A rapid preparation method of a colored sericin hydrogel and a product

Abstract

The present application provides a kind of quick preparation method and product of colored cocoon sericin protein hydrogel, and the preparation method is: by boiling water heating treatment extraction regenerated silk fibroin aqueous solution, after pre-cooling, freeze drying to obtain colored cocoon regenerated silk fibroin powder;Finally, colored cocoon regenerated silk fibroin powder is placed in water and heated to dissolve and continue heating to evaporate moisture to set value, after cooling and standing, it can be used.The whole preparation process does not add any chemical reagent, green environmental protection, low energy consumption, high biological safety, low price, simple operation is convenient, fast, efficient.The concentration of colored cocoon silk fibroin hydrogel prepared by the preparation method can reach more than 5%, has excellent mechanical properties, can better retain colored cocoon functional pigment ingredients, so that the hydrogel has antibacterial, antioxidant and other effects, further improve the application value of silk fibroin hydrogel.The colored cocoon silk fibroin hydrogel prepared by the present application can be applied to drug carrier, wound dressing, bone tissue repair and the like.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of biological medical materials, and particularly relates to a rapid preparation method of a colored cocoon sericin protein hydrogel and a product. BACKGROUND

[0002] Sericin is an important component of silk fibers, which plays a role in binding and protecting fibroin fibers, and its proportion in silk is about 20-30%. In addition, sericin also has the functions of moisturizing, anti-ultraviolet, antioxidant, etc., and is widely used in the fields of cosmetics, food, biological medicine, etc.

[0003] Hydrogel is a three-dimensional network structure gel with high hydrophilicity, and has excellent mechanical toughness, biocompatibility, degradability, etc. In recent years, it is favored in the fields of tumor treatment, cartilage repair, wound dressing, etc. At present, due to the high molecular weight characteristics of domestic silk fibroin, hydrogels can be prepared by physical, chemical, biological and other ways. However, due to the characteristics of high proportion of hydrophilic amino acids and uneven molecular weight distribution, the silk sericin of domestic silkworm often forms cross-linking at a low concentration (0.1%), which leads to poor mechanical properties of the gel and is difficult to shape. Therefore, the reported silk sericin hydrogels are mostly prepared by compounding with other polymers or chemical cross-linking, which limits their use.

[0004] On the other hand, natural colored cocoon is a new variety of multi-color cocoon produced by silkworms due to genetic variation and artificial breeding. Compared with traditional white cocoon, colored cocoon contains a higher content of flavonoids, carotenoids (such as lutein), etc. Current studies have shown that flavonoids are a very strong antioxidant and antibacterial agent, which can effectively scavenge oxygen free radicals in the body, thereby preventing cell degeneration and aging; in addition to the same antioxidant effect, carotenoids also have good anti-ultraviolet effect. Therefore, colored cocoon silk protein has more functions. However, there is no report on the rapid and effective preparation of hydrogel based on colored cocoon extracted silk sericin. SUMMARY

[0005] In order to overcome the deficiencies in the prior art that the preparation of silk sericin-based hydrogel destroys the natural structure of silk sericin, is difficult to shape, and has a complex process, and to expand the application of natural colored cocoon silk sericin, the present application provides a simple and rapid method for preparing colored cocoon silk sericin hydrogel. The preparation method not only has a simple process, mild and environmentally friendly reaction conditions; the silk sericin hydrogel prepared by the preparation method has controllable mechanical properties, good biocompatibility, and retains functional pigment components of colored cocoon, and can be used in drug carriers, wound dressings and other medical fields.

[0006] A rapid preparation method of colored cocoon silk sericin hydrogel, comprising the following steps:

[0007] (1) The natural colored cocoon shell is cut, washed, heated in boiling water, cooled, filtered, centrifuged, and the supernatant is taken to obtain a regenerated silk fibroin aqueous solution;

[0008] (2) The regenerated silk fibroin aqueous solution is pre-cooled and then freeze-dried to obtain a colored cocoon regenerated silk fibroin powder.

