KR20180099569A

KR20180099569A - Composition for preventing, improving or treating skin wrinkle comprising Oxya chinensis sinuosa extract or compound isolated from the extract as effective component

Info

Publication number: KR20180099569A
Application number: KR1020180024840A
Authority: KR
Inventors: 채성욱; 임아랑; 나민균
Original assignee: 한국 한의학 연구원; 충남대학교산학협력단
Priority date: 2017-02-28
Filing date: 2018-02-28
Publication date: 2018-09-05
Also published as: KR101995220B1; WO2018160021A1

Abstract

The present invention relates to a composition for preventing, ameliorating, or treating skin wrinkles containing an Oxya chinensis sinuosa extract as an active ingredient, comprising a compound represented by chemical formula 1 or an acceptable salt thereof; a compound represented by chemical formula 2 or an acceptable salt thereof; a compound represented by chemical formula 3 or an acceptable salt thereof; or at least one compound selected among compounds represented by chemical formula 1 to 3. The composition of the present invention prevents cell damage caused by ultraviolet irradiation, and significantly suppresses expression of matrix metalloproteinase-1 (MMP-1), thereby being applied as drugs or health functional foods for preventing, ameliorating, or treating skin wrinkles.

Description

[0001] The present invention relates to a composition for preventing, improving or treating skin wrinkles containing an extract of a rice plant or a compound isolated therefrom as an active ingredient.

The present invention relates to a rice plant chinensis The present invention relates to a composition for preventing, ameliorating or treating skin wrinkles containing an extract or a compound isolated therefrom as an active ingredient.

Skin protects the organs in the body from external stimuli and plays an important role in the maintenance of body homeostasis, such as body temperature control. Such skin is aged by various internal and external factors and is divided into endogenous aging by genetic cause and exogenous aging by environmental cause. Among them, photoaging refers to aging by ultraviolet (UV). Recently, ozone depletion due to environmental pollution has increased the amount of ultraviolet rays, and accordingly, research on photoaging has been attracting attention. In the photoaged skin, appearance characteristics such as roughness, loss of elasticity, wrinkles and irregular pigmentation are observed. Among them, the main research field of photoaging is the change of the skin wrinkles. There have been reported many research results on basic physiological metabolism changes such as synthesis, degradation and moisture content of collagen which is a main constituent of skin in the formation of skin wrinkles by the photoaging. Particularly, ultraviolet rays increase the production of reactive oxygen species, collapse of enzymatic and nonenzymatic antioxidant defenses of skin, decrease collagen decomposition and biosynthesis, and collagen in the dermis layer is remarkably reduced. Important collagen degradation factors are matrix metalloproteinases (MMPs), which are involved in the degradation of extracellular matrix and basement membrane. Studies have been reported that the enzyme increases activity by ultraviolet rays and inhibits it, thereby increasing skin thickness and wrinkling induced by ultraviolet light. Therefore, it is known that effective control of MMPs is required for prevention and treatment of photoaging.

On the other hand, the grasshopper is 30 ~ 38cm in length, body color is yellow-green, and head and chest are yellowish brown. The compound eyes are egg-shaped and lustrous grayish brown. There are three thin lateral grooves on the pronotum, with brown vertical stripes on both sides. The wing is yellowish brown and longer than the tip of the wing but does not fly much. Inhabits grassy areas near rice fields and cultivated areas. It occurs once a year, and it drifts with a bunch of ground in the ground. The alum is wrapped in a thin film of collagen. The larvae grow up to the fourth ventral wing and the body length is about 30mm. The use of pesticides as a major pest of rice has decreased, but it is gradually increasing. In 1930, it originated in Hwanghae Province and bought eggs from the government. It is distributed in Korea, Japan, and China.

Korean Patent No. 1648812 discloses a method for producing a diet food composition containing a rice-grasshopper ingredient, Korean Patent Publication No. 2010-0002661 discloses a functional complex comprising hyaluronic acid and a method for producing the same, There is no disclosure of a technique relating to the prevention, improvement or treatment of skin wrinkles containing the rice grasshopper extract of the invention or a compound isolated therefrom as an active ingredient.

SUMMARY OF THE INVENTION The present invention has been achieved by the above-mentioned needs, and the present invention provides a compound of the formula 1 or an acceptable salt thereof; A compound of formula 2 or an acceptable salt thereof; A compound of formula (III) or an acceptable salt thereof; Or byeomettugi (Oxya comprising at least one compound selected from compounds of formula 1-3 chinensis The present invention relates to a composition for preventing, ameliorating or treating skin wrinkles containing an extract of MMP-1 as an active ingredient, wherein the compound of Chemical Formulas 1 to 3 and the extract of rice hogs are cytotoxic and the expression level of MMP- , The present invention has been completed.

