JP2020193196A - Ultra-high molecular weight sericin composition capable of forming a film and a method for producing the same - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a simple and industrializable method for producing a high molecular weight sericin protein, a high molecular weight sericin protein having an excellent storage stability and a film forming ability, and a skin moisturizer. The production method includes (1) a step of preparing a high molecular weight sericin aqueous solution, and (2) a step of mixing the high molecular weight sericin aqueous solution with glycerin or glycols. The high molecular weight sericin protein contains high molecular weight sericin and glycerin or glycols. [Selection diagram] None

Description

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

The silk cocoon thread made by silkworms and wasps has a two-layer structure consisting of fibroin protein in the center and sericin protein surrounding fibroin. Sericin acts like glue to bind fibroin fibers to build cocoons and make them firm. There is a description that silk was used as a thread (suture) for sewing wounds in surgical books 2500 years ago, and it is still used as a surgical suture. Silk has high biocompatibility and can be said to be a safe material. In recent years, as a new utilization method, it is expected to be used as a regenerative medical material that promotes tissue regeneration by attaching silk as a scaffold for cell regeneration to damaged or defective parts of the living body by taking advantage of its high biocompatibility and processability. There is.

The use of silk in the field of topical skin application preparations such as cosmetics and external medicines is generally used as an aqueous moisturizer because the amino acid composition is similar to that of human skin. However, most of the silk contained in them becomes hydrolyzed silk. Hydrolyzed silk aqueous solution is soluble silk obtained by hydrolyzing the proteins constituting silk fibers under acid, alkali or proteolytic enzyme, and the molecular weight distribution is mostly low molecular weight and contains high molecular weight protein. (Patent Document 1). Therefore, since the molecular chain is broken in the process of hydrolysis, 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 protein, and if high molecular weight silk protein can be extracted, it will promote the protection of the skin by having a good film-forming ability derived from silk protein. Development of new applications can be expected as a moisturizer and a protective agent that supplements the barrier function.

In order to obtain a high molecular weight silk protein, a method of preparing an aqueous solution by dissolving a cocoon in a high-concentration salt solution such as lithium bromide and then removing the salt by dialysis is generally used (Patent Document 2). However, the silk protein aqueous solution obtained by dialysis has poor stability, and there is a problem that long-term storage is difficult due to gelation, especially when a high molecular weight silk protein is contained. In addition, the conventional technique using dialysis requires various processes and time, and there is a problem that it is difficult to scale up for industrialization. Lithium bromide, which is generally used for dissolving silk proteins, may remain in a small amount after dialysis. Lithium bromide has no precedent for use on the skin, and there is concern about its safety to the living body. Therefore, since the silk film forming ability increases depending on the molecular weight, a stable solution preparation method containing a high molecular weight silk protein and a simple and industrializable manufacturing method (material) using a safe raw material in the manufacturing process. Chemical method) is desired.

Japanese Unexamined Patent Publication No. 2005-002063 Japanese Unexamined Patent Publication No. 2006-11166

The subject of the present invention is to solve the above-mentioned drawbacks of the prior art, to develop a simple and industrializable method for producing a high molecular weight sericin protein, and to develop a high molecular weight sericin protein having excellent storage stability and a film-forming ability and skin moisturizing. To provide the agent.

As a result of intensive research for the purpose of solving the above-mentioned problems, the present inventors have developed a technique for stably dispersing sericin in a high-concentration glycerin aqueous solution or a high-concentration glycol aqueous solution, and have developed this sericin composition. It was found that when applied to the skin, it exhibits a high inhibitory effect on the amount of transepidermal water evaporation, which is the amount of water volatilized through the stratum corneum of the skin (Fig. 2). It was also confirmed that when a skin moisturizer (cream) containing the sericin composition was applied to the skin, the tendency to suppress the transepidermal water loss was higher than that of placebo (FIGS. 3 to 5).

