JP2009203167A - Cytokine production inhibitor and cosmetic composition comprising the same - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a cytokine production inhibitor that inhibits inflammation while retaining beneficial actions of IL-1α or TNFα for cells by selectively controlling IL-6 and IL-8 with respect to inhibition or alleviation of skin inflammation caused by external stimulation including irradiation with ultraviolet rays, and a cosmetic composition containing the same. <P>SOLUTION: The cytokine production inhibitor comprises sericin and inhibits formation of interleukin-6 and/or interleukin-8 without influences on the formation of interleukin-1α and a tumor necrosis factor in an epidermal keratinocyte after irradiated with ultraviolet rays. <P>COPYRIGHT: (C)2009,JPO&INPIT

Description

The present invention improves inflammation by inhibiting the production of cytokines, interleukin-6 (IL-6) and / or interleukin-8 (IL-8), in epidermal keratinocytes after ultraviolet irradiation, and The present invention relates to a cytokine production inhibitor that suppresses onset and a cosmetic composition containing the same.

External stimuli such as UV irradiation, drying, and chemical exposure can cause various damage to the skin. In particular, when the skin becomes red due to inflammation caused by UV irradiation (sunburn), it not only causes pain and hot flashes, but in severe cases it may cause burns similar to burns.

One of the causes of inflammation caused by various external stimuli including ultraviolet rays is the production of active oxygen and free radicals. When active oxygen and free radicals are generated in the skin, the cells are damaged, sunburn cells (sunburn cells) are formed, genes are damaged (DNA damage), and DNA damage is accumulated over a long period of time. This can cause skin cancer. For this reason, anti-oxidant drugs and herbal medicines are often used for the purpose of suppressing skin inflammation. In addition, steroids having anti-inflammatory activity, various moisturizing agents that exhibit a moisturizing effect, and animal / plant extracts are also used for the purpose of suppressing inflammation.

On the other hand, when a person receives external stimuli such as ultraviolet irradiation, the production amount of interleukin-1α (IL-1α) and tumor necrosis factor (TNFα) called inflammatory cytokines increases in the keratinocytes. It is known to do. Until recently, suppression of the production of IL-1α and TNFα has been considered to lead to anti-inflammation. However, since these IL-1α and TNFα also have beneficial effects for cells such as cell activation, it is now considered desirable as an anti-inflammatory effect to control a signal transduction substance that is closer to inflammation. IL-6 and IL-8 are cytokines that are involved in the migration of neutrophils and eosinophils, and have been reported to be directly involved in the development of inflammation. Recently, IL-6 and IL-8 have been reported. Regarding the production of IL-8, a route that does not pass through the production of IL-1α or TNFα has been discovered.
Japanese National Patent Publication No. 11-515020 Ishida T. and Sakaguchi I., Protection of human keratinocytes from UVB-iuduced inflammation using root extract of lithospermum erythrorhizon, Biol. Pharm. Bull. 30 (5), 928-934, 2007 Kunsch C. and Rosen CA, NF-kB subunit-specific regulation of the interleukin-8 promoter, Molecular and Cellular Biology 13 (10), 6137-6146, 1993

As described above, when the skin is irradiated with ultraviolet rays, it is known that IL-1α and TNFα are generated in the epidermis. Although these cytokines certainly induce inflammation indirectly, cell proliferation action, etc. It also has the effect of activating skin immunity. If the production of IL-1α and TNFα is suppressed, although there is an anti-inflammatory effect, skin immunity is not activated, so it is necessary to control cytokines that suppress inflammation more directly.

The present invention focuses on the fact that IL-6 and IL-8 have a direct effect on the occurrence of inflammation with respect to the suppression and improvement of skin inflammation caused by external stimuli such as ultraviolet irradiation.
The present invention aims to provide a cytokine production inhibitor that suppresses inflammation while leaving beneficial effects for cells of IL-1α and TNFα by selectively controlling -6 and IL-8, and a cosmetic composition containing the same. To do.

