KR102237543B1 - Method of Producing DNA Derived from Aloe Genus Plant, and Composition Containing DNA Derived from Aloe Genus Plant for Anti-Aging and Anti-Inflammation - Google Patents

Abstract

The present invention relates to a method for preparing DNA from plants of the genus Aloe, and an anti-aging and anti-inflammatory composition comprising DNA from plants of the genus Aloe as an active ingredient, and in more detail, obtained after treating a plant of the genus Aloe with a hydrolase. A method for effectively extracting the DNA of the Aloe plant by adjusting the pH of the lysate formed by mixing a hydrolyzate with a surfactant and deep ocean water extraction minerals, and then precipitating it with an organic solvent, and a plant of the genus Aloe prepared by the above method. It relates to an anti-aging and anti-inflammatory composition comprising DNA as an active ingredient.

Description

A method of preparing DNA derived from plants of the genus Aloe, and a composition for anti-aging and anti-inflammatory containing DNA derived from plants of the genus Aloe as an active ingredient TECHNICAL FIELD [Method of Producing DNA Derived from Aloe Genus Plant, and Composition Containing DNA Derived from Aloe Genus Plant for Anti -Aging and Anti-Inflammation}

The present invention relates to a method for preparing DNA from plants of the genus Aloe, and an anti-aging and anti-inflammatory composition comprising DNA from plants of the genus Aloe as an active ingredient, and in more detail, obtained after treating a plant of the genus Aloe with a hydrolase. A method for effectively extracting the DNA of the Aloe plant by adjusting the pH of the lysate formed by mixing a hydrolyzate with a surfactant and deep ocean water extraction minerals, and then precipitating it with an organic solvent, and a plant of the genus Aloe prepared by the above method. It relates to an anti-aging and anti-inflammatory composition comprising DNA as an active ingredient.

DNA is a polymer in which a polymer of nucleotide monomers composed of deoxyribose, phosphoric acid, and bases (adenine, thymine, guanine, and cytosine) is double-bonded according to the complementarity of the base structure, and contains the genetic information of all living organisms.

Since DNA has the effect of activating the adenosine A2 receptor in cell membranes, it is used in medicines for the purpose of treating and improving wounds and scars, skin regeneration, degenerative arthritis, and ligament damage. However, plant cells, which are the sources of DNA, have a cell wall structure composed of cellulose outside the cell membrane, which makes it difficult to extract DNA from cells, so its use has been limited.

In the case of separating DNA from microbial and animal cells, since the microbial and animal cells are cell membranes composed of phospholipids, the separation and purification process can be easily extracted only by chemical reactions using organic solvents such as phenol and chloroform without physical pulverization. I did.

On the other hand, in the case of plants, cell lysis does not occur only with a simple chemical reaction due to the cell wall composed of cellulose, and the plants must be pulverized by using physical and chemical methods sequentially or simultaneously, such a multi-stage extraction method. There is a disadvantage in that the DNA yield is low.

Although a method for preparing a mixture containing DNA having a specific molecular weight range, that is, 3,000 to 10,000 kDa, from animal raw materials (eg, testis of fish) is known (see Patent Document 1 below), it has a strong cell wall. There have been no reports of methods of extracting DNA from plants, particularly plants of the genus Aloe, in high yield, and methods of producing DNA fragments having a molecular weight of about 0.5 to 11 kDa by remarkably reducing the molecular weight of the extracted DNA.

On the other hand, there are a number of prior art for a composition for external application for skin containing an extract prepared using a plant of the genus Aloe as a raw material (see Patent Document 2-3 below).

However, in the past, extracts of the genus Aloe plant have been simply added to skin-applied products, and in fact, there has been no case of identifying DNA derived from plants of the genus Aloe that has particularly high activity and great industrial utility. Under the background, the present inventors have made diligent efforts to develop a composition for anti-aging and anti-inflammatory containing DNA from plants of the genus Aloe as an active ingredient, and as a result, DNA from plants of the genus Aloe prepared using minerals extracted from deep ocean water, in particular about 0.5~ Aloe plant-derived DNA having a molecular weight of 11 kDa and a composition containing the same inhibits the expression of collagen-degrading enzyme in skin cells to prevent skin aging (improving skin wrinkles and improving skin elasticity), and has an antioxidant effect by eliminating ROS, and It was confirmed that the effect of relieving skin inflammation was excellent, and the present invention was completed.

Korean Patent Application Publication No. 10-2013-0044195 (May 2, 2013) Korean Patent Application Publication No. 10-2015-0104754 (September 16, 2015) Korean Patent Application Publication No. 10-2009-0110727 (October 22, 2009)

An object of the present invention is a method for producing DNA derived from plants of the genus Aloe having excellent anti-aging and anti-inflammatory activity, DNA from plants of the genus Aloe prepared by the above method, and for anti-aging and anti-inflammatory including DNA from plants of the genus Aloe It is to provide a composition.

In order to achieve the above object, the present invention,

(a) adding purified water to plants of the genus Aloe, homogenizing, and treating a hydrolyzate to produce a hydrolyzate;

(b) adding and dissolving a surfactant and minerals extracted from deep sea water to the hydrolyzate produced in step (a) to generate a dissolved product;

(c) adjusting the pH of the lysate produced in step (b); And

(d) obtaining a DNA precipitate by adding an organic solvent to the pH-adjusted lysate obtained in step (c).

The present invention also provides a DNA derived from a genus Aloe plant prepared by the method for producing DNA derived from a genus Aloe plant.

The present invention also provides an anti-aging composition containing the DNA derived from a plant of the genus Aloe as an active ingredient, preferably a composition for external application for skin for improving skin wrinkles and skin elasticity having a pharmaceutical or cosmetic use, most preferably skin wrinkles It provides a cosmetic composition for improving and improving skin elasticity.

The present invention also provides an anti-inflammatory composition containing DNA derived from a plant of the genus Aloe as an active ingredient, preferably an anti-inflammatory skin external preparation composition for pharmaceutical or cosmetic use, and most preferably an anti-inflammatory cosmetic composition. to provide.