In order to accomplish the above object, the present invention provides a compound of formula 1 or an acceptable salt thereof; A compound of formula 2 or an acceptable salt thereof; A compound of formula (III) or an acceptable salt thereof; Or byeomettugi (Oxya comprising at least one compound selected from compounds of formula 1-3 chinensis The present invention provides a health functional food composition for preventing or ameliorating skin wrinkles containing an extract of Aspergillus sinuosa as an active ingredient.

The present invention also relates to a compound of formula I or an acceptable salt thereof; A compound of formula 2 or an acceptable salt thereof; A compound of formula (III) or an acceptable salt thereof; Or byeomettugi (Oxya comprising at least one compound selected from compounds of formula 1-3 chinensis The present invention also provides a pharmaceutical composition for preventing or treating skin wrinkles containing, as an active ingredient,

The present invention relates to a compound of formula (I) or an acceptable salt thereof; A compound of formula 2 or an acceptable salt thereof; A compound of formula (III) or an acceptable salt thereof; Or as the active ingredient for the prevention, improvement or treatment composition for the skin wrinkles containing byeomettugi (Oxya chinensis sinuosa) extract comprising one or more compounds selected from compounds of formula 1-3 as an active ingredient it has no cytotoxicity , The effect of inhibiting the expression of MMP-1 is remarkable, and thus it can be usefully used for health functional foods or medicines for prevention, improvement or treatment of skin wrinkles.

FIG. 1 shows the results of confirming cell viability (%) after treatment of rice plant extracts of the present invention by concentration.
2 shows the results of confirming the cell viability (%) after treating the compounds derived from the rice-grasshopper extract of the present invention (Compound A (Formula 1), Compound B (Formula 2) and Compound C (Formula 3)) by concentration.
FIG. 3 shows the results of confirming changes in the expression level of MMP-1 after treatment of the rice-grasshopper extract of the present invention at various concentrations.
4 is a result of examining changes in the expression level of MMP-1 after treatment of the compounds (Compound A (Formula 1), Compound B (Formula 2) and Compound C (Formula 3)) derived from the rice grasshopper extract of the present invention by concentration .
FIG. 5 shows the results of confirming that the skin thickness increased by UV irradiation was reduced by administration of the rice grasshopper extract of the present invention. #### indicates that the epidermal thickness of the ultraviolet irradiated group was increased compared to the normal group, and p <0.0001. **** indicates that the epidermal thickness of the group treated with the rice plant extract of the present invention was decreased compared to the ultraviolet irradiation group, and p < 0.0001.
FIG. 6 is a result of confirming the increase of the epidermal thickness and the collagen content by administering the rice grasshopper extract of the present invention after confirming the increase of the skin thickness and the reduced collagen content by UV irradiation. A is the result of H & E staining, and B is the result of Masson's trichome staining.
FIG. 7 shows the results of analysis of changes in the expression levels of MMP-1 and MMP-9, which are involved in the wrinkles of the skin upon administration of the rice-grasshopper extract of the present invention. ### and #### showed a significant increase in the expression levels of MMP-1 and MMP-9 in the ultraviolet irradiated group compared to the normal group. ### is p <0.001 and #### is p < 0.0001. ** and *** indicate that the expression levels of MMP-1 and MMP-9 in the group treated with the rice plant extract of the present invention were significantly decreased compared to the ultraviolet irradiation group, p < 0.001.

The present invention relates to compounds of the general formula (I) or acceptable salts thereof; Claims 1. A compound of formula (2) or an acceptable salt thereof; Claims 1. A compound of formula (3): Or one or more compounds selected from the group consisting of compounds represented by the following formulas (1) to (3): Oxya chinensis The present invention relates to a health functional food composition for preventing or ameliorating skin wrinkles containing, as an active ingredient,

The compounds of the above formulas (1) to (3) are preferably extracted from rice hermit, but the present invention is not limited thereto, and extraction or synthesis from other natural products is possible.

The rice grasshopper extract may be produced by a method including, but not limited to, the following steps:

(1) extracting the rice seedlings with an extraction solvent;

(2) filtering the extract of step (1); And

(3) The step of extracting the filtered extract of step (2) by concentration under reduced pressure and drying.

In step (1), the extraction solvent is preferably selected from water, a C ₁ -C ₄ lower alcohol or a mixture thereof, more preferably ethanol, even more preferably 70% (v / v) ethanol But is not limited thereto.

