CN115340589A

CN115340589A - Purification and application of lamb abomasum active ingredient

Info

Publication number: CN115340589A
Application number: CN202211153602.1A
Authority: CN
Inventors: 阿布力米提·伊力; 阿吉艾克拜尔·艾萨; 阿米娜·艾尔肯
Original assignee: Xinjiang Technical Institute of Physics and Chemistry of CAS
Current assignee: Xinjiang Technical Institute of Physics and Chemistry of CAS
Priority date: 2022-09-21
Filing date: 2022-09-21
Publication date: 2022-11-15

Abstract

The invention relates to purification of active ingredients of lamb abomasums and application thereof, the method comprises the steps of taking a fresh lamb abomasum, freezing, shearing, freeze-drying, crushing by liquid nitrogen to obtain lamb abomasum powder, degreasing by using petroleum ether with a magnetic stirring method to obtain lamb abomasum degreased powder, stirring by using phosphoric acid slow solution containing serine enzyme inhibitor, diethylamine tetraacetic acid and NaCl, centrifuging, dialyzing, freeze-drying to obtain an extract, separating and purifying by anion exchange resin, desalting, separating and purifying by using a gel column, and freeze-drying to obtain high-purity compounds YGP-1, YGP-2, YGP-3 and YGP-4 glycoprotein. The anti-inflammatory activity results show that: YGP-2 has significant LOX-15 enzyme inhibitory activity; YGP-1, YGP-2, YGP-3, YGP-4 have COX-2 enzyme inhibitory activity; the result of the inhibitory activity on human colon cancer cells HT-29 shows that: YGP-2, YGP-3, YGP-4 has the activity of inhibiting HT-29 of human colon cancer cells; the method has simple and mature steps and simple and uncomplicated operation, and the obtained active ingredients of the lamb abomasums can be used in the fields of medicines and health care products.

Description

Purification and application of lamb abomasum active ingredient

Technical Field

The invention relates to purification of active ingredients of lamb abomasums and application thereof.

Background

Glycoproteins (glycoproteins) are complex macromolecules formed by covalent linkage of oligosaccharide chains and polypeptide chains, and are widely found in animals, plants and microorganisms. The natural glycoprotein has the activities of regulating immunity, inhibiting tumor, reducing blood sugar, reducing blood fat, resisting oxidation, resisting fatigue, resisting radiation and the like, and has remarkable medicinal effect and health-care function. Currently, most of pharmaceutical protein preparations used in clinic and having high immunological activity are glycoproteins, and the functional diversity thereof is the most active field in glycobiology research.

The lamb abomasums are the only stomachs with chemical and enzymatic digestion functions in the lamb gastric digestive system, contain more than 10 secretory cells and contain rich glycoprotein. The abomasum of the lamb is not only a traditional medicinal material used as both medicine and food, but also the history of modern clinical medicine can be traced back to 60 years in the last century. The lamb abomasums have remarkable efficacy on treating chronic gastritis and dyspepsia, are low in price and are hot spots from digestion to disease treatment and research on related active ingredients. However, only the active ingredients such as chymosin, pepsin and other digestive enzymes, antioxidant active polypeptide protein, probiotic active glycoprotein and the like are researched in the recorded key technology, and the drug effects of the raw materials and the total extract on treating chronic gastritis and dyspepsia are reported. There is no report on the chemical components related to anti-inflammation and anti-tumor based on the clinical efficacy and the prior art. The anti-inflammatory and anti-tumor active ingredients in the abomasums of the lambs are necessarily researched, comprehensive structural characterization is carried out on the anti-inflammatory and anti-tumor active ingredients, and the medicinal ingredients are explained.

The method comprises searching 88 related documents of glycoprotein obtained from natural raw materials with glycoprotein as key word, and extracting and separating and purifying 14 documents.

The probiotic active glycoprotein is extracted from lamb abomasums serving as raw materials in patent application No. 202110922724.1, the hyaluronidase inhibition active glycoprotein is extracted from the abomasums of Tianshan red deer serving as raw materials in application No. 202111360384.4, both applications are preliminary studies of the subject, the probiotic active glycoprotein is obtained from lamb abomasums serving as raw materials in application No. 202110922724.1, the probiotic active glycoprotein is extracted by an ultrasonic-assisted extraction method at the high temperature of 60 ℃, the glycoprotein is obtained after ethanol precipitation and savage reagent deproteinization, and the yield is 13.8%. Application No. 202111360384.4 is the extraction of glycoproteins at room temperature with sodium chloride as the extractant.

The present invention is further developed based on the previous work of the present subject, and the difference between them is that: the extraction method of the invention is that the extraction is carried out in a phosphate buffer solution with the temperature of 25-40 ℃ and the pH value of 0.1Mm-1mM of serine protease inhibitor, 5mM of diethylamine tetraacetic acid and 0.4-0.6 of sodium chloride by magnetic stirring, the phosphate buffer solution can keep the pH value stability of the extracting solution and avoid the protein denaturation caused by the sudden change of the pH value of the solution, the serine protease inhibitor and the diethylamine tetraacetic acid are commonly used enzyme inhibitors and metal ion chelating agents and can avoid the active protein, glycoprotein and other components in the solution from being degraded or inactivated in the extraction process; the extraction at the temperature of 25-40 ℃ is a mild condition close to normal physiological temperature, which is favorable for fully dissolving out active ingredients in the raw materials and keeping stable configuration, the reasonable increase of the concentration of sodium chloride can increase the extraction rate of the activity in the raw materials, because the higher the salt concentration of the protein and the derivatives in a certain concentration range is, the higher the solubility is, the more the reasonable range is, the solubility of the protein and the derivatives is reduced, and the hydrophilicity of the glycoprotein is higher than that of the protein, so the lower salt solution can effectively extract the glycoprotein; secondly, the active ingredients of the lamb abomasum glycoprotein are side-by-side products, most of glycoprotein in the animal body takes a protein chain as a main skeleton, the proportion of the glycoprotein is higher, and the protein is easy to lose structural stability in an organic reagent to cause inactivation; the high-purity glycoprotein obtained by the method only uses DEAE-52 anion exchange resin and SaphadexG-50 for separation and purification, and the used instruments and fillers are low in cost compared with HPLC instruments and columns.

After a large number of searches: the glycoprotein protein site is defined as a product obtained after one or two deproteinizing treatment extracts of methods such as trichloroacetic acid deproteinization, acetone precipitation, ethanol precipitation, savage reagent deproteinization and the like are used in almost all related patents and articles of naturally-derived glycoproteins in documents with application numbers of 202111360384.4, 201110138032.4, 201110403339.2, 200910089580.5, 201110363289.X,201410129587.6, 201610645106.6, 201110363289.X, 201310864.7, 201611261450.1, 201810701088.1, 201810982435.9, 201811300319.0, 201811300319.0, 202110562376.1, 202111360384.4 and the like.

In 202110562376.1, ethylenediaminetetraacetic acid (EDTA) is used at all times but in a relatively high amount, 80mM to 120mM of EDTA is used as an extractant in order to extract EDTA-soluble calcium-binding glycoproteins from the starting material, and therefore the purpose of use determines the concentration and thus the difference in the resulting product; in the searched documents, DEAE-52 and Sephadex G series packing are used for separating and purifying glycoprotein; the concentration of the buffer and buffer solution used in the purification process, the pH of the buffer solution, and the salt concentration used are all selected according to the characteristics of the glycoprotein in the starting material.

The innovation point of the invention is that the serine enzyme inhibitor and EDTA are used in the previous step of extracting and purifying the gel column, and the concentration of the EDTA is 5mM, so that the inactivation caused by the combination of the active ingredients and heavy metals is prevented, the activity of the active ingredients is better reserved, and the reasonability and the high efficiency are realized.