The composition containing the plant DNA of the genus Aloe produced by the method for producing DNA derived from a plant of the genus Aloe according to the present invention as an active ingredient suppresses the expression of collagen-degrading enzyme in skin cells to improve skin elasticity and improve skin wrinkles, It can be manufactured and applied as a drug or cosmetics having an antioxidant effect and an anti-inflammatory effect that suppresses the expression of inflammatory cytokines.

1 is a photograph showing the size of DNA derived from Aloe vera according to Example 1 of the present invention on an electrophoresis gel.
2 is a graph showing the effect of inhibiting the expression of MMP-1, a collagen-degrading enzyme in skin cells, by Aloe vera-derived DNA according to Example 1 of the present invention.
3 is a graph showing the effect of inhibiting the expression of IL-6, an inflammatory cytokine in skin cells, by Aloe vera-derived DNA according to Example 1 of the present invention.
Figure 4 is a graph showing the antioxidant effect (ROS removal effect) by Aloe vera-derived DNA according to Example 1 of the present invention.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by an expert skilled in the art to which the present invention belongs. In general, the nomenclature used in this specification is well known and commonly used in the art.

Hereinafter, the present invention will be described in more detail.

In this specification, the term "about" used to express volume, area, length, time (period), concentration, capacity (content, etc.), temperature, pH, etc. has a tolerance of up to 10% in the corresponding value or numerical range. Means to do.

In one aspect, the present invention is a method for producing DNA derived from plants of the genus Aloe,

(a) adding purified water to plants of the genus Aloe, homogenizing, and treating a hydrolyzate to produce a hydrolyzate;

(b) adding and dissolving a surfactant and minerals extracted from deep sea water to the hydrolyzate produced in step (a) to generate a dissolved product;

(c) adjusting the pH of the lysate produced in step (b); And

(d) obtaining a DNA precipitate by adding an organic solvent to the pH-adjusted lysate obtained in step (c).

The plant of the genus Aloe in step (a) is Aloe Barbadensis ( Aloe Vera ) , Aloe Aculeata ( Aloe aculeata ), Aloe Africana ( Aloe africana) , Aloe albida (Aloe albida), Aloe Albiflora. (Aloe albiflora), aloe ahreubo Les kenseu (Aloe arborescens), aloe Arezzo Nicolas (Aloe arenicola), aloe are Gen tikka Uda (Aloe argenticauda), Aloe bar Kerry (Aloe bakeri), aloe balriyi (Aloe ballii), aloe balriyi ( Aloe ballyi), aloe breather non polyamic (Aloe brevifolia), aloe bromo ohmiyi (Aloe broomii), aloe portion eth- Neri (Aloe buettneri), aloe Kam Perry (Aloe camperi), aloe copy of tartar (Aloe capitata), aloe nose Simulation ( Aloe comosa), aloe kooh Perry (Aloe cooperi), aloe Coral Lina (Aloe corallina), aloe Devin Terry (Aloe dewinteri), aloe Erina Kea (Aloe erinacea), aloe X kelsa (Aloe excelsa), aloe page rocks (Aloe ferox ), aloe formate besiyi (Aloe forbesii), aloe helrenayi (Aloe helenae), aloe Herrera as the cis (Aloe hereroensis), aloe enabled Le Miss (Aloe inermis), aloe yiniang gen system (Aloe inyangensis), aloe coarseness ion (Aloe jawiyon ), Aloe jucunda , Aloe khamiesensis , Aloe kilifiensis , Aloe maculata , Aloe marlothii, Aloe mubendiensis ( Aloe mubendiensis), aloe Nami Ben sheath (Aloe namibensis), aloe Nyeri N-Sys (Aloe nyeriensis), aloe pages are soniyi (Aloe pearsonii), aloe pegeul rerayi (Aloe peglerae), aloe Pere poly-Ata (Aloe perfoliata), aloe periyi ( Aloe perryi), aloe Petri coke (Aloe petricola), aloe poly pillar (Aloe polyphylla), aloe Lau hiyi (Aloe rauhii), aloe Reynolds deusiyi (Aloe reynoldsii), aloe Scoring beanie polyamic (Aloe scobinifolia), aloe Singh Katana ( Aloe sinkatana), aloe Squash Rosa (Aloe squarrosa), aloe registry Ata (Aloe striata), aloe can coat Lena (Aloe succotrina), aloe sujannayi (Aloe suzannae), aloe Trapani seukiyi (Aloe thraskii), aloe corruption di Flora (Aloe viridiflora ) and Aloe wildii may be one or more selected from the group consisting of, and preferably, aloe barbadensis, but is not limited thereto.

The site of use of a plant of the genus Aloe used in the production of DNA derived from a plant of the genus Aloe of the present invention may be a flower, a fruit, a seed, a leaf, a stem or an outpost, preferably a leaf.

The hydrolase of step (a) is pectinase, α,β-glucosidase, amyloglucosidase, and β-glucanase. ), cellulase, naringinase, α-amylase, hemicellulase, arabinase, xylanase, and tannase. It may be one or more selected from the group consisting of, preferably cellulase, but is not limited to the above enzymes.

In the step (a), the hydrolase treatment is performed by mixing purified water in the Aloe plant in the same amount as the weight of the plant in the genus Aloe, and then homogenizing the prepared pulverized product with a hydrolase of 0.01 to 0.5 based on the weight of the pulverized product. It may be characterized in that it is made at an appropriate pH, temperature, and treatment time after adding by weight%, preferably 0.05 to 0.1% by weight.

When the hydrolase is treated, the appropriate pH is pH 3.0 to 7.0, preferably pH 3.0 to 5.0, the appropriate temperature is 20 to 60°C, preferably 25 to 55°C, and the appropriate treatment time is 1 to 5 hours, preferably It may be characterized in that it is 2 to 3 hours.

In the step (b), the lysate is heated to a temperature of 60 to 80°C, preferably 60 to 70°C for 30 to 40 minutes to inactivate the hydrolyzate contained in the hydrolyzate, and then 0.2 It may be characterized in that it is obtained by dissolving by adding and dissolving a surfactant and a mineral extracted from deep ocean water after filtering through a ~2.0μm filter, preferably a 0.45~0.8μm filter.

Deep Ocean Water used in the present invention can be used in the East Sea of Korea, the sea near Kochi Prefecture in Japan, the sea near Hawaii in the United States, preferably in Goseong, Gangwon-do, and post-treatment through processes such as sterilization and desalination. It is desirable to use one.

