CN116676361A

CN116676361A - Method for preparing solution containing osteogenic growth peptide and C-terminal pentapeptide by enzymatic degradation of animal tissues

Info

Publication number: CN116676361A
Application number: CN202310632212.0A
Authority: CN
Inventors: 刘建利; 艾芸
Original assignee: Xian International Medical Center Co Ltd
Current assignee: Xian International Medical Center Co Ltd
Priority date: 2023-05-31
Filing date: 2023-05-31
Publication date: 2023-09-01

Abstract

A method for preparing solution containing osteogenic growth peptide and C-terminal pentapeptide by enzymatic degradation of animal tissue, which is a method for obtaining osteogenic growth peptide and C-terminal pentapeptide in meat by enzymolysis, wherein the meat is rich in protein, OGP is obtained by enzymatic degradation, so that a natural source is provided for OGP, and the osteogenic growth peptide can be developed into functional food or health food for more people.

Description

Method for preparing solution containing osteogenic growth peptide and C-terminal pentapeptide by enzymatic degradation of animal tissues

Technical Field

The invention belongs to the technical field of enzymolysis, and particularly relates to a method for preparing a solution containing osteogenic growth peptide and C-terminal pentapeptide by enzymatic degradation of animal tissues.

Background

Osteogenic growth peptide (osteogenic growth peptide, OGP) is a small molecule polypeptide substance with significant physiological activity. The propeptide consists of fourteen amino acids and has a primary structure of ALKRQGRTLYGFGG. Can promote the expression of cell type I collagen mRNA, osteocalcin (BGP) and alkaline phosphatase (ALpase), so as to increase the cell collagen synthesis, osteocalcin content and alkaline phosphatase activity. Can be clinically used for treating diseases related to bone metabolism unbalance, bone injury, bone marrow transplantation and the like, and can improve the hematopoietic activity of bones and bone marrow, and can be used as a potential application of hematopoietic agonists. The C-terminal pentapeptide OGP (10-14) of the osteogenic growth peptide is the minimum amino acid sequence for keeping the total activity of the propeptide, has obvious proliferation effect on osteoblasts (MC 3T3E 1) and fibroblasts (NIH 3T 3) and also shows obvious osteogenic and hematopoietic activities in animals, and is expected to replace the propeptide to become a new clinical candidate medicament. Although human and animal blood contains osteogenic growth peptides, the content is too small and chemical synthesis is already available in a scale.

Disclosure of Invention

The invention aims to provide a method for preparing a solution containing osteogenic growth peptide and C-terminal pentapeptide by enzymatic degradation of animal tissues, which is used for obtaining the osteogenic growth peptide and the C-terminal pentapeptide in meat by an enzymolysis mode.

In order to achieve the above purpose, the technical scheme adopted by the invention is as follows:

a method for preparing a solution containing osteogenic growth peptide and C-terminal pentapeptide by enzymatic degradation of animal tissue, comprising the steps of:

1. adding 1% NaHCO into proteinase ₃ The solution was prepared to contain 1. Mu.g of protease per 1. Mu.L;

2. placing the raw materials into a reaction container, adding a buffer solution with the concentration of 1% -3%, carrying out ultrasonic treatment for 20-40min, shaking up after the volume is fixed by using the buffer solution with the concentration of 1% -3%, filtering by a microporous filter membrane, placing the obtained filtrate into a microscale sample bottle, adding an enzyme solution, shaking up, carrying out enzymolysis at a constant temperature for 10-15 h, and obtaining a solution containing Osteogenic Growth Peptide (OGP) and carbon end pentapeptide OGP (10-14).

Such enzymes include, but are not limited to, pepsin, trypsin, papain, and other enzymes capable of degrading proteins.

The filter membrane is a microporous filter membrane with the thickness of 0.22 mu m.

The buffer solution is NH ₄ HCO ₃ ,NaHCO ₃ ,NH ₄ Cl,NaH ₂ PO ₄ /K ₂ HPO ₄ One or a mixture of any of the above.

The constant temperature is one of 20-80 ℃.

The solution containing the Osteogenic Growth Peptide (OGP) and the carbon end pentapeptide OGP (10-14) is applied to health-care food or functional food.

