecting components with the hyaluronidase inhibition activity;
i. and (4) carrying out secondary purification on the components with the hyaluronidase inhibition activity collected in the step h, dissolving the components with the hyaluronidase inhibition activity by using distilled water, separating and purifying the components by using a gel column HW-55F to obtain a pure red deer abomasum glycoprotein solution, and carrying out concentration under the pressure of 35-60mbar and the temperature of 50 ℃, and freeze-drying under the temperature of-80 ℃ and the pressure of 10Pa to obtain the Tianshan red deer abomasum glycoprotein.
2. Use of abomasum glycoprotein obtained by the method according to claim 1, for the preparation of an anti-inflammatory medicament.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113444141A (en) * | 2021-08-12 | 2021-09-28 | 中国科学院新疆理化技术研究所 | Method for extracting, separating and purifying lamb abomasum glycoprotein and application thereof |
| CN115353545A (en) * | 2022-09-22 | 2022-11-18 | 中国科学院新疆理化技术研究所 | Method for preparing lamb abomasum glycoprotein part by response surface method and application |
| CN116178482A (en) * | 2022-12-07 | 2023-05-30 | 中国科学院新疆理化技术研究所 | Method for optimizing ultrasonic-assisted extraction of Tianshan red deer abomasum glycoprotein based on response surface method and application |
Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| GB575457A (en) * | 1944-02-10 | 1946-02-19 | Francisco Spitzer | Process for treating abomasum for manufacture of rennet |
| US20030165487A1 (en) * | 2002-02-12 | 2003-09-04 | Juan Tricarico | Amylase feed supplements for improved ruminant nutrition |
| CN1724530A (en) * | 2005-07-13 | 2006-01-25 | 东北林业大学 | Method of chromatography preparing high purity EGCG by continous medium-pressure column |
| WO2021109714A1 (en) * | 2019-12-06 | 2021-06-10 | 华南理工大学 | Millettia speciosa polysaccharides, preparation method therefor, and application thereof in antibacterial aspects |
| CN113278047A (en) * | 2021-06-28 | 2021-08-20 | 深圳中正生物技术有限责任公司 | Processing method for extracting composite active polypeptide from fresh pilose antler |
| CN113444141A (en) * | 2021-08-12 | 2021-09-28 | 中国科学院新疆理化技术研究所 | Method for extracting, separating and purifying lamb abomasum glycoprotein and application thereof |
| CN113527411A (en) * | 2021-07-24 | 2021-10-22 | 中国科学院新疆理化技术研究所 | Separation and purification method of dioscorea opposita glycopeptide |
-
2021
- 2021-11-17 CN CN202111360384.4A patent/CN113956341B/en active Active
Patent Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| GB575457A (en) * | 1944-02-10 | 1946-02-19 | Francisco Spitzer | Process for treating abomasum for manufacture of rennet |
| US20030165487A1 (en) * | 2002-02-12 | 2003-09-04 | Juan Tricarico | Amylase feed supplements for improved ruminant nutrition |
| CN1724530A (en) * | 2005-07-13 | 2006-01-25 | 东北林业大学 | Method of chromatography preparing high purity EGCG by continous medium-pressure column |
| WO2021109714A1 (en) * | 2019-12-06 | 2021-06-10 | 华南理工大学 | Millettia speciosa polysaccharides, preparation method therefor, and application thereof in antibacterial aspects |
| CN113278047A (en) * | 2021-06-28 | 2021-08-20 | 深圳中正生物技术有限责任公司 | Processing method for extracting composite active polypeptide from fresh pilose antler |
| CN113527411A (en) * | 2021-07-24 | 2021-10-22 | 中国科学院新疆理化技术研究所 | Separation and purification method of dioscorea opposita glycopeptide |
| CN113444141A (en) * | 2021-08-12 | 2021-09-28 | 中国科学院新疆理化技术研究所 | Method for extracting, separating and purifying lamb abomasum glycoprotein and application thereof |
Non-Patent Citations (1)
| Title |
|---|
| 熊和丽等: "梅花鹿鹿茸蛋白聚糖的提取与分离", 西北农业学报, vol. 16, no. 04, pages 80 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113444141A (en) * | 2021-08-12 | 2021-09-28 | 中国科学院新疆理化技术研究所 | Method for extracting, separating and purifying lamb abomasum glycoprotein and application thereof |
| CN115353545A (en) * | 2022-09-22 | 2022-11-18 | 中国科学院新疆理化技术研究所 | Method for preparing lamb abomasum glycoprotein part by response surface method and application |
| CN116178482A (en) * | 2022-12-07 | 2023-05-30 | 中国科学院新疆理化技术研究所 | Method for optimizing ultrasonic-assisted extraction of Tianshan red deer abomasum glycoprotein based on response surface method and application |
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