The present invention
[1] (1) Step of preparing high molecular weight sericin aqueous solution,
(2) A method for producing a high molecular weight sericin composition, which comprises a step of mixing the high molecular weight sericin aqueous solution with glycerin or glycols.
[2] (3) The production method according to [1], further comprising a step of washing the mixed solution obtained in the step (2) by centrifugation and decantation using an aqueous glycerin solution or an aqueous glycol glycol solution.
[3] The production method of [1] or [2], wherein the high molecular weight sericin aqueous solution prepared in the step (1) is prepared by dissolving the cocoon layer or the cocoon, or the silk gland derived from the silkworm in a salt solution.
[4] A high molecular weight sericin composition containing high molecular weight sericin and glycerin or glycols.
[5] The high molecular weight sericin composition of [4] obtained by any of the production methods of [1] to [3].
[6] The present invention relates to a skin moisturizer containing the high molecular weight sericin composition of [4] or [5].

According to the present invention, it is possible to provide a simple and industrialized method for producing a high molecular weight sericin protein, and to provide a high molecular weight sericin protein having excellent storage stability and a film-forming ability and a skin moisturizer. .. Further, in a preferred embodiment of the present invention, it is possible to provide a skin moisturizer having excellent safety when applied to the skin.

6 is an electrophoresis image showing the result of subjecting a sericin solution obtained by dissolving a cocoon layer in an aqueous solution of calcium chloride or an aqueous solution of lithium bromide at a temperature of 50 ° C. to 80 ° C. by SDS-polyacrylamide gel electrophoresis (PAGE). It is a graph which shows the result of having measured the transepidermal water loss amount when the composition of this invention was applied to the skin. It is a graph which shows the result of having measured the transepidermal water loss amount at the time of applying the skin moisturizer (cream agent) of this invention to the skin. It is a graph which shows the result of having measured the transepidermal water loss amount at the time of applying another skin moisturizer (cream agent) of this invention with a different base to the skin. It is a graph which shows the result of having measured the transepidermal water loss amount when the composition of this invention was applied to the skin.

<< Manufacturing method of the present invention >>
The manufacturing method of the present invention
(1) Step of preparing high molecular weight sericin aqueous solution (preparation step),
(2) Step of mixing the high molecular weight sericin aqueous solution with glycerin or glycols (mixing step)
Including, if desired, further
(3) A step of washing the mixed solution obtained in the mixing step (2) by centrifugation and decantation using an aqueous glycerin solution or an aqueous glycol glycol solution (washing step).
Can be included.

In the preparation step in the production method of the present invention, an aqueous solution containing high molecular weight sericin is prepared. As used herein, the term "high molecular weight sericin" refers to a substance that is confirmed to spontaneously gel (solidify) and lose its fluidity as a solution if left in an aqueous solution. As shown in Examples described later, the molecular weight corresponds to about 200,000 to 400,000, but is not limited to this range (FIG. 1). For reference, the average molecular weight of low molecular weight hydrolyzed sericin is about tens of thousands.

The high molecular weight sericin aqueous solution can be prepared, for example, by dissolving, but not limited to, a cocoon layer or a cocoon, or a silkworm-derived silk gland in a salt solution. Here, the cocoon is a structure formed by the silkworm larvae secreting silk protein in the silk gland during pupation, and the cocoon layer is a layer of silk protein other than pupa in the cocoon. Means. The silkworm-derived silk gland means a pair of exocrine glands that secrete silk thread in the silkworm. The silkworm silk gland is composed of anterior, middle, and posterior parts, and sericin is secreted from the central silk gland. Therefore, the central silk gland can be used as the silkworm-derived silk gland.

The silk gland or the central silk gland can be isolated by incising the 5th instar larva of the silkworm and removing it from the body. The cocoon layer can also be isolated by separating the cocoons from the cocoons produced by the 5th instar larvae of the silkworm during pupation. Furthermore, the cocoons produced by silkworms can be used as they are.