The solution containing the Osteogenic Growth Peptide (OGP) and the carbon end pentapeptide OGP (10-14) is applied to cosmetics.

The solution containing the Osteogenic Growth Peptide (OGP) and the carbon end pentapeptide OGP (10-14) is applied to medicines.

The raw materials are meat, animal skin, cornu Cervi Pantotrichum and colla Plastri Testudinis.

The beneficial effects of the invention are as follows:

the meat is rich in protein, and is subjected to enzymatic degradation to obtain OGP, so that a natural source is provided for OGP, and the OGP is obtained from food materials, so that the osteogenic growth peptide can be developed into functional foods or health-care foods for more people.

Drawings

FIG. 1 is an ion chromatogram of the osteogenic growth peptide OGP (10-14) standard and mutton MRM extract of example 1.

FIG. 2 is an ion chromatogram of osteogenic growth peptide OGP (10-14) standard and duck MRM extract of example 2.

FIG. 3 is an ion-extracting chromatogram of the osteogenic growth peptide OGP (10-14) standard and beef MRM in example 3.

FIG. 4 is an ion-extracting chromatogram of osteogenic growth peptide OGP (10-14) standard and beef MRM in example 3.

FIG. 5 is an ion chromatogram of the osteogenic growth peptide OGP (10-14) standard and deer horn gum MRM extract of example 4.

FIG. 6 is an ion chromatogram of the osteogenic growth peptide OGP (10-14) standard and deer horn gum MRM extract of example 4.

FIG. 7 is an ion chromatogram of the osteogenic growth peptide OGP (10-14) standard and tortoise plastron glue MRM extraction in example 5.

FIG. 8 is an ion chromatogram of the osteogenic growth peptide OGP (10-14) standard and tortoise plastron glue MRM extraction in example 5.

FIG. 9 is an ion chromatogram of the osteogenic growth peptide OGP (10-14) standard and donkey-hide gelatin MRM extract of example 6.

FIG. 10 is an ion chromatogram of the osteogenic growth peptide OGP (10-14) standard and donkey-hide gelatin MRM extract of example 6.

FIG. 11 is an ion-extracting chromatogram of the osteogenic growth peptide OGP (10-14) standard and pigskin MRM in example 7.

Detailed Description

The present invention will be further described with reference to the following examples, but the present invention is not limited to the following examples.

Detection of osteogenic growth peptide:

1. experimental instrument:

island UFLC Nexera X2 series ultra-high performance liquid chromatograph (equipped with DGU-20A5R degasser, LC-30AD liquid pump, CBM-20A system controller, SIL-30AC autosampler, CTO-20A column incubator), 8060 triple four-bar tandem mass spectrometer (Shimadzu Corp., japan); island Labsolution Ver.5.93 workstation;

chromatographic column: syncronis HILIC (100 mm. Times.2.1 mm,1.7 μm, thermo Inc. of America)

Milli-Q ultra-pure water machine (Millipore Co., U.S.A.); BT25S electronic balance (precision 0.01mg, sartorius company, germany); KQ-700VDB ultrasonic cleaner (Kunshan ultrasonic instruments Co., ltd.); BI-250A incubator (Shanghai Shi Doukai instruments and equipment Co., ltd.).

2. Experimental materials:

2.1 Standard and control herbs:

the osteogenic growth peptide OGP (10-14) was offered by Shaanxi Huikang Biotechnology Limited liability company.

2.2, reagent:

4-bromophenyl isothiocyanate (purity > 97%, lot C202706, shanghai alading Co., ltd.), trypsin (SEQ ID NO: 0000344639, promega Co., U.S.A.); acetonitrile, methanol (chromatographic grade, merk, germany); formic acid (chromatographic grade, shanghai alading company), ammonium bicarbonate is domestic analytical pure; the water is ultrapure water.

2.3, preparation of standard substance solution:

accurately weighing 1.62mg of osteogenic growth peptide OGP (10-14), placing into a 10mL volumetric flask, adding acetonitrile, dissolving, fixing volume to scale, shaking, and preparing into standard stock solution (concentration of 0.162 mg/mL) of osteogenic growth peptide OGP (10-14) in a refrigerator at 4deg.C.

