Method for detecting protein content in silicone patch containing collagen

Abstract

The invention discloses a method for detecting the protein content in a silicone patch containing collagen, which comprises the following steps: adding the prepared silicone patch containing collagen into dichloromethane, carrying out water bath reflux treatment, and cooling to room temperature after the treatment is completed; then adding purified water for extraction, taking a water layer for concentration to obtain a concentrated solution, and finally testing the protein content of the concentrated solution. According to the method provided by the invention, the physical barrier of a silicone matrix can be overcome, and efficient and complete release of collagen is realized; meanwhile, the biochemical characteristics are not damaged, so that a key and reliable technical basis is provided for research and development, quality control and registration and declaration of products.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of medical device detection, and particularly relates to a method for detecting the protein content of a silicone-based patch containing collagen. BACKGROUND

[0002] Silicone-based patches (such as scar patches) as a mature medical dressing have been widely used in the prevention and treatment of hypertrophic scars and keloids. Its mechanism of action is mainly based on the closing of hydration, the regulation of oxygen tension and the provision of moderate mechanical pressure, thereby softening, smoothing and improving the appearance of scar tissue. In recent years, in order to further enhance the active repair function and market competitiveness of the product, there has been a trend of combining silicone patches with biologically active ingredients (such as medical recombinant collagen) in the industry. In particular, human recombinant type III collagen is considered as an ideal additive ingredient for enhancing the biological activity of silicone patches due to its excellent cell adhesion and function of promoting epithelial cell regeneration and repair. Therefore, some research institutions have developed a new type of high-end medical device product by compounding it in a silicone gel matrix, which has both physical protection and active biological repair functions.

[0003] However, the physicochemical properties of silicone-based patches are very stable, and the commonly used protein content detection methods cannot directly test the content of the product. In the research and development and quality control process of such composite products, a long-standing technical problem has seriously hindered their development: how to accurately and efficiently extract and quantitatively detect the content of human recombinant type III collagen in the solidified silicone gel matrix. This problem is mainly due to the huge difference in the inherent physicochemical properties of silicone matrix and collagen, which are as follows: (1) Matrix incompatibility: silicone gel is a hydrophobic, water-insoluble solid elastic network formed by cross-linking solidification, while recombinant collagen is a typical water-soluble macromolecular raw material.

[0004] (2) Extraction difficulty: Traditional protein extraction methods (such as buffer soaking, homogenization, and shaking) have little effect on silicone gel. The hydrophobic silicone network has a strong "locking" effect on water-soluble proteins, making it difficult for collagen to be extracted into the water phase extract. The extraction process often has the problems of low extraction efficiency, long time consumption, and poor repeatability of results.

[0005] (3) Difficulty in traditional digestion reaction: Kjeldahl nitrogen determination is a common method for detecting protein content. However, due to the presence of a large amount of carbon elements in silicone-based products and the relatively small amount of collagen contained in the products, carbonization is serious during the digestion process, which cannot be continued.

[0006] At present, there is a lack of a standardized and efficient protein content identification method for "silicone-protein" composite patches in the industry. The limitations of the prior art directly lead to the following problems: (1) Research and development stage: unable to accurately evaluate the influence of the formulation process on the protein load and stability, hindering the optimization of product formulation.

[0007] (2) Quality control stage: it is difficult to establish reliable quality standards, and it is impossible to effectively monitor the active collagen content in each batch of product, which poses a huge quality risk and regulatory risk.

[0008] (3) Product declaration: due to the inability to provide a collagen detection report, the product lacks solid data support for the designation of "recombinant collagen" scar patches, thereby affecting the normal declaration and marketing of the product.

[0009] Therefore, there is an urgent need in the art to develop a new treatment method specifically for the identification of water-soluble protein content in silicone-based patch products. Therefore, in view of the above problems, the present application is proposed. SUMMARY

[0010] In view of the above technical problems, the purpose of the present application is to provide a method for detecting the protein content in silicone-based patches containing collagen, which can overcome the physical barrier of the silicone matrix, realize the efficient and complete release of collagen, and at the same time, without destroying its biochemical properties, thereby providing key and reliable technical basis for the research and development, quality control and registration of the product.

