TWI488655B - Aqueous solution of silybin glycocides and external skin treatment composition - Google Patents

Description

Silibinin aqueous solution and skin external composition

The present invention relates to a dissolution technique of silybin and a utilization technique of the solution.

In the past, it is known that silymarin has a type I collagen production-promoting action and an elastin-promoting action (Patent Document 1: WO2004/85429) and epidermal cell differentiation inhibitory action (Patent Document 2: JP-A-2004-91397) (Japanese Unexamined Patent Publication No. Hei. No. Hei. No. Hei. No. Hei. No. Hei. No. Hei.

On the other hand, since the silybin is hardly dissolved in water or oil, there is a problem that it is difficult to prepare a composition such as a skin external preparation, and a technique of dispersing using a polyol, a surfactant, or a strong alkali has been disclosed ( Patent Document 5: JP-A-2006-89418) A technique of dispersing a phospholipid, a polyglycerin fatty acid ester, or glycerin (Patent Document 6: JP-A-2006-282568).

However, the use of these techniques has constraints on prescription design.

Glucosinolates, galactosides, lactosides, and maltosides of silibinin are known to have excellent water solubility compared to silibinin. (Refer to Non-Patent Document 1: Kosina P. et al., Phytother. Res. 16, S33-S39 (2002)).

The present applicant has been studying silybin glycosides, and has applied silybin glycosides, which have wrinkle formation inhibitory effects, epidermal keratinocyte stimulating effects, and type I gels. A patent for a skin external composition which produces a pro-protein effect (Japanese Patent Application No. 2008-14361).

The problem to be solved by the present inventors is the problem of the browning of the silybin glycoside solution over time when the silybin glycoside is compounded to the external preparation for skin.

[Patent Document 1] WO2004/85429

[Patent Document 2] Japanese Patent Laid-Open Publication No. 2004-91397

[Patent Document 3] Japanese Patent Laid-Open Publication No. 2006-265186

[Patent Document 4] Japanese Patent Laid-Open Publication No. 2007-99650

[Patent Document 5] Japanese Patent Laid-Open Publication No. 2006-89418

[Patent Document 6] Japanese Patent Laid-Open Publication No. 2006-282568

[Non-Patent Document 1] Kosina P. et al., Phytother. Res. 16, S33-S39 (2002)

The present invention has been made in the development of a silybin glycoside solution excellent in coloring stability.

The main constitution of the present invention is as follows.

An aqueous solution of silybin glycoside, characterized by comprising polyoxyalkylene diglyceryl ether, dipropylene glycol, 1,2-hexanediol, 1,2-pentanediol, polyethylene glycol, polyoxygen One or two or more selected from the group consisting of ethylene methyl glycoside and 1,3-butylene glycol, and silybin glycoside.

2. An aqueous solution of silibinin, characterized in that silibinin is dissolved in an aqueous solution containing a compound having a polyoxypropyl group.

3. The aqueous solution of silybin glycoside according to 2, characterized in that the compound having a polyoxypropyl group is polyoxypropyl glyceryl ether, dipropylene glycol, polypropylene glycol, polyoxypropyl methyl glycoside, polyoxygen One or more selected from the group consisting of vinyl polyoxypropyl polyoxybutyl glycerol, polyoxypropyl glyceryl ether, polyoxyethylene polyoxypropyl alkyl ether, and polyoxypropyl sorbitol substance.

4. The aqueous silybin glycoside solution according to any one of 1 to 3, wherein the silybin glycoside is silybin maltoside.

A skin external preparation containing the aqueous solution of silybin glycoside according to any one of 1 to 4.

A wrinkle formation inhibitor comprising the aqueous solution of silybin glycoside according to any one of 1 to 4.

By polyoxyalkylene diglyceryl ether, dipropylene glycol, 1,2-hexanediol, 1,2-pentanediol, polyethylene glycol, polyoxyethylene methyl glycoside, 1,3-butanediol By blending, it is possible to realize a stable aqueous solution of silybin glycoside which can suppress browning over time. In addition, by mixing polyoxypropyl diglyceryl ether, dipropylene glycol, polypropylene glycol, polyoxypropyl methyl glycoside, polyoxyethylene polyoxypropyl polyoxybutyl glycerol, polyoxypropyl glyceryl ether, polyoxyethylene A polyoxypropyl compound containing polyoxypropyl alkyl ether or polyoxypropyl sorbitol can realize a stable aqueous solution of silybin glycoside which can suppress browning over time.

Since a silybin high-concentration aqueous solution excellent in long-term storage stability and transparency can be realized, a practical application range can be expanded for a dosage form such as a skin external preparation or a cosmetic using an aqueous solution.

that is,

1. By using a highly soluble silybin glycoside, it is possible to provide a skin external composition, a wrinkle formation inhibitor, an epidermal keratinocyte differentiation inhibitor, and an aging prevention skin external composition for improving the action mechanism of silibinin. A type I collagen production promoter and a skin roughness improving agent caused by sun exposure. In particular, the water solubility is improved, the safety is improved, the irritation is low, and the scope of use is expanded.

