Polypeptide extract, preparation method and application

Abstract

The invention provides a polypeptide extract, a preparation method and application, and belongs to the technical field of polypeptide extraction. In the method, the human amniotic membrane tissue is pretreated by adopting a low-temperature plasma technology, so that the problem of protein denaturation caused by a traditional freeze-thawing method is avoided, the enzymolysis efficiency is improved by about 30%, and the extraction rate of the polypeptide extract is further improved. Besides, through a two-stage enzymolysis mode, a human amnion collagenous fiber network can be dissociated in sequence, and human amnion epithelial intercellular connexin can be cut in a targeted manner, so that fibronectin and active peptide fragments are released, further, active peptides in human amnion tissues are prevented from being excessively degraded, and the extraction rate of the polypeptide extract is increased. The polypeptide extract extracted by the invention can be used for skin tissue repair.

Description

Technical Field

[0001] The present invention belongs to the technical field of polypeptide extraction, and relates to a polypeptide extract, a preparation method and an application. Background Art

[0002] Placental tissue is rich in mesenchymal stem cells and tissue repair stem cells, which possess potent proliferation and differentiation potential and are potentially useful for therapeutic research on a wide range of diseases. Placental tissue also contains a variety of growth factors, cytokines, and extracellular matrix components that promote tissue repair, have anti-inflammatory, and immunomodulatory effects. Its derivatives show promising applications in anti-aging and drug delivery systems. Current research is exploring the potential of placental tissue in various medical fields, including ophthalmology, plastic surgery, and wound repair. With the advancement of biotechnology, the potential value of placental tissue will continue to be explored.

[0003] Human amniotic membrane (hAM), the inner layer of the placenta, possesses low immunogenicity, multiple active components, and potent anti-inflammatory and anti-scarring properties, making it of great research value and potential for clinical application. With the advancement of 3D bioprinting and organoid technologies, hAM is poised to become an ideal scaffold material for personalized regenerative therapies.

[0004] Enzymatic hydrolysis of human amniotic membrane tissue can yield a variety of active substances, such as active peptides that promote red blood cell migration and elastin peptides that enhance skin elasticity. Furthermore, EGF within human amniotic membrane tissue can promote epithelial cell growth, bFGF can stimulate fibroblast growth, and HGF can promote angiogenesis and tissue regeneration. However, currently, the extraction rate of peptides from human amniotic membrane tissue is low. Summary of the Invention

[0005] In order to solve the above technical problems, the present invention provides a polypeptide extract, a preparation method and an application.

[0006] To achieve the above object, the present invention adopts the following technical solutions: The present application provides a polypeptide extract, and the preparation method of the polypeptide extract comprises: S01: Collagenase IV and hyaluronidase are added to the washed and minced human amniotic membrane tissue, and enzymatic hydrolysis is performed at 4° C. and pH 7.4 to obtain enzymatic hydrolysis solution a.

[0007] In a low-temperature plasma treatment instrument, human amniotic membrane tissue was cleaned with 4°C saline and cut into 1-2 mm 2The tissue blocks are then processed to destroy the cell membrane structure of the human amniotic tissue while preserving the ECM (extracellular matrix) components of the human amniotic tissue, thereby enhancing the efficiency of subsequent enzymatic hydrolysis and increasing the extraction rate of the peptide extract. In this application, the gas atmosphere of the low-temperature plasma treatment instrument is argon, the power is 50W, and the treatment time is 3 minutes.

[0008] Based on the volume and mass of human amniotic membrane tissue, 0.1%-0.2% w / v collagenase IV and 100-120 U / g hyaluronidase are added to the minced human amniotic membrane tissue blocks. Enzymatic hydrolysis is performed at 4°C and pH 7.4 for 2 hours to gently dissociate the human amniotic membrane collagen fiber network and release fibronectin, thereby obtaining enzymatic hydrolysis solution A. Preferably, the amounts of collagenase IV and hyaluronidase added are 0.1% w / v and 100 U / g, respectively.

[0009] S02: adding trypsin and bromelain to the enzymatic hydrolyzate a, and performing enzymatic hydrolysis at 37° C. and pH 8.0 to obtain an enzymatic hydrolyzate b.

