JP5570749B2 - Functional skin external preparation with high moisturizing effect - Google Patents

Description

The present invention relates to a functional skin external preparation containing phosphorylated saccharide and phospholipid.

The skin loses its original moisturizing property due to the effects of aging, ultraviolet rays, active oxygen, etc., and further shrinkage and flexibility. When the moisture retention of the skin is lowered, wrinkles are generated in the skin, and not only the appearance beauty is lowered, but also dry skin and rough skin are formed. Furthermore, it is very important in the healing method to increase the moisture retention of the skin even in the case of allergies and atopic dermatitis. Conventionally, humectants such as sorbitol, glycerol, propylene glycol, 1,3-butylene glycol, ethylene glycol, polypropylene glycol, polyethylene glycol, xylitol, and maltitol have been used to determine the skin moisturizing effect. It was.

The topical skin preparation containing phosphorylated saccharide has already been disclosed in Patent Documents 1 and 2. However, the phosphorylated saccharide described in Patent Document 1 includes glucan, reduced glucan reduced by hydrogenation of glucan, mannan, dextran, agar, cyclodextrin, fucoidan, gellan gum, locust bean gum, guar gum, tamarind gum And sugars selected from xanthan gum, which are different from the monosaccharides described in the present invention. Furthermore, Patent Document 2 discloses that by applying a phosphorylated saccharide typified by mannose-6-phosphate, it gently promotes skin desquamation without irritation, and synthesizes glycosaminoglycans in the skin. A method of enhancing is described. However, the individually described methods only describe the action of the composition containing the phosphorylated saccharide alone.

JP2006-249077 Special table 2005-501062

The Journal of Cell Biology, Vol. 180, No. 3, 451-458, 2008 Nature Cell Biol. 9: 666-674, 2007

However, these generally used wetting agents are excellent in wetting action, but have a sticky feeling when applied and only show a temporary moisturizing effect. It did not produce an effect that lasted. NMF (natural moisturizing factor), a so-called natural moisturizing factor, is a moisturizing substance indispensable for maintaining moisture in the skin. It has been reported that NMF is produced when filaggrin produced by degradation of profilagrin secreted from keratohyalin granules in keratinocytes is degraded by an enzyme called caspase-14. (Non-patent document 3) For this reason, it is considered that activating Caspase-14 promotes the production of NMF in the skin and significantly improves the water retention capacity of the skin. In fact, it has been shown that in the skin of Caspase-14 knockout mice, transdermal water transpiration (TEWL) is markedly increased and skin moisture is reduced. (Non-patent document 4) From the above, the moisture content in the skin is maintained by activating Caspase-14 present in the skin rather than simply applying a moisturizing agent to the skin as in the past. It is considered useful to lead to moisturized skin.

Under such circumstances, the present inventors have conducted intensive research to search for a material having a sustained moisturizing effect, and as a result, the addition of phosphorylated saccharide and / or phospholipid improves caspase-14 activity. The present inventors have found that it is more effective for maintaining the moisture content of the skin and completed the present invention.
That is, the present invention relates to a functional skin external preparation characterized by containing a phosphorylated saccharide and a phospholipid.

Phospholipid is a generic term for lipids that have a phosphate ester moiety in the structure. It has amphipathic properties, forms a lipid bilayer, and becomes a major component of cell membranes together with glycolipids and cholesterol. It has been widely applied to external preparations for skin as a liposome preparation with attention paid to its properties. The phospholipid used in the present invention is particularly preferably phosphatidic acid or lysophosphatidic acid. A molecule in which glycerin has an ester bond with a fatty acid at the C1 and C2 positions and phosphoric acid at the C3 position is called phosphatidic acid, and a molecule from which the fatty acid at the C2 position is removed from phosphatidic acid is called lysophosphatidic acid. Recently, lysophosphatidic acid is known to have various pharmacological effects such as cell proliferation effect, platelet aggregation effect, cell motility enhancement, neurite retraction, anti-apoptotic effect, smooth muscle contraction effect, cancer invasion promoting effect, etc. It is a substance that is attracting attention as a lipid mediator. However, the present invention newly found a synergistic effect on Caspase-14 activation that was not obtained by the combination of phosphorylated sugar and phospholipid alone by containing phosphorylated sugar and phospholipid, and the skin external preparation of the present invention This has led to the discovery.

