High molecular weight sericin composition having film-forming ability and method for producing same

Abstract

To provide a method for producing high-molecular-weight sericin protein that can be used conveniently for industry, and a high-molecular-weight sericin protein having a film formation ability and excellent storage stability, and a skin humectant.SOLUTION: A production method has the steps of (1) preparing high-molecular-weight sericin solution and (2) mixing the high-molecular-weight sericin solution with glycerol or glycol. The high-molecular-weight sericin protein contains high-molecular-weight sericin and glycerol or glycol.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to a high molecular weight sericin composition having film-forming ability, a skin moisturizing agent, and a method for producing the same. [Background technology]

[0002] Silk, the cocoon thread made by silkworms and wasps, has a two-layer structure consisting of fibroin protein in the center and sericin protein surrounding the fibroin. Sericin acts like glue to bind the fibroin fibers together, building and strengthening the cocoon. Silk is described as being used as a thread (suture) to stitch wounds in a surgical book from 2,500 years ago, and it is still used as a surgical suture today. Silk has high biocompatibility and can be said to be a safe material. In recent years, a new way of using silk is to take advantage of its high biocompatibility and processability and use it as a regenerative medical material to promote tissue regeneration by attaching it to damaged or defective areas of the body as a scaffold for cell regeneration.

[0003] In the field of topical skin application preparations such as cosmetics and topical drugs, silk is generally used as an aqueous moisturizing agent because its amino acid composition is similar to that of human skin. However, most of the silk contained in them is hydrolyzed silk. Aqueous solutions of hydrolyzed silk are soluble silk obtained by hydrolyzing the proteins that make up silk fibers under acid, alkali, or proteolytic enzymes, and the molecular weight distribution is mostly low molecular weight, with no high molecular weight proteins (Patent Document 1). Therefore, since the molecular chains are broken during the hydrolysis process, the original characteristics of silk cannot be utilized in a low molecular weight hydrolyzed silk solution. In order to utilize the original characteristics of silk, it is necessary to extract high molecular weight silk proteins, and if high molecular weight silk proteins can be extracted, the good film-forming ability derived from silk proteins can be expected to lead to the development of new applications such as moisturizing agents that promote skin protection and protective agents that supplement barrier function.

[0004] To obtain high molecular weight silk protein, a method is generally used in which cocoons are dissolved in a high concentration salt solution such as lithium bromide, and then the salt is removed by dialysis to prepare an aqueous solution (Patent Document 2). However, the silk protein aqueous solution obtained by dialysis has poor stability, and there is a problem that it is difficult to store for a long time due to gelation, especially when it contains high molecular weight silk protein. In addition, conventional techniques using dialysis require various steps and time, and there is also a problem that it is difficult to scale up for industrialization. Lithium bromide, which is generally used to dissolve silk protein, may remain in trace amounts after dialysis. There is no precedent for lithium bromide being used on the skin, and there are concerns about its safety to the living body. Therefore, since the film-forming ability of silk increases depending on the molecular weight, a method for preparing a stable solution containing high molecular weight silk protein and a simple and industrializable manufacturing method (materialization method) using safe raw materials in the manufacturing process are desired. [Prior art documents] [Patent documents]

[0005] [Patent Document 1] JP 2005-002063 A [Patent Document 2] JP 2006-111667 A Summary of the Invention [Problem to be solved by the invention]

[0006] The object of the present invention is to eliminate the above-mentioned drawbacks of the conventional technology, to develop a simple and industrially feasible method for producing high molecular weight sericin protein, and to provide a high molecular weight sericin protein and a skin moisturizer having excellent storage stability and film-forming ability. [Means for solving the problem]

[0007] As a result of intensive research aimed at solving the above problems, the present inventors developed a technique for stably dispersing sericin in a high-concentration aqueous glycerin solution or a high-concentration aqueous glycol solution, and discovered that when this sericin composition was applied to the skin, it exhibited a high inhibitory effect on transepidermal water loss, which is the amount of water that evaporates through the stratum corneum of the skin (Figure 2). In addition, it was confirmed that when a skin moisturizer (cream) containing the sericin composition was applied to the skin, it tended to inhibit transepidermal water loss more than a placebo (Figures 3 to 5).

