KR20230070839A - Composition for preventing, improving or treating atopic dermatitis comprising nanoemulsion of miR126 and Chamaecyparis obutsa oil as active ingredients - Google Patents

Abstract

The present invention relates to an emulsion of microRNA and cypress essential oil having the effect of preventing, treating, and improving dermatitis. Since it reduces phosphorus IL-4 and IL-13 and rapidly reduces the immune response of IgE, it can be usefully used as a composition for treating atopic dermatitis.

Description

Composition for preventing, improving or treating atopic dermatitis comprising nanoemulsion of miR126 and Chamaecyparis obutsa oil as active ingredients}

The present invention relates to a nanoemulsion composed of miRNA and cypress oil having a therapeutic effect on atopic dermatitis, and more specifically, to a nanoemulsion composition for treating atopic dermatitis using active ingredients of microRNA-126 and cypress essential oil having physiological activity. it's about

Atopic dermatitis is a chronic skin disease of unknown medical cause that frequently occurs in modern people. Atopic dermatitis is generally known as a disease caused by an imbalance of the human immune system.

Atopic dermatitis, a chronic and recurrent inflammatory skin disease in which abnormalities occur in the stratum corneum, the protective wall of the skin, shows symptoms of red rash, dry skin, thickening of the skin, and scaling.

In addition, atopic dermatitis is a chronic or recurrent dermatitis that tends to occur in infants and children, and is characterized by symptoms such as itching, dry skin, perifollicular hyperactivity, cheilitis, lichenification, and eczema-like lesions. Their causes have been precisely identified. However, people with atopic dermatitis usually show allergic reactions to foreign substances in the external environment such as house dust, mites, animal hair, food, pollen, mold, etc., and atopic dermatitis is associated with allergic rhinitis, asthma, It is regarded as one of the allergic diseases in that it is accompanied by conjunctivitis.

In addition, other immunological factors have been reported such as abnormal increase in IgE, decrease in the number and function of T cells that play a central role in cellular immunity, and Th1/Th2 imbalance due to an increase in the number of Th2 cells compared to Th1 cells. there is.

There are two types of Th-1 and Th-2 among T cells involved in cellular immunity, and when the balance is broken and the amount of Th-2 suddenly increases and the amount of Th-1 decreases, diseases such as atopic dermatitis this happens Hypersensitivity of the Th-2 immune response is a key factor in atopic skin disease. This immune response is an immediate hypersensitivity reaction and is mediated by the secretion of IL-4, IL-5, and IL-13 cytokines, and IL-4 and IL-13 regulate the production of IgE.

Natural products have excellent pharmacological effects and have no side effects on the human body, so they are used as raw materials for pharmaceuticals and cosmetics, and are considered industries with high added value. Among these natural substances, essential oil components extracted from cypress trees are effective in relieving stress and psychological stability, and are effective in allergic skin diseases such as atopy through strong sterilization, and are known to neutralize harmful substances and soothe skin.

Plant essential oil is a volatile substance separated from a plant by a physical method. Its essential oil is a fat-soluble compound that dissolves well in fats and oils but does not mix with water. Emulsification is a method for increasing the absorption rate while stabilizing the essential oil having these characteristics. Emulsion refers to a system in which one liquid is dispersed in another liquid as fine particles. It is divided into two types (W/O).

To date, there has been no report on using plant essential oils and microRNAs that control the secretion of inflammatory cytokines and IgE to treat atopic dermatitis.

(001) Korea registered patent KR10-1878069 (002) Republic of Korea Patent Publication KR10-2019-00051126

Therefore, in order to overcome the problems of the prior art, the present inventors have made efforts to develop microRNA that regulates the secretion of inflammatory cytokines and the production of IgE and to search for natural extracted essential oil. As a result, the nanoemulsion of microRNA-126 and cypress oil is atopic In the dermatitis-induced experimental group, it was confirmed that the inflammatory cytokines IL-4 and IL-13 could be reduced and the IgE immune response rapidly reduced, and the present invention was completed.

An object of the present invention is to provide a pharmaceutical composition for preventing or treating atopic dermatitis comprising a nanoemulsion composed of micro RNA-126 represented by the nucleotide sequence of SEQ ID NO: 1 and cypress essential oil.

Another object of the present invention is to provide a cosmetic composition for preventing or improving atopic dermatitis comprising a nanoemulsion composed of micro RNA (micro RNA) -126 represented by the nucleotide sequence of SEQ ID NO: 1 and cypress essential oil.

