CN114350735A - Method for extracting small molecular polypeptide from chick embryo - Google Patents
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Abstract
The invention discloses a method for extracting small molecular polypeptides from chick embryos. The method utilizes the characteristics that resistant starch has large molecular weight and high polymerization degree and can spontaneously form a cavity structure with larger internal volume, and the small molecular polypeptide in the chicken embryo is obtained by including the chicken embryo protein subjected to enzymolysis with the resistant starch solution, performing ultra-filtration and tube centrifugation after ultrasonic treatment. Compared with a crushing impregnation extraction method and an enzymolysis impregnation extraction method, the extraction method of the micromolecule polypeptide disclosed by the invention has higher extraction rate. The micromolecular polypeptide extracted by the method has the molecular weight of 11-67 kDa, is simple in structure, has the purity of 89.2 +/-3.1 percent, has the extraction yield of 67 +/-2.2 percent, can quickly permeate a biological barrier, and has a remarkable anti-inflammatory effect and a better treatment effect on osteoarthritis through animal experiments.
Description
Technical Field
The invention relates to the field of separation and extraction of traditional Chinese medicinal materials, and in particular relates to a method for extracting small molecular polypeptides in chick embryos.
Background
The chick embryo is a dried chick embryo (English name: chick embryo or egg embryo) of a phasianidae animal, the chick egg is incubated for 17-18 days at 37.9 +/-0.5 ℃, the eggshell is removed, the chick egg is dried, has sweet, salty, fishy and warm in nature, enters spleen and stomach channels, has the effects of warming middle-jiao and replenishing qi, and replenishing essence and marrow, is used for treating body deficiency and emaciation, fracture, tendon injury and pain and the like, and is recorded in ' quality standard of traditional Chinese medicinal materials in Guizhou province ' version ' 2019. In addition, the main component in the chick embryo is protein, and is similar to some components in human placenta, such as small molecule polypeptide. Research shows that the active peptide has the function of eliminating free radicals and is one of the main components for treating fracture and muscle injury pain. In addition, the peptides also have antibacterial, antiviral, antioxidant, antitumor, antiaging and immunity regulating effects. Due to many characteristics, the material is receiving wide attention. At present, related documents report extraction and methods of small molecular polypeptides in various animals and plants, but the extraction purity is low, the extraction rate is low, and the like. How to improve the extraction rate and purity of the small molecular polypeptide has profound significance for the research of the chick embryo.
The polygonum multiflorum Resistant Starch (RS) is a novel resistant starch, mainly belongs to RS3 resistant starch and has the following characteristics: the molecular weight is large, the polymerization degree is high, and a V-shaped cavity structure with larger internal volume can be spontaneously formed. On the basis of the original enzymolysis extraction, the method obtains the small molecular polypeptide from the chick embryo by utilizing the characteristics of the polygonum multiflorum resistant starch, has high extraction rate and purity, and has great significance for the study of the chick embryo.
Disclosure of Invention
In order to solve the technical problems, the invention provides a method for extracting small molecular polypeptides from chicken embryos, which solves the problem of how to efficiently extract the small molecular polypeptides from the chicken embryos. In order to achieve the purpose, the invention is realized by the following technical scheme.
The extraction method of the small molecular polypeptide in the chick embryo is a method for preparing the small molecular polypeptide in the chick embryo by spontaneously forming a V-shaped cavity structure with a larger internal volume by resistant starch, clathrating the enzymolyzed chick embryo protein with a resistant starch solution, and centrifuging an ultrafiltration tube after ultrasonic treatment.
The extraction method of the small molecular polypeptide in the chick embryo is carried out according to the following steps:
a. dissolving Polygoni Multiflori radix resistant starch in 2mol/L KOH aqueous solution, adding HCl at room temperature to adjust pH to 6.5, adding water to make the resistant starch concentration 1mg/mL to obtain resistant starch solution, i.e. product A;
b. oven drying chicken embryo under reduced pressure, pulverizing into fine powder, adding papain in water as solvent, and performing enzymolysis to obtain product B;
c. adding neutral protease into the product B, and performing enzymolysis to obtain product C;
d. adding alkaline protease into product C, performing enzymolysis, centrifuging, and collecting supernatant to obtain micromolecular polypeptide crude extract, i.e. product D;
e. stirring the product D, dropwise adding the product D into the product A in a volume ratio of 4: 1-6: 1, and stirring for inclusion to obtain a product E;
f. and (3) centrifuging the product E at 5000rpm for 10 minutes, removing supernatant, adding a proper amount of water into the precipitate for dispersion, performing ultrasonic treatment at 25kHz for 30 minutes, putting the solution after ultrasonic crushing into an 80kDa ultrafiltration centrifugal tube, performing centrifugation at 12000rpm for 10 minutes, and obtaining filtrate which is the chick embryo small molecule polypeptide concentrate.