Disclosure of Invention

The invention aims to provide purification and application of active ingredients of lamb abomasumsPulverizing under nitrogen to obtain lamb stomach powder, magnetic stirring, defatting with petroleum ether to obtain lamb stomach powder, extracting glucoprotein with slow phosphoric acid solution of NaCl, centrifuging, dialyzing, freeze drying to obtain extract, purifying with anion exchange resin, desalting, purifying with gel column, and freeze drying to obtain high-purity compounds YGP-1, YGP-2, YGP-3, and YGP-4 glucoprotein. The anti-inflammatory activity results of the four compounds show that YGP-1, YGP-2, YGP-3 and YGP-4 all show stronger COX-2 enzyme inhibition activity, and the IC50 values are respectively 18.45 + -0.679 μ g/mL,17.64 + -1.25 μ g/mL,16.14 + -1.11 μ g/mL and 65.10 + -11.57 μ g/mL; while only YGP-2 had significant LOX-15 enzyme inhibitory activity, its IC ₅₀ IC with value of 2.79 +/-0.05 mug/mL greater than positive control quercetin ₅₀ The value was 6.67. + -. 0.22. Mu.g/mL; the results of the inhibitory activity on human colon cancer cells HT-29 showed that EC of YGP-2, YGP-3, YGP-4 ₅₀ 164.4 +/-15.7 mu g/mL,19.9 +/-1.46 mu g/mL and 184.9 +/-5.6 mu g/mL respectively; the results of cytotoxicity on normal human hepatocytes L02 showed that YGP-2 and YGP-3 had certain cytotoxicity, EC ₅₀ 47.77. Mu.g/mL and 40.23. Mu.g/mL, respectively, whereas YGP-4 showed no cytotoxicity;

the purification of the active ingredients of the lamb abomasums is carried out according to the following steps:

a. cleaning stomach cavity contents of fresh lamb abomasums with tap water, removing fat and connective tissues, freezing at-80 deg.C for 3h, shearing, freeze-drying, and pulverizing with liquid nitrogen to obtain lamb abomasums powder;

b. b, magnetically stirring the lamb fourth stomach powder obtained in the step a, degreasing for 3 times by using petroleum ether to obtain lamb fourth stomach degreased powder, and freezing and storing at the temperature of-20 ℃;

c. b, extracting the lamb stomach degreasing powder obtained in the step b for 1.5 to 2.5 hours in a phosphate buffer solution with the concentration of 50mM at the temperature of 20 to 45 ℃ and the pH value of 7.0 according to the feed-liquid ratio of 24 to 36mL/g, wherein the phosphate buffer solution contains 0.1 to 1mM of serine enzyme inhibitor, 5mM of ethylene diamine tetraacetic acid and 0.4 to 0.6M of sodium chloride;

d. centrifuging the extracting solution obtained in the step c for 10 minutes at 10000 rpm to obtain an extracting solution;

e. d, dialyzing the extracting solution obtained in the step d by using a dialysis bag for desalting for 60 hours, freezing and drying to obtain a lamb abomasum extract, weighing to calculate the extraction rate, and storing at the temperature of-20 ℃;

and (3) separating and purifying the active ingredient extract of the lamb abomasums by using ion exchange resin:

f. d, dialyzing the extract obtained in the step d for 60 hours by using a dialysis bag by using 50mM phosphate buffer solution containing 0.1-1mM serine enzyme inhibitor and 5mM ethylene diamine tetraacetic acid and taking pH value of 7.0 as dialysis external solution;

g. diluting the dialyzed extract obtained in the step f to 800mL by using the buffer solution obtained in the step f, loading the diluted extract to DEAE-52 anion exchange resin with the pH value of 7.0 and pre-balanced by a phosphate buffer solution with the concentration of 0.1-1mM containing a serine enzyme inhibitor and 5mM of ethylenediamine tetraacetic acid being 50mM, eluting the column by using the same buffer solution and NaCl solutions with the concentrations of 0.1M,0.2M,0.3M,0.4M,0.5M,0.6M and 0.8M in sequence, measuring a protein elution curve of the eluent at 280nm of ultraviolet (ultraviolet), and measuring a sugar elution curve at 490nm of ultraviolet by using a phenol-sulfuric acid method;

h. d, dialyzing and desalting NaCl elution parts with the concentrations of 0.1M,0.2M,0.3M,0.4M,0.5M,0.6M and 0.8M in the step g for 60 hours by using dialysis bags, respectively freeze-drying and weighing, and storing at the temperature of-20 ℃ after sequentially naming F1, F2, F3, F4, F5, F6, F7 and F8;

i. performing SDS-PAGE analysis on the F1, F2, F3, F4, F5, F6, F7 and F8 obtained in the step h, and respectively performing dyeing by using Coomassie brilliant blue and PAS-Coomassie dyeing method;

and (3) separating and purifying the active ingredients of the lamb abomasums by using a gel column:

j. separating and purifying the F2 part obtained in the step i by Sephadex G-50 in a low-temperature chromatography system by taking deionized water as an eluent, detecting the absorbance of the eluent at 280nm at the flow rate of 0.5mL/min, collecting one tube every 8min to obtain two elution peaks, namely single glycoprotein named YGP-1 and YGP-2 respectively, and then respectively freezing and drying the single glycoprotein, and storing the single glycoprotein at the temperature of-20 ℃;

k. and (3) separating and purifying the F8 obtained in the step (i) by Sephadex G-50 in a low-temperature chromatography system by using deionized water as an eluent, detecting the absorbance of the eluent at 280nm at the flow rate of 0.4mL/min, collecting one tube every 8min to obtain two elution peaks, namely single glycoprotein named YGP-3 and YGP-4 respectively, freeze-drying the single glycoprotein respectively, and storing the single glycoprotein at the temperature of-20 ℃.

The lamb abomasum active glycoprotein YGP-1, YGP-2, YGP-3 and YGP-4 obtained by the method are used for preparing anti-inflammatory active medicaments.

The lamb abomasum active glycoprotein YGP-1, YGP-2, YGP-3 and YGP-4 obtained by the method are used for preparing anti-inflammatory active health care products.

The lamb abomasum active glycoprotein YGP-1, YGP-2, YGP-3 and YGP-4 obtained by the method are used for preparing the medicine for inhibiting HT-29 human colon cancer.

The lamb abomasum active glycoprotein YGP-1, YGP-2, YGP-3 and YGP-4 obtained by the method can be used for preparing health products for inhibiting HT-29 human colon cancer.

The glycoprotein YGP-2, YGP-YGP-4 with anti-inflammatory and anti-tumor activities obtained by the method has low or no cytotoxicity to human normal liver cells L02, and supports the practicability of the application of the glycoprotein obtained by the invention in the application of medicines.

The extract of the active ingredients of the lamb abomasums obtained by the method of the invention is at the part obtained by DEAE-52 anion exchange resin separation and purification, the F1, F2, F3, F4, F5, F6, F7 and F8 parts of SD-PAGE electrophoresis gel after PAS-Coomassie dyeing contain the ingredients dyed into pink, a pink product can be generated after glycoprotein PAS dyeing, and YGP-1, YGP-2, YGP-3 and YGP-4 lamb abomasum active glycoprotein are obtained by gel column separation and purification.