[0008] The present invention relates to [1] (1) preparing an aqueous solution of high molecular weight sericin; (2) A step of mixing the aqueous high molecular weight sericin solution with glycerin or glycols. A method for producing a high molecular weight sericin composition, comprising: [2] (3) A step of washing the mixture obtained in the step (2) with an aqueous glycerin solution or an aqueous glycol solution by centrifugation and decantation. The method of producing [1], [3] The method according to [1] or [2], wherein the aqueous high molecular weight sericin solution prepared in the step (1) is prepared by dissolving a cocoon shell or a cocoon, or a silk gland derived from a silkworm, in a salt solution; [4] A high molecular weight sericin composition comprising high molecular weight sericin and glycerin or glycols. [5] A high molecular weight sericin composition according to [4], obtained by any one of the production methods according to [1] to [3]. [6] A skin moisturizer comprising the high molecular weight sericin composition according to [4] or [5]. Regarding. Effect of the Invention

[0009] According to the present invention, a method for producing a high molecular weight sericin protein that is simple and industrially feasible can be provided, and a high molecular weight sericin protein and a skin moisturizer having a film-forming ability with excellent storage stability can be provided. In addition, in a preferred embodiment of the present invention, a skin moisturizer that is safe when applied to the skin can be provided. [Brief description of the drawings]

[0010] [Figure 1] This is an electrophoretic image showing the results of SDS-polyacrylamide gel electrophoresis (PAGE) of a sericin solution obtained by dissolving a cocoon shell in an aqueous calcium chloride solution or an aqueous lithium bromide solution at a temperature of 50°C to 80°C. [Diagram 2] 1 is a graph showing the results of measuring transepidermal water loss when the composition of the present invention was applied to the skin. [Diagram 3] 1 is a graph showing the results of measuring transepidermal water loss when the skin moisturizer (cream) of the present invention is applied to the skin. [Figure 4] 1 is a graph showing the results of measuring transepidermal water loss when another skin moisturizer (cream) of the present invention having a different base was applied to the skin. [Diagram 5] 1 is a graph showing the results of measuring transepidermal water loss when the composition of the present invention was applied to the skin. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0011] <<Production method of the present invention>> The production method of the present invention comprises the steps of: (1) preparing an aqueous solution of high molecular weight sericin (preparation step); (2) A step of mixing the aqueous high molecular weight sericin solution with glycerin or glycols (mixing step) and optionally further comprising (3) A step of washing the mixture obtained in the mixing step (2) by centrifugation and decantation using an aqueous glycerin solution or an aqueous glycol solution (washing step). may include.

[0012] In the preparation step of the manufacturing method of the present invention, an aqueous solution containing high molecular weight sericin is prepared. In this specification, "high molecular weight sericin" refers to a substance that has been confirmed to spontaneously gel (solidify) and lose fluidity as a solution when left in the form of an aqueous solution. As shown in the examples described later, this corresponds to a molecular weight of about 200,000 to 400,000, but is not limited to this range (Figure 1). For reference, the average molecular weight of low molecular weight hydrolyzed sericin is about several tens of thousands.

[0013] The high molecular weight sericin aqueous solution can be prepared, for example, by dissolving a cocoon layer or cocoon, or a silkworm-derived silk gland in a salt solution, although the present invention is not limited thereto. Here, the term "cocoon" refers to a structure that silkworm larvae create by secreting silk proteins in the silk gland during pupation, and the term "cocoon layer" refers to the layer of the cocoon that is made of silk proteins other than the pupa. Furthermore, the term "silk gland derived from silkworms" refers to a pair of exocrine glands that secrete silk threads in silkworms. The silk gland of silkworms is composed of anterior, middle, and posterior parts, and sericin is secreted from the middle silk gland. Therefore, the middle silk gland can be used as the silkworm-derived silk gland.

[0014] The silk gland or middle silk gland can be isolated by dissecting a 5th instar silkworm larva and removing it from the body. The cocoon shell can be isolated by separating the pupa from the cocoon produced by the 5th instar silkworm larva during pupation. Furthermore, the cocoon produced by the silkworm can be used as it is.

[0015] The silkworm variety from which the cocoon shell or cocoon, or the silk gland of the silkworm used in the production method of the present invention is derived is not particularly limited, but examples thereof include a cocoon-spinning sericin silkworm lineage, such as Sericin Hope (Sericin C), Sericin N, Nd silkworm, Nd-s and Nd-sD silkworm, and the like, with Sericin Hope being preferred. The cocoon-spinning sericin silkworm lineage is a silkworm lineage that has a special characteristic of being able to spin a cocoon, even though it is a sericin silkworm lineage that is not capable of spinning a cocoon due to a mutation in the fibroin synthesis pathway. The silkworm variety Sericin Hope (Sericin C) is a variety that was created by crossing the Chinese variety "CS83" lineage with the variety "Nd lineage" (a naked pupa, a mutant species that does not synthesize fibroin but only synthesizes sericin), and then backcrossing, etc. (JP Patent Publication No. 2001-245550). The silkworm cultivar Sericin Hope (Sericin C) has a degenerated ability to synthesize fibroin and produces a large amount of sericin. A polyphagous sericin silkworm strain obtained by crossbreeding a cocoon-spinning sericin silkworm strain with a polyphagous silkworm strain and then backcrossing the sericin Hope silkworm strain can also be used (WO 2014 / 065217).