The deep sea water is a sterilization filter, preferably a 0.2 μm filter, after removing impurities such as microorganisms in the seawater collected, and then a conventionally known desalination method such as flash evaporation method, seawater freezing method, reverse osmosis method, ion exchange resin method. , Electrodialysis (ED) or a commercially available electrodialysis machine or demineralizer may be used to demineralize, and preferably, the electrodialysis method demineralized the mineral concentration and hardness of the desalted seawater by varying the electrical conductivity value. It is preferable because it can be adjusted. For example, desalted seawater may be prepared through a desalting process of removing monovalent cations and divalent anions by adjusting the electrical conductivity values to 8mS/cm, 10mS/cm, 12mS/cm, 15mS/cm, etc.

In the present invention, the deep sea water extraction mineral is a method of drying seawater desalted by the above-described desalination method well known in the art, for example, drying methods such as heat evaporation, vacuum evaporation, sun drying or ventilation drying method of deep sea water A mineral obtained through a functional salt or high-temperature melting deep sea water mineral, specifically, a mineral obtained by melting the deep sea water mineral at 600 to 1,000°C, preferably 700 to 900°C for 4 to 12 hours, preferably 6 to 10 hours to be.

The mineral extracted from deep sea water may contain not only sodium, calcium, magnesium, potassium, but also inorganic components such as selenium and zinc, and metal ions such as iron, manganese, and stronium, and preferably Contains sodium chloride as the main component.

In the step (b), the lysate is added to the hydrolyzate by adding a surfactant and minerals extracted from deep ocean water, and then by stirring, preferably about 10 to 30 times, and slowly inverting the hydrolyzate, the surfactant, and the ocean. After allowing the deep water extracted minerals to be evenly mixed, it may be characterized in that it is produced by standing at room temperature, preferably at a temperature of 18 to 25°C for 0.1 to 1.0 hours, preferably 0.5 to 0.8 hours.

In the step (b), the surfactant and the mineral extracted from deep sea water are mixed in a combination ratio of 0.5 to 3.0: 1.0 to 3.0, preferably 0.5 to 2.0: 1.0 to 2.0 by weight.

The content of the surfactant may be 0.5 to 3.0% by weight, preferably 0.5 to 2.0% by weight of the composition, while maintaining the combination ratio of the surfactant and the mineral extracted from deep sea water, and the content of the surfactant is 0.5 If it is less than wt%, there may be a problem of lowering the dissolution efficacy, and if it exceeds 3.0 wt%, the surfactant is not sufficiently removed, so there is a concern about safety for use in a composition applicable to the human body (for example, a composition for external application for skin). .

The content of the minerals extracted from deep sea water may be 1.0 to 3.0% by weight, preferably 1.0 to 2.0% by weight of the composition, while maintaining the combination ratio of the surfactant and the minerals extracted from deep sea water, and the minerals extracted from deep sea water If the content of is less than 1.0% by weight, the dissolution and precipitation efficiency may be deteriorated, and if it exceeds 3.0% by weight, the increase of the effect may not be significant compared to the increase in the content.

The surfactant is a negatively charged surfactant, preferably sodium dodecyl sulfate (SDS), ammonium dodecyl sulfate (ALS), sodium myreth sulfate, sodium pares sulfate. (sodium pareth sulfate), sodium laureth sulfate (SLES) and potassium lauryl sulfate (potassium lauryl sulfate) may be one or more selected from the group consisting of, preferably sodium dodecyl sulfate, It is not limited thereto.

In the step (c), the pH of the solution is firstly pH 7.0 to 13.0, preferably pH 8.0 to 12.0, most preferably pH 9.0 to 11.0, after adding a basic pH adjuster, then 40 to 80°C, preferably Is stirred at a temperature of 55 to 75° C. for 1 to 5 hours, preferably 1 to 3 hours, and then pH 6.0 to 8.0, preferably pH 6.5 to 7.5, most preferably pH 6.7 to 7.3. It can be adjusted by adding a modulator.

The basic pH adjusting agent is _{1 selected from the group consisting of ammonium hydroxide (NH 4} OH), AMP (2-amino-2-methyl-1-propanol)-95, sodium carbonate (Na ₂ CO ₃ ), and sodium hydroxide (NaOH). It may be more than one species, or a mixture thereof, preferably sodium hydroxide.

The acidic pH adjuster is from the group consisting of citric acid, lactic acid, malic acid, hydrochloric acid, acetic acid, phosphoric acid, and tartaric acid. It may be one or more selected, or a mixture thereof, preferably citric acid.

Adjusting the pH of the lysate in step (c) may be characterized by reducing the molecular weight of the DNA contained in the lysate.

The molecular weight of the DNA derived from the genus Aloe plant obtained by adjusting the pH of the lysate may be 0.1 to 400 kDa, preferably 0.5 to 100 kDa, more preferably 0.5 to 50 kDa, most preferably 0.5 to 11 kDa, and When the molecular weight is less than 0.1kDa, the desired skin elasticity improvement effect, skin wrinkle improvement effect, antioxidant effect, and anti-inflammatory effect cannot be exhibited, and when the molecular weight exceeds 400kDa, the effect may not be as great as the increase in molecular weight.

The pH-adjusted lysate in step (c) may be a supernatant of a pH-adjusted lysate obtained by centrifugation at 3,800 to 10,000 rpm or 1,500 to 15,000 x g using a centrifuge.

In the (d) step, the precipitate is based on the volume of the organic solvent in the dissolved product: organic solvent = 20 ~ 40: 60 ~ 80, preferably dissolved material: organic solvent = 25 ~ 35: 65 ~ 75 After addition, it may be characterized in that it is produced by centrifugation at 3,800 to 10,000 rpm or 1,500 to 15,000 xg using a centrifuge.

After dissolving the DNA precipitate obtained in step (d) with an aqueous solution containing an RNA degrading enzyme, RNA contained in the precipitate may be removed. The aqueous solution containing the RNA degrading enzyme may be a buffer solution (eg, 25 mM Tris-HCl pH 8.0, etc.) or purified water containing RNase A at a concentration of 1 to 20 mg/ml. It is not limited thereto.