The purification and application of the active ingredient of the abomasum lamb are characterized in that fresh fourth stomach of the lamb is taken, and is frozen, sheared, freeze-dried and crushed by liquid nitrogen to obtain fourth stomach powder of the lamb, glycoprotein is extracted by stirring with a phosphoric acid slow solution containing a serine enzyme inhibitor, diethylamine tetraacetic acid and sodium chloride, and the extraction rate is 24.2 percent after centrifugation, dialysis and freeze-drying; separating and purifying with anion exchange resin, desalting, separating and purifying with gel column, and lyophilizing to obtain high-purity active glycoproteins YGP-1, YGP-2, YGP-3, YGP-4 with molecular weights of 66kDa, 169da, 15.6kDa and 6.4kDa respectively as determined by SDS-PAGE electrophoresis; it has significant anti-inflammatory activityIt is shown that YGP-2 has significant inhibitory effect on LOX-15 enzyme, IC thereof ₅₀ IC with value of 2.79 + -0.05 μ g/mL and greater than positive control quercetin ₅₀ The value was 6.67. + -. 0.22. Mu.g/mL; YGP-1, YGP-2, YGP-3 and YGP-4 all showed strong COX-2 enzyme inhibitory activity, IC ₅₀ The values are respectively 18.45 +/-0.679 mu g/mL,17.64 +/-1.25 mu g/mL,16.14 +/-1.11 mu g/mL and 65.10 +/-11.57 mu g/mL; the results of the inhibitory activity against human colon cancer cell HT-29 showed that EC was found in YGP-2, YGP-3, YGP-4 ₅₀ 164.4 +/-15.7 mu g/mL,19.9 +/-1.46 mu g/mL and 184.9 +/-5.6 mu g/mL respectively; the results of cytotoxicity on normal human hepatocytes L02 showed that YGP-2 and YGP-3 had a certain cytotoxicity, and their EC ₅₀ 47.77. Mu.g/mL and 40.23. Mu.g/mL, respectively, whereas YGP-4 showed no cytotoxicity; the YGP-3 and YGP-4 are structurally analyzed in view of high activity and low toxicity, and the monosaccharide analysis result shows that both are ribose-bound eggs and are rich in essential amino acids for human body; the YGP-3 and YGP-4 are O-glycosylated and N-glycosylated glycoproteins respectively obtained by periodate oxidation-beta elimination reaction analysis; LC-MS/MS glycosylation site analysis was performed on YGP-2 in view of high yield, which was identified as a viscous glycoprotein having a novel polypeptide sequence, containing 1N-glycosylation site and 14O-glycosylation sites.

The technical problem to be solved by the invention is to fill the research on the anti-inflammatory and anti-tumor active ingredients in the abomasum of the lamb, effectively extract the anti-inflammatory and anti-tumor ingredients, perform high-efficiency and rapid separation and purification, and perform activity evaluation on the high-yield and high-activity ingredients. The existing application way of the lamb abomasums is broken through, new activity and components are excavated, a new application way of the lamb abomasums is expanded, and the economic efficiency is improved. The high-purity active glycoprotein obtained from lamb abomasums is comprehensively characterized in terms of polypeptide chain and oligosaccharide chain composition, polypeptide chain sequence, glycosylation type, carbohydrate peptide bond type, secondary structure of complete glycoprotein, element composition and the like for the first time.

Compared with the prior art, the invention has the beneficial effects that:

according to the purification method and the application of the active ingredients of the lamb abomasums, the enzyme inhibitor is used in the extraction, separation and purification processes, the activity of the compound is better reserved, and the high-purity compound product is obtained by two-step rapid and efficient separation and purification method. The use of organic solvent is avoided in the whole preparation process. Not only the activity of the product is kept, but also the production cost and the pollution are reduced. The purity of YGP-2, YGP-4 was higher than 97%, and the purity of YGP-3 was higher than 90%. Has the advantages of simple process, high stability, simple operation, easy amplification and preparation, and the like.

In the method, 4 compounds obtained by purifying through a gel column are analyzed into a uniform band through electrophoresis, and in the method, the lamb abomasums contain anti-inflammatory active ingredients. The compound YGP-2 obtained by purification has significant inhibitory effect on LOX-15 enzyme. The method carries out the antineoplastic activity evaluation on the active ingredients of the abomasums of the lambs for the first time, and carries out the activity determination. A comprehensive structural characterization of high purity components with anti-inflammatory and anti-tumor activity was performed. The method comprises the steps of protein content measurement by a Bradford method, neutral sugar content measurement by a phenol-sulfuric acid method, total amino sugar content measurement, galactosamine content measurement, uronic acid content measurement, molecular weight measurement by an SDS-PGE method, PAS dyeing of glycoprotein, amino acid composition analysis by a PITC derivatization method, monosaccharide composition analysis by a PMP derivatization method, periodate oxidation-beta elimination, sulfate content measurement, FT-IR, CD, NMR, LC-MS/MS molecular weight, EDX element analysis and the like.

Drawings

FIG. 1 is a DEAE-52 anion exchange resin separation and purification diagram of the present invention;

FIG. 2 is a Coomassie blue stained image of SDS-PAGE electrophoresis of F1-F8 portions separated and purified by DEAE-52 anion exchange resin according to the present invention;

FIG. 3 is a Coomassie brilliant blue staining pattern after SDS-PAGE PAS staining of F1-F8 parts separated and purified by DEAE-52 anion exchange resin according to the present invention;

FIG. 4 is a diagram showing the Sephadex-G50 separation and purification of the F2 region of the present invention, in which YGP-1 and YGP-2 represent the first and second elution peaks, respectively.

FIG. 5 is a diagram of Sephadex-G50 separation and purification of the F8 region of the present invention, in which YGP-3 and YGP-4 represent the first and second elution peaks, respectively.

FIG. 6 is a DSD-PAGE molecular weight determination chart of the glycoproteins YGP-1, YGP-2, YGP-3 and YGP-4 of the present invention;

FIG. 7 shows the result of HPLC monosaccharide analysis by pre-column derivatization of the glycoprotein YGP-1 of the present invention;

FIG. 8 shows the result of HPLC monosaccharide analysis by PMP pre-column derivatization of glycoprotein YGP-2 of the present invention;

FIG. 9 shows the result of HPLC monosaccharide analysis by PMP pre-column derivatization of the glycoprotein YGP-3 according to the present invention;

FIG. 10 shows the result of HPLC monosaccharide analysis by PMP pre-column derivatization of glycoprotein YGP-4 of the present invention;

FIG. 11 is an HPLC purity measurement chromatogram of the glycoprotein YGP-1 of the present invention;

FIG. 12 is a HPLC purity measurement chromatogram of the glycoprotein YGP-2 of the present invention;

FIG. 13 is a HPLC purity measurement chromatogram of the glycoprotein YGP-3 of the present invention;

FIG. 14 is a HPLC purity measurement chromatogram of the glycoprotein YGP-4 of the present invention;

FIG. 15 is a graph showing the comparison of the COX-2 enzyme activities of the glycoproteins YGP-1, YGP-2, YGP-3 and YGP-4 of the present invention with a positive control Celecoxib;

FIG. 16 is a graph comparing the results of the YGP-2, YGP-3 and YGP-4 inhibitory activities on human colon cancer cells HT-29 according to the present invention;

FIG. 17 is a graph comparing the cytotoxicity results of YYGP-2, YGP-3 and YGP-4 in human normal stem cells L02 according to the invention;

FIG. 18 is an infrared chromatogram of the glycoprotein YGP-3 of the present invention;

FIG. 19 is an infrared chromatogram of glycoprotein YGP-4 of the present invention;

FIG. 20 is a full-UV wavelength scan of the periodic acid oxidation-beta elimination reaction of glycoprotein YGP-3 of the present invention;

FIG. 21 is a full-UV wavelength scan of the periodic acid oxidation-beta elimination reaction of glycoprotein YGP-4 of the present invention;

FIG. 22 is a diagram of the circular dichromatic secondary structure analysis of the glycoprotein YGP-3 of the present invention;

FIG. 23 is a diagram of circular dichroism secondary structure analysis of the glycoprotein YGP-4 of the present invention;

FIG. 24 is a photograph showing LC-MS/MS analysis of the glycoprotein YGP-3 of the present invention;

FIG. 25 is a schematic view of the glycoprotein YGP-3 of the present invention ¹ An H-NMR spectrum;

FIG. 26 shows a schematic view of YGP-3 glycoprotein according to the present invention ¹³ C-NMR spectrum.