The cocoon layer or cocoon used in the production method of the present invention, or the silkworm cultivar from which the silkworm gland of the silkworm is derived is not particularly limited, but for example, a cocoon-bearing sericin silkworm strain, for example, sericin hope (sericin C). ), Sericin N, Nd silkworm, Nd-s, Nd-sD silkworm and the like, and sericin hope is preferable. The cocoon-bearing sericin silkworm line is a silkworm line having a special trait that it can be cocooned in the sericin silkworm line that cannot be cocooned originally because the synthetic pathway of fibroin is mutated. The silkworm variety Sericin Hope (Sericin C) is produced by crossing the Chinese variety "CS83" with the variety "Nd" (a mutant species that does not synthesize bare frogs and fibroin but synthesizes only sericin), and then backcrosses. It is a variety produced by performing (Japanese Patent Laid-Open No. 2001-245550). The silkworm variety 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 broad-eating sericin silkworm line obtained by backcrossing the sericin hope silkworm line after mating the broad-eating silkworm line with the cocoon-bearing sericin silkworm line (International Publication No. 2014/065217).

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

The salt solution used to dissolve the cocoon layer or cocoon or silk gland derived from silkworm is not particularly limited as long as high molecular weight sericin can be extracted from the cocoon layer or cocoon or silk gland derived from silkworm. However, for example, a calcium chloride aqueous solution, a lithium bromide aqueous solution, a urea aqueous solution and the like can be mentioned, and from the viewpoint of safety when applied to the skin, a calcium chloride aqueous solution or a urea aqueous solution is preferable, and a calcium chloride aqueous solution is particularly preferable.

The concentration of the salt solution can 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. This is because if it is less than 2.5 mol / L or more than 5.0 mol / L, sericin may not be sufficiently dissolved, and if it exceeds 5.0 mol / L, gelation may not occur in the mixing step described later. .. The amount of the salt solution used for dissolution can be, for example, 10 to 100 times, preferably 20 to 40 times, the weight ratio with respect to the cocoon layer or the like. The upper limit and the lower limit can be arbitrarily combined as desired.

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

The sericin concentration in the obtained high molecular weight sericin aqueous solution can be, for example, 1 to 10% by weight, preferably 2 to 5% by weight. The sericin concentration can be adjusted to the above concentration by adjusting the amount of the salt solution with respect to the cocoon layer or the like, or the obtained high molecular weight sericin aqueous solution is appropriately diluted with water or a salt solution or the like, or It can also be adjusted by concentrating. The upper limit and the lower limit can be arbitrarily combined as desired.

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

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, preferably 0.3 to 1.0% by weight. Further, 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, preferably 50 to 80% by weight, when glycerin is used. This is because if the glycerin concentration is less than 40%, gelation may not occur, and if it exceeds 90%, water is brought in from the sericin aqueous solution, so that it cannot be carried out. As the gelling dispersion solvent, one type of glycerin or glycols may be used alone, or two or more types may 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 mixed with the high molecular weight sericin aqueous solution. The upper limit and the lower limit can be arbitrarily combined as desired.

The cleaning 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 centrifuged and separated using an aqueous glycerin solution or an aqueous glycol glycol solution (hereinafter, may be referred to as a cleaning solution). Clean by decantation. By this washing, the salt 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.

The concentration of glycerin or glycols in the cleaning liquid can be, for example, 50 to 90% by weight, preferably 70 to 80% by weight in the case of an aqueous glycerin solution. A smooth and uniform composition can be obtained by using 70 to 80% by weight. As the cleaning liquid, one type of the glycerin aqueous solution or the glycol aqueous solution may be used alone, or two or more types may be used in combination. Further, the gelling dispersion solvent (that is, 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. When the washing in the washing step is performed a plurality of times, the washing may be performed with one type of aqueous solution or a plurality of types of aqueous solutions, and the concentrations of the aqueous solutions may be changed even if they are all the same. May be good. The upper limit and the lower limit can be arbitrarily combined as desired.

The conditions for centrifugation are not particularly limited as long as they can be precipitated by centrifugation and uniformly dispersed in the cleaning liquid after decantation, but can be, for example, 2,000 × g to 20,000 × g. It is preferably 000 × g to 15,000 × g. The upper limit and the lower limit can be arbitrarily combined as desired.