The organic solvent may be an alcohol solvent, preferably an alcohol having 1 to 4 carbon atoms, more preferably one or more selected from the group consisting of ethanol, propanol, isopropanol, and butanol. And, most preferably, ethanol or isopropanol, but is not limited thereto.

In another aspect, the present invention is an anti-aging composition containing as an active ingredient a DNA derived from a plant of the genus Aloe produced by the method for producing DNA derived from a plant of the genus Aloe, preferably improving skin wrinkles and skin having a pharmaceutical or cosmetic use It relates to a composition for external application for skin for improving elasticity, most preferably, to a cosmetic composition for improving skin wrinkles and improving skin elasticity.

The anti-aging composition may be characterized in that it inhibits the expression of collagen-degrading enzyme in skin cells.

The anti-aging composition may be characterized in that it has antioxidant activity.

In another aspect, the present invention is an anti-inflammatory composition containing, as an active ingredient, an Aloe plant-derived DNA prepared by the method for producing DNA derived from the genus Aloe plant, preferably for anti-inflammatory use, having a pharmaceutical or cosmetic use It relates to a composition for external application for skin, most preferably an anti-inflammatory cosmetic composition.

The composition may be characterized in that it contains DNA from a plant of the genus Aloe in an amount of 0.01 to 10.0% by weight, preferably 0.01 to 1.0% by weight, and most preferably 0.01 to 0.02% by weight, based on the total weight of the composition. have. If the content of DNA derived from plants of the genus Aloe is less than 0.01% by weight, there is little effect of preventing skin aging and alleviating skin inflammation, and if the content of DNA derived from plants of the genus Aloe exceeds 10.0% by weight, formulation stability may be affected. .

In the composition according to the present invention, the extract of the genus Aloe plant, preferably the leaf extract of the genus Aloe plant, most preferably the pulverized product or gel of the leaves of the genus Aloe plant, is the desired DNA of the genus Aloe plant. It may be contained as an auxiliary ingredient to further improve the skin condition improvement effect (skin wrinkle improvement and skin elasticity improvement effect).

In the composition, the auxiliary component and DNA derived from plants of the genus Aloe may be contained in the same amount, but are not limited thereto.

The composition according to the present invention is generally a composition for external application for skin, and may be a cosmetic composition or a pharmaceutical composition, but is preferably a cosmetic composition, and the external preparation for skin is a generic term that may include anything applied from the outside of the skin. This may include cosmetics and pharmaceuticals.

The cosmetic composition may include, for example, a basic cosmetic, a makeup cosmetic, a hair cosmetic, a body cosmetic, and the like, and the formulation is not particularly limited, and may be appropriately selected according to the intended purpose. For example, the cosmetic composition may be formulated as a solution, a suspension, an emulsion, a paste, a gel, a cream, a lotion, a powder, a soap, a surfactant-containing cleansing, an oil, a powder foundation, an emulsion foundation, a wax foundation, and a spray. , But is not limited thereto. More specifically, softening lotion, nourishing lotion, lotion, body lotion, nourishing cream, massage cream, moisture cream, hand cream, essence, eye cream, cleansing cream, cleansing foam, cleansing water, pack, gel, patch, oil-in-water Basic cosmetics such as (O/W) type and water-in-oil (W/O) type, color cosmetics such as lipstick, makeup base or foundation, cleaning agents such as shampoo, conditioner, body cleanser, toothpaste, feminine detergent or mouthwash, Hair tonic, hairdressing agents such as gels or mousses, hair dyes, hair dyes, etc. may be formulated as cosmetic compositions for hair.

The composition is a moisturizing agent (polyol, etc.), oil, wax, viscosity adjuster, thickener, pH adjuster (pH adjuster), fat, organic solvent, solubilizer, thickener , Gelling agent, softening agent, antioxidant, suspending agent, stabilizer, foaming agent, fragrance, surfactant, water, ionic emulsifier, nonionic emulsifier, filler, sequestering agent, chelating agent, preservative, vitamin , Wetting agents, dyes, pigments, hydrophilic activators, lipophilic activators, lipid vesicles or any other ingredients commonly used in cosmetics. In addition, the composition of the present invention may further contain a substance that promotes skin absorption in order to increase the effect of improving the skin condition.

Pharmaceutical compositions containing DNA derived from plants of the genus Aloe of the present invention include pharmaceutical adjuvants such as preservatives, stabilizers, hydrating agents or emulsification accelerators, salts and/or buffers for regulating osmotic pressure, and other therapeutically useful substances (carriers, excipients, Diluents, etc.) may be additionally contained, and may be formulated in the form of various parenteral administration preparations (coating agents) according to a conventional method.

The term “carrier” is defined as a compound that facilitates the addition of the compound into a cell or tissue. For example, dimethyl sulfoxide (DMSO) is a commonly used carrier that facilitates the incorporation of many organic compounds into cells or tissues of an organism.

The term "excipient" is a substance that is added to make it easier to eat or has a certain color and shape when the amount of the main drug is small in the preparation process of tablets or pills, and lactose or starch is mainly used. do.

The term “diluent” is defined as a compound that is diluted in water that will dissolve the compound as well as stabilize the biologically active form of the compound of interest. Salts dissolved in buffer solutions are used as diluents in the art. A commonly used buffer solution is phosphate buffered saline, because it mimics the salt state of human solutions. Because buffer salts can control the pH of a solution at low concentrations, buffer diluents rarely alter the biological activity of a compound.

The composition comprising DNA from a plant of the genus Aloe used herein is administered (applied) to a human patient as such, or as a pharmaceutical composition mixed with other active ingredients or with a suitable carrier or excipient, such as in combination therapy. Can be.

Carriers, excipients, and diluents that may be included in the pharmaceutical composition include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum acacia, alginate, gelatin, calcium phosphate, calcium silicate, cellulose , Methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oils.

The parenteral administration agent (coating agent) may be, for example, injection, drop, ointment, lotion, gel, cream, spray, suspension, emulsion, suppository, patch, etc., but is limited thereto. It does not become.

