Skin-moisturising or wrinkle-improving external composition and cosmetic composition

Abstract

The present invention relates to a skin-moisturizing or wrinkle-improving external composition or cosmetic composition which contains, as an active ingredient, a compound represented by Formula 1, which can be extracted from Anemarrhena asphodeloides Bunge, or a pharmaceutically acceptable salt thereof. The composition prevents skin moisture loss and thus suppresses skin tissue damage induced by UV rays and so has a skin-wrinkle preventing or alleviating effect.

Claims

1. A method for preventing transdermal water loss or suppressing skin wrinkles comprising applying a composition to skin of a subject having UV-induced skin aging, wherein the composition comprises a chromatographically purified compound from Anemarrhena asphodeloides Bunge represented by Formula 1, or a pharmaceutically acceptable salt thereof as an active ingredient: ##STR00002##

Description

DESCRIPTION OF DRAWINGS

(1) FIG. 1 is an image showing the degree of skin-wrinkle formation of hairless mice using a skin replica according to one embodiment of the present invention.

(2) FIG. 2 shows a graph illustrating the changes in the mean length of a skin wrinkle of hairless mice according to one embodiment of the present invention.

(3) FIG. 3 shows a graph illustrating the changes in the mean depth of a skin wrinkle of hairless mice according to one embodiment of the present invention.

(4) FIG. 4 is an image showing the measurement result of thickness of the cornified layers of hairless mice skin tissues via H&E staining according to one embodiment of the present invention.

(5) FIG. 5 is an image showing the observation result of the collagen fibers in hairless mice skin tissues via Masson's trichome staining according to one embodiment of the present invention.

(6) FIG. 6 is a graph showing the measurement result on epidermal thickness in H&E-stained skin tissues of hairless mice according to one embodiment of the present invention.

(7) FIG. 7 is a graph showing the measurement result of transepidermal water loss of hairless mice according to one embodiment of the present invention.

BEST MODE

(8) Hereinafter, the present invention will be described in further detail with reference to examples and preparation examples. It is to be understood, however, that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention.

EXAMPLE

Extraction of the Compound Represented by Formula 1 from Anemarrhena asphodeloides Bunge

(9) Anemarrhena asphodeloides Bunge was purchased from Omni Hub, and was stored in a refrigerator for herb storage at 5.5±0.3° C. with 55±5% humidity. 3 kg of Anemarrhena asphodeloides Bunge was immersed in 70% ethanol of 10 times by volume thereof, and underwent a reflux cooling extraction three times for three hours to prepare the extract. The solvents were removed from the filtrate obtained using a filter paper by a vacuum evaporator to prepare Anemarrhena asphodeloides Bunge ethanol extract. The Anemarrhena asphodeloides Bunge ethanol extract was sequentially treated with n-hexane, ethyl acetate, and n-butanol to obtain a n-butanol fraction. The thus-obtained n-butanol fraction was separated into 5 small fractions using Diaion hp-20 resin via gradient elution column chromatography using a mixture containing water and methanol. In particular, the ratio of water to methanol used was 10:0 (v/v) to 7:3 (v/v). Among the 5 small fractions, the fourth small-fraction was separated again using RP-18 reverse resin (LiChroprep) via gradient elution column chromatography using a mixture containing water and methanol to obtain a compound represented by Formula 1. In particular, the ratio of water to methanol used was 10:0 (v/v) to 5:5 (v/v).

(10) .sup.1H NMR(400 MHz, Pyridine-d.sub.5) δ: 0.83 (3H, s, H-18), 0.98 (3H, s, H-19), 1.09 (3H, d, J=6.8 Hz, H-21), 1.17 (3H, d, J=6.4 Hz, H-27), 3.39 (1H, d, J=10.8 Hz, H-26β), 4.94 (1H, d, J =7.6 Hz, H-1′), 5.31 (1H, d, J=7.6 Hz, H-1″).

(11) .sup.13C NMR (100 MHz, Pyridine-d.sub.5) δ: 110.0 (C-22), 106.5 (C-1″), 102.9 (C-1′), 82.2 (C-2′), 81.7 (C-16), 78.7 (C-5″), 78.3 (C-3″), 77.3a (C-2″), 76.9 (C-5′), 75.8a (C-3), 75.5a (C-3′), 72.0 (C-4″), 70.2 (C-4), 65 4 (C-26), 63.3 (C-17), 63.1 (C-6″), 62.5 (C-6′), 56.8 (C-14), 42.8 (C-20), 41.2 (C-13), 40.6b (C-12), 40.5b (C-9), 37.3 (C-5), 35.8c (C-10), 35.6c (C-8), 32.5 (C-15), 31.3 (C-1 and C-4), 27.9 (C-25), 27.3 (C-2), 27.1 (C-6 and C-7), 26.7 (C-23), 26.5 (C-24), 24.3 (C-19), 21.5 (C-11), 16.9 (C-18), 16.6 (C-27), 15.2 (C-21).