The present invention contains sericin and produces interleukin-6 and / or interleukin-8 in the keratinocytes after ultraviolet irradiation without affecting the production of interleukin-1α and tumor necrosis factor. It is a cytokine production inhibitor characterized by inhibiting, and a cosmetic composition comprising the same.

According to the present invention, IL-6 and IL-8, which have a direct effect on the occurrence of inflammation, are selectively controlled with respect to the suppression and improvement of skin inflammation caused by external stimulation including ultraviolet irradiation. It is possible to provide a cytokine production inhibitor that suppresses inflammation while leaving a beneficial effect on cells possessed by -1α and TNFα, and a cosmetic composition containing the same. As a result, it becomes possible to prevent, treat, and improve inflammation by ultraviolet rays more effectively than conventional techniques, for example, without any side effects and more effective.

The cytokine production inhibitor used in the present invention is preferably a protein containing serine, and sericin is a typical protein. The molecular weight of sericin is preferably 50 kDa or less in consideration of absorption into the skin. More specifically, 30 kDa or less is more preferable. If the molecular weight of sericin is too small, there is a concern of promoting a skin differential reaction due to protein, which is not preferable, and 500 Da or more is preferable.

The cosmetic composition obtained by the present invention may be any conventional composition such as ointments, creams, emulsions, lotions, packs, bath preparations and the like, and the dosage form is not particularly limited.

The amount of the cytokine production inhibitor added to the cosmetic composition obtained according to the present invention is not particularly limited, but is, for example, 0.005% to 5%, preferably 0.05% to 0.5% due to the properties of the cosmetic composition. % Concentration range. In addition, when preparing the chemical | medical agent based on this invention as a bath agent, since it is normally diluted about 100 to 1000 times at the time of use, it is preferable that a mixing | blending is prescribed | regulated by the high density | concentration which considered it.

Further, the cosmetic composition obtained according to the present invention contains components other than the above-described essential component, ie, cytokine production inhibitor, which are usually used in cosmetics, such as other whitening agents, moisturizers, antioxidants, oily components, ultraviolet absorbers. , Surfactants, thickeners, alcohols, powder components, colorants, aqueous components, water, various skin nutrients, and the like can be appropriately blended as necessary.

For example, metal sequestering agents such as disodium edetate, trisodium edetate, sodium citrate, sodium polyphosphate, sodium metaphosphate, gluconic acid, caffeine, tannin, verapamil, tranexamic acid and Derivatives, licorice extract, glabrizine, hot water extract of karin fruit, various herbal medicines, tocopherol acetate, glycyrrhizic acid and its derivatives or salts thereof, vitamin C, magnesium ascorbate phosphate, glucoside ascorbate, arbutin, Other whitening agents such as kojic acid, saccharides such as glucose, fructose, mannose, sucrose, and trehalose, vitamin A such as retinoic acid, retinol, retinol acetate, and retinol palmitate can be appropriately blended.

Next, the present invention will be described in more detail with reference to examples.

(Materials and methods)
(1) Preparation of sericin 100 g of boiling water is added to 1 L of boiling purified water, boiled for 1 hour, allowed to cool to room temperature, filtered to obtain an extract, and this is subjected to gel filtration chromatography to a molecular weight of 10 kDa to 50 kDa. Were fractionated and freeze-dried to obtain a sericin solid having a weight average molecular weight of 30 kDa.

(2) Culture of normal human epidermal keratinocytes A commercially available epidermal keratinocyte (manufactured by Kurabo Industries) was purchased and cultured in an epidermal keratinocyte growth medium (Humdia-KG2). Cells were seeded in a 60 mm culture chalet at 1.5 × 10 ⁵ per chalet.