The pharmaceutical composition according to an embodiment of the present invention may be administered (applied) parenterally, topically, transdermally, or subcutaneously. The pharmaceutical composition according to an embodiment of the present invention may be topically administered (applied) to the scalp, for example.

In addition, the pharmaceutically acceptable dose, that is, the dosage (application amount) of the active ingredient is the age, sex, and weight of the subject to be treated, the specific disease or pathological condition to be treated, the severity of the disease or pathological condition, and the route of administration (application route ) And the judgment of the prescriber. Determination of the dosage (application amount) based on these factors is within the level of those skilled in the art. The dosage is, for example, 0.01 to 5000 mg per 1 kg of body weight per day, preferably 0.1 to 2000 mg, more preferably 0.5 to 500 mg, and most preferably 1 to 100 mg to be administered (applied). Although it can be administered (applied) in one to several times, the dosage is not intended to limit the scope of the present invention in any way.

Since the DNA derived from a plant of the genus Aloe of the present invention is a natural substance, it is not toxic at all, so it can be continuously used in large amounts as a medicine.

Pharmaceutical compositions suitable for use in the present invention include compositions in which the active ingredients including DNA derived from plants of the genus Aloe are contained in an amount effective to achieve their intended purpose. More specifically, a therapeutically effective amount means an amount of a compound effective to prolong the survival of the subject to be treated or to prevent, alleviate or alleviate the symptoms of the disease. Determination of a therapeutically effective amount is within the capabilities of one of ordinary skill in the art, particularly in light of the detailed disclosure provided herein.

A therapeutically effective amount of DNA derived from a plant of the genus Aloe used in the methods of the present invention, and a composition (compounds) containing the same as an active ingredient can be determined initially from cell culture analysis. For example, the dose can be calculated in an animal model to obtain a range of circulating concentrations including the half maximal inhibitory concentration ( _{IC 50 ) or half maximal effective concentration (EC 50) determined in the cell culture.} Such information can be used to more accurately determine useful doses in humans.

The toxicity and therapeutic efficiency of DNA derived from plants of the genus Aloe described herein, or compositions (compounds) containing the same as an active ingredient, are, for example, LD ₅₀ (lethal dose for 50% of the _{population), ED 50} (cluster To determine the dose having a therapeutic effect for 50% of), IC ₅₀ (a dose having a therapeutic inhibitory effect for 50% of the population), it can be estimated by cell culture or standard pharmaceutical procedures in experimental animals. . The dose ratio between toxic and therapeutic effect is the therapeutic index and it can be expressed as the ratio between _{LD 50} and ED ₅₀ (or IC _{50 ).} Compounds showing a high therapeutic index are preferred. Data obtained from these cell culture assays can be used to estimate a range of doses for use in humans. The dosage or application of such compounds is preferably within a range of circulating concentrations comprising _{ED 50} (or IC _{50) in a state of little or no toxicity.}

Hereinafter, the present invention will be described in more detail through examples. These examples are for illustrative purposes only, and it will be apparent to those of ordinary skill in the art that the scope of the present invention is not construed as being limited by these examples. Therefore, it will be said that the practical scope of the present invention is defined by the appended claims and their equivalents.

<Production of Aloe Vera-derived DNA>

Raw materials : Aloe vera gel and DNA derived from aloe vera were prepared using aloe vera, purified water and general brine obtained from retail stores, and sodium chloride extracted from deep ocean water taken from Goseong, Gangwon-do.

[Reference Example 1] Preparation of Aloe Vera Gel

After mixing 100 g of aloe vera leaves and 100 g of purified water, put in a homogenizer and homogenized to prepare a pulverized aloe vera (aloe vera gel) (concentration of a pulverized Aloe vera sample: 10,000 μg/ml).

[Example 1] Preparation of DNA derived from Aloe Vera using deep ocean water

(a) Hydrolysis step : After mixing 100g of leaves of Aloe vera and 100g of purified water, putting it in a homogenizer and homogenizing to prepare a pulverized Aloe vera, and then pulverizing cellulase (Novozymes) in the pulverized product. The hydrolysis reaction was performed by adding 0.05 to 0.1% by weight based on the weight of water, and stirring for about 2 to 3 hours under conditions of about pH 4.5 and about 25 to 30°C.

(b) dissolving step (lysis step): in which cellulase is added to the pulverized polysaccharide inactivate the cellulase for hydrolyzing the reaction product to a temperature of 60 ~ 70 ℃, then filtered through a 0.45μm filter to obtain a filtrate. Thereafter, SDS and sodium chloride extracted from deep sea water were added so that the final concentration of SDS in the filtrate was about 1% by weight and the final concentration of sodium chloride extracted from deep sea water was about 1.25% by weight so that DNA could be extracted from the filtrate. After that, inverting slowly about 10 to 30 times so that the filtrate, SDS, and sodium chloride extracted from deep sea water are evenly mixed, and then allowed to stand at room temperature (for example, 18 to 25°C) for about 1 hour to dissolve. Was obtained.

(c) pH calibration : Sodium hydroxide was added to the lysate to make about pH 9.5 to 10.5, followed by stirring at about 60 to 75° C. for about 1 to 2 hours. Thereafter, citric acid was added to adjust the final pH to about pH 6.7 to 7.3, and then centrifuged at about 10,000 rpm using a centrifuge to obtain a lysate (supernatant) whose pH was adjusted.

(d) Precipitation step : After adding ethanol to the pH-adjusted lysate so that the lysate by volume: ethanol = 25 to 35: 65 to 75, using a centrifugal separator, at about 7,000 rpm. The precipitate was obtained by centrifugation.

Then, the precipitate was dissolved in 25 mM Tris-HCl pH 8.0 containing an appropriate amount of RNase A to prepare a DNA sample (DNA sample concentration: 10,000 μg/ml).

[Comparative Example 1] Preparation of DNA derived from Aloe Vera using normal saline

Aloe vera leaves were used as a raw material in the same manner as in the preparation method of Example 1, and a precipitate was obtained in the precipitation step using general brine salt (edible refined salt) instead of deep sea water.

Then, the precipitate was dissolved in 25 mM Tris-HCl pH 8.0 containing an appropriate amount of RNase A to prepare a DNA sample (DNA sample concentration: 10,000 μg/ml).