2. As silybin glycosides, especially silybin maltoside represented by the chemical formula (1) is effective.

3. The silybin glycoside used in the present invention has high water solubility and can be utilized as an aqueous solution type. It can be used as a skin external composition such as a lotion, a creamy lotion, an emulsion, a cream or a mask, a makeup skin external composition such as a cosmetic base lotion, a cosmetic cream, a lotion or a creamy foundation. Body skin external composition such as hand cream, leg warmer, body lotion, bathing agent, and the like.

4. The silybin glycoside used in the present invention can be highly blended even in an emulsion or a cream.

The silybin glycoside aqueous solution of the present invention dissolves silybin glycoside containing polyoxyalkylene diglyceryl ether, dipropylene glycol, 1,2-hexanediol, 1,2-pentanediol, polyethylene glycol A solution in an aqueous solution of any one of an alcohol, a polyoxyethylene methyl glycoside, and a 1,3-butylene glycol.

Further, the aqueous silybin glycoside solution of the present invention is a solution in which silybin is dissolved in an aqueous solution containing a polyoxypropyl compound. As a polyoxypropyl group The compound may, for example, be exemplified by polyoxypropyl diglyceryl ether, dipropylene glycol, polypropylene glycol, polyoxypropyl methyl glycoside, polyoxyethylene polyoxypropyl polyoxybutyl glycerol, polyoxypropyl glyceryl ether. , polyoxyethylene polyoxypropyl alkyl ether, polyoxypropyl sorbitol.

Silybin (CAS No. 22888-70-6) is from the genus Sylvestris sylvestris (scientific name Silibum marianum Gaertn, alias big cockroach, big-winged pheasant, chyle; CAS No. 84604-20-6) One of the extracted flavonoid lignans, the flavonol lignan extracted from the milk thistle is called silymarin (CAS No.65666-07-1), in addition to the water scorpion, it also contains silymarin. (Silydianin; CAS No. 29782-68-1), Silychristin (CAS No. 33889-69-9), Isosilybin (CAS No. 72581-71-6), and the like.

Silibinin can be separated from silymarin by chromatography, or it can be obtained by purchasing a reagent.

The silybin glycoside used in the present invention can be obtained by using Lewis acid as a catalyst according to the literature (Kren V. et al., J. Chem. Soc., Perkin Trans 1, 2467-2474 (1997)). The silybin is combined with a sugar which protects the hydroxyl group with an ethyl hydrazine group, and is prepared by deacetylation. In the reaction system, the sugar selectively forms a glycosidic bond with the hydroxyl group of the primary alcohol of silymarin.

Using Lewis acid as a catalyst, a glycosidic bond is formed by reacting silibinin and acetylated maltose to obtain a glycosidic bond, thereby obtaining a silibinin maltoside of the formula (1).

[Chemical Formula 1]

Specifically, silybin maltoside can be synthesized by the following procedure.

[Synthesis of silybin maltoside]

The silybin maltoside was synthesized according to the method of Helferich.

Solvents of silybin (3.0 g, 6.2 mol) and octyl-O-ethinyl-D-maltose (6.3 g, 9.2 mol) in 180 ml of dichloromethane-acetonitrile (1:1, v/v) The reaction was stirred with a boron trifluoride dimethyl ether complex (1.14 ml, 12.4 mmol) in the presence of nitrogen at room temperature for 19 hours. After completion of the reaction, the mixture was cooled with ice, and a saturated aqueous solution of sodium hydrogencarbonate was added, and extracted twice with 150 ml of dichloromethane.

Triethylamine-methanol-water (1:8:1) was reacted at 35 ° C for 30 hours, and then the solvent was removed by an evaporator. Purification was carried out using BONDESIL-C18 (Varian) to obtain silybin maltoside (1.0 g, yield 20%). The obtained silybin maltoside was confirmed by MS spectrum, and a mass spectrum peak of [M+H]+: 807.5 was detected.

Using Lewis acid as a catalyst, a glycosidic bond is formed by reacting silibinin with total acetylated lactose to deacetylate to obtain a silibinin of formula (2).

Specifically, silybin lactosides can be synthesized by the following procedure.

[Synthesis of silibinin]

The silybin lactosides were synthesized according to the method of Helferich.

a solution of silybin (3.0 g, 6.2 mol) and octyl-O-ethinyl-D-lactose (6.3 g, 9.2 mol) in 180 ml of dichloromethane-acetonitrile (1:1, v/v) The reaction was stirred with a boron trifluoride dimethyl ether complex (1.14 ml, 12.4 mmol) in the presence of nitrogen at room temperature for 19 hours. After completion of the reaction, the mixture was cooled with ice, and a saturated aqueous solution of sodium hydrogencarbonate was added, and extracted twice with 150 ml of dichloromethane.

Triethylamine-methanol-water (1:8:1) was reacted at 35 ° C for 30 hours, and then the solvent was removed by an evaporator. Purification was carried out using BONDESIL-C18 (Varian) to obtain silibinin (1.0 g, yield 20%). The obtained silibinin was confirmed by MS spectrum, and a mass spectrum peak of [M+H] ⁺ : 807.5 was detected.

Inhibition of wrinkle formation as an active ingredient of the silybin glycoside of the present invention For the agent, a skin external composition for wrinkle improvement such as a lotion, an emulsion, a cream, and a mask, a medical product for wrinkle improvement, a pharmaceutical for wrinkle improvement, and the like can be exemplified. Even in emulsions or creams, it can be combined with silybin glycosides in high concentrations in aqueous parts.