2.4, preparation of derivatizing reagent:

99.20mg of 4-bromophenyl isothiocyanate is precisely weighed, placed in a 200mL volumetric flask, dissolved by adding acetonitrile, fixed to a scale, and shaken uniformly to obtain a derivative reagent solution (the concentration is 0.48 mg/mL).

Derivatization of series OGP (10-14) standard solutions:

precisely sucking 5mL of OGP (10-14) standard stock solution, placing in a 10mL measuring flask, and diluting with acetonitrile to scale to obtain intermediate stock solution (concentration of 0.081 mg/mL) of OGP (10-14) standard. A serial standard substance solution of OGP (10-14) is prepared by adopting a progressive dilution method, and the derivatization reaction of the OGP (10-14) and the 4-bromophenyl isothiocyanate is carried out according to the volume ratio of 1:1.

Precisely sucking 1mL of OGP (10-14) standard intermediate stock solution 1, placing in a 100mL measuring flask, adding acetonitrile to dilute to a scale, and taking the mixture as No. 7 standard solution (the concentration is 810 ng/mL); precisely sucking 5mL of No. 7 standard solution, placing the solution into a 10mL measuring flask, adding acetonitrile, and diluting to a scale to obtain No. 6 standard solution (with the concentration of 405 ng/mL); precisely sucking 5mL of No. 6 standard solution, placing the solution into a 10mL measuring flask, adding acetonitrile, and diluting to a scale to obtain No. 5 standard solution (the concentration is 202.5 ng/mL); precisely sucking 5mL of No. 5 standard solution, placing the solution into a 10mL measuring flask, adding acetonitrile, and diluting to a scale to obtain No. 4 standard solution (the concentration is 101.25 ng/mL); precisely sucking 4mL of the No. 4 standard solution, placing the solution into a 10mL measuring flask, adding acetonitrile, and diluting to a scale to obtain a No. 3 standard solution (the concentration is 40.5 ng/mL); precisely sucking 5mL of No. 3 standard solution, placing the solution into a 10mL measuring flask, adding acetonitrile, and diluting to a scale to obtain No. 2 standard solution (the concentration is 20.25 ng/mL); 5mL of the No. 2 standard solution was precisely aspirated and placed in a 10mL measuring flask, and acetonitrile was added to dilute to a scale to obtain a No. 1 standard solution (concentration: 10.125 ng/mL).

The derivatization reaction of the OGP (10-14) series standard substance solution and the 4-bromophenyl isothiocyanate is carried out according to the volume ratio of 1:1: precisely sucking 5mL of each of the 1-7 OGP (10-14) serial standard substance solutions, precisely adding 5mL of the derivatization reagent solution respectively, and shaking for 10min to obtain serial standard substance derivatization solutions. The concentrations were 5.06ng/mL, 10.13ng/mL, 20.25ng/mL, 50.63g/mL, 101.25ng/mL, 202.5ng/mL, 405ng/mL, respectively.

Example 1

Taking mutton as an example:

preparation of trypsin solution: adding 1% NH into sequence analysis grade trypsin ₄ HCO ₃ The solution was prepared to contain 1. Mu.g of solution per 1. Mu.L, which was prepared immediately before use.

10g of mutton is weighed, precisely weighed and placed in a 50mL measuring flask. Adding 1% NH ₄ HCO ₃ 40mL of solution, sonicated for 30min with 1% NH ₄ HCO ₃ The solution is fixed to the scale and shaken up. Filtering with 0.22 μm microporous membrane, collecting 100 μl of the filtrate, placing into microscale sample bottle, adding trypsin solution 10 μl, and shaking. And carrying out enzymolysis at a constant temperature of 37 ℃ for 12 hours to obtain a mutton sample solution.

Derivatization of test solutions:

30 mu L of mutton sample solution is added with 90 mu L of derivatization reagent solution and is shaken for 10min to obtain beef sample derivatization solution.

UHPLC-MS/MS conditions:

chromatographic conditions:

mobile phase a was 0.1% formic acid solution, mobile phase B was acetonitrile, isocratic eluted with 60% B; the flow rate was 0.3 ml.min ^-1 The method comprises the steps of carrying out a first treatment on the surface of the Column temperature: 30 ℃; sample injection amount: 2. Mu.L.