Detailed Description

Example 1

c. weighing 10g of lamb stomach degreasing powder obtained in the step b, extracting for 1.5h at the temperature of 30 ℃ by using a material-liquid ratio of 30mL/g and using a phosphate buffer solution with the pH value of 7.0 and the concentration of 50mM as an extracting agent, wherein the phosphate buffer solution contains 0.1mM serine enzyme inhibitor, 5mM ethylene diamine tetraacetic acid and 0.6M sodium chloride;

d. centrifuging the extract obtained in the step c at 10000 rpm for 10 minutes to obtain an extracting solution;

e. d, dialyzing the extracting solution obtained in the step d by using a dialysis bag for desalting for 60 hours, freezing and drying to obtain an extract, weighing to calculate the extraction rate, and storing at the temperature of-20 ℃;

through the measurement of protein content and sugar content, the extracted protein content is 21.3%, the sugar content is 5.4%, and the extraction rate is 22.2%;

f. d, dialyzing the extract obtained in the step d for 60 hours by using a dialysis bag by using 50mM phosphate buffer solution containing 0.1mM serine enzyme inhibitor and 5mM ethylene diamine tetraacetic acid and taking pH value of 7.0 as dialysis external liquid;

g. diluting the dialyzed extract obtained in the step f to 800mL by using the buffer solution obtained in the step f, loading the diluted extract to DEAE-52 anion exchange resin with the pH value of 7.0 and pre-balanced by a phosphate buffer solution with the concentration of 0.1-1mM containing a serine enzyme inhibitor and 5mM of ethylenediamine tetraacetic acid being 50mM, eluting the column by using the same buffer solution and NaCl solutions with the concentrations of 0.1M,0.2M,0.3M,0.4M,0.5M,0.6M and 0.8M in sequence, measuring a protein elution curve of the eluent at 280nm of ultraviolet, and measuring a sugar elution curve at 490nm of ultraviolet by using a phenol-sulfuric acid method;

h. d, dialyzing and desalting NaCl elution parts with concentrations of 0.1M,0.2M,0.3M,0.4M,0.5M,0.6M and 0.8M in the step g for 60 hours by using dialysis bags, respectively freeze-drying and weighing, and storing at the temperature of-20 ℃;

i. performing SDS-PAGE analysis on the F1, F2, F3, F4, F5, F6, F7 and F8 obtained in the step h, and dyeing by Coomassie brilliant blue and PAS dyeing respectively;

the glycoprotein extract is separated and purified by anion exchange resin to obtain 8 components of F1-F8, an elution curve chart 1 can be obtained, almost all parts of the extract have protein and sugar elution curves at the same time, wherein the elution curves of F2 and F8 have the best overlapping property, and the PAS-Coomassie dyeing and PAS-Coomassie dyeing electrophoresis analysis of F1, F2, F3, F4, F5, F6, F7 and F8 which are separated and purified by the anion exchange resin are higher than those of F2, F3, F4, F5, F6, F7 and F8 which are separated and purified by the anion exchange resin respectively if as shown in a figure 2 and a figure 3, PAS dyeing is a common method for dyeing glycoproteins in tissues, aldehyde groups formed after oxidizing oligosaccharide chains in the glycoproteins by periodate react with Schiff reagents and then show pink to purple, so that the resolution of the glycoproteins can be improved by Coomassie brilliant blue dyeing after PAS dyeing, and bands which are simultaneously dyed blue and purple exist in F1-F8 in an electrophoresis chart after PAS-Coomassie dyeing indicate that the parts contain the glycoproteins; because the yield is high, F2 and F8 are used for continuously separating and purifying to obtain high-purity and high-activity glycoprotein;

and (3) carrying out gel column separation and purification on active ingredient glycoprotein of abomasum lamb:

the molecular weights of the Sephadex G-50 gel column separation and purification elution curves of the F2 part are 66kDa and 16kDa respectively as shown in the figure;

k. separating and purifying the F8 obtained in the step i by Sephadex G-50 in a low-temperature chromatography system by using deionized water as an eluent, detecting the absorbance of the eluent at 280nm at the flow rate of 0.4mL/min, collecting one tube every 8min to obtain two elution peaks which are named as YGP-3 and YGP-4 respectively, freezing and drying the two elution peaks respectively, and storing the elution peaks at the temperature of-20 ℃;

the Sephadex G-50 gel column separation and purification elution curve of the F8 site is shown in FIG. 5, and the active ingredients YGP-3 and YGP-4 of the glycoproteins are obtained by purification, and YGP-3 and YGP-4 are single glycoproteins shown in the electrophoresis chart of FIG. 6, and the molecular weights are 15.6kDa and 6.4kDa, respectively. 4, YGP-1 and YGP-2, which are glycoprotein active ingredients, are obtained by purification, and YGP-1 and YGP-2 are single glycoprotein as shown in electrophoresis chart of FIG. 6,

example 2

c. weighing 10g of lamb stomach degreasing powder obtained in the step b, extracting for 2.0h in 50mM phosphate buffer solution with the pH value of 7.0 and containing 0.5mM serine enzyme inhibitor, 5mM ethylene diamine tetraacetic acid and 0.5M sodium chloride at the temperature of 36 ℃ and the feed-liquid ratio of 24 mL/g;

by measuring the content of protein and sugar, the extracted protein content is 21.8 percent, the sugar content is 6.3 percent, and the extraction rate is 22.8 percent;

f. d, dialyzing the extract obtained in the step d for 60 hours by using a dialysis bag by using 50mM phosphate buffer solution containing 0.5mM serine enzyme inhibitor and 5mM ethylene diamine tetraacetic acid and taking pH value of 7.0 as dialysis external solution;

h. d, dialyzing and desalting NaCl elution parts with concentrations of 0.1M,0.2M,0.3M,0.4M,0.5M,0.6M and 0.8M in the step g for 60 hours by using dialysis bags, freeze-drying and weighing the NaCl elution parts respectively, and storing the NaCl elution parts at the temperature of-20 ℃ sequentially named as F1, F2, F3, F4, F5, F6, F7 and F8;

the resulting fractions were identified and analyzed as in example 1;

and (3) carrying out gel column separation and purification on active ingredient glycoprotein of abomasum of lamb:

the Sephadex G-50 gel column separation and purification elution curve of the F2 part is shown in FIG. 4, and the active ingredients YGP-1 and YGP-2 of the glycoproteins are obtained by purification, and the YGP-1 and YGP-2 are single glycoproteins shown in the electrophoresis chart of FIG. 6, and the molecular weights are 66kDa and 16kDa respectively;

the Sephadex G-50 gel column separation and purification elution curve of the F8 site is shown in FIG. 5, and the active ingredients YGP-3 and YGP-4 of the glycoproteins are obtained by purification, and YGP-3 and YGP-4 are single glycoproteins shown in the electrophoresis chart of FIG. 6, and the molecular weights are 15.6kDa and 6.4kDa, respectively.

Example 3

c. weighing 10g of lamb stomach degreasing powder obtained in the step b, extracting for 2.0h by using 1mM serine inhibitory enzyme, 5mM ethylene diamine tetraacetic acid and 0.6M sodium chloride in 50mM phosphate buffer solution with the pH value of 7.0 at the temperature of 36 ℃ and the feed-liquid ratio of 24 mL/g;

by determining the protein content and the sugar content, the extracted protein content is 22.8 percent, the sugar content is 6.8 percent, and the extraction rate is 23.8 percent;

h. d, dialyzing and desalting NaCl elution parts with the concentrations of 0.1M,0.2M,0.3M,0.4M,0.5M,0.6M and 0.8M in the step g for 60 hours by using dialysis bags, respectively freeze-drying and weighing, and storing at the temperature of-20 ℃ sequentially named as F1, F2, F3, F4, F5, F6, F7 and F8;

the resulting ingredients were identified and analyzed as in example 1;