[0016] When using normal silkworms capable of synthesizing both fibroin and sericin, sericin can be preferentially extracted by using an aqueous calcium chloride solution, which will be described later. This is because a mixed solvent of an aqueous calcium chloride solution and ethanol is generally used as a solvent for extracting fibroin, and the efficiency of fibroin extraction is reduced when an aqueous calcium chloride solution is used alone.

[0017] The salt solution used to dissolve the cocoon shell or cocoon, or the silk gland derived from a silkworm is not particularly limited as long as it can extract high molecular weight sericin from the cocoon shell or cocoon, or the silk gland derived from a silkworm. Examples of the salt solution include an aqueous calcium chloride solution, an aqueous lithium bromide solution, and an aqueous urea solution. From the viewpoint of safety when applied to the skin, an aqueous calcium chloride solution or an aqueous urea solution is preferred, and an aqueous calcium chloride solution is particularly preferred.

[0018] The concentration of the salt solution may be, for example, 2.5 to 5.0 mol / L, preferably 3.0 to 4.0 mol / L, when an aqueous calcium chloride solution is used. If the concentration is less than 2.5 mol / L or more than 5.0 mol / L, sericin may not be sufficiently dissolved, and if the concentration is more than 5.0 mol / L, gelation may not occur in the mixing step described below. The amount of the salt solution used for dissolution may be, for example, 10 to 100 times, preferably 20 to 40 times, the amount of the cocoon shell, etc., in weight ratio. The upper and lower limits described above may be combined arbitrarily as desired.

[0019] The temperature during dissolution is not particularly limited as long as it is a temperature that can suppress the decomposition of sericin, and can be, for example, room temperature to 100° C. The dissolution time can be determined taking into consideration the temperature, and for example, in the case of 70° C., dissolution can be achieved in 1 to 3 hours without reducing the molecular weight.

[0020] The sericin concentration in the obtained aqueous high molecular weight sericin solution may be, for example, 1 to 10% by weight, and preferably 2 to 5% by weight. The sericin concentration can be adjusted to the above concentration by adjusting the amount of salt solution relative to the cocoon shell, etc., or by appropriately diluting the obtained aqueous high molecular weight sericin solution with water or a salt solution, etc., or by concentrating it. The above upper and lower limits can be arbitrarily combined as desired.

[0021] In the mixing step in the production method of the present invention, the high molecular weight sericin composition of the present invention can be obtained by mixing the high molecular weight sericin aqueous solution with glycerin or glycols (hereinafter sometimes referred to as gelling dispersion solvent). The gelling dispersion solvent is not particularly limited as long as it becomes a cloudy sol without forming a gel-like mass when mixed with the high molecular weight sericin aqueous solution. It is considered that in the mixed liquid, fine particulate gel of high molecular weight sericin is smoothly and uniformly dispersed in the gelling dispersion solvent. Examples of glycols include various diols, such as 1,3-butylene glycol (1,3-butanediol), isoprene glycol (3-methyl-1,3-butanediol), and propylene glycol (1,2-propanediol).

[0022] The sericin concentration in the high molecular weight sericin aqueous solution at the time of mixing can be, for example, 0.1 to 3.0% by weight, and is preferably 0.3 to 1.0% by weight. The concentration of the gelling dispersion solvent in the mixed solution at the time of mixing can be, for example, 40 to 90% by weight, and is preferably 50 to 80% by weight, when glycerin is used. If the glycerin concentration is less than 40%, gelation may not occur, and if it exceeds 90%, water may be brought in from the sericin aqueous solution, making it impossible to carry out the method. The gelling dispersion solvent may be one type of glycerin or glycols, or two or more types may be used in combination. The gelling dispersion solvent may be mixed with the high molecular weight sericin aqueous solution as it is, or may be diluted with a suitable solvent (for example, water) and then mixed with the high molecular weight sericin aqueous solution. The upper and lower limits described above may be combined as desired.

[0023] The washing step in the production method of the present invention can be carried out as desired, and the mixed solution obtained in the mixing step is washed by centrifugation and decantation with an aqueous glycerin solution or an aqueous glycol solution (hereinafter sometimes referred to as a washing solution). By this washing, salts in the mixed solution derived from the salt solution can be removed from the mixed solution, and the high molecular weight sericin composition of the present invention having a reduced salt concentration can be obtained.