And in the step B, drying the chicken embryo at 60-70 ℃ under reduced pressure, crushing the chicken embryo into fine powder, taking water as a solvent, uniformly stirring the chicken embryo and the water in a ratio of 1: 9-10, adding papain, controlling the pH value to be 3-6, controlling the enzyme addition amount to be 3-6%, controlling the temperature to be 40-60 ℃, and carrying out enzymolysis for 1.0-2.0 hours to obtain a product B.
More specifically, in the step B, drying the chicken embryo under reduced pressure at 60-70 ℃, crushing the chicken embryo into fine powder, adding 75% ethanol, wherein the ratio of the chicken embryo to water is 1: 5-10, uniformly stirring, adding papain, wherein the pH value is 5, the enzyme addition amount is 5%, the temperature is 53 ℃, and the enzymolysis time is 1.0-2.0 hours, so as to obtain the product B.
And C, adding neutral protease into the product B, wherein the pH value of the neutral protease is 5-7, the enzyme adding amount is 3% -4%, the temperature is 45-55 ℃, and the enzymolysis time is 1.0-1.5 hours to obtain a product C.
More specifically, in the step C, the product B is added with neutral protease with the pH value of 6, the enzyme adding amount of 4 percent, the temperature of 52 ℃ and the enzymolysis time of 1.0-1.5 hours to obtain the product C.
The extraction method of the small molecular polypeptide in the chick embryo is characterized by comprising the following steps: and in the step D, adding the product C into alkaline protease with the pH of 7.8-8.2 and the enzyme addition of 5-6% at the temperature of 49-53 ℃, centrifuging for 5 minutes at 1000rpm after enzymolysis, and taking supernatant to obtain a micromolecular polypeptide crude extract, namely a product D.
More specifically, in the step D, the product C is taken and added with alkaline protease pH8.2, the enzyme adding amount is 5%, the temperature is 49 ℃, after enzymolysis, the product C is centrifuged for 5 minutes at 1000rpm, and the supernatant is taken to obtain the micromolecular polypeptide crude extract, namely the product D.
In the step E, the product D is taken, stirred at the temperature of 60-70 ℃ and the rpm of 200-300, and is dropwise added into the product A, the volume ratio of the product A to the product B is 4: 1-6: 1, and the product A and the product B are stirred for 3-6 hours to carry out inclusion, so that a product E is obtained;
more specifically, in the step E, the product D is taken, stirred at the temperature of 60 ℃ and the rpm of 200-300, and is dropwise added into the product A, the volume ratio of the product A to the product B is 6:1, and the product A and the product B are stirred for 3 hours to carry out inclusion, so that a product E is obtained;
the invention has the beneficial effects that:
the method optimizes the optimal enzymolysis conditions for extracting the micromolecule polypeptide in the chick embryo based on the traditional method, and then utilizes the V spiral cavity with larger internal volume which is peculiar to the polygonum multiflorum resistant starch to perform inclusion, so as to obtain the micromolecule polypeptide with higher purity, and the yield of the micromolecule polypeptide is obviously higher than that of the micromolecule polypeptide prepared by an enzymolysis method.
Drawings
FIG. 1 liquid chromatogram of pulverization and maceration extraction;
FIG. 2 is a liquid phase diagram of an enzymatic maceration extraction process;
FIG. 3 is a liquid phase diagram of the pulverization-enzymolysis-resistant starch inclusion ultrasonic method;
FIG. 4 is a molecular weight distribution diagram of a small-molecule polypeptide in chick embryos;
FIG. 5 results of HE staining of rat cartilage;
FIG. 6 resistant starch electron microscope image of small molecule polypeptide coated chicken embryo
In order to make the technical solutions of the present invention better understood and enable those skilled in the art to practice the present invention, the following embodiments are further described, but the present invention is not limited to the following embodiments.