The inhibitor of epidermal keratinocyte differentiation with the silybin glycoside of the present invention as an active ingredient can inhibit the differentiation of epidermal keratinocytes, maintain proliferation, prevent, prevent, and improve the delay of metabolic renewal, and prevent the growth due to aging or ultraviolet radiation. Since the epidermis is flattened and has an action of regenerating aged skin, it can be used as a skin external composition for preventing aging.

The type I collagen production promoter containing the silybin glycoside of the present invention as an active ingredient improves the tension and elasticity of the skin, and can prevent, prevent, and improve the effect of wrinkles and slack, and thus can be used as an external skin for preventing aging. The composition is used.

The polyoxyalkylene diglyceryl ether used in the present invention is a compound obtained by addition polymerization of an alkylene oxide to diglycerin.

The polyoxyalkylene diglyceryl ether may, for example, be a polyoxypropyl diglyceryl ether in which propylene oxide is added and polymerized in diglycerin, and polyoxyethyl diglycerol in which ethylene oxide is added and polymerized in diglycerin. ether.

A commercially available product can be used as the polyoxypropyl glyceryl ether, and SY-DP4 (name: PPG-4 diglycerin: diglycerin) 4), SY-DP9 (representing the name PPG-9 diglycerol: diglycerol has a glycidyl group average addition mole number of 9), SY-DP14T (representing the name PPG-14 diglycerol: diglycerol of propylene glycol Base average addition mole number is 14), SC-P400, SC-P750, SC-P1000, UNILUB DGP-700 manufactured by NOF Corporation (Indicating the name PPG-9 diglycerin: the average addition mole number of the glycidyl group of diglycerol is 9), DGP-950 (representing the name PPG-14 diglycerol: the average addition mole number of the glycidyl group of diglycerol is 14).

Commercially available products can be used as the polyoxyethyl glyceryl ether, and as a commercial product, SC-E450, SC-E750, SC-E1000, etc., manufactured by Sakamoto Pharmaceutical Co., Ltd. can be exemplified.

The polyoxypropylmethylglycoside used in the present invention is a polyoxypropyl glycol ester of methyl glycoside. It can be represented by CH ₃ (C ₆ H ₁₀ O ₅ )(OC(CH ₃ )HCH ₂ )nOH. A commercially available product can be used as the polyoxypropylmethylglycoside, for example, MAC BIO BRIDE MG-10P (POP (10) methyl glycoside represents the name PPG-10 methyl glucose: the average chain of the polyether diol bound to the ether The length n is 10), MAC BIO BRIDE MG-20P (POP(20) methyl glycoside means the name PPG-20 methyl glucose: the average chain length n of the ether-bound polypropyl diol is 20), GLUCAM P-made by NOVEON 10 (POP(10) methyl glycoside represents the name PPG-10 methyl glucose), GLUCAM P-20 (POP (20) methyl glycoside represents the name PPG-20 methyl glucose).

The polyoxyethylene polyoxypropyl polyoxybutyl glycerol used in the present invention is a compound obtained by adding a polyoxyethyl group, a polyoxypropyl group or a polyoxybutyl group to all the hydroxyl groups of glycerin, and a commercially available product can be used. For example, the daily oil preparation WILBRIDE S-753 (representing the name PEG/PPG/polybutylene glycol-8/5/3 diol).

The polyoxypropyl glyceryl ether used in the present invention is a polypropyl glycol ether of glycerin, which can be represented by C ₃ H ₇ O ₂ (OC(CH ₃ )HCH ₂ )nOH. As the polyoxypropyl glyceryl ether, a commercially available product such as Nippon Oil UNIOLTG-1000 (representing the name PPG-16 glycerol: the average chain length n of the polyglycol diol of the bonded ester is 16), UNIOLSGP-65 (representing the name PPG) can be used. -8 Glycerol: The ester chain-bound polypropyl diol has an average chain length n of 8), UNIOLTG-1500 (representing the name PPG-24 glycerol: the ester chain-bound polypropyl diol has an average chain length n of 24).

The polyoxyethylene polyoxypropyl alkyl ether used in the present invention is an ether in which an ethoxy group and a propoxy group are added to a higher alcohol, and R-(OC(CH ₃ )CH ₂ ) _X (OCH ₂ CH ₂ ) can be used. _Y OH indicates. Commercial products can be used. For example, PEN-4620 (POE(20) POP(6) decyltetradecyl ether represented by Nikko Chemical Co., Ltd. stands for the name PPG-6 decyltetradecene-20: addition polymerization average on thiol-14 fluorenyl group 6 a compound in which a molar propoxy group and an average of 20 moles of an ethoxy group), PEN-4612 (POE(12) POP(6)decyltetradecyl ether represents the name PPG-6 mercaptotetradecene-12 : a compound obtained by adding an average of 6 moles of a propoxy group to an average of 12 moles of an ethoxy group on a fluorenyltetradecyl group, PEN-4630 (POE(30)POP(6)decyltetradecane) The ether represents the name PPG-6-decyltetradecene-30: a compound obtained by addition polymerization of an average of 6 moles of a propoxy group to an average of 30 moles of an ethoxy group on a fluorenyltetradecyl group, manufactured by Nippon Oil Co., Ltd. UNILUB50MT-2200B (POE(24)POP(13)decyltetradecyl ether represents the name PPG-13 mercaptotetradecene-24: addition polymerization on the mercaptotetradecyl group average 13 moles of propoxy With an average of 24 moles of ethoxylated compound), UNILUB50MT-2000B (POE(10) POP(20) decyltetradecyl ether represents the name PPG-20 decyltetradecene-10: in decyl decyl Addition polymerization on a tetradecyl group to average 20 moles of propoxy groups with an average of 10 moles of ethoxylated From the compound).