Example 4

c. weighing 10g of lamb stomach degreasing powder obtained in the step b, extracting for 2.5h by using 50mM phosphate buffer solution at the pH value of 7.0, wherein the phosphate buffer solution contains 0.1mM serine inhibitory enzyme, 5mM ethylene diamine tetraacetic acid and 0.6M sodium chloride according to the feed-liquid ratio of 30mL/g at the temperature of 30 ℃;

e. d, dialyzing the extracting solution obtained in the step d by using a dialysis bag for desalting for 60 hours, freezing and drying to obtain an extract, weighing to calculate the extraction rate, and storing at the temperature of minus 20 ℃;

f. d, dialyzing the extract obtained in the step d for 60 hours by using a dialysis bag by using a phosphate buffer solution containing 0.1mM of serine inhibiting enzyme and 5mM of diethylamine tetraacetic acid with the concentration of 50mM and the pH value of 7.0 as a dialysis external solution;

i. performing SDS-PAGE analysis on the F1, F2, F3, F4, F5, F6, F7 and F8 obtained in the step h, and dyeing by using Coomassie brilliant blue and PAS dyeing methods respectively;

the resulting fractions were identified and analyzed as in example 1;

j. separating and purifying the F2 part obtained in the step i in a low-temperature chromatography system by using deionized water as an eluent and Sephadex G-50, detecting the absorbance of eluent at 280nm at the flow rate of 0.5mL/min, collecting one tube every 8min to obtain two elution peaks which are respectively named as single glycoprotein YGP-1 and single glycoprotein YGP-2, and respectively freezing and drying the single glycoprotein, and storing the single glycoprotein at the temperature of-20 ℃;

the Sephadex G-50 gel column separation and purification elution curve of the F2 site is shown in FIG. 4, and YGP-1 and YGP-2 sites are obtained by purification, and the single glycoproteins of YGP-1 and YGP-2 are shown in FIG. 6, the molecular weights are 66kDa and 16kDa respectively;

k. separating and purifying the F8 obtained in the step i in a low-temperature chromatography system by using Sephadex G-50 with deionized water as an eluent, detecting the absorbance of the eluent at 280nm at the flow rate of 0.4mL/min, collecting one tube every 8min to obtain two elution peaks which are named as YGP-3 and YGP-4 respectively, freezing and drying the two elution peaks respectively, and storing the two elution peaks at the temperature of-20 ℃;

the Sephadex G-50 gel column separation and purification elution curve of the F8 site is shown in FIG. 5, and YGP-3 and YGP-4 sites are obtained by purification, and the single glycoproteins of YGP-3 and YGP-4 have molecular weights of 15.6kDa and 6.4kDa, respectively, as shown in the electrophoresis chart of FIG. 6.

Example 5

c. weighing 10g of lamb stomach degreasing powder obtained in the step b, and extracting for 2.5h at the temperature of 40 ℃ by using a feed-liquid ratio of 35mL/g and using a phosphate buffer solution with the pH value of 7.0 and the concentration of 50mM, wherein the phosphate buffer solution contains 1mM of serine enzyme inhibitor, 5mM of ethylene diamine tetraacetic acid and 0.6M of sodium chloride as an extracting agent;

e. d, dialyzing the extracting solution obtained in the step d by using a dialysis bag for desalination for 60 hours, freezing and drying to obtain an active ingredient extract of the abomasum of the lamb, weighing and calculating the extraction rate, and storing at the temperature of-20 ℃;

f. d, dialyzing the extract obtained in the step d for 60 hours by using a dialysis bag by using 50mM phosphate buffer solution containing 1mM serine enzyme inhibitor and 5mM ethylene diamine tetraacetic acid and taking pH value of 7.0 as dialysis external solution;

the resulting fractions were identified and analyzed as in example 1;

the Sephadex G-50 gel column separation and purification elution curve of the F2 site is shown in FIG. 4, and active glycoproteins YGP-1 and YGP-2 are obtained by purification, and YGP-1 and YGP-2 are single glycoproteins shown in the electrophoresis chart of FIG. 6, and the molecular weights are 66kDa and 16kDa respectively;

the Sephadex G-50 gel column separation and purification elution curve of the F8 site is shown in FIG. 5, and active down glycoproteins YGP-3 and YGP-4 are obtained by purification, and YGP-3 and YGP-4 are single glycoproteins shown in the electrophoresis chart of FIG. 6, and the molecular weights are 15.6kDa and 6.4kDa, respectively.

Example 6

Oligosaccharide chain sugar composition analysis: respectively measuring the content of neutral sugar, the content of uronic acid and the content of amino sugar;

and (3) measuring the content of neutral sugar: the content of neutral sugar is determined by a conventional benzene sulfuric acid method, and a determined standard curve is as follows: y =26.412x-1.2936 ² =0.9561, and the obtained glycoproteins YGP-12.8%, YGP-22.25%, YGP-3.9% and YGP-41.1% neutral sugar content;

measurement of uronic acid content: the content of uronic acid is determined by conventional m-phenylphenol color development method, and the standard curve is y =26.495x-0.6767 ² =0.9915, the resulting glycoprotein YGP-1.6%1, YGP-20.7%, YGP-30.9% and YGP-40.3% uronic acid content;

determination of the amino sugar content by the acetylacetone reagent method:

and (3) determining the content of the total amino sugar: the standard curve is y =805.93x-48.001 ² =0.9918, total amino sugar content, YGP-15.52%, YGP-22.21%, YGP-33.4%, and YGP-41.1% calculated from standard curve;

and (3) measuring the content of the galactosamine: the standard curve is y =1076.3x-50.884 ² =0.9945, and the contents of galactosamine were calculated according to the standard curve to obtain the contents of galactosamine of glycoprotein YGP-1.75%, YGP-2.01%, YGP-3.1% and YGP-4.7%; glucosamine content is the difference between the total hexosamine content and the galactosamine content, YGP-1.8%, YGP-2.1%, YGP-3.0% and YGP-4.7%.

Example 7

1-phenyl-3-methyl-5-pyrazolone (PMP) -pre-column derivatization for monosaccharide analysis:

HPLC monosaccharide analyzer instrument and experimental conditions were as follows: the instrument comprises: an HPLC analysis condition Shimaduz high performance liquid ultraviolet detection system; a chromatographic column: ZORBAX Ecplise XDB-C18-C18 (5 μm,250mm X4.6 mm); mobile phase A:0.1M phosphate buffer solution at pH 6.8/acetonitrile =83% 17%; and (3) mobile phase B:10% acetonitrile/deionized water solution; flow rate: 1mL/min; detection wavelength: λ =245nm; the gradient elution procedure is shown in table 5:

TABLE 1 gradient program Table for HPLC analysis of monosaccharide composition

PMP pre-column derivatization-HPLC analysis of YGP-1, YGP-2, YGP-3 and YGP-4 gave the monosaccharide composition as shown in 7,8,9,10; as a result, YGP-1, YGP-2, YGP-3 and YGP-4 all have specific monosaccharides such as glucosamine, galactosamine, acetylglucosamine and L-fucose among glycoproteins, and thus four compounds can be preliminarily judged as glycoproteins; YGP-3 and YGP-4 contained higher ribose content, indicating that both are ribose-bound glycoproteins; the role of ribose-binding glycoproteins in apoptosis and in the response to acute oxidation suggests, to a certain extent, the relationship between the antitumor activity and structure of YGP-3 and YGP-4.

Example 8

Because ethylenediamine tetraacetic acid is added in the extracting solution and the separation and purification process, the protein content of the sample is measured by a Bradford method: the standard curve is: y =0.3255x-0.0467, R ² =0.9879; the protein contents of YGP-1, YGP-2, YGP-3 and YGP-4 determined according to the standard curve were 70.5%,41.3%,53.9% and 29.3%, respectively.

