JP2008169171A - Composition containing silk fibroin and method for producing the same - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a silk fibroin-containing composition containing a silk fibroin having high molecular weight at high concentration and to provide a method for producing the composition. <P>SOLUTION: A composition containing a silk fibroin having a molecular weight of ≥100,000 is produced by dissolving an alkali metal halide in a mixture of a lower aliphatic alcohol and water, dissolving a silk fibroin in the resultant mixed solvent, dialyzing the resultant solution and dissolving a protein modifying agent in the dialyzed solution. Preferably, the concentration of the silk fibroin is 5-25 wt.% and that of the protein modifying agent is from 5 wt.% to the saturated concentration of the agent. <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

  The present invention relates to cosmetics, quasi-drugs, pharmaceuticals, and the like containing silk fibroin, and more specifically, a silk fibroin-containing composition excellent in skin moisture retention function (moisturizing effect) and ultraviolet ray blocking function (UV cut effect) And its manufacturing method.

Conventionally, fibroin, which is the main protein component of silk fiber, has characteristics such as moderate moisture absorption and moisture release, affinity for skin and hair, and UV absorption, so it is widely used in the form of powder, liquid, or derivatives thereof. It is used for. For example, Japanese Unexamined Patent Publication (Kokai) No. 55-66929 proposes high purity fine powdery silk fibroin. JP-A-11-181399 proposes a UV cut material using silk.
JP 55-66929 A JP-A-11-181399

  Various end products containing silk fibroin, such as cosmetics using liquid silk fibroin considering the diversification of dosage forms such as lotions, creams, foundations, etc. and compatibility with the skin during use It is preferable to manufacture quasi-drugs, pharmaceuticals and the like.

  Examples of the solvent for obtaining such liquid silk fibroin include the following. 1) Saturated aqueous solution of calcium chloride, 2) Saturated aqueous solution of lithium bromide, 3) Saturated aqueous solution of sodium thiocyanate, 4) Saturated aqueous solution of halide of alkali metal or alkaline earth metal, Schweitzer solution. However, these solvents have a low dissolution rate and a dissolution concentration of about several percent, and in order to recycle them, it was necessary to improve the dissolution concentration by some method.

  In order to avoid such inconvenience, a mixed solvent (hereinafter abbreviated as MU solvent) in which an alkali metal halide is dissolved in a mixed solvent of a lower aliphatic alcohol and water may be used. In the case of this MU solvent, it shows much better solubility than existing solvents, and exhibits a silk fibroin dissolution concentration of about 28%. Thus, if a MU solvent is used, it is possible to obtain a silk fibroin solution with a high concentration. However, since a high concentration of alkali metal halide is harmful to the human body, it cannot be used as it is in cosmetics, quasi drugs, pharmaceuticals, and the like. Therefore, it is necessary to remove the alkali metal halide by dialysis. However, when the alkali metal halide is removed, silk fibroin cannot be dissolved and solidifies.

  On the other hand, a silk fibroin solution obtained by dialysis and concentration of a silk fibroin solution using a calcium chloride aqueous solution or the like as a solvent has a drawback that it is difficult to obtain an intended effect when used as a cosmetic or the like. In other words, hydrolysis occurs during the dissolution process, and the degree of polymerization of the protein is extremely reduced and the molecular weight is reduced (generally, the average molecular weight is about 10,000 to 30,000. For example, a dialysis membrane having a molecular weight cutoff of 10,000. About half of the silk fibroin will leak from the dialysis membrane. That is, when used on the human body, it is difficult to form a water-insoluble film, and the durability and sustainability of the skin moisturizing effect and UV-cutting effect are reduced.

  Accordingly, an object of the present invention is to provide a silk fibroin-containing composition containing silk fibroin having a high concentration and a high molecular weight.

  The inventors of the present invention have intensively studied to solve the above-mentioned problems. As a result, a solution in which silk fibroin is dissolved in a MU solvent is dialyzed to obtain a solution from which alkali metal halide has been removed. More specifically, the inventors have obtained the knowledge that coagulation of silk fibroin is suppressed when urea or a urea derivative is added, and the present invention has been completed based on this finding. In other words, after dialysis of high-concentration silk fibroin solution dissolved in MU solvent, it is possible to break hydrogen bonds between the molecular chains of silk fibroin by adding urea or urea derivatives. The inventor has obtained knowledge that it can be prevented, and has completed the present invention.