The number of washings is not particularly limited as long as the salt can be sufficiently removed, but can be, for example, 1 to 5 times, preferably 4 to 5 times. The precipitate after washing (that is, after the last 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. You can also get things.

<< 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 to this, 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 can be, for example, 0.01-5.0% by weight, 0.1-3.0% by weight, 0.5-2.0. It is preferably by weight%. The concentration of glycerin or glycols in the high molecular weight sericin composition (the total concentration thereof when two or more compounds are contained) can be, for example, 50 to 90% by weight, and 70 to 80% by weight. Is preferable. The upper limit and the lower limit can be arbitrarily combined as desired.

The high molecular weight sericin composition of the present invention is considered to be a suspension in which the high molecular weight sericin becomes a fine particulate gel and is smoothly and uniformly dispersed in a gelling dispersion solvent or a washing liquid, and storage stability. Is excellent. The conventional method is obtained by dissolving in a high-concentration salt solution of lithium bromide and then removing the salt by dialysis. The known high molecular weight sericin aqueous solution is a transparent aqueous solution immediately after dissolution, but has a problem that it is poor in stability and it is difficult to store it for a long period of time due to gelation. The high molecular weight sericin composition of the present invention solves this problem.

Further, while the known aqueous solution of high molecular weight sericin uses lithium bromide, which has never been used for skin, in a preferred embodiment of the high molecular weight sericin composition of the present invention, when it is applied to skin. It is excellent in safety because it uses only salts whose safety has been confirmed (for example, calcium chloride). Further, in the high molecular weight sericin composition obtained by carrying out the washing step, which is a preferred embodiment of the present invention, the salt concentration can be significantly reduced, and the safety is further excellent.

Further, since the above-mentioned known high molecular weight sericin aqueous solution removes salts by dialysis, it is difficult to scale up for industrialization, whereas the high molecular weight sericin composition for carrying out the washing step which is a preferred embodiment of the present invention. Since the salt is removed by centrifugation and decantation, it can be easily industrialized.

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

The concentration of sericin contained in the skin moisturizer of the present invention can be, for example, 0.01 to 5.0% by weight, 0.025 to 4.0% by weight, and 0.1 to 3%. It can be 0.0% by weight, 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 thereof when two or more kinds of compounds are contained) can be, for example, 50 to 90% by weight, and 70 to 80% by weight. Is preferable. The upper limit and the lower limit can be arbitrarily combined as desired.

In addition to the high molecular weight sericin composition, the skin moisturizer of the present invention contains various additives that can be applied to the skin moisturizer, pharmaceuticals that can be applied to the skin, and active ingredients of quasi-drugs, if desired. Can be added and used.

For example, as additives, fats and oils, waxes, fatty acids, organic acids, hydrocarbons, alcohols, polyhydric alcohols, ethers, esters, silicone oils, sugars, polymers, surfactants, powders, coloring materials, etc. Extracts, amino acids, peptides, vitamins, UV absorbers, UV scatterers, bactericidal / antiseptic agents, antioxidants, chelating agents, fragrances, water, and active ingredients include antipruritic agents, anti-inflammatory agents, and topical agents. Drugs such as anesthetics, antihistamines, blood circulation promoters, antibacterial / bactericidal agents, tissue repair agents, anti-inflammatory analgesics, and skin softeners can be added and used.

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, canderia wax, lanolin, beeswax, jojoba oil and the like.

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

Examples of the hydrocarbon include squalane, vegetable squalane, light isoparaffin, petrolatum, liquid paraffin, α-olefin oligomer and the like.
Examples of alcohols include ethanol, isopropyl alcohol, cetanol, behenyl alcohol, stearyl alcohol, cholesterol, phytosterol, benzyl alcohol and the like.
Examples of the polyhydric alcohol include glycerin, butylene glycol, propylene glycol, ethylene glycol, isoprene glycol, dipropylene glycol, polyglycerin and the like.
Examples of the ether include diethylene glycol ether, dipropylene glycol ether, alkylglyceryl ether, batyl alcohol, kimil alcohol, and ceracyl alcohol.
Examples of the ester include isopropyl myristate, isopropyl palmitate, myristyl myristate, glyceryl tri-2-ethylhexanoate, glyceryl tri (capryl capric acid), and cholesteryl stearate.

