KR102070799B1 - Composition for ameliorating skin wrinkle or skin moisturizing comprising Tenebrio molitor extract as effective component - Google Patents

Abstract

The present invention relates to a composition for improving skin wrinkles or skin moisturizing containing brown gourd extract as an active ingredient, and in particular, brown gourd extract has little cytotoxicity and enhances the content of hyaluronic acid that can promote skin moisturizing. In addition to reducing percutaneous moisture loss, increasing skin moisturization, decreasing skin thickness, decreasing expression levels of MMP-1 and MMP-9 proteins, and increasing wrinkle-related protein expression To adjust the amount. Therefore, the composition of the present invention can be used as a pharmaceutical composition for preventing or treating skin wrinkles or health functional food composition for skin wrinkle improvement or skin moisturizing.

Description

Composition for ameliorating skin wrinkle or skin moisturizing comprising Tenebrio molitor extract as effective component}

The present invention is brown meal ( Tenebrio molitor ) relates to a composition for improving skin wrinkles or moisturizing skin containing the extract as an active ingredient.

The skin is an organ that is always in contact with the external environment. It mainly protects the human body from external physical damage and chemicals, prevents the invasion of bacteria, fungi, viruses, etc. into the skin, and acts as a protective barrier against moisture loss. The structure of the skin consists of three layers of epidermis, dermis and subcutaneous fat. The epidermis is the thinnest of the three layers and plays an important role in moisturizing and protecting the skin. In particular, the moisture in the stratum corneum of the epidermis makes the skin elastic and soft. It is known that moisture content is essential for maintaining the elasticity of the stratum corneum (OK Jacobi, Proc. Sci. Sec. Toilet Goods Assoc. 1959, 31 , 22).

In recent years, exposure to strong ultraviolet rays due to environmental pollution and the like has increased in many cases, skin aging is accelerated as the water loss of the skin increases, and as the incidence of related diseases caused by skin damage increases, exposure to ultraviolet rays is caused. There is an increasing demand for compositions that maintain the moisturizing and wrinkle improvement effects of the skin for a long time.

On the other hand, the brown gourd is about 15mm long and its body color is similar to Kowloon gourd (Kowloon gourd), which is dark brown and glossy. There are spots on the head, but they are not compact, and the end of the tactile (antennae) is the same length and butterfly. Pronotum square, with vertical grooves on hind rim. The middle is a pentagram with a wide butterfly. The scab wings are brown with 8 rows of vertical grooves, the trunk is convex, and there are small dots. Adults lay eggs mainly in cereals. Eggs are so small that they cannot be easily found by the eye and hatch after 1-2 weeks. Larvae are called worms (mealworms), which are used for feeding insects or pets, and the larvae are about 10 weeks long, but about 2 weeks for artificial breeding. The reason why worms are used for pets is that they can easily experience insects due to their relatively short metamorphosis and are very clean insects that feed on grain. Caterpillars molt 9-20 times depending on food and temperature until they become pupa. After two to three weeks of pupae, it becomes an adult, and when it is the first allegory, it becomes light brown but gradually turns black. Adults are nocturnal, hiding in holes during the day and active at night. It is usually overwintered as a larva in a grain bag near Gaeng, and then transforms into a pupa and adult in spring. It is famous for its pests in grains, and has recently been raised in zoos by small animals such as birds and hedgehogs. It is distributed all over the world including Korea.

An example of the technology known to be known as brown gourd is a 2016 master's dissertation at Daegu Haany University in 2016. 'Verifying the NF-kB, MAPK pathway inflammation and whitening mechanism of CopA3 peptide derived from brown gourd water extract and baby dung beetle. And a study on the application of WOW formulations are disclosed, and the melanin production inhibitory effect of the brown ethanol extract, which was presented at the 2013 Annual Meeting of the Korean Society of Insects, has been disclosed. There is no disclosure about skin wrinkle improvement or skin moisturizing compositions.

