KR102070801B1 - Composition for improving skin wrinkle or skin moisturizing comprising Allomyrina dichotoma larva extract as effective component - Google Patents

Abstract

The present invention relates to a composition for improving skin wrinkles or skin moisturizing containing longevity extract as an active ingredient, in particular, the longevity extract has little cytotoxicity, as well as improving the content of hyaluronic acid that can enhance skin moisturizing Rather, it is to reduce the amount of transdermal moisture loss increased by ultraviolet rays, to increase the amount of skin moisturizing, to reduce the expression of MMP-1 and MMP-9, and to control the expression of wrinkle-related proteins. 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 improving skin wrinkle or skin moisturizing comprising Allomyrina dichotoma larva extract as effective component}

The present invention ( Allomyrina) dichotoma It relates to a composition for improving skin wrinkles or moisturizing skin containing larva) 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, longevity is a beetle larvae, Allomyrina dichotoma ) is an insect belonging to the Coleoptera, Family Dynastidae.

As folk remedies have been known to be effective for liver disease, slugs, the larvae of beetles, are increasingly being used as health supplements. Proteins isolated from beetles show strong inhibitory activity against bacteria, and lectins extracted from beetles show strong anticancer activity and can be expected to have anticancer effects. In addition, hepatoprotective effects have been reported in rats inducing hepatotoxicity with carbon tetrachloride. However, the research data on its physiological function is very insufficient compared to the wild beetle and its larvae in folk remedies.

As an example of the longevity (long beetle larvae) related technology known to date, Korean Patent No. 1702046 discloses a liver protection and anticancer composition containing an active beetle larvae or a fraction thereof as an active ingredient, Korean Patent No. 1533600 A method for preparing a beetle larvae extract having an anti-obesity effect and an anti-obesity composition comprising the same have been disclosed. However, the composition for improving skin wrinkles or skin moisturizing containing the long-life extract of the present invention as an active ingredient is disclosed. none.

The present invention is derived from the above requirements, the present invention provides a composition for improving skin wrinkles or moisturizing skin containing the extract of longevity love as an active ingredient, collagen in which the active ingredient is involved in the decomposition of the extracellular matrix and the basement membrane Reducing the expression level of MMP-1, a matrix metalloproteinase (MMPs), and enhancing the content of hyaluronic acid to prevent skin moisturizing function and skin barrier damage, and reduced transdermal moisture loss using animal models. The present invention was completed by confirming that there is an effect of controlling the amount of skin moisturizing, the amount of MMP-1 and MMP-9 and the amount of protein 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 the extract as an active ingredient.

The present invention also provides a pharmaceutical composition for the prevention or treatment of skin wrinkles containing the extract of Jangsuaeae as an active ingredient.