[0009] (3) The colored cocoon regenerated silk fibroin powder is dissolved in deionized water by heating and stirring until the solution is clear, and the solution is heated until the volume reaches a set value, cooled and left to stand to obtain the colored cocoon silk fibroin hydrogel.

[0010] In the above step (1), the boiling water used is boiling deionized water.

[0011] In the above step (3), after the colored cocoon regenerated silk fibroin powder is completely dissolved in deionized water by heating and stirring, the solution is continuously heated until the volume of the solution is reduced to a concentration of silk fibroin in the solution reaching a set value, the heating is stopped and the solution is naturally cooled, and then left to stand at room temperature for 1-2 h to obtain the colored cocoon silk fibroin hydrogel. In this process, by controlling the amount of water evaporation, a high-concentration natural colored cocoon silk fibroin hydrogel with a concentration of 2-10% can be obtained, and the obtained hydrogel can be used as a tumor drug carrier, cartilage repair, and wound dressing. By calculating the amount of colored cocoon regenerated silk fibroin powder added and the set value of the final solution volume, the concentration of silk fibroin in the hydrogel can be determined.

[0012] In the above preparation method, the regenerated silk fibroin aqueous solution extracted by water bath heating is freeze-dried to prepare a colored cocoon regenerated silk fibroin powder, and then the silk fibroin powder is dissolved in deionized water to form a silk fibroin solution, and the water is continuously evaporated by heating and the solution is cooled and left to stand to form a colored cocoon silk fibroin hydrogel. The concentration of silk fibroin in the final hydrogel is controlled by controlling the amount of water evaporated during the continuous heating process. This preparation method can obtain a hydrogel with high and controllable silk fibroin concentration, which has good forming and excellent mechanical properties; and no crosslinking agent is needed in the preparation process, the process is simple, the reaction conditions are mild and environmentally friendly.

[0013] Preferably, in step (1), the natural colored cocoon is one or more of yellow cocoon, green cocoon, pink cocoon, and red cocoon. Further preferably, it is one of yellow cocoon, green cocoon, pink cocoon, and red cocoon. More preferably, it is one of yellow cocoon and pink cocoon.

[0014] Preferably, in step (1), the mass-to-volume ratio of the natural colored cocoon shell to water is 1:(50-200) g / mL.

[0015] As further preferred, when the natural colored cocoon is green cocoon, the mass-volume ratio of the green cocoon shell to water is 1:200 g / mL; when the natural colored cocoon is yellow cocoon, the mass-volume ratio of the yellow cocoon shell to water is 1:(50-200) g / mL; when the natural colored cocoon is pink or red cocoon, the mass-volume ratio of the pink or red cocoon shell to water is 1:50 g / mL.

[0016] As preferred, in step (1), the heating treatment time of the natural colored cocoon shell in boiling water is 0.5-1.5 h.

[0017] As further preferred, when the natural colored cocoon is green cocoon, the heating treatment time of the green cocoon shell in boiling water is 1-1.5 h; when the natural colored cocoon is yellow cocoon, the heating treatment time of the yellow cocoon shell in boiling water is 0.5-1.5 h; when the natural colored cocoon is pink or red cocoon, the heating treatment time of the pink or red cocoon shell in boiling water is 0.5 h.

[0018] In actual preparation, the regenerated silk fibroin aqueous solution with different concentrations can be obtained by adjusting the mass-volume ratio of the cocoon shell to boiling water and the boiling time.

[0019] As preferred, in step (1), the centrifugal speed is 6000-10000 rpm and the centrifugal time is 5-15 min. As further preferred, in step (1), the centrifugal speed is 8000 rpm and the centrifugal time is 10 min.

[0020] As preferred, in step (2), the temperature of the pre-cooling treatment is -60--100℃. Further preferred is -80℃. The pre-cooling treatment can be carried out in a refrigerator.

[0021] As preferred, in step (2), the pre-cooling treatment time is 12-24 h.

[0022] As preferred, in step (2), the freeze-drying time is 40-60 h. Further preferred is 48 h.