As the phosphorylated saccharide according to the present invention, glucose-1-phosphate, glucose-6-phosphate, phosphoenolpyruvate, or the like is used. Examples of the salt include salts of alkali metals, alkaline earth metals, etc. Among them, sodium, potassium, calcium and the like are preferable.
The above phosphorylated saccharide may be a commercially available reagent, synthesized from phosphorylase such as glucokinase or hexokinase from glucose, or transferase such as glucophosphomutase, or a microorganism such as yeast. Alternatively, those extracted and purified from cells such as animals and plants can be used in the present invention.

As the phospholipid according to the present invention, phosphatidic acid and lysophosphatidic acid are used. Examples of the salt include salts of alkali metals, alkaline earth metals, etc. Among them, sodium, potassium, calcium and the like are preferable.
As the phospholipid, a commercially available reagent may be used, or a phospholipid extracted from soybean or egg white is decomposed with an enzyme such as phospholipase A ₂ and then extracted and purified. it can.

The form of the phosphorylated saccharide and phospholipid used in the present invention may be any form such as liquid, solid, powder, paste, gel, etc., and the optimum shape for constituting the final product. Can be arbitrarily selected.

The dosage form of the external preparation for skin of the present invention is arbitrary, and is capsule, powder, granule, pill, tablet, solid, liquid, gel, foam, emulsion, cream, ointment, sheet The shape can be in the form of an aerosol or aerosol. Furthermore, it can mix | blend and use for pharmaceuticals, quasi-drugs, and cosmetics. In particular, it is applied to external preparation compositions such as pharmaceuticals, quasi-drugs, and cosmetic compositions applied to the outer skin.

Specific use forms of the present invention include aqueous components, oily components, plant extracts, animal extracts, powders, excipients, surfactants, oils, alcohols, pH adjusters, preservatives, antioxidants, A lotion, a milky lotion, a cream, a pack, a powder, a spray, an ointment, a dispersion, and a liquid form of the composition for external use by mixing thickeners, sweeteners, pigments, fragrances, and the like as necessary. It can be made into various dosage forms such as paste and powder.

The amount of phosphorylated saccharide added to the skin external preparation of the present invention is slightly different depending on the expected effect and is not particularly limited, but is usually 0.0001% by mass or more in terms of solid content in the total amount of the preparation, preferably Is effective in a concentration range of 0.01 to 10.0% by mass.
Further, the amount of phospholipid to be added to the external preparation for skin of the present invention is slightly different depending on the expected degree of action and is not particularly limited, but is usually 0.0001% by mass or more in terms of solid content in the total amount of the preparation, It is effective to set the concentration range to 0.01 to 10.0% by mass.

Hereinafter, test examples relating to phosphorylated saccharides and phospholipids according to the present invention will be shown and application examples of external preparations using the materials will be described, but it goes without saying that the examples are not limited thereto. .

In Example 1, human skin keratinocytes were cultured, and each sample was added to the cultured keratinocytes, and then Caspase-14 activity in the cell extract was measured. Caspase-14 activity was measured by the method described in Reference 5. That is, it is a method of measuring the absorbance of pNitro aniline produced when WEHD-pNitro aniline as a substrate is decomposed by Caspase-14.
Reference 5: FEBS Letters 577,
2004, 446-450

[Sample preparation]
Phosphorylated sugars glucose-1-phosphate, glucose-6-phosphate, and phosphoenolpyruvate were added to the culture medium in an aqueous solution using commercially available reagents. A commercially available egg yolk-derived sodium phosphatidate was used as the phosphatidic acid, and 1-oleoyl lysophosphatidic acid was used as the lysophosphatidic acid, and a 0.1% aqueous solution was prepared and added to the culture medium.
(Preparation of cells)
Normal human keratinocytes (NHEK) were cultured in Epi-Life KG2 medium (Kurabo) on 50cm ² plate, and each sample was added at about 80% confluence, and cells were collected 24 hours after the addition. .
[Measurement of Caspase-14 activity]
The activity was measured using an assay kit manufactured by Calbiochem. 200 cells collected
μl of Cell Lysis Buffer was added to obtain a cell extract. The amount of protein in the cell extract was measured, and a cell extract with a protein amount of 200 μg was prepared as a measurement sample. After adding 5 μl of WEHD-pNA Substrate to this measurement sample and incubating at 37 ° C. for 6 hours,
The absorbance was measured at 405 m, and the activity was measured. The degree of Caspase-14 activation was calculated according to the following formula.

