CN111812333A - Method for comparing activity of ligamentum cervi natural peptide and enzymatic hydrolysis peptide on resisting rheumatoid arthritis - Google Patents
Method for comparing activity of ligamentum cervi natural peptide and enzymatic hydrolysis peptide on resisting rheumatoid arthritis Download PDFInfo
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
- G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
- G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
- G01N33/5044—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics involving specific cell types
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6863—Cytokines, i.e. immune system proteins modifying a biological response such as cell growth proliferation or differentiation, e.g. TNF, CNF, GM-CSF, lymphotoxin, MIF or their receptors
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6863—Cytokines, i.e. immune system proteins modifying a biological response such as cell growth proliferation or differentiation, e.g. TNF, CNF, GM-CSF, lymphotoxin, MIF or their receptors
- G01N33/6869—Interleukin
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/10—Musculoskeletal or connective tissue disorders
- G01N2800/101—Diffuse connective tissue disease, e.g. Sjögren, Wegener's granulomatosis
- G01N2800/102—Arthritis; Rheumatoid arthritis, i.e. inflammation of peripheral joints
Abstract
The invention discloses a method for comparing the activity of natural ligamentum cervi peptide and zymolytic peptide against rheumatoid arthritis, which belongs to the technical field of the comparative analysis of anti-inflammatory active ingredients of animal drugs, and in vitro anti-inflammatory experiments prove that the natural ligamentum cervi peptide and the zymolytic peptide with different mass concentrations can inhibit the proliferation of MH7A cells and reduce the content levels of NO, IL-6 and TNF-alpha, thereby verifying that the natural ligamentum cervi peptide and the zymolytic peptide both have certain anti-RA activity, and comparing the cell proliferation inhibition rate and the release amount of cell inflammatory factors, the invention proves that the natural ligamentum cervi peptide has stronger capability of inhibiting the proliferation of MH7A cells and has better effect of inhibiting the MH7A cells from releasing inflammatory factors.
Description
Technical Field
The invention relates to the technical field of contrastive analysis of anti-inflammatory active ingredients of animal medicaments, in particular to a method for contrasting the activity of natural deer sinew peptide and enzymolysis peptide on rheumatoid arthritis resistance.
Background
The ligamentum Cervi is dry muscle of extremity of Cervus nippon Temminck, has high medicinal value, is a rare animal medicine in China, and is mainly used for treating rheumatic arthralgia, spasm, etc. The main chemical components of the deer sinew comprise amino acids, peptides, collagen, vitamins, trace elements and the like, wherein the collagen is rich and is more than 80 percent, and the deer sinew collagen has the effects of anti-inflammation (Sunxydi, Liyinjing, Zhaoyu and the like, deer sinew collagen anti-inflammatory immune action research [ J ]. Shizhen Chinese medical and national medicine, 2010, 21(04): 853-.
The release of the cell inflammatory factors plays an important role in the generation process of inflammatory reaction, and Nitric Oxide (NO), interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-alpha) are key effector factors in the inflammatory reaction, and the excessive secretion of the nitric oxide, the interleukin-6 and the tumor necrosis factor-alpha can generate inflammatory injury, so the content level of the released cell inflammatory factors in the inflammatory activity screening can be used as an important index of the anti-inflammatory activity of a tested medicament.
Disclosure of Invention
The invention aims to: aiming at the problems in the prior art, a method for comparing the activity of the ligamentum cervi natural peptide and the enzymolysis peptide on the rheumatoid arthritis is provided, specifically, thiazole blue (MTT) is adopted to test and research the influence of the ligamentum cervi natural peptide and the enzymolysis peptide on the MH7A cell proliferation inhibition activity, an enzyme-linked immunosorbent assay method is selected to test the capability of the ligamentum cervi natural peptide and the enzymolysis peptide on inhibiting the MH7A cell from releasing inflammatory factors, and the activity of the Rheumatoid Arthritis (RA) is compared.
