Method for detecting content of collagen in collagen-hyaluronic acid cross-linked gel

Abstract

The invention discloses a method for detecting the content of collagen in collagen-hyaluronic acid cross-linked gel. The detection method comprises the following steps: testing the content of collagen in collagen-hyaluronic acid cross-linked gel by adopting a folin-phenol method in combination with an external standard method, specifically, mixing the gel and an alkaline copper test solution for reaction at the temperature of 50-85 DEG C to prepare a test solution; wherein the concentration of sodium hydroxide in the alkaline copper test solution is 50-100 mg / mL; the concentration of the gel in the test solution is 0.02-0.1 g / mL; and mixing the test solution with the folin-phenol test solution, detecting an absorption value by using an ultraviolet and visible spectrophotometer, and calculating the collagen content in the gel through a standard curve. According to the collagen content detection method, a folin-phenol method in pharmacopoeia is improved, the operation is simple, and the detection accuracy, repeatability and durability all meet the requirements.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of analysis, in particular to a method for detecting the content of collagen in collagen-hyaluronic acid cross-linked gel. BACKGROUND

[0002] Hyaluronic acid and collagen are important substances in the human body and are important components of the extracellular matrix. Collagen can provide a spatial structure for cells, and hyaluronic acid can provide a hydrated environment for cells. The medical and beauty field attempts to cross-link the two substances with cosmetic effects, collagen and hyaluronic acid, to prepare collagen-hyaluronic acid cross-linked gel. The gel has the effects of hyaluronic acid and collagen, such as ideal moisturizing, brightening skin color, reducing pores, and reducing wrinkles. The content of collagen in the gel is an important monitoring index in the production process.

[0003] The field often uses the Folin phenol method to determine the protein content in the test substance, which has high sensitivity. However, when using the Folin phenol method to test the collagen content in collagen-hyaluronic acid cross-linked gel, there are many problems. On the one hand, part of the collagen is covered inside the gel, which cannot combine with the test reagent to develop color, thereby affecting the detection result. On the other hand, hyaluronic acid is a polysaccharide substance, and the Chinese Pharmacopoeia mentions that sugar is one of the interference factors for testing protein content by the Folin phenol method. The Pharmacopoeia also mentions that when there are interfering substances, it is recommended to first use deoxycholate-trichloroacetic acid to precipitate the protein to remove the interference. However, for collagen-hyaluronic acid cross-linked gel, the problem of collagen covered inside the gel cannot be detected, resulting in inaccurate detection results, cannot be solved.

[0004] Therefore, there is an urgent need in the field to develop a collagen content detection method that is simple to operate, accurate, reproducible, and durable. SUMMARY

[0005] The technical problem to be solved by the present application is that when using the Folin phenol method in the Pharmacopoeia to test the collagen content in collagen-hyaluronic acid cross-linked gel, part of the collagen cannot combine with the test reagent, resulting in a low detection result, and hyaluronic acid interferes with the detection result, thereby causing inaccurate detection results. The present application provides a method for detecting the content of collagen in collagen-hyaluronic acid cross-linked gel. The present application improves the Folin phenol method in the Pharmacopoeia and provides a collagen content detection method that is simple to operate, accurate, reproducible, and durable.

[0006] The present application solves the above technical problems through the following technical solutions.

[0007] The application provides a method for detecting the content of collagen in collagen-hyaluronic acid cross-linked gel, comprising the following steps: using the Folin phenol method combined with the external standard method to test the content of collagen in collagen-hyaluronic acid cross-linked gel, specifically:

[0008] (1) the gel is mixed with an alkaline copper test solution at a temperature of 50-85°C to prepare a test sample solution; wherein the concentration of sodium hydroxide in the alkaline copper test solution is 50-100 mg / mL; and the concentration of the gel in the test sample solution is 0.02-0.1 g / mL;

[0009] (2) the test sample solution is mixed with a Folin phenol test solution, and the absorption value is detected by a UV-visible spectrophotometer, and the content of collagen in the gel is calculated by a standard curve.

[0010] In some embodiments, the concentration of the gel in the test sample solution is 0.03-0.07 g / mL, for example, 0.02 g / mL, 0.03 g / mL, 0.04 g / mL, 0.05 g / mL, 0.06 g / mL, 0.07 g / mL, 0.08 g / mL, 0.09 g / mL or 0.1 g / mL.

[0011] In some embodiments, the concentration of sodium hydroxide in the alkaline copper test solution is preferably 60-80 mg / mL, for example, 50 mg / mL, 55 mg / mL, 60 mg / mL, 65 mg / mL, 70 mg / mL, 75 mg / mL, 80 mg / mL, 85 mg / mL, 90 mg / mL, 95 mg / mL or 100 mg / mL. It is found in the research process that when the concentration of sodium hydroxide is low, the detection result is low. When the concentration of sodium hydroxide is too high, it will also affect the color reaction, resulting in inaccurate detection results.

