JP2014033881A - Collagen artificial skin comprising collagen fiber and evaluation method of ultraviolet damage using the same - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a testing method that can further correctly investigate screening effect of ultraviolet and can suitably correspond to even an object that investigates the screening effect in which difference between races, difference between individuals or the like is considered.SOLUTION: Provided are a collagen artificial skin that has a nonwoven cloth shape structure comprising a collagen fiber that is obtained by reconstituting a solubilization collagen; and an evaluation method of screening effect of ultraviolet using the same.

Description

  The present invention relates to a collagen artificial skin comprising collagen fibers reconstituted from solubilized collagen, and a method for evaluating ultraviolet damage using the same.

  If you continue to be exposed to sunlight for a long time, various skin disorders will be caused, causing wrinkles and spots called photoaging. These phenomena due to photoaging are considered to be quantitative and qualitative changes of collagen due to ultraviolet rays.

In order to avoid the occurrence of these obstacles, cosmetics containing various UV shielding properties have been developed, and SPF which is an ultraviolet protection index caused by UV-B as a method for evaluating their UV shielding properties (Sun Protection
PA (Protection), which is the UV protection index caused by the Factor value and UV-A
grade of UV-A) value. This evaluation method is defined by the Japan Cosmetic Industry Association and widely used internationally (Non-patent Document 1), but these methods are methods using a subject, such as the genetic, age or physical condition of the subject. Since differences due to individual differences affect the validity of the evaluation, it is not necessarily a suitable method for accurate evaluation of sunclean agents and the like based on genetic characteristics.

  On the other hand, a method for reconstituting solubilized collagen is known as a method for producing artificial skin. (Patent Documents 1 and 2)

JP 2009-5814 A JP 2012-1859 A

ISO-24444

  It is desired to develop an ultraviolet damage evaluation method that can cope with the purpose of examining the shielding effect of ultraviolet rays more accurately and taking into account individual differences, which is difficult with the conventional method.

  The present inventor has intensively studied to solve the above problems, and has found that the above problems can be solved by using artificial skin made of specific collagen, and has completed the present invention.

  That is, the present invention is a collagen artificial skin which is a non-woven multi-element structure composed of collagen fibers obtained by reconstituting solubilized collagen.

  The present invention further relates to a collagen artificial skin formed into a non-woven multi-component structure after adding 0.01% to 70% of eumelanin to collagen fibers (the total of collagen fibers and eumelamine is taken as 100%).

  The present invention further provides a collagen artificial skin formed by immersing collagen fibers in an aqueous solution of 0.005 mM to 30 mM saccharide and reacting at 32.0 ° C. to 40.0 ° C. for saccharification and crosslinking to form a non-woven multi-component structure.

  The present invention is also an ultraviolet damage evaluation method characterized by using the collagen artificial skin.

  The present invention is also an ultraviolet damage evaluation method for evaluating the degree of damage caused by ultraviolet rays based on the difference in terminal amino group concentration and total protein amount in the extract of acetic acid aqueous solution of the collagen artificial skin before and after ultraviolet irradiation.

  The artificial skin of the present invention can be suitably used for evaluation of ultraviolet damage and is extremely useful industrially. In particular, the use of the artificial skin of the present invention is of great value in that the effects of sunclean agents and the like on ultraviolet damage due to individual differences can be evaluated.

2 is a photograph taken with a scanning electron microscope of a cross section of an artificial skin produced in Example 1. FIG.

  In the present invention, solubilized collagen is a telopeptide of tropocollagen that forms a collagen fiber through the action of a proteolytic enzyme such as pepsin containing a large amount of type I collagen such as pork leg. The collagen molecules are broken into pieces (atelocollagen) by decomposing parts, and those of various origins are available on the market. By dissolving so as to become a uniform aqueous solution of collagen.

  The collagen aqueous solution is neutralized at a low temperature using a buffer solution such as a mixed aqueous solution of sodium carbonate and sodium hydrogen carbonate of about 0.1 to 1 mol / L, and then the temperature is raised (about 30 ° C. or higher). Fibrous collagen can be reconstituted (for example, Patent Document 2).

  Further, the aqueous solution of solubilized collagen can be made into artificial skin having a desired structure by reconstituting collagen fibers by neutralizing with phosphate buffered saline and heat-treating and further crosslinking or depositing them. (For example, Patent Document 1)

  Here, when reconstituting collagen fibers by heat treatment, as a method for forming a non-woven multi-element structure, an aqueous dispersion of collagen fibers is gently stirred with a stirring blade to form a uniform dispersion, and then a filter paper A lamination method such as repeating suction filtration with a Buchner funnel or the like equipped with can be exemplified. An artificial skin can be obtained by drying or freeze-drying the sheet of the non-woven multi-component structure thus obtained.

