Anti-aging and firming medical silk fibroin gel dressing and preparation method thereof

Abstract

The invention relates to an anti-aging and firming medical silk fibroin gel dressing and a preparation method thereof, and belongs to the technical field of dressings. According to the invention, a certain amount of deionized water is creatively added into a deep eutectic solvent prepared by using a surfactant hexadecyl trimethyl ammonium chloride as a hydrogen bond acceptor and xylitol as a hydrogen bond donor for dilution, so that a pretreatment agent is prepared and is applied to extraction of silk fibroin; silk is sequentially subjected to pretreatment of a pretreatment agent, degumming of a sodium carbonate aqueous solution, dissolution of a calcium chloride / ethanol / deionized water ternary dissolving agent, dialysis, centrifugation and drying operation, silk fibroin is obtained, the extraction rate is high, and the biological activity is good; irreversible damage and damage caused by high temperature of the silk fibroin in the traditional alkaline degumming process can be effectively reduced, and correspondingly, the obtained silk fibroin can also play more excellent anti-aging and firming effects after being prepared into the dressing.

Description

Technical Field

[0001] The invention belongs to the technical field of dressings, and in particular relates to an anti-aging and firming medical silk fibroin gel dressing and a preparation method thereof. Background Art

[0002] Fibroin, also known as silk protein, is a natural high-molecular fiber protein extracted from silk, accounting for about 70%-80% of silk. A large number of studies have shown that fibroin can stimulate the synthesis of collagen in skin cells to a certain extent, thereby maintaining and enhancing the firmness of the skin, reducing the appearance of wrinkles, and resisting aging. Based on this, many dressings will add fibroin to have anti-aging and firming effects. However, these dressings still have certain disadvantages that limit their actual application effects. This is because in the current industrial production, the extraction of silk fibroin mostly needs to undergo degumming first, that is, the sericin wrapped on the surface of the silk fibroin is removed, and this degumming process is basically based on simple and low-cost alkaline degumming, which needs to be carried out at a higher temperature (about 100°C). This leads to the alkaline degumming that inevitably causes certain irreversible damage and damage to the silk fibroin, which in turn causes the biological activity of the extracted silk fibroin to decrease, and ultimately seriously affects its anti-aging and firming effect in the dressing. Summary of the invention

[0003] In view of the problems existing in the prior art, the purpose of the present invention is to provide an anti-aging and firming medical silk fibroin gel dressing and a preparation method thereof. The present invention creatively adds a certain amount of deionized water to a low eutectic solvent prepared by using a surfactant, cetyltrimethylammonium chloride, as a hydrogen bond acceptor and xylitol as a hydrogen bond donor for dilution, thereby preparing a pretreatment agent, which is applied to the extraction of silk fibroin. Silk is pretreated with a pretreatment agent, degummed with an aqueous sodium carbonate solution, dissolved with a ternary solvent of calcium chloride / ethanol / deionized water, dialyzed, centrifuged, and dried in sequence to obtain silk fibroin, which has not only a high extraction rate but also good biological activity, and can effectively reduce the irreversible destruction and damage of silk fibroin caused by high temperature during traditional alkaline degumming. Correspondingly, the obtained silk fibroin can also exert a more excellent anti-aging and firming effect after being prepared into a dressing.

[0004] The purpose of the present invention can be achieved through the following technical solutions:

[0005] An anti-aging and firming medical silk fibroin gel dressing comprises deionized water, silk fibroin and carbomer.

[0006] As a preferred technical solution of the present invention, the mass ratio of the deionized water, the silk fibroin and the carbomer is 98-100:0.06:0.2-0.3.

[0007] As a preferred technical solution of the present invention, the mass ratio of the deionized water, the silk fibroin and the carbomer is 99.74:0.06:0.2.

[0008] As a preferred technical solution of the present invention, the silk fibroin is prepared by the following steps:

[0009] Step A: mixing silk and a pretreatment agent at a mass ratio of 1:1.3-1.5 at room temperature to obtain component A;

[0010] Step B: adding a sodium carbonate aqueous solution to the component A, and then stirring at 50-60° C. for 20-30 minutes to degumming, filtering, taking the filter residue, washing with deionized water, and finally vacuum drying at 50-60° C. until constant weight, to obtain component B;

[0011] Step C: adding a ternary solvent to the component B, and then stirring and mixing at 80-85° C. for 30 minutes to obtain component C;

[0012] Step D: transferring the component C into a dialysis bag, dialyzing, and centrifuging to obtain component D;

[0013] Step E: freeze-drying the component D in vacuum to complete the preparation.