[0012] In some embodiments, the alkaline copper test solution further comprises sodium carbonate, potassium sodium tartrate and copper sulfate.

[0013] In some embodiments, the concentration of sodium carbonate in the alkaline copper test solution is 70-130 mg / mL, preferably 90-110 mg / mL, for example, 70 mg / mL, 75 mg / mL, 80 mg / mL, 85 mg / mL, 90 mg / mL, 95 mg / mL, 100 mg / mL, 105 mg / mL, 110 mg / mL, 115 mg / mL, 120 mg / mL, 125 mg / mL or 130 mg / mL.

[0014] The concentration of the potassium sodium tartrate in the alkaline copper reagent solution is 1.0-3.0 mg / mL, preferably 1.5-2.5 mg / mL, for example 1 mg / mL, 1.2 mg / mL, 1.4 mg / mL, 1.6 mg / mL, 1.8 mg / mL, 2.0 mg / mL, 2.2 mg / mL, 2.4 mg / mL, 2.6 mg / mL, 2.8 mg / mL or 3.0 mg / mL.

[0015] The concentration of the copper sulfate in the alkaline copper reagent solution is 0.3-0.8 mg / mL, preferably 0.4-0.6 mg / mL, for example 0.3 mg / mL, 0.4 mg / mL, 0.5 mg / mL, 0.6 mg / mL, 0.7 mg / mL or 0.8 mg / mL.

[0016] In step (1) of a preferred embodiment, the alkaline copper reagent solution comprises 60 mg / mL of sodium hydroxide, 100 mg / mL of sodium carbonate, 2 mg / mL of potassium sodium tartrate, 0.5 mg / mL of copper sulfate and the rest of water.

[0017] In step (1) of a preferred embodiment, the alkaline copper reagent solution comprises 80 mg / mL of sodium hydroxide, 100 mg / mL of sodium carbonate, 2 mg / mL of potassium sodium tartrate, 0.5 mg / mL of copper sulfate and the rest of water.

[0018] In step (1) of a preferred embodiment, the alkaline copper reagent solution comprises 100 mg / mL of sodium hydroxide, 100 mg / mL of sodium carbonate, 2 mg / mL of potassium sodium tartrate, 0.5 mg / mL of copper sulfate and the rest of water.

[0019] In step (1) of a preferred embodiment, the preparation method of the alkaline copper reagent solution comprises the following steps: mixing the sodium hydroxide, the sodium carbonate and a first part of water to prepare a liquid A; mixing the copper sulfate aqueous solution and the potassium sodium tartrate aqueous solution to prepare a liquid B; and mixing the liquid A, the liquid B and the rest of water when in use.

[0020] The volume ratio of the liquid A to the liquid B is (3-7): 1, preferably (4-6): 1, for example 5: 1.

[0021] The volume ratio of the copper sulfate aqueous solution to the potassium sodium tartrate aqueous solution is (0.2-1.0): 1, preferably (0.4-0.8): 1, for example 0.6: 1.

[0022] In step (1) of some embodiments, the temperature for mixing and reacting the gel with the alkaline copper reagent solution is 60-80°C.

[0023] In step (1) of some embodiments, the mixing reaction time of the gel with the alkaline copper reagent is 45-75 min, preferably 55-65 min, for example 60 min.

[0024] In step (1) of a preferred embodiment, the mixing reaction is performed with intermittent stirring, and more preferably, the mixing reaction is stirred every 10 min after 30 min.

[0025] In step (1) of some embodiments, the mass ratio of sodium hyaluronate in the gel to sodium hydroxide in the alkaline copper reagent is 1:(40-100), preferably 1:(44-95), for example 1:40, 1:42, 1:44, 1:47, 1:48, 1:49, 1:50, 1:52, 1:54, 1:55, 1:56, 1:57, 1:59, 1:60, 1:62, 1:64, 1:66, 1:67, 1:69, 1:72, 1:74, 1:76, 1:78, 1:80, 1:82, 1:84, 1:86, 1:88, 1:90, 1:91, 1:93, 1:95, 1:97, or 1:100.

[0026] In step (1) of some embodiments, the preparation method of the gel comprises the following steps: cross-linking sodium hyaluronate and collagen.

[0027] In the cross-linking, the cross-linking agent is selected from BDDE or DMTMM (4-(4,6-dimethoxytriazin-2-yl)-4-methylmorpholinium hydrochloride).