  According to the present invention, it is easy to form a water-insoluble film when used on the human body because of its high molecular weight, that is, a silk fibroin-containing composition having a large skin moisturizing effect and UV-cutting effect can be obtained. In addition, combined with the moisturizing effect of urea or urea derivatives, the composition has a large moisturizing effect.

  Furthermore, it has the effect that generation | occurrence | production of mold | fungi can be prevented over a long term by the antiseptic action of urea or a urea derivative.

  The silk fibroin according to the present invention refers to the main protein component of silk thread made by rabbits and the like. Raw materials such as eyebrows, raw silk, scraps, raw silk waste, yarn waste, fried cotton, silk cloth waste, boulet, fluff, etc. in the presence of an activator, in warm water or in the presence of an enzyme as required Among them, sericin is removed and a dried product can be used, but is not limited thereto, and any silk fibroin raw material that is usually available can be used.

  In the present invention, the silk fibroin described above is dissolved in a MU solvent, dialyzed, and then urea or a urea derivative is dissolved. This will be described in detail below.

  The MU solvent is obtained by dissolving an alkali metal halide in a mixed solvent of a lower aliphatic alcohol and water. More specifically, it is a silk fibroin solvent in which 20% or more of an alkali metal halide is dissolved in a mixed solution in which the mixing ratio of water and lower aliphatic alcohol is alcohol / water> 1. The lower aliphatic alcohol herein is represented by CnH2n + 1OH, preferably n = 1 to 4, and particularly preferably methyl alcohol, ethyl alcohol, or propyl alcohol, but is not limited thereto. Of the alkali metals mentioned here, lithium and sodium are particularly preferred, but not limited thereto. Of the halogens mentioned here, particularly preferred are chlorine and bromine, but not limited thereto. Of these alkali metal halides, lithium bromide and lithium chloride are particularly preferred, but are not limited thereto. The concentration of the alkali metal halide is 20% to a saturated concentration, particularly preferably 40 to 50%, but is not limited thereto. The alcohol / water mixing ratio is preferably greater than 1, but is particularly preferably 5/5 to 10/0, but is not limited thereto.

  Next, silk fibroin is dissolved from above 0% to 28 wt%, more preferably 5 wt% to 28 wt% (absolute dry weight) in the MU solvent configured as described above.

  Next, after adding an appropriate amount of water and removing the alcohol by distillation or without removing it, it is sealed in a regenerated cellulose dialysis tube and dialyzed with purified water. As a result of this dialysis, about 90% of the silk fibroin remains without flowing out as shown in Table 3. That is, when MU solvent is used, it can be said that it has melt | dissolved in the state hardly hydrolyzed. The molecular weight of silk fibroin remaining without flowing out is estimated to be several hundred thousand to one million. The experimental results of the solubility and molecular weight of silk fibroin when using MU solvent in Table 1, and the solubility of silk fibroin when using a solvent in which calcium chloride is dissolved instead of lithium bromide as a comparative example in Table 2 And the experimental result of molecular weight is shown.

  As shown in Table 1, when the mixing ratio of ethyl alcohol and water exceeds ethyl alcohol / water> 1, the solubility is greatly increased, and the molecular weight of silk fibroin is very large at about 240,000. On the other hand, as shown in Table 2, when calcium chloride is used, the solubility is low. However, the solubility is greatly increased only when the mixing ratio of ethyl alcohol and water is 30:70, and the molecular weight of silk fibroin at this time is about 180,000. This seems to be because the silk used was in the state immediately after being collected from the cocoon. The measuring method is based on SDS-PAGE. The measurement conditions were a separation gel concentration of 10% and an electrophoresis condition of 250 V / 20 mA / 115 minutes. The staining method is CBB staining, and the final sample concentration is about 1 g / L. The addition amount is 20 μL. The reducing agent is dithiothreitol. As the sample treatment solution, 2% SDS (sodium lauryl sulfate), 100 mM DTT (dithiothreitol), 0.125 M Tris-hydrochloric acid (pH 6.8), and 20% glycerin are used. For the sample, 100 μL of fibroin solution (immediately after dialysis) +100 μL of sample treatment solution is boiled for 2 minutes after stirring, and added before cooling.

  Thus, when the MU solvent is used, the molecular weight of silk fibroin is too large, and when the alkali metal halide is removed by dialysis, it solidifies within a few days and becomes an eraser-like substance. Even if it is made into a cosmetic before solidification, it will solidify after a few days. Therefore, coagulation is prevented by dissolving urea or a urea derivative. At this time, the amount of urea or urea derivative to be dissolved must be at least about 5 wt%. At most, about 50 wt%, which is the saturated concentration of urea or urea derivative, can be added. In this case, the silk fibroin concentration is relatively lowered. A practically preferable concentration is about 5 to 15 wt%. Experimental examples showing the inhibition of coagulation by urea or urea derivatives are shown below.