In the above production method, any conventional method known in the art such as filtration, hot water extraction, immersion extraction, reflux cooling extraction, and ultrasonic extraction may be used. The extraction solvent is preferably added by 1 to 20 times the weight of the dried rice hogger, more preferably 5 to 15 times. The extraction temperature is preferably 4 to 50 DEG C, but is not limited thereto. The extraction time is preferably 0.5 to 10 hours, more preferably 0.5 to 5 hours, most preferably 1 hour, but not always limited thereto. In the above method, it is preferable to use a vacuum decompression concentrator or a vacuum rotary evaporator for the decompression concentration in the step (3), but it is not limited thereto. The drying is preferably performed under reduced pressure, vacuum drying, boiling, spray drying or freeze drying, but not always limited thereto.

The health functional food composition is characterized by inhibiting the expression of MMP-1 or MMP-9.

The health functional food composition may be prepared in any form of powder, granule, ring, tablet, capsule, candy, syrup and beverage, but is not limited thereto.

The health functional food composition of the present invention may be prepared by directly adding one or more selected from the compounds of the general formulas (1) to (3) derived from a rice plant extract or a rice plant extract or by blending it with another food or food ingredient, . Examples of the food to which at least one selected from the compounds of Chemical Formulas 1 to 3 derived from the rice grasshopper extract can be added include caramel, meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, Beverages, tea, drinks, alcoholic beverages, and vitamin complexes, all of which include health functional foods in a conventional sense. That is, there is no particular limitation on the kind of the food. The health functional food composition may contain various nutrients, vitamins, minerals (electrolytes), synthetic and natural flavors, colorants and enhancers (cheese, chocolate etc.), pectic acid and its salts, alkynic acid and its salts, Thickening agents, pH adjusting agents, stabilizers, preservatives, glycerin, alcohols, carbonating agents used in carbonated drinks, and the like. It may also contain flesh for the production of natural fruit juices and vegetable drinks. The above components can be used independently or in combination. In addition, the health functional food composition of the present invention may contain various flavors or natural carbohydrates as an additional ingredient. The natural carbohydrates may be selected from the group consisting of glucose, monosaccharides such as fructose, disaccharides such as maltose and sucrose, Polysaccharides such as cyclodextrin, and sugar alcohols such as xylitol, sorbitol and erythritol. The ratio of the natural carbohydrate is not critical, but is preferably 0.01 to 0.04 g, more preferably 0.02 to 0.03 g per 100 g of the composition of the present invention, but is not limited thereto. Examples of sweeteners include natural sweeteners such as tau martin and stevia extract, synthetic sweeteners such as saccharin and aspartame, and the like.

The present invention also relates to a compound of formula (I) or an acceptable salt thereof; A compound of formula 2 or an acceptable salt thereof; A compound of formula (III) or an acceptable salt thereof; Or byeomettugi (Oxya comprising at least one compound selected from compounds of formula 1-3 chinensis The present invention relates to a pharmaceutical composition for prevention or treatment of skin wrinkles containing an extract of Aspergillus sinuosa as an active ingredient.

[Chemical Formula 1]

(2)

(3)

The pharmaceutical composition of the present invention may further comprise a pharmaceutically acceptable carrier, excipient or diluent. The pharmaceutically acceptable carrier to be contained in the pharmaceutical composition of the present invention is one which is usually used at the time of formulation and includes saline solution, sterilized water, Ringer's solution, buffered saline solution, dextrose solution, maltodextrin solution, glycerol, ethanol, But are not limited to, sucrose, sucrose, sucrose, sorbitol, mannitol, starch, acacia, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, syrup, methylcellulose, methylhydroxybenzoate, Hydroxybenzoate, talc, magnesium stearate and mineral oil, and the like. In addition, the composition may further contain an antioxidant, a buffer, a bacteriostatic agent, a diluent, a surfactant, a binder, a lubricant, a wetting agent, a sweetener, a flavoring agent, an emulsifier, a suspending agent or a preservative. The pharmaceutical composition of the present invention can be administered orally or parenterally, and parenteral administration can be carried out by injection or application to skin. A suitable dose of the pharmaceutical composition of the present invention may be variously prescribed by factors such as the formulation method, the administration method, the age, body weight, sex, pathological condition, food, administration time, administration route, excretion rate and responsiveness of the patient .

Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these embodiments are merely illustrative of the present invention and that the scope of the present invention is not limited thereto.

[ Materials and Methods]

1. Manufacture of rice-grasshopper extract

The rice seedlings (4.87 kg) were extracted with ethanol containing 1% (v / v) acetic acid and then distilled under reduced pressure to obtain 421.8 g of a brown rice seed extract ethanol extract.