[0010] Based on the volume and mass of human amniotic membrane tissue, 0.04%-0.08% w / v trypsin and 0.01%-0.03% w / v bromelain are added to enzymatic hydrolysis solution a. Enzymatic hydrolysis is carried out at 37°C and pH 8.0 for 4 hours to facilitate targeted cleavage of the human amniotic epithelial cell-intercellular junction proteins by trypsin and bromelain, releasing active peptides, thereby obtaining enzymatic hydrolysis solution b. Preferably, the amounts of trypsin and bromelain added are 0.05% w / v and 0.02% w / v, respectively.

[0011] S03: After the enzymatic hydrolysate b is concentrated, the temperature is cooled to 4° C., trehalose is added, the temperature is slowly raised to 25° C., and the mixture is centrifuged to obtain a micellar phase containing the polypeptide.

[0012] After concentrating the enzymatic hydrolysate b using a 10 kDa ultrafiltration membrane, the temperature is lowered to 4°C. Trehalose is added at 4%-8% w / v, based on the volume of the enzymatic hydrolysate b, and the temperature is then slowly raised to 25°C. During the slow heating process, the mixture of the enzymatic hydrolysate b and trehalose forms liquid crystalline micelles. Centrifugation at 8000-9000 g for 15-20 minutes yields a micellar phase containing the polypeptide. Preferably, the slow heating rate is 1°C / min.

[0013] S04: The micellar phase is dissolved in a citric acid buffer solution, loaded onto a cation exchange column, eluted using a pH gradient, and the eluate with a pH of 5.5-6.5 is collected.

[0014] The micellar phase was dissolved in a citric acid buffer having a pH of 4.0, loaded onto a cation exchange column, and eluted using a pH gradient elution method of washing with a pH 4.0 citric acid buffer for 5 min, washing with a pH 5.0 citric acid buffer for 5 min, washing with a pH 5.5 citric acid buffer for 5 min, washing with a pH 6.0 citric acid buffer for 5 min, and washing with a pH 6.5 citric acid buffer for 5 min, and collecting the eluate with a pH of 5.5-6.5.

[0015] S05: adding mannitol to the eluate, and freeze-drying to obtain a powdered polypeptide extract.

[0016] Based on the volume of the eluate, mannitol at a concentration of 2%-4% w / v was added to the eluate as a protective agent, and the eluate was pre-frozen at -80°C for 12 hours, and then freeze-dried at -50°C for 24 hours to obtain a powdered polypeptide extract.

[0017] In addition, the polypeptide extract prepared by the above preparation method of the present application is used for skin tissue repair.

[0018] The present invention has the following beneficial effects: (1) In this application, low-temperature plasma technology is used to pretreat human amniotic membrane tissue, which avoids the protein denaturation problem caused by the traditional freeze-thaw method, increases the enzymatic hydrolysis efficiency by about 30%, and thus improves the extraction rate of polypeptide extracts.

[0019] (2) In this application, a two-stage enzymatic hydrolysis method is used to successively dissociate the human amniotic collagen fiber network and target the cleavage of the junction protein between human amniotic epithelial cells to release fibronectin and active peptides, thereby avoiding excessive degradation of active peptides in human amniotic tissue and improving the extraction rate of polypeptide extracts. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 These are pictures of wound healing on the back of mice, where A is the model group on day 0, B is the treatment group on day 0, C is the model group on day 15, and D is the treatment group on day 15. DETAILED DESCRIPTION

[0021] The technical solution of the present invention is further explained and illustrated by means of specific embodiments below.

[0022] Example 1 The present invention provides a polypeptide extract, and the preparation method of the polypeptide extract includes: S101: Select healthy donated human amniotic tissue, place it in a low-temperature plasma treatment device at 4°C, argon atmosphere, and 50W power, and wash it three times with normal saline to remove blood stains. Then cut it into 1mm pieces. 2Based on the volume and weight of human amniotic membrane tissue, 0.1% w / v collagenase IV and 100 U / g hyaluronidase were added to the minced human amniotic membrane tissue blocks, and enzymatic hydrolysis was performed at 4°C and pH 7.4 for 2 h to obtain enzymatic hydrolysis solution a.

[0023] S102: Based on the volume and mass of human amniotic membrane tissue, 0.05% w / v trypsin and 0.02% w / v bromelain were added to the enzymatic hydrolyzate a, and enzymatic hydrolysis was carried out at 37°C and pH 8.0 for 4 h to obtain enzymatic hydrolyzate b.