Detailed Description
Example 1:
a. dissolving Polygoni Multiflori radix resistant starch in 2mol/L KOH aqueous solution, adding HCl at room temperature to adjust pH to 6.5, adding water to make the resistant starch concentration 1mg/mL to obtain resistant starch solution, i.e. product A;
b. drying chicken embryo at 60-70 deg.C under reduced pressure, pulverizing into fine powder, taking water as solvent, the ratio of chicken embryo to water is 1:9, stirring well, adding papain, pH value is 5, enzyme addition amount is 5%, temperature is 53 deg.C, and enzymolysis time is 1.5 hr to obtain product B;
c. adding neutral protease into the product B, wherein the pH value is 6, the enzyme addition amount is 4%, the temperature is 52 ℃, and the enzymolysis time is 1.2 hours, thus obtaining a product C;
d. adding alkaline protease pH8.2, enzyme adding amount 5%, and temperature 49 deg.C into product C, performing enzymolysis, centrifuging at 1000rpm for 5min, and collecting supernatant to obtain micromolecular polypeptide crude extract, i.e. product D;
e. taking the product D, stirring at 60 ℃ and 200-300 rpm, dropwise adding the product D into the product A, wherein the volume ratio of the product A to the product B is 6:1, and stirring for 3 hours to perform inclusion to obtain a product E;
f. and (3) centrifuging the product E at 5000rpm for 10 minutes, removing supernatant, adding a proper amount of water into the precipitate for dispersion, performing ultrasonic treatment at 25kHz for 30 minutes, putting the solution after ultrasonic crushing into an 80kDa ultrafiltration centrifugal tube, performing centrifugation at 12000rpm for 10 minutes, and obtaining filtrate which is the chick embryo small molecule polypeptide concentrate.
The preparation process comprises the following steps: taking 100g of chick embryo small molecule polypeptide concentrate, adding 10% of prepared soluble starch, uniformly mixing, granulating with 75% of ethanol, drying, grading, and filling into capsules to obtain capsules.
The usage and dosage are as follows: it is taken with boiled water 3g each time and 3 times a day.
The efficacy is as follows: can be used for treating osteoarthritis.
Example 2
a. Dissolving Polygoni Multiflori radix resistant starch in 2mol/L KOH aqueous solution, adding HCl at room temperature to adjust pH to 6.5, adding water to make the resistant starch concentration 1mg/mL to obtain resistant starch solution, i.e. product A;
b. drying chicken embryo at 60-70 deg.C under reduced pressure, pulverizing into fine powder, taking water as solvent, the ratio of chicken embryo to water is 1:5, stirring well, adding papain, pH 3, enzyme adding amount 3%, temperature 40 deg.C, and enzymolysis for 1.0 hr to obtain product B;
c. adding neutral protease into the product B, wherein the pH value is 5, the enzyme addition amount is 3%, the temperature is 45 ℃, and the enzymolysis time is 1.0 hour to obtain a product C;
d. adding alkaline protease with pH of 7.8 and enzyme addition of 5.5% into product C at 52 deg.C, performing enzymolysis, centrifuging at 1000rpm for 5min, and collecting supernatant to obtain micromolecular polypeptide crude extract, i.e. product D;
e. taking the product D, stirring at 65 ℃ and 200-300 rpm, dropwise adding the product D into the product A, wherein the volume ratio of the product A to the product B is 4:1, and stirring for 4 hours to perform inclusion to obtain a product E;
f. and (3) centrifuging the product E at 5000rpm for 10 minutes, removing supernatant, adding a proper amount of water into the precipitate for dispersion, performing ultrasonic treatment at 25kHz for 30 minutes, putting the solution after ultrasonic crushing into an 80kDa ultrafiltration centrifugal tube, performing centrifugation at 12000rpm for 10 minutes, and obtaining filtrate which is the chick embryo small molecule polypeptide concentrate.
The preparation process comprises the following steps: mixing chick embryo small molecule polypeptide concentrate 100g with 10% starch, granulating with 65% ethanol as humectant, drying, tabletting, and film coating to obtain tablet.
The usage and dosage are as follows: it is taken with boiled water 3g each time and 3 times a day.
The efficacy is as follows: can be used for treating osteoarthritis.