Example 9

The amino acid compositions of the glycoproteins YGP-1, YGP-2, YGP-3 and YGP-4 obtained by the present invention were analyzed by phenylisothiocyanate-pre-column derivatization, and the results are shown in the following table:

TABLE 2 amino acid composition of glycoproteins YGP-2, YGP-3, YGP-4

As can be seen from table 2: YGP-2, YGP-3, YGP-4 have substantially the same amino acid composition, and all have higher serine contents of 14.1%,9.04% and 9.8%, respectively, and are the composition that is deficient in the O-glycosylation site or N-glycosylation step of glycoprotein; YGP-2 and YGP-3 have a higher content of acidic amino acids, while YGP-4 has a higher content of basic amino acids; wherein gYGP-and YGP-4 contain a small amount of Cys-Cys and thus exist in solution as a dimer or multimer;

example 10

Detecting the purity by using a high performance liquid chromatograph:

analyzing the purity by adopting an HPLC analysis condition Shimaduz high performance liquid chromatography system; the detector is a Shimaduz SPD 20-A ultraviolet detector; mobile phase ultrapure water; flow rate: 1mL/min; detection wavelength: 280nm; FIGS. 11, 12, 13 and 14 are HPLC purity measurement chromatograms of glycoproteins YGP-1, YGP-2, YGP-3 and YGP-4, respectively, with the purities of YGP-2 and YGP-4 being higher than 97% and the purities of YGP-3 being higher than 90% according to integral analysis, and YGP-1 being a mixture of single molecular weights.

Example 11

SDS-PAGE electrophoresis:

and (3) processing of a sample: weighing 2mg, dissolving in 500 μ L distilled water to prepare 4mg/mL sample solution, adding 25 μ L denatured non-reducing sample buffer solution into 100 μ L sample solution, shaking, heating at 95 deg.C for 10min, and centrifuging at 10,000rpm for 2min; electrophoresis conditions: constant pressure 75V,30min, then constant pressure 150V,90min; fixing liquid: 25% of isopropanol, 10% of glacial acetic acid and 65% of water;

the Coomassie brilliant blue dyeing method and the PAS dyeing method are carried out according to a conventional experimental method; PAS-Coomassie staining is to carry out the Coomassie staining on the electrophoresis gel after PAS staining;

example 12

Determination of cyclooxygenase 2 (COX-2) inhibitory Activity:

sample preparation: YGP-1, YGP-2, YGP-and YGP-4 and dissolved with Milli-Q grade pure water for use; preparing cyclooxygenase-2 Probe, cyclooxygenase-2 Cofactor (50X) and cyclooxygenase-2 Substrate (50X) in DMSO, and heating in water bath at 37 deg.C for 0.5-2min to promote melting; preparing a working solution of cyclooxygenase 2 Cofactor: preparing according to the proportion that each sample needs 5 microliters of cyclooxygenase 2Cofactor working solution, taking a proper amount of cyclooxygenase 2Cofactor (50X), and diluting with cyclooxygenase 2Assay Buffer according to the proportion of 1; preparing a working solution of cyclooxygenase 2: preparing each sample according to the proportion that 5 microliter of a working solution of the cyclooxygenase-2 is needed, taking a proper amount of rhCOX-2 (25X), and diluting the rhCOX-2 with the cyclooxygenase-2 Assay Buffer according to the proportion of 1; preparing a cyclooxygenase 2Substrate working solution: preparing according to the proportion that each sample needs 5 microliters of cyclooxygenase 2Substrate working solution, taking a proper amount of cyclooxygenase 2Substrate (50X), adding an equal volume of Substrate Buffer, fully and uniformly mixing by vortex, diluting by Milli-Q grade pure water or redistilled water according to the volume ratio of 1. Note: the Substrate working solution can also be prepared in the process of incubating at 37 ℃ for 10 minutes during sample detection;

and (3) detecting COX-2 enzyme inhibitory activity of a sample:

referring to table 3, a 96-well blackboard was used to set control wells and sample wells, and the samples and the solutions were added in sequence according to table 7, mixed after the addition of the samples, and incubated at 37 ℃ for 10 minutes; note: the incubation after the sample to be tested is added can also be carried out at the temperature of 25 ℃ or room temperature; most inhibitors are time-dependent on inhibition of cyclooxygenase-2 activity, and altering the duration of action of the inhibitor can significantly alter the IC of the compound ₅₀ Values suggesting that the incubation time for which the unknown inhibitor is more suitable is determined by the test;

TABLE 3 test sample detection method for glycoprotein COX-2 enzyme inhibitory activity

IC of COX-2 enzyme inhibitory Activity of YGP-1, YGP-2, YGP-3 and YGP-4 ₅₀ The values are respectively 18.45 +/-0.679 mu g/mL,17.64 +/-1.25 mu g/mL,16.14 +/-1.11 mu g/mL and 65.10 +/-11.57 mu g/mL; the activity ratio of the four compounds to 100nM positive control cisplatin is shown in FIG. 15, and the cyclooxygenase-2 enzyme inhibitory activity of the four glycoproteins at the concentrations of 160. Mu.g/mL, 120. Mu.g/mL, 210. Mu.g/mL and 90. Mu.g/mL is higher than that of the positive control, i.e., the four glycoproteins have stronger anti-inflammatory activity.

Example 13

Determination of fat synthase (LOX-15) inhibitory Activity

Reagent preparation: tris-HCl buffer, 50mM, pH =7.4; FOX reagent: 30mM of sulfuric acid, 100 mu M of xylenol orange and 200 mu M of ferrous sulfate; sample solution: YGP-1, YGP-2, YGP-3 and YGP-4 were dissolved in the above Tris-HCl buffer solution;

the detection method comprises the following steps: 2 mul of sample with the total reaction volume of 200 mul, 58 mul of Tris-HCl buffer solution containing lipoxygenase (LOX-15) with the final concentration of 9.6U/mL, the enzyme activity control group is Tris-HCl buffer solution without enzyme, the temperature is incubated for 5min at the room temperature of 25 ℃, then 40 mul of linoleic acid (with the final concentration of 140 mul) is added, after the reaction is carried out for 20min in a dark place at the room temperature, 100 mul of FOX reagent is added into each hole, the oscillation and the mixing are carried out, the absorption value under 560nm is measured, and quercetin is taken as the positive control;

table 4 lox-15 enzyme inhibitory activity assay system:

inhibition rate (%) = [ (enzyme activity group-enzyme activity control group) - (drug group-drug control group)/(enzyme activity group-enzyme activity control group) ] × 100%

The results of the inhibition activity of YGP-1, YGP-2, YGP-3, YGP-4 for fat synthase (LOX-15) showed that YGP-1, YGP-3, YGP-4 had no fat synthase (LOX-15) inhibitory activity, while YGP-2 had significant fat synthase (LOX-15) inhibitory activity, and IC thereof ₅₀ The value is 2.79 + -0.05 μ g/mL and is greater than the IC of the positive control quercetin ₅₀ The value was 6.67. + -. 0.22. Mu.g/mL.

Example 14

HT-29 in vitro inhibitory Activity assay of human Colon cancer cells:

sample pretreatment: YGP-2, YGP-3 and YGP-4 which were weighed correctly were dissolved in DMEM medium and filtered for further use;

culturing HT-29 human colon cancer cells in DMEM broth to logarithmic phase, removing culture medium by suction, and washing with PBSOne time, pancreatin digestion, addition of medium to stop digestion, gentle pipetting, counting, inoculation into 96-well plates (100. Mu.l/well) at the corresponding cell density, CO at 37 ℃ ₂ Culturing overnight in an incubator, adding the prepared sample solution into a 96-well plate of HT29 human colon cancer cells, removing edge effect, adding 20 microliters of glycoprotein of the invention into each small hole, respectively measuring 10 series concentrations, repeating for three times, continuing culturing for 48 hours after adding the sample, measuring cell activity by using an MTT method, removing old culture medium, adding 100 microliters of MTT, incubating at 37 ℃ for 2 hours, and measuring the light absorption value (OD) at 570nm by using an MB microplate reader; doxorubicin hydrochloride is used as a positive control, a DMEM culture medium without a sample is used as a blank control, and the determination method is the same as that of the patent glycoprotein; finally, the cell viability was used to evaluate the inhibitory activity of compounds against HT-29 human colon cancer cells, and the calculation formula was as follows:

percent cell viability% = { (Compound OD-blank OD)/(control OD-blank OD) } × 100%

Cell inhibition rate% = 1-cell viability% = {1- (compound OD-blank OD)/(control OD-blank OD) } × 100%, and IC is obtained by fitting formula with graphpad software ₅₀ 。；

The concentration-dependent relationship between the inhibitory activities of YGP-2, YGP-3, YGP-4 on human colon cancer cell HT-29 of the glycoprotein of the present invention is shown in FIG. 16, which illustrates that YGP-2, YGP-3, YGP-4 have a concentration-dependent inhibitory relationship on the inhibitory activity of HT-29 on human colon cancer cell, and the inhibitory effect is enhanced with the increase of concentration, and the EC of the inhibitory activity of HT-29 on human colon cancer cell is shown ₅₀ 164.4. Mu.g/mL, 19.9. Mu.g/mL and 184.9. Mu.g/mL, respectively.