[0024] The concentration of glycerin or glycols in the washing liquid may be, for example, 50 to 90% by weight in the case of a glycerin aqueous solution, and is preferably 70 to 80% by weight. By using 70 to 80% by weight, a smooth and uniform composition can be obtained. The washing liquid may be a single type of glycerin aqueous solution or a glycol aqueous solution, or a combination of two or more types. In addition, the gelling dispersion solvent (i.e., glycerin or glycols) used in the mixing step and the washing liquid used in the washing step may be the same compound or different compounds. In addition, when washing is performed multiple times in the washing step, washing may be performed with one type of aqueous solution or multiple types of aqueous solutions, and the concentrations of the aqueous solutions may be the same or different. The upper and lower limits described above can be combined arbitrarily as desired.

[0025] The conditions for centrifugation are not particularly limited as long as a precipitate is generated by centrifugation and can be uniformly dispersed in the washing liquid after decantation, but for example, the conditions can be 2,000×g to 20,000×g, and preferably 3,000×g to 15,000×g. The above upper and lower limits can be arbitrarily combined as desired.

[0026] The number of washings is not particularly limited as long as the salt can be sufficiently removed, but may be, for example, 1 to 5 times, and preferably 4 to 5 times. The precipitate after washing (i.e., after the final decantation) can be used as it is as the high molecular weight sericin composition of the present invention, or the high molecular weight sericin composition of the present invention can be obtained by suspending the precipitate in a washing solution.

[0027] <High molecular weight sericin composition of the present invention> The high molecular weight sericin composition of the present invention contains high molecular weight sericin and glycerin or glycols. The high molecular weight sericin composition of the present invention is not limited thereto, but can be obtained by the production method of the present invention, and is preferably obtained by the production method of the present invention. The sericin concentration in the high molecular weight sericin composition may be, for example, 0.01 to 5.0% by weight, 0.1 to 3.0% by weight, and preferably 0.5 to 2.0% by weight. The glycerin or glycols concentration (when two or more types of compounds are contained, the total concentration of these compounds) in the high molecular weight sericin composition may be, for example, 50 to 90% by weight, and preferably 70 to 80% by weight. The upper and lower limits may be combined in any combination as desired.

[0028] The high molecular weight sericin composition of the present invention is considered to be a suspension in which high molecular weight sericin is smoothly and uniformly dispersed in a gelling dispersion solvent or washing solution as a fine particulate gel, and has excellent storage stability. Conventional methods involve dissolving the sericin in a high-concentration salt solution of lithium bromide, followed by dialysis to remove the salt. Known aqueous solutions of high molecular weight sericin are transparent immediately after dissolution, but have poor stability and have the problem that gelation makes it difficult to store for long periods of time. This problem has been solved by the high molecular weight sericin composition of the present invention.

[0029] Furthermore, while the above-mentioned known aqueous high molecular weight sericin solution uses lithium bromide, which has never been used on the skin, the preferred embodiment of the high molecular weight sericin composition of the present invention uses only salts (e.g., calcium chloride) whose safety when applied to the skin has been confirmed, and therefore is excellent in safety. Furthermore, in the preferred embodiment of the high molecular weight sericin composition of the present invention, which has been subjected to a washing step, the salt concentration can be significantly reduced, and it is even safer.

[0030] Furthermore, the above-mentioned known aqueous high molecular weight sericin solution requires dialysis to remove salts, making it difficult to scale up for industrial use. In contrast, the high molecular weight sericin composition which undergoes the washing step, which is a preferred embodiment of the present invention, requires centrifugation and decantation to remove salts, making it easy to industrialize.

[0031] <Skin moisturizing agent of the present invention> The high molecular weight sericin composition of the present invention has a film-forming ability when applied to the skin, and therefore can be used as an active ingredient of a skin moisturizer. The moisturizer to be applied to the skin may be used for cosmetics, quasi-drugs, medicines, miscellaneous goods, medical devices, or medical materials. The dosage form of the skin moisturizer of the present invention is not limited to the following, but examples thereof include creams, ointments, gels, lotions, sprays, and the like.

[0032] The concentration of sericin contained in the skin moisturizer of the present invention can be, for example, 0.01 to 5.0 wt%, 0.025 to 4.0 wt%, 0.1 to 3.0 wt%, and preferably 0.5 to 2.0 wt%. The concentration of glycerin or glycols contained in the skin moisturizer of the present invention (when two or more types of compounds are contained, the total concentration of these compounds) can be, for example, 50 to 90 wt%, and preferably 70 to 80 wt%. The upper and lower limits can be arbitrarily combined as desired.

[0033] The skin moisturizer of the present invention can be used by adding, in addition to the high molecular weight sericin composition, various additives that can be applied to skin moisturizers, and active ingredients of pharmaceuticals and quasi-drugs that can be applied to the skin, if desired.