[0014] Furthermore, the pretreatment agent in step A is prepared by the following steps:

[0015] Step a: stir and mix hexadecyltrimethylammonium chloride and xylitol at a molar ratio of 1:2-2.5 at 80-90° C. for 3-4 hours, cool naturally to room temperature, then add deionized water until the water content is 20-25wt%, and finally stir and mix at room temperature for 10-15 minutes to complete the preparation.

[0016] Furthermore, the stirring time in step A is 30-40 minutes.

[0017] Furthermore, the concentration of the sodium carbonate aqueous solution in step B is 2-4 g / L at 25° C.; the mass ratio of component A to the sodium carbonate aqueous solution is 1:9-10.

[0018] Furthermore, the ternary solvent in step C refers to a solution obtained by mixing calcium chloride, ethanol and deionized water in a molar ratio of 1:2-3:8-8.5 at room temperature and stirring for 15-30 minutes; the mass ratio of component B to the ternary solvent is 1:10-12.

[0019] Furthermore, the molecular weight cutoff of the dialysis bag in step D is 3500Da; the dialysis refers to dialysis using deionized water at a temperature of 5-10°C for 48-72 hours, during which the deionized water is replaced every 4-6 hours.

[0020] Furthermore, the centrifugation in step D refers to centrifugation at a speed of 10000 r / min for 5 min and taking the supernatant.

[0021] Furthermore, the vacuum freeze drying in step E refers to vacuum freeze drying at -15-20°C until constant weight is achieved.

[0022] A method for preparing an anti-aging and firming medical silk fibroin gel dressing, the method comprising the following steps:

[0023] Add carbomer to deionized water, then stir and mix at 80-90°C at 25-50 r / min for 20-30 min, cool naturally to 35-45°C and keep warm, add silk fibroin, stir and mix at 25-50 r / min for 10-15 min, cool naturally to room temperature, and the preparation is complete.

[0024] Beneficial effects of the present invention:

[0025] (1) The present invention creatively adds a certain amount of deionized water to a low eutectic solvent prepared by using the surfactant hexadecyltrimethylammonium chloride as a hydrogen bond acceptor and xylitol as a hydrogen bond donor for dilution, thereby preparing a pretreatment agent for use in the extraction of silk fibroin. The silk is pretreated with the pretreatment agent, degummed with an aqueous sodium carbonate solution, dissolved with a ternary solvent of calcium chloride / ethanol / deionized water, dialyzed, centrifuged, and dried to obtain silk fibroin. The silk fibroin has a high extraction rate and good biological activity, and can effectively reduce the irreversible destruction and damage of the silk fibroin caused by high temperature during traditional alkaline degumming. Correspondingly, the obtained silk fibroin can also exert a more excellent anti-aging and firming effect after being prepared into a dressing.

[0026] (2) The present invention creatively adds a certain amount of deionized water to a low eutectic solvent prepared by using the surfactant hexadecyltrimethylammonium chloride as a hydrogen bond acceptor and xylitol as a hydrogen bond donor for dilution, thereby preparing a pretreatment agent for use in the extraction of silk fibroin. On the one hand, the low eutectic solvent contains a large amount of hydrogen bond network, which can be combined with sericin through hydrogen bonds, thereby weakening the interaction between the molecular structures of the sericin itself, and then forming a more "relaxed" and "loose" protein structure, which is convenient for subsequent degumming. On the other hand, since the low eutectic solvent uses the surfactant as a hydrogen bond acceptor, when the sodium carbonate aqueous solution is directly added for degumming, the structure of the low eutectic solvent will be destroyed due to the presence of a large amount of deionized water, thereby achieving the dissociation of the surfactant. At this time, the dissociated surfactant can play a role in reducing the surface tension of the medium in the entire system, thereby obtaining a better degumming effect. At the same time, it can also achieve the purpose of removing the oil contained in the silk itself, further promoting the degumming of the silk by sodium carbonate.