The present invention is derived from the above requirements, the present invention provides a composition for improving skin wrinkles or skin moisturizing containing brown gourd extract as an active ingredient, the active ingredient is involved in the decomposition of the extracellular matrix and the basement membrane Reduced expression of collagen degrading enzymes (MMPs, matrix metalloproteinases), MMP-1 protein, and enhancement of hyaluronic acid to prevent skin moisturizing function and skin barrier damage. The present invention was completed by confirming that there is an effect of reducing loss, increasing skin moisturizing amount, decreasing skin thickness, expressing amounts of MMP-1 and MMP-9 proteins, and expressing proteins related to wrinkle improvement.

In order to achieve the above object, the present invention provides a health functional food composition for improving skin wrinkles or moisturizing skin containing brown gourd extract as an active ingredient.

In another aspect, the present invention provides a pharmaceutical composition for the prevention or treatment of skin wrinkles containing the brown gourd extract as an active ingredient.

The present invention relates to a composition for improving skin wrinkles or skin moisturizing containing the brown gourd extract as an active ingredient, the active ingredient of the present invention has little cytotoxicity, and is effective in improving skin wrinkles and skin moisturizing. Therefore, the composition of the present invention for improving skin wrinkles or skin moisturizing health functional food; It can be applied to medicines for preventing or treating skin wrinkles.

1 is an MTS assay result confirming the cell survival rate (%) of HaCaT cells according to the concentration-specific treatment (10, 50, 100, 200 and 250㎍ / ㎖) of the extract of the present invention.
Figure 2 shows the results of confirming the change in the amount of expression of MMP-1 according to the treatment of brown rice wine extract of the present invention. # Indicates that the MMP-1 expression level of the UVB irradiation group was significantly increased compared to the Control group, which means p <0.05, and * indicates the MMP-1 expression level of the experimental group treated with the brown edible extract and UVB irradiation. Statistically significant reduction compared to the MMP-1 expression level of the group, which means p <0.05.
Figure 3 is a result of analyzing the change in hyaluronic acid (HA) content according to the UV irradiation and the treatment according to the concentration of the brown rice wine extract of the present invention. Control is a normal group that does not process anything, UVB is a control group irradiated with only ultraviolet light, without treatment of the brown gourd extract of the present invention. # Indicates that the hyaluronic acid content in the UVB irradiated group was statistically significantly reduced compared to the control group, which means p <0.05, and * indicates that the hyaluronic acid content of the experimental group treated with the brown gourd extract and UVB irradiated was Statistically significant increase, which means that p <0.05.
Figure 4 is the result of confirming the percutaneous moisture loss amount (TEWL) of the brown rice wine extract administration group of the present invention. Nor is a normal group not irradiated with ultraviolet rays, Veh is a group inducing transdermal moisture loss by irradiating ultraviolet rays, GGR is a group administered with ultraviolet irradiation (UVB) and the brown mealworm extract of the present invention. # Is a statistically significant increase in the amount of transdermal moisture loss in the UV irradiation group compared to the normal group, p <0.05, * is a statistically significant in the transdermal moisture loss amount of the brown meal extract extract group of the present invention compared to the ultraviolet irradiation group. Decreased, p <0.05.
Figure 5 is the result of confirming the skin moisture content of the brown rice wine extract administration group of the present invention. Nor is a normal group not irradiated with ultraviolet rays, Veh is a group irradiated with ultraviolet rays, and GGR is a group administered with ultraviolet irradiation and the brown mealworm extract of the present invention. ## is a statistically significant decrease in the skin moisture content of the ultraviolet irradiation group compared to the normal group, p <0.01, * * is a statistically significant increase in the skin moisture content of the brown rice wine extract administration group compared to the ultraviolet irradiation group. P <0.05.
Figure 6 is the result of confirming the change in the skin thickness of the brown rice wine extract administration group of the present invention. Nor is a normal group not irradiated with ultraviolet rays, Veh is a group irradiated with ultraviolet rays, and GGR is a group administered with ultraviolet irradiation and the brown mealworm extract of the present invention. #### is a statistically significant increase in the skin thickness of the ultraviolet irradiation group compared to the normal group, p <0.0001, *** *** is a statistically significant skin thickness of the brown edible extract administration group compared to the ultraviolet irradiation group. Significantly decreased, p <0.001.
Figure 7 is a result of confirming the effect of improving wrinkles through the H & E staining of tissues collected from the hairless mouse group administered the brown edible extract of the present invention. Normal is a normal group not irradiated with ultraviolet rays, Vehicle is a group irradiated with ultraviolet rays, GGR is a group administered with ultraviolet irradiation and the brown mealworm extract of the present invention.
8 is a result of confirming the change in the expression amount of MMP-1 and MMP-9 protein according to the administration of the brown rice wine extract of the present invention. Nor is a normal group not irradiated with ultraviolet rays, Veh is a group irradiated with ultraviolet rays, and GGR is a group administered with ultraviolet irradiation (UVB) and the brown mealworm extract of the present invention. # Is a statistically significant increase in the expression level of MMP-1 and MMP-9 protein in the UV irradiation group compared to the normal group, p <0.05, * is MMP- The expression levels of 1 and MMP-9 proteins were statistically significant, p <0.05.
9 is a result of confirming the change in the expression level of pERK, pMEK and pp38 protein according to the administration of the brown rice wine extract of the present invention.