The polyoxypropyl sorbitol used in the present invention is a polypropyl glycol ester of sorbitol. A commercially available product such as UNIOLHS-1600D (POP (25) sorbitol represents the name PPG-25 sorbitol: the average molar mole of the propoxy group is 25) can be used.

Dipropylene glycol (DPG), 1,2-hexanediol (1,2-HD), 1,2- used in the present invention Pentylene glycol (1,2-PD), polyethylene glycol (PEG 1540, PEG 4000), polyoxyethylene methyl glycoside (methyl glycoside-10), and 1,3-butylene glycol are all used as cosmetic raw materials.

For example, KMO-6 manufactured by Photoelectric Co., Ltd., or the like can be used as the 1,2-hexanediol. As the 1,2-pentanediol, Hydro-Light-5 manufactured by symrise K.K. As the polyethylene glycol, PEG 1540, PEG 4000, or the like manufactured by Toho Chemical Co., Ltd. can be used. As the polyoxyethylene methyl glycoside, GLUCAM E-10 manufactured by LUBRIZOL Co., Ltd., MACBIO BRIDE MG-10E manufactured by Nippon Oil Co., Ltd., or the like can be used.

The skin external preparation containing the aqueous silybin glycoside solution of the present invention can be used as a cosmetic raw material, a medical external product, or a pharmaceutical. The external preparation for skin of the present invention may be not only a solution but also an emulsified composition. The emulsified composition can be applied to both the oil type and the oil type. Specifically, it can be used as a dosage form of a lotion, an emulsion, a cream, a cosmetic liquid, a mask, or the like.

The external preparation for skin of the present invention may contain an oil agent, a surfactant, a preservative, a polyol, an alcohol, a saccharide, a metal ion blocking agent, a water-soluble polymer polymer, a tackifier, a powder component, and an ultraviolet absorber. , UV blocking agent, moisturizer, fragrance, pH adjuster, etc. Further, it may contain other medicinal ingredients and physiologically active ingredients such as vitamins, skin activating agents, blood flow promoting agents, resident bacteria controlling agents, active oxygen scavengers, anti-inflammatory agents, whitening agents, and bactericides.

[Example 1]

1.0 mass% of a large amount of silybin maltoside (manufactured by Iwao Pharmaceutical Co., Ltd.) in a mass of 50 mass% of the following 17 kinds of hydrophilic solvents (glycerol, diglycerin, polyglycerol tetramerization) , polyglycerol decamer, polyglycerol hexamer, 1,3-propanediol, propylene glycol, ethanol, 1,3-butanediol, POE (10) methyl glycoside (polyoxyethyl methyl glycoside), PEG 1540 (polyethylene glycol), PEG4000 (polyethylene glycol), 1,2-pentanediol, 1,2-hexanediol, dipropylene glycol, SY-DP14T (PPG-14 diglycerol: polyoxyethyl diglycerol A compound obtained by co-addition polymerization of 14 moles of oxypropyl group on the epoxy propyl group of diglycerol), SY-DP9 (PPG-9 diglycerol: polyoxyethyl diglycidyl ether in the glycidyl group of diglycerol) After dispersing in a compound obtained by co-addition polymerization of 9 mol of the oxidized propyl group), it was dissolved in purified water, and the pH was adjusted to 5.5 with a 1% KOH aqueous solution to obtain 100% by mass.

Using a KONICA MINOLTA spectrophotometer CM-3500d, 17 kinds of silybin maltoside aqueous solution were added to a glass tank having a thickness of 2 mm (CM-A97) to measure color by a transmission method.

17 aqueous solutions of silybin maltoside were stored at 50 ° C for 5 months, and color was measured in the same manner to obtain a color difference before storage at 50 ° C. Further, the degree of browning after storage at 50 ° C for 5 months was visually evaluated by the following criteria.

Visual evaluation criteria

○: Very little yellowing, almost no change

△: slightly yellowing

×: browning

The results are shown in Table 1. The results of Table 1 were made into the graph of Fig. 1.

Table 1 Spectrophotometer measurement results of silybinin maltoside aqueous solution

[Example 2]

1.0 mass% of a large amount of silybin maltoside (manufactured by Iwao Pharmaceutical Co., Ltd.) in a mass of 50% by mass of the following five kinds of hydrophilic solvents (diglycerol, SY-DP4 (PPG-4 diglycerol: in the ring of diglycerin) a compound obtained by co-addition polymerization of 4 moles of oxypropyl group on oxypropyl group), SY-DP9 (PPG-9 diglycerol: co-addition polymerization of 9 moles of oxypropyl group on the epoxy propyl group of diglycerol) Compound), SY-DP14T (PPG-14 diglycerol: a compound obtained by co-addition polymerization of 14 moles of oxypropyl group on a glycidyl group of diglycerol And disperse in SC-E750 (a compound obtained by co-addition polymerization of 13 moles of oxidized ethyl group on the epoxy propyl group of diglycerol)), and dissolve it in purified water to adjust the pH to 5.5 with 1% KOH aqueous solution. , as 100% by mass.