[0024] S103: After concentrating the enzymatic hydrolysate b using a 10 kDa ultrafiltration membrane, the solution was cooled to 4°C. Based on the volume of the enzymatic hydrolysate b, 5% w / v trehalose was added to the solution. The solution was then slowly heated to 25°C at a rate of 1°C / min. The solution formed liquid crystalline micelles. The solution was centrifuged at 8000 g for 15 minutes to obtain a micellar phase containing the polypeptide.

[0025] S104: The micellar phase was dissolved in a citric acid buffer having a pH of 4.0, and loaded onto a cation exchange column. The column was eluted using a pH gradient elution method of washing with a pH 4.0 citric acid buffer for 5 min, washing with a pH 5.0 citric acid buffer for 5 min, washing with a pH 5.5 citric acid buffer for 5 min, washing with a pH 6.0 citric acid buffer for 5 min, and washing with a pH 6.5 citric acid buffer for 5 min, and the eluate with a pH of 5.5-6.5 was collected.

[0026] S105: Based on the volume of the eluate, add mannitol at a concentration of 3% w / v to the eluate, pre-freeze at -80°C for 12 hours, and then freeze-dry at -50°C for 24 hours to obtain a powdered polypeptide extract.

[0027] Example 2 The present invention provides a polypeptide extract, and the preparation method of the polypeptide extract includes: S201: Select healthy donated human amniotic tissue, place it in a low-temperature plasma treatment device at 4°C, argon atmosphere, and 50W power, and wash it three times with normal saline to remove blood stains. Then cut it into 2mm pieces. 2 Based on the volume and weight of human amniotic membrane tissue, 0.2% w / v collagenase IV and 120 U / g hyaluronidase were added to the minced human amniotic membrane tissue block, and enzymatic hydrolysis was performed at 4°C and pH 7.4 for 2 h to obtain enzymatic hydrolysis solution a.

[0028] S202: Based on the volume and mass of human amniotic membrane tissue, 0.04% w / v trypsin and 0.01% w / v bromelain were added to the enzymatic hydrolyzate a, and enzymatic hydrolysis was carried out at 37° C. and pH 8.0 for 4 h to obtain enzymatic hydrolyzate b.

[0029] S203: Enzyme hydrolysate b is concentrated using a 10 kDa ultrafiltration membrane and cooled to 4°C. 4% w / v trehalose is added to the hydrolysate b, based on its volume. The solution is then slowly heated to 25°C at a rate of 1°C / min. The solution forms liquid crystalline micelles. Centrifuge at 9000 g for 20 minutes to obtain a micellar phase containing the polypeptide.

[0030] S204: The micellar phase was dissolved in a citric acid buffer having a pH of 4.0, and loaded onto a cation exchange column. The column was eluted using a pH gradient elution method of washing with a pH 4.0 citric acid buffer for 5 min, washing with a pH 5.0 citric acid buffer for 5 min, washing with a pH 5.5 citric acid buffer for 5 min, washing with a pH 6.0 citric acid buffer for 5 min, and washing with a pH 6.5 citric acid buffer for 5 min, and the eluate with a pH of 5.5-6.5 was collected.

[0031] S205: Based on the volume of the eluate, add mannitol at a concentration of 2% w / v to the eluate, pre-freeze at -80°C for 12 hours, and then freeze-dry at -50°C for 24 hours to obtain a powdered polypeptide extract.

[0032] Example 3 The present invention provides a polypeptide extract, and the preparation method of the polypeptide extract includes: S301: Select healthy donated human amniotic tissue, place it in a low-temperature plasma treatment device at 4°C, argon atmosphere, and 50W power, and wash it three times with normal saline to remove blood stains. Then cut it into 1.5mm pieces. 2 Based on the volume and weight of human amniotic membrane tissue, 0.15% w / v collagenase IV and 110 U / g hyaluronidase were added to the minced human amniotic membrane tissue blocks, and enzymatic hydrolysis was performed at 4°C and pH 7.4 for 2 h to obtain enzymatic hydrolysis solution a.

[0033] S302: Based on the volume and mass of human amniotic membrane tissue, 0.08% w / v trypsin and 0.03% w / v bromelain were added to the enzymatic hydrolyzate a, and enzymatic hydrolysis was carried out at 37°C and pH 8.0 for 4 hours to obtain enzymatic hydrolyzate b.

[0034] S303: Enzyme hydrolysate b is concentrated using a 10 kDa ultrafiltration membrane and cooled to 4°C. 8% w / v trehalose, based on the volume of enzymatic hydrolysate b, is added to the solution. The solution is then slowly heated to 25°C at a rate of 1°C / min. The solution forms liquid crystalline micelles. Centrifuge at 8000 g for 20 minutes to obtain a micellar phase containing the polypeptide.