(3) Measurement of cytokine mRNA expression level (IL-1α, TNFα, IL-6, IL-8) by recovering cells Before the seeded cells became 80% confluent, the Humdia-KG2 medium was discarded and the epidermis angle 2 mL of the test cell solution dissolved in the modified cell basal medium (Humedia-KB2) or Humedia-KB2 was added, and the culture supernatant was collected after 24 hours. At the same time, the cells were collected, and the mRNA expression levels of IL-1α, TNFα, IL-6, and IL-8 in the cells were measured by the RT-PCR method. In addition, IL-6 and IL-8 in the collected culture supernatant were measured using a commercially available ELISA kit (R & D systems, USA).

(-) (4) PBS after discarding the test solution dissolved in condition Humedia-KB2 or Humedia-KB2 ultraviolet irradiation put was irradiated with UVB so that intensity 11 mJ / cm ^2. After irradiation, PBS (-) was discarded, 22 mL of fresh Humeria-KB was added, and the culture supernatant was collected 24 hours later.

(5) Preparation of RNA and preparation of cDNA Cells 24 hours after irradiation were used. Cell tRNA was extracted using mirVana miRNA Isolation Kit (Ambion) according to the procedure. cDNA was prepared using reverse transcriptase kit (AB) using tRNA as a template.

(6) Quantification of gene expression by RT-PCR method (Taqman-PCR method) using a fluorescent probe The obtained cDNA is quantified by TaqMan method according to the equipment operation manual of ABI PRISM 7700 Sequence Detector (AB). Analysis. The obtained results were corrected by the expression level of the internal standard human GAPDH (glyceraldehyde 3-phosphate dehydrogenase).

Example 1
(1) Measurement of mRNA amount of IL-1α, TNFα, IL-6, IL-8 in epidermal keratinocytes after UV irradiation Cultured cells were collected 24 hours later, and IL-1α, TNFα, IL-6 and IL-8 mRNA expression levels were measured. The result is shown in FIG. As shown in FIG. 1, the IL-1α and TNFα mRNA expression levels of the cells were not significantly changed by adding sericin, whereas the IL-6 and IL-8 mRNA expression levels were significant. Decreased. The vertical axis represents the expression level of each cytokine before UV irradiation with respect to the IL-1α, TNFα, IL-6, and IL-8 gene expression levels of the cells corrected by the GAPDH gene expression level.
The degree of expression (relative value) is shown.

Example 2 (IL-6, IL-8 production inhibition test)
(1) Measurement of IL-6 secretion in culture supernatant after UV irradiation The culture supernatant after 24 hours was collected, and the amount of IL-6 was measured using an ELISA measurement kit. The result is shown in FIG. As shown in FIG. 2, the amount of IL-6 in the culture supernatant significantly increased after UV irradiation. By adding sericin before ultraviolet irradiation and pre-culturing for 24 hours, the increase in IL-6 secretion due to ultraviolet irradiation was significantly suppressed by the addition of sericin.

(2) Measurement of IL-8 secretion in culture supernatant after UV irradiation Culture supernatant after 24 hours was collected, and IL-8 was measured using an ELISA measurement kit. The result is shown in FIG. As shown in FIG. 3, the amount of IL-8 in the culture supernatant significantly increased after UV irradiation. By adding sericin before ultraviolet irradiation and pre-culturing for 24 hours, the increase in IL-8 secretion due to ultraviolet irradiation was significantly suppressed by the addition of sericin.

Example 3 (Erythema suppression test of human skin)
UV sensitive (SkinTypeI and II), and directed to ten healthy women 20 to 40 years old, after UV irradiation of (1.2MED) sericin aqueous solution (0.1%) 2mg / cm ² applied to erythema The influence was measured with a color difference meter manufactured by Minolta.

As shown in FIG. 4, in the 0.1% sericin solution, a significant erythema suppressing effect was confirmed immediately after UV irradiation. Furthermore, the tendency to suppress erythema was also observed after 24 hours.

