Silk protein-lecithin inclusion and preparation method thereof

Abstract

The invention relates to the technical field of cosmetic raw materials, in particular to a fibroin-lecithin inclusion and a preparation method thereof.The preparation method is characterized by comprising the following steps that S1, a fibroin aggregate aqueous solution and lecithin are obtained, the lecithin and the fibroin aggregate aqueous solution are mixed, heating and slow stirring are conducted, and a fibroin-lecithin inclusion solution is obtained; obtaining a first mixed solution; s2, adding an active matter into the cooled first mixed solution, and stirring to a transparent state to obtain a target product, wherein the melting point of the lecithin is not higher than 100 DEG C, the mass ratio of the silk fibroin aggregate to the lecithin in the first mixed solution is (6: 1)-(1: 6), the mass ratio of the active matter to the first mixed solution is (1: 1000)-(1: 1), and the target product is of a multi-cavity structure. The composite material has the advantages of being stable in structure, good in water dispersibility and high in wrapping performance.

Description

Technical Field

[0001] The invention relates to the technical field of cosmetic raw materials, and more specifically, to a silk protein-lecithin inclusion body and a preparation method thereof. Background Art

[0002] Nanocarriers are an important component in the field of drug controlled release and functional skin care. Highly crystalline silk protein aggregates have special hydrophilicity and hydrophobicity and a nano-morphology that is easy for drugs to adhere and fix. Therefore, optimizing and improving the drug encapsulation performance and active and passive targeted controlled release capabilities through the nanostructure of silk protein aggregates is a feasible idea for developing new silk protein carriers. Lecithin, as a key structural component, is widely used in the preparation of liposome inclusions.

[0003] Most of the current liposome inclusions are single-cavity structures with relatively poor stability. They are easy to break under conditions such as high-speed homogenization, microfluidization, high temperature and high pressure, and are difficult to be widely used. Previously, researchers developed a technology for preparing nano-inclusions by reassembling high-crystalline silk protein aggregates mediated by emulsifiers. The inclusions were prepared by emulsifiers and silk protein, but they were still single-cavity structures, and emulsifiers would reduce the biocompatibility of the system and cause irritation to the skin. Therefore, the development of a new silk protein-lecithin system that does not contain traditional emulsifiers is the key to further increase the number of active ingredients encapsulated and the biocompatibility, which is beneficial to its application in different fields such as functional skin care and tissue repair. Summary of the invention

[0004] In view of the shortcomings of the prior art, the object of the present invention is to provide a silk protein-lecithin inclusion body and a preparation method thereof, which has the advantages of stable structure, good water dispersibility and strong inclusion property.

[0005] The above technical purpose of the present invention is achieved through the following technical scheme: a method for preparing a silk protein-lecithin inclusion body, comprising the following steps: S1, obtaining a fibroin aggregate aqueous solution and lecithin, mixing the lecithin and the fibroin aggregate aqueous solution, heating and slowly stirring to obtain a first mixed solution; S2, adding the active substance to the first mixed solution after cooling, stirring until it becomes transparent, and obtaining the target product; Among them, the melting point of the lecithin is not higher than 100°C, the mass ratio of the silk fibroin aggregate and the lecithin in the first mixed solution is 6:1-1:6, the mass ratio of the active substance and the first mixed solution is 1:1000-1:1, and the target product is a multi-cavity structure.

[0006] In one embodiment, the concentration of the fibroin aggregates in the fibroin aggregate aqueous solution is 0.1-6 wt %.

[0007] In one embodiment, the lecithin is hydrogenated lecithin.

[0008] In one embodiment, the beta-sheet content of the silk fibroin aggregate is greater than 40%, and the size range is 20-4000nm.

[0009] In one embodiment, the active ingredient includes at least one of a liquid active ingredient and a solution active ingredient, and the solution active ingredient can be dissolved in water, alcohol, or oil phase.

[0010] In one embodiment, the stirring rate of S2 is 300-3000 rpm, and the stirring time is 1-120 min.

[0011] A silk protein-lecithin inclusion body is prepared by the silk protein-lecithin inclusion body preparation method.