[0028] In a preferred embodiment, the preparation method of the gel comprises the following steps:

[0029] S1. Dissolve sodium hyaluronate and collagen in a phosphate buffer, respectively, to prepare a sodium hyaluronate solution and a collagen solution;

[0030] S2. Mix the sodium hyaluronate solution and the collagen solution prepared in step S1 to obtain a mixed solution, add a cross-linking agent to the mixed solution, and perform a cross-linking reaction to obtain a collagen-sodium hyaluronate cross-linked gel.

[0031] In the cross-linking, the cross-linking agent is selected from BDDE or DMTMM (4-(4,6-dimethoxytriazin-2-yl)-4-methylmorpholinium hydrochloride).

[0032] In the cross-linking, the cross-linking agent is selected from BDDE or DMTMM (4-(4,6-dimethoxytriazin-2-yl)-4-methylmorpholinium hydrochloride).

[0033] The collagen includes recombinant collagen and / or natural collagen.

[0034] The sodium hyaluronate solution contains sodium hyaluronate at a mass percentage of 2.5% to 10.0%, preferably 5.0% to 8.0%.

[0035] The mass ratio of the crosslinking agent to the sodium hyaluronate is 1:(0.5~3), preferably 1:(1~2).

[0036] The concentration of the phosphate buffer solution is 0.02~0.2 mol / L, preferably 0.05~0.15 mol / L.

[0037] The pH value of the phosphate buffer solution is 6-8, preferably 7-8.

[0038] The cross-linking reaction takes 12 to 72 hours, for example, 36 hours.

[0039] The cross-linking reaction is carried out at room temperature.

[0040] The mass ratio of sodium hyaluronate to the gel is (1~10):100, preferably (2~5):100, for example 3:100.

[0041] In step (2) of some embodiments, the volume ratio of the test solution to the phenol test solution is 1:(2~8), preferably 1:(4~6), for example 1:5.

[0042] In step (2) of some embodiments, the Folin-Ciocalteu solution is prepared by diluting a 2 mol / L acid concentration phosphomolybdic acid-phosphotungstic acid complex solution by 16 times.

[0043] In step (2) of some embodiments, the mixing temperature is room temperature.

[0044] In step (2) of some embodiments, the mixing time can be conventional in the art, generally 20 to 50 minutes, for example 30 minutes.

[0045] In step (2) of some embodiments, the detection wavelength of the ultraviolet-visible spectrophotometer is 645~655nm, for example 648nm, 650nm or 652nm.

[0046] Based on common knowledge in the field, the above-mentioned preferred conditions can be combined arbitrarily to obtain various preferred embodiments of this application.

[0047] All reagents and raw materials used in this application are commercially available.

[0048] The positive advancements of this application are as follows: Research has shown that the Folin-Ciocalteu method for determining protein content in pharmacopoeias is unsuitable for detecting collagen content in collagen-hyaluronic acid crosslinked gels. Firstly, the pharmacopoeia mentions polysaccharides as interfering factors; the presence of hyaluronic acid in the system may affect the detection results. Generally, protein precipitation is required before detection, but this method is complex. Secondly, some collagen is encapsulated within the gel; even after removing polysaccharide interference, it cannot be guaranteed that all collagen will bind to the detection reagent for a colorimetric reaction, leading to inaccurate results. This application improves the Folin-Ciocalteu method for determining protein content in pharmacopoeias by adjusting the concentration of sodium hydroxide in the alkaline copper solution and the processing temperature, addressing the aforementioned problems. The improved method is not only simple to operate but also meets the requirements for accuracy, repeatability, and robustness, providing strong support for quality control during the production of such products. Detailed Implementation

[0049] The present application is further illustrated below by way of embodiments, but these embodiments are not intended to limit the scope of the present application. Experimental methods not specifically described in the following embodiments are performed according to conventional methods and conditions, or as selected in accordance with the product instructions.

[0050] The reagents and raw materials used in the following examples are all commercially available, and the reagents used are of analytical grade.

[0051] The preparation methods for the collagen-hyaluronic acid crosslinked gels of the test samples in the following examples and comparative examples are as follows: A phosphate buffer solution with a pH of 7 and a concentration of 0.1 mol / L was prepared using sodium dihydrogen phosphate monohydrate and disodium hydrogen phosphate dodecahydrate; 1 g of sodium hyaluronate and 20.0 g of phosphate buffer were mixed and stirred until completely dissolved to obtain a sodium hyaluronate solution; 6 g of phosphate buffer and 0.6 g of recombinant collagen were mixed to obtain a recombinant collagen solution; 3.685 g of the recombinant collagen solution and the above-prepared solution were then mixed... The sodium hyaluronate solution was mixed and stirred thoroughly to disperse it evenly. 1.0g of cross-linking agent DMTMM was added and stirred until a gel was formed. After sealing, the gel was cross-linked at room temperature for 36 hours. It was then placed in a dialysis bag and dialyzed for 4 days. During dialysis, the recombinant collagen remained in the dialysis bag without loss. Dialysis was only used to remove small molecules such as the cross-linking agent. After dialysis, the gel was diluted to 50g with phosphate buffer to obtain a collagen-hyaluronic acid cross-linked gel. The mass percentage of recombinant collagen was 0.67%.