Examples of the silicone oil include dimethicone, diphenyldimethicone, methylcyclopolysiloxane, and polyoxyethylene / methylpolysiloxane copolymer.
Examples of sugars include xylitol, mannitol, sorbitol, inositol, trehalose and the like.

Examples of the polymer include moisturizing components 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 the surfactant include anionic surfactants such as N-acylamino acid salt, N-acylmethyltaurine salt and alkylsulfate, cationic surfactants such as benzalkonium chloride and benzethonium chloride, carboxybetaine and imidazole. Examples thereof include amphoteric surfactants such as linium salts, nonionic surfactants such as ethylene glycol monofatty acid esters, polyglycerin fatty acid esters, sucrose fatty acid esters, alkyl polyglucosides, and polyoxyethylene hydrogenated castor oil.

Examples of the powder include magnesium aluminum silicate, talc, kaolin, mica and the like.
Examples of the coloring material include legal pigments such as fluorescein and indigo.

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

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

Examples of the bactericidal / preservative include isopropylmethylphenol, methyl paraoxybenzoate, ethyl paraoxybenzoate, propyl paraoxybenzoate, butyl paraoxybenzoate, and silver oxide.
Examples of the antioxidant include tocopherol, dibutylhydroxytoluene, ascorbyl palmitate and the like.
Examples of the chelating agent include disodium edetate, caprylic hydroxamic acid, and tetrasodium etidronate.
Examples of fragrances include synthetic fragrances and essential oils.

Active ingredients include, for example, corticosteroids (steroids), lidocaine, dibucaine hydrochloride, chlorpheniramine maleate, glycyrrhetinic acid, dipotassium glycylicylate, allantin, urea, diphenhydramine, crotamitone, ascorbic acid derivatives, tocopherol acetate. Examples thereof include esters, isopropylmethylphenol, l-menthol, dl-camfur, indomethacin, ufenamate, glycol salicylate, methyl salicylate, sodium loxoprofen, sodium diclofenac, fervinac, zinc oxide, panthenol, heparin analogs, pyridoxin hydrochloride and the like.

Hereinafter, the present invention will be specifically described with reference to Examples, but these do not limit the scope of the present invention.

<< Example 1: Examination of Silk Dispersion Solvent in Manufacturing Method >>
In this example, the dispersion medium of silk protein was examined in the production method of the present invention. As a procedure, 1.0 g of a silkworm variety sericin hope (sericin C) cocoon layer is added to 20 mL of a 3.5 mol / L calcium chloride aqueous solution (with a specific gravity of about 1.3, the weight ratio to the cocoon layer is about 26 times) at 70 ° C. To prepare a solution having a sericin concentration of about 3.6% by weight. Using the various solvents and purified water shown in Table 1, the sericin concentration was 0.73% by weight, the various solvents were 20, 35, and 50% by weight, and the mixture was allowed to stand overnight in a refrigerator, and then the composition was prepared. The state of was observed. As a result, when glycols (PG, IPG, BG) or glycerin (GC) were used as the solvent, a uniform dispersion was obtained without forming large lumps, and the composition with the highest homogeneity was obtained particularly when glycerin was used. I got the thing.

<< Example 2: Examination of the number of times the composition was washed in the manufacturing method >>
In this example, the number of times the composition was washed was examined in the production method of the present invention. As a procedure, 1.0 g of a sericin hope cocoon layer is dissolved in 20 mL of a 3.5 mol / L calcium chloride aqueous solution at 35 ° C., diluted 5-fold with purified water, and then various dispersion solvents shown in Table 2 and the sericin solution are dissolved. The composition prepared by mixing the above in a volume ratio of 1: 1 was washed by centrifugation (2,000 × g) and decantation using various washing solvents same as the dispersion solvent as a 50% aqueous solution. , The calcium ion concentration of the supernatant was measured, and the state of the composition was observed. As a result, in any solvent of glycols (PG, IPG, BG) or glycerin (GC), a decrease in calcium ion concentration was confirmed after 5 washings, and particularly when 5 washings with an aqueous glycerin solution, the calcium ion concentration was confirmed. Was reduced to give the smoothest and most homogeneous composition.