[0012] In one embodiment, the size of the silk protein-phosphatidylcholine inclusions ranges from 10 to 2000 nm.

[0013] The application of the above silk protein-lecithin inclusions in functional skin care products.

[0014] The above-mentioned silk protein-lecithin inclusion body and preparation method thereof have the following beneficial effects: First, the silk fibroin aggregates, lecithin and active ingredients are mixed and stirred. Through the interaction between the silk fibroin aggregates and lecithin, a stable silk fibroin-lecithin inclusion body with a multi-chamber structure is formed. This can stably encapsulate the active ingredient and has good and stable water dispersibility, thereby improving the silk fibroin nanocarrier function and the transdermal properties of the active ingredient.

[0015] Secondly, the entire preparation process is simple and the conditions are mild, which is conducive to large-scale production and future transformation. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 is a flow chart of the steps of the present invention; Figure 2 It is a macroscopic picture of Example 1, Example 2, and Comparative Example 1; Figure 3 1 is a scanning electron microscope image of Example 1, Example 2, and Comparative Example 3; Figure 4 is a transmission electron microscope image of Example 1; Figure 5 is the particle size result diagram of Example 1; Figure 6 The fluorescence staining test diagrams of Example 1, Comparative Example 1 and Comparative Example 1 are shown; Figure 7It is the cck-8 detection diagram of Example 1, Comparative Example 1 and Comparative Example 2; Figure 8 This is a macroscopic comparison diagram of Example 1 and Comparative Example 3 in the control group and the experimental group; Fig. 9 The infrared spectra of Example 1, Comparative Example 1 and Comparative Example 3 are shown in FIG. Fig.10 The XRD diagrams of Example 1, Comparative Example 1 and Comparative Example 3 are shown; Fig.11 It is the zeta point diagram of Example 1 and Comparative Example 3; Fig.12 It is a comparison chart of the particle size results of Example 1 and Comparative Example 3 before and after heating at 60°C; Fig.13 It is a macroscopic comparison diagram of Example 5 and Comparative Example 4. DETAILED DESCRIPTION

[0017] The present invention is described in further detail below in conjunction with the embodiments. It should be noted that the embodiments described below are intended to facilitate the understanding of the present invention and do not have any limiting effect on it. Unless otherwise specified, all technical and scientific terms used herein have the same meanings as those of ordinary skill in the art to which the present invention belongs. When there is a conflict, the definitions in this specification shall prevail.

[0018] As used herein, the term "prepared from" is synonymous with "comprising." As used herein, the terms "comprising," "including," "having," "containing," or any other variation thereof, are intended to cover a non-exclusive inclusion. For example, a composition, process, method, article, or apparatus that comprises the listed elements is not necessarily limited to only those elements but may include other elements not expressly listed or inherent to such composition, process, method, article, or apparatus.

[0019] The conjunction "consisting of excludes any unspecified element, step, or component. If used in a claim, this phrase renders the claim closed-ended so that it does not include materials other than those described, except for conventional impurities associated therewith. When the phrase "consisting of" appears in a clause of the body of a claim rather than immediately following the subject matter, it limits only the elements described in that clause; other elements are not excluded from the claim as a whole.

[0020] When amount, concentration or other value or parameter is expressed as range, preferred range or a series of upper preferred value and lower preferred value limit range, this should be understood as specifically disclosing all ranges formed by any pairing of any range upper limit or preferred value and any range lower limit or preferred value, regardless of whether the range is disclosed separately. For example, when disclosing range "1 to 5", described range should be interpreted as including range "1 to 4", "1 to 3", "1 to 2", "1 to 2 and 4 to 5", "1 to 3 and 5" etc. When numerical range is described in this article, unless otherwise stated, the range is intended to include its end value and all integers and fractions within the range.

[0021] The present invention is described in detail below in conjunction with the accompanying drawings and embodiments.