Example 3
a. Dissolving Polygoni Multiflori radix resistant starch in 2mol/L KOH aqueous solution, adding HCl at room temperature to adjust pH to 6.5, adding water to make the resistant starch concentration 1mg/mL to obtain resistant starch solution, i.e. product A;
b. oven drying chicken embryo at 70 deg.C under reduced pressure, pulverizing into fine powder, taking water as solvent, the ratio of chicken embryo to water is 1:10, stirring, adding papain, adjusting pH to 6, adding enzyme at 60 deg.C, and performing enzymolysis for 2.0 hr to obtain product B;
c. adding neutral protease into the product B, wherein the pH value is 7, the enzyme addition amount is 4%, the temperature is 55 ℃, and the enzymolysis time is 1.2 hours, thus obtaining a product C;
d. adding alkaline protease with pH of 8.0 and enzyme addition of 6% into product C at 53 deg.C, performing enzymolysis, centrifuging at 1000rpm for 5min, and collecting supernatant to obtain micromolecular polypeptide crude extract, i.e. product D;
e. taking the product D, stirring at 70 ℃ and 200-300 rpm, dropwise adding the product D into the product A, wherein the volume ratio of the product A to the product B is 6:1, and stirring for 6 hours to perform inclusion to obtain a product E;
f. and (3) centrifuging the product E at 5000rpm for 10 minutes, removing supernatant, adding a proper amount of water into the precipitate for dispersion, performing ultrasonic treatment at 25kHz for 30 minutes, putting the solution after ultrasonic crushing into an 80kDa ultrafiltration centrifugal tube, performing centrifugation at 12000rpm for 10 minutes, and obtaining filtrate which is the chick embryo small molecule polypeptide concentrate.
The preparation process comprises the following steps: taking 100g of chick embryo small molecule polypeptide concentrate, adding 2 times of sucrose, mixing, granulating with 80% ethanol as wetting agent, drying at 80 deg.C, sieving with 16 mesh sieve for 1 time, and sieving with 60 mesh sieve to obtain fine powder, i.e. granule.
The usage and dosage are as follows: it is administered with 15g of boiled water for one time, 3 times a day.
The efficacy is as follows: can be used for treating osteoarthritis.
The inventors carried out a number of experiments and the following were studies of the extraction method of the present invention:
1 instruments and materials
1.1 instruments
PHS-3C pH meter (Shanghai apparatus, electrosciences instruments, Inc.); TU-1810 ultraviolet spectrophotometer (Beijing Pujingyo general instruments, LLC); an AL204 electronic balance (mettlerlotordo, usa); FD freeze-drying machines (shanghai pioneer mechanical equipment ltd); a pulverizer (Zhejiang Roc machinery, Inc.); magnetic stirrers (ai ka (IKA) corporation); an autoclave (Shandong Xinhua medical instruments Co., Ltd.); high speed centrifuges (Eppendorf corporation); ultrasonograph (Shanghaineth science and technology Co., Ltd.); olympus optical microscope (Olympus, Japan, model: BX43+ DP 26); EG1150 paraffin embedding machine (Leica, germany); RM2245 rotary microtomes (Leica, germany); TKY-TKA sheet spreading and baking machine (Hubeitaikang).
1.2 drugs and reagents
Chick embryo (Guizhou aquarium drug development, LLC); polygonum multiflorum (Beijing herbal medicine); pullulanase (alatin reagent (shanghai) ltd); papain (pombo bio ltd, south-Guangxi-Ning); neutral proteases (Shanghai Piano Cyanine industries, Ltd.); alkaline protease (zu zhuanxin biotechnology ltd); xylene (Tianjin Hengxing reagent, Inc.); animal SD mouse, SPF grade, weight 18-22g (provided by the third military medical experiment animal center of China people's liberation force, the license number: SCXK 2020-; chloral hydrate (Tianjin, Kemiou, Lot: 20160913).
2 preparation method
2.1 preparation of Polygonum Multiflorum resistant starch
2.1.1 preparation of Polygonum Multiflorum starch
Taking coarse powder of a polygonum multiflorum medicinal material, taking water as an extraction solvent, stirring the coarse powder at the room temperature of 100rpm for 3 hours at the liquid-liquid ratio of 1:10 (the polygonum multiflorum accounts for 0.1), filtering, removing supernatant, collecting precipitate, washing the precipitate twice, and drying the precipitate under reduced pressure at the temperature of 60 ℃.
2.1.2 preparation of Polygonum Multiflorum resistant starch
Preparing 15% starch solution with water as solvent, adjusting pH to 4.5 with hydrochloric acid, stirring in boiling water bath for 30min for pre-gelatinization, cooling to 60 deg.C after 15min at 121 deg.C under high pressure, adding pullulanase 4NPUN/g for enzymolysis for 6h, inactivating enzyme in boiling water bath for 20min after enzymolysis, slowly cooling to room temperature, refrigerating in refrigerator for 24h, and freeze drying.