[Experimental Example 1] Confirmation of the state of Aloe vera-derived DNA

The DNA samples prepared in each of Example 1 and Comparative Example 1 were subjected to electrophoresis on a 1% agarose gel at the same concentration, and then stained with EtBr solution to confirm the DNA status of each of Example 1 and Comparative Example 1.

As a result, as shown in FIG. 1 below, the Aloe vera-derived DNA of each of Example 1 and Comparative Example 1 was about 0.5 to 11 kDa based on a marker (MK: 50 to 10,000 bp) upon electrophoresis on a 1% agarose gel. It was confirmed to have a size of. In particular, it was confirmed that the DNA extraction efficiency of Aloe vera of Example 1 was higher than that of the Aloe vera-derived DNA of Comparative Example 1 at a molecular weight value of about 0.5 to 4 kDa, which had higher DNA yields.

[ Experimental example 2] Evaluation of Aloe Vera-derived DNA to Inhibit Expression of Collagen Degrading Enzyme in Skin Cells

Inhibitory efficacy of the Aloe vera-derived DNA prepared in Example 1, the Aloe vera-derived DNA prepared in Comparative Example 1, and the Aloe vera gel prepared in Reference Example 1 to inhibit MMP-1 (Matrix metalloproteinase-1), a collagen-degrading enzyme in skin cells To confirm, human dermal fibroblasts (HDF) were irradiated with UV B (UVB) of 220 J/cm 2 in a UV chamber, and then the Aloe vera-derived DNA of Example 1, the Aloe vera-derived DNA of Comparative Example 1, or After the treatment of the aloe vera gel of Reference Example 1, MMP-1 inhibitory ability was confirmed.

Specifically, HDF was added to a 12-well plate using DMEM (Dulbecco Modified Eagle Medium, Gibco) to which penicillin/streptomycin and 10% FBS (Fetal Bovine Serum, Gibco) were added to 7.5×10 ⁴ cells/well (cells/ well), and incubated for about 24 hours in a 37°C/5% CO _{2 incubator.} Then, the HDF was treated under the following (i) to (vi) treatment conditions (triplicate samples were prepared for each sample), and then further incubated for about 72 hours.

Then, using an ELISA kit (MMP-1, Human, Biotrak Assay, RPN2610, GE Healthcare Life Sciences) that can measure the expression level of MMP-1, the expression level of MMP-1 contained in the HDF medium was measured. (Measured at 3 for each sample, and an average value was calculated).

Samples and treatment conditions used to measure the expression level of MMP-1 were as follows.

(i) The positive control group was a 12-well plate seeded with HDF was irradiated with UVB of 220J/cm2 in a UV chamber, and then treated by diluting RA (retinoic acid) in DMEM at a concentration of 10 μg/ml.

(ii) Comparative group 1 is a 12-well plate seeded with HDF was irradiated with UVB of 220J/cm2 in a UV chamber, and then the aloe vera gel of Reference Example 1 was diluted in DMEM at a concentration of 200 μg/ml and treated.

(iii) In Comparative Group 2, a 12-well plate seeded with HDF was irradiated with 220 J/cm 2 UVB in a UV chamber, and then the Aloe vera-derived DNA of Comparative Example 1 was diluted in DMEM at a concentration of 200 μg/ml and treated.

(iv) In the experimental group, a 12-well plate seeded with HDF was irradiated with UVB of 220 J/cm 2 in a UV chamber, and then the Aloe vera-derived DNA of Example 1 was diluted in DMEM at a concentration of 200 μg/ml and treated.

(v) In the untreated group, a 12-well plate seeded with HDF was wrapped in silver paper and irradiated with 220J/cm2 UVB in a UV chamber.

(vi) The negative control group was a 12-well plate seeded with HDF was irradiated with 220J/cm2 UVB in a UV chamber.

Method of measuring the expression level of MMP-1

First, the primary antibodies MMP-1 (Ab-5) monoclonal antibody and MMP-2 (Ab-3) monoclonal antibody were added to a 96-well plate for measurement of MMP-1 expression level at about 37°C. Attached for about 60 minutes at. Then, the culture medium recovered from the HDF treated under the conditions (i) to (vi) was treated for about 60 minutes in a 96-well plate to which the monoclonal antibodies were attached, and then the secondary antibody, alkaline phosphatase, was added. The conjugated anti-mouse IgG (whole mouse, alkaline phosphatase conjugated) was treated and reacted for about 60 minutes, and then an alkaline phosphatase substrate solution (1mg/ml ρ-nitrophenyl phosphate in diethanolamine buffer) was treated for about 30 minutes and at room temperature. After reacting for about 30 minutes at, the absorbance was measured at 405 nm with a microplate reader.

As a result, as shown in FIG. 2 below, the experimental group (aloe vera-derived DNA treatment group of Example 1) had the effect of reducing the increased MMP-1 expression level due to UVB irradiation of HDF compared to Comparative Group 1 (Reference Example 1). Of Aloe vera gel treatment group) and Comparative Group 2 (aloe vera-derived DNA treatment group of Comparative Example 1) was significantly higher than that, it was confirmed that the most excellent skin wrinkle improvement and skin elasticity improvement effect can be exhibited.

[ Experimental example 3] Evaluation of Aloe Vera-derived DNA to Inhibit Expression of Inflammatory Cytokines in Skin Cells

To confirm the efficacy of the Aloe vera-derived DNA prepared in Example 1, the Aloe vera-derived DNA prepared in Comparative Example 1, and the Aloe vera gel prepared in Reference Example 1 to inhibit the production of inflammatory cytokines due to ultraviolet rays of skin cells , Human keratinocytes (HaCaT) were seeded in a 24-well plate to become 4.5×10 ⁴ cells/well _{using DMEM, and cultured in a 37° C./5% CO 2} incubator for 16 hours, followed by (i ) to (v) treatment conditions. After 24 hours of treatment, the HaCaT cells were irradiated ^{with UVB of 200 mJ/cm 2} in a UV chamber, and then cultured at 37° C. for 48 hours, and the culture medium was collected to measure the expression level of the inflammatory cytokine IL-6 ( IL-6 Human ProQuantum Immunoassay kit, A35573, ThermoFisher Scientific) was used to measure the expression level of IL-6 contained in the medium of HaCaT cells (triplicate samples were measured for each sample to obtain an average value).