[0034] For example, additives include oils and fats, waxes, fatty acids, organic acids, hydrocarbons, alcohols, polyhydric alcohols, ethers, esters, silicone oils, sugars, polymers, surfactants, powders, colorants, extracts, amino acids, peptides, vitamins, UV absorbers, UV scattering agents, germicides and preservatives, antioxidants, chelating agents, fragrances, and water. Active ingredients include antipruritics, anti-inflammatory drugs, local anesthetics, antihistamines, blood circulation promoters, antibacterial and germicide drugs, tissue repair drugs, anti-inflammatory and analgesic drugs, skin softeners, and other drugs that can be added and used.

[0035] Examples of fats and oils include vegetable oils such as olive oil, soybean oil, rapeseed oil, palm kernel oil, castor oil, and coconut oil, and animal oils such as horse oil and mink oil. Examples of waxes include carnauba wax, candelilla wax, lanolin, beeswax, and jojoba oil.

[0036] Examples of fatty acids include stearic acid, palmitic acid, linoleic acid, linolenic acid, oleic acid, palmitoleic acid, myristic acid, behenic acid, lauric acid, and isostearic acid. Examples of organic acids include citric acid, lactic acid, glycolic acid, and tartaric acid.

[0037] Examples of the hydrocarbon include squalane, vegetable squalane, light isoparaffin, petrolatum, liquid paraffin, and α-olefin oligomers. Examples of alcohols include ethanol, isopropyl alcohol, cetanol, behenyl alcohol, stearyl alcohol, cholesterol, phytosterol, and benzyl alcohol. Examples of polyhydric alcohols include glycerin, butylene glycol, propylene glycol, ethylene glycol, isoprene glycol, dipropylene glycol, and polyglycerin. Examples of the ether include diethylene glycol ether, dipropylene glycol ether, alkyl glyceryl ether, batyl alcohol, chimyl alcohol, and selachyl alcohol. Examples of esters include isopropyl myristate, isopropyl palmitate, myristyl myristate, glyceryl tri-2-ethylhexanoate, caprylic / capric triglyceride, and cholesteryl stearate.

[0038] Examples of silicone oils include dimethicone, diphenyldimethicone, methylcyclopolysiloxane, and polyoxyethylene-methylpolysiloxane copolymer. Examples of sugars include xylitol, mannitol, sorbitol, inositol, and trehalose.

[0039] Examples of polymers include moisturizing ingredients such as sodium hyaluronate and collagen, and thickeners such as carboxyvinyl polymer, acrylic acid / alkyl methacrylate copolymer, sodium alginate, carrageenan, and xanthan gum. Examples of surfactants include anionic surfactants such as N-acylamino acid salts, N-acylmethyl taurine salts, and alkyl sulfate salts; cationic surfactants such as benzalkonium chloride and benzethonium chloride; amphoteric surfactants such as carboxybetaine and imidazolinium salts; and nonionic surfactants such as ethylene glycol mono fatty acid esters, polyglycerin fatty acid esters, sucrose fatty acid esters, alkyl polyglucosides, and polyoxyethylene hydrogenated castor oil.

[0040] Examples of powders include magnesium aluminum silicate, talc, kaolin, and mica. Examples of coloring materials include legal dyes such as fluorescein and indigo.

[0041] Examples of extracts include aloe extract, perilla extract, oil-soluble chamomile extract, and licorice extract. Examples of amino acids and peptides include glycine, ε-aminocaproic acid, tranexamic acid, hydrolyzed collagen, hydrolyzed elastin, and casein. Examples of vitamins include retinol, riboflavin, pyridoxine hydrochloride, sodium ascorbate, and cyanocobalamin.

[0042] Examples of ultraviolet absorbents include tetrahydroxybenzophenone, 2-ethylhexyl paramethoxycinnamate, and ferulic acid. Examples of ultraviolet scattering agents include zinc oxide and titanium oxide.

[0043] Examples of the disinfectant / preservative include isopropylmethylphenol, methyl parahydroxybenzoate, ethyl parahydroxybenzoate, propyl parahydroxybenzoate, butyl parahydroxybenzoate, and silver oxide. Examples of the antioxidant include tocopherol, dibutylhydroxytoluene, and ascorbyl palmitate. Chelating agents include, for example, disodium edetate, caprylhydroxamic acid, tetrasodium etidronate, and the like. Examples of the fragrance include synthetic fragrances and essential oils.

[0044] Examples of active ingredients include adrenal cortical hormones (steroids), lidocaine, dibucaine hydrochloride, chlorpheniramine maleate, glycyrrhetinic acid, dipotassium glycyrrhizinate, allantoin, urea, diphenhydramine, crotamiton, ascorbic acid derivatives, tocopherol acetate, isopropylmethylphenol, l-menthol, dl-camphor, indomethacin, ufenamate, glycol salicylate, methyl salicylate, loxoprofen sodium, diclofenac sodium, felbinac, zinc oxide, panthenol, heparinoids, and pyridoxine hydrochloride. EXAMPLES

[0045] The present invention will be specifically described below with reference to examples, but the scope of the present invention is not limited thereto.