The present invention relates to a skin wrinkle improvement or skin moisturizing health functional food composition containing the brown edible extract as an active ingredient.

The brown rice wine extract may be prepared by a method comprising the following steps, but is not limited thereto:

(1) extracting by adding an extraction solvent to the brown meal dried powder;

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

(3) concentration of the filtered extract of step (2) under reduced pressure and drying to prepare an extract.

The extraction solvent in the step (1) is preferably selected from water, lower alcohols of C ₁ ~ C ₄ or mixtures thereof, more preferably ethanol, even more preferably 70% (v / v) ethanol It is not limited to this.

In the above production method, the extraction method may use all conventional methods known in the art, such as filtration, hot water extraction, immersion extraction, reflux cooling extraction and ultrasonic extraction. The extraction solvent is preferably extracted by adding 1 to 20 times the weight of dried brown rice wine, more preferably 5 to 15 times. Extraction temperature is preferably 4 to 50 ℃ but is not limited thereto. In addition, the extraction time is preferably 0.5 to 10 hours, more preferably 0.5 to 5 hours, most preferably 1 hour, but is not limited thereto. In the above method, the vacuum concentration in step (3) is preferably used as a vacuum vacuum concentrator or vacuum rotary evaporator, but is not limited thereto. In addition, the drying is preferably reduced pressure drying, vacuum drying, boiling drying, spray drying or freeze drying, but is not limited thereto.

In the present invention, wrinkle generation occurs due to a decrease in collagen, and the decrease in collagen may be caused by excessive oxidative stress or ultraviolet irradiation.

The brown rice wine extract may reduce the expression level of MMP-1 or MMP-9 protein and increase the content of hyaluronic acid.

When the health functional food composition of the present invention is used as a food additive, the health functional food composition may be added as it is or used with other foods or food ingredients, and may be appropriately used according to a conventional method. Mixed amounts of active ingredients can be appropriately used depending on the purpose of use (prevention or improvement). In general, in the preparation of food or beverages, the nutraceutical composition of the present invention is added in an amount of 15 parts by weight or less, preferably 10 parts by weight or less based on the raw materials. However, in the case of long-term intake for health purposes, the amount may be below the above range, and since there is no problem in terms of safety, the active ingredient may be used in an amount above the above range.

There is no particular limitation on the type of dietary supplement. Examples of foods to which the health functional food composition may be added include meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gums, dairy products including ice cream, various soups, drinks, tea Drinks, alcoholic beverages and vitamin complexes, and the like, includes all of the health food in the conventional sense.