Samples and treatment conditions used to measure the expression level of IL-6 were as follows.

(i) In the untreated group, a 24-well plate seeded with HaCaT cells was wrapped in silver paper and irradiated with 220 J/cm 2 UVB in a UV chamber.

(ii) The negative control group was a 24-well plate seeded with HaCaT cells irradiated with 220 J/cm 2 UVB in a UV chamber.

(iii) Comparative group 1 is a 24-well plate seeded with HaCaT cells was irradiated with 220J/cm2 UVB in a UV chamber, and then the aloe vera gel of Reference Example 1 was diluted in DMEM at a concentration of 200 μg/ml and treated.

(iv) Comparative group 2 is a 24-well plate seeded with HaCaT cells was irradiated with 220 J/cm 2 UVB in a UV chamber, and then the Aloe vera-derived DNA of Comparative Example 1 was diluted in DMEM at a concentration of 200 μg/ml and treated. .

(v) In the experimental group, a 24-well plate seeded with HaCaT cells was irradiated with 220 J/cm 2 UVB in a UV chamber, and then the Aloe vera-derived DNA of Example 1 was diluted in DMEM at a concentration of 200 μg/ml and treated.

As a result, as shown in FIG. 3 below, the experimental group (aloe vera-derived DNA treatment group of Example 1) had the effect of reducing the increased IL-6 expression level due to UVB irradiation of HaCaT cells in Comparative Group 1 (Reference Example It was confirmed that it was significantly higher than that of the aloe vera gel treatment group of 1) and the comparative group 2 (the aloe vera-derived DNA treatment group of Comparative Example 1) exhibiting the most excellent anti-inflammatory effect.

[ Experimental example 4] Aloe Vera-derived DNA Reactive oxygen species (reactive oxygen species, ROS) scavenging ability evaluation

<ROS scavenging ability due to oxidative stress>

In order to measure the ROS removal ability of the Aloe vera-derived DNA prepared in Example 1, the Aloe vera-derived DNA prepared in Comparative Example 1, and the aloe vera gel prepared in Reference Example 1, a method of measuring fluorescence using DCFDA was used. .

First, human keratinocytes (HaCaT) were seeded in a 96-well black microplate for fluorescence measurement at 1x10 ⁴ cells/well using DMEM added with penicillin/streptomycin and 10% FBS, and then 37°C. Incubated for about 24 hours in /5% CO _{2 incubator.} Thereafter, the samples (Example 1, Comparative Example 1, and Reference Example 1) were diluted in each serum-free DMEM, and further cultured for about 24 hours (positive control: 0.2mM Trolox[6-hydroxy-2,5,7, 8-tetramethylchroman-2-carboxylic acid] was treated with HaCaT and incubated for about 24 hours).

After 24 hours of incubation, the medium used for HaCaT culture was removed, and HaCaT was washed with HBSS (Hanks' Balanced Salt Solution, Welgene, Korea), and 50 μM DCFDA (dilution solvent: HBSS) was added to 100 μL. And incubated for about 1 hour in a 37° _{C./5% CO 2} incubator, and then washed with HBSS. Then, after treatment with 100 μM H ₂ O ₂ (dilution solvent: HBSS) (non-treatment group: sample treated with only HBSS; negative control group: _{sample treated with only H 2} O ₂ ), treated with DCFDA and H ₂ O ₂ Fluorescence generated from HaCaT was measured with a fluorescent plate reader (Tecan plate reader, Switzerland) at Excitation/Emission = 485nm/535nm (three triplicate samples were measured for each sample to obtain an average value).

As a result, as shown in FIG. 4 below, the experimental group (aloe vera-derived DNA treatment group of Example 1) was H ₂ O _{2 of HaCaT cells.} The effect of reducing the increased ROS due to the treatment was significantly higher than that of Comparative Group 1 (Aloe vera gel treatment group of Reference Example 1) and Comparative Group 2 (Aloe vera-derived DNA treatment group of Comparative Example 1). It was confirmed that it can be exerted.

Pharmaceutical compositions and cosmetic compositions containing DNA derived from aloe genus of the present invention as an active ingredient may be commercialized (medicines, cosmetics) in various formulations of Formulation Examples 1 to 4, but are not limited thereto, and the functionality of the product to be implemented It can be applied in various formulations and formulations containing DNA derived from plants of the genus Aloe in consideration of cost and other conditions.

[Formulation Example 1] Preparation of ointment

The ointment containing the DNA derived from the Aloe genus plant of the present invention was prepared by mixing the components of Table 1 including the oil phase component and the water phase component.

Ingredients Content (% by weight) Purified water Balance DNA from plants of the genus Aloe 10.0 Caprin/Capril Triglic Seride 10.0 Liquid paraffin 10.0 Solbitansesuquioleate 6.0 Octyldodeces-25 9.0 Cetylethylhexanoate 10.0 Squalane 1.0 Salicylic acid 1.0 glycerin 15.0 Sorbitol 10.0

[ Formulation example 2] Preparation of nutrient cosmetic water

By mixing the components of Table 2 including the oil phase component and the water phase component to prepare a nutrient lotion containing DNA derived from the Aloe genus plant of the present invention.

Ingredients Content (% by weight) Purified water Balance DNA from plants of the genus Aloe 0.1 Beeswax 4.0 Polysorbate 60 1.5 Sorbitansquioleate 1.5 Floating paraffin 0.5 Montana 202 (Manufacturer:Seppic) 5.0 glycerin 3.0 Butylene glycol 3.0 Propylene glycol 3.0 Carboxyvinyl polymer 0.1 Triethanolamine 0.2 Preservatives, colors, flavors Appropriate amount

[ Formulation example 3] Manufacturing of massage cream

A massage cream containing DNA derived from plants of the genus Aloe of the present invention was prepared by mixing the components of Table 3 including the oily component and the water phase component.