[0011] In order to achieve the above purpose, the technical scheme adopted by the present application is as follows: The present application provides a method for detecting the protein content in silicone-based patches containing collagen, which comprises the following steps: adding the prepared silicone-based patches containing collagen to dichloromethane, and performing water bath reflux treatment, and then cooling to room temperature after the treatment is completed; then adding purified water for extraction, taking the water layer for concentration to obtain a concentrated solution, and finally testing the protein content of the concentrated solution.

[0012] The above technical scheme is not directly determining the content of collagen in the product, but finding a suitable method for extracting collagen in the silicone patch product containing collagen, extracting the collagen, and then testing the content. The change in thinking is adjusted by the applicant after repeated exploration. Based on the preliminary investigation, considering that the product is not dissolved in most organic solvents, and considering that various testing methods of protein content have their specific requirements and limitations, the applicant first directly used the Kjeldahl method for testing. The results show that by adjusting the reagent and changing the catalyst, the digestion of the product cannot be completed, which leads to the inability to proceed to the next step of Kjeldahl detection, so the thinking is changed, and the collagen in the product is extracted, and then the content of collagen is considered. In the determination process, the color developing principle of BCA method (protein molecules in alkaline solution reduce Cu 2+ to Cu + , 2, 2'-biquinoline-4, 4'-dicarboxylic acid (BCA) and Cu + form a purple complex, and the color depth is proportional to the protein concentration within a certain range) is used to preliminarily determine whether the protein is extracted, and the Kjeldahl instrument is used to test the protein content in the later period. The evaluation method of the extraction rate of protein.

[0013] As a preferred embodiment of the present application, the ratio of the silicone patch containing collagen to dichloromethane is 1:10 (w / v).

[0014] As a preferred embodiment of the present application, the water bath reflux temperature is 45-55℃, and the water bath reflux treatment time is 0.5-2h.

[0015] As a preferred embodiment of the present application, the purified water extraction times are 1-6 times. Preferably, the extraction times are 4-5 times.

[0016] As a preferred embodiment of the present application, the amount of purified water added each time is 60-150mL.

[0017] As a preferred embodiment of the present application, the purified water extraction time is 10-60min / time. Preferably, the extraction time is 30min each time, and the treatment is performed by shaking.

[0018] As a preferred embodiment of the present application, the concentration is performed by water bath concentration.

[0019] As a preferred embodiment of the present application, the protein content is tested by Kjeldahl instrument, and the test is performed according to the third method of nitrogen determination method in Chinese Pharmacopoeia (2025 edition) 0704.

[0020] As a preferred technical scheme of the present application, the silicone-based patch containing collagen is composed of a silicone gel layer, an anti-adhesion layer and a backing layer.

[0021] As a preferred technical scheme of the present application, the silicone gel layer is composed of a polydimethylsiloxane cross-linked polymer, recombinant collagen, purified water and fumed silica; the anti-adhesion layer is a polyethylene anti-adhesion film; and the backing layer is a polyurethane composite film.

[0022] Further, the recombinant collagen mentioned above is recombinant human type III collagen.

[0023] For example, the amounts of the substances in the silicone gel layer are as follows in terms of weight parts: .

[0024] Compared with the prior art, the present application has the following beneficial effects: (1) The method for detecting the protein content in the silicone-based patch containing collagen provided by the present application can efficiently and sufficiently release the recombinant human type III collagen embedded in the silicone solid patch into the extraction liquid through an innovative pretreatment step, ensuring that the amount of the extracted protein can truly reflect the actual collagen protein existing in the product, improving the accuracy of quantitative detection, and overcoming the deficiency of the prior art that the cross-linked network of the silicone gel cannot effectively break the "locking" effect of the water-soluble collagen protein, resulting in extremely low protein extraction efficiency.

[0025] (2) The method for detecting the protein content in the silicone-based patch containing collagen provided by the present application establishes a processing method that is standardized in operation, process and results, and is stable and reliable, and provides a consistent technical basis for product quality control, overcoming the deficiency of the current lack of a standardized pretreatment method, which results in large fluctuations in the extraction results obtained by different batches and different operators, and poor repeatability and stability.

[0026] (3) The method for detecting the protein content in the silicone-based patch containing collagen provided by the present application shortens the pretreatment time under the premise of ensuring accuracy by optimizing the processing steps, establishes a standardized process that can be applied to the rapid quality control of the production line, and overcomes the deficiency of the current exploratory extraction method that is often time-consuming and cannot meet the needs of rapid and high-throughput detection of raw materials, intermediate products and finished products in industrial production.

