WO2017195951A1 - Cosmetic and nutritional supplement using aptamer, and method for producing same - Google Patents

Abstract

The present invention relates to a cosmetic and nutritional supplement using an aptamer and a method for producing same. More specifically, the aptamer according to the present invention has the effect of preventing the oxidation of an antioxidant, such as vitamin C. The composite of an aptamer and an antioxidant according to the present invention uses an aptamer selectively binding to vitamin C (ascorbic acid) for instance, so as to maintain the reduced state of vitamin C and thereby maintain the antioxidation function thereof for a long period of time, and thus can be used in functional cosmetics of various formulations, oral nutritional supplements (dietary supplements), and the like.

Description

Cosmetics and Nutritional Aids Using Aptamers and Manufacturing Method Thereof

The present invention relates to cosmetics and nutritional supplements using aptamers and to a method of manufacturing the same, and more particularly, to cosmetics and nutritional supplements using long-term maintenance and antioxidant properties of antioxidants such as reduced vitamin C by aptamers. Is to apply.

There are many causes of aging, but the active oxygen species (ROS) are considered to be one of the major causes. This active oxygen is indispensable in energy metabolism, immune response, etc., and is an unavoidable stimulus caused by external harmful environment. Free radicals are highly reactive and cause DNA denaturation, excessive signaling, and protein denaturation in the body, causing a series of reactions that accumulate harmful effects. However, these harmful reactions are elaborately maintained by antioxidants (uric acid, vit.C, vit.E, etc.) or antioxidant enzymes (Glutathione peroxidase, superoxide dismutase, catalse, etc.) present in vivo. However, the deterioration of antioxidant system due to endogenous aging and the accumulation of free radicals caused by persistent harmful stimuli breaks this balance, impairs health, promotes aging, and causes various diseases such as skin diseases, skin cancer, arteriosclerosis and blood clots. (Laure Rittie et al., Ageing Research Reviews, 1, 705-720, 2002; Cutler RG, Annals of the New York Academy of Sciences, 1055, 93-135, 2005).

Therefore, there is an increasing interest in antioxidants that inhibit the formation of active oxygen systems or remove the formed active oxygen systems. Antioxidants can be divided into those that exist naturally in the human body and those that are administered externally.

Antioxidants that are naturally present in the human body include enzymes such as superoxide dismutase (SOD), glutathione, peroxidase and catalase. phytochemicals such as kaempferol, catechin and genistein; Vitamin E, vitamin C and beta carotene; And minerals such as selenium.

Vitamin C (L-ascorbic acid, ascorbate) is an essential influencer for humans and animals. It is important for collagen synthesis and is a cofactor for at least eight enzymes. Therefore, vitamin C deficiency will cause diseases such as scurvy (Scurvy). Vitamin C in animals is an important factor in preventing wound healing and capillary bleeding, and is an important antioxidant in the body along with Vitamin A and Vitamin E.

Cells are attacked by free radicals and oxygen free radicals generated by UVA (UVA) and ultraviolet B (UVB), pollutants, stress, smoking, drinking, fatty foods, etc. In the absence of proper protection from these substances, cells may age or die. In the case of skin, these substances reduce or degenerate the production of collagen and elastin, causing the skin to lose elasticity and wrinkles. In order to prevent this, it is known that it is important to prevent the aging of the skin by preventing the oxidation by the harmful active substances by applying the formulation containing antioxidants such as vitamin A, C, E to the skin, absorbed into the skin. However, due to the problem that the synthetic vitamin C is easily oxidized in the air and its antioxidant action disappears, there is a problem in preparing a variety of formulations with a long shelf life.

[Previous Patent Document]

Republic of Korea Patent Publication No. 10-2015-0079177

The present invention has been made in view of the above necessity, and an object of the present invention is to provide a substance which acts as an antioxidant for antioxidant substances.

It is another object of the present invention to provide a cosmetic and nutritional supplement for a substance that acts as an antioxidant for antioxidants.

In order to achieve the above object, the present invention provides an aptamer for preventing oxidation of an antioxidant substance.