[0046] Example 1: Examination of silk dispersion solvent in manufacturing method In this example, the dispersion medium of silk protein was examined in the manufacturing method of the present invention. The procedure was as follows: 1.0 g of the cocoon shell of the silkworm variety Sericin Hope (Sericin C) was dissolved in 20 mL of 3.5 mol / L calcium chloride aqueous solution (specific gravity of about 1.3, weight ratio to the cocoon shell is about 26 times) at 70 ° C. to prepare a solution with a sericin concentration of about 3.6 wt %. Using various solvents and purified water shown in Table 1, the mixture was mixed so that the sericin concentration was 0.73 wt % and the various solvents were 20, 35, and 50 wt %, and the mixture was left to stand overnight under refrigeration, and the state of the composition was observed. As a result, a uniform dispersion was obtained without large lumps when glycols (PG, IPG, BG) or glycerin (GC) were used as the solvent, and the most homogeneous composition was obtained when glycerin was used in particular.

[0047] [Table 1]

[0048] Example 2: Study of the number of times the composition was washed during the manufacturing method In this example, the number of washings of the composition was examined in the manufacturing method of the present invention. The procedure was as follows: 1.0 g of sericin hope cocoon shell was dissolved in 20 mL of 3.5 mol / L calcium chloride aqueous solution at 35°C, and then diluted 5 times with purified water. The composition was prepared by mixing the various dispersion solvents shown in Table 2 with the sericin solution in a volume ratio of 1:1. The composition was then washed by centrifugation (2,000 x g) and decantation using the same various washing solvents as the dispersion solvent as a 50% aqueous solution, and the calcium ion concentration of the supernatant was measured and the state of the composition was observed. As a result, it was confirmed that the calcium ion concentration decreased after five washings in either glycols (PG, IPG, BG) or glycerin (GC). In particular, when the composition was washed five times with a glycerin aqueous solution, the calcium ion concentration decreased and the smoothest and most homogeneous composition was obtained.

[0049] [Table 2]

[0050] Example 3: Examination of the concentration of the glycerin aqueous solution used during centrifugation washing in the manufacturing method In this example, the concentration of the glycerin aqueous solution used in the washing by centrifugation and decantation (Example 2) in the manufacturing method of the present invention was examined. As a procedure, various concentrations of glycerin aqueous solutions shown in Table 3 were added as washing solutions to a composition (glycerin concentration: 50% by weight) prepared according to the procedure shown in Example 1, and the properties of the composition were observed when the glycerin aqueous solution concentration was 50% by weight to 80% by weight when the washing process by centrifugation was repeated five times. In addition, the calcium ion concentration of the supernatant liquid at the fifth washing was measured. The centrifugation was performed under conditions of 4,000 to 13,000×g. Specifically, the centrifugation was performed at 4,000×g first, and if separation did not occur, the centrifugation was performed again at 9,500×g, and if separation still did not occur, the centrifugation was performed at 13,000×g. As a result, the smoothest and most uniform composition was obtained when an 80% by weight glycerin aqueous solution was used as the washing solution.

[0051] [Table 3]

[0052] Example 4: Examination of glycerin concentration during gel formation in the production method In this example, the glycerin concentration at the time of gel formation in the manufacturing method of the present invention and the yield of sericin contained in the resulting composition were examined. The procedure was as follows: sericin hope cocoon layers were dissolved in a 3.5 mol / L calcium chloride aqueous solution, and sericin solutions prepared at various concentrations were mixed with glycerin or glycerin aqueous solutions prepared at various concentrations in appropriate volume ratios, and the gels formed were centrifuged five times using an 80 wt% glycerin aqueous solution and washed by decantation, and the sericin concentration was measured to calculate the sericin yield. The sericin solution was mixed with glycerin or glycerin aqueous solution so that the sericin concentration after mixing was 0.36 wt% or 0.73 wt%, and the glycerin concentration after mixing was 10 wt%, 20 wt%, 30 wt%, 40 wt%, 50 wt%, 60 wt%, 70 wt%, and 80 wt%. The results are shown in Table 4.

[0053] When the glycerin concentration after mixing was 10%, 20%, or 30%, no gel was formed. When the glycerin concentration after mixing was 40% to 80%, a smooth and homogeneous composition (skin moisturizer) was obtained. A tendency for the yield to improve was observed when the glycerin concentration was 40% by weight, 50% by weight, and 80% by weight.