2.2 determination of enzymolysis pH, enzyme addition amount, temperature and enzymolysis time in the extraction method of chick embryo micromolecule polypeptide
2.2.1 type of chick embryo enzymolysis, pH of enzymolysis, enzyme addition, temperature and enzymolysis time
According to the result of preliminary test, the enzymolysis time is optimal for 3 hours, the chicken embryo is dried under reduced pressure at 60 ℃, crushed into fine powder, added with water and stirred uniformly, and the ratio of material to liquid is 1:10, adding papain, bacillus subtilis neutral protease and alkaline protease respectively and sequentially for enzymolysis, and taking 1ml to calculate the content of the micromolecular polypeptide by adopting a BCA method. So as to obtain the optimal addition of the papain, the subtilisin and the alkaline protease under different temperatures, pH values and enzyme addition amounts.
TABLE 1 Effect of papain on the extraction of small-molecule polypeptides from chick embryos at different temperatures, pH values, and enzyme dosages
TABLE 2 influence of subtilisin neutral protease at different temperatures, pH and enzyme dosages on the extraction of small-molecule polypeptides from chick embryos
TABLE 3 influence of alkaline protease on extraction of small polypeptides from chicken embryos at different temperatures, pH values and enzyme dosages
Finally, determining the pH value of the papain to be 5, the enzyme adding amount to be 5 percent and the temperature to be 53 ℃; the pH value of the bacillus subtilis neutral protease is 6, the enzyme adding amount is 4 percent, and the temperature is 52 ℃; the pH of the alkaline protease was 8.2, the amount of enzyme added was 5%, and the temperature was 49 ℃.
3 comparative experiment of results
3.1 Effect of extraction method on extraction yield and purity
3.1.1 Experimental methods
The chick embryo micromolecule polypeptide obtained by the method is subjected to content determination by adopting a pre-column derivative reagent method. Taking 0.2g of chick embryo micromolecule polypeptide obtained in the patent, adding 5ml of water for dissolving, adding 6mol/L hydrochloric acid, drying for 6 hours at 105 ℃, volatilizing, adding 2ml of 0.1mol/L hydrochloric acid for dissolving, sucking 800 mu L of solution, adding 400 mu L of each of 1mol/L triethylamine acetonitrile solution and 0.1mol/L PITC solution, reacting for 1 hour in a dark place, extracting for 2 times by using n-hexane, removing the n-hexane layer each time by using 3ml, and obtaining a sample solution. The detection wavelength is as follows: 214nm, the sample size is: 10 μ l, chromatography conditions are shown in Table 1: and (3) determining the amino acid of the small molecular polypeptide in the chick embryo. The same method is used for determination of the crushing, dipping and extracting method and the enzymolysis, dipping and extracting method.
TABLE 4 chromatographic conditions for small molecule polypeptides
3.1.2 results of the experiment
On the basis of the traditional method, the invention optimizes the optimal enzymolysis condition for extracting the micromolecule polypeptide in the chick embryo, and then utilizes the V spiral cavity with larger internal volume which is peculiar to the polygonum multiflorum resistant starch to perform inclusion, thereby obtaining the micromolecule polypeptide with higher purity, and the yield is obviously higher than that of the micromolecule polypeptide prepared by an enzymolysis method.
And (3) performing content determination on the obtained chick embryo small molecular polypeptide by adopting a pre-column derivative reagent method. And (3) determining the amino acid of the small molecular polypeptide in the chick embryo by adopting a pre-column derivatization method. By comparing the liquid phase spectra of the three extracting solutions of the crushing and dipping extraction method, the enzymolysis and dipping extraction method and the crushing-enzymolysis-resistant starch inclusion ultrasonic method (the invention) and carrying out content measurement, the method related by the invention is proved to have obviously improved extraction yield and purity compared with the traditional method.
The results of the comparison are shown in FIGS. 1, 2, 3 and Table 5 below:
TABLE 5 Experimental results of different extraction methods
3.2 Artificial biofilm Permeability
The chick embryo has the effects of warming the middle-jiao and replenishing qi, replenishing essence and filling marrow, is used for body weakness and emaciation, fracture, muscle injury and pain and the like, mainly contains protein, and adopts a single-chamber Franz diffusion cell method to perform an in-vitro transdermal release test for researching the transdermal condition of the protein in the chick embryo. A certain amount of micromolecule polypeptide extracted by the method is coated on a Strat-M membrane, and 2ml of micromolecule polypeptide is taken after being subjected to transdermal for 24 hours to measure the transdermal accumulated permeation amount of the micromolecule polypeptide by a BCA method. The small molecular polypeptide extracted by the method can reach 403.41 +/-9.58 mu g/cm2 after 24 hours, and the transdermal accumulated permeation quantity is the largest within 24 hours compared with the small molecular polypeptide prepared by a crushing impregnation extraction method, an enzymolysis impregnation extraction method and a crushing-enzymolysis-resistant starch inclusion ultrasonic method (the invention), which shows that the small molecular polypeptide prepared by the method easily permeates skin barriers. The results are given in Table 6 below.