  Fibroin 10.0% by weight was dissolved in solution A and B having the composition shown in Table 3, sealed in a dialysis tube made of cellulose (fractional molecular weight: 12000 to 14000), and dialyzed in deionized water. Every 24 hours, the deionized water outside the dialysis tube is replaced, and after confirming that no white precipitate is formed even when an aqueous silver nitrate solution is dropped into the water, the fibroin solution in the dialysis tube Was taken into a weighing bottle. This was dried to a constant weight at 105 ° C. and then weighed accurately. Further, when the above solution was subjected to the above dialysis treatment again after adding 10 wt% urea immediately after dialysis of the solution of the former, the residual rate was about 89%. This revealed the fact that coagulation can be suppressed by urea without lowering the molecular weight.

EXAMPLES The present invention will be specifically described below with reference to examples, but the scope of the present invention is not limited to these examples. In the following description, “%” means mass percent.
Example 1 Cream formulation * 1 Fibroin mixed solution 8.0 to 30.0%
Base Stearic acid 2.0%
Base Stearyl alcohol 5.0%
Moisturizer Squalane 5.0%
Base Liquid paraffin 5.0%
Emulsifier Octyldodecanol 6.0%
Emulsifier Polyoxyethylene cetyl ether (25E.O) 3.0%
Emulsifier glyceryl monostearate 2.0%
Moisturizer 1,3-butylene glycol 5.0%
Preservatives Methyl paraoxybenzoate 0.2%
Base Purified water Appropriate amount

* 1 The components of the fibroin mixed solution are as follows.

Fibroin 10.0-11.0% (LiBr dialyzed)
Urea 9.90%
Appropriate amount of water

The ultraviolet absorption effect of the cream formulated as described above is shown in Table 4 in comparison with the blank (direct light). The measuring model is a self-recording spectrophotometer UV-3100 manufactured by Shimadzu Corporation, and the sample is thinly coated on quartz glass and measured. Specifically, the transmittance of 190 to 700 nm including the UV-A region (320 to 400 nm), the UV-B region (280 to 320 nm), and the UV region (280 to 400 nm) was determined. The transmittance in each wavelength region was obtained by expressing the ratio of the integral value (area) of the spectral transmittance of the sample to the integral value (area) of the 100% spectral transmittance in that wavelength region in%. Here, the UV-A region is called long-wave ultraviolet light and causes human skin darkening and skin cancer. The UV-B region is called a medium wavelength ultraviolet ray, which denaturates nucleic acids and proteins in human skin epidermis cells to cause erythema and then secondary blackening. As shown in Table 3, the UV-A region has a transmittance of about 86.6% compared to the blank, and the UV-B region has a transmittance of about 76.6% compared to the blank. Indicated.

Example 2 Emulsion formulation * 2 Fibroin mixed solution 8.0-15.0%
Base Stearic acid 0.2%
Base ethanol 1.5%
Base liquid paraffin 8.0%
Emulsifier Octyldodecanol 6.0%
Emulsifier Polyoxyethylene sorbitan monostearate (20E.O) 3.0%
Emulsifier glyceryl monostearate 1.0%
Moisturizer Glycerin 10.0%
Moisturizer 1,3-butylene glycol 5.0%
Thickener xanthan gum 0.1%
Preservative Methyl paraoxybenzoate 0.1%
pH adjuster Triethanolamine 0.8%
Base Purified water Appropriate amount

* 2 The components of the fibroin mixed solution are as follows.

Fibroin 10.0-11.0% (LiBr dialyzed)
Urea 9.90%
Appropriate amount of water

Claims (4)

A silk fibroin-containing composition in which a protein denaturant is dissolved in a silk fibroin solution having a molecular weight of 100,000 or more. The silk fibroin-containing composition according to claim 1, wherein the concentration of the silk fibroin is 5 wt% to 25 wt%. The silk fibroin-containing composition according to claim 1 or 2, wherein the concentration of the protein denaturant is from 5 wt% to a saturated concentration. A method for producing a silk fibroin-containing composition, comprising dissolving a silk fibroin in a mixed solvent obtained by dissolving an alkali metal halide in a mixed solution of a lower aliphatic alcohol and water and then dissolving the protein denaturant in a solution obtained by dialysis.