Example 15

The cytotoxicity of YGP-2, YGP-3 and YGP-4 was determined by MTT staining method:

MTT is reduced by succinyl epoxidase in living cells, with color intensity proportional to cell viability, whereas dead cells cannot. SAGP-I and SAGP-II cytotoxicity was tested on a normal human liver cell line L02. Cells were cultured for 48 hours after administration, treated with MTT for 2 hours, and then absorbance was measured at 562nm with a microplate reader;

cytotoxicity is cell death caused by cells or chemicals, is independent of apoptosis or necrosis, reflects toxic damage of the drug to normal cells, MTT measures cell viability through metabolic activity, the darker the color indicates the stronger the cell viability, the lower the cytotoxicity, fig. 17 shows the cell viability of YGP-2, YGP-3 and YGP-4 to human normal hepatocytes L-02 at the range of 6.6-50 μ g/mL; the results showed that YGP-2 and YGP-3 cells showed dose-dependent toxicity, and the toxicity increased with increasing administration concentration, and EC of YGP-2 and YGP-3 cytotoxicity ₅₀ 47.77 mu g/mL and 40.23 mu g/mL respectively; the cell viability of YGP-4 in the concentration range is always more than 65%, and the lowest value of YGP-4 in 12.5 mu g/mL is 67.26%, which shows lower cytotoxicity; YGP-3 and YGP-4 all had higher tumor-inhibiting activity and lower cytotoxicity.

Example 16

FT-IR characteristic peak analysis: and (3) measuring an infrared spectrogram by a potassium bromide tabletting method.

The IR chromatograms of YGP-3 and YGP-4 are shown in FIG. 18 and FIG. 19, respectively, which show the absorption characteristics of typical functional groups of proteins and polysaccharides; at 3000-3670cm ^-1 The wide and strong absorption peak is the combined absorption peak of O-H and N-H stretching vibration; 1630-1670cm ^-1 The strong absorption peak of (a) is a characteristic absorption peak of C = O (amide I) stretching vibration; 1650-1620cm ^-1 Bending vibration of N-H (amide II) in the primary amide of (1), and 1570-1515cm ^-1 Characteristic absorption peak of the free secondary amide of (1); at 1420-1400cm ^-1 And 1300-1260cm ^-1 The medium-intensity absorption band of (a) is the mixed frequency of the N-H bending vibration and the C-N stretching vibration (amide III); at 1098cm ^-1 And 813cm ^-1 Is a characteristic region of the pyran ring, and 913cm ^-1 、1071cm ^-1 A characteristic absorption zone of the furan ring of the saccharide; the absorbance of 1063.7 in YGP-3 and 1090.2cm-1 in YGP-4 was the characteristic absorbance of S = O, and 823.1cm in YGP-3 ^-1 And 854cm in YGP-4 ^-1 Is the characteristic absorbance of C-S vibration, indicating the presence of SO in both YGP-3 and YGP-4 ₄ ^-2 The results were consistent with those of elemental analysis (EDX) and sulfate content determination; 1240.2cm in YGP-3 ^-1 And 918.1cm ^-1 The sum of absorption of (A) and (B) 1259.6cm ^-1 And 920.5cm ^-1 Is the characteristic absorption of P = O and C-O-P vibrations, indicating the presence of PO ₄ ^3- The results are consistent with the results of analysis of monosaccharide components, i.e., glycoproteins with higher ribose content.

Example 17

The periodic acid oxidation-beta-elimination reaction is carried out according to the usual methods:

the glycosylation pattern was determined by hydrogen peroxide oxidation and β -elimination in combination with UV analysis and the UV spectra of YGP-3 (FIG. 20) and YGP-4 (FIG. 21) are shown; the UV absorption at 220-240nm of the alkali-treated YGP-3 was significantly enhanced compared to that of the YGP-3 which had not been alkali-treated, indicating the presence of O-glycosylated oligosaccharide chains in YGP-3; the alpha-amino group of serine or threonine at the O-glycosylation site can be converted into alpha-amino acrylic acid or alpha-amino butyric acid after alkaline dissociation, and the unsaturated amino acid has absorption at about 240 nm; for YGP-4, there was no change in absorbance around 220-240nm before and after the alkali treatment, indicating no O-glycosylated oligosaccharide chain; YGP-3 and YGP-4 both have enhanced UV absorption between 200 and 220nm and a higher degree of enhancement after heating than the alkali treated samples, indicating that carboxyl groups may be formed after the alkali treatment; sialic acid is the terminal unit of the glycoprotein oligosaccharide chain; b-elimination of sialic acid by periodate oxidation can result in a-, b-unsaturated lactone pyran rings, which can lead to enhanced UV absorbance at around 210-220nm and 260 nm; in summary, the results indicate that SAGP-I has more oligosaccharide chains than SAGP-II and that YGP-3 has an o-glycosylation site, which is not present in YGP-4.

Example 18

Sulfate radical content determination with anhydrous Na ₂ SO ₄ And (3) performing content determination as a standard substance: the standard curve is: y =1938.1x-262.56 ² =0.9814; SO of YGP-3 was measured according to the standard curve ₄ ^2- The content was 16.57%, while YGP-4 did not detect the sulfate group content but had SO in the infrared chromatogram ₄ ^2- Characteristic absorption of (1). The elemental analysis also contains S elements.

Example 19

Circular Dichroism (CD) analysis secondary structure: secondary structure of glycoprotein sample was determined using a chiralscan CD spectrometer (Applied Photophysics, UK), 1mg/mL glycoprotein sample was prepared with 50mM sodium phosphate buffer at pH 7.0, CD spectra were recorded at 180-260nm, with buffer as background, triplicate scans were repeated, CD spectra were determined by using K2D3 on-line tool after averaging and smoothing;

the results of the two-color chromatography of YGP-3 and YGP-4 are shown in FIGS. 22 and 23: YGP-3 has a stronger CD curve than YGP-4, and YGP-3 has a higher protein content and higher molecular weight than YGP-4, so YGP-3 has a more complex secondary structure than YGP-4; YGP-3 showed a positive cotton effect around 195nm, negative cotton effects around 208 and 222nm, and a positive to negative crossover around 202nm, which is characteristic of α -helical proteins, while the cotton effect at 218nm and the positive cotton effect at 195nm are characteristic of β -helical structures; YGP-4 has a very low positive ovality cotton curve near 208nm and a negative random coil characteristic near 195 nm. SAGP-I contained 5.04% alpha helix and 34.03% beta-strand and SAGP-II contained 0.83% alpha helix and 26.97% beta-strand, calculated according to the K2D3 on-line tool.

Example 20

Elemental analysis: tables 5YGP-3 and YGP-4 are analyzed by atomic energy chromatography and the results are shown;

TABLE 5 results of elemental analysis of YGP-3 and YGP-4

As can be seen from the table: YGP-3 and YGP-4 both contain P and S and are high in content, and according to the results of monosaccharide analysis, YGP-3 and YGP-4 are sulfated and phosphated glycoproteins by comprehensive analysis such as infrared chromatography.