TABLE 6 measurement results of transdermal efficiency of extracts by different extraction methods
3. Determination of molecular weight
In order to further research the chick embryo small molecule polypeptide, the molecular weight distribution of the chick embryo small molecule polypeptide is determined. An appropriate amount of the chick embryo small molecule polypeptide extracted by the method is fully and uniformly mixed on a vortex mixer and centrifuged, a multifunctional gradient PCR instrument denatures for 5 minutes at 95 ℃, 4.5% concentrated gel and 12.5% separation gel are used for carrying out SDS-PAGE electrophoresis, the molecular weight of the chick embryo polypeptide obtained by the method is 11-67 kDa, and the test result is shown in figure 4 (in the figure, 1 is marker; and 2, 3, 4, 5 and 6 are chick embryo small molecule polypeptide SDS-PAGE gel electrophoresis.).
4. Pharmacodynamic test of chick embryo polypeptide
4.1 Effect of chick embryo Polypeptides on xylene-induced ear swelling in mice
4.1.1 Experimental methods
Taking 60 SPF mice, randomly dividing the mice into a chick embryo polypeptide high dose group, a chick embryo polypeptide medium dose group, a chick embryo polypeptide low dose group, a hibirine group, a model group and a blank group, wherein each group comprises 10 mice. (distilled water solvent is adopted for the chick embryo). The administration was performed daily for 2 days, and after 1 hour from the last administration, 40 μ l of xylene was uniformly applied to both surfaces of the right ear of each group of mice to cause inflammation, the left ear of the mice was not applied as a control, the cervical vertebrae were removed after 30min to be sacrificed, both ears were cut off, the ears were punched out at symmetrical portions of the left and right ears using a 6mm diameter punch, the mass of each mouse ear was weighed using a precision electronic balance, and the mouse ear swelling rate [ swelling rate ═ mass of right ear-mass of left ear)/mass of left ear × 100% ] was calculated. The ear application dose is obtained.
4.1.2 test results
The experimental results show that the model group has very significant difference (p <0.01) from the blank group, which indicates that the molding is successful. Compared with the swelling degree of a model group, the chicken embryo polypeptide low-dose group has significant difference (p is less than 0.05), the sitagliptin group has very significant difference (p is less than 0.01), and the swelling degree of other administration groups is lower than that of the model group, so that the chicken embryo polypeptide has certain anti-inflammatory effect, and the details are shown in table 7.
TABLE 7 Effect of chick embryo Polypeptides on xylene-induced ear swelling in mice
(n＝10)
P <0.05 compared to model group; p <0.01 in comparison with model groups
4.2 Effect of chick embryo Polypeptides on carrageenan-induced foot swelling in rats
4.2.1 Experimental methods
The mice were randomly divided into 6 groups of 20 mice each. The test method comprises the following steps of randomly dividing the test result into a blank group, a chick embryo polypeptide high dose group, a chick embryo polypeptide medium dose group, a chick embryo polypeptide low dose group, a sitalin group and a model group, adopting 150 mu l of carrageenan with the content of 1% injected subcutaneously into the right paw of a mouse to cause inflammation, adopting the chick embryo polypeptide high, medium and low dose groups (the administration dose of the chick embryo polypeptide is the same as that of 2.4.3.1) and the chick embryo polypeptide group to be administered after the chick embryo polypeptide is caused to cause inflammation for 0.5h, 1h and 2h, adopting a volume measuring instrument to measure the volume (ml) of the claw causing inflammation, adopting a micrometer to measure the thickness (mm) of the claw causing inflammation, and then measuring once every 1h, and measuring 6 times in total. The inhibition rate of each medicine group on swelling is obtained by calculating the swelling degree of the difference value of the volume and the thickness of the paw before and after inflammation. The formula for calculating the inhibition rate is as follows:
inhibition (%). ratio-negative control value-drug administration value/negative control value X100%
4.2.2 results of the experiment
As can be seen from Table 8, compared with the blank group, the swelling degree of foot of the model group is obviously increased, and the difference is very significant (P <0.01), which indicates that the molding is successful. Compared with the model group, the high, medium and low dose groups of the chick embryo polypeptide and the sitagliptin group have obvious trend of decreasing the foot swelling degree when the chick embryo polypeptide causes inflammation for 0.5h, 1h and 2h, and have significant difference, which shows that the chick embryo polypeptide has the function of inhibiting the swelling of the feet of the rat caused by carrageenan.