[0035] S304: The micellar phase was dissolved in a citric acid buffer having a pH of 4.0, and loaded onto a cation exchange column. The column was eluted using a pH gradient elution method of washing with a pH 4.0 citric acid buffer for 5 min, washing with a pH 5.0 citric acid buffer for 5 min, washing with a pH 5.5 citric acid buffer for 5 min, washing with a pH 6.0 citric acid buffer for 5 min, and washing with a pH 6.5 citric acid buffer for 5 min, and the eluate with a pH of 5.5-6.5 was collected.

[0036] S305: Based on the volume of the eluate, add mannitol at a concentration of 4% w / v to the eluate, pre-freeze at -80°C for 12 hours, and then freeze-dry at -50°C for 24 hours to obtain a powdered polypeptide extract.

[0037] Example 4 The present invention provides a polypeptide extract, and the preparation method of the polypeptide extract includes: S401: Select healthy donated human amniotic tissue, place it in a low-temperature plasma treatment device at 4°C, argon atmosphere, and 50W power, and wash it three times with normal saline to remove blood stains. Then cut it into 2mm pieces. 2 Based on the volume and weight of human amniotic membrane tissue, 0.2% w / v collagenase IV and 110 U / g hyaluronidase were added to the minced human amniotic membrane tissue block, and enzymatic hydrolysis was performed at 4°C and pH 7.4 for 2 h to obtain enzymatic hydrolysis solution a.

[0038] S402: Based on the volume and mass of human amniotic membrane tissue, 0.06% w / v trypsin and 0.02% w / v bromelain were added to the enzymatic hydrolyzate a, and enzymatic hydrolysis was carried out at 37°C and pH 8.0 for 4 hours to obtain enzymatic hydrolyzate b.

[0039] S403: Enzyme hydrolysate b is concentrated using a 10 kDa ultrafiltration membrane and cooled to 4°C. 6% w / v trehalose is added to the solution, based on the volume of enzymatic hydrolysate b. The solution is then slowly heated to 25°C at a rate of 1°C / min. The solution forms liquid crystalline micelles. Centrifuge at 9000 g for 15 minutes to obtain a micellar phase containing the polypeptide.

[0040] S404: The micellar phase was dissolved in a citric acid buffer having a pH of 4.0, and loaded onto a cation exchange column. The column was eluted using a pH gradient elution method of washing with a pH 4.0 citric acid buffer for 5 min, washing with a pH 5.0 citric acid buffer for 5 min, washing with a pH 5.5 citric acid buffer for 5 min, washing with a pH 6.0 citric acid buffer for 5 min, and washing with a pH 6.5 citric acid buffer for 5 min, and the eluate with a pH of 5.5-6.5 was collected.

[0041] S405: Based on the volume of the eluate, add mannitol at a concentration of 2.5% w / v to the eluate, pre-freeze at -80°C for 12 hours, and then freeze-dry at -50°C for 24 hours to obtain a powdered polypeptide extract.

[0042] Comparative Example 1 Comparative Example 1 provides a polypeptide extract. The preparation method of the polypeptide extract is the same as that of Example 1, except that the human amniotic membrane tissue is directly cleaned with physiological saline and is not treated with low-temperature plasma technology.

[0043] The extraction rates in Example 1 and Comparative Example 1 were calculated based on the weight of the human amniotic tissue before treatment and the weight of the extracted polypeptide extracts, and Table 1 was obtained.

[0044] Table 1: Extraction rate of polypeptide extracts in Example 1 and Comparative Example 1 Note: Comparison between Example 1 and Comparative Example 1, * P <0.05; As can be seen from Table 1, compared with Comparative Example 1, the extraction rate of the amniotic membrane polypeptide product in Example 1 is 36.73% higher than that in Comparative Example 1, which indicates that the low-temperature plasma technology reduces the denaturation problem of the protein in the enzymatic hydrolysis of the amniotic membrane tissue and improves the extraction rate of the polypeptide.

