JP7634814B2 - High molecular weight sericin composition having film-forming ability and method for producing same - Google Patents

Description

The present invention relates to a high molecular weight sericin composition having film-forming ability, a skin moisturizer, and a method for producing the same.

The silk threads known as silk, made by silkworms and wasps, have a two-layer structure with 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. There are records of silk being used as a thread (suture) for sewing up wounds in surgical texts from 2,500 years ago, and it is still used as a surgical suture today. Silk has high biocompatibility and can be considered a safe material. In recent years, a new way of using silk is to take advantage of its high biocompatibility and workability 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.

In the field of cosmetics, topical drugs, and other topical skin preparations, silk is generally used as an aqueous moisturizer because its amino acid composition is similar to that of human skin. However, most of the silk contained in these preparations 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. 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 moisturizers that promote skin protection and protective agents that supplement barrier function.

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 small 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.

JP 2005-002063 A JP 2006-111667 A

The objective of the present invention is to overcome the above-mentioned drawbacks of the conventional technology, develop a simple and industrially feasible method for producing high molecular weight sericin protein, and provide a high molecular weight sericin protein and a skin moisturizer that have excellent storage stability and film-forming ability.

As a result of intensive research aimed at solving the above problems, the 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).

The present invention relates to
[1] (1) preparing an aqueous solution of high molecular weight sericin;
(2) A method for producing a high molecular weight sericin composition, comprising a step of mixing the high molecular weight sericin aqueous solution with glycerin or glycols.
[2] (3) The method according to [1], further comprising the step of washing the mixture obtained in the step (2) with an aqueous glycerin solution or an aqueous glycol solution by centrifugation and decantation;
[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] The 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 of [4] or [5].

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 that have excellent film-forming ability and 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.

This is an electrophoretic image showing the results of subjecting 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. to SDS-polyacrylamide gel electrophoresis (PAGE). 1 is a graph showing the results of measuring transepidermal water loss when the composition of the present invention was applied to the skin. 1 is a graph showing the results of measuring the transepidermal water loss when the skin moisturizer (cream) of the present invention is applied to the skin. 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. 1 is a graph showing the results of measuring transepidermal water loss when the composition of the present invention was applied to the skin.

<<Production method of the present invention>>
The manufacturing 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.

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 an aqueous solution state. As shown in the examples below, this corresponds to a molecular weight of approximately 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 approximately several tens of thousands.

The high molecular weight sericin aqueous solution can be prepared, for example and not limited to, by dissolving a cocoon layer or cocoon, or a silkworm-derived silk gland in a salt solution. Here, a cocoon is a structure that silkworm larvae create by secreting silk proteins in the silk gland during pupation, and a cocoon layer refers to the layer of the cocoon that is made of silk proteins other than the pupa. A silkworm-derived silk gland refers to a pair of exocrine glands that secrete silk threads in silkworms. The silkworm silk gland 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.

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 that the 5th instar silkworm larva produces during pupation. Furthermore, the cocoon produced by the silkworm can be used as is.

The silkworm cultivar from which the cocoon layer or cocoon or silkworm silk gland used in the manufacturing method of the present invention is derived is not particularly limited, but examples include spinning sericin silkworm strains, such as Sericin Hope (Sericin C), Sericin N, Nd silkworms, Nd-s and Nd-sD silkworms, with Sericin Hope being preferred. The spinning sericin silkworm strain is a silkworm cultivar that has a special trait of being able to spin a cocoon, even though it is a sericin silkworm cultivar that is not capable of spinning a cocoon due to a mutation in the fibroin synthesis pathway. The silkworm cultivar Sericin Hope (Sericin C) was created by crossing the Chinese cultivar "CS83" with the cultivar "Nd" (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. It is also possible to use a polyphagous sericin silkworm strain obtained by crossing a cocoon-spinning sericin silkworm strain with a polyphagous silkworm strain and then backcrossing the sericin Hope silkworm strain (WO 2014/065217).

When using normal silkworms that can synthesize 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.

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.

When using an aqueous calcium chloride solution, the concentration of the salt solution can be, for example, 2.5 to 5.0 mol/L, and preferably 3.0 to 4.0 mol/L. 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 exceeds 5.0 mol/L, gelation may not occur in the mixing step described below. The amount of salt solution used for dissolution can be, for example, 10 to 100 times, and preferably 20 to 40 times, the amount of the cocoon layer, etc., by weight. The upper and lower limits mentioned above can be combined as desired.