Caspase-14 activation degree (%) = (absorbance value at 405 nm in the sample added group / absorbance value at 405 nm in the sample-free group) × 100

The results are shown in Table 1. From Table 1, sodium phosphatidate showed 135% Caspase-14 activation and 135% 1-oleoyllysophosphatidic acid. Glucose-1-phosphate, phosphoenolpyruvate, and glucose-6-phosphate showed a high degree of activation even when added alone at 158%, 139%, and 111%, respectively. Furthermore, when glucose-1-phosphate and sodium phosphatidate were used in combination, the Caspase-14 activity showed a very high value of 468%. In addition, the combination of glucose-1-phosphate and 1-oleoyllysophosphatidic acid also showed a very high Caspase-14 activity of 600%.
By using glucose-1-phosphate and sodium phosphatidate in combination, Caspase-14 activity showed a very high value of 468%. Glucose-1-phosphate has a Caspase-14 activity of 158% and sodium phosphatidate has a Caspase-14 activity of 135%. Theoretically, when these two samples are added, the activity shows 213% activity expressed as the product of 158% and 135%, but here it shows 468% activity and glucose-1 -A synergistic effect of the combined use of phosphoric acid and sodium phosphatidate was observed.
Furthermore, glucose-1-phosphate has a Caspase-14 activity of 158% and 1-oleoyllysophosphatidic acid has a Caspase-14 activity of 155%. Theoretically, when these two types of samples are added, the activity shows 245% activity expressed by the product of 158% and 155%, but here it shows 600% activity, and glucose-1- A synergistic effect was obtained by the combined use of phosphoric acid and 1-oleoyllysophosphatidic acid.
Similarly, a combination of phosphoenolpyruvate and glucose-6-phosphate with sodium phosphatidate and 1-oleoyllysophosphatidic acid resulted in a synergistic effect.
As described above, the combined use of phosphorylated saccharide, sodium phosphatidate, and 1-oleoyllysophosphatidic acid clearly showed a synergistic effect as compared with the combination of each of them.

In order to confirm that the above-mentioned Caspase-14 activation effect of phosphatidic acid and lysophosphatidic acid is not a mere phospholipid surface-active effect, studies were conducted using surfactants widely used for external preparations for skin. Commonly used surfactants include Rhedol TWL-120 (Polysorbate 20), HCO-50 (Polyoxyethylene hydrogenated castor oil), Sunfact 5GMM-Z (Polyglyceryl monomyristate), Sunsoft Q-12Y (Polyglyceryl laurate) -10), Caspase-14 activity was measured using 5 types of surfactants, PEN4630 (PPG-decyltetradeces-30).

The results are shown in Table-2. As is clear from Table-2, Caspase-14 activity is suppressed in any surfactant, and the Caspase-14 activation effect of phosphatidic acid and lysophosphatidic acid is not the general effect of surfactants. It turns out that it is clear that it is a specific effect of phospholipid.