The technical scheme adopted by the invention for realizing the purpose is as follows: a method for comparing the anti-rheumatoid arthritis activity of ligamentum cervi natural peptide and enzymolysis peptide is characterized by comprising the following steps:
(1) weighing 5g of raw deer sinew products, crushing the raw deer sinew products into particles with the particle size of 60 meshes, adding the particles into distilled water with the weight of 10-30 times of the raw deer sinew products, magnetically stirring and extracting the mixture for 4-8 h in a constant-temperature water bath at the extraction temperature of 70-100 ℃, collecting an extracting solution, centrifuging the extracting solution at the rotation speed of 4000r/min for 10-20 min, taking supernatant, carrying out microfiltration on the supernatant by a 0.45 mu m filter membrane, and freeze-drying the supernatant for later use to obtain the natural deer sinew peptides;
accurately weighing 0.02 g-0.05 g of the ligamentum cervi natural peptide, adding 5 mL-6 mL of water, ultrasonically dissolving, heating in a water bath at 100 ℃ for 15min, stirring in a constant-temperature magnetic stirrer when the temperature is reduced to the temperature required by enzymolysis, adjusting the enzymolysis temperature and the pH value, carrying out enzymolysis, after the hydrolysis is finished, boiling in a boiling water bath for 15min, inactivating enzymes, cooling to room temperature, centrifuging at 3600r/min for 20min, carrying out microfiltration on supernatant to obtain filtrate, and freeze-drying for later use to obtain the ligamentum cervi enzymolysis peptide;
(2) an in vitro inflammation model is constructed by inducing MH7A cells by 60ng/mL TNF-alpha, and the influence of deer sinew natural peptide with different mass concentrations on MH7A cell proliferation inhibition activity is determined by an MTT method; detecting the levels of NO, IL-6 and TNF-alpha of culture supernatant by using an ELISA kit, and evaluating the anti-inflammatory activity of the ligamentum cervi natural peptide by taking the capability of the ligamentum cervi natural peptide with different mass concentrations for inhibiting the release of NO, IL-6 and TNF-alpha as a screening index;
(3) an in vitro inflammation model is constructed by inducing MH7A cells by 60ng/mL TNF-alpha, and the influence of the deer sinew enzymolysis peptide with different mass concentrations on the MH7A cell proliferation inhibition activity is determined by an MTT method; the ELISA kit is used for detecting the levels of NO, IL-6 and TNF-alpha of culture supernatant, and the anti-inflammatory activity of the deer sinew enzymolysis peptide is evaluated by taking the capability of the deer sinew enzymolysis peptide with different mass concentrations for inhibiting the release of NO, IL-6 and TNF-alpha as a screening index;
(4) the determination experiment of the cell inflammatory factors adopts SPSS 25.0 software to perform significance analysis of data, and the data is metered to
Shown, comparisons between groups were performed using one-way anova and t-test.
Wherein, the concentration range of the deer sinew natural peptide in the step (2) is 25 mug/mL-200 mug/mL.
Wherein the concentration range of the deer sinew enzymolysis peptide in the step (3) is 12.5-100 mug/mL.
Through the design scheme, the invention can bring the following beneficial effects: the in-vitro anti-inflammatory experiment proves that the ligamentum cervi natural peptide and the enzymolysis peptide with different mass concentrations can inhibit the proliferation of MH7A cells and reduce the content levels of NO, IL-6 and TNF-alpha, and the invention verifies that the ligamentum cervi natural peptide and the enzymolysis peptide both have certain anti-RA activity, and by comparing the cell proliferation inhibition rate and the cell inflammatory factor release amount of the two peptides, the invention proves that the ligamentum cervi natural peptide has stronger proliferation inhibition capability on MH7A cells, and the effect of the ligamentum cervi enzymolysis peptide on inhibiting the MH7A cells from releasing inflammatory factors is better.
Drawings
The accompanying drawings, which are included to provide a further understanding of the invention and are incorporated in and constitute a part of this application, illustrate embodiment(s) of the invention and together with the description serve to explain the invention without limiting the invention to the right, and in which:
FIG. 1 shows the result of measuring the NO content in the cell culture fluid of each group of deer sinew natural peptide;
FIG. 2 shows the result of measuring the content of TNF-alpha in the cell culture fluid of each group of deer sinew natural peptide;
FIG. 3 shows the measurement results of IL-6 content in cell culture fluid of each group of deer sinew natural peptide;
FIG. 4 shows the result of measuring the NO content in the cell culture fluid of each group of deer sinew enzymolysis peptide;
FIG. 5 shows the result of measuring the content of TNF-alpha in the cell culture fluid of each group of deer sinew enzymolysis peptide;
FIG. 6 shows the results of determination of IL-6 content in cell culture solutions of each group of deer sinew enzymolysis peptides.