[0052] The recombinant collagen used in the following examples and comparative examples has a protein content of 94%.

[0053] Example 1

[0054] Alkaline copper test solution: Dissolve 30g of sodium hydroxide and 50g of sodium carbonate in 400mL of water to obtain solution A; dissolve 1.0g of potassium sodium tartrate in 50mL of water to obtain an aqueous solution of potassium sodium tartrate; separately dissolve 0.25g of copper sulfate in 30mL of water to obtain an aqueous solution of copper sulfate; mix the aqueous solution of potassium sodium tartrate and the aqueous solution of copper sulfate to obtain solution B. Before use, combine solutions A and B and add water to a final volume of 500mL; the concentration of sodium hydroxide in the alkaline copper test solution is 60mg / mL.

[0055] Recombinant collagen standard stock solution: Accurately weigh recombinant collagen (dry product), place it in a volumetric flask, add the alkaline copper test solution prepared above to dilute to the mark, and incubate in a 60℃ water bath for 60 min (vortex once every 10 min after 30 min) to obtain a recombinant collagen protein standard stock solution with a recombinant collagen protein concentration of approximately 0.7 mg / mL.

[0056] Blank solution: Take the alkaline copper test solution prepared above and keep it in a 60℃ water bath for 60 min (vortex once every 10 min after 30 min).

[0057] Recombinant collagen standard series solutions: Accurately measure 0.2 mL, 0.4 mL, 0.6 mL, 0.8 mL and 1.0 mL of recombinant collagen standard stock solution into test tubes respectively, and add blank solution to each to 1.0 mL.

[0058] Test solution: Take about 0.5g of the collagen-hyaluronic acid crosslinked gel prepared above as the test sample, accurately weigh it, add the alkaline copper test solution prepared above to 10mL, and incubate in a 60℃ water bath for 60min (vortex once every 10min after 30min) to prepare the test solution. Perform 6 parallel tests, which are numbered as test solutions S1~S6 respectively. The mass ratio of sodium hyaluronate in the collagen-hyaluronic acid crosslinked gel to sodium hydroxide in the alkaline copper test solution is 1:57, 1:49, 1:52, 1:53, 1:54, and 1:47 respectively.

[0059] Blank preparation: Weigh approximately 0.5g of a 2% sodium hyaluronate aqueous solution, add the prepared alkaline copper test solution to 10mL, and incubate in a 60℃ water bath for 60min (vortex once every 10min after 30min) to obtain the blank preparation; the mass ratio of sodium hyaluronate in the sodium hyaluronate aqueous solution to sodium hydroxide in the alkaline copper test solution is 1:57.

[0060] Folin-Ciocalteu test solution: Take a 2 mol / L Folin-Ciocalteu stock solution and dilute it by dilution factor from 1 mL to 16 mL to prepare the Folin-Ciocalteu test solution.

[0061] Assay: Take 1.0 mL of the test solution, recombinant collagen standard series solution, blank solution, and blank preparation into test tubes, add 5.0 mL of Folin-Ciocalteu solution, and incubate at room temperature for 30 min. Measure the absorbance at 650 nm using a UV-Vis spectrophotometer (the value after subtracting the blank solution). Plot a standard curve using the concentration of the recombinant collagen standard series solution and the corresponding absorbance. The results of the standard curve are shown in Table 1 below. Based on the standard curve, calculate the recombinant collagen content in the test tube, and then calculate the collagen content, recovery rate, and relative standard deviation of the recovery rate in the collagen-hyaluronic acid crosslinked gel. The results are shown in Table 2.

[0062] Recovery rate = Test concentration of collagen / Theoretical concentration of collagen 100%.

[0063] Table 1

[0064]

[0065] Table 2

[0066]

[0067] Based on the above results, the theoretical concentration and the test concentration of recombinant collagen in the six test solutions are very close, and the RSD of the six parallel experiments is less than 5%, indicating that the method for detecting the content of recombinant collagen in collagen-hyaluronic acid crosslinked gel in this application has high accuracy and good repeatability, and meets the requirements.

[0068] This application also tested the absorbance of the blank preparation, which was 0.002. It can be seen that when the method of this application is used, the raw material sodium hyaluronate has almost no effect on the detection results and does not cause interference. It is evident that the detection method of this application has ideal specificity.

[0069] Example 2: Study on the durability of detection wavelength

[0070] Compared with Example 1, the only difference is that the detection wavelength was adjusted to 648nm and 652nm respectively, and the absorbance values ​​were tested. The results are shown in Table 3 below.