[0054] [Table 4]

[0055] Example 5: Study on the temperature at which cocoons are dissolved in the manufacturing method In this example, the temperature at which the cocoon layer is dissolved in the manufacturing method of the present invention was examined. As a procedure, the cocoon layer of Sericin Hope was dissolved in a 3.5 mol / L calcium chloride aqueous solution or an 8.0 mol / L lithium bromide aqueous solution, and the temperature was changed to 50°C to 80°C, and the degree of dissolution of the protein was confirmed by the absorbance at 280 nm. In addition, the molecular weight of the protein in the dissolution solution was measured using SDS-polyacrylamide gel electrophoresis (PAGE). The results are shown in Table 5 and Figure 1. Of the two bands detected as the main ones, the molecular weight of the larger band is about 400,000, and the molecular weight of the smaller band is about 200,000. As a result, when the dissolution time was set to 1 to 3 hours at 70°C, the protein was dissolved without decreasing its molecular weight.

[0056] [Table 5]

[0057] Example 6: Manufacturing method using normal cocoons In this example, a composition was produced from normal cocoons containing fibroin. 20 mL of 3.5 mol / L calcium chloride aqueous solution was added to 1.0 g of cocoon shell from which pupae had been removed, and heated at 70°C for 2 hours. The supernatant was separated, and glycerin was added to the mixture so that the mixture was 50% by weight. The resulting composition was washed five times with an 80% by weight glycerin aqueous solution. Centrifugation was performed under conditions of 9,500 x g to 13,000 x g. Specifically, the mixture was first centrifuged at 9,500 x g, and if separation was not achieved, it was centrifuged at 13,000 x g. The yield of sericin was about 21%, and the sericin concentration was about 1.3% by weight.

[0058] Example 7: Measurement of transepidermal water loss when the composition obtained by the manufacturing method was applied In this example, the transepidermal water loss was measured when the composition of the present invention obtained by the manufacturing method of the present invention was applied to the skin. With the cooperation of subjects, Examples A and B of the present invention prepared according to the compositions in Table 6 and Comparative Examples a, b, c, d, and e were applied to the skin, and the transepidermal water loss was measured.

[0059] [Table 6]

[0060] In Example A, 5 g of sericin hope cocoon shell is dissolved in 100 mL of 3.5 mol / L calcium chloride aqueous solution with slow stirring at 35°C for 3 hours, and the insoluble fraction is removed by centrifugation. The supernatant is diluted 5 times with purified water to obtain a sericin solution, and glycerin is mixed to a concentration of 50%. The composition is stored in a refrigerator overnight. The composition is washed under the best washing conditions in Example 3 (80% by weight glycerin, 9,500 x g to 13,000 x g), and the composition (skin moisturizer) of the present invention is obtained by washing the composition, and the sericin concentration is about 1%. In Example B, the composition of Example A is diluted 10 times with purified water, and the sericin concentration is about 0.1%. In Comparative Example A, purified water is used. In Comparative Example B, glycerin is used (standard according to the 17th revised Japanese Pharmacopoeia). In Comparative Example C, a high molecular weight sericin aqueous solution obtained by a conventional preparation method (dissolving in potassium bromide aqueous solution, followed by dialysis) is used, and the sericin concentration is about 1%. Comparative Example d is a solution of hydrolyzed sericin in purified water, with a sericin concentration of 10%, and Comparative Example e is a control (no application). The compositions of the present invention (Examples A and B) have fine particulate gel dispersed smoothly and uniformly in the glycerin aqueous solution, and are opaque, whereas Comparative Example c is a transparent aqueous solution.

[0061] The procedure was as follows: the subjects were allowed to enter a laboratory with a room temperature of 20°C and a relative humidity of 50%, and both arms were exposed and allowed to acclimate for 20 minutes. A 2 cm square area was marked on the anterior part of both forearms as the sample application site. The transepidermal water loss of the marked area before application was measured without any application. A Tewameter CM300 (Courage+Khazaka) was used for the measurement. 10 μL of each sample was applied to a 2 cm square area marked on the anterior part of the forearm. The transepidermal water loss of the application site was measured at each time point, and the measurement results between the samples were compared. The experiment was conducted on seven subjects, and the average value was calculated. The results are shown in Table 7 and Figure 2.

[0062] [Table 7]

[0063] As a result, Example A (the composition of the present invention) suppressed the amount of transepidermal water loss compared to Comparative Example b (aqueous glycerin solution), Comparative Example c (aqueous potassium bromide solution, a conventional sericin solution prepared by dialysis), and Comparative Example d (aqueous hydrolyzed sericin solution). Example B suppressed the amount of transepidermal water loss compared to Comparative Example d (aqueous hydrolyzed sericin solution). The composition of the present invention can be used as a film-forming agent that suppresses human transepidermal water loss and supplements the skin barrier function.