In order to achieve the above object, to provide a pharmaceutical composition for preventing or treating atopic dermatitis comprising a nanoemulsion consisting of micro RNA (micro RNA) -126 represented by the nucleotide sequence of SEQ ID NO: 1 and cypress essential oil.

According to one embodiment, the composition can inhibit the expression of IgE.

According to one embodiment, the composition can inhibit the expression of IL-4.

According to one embodiment, the composition can inhibit the expression of TNF-a.

It can be formulated into a pharmaceutical unit dosage form by adding pharmaceutically acceptable carriers, excipients or diluents to the nanoemulsion composed of micro RNA-126 represented by the nucleotide sequence of SEQ ID NO: 1 and cypress essential oil. there is.

The composition of the pharmaceutical unit dosage form of the present invention is a powder, granule, tablet, capsule, suspension, ointment, gel, cream, patch, spray emulsion, syrup, aerosol, etc. for oral administration, external preparation, Sterile injection solutions, suppositories, and preparations for transdermal administration may be formulated and used in a conventional manner.

According to another embodiment of the present invention, a cosmetic composition for preventing or improving atopic dermatitis comprising a nanoemulsion composed of micro RNA-126 represented by the nucleotide sequence of SEQ ID NO: 1 and cypress essential oil to provide.

According to one side, the emulsion may be a nano- or micro-sized emulsion, and the formulation of the emulsion selected from the double emulsion group in the form of O / W or W / O or O / W / O, W / O / W can have

The cosmetic composition of the present invention can be prepared in any dosage form conventionally prepared in the art, for example, a solution, suspension, emulsion, paste, gel, cream, lotion, powder, soap, surfactant-containing cleansing, It may be formulated as a hair cosmetic, oil, etc., but is not limited thereto. More specifically, the cosmetic composition of the present invention is lotion, skin lotion, skin softener, skin toner, astringent, lotion, milk lotion, moisture lotion, nutrient lotion, massage cream, nutrient cream, moisture cream, hand cream, It includes formulations of sunscreen, essence, nutritional essence, pack, soap, cleansing foam, cleansing lotion, cleansing cream, body lotion and body cleanser, emulsion, bath additives and sprays.

When the formulation of the present invention is a paste, cream or gel, animal oil, vegetable oil, wax, paraffin, starch, cellulose derivative, polyethylene glycol, bentonite, talc or zinc oxide may be used as an excipient component.

When the formulation of the present invention is powder or spray, lactose, talc, aluminum hydroxide, calcium silicate or polyamide powder may be used as an excipient component, and in particular, in the case of spray, a propellant such as propane/butane or dimethyl ether may be additionally included. can do.

When the formulation of the present invention is a solution or emulsion, a solvent, solubilizing agent or emulsifying agent is used as an excipient component,

Examples include water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butyl glycol oil, fatty acid esters of glycerol, polyethylene glycol or sorbitan.

When the dosage form of the present invention is a suspension, liquid diluents such as water, ethanol or propylene glycol, suspension agents such as ethoxylated isostearyl alcohol, polyoxyethylene sorbitol esters and polyoxyethylene sorbitan esters, microcrystals as excipient components Star cellulose, aluminum metahydroxide, bentonite, agar or tracanth and the like may be used.

When the formulation of the present invention is surfactant-containing cleansing, as excipient components, aliphatic alcohol sulfate, aliphatic alcohol ether sulfate, sulfosuccinic acid monoester, isethionate, imidazolinium derivative, methyl taurate, sarcosinate, fatty acid amide ether Sulfates, alkylamidobetaines, fatty alcohols, fatty acid glycerides, fatty acid diethanolamides, vegetable oils, lanolin derivatives or ethoxylated glycerol fatty acid esters and the like can be used.

Since the nanoemulsion made of microRNA-126 and cypress oil has a significant inhibitory effect on mitigating atopic inflammation, it can be usefully used as a treatment for atopic dermatitis.