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

The sericin concentration in the obtained aqueous high molecular weight sericin solution can 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 layer, etc., or by diluting the obtained aqueous high molecular weight sericin solution with water or a salt solution, etc., as appropriate, or by concentrating it. The above upper and lower limits can be combined in any desired manner.

In the mixing step of the manufacturing 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).

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

In the production method of the present invention, the washing step 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). This washing makes it possible to remove salts in the mixed solution derived from the salt solution from the mixed solution, and a high molecular weight sericin composition of the present invention having a reduced salt concentration can be obtained.

The concentration of glycerin or glycols in the cleaning solution can 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 cleaning solution can 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 cleaning solution used in the cleaning step can be the same compound or different compounds. In addition, when cleaning is performed multiple times in the cleaning step, cleaning can be performed with one type of aqueous solution or multiple types of aqueous solutions, and the concentrations of the aqueous solutions can be the same or different. In addition, the upper and lower limits described above can be combined arbitrarily as desired.

The conditions for centrifugation are not particularly limited as long as a precipitate is formed by centrifugation and can be uniformly dispersed in the washing solution after decantation. 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 combined in any desired manner.

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.

<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 above upper and lower limits may be arbitrarily combined as desired.

The high molecular weight sericin composition of the present invention is considered to be a suspension in which high molecular weight sericin has become a fine particulate gel and is smoothly and uniformly dispersed in a gelling dispersion solvent or cleaning solution, and has excellent storage stability. In the conventional method, it is obtained by dissolving in a high concentration salt solution of lithium bromide and then removing the salt by dialysis. Although known aqueous solutions of high molecular weight sericin are transparent immediately after dissolution, they 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.

In addition, the known high molecular weight sericin aqueous solution uses lithium bromide, which has never been used on the skin, whereas the preferred embodiment of the high molecular weight sericin composition of the present invention uses only salts (e.g., calcium chloride) whose safety has been confirmed when applied to the skin, and therefore has excellent safety. In addition, in the preferred embodiment of the high molecular weight sericin composition of the present invention, which has been subjected to a washing process, the salt concentration can be significantly reduced, and it has even greater safety.

Furthermore, the known aqueous high molecular weight sericin solution described above has difficulty in scaling up for industrialization because salts are removed by dialysis, whereas the high molecular weight sericin composition that undergoes the washing process, which is a preferred embodiment of the present invention, has salts removed by centrifugation and decantation, making it easy to industrialize.

<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.
Furthermore, 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.

The concentration of sericin contained in the skin moisturizer of the present invention may be, for example, 0.01 to 5.0% by weight, 0.025 to 4.0% by weight, 0.1 to 3.0% by weight, and preferably 0.5 to 2.0% by weight. The concentration of glycerin or glycols contained in the skin moisturizer of the present invention (the total concentration when two or more types of compounds are contained) may be, for example, 50 to 90% by weight, and preferably 70 to 80% by weight. The above upper and lower limits may be combined in any combination as desired.

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.

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

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.

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.

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, glyceryl tri(capryl/capric acid), and cholesteryl stearate.

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

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.

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

Examples of extracts include aloe extract, perilla extract, oil-soluble chamomile extract, and licorice extract.
Examples of the 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.

Examples of the ultraviolet absorbing agent include tetrahydroxybenzophenone, 2-ethylhexyl paramethoxycinnamate, and ferulic acid.
Examples of ultraviolet scattering agents include zinc oxide and titanium oxide.

Examples of the disinfectant/antiseptic 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.
The fragrances include, for example, synthetic fragrances and essential oils.

Active ingredients include, for example, 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.

The present invention will be explained in detail below with reference to examples, but these are not intended to limit the scope of the present invention.

Example 1: Study 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 layer 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 layer is about 26 times) at 70°C to prepare a solution with a sericin concentration of about 3.6% by weight. Using various solvents and purified water shown in Table 1, the sericin concentration was 0.73% by weight, and the various solvents were mixed to 20, 35, and 50% by weight, and the mixture was left to stand overnight under refrigeration, after which the state of the composition was observed. As a result, when glycols (PG, IPG, BG) or glycerin (GC) were used as the solvent, a uniform dispersion was obtained without large lumps, and the most homogeneous composition was obtained when glycerin was used in particular.