Example 4 (skin lotion)
Ingredient name Compounding amount (%)
(1) Ethanol 10.0
(2) POE (60) hydrogenated castor oil 0.5
(3) Soy lecithin 0.01
(4) Glycerin 3.0
(5) 1,3-butylene glycol 2.0
(6) Dipropylene glycol 3.0
(7) Polyethylene glycol 1500 1.0
(8) Sericin (average molecular weight 5 kDa) 0.01
(9) Na citrate 0.02
(10) Phenoxyethanol 0.3
(11) Edetate tetrasodium 0.01
(12) Purified water balance ――――――――――――――――――――――――――――――――――――――
Total 100.0

(Preparation method)
1. Components (1) to (3) are mixed and dissolved (a).
2. Components (4) to (12) are mixed and dissolved (b).
3. (A) was added to (b) and stirred to obtain a skin lotion.

Example 5 (cream)
Ingredient name Compounding amount (%)
(1) Stearic acid 2.0
(2) Sorbitan monoisostearate 2.0
(3) Cetanol 3.0
(4) Cholesterol 0.5
(5) Squalane 5.0
(6) Liquid paraffin 5.0
(7) Octyldodecyl myristate 5.0
(8) Methylcyclopolysiloxane 5.0
(9) Hydrogenated soybean lecithin 2.0
(10) Sericin (average molecular weight 30 kDa) 0.1
(11) Xanthan gum 0.1
(12) Glycerin 5.0
(13) 1,3-butylene glycol 3.0
(14) Phenoxyethanol 0.3
(15) Dipotassium edetate 0.05
(16) Residual amount of purified water ――――――――――――――――――――――――――――――――――――――
Total 100.0

(Preparation method)
1. Components (1) to (9) are mixed and dissolved at 80 ° C. (a).
2. A part of the components (11) to (15) and (16) is mixed and dissolved at 80 ° C. (b).
3. (B) is added to (a), stirred and cooled (c).
4). To (c), the component (16) dissolved in the remainder of the component (16) was added, and after stirring, a cream was obtained.

Example 6 (Sunscreen)
Ingredient name Compounding amount (%)
(1) Ethanol 10.0
(2) Octyl methoxycinnamate 7.0
(3) POE / POP-modified dimethylpolysiloxane 2.0
(4) Fine particle titanium oxide 5.0
(5) Zinc oxide 5.0
(6) Methylcyclopolysiloxane 10.0
(7) Dimethylpolysiloxane 5.0
(8) Egg yolk lecithin 0.1
(9) SEPIPULS 265 (*) 0.1
(10) Sericin (average molecular weight 30 kDa) 0.2
(11) Purified water balance ――――――――――――――――――――――――――――――――――――――
Total 100.0
*: SEPPIC 265 manufactured by SEPPIC

(Preparation method)
1. Components (2) to (8) are mixed and dissolved at 80 ° C. (a).
2. A part of the components (9) and (11) is mixed at 80 ° C. (b).
3. (B) is added to (a), stirred and cooled (c).
4). To (c), the component (11) dissolved in component (10) and (1) were added, and after stirring, a sunscreen was obtained.

In any of the cosmetics of Example 4 (skin lotion), Example 5 (cream), and Example 6 (sunscreen), erythema was suppressed.

It is a graph which shows the change of IL-1 (alpha), TNF- (alpha), IL-6, and IL-8 mRNA of a keratinocyte after ultraviolet irradiation. It is a graph which shows the change of the secretion amount of IL-6 in a culture medium after ultraviolet irradiation. It is a graph which shows the change of the secretion amount of IL-8 in a culture medium after ultraviolet irradiation. It is a graph which shows the erythema suppression effect by the ultraviolet-ray in the human skin of sericin.

Claims (3)

Suppresses the production of interleukin-6 and / or interleukin-8 without affecting the production of interleukin-1α and tumor necrosis factor in keratinocytes after UV irradiation, containing sericin A cytokine production inhibitor characterized by the above. The cytokine production inhibitor according to claim 1, wherein the average molecular weight of sericin is 50 kDa or less. A cosmetic composition comprising the cytokine production inhibitor according to claim 1 or 2.