EXPERIMENTAL EXAMPLE

Analysis of Moisturizing and Wrinkle Improving Effects

(12) Skin-moisturizing and wrinkle-improving effects were confirmed by spreading the compound represented by Formula 1, prepared from the Example, onto the skins of hairless mice.

(13) 1) Experimental Animals and Sample Administration

(14) 7-week old male hairless mice (male HR-1, hairless mice, Japan SLC, Inc.), the experimental animals, were purchased from Central Lab. Animal Inc (Seoul, Korea). and used after an adaption period of a week. Only healthy animals were used in the experiments by observing general conditions during the adaption period. The breeding environment was maintained at 23±3° C. with 50±5% humidity with a light-dark cycle of 12 hours (7:00-19:00/lighting time). During the experiments, seven experimental animals per each experimental group were raised in a polycarbonate cage (200×320×145 mm, Three-shine Co., Daejeon, Korea), and the animals were given both mouse feed 5L79 (Charles river, USA) and tap water for drinking sterilized with UV rays to be consumed freely.

(15) The experiments were carried out on the following groups: a control group (Con), a UV-treated group (UV), an excipient-treated group (Vehicle), and a group treated by the compound represented by Formula 1 (TM), and the samples were administered thereto. The sample administration was performed for 12 weeks in total by spreading the samples onto skins. For the compound treated group (TM), the compound was dissolved in dimethyl sulfoxide (DMSO), and subsequently in a mixed solution containing propylene glycol, ethanol, and poly ethylene glycol with a 1:1:1 ratio, and the dissolved formulation (the final concentration of the compound was 0.25% (w/v)) was applied to skins with the amount of 40 uL per 6 cm.sup.2 once a day for 5 days a week. The excipient-treated group (Vehicle) were treated in the same manner as the compound treated group (TM), except the compound omitted.

(16) Ultraviolet irradiation was performed on the experimental groups except for the control group using a UVB lamp (Mineralight UV Display lamp, UVP, USA) three times a week for 8 weeks. The amount of UV irradiation for the 8 weeks was 60 mJ/cm.sup.2 for the first 1 to 2 weeks, 90 mJ/cm.sup.2 for the subsequent 3 to 5 weeks, and 120 mJ/cm.sup.2 for the subsequent 6 to 8 weeks. The UV irradiance was measured by a radiometry instrument (Delta OHM, Italy), and then, was adjusted according to irradiation time. The UV irradiation variable and the sample amount for each experimental group are shown in Table 1 below.

(17) TABLE-US-00001 TABLE 1 UV irradiation Sample amount Control Group (Con) x UV-treated Group (UV) ∘ Excipient-treated Group (Vehicle) ∘ Group treated by compound represented ∘ 0.25%(w/v) by Formula 1 (TM)

(18) 2) Skin Wrinkle Measurement

(19) The degree of wrinkle formation was measured with a silicone skin replica of the back skins of the hairless mice of each experimental group. The skin replica was prepared using a Repliflo Cartridge Kit (CuDerm Corporation, USA) by thinly applying the cartridge to the back and completely drying it, followed by peeling off the disk carefully. The preparation of skin replica was carried out under constant temperature and humidity at 20 to 22° C. with 40 to 50% humidity. Then, the skin replica was inserted into a cartridge, which was prepared so that a special light source could pass through, and the light was passed through the replica at an incidence angle of 35°. A shadow image, generated according to the thickness of the skin replica, was filed using a CCD camera, and the depth and length of wrinkles were measured using the Skin Visiometer VL 650 software, which is a computer image analysis system. The results are illustrated in FIGS. 1 to 3.

(20) As shown in FIG. 1, thick, deep wrinkles were induced in the UV-treated group (UV) compared to the control group (CON), whereas thick wrinkles were alleviated in the group treated by compound represented by Formula 1 (TM) compared to the UV-treated group (UV).

(21) As shown in FIG. 2, it was confirmed that the mean wrinkle length was increased in the UV-treated groups (UV) compared to the control group (CON) (p<0.0001), whereas the wrinkle mean length was significantly reduced in the group treated by compound represented by Formula 1 (TM) compared to the UV-group (UV) and the excipient-treated group (Vehicle) (p<0.0001).