[0027] (4) The method for detecting the protein content in the silicone-based patch containing collagen provided by the present application can obtain reliable detection data, providing solid, scientific and verifiable evidence for the filing and approval of products, and overcoming the deficiency of the lack of data support in the naming of similar collagen scar patches due to the inability to qualitatively determine the collagen protein.

[0028] (5) The application provides a method for detecting the protein content of a silicone-based patch containing collagen, which significantly improves the swelling degree of collagen in the scar patch by optimizing the solvent selection and extraction conditions, realizes efficient extraction of collagen from the silicone gel crosslinked network, and even when the addition amount of collagen is extremely low (only 0.001%), a high extraction rate (> 50%) can be realized, so that the collagen can be accurately determined by means of conventional detection means. BRIEF DESCRIPTION OF DRAWINGS

[0029] Figure 1 The left side is a product diagram before dichloromethane swelling in Example 3; and the right side is a product diagram after dichloromethane swelling in Example 3.

[0030] Figure 2 The left side is a product diagram before ultrasonic treatment in dichloromethane solvent in Comparative Example 2; and the right side is a product diagram after ultrasonic treatment in dichloromethane solvent in Comparative Example 2. DETAILED DESCRIPTION

[0031] The technical solutions in the embodiments of the application will be clearly and completely described below with reference to the embodiments of the application. Obviously, the described embodiments are only part of the embodiments of the application, rather than all the embodiments of the application. Based on the embodiments in the application, all other embodiments obtained by those skilled in the art without creative labor fall within the scope of protection of the application.

[0032] Unless otherwise specified, the raw materials used in the application can be purchased from the market, and the specifications meet the detection and use standards.

[0033] The recombinant collagen used in the application is purchased from Jiangsu Jiangshan Juyuan Biotechnology Co., Ltd. The polydimethylsiloxane crosslinked polymer used in the application has a trade name of Liveo MG-7-9960 and is purchased from DuPont Company. The fumed silica used in the application is purchased from Dow Corning Company. The polyethylene release film used in the application is purchased from Gaoming Medical Instrument Co., Ltd. The polyurethane composite film used in the application is purchased from Shenzhen Deguo Sai Technology Co., Ltd.

[0034] Example 1 A silicone-based patch containing collagen is prepared from the following raw materials, based on the weight and 100%: In this embodiment, a preparation method of the above-mentioned silicone-based patch is also provided, which comprises the following steps: (1) A phase stirring dissolution: the recombinant type III humanized collagen is weighed into a beaker and purified water is added, and stirring is performed until complete dissolution, to obtain a recombinant type III humanized collagen aqueous solution; (2) ABC phase stirring mixing process: fumed silica, the recombinant type III humanized collagen aqueous solution, and dimethicone cross-linked polymer are sequentially added into a stirring barrel, and stirring is performed until uniform; (3) The stirring-qualified ABC mixed phase is poured into a film, and standing, defoaming, drying, and cutting are performed, to obtain the medical recombinant collagen scar patch.

[0035] Example 2 A silicone-based patch containing collagen is prepared from the following raw materials, based on weight and 100%: In this example, a preparation method of the above silicone-based patch is also provided, comprising the following steps: (1) A phase stirring dissolution: the recombinant type III humanized collagen is weighed into a beaker and purified water is added, and stirring is performed until complete dissolution, to obtain a recombinant type III humanized collagen aqueous solution; (2) ABC phase stirring mixing process: fumed silica, the recombinant type III humanized collagen aqueous solution, and dimethicone cross-linked polymer are sequentially added into a stirring barrel, and stirring is performed until uniform; (3) The stirring-qualified ABC mixed phase is poured into a film, and standing, defoaming, drying, and cutting are performed, to obtain the medical recombinant collagen scar patch.

[0036] Example 3 Since the product does not change significantly in state (see the test results in the comparative example part) under normal temperature conditions, shaking, or ultrasonic, the temperature is now considered to be increased for testing. Considering the volatility of the solvent, the scheme is adjusted to heating reflux extraction. The sample of Example 1 treated with dichloromethane is extracted twice with purified water (100 mL / time, shaking for 30 min / time), and after the water layer is concentrated to about 10 mL, 0.5 mL is taken and observed for the presence of collagen by the color development degree using the BCA method; after the other reagent-treated filtrate is evaporated to dryness in a water bath, 2 mL of water is added for dissolution, and 0.5 mL is taken and observed for the presence of collagen by the color development degree using the BCA method. The results are shown in Table 1, which shows that the product swells significantly in dichloromethane, slightly swells in methanol, and does not change significantly in state in other solvents. The sample treated with dichloromethane has a significant color reaction.