The present invention relates to a skin wrinkle improvement or skin moisturizing composition containing the extract of Jangaeae Ae or a compound separated therefrom as an active ingredient, the active ingredient of the present invention has little cytotoxicity, and is effective in improving skin wrinkles and moisturizing skin. It is. 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 a 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.
2 is a cell protection effect of HaCaT cells of the longevity extract of the present invention against the cell damage caused by UVB irradiation, each sample for 24 hours in HaCaT cells (human keratinocytes) by concentration (10, 50) , 100 and 200 μg / ml), UV irradiation with UV Crosslinker (UltR5 Lum) for 30 minutes, and MTS assay. # Indicates that the cell survival rate of the UVB irradiated group was statistically significantly lower than that of the Control group, which means p <0.05, and * indicates that the cell viability of the experimental group treated with longevity extract and UVB irradiated on the cell viability of the UVB irradiated group. The statistically significant increase in comparison means p <0.05.
Figure 3 is a result confirming the change in the expression amount of MMP-1 according to the treatment of Jangsuae extract of the present invention. # Indicates that the MMP-1 expression level of the UVB irradiated group was significantly increased compared to the control group, which means p <0.05, and * indicates that the MMP-1 expression level of the experimental group treated with longevity extract and UVB irradiated UVB irradiated group It was statistically significant compared to the MMP-1 expression of the mean, p <0.05.
Figure 4 is a result of analyzing the change in the content of hyaluronic acid (HA) according to the UV irradiation and the treatment according to the concentration of Jangsuae extract of the present invention. Control is a normal group that does not process anything, UVB is a control group irradiated with ultraviolet light only without treating the extract of Jangsuae of the present invention. # Indicates that the hyaluronic acid content of 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 Jangsuae extract and UVB irradiated was higher than that of the UVB irradiated group. Statistically significant increase means p <0.05.
5 is a result of confirming the transdermal moisture loss amount (TEWL) of the longevity extract administration group of the present invention. Nor is a normal group not irradiated with ultraviolet rays, Veh is a group that induced transdermal moisture loss by irradiating ultraviolet rays, JSA is a group to which the UVA (UVB) and the long-life extract of the present invention was administered. #### is a statistically significant increase in the transdermal moisture loss of the ultraviolet irradiation group compared to the normal group, p <0.0001, **** is a transdermal moisture loss of the longevity extract administration group of the present invention compared to the ultraviolet irradiation group This statistically significant decrease is p <0.0001.
6 is a result of confirming the skin moisture content of the longevity extract administration group of the present invention. Nor is a normal group not irradiated with ultraviolet rays, Veh is an ultraviolet irradiated group, and JSA is a group administered with ultraviolet irradiation and the longevity extract of the present invention. ### is a statistically significant decrease in the skin moisture content of the UV irradiation group compared to the normal group, p <0.001, * is a statistically significant increase in the skin moisture content of the longevity extract administration group of the present invention compared to the UV irradiation group. P <0.05.
Figure 7 is a result of confirming the wrinkle improvement effect through the H & E staining of tissue collected from the hairless mouse group administered the longevity extract of the present invention. Normal is a normal group not irradiated with ultraviolet rays, Vehicle is a group irradiated with ultraviolet rays, JSA is a group administered with ultraviolet irradiation and longevity extract of the present invention.
8 is a result of confirming the wrinkle improvement effect through Masson's trichrome staining of tissue collected from the hairless mouse group administered the longevity extract of the present invention. Normal is a normal group not irradiated with ultraviolet rays, Vehicle is a group irradiated with ultraviolet rays, JSA is a group administered with ultraviolet irradiation and longevity extract of the present invention.
9 is a result of confirming the change in the expression amount of MMP-1 and MMP-9 protein according to the administration of longevity extract of the present invention. Nor is a normal group not irradiated with ultraviolet rays, Veh is a group irradiated with ultraviolet rays, and JSA is a group administered with ultraviolet irradiation (UVB) and the longevity 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 <0.0001, **** is compared to the UV irradiation group The expression levels of MMP-1 and MMP-9 proteins in the longevity extract administration group were statistically significant, p <0.0001.
10 is a result of confirming the change in the expression level of pERK, pMEK, pJNK and pp38 protein according to the administration of longevity extract of the present invention.

The present invention relates to a functional skin composition for improving skin wrinkles or moisturizing the skin containing the extract as an active ingredient.

The Jangsuae 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 long-life dry 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 longevity, 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 extract of longevity can reduce the expression level of MMP-1 and MMP-9 proteins 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.

The present invention also relates to a pharmaceutical composition for the prevention or treatment of skin wrinkles containing the extract of Jangsuaeae 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  One. Longevity  Preparation of Extract

It was refluxed for 3 hours using 70% ethanol in long dry powder. A 70% ethanol extract was obtained and distilled under reduced pressure to prepare a Jangsuae ethanol extract.

Example  2. Longevity  Analysis of Cell Viability by Treatment of Extracts

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 extract of longevity of the present invention, there was almost no change in cell viability (%) relative to the control group treated with nothing, the extract of longevity of the present invention showed no cytotoxicity Confirmed.