[0022] A method for preparing a silk protein-lecithin inclusion body, Figure 1 As shown, the following steps are included: S1, obtaining a fibroin aggregate aqueous solution and solid lecithin, mixing the lecithin and the fibroin aggregate aqueous solution, heating and slowly stirring to obtain a first mixed solution; S2, adding the active substance to the cooled first mixed solution, stirring until it becomes transparent, and obtaining the target product; Among them, the melting point of lecithin is not higher than 100°C, the mass ratio of silk fibroin aggregates and lecithin in the first mixed liquid is 6:1-1:6, the mass ratio of the active substance and the first mixed liquid is 1:1000-1:1, and the target product is a multi-cavity structure.

[0023] Silk fibroin aqueous solution cannot be heated to above 100°C, so lecithin with a melting point below 100°C is selected, mixed with the silk fibroin aqueous solution, heated and slowly stirred until the lecithin melts. Lecithin with a melting point above 100°C can also be selected, dissolved in alcohol, mixed with the silk fibroin aqueous solution and stirred, but the composition is not as pure as the first mixed solution obtained in S1 above. The mass ratio of the active substance to the first mixed solution ranges from 1:1000 to 1:1, that is, the first mixed solution contains up to 50% of the active substance in the mixed system. Lecithin has special hydrophilicity and hydrophobicity, similar to the aggregated state performance of high-crystalline silk fibroin. The comprehensive performance of silk fibroin nanocarriers can be improved by using lecithin to mediate the reassembly of silk fibroin.

[0024] The silk fibroin aggregates, lecithin and active ingredients are mixed and stirred to form a stable silk fibroin-lecithin inclusion body with a multi-chamber structure through the interaction between the silk fibroin aggregates and lecithin. The silk fibroin-lecithin inclusion body can stably encapsulate the active ingredient and has good and stable water dispersibility, thereby improving the silk fibroin nanocarrier function and the transdermal transdermal properties of the active ingredient.

[0025] Specifically, the concentration of the fibroin aggregates is 0.1-6 wt %. When the concentration is greater than or equal to 2 wt %, the fibroin aggregate aqueous solution is in a gel state.

[0026] Specifically, the lecithin includes hydrogenated lecithin. The hydrogenated lecithin with a melting point below 100°C is selected, preferably, the hydrogenated lecithin with a melting point of about 60°C is selected. After the lecithin and the fibroin aqueous solution are mixed, the heating temperature is too high, which will cause the water in the fibroin aqueous solution to boil.

[0027] Specifically, the beta-sheet content of the silk fibroin aggregate is greater than 40%, and the size range is 20-4000nm. The silk fibroin aggregate used in the present invention is a high-crystalline silk fibroin aggregate, containing more than 40% of the beta-folded structure, having good structural stability and mechanical properties, so as to better achieve the attachment of active substances.

[0028] Specifically, the active substance includes at least one of a liquid active substance and a solution active substance, and the solution active substance can be dissolved in water, alcohol, or oil phase. The types of active substances include antioxidants, whitening agents, oils, drugs, proteins, and polysaccharides, such as retinol, ceramide, asiatica glycoside, and essential oils in the solution active substance, and plant oils in the liquid active substance. The types of active substances are not limited here, and active substances that are difficult to penetrate the skin are included in the scope of the present invention.

[0029] Specifically, the stirring rate of S2 is 300-3000rpm, and the stirring time is 1-120min. It can also be homogenized, and the homogenization rate is 500-5000rpm, and the homogenization time is 1-240min. When the mixed solution is observed to change from milky white to translucent, stirring can be stopped. When the stirring rate is too low, it will cause the silk protein-lecithin inclusions and the active substance to be difficult to form uniform nano-aggregates. When the stirring rate is too high, the aggregation of the two will be too strong, and phase separation or precipitation will occur.

[0030] A silk protein-lecithin inclusion body is prepared by the silk protein-lecithin inclusion body preparation method.

[0031] Furthermore, the size of the silk protein-phosphatidylcholine inclusions ranges from 10 to 2000 nm.