[0037] Table 1 Product heating reflux extraction in different solvents Example 4 According to the embodiment 3, the product is confirmed to change when heated and refluxed in the solvent dichloromethane, the protein is released, the test scheme is designed, the reflux time is adjusted, the water extraction times are adjusted, the protein content is determined by the Kjeldahl apparatus, the extraction rate is calculated, the optimal extraction condition is determined, and the results are shown in Table 2. The data prove that the extraction rate can reach more than 50% by using the dichloromethane water bath extraction for 60 min, the water extraction for 4 times, the Kjeldahl apparatus for the protein content test after concentration.

[0038] Table 2 Extraction of the product with different extraction times Comparative example 1 10 g of the sample in the embodiment 1 is taken, 100 mL of solvent is added, and the sample is shaken at room temperature for 30 min, 60 min and 120 min. After filtration, the liquid is evaporated in a water bath, 2 mL of water is added for dissolution, 0.5 mL of the solution is taken, and the BCA method is used to observe whether the collagen protein is contained by the color development degree. The results are shown in Table 3. The product state has no obvious change, and there is no obvious color reaction, so it is considered that the collagen protein in the product is not extracted by the method.

[0039] Table 3 Extraction of the product in different solvents by shaking at room temperature Comparative example 2 10 g of the sample in the embodiment 1 is taken, 100 mL of solvent is added, and the sample is ultrasonically treated at room temperature for 30 min, 60 min and 120 min. After filtration, the liquid is evaporated in a water bath, 2 mL of water is added for dissolution, 0.5 mL of the solution is taken, and the BCA method is used to observe whether the collagen protein is contained by the color development degree. The results are shown in Table 4. The product state has no obvious change, and there is no obvious color reaction, so it is considered that the collagen protein in the product is not extracted by the method.

[0040] Table 4 Ultrasonic extraction of the product in different solvents The applicant declares that the present application is illustrated by the above embodiments, but the present application is not limited to the above embodiments, that is, it does not mean that the present application must rely on the above embodiments to be implemented. It should be understood by those skilled in the art that any improvement of the present application, equivalent replacement of individual raw materials of the product of the present application, addition of auxiliary ingredients, selection of specific modes, etc. fall within the protection scope and disclosure scope of the present application.

Claims

1. A method for detecting the protein content in a silicone patch containing collagen, characterized in that, The process includes the following steps: the prepared silicone patch containing collagen is added to dichloromethane and refluxed in a water bath. After the treatment is completed, it is cooled to room temperature. Then, purified water is added for extraction, the aqueous layer is collected and concentrated to obtain a concentrate. Finally, the protein content of the concentrate is tested.

2. The method for detecting protein content in a silicone patch containing collagen according to claim 1, characterized in that, The w / v ratio of the collagen-containing silicone patch to dichloromethane is 1:

10.

3. The method for detecting protein content in a silicone patch containing collagen according to claim 1, characterized in that, The water bath reflux temperature is 45~55℃, and the water bath reflux treatment time is 0.5~2h.

4. The method for detecting protein content in a silicone patch containing collagen according to claim 1, characterized in that, The number of times for purified water extraction is 1 to 6.

5. The method for detecting protein content in a silicone patch containing collagen according to claim 4, characterized in that, The amount of purified water added each time is 60~150mL.

6. The method for detecting protein content in a silicone patch containing collagen according to claim 4, characterized in that, The extraction time for purified water is 10-60 min per extraction.

7. The method for detecting protein content in a silicone patch containing collagen according to claim 1, characterized in that, Concentration is carried out using a water bath.

8. The method for detecting protein content in a silicone patch containing collagen according to claim 1, characterized in that, Protein content was determined using a Kjeldahl nitrogen analyzer.

9. The method for detecting protein content in a silicone patch containing collagen according to claim 1, characterized in that, Collagen-containing silicone patches consist of a silicone gel layer, an anti-adhesive layer, and a backing layer.

10. The method for detecting protein content in a silicone patch containing collagen according to claim 9, characterized in that, The silica gel layer is composed of polydimethylsiloxane crosslinked polymer, recombinant collagen, purified water, and fumed silica.

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