Example  3. Confirmation of cell protection effect by UV irradiation

After diluting each sample with concentration for 24 hours in HaCaT cells and irradiating with UV crosslinker (Ultra Lum) for 30 minutes, MTS assay (CellTiter Aqueous One Solution Cell proliferation assay kit, 3- (4,5-) Dimethylthiazol-2-yl) -5- (3-carboxymethoxyphenyl) -2- (4-sulfophynyl) -2H-tetrazolium, inner salt; MTS, Promega Co. Madison, WI, USA) technique for cell viability at 490 nm Test by measuring with a reader (Molecular Devices, Sunnyvale, CA, USA).

As a result, as shown in FIG. 2, the cell survival rate (%) was confirmed to be reduced by about 50% by UVB irradiation. After UV irradiation, the cell survival rate (%) was increased when the longevity extract of the present invention was treated. It was confirmed that the increase in dependence was determined that the longevity extract of the present invention has a cytoprotective effect.

Example  4. 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 longevity extract of the present invention was treated by concentration (10, 50, 100 and 200 µg / ml), respectively. Thereafter, ultraviolet (UV) irradiation, 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. 3, it was confirmed that the expression level of MMP-1 was remarkably increased in the group irradiated with ultraviolet rays, compared to the normal group not irradiated with ultraviolet rays. It was confirmed that the expression level of P-1 was significantly reduced in concentration-dependently compared to the ultraviolet irradiation group.

Example  5. 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 Figure 4, by the ultraviolet irradiation, the content of hyaluronic acid was reduced, but the content of hyaluronic acid was increased in each experimental group treated with 100 and 200 ㎍ / ㎖ long-life extract of the present invention It was confirmed (FIG. 4).

Example  6. 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 fed free feed of 5L79 (Charles river, USA).

Sample administration was performed by dividing into the normal group (Nor), ultraviolet treatment group (UVB + Veh), ultraviolet irradiation and longevity extract (UVB + JSA) 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. 5, it was confirmed that the skin barrier was damaged by UV irradiation, and the skin moisturizing function was decreased. In the case of the longevity extract administration group, the skin moisture loss caused by UV was statistically significantly prevented. It was confirmed.

(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. 6, the UV irradiated group (Veh) decreased the skin moisture content compared to the normal group (Nor) which did not irradiate the UV light, and the longevity extract administration group (JSA) had a skin moisture content compared to the UV irradiated group. It was high. Based on these results, it was determined that Jangsuae extract has 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 and fixed in 10% neutral formalin solution, washed with water, dehydrated, transparent, infiltrated, embedded with paraffin, sections were made into 4μm thickness, and H & E (Hematoxylin & Eosin) staining and Masson's trichome staining were 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. 7, the JSA administration group was alleviated the stratum corneum compared to the UV irradiation group, and it was confirmed that the collagen was decreased by UV from Masson's trichome staining results. Confirmed recovery (Fig. 8).

(7) Confirmation of MMP-1 or MMP-9 Expression Change

MMP-1 or MMP-9 is a protein associated with skin aging and is known to be increased by oxidative stress such as ultraviolet rays. Therefore, extract of longevity extract to measure the inhibitory effect of MMP-1 or MMP-9 derived from oxidative damage After oral administration of (JSA), skin tissues were extracted, and MMP-1 or MMP-9 was measured using Quantikine ELISA pro MMP-1 or MMP-9 kit (R & D systems, Minneapolis, MN, USA).

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 when the longevity extract was administered, it was confirmed that the amount of expression decreased (Fig. 9).

(6) 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. 10, the expression levels of pERK, pMEK, pJNK, and pp38 proteins, known as proteins that play an important role in wrinkle formation in the skin, were increased in the UV-irradiated group, compared to the unirradiated group. In it was confirmed that the expression level of pERK, pMEK, pJNK and pp38 protein is reduced.

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 skin wrinkle improvement or skin moisturizing containing longevity water or ethanol extract as an active ingredient. delete According to claim 1, wherein the longevity water or ethanol extract is a skin functional improvement or skin moisturizing health functional food composition, characterized in that for reducing the expression of MMP-1 and MMP-9 protein. According to claim 1, wherein the longevity 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. Pharmaceutical composition for the prevention or treatment of skin wrinkles containing longevity water or ethanol extract as an active ingredient.

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