  Further, in order to evaluate the ultraviolet damage under a specific condition, a corresponding condition is set when manufacturing the artificial skin. For example, in order to confirm the effect of the amount of melamine pigment, by changing the amount ratio of eumelamine to collagen by coexisting eumelamine during deposition, it shows the difference in UV damage due to differences in race etc. Such artificial skin can be obtained.

  Further, in order to change the degree of cross-linking between collagen fibers or collagen fine fibers, a predetermined amount of a cross-linking agent such as glyoxal can be allowed to coexist at a predetermined timing to make the degree of cross-linking desired. By doing so, an artificial skin corresponding to skin aging can be obtained.

  Since artificial skin under various conditions can be manufactured by the above-described method, it is possible to know the relationship between ultraviolet damage and the state of artificial skin by irradiating ultraviolet rays using such a material and observing the effect. it can.

  In the present invention, the amount of eumelanin added to highlight the skin characteristics is about 0.01% to 70%, preferably 0.8% to 50%, more preferably 100% of the total of collagen fibers and eumelamine. It is common to make a nonwoven fabric after adding 3% to 20%.

  In the present invention, collagen fibers are dispersed in an aqueous solution of saccharides in which glyoxal is dissolved in an amount of 0.005 mM to 30 mM, preferably 0.008 mM to 12 mM, more preferably 0.5 mM to 5.0 mM, in order to highlight the degree of skin aging. It is common to carry out a saccharification crosslinking by reacting at 32.0 ° C to 40.0 ° C, preferably 35.5 ° C to 38.5 ° C for 6 hours or more, and then forming a nonwoven fabric.

  In the present invention, the method for evaluating the ultraviolet damage of the artificial skin is preferably performed by comparing the amount of terminal amino groups in the extract of the artificial skin before and after the ultraviolet irradiation.

  Specifically, the artificial skin used for the evaluation was extracted with an acetic acid aqueous solution, and the terminal amino group can be quantified by reacting the extract with a ninhydrin solution. The degree of damage caused by can be evaluated.

  In addition, the amount of total protein in the collagen extract can be quantified by reacting the extract with a burette reagent, and the degree of damage caused by UV can be evaluated by the change in the total protein in the extract before and after UV irradiation. .

  EXAMPLES The present invention will be further described below with reference to examples, but the present invention is not limited to the examples.

  How to adjust sun cream-1 (P3. Standard sample for high SPF)

  The following “Part I” is heated to 75-80 ° C. Heat “Part II” to 80 ° C. Add "Part I" to "Part II" with stirring. "Part III" disperses carbomer in water while stirring with a stirrer. Furthermore, it neutralizes with sodium hydroxide aqueous solution. While stirring the mixture of “Part I” and “Part II”, add “Part III” and emulsify for 3 minutes. Adjust the pH with sodium hydroxide or lactic acid and cool completely with stirring. Emulsify after correcting the weight of water.

Ingredient Weight%
Part I cetostearyl alcohol * 1 2.205
Polyoxyethylene castor oil (4O EO) * 2 0.63
Sodium cetostearyl sulfate * 3 0.315
Decyl oleate 15.0
2-Ethylhexyl paramethoxycinnamate 3.0
4-tert-Butyl-4'-methoxydibenzoylmethane 0.5
Propyl paraoxybenzoate 0.1
Note: Emulgate F = * 1 + * 2 + * 3: 3.15%

Part II purified water 53.57
Phenylbenzimidazolesulfonic acid 2.78
Sodium hydroxide aqueous solution (45%) 0.9
Methyl paraoxybenzoate 0.3
Edetate disodium 0.1

Part III purified water 20.0
Carboxyvinyl polymer 0.3
Sodium hydroxide aqueous solution (45%) 0.3

  How to adjust sun cream -2 (P7. Standard sample for low SPF)

  Heat "Part I" and "Part II" from 77 ° C to 82 ° C and stir until they are completely dissolved. Slowly add "Part I" to "Part II" with stirring. Cool to room temperature with continued stirring. Add purified water to correct the weight.

Ingredient Weight%
Part I Lanolin 5.0
Homomentil salicylate 8.0
Vaseline 2.5
Stearic acid 4.0
Propyl paraoxybenzoate 0.05

Part II Methyl paraoxybenzoate 0.10
Edetate disodium 0.05
Propylene glycol 5.0
Triethanolamine 1.0
Purified water 74.3

  How to apply sunscreen cream

A method was used in which a sunscreen cream was evenly applied to a polyethylene wrap film (thickness: 12.16 μm) having a relatively high ultraviolet transmittance and adhered to artificial collagen skin. Each sunscreen cream was evenly applied to the polyethylene wrap film with fingers so that the amount was about 2.00 mg / cm ² . At that time, by applying a very small amount of sunscreen cream to the finger in advance, the sunscreen cream was absorbed into the skin of the finger to be applied and the amount of application was prevented from changing. After drying for 30 minutes, a polyethylene wrap film was applied, and this was overlaid on the collagen artificial skin. Then, ultraviolet irradiation and exposure experiments were performed.