Figure 1 is It is a graph showing the results of particle size analysis of miR-126/cypress oil nanoemulsion.
Figure 2 is a photograph showing the ears of mice according to the treatment of miR-126 / cypress oil nanoemulsion in atopy-induced animal models.
Figure 3 is a picture of H&E staining of mouse ear photographic tissue according to the treatment of miR-126 / cypress oil nanoemulsion in an atopic induced animal model.
Figure 4 is a graph showing the change in thickness of the mouse ear skin according to the treatment of miR-126 / cypress oil nanoemulsion in an atopic induced animal model.
Figure 5 is a graph showing the effect of suppressing IgE in serum according to the treatment of miR-126 / cypress oil nanoemulsion in atopy-induced animal models.
Figure 6 is a graph showing the TNF-α inhibitory effect in serum according to the treatment of miR-126 / cypress oil nanoemulsion in atopy-induced animal models.
Figure 7 is a graph showing the IL-4 inhibitory effect in serum according to the treatment of miR-126 / cypress oil nanoemulsion in atopy-induced animal models.

Hereinafter, the present invention will be described in more detail based on the following specific experimental methods and examples. However, the following specific experimental methods and examples are only for exemplifying the present invention, and the scope of the present invention is not limited thereto.

<Example 1> Test method

<1-1> Production of microRNA

MicroRNA 126 and control microRNA were synthesized and used by Genolution (Seoul, Korea), and their sequence numbers are shown in Table 1 below.

name order sequence number miR-126 hsa-miR-126-3P 5'-ucguaccgugaguaauaaugcg-3' One miR-control hsa-miRNA control 5'-uucuccaacgugucacgutt-3' 2

<1-2> Extraction of cypress oil

Cypress (Chamaecyparis obtuse) leaves were collected in Jangseong, Jeollanam-do in August 2020, and essential oil extracted using a supercritical extraction method was used. In detail, it was extracted with a supercritical fat extraction device (SCCO2 Extraction System, IL Sin, Korea). 200 g of cypress leaves were put into a sample extractor, and cypress oil was extracted by extracting for 5 hours at a pressure of 420 atm and an extraction temperature of 45 ° C.

<1-3> Preparation of complex nanoemulsion of cypress oil and miR-126

To prepare the nanoemulsion, gelatin (sigma aldrich) was prepared at a concentration of 0.5% (w/v) using RNase free water. 100 ul of miR-126 and miR-control at a concentration of 0.1 ug/ul were added to 10 mL of the gelatin aqueous solution, respectively, and mixed by stirring at 4° C. for 1 hour. Then, 1.0 mL of cypress oil and 3.0 mL of emulsifier Tween80 were mixed and stirred at 4 ° C. at a speed of 300 rpm for 12 hours to prepare an oil in water (O / W) emulsion of cypress essential oil containing miR-126.

<1-4> Particle size analysis of nanoemulsion

Particle size was measured to measure the stability of the nanoemulsion. The particle size was measured using a light-scattering particle size analyzer (Nanotrac TM250, Microtrac Inc., USA) capable of measuring sizes from 3 nm to 6 μm.

<1-5> Measurement of thermal stability of nanoemulsion

The prepared nanoemulsion was heat-treated at 10 ° C, 30 ° C, and 60 ° C for 10 minutes, respectively, and then measured using a light-scattering particle size analyzer (Nanotrac TM250, Microtrac Inc., USA).

<1-6> Measurement of pH stability of nanoemulsion

The pH of the prepared emulsion was adjusted to 3,4,5,6,7,8,9 using HCl and NaOH, and incubated at room temperature for 24 hours, and the stability was measured by measuring the size of the particles.

<1-7> Atopic dermatitis model and dermatitis analysis for animal testing

In an aseptic environment, 5-week-old male ICR mice were purchased from Central Experimental Animals Co., Ltd. (Seoul City, Korea), and were acclimatized for one week while sufficiently supplied with food and water, and then used in the experiment. The rearing environment was a day and night cycle of 12 hours each, temperature (20 ~ 22 ℃) and humidity (50 ~ 60%) were kept constant, and experiments were conducted in accordance with the regulations of the Laboratory Animal Committee. After dividing the ICR mice into two groups, a control group and an experimental group, 5 mice each, 100 μl of DNCB, a skin disease-inducing substance at a concentration of 0.3%, was applied to the ears of the experimental animals for 1 week to induce skin disease similar to atopy. Thereafter, each miR-126/cypress oil nanoemulsion was applied to the skin of the rats in the experimental group.

After the end of the experiment, the change in skin lesions at the time of the test on the application date was taken and examined, and the thickness of the ear was measured using a digital caliper.

Serum was extracted from each rat by orbital sampling at the time of application, and ELISA was performed to determine the titer concentrations of IL-4, TNF-α, and IgE in the serum, which are indicators of atopic dermatitis.