Example 2: Study on the number of times the composition was washed during the production 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 the mixture was diluted 5 times with purified water. The compositions were then mixed with the various dispersion solvents shown in Table 2 in a volume ratio of 1:1. The compositions were then washed by centrifugation (2,000×g) and decantation using the same washing solvents as the dispersion solvents as 50% aqueous solutions, 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.

Example 3: Examination of the concentration of the glycerin aqueous solution used during centrifugation washing in the production 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 set to 50% by weight to 80% by weight when the washing step 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 x g. Specifically, the mixture was first centrifuged at 4,000 x g, and if separation did not occur, it was centrifuged again at 9,500 x g, and if separation still did not occur, it was centrifuged at 13,000 x 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.

Example 4: Study 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 layer was 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 gel formed was 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.

When the glycerin concentration after mixing was 10%, 20%, or 30%, no gel formation occurred. 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%, 50%, and 80% by weight.

Example 5: Study on the temperature at which cocoons are dissolved in the production method
In this example, the temperature at which the cocoon layer is dissolved in the manufacturing method of the present invention was examined. The procedure involved dissolving the Sericin Hope cocoon layer in a 3.5 mol/L calcium chloride aqueous solution or an 8.0 mol/L lithium bromide aqueous solution, changing the temperature conditions from 50°C to 80°C, dissolving the cocoon layer, and confirming the degree of dissolution of the protein by 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 main bands detected, the larger band has a molecular weight of about 400,000, and the smaller band has a molecular weight of about 200,000. As a result, when the dissolution time was set to 1 to 3 hours at 70°C, the protein could be dissolved without decreasing its molecular weight.

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 the mixture was heated at 70°C for 2 hours. The supernatant was separated, and glycerin was added to the mixture so that the content 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.

Example 7: Measurement of transepidermal water loss when the composition obtained by the production 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.

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). The composition (skin moisturizer) of the present invention is obtained by washing the composition under the best washing conditions in Example 3 (80% by weight glycerin, 9,500 x g to 13,000 x g). 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 and then 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%. 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 cloudy, whereas Comparative Example c is a transparent aqueous solution.

The procedure involved subjects entering a laboratory with a room temperature of 20°C and a relative humidity of 50%, and acclimating both arms for 20 minutes with both arms exposed. 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 was measured before application 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 samples were compared. The experiment was performed on seven subjects, and the average value was calculated. The results are shown in Table 7 and Figure 2.

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 using 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 the amount of transepidermal water loss in humans and supplements the skin barrier function.

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) based on different bases (base C or base D) and containing the composition of the present invention (Example A1) and the corresponding placebos (creams; Comparative Examples c1 and d1) were prepared, and the transepidermal water loss was measured when applied to the skin. In addition, the transepidermal water loss was also measured for the composition of the present invention (Examples A1 and B1) and white petrolatum (Comparative Example e1), a conventionally known skin moisturizer.

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 with 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 with 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 coating).

The procedure was as follows: subjects (five healthy subjects) were allowed to enter a laboratory at room temperature of 20°C and 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 the sample application sites were used. The transepidermal water loss of the marked areas was measured before application 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 following 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 the 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).

Table 11 shows the measurement results (absolute values) based on the transepidermal water loss, and Table 12 shows the conversion results (relative values) when the transepidermal water loss (TEWL) before application is set to 100%. Based on the values 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.

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

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

Claims (5)

(1) preparing an aqueous solution of high molecular weight sericin;
(2) A method for producing a high molecular weight sericin composition, comprising a step of mixing the high molecular weight sericin aqueous solution with a compound selected from the group consisting of 1,3-butanediol, isoprene glycol, and propylene glycol, or with glycerin,
The production method as described above, wherein the molecular weight of the high molecular weight sericin is 200,000 or more. The method according to claim 1, further comprising the step of: (3) washing the mixture obtained in the step (2) with an aqueous solution of a compound selected from the group consisting of 1,3-butanediol, isoprene glycol, and propylene glycol, or with an aqueous solution of glycerin by centrifugation and decantation. The method according to claim 1 or 2, wherein the aqueous high molecular weight sericin solution prepared in step (1) is prepared by dissolving a cocoon shell or cocoon, or a silk gland derived from a silkworm, in a salt solution. 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. A skin moisturizer comprising the high molecular weight sericin composition according to claim 4 .