[0027] (3) Compared with the prior art, the present invention can still obtain silk fibroin with high extraction rate and good biological activity even if alkaline degumming is performed at a lower temperature and in a shorter time, thereby effectively reducing the irreversible destruction and damage of silk fibroin caused by high temperature during degumming. DETAILED DESCRIPTION

[0028] In order to further illustrate the technical means and effects adopted by the present invention to achieve the predetermined invention purpose, the specific implementation methods, structures, features and effects of the present invention are described in detail below in combination with the embodiments.

[0029] The silk in all the embodiments and comparative examples of the present invention is mulberry silk; the carbomer is purchased directly from the market, purchased from Hubei Xindesheng Material Technology Co., Ltd., and the model is Carbomer 940.

[0030] Example 1

[0031] An anti-aging and firming medical silk fibroin gel dressing comprises deionized water, silk fibroin and carbomer.

[0032] The mass ratio of the deionized water, the silk fibroin and the carbomer is 98:0.06:0.2.

[0033] The silk fibroin is prepared by the following steps:

[0034] Step A: mixing silk and a pretreatment agent at a mass ratio of 1:1.3 at room temperature to obtain component A;

[0035] Step B: adding a sodium carbonate aqueous solution to the component A, and then stirring at 50° C. for 20 minutes to degumming, filtering, taking the filter residue, washing with deionized water, and finally vacuum drying at 50° C. until constant weight, to obtain component B;

[0036] Step C: adding a ternary solvent to the component B, and then stirring and mixing at 80° C. for 30 minutes to obtain component C;

[0037] Step D: transferring the component C into a dialysis bag, dialyzing, and centrifuging to obtain component D;

[0038] Step E: freeze-drying the component D in vacuum to complete the preparation.

[0039] The pretreatment agent described in step A is prepared by the following steps:

[0040] Step a: stir and mix hexadecyltrimethylammonium chloride and xylitol at a molar ratio of 1:2 at 80° C. for 3 h, cool naturally to room temperature, then add deionized water until the water content is 20 wt %, and finally stir and mix at room temperature for 10 min to complete the preparation.

[0041] The stirring time in step A is 30 min.

[0042] The concentration of the sodium carbonate aqueous solution in step B is 2 g / L at 25° C.; the mass ratio of component A to the sodium carbonate aqueous solution is 1:9.

[0043] The ternary solvent in step C refers to a solution obtained by mixing calcium chloride, ethanol and deionized water in a molar ratio of 1:2:8 at room temperature and stirring for 15 minutes; the mass ratio of component B to the ternary solvent is 1:10.

[0044] The molecular weight cutoff of the dialysis bag in step D is 3500Da; the dialysis refers to dialysis using deionized water at a temperature of 5°C for 48 hours, during which the deionized water is replaced every 4 hours.

[0045] The centrifugation in step D refers to centrifugation at a speed of 10000 r / min for 5 min and taking the supernatant.

[0046] The vacuum freeze drying in step E refers to vacuum freeze drying at -15°C until constant weight.

[0047] A method for preparing an anti-aging and firming medical silk fibroin gel dressing, the method comprising the following steps:

[0048] Carbomer was added to deionized water, and then stirred at 25 r / min for 20 min at 80° C., and then naturally cooled to 35° C. and kept warm. Silk fibroin was added, and then stirred at 25 r / min for 10 min. The mixture was naturally cooled to room temperature, and the preparation was completed.

[0049] Example 2

[0050] An anti-aging and firming medical silk fibroin gel dressing comprises deionized water, silk fibroin and carbomer.

[0051] The mass ratio of the deionized water, the silk fibroin and the carbomer is 100:0.06:0.3.

[0052] The silk fibroin is prepared by the following steps:

[0053] Step A: mixing silk and a pretreatment agent at a mass ratio of 1:1.5 at room temperature to obtain component A;

[0054] Step B: adding a sodium carbonate aqueous solution to the component A, and then stirring at 60° C. for 30 minutes to degumming, filtering, taking the filter residue, washing with deionized water, and finally vacuum drying at 60° C. until constant weight, to obtain component B;

[0055] Step C: adding a ternary solvent to the component B, and then stirring and mixing at 85° C. for 30 minutes to obtain component C;

[0056] Step D: transferring the component C into a dialysis bag, dialyzing, and centrifuging to obtain component D;

[0057] Step E: freeze-drying the component D in vacuum to complete the preparation.