Mass spectrometry conditions:

ESI negative ion detection and multi-reaction monitoring mode (MRM) measurement are adopted, and main mass spectrum parameters are as follows: ion source temperature 300 ℃; desolventizing tube temperature: 250 ℃; heating block temperature: 400 ℃; atomized air flow rate was 3.0 L.min ^-1 The method comprises the steps of carrying out a first treatment on the surface of the Dry air flow: 10.0L min ^-1 The method comprises the steps of carrying out a first treatment on the surface of the Heating air flow: 10.0L min ^-1 The method comprises the steps of carrying out a first treatment on the surface of the Collision gas: argon gas.

Table 1 mass spectral parameters of two compounds

Note that is a quantitative ion pair

Preparation of derivative standard curve:

serial standard substance derivatization solutions with 7 different concentrations in derivatization of serial OGP (10-14) standard substance solutions are measured according to the method of the OGP (10-14) derivatization products in UHPLC-MS/MS conditions, each sample is subjected to parallel sample injection measurement for 2 times, and the peak area of the serial standard substance derivatization solutions is recorded. And (3) carrying out linear regression by taking the standard substance concentration as an abscissa and taking the average peak area measured for 2 times as an ordinate, and calculating a regression equation and a correlation coefficient of a standard curve.

Determination of the detection Limit (LOD) and the quantification Limit (LOQ):

continuously diluting and sampling the standard substance derivatization solution, wherein when the signal to noise ratio is more than or equal to 3 (S/N is not less than 3), the concentration of the corresponding OGP (10-14) is the lowest detection limit; when the signal-to-noise ratio is more than or equal to 10 (S/N is equal to or greater than 10), the concentration of the corresponding OGP (10-14) is the quantitative limit. Determination of 3.7.3 precision, stability, reproducibility and recovery:

measuring an OGP (10-14) derivative solution with the concentration of 5.06ng/mL by an injection liquid mass spectrometer, continuously and repeatedly sampling for 6 times, recording the corresponding peak area and retention time, and respectively calculating the relative standard deviation RSD (n=6) of the peak area and retention time, namely the daily sampling precision; the measurement was continuously performed for 3 days, sampling was repeated 6 times per day, the corresponding peak area and retention time were recorded, and the relative standard deviation RSD% (n=18) of the peak area and retention time was calculated, which is the daytime precision.

OGP (10-14) derivatization solutions at a concentration of 101.25ng/mL were injected into a UHPLC-MS/MS instrument for measurement at 0 hours, 2 hours, 4 hours, 8 hours, 12 hours, 16 hours, 20 hours, 24 hours, respectively, corresponding peak areas were recorded, and the relative standard deviation RSD of the peak areas was calculated to examine the stability of the derivatization products under the same analytical measurement conditions.

Taking the reference medicinal materials of the mutton sample solution after enzymolysis, preparing 6 parts of sample solutions after the derivatization of the mutton in parallel according to the derivatization method of the sample solutions, and respectively injecting each part of sample solution into a liquid chromatograph-mass spectrometer for measurement. Each sample solution was assayed 2 times in parallel, the corresponding peak area was recorded, the content of OGP (10-14) derivative was calculated, and the relative standard deviation RSD was calculated for evaluating the reproducibility of the samples after derivatization.

Taking 3 parts of the enzymatic hydrolysate of mutton, adding 20 mu L of OGP (10-14) standard substance solution with the concentration of 50.6ng/mL into each 20 mu L of the enzymatic hydrolysate of mutton, adding 40 mu L of a derivative reagent into each 20 mu L of the enzymatic hydrolysate of mutton, and shaking for 10min. All samples were injected into a UHPLC-MS/MS instrument for measurement, the corresponding peak areas were recorded, the content thereof was measured, and the recovery rate of the added samples and the relative standard deviation RSD% were calculated for each sample.

2 parts of sample solution is prepared in parallel by mutton, and 4-bromophenyl isothiocyanate is respectively added for derivatization. And (5) injecting each derived gum sample solution into a liquid chromatograph-mass spectrometer for measurement. Each sample solution was measured 2 times in parallel, the corresponding peak area was recorded, and the content of OGP (10-14) was calculated, and the MRM extract ion chromatograms of OGP (10-14) standards and samples were shown in fig. 1. The results of the sample measurements are shown in Table 2.