(22) Further, as shown in FIG. 3, it was confirmed that the mean wrinkle depth was increased in the UV ray treated group (UV) compared to the control group (CON) (p<0.0001), whereas the mean wrinkle depth was significantly reduced in the group treated by compound represented by Formula 1 (TM) compared to the UV-treated group (UV) (p<0.0001).

(23) From the above results, it was confirmed that the mean wrinkle length and depth, which were increased by UV rays, were significantly reduced by treating the wrinkles with the compound represented by Formula 1 prepared in Example 1. Accordingly, it was confirmed that the compound represented by Formula 1 is effective in improving skin wrinkles, and especially, effective in alleviating the wrinkles caused by UV rays.

(24) 3) Histological Observation of Skin

(25) To confirm the wrinkle-suppressing effect, the back skin tissues of hairless mice from each experimental group were collected, fixed in a 10% neutral formalin solution followed by washing, dehydrating, clearing, and infiltrating processes, and embedded with paraffin. After being cut into a 4 μm section, the skin tissues were stained with Hematoxylin & Eosin (H&E) and Masson's trichome. The results are shown in FIGS. 4 and 5.

(26) As shown in FIG. 4, when the H&E staining was performed, it was confirmed that cornified layers were induced in the UV-treated group (UV) and the excipient-treated group (Vehicle) compared to the control group (CON), and thicker epidermises were also observed. Meanwhile, cornified layers were alleviated in the group treated by a compound represented by Formula 1 (TM) compared to the UV-treated group (UV) and excipient-treated group (Vehicle), and thinner epidermises were observed. Accordingly, it can be inferred that the compound represented by Formula 1 is effective in alleviating the cornified layer and reducing the epidermis thickness.

(27) Further, as shown in FIG. 5, when Masson's trichome staining was performed, it was observed that the tissues in the control group (CON), which were mostly composed of the dermis, which collagen fibers were observed throughout, whereas the collagen fibers were not observed in the UV-treated group (UV) and the excipient-treated group (Vehicle). Meanwhile, it was confirmed that collagen fibers were increased in the group treated by compound represented by Formula 1 (TM) compared to the UV-treated group (UV). Accordingly, it can be inferred that the compound represented by Formula 1 is effective in suppressing the destruction of collagen tissues caused by UV rays.

(28) 4) Analysis of Wrinkle-Suppressing Effect by Differences in Epidermis Thickness

(29) The epidermis thickness was measured from the keratin layer to the epidermal basement membrane of tissues, which were stained with H&E in Experimental Example 3), by a ruler installed in a microscope, and the result is shown in FIG. 6.

(30) As shown in FIG. 6, it was confirmed that the epidermis thickness was increased in the UV-treated group (UV) due to the exposure to UV rays (p<0.0001). Further, the epidermis thickness was significantly reduced in the group treated by compound represented by Formula 1 (TM) (p<0.05).

(31) 5) Transepidermal Water Loss (TEWL) Analysis

(32) To confirm the skin-moisturizing effect, the transepidermal water loss analysis was carried out with back skins of hairless mice in each experimental group. The transepidermal water loss level refers to the water quantity diffused from a skin, and if the level is high, it would mean that the skin moisturizing function is weakened, and that the function of innate skin barrier is damaged. To measure the transepidermal water loss, the quantity (g/m.sup.2/hr) of water evaporated from the skins was calculated according to the area and time using Tewameter (Tewameter Courage & Khazaka, Germany) under constant temperature and humidity (at 23° C., relative humidity: 50%). The evaporated water quantity was then measured using Tewameter, and the skin moisturizing-ability was calculated. The result is shown in FIG. 7.

(33) As shown in FIG. 7, it was confirmed that the skin moisturizing-ability was reduced due to damage in the skin bather resulted from UV irradiation (p<0.0001), and that the skin water loss induced by UV rays was significantly prevented (p<0.01) when the skins were treated with the compound represented by Formula 1. Accordingly, the compound represented by Formula 1 is effective in moisturizing skin.

(34) In all experimental results, significance between the control group and the experimental groups was determined using a one-way ANOVA and a Student t-test (p<0.05).

(35) Hereinafter, Formulation Examples of the cosmetic composition including the compound represented by Formula 1 will be described. However, these examples are shown for illustrative purposes only and are not intended to limit formulation of the cosmetic composition of the present invention.