Five kinds of silybin maltoside aqueous solutions were added to a glass tank having a thickness of 2 mm (CM-A97) by a KONICA MINOLTA spectrophotometer CM-3500d, and the color was measured by a transmission method.

Five kinds of silymarin aqueous solution of silymarin was stored at 50 ° C for 7 days, 14 days, and 28 days, and the color was measured in the same manner to obtain a color difference before storage at 50 ° C.

The result is made into the graph of Fig. 2.

[Example 3]

1.0 mass% of a large amount of silybin maltoside (made by Iwao Pharmaceutical Co., Ltd.) in a mass of 50 mass% of the following seven kinds of hydrophilic solvents (POP(10) methyl glycoside (polyoxypropylmethylglycoside MACBIO BRIDE MG-10P), PPG-14 diglycerol (polypropylene glycol diglyceryl ether SY-DP14T manufactured by Sakamoto Pharmaceutical Co., Ltd., a compound obtained by co-addition polymerization of 14 moles of oxypropyl group on a glycidyl group of diglycerol), PEG /PPG/polybutylene glycol-8/5/3 diol (polyoxyvinyl polyoxypropyl polyoxybutyl glycerol (ether) daily oil made WILLBRIDES-753), PPG-16 glycerol (polyoxypropyl Glycerol ether daily oil UNIOLTG-1000), PPG-9 diglycerol (polypropylene glycol diglyceride sakamoto Pharmaceutical Co., Ltd. SY-DP9 co-addition polymerization of 9 moles of oxidized propyl group on the propylene glycol diglyceride ), POE (20) POP (16) mercaptotetradecyl ether (polyoxyethylene polyoxypropyl alkyl ether PEN-4620 manufactured by Nikko Chemical Co., Ltd.), PPG-25 sorbitol (polyoxypropyl sorbose) Dissolved in pure water after dispersing in UNIOL HS-1600D) The solution was adjusted to pH 5.5 with a 1% aqueous KOH solution as 100% by mass.

Seven kinds of silybin maltoside aqueous solutions were added to a glass tank having a thickness of 2 mm (CM-A97) by a spectrophotometer CM-3500d manufactured by KONICA MINOLTA, and the color was measured by a transmission method.

Seven aqueous solutions of silybin maltoside were stored at 50 ° C for 3 months, and color was measured in the same manner to obtain a color difference before storage at 50 ° C. Further, the degree of browning after storage at 50 ° C for 3 months was visually evaluated by the following criteria.

Visual evaluation criteria

○: Very little yellowing, almost no change

△: slightly yellowing

×: browning

The results are shown in Table 5. The results of Table 5 were made into the graph of Fig. 3.

In Example 1, the color difference of PPG-14 diglycerol after storage at 50 ° C for 5 months was 10.8, but in Example 3, the color difference after storage at 50 ° C for 3 months was 14.5. On the other hand, in Example 1, the color difference of PPG-9 diglycerin after storage at 50 ° C for 5 months was 9.1, but in Example 3, the color difference after storage at 50 ° C for 3 months was 7.3.

This difference was caused by experiments with different batches of silybin maltoside at different times.

As shown in Example 1 and Example 3, PPG-14 diglycerin and PPG-9 diglycerin were the most excellent in inhibiting the browning of the aqueous solution of silybin maltoside over time, but POP (10) also having polyoxyethyl group. )methylglycoside (polyoxypropylmethylglycoside), PEG/PPG/polybutylene glycol-8/5/3 diol (polyoxyethylene polyoxypropyl polyoxybutyl glycerol), PPG-16 Glycerol (polyoxypropyl glyceryl ether), POE (20) POP (16) mercaptotetradecyl ether (polyoxyethylene polyoxypropyl alkyl ether), PPG-25 sorbitol (polyoxypropyl sorbitol) Sugar alcohols and the like can be seen to have an excellent effect of suppressing browning of the aqueous solution of silybin maltoside over time.

[Test Example 1]

Epidermal keratinocyte differentiation inhibition test, proliferation maintenance

Experimental material

1.1 normal human epidermal keratinocytes

Normal human epidermal keratinocytes, NHEK (Asahi Techno Glass), were cultured in a culture medium of epidermal keratin cells: KGM (Asahi Techno Glass) in a 37 ° C - 5% CO ₂ incubator. In this experiment, cells with sub-algebras of 3 to 5 generations were used.

1.2 KGM (the medium for epidermal keratinocytes)

KGM is added to human epithelial cell proliferation factor (0.1 ng/ml), insulin (5.0 μg/ml), hydrocortisone (0.5 μg/ml), gentamicin (50 μg/ml) on epidermal keratinocyte basal medium. Medium after amphotericin B (50 μg/ml) and bovine pituitary extract (2 ml). When a sample represented by silibinin was added to the cells, the experiment was carried out using KGM medium in which only the bovine pituitary extract was removed.

1.3 Adding samples

Silibinin (SB), silybin maltoside (SBM), and silibinin (SBL) were dissolved in DMSO (dimethyl hydrazine: Wako Pure Chemical Industries, Ltd.) and added at various concentrations.