2. Isolation and Structure Determination of Compounds of Formulas 1 to 3

Ethanol extract (421.8 g) was purified by vacuum liquid chromatography (VLC) using hexane / ethyl acetate 100: 0 (6 L), 80:20 (6 L), 60:40 (6 L), 40: 80 (6 L) (v: v); Concentration gradient elution conditions of chloroform / methanol 100: 0 (6 L), 80:20 (6 L), 60:40 (6 L), 40:60 (6 L) and 20:80 (6 L) Six small fractions were obtained from the conditions (AF).

D fraction (35.7 g) was purified by VLC (vacuum liquid chromatography) using hexane / ethyl acetate 100: 0 (6 L), 85:15 (6 L), 75:25 (6 L) and 50: v); The concentration gradient elution and methanol washing conditions of chloroform / methanol 85: 15 (6 L), 80:20 (6 L), 67:33 (6 L) and 50:50 (6 L) (D-1 to D-7).

D-5 fraction (18.3 g) was purified by gradient elution of acetonitrile / water (10:90 to 60:40, 8160 ml) using MPLC (medium pressure liquid chromatography) and acetonitrile (D-5-1 to D-5-6).

D-5-2 fraction (1.9g) prep HPLC (Phenomenex Kinetex C 18 250 × 21mm, 5μm, Kinetex Biphenyl 250 × 21mm, 5μm) proceeds to the compound A with respect to (t _R 52 bun: 331.2mg), Compound B (t _R, 47 bun: 389.2mg), compound C (t _R 64 bun: 37.2mg) was obtained.

3. Cell culture

HaCaT cells were cultured in Dulbecco's Modified Eagle Medium (DMEM, Gibco) supplemented with 10% fetal bovine serum (FBS, Gibco) and 1% penicillin-streptomycin And cultured in medium containing penicillin-streptomycin (PS, Gibco) at 37 ° C and 5% CO ₂ .

4. Experimental animal and sample administration

Reared mice were purchased from 6-week-old male rats (male HR-1, hairless mice, Japan SLC, Inc.) from a central laboratory animal and adapted for 1 week before use. Animals in healthy state were used for the test by observing the general condition during the adaptation period. The breeding environment was maintained at a temperature of 23 ± 3 ° C, a humidity of 50 ± 5%, and a light period of 12 hours (07: 00-19: 00 / lighting time). During the test period, the animals were housed in a polycarbonate cage (200 × 320 × 145 mm, Three-shine Co., Daejeon, Korea) at a rate of 6 per group. Feeds were fed free of mouse 5L79 (Charles river, USA) , And negative water was free of tap water which was sterilized by ultraviolet rays.

The experimental groups were divided into three groups: control, UV-vehicle, and rice (BMG). Samples were administered orally using a mouse mouse. The administration period was 12 weeks for a total of 5 days.

5. UV irradiation

Ultraviolet irradiation was performed three times a week for 12 weeks in the experimental group except for the control group. UVB lamp (Mineralight UV Display lamp, UVP, USA) was used for ultraviolet rays. The ultraviolet dose was 60 mJ / cm ² for 1-4 weeks, 90 mJ / cm ^{2 for} 5-8 weeks, 9-12 weeks were irradiated at 120 mJ / cm ² for 12 weeks. Ultraviolet radiation was measured by measuring the amount of ultraviolet light using a photometer (Delta OHM, Italy) and then adjusting the irradiation time.

6. Histological analysis of skin

To confirm the efficacy of wrinkle suppression, skin tissues of each experimental group were extracted and fixed in 10% neutral formalin solution, washed, dehydrated, transparent, permeated, embedded in paraffin and cut to a thickness of 4 μM. Hematoxylin & Eosin H & E) staining and Masson's trichome staining. The thickness from the keratin layer to the epidermal basement membrane of H & E stained tissue was measured using a microscope.

Example One. Cell survival rate analysis

The HaCaT cells were cultured for 24 hours in a concentration-dependent manner, and the cells were treated with MTS assay (CellTiter Aqueous One Solution Cell proliferation assay kit, 3- (4,5-dimethylthiazol-2-yl) Cell viability was measured by a microplate reader (Molecular Devices, Sunnyvale, Calif., USA) at 490 nm by the method of MTS, Promega Co. Madison, WI, ).

As a result, the cell survival rate in the UVB irradiated group was lower than that in the control group treated with nothing, and the relative cell survival rate (%) after treatment with the samples of the present invention (the rice grasshopper extract and the compounds of Formulas 1 to 3) ) Were slightly increased or remained in Fig. 1 and Fig. 2, respectively. Therefore, it was found that the rice plant extract of the present invention and the compounds of the formulas (1) to (3) had little cytotoxicity.