In addition, the health functional food composition of the present invention may be prepared as food, in particular functional food. Functional foods of the present invention include ingredients that are commonly added in food production, and include, for example, proteins, carbohydrates, fats, nutrients and seasonings. For example, when prepared with a drink, in addition to the active ingredient, natural carbohydrates or flavoring agents may be included as additional ingredients. The natural carbohydrates may be monosaccharides (e.g. glucose, fructose, etc.), disaccharides (e.g. maltose, sucrose, etc.), oligosaccharides, polysaccharides (e.g., dextrins, cyclodextrins, etc.), or sugar alcohols ( For example, xylitol, sorbitol, erythritol, etc.) is preferable. The flavourant may be a natural flavourant (eg, taumartin, stevia extract, etc.) and a synthetic flavourant (eg, saccharin, aspartame, etc.).

Various nutritional supplements, vitamins, electrolytes, flavors, coloring agents, pectic acid and salts thereof, alginic acid and salts thereof, organic acids, protective colloid thickeners, pH regulators, stabilizers, preservatives, glycerin, alcohols, carbonic acid Carbonation agent etc. which are used for a drink can be contained further. Although the ratio of the above added components is not critical, it is generally selected from the range of 0.01 to 0.1 parts by weight based on 100 parts by weight of the nutraceutical composition of the present invention.

In addition, the present invention relates to a pharmaceutical composition for the prevention or treatment of skin wrinkles containing the brown gourd extract as an active ingredient.

The pharmaceutical composition of the present invention may further comprise a pharmaceutically acceptable carrier, excipient or diluent. Pharmaceutically acceptable carriers included in the pharmaceutical composition of the present invention are conventionally used in the preparation, and include saline solution, sterile water, Ringer's solution, buffered saline solution, dextrose solution, maltodextrin solution, glycerol, ethanol, lactose, dex Straw, sucrose, sorbitol, mannitol, starch, acacia rubber, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, syrup, methyl cellulose, methylhydroxybenzoate, propyl Hydroxybenzoate, talc, magnesium stearate and mineral oil, and the like. In addition to the above components, antioxidants, buffers, bacteriostatic agents, diluents, surfactants, binders, lubricants, wetting agents, sweeteners, flavoring agents, emulsifiers, suspending agents or preservatives may be further included. The pharmaceutical composition of the present invention may be administered orally or parenterally, and the parenteral administration may be administered by injection or application to the skin. Suitable dosages of the pharmaceutical compositions of the present invention may be prescribed in various ways depending on such factors as the formulation method, mode of administration, age, weight, sex, morbidity, condition of food, time of administration, route of administration, rate of excretion and response to reaction. Can be.

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only for explaining the present invention in more detail, it is obvious to those skilled in the art that the scope of the present invention is not limited thereto.

Example  1. Preparation of brown bran extract

70% (v / v) ethanol was used for the reflux extraction for 3 hours on the brown rice wine dried powder. A 70% (v / v) ethanol extract was obtained and distilled under reduced pressure to prepare a brown rice ethanol extract.

Example  2. Analysis of Cell Viability by Treatment of Brown Rice Wine Extract

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

The cultured HaCaT cells (human keratinocytes) were treated by diluting each sample by concentration for 24 hours, followed by MTS assay (CellTiter Aqueous One Solution Cell proliferation assay kit 3- (4,5-dimethylthiazol-2-yl) Microplate reader (Molecular Devices, Sunnyvale) at 490 nm using -5- (3-carboxymethoxyphenyl) -2- (4-sulfophynyl) -2H-tetrazolium, inner salt; MTS, Promega Co. Madison, WI, USA , CA, USA) and tested.

As a result, as shown in Figure 1, even if treated with the brown gourd extract of the present invention almost no change in cell viability (%) based on the control group treated nothing, the brown gourd extract of the present invention shows no cytotoxicity It was confirmed.

Example  3. MMP Analysis of Changes in Expression Level of -1 Protein

Each sample was treated with HaCaT cells for 24 hours and irradiated with UV crosslinker (Ultra Lum) at 20 mJ / cm ² , followed by MMP-1 ELISA kit (R & D Systems, Inc., Minneapolis, MN, USA). The expression level of MMP-1 was measured.