[0023] As preferred, in step (3), the mass-volume ratio of the colored cocoon regenerated silk fibroin powder to deionized water is 1:(40-100) g / mL. Further preferred is 1:(40-60) g / mL.

[0024] As preferred, in step (3), the heating temperature is 50-100℃.

[0025] As further preferred, when the natural colored cocoon is green or yellow cocoon, the heating temperature is 80-100℃; when the natural colored cocoon is pink or red cocoon, the heating temperature is 50-60℃.

[0026] In step (3), the specific heating time can be adjusted according to the concentration of sericin in the desired hydrogel.

[0027] The rapid preparation method of the colored cocoon sericin hydrogel of the present application comprises the following steps:

[0028] The natural colored cocoon shells are cut, washed, and subjected to boiling and heating treatment in deionized water, and the supernatant is obtained by filtration and centrifugation to obtain a regenerated sericin aqueous solution; the regenerated sericin aqueous solution is subjected to pre-cooling treatment at -80 DEG C, and then freeze-dried to obtain a colored cocoon sericin powder; the colored cocoon sericin powder is placed in deionized water and heated, and after the sericin is completely dissolved into a clear and transparent solution, the water is further evaporated by heating to adjust the concentration of sericin in the solution; and finally, after cooling and standing, a high-concentration colored cocoon sericin hydrogel is obtained.

[0029] The preparation method of the present application is simple, convenient to operate, and free of any chemical reagents, and is green and environmentally friendly. The hydrogel prepared by the preparation method has a controllable concentration, excellent mechanical properties, and retains functional components of colored cocoon pigments, and has potential application value in the fields of drug carriers and tissue repair.

[0030] A colored cocoon sericin hydrogel is prepared by the rapid preparation method of the colored cocoon sericin hydrogel of any one of the above.

[0031] Compared with the prior art, the present application has the following beneficial effects:

[0032] The rapid preparation method of the colored cocoon sericin hydrogel of the present application extracts a regenerated sericin aqueous solution by boiling water heating treatment, and then obtains a colored cocoon regenerated sericin powder by pre-cooling and freeze-drying; finally, the colored cocoon regenerated sericin powder is placed in water and heated to dissolve, and the water is further evaporated by heating to a set value, and then cooled and stood. The entire preparation process does not add any chemical reagents, is green and environmentally friendly, has low energy consumption, high biological safety, low price, and is simple, convenient, fast, efficient. The colored cocoon sericin hydrogel prepared by the preparation method has a concentration of more than 5%, excellent mechanical properties, and can well retain functional pigment components of colored cocoon, so that the hydrogel has the effects of antibiosis and oxidation resistance, and further improves the application value of the sericin hydrogel. The colored cocoon sericin hydrogel prepared by the present application can be applied to drug carriers, wound dressings, bone tissue repair, etc. BRIEF DESCRIPTION OF DRAWINGS

[0033] Figure 1 The physical pictures of the yellow cocoon sericin hydrogel prepared in Example 1 and the white cocoon sericin hydrogel prepared in the comparative example;

[0034] Figure 2Figure for rheological mechanics performance test of the yellow cocoon silk fibroin hydrogel prepared in Example 1;

[0035] Figure 3 Figure for lutein release curve of the yellow cocoon silk fibroin hydrogel with different concentrations;

[0036] Figure 4 Figure for scanning electron microscope cross-section of the powder cocoon silk fibroin hydrogel prepared in Example 2;

[0037] Figure 5 Figure for rheological mechanics performance test of the powder cocoon silk fibroin hydrogel prepared in Example 2. DETAILED DESCRIPTION

[0038] The technical solutions of the present application are further described below in combination with specific examples:

[0039] Example 1

[0040] 10 g of natural yellow cocoon shell was weighed, cut, washed, and then poured into 1000 mL of boiling deionized water, and continued to be heated and boiled for 1 h. After cooling, the filtrate was filtered with gauze. The filtrate was centrifuged at 8000 rpm for 10 min, and the supernatant was taken to obtain a regenerated silk fibroin aqueous solution.

[0041] The regenerated silk fibroin aqueous solution was pre-cooled at -80°C for 12 h, and then placed in a freeze-drying instrument for 48 h to obtain a natural yellow cocoon silk fibroin powder.