<< Example 3: Examination of the concentration of the glycerin aqueous solution used for centrifugal 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 production method of the present invention was examined. The procedure is as follows: when the cleaning step of adding various concentrations of glycerin aqueous solution shown in Table 3 as a cleaning solution to the composition (glycerin concentration: 50% by weight) prepared according to the procedure shown in Example 1 and centrifuging is repeated 5 times. The properties of the composition when the glycerin aqueous solution concentration was 50% by weight to 80% by weight were observed. In addition, the calcium ion concentration of the supernatant at the time of the fifth washing was measured. Centrifugation was performed under the condition of 4,000 to 13,000 × g. Specifically, it was first centrifuged at 4,000 xg, if it was not separated, it was centrifuged again at 9,500 xg, and if it was still not separated, it was centrifuged at 13,000 xg. As a result, the smoothest and most uniform composition was obtained when an 80% by weight aqueous solution of glycerin was used as a cleaning solution.

<< Example 4: Examination of glycerin concentration at the time of gel formation in the production method >>
In this example, the concentration of glycerin at the time of gel formation in the production method of the present invention and the yield of sericin contained in the obtained composition were examined. As a procedure, the sericin hope cocoon layer is dissolved in a 3.5 mol / L calcium chloride aqueous solution, and the sericin solution prepared at various concentrations and the glycerin or the glycerin aqueous solution prepared at various concentrations are prepared in appropriate volume ratios. The mixture was mixed, and the gel produced was centrifuged with an 80 wt% glycerin aqueous solution and washed by decantation 5 times, and then the sericin concentration was measured to calculate the yield of sericin. In the above mixing of the sericin solution and the glycerin or the glycerin aqueous solution, the sericin concentration after mixing was 0.36% by weight or 0.73% by weight, and the glycerin concentration after mixing was 10% by weight, 20% by weight, 30% by weight. It was carried out so that it became weight%, 40% by weight, 50% by weight, 60% by weight, 70% by weight, 80% by weight.
The results are shown in Table 4.

When the glycerin concentration after mixing was 10%, 20%, and 30%, gel formation did not occur. When the glycerin concentration after mixing was 40% to 80%, a smooth and homogeneous composition (skin moisturizer) was obtained. When the glycerin concentration was 40% by weight, 50% by weight, and 80% by weight, the yield tended to improve.

<< Example 5: Examination of temperature at the time of cocoon melting in the manufacturing method >>
In this example, the temperature at the time of melting the cocoon layer in the production method of the present invention was examined. As a procedure, when the sericin hope cocoon layer is dissolved in a 3.5 mol / L calcium chloride aqueous solution or an 8.0 mol / L lithium bromide aqueous solution, the temperature is changed to 50 ° C to 80 ° C, and the cocoon layer is dissolved to dissolve the protein. The degree of dissolution of was confirmed by the absorbance at 280 nm. In addition, the molecular weight of the protein in the lysate was measured using SDS-polyacrylamide gel electrophoresis (PAGE). The results are shown in Table 5 and FIG. Of the two bands detected as the main, the one with the larger molecular weight has a molecular weight of about 400,000, and the one with the smaller molecular weight 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 lowering the molecular weight.

<< Example 6: Manufacturing method using a normal cocoon >>
In this example, the composition was prepared from a normal cocoon containing fibroin. To 1.0 g of the cocoon layer from which the pupae had been removed, 20 mL of a 3.5 mol / L calcium chloride aqueous solution was added, and the mixture was heated at 70 ° C. for 2 hours. The supernatant portion was separated and glycerin was added so as to be 50% by weight. The resulting composition was washed 5 times with 80 wt% aqueous glycerin solution. Centrifugation was performed under the conditions of 9,500 × g to 13,000 × g. Specifically, it was first centrifuged at 9,500 xg, and if it was not separated, it was centrifuged at 13,000 xg. The yield of sericin was about 21% and the concentration of sericin was about 1.3% by weight.