The present invention also provides an aptamer and an antioxidant complex obtained by combining an antioxidant with the aptamer of the present invention.

In one embodiment of the invention, the antioxidant is preferably a substance selected from the group consisting of vitamin C, vitamin A, retinol, vitamin E, astaxanthin, resveratinol, polyphenols, coenzyme Q10, peptides and oils. One is not limited thereto.

In another aspect, the present invention provides a cosmetic composition comprising the aptamer and antioxidant complex of the present invention as an active ingredient.

In one embodiment of the present invention, the cosmetic composition preferably further comprises one or more of collagen, elastin, hyaluronic acid, and a peptide, but is not limited thereto.

In another embodiment of the present invention, the cosmetic composition is to release the bound antioxidant (eg, vitamin C) when the structure of the aptamer changes according to the ultraviolet irradiation of the skin, or ATP according to the change in the temperature or acidity of the skin It is preferable to release vitamin C as the structure of the aptamer which binds thereto when the amount is changed, but is not limited thereto.

The cosmetic composition of the present invention can use any raw material used in cosmetics regardless of any kind of extracts, active substances. For example, green tea extract, licorice extract, medicinal extract, lettuce extract, golden extract, pueraria extract, red ginseng extract and apricot extract, oil extract, orange extract, lemon extract, bamboo extract guava extract Rosemary Extract Cornus Extract, Ganoderma Lucidum Extract Ginkgo Extract Seoxiok-Yuksan Extract Consonant Extract, Moisturizing Chinese Quince Extract, White Lotus Extract, Paprika Extract, Aloe Extract, Scrubber Extract, Seaweed Extract, Antioxidant Carrot Extract, Soybean Extract, Grapefruit Extract Grape Seed Extract, Portulaca Extract, Caviar to Improve Wrinkles, Pomegranate, Ginseng Extract, Peach Extract for Skin Regeneration, Cheongung Extract, Centella asiatic Extract, Chamomile Extract, Licorice Extract, Ginseng Extract, Angelica Extract, Acne Good peppermint extract, triticale extract, eosungcho extract , Peony extracts, anti-inflammatory and antibacterial herb extracts, dandelion extracts, calendula extracts, yellow white skin extracts, tanza extracts, golden extracts, fennel extracts, extract extracts, tea extracts, green tea extracts, moisturizing functions Glycerin, Panthenol, Hyaluronic Acid, Ceramide, Beta-Glucan, Whitening Arbutin, Vitamin C, Preference, Retinol, Astaxanthin, Resveratinol, Polyphenols, Elastic Elastin, Collagen, Coenzyme Q10, Epectin, EGF Anti-inflammatory antibacterial propolis, allantoin, phytostane, infra acid, antioxidant vitamin E (natural tocopherol) ROE (rose marioyl extract), grapefruit seed extract is applied.

In another aspect, the present invention provides a nutritional supplement composition comprising the aptamer and antioxidant complex of the present invention as an active ingredient.

Examples of suitable vitamins for the nutritional supplements of the present invention include vitamin A, vitamin C, vitamin D, vitamin E, vitamin K, vitamin B 6, vitamin B 12, thiamine, riboflavin, biotin, folic acid, niacin, pantothenic acid, mixtures thereof, and the like. to be. Examples of suitable mineral nutrients to be included in the dietary supplement composition are selected from sodium, potassium, calcium, magnesium, phosphorus, sulfur, chlorine, iron, copper, iodine, zinc, selenium, manganese, chromium, molybdenum, fluorine, cobalt, and compounds thereof They hold more than one element.

Various herbs can also be used as nutritional supplements. In general, herbs are selected from those having various medicinal or dietary supplement properties. Generally, herbs are aromatic plants or parts of plants that can be used for medicinal or flavoring purposes.

In another aspect, the present invention provides a method for binding an aptamer to an antioxidant to maintain the reduced state of the antioxidant and prevent oxidation.

Hereinafter, the present invention will be described.