2. Experimental methods

2.1 Epidermal keratinocyte differentiation inhibition test

The NHEK was suspended in KGM to make it 5 × 10 ⁴ /ml, and seeded on a 6-well culture plate at 4 ml/well, and cultured for 24 hours to bind the cells to the culture plate. KGM supplemented with each compound was removed at 4 ml/well, and the medium was changed every 2 days for 8 to 10 days. The morphology was observed with a microscope every day, and when the DMSO-treated control cells showed a morphological change (flattening) of signs of differentiation, photographing was performed to terminate the culture.

2.2 Epidermal keratinocyte proliferation maintenance test

The cells obtained in the above experiment were peeled off from the culture plate by trypsin treatment, and then suspended in KGM to make it 2.5 × 10 ⁴ /ml. The cell suspension was seeded in a 24-well culture plate at 2 ml/well, and the medium was changed every 2 days for 8 days. After the culture, NHEK was peeled off from the culture plate by trypsin treatment, and the number of cells was measured with a Coulter counter (Beckman-Coulter).

3. Experimental results

3.1 Epidermal keratinocyte differentiation inhibition test

In the DMSO-treated comparison control group and SB 3 μM (cultured in a medium supplemented with silybin 3 μM), epidermal keratinocytes were flattened, showing morphological changes in signs of differentiation. On the other hand, in the SBM 3 μM and SBL 3 μM, no signs of differentiation appeared. When cultured in a medium supplemented with 10 μM of SB, SBL, or SBM, the differentiation of epidermal keratinocytes was all inhibited. SB, SBL, and SBM all have differentiation inhibitory effects on epidermal keratinocytes, but SBM and SBL have superior epidermal keratinocyte differentiation inhibitory effects compared with SB.

3.2 Epidermal keratinocyte proliferation maintenance test

In the above-described epidermal keratinocyte differentiation inhibition test, if the differentiation is inhibited, the cells should maintain proliferative ability and can be sequentially propagated by subculture. Cells that are induced to differentiate cannot proliferate because they are irreversible reactions.

Then, the cells obtained in the above test were subcultured, and the proliferative ability maintained was examined by measuring the number of proliferating cells.

As a result, as shown in Fig. 4, when the sample concentration was 3 μM, the cell proliferation ability after the addition of SB was hardly maintained, and the number of cells increased to 1.8 times as compared with the case where no sample was added, and SBL was not added with the sample. In comparison, the number of cells was increased to 1.4 times, and the maintenance effect of cell proliferation ability was confirmed. When the sample concentration is 10μM, SB, SBM, SBL The number of cells increased, SB increased the number of cells by 1.5 times compared with no sample, and the number of cells increased to 2.1 times compared with the sample without SBM. The number of cells increased to SBL compared with no sample added. 1.8 times, it was confirmed that the cell proliferation ability was maintained. Compared with SB, it can be seen that the maintenance effect of cell proliferation ability of SBM and SBL is high.

[Experimental Example 2]

Type I collagen production promoting effect

Experimental material

1.1 Human skin fibroblasts

Human skin fibroblasts CCD1074SK (Daichi Sumitomo Pharmaceutical Co., Ltd.) were cultured in D-MEM in a 37 ° C - 5% CO ₂ incubator. In this experiment, cells with a passage number of 10 to 15 passages were used.

1.2 D-MEM

D-MEM, 10% of bovine fetal serum (Hyclone) was added to D-MEM basal medium (GIBCO) and used. Further, when the sample was processed, the experiment was carried out using D-MEM without adding bovine fetal serum.

2. Experimental methods

The human skin fibroblast CCD1074SK was suspended in D-MEM medium containing 10% fetal bovine serum to make it 3×10 ⁵ /ml, inoculated 1 ml in a 10 cm culture dish, and cultured for 24 hours to bind the cells to the culture plate. on. The medium was changed to D-MEM medium in which each sample dissolved in DMSO was added at each concentration without adding bovine fetal serum. After the medium was changed for 48 hours, the cell culture solution was recovered, and the culture solution was concentrated using an ultrafiltration apparatus. After concentration to about 500 ml or less, protein quantification was carried out, and after collecting the protein, the sample was concentrated as a cell culture solution and used for Western blot analysis.

10 μg of protein per lane was used, separated by SDS-PAGE, and transferred to a nitrocellulose membrane. The transferred nitrocellulose membrane was immersed in a blocking solution (solution of 5% concentration of skim milk powder in PBS containing 0.1% polyoxyethylene (20) sorbitan monolaurate), and sealed at 4 ° C. day and night. After washing with a washing solution (PBS containing 0.1% polyoxyethylene (20) sorbitan monolaurate), immersed in a primary antibody [polyclonal antibody prepared as a 500 ng/ml type I collagen with washing solution (Rockland) In the reaction, the reaction was carried out for 1 hour at room temperature. After washing, it was immersed in a secondary antibody (horseradish peroxidase-labeled anti-immunoglobulin G prepared by washing with 250 ng/ml), and reacted at room temperature for 1 hour. After washing, detection was carried out using ECL Plus Western Blotting Detection Reagent (Amersham Biosciences).