[0058] The pretreatment agent described in step A is prepared by the following steps:

[0059] Step a: stir and mix hexadecyltrimethylammonium chloride and xylitol at a molar ratio of 1:2.5 at 90° C. for 4 h, cool naturally to room temperature, then add deionized water until the water content is 25 wt %, and finally stir and mix at room temperature for 15 min to complete the preparation.

[0060] The stirring time in step A is 40 min.

[0061] The concentration of the sodium carbonate aqueous solution in step B is 4 g / L at 25° C.; the mass ratio of component A to the sodium carbonate aqueous solution is 1:10.

[0062] The ternary solvent in step C refers to a solution obtained by mixing calcium chloride, ethanol and deionized water in a molar ratio of 1:3:8.5 at room temperature and stirring for 30 minutes; the mass ratio of component B to the ternary solvent is 1:12.

[0063] The molecular weight cutoff of the dialysis bag in step D is 3500Da; the dialysis is performed using deionized water at a temperature of 10°C for 72 hours, during which the deionized water is replaced every 6 hours.

[0064] The centrifugation in step D refers to centrifugation at a speed of 10000 r / min for 5 min and taking the supernatant.

[0065] The vacuum freeze drying in step E refers to vacuum freeze drying at -20°C until constant weight.

[0066] A method for preparing an anti-aging and firming medical silk fibroin gel dressing, the method comprising the following steps:

[0067] Carbomer was added to deionized water, and then stirred at 50 r / min for 30 min at 90° C., and then naturally cooled to 45° C. and kept warm. Silk fibroin was added, and then stirred at 50 r / min for 15 min. The mixture was naturally cooled to room temperature, and the preparation was completed.

[0068] Example 3

[0069] An anti-aging and firming medical silk fibroin gel dressing comprises deionized water, silk fibroin and carbomer.

[0070] The mass ratio of the deionized water, the silk fibroin and the carbomer is 99.74:0.06:0.2.

[0071] The silk fibroin is prepared by the following steps:

[0072] Step A: mixing silk and a pretreatment agent at a mass ratio of 1:1.4 at room temperature to obtain component A;

[0073] Step B: adding a sodium carbonate aqueous solution to the component A, and then stirring at 55° C. for 25 minutes to degumming, filtering, taking the filter residue, washing with deionized water, and finally vacuum drying at 55° C. until constant weight, to obtain component B;

[0074] Step C: adding a ternary solvent to the component B, and then stirring and mixing at 83° C. for 30 minutes to obtain component C;

[0075] Step D: transferring the component C into a dialysis bag, dialyzing, and centrifuging to obtain component D;

[0076] Step E: freeze-drying the component D in vacuum to complete the preparation.

[0077] The pretreatment agent described in step A is prepared by the following steps:

[0078] Step a: stir and mix hexadecyltrimethylammonium chloride and xylitol at a molar ratio of 1:2.3 at 85° C. for 3.5 hours, cool naturally to room temperature, then add deionized water until the water content is 23 wt %, and finally stir and mix at room temperature for 13 minutes to complete the preparation.

[0079] The stirring time in step A is 35 min.

[0080] The concentration of the sodium carbonate aqueous solution in step B is 3 g / L at 25° C.; the mass ratio of component A to the sodium carbonate aqueous solution is 1:9.5.

[0081] The ternary solvent in step C refers to a solution obtained by mixing calcium chloride, ethanol and deionized water in a molar ratio of 1:2.5:8.3 at room temperature and stirring for 20 minutes; the mass ratio of component B to the ternary solvent is 1:11.

[0082] The molecular weight cutoff of the dialysis bag in step D is 3500Da; the dialysis is performed using deionized water at a temperature of 8°C for 60 hours, during which the deionized water is replaced every 5 hours.

[0083] The centrifugation in step D refers to centrifugation at a speed of 10000 r / min for 5 min and taking the supernatant.