The present invention, like Vitamin C, vitamin A (Retinol), Vitamin E, Astaxanthin, Resveratinol, Polyphenol, Coenzyme Q10, Peptide, Oil, etc. Can be used to manufacture. This prevents the oxidation (corrosion) of the materials to ensure that they maintain the maximum effect they require. In addition, by releasing it under a target condition through apta-sensing, the effect can be increased as much as possible.

Maintaining the reduced state of vitamin C Aptamer

Aptamer is a method of detecting a specific substance by using the three-dimensional structure of single strand DNA or RNA, which is similar to an antigen-antibody reaction, but has a much smaller substance, and can be controlled in various ways. This is an easy advantage. In addition, unlike antibodies, it is possible to synthesize aptamers that bind to very small chemicals (vitamins) and the like.

Vitamin C is a water-soluble six carbon compound, which includes the 3-, 4- and 3-hydrocarbons of the furan ring and the semidehydroascorbic acid and dehydroascorbic acid, which are oxidized in the reduced form and biparticulate, respectively.

As can be seen in Figure 1 through the hydrogen bonding of the hydroxyl group of the base and vitamin C constituting the aptamer (RNA or DNA) to maintain the reduced state of Vitamin C.

With aptamers  Various formulations using vitamin C in combination

Vitamin C, which is combined with aptamers and maintains a reduced state, may be used in skin care compositions and nutritional supplements of various types of cream or hydrogel types including collagen, elastin, hyaluronic acid and peptides.

Control of Vitamin C Release According to Skin Condition or External Stimulation

The present invention also includes a method of slowly releasing Vitamin C according to various conditions of the skin (Aptasensing) through aptamers that react differently depending on the condition of the skin or external stimuli (eg ultraviolet rays or the temperature or acidity of the skin). For example, when the structure of the aptamer is changed by UV irradiation of the skin, the bound antioxidant (e.g., vitamin C) is released or the structure of the aptamer that binds when the amount of ATP changes according to the temperature or acidity of the skin is changed. And a method of releasing Vitamin C as a substitute.

As can be seen through the present invention, the aptamer of the present invention has an antioxidant effect of an antioxidant substance such as vitamin C, and the complex of the aptamer and antioxidant of the present invention may be, for example, contained in Vitamin C (ascorbic acid). Aptamers that selectively bind to maintain the reduced state of Vitamin C to maintain its antioxidant function for a long time can be used in functional cosmetics and oral dietary supplements of various formulations.

1 is a diagram of maintaining the reduced state of vitamin C through hydrogen bonding of the hydroxyl group of the base and vitamin C constituting the aptamer (RNA or DNA),

Figures 2 to 4 show that the three aptamers of the present invention prevent vitamin C oxidation by hydrogen peroxide solution,

5 to 10 are graphs of dissociation constants (KD) for AA (ascorbic acid) and DHA (dehydroascorbic acid) of the aptamer of the present invention.

Hereinafter, the present invention will be described in detail through non-limiting examples. However, the following examples are intended to illustrate the invention and the scope of the present invention is not to be construed as limited by the following examples.

Example  1: bound to reduced vitamin C Aptamer  Military building

SELEX (systematic evolution of ligands by exponential enrichment) was performed to find aptamers that selectively bind to reduced ascorbic acid from 10 ¹³ DNA aptamer libraries under the following conditions. In order to proceed with the SELEX while maintaining the reduced state of ascorbic acid, glutathione was added while maintaining the pH around 5.5.

Under this condition, more than 99% of vitamin C remained in reduced L-ascorbic acid state, not oxidized dehydro ascorbic acid (DHA). Under the above reaction conditions, SELEX was carried out and the entire selected aptamer was subjected to Next Generation Sequencing and analyzed. As a result, an aptamer composed of more than 3000 secondary structures was obtained.