  UV irradiation method

As the ultraviolet rays, ultraviolet irradiation was performed for a predetermined time using a tabletop xenon accelerated exposure apparatus Suntest CPS manufactured by Shimadzu Corporation. At that time, when the irradiation intensity was measured using a digital UVX ultraviolet intensity meter manufactured by Ultraviolet Products, 4,120 μW / cm ² (365 nm), 5,540 μW / cm ² (310 nm), 2,580 μW / cm ² ( 254 nm). In other words, the cumulative amount of UV energy for 60 minutes is 3.33 × 10 ^{5 at} 365 nm.
It becomes 3.03 × 10 ⁵ J / m ^{2 at} J / m ² and 310 nm, and 4.65 × 10 ⁴ J / m ^{2 at} 254 nm.

  Analysis method of collagen artificial skin after UV irradiation

  The artificial skin after irradiation for 25 hours and before irradiation was analyzed and evaluated by the following methods. First, the collagen artificial skin after ultraviolet irradiation was cut into a size of about 3 × 3 mm, and about 0.1 g was put into a weighing bottle, and a 50-fold volume of 50 mM acetic acid aqueous solution was added and immersed. The weighing bottle was capped and shaken with a shaker at a shaking speed of 120 rpm for 24 hours. Thereafter, only the supernatant was collected to obtain a collagen extract used for analysis.

Also, 1.83 × 10 ^-2
80 ml of 9.49 mol / l sodium hydroxide aqueous solution was added to 42 ml of mol / l copper sulfate aqueous solution and stirred to prepare a burette reagent. 500 μl of the prepared burette reagent was added to 1,000 μl of the collagen extract and stirred, and left for 10 minutes. Absorbance at 310 nm was measured using a spectrophotometer, and the total amount of protein in the collagen extract was calculated. In addition, different collagen standard solutions (0.01-0.1%) were used for quantification of the total protein amount.

  Place 0.1 ml of collagen extract obtained by the above method into a test tube, 1 ml of ninhydrin solution (propylene glycol monomethyl ether, ninhydrin) manufactured by Wako Pure Chemical Industries, Ltd., buffer solution manufactured by Wako Pure Chemical Industries, Ltd. ( 1 ml of propylene glycol monomethyl ether, lithium acetate dihydrate) was added. After stirring, a glass bulb was placed on the test tube and heated at 100 ° C. for 10 minutes. Then, it was left for about 3 hours at room temperature. The absorbance of the sample liquid at a wavelength of 570 nm was measured using Shimadzu Corporation spectrophotometer UV-3100 manufactured by Shimadzu Corporation. Similarly to collagen molecular chains, solutions with different concentrations of bovine serum albumin having one terminal amino group in one molecule were prepared, and the absorbance was measured by the same treatment. Assuming that ninhydrin reacts only with the terminal amino group, a calibration curve of terminal amino group concentration and absorbance was prepared. Using this calibration curve, the terminal amino group concentration in the liquid was calculated from the absorbance of the sample liquid. Since the increased terminal amino group concentration corresponds to the number of sites damaged by ultraviolet rays, the degree of damage due to ultraviolet rays can be estimated from the difference in terminal amino group concentrations before and after the call irradiation.

Example 1
The porcine dermis-derived pepsin-solubilized collagen aqueous solution was neutralized with phosphate buffered saline and heated at 37 ° C. to reconstitute collagen fibers. Next, after gently stirring, a predetermined amount of eumelanin (manufactured by Toyo Ink Manufacturing Co., Ltd.) was added and stirred. The obtained fiber dispersion was deposited on a quantitative filter paper (manufactured by ADVANTEC) having an inner diameter of 50 mm using a Buchner funnel, and the collagen fibers in sheet form were freeze-dried to obtain a collagen artificial skin. The result of observing the surface of the artificial skin thus obtained with a scanning electron microscope is shown in FIG. As a result, a three-dimensional fine fiber structure was observed, and it was found that the structure was a sponge-like structure.

  Two types of sunscreen standard samples described below were applied to the artificial skin thus obtained, and irradiated with ultraviolet rays. As standard samples, a standard sample for P7 low SPF (SPF: 5.1) and a standard sample for P3 high SPF (SPF: 16.2) were used.

(Examples 2 to 4)
A collagen artificial skin to which a predetermined amount of eumelanin was added was used in consideration of differences according to race. Others are the same as in the first embodiment.