[0042] 1 g of the obtained natural yellow cocoon silk fibroin powder was taken and placed in 50 mL of deionized water, and heated to control the temperature at 90°C. The natural yellow cocoon silk fibroin powder was continuously stirred during the heating process until it was completely dissolved. Then the heating was continued to evaporate the water, until the volume of the solution was 25 mL. The heating was stopped and the solution was naturally cooled, and was left to stand for 1 h to obtain a natural yellow cocoon silk fibroin hydrogel with a concentration of 4%.

[0043] Comparative Example

[0044] According to the steps in Example 1, a white cocoon silk fibroin hydrogel was prepared, with the difference that the natural yellow cocoon in Example 1 was replaced by white cocoon, to obtain a white cocoon silk fibroin hydrogel with a concentration of 2%.

[0045] The 4% natural yellow cocoon silk fibroin hydrogel prepared in Example 1 and the 2% white cocoon silk fibroin hydrogel prepared in the comparative example were respectively placed in a container and allowed to stabilize. Then the container was inverted, and the forming stability of the two was observed, and the results are shown in Figure 1 Figure 1 The left side of Figure in Example 2 is a real object diagram of the white cocoon silk fibroin hydrogel prepared in Comparative Example 1, Figure 1 ​The white cocoon sericin hydrogel is milky white; since the solubility of the white cocoon sericin freeze-dried powder in deionized water is poor, the preparation method in Example 1 can only obtain a white cocoon sericin gel solution with a concentration of 2%, and the gel stability is poor, resulting in visible solution dripping. Figure 1 The right side of the figure is the yellow cocoon sericin hydrogel prepared in Example 1. Figure 1 It can be seen that the yellow cocoon sericin hydrogel is yellow in color and has good stability, and remains stable after being inverted.

[0046] In addition, a rheometer was used to test the mechanical properties of the yellow cocoon sericin hydrogel prepared in Example 1. Before cooling and standing, the 4% yellow cocoon sericin hydrogel was poured into a cylindrical mold with an inner diameter of 25 mm and a height of 1 mm. After cooling and standing, the gel was placed on the rheometer test platform. A PP25 rotor was used to perform strain scanning at a temperature of 25°C and a strain range of 0.1% to 100%. The elastic / viscous modulus-strain curve was recorded. The rheological test results are shown in Figure 2. Figure 2 As shown. Figure 2 It can be seen that the storage modulus G' of 4% yellow cocoon sericin hydrogel can reach 10 4 Pa, it can still maintain the gel state under the condition of 10% deformation (G'>G"), and after the deformation is greater than 10%, the storage modulus G' gradually decreases and the loss modulus G" gradually increases, indicating that the 4% yellow cocoon sericin hydrogel prepared in Example 1 has excellent mechanical properties and rheological properties.

[0047] An in vitro simulated release experiment was used to verify whether the colored cocoon sericin hydrogel preserved the functional components of sericin: 1 mL of the colored cocoon sericin hydrogel (hydrogel concentrations of 4% and 6%) before cooling and standing was taken with a pipette and injected into a 2 ml centrifuge tube, which was sealed tightly. After the gel was cooled and allowed to stand, PBS was used as the dissolution medium to simulate the drug release process of the colored cocoon sericin hydrogel after injection into the body. During this period, the centrifuge tube was placed at room temperature and in a 37°C constant temperature box under continuous shaking. Samples were taken at regular intervals (3h, 6h, 9h, 12h, 24h, 36h) and added to a 96-well plate. The absorbance was measured using an enzyme marker to calculate the release of the main functional component, lutein. The results are as follows: Figure 3 As shown. Figure 3 It can be seen that as time goes by, the lutein in the hydrogel can be released stably, and the total release amount can reach 1 mg / mL after 24 hours of release, and the 6% gel has a higher release amount than the 4% gel. Figure 1 All of these indicate that the colored cocoon sericin hydrogel maintains the original functional pigment components of the colored cocoon during the preparation process.