Furthermore, in order to confirm the specificity with phosphorylated saccharide, dextran (molecular weight 200,000-300,000), which is a non-phosphorylated polysaccharide, was compared with the amount of skin moisture by continuous application. In addition, in order to confirm the specificity of phospholipids, we compared the moisture content of the skin by continuous application using polyoxyethylene hydrogenated castor oil (HCO-50), a surfactant widely used in cosmetics. In the test, each sample shown in Table 3 was impregnated on the inner skin of the upper arm with a paper disk for a fin chamber (Taisho Toyama Pharmaceutical Co., Ltd.) and applied for 10 minutes. 24 hours after application, the skin moisture content at the application site was measured with SKICON-200EX (I.B.S.CO., LTD.). This operation was repeated for 5 days. From Table 3, dextran, which is a polysaccharide, showed a high skin moisture value of 130% for about 1 hour after application compared to the non-application site, but it changed significantly from the first day after the next day. There was no. In addition, HCO-50, a surfactant widely used in cosmetics, was not significantly different from the initial skin moisture content. In contrast, when glucose-1-phosphate, which is a phosphorylated sugar, and 1-oleoyllysophosphatidic acid, a phospholipid, were applied, it was confirmed that the amount of skin moisture increased with application. . This is because the application of Caspase-14 activity in the skin increased, and the production of NMF (natural moisturizing factor), which is said to be a natural moisturizing factor in the skin, was promoted, resulting in an increase in sustained skin moisture. it was thought.

Next, in order to confirm the effect on humans, the skin moisture content was measured using the external preparation for skin shown in (Table-4.5). In the test, glucose-1-phosphate and phosphoenolpyruvate were used as phosphorylated sugar, phospholipid was 1-oleoyllysophosphatidic acid, sodium phosphatidate, and Leodol TWL as a surfactant widely used for skin external preparations. -120 (polysorbate 20) was tested in combination.
A 10% aqueous solution of sodium lauryl sulfate: ethanol = 30: 70 was obstructed and applied to the inner side of the upper arm for 2 hours. Affixing was performed by adding 20 μl of sodium lauryl sulfate solution to a paper disk of a fin chamber (Taisho Toyama Pharmaceutical Co., Ltd.). After 2 hours, the fin chamber is peeled off, the application site is washed with purified water, and the external preparation for skin shown in (Table-4.5) is applied for 7 days. Thereafter, the skin moisture content of the application site was measured using SKICON-200EX (IBSCO., LTD.).

Table 6 shows the evaluation criteria for the amount of skin moisture, and Table 7 shows the results based thereon. As is apparent from Table 7, the skin moisture content of the topical skin preparation using glucose-1-phosphate and phosphoenolpyruvate, which are phosphorylated sugars, and 1-oleoyllysophosphatidic acid and sodium phosphatidate, which are phospholipids, is It was confirmed that the increase was highest and the effect of increasing the amount of skin moisture was high. In addition, skin external preparations containing Rhedol TWL-120, a surfactant widely used for skin external preparations, have a low increase in skin water content when combined with either phosphorylated saccharide or phospholipid. there were.

(Manufacture of various compositions)
Various compositions according to the present invention were prepared. Although the formulation example is shown below, this invention is not necessarily limited to these. In addition, a compounding quantity is shown by weight%.

(1) Cream composition
a) Beeswax: 3.0
b) Stearyl alcohol: 5.0
c) Stearic acid: 8.0
d) Squalane: 1.0
e) Self-emulsifying glyceryl monostearate: 3.0
f) Polyoxyethylene cetyl ether (20E.O.): 1.0
g) Phosphatidic acid: 10.0
h) Glucose-1-phosphate: 10.0
i) 1,3-butylene glycol: 5.0
j) Potassium hydroxide: 0.3
k) Preservatives and antioxidants: appropriate amount
l) Purified water: Remainder manufacturing method: Heat up to a) to g) and keep at 80 ° C. h) to l) are heated and dissolved,
Maintain at 80 ° C., emulsify in addition to a) to g), cool to 40 ° C. with stirring.

(2) Cream composition
a) Beeswax: 2.0
b) Stearyl alcohol: 5.0
c) Stearic acid: 8.0
d) Squalane: 10.0
e) Self-emulsifying glyceryl monostearate: 3.0
f) Polyoxyethylene cetyl ether (20E.O.): 1.0
g) Lysophosphatidic acid: 1.0
h) Phosphoenolpyruvate: 2.0
i) 1,3-butylene glycol: 5.0
j) Potassium hydroxide: 0.3
k) Preservatives and antioxidants: appropriate amount
l) Purified water: Remainder manufacturing method: Heat up to a) to f) and keep at 80 ° C. g) to l) are heated and dissolved,
Maintain at 80 ° C., emulsify in addition to a) to f), and cool to 40 ° C. with stirring.