[0064] Example 8: Measurement of transepidermal water loss upon application of a skin moisturizer containing a composition In this example, based on the compositions shown in Tables 8 to 10, two types of skin moisturizers (creams; Examples C1 and D1) containing different bases (base C or base D) and the composition of the present invention (Example A1) were prepared, along with the corresponding placebos (creams; Comparative Examples c1 and d1), and the transepidermal water loss was measured when applied to the skin. The transepidermal water loss was also measured for the compositions of the present invention (Examples A1 and B1) and white petrolatum (Comparative Example e1), a conventionally known skin moisturizer.

[0065] [Table 8]

[0066] [Table 9]

[0067] [Table 10]

[0068] Examples A1 and B1 were prepared in the same manner as Examples A and B of Example 7. The sericin concentration of Example A1 was about 1% by weight, and the sericin concentration of Example B1 was about 0.1% by weight. Example C1 is a skin moisturizer of the present invention obtained by blending 2.5% by weight of the composition of Example A1 into Base C having the composition shown in Table 10, and the sericin concentration is about 0.025% by weight. Comparative Example c1 is a placebo corresponding to Example C1 obtained by blending 2.5% by weight of purified water into Base C having the composition shown in Table 10. Example D1 and Comparative Example d1 were prepared in the same manner as Example C1 and Comparative Example c1, except that Base D having the composition shown in Table 10 was used. Comparative Example e1 is a control (no application).

[0069] The procedure was as follows: subjects (five healthy subjects) were allowed to enter a laboratory with a room temperature of 20°C and a relative humidity of 50%, and were asked to rest for 30 minutes with both arms exposed, to acclimate to the test environment. Four 2 cm square areas were marked on the anterior part of both forearms, and these were used as the sample application sites. The transepidermal water loss was measured before application of the marked areas without any application. The measurement was performed using the Tewameter (Courage+Khazaka) MPA580 multi-skin measuring device. The measuring device measures at 1 second intervals, and the average value is calculated every 5 seconds. The stabilization time was 20 seconds after the start of measurement, and the average value was adopted when the standard deviation of the measured values ​​for the next 5 seconds was 0.2 or less. 10 μL of each sample was measured with a syringe and applied with a spatula to a 2 cm square area marked on the front of the forearm. The transepidermal water loss at the application site was measured at each time point (30 minutes, 60 minutes, and 120 minutes after application), and the measurement results were compared between samples. The measurement results were subjected to multiple comparison tests using the Tukey-Kramer method for each time point, and a significant difference test was performed with respect to the non-application (control, comparative example e1).

[0070] The measurement results (absolute values) based on the transepidermal water loss are shown in Table 11, and the conversion results (relative values) when the transepidermal water loss (TEWL) before application is set to 100% are shown in Table 12. Based on the values ​​shown in Table 12, the results of Example C1, Comparative Example c1, and Comparative Example e1 (control, no application) are shown in Figure 3, the results of Example D1, Comparative Example d1, and Comparative Example e1 (control, no application) are shown in Figure 4, and the results of Example A1, Comparative Example f1, and Comparative Example e1 are shown in Figure 5.

[0071] Transepidermal water loss relative value (%) As shown in Table 11, the only samples that showed significant differences compared to no application (control, comparison example e1) were pure sericin-glycerin gel (Example A1) and the samples containing pure sericin-glycerin gel (Examples B1, C1, and D1) (n=5). Furthermore, as shown in Figures 3 and 4, Examples C1 and D1, which contained 2.5% of the composition of the present invention (sericin concentration of approximately 0.025% by weight), both showed a tendency to suppress transepidermal water loss more than the placebo.

[0072] [Table 11]

[0073] [Table 12] [Industrial Applicability]

[0074] The high molecular weight sericin composition of the present invention can be used as a skin moisturizer for medicines, quasi-drugs, cosmetics, etc.

Claims

1. (1) preparing an aqueous solution of high molecular weight sericin; (2) Mixing the aqueous high molecular weight sericin solution with a compound selected from the group consisting of 1,3-butanediol, isoprene glycol, and propylene glycol, or with glycerin. A method for producing a high molecular weight sericin composition, comprising: The production method as described above, wherein the molecular weight of the high molecular weight sericin is 200,000 or more.

2. (3) A step of washing the mixture obtained in the step (2) by centrifugation and decantation with an aqueous solution of a compound selected from the group consisting of 1,3-butanediol, isoprene glycol, and propylene glycol, or an aqueous solution of glycerin. The method of claim 1 further comprising:

3. 3. The method according to claim 1, wherein the aqueous high molecular weight sericin solution provided in step (1) is prepared by dissolving a cocoon shell or cocoon, or a silk gland derived from a silkworm, in a salt solution.

4. A high molecular weight sericin composition comprising high molecular weight sericin having a molecular weight of 200,000 or more and a compound selected from the group consisting of 1,3-butanediol, isoprene glycol, and propylene glycol, or glycerin.

5. A skin moisturizer comprising the high molecular weight sericin composition according to claim 4.

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