[0032] The application of the above silk protein-lecithin inclusions in functional skin care products. Example 1

[0033] This embodiment provides a silk protein-lecithin inclusion body, and the specific preparation steps are as follows: S1, mixing 0.2 g of hydrogenated lecithin and 20 g of a high-crystalline silk protein nanofiber aggregate solution having a length of 1000 nm and a concentration of 2 wt%, heating to 70-80° C., and stirring at a speed of 100 rpm until uniform, to obtain a first mixed solution; S2. Cool the first mixed solution to room temperature and stir at 1500 rpm for 15 min to obtain a silk protein-lecithin inclusion body in a liquid state. Example 2

[0034] This embodiment provides a silk protein-lecithin inclusion body, and the specific preparation steps are as follows: S1, mixing 0.6 g of hydrogenated lecithin and 10 g of a high-crystalline silk protein nanofiber aggregate solution having a length of 1500-2000 nm and a concentration of 1 wt%, heating to 60-90° C., and stirring at a speed of 200 rpm until uniform, to obtain a first mixed solution; S2. The first mixed solution was cooled to 4° C., 1 g of a 50 wt % retinol-Tween solution was added, and the mixture was homogenized at 3000 rpm for 10 min to obtain a silk protein-phosphatidylcholine inclusion body encapsulating retinol. Example 3

[0035] This embodiment provides a silk protein-lecithin inclusion body, and the specific preparation steps are as follows: S1, mixing 0.2 g of hydrogenated lecithin and 40 g of a high-crystalline silk protein nanofiber aggregate solution with a length of 100 nm and a concentration of 3 wt%, heating to 90-99° C., and stirring at a speed of 400 rpm until uniform, to obtain a first mixed solution; S2. Cool the first mixed solution to 40° C., add 0.4 g of bisabolol oil, and stir at 2000 rpm for 20 min to obtain a silk protein-lecithin inclusion body encapsulating bisabolol. Example 4

[0036] This embodiment provides a silk protein-lecithin inclusion body, and the specific preparation steps are as follows: S1, mixing 0.2 g of hydrogenated lecithin and 20 g of high-crystalline silk protein nanofiber aggregate gel with a length of 1000-2000 nm and a concentration of 2 wt%, heating to 60-70° C., stirring at 80 rpm until uniform, to obtain a first mixed solution; S2. The first mixed solution was cooled to 4° C., 2 g of vitamin C powder was added, and the mixture was stirred at 1200 rpm for 60 min to obtain a silk protein-lecithin inclusion body encapsulating vitamin C. Example 5

[0037] This embodiment provides a silk protein-lecithin inclusion body, and the specific preparation steps are as follows: S1, mixing 0.2 g of hydrogenated lecithin and 20 g of high-crystalline silk protein nanofiber aggregate gel with a length of 1000-2000 nm and a concentration of 2 wt%, heating to 60-70° C., stirring at 80 rpm until uniform, to obtain a first mixed solution; S2. Cool the first mixed solution to 40° C., add 0.4 g of jojoba oil, and stir at 2000 rpm for 20 min to obtain a silk protein-lecithin inclusion body encapsulating jojoba oil.

[0038] The present invention also provides a comparative example for comparing the performance of the above embodiments.

[0039] Comparative Example 1 is a silk protein-emulsifier inclusion body, and the specific preparation steps are as follows: S1, 0.2g of Charming Bee emulsifier and 20g of high-crystalline silk protein nanofiber aggregate solution with a length of 1000nm and a concentration of 2wt% are mixed, heated to 70-80°C, and stirred at a speed of 100rpm until uniform to obtain a first mixed solution; S2. Cool the first mixed solution to room temperature and stir at 1500 rpm for 15 min to obtain a silk protein-emulsifier inclusion body in a liquid state.

[0040] Comparative Example 2 is a 2wt% aqueous solution of high-crystalline silk protein nanofiber aggregates, wherein the length of the high-crystalline silk protein nanofiber aggregates is 1000-2000nm.

[0041] Comparative Example 3 is commercially available lecithin.

[0042] Comparative Example 4 is a liposome-jojoba oil inclusion body, and the specific preparation steps are as follows: 0.4 g of jojoba oil was added to 20 g of hydrogenated lecithin, and the mixture was stirred at 2000 rpm for 20 min to obtain the target product.

[0043] Experimental analysis and testing were carried out on the products of each embodiment and comparative example.

[0044] Test 1: Macro state analysis The products obtained in Example 1, Example 2 and Comparative Example 1 were used as detection raw materials, and macroscopic analysis was performed at room temperature.