Detailed Description
The invention uses TNF-alpha to induce human rheumatoid arthritis fibroblast synovial cells (MH7A) to establish an in vitro inflammation model, uses an ELISA kit to detect the content levels of NO, IL-6 and TNF-alpha in culture supernatant, uses the capability of the ligamentum cervi natural peptide and the enzymolysis peptide with different mass concentrations for inhibiting the release of NO, IL-6 and TNF-alpha as screening indexes, and compares the anti-RA activity of the ligamentum cervi natural peptide and the enzymolysis peptide.
The preferred embodiments of the present invention will be described in conjunction with the accompanying drawings, and it is to be understood that the preferred embodiments described herein are merely for purposes of illustration and explanation and are not intended to limit the present invention.
Example 1:
weighing 5g of crude deer sinew products, crushing the crude deer sinew products into particles with the particle size of 60 meshes, adding the particles into distilled water with the weight of 10 times of that of the crude deer sinew products, magnetically stirring and extracting the mixture for 4 hours in a constant-temperature water bath at the extraction temperature of 70 ℃, collecting an extracting solution, centrifuging the extracting solution at the rotating speed of 4000r/min for 10 minutes, taking supernate, carrying out microfiltration on the supernate by a 0.45 mu m filter membrane, and freeze-drying the supernate for later use to obtain the natural deer sinew peptides;
accurately weighing 0.02g of the ligamentum cervi natural peptide, adding 5mL of water, carrying out ultrasonic dissolution, heating in a water bath at 100 ℃ for 15min, placing in a constant-temperature magnetic stirrer for stirring when the temperature is reduced to the temperature required by enzymolysis, adjusting the enzymolysis temperature and the pH value, carrying out enzymolysis, after the hydrolysis is finished, transferring into a boiling water bath for boiling for 15min, inactivating enzyme, cooling to room temperature, centrifuging at 3600r/min for 20min, carrying out microfiltration on supernatant to obtain filtrate, and freeze-drying to obtain the ligamentum cervi enzymolysis peptide.
Example 2:
weighing 5g of crude deer sinew products, crushing the crude deer sinew products into particles with the particle size of 60 meshes, adding the particles into distilled water with the mass of 20 times of that of the crude deer sinew products, magnetically stirring and extracting the mixture for 6 hours in a constant-temperature water bath at the extraction temperature of 85 ℃, collecting an extracting solution, centrifuging the extracting solution at the rotating speed of 4000r/min for 15 minutes, taking supernate, carrying out microfiltration on the supernate by a 0.45 mu m filter membrane, and freeze-drying the supernate for later use to obtain the natural deer sinew peptides;
accurately weighing 0.035g of ligamentum Cervi natural peptide, adding 5.5mL of water, ultrasonically dissolving, heating in 100 ℃ water bath for 15min, placing in a constant temperature magnetic stirrer for stirring when the temperature is reduced to the temperature required by enzymolysis, adjusting the enzymolysis temperature and pH value, carrying out enzymolysis, after the hydrolysis is finished, transferring into boiling water bath for boiling for 15min, inactivating enzyme, cooling to room temperature, centrifuging at 3600r/min for 20min, microfiltering the supernatant, and freeze-drying to obtain the ligamentum Cervi enzymolysis peptide;
example 3:
weighing 5g of crude deer sinew products, crushing the crude deer sinew products into particles with the particle size of 60 meshes, adding the particles into distilled water with the mass of 30 times of that of the crude deer sinew products, magnetically stirring and extracting the mixture for 8 hours in a constant-temperature water bath at the extraction temperature of 100 ℃, collecting an extracting solution, centrifuging the extracting solution at the rotating speed of 4000r/min for 20 minutes, taking supernate, carrying out microfiltration on the supernate by a 0.45 mu m filter membrane, and freeze-drying the supernate for later use to obtain the natural deer sinew peptides;
accurately weighing 0.05g of the ligamentum cervi natural peptide, adding 6mL of water, carrying out ultrasonic dissolution, heating in a water bath at 100 ℃ for 15min, placing the mixture in a constant-temperature magnetic stirrer for stirring when the temperature is reduced to the temperature required by enzymolysis, adjusting the enzymolysis temperature and the pH value, carrying out enzymolysis, after the hydrolysis is finished, transferring the mixture into a boiling water bath for boiling for 15min, inactivating enzymes, cooling to room temperature, centrifuging at 3600r/min for 20min, carrying out microfiltration on supernatant to obtain filtrate, and freeze-drying to obtain the ligamentum cervi enzymolysis peptide;