[0048] The 4% gel is the 4% yellow cocoon sericin hydrogel prepared in Example 1.

[0049] 6% gel was prepared in the same way as in Example 1, except that after the natural yellow cocoon silk sericin powder was completely dissolved in deionized water, heating was continued until the volume of the solution was 16.6 mL, heating was stopped and the solution was naturally cooled, and the solution was left to stand for 1 h to obtain a 6% yellow cocoon silk sericin hydrogel.

[0050] Example 2

[0051] A natural pink cocoon shell was weighed at 10 g, cut, washed, and then added to 500 mL of boiling deionized water, and boiling was continued for 0.5 h. After the solution was cooled, it was filtered with gauze. The filtrate was centrifuged at 8000 rpm for 10 min, and the supernatant was taken as a silk sericin aqueous solution.

[0052] The above regenerated silk sericin aqueous solution was pre-cooled at -80°C for 24 h, and then placed in a freeze-drying instrument for 48 h to obtain a natural pink cocoon silk sericin powder.

[0053] A natural pink cocoon silk sericin powder was weighed at 2 g, and placed in 80 mL of deionized water, and heated to dissolve the powder at 55°C. The water was evaporated, and the remaining volume of deionized water was controlled at 20 mL. The solution was left to stand for 1 h after being naturally cooled to obtain a 10% natural pink cocoon silk sericin hydrogel.

[0054] The scanning electron microscope image of the pink cocoon silk sericin hydrogel prepared in this example is shown in Figure 4 From Figure 4 it can be seen that the natural pink cocoon silk sericin hydrogel prepared in this example has a clear hydrogel characteristic of a three-dimensional porous structure.

[0055] At the same time, the same method as in Example 1 was used to test the mechanical properties of the hydrogel, and the results showed (as shown in Figure 5 ) that the pink cocoon silk sericin hydrogel also had excellent mechanical properties and rheological properties.

[0056] Finally, it should be noted that the above examples are only specific examples of the present application. Obviously, the present application is not limited to the above examples, and many variations are possible. All variations that can be directly derived or inferred from the disclosure of the present application by those of ordinary skill in the art should be considered to be within the scope of the present application.

Claims

1. A method for rapid preparation of a coloured sericin hydrogel, characterized in that, The method comprises the following steps: (1) cutting and washing natural colored cocoon shells, heating treating in boiling water, cooling, filtering, centrifuging, taking supernatant, and obtaining regenerated sericin protein aqueous solution; (2) pre-cooling the regenerated sericin protein aqueous solution, and freeze-drying to obtain colored cocoon regenerated sericin protein powder; (3) putting the colored cocoon regenerated sericin protein powder into deionized water, heating and stirring to dissolve until the solution is clear, continuing to heat until the volume of the solution reaches a set value, cooling and standing to obtain the colored cocoon sericin hydrogel. In step (3), the mass-volume ratio of the colored cocoon regenerated sericin protein powder to deionized water is 1: (40-100) g / mL.

2. The process for rapid production of colored sericin protein hydrogel according to claim 1, characterized in that, In step (1), the natural colored cocoon is one or more of yellow cocoon, green cocoon, pink cocoon and red cocoon.

3. The process for rapid production of colored sericin protein hydrogel according to claim 1, characterized in that, In step (1), the mass-volume ratio of the natural colored cocoon shell to water is 1: (50-200) g / mL.

4. The process for rapid preparation of colored sericin protein hydrogel according to claim 1, characterized in that, In step (1), the heating treatment time of the natural colored cocoon shell in boiling water is 0.5-1.5 h.

5. The process for rapid production of colored sericin protein hydrogel according to claim 1, characterized in that, In step (2), the pre-cooling temperature is-60--100 ℃. The freeze-drying time is 40-60 h.

6. The process for rapid preparation of colored sericin protein hydrogel according to claim 1, characterized in that, In step (3), the heating temperature is 50-100 ℃.

7. A coloured sericin hydrogel, characterized in that, Prepared by the rapid preparation method of the colored cocoon sericin hydrogel according to any one of claims 1-6.

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