[0045] like Figure 2 As shown, Figure 2 (a), (b), and (c) are macroscopic schematic diagrams of the products obtained in Example 1, Example 2, and Comparative Example 1, respectively, wherein Example 1 is a translucent colorless solution, Example 2 is an opaque light yellow solution, and Comparative Example 1 is a translucent white solution.

[0046] Test 2: Morphology and particle size test The products obtained in Example 1, Example 2 and Comparative Example 3 were tested and analyzed using a scanning electron microscope.

[0047] like Figure 3 As shown, Figure 3 (a) and (b) are scanning electron microscope images of the product obtained in Example 1, (c) is a scanning electron microscope image of the product obtained in Example 2, and (d) is a scanning electron microscope image of Comparative Example 3. Comparative Example 3 has obvious aggregation phenomenon and uneven structure. Compared with Comparative Example 3, the structure of Example 1 is more uniform and has better stability, and the structure of Example 2 is relatively uniform. The silk protein-lecithin inclusion body has a uniform structure and good stability.

[0048] The product obtained in Example 1 was subjected to transmission electron microscopy and particle size testing.

[0049] like Figure 4 and Figure 5 As shown, Example 1 is a particle of about 100 nm, which is formed by reassembling smaller particles and has a multi-cavity structure inside.

[0050] Test 3: Skin penetration test The products obtained in Example 1, Comparative Example 1 and Comparative Example 2 were used as test materials to conduct skin permeability test analysis at room temperature.

[0051] After FITC staining was performed on Example 1, Comparative Example 1 and Comparative Example 2, the samples were quantitatively applied on the surface of mouse ex vivo skin and placed in a permeameter for 24 hours to obtain test results.

[0052] Test analysis: Figure 6 (a), (b), and (c) are the fluorescence staining test result diagrams of Example 1, Comparative Example 1, and Comparative Example 2, respectively. The dyed silk protein-phosphatidylcholine inclusions penetrated into the mouse ex vivo skin. Example 1 has the strongest permeability, while Comparative Example 1 has the weakest permeability.

[0053] Test 4: Irritation test The cck-8 test was performed using the products obtained in Example 2, Comparative Example 1, and Comparative Example 2 and Hacat cells as detection raw materials.

[0054] The products obtained in Example 1, Comparative Example 1 and Comparative Example 2 diluted to the same multiple were co-cultured with Hacat cells, and CCK-8 detection was performed on the 1st, 2nd and 3rd day, respectively.

[0055] Test analysis: Figure 7The cck-8 detection diagram of the products obtained from Example 1 (SLC-150), Comparative Example 1 (SLNP-200), Comparative Example 2 (SNF-100) and Hacat cells (Control) shows that Example 1, Comparative Example 1 and Comparative Example 2 are all non-irritating, but Example 1 is more beneficial to promoting cell growth.

[0056] Test 5: Stability test (1) The product obtained in Example 1 and hydrogenated lecithin were used as test materials for stability comparison analysis.

[0057] Set up a control group: two groups of hydrogenated lecithin, the first group was placed at room temperature, and the second group was heated to 60°C, cooled to room temperature, and left to stand for a period of time.

[0058] An experimental group was set up: the product obtained in Example 1 and hydrogenated lecithin were dispersed in water to the same extent, then both were heated to 60° C., cooled to room temperature, and left to stand for a period of time.

[0059] Test analysis: Figure 8 As shown, the hydrogenated lecithin of the control group was insoluble in water without heating; the hydrogenated lecithin of the control group 2 was dissolved in water when heated to above 60°C, and partially precipitated after being cooled to room temperature for a period of time; there was no precipitation after Example 1 of the experimental group was heated and cooled to room temperature, indicating that combining with silk protein can improve its stability and inhibit precipitation.

[0060] (2) The products obtained in Example 1, Comparative Example 2 and Comparative Example 3 were subjected to infrared spectroscopy and XRD analysis.