Example  2: Aptamer  Quantitative Analysis of Antioxidant Vitamin C

According to the type of secondary structure, 20 individual aptamers were selected and subjected to the anti-oxidation experiment of vitamin C. After heating the aptamer dissolved in the annealing buffer to 95 ^o C and slowly lowering the temperature to room temperature, the secondary structure of the aptamer is mixed. The reaction was carried out for about 30 minutes. After oxidizing conditions by adding hydrogen peroxide solution, oxidation of L-ascorbic acid was measured by adding OPDA (o-phenylenediamine) as a fluorescent dye. DHA, the oxide of L-ascorbic acid, can be quantitatively analyzed by measuring the amount of fluorescence from DHA-OPDA produced by reaction with OPDA. Under the above conditions, the amount of fluorescence of DHA-OPDA was measured every 34 seconds for 25 minutes.

All three aptamers prevented vitamin C oxidation by hydrogen peroxide. Aptamer 12 prevented about 40% oxidation, 8 times about 20%, and 10 times about 40%. Based on these experiments and other experiences, it can be concluded that the three aptamers respond directly to vitamin C, preventing the oxidation of vitamin C (see FIGS. 2-4).

Example  3: of the present invention Aptamer  ascorbic acid (AA) and dehydroascorbic  acid ( DHA Steady-state solution dissociation constant (KD)

Dissociation constants were determined using microscale thermophoresis (MST).

This example includes the MST data and the calculated KD for the three aptamers in the assay buffer for both targets. All reagents containing ascorbic acid and dehydroascorbic acid were purchased from Sigma-Aldrich (St. Louis, MO) and deionized water was treated with Chelex-100 resin for 1 hour to remove accidental metals prior to buffer preparation. After Chelex treatment, the water was sprayed with nitrogen gas for 10 minutes to minimize oxygen and then sealed and stored. This water was used for all aqueous solutions. Final buffer is 50 mM sodium acetate, pH 5.5, 1 mM MgCl 2, and 0.05% Tween-20, both AA and DHA were 153 pM (final) at e5 uM in buffer (for aptamers # 10 &# 12) Or at 1: 1 passage dilution at 1.53 nM (final) (Aptamer # 8) at 50 uM. The final concentration of each Cy5-conjugated aptamer is 20 nM.

Each technical double dilution was measured twice on a Monolith NT.115 MST instrument from NanoTemper Technologies GmbH (Munich, Germany).

The results are shown in Figures 5 to 10. Each data point is shown in the figure with an average and a fitted curve (black line). Vertical dashed lines in each graph represent KD.

The following conclusions were inferred from the above results.

1) Aptamers # 10 & 12 have better selectivity for AA vs DHA, while # 8 has slightly better selectivity than AA for DHA.

2) Aptamer # 10 Three aptamers have the best selectivity for AA vs DHA.

3) Aptamer # 12 was best for protection of AA from oxidation but had minimal selectivity of AA vs DHA.

Claims (8)

Aptamers to prevent oxidation of antioxidants. The method according to claim 1, wherein the antioxidant substance is selected from the group consisting of vitamin C, vitamin A, retinol, vitamin E, astaxanthin, resveratinol, polyphenol, coenzyme Q10, peptide and oil. Tamer. An aptamer and an antioxidant complex obtained by combining an antioxidant with the aptamer of claim 1. The method of claim 3, wherein the antioxidant substance is selected from the group consisting of vitamin C, vitamin A, retinol, vitamin E, astaxanthin, resveratinol, polyphenols, coenzyme Q10, peptides and oils. Tamer and Antioxidant Complex. Cosmetic composition comprising the aptamer and antioxidant complex of claim 3 or 4 as an active ingredient. The cosmetic composition of claim 5, wherein the cosmetic composition further comprises one or more of collagen, elastin, hyaluronic acid, and a peptide. The aptamer of claim 5, wherein the cosmetic composition releases bound antioxidants when the structure of the aptamer changes according to ultraviolet irradiation of the skin, or binds to the ATP when the amount of ATP changes according to a change in skin temperature or acidity. Cosmetic composition, characterized in that to release the antioxidant substance by changing the structure of. Nutritional supplement composition comprising the aptamer and antioxidant complex of claim 3 as an active ingredient.

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