[0071] Table 3

[0072]

[0073] According to the results in Table 3, when the detection wavelength is... With a 2nm variation, the absorbance value hardly changes, and the change rate is much less than 5%, indicating that the method of this application is robust enough to ensure the reliability and transferability of the method and will not fail due to wavelength calibration error.

[0074] Example 3

[0075] The only difference from Example 1 was the adjustment of the water bath temperature during the preparation of the recombinant collagen standard stock solution, blank solution, and test solution, to verify the effect of temperature on the stability of the detection method. Specifically, the water bath temperature in Example 1 was adjusted from 60℃ to 55℃, while all other conditions and parameters remained the same as in Example 1. Two parallel experiments were conducted on the test solution, numbered S7 to S8, with the mass ratio of sodium hyaluronate in the collagen-hyaluronic acid crosslinking gel to sodium hydroxide in the alkaline copper solution being 1:55.

[0076] The absorbance at 650 nm was measured using a UV-Vis spectrophotometer (the value after subtracting the blank solution). A standard curve was plotted using the concentrations of the recombinant collagen standard series solutions and their corresponding absorbances. The results of the standard curve are shown in Table 4 below. Based on the standard curve, the recombinant collagen content in the test tubes was calculated, and subsequently, the collagen content, recovery rate, and average recovery rate in the collagen-hyaluronic acid crosslinked gel were calculated. The results are shown in Table 5.

[0077] Recovery rate = Test concentration of collagen / Theoretical concentration of collagen 100%.

[0078] Table 4

[0079]

[0080] Table 5

[0081]

[0082] Example 4

[0083] The only difference from Example 1 is the adjustment of the water bath temperature during the preparation of the recombinant collagen standard stock solution, blank solution, and test solution, to verify the effect of temperature on the stability of the detection method. Specifically, the water bath temperature in Example 1 was adjusted from 60℃ to 65℃, while other conditions and parameters remained the same as in Example 1. Two parallel experiments were conducted on the test solution, numbered S9 to S10. The mass ratio of sodium hyaluronate in the collagen-hyaluronic acid crosslinking gel to sodium hydroxide in the alkaline copper solution was 1:48.

[0084] The absorbance at 650 nm was measured using a UV-Vis spectrophotometer (the value after subtracting the blank solution). A standard curve was plotted using the concentrations of the recombinant collagen standard series solutions and their corresponding absorbances. The results of the standard curve are shown in Table 6 below. Based on the standard curve, the recombinant collagen content in the test tubes was calculated, and subsequently, the collagen content, recovery rate, and average recovery rate in the collagen-hyaluronic acid crosslinked gel were calculated. The results are shown in Table 7.

[0085] Recovery rate = Test concentration of collagen / Theoretical concentration of collagen 100%.

[0086] Table 6

[0087]

[0088] Table 7

[0089]

[0090] Example 5

[0091] The only difference from Example 1 is the adjustment of the water bath temperature during the preparation of the recombinant collagen standard stock solution, blank solution, and test solution, to verify the effect of temperature on the stability of the detection method. Specifically, the water bath temperature in Example 1 was adjusted from 60℃ to 80℃, while other conditions and parameters remained the same as in Example 1. Two parallel experiments were conducted on the test solution, numbered S11 to S12. The mass ratio of sodium hyaluronate in the collagen-hyaluronic acid crosslinking gel to sodium hydroxide in the alkaline copper solution was 1:49.

[0092] The absorbance at 650 nm was measured using a UV-Vis spectrophotometer (the value after subtracting the blank solution). A standard curve was plotted using the concentrations of the recombinant collagen standard series solutions and their corresponding absorbances. The results of the standard curve are shown in Table 8 below. Based on the standard curve, the recombinant collagen content in the test tubes was calculated, and subsequently, the collagen content, recovery rate, and average recovery rate in the collagen-hyaluronic acid crosslinked gel were calculated. The results are shown in Table 9.

[0093] Recovery rate = Test concentration of collagen / Theoretical concentration of collagen 100%.

[0094] Table 8

[0095]

[0096] Table 9

[0097]

[0098] Comparative Example 1

[0099] The only difference from Example 1 was the adjustment of the water bath temperature during the preparation of the recombinant collagen standard stock solution, blank solution, and test solution, to verify the effect of temperature on the stability of the detection method. Specifically, the water bath temperature in Example 1 was adjusted from 60℃ to 40℃, while all other conditions remained the same as in Example 1. Two parallel experiments were conducted on the test solution, numbered S13 to S14. The mass ratio of sodium hyaluronate in the collagen-hyaluronic acid crosslinking gel to sodium hydroxide in the alkaline copper solution was 1:55.