Example 2. MMP -1 expression level

Each sample was treated with HaCaT cells for 24 hours and irradiated with ultraviolet light (UV Crosslinker, Ultra Lum) at 20 mJ / cm ^{2. Then} , using an MMP-1 ELISA kit (R & D Systems, Inc., Minneapolis, The expression level of MMP-1 was measured.

Specifically, HaCaT cells were plated on a 96-well plate, and then the rice-grasshopper extract of the present invention and the compounds of Formulas 1 to 3 were treated at respective concentrations. Then, ultraviolet (UV) irradiation was performed, and the supernatant of the culture was collected and centrifuged at 13,000 rpm for 5 minutes. The supernatant was then subjected to fluorescent assay using an ELISA kit to quantitatively analyze the expression level of MMP-1.

As a result, it was confirmed that the expression level of MMP-1 remarkably increased in the group irradiated with ultraviolet light as compared with the negative control group not irradiated with ultraviolet light as disclosed in Figs. 3 and 4, and the sample of the present invention 1 to 3) of MMP-1 was remarkably decreased compared with that of ultraviolet irradiation group.

Example 3. Assessment of Efficacy by Change of Epidermal Thickness of Animal Model

The effect of wrinkle suppression was also confirmed by measurement of skin thickness change. The thickness of the epidermis was measured using the thickness of the keratin layer from the H & E stained tissue to the epidermal basement membrane using a microscope.

As a result, as shown in Fig. 5, it was confirmed that the epidermal thickness was increased at the time of ultraviolet irradiation, and it was confirmed that the epidermal thickness was decreased by the rice group extract (BMG) administration group.

Example 4. Evaluation of efficacy by tissue staining of animal models (H & E staining)

To confirm the efficacy of wrinkle suppression induced by ultraviolet rays, skin tissue of hairless mice was detached and then confirmed by tissue staining.

As a result, as shown in Fig. 6 (A), it was confirmed that the stratum corneum layer and the skin layer thickness were increased in the ultraviolet irradiation group compared to the control group. However, in the group treated with the rice grasshopper extract (BMG) And the skin thickness was also decreased.

Example 5. Evaluation of efficacy by tissue staining Masson's trichome dyeing)

To confirm the efficacy of wrinkle suppression induced by ultraviolet rays, skin tissue of hairless mice was detached and confirmed by collagen tissue staining.

As a result, as shown in Fig. 6 (B), collagen was reduced by ultraviolet rays, and it was confirmed that the amount of collagen was recovered in the rice-grasshopper extract-treated group of the present invention.

Example 6. MMP -1 and MMP -9 expression level

Collagen degradation enzymes (MMPs) are involved in the degradation of extracellular matrix and basement membrane, thereby affecting collagen degradation. The changes in the expression levels of MMP-1 and MMP-9, which are involved in the wrinkles of the skin, were analyzed to confirm whether the wrinkles were improved.

As a result, as shown in FIG. 7, both the amounts of expression of MMP-1 and MMP-9 were increased by ultraviolet irradiation compared to the cells not irradiated with ultraviolet light. It was confirmed that the expression amount of MMP-1 and MMP- Respectively.

Claims

Claims 1. Compounds of the general formula (I) or acceptable salts thereof; Claims 1. A compound of formula (2) or an acceptable salt thereof; Claims 1. A compound of formula (3): Or one or more compounds selected from the group consisting of compounds represented by the following formulas (1) to (3): Oxya chinensis sinuosa extract as an active ingredient.
[Chemical Formula 1]

(2)

(3)

The health functional food composition according to claim 1, wherein the rice plant extract is extracted using at least one solvent selected from water, C ₁ -C ₄ lower alcohol and mixtures thereof.

The health functional food composition according to claim 1, wherein the composition inhibits the expression of MMP-1 or MMP-9.

The health functional food composition according to claim 1, wherein the composition is prepared from any one of powder, granule, ring, tablet, capsule, candy, syrup and beverage.

Claims 1. Compounds of the general formula (I) or acceptable salts thereof; Claims 1. A compound of formula (2) or an acceptable salt thereof; Claims 1. A compound of formula (3): Or one or more compounds selected from the group consisting of compounds represented by the following formulas (1) to (3): Oxya chinensis The present invention relates to a pharmaceutical composition for the prevention or treatment of skin wrinkles,
[Chemical Formula 1]

(2)

(3)

[Claim 7] The pharmaceutical composition according to claim 5, which further comprises a pharmaceutically acceptable carrier, excipient or diluent in addition to the active ingredient.