<< Example 7: Measurement of transepidermal water loss amount when the composition obtained by the production method is applied >>
In this example, the transepidermal water loss when the composition of the present invention obtained by the production method of the present invention was applied to the skin was measured. With the cooperation of the subjects, Examples A and B of the present invention prepared with the compositions shown in Table 6 and Comparative Examples a, b, c, d, and e were applied to the skin, and the transepidermal water loss was measured. went.

Example A was obtained by dissolving 5 g of a sericin hope cocoon layer in 100 mL of a 3.5 mol / L calcium chloride aqueous solution with slow stirring at 35 ° C. for 3 hours, and then centrifuging to remove the insoluble fraction. The composition obtained by diluting Qing with purified water 5 times to obtain a sericin solution, mixing glycerin to 50% and storing it in a refrigerator overnight, was obtained with the best washing conditions in Example 3 (the best cleaning conditions in Example 3). The composition (skin moisturizer) of the present invention obtained by washing with 80% by weight glycerin (9,500 × g to 13,000 × g), and has a sericin concentration of about 1%. In Example B, the composition of Example A was diluted 10-fold with purified water, and the sericin concentration was about 0.1%. Comparative example a is purified water. Comparative example b is glycerin (standard based on the 17th revised Japanese Pharmacopoeia). Comparative Example c is a high-molecular-weight sericin aqueous solution obtained by a conventional preparation method (dissolved in a potassium bromide aqueous solution and then dialyzed), and the sericin concentration is about 1%. In Comparative Example d, the reagent hydrolyzed sericin was dissolved in purified water, and the sericin concentration was 10%. Comparative example e is a control (no coating).
In the composition of the present invention (Examples A and B), the fine particulate gel is smoothly and uniformly dispersed in the glycerin aqueous solution and becomes cloudy, whereas Comparative Example c is a transparent aqueous solution. ..

As a procedure, the subject was admitted to a laboratory at room temperature of 20 ° C. and relative humidity of 50%, and acclimatized for 20 minutes with both arms exposed. The anterior parts of both forearms were labeled so as to enclose a 2 cm square area as a sample application site. The amount of transepidermal water loss before application of the labeled portion was measured in the state where nothing was applied. A Tewameter CM300 (Courage + Khazaka) was used for the measurement. Each 10 μL sample was applied to a 2 cm square area labeled on the anterior part of the forearm. The transepidermal water loss at the application site was measured at each time point, and the measurement results between the samples were compared. Experiments were conducted on 7 subjects, and the average value was calculated. The results are shown in Table 7 and FIG.

As a result, Example A (composition of the present invention) includes Comparative Example b (glycerin aqueous solution), Comparative Example c (potassium bromide aqueous solution, conventional sericin solution using dialysis method), and Comparative Example d (hydrolyzed sericin aqueous solution). Compared with this, the amount of transepidermal water evaporation was suppressed. In Example B, the amount of transepidermal water evaporation was suppressed as compared with Comparative Example d (hydrolyzed sericin aqueous solution). The composition of the present invention can be used as a film-forming agent that suppresses the amount of transepidermal water evaporation in humans and supplements the skin barrier function.

<< Example 8: Measurement of transepidermal water loss when a skin moisturizer containing the composition is applied >>
In this example, based on the compositions shown in Tables 8 to 10, two types are based on different bases (base C or base D) and contain the composition of the present invention (Example A1). Skin moisturizers (creams; Examples C1 and D1) and corresponding placebos (creams; Comparative Examples c1 and d1) were prepared and the amount of transepidermal water loss when applied to the skin was measured. In addition, the transepidermal water loss amount was also measured for the composition of the present invention (Example A1, Example B1) and white petrolatum (Comparative Example e1), which is a conventionally known skin moisturizer.