[0100] The absorbance at 650 nm was measured using a UV-Vis spectrophotometer (the value after subtracting the blank solution). A standard curve was plotted using the concentrations of the recombinant collagen standard series solutions and their corresponding absorbances. The results of the standard curve are shown in Table 10 below. Based on the standard curve, the recombinant collagen content in the test tubes was calculated, and subsequently, the collagen content, recovery rate, and average recovery rate in the collagen-hyaluronic acid crosslinked gel were calculated. The results are shown in Table 11.

[0101] Recovery rate = Test concentration of collagen / Theoretical concentration of collagen 100%.

[0102] Table 10

[0103]

[0104] Table 11

[0105]

[0106] Comparative Example 2

[0107] The only difference from Example 1 was the adjustment of the water bath temperature during the preparation of the recombinant collagen standard stock solution, blank solution, and test solution, to verify the effect of temperature on the stability of the detection method. Specifically, the water bath temperature in Example 1 was adjusted from 60℃ to 100℃, while all other conditions and parameters remained the same as in Example 1. Two parallel experiments were conducted on the test solution, numbered S15~S16. The mass ratio of sodium hyaluronate in the collagen-hyaluronic acid crosslinking gel to sodium hydroxide in the alkaline copper solution was 1:56 and 1:52, respectively.

[0108] The absorbance at 650 nm was measured using a UV-Vis spectrophotometer (the value after subtracting the blank solution). A standard curve was plotted using the concentrations of the recombinant collagen standard series solutions and their corresponding absorbances. The results of the standard curve are shown in Table 12 below. Based on the standard curve, the recombinant collagen content in the test tubes was calculated, and subsequently, the collagen content, recovery rate, and average recovery rate in the collagen-hyaluronic acid crosslinked gel were calculated. The results are shown in Table 13.

[0109] Recovery rate = Test concentration of collagen / Theoretical concentration of collagen 100%.

[0110] Table 12

[0111]

[0112] Table 13

[0113]

[0114] Based on the results of Examples 1, 3-5 and Comparative Examples 1-2 above, it can be seen that the water bath temperature during the preparation of the test solution has a significant impact on the detection results. When the water bath temperature is within the range specified in this application, the detection value and the theoretical value are extremely close. However, when the water bath temperature is too low or too high, the accuracy of the detection results cannot be guaranteed.

[0115] Example 6

[0116] Compared with Example 1, the only difference is that when preparing the alkaline copper test solution, the amount of sodium hydroxide added was adjusted from 30g to 40g, and the concentration of sodium hydroxide in the alkaline copper test solution was 80mg / mL. Two parallel tests were performed on the test solution, numbered S17~S18 respectively. In the prepared test solutions, the mass ratio of sodium hyaluronate in the collagen-hyaluronic acid crosslinking gel to the mass ratio of sodium hydroxide in the alkaline copper test solution was 1:72 and 1:69, respectively. All other conditions and parameters were the same as in Example 1.

[0117] The absorbance at 650 nm was measured using a UV-Vis spectrophotometer (the value after subtracting the blank solution). A standard curve was plotted using the concentrations of the recombinant collagen standard series solutions and their corresponding absorbances. The results of the standard curve are shown in Table 14 below. Based on the standard curve, the recombinant collagen content in the test tubes was calculated, and subsequently, the collagen content, recovery rate, and average recovery rate in the collagen-hyaluronic acid crosslinked gel were calculated. The results are shown in Table 15.

[0118] Recovery rate = Test concentration of collagen / Theoretical concentration of collagen 100%.

[0119] Table 14

[0120]

[0121] Table 15

[0122]

[0123] Example 7

[0124] Compared with Example 1, the only difference is that when preparing the alkaline copper test solution, the amount of sodium hydroxide added was adjusted from 30g to 50g, and the concentration of sodium hydroxide in the alkaline copper test solution was 100mg / mL. Two parallel tests were performed on the test solution, numbered S19 to S20. In the prepared test solutions, the mass ratio of sodium hyaluronate in the collagen-hyaluronic acid crosslinking gel to the mass of sodium hydroxide in the alkaline copper test solution was 1:95 and 1:88, respectively. All other conditions and parameters were the same as in Example 1.

[0125] The absorbance at 650 nm was measured using a UV-Vis spectrophotometer (the value after subtracting the blank solution). A standard curve was plotted using the concentrations of the recombinant collagen standard series solutions and their corresponding absorbances. The results of the standard curve are shown in Table 16 below. Based on the standard curve, the recombinant collagen content in the test tubes was calculated, and subsequently, the collagen content, recovery rate, and average recovery rate in the collagen-hyaluronic acid crosslinked gel were calculated. The results are shown in Table 17.

[0126] Recovery rate = Test concentration of collagen / Theoretical concentration of collagen 100%.