[0084] The vacuum freeze drying in step E refers to vacuum freeze drying at -18°C until constant weight.

[0085] A method for preparing an anti-aging and firming medical silk fibroin gel dressing, the method comprising the following steps:

[0086] Carbomer was added to deionized water, and then stirred at 40 r / min for 25 min at 85° C., and then naturally cooled to 40° C. and kept warm. Silk fibroin was added, and then stirred at 30 r / min for 13 min. The mixture was naturally cooled to room temperature, and the preparation was completed.

[0087] Comparative Example 1

[0088] On the basis of Example 3, the water content in step a was changed from 23 wt % to 30 wt %, and the rest remained unchanged.

[0089] Comparative Example 2

[0090] Based on Example 3, deionized water was not added in step a, and the rest remained unchanged.

[0091] Comparative Example 3

[0092] On the basis of Example 3, step A is changed to adding a pretreatment agent to the silk at a mass ratio of 1:1.4 to obtain component A; the rest remain unchanged.

[0093] Comparative Example 4

[0094] On the basis of Example 3, step A is changed to stirring and mixing silk and deionized water at a mass ratio of 1:1.4 at room temperature to obtain component A; the rest remain unchanged.

[0095] Comparative Example 5

[0096] On the basis of Example 3, the degumming process of stirring for 25 min in step B was changed to stirring for 40 min, and the rest remained unchanged.

[0097] Comparative Example 6

[0098] On the basis of Example 3, step A is changed to stirring and mixing silk and deionized water at a mass ratio of 1:1.4 at room temperature to obtain component A; at the same time, the degumming process at 55°C for 25 min in step B is changed to degumming process at 95°C for 25 min, and the rest remain unchanged.

[0099] Test Example 1

[0100] Extraction rate statistics:

[0101] The extraction rates of the silk fibroin prepared in Example 3 and Comparative Examples 1-6 were respectively counted.

[0102] Table 1. Statistical results of extraction rate

[0103]

[0104]

[0105] Test Example 2

[0106] Skin firmness test:

[0107] Here are the steps:

[0108] (1) A total of 140 volunteers (aged 40-45 years, female, with nasolabial folds graded at level 4 according to the WSRS) were randomly divided into 7 groups, with 20 participants in each group.

[0109] (2) Each group was smeared with the medical silk fibroin gel dressing prepared in Example 3 and Comparative Examples 1-6, and the smearing position was all on the left side of the face (the right side of the face was the blank group, and deionized water was applied instead), and the smearing amount was 5 mg / cm 2 , apply continuously for 90 days, once a day.

[0110] (3) Five professionals were selected to evaluate each volunteer based on the improvement effect of nasolabial folds. The full score was 5 points, and the score was rounded to the nearest integer. 5 points corresponded to very obvious, 4 points corresponded to relatively obvious, 3 points corresponded to average, 2 points corresponded to not very obvious, and 1 point corresponded to not obvious (the score of each professional in the same group was first averaged separately, and then the average of the five means was continued to be taken to obtain the final evaluation result, retaining one decimal place).

[0111] Table 2. Skin firmness test results

[0112] Evaluation results Example 3 4.3 Comparative Example 1 4.1 Comparative Example 2 4.1 Comparative Example 3 4.2 Comparative Example 4 4.3 Comparative Example 5 3.7 Comparative Example 6 3.8

[0113] Comparison between Example 3 and Comparative Examples 1-6 shows that:

[0114] The difference between Comparative Example 1 and Example 3 is that the water content of the pretreatment agent is increased.

[0115] The difference between Comparative Example 2 and Example 3 is that the pretreatment agent does not contain water.

[0116] The difference between Comparative Example 3 and Example 3 is that the subsequent steps are directly carried out after the pretreatment agent is added to the silk.

[0117] The difference between Comparative Example 4 and Example 3 is that the pretreatment agent is replaced by an equal weight of deionized water.

[0118] The difference between Comparative Example 5 and Example 3 is that the degumming time is prolonged.

[0119] The difference between Comparative Example 6 and Example 3 is that the pretreatment agent is changed to deionized water of equal weight, and at the same time, the degumming temperature is increased.