Specifically, after the HaCaT cells were plated in 96-well plates, the brown gourd extracts of the present invention were treated at different concentrations (10, 50, 100, and 200 µg / ml), respectively. Thereafter, ultraviolet (UV) light was irradiated, the supernatant of the culture was collected and centrifuged for 5 minutes at 13,000 rpm. Subsequently, the supernatant was taken and subjected to fluorescence assay using an ELISA kit to quantitatively analyze the expression level of MMP-1.

As a result, as shown in FIG. 2, it was confirmed that the expression level of MMP-1 was significantly increased in the group irradiated with ultraviolet rays, compared to the negative control group not irradiated with ultraviolet rays. It was confirmed that the expression level of MMP-1 was significantly reduced compared to the ultraviolet irradiation group.

Example  4. Hyaluronic acid ( Hyaluronic  Acid; Analysis of content change of HA)

Each HaCaT cell was treated with each sample for 24 hours and irradiated with UV (UV Crosslinker, Ultra Lum) at 20 mJ / cm ² for 30 minutes, and then the supernatant of the culture was collected and a hyaluronic acid ELISA kit (R & D Systems, Inc., Minneapolis, MN, USA) was used to determine the content of HA. The reaction was subjected to an absorbance assay.

As a result, as shown in FIG. 3, the content of hyaluronic acid was decreased by ultraviolet irradiation, but in each experimental group treated with 10, 50, 100, and 200 µg / ml brown gourd extract of the present invention, hyaluronic acid It was confirmed that the content of increased.

Example  5. Using animal models Skin hydration  And wrinkle improvement

(1) Experimental animal and sample administration

Hairless mice were used after 6 week old male hairless mice (male HR-1, hairless mice, Japan SLC, Inc.) were purchased from a central laboratory animal and adapted for 1 week. Healthy animals were used for testing by observing general conditions during the adaptation period. The breeding environment was maintained at a temperature of 23 ± 3 ℃, a humidity of 50 ± 5%, and a contrast cycle of 12 hours (07: 00-19: 00 / lighting time). During the test period, 6 animals were raised per group, and the feed was free of 5L79 (Charles river, USA) feed for the mouse, and the free water was negatively treated with UV-water-treated tap water.

Sample administration was performed by dividing into the normal group (Nor), the ultraviolet treatment group (UVB + Veh), the ultraviolet irradiation and the brown gourd extract (UVB + GGR) administration group not irradiated with ultraviolet rays. Sample administration was performed by oral administration using a mouse zone. The administration period was a total of 12 weeks for 5 days a week.

(2) UV irradiation

Ultraviolet irradiation was carried out three times a week for 12 weeks in the experimental group except the control group, the UVB lamp (Mineralight UV Display lamp, UVP, USA) was used. 1-4 to 60mJ / cm ^2, 5 ~ 8 to 90mJ / cm ^2, 9-12 weeks of 120mJ / cm ² size, was irradiated with ultraviolet light for 12 weeks. The amount of ultraviolet radiation was adjusted to the irradiation time after measuring the amount of light using a photometer (Delta OHM, Italy).

(3) Evaluation of transepidermal water loss (TEWL)

Percutaneous moisture loss amount is the amount of moisture emitted from the skin, the higher the value means that the skin's moisturizing function is off, indicating that the inherent barrier function of the skin is impaired.

Percutaneous moisture loss is calculated by the amount of moisture evaporated from the skin by area and time using a Tewameter, Courage & Khazaka, Germany at constant temperature and humidity conditions (23 ℃, 50% relative humidity). The amount of water evaporated (g / m ² / hr) was read with a moisturizing electronic sensor, it was numerically measured to measure the skin's moisturizing power.

As a result, as shown in FIG. 4, it was confirmed that the skin barrier was damaged by UV irradiation, and the skin moisturizing function was decreased. In the case of the brown rice wine extract administration group, statistically significant prevention of skin moisture loss caused by ultraviolet light was prevented. It confirmed that.