[0127] Table 16

[0128]

[0129] Table 17

[0130]

[0131] Comparative Example 3

[0132] Compared with Example 1, the only difference is that when preparing the alkaline copper test solution, the amount of sodium hydroxide added was adjusted from 30g to 20g, and the concentration of sodium hydroxide in the alkaline copper test solution was 40mg / mL. Two parallel tests were performed on the test solution, numbered S21~S22. In the prepared test solutions, the mass ratio of sodium hyaluronate in the collagen-hyaluronic acid crosslinking gel to the mass of sodium hydroxide in the alkaline copper test solution was 1:33 and 1:35, respectively. All other conditions and parameters were the same as in Example 1.

[0133] The absorbance at 650 nm was measured using a UV-Vis spectrophotometer (the value after subtracting the blank solution). A standard curve was plotted using the concentrations of the recombinant collagen standard series solutions and their corresponding absorbances. The results of the standard curve are shown in Table 18 below. Based on the standard curve, the recombinant collagen content in the test tubes was calculated, and then the collagen content, recovery rate, and average recovery rate in the collagen-hyaluronic acid crosslinked gel were calculated. The results are shown in Table 19.

[0134] Recovery rate = Test concentration of collagen / Theoretical concentration of collagen 100%.

[0135] Table 18

[0136]

[0137] Table 19

[0138]

[0139] Based on the results of Examples 1, 6-7, and Comparative Example 3, it is evident that the concentration of sodium hydroxide in the alkaline copper test solution has a significant impact on the detection results. When the concentration of the alkaline solution is too low, the detection results are inaccurate. When the concentration of sodium hydroxide in the alkaline copper test solution is 100 mg / mL, the colors of the test sample solution and the recombinant collagen standard series solutions are already quite light, and the absorbance value of the standard curve has reached the compliance limit. Therefore, it is clear that when the concentration of sodium hydroxide is too high, the detection results will be inaccurate.

[0140] Example 8

[0141] The concentration of collagen-hyaluronic acid crosslinked gel in the test solution was adjusted to detect the effect of different concentrations on the accuracy of the test results.

[0142] Alkaline copper test solution: Dissolve 30g of sodium hydroxide and 50g of sodium carbonate in 400mL of water to obtain solution A; dissolve 1.0g of potassium sodium tartrate in 50mL of water to obtain an aqueous solution of potassium sodium tartrate; separately dissolve 0.25g of copper sulfate in 30mL of water to obtain an aqueous solution of copper sulfate; mix the aqueous solution of potassium sodium tartrate and the aqueous solution of copper sulfate to obtain solution B. Before use, combine solutions A and B and add water to a final volume of 500mL; the concentration of sodium hydroxide in the alkaline copper test solution is 60mg / mL.

[0143] Recombinant collagen standard stock solution: Accurately weigh recombinant collagen (dry product), place it in a volumetric flask, dilute to the mark with the alkaline copper test solution prepared above, and incubate in a 60℃ water bath for 60 min (vortex once every 10 min after 30 min) to obtain a recombinant collagen protein standard stock solution with a recombinant collagen protein concentration of approximately 0.7 mg / mL.

[0144] Blank solution: Take the alkaline copper test solution prepared above and keep it in a 60℃ water bath for 60 min (vortex once every 10 min after 30 min).

[0145] Recombinant collagen standard series solutions: Accurately measure 0.2 mL, 0.4 mL, 0.6 mL, 0.8 mL and 1.0 mL of recombinant collagen standard stock solution into test tubes respectively, and add blank solution to each to 1.0 mL.

[0146] Test solutions: Take approximately 0.5 g, 0.4 g, and 0.6 g of the collagen-hyaluronic acid crosslinked gel prepared above as test samples, respectively, accurately weigh them, add the alkaline copper test solution prepared above to 10 mL, and incubate in a 60 °C water bath for 60 min (vortex once every 10 min after 30 min). The test solutions are numbered as test solutions S23~S25 respectively. The mass ratio of sodium hyaluronate in the collagen-hyaluronic acid crosslinked gel to sodium hydroxide in the alkaline copper test solution is 1:69, 1:56, and 1:44 respectively.

[0147] Folin-Ciocalteu test solution: Take a 2 mol / L Folin-Ciocalteu stock solution and dilute it by dilution factor from 1 mL to 16 mL to prepare the Folin-Ciocalteu test solution.

[0148] Assay: Take 1.0 mL of the test solution, recombinant collagen standard series solution, and blank solution into test tubes, add 5.0 mL of Folin-Ciocalteu solution, and incubate at room temperature for 30 min. Measure the absorbance at 650 nm using a UV-Vis spectrophotometer (the value after subtracting the blank solution). Plot a standard curve using the concentration of the recombinant collagen standard series solution and the corresponding absorbance. The results of the standard curve are shown in Table 20 below. Based on the standard curve, calculate the recombinant collagen content in the test tube, and then calculate the collagen content, recovery rate, and relative standard deviation of the recovery rate in the collagen-hyaluronic acid crosslinked gel. The results are shown in Table 21.