Example 21

LC-MS/MS analysis:

QE sequence identification and molecular weight determination of protein chains:

and (3) enzymolysis treatment: carrying out reductive alkylation on YGP-3, adding trypsin for enzymolysis, reacting at 37 ℃ overnight, desalting, and carrying out LC-MS (liquid chromatography-mass spectrometry) and LC-MS/MS (liquid chromatography-mass spectrometry) analysis, wherein the elution conditions are as follows: the solution A is 0.1% formic acid aqueous solution, and the solution B is 0.1% formic acid acetonitrile solution; and (3) after the enzymolysis peptide segment is subjected to reversed phase ultra-high performance liquid chromatography, performing mass spectrum detection and analysis by using a Q-active mass spectrometer, wherein the Q-active mass spectrometer comprises the following steps: analysis duration: 75min, detection mode: positive ion, parent ion scan range: 300-1800m/z, first order mass spectrum, resolution: 70,000-m/z 200, second order mass spectral resolution: 17,500-m/z 200;

analyzing liquid quality detection data: carrying out data retrieval analysis on a raw data file by using PD software, and carrying out sequence analysis on a protein in the glycoprotein by using a theoretical protein sequence obtained by transcription of an annotated gene; identifying to obtain the peptide segment of the main protein and the amino acid sequence coverage rate;

analysis of glycoprotein N// O site sequence:

and (3) enzymolysis treatment: after the glycoprotein sample is subjected to reductive alkylation, PNGase F/sialidase is added, trypsin is added for enzymolysis, the reaction is carried out at 37 ℃ overnight, and LC-MS/MS analysis are carried out after desalination; performing data retrieval analysis on a raw data file by using PD software, and performing sequence analysis on the protein of the glycoprotein by using a theoretical protein sequence obtained by transcription of an annotated gene; b, the protein of the top10 enters a bioharmafinder library for site confirmation, a peptide fragment of the main protein is identified and obtained, whether the main protein has an N/O sugar chain is confirmed, and N/O site analysis is carried out; the results show that: glycoprotein YGP-3 is composed of oligosaccharide chains with 1N-glycosylation site and 14O-glycosylation sites composed of new polypeptide sequence, and is a typical viscous glycoprotein;

example 22

NMR analysis: SAGP-I sample (35 mg) was dissolved in D ₂ In O (99.9%), recorded on an Avance 600MHz NMR spectrometer (Bruker, germany) ¹ H NMR and ¹³ c NMR (temperature 25 ℃ C.), tetramethylsilane (TMS) as an internal standard, and the unit of chemical shift is ppm, ¹ the number of H-MR scans was 32, ¹³ C-NMR is 70000;

of SAGP-I ¹ The H-NMR spectrum showed that, as shown in FIG. 25, the strong signal at δ 4.79 can be attributed to D ₂ A proton of O; in the range of 3.53-5Multiple signals in the 87ppm range may indicate the presence of similar monosaccharide residues; the signal combination of the prominent peaks centered at about 2.03ppm was-COCH in N-acetylglucosamine, N-acetylgalactosamine (GalNAc) and N-acetylneuraminic acid (Neu 5 Ac) ₃ A characteristic signal of the group; the complex signal around 6.80-8.10ppm may be related to the protons of the-CONH-groups in the protein; chemical shifts of 4.41, 4.47, 4.86, 5.32, 5.78 and 5.88ppm are isomeric regions, and chemical shifts below and above 5ppm indicate the presence of β and α configurations in the polysaccharide, consistent with FT-IR and CD results; chemical shifts ranging from 1.91ppm to 2.87ppm are signals of aliphatic and methyl protons;

in that ¹³ C NMR FIG. 26, the strong signal at 182.96ppm and the smaller signals near 181.41, 177.81 and 177.02, 168.73ppm are the chemical shift of-CONH-in the protein backbone; 5363 the chemical shifts at 159.51, 148.81, 133.61, 133.21 and 1331.95 are the amino skeletons of Trp, tyr and Phe; signals of 18.81, 114.32, 106.17 and 105.19ppm are the alpha configuration, while signals of 99.60, 98.76 and 96.64ppm are the chemical shifts of the beta configuration of the sugar backbone in the polysaccharide; the complex signal between 78.80-11.38ppm is CH, CH in the protein and oligosaccharide backbone ₂ And CH ₃ Signal of the group.

Claims

1. The purification of active ingredients of the abomasum of the lamb is characterized by comprising the following steps:

c. b, extracting the lamb stomach degreasing powder obtained in the step b for 1.5 to 2.5 hours in phosphate buffer solution with the material-to-liquid ratio of 24 to 36mL/g, the temperature of 20 to 45 ℃, the pH value of 7.0 and the concentration of 50mM, wherein the phosphate buffer solution contains 0.1 to 1mM of serine enzyme inhibitor, 5mM of ethylene diamine tetraacetic acid and 0.4 to 0.6M of sodium chloride;

f. d, dialyzing the extract obtained in the step d for 60 hours by using a dialysis bag by using a phosphate buffer solution which contains 0.1-1mM of serine enzyme inhibitor and 5mM of ethylenediamine tetraacetic acid and has the concentration of 50mM and the pH value of 7.0 as an external dialysate;

g. diluting the dialyzed extract obtained in the step f to 800mL by using the buffer solution of the step f, loading the extract to DEAE-52 anion exchange resin with pH value of 7.0 and pre-balanced by phosphoric acid buffer solution containing 0.1-1mM serine enzyme inhibitor and 5mM ethylene diamine tetraacetic acid with concentration of 50mM, eluting the column by using the same buffer solution and NaCl solution with concentration of 0.1M,0.2M,0.3M,0.4M,0.5M,0.6M and 0.8M in sequence, measuring a protein elution curve at ultraviolet 52280 zxft 5283, and measuring a sugar elution curve at ultraviolet nm by using a phenol-sulfuric acid method;

h. d, dialyzing and desalting NaCl elution parts of 0.1M,0.2M,0.3M,0.4M,0.5M,0.6M and 0.8M in the step g by using dialysis bags respectively to obtain 60h, and then respectively freeze-drying and weighing the NaCl elution parts, wherein the NaCl elution parts are sequentially named as F1, F2, F3, F4, F5, F6, F7 and F8 and stored at the temperature of-20 ℃;

j. separating and purifying the F2 part obtained in the step i by Sephadex G-50 in a low-temperature chromatography system by taking deionized water as an eluent, detecting the absorbance of the eluent at 280nm at the flow rate of 0.5mL/min, collecting one tube every 8min to obtain two elution peaks, namely single glycoprotein named YGP-1 and YGP-2 respectively, freeze-drying the single glycoprotein respectively, and storing the single glycoprotein at the temperature of-20 ℃;

k. and (3) separating and purifying the F8 obtained in the step (i) by using Sephadex G-50 in a low-temperature chromatography system by using deionized water as an eluent, detecting the absorbance of the eluent at 280nm at the flow rate of 0.4mL/min, collecting one tube every 8min to obtain two elution peaks, namely single glycoprotein named YGP-3 and YGP-4, respectively freezing and drying the single glycoprotein, and storing the single glycoprotein at the temperature of-20 ℃.

2. Use of the lamb abomasum active glycoprotein YGP-1, YGP-2, YGP-3, YGP-4 obtained by the method of claim 1 in the preparation of anti-inflammatory active drugs.

3. Use of the lamb abomasum active glycoprotein YGP-1, YGP-2, YGP-3, YGP-4 obtained by the method according to claim 1 in the preparation of anti-inflammatory active health products.

4. Use of the lamb abomasum active glycoprotein YGP-1, YGP-2, YGP-3, YGP-4 obtained by the method of claim 1 in the preparation of a medicament for inhibiting HT-29 human colon cancer.

5. Use of the lamb abomasum active glycoproteins YGP-1, YGP-2, YGP-3, YGP-4 obtained by the method of claim 1 in the preparation of health products for inhibiting HT-29 human colon cancer.