TABLE 8 Effect of chick embryo Polypeptides on the swelling of the foot of rats at different times: (
n＝10)
Compared with blank group, # P < 0.01; p <0.01, P <0.05 compared to model group
5 protective action of chick embryo polypeptide on rat osteoarthritis induced by papain
5.1 Experimental methods
SPF-grade 40 mice were randomly divided into 6 groups, 10 mice each of blank control group, model group, chick embryo polypeptide group, and positive control group. Model group (10% papain +0.03mol/L L-cysteine), chick embryo polypeptide group 5.5 g/kg. Mixing 10% of papain with 0.03mol/L of L-cysteine according to the ratio of 1:1, and standing for 30min for later use. On the 0, 4 and 8 days of the experiment, 0.3mL of mixed solution is injected into the knee joint cavity of the rats in the model group and the chick embryo polypeptide group administration group to induce the osteoarthritis model, and the same volume of normal saline is injected into the blank control group. After the papain mixed solution is injected for the last time, the chick embryo polypeptide test medicine is respectively given, and the model group and the normal control group are given with physiological saline with the same volume once a day for 2 weeks. The rats were anesthetized with 7% chloral hydrate for the last time, and tissues were stained with hematoxylin-eosin (HE) for qualitative analysis of cartilage.
5.2 qualitative analysis of cartilage pathological results:
blank control group: the joint surface was covered with a thin layer of hyaline cartilage, the joint capsule cavity was smooth, no obvious tissue proliferation, inflammatory cell infiltration, and no other obvious pathological changes were seen (fig. 5A).
Model group: hyaline cartilage was destroyed on the joint surface and fibrous tissue was proliferated to fibrosis in a large part of the joint, with massive inflammatory cell infiltration (fig. 5B).
Chick embryo polypeptidyl: the joint surface was covered with a thin layer of hyaline cartilage with no apparent tissue proliferation and occasional inflammatory cell infiltration (fig. 5C).
Positive control group: the hyaline cartilage on the joint surface occasionally showed fibrous tissue hyperplasia, and a small amount of inflammatory cell infiltration was observed, as shown in fig. 5(a. blank control group b. model group c. chick embryo polypeptide group d. positive control group).
5.3 cartilage histopathological grading Mankin's score results
The results and scoring criteria for the cartilage histopathology grading Mankin's are shown in tables 9 and 10;
table 9: cartilage histopathological grading Mankin's scoring results
| Group and number | Structure of the product | Cells | Substrate dyeing | Integrity of tide line |
| Blank control group-1 | 0 | 0 | 0 | 0 |
| Blank control group-2 | 0 | 0 | 0 | 0 |
| Blank control group-3 | 1 | 0 | 0 | 0 |
| Model set-2 | 4 | 2 | 3 | 1 |
| Model group-3 | 1 | 1 | 1 | 1 |
| Model group-4 | 5 | 2 | 2 | 1 |
| High dose group-1 | 0 | 0 | 1 | 0 |
| High dose group-2 | 0 | 0 | 0 | 0 |
| High dose group-3 | 1 | 0 | 0 | 0 |
| Medium dose group-1 | 1 | 1 | 0 | 0 |
| Medium dose group-2 | 1 | 1 | 0 | 1 |
| Medium dose group-3 | 1 | 1 | 1 | 1 |
| Low dose group-1 | 2 | 1 | 1 | 0 |
| Low dose group-2 | 2 | 1 | 1 | 1 |
| Low dose group-3 | 3 | 2 | 2 | 1 |
| Positive control group-1 | 3 | 1 | 2 | 1 |
| Positive control group-2 | 4 | 1 | 2 | 1 |
| Positive control group-3 | 3 | 1 | 2 | 1 |
Table 10; mankin's scoring criteria
Claims (10)
1. A method for extracting small molecular polypeptides in chick embryos is characterized by comprising the following steps: the extraction method of the micromolecular polypeptide in the chicken embryo is a method for preparing the micromolecular polypeptide in the chicken embryo by spontaneously forming a V-shaped cavity structure with larger internal volume by resistant starch, clathrating the chicken embryo protein subjected to enzymolysis with resistant starch solution, and centrifuging an ultrafiltration tube after ultrasonic treatment.