(3) Emulsion composition
a) Beeswax: 0.5
b) Petrolatum: 2.0
c) Squalane: 8.0
d) Sorbitan sesquioleate: 0.8
e) Polyoxyethylene oleyl ether (20E.O.): 1.2
f) Glucose-6-phosphate: 0.001
g) Lysophosphatidic acid: 0.001
h) 1,3-butylene glycol: 7.0
i) Carboxyvinyl polymer: 0.2
j) Potassium hydroxide: 0.1
k) Purified water: balance
l) Preservatives and antioxidants: appropriate amount
m) Ethanol: 7.0
Production method: Heat up to a) to e) and keep at 80 ° C. f) to l) are heated and dissolved,
Maintain at 80 ° C., emulsify in addition to a) to e), and cool to 50 ° C. with stirring.
Add m) at 50 ° C and cool to 40 ° C.

(4) Emulsion composition
a) Beeswax: 0.5
b) Petrolatum: 2.0
c) Squalane: 8.0
d) Sorbitan sesquioleate: 0.8
e) Polyoxyethylene oleyl ether (20E.O.): 1.2
f) Fructose-1,6-2 phosphate: 0.1
g) Phosphatidic acid: 0.1
h) Glucose-6-phosphate: 0.2
i) 1,3-butylene glycol: 7.0
j) Carboxyvinyl polymer: 0.2
k) Potassium hydroxide: 0.1
l) Purified water: balance
m) Preservatives and antioxidants: appropriate amount
n) Ethanol: 7.0
Production method: Heat up to a) to e) and keep at 80 ° C. f) to m) are heated and dissolved,
Keep at 80 ° C, emulsify in addition to a) to e), stir to 50 ° C,
Add n) at 50 ° C and cool to 40 ° C.

(5) Lotion-like composition
a) Phosphatidic acid: 0.0001
b) Glucose-1-phosphate: 0.0001
c) Phosphoenolpyruvate: 0.001
d) Glycerin: 5.0
e) Polyoxyethylene sorbitan monolaurate (20E.O.): 1.0
f) Ethanol: 6.0
g) Fragrance: appropriate amount
h) Preservatives and antioxidants: appropriate amount
i) Purified water: The remaining manufacturing method: a) to i) are mixed and dissolved uniformly.

(6) Gel-like composition
a) Lysophosphatidic acid: 0.1
b) Glycerin: 5.0
c) Polyoxyethylene sorbitan monolaurate (20E.O.): 1.0
d) Fructose-1,6-2 phosphate: 0.01
e) Phosphoenolpyruvate: 0.1
f) Carboxyvinyl polymer: 0.2
g) Ethanol: 6.0
h) Fragrance: appropriate amount
i) Preservatives, antioxidants, neutralizers: appropriate amount
j) Purified water: The remaining manufacturing method: a) to j) are mixed and dissolved uniformly.

(7) Packing agent
a) Lysophosphatidic acid: 1.0
b) Phosphoenolpyruvate: 2.0
c) Vinyl acetate resin emulsion: 15.0
d) Polyvinyl alcohol: 10.0
e) Glucose-6-phosphate: 3.0
f) Glycerin: 5.0
g) Titanium oxide: 8.0
h) Kaolin: 7.0
i) Ethanol: 8.0
j) Fragrance: appropriate amount
k) Preservatives and antioxidants: appropriate amount
l) Purified water: Remaining manufacturing method: Mix up to a) to l), stir and disperse well to make uniform.

Claims (2)

An external preparation for moisturizing skin containing Component A and Component B.
Component A: One or more selected from glucose-1-phosphate, phosphoenolpyruvate, glucose-6-phosphate Component B: One or two selected from sodium phosphatidate and lysophosphatidic acid A caspase-14 activity promoter comprising component A and component B as active ingredients.
Component A: One or more selected from glucose-1-phosphate, phosphoenolpyruvate, glucose-6-phosphate Component B: One or two selected from sodium phosphatidate and lysophosphatidic acid