example 4: in vitro anti-inflammatory Activity assay
MH7A cell is a research object usually selected in vitro anti-inflammatory activity experiments, and NO, IL-6 and TNF-alpha are key effector factors of inflammatory reaction. MH7A cells were cultured with 10% fetal bovine serum, 100mg/L streptomycin, and 1X 10 cells5The DMEM high-sugar culture solution used in the invention is a DMEM high-sugar culture solution commonly used in the market, and the preferred DMEM high-sugar culture solution is selected from Hyclone company of America, contains 4500mg/L D-glucose, 584mg/L L-glutamine, 110mg/L sodium pyruvate and 3700mg/L sodium bicarbonateAnd pH 7.0-7.4, culturing in 5% CO2 incubator at 37 deg.C, and selecting cells in logarithmic phase for experiment. The experiment was performed by setting a blank group, a model group, and a dosing group, each well was 100. mu.L, and each concentration was set to 5 duplicate wells. The cells were cultured at 5X 103Adding 100 mu L of 96-well plate into each well, culturing in a 5% CO2 incubator at 37 ℃ for 24h, adding 60ng/mL TNF-alpha into each group except a blank group (adding DMEM high-sugar culture solution) for molding, and continuously culturing for 24h, wherein each group is subjected to administration intervention (ligamentum Cervi natural peptide: 25 mu g/mL, 50 mu g/mL, 100 mu g/mL, 200 mu g/mL, ligamentum Cervi enzymolysis peptide: 12.5 mu g/mL, 25 mu g/mL, 50 mu g/mL, 100 mu g/mL). After 24h of culture, adding 20 mu L of MTT solution with the mass concentration of 5g/L into each hole, continuing to culture for 4h, detecting the absorbance OD value at the wavelength of 490nm by using an enzyme-labeling instrument, and calculating the proliferation inhibition rate of each group of cells;
the experimental operation and grouping are as above, and after the cells are administrated and cultured for 24h, the supernatant is respectively sucked. Measuring the content levels of NO, IL-6 and TNF-alpha by using an ELISA kit, measuring an OD value under an enzyme-linked immunosorbent assay (ELISA) instrument, wherein the measuring wavelength is 490nm, and independently repeating the experiment for 3 times;
TABLE 1 measurement results of cell proliferation inhibition rates of various groups of ligamentum Cervi Natural peptides: (
n＝3)
Note: # indicates p <0.01 (compared to blank); denotes p < 0.05; denotes p < 0.01; denotes p <0.001 (compared to model group).
TABLE 2 measurement results of cell proliferation inhibition rates of each group of deer sinew enzymolysis peptides (
n＝3)
Compared with a blank group, the cell proliferation of the TNF-alpha model group is obvious (p is less than 0.01), and the success of experimental modeling is proved; compared with a TNF-alpha model group, each administration group has certain inhibition effect on the proliferation of MH7A cells, and the corresponding proliferation inhibition rate is shown in tables 1 and 2;
under the mass concentration of 200 mug/mL, the inhibition effect of the ligamentum cervi natural peptide is most obvious, the proliferation inhibition rate is 78.90%, and the difference is significant compared with a model (p is less than 0.01). In the mass concentration range of 50-100 mug/mL, the cell proliferation inhibition rate is increased along with the increase of the sample concentration, the concentration dependence is presented, and each sample group has significant difference compared with the model group. The results of the activity of the deer tendon natural peptide on MH7A cell proliferation inhibition are as follows: 200 μ g/mL >100 μ g/mL >50 μ g/mL >25 μ g/mL;
in the mass concentration range of 12.5-100 mug/mL, the proliferation inhibition rate of cells shows the trend of increasing first and then decreasing along with the increase of the sample concentration, each sample group has significant difference compared with a model group, when the mass concentration is 50 mug/mL, the inhibition effect of the cervus tendon enzymolysis peptide is strongest, the proliferation inhibition rate is 47.36%, and the difference is the most significant compared with the model (p is less than 0.01). In conclusion, the results of the inhibitory activity of the deer sinew enzymolysis peptide on MH7A cell proliferation are as follows: 50 μ g/mL >100 μ g/mL >25 μ g/mL >12.5 μ g/mL.