2. The method for extracting small molecular polypeptide from chick embryo according to claim 1, wherein: the extraction method comprises the following steps:
a. dissolving Polygoni Multiflori radix resistant starch in 2mol/L KOH aqueous solution, adding HCl at room temperature to adjust pH to 6.5, adding water to make the resistant starch concentration 1mg/mL to obtain resistant starch solution, i.e. product A;
b. oven drying chicken embryo under reduced pressure, pulverizing into fine powder, adding ethanol, adding papain, and performing enzymolysis to obtain product B;
c. adding neutral protease into the product B, and performing enzymolysis to obtain product C;
d. adding alkaline protease into product C, performing enzymolysis, centrifuging, and collecting supernatant to obtain micromolecular polypeptide crude extract, i.e. product D;
e. stirring the product D, dropwise adding the product D into the product A in a volume ratio of 4: 1-6: 1, and stirring for inclusion to obtain a product E;
f. and (3) centrifuging the product E at 5000rpm for 10 minutes, removing supernatant, adding a proper amount of water into the precipitate for dispersion, performing ultrasonic treatment at 25kHz for 30 minutes, putting the solution after ultrasonic crushing into an 80kDa ultrafiltration centrifugal tube, performing centrifugation at 12000rpm for 10 minutes, and obtaining filtrate which is the chick embryo small molecule polypeptide concentrate.
3. The method for extracting small molecular polypeptide from chick embryo according to claim 2, wherein: and in the step B, drying the chicken embryo at 60-70 ℃ under reduced pressure, crushing the chicken embryo into fine powder, taking water as a solvent, mixing the chicken embryo and the water at a ratio of 1: 9-10, uniformly stirring, adding papain, controlling the pH value to be 3-6, controlling the enzyme addition amount to be 3-6%, controlling the temperature to be 40-60 ℃, and carrying out enzymolysis for 1.0-2.0 hours to obtain a product B.
4. The method for extracting small molecular polypeptide from chick embryo according to claim 3, wherein: and in the step B, drying the chicken embryo at 60-70 ℃ under reduced pressure, crushing the chicken embryo into fine powder, adding 75% ethanol, uniformly stirring the mixture with the ratio of the chicken embryo to the ethanol being 1: 5-10, adding papain with the pH value of 5, the enzyme addition amount of 5%, the temperature of 53 ℃ and the enzymolysis time of 1.0-2.0 hours to obtain a product B.
5. The method for extracting small molecular polypeptide from chick embryo according to claim 2, wherein: and in the step C, adding neutral protease into the product B, wherein the pH value of the neutral protease is 5-7, the enzyme adding amount is 3% -4%, the temperature is 45-55 ℃, and the enzymolysis time is 1.0-1.5 hours, so as to obtain a product C.
6. The method for extracting small molecular polypeptide from chick embryo according to claim 5, wherein: and in the step C, adding neutral protease with the pH of 6 and the enzyme addition of 4% into the product B, keeping the temperature at 52 ℃, and carrying out enzymolysis for 1.0-1.5 hours to obtain a product C.
7. The method for extracting small molecular polypeptide from chick embryo according to claim 2, wherein: and in the step D, adding the product C into alkaline protease with the pH of 7.8-8.2 and the enzyme addition of 5-6% at the temperature of 49-53 ℃, centrifuging for 5 minutes at 1000rpm after enzymolysis, and taking supernatant to obtain a micromolecular polypeptide crude extract, namely a product D.
8. The method for extracting small molecular polypeptide from chicken embryo as claimed in claim 7, wherein the method comprises the following steps: and in the step D, adding the product C into alkaline protease pH8.2, adding 5% of enzyme, keeping the temperature at 49 ℃, centrifuging for 5 minutes at 1000rpm after enzymolysis, and taking supernatant to obtain a micromolecule polypeptide crude extract, namely a product D.
9. The method for extracting small molecular polypeptide from chick embryo according to claim 2, wherein: and in the step E, taking the product D, stirring at 60-70 ℃ and 200-300 rpm, dropwise adding the product D into the product A, stirring for 3-6 hours to perform inclusion to obtain a product E, wherein the volume ratio of the product A to the product B is 4: 1-6: 1.
10.The method for extracting small molecule polypeptide from chick embryo according to claim 9, wherein: in the step e, the step (c), taking the product D, stirring at 60 ℃ and 200-300 rpm, dropwise adding the product D into the product A, wherein the volume ratio of the product A to the product B is 6:1, and stirring Stirring for 3 hr for inclusion to obtain product E.
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