Ingredients Content (% by weight) Purified water Balance DNA from plants of the genus Aloe 0.1 Beeswax 10.0 Polysorbate 60 1.5 PEG 60 Hardened Castor Oil 2.0 Sorbitansquioleate 0.8 Floating paraffin 40.0 Squalane 5.0 Montana 202 (Manufacturer: Seppic) 4.0 glycerin 5.0 Butylene glycol 3.0 Propylene glycol 3.0 Triethanolamine 0.2 Preservatives, colors, flavors Appropriate amount

[ Formulation example 4] Preparation of the pack

A pack containing the DNA derived from the Aloe genus plant of the present invention was prepared by mixing the components of Table 4 including the oil phase component and the water phase component.

Ingredients Content (% by weight) Purified water Balance DNA from plants of the genus Aloe 0.1 Polyvinyl alcohol 13.0 Sodium carboxymethyl cellulose 0.2 glycerin 5.0 Allantoin 0.1 ethanol 6.0 PEG-12 nonylphenyl ether 0.3 Polysorbate 60 0.3 Preservatives, colors, flavors Appropriate amount

As described above, specific parts of the present invention have been described in detail, and it will be apparent to those of ordinary skill in the art that these specific techniques are only preferred embodiments, and the scope of the present invention is not limited thereby. will be. Accordingly, it will be said that the substantial scope of the present invention is defined by the appended claims and their equivalents.

Claims (19)

As a method for producing DNA derived from plants of the genus Aloe using minerals extracted from deep ocean water,
(a) 0.01 to 0.5% by weight of a hydrolase is added to the pulverized product prepared by mixing and homogenizing the plant of the genus Aloe and purified water at the same amount, followed by pH 3.0 to 5.0 and Producing a hydrolyzate under conditions;
(b) heating the hydrolyzate to a temperature of 65 to 75° C. to inactivate the hydrolyzate contained in the hydrolyzate, and then filtering through a 0.2 to 2.0 μm filter;
(c) adding negatively charged surfactants and minerals extracted from deep ocean water to the filtered hydrolyzate in a weight ratio of 0.5 to 3.0: 1.0 to 3.0, stirring, and standing at room temperature to generate a dissolved product;
(d) adjusting the pH of the lysate to pH 9.0 to 11.0, stirring at 55 to 75°C for 1 to 3 hours, and then adjusting the pH to 6.0 to 8.0 to obtain a pH-adjusted lysate ;
(e) adding an organic solvent to the pH-adjusted lysate on a volume basis so that the lysate: organic solvent = 20 to 40: 60 to 80 to obtain a DNA precipitate; And
(f) removing RNA contained in the obtained DNA precipitate to obtain DNA derived from plants of the genus Aloe; including,
The hydrolase is pectinase, α,β-glucosidase, amiloglucosidase, beta-glucanase, cellulase, naringinase, α-amylase, hemicellulase, arabinase, xylanase and It is one or more selected from the group consisting of tannases,
The negatively charged surfactant is at least one selected from the group consisting of sodium dodecyl sulfate, amonium dodecyl sulfate, sodium myreth sulfate, sodium pareth sulfate, sodium laureth sulfate, and potassium lauryl sulfate,
The organic solvent is characterized in that at least one selected from the group consisting of ethanol, propanol, isopropanol, and butanol. The method of claim 1, wherein the RNA contained in the DNA precipitate is removed through an RNA degrading enzyme treatment. The method of claim 1, wherein the plant of the genus Aloe is Aloe Barbadensis ( Aloe Vera ) , Aloe aculeata (Aloe aculeata), Aloe africana (Aloe africana) , Aloe albida (Aloe albida), Aloe albi. Flora (Aloe albiflora), aloe ahreubo Les kenseu (Aloe arborescens), aloe Arezzo Nicolas (Aloe arenicola), aloe are Gen tikka Uda (Aloe argenticauda), Aloe bar Kerry (Aloe bakeri), aloe balriyi (Aloe ballii), aloe balriyi (Aloe ballyi), aloe breather non polyamic (Aloe brevifolia), aloe bromo ohmiyi (Aloe broomii), aloe portion eth- Neri (Aloe buettneri), aloe Kam Perry (Aloe camperi), aloe copy of tartar (Aloe capitata), aloe nose Simulation (Aloe comosa), aloe kooh Perry (Aloe cooperi), aloe Coral Lina (Aloe corallina), aloe Devin Terry (Aloe dewinteri), aloe Erina Kea (Aloe erinacea), aloe X kelsa (Aloe excelsa), aloe page rocks (Aloe ferox), aloe formate besiyi (Aloe forbesii), aloe helrenayi (Aloe helenae), cis (Aloe hereroensis), aloe enabled Le Miss (Aloe inermis), aloe yiniang gen system (Aloe inyangensis), aloe coarseness ion Aloe Herrera ( Aloe jawiyon ), Aloe jucunda , Aloe khamiesensis , Aloe kilifiensis , Aloe maculata , Aloe marlothii, Aloe marlothii Aloe mubendiensis), aloe Nami Ben sheath (Aloe namibensis), aloe Nyeri N-Sys (Aloe nyeriensis), aloe pages are soniyi (Aloe pearsonii), aloe pegeul rerayi (Aloe peglerae), aloe Pere poly-Ata (Aloe perfoliata), aloe periyi ( Aloe perryi), aloe Petri coke (Aloe petricola), aloe poly pillar (Aloe polyphylla), aloe Lau hiyi (Aloe rauhii), aloe Reynolds deusiyi (Aloe reynoldsii), aloe Scoring beanie polyamic (Aloe scobinifolia), aloe Singh Katana ( Aloe sinkatana), aloe Squash Rosa (Aloe squarrosa), aloe registry Ata (Aloe striata), aloe can coat Lena (Aloe succotrina), aloe sujannayi (Aloe suzannae), aloe Trapani seukiyi (Aloe thraskii), aloe corruption di Flora (Aloe viridiflora ) and Aloe wildii (Aloe wildii), characterized in that at least one selected from the group consisting of, a method for producing DNA derived from aloe genus using minerals extracted from deep ocean water. The method of claim 1, wherein the hydrolase is a cellulase. The method of claim 1, wherein the negatively charged surfactant is sodium dodecyl sulfate. The method of claim 1, wherein the organic solvent is ethanol. The method of claim 1, wherein the molecular weight of the DNA derived from plants of the genus Aloe is 0.1 to 400 kDa. delete delete delete delete delete delete delete delete delete delete delete delete

Images