FORMULATION EXAMPLE 1:

Preparation of Softening Cosmetic Water (Toner)

(36) Among the cosmetics containing the compound represented by Formula 1 of the present invention, a Formulation Example of softening cosmetic water (toner) is shown in Table 2 below.

(37) TABLE-US-00002 TABLE 2 NO. Raw Materials Unit (weight %) 1 Compound represented by Formula 1 0.1 2 Glycerin 3.0 3 Butylene glycol 2.0 4 Propylene glycol 2.0 5 Polyoxyethylene (60) hydrogenated castor oil 1.00 6 Ethanol 10.0 7 Triethanolamine 0.1 8 Preservative Small amount 9 Coloring Small amount 10 Flavoring Small amount 11 Purified water Residual amount Total 100

(38) Among the raw materials of Table 2, NOS: 2, 3, 4 and 8 were added to NO: 11 in sequence, and the resultant was stirred and dissolved. And then, NO: 5 was dissolved by being heated at around 60° C. and added with NO: 10, and the resultant was stirred and added to the above mixture of NO: 11 containing NOS: 2, 3, 4, and 8. Lastly, NOS: 1, 6, 7, and 9 were added to the mixture of NO: 11, stirred sufficiently, and passed through a microfluidizer to age the mixture.

FORMULATION EXAMPLE 2:

Preparation of Nutritive Cosmetic Water (Milky Lotion)

(39) Among the cosmetics containing the compound represented by Formula 1 of the present invention, a Formulation Example of nutritive cosmetic water (milky lotion) is shown in Table 3 below.

(40) TABLE-US-00003 TABLE 3 NO. Raw Materials Unit (weight %) 1 Compound represented by Formula 1 0.1 2 Sitosterol 1.70 3 Polyglyceryl-2 oleate 1.50 4 Ceteareth-4 1.2 5 Cholesterol 1.5 6 DEA-Cetyl Phosphate 0.4 7 Concentrated Glycerin 5.0 8 Sunflower oil 10.0 9 Carboxy vinyl polymer 0.2 10 Xanthan Gum 0.3 11 Preservative Small amount 12 Flavoring Small amount 13 Purified water Residual amount Total 100

(41) Among the raw materials of Table 3, NOS: 2, 3, 4, and 5 were homogenized under a constant temperature, and the resultant was named non-ionic amphipathic lipids, which were mixed with NOS: 1, 6, 7, and 13, and the resultant was homogenized at a constant temperature, passed through a microfluidizer, and homogenized by being slowly added with NO: 8 at a constant temperature followed by being passed through a microfluidizer once again. The resultant was added with NOS: 9, 10, 11, and 12, dispersed to be stabilized, and aged.

FORMULATION EXAMPLE 3:

Preparation of Nourishing Cream

(42) Among the cosmetics containing the compound represented by Formula 1 of the present invention, a Formulation Example of nourishing cream is shown in Table 4 below.

(43) TABLE-US-00004 TABLE 4 NO. Raw Materials Unit (weight %) 1 Compound represented by Formula 1 0.1 2 Sitosterol 4.0 3 Polyglyceryl-2 oleate 3.0 4 Ceteareth-4 2.0 5 Cholesterol 3.0 6 DEA-Cetyl Phosphate 0.4 7 Concentrated Glycerin 5.0 8 Sunflower oil 22.0 9 Carboxy vinyl polymer 0.5 10 Triethanolamine 0.5 11 Preservative Small amount 12 Flavoring Small amount 13 Purified water Residual amount Total 100

(44) Among the raw materials of Table 4, NOS: 2, 3, 4, and 5 were homogenized under a constant temperature, and the resultant was named non-ionic amphipathic lipids, which were mixed with NOS: 1, 6, 7, and 13, and the resultant was homogenized at a constant temperature, passed through microfluidizer, and homogenized by being slowly added with NO: 8 at a constant temperature followed by being passed through a microfluidizer once again. The resultant was added with NOS: 9, 10, 11 and 12, dispersed to be stabilized, and aged.

INDUSTRIAL APPLICABILITY

(45) The composition containing the compound represented by Formula 1 extracted from Anemarrhena asphodeloides Bunge according to the present invention or the pharmaceutically acceptable salt thereof is effective in moisturizing skin and improving wrinkles, especially, preventing or alleviating wrinkles by preventing the skin moisture loss and suppressing the skin tissue damages induced by UV rays.

(46) Accordingly, the composition may be usefully applied to an external skin application or cosmetics having skin-moisturizing and wrinkle-improving effects.