TABLE 2 OGP (10-14) content in mutton

As is clear from Table 2, OGP (10-14) was detected in mutton.

Example 2

Taking duck meat as an example, preparing a duck meat test solution and a derivatization test solution by the same method, detecting in the same way, taking an enzymolysis duck meat reference medicinal material, taking 3 parts of enzymolysis liquid of the enzymolysis duck meat, adding 20 mu L of OGP (10-14) standard solution with the concentration of 50.6ng/mL into each 20 mu L of enzymolysis liquid of the enzymolysis duck meat, adding 40 mu L of derivatization reagent into each 20 mu L of enzymolysis liquid, and shaking for 10min. All samples were injected into a UHPLC-MS/MS instrument for measurement, the corresponding peak areas were recorded, the content thereof was measured, and the recovery rate of the added samples and the relative standard deviation RSD% were calculated for each sample. 2 parts of sample solution is prepared in parallel from duck meat, and 4-bromophenyl isothiocyanate is respectively added for derivatization. And (5) injecting each derived gum sample solution into a liquid chromatograph-mass spectrometer for measurement. Each sample solution was assayed 2 times in parallel, the corresponding peak area was recorded, the content of OGP (10-14) was calculated, the MRM extracted ion chromatograms of OGP (10-14) standard and sample were shown in fig. 2, and the results of the sample assays are shown in table 3.

TABLE 3 OGP (10-14) content in Duck meat

As is clear from Table 3, OGP (10-14) was detected in duck meat.

Example 3

Taking beef as an example, preparing beef sample solution and derivatization sample solution by the same method, detecting in the same way, taking beef reference medicinal materials after enzymolysis, preparing 6 parts of beef sample solution after derivatization in parallel according to the derivatization method of the sample solution, and respectively injecting each part of sample solution into a liquid chromatograph-mass spectrometer for measurement. Each sample solution was assayed 2 times in parallel, the corresponding peak area was recorded, the content of OGP (10-14) derivative was calculated, and the relative standard deviation RSD was calculated for evaluating the reproducibility of the samples after derivatization.

Taking 3 parts of beef subjected to enzymolysis, adding 20 mu L of OGP (10-14) standard substance solution with the concentration of 50.6ng/mL into each 20 mu L, adding 40 mu L of derivative reagent into each mu L, and shaking for 10min. All samples were injected into a UHPLC-MS/MS instrument for measurement, the corresponding peak areas were recorded, the content thereof was measured, and the recovery rate of the added samples and the relative standard deviation RSD% were calculated for each sample.

2 parts of test solution are prepared in parallel by beef, and 4-bromophenyl isothiocyanate is respectively added for derivatization. And (5) injecting each derived beef sample solution into a liquid chromatograph-mass spectrometer for measurement. Each sample solution was measured 2 times in parallel, the corresponding peak area was recorded, and the content of OGP (10-14) was calculated, and the MRM extract ion chromatograms of OGP (10-14) standard and beef sample were shown in fig. 3 and 4. The results of the sample measurements are shown in Table 4.

TABLE 4 OGP (10-14) content in beef

Example 4

Taking deer horn glue as an example: and (3) preparing a deer-horn glue test solution and a derivatization test solution by the method, detecting in the same manner, taking an enzymolysis deer-horn glue reference medicinal material, preparing 6 parts of deer-horn glue derivatized test solution in parallel according to the derivatization method of the test solution, and respectively injecting each part of test solution into a liquid chromatograph-mass spectrometer for measurement. Each sample solution was assayed 2 times in parallel, the corresponding peak area was recorded, the content of OGP (10-14) derivative was calculated, and the relative standard deviation RSD was calculated for evaluating the reproducibility of the samples after derivatization.

Taking 3 parts of the deer-horn glue after enzymolysis, adding 20 mu L of OGP (10-14) standard substance solution with the concentration of 50.6ng/mL into each 20uL, adding 40 mu L of derivative reagent into each mu L, and shaking for 10min. All samples were injected into a UHPLC-MS/MS instrument for measurement, the corresponding peak areas were recorded, the content thereof was measured, and the recovery rate of the added samples and the relative standard deviation RSD% were calculated for each sample.