Experimental result

As a result of the experiment, it was confirmed that, like SB, both SBL and SBM have a type I collagen production promoting effect. The experimental results are shown in Figure 5.

Table 2 shows the results obtained by digitizing the intensity of the obtained band by the program image Image J and the amount of production of type I collagen when the DMSO treatment was 1.

Table 2

It is indicated that SB, SBM and SBL all have type I collagen production promoting effects. Compared with SB, SBM and SBL have a strong type I collagen production promoting effect. In particular, when the sample concentration is 10 μM, the effects of SBM and SBL are large, and a significant effect is obtained when a high concentration is confirmed.

[Experimental Example 3]

Inhibition of water evaporation caused by ultraviolet irradiation, inhibition of wrinkle formation

1. Experimental materials, appliances

Experimental animal hairless mouse Hos; HR1♀5 weeks old (Hoshino experimental material)

1.2 UV irradiation device

Ultraviolet A wave (FL32SBL/DMR: (company) clinicalsupply)

Ultraviolet B wave (FL32SE/DMR: (company) clinicalsupply)

1.3 Percutaneous water evaporation measuring device

Vapometer (k-science company)

1.4 Replica collection equipment and replica analysis system

(Yes) Reflective Replica Collection Kit and Replica Analysis System made by Asahibiomed ASA-03RXD

2. Experimental methods

2.1 Feeding environment

The feed can be fed at a temperature of 25 ° C ± 2 ° C and a humidity of 50% ± 5% in a conventional feeding environment in which the feed MR and the tap water are respectively taken as a prey.

Each component was 5 and kept in the same cage.

The sunshine is from 7 am to 7 pm, and the day and night are set every 12 hours.

2.2 UV irradiation

The hairless mouse Hos; HR1 was acclimated for 1 week and began to irradiate ultraviolet rays. The hairless mice were transferred to a special cage under ultraviolet irradiation, and each group was irradiated with ultraviolet rays of UVB 20 mJ/cm ² and UVA 10 J/cm ² . The irradiation was performed on Monday, Wednesday, and Friday, and was circled three days a week for 10 weeks, and a total of 30 ultraviolet rays were irradiated.

2.3 Group settings

After ultraviolet irradiation, 100 μL of SB, SBL, SBM methanol solution or solvent (methanol) was treated on the entire surface of the back skin of the mouse within 30 minutes. The concentrations of SB, SBL, and SBM coating were set to 1.0%, 0.3%, and 0.1%, respectively, and ultraviolet irradiation was performed for all the groups. For the mice to which only the solvent methanol was applied, the ultraviolet non-irradiated group and the ultraviolet irradiation group were used. The use of methanol as a solvent is to dissolve the SB. In the case of methanol, SBM and SBL are completely dissolved at 1% concentration, but SB is only 0.1%. When it was completely dissolved, there was some precipitation at 0.3%, and insoluble matter was found at 1%. Even when insoluble matter was found in the SB methanol solution, it was directly used for the experiment.

2.4 Determination of transepidermal water evaporation

The amount of percutaneous water evaporation was measured three times using a VapoMeter (manufactured by Keystone Scientific Co., Ltd.) from the base of the back to the head in the direction of the head and 2 cm from the lumbar vertebra to the right side, and the average value was determined. The Nail model was used for the opening at the end of the measurement (the opening portion was narrowed to correspond to the narrow range of the mouse skin), and it took about 19 seconds per measurement time. The measurement day was carried out 10 weeks after the ultraviolet irradiation.

2.5 Replica image analysis

In order to correctly grasp the formation of wrinkles, a replica is collected. The replica image analysis was carried out using a reflection replica analysis system ASA-03RXD (manufactured by Asahibiomed). Using the ASA-03RXD, by applying parallel light (LED light source) to the captured replica from an angle of 27 degrees, the shadow image corresponding to the obtained wrinkle shape is imaged by a CCD camera, input to a computer, and image processed. The wrinkle volume ratio (μm ³ /mm ² /100) of the surface of the replica was measured.

2.6 Statistical analysis

The test results are expressed as mean ± standard deviation (S.D.), and the significant difference test is performed by Bartlett test for homoskedasticity test. When the homosexual hypothesis was not rejected, the Dunnett multiple test was performed, and the Dunnett multiple test was performed as a reference when rejecting the same variance hypothesis.

3. Test results

3.1 Weight change, appearance observation

After the end of the 10 weeks period, there was no significant difference in body weight between the groups. There were also no mice showing severe lesions.

As a result of observing the appearance of the mouse skin, it was found that the ultraviolet-irradiated methanol-treated group of group 2 had wrinkles, and the group of 0.3% silybin coating group, group 6-group 8 all SBM coating group, group 9 In all of the SBL-coated groups of the group 11 , wrinkle suppression was observed.

3.2 Percutaneous water evaporation

As an indicator of skin roughness, the amount of water evaporation of each group was measured using VapoMeter after 10 weeks of ultraviolet irradiation. The results are shown in Table 3.

The amount of transepidermal water evaporation in the ultraviolet irradiation group of the group 2 was increased as compared with the group 1 ultraviolet non-irradiated group. Groups 3 to 11 of the silybin (SB) and silybin glycosides (SBM, SBL) coating groups, the significant level of 1% or less significantly inhibited the increase in transepidermal water evaporation.