Examples A1 and B1 were prepared in the same manner as in Example A and Example B of Example 7. The sericin concentration of Example A1 is about 1% by weight, and the sericin concentration of Example B1 is 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 a base C having the composition shown in Table 10, and has a sericin concentration of about 0. It is 025% by weight. Comparative Example c1 is a placebo corresponding to Example C1 obtained by blending 2.5% by weight of purified water with a base C having the composition shown in Table 10.
Example D1 and Comparative Example d1 were prepared in the same manner as in Example C1 and Comparative Example c1 except that the base D having the composition shown in Table 10 was used.
Comparative example e1 is a control (no coating).

As a procedure, the subjects (five healthy subjects) were admitted to a laboratory at room temperature of 20 ° C. and relative humidity of 50%, and were allowed to rest for 30 minutes with both arms exposed to acclimatize to the test environment. A 2 cm square area was marked on both arms at four locations on each of the anterior parts of the forearms to serve as sample application sites. The amount of transepidermal water loss before application of the labeled portion was measured in the state where nothing was applied. For the measurement, a Tewameter (Courage + Khazaka) of the MPA580 multi-skin measuring machine was used. In the measuring machine, measurement is performed at 1-second intervals, and an average value is calculated every 5 seconds. The stabilization time was set to 20 seconds after the start of measurement, and the average value when the standard deviation of the measured values for 5 seconds was 0.2 or less was adopted.
Each 10 μL sample was measured with a syringe and applied with a spatula to a 2 cm square area labeled on the anterior part of the forearm. The transepidermal water loss at the application site was measured at each time point (30 minutes, 60 minutes, 120 minutes after application), and the measurement results between the samples were compared. The measurement results were subjected to a multiple comparison test by the Tukey-Kramer method for each time point, and a significant difference test with no coating (control, comparative example e1) was performed.

Table 11 shows the measurement results (absolute values) based on the transepidermal moisture evaporation amount, and Table 12 shows the conversion results (relative values) when the transepidermal moisture evaporation amount (TEWL) before application is 100%. .. Further, based on the numerical values shown in Table 12, the results of Example C1, Comparative Example c1, and Comparative Example e1 (control, no coating) are shown in FIG. 3, Example D1, Comparative Example d1, and Comparative Example e1 (Control, None). The results of coating) are shown in FIG. 4, and the results of Example A1, Comparative Example f1, and Comparative Example e1 are shown in FIG. 5, respectively.

Transepidermal water loss Relative value (%)
As shown in Table 11, a significant difference was observed with respect to the non-applied product (control, Comparative Example e1), in which the pure sericin-glycerin gel product (Example A1) and the pure sericin-glycerin gel product were blended. Only the samples (Example B1, Example C1, Example D1) were used (n = 5).
Further, as shown in FIGS. 3 and 4, both Example C1 and Example D1 (about 0.025% by weight as a sericin concentration) containing 2.5% of the composition of the present invention had transepidermis more than placebo. There was a tendency to suppress the amount of water evaporation.

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

Claims (6)

(1) Step of preparing high molecular weight sericin aqueous solution,
(2) A method for producing a high molecular weight sericin composition, which comprises a step of mixing the high molecular weight sericin aqueous solution with glycerin or glycols. (3) The production method according to claim 1, further comprising a step of washing the mixed solution obtained in the step (2) by centrifugation and decantation using an aqueous glycerin solution or an aqueous glycol glycol solution. The production method according to claim 1 or 2, wherein the high-molecular-weight sericin aqueous solution prepared in the step (1) is prepared by dissolving a cocoon layer or a cocoon or a silkworm-derived silk gland in a salt solution. A high molecular weight sericin composition containing a high molecular weight sericin and glycerin or glycols. The high molecular weight sericin composition according to claim 4, which is obtained by the production method according to any one of claims 1 to 3. A skin moisturizer containing the high molecular weight sericin composition according to claim 4 or 5.