Preparing 2 parts of sample solution in parallel from deer antler glue, and respectively adding 4-bromophenyl isothiocyanate for derivatization. And (5) injecting each part of the derived deer horn gum sample solution into a liquid chromatograph-mass spectrometer for measurement. Each sample solution was measured 2 times in parallel, the corresponding peak area was recorded, and the content of OGP (10-14) was calculated, and the MRM extract ion chromatograms of OGP (10-14) standard and deer horn gum samples were shown in fig. 5 and 6. The results of the sample measurements are shown in Table 5.

TABLE 5 OGP (10-14) content in deer antler glue

As is clear from Table 5, OGP (10-14) was detected in deer horn glue.

Example 5

Taking tortoise plastron as an example: and preparing a tortoise plastron solution to be tested and a derivatization solution to be tested by the method, detecting in the same manner, taking a tortoise plastron reference medicinal material after enzymolysis, preparing 6 tortoise plastron solution to be tested after derivatization in parallel according to the derivatization method of the solution to be tested, and respectively injecting each solution to be tested into a liquid chromatograph to be tested. Each sample solution was assayed 2 times in parallel, the corresponding peak area was recorded, the content of OGP (10-14) derivative was calculated, and the relative standard deviation RSD was calculated for evaluating the reproducibility of the samples after derivatization.

Taking 3 parts of tortoise plastron after enzymolysis, adding 20 mu L of OGP (10-14) standard substance solution with the concentration of 50.6ng/mL into each 20 mu L, adding 40 mu L of derivative reagent into each mu L, and shaking for 10min. All samples were injected into a UHPLC-MS/MS instrument for measurement, the corresponding peak areas were recorded, the content thereof was measured, and the recovery rate of the added samples and the relative standard deviation RSD% were calculated for each sample.

2 parts of sample solution is prepared by parallel preparation of tortoise plastron, and 4-bromophenyl isothiocyanate is respectively added for derivatization. And (5) injecting each derived tortoise plastron sample solution into a liquid chromatograph-mass spectrometer for measurement. Each sample solution was measured 2 times in parallel, the corresponding peak area was recorded, the content of OGP (10-14) was calculated, and the MRM extracted ion chromatograms of OGP (10-14) standard and tortoise plastron samples were shown in fig. 7 and 8. The results of the sample measurements are shown in Table 6.

TABLE 6 OGP (10-14) content in tortoise plastron glue

As can be seen from Table 6, OGP (10-14) was detected in tortoise-plastron glue.

Example 6

Taking donkey-hide gelatin as an example, preparing an donkey-hide gelatin sample solution and a derivatization sample solution by the same method, detecting the donkey-hide gelatin sample solution in the same manner, performing derivatization on 6 parts of donkey-hide gelatin sample solution in parallel according to the derivatization method of the sample solution, and respectively injecting each part of donkey-hide gelatin sample solution into a liquid chromatograph-mass spectrometer for measurement. Each sample solution was assayed 2 times in parallel, the corresponding peak area was recorded, the content of OGP (10-14) derivative was calculated, and the relative standard deviation RSD was calculated for evaluating the reproducibility of the samples after derivatization.

Taking 3 parts of the donkey-hide gelatin control medicinal material after enzymolysis, adding 20 mu L of OGP (10-14) standard substance solution with the concentration of 50.6ng/mL into each 20 mu L, adding 40 mu L of derivative reagent into each mu L, and shaking for 10min. All samples were injected into a UHPLC-MS/MS instrument for measurement, the corresponding peak areas were recorded, the content thereof was measured, and the recovery rate of the added samples and the relative standard deviation RSD% were calculated for each sample. 2 parts of donkey-hide gelatin are prepared in parallel into test solution, and 4-bromophenyl isothiocyanate is respectively added for derivatization. And (5) injecting each derived gum sample solution into a liquid chromatograph-mass spectrometer for measurement. Each sample solution was measured 2 times in parallel, the corresponding peak area was recorded, the content of OGP (10-14) was calculated, the MRM extraction ion chromatograms of OGP (10-14) standard and donkey-hide gelatin samples were shown in fig. 9 and 10, and samples of yellow gelatin and new donkey-hide gelatin were prepared, and the measurement results of the samples were shown in table 7.