[0120] From the comparison of test examples 1-2, embodiment 3 and comparative examples 1-6, it can be seen that only the silk fibroin prepared by the present invention not only has a high extraction rate but also has good biological activity. After being prepared into a dressing, it can better exert the effect of tightening the skin, improving wrinkles and resisting aging.

[0121] The above description is only a preferred embodiment of the present invention and does not limit the present invention in any form. Although the present invention has been disclosed as a preferred embodiment as above, it is not used to limit the present invention. Any technical personnel in this field can make some changes or modify the technical contents disclosed above into equivalent embodiments without departing from the scope of the technical solution of the present invention. However, any brief modifications, equivalent changes and modifications made to the above embodiments based on the technical essence of the present invention without departing from the content of the technical solution of the present invention are still within the scope of the technical solution of the present invention.

Claims

1. An anti-aging and firming medical silk fibroin gel dressing, characterized in that: The medical silk fibroin gel dressing comprises deionized water, silk fibroin and carbomer.

2. The anti-aging and firming medical silk fibroin gel dressing according to claim 1, characterized in that: The mass ratio of the deionized water, the silk fibroin and the carbomer is 98-100:0.06:0.2-0.

3.

3. The anti-aging and firming medical silk fibroin gel dressing according to claim 1, characterized in that: The mass ratio of the deionized water, the silk fibroin and the carbomer is 99.74:0.06:0.

2.

4. The anti-aging and firming medical silk fibroin gel dressing according to claim 1, characterized in that: The silk fibroin is prepared by the following steps: Step A: mixing silk and a pretreatment agent at a mass ratio of 1:1.3-1.5 at room temperature to obtain component A; Step B: adding a sodium carbonate aqueous solution to the component A, and then stirring at 50-60° C. for 20-30 minutes to degumming, filtering, taking the filter residue, washing with deionized water, and finally vacuum drying at 50-60° C. until constant weight, to obtain component B; Step C: adding a ternary solvent to the component B, and then stirring and mixing at 80-85° C. for 30 minutes to obtain component C; Step D: transferring the component C into a dialysis bag, dialyzing, and centrifuging to obtain component D; Step E: freeze-drying the component D in vacuum to complete the preparation.

5. The anti-aging and firming medical silk fibroin gel dressing according to claim 4, characterized in that: The pretreatment agent described in step A is prepared by the following steps: Step a: stir and mix hexadecyltrimethylammonium chloride and xylitol at a molar ratio of 1:2-2.5 at 80-90° C. for 3-4 hours, cool naturally to room temperature, then add deionized water until the water content is 20-25wt%, and finally stir and mix at room temperature for 10-15 minutes to complete the preparation.

6. The anti-aging and firming medical silk fibroin gel dressing according to claim 4, characterized in that: The concentration of the sodium carbonate aqueous solution in step B is 2-4 g / L at 25° C.; the mass ratio of component A to the sodium carbonate aqueous solution is 1:9-10.

7. The anti-aging and firming medical silk fibroin gel dressing according to claim 4, characterized in that: The ternary solvent in step C refers to a solution obtained by mixing calcium chloride, ethanol and deionized water at a molar ratio of 1:2-3:8-8.5 at room temperature and stirring for 15-30 minutes; the mass ratio of component B to the ternary solvent is 1:10-12.

8. The anti-aging and firming medical silk fibroin gel dressing according to claim 4, characterized in that: The molecular weight cutoff of the dialysis bag in step D is 3500Da; the dialysis refers to dialysis using deionized water at a temperature of 5-10°C for 48-72 hours, during which the deionized water is replaced every 4-6 hours.

9. The anti-aging and firming medical silk fibroin gel dressing according to claim 4, characterized in that: The vacuum freeze drying in step E refers to vacuum freeze drying at -15-20°C until constant weight.

10. A method for preparing the anti-aging and firming medical silk fibroin gel dressing according to any one of claims 1 to 9, characterized in that: The preparation method comprises the following steps: Add carbomer to deionized water, then stir and mix at 80-90°C at 25-50 r / min for 20-30 min, cool naturally to 35-45°C and keep warm, add silk fibroin, stir and mix at 25-50 r / min for 10-15 min, cool naturally to room temperature, and the preparation is complete.

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