[0149] Recovery rate = Test concentration of collagen / Theoretical concentration of collagen 100%.

[0150] Table 20

[0151]

[0152] Table 21

[0153]

[0154] Based on the above results, it can be seen that when the concentration of collagen-hyaluronic acid crosslinking gel in the test sample solution is within a certain range... When the concentration varies within a 20% range, the ratio of the collagen test concentration to the theoretical concentration changes little, indicating that the method of this application is robust enough, ensuring the reliability of the method and that the method will not fail due to fluctuations in the concentration of the test solution.

[0155] Finally, it should be noted that the terms “comprising,” “including,” or any other variations thereof in this application are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such process, method, article, or apparatus.

[0156] Although this application has been disclosed above through the description of specific embodiments, it should be understood that those skilled in the art can devise various modifications, improvements, or equivalents to this application within the spirit and scope of the appended solutions. Such modifications, improvements, or equivalents should also be considered to be included within the scope of protection claimed in this application.

Claims

1. A method for detecting collagen content in a collagen-hyaluronic acid cross-linked gel, characterized in that, The steps include: The collagen content in the collagen-hyaluronic acid cross-linked gel is tested using the Folin-Ciocalteu method combined with the external standard method, specifically: (1) The gel is mixed and reacted with alkaline copper test solution at a temperature of 50~85℃ to prepare a test solution; wherein the concentration of sodium hydroxide in the alkaline copper test solution is 50~100mg / mL; and the concentration of the gel in the test solution is 0.02~0.1g / mL. (2) Mix the test solution with Folin-Ciocalteu solution, detect the absorbance using a UV-Vis spectrophotometer, and calculate the collagen content in the gel using a standard curve.

2. The detection method as described in claim 1, characterized in that, The detection method satisfies at least one of the following conditions (1) to (3): (1) The concentration of the gel in the test solution is 0.03~0.07 g / mL; (2) The concentration of sodium hydroxide in the alkaline copper test solution is 60~80 mg / mL; (3) The alkaline copper test solution also includes sodium carbonate, potassium sodium tartrate and copper sulfate.

3. The detection method as described in claim 2, characterized in that, The detection method satisfies at least one of the following conditions (1) to (3): (1) The concentration of sodium carbonate in the alkaline copper test solution is 70~130 mg / mL; (2) The concentration of potassium sodium tartrate in the alkaline copper test solution is 1.0~3.0 mg / mL; (3) The concentration of copper sulfate in the alkaline copper test solution is 0.3~0.8 mg / mL.

4. The detection method as described in claim 3, characterized in that, The detection method satisfies at least one of the following conditions (1) to (3): (1) The concentration of sodium carbonate in the alkaline copper test solution is 90~110 mg / mL; (2) The concentration of potassium sodium tartrate in the alkaline copper test solution is 1.5~2.5 mg / mL; (3) The concentration of copper sulfate in the alkaline copper test solution is 0.4~0.6 mg / mL.

5. The detection method according to any one of claims 1 to 4, characterized in that, The detection method satisfies at least one of the following conditions (A) to (H): (A) In step (1), the temperature of the mixing reaction is 60~80℃; (B) In step (1), the mixing reaction time is 45~75 min; (C) In step (1), the mixing reaction is carried out intermittently; (D) In ​​step (1), the preparation method of the gel includes the following steps: sodium hyaluronate and collagen are cross-linked to obtain the gel; (E) In step (2), the volume ratio of the test solution to the Folin-Ciocalteu test solution is 1:(4~6). (F) In step (2), the temperature at which the test solution is mixed with the Folin-Ciocalteu test solution is room temperature; (G) In step (2), the time for mixing the test solution with the Folin-Ciocalteu solution is 20-50 min; (H) In step (2), the detection wavelength of the ultraviolet-visible spectrophotometer is 645~655nm.

6. The detection method as described in claim 5, characterized in that, The detection method satisfies at least one of the following conditions (A) to (F): (A) In step (1), the temperature of the mixing reaction is 55~65℃. (B) In step (1), the mixing reaction time is 55~65 min; (C) In step (1), after the mixing reaction has been going on for 30 minutes, the mixture is stirred every 10 minutes. (D) In ​​step (1), the stirring method is vortexing and / or mechanical stirring; (E) In step (1), the crosslinking agent used for crosslinking is selected from BDDE or DMTMM; (F) In step (2), the volume ratio of the test solution to the Folin-Ciocalteu test solution is 1:(4~6).

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