When the evaporation amount of water in one group (ultraviolet non-irradiation and methanol coating) is 0%, and the amount of water evaporation in two groups (ultraviolet irradiation or methanol coating) is 100%, 3 to 5 groups (ultraviolet irradiation, SB 0.1 to 1) The water evaporation of % coating is 26~76%, and the moisture content of 6~8 groups (UV irradiation, SBM 0.1~1% coating) is 11~21%, 9~11 group (UV irradiation, SBL 0.1~1%) The water content of the coating is 24 to 27%, and the addition of SB, SBM, and SBL suppresses an increase in the amount of water evaporation. In particular, SBM and SBL of silybin glycoside have a high effect of suppressing an increase in water evaporation. In short, it has the effect of improving the skin roughness caused by the sun.

table 3

3.3 wrinkle volume rate

After the end of the irradiation for 10 weeks, in order to accurately grasp the formation of wrinkles, a replica was collected, and the wrinkle volume ratio (μm ³ /mm ² /100) on the surface of the replica was measured by image analysis. The results are shown in Table 4.

The results of the significant difference test of Dunnett were carried out, with respect to group 2 (ultraviolet irradiation of methanol coating), group 1 (ultraviolet irradiation without methanol coating), group 4 (ultraviolet irradiation SB 0.3% coating), group 7 (ultraviolet irradiation SBM 0.3%) Coating), Group 11 (UV-irradiated SBL 1.0% coating), wrinkle volume was significantly suppressed at a significance level of 5% or less.

Table 4

[Prescription example 1 lotion]

(method of law)

Dissolve 1~5 in 6.

[Prescription Example 2 Emulsion]

(method of law)

Heat 1 to 4 and 7 to 12 at 80 ° C to dissolve. 5 and 6 which had been warmed to about 80 ° C were added thereto, stirred and mixed with a homogenizer, and cooled to 30 ° C to obtain an emulsion.

[Prescription Example 3 Moisturizing Beauty Solution]

(method of law)

Stir and dissolve 1 and 11~13, add 4~6, 10, and then heat to 80 °C to dissolve. A moisturizing cosmetic liquid was obtained by adding 2, 3, 7 to 9 which had been heated to about 80 ° C and cooling to 30 ° C.

[Prescription Example 4 Moisturizer]

(method of law)

1 and 16~18 were stirred and dissolved, and after adding 2 to 5, the mixture was heated to about 80 ° C to dissolve. A moisturizer was obtained by adding 6 to 14 which had been heated to about 80 ° C and cooling to 30 ° C.

[Prescription Example 5 Body lotion]

(method of law)

1 and 10~12 were stirred and dissolved, and after adding 2~5, 9, it was heated to about 80 °C to dissolve. 6 to 8 which had been heated to about 80 ° C was added thereto, cooled to 30 ° C, and 13 was added to obtain a body lotion.

[Prescription Example 6 Massage Cream]

(method of law)

1 and 11 to 14 were stirred and dissolved, and after adding 2 to 4, the mixture was heated to 80 ° C to dissolve. 5 to 9 which had been heated to about 80 ° C was added thereto, cooled to 30 ° C, and 10 was added to obtain a massage cream.

[Prescription Example 7 Emulsified Foundation]

20. Pure water remaining

(method of law)

1 and 18~20 were stirred and dissolved, and after adding 2~5, it was heated to about 70 ° C to dissolve. Then, the fully pulverized 13-17 was added and stirred and mixed. Emulsified foundation was obtained by adding 6 to 12 which had been heated to about 80 ° C and cooling to 30 ° C.

[Prescription Example 8 Creamy Beauty Solution]

(method of law)

Let 7 dissolve in 9 and stir to dissolve 1~6. Add 8 to obtain a creamy beauty lotion.

[Prescription Example 9 Thin Mask]

(method of law)

Add 5~8 to 80 °C to dissolve, add 1~4 and 9, 10/12 which are heated and dissolved, stir and mix. After adding 11 to neutralize, it was cooled to 30 °C. Thereafter, it was infiltrated into the nonwoven fabric to obtain a thin mask.

Fig. 1 is a graph showing the color change of the experiment of Example 1 at 50 ° C for 5 months.

Fig. 2 is a graph showing a change in color change at 50 ° C in Example 2 in 30 days.

Fig. 3 is a graph showing the color change of the experiment of Example 3 at 50 ° C for 3 months.

Fig. 4 is a graph showing the results of an epidermal keratinocyte proliferation maintenance experiment.

Fig. 5 is a graph showing the results of a type I collagen production promoting experiment.

Claims (4)

An aqueous solution of silybin glycoside containing a compound having a polyoxypropyl group, characterized in that the compound having a polyoxypropyl group is a polyoxypropyl diglyceride, a polyoxypropylmethyl glycoside, a polyoxyethylene group One or two or more selected from the group consisting of oxypropyl polyoxybutyl glycerol, polyoxypropyl glyceryl ether, polyoxyethylene polyoxypropyl alkyl ether, and polyoxypropyl sorbitol. An aqueous solution of silybin glycoside according to claim 1, wherein the silybin glycoside is silybin maltoside. An external preparation for skin containing an aqueous solution of silybin glycoside as described in claim 1 or 2. A wrinkle formation inhibitor comprising an aqueous solution of silybin glycoside as described in claim 1 or 2.