[0045] In addition, in order to verify that the polypeptide extract prepared in the examples of the present application has the function of repairing skin tissue, 8-week-old SD rats were divided into 3 groups in the examples of the present application, namely, a control group, a model group and a treatment group, with 6 rats in each group. The rats in the model group and the treatment group were anesthetized with isoflurane, and the back hair was removed, and a circular full-thickness skin with a diameter of about 1 cm was excised on the back of the rats to form a skin wound. The rats in the control group were not treated. The rats in the treatment group were smeared with the polypeptide extract prepared in Example 1 every 3 days, with a dose of 5 mg / time / rat, and a total of 4 treatments. The wounds of the rats in the model group were not treated and healed naturally. 15 days after modeling, photos of the wounds of the rats in the model group and the treatment group were taken, and the attached data were obtained. Figure 1 After the experiment, the levels of serum inflammatory factors IL-6 and TNF-α in the rats of the control group, model group and treatment group were detected, and the results were shown in Table 2.

[0046] Table 2: Expression levels of inflammatory factors in rat serum (n=6, ±s) Note: Compared with the control group, the model group* P <0.05; compared with the model group, * P <0.05.

[0047] As can be seen from Table 2, compared with the control group, the levels of serum inflammatory factors IL-6 and TNF-α in the rats of the model group and the treatment group were significantly increased, and the content of serum inflammatory factors in the rats of the model group was the highest, which indicates that the polypeptide extract prepared in Example 1 can significantly improve the tissue repair ability of rats.

[0048] In addition, by the attached Figure 1 It can be seen that the back wounds of the rats in the treatment group were significantly smaller than those in the rats in the model group, further verifying that the polypeptide extract prepared in Example 1 can significantly improve the tissue repair ability of rats.

[0049] The above description is merely a preferred embodiment of the present application and is not intended to limit the present application. Various modifications and variations are possible for those skilled in the art. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present application shall be included within the scope of protection of the present application.

Claims

1. A method for preparing a polypeptide extract, characterized in that: include: Add collagenase IV and hyaluronidase to the washed and minced human amniotic membrane tissue, and perform enzymatic hydrolysis at 4°C and pH 7.4 to obtain enzymatic hydrolyzate solution a; adding trypsin and bromelain to the enzymatic hydrolyzate a, and performing enzymatic hydrolysis at 37° C. and pH 8.0 to obtain an enzymatic hydrolyzate b; The enzymatic hydrolysate b was concentrated and then cooled to 4°C, trehalose was added, the temperature was slowly raised to 25°C, and centrifuged to obtain a micellar phase containing polypeptides; The micellar phase is dissolved in a citric acid buffer, loaded onto a cation exchange column, eluted using a pH gradient, and the eluate with a pH of 5.5-6.5 is collected; Mannitol is added to the eluate, and the mixture is freeze-dried to obtain a powdered polypeptide extract.

2. The method for preparing the polypeptide extract according to claim 1, wherein In a low-temperature plasma treatment instrument, human amniotic membrane tissue was cleaned with 4°C saline and cut into 1-2 mm 2 .

3. The method for preparing the polypeptide extract according to claim 1, wherein: Based on the volume and mass of the human amniotic membrane tissue, the added amounts of the collagenase IV and the hyaluronidase are 0.1%-0.2% w / v and 100-120 U / g, respectively.

4. The method for preparing the polypeptide extract according to claim 1, wherein Based on the volume and mass of the human amniotic membrane tissue, the added amounts of the trypsin and the bromelain are 0.04%-0.08% w / v and 0.01%-0.03% w / v, respectively.

5. The method for preparing the polypeptide extract according to claim 1, wherein: The enzymatic hydrolysate b was concentrated using a 10 kDa ultrafiltration membrane.

6. The method for preparing the polypeptide extract according to claim 1, wherein: Based on the volume of the enzymatic hydrolysate b, the amount of trehalose added is 4%-8% w / v.

7. The method for preparing the polypeptide extract according to claim 1, wherein: The pH gradient elution includes: washing with a pH 4.0 citric acid buffer for 5 minutes, washing with a pH 5.0 citric acid buffer for 5 minutes, washing with a pH 5.5 citric acid buffer for 5 minutes, washing with a pH 6.0 citric acid buffer for 5 minutes, and washing with a pH 6.5 citric acid buffer for 5 minutes.

8. The method for preparing the polypeptide extract according to claim 1, wherein: The freeze-drying comprises: pre-freezing at -80°C for 12 hours and then freeze-drying at -50°C for 24 hours.

9. A polypeptide extract, characterized in that The method is prepared by any one of claims 1 to 8.

10. The polypeptide extract prepared by the preparation method according to any one of claims 1 to 8 is used for repairing skin tissue.

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