(4) skin moisture measurement

Skin moisture content was measured by measuring the moisture contained in the skin using a coronometer (Corneometer, Courage & Khazaka, Germany) equipment at constant temperature, constant humidity (23 ℃, 50% relative humidity). The amount of moisture present in the epidermis of the skin was measured by using a sensor to measure the ion level of the moisture, and the moisture content was measured by numerically calculating the amount of moisture.

As a result, as shown in FIG. 5, the UV irradiation group (Veh) reduced the skin moisture content compared to the normal group (Nor) that did not irradiate the ultraviolet rays, and the brown gourd extract administration group (GGR) compared to the UV irradiation group. Appeared high. Based on these results, it was judged that brown gourd extract had a moisturizing effect on the skin.

(5) Histological observation of skin

In order to confirm the antiwrinkle effect, skin tissue of each experimental group was extracted, fixed in 10% neutral formalin solution, washed with water, dehydrated, transparent, and infiltrated, embedded with paraffin, sections were made into 4μm thickness, and H & E (Hematoxylin & Eosin) staining was performed. The thickness from the keratin layer of the tissue subjected to H & E staining to the epidermal basement membrane was measured using a microscope mounted on a microscope.

As a result, as shown in FIG. 6, when the epidermal thickness increased by ultraviolet irradiation was administered the brown gourd extract of the present invention, the epidermal thickness was decreased, and from the H & E staining results, the UV irradiation group was a normal group. Compared to (Nor), the stratum corneum occurred, it was confirmed that the epidermal thickness also increased. The brown gourd extract (GGR) administration group was confirmed that the stratum corneum was alleviated and the epidermal thickness was also reduced compared to the ultraviolet irradiation group (Fig. 7).

(6) Confirmation of change in expression level of MMP-1 or MMP-9 protein

The MMP-1 or MMP-9 protein is a protein associated with skin aging and is known to be increased by oxidative stress such as ultraviolet light. Therefore, in this experiment, to determine the inhibitory effect of MMP-1,9 induced from oxidative damage, The skin tissue was extracted after oral administration of brown rice bran extract (GGR), and the MMP-1 or MMP-9 was measured using Quantikine ELISA pro MMP-1 or MMP-9 kit (R & D systems, Minneapolis, MN, USA). It was.

As a result, the amount of expression of MMP-1 and MMP-9 by ultraviolet irradiation was increased compared to the cells not irradiated with ultraviolet rays, and it was confirmed that the amount of expression decreases when the brown goose extract was administered (FIG. 8).

(7) Electrophoresis and Western blotting

The protein was extracted and pulverized by adding a solution extracting protein to 20-30 mg of skin tissue, and the same amount of protein was electrophoresed to a PVDF membrane, and then reacted with an antibody to measure the expression level of the protein.

As shown in FIG. 9, the expression levels of pERK, pMEK and pp38 proteins, known as proteins that play an important role in wrinkle formation in the skin, were increased in the UV irradiation group compared with the group not irradiated, and in the brown worm extract (GGR) administration group. It was confirmed that the expression levels of pERK, pMEK and pp38 proteins decreased.

Statistical method

The results obtained in the present invention were tested for statistical significance between the control group and the experimental group using one-way ANOVA and Tukey's multiple comparison analysis.

Claims (5)

Health functional food composition for improving skin wrinkles or moisturizing skin containing brown edible water or ethanol extract as an active ingredient. delete The method according to claim 1, wherein the brown mealy water or ethanol extract is a functional food composition for skin wrinkle improvement or skin moisturizing, characterized in that to reduce the expression amount of MMP-1 or MMP-9 protein. The method according to claim 1, wherein the brown mealy water or ethanol extract is a functional food composition for skin wrinkle improvement or skin moisturizing, characterized in that to increase the content of hyaluronic acid. A pharmaceutical composition for the prevention or treatment of skin wrinkles, which contains brown skin water or ethanol extract as an active ingredient.

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