  As a result, the extract obtained from the collagen artificial skin after ultraviolet irradiation showed a difference in the terminal amino group concentration and the total protein amount due to the application of the sunscreen cream. In other words, it was possible to evaluate the protective effect of sunscreen cream on the skin of different melanin amounts (difference depending on race).

(Example 5)
The effect of sunscreen became difficult to see when the same procedure as in Example 1 was performed except that the amount of eumelanin was increased and collagen artificial skin was used.

(Examples 6 to 8)
Assuming differences depending on age, collagen artificial skin glycated and cross-linked with different concentrations of glyoxal solution was used. Others are the same as in the first embodiment.

  As a result, the extract obtained from the collagen artificial skin after ultraviolet irradiation showed a difference in the terminal amino group concentration and the total protein amount due to the application of the sunscreen cream. In other words, it was possible to evaluate the protective effect of sunscreen cream on the skin with different cross-linking degrees (difference depending on age).

(Examples 9 to 11)
Collagen artificial skin was used under different saccharification and crosslinking conditions. Others are the same as in the first embodiment.

  As a result, it was difficult to evaluate the extract obtained from the collagen artificial skin after ultraviolet irradiation because differences in terminal amino group concentration and total protein amount due to the application of sunscreen cream did not easily appear.

(Examples 12 to 13)
The same collagen artificial skin as in Example 1 was used. Taking into account the difference in the amount of ultraviolet light, the irradiation time of the ultraviolet light was changed. Others are the same as in the first embodiment.

  As a result, the extract obtained from the collagen artificial skin after ultraviolet irradiation showed a difference in the terminal amino group concentration and the total protein amount due to the application of the sunscreen cream. That is, it was possible to evaluate the protective effect of sunscreen cream on the skin due to the difference in the amount of UV irradiation.

  FIG. 1 is a photograph (200 times) of a cross-sectional structure of a collagen artificial skin by a scanning electron microscope.

(Comparative Example 1)
Hair and epidermis were removed from the fetal bovine skin tissue using a razor and a scalpel to prepare a tissue piece. Others are the same as in the first embodiment.

  As a result, a collagen extract was not obtained from the fetal bovine skin tissue piece after UV irradiation, and the terminal amino group concentration and total protein amount due to application of sunscreen cream could not be evaluated.

(Comparative Example 2)
Hair and epidermis were removed from the fetal bovine skin tissue using a razor and a scalpel to prepare a tissue piece. Further, the tissue piece was immersed in an aqueous solution of 0.1 mM to 30 mM glyoxal and reacted at 37 ° C. for 24 hours for saccharification and crosslinking. Others are the same as in the first embodiment.

  As a result, a collagen extract was not obtained from the fetal bovine skin tissue piece after UV irradiation, and the terminal amino group concentration and total protein amount due to application of sunscreen cream could not be evaluated.

(Comparative Example 3)
Human epidermal cells were cultured on collagen gel in plastic intercell. By culturing at the gas-liquid interface after the immersion culture process, a regenerated epidermis of a three-dimensional cultured skin having a stratum corneum equivalent to human skin was formed through a normal keratinization process. Others are the same as in the first embodiment.

  As a result, no collagen extract was obtained from the regenerated epidermis of cultured skin after UV irradiation, and the terminal amino group concentration and total protein amount due to the application of sunscreen cream could not be evaluated.

(Comparative Example 4)
A fine fibrous sheet-like non-woven fabric was produced from high-purity aminopolysaccharide chitin extracted and purified from shells of crustaceans (Spis crab). Others are the same as in the first embodiment.

  As a result, a collagen extract was not obtained from the nonwoven fabric after irradiation with ultraviolet rays, and the terminal amino group concentration and the total protein amount by application of sunscreen cream could not be evaluated.

Claims (5)

  A collagen artificial skin which is a non-woven multi-element structure composed of collagen fibers obtained by reconstituting solubilized collagen.   The collagen artificial skin according to claim 1, which is formed into a non-woven multi-element structure after adding eumelanin to collagen fibers in an amount of 0.01% to 70% (summing the total of collagen fibers and eumelamine as 100%).   The collagen artificial skin according to claim 1 or 2, wherein collagen fibers are immersed in an aqueous solution of 0.005 mM to 30 mM saccharide, reacted at 32.0 ° C to 40.0 ° C to be saccharified and crosslinked, and then formed into a non-woven multi-component structure.   A method for evaluating ultraviolet damage, wherein the collagen artificial skin according to any one of claims 1 to 3 is used.   The method of evaluating ultraviolet damage according to claim 4, wherein the degree of damage due to ultraviolet rays is evaluated by the difference in terminal amino group concentration and total protein amount in an extract solution of acetic acid in collagen artificial skin before and after ultraviolet irradiation.