TABLE 7 OGP (10-14) content in donkey-hide gelatin

As is clear from Table 7, OGP (10-14) was detected in donkey-hide gelatin. OGP was not detected in yellow gelatin and new donkey-hide gelatin (10-14).

Example 7

Taking pigskin as an example, preparing an Ajiao test solution and a derivatization test solution by the same method, detecting in the same way, performing enzymolysis on the pigskin reference medicinal material, preparing 6 parts of pigskin derivatization test solutions in parallel according to the derivatization method of the test solution, and respectively injecting each part of test solution into a liquid chromatograph-mass spectrometer for measurement. Each sample solution was assayed 2 times in parallel, the corresponding peak area was recorded, the content of OGP (10-14) derivative was calculated, and the relative standard deviation RSD was calculated for evaluating the reproducibility of the samples after derivatization.

Taking 3 parts of pig skin control medicinal materials after enzymolysis, adding 20 mu L of OGP (10-14) standard substance solution with the concentration of 50.6ng/mL into each 20 mu L, adding 40 mu L of derivative reagent into each mu L, and shaking for 10min. All samples were injected into a UHPLC-MS/MS instrument for measurement, the corresponding peak areas were recorded, the content thereof was measured, and the recovery rate of the added samples and the relative standard deviation RSD% were calculated for each sample. 2 parts of sample solutions are prepared in parallel from pigskin, and 4-bromophenyl isothiocyanate is added for derivatization. And (5) injecting each derived gum sample solution into a liquid chromatograph-mass spectrometer for measurement. Each sample solution was measured 2 times in parallel, the corresponding peak area was recorded, the content of OGP (10-14) was calculated, the MRM extraction ion chromatograms of OGP (10-14) standard and pigskin samples were shown in fig. 11, and samples in yellow gelatin and new pigskin were prepared, and the measurement results of the samples were shown in table 8.

TABLE 8 OGP (10-14) content in pigskin and pork

As is clear from Table 8, OGP (10-14) was detected in pigskin, but not in pork.

Claims

1. A method for preparing a solution containing osteogenic growth peptide and C-terminal pentapeptide by enzymatic degradation of animal tissue, comprising the steps of:

2. The method of claim 1, wherein the enzyme comprises, but is not limited to, pepsin, trypsin, papain and other enzymes capable of degrading proteins.

3. The method for preparing a solution containing osteogenic growth peptide and C-terminal pentapeptide by enzymatic degradation of animal tissue according to claim 1, wherein the filter membrane is a microporous filter membrane of 0.22 μm.

4. The method for preparing a solution containing osteogenic growth peptide and C-terminal pentapeptide by enzymatic degradation of animal tissue according to claim 1, wherein the buffer solution is NH ₄ HCO ₃ ,NaHCO ₃ ,NH ₄ Cl,NaH ₂ PO ₄ /K ₂ HPO ₄ One or a mixture of any of the above.

5. The method of claim 1, wherein the constant temperature is one of 20-80 ℃.

6. The method for preparing the solution containing the osteogenic growth peptide and the C-terminal pentapeptide from the enzymatically degraded animal tissue according to claim 1, wherein the solution containing the Osteogenic Growth Peptide (OGP) and the C-terminal pentapeptide OGP (10-14) is applied to health foods or functional foods.

7. The method for preparing a solution containing osteogenic growth peptide and C-terminal pentapeptide according to claim 1, wherein the solution containing Osteogenic Growth Peptide (OGP) and C-terminal pentapeptide OGP (10-14) is applied in cosmetics.

8. The method for preparing a solution containing osteogenic growth peptide and C-terminal pentapeptide according to claim 1, wherein the solution containing Osteogenic Growth Peptide (OGP) and carbon-terminal pentapeptide OGP (10-14) is used in pharmaceutical products.

9. The method for preparing a solution containing osteogenic growth peptide and C-terminal pentapeptide by enzymatic degradation of animal tissue according to claim 1, wherein the raw material is meat, animal skin, cornu Cervi Pantotrichum, and colla Plastri Testudinis.