The result of cell proliferation inhibition rate comparison shows that the deer sinew natural peptide has stronger proliferation inhibition effect on MH 7A;
FIGS. 1-6 show the effect of ligamentum Cervi natural peptide and ligamentum Cervi enzymolysis peptide on the release of NO, IL-6, TNF-alpha from MH7A cells;
after administration, the contents of NO, IL-6 and TNF-alpha in MH7A cell supernatant are reduced in different degrees, the release amounts of the three cell inflammatory factors reach the minimum when the mass concentrations of the ligamentum cervi natural peptide and the ligamentum cervi enzymolysis peptide reach 100 mu g/mL and 50 mu g/mL respectively, the anti-inflammatory effects of the ligamentum cervi natural peptide and the ligamentum cervi enzymolysis peptide are closely related to the release of the inhibition inflammatory factors, and the ligamentum cervi natural peptide and the ligamentum cervi enzymolysis peptide are further verified to have certain anti-RA activity.
By comparing the content levels of the cell inflammatory factors of the ligamentum Cervi natural peptide and the ligamentum Cervi enzymolysis peptide at the optimum inhibitory concentration, the ligamentum Cervi enzymolysis peptide has stronger ability of inhibiting MH7A cell from secreting inflammatory factors.
Claims (3)
1. A method for comparing the anti-rheumatoid arthritis activity of ligamentum cervi natural peptide and enzymolysis peptide is characterized by comprising the following steps:
(1) weighing 5g of raw deer sinew products, crushing the raw deer sinew products into particles with the particle size of 60 meshes, adding the particles into distilled water with the weight of 10-30 times of the raw deer sinew products, magnetically stirring and extracting the mixture for 4-8 h in a constant-temperature water bath at the extraction temperature of 70-100 ℃, collecting an extracting solution, centrifuging the extracting solution at the rotation speed of 4000r/min for 10-20 min, taking supernatant, carrying out microfiltration on the supernatant by a 0.45 mu m filter membrane, and freeze-drying the supernatant for later use to obtain the natural deer sinew peptides;
accurately weighing 0.02 g-0.05 g of the ligamentum cervi natural peptide, adding 5 mL-6 mL of water, ultrasonically dissolving, heating in a water bath at 100 ℃ for 15min, stirring in a constant-temperature magnetic stirrer when the temperature is reduced to the temperature required by enzymolysis, adjusting the enzymolysis temperature and the pH value, carrying out enzymolysis, after the hydrolysis is finished, boiling in a boiling water bath for 15min, inactivating enzymes, cooling to room temperature, centrifuging at 3600r/min for 20min, carrying out microfiltration on supernatant to obtain filtrate, and freeze-drying for later use to obtain the ligamentum cervi enzymolysis peptide;
(2) an in vitro inflammation model is constructed by inducing MH7A cells by 60ng/mL TNF-alpha, and the influence of deer sinew natural peptide with different mass concentrations on MH7A cell proliferation inhibition activity is determined by an MTT method; detecting the levels of NO, IL-6 and TNF-alpha of culture supernatant by using an ELISA kit, and evaluating the anti-inflammatory activity of the ligamentum cervi natural peptide by taking the capability of the ligamentum cervi natural peptide with different mass concentrations for inhibiting the release of NO, IL-6 and TNF-alpha as a screening index;
(3) an in vitro inflammation model is constructed by inducing MH7A cells by 60ng/mL TNF-alpha, and the influence of the deer sinew enzymolysis peptide with different mass concentrations on the MH7A cell proliferation inhibition activity is determined by an MTT method; the ELISA kit is used for detecting the levels of NO, IL-6 and TNF-alpha of culture supernatant, and the anti-inflammatory activity of the deer sinew enzymolysis peptide is evaluated by taking the capability of the deer sinew enzymolysis peptide with different mass concentrations for inhibiting the release of NO, IL-6 and TNF-alpha as a screening index;
(4) the determination experiment of the cell inflammatory factors adopts SPSS 25.0 software to perform significance analysis of data, and the data is metered to
Shown, comparisons between groups were performed using one-way anova and t-test.
2. The method of comparing the anti-rheumatoid arthritis activities of the ligamentum cervi natural peptide and the zymolytic peptide according to claim 1, wherein the method comprises the following steps: the concentration range of the deer sinew natural peptide in the step (2) is 25 mug/mL-200 mug/mL.
3. The method of comparing the anti-rheumatoid arthritis activities of the ligamentum cervi natural peptide and the zymolytic peptide according to claim 1, wherein the method comprises the following steps: the concentration range of the deer sinew enzymolysis peptide in the step (3) is 12.5 to 100 mu g/mL.
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