[0061] like Fig. 9 As shown, the characteristic peaks of lecithin and high-crystalline silk protein nanoaggregates exist in the silk protein-lecithin inclusions of Example 1, and no new characteristic peaks appear, indicating that there is no chemical reaction between lecithin and high-crystalline silk protein nanoaggregates. Fig.10 The XRD diagrams of Example 1 (SLC), Comparative Example 2 (SNF) and Comparative Example 3 (LH) show that in Example 1, after lecithin and high-crystalline silk protein nanoaggregates are combined, the crystallization peak of lecithin decreases significantly, proving that lecithin and high-crystalline silk protein nanoaggregates inhibit the crystallization of lecithin through interaction, and there is a strong interaction between the two.

[0062] (3) Zeta point analysis was performed using Example 1 and Comparative Example 3.

[0063] Fig.11This is a zeta point test diagram of Example 1 (SLC) and Comparative Example 3 (Liposomes). Compared with Comparative Example 3, the zeta potential of Example 1 is below -40mv, which proves that it has a strong electrostatic repulsion, which is conducive to the uniform dispersion of Example 1 in the system without aggregation and has strong stability.

[0064] (4) A stability comparison was performed using Example 1 and Comparative Example 3.

[0065] The specific steps are as follows: take the same amount of the test object, disperse it in water to the same extent, heat it to 60°C, keep it warm for a period of time, and then perform a particle size test.

[0066] like Fig.12 As shown, after Example 1 and Comparative Example 3 were heated at 60°C, the particle size of Comparative Example 3 became larger, while there was no obvious change in Example 1. Example 1 was more stable than Comparative Example 3.

[0067] (5) A stability comparison was performed using Example 5 and Comparative Example 4.

[0068] The specific steps are as follows: take the same amount of the test object and disperse it in water to the same extent, and then use ultrasonic treatment at a power of 500w for 10 minutes.

[0069] like Fig.13 As shown, the fluidity of Example 5 is significantly increased, and no demulsification occurs, while the two-phase separation phenomenon occurs in Comparative Example 4, and the stability of Example 5 is better.

[0070] The above-mentioned embodiments only express several implementation methods of the present invention, and the description thereof is relatively specific and detailed, but it cannot be understood as limiting the scope of the patent of the present invention. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, which all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims.

Claims

1. A method for preparing a silk protein-lecithin inclusion body, characterized in that: The following steps are involved: S1, obtaining a fibroin aggregate aqueous solution and lecithin, mixing the lecithin and the fibroin aggregate aqueous solution, heating and slowly stirring to obtain a first mixed solution; S2, adding the active substance to the first mixed solution after cooling, stirring until it becomes transparent, and obtaining the target product; Among them, the melting point of the lecithin is not higher than 100°C, the mass ratio of the silk fibroin aggregate and the lecithin in the first mixed solution is 6:1-1:6, the mass ratio of the active substance and the first mixed solution is 1:1000-1:1, and the target product is a multi-cavity structure.

2. The method for preparing a silk protein-lecithin inclusion body according to claim 1, characterized in that: The concentration of the fibroin aggregates in the fibroin aggregate aqueous solution is 0.1-6wt%.

3. The method for preparing a silk protein-lecithin inclusion body according to claim 1, characterized in that: The lecithin is hydrogenated lecithin.

4. The method for preparing a silk protein-lecithin inclusion body according to claim 1, characterized in that: The beta-sheet content of the silk fibroin aggregate is greater than 40%, and the size range is 20-4000nm.

5. The method for preparing a silk protein-lecithin inclusion body according to claim 1, characterized in that: The active substance includes at least one of a liquid active substance and a solution active substance, and the solution active substance can be dissolved in water, alcohol, or oil phase.

6. The method for preparing a silk protein-lecithin inclusion body according to claim 1, characterized in that: The stirring rate of S2 is 300-3000 rpm, and the stirring time is 1-120 min.

7. A silk protein-lecithin inclusion body, characterized in that: The silk protein-lecithin inclusion body is prepared according to the preparation method of any one of claims 1-6.

8. The silk protein-lecithin inclusion body according to claim 7, characterized in that: The size of the silk protein-lecithin inclusions ranges from 10 to 2000 nm.

9. Use of the silk protein-lecithin inclusion body according to claim 7 in functional skin care products.

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