<1-8> Skin tissue H&E staining

For histopathological analysis, H&E staining (Hematoxylin & Eosin staining) was performed. Atopic dermatitis-induced mouse model and miR-126/cypress oil nanoemulsion-treated mouse model were sacrificed to remove ear tissue, respectively. Thereafter, after washing with saline, water was removed, fixed with 4% paraformaldehyde (pH 7.4), and a paraffin block was produced through a series of processes. The cut ear tissue sections were subjected to deparaffinization and hydration, then stained with H&E and observed under a microscope (×100, Olympus, Tokyo, Japan).

<1-9> Measurement of serum IgE (immunoglobulin E), IL-4, and TNF-α cytokines

IL-4 and IgE measurements of atopic dermatitis model mice were measured in the serum of a mouse model that induced atopic dermatitis, a mouse model treated with miR-control group/cypress oil nanoemulsion, and a mouse model treated with miR-126/cypress oil nanoemulsion. IgE, TNF-α, and IL-4 were measured. IL-4, TNF-α and IgE, etc., were prepared using ELISA kits (Goma Biotech, Korea) that act specifically for each using antibodies such as anti-mouse IL-4, TNF-α and anti-mouse IgE. It was measured and quantified according to the method provided by For quantification of each substance, each substance was treated and reacted by concentration to establish a standard curve and the amount of substance contained in serum was calculated.

<1-10> Statistical processing

All experimental values were expressed as mean ± standard error, statistical analysis was performed by ANOVA and Student's t-test, and statistical significance limits were indicated by *, ** and ***, * means p<0.05, and * * means p<0.01, *** means p<0.001.

<Example 2> Experimental results

<2-1> Confirmation of particle size analysis of nanoemulsion

The prepared nanoemulsion showed that an emulsion having an average particle size of 234±11 nm was formed, and a formulation having a relatively narrow distribution curve was stably prepared (FIG. 1).

<2-2> Confirmation of stability of nanoemulsion by heat treatment

There was no significant change from 10 to 50 ℃ by heat treatment. At a relatively high temperature of 70 ° C, the particle size increased to 295 ± 23 nm, but no specific emulsion phase separation was observed. These results suggest that the prepared miR-126 / cypress oil nanoemulsion has a stability phase from low to relatively high temperatures.

temperature 10℃ 30℃ 50℃ 70℃ Average size (nm) 224±11 229±11 246±14 295±23

<2-3> Confirmation of stability of nanoemulsion according to pH change

In the evaluation of the pH stability of the prepared miR-126/cypress oil nanoemulsion, it was stable under conditions ranging from pH3 to pH9, and phase separation was not observed. These results suggest that the prepared miR-126/cypress oil nanoemulsion has relatively stability at pH and can be used as a composition for use in various formulations such as lotion, cream, and shampoo in the future.

pH 3 4 5 6 7 8 9 Average size (nm) 234±11 223±9 236±10 239±13 235±11 235±12 265±7

<2-4> Confirmation of atopy improvement effect in atopic animal model

As a result of visual observation, skin rashes intensified in the atopy-induced experimental group, and in particular, skin spots, erythema, skin dryness, edema, and bleeding were severely observed. When miR-126/cypress oil nanoemulsion was continuously applied to the animal model in this state for 2 weeks, the dermatitis of the affected area was recovered. It was restored to a state with a higher degree of relief (FIG. 2).

In addition, to investigate the effect of miR-126/cypress oil nanoemulsion on histological changes on atopic inflammatory disease, hematoxylin-eosin staining was performed to examine the tissue thickness of the back. It was confirmed that the thickness of the dermis and dermis increased significantly compared to the normal group. On the other hand, in the miR-126/cypress oil nanoemulsion-treated group, the thickness of the epidermis and dermis was relatively reduced compared to the control group, so miR-126/cypress oil nanoemulsion is effective in reducing the tissue thickness of the DNCB-induced atopic dermatitis animal experimental group. was confirmed (Fig. 3).

The miR-126 / cypress oil nanoemulsion prepared as a result of the above experiment showed that the ear tissue thickened by atopy induction was 0.58 ± 0.12 mm, which slightly decreased from the miR-control group / cypress oil nanoemulsion at 2 weeks. However, no significant decrease was seen. However, miR-126/cypress oil nanoemulsion treatment group showed statistically significant reduction effect up to 0.36±0.14 mm. It can be seen that the effect of the emulsion prepared by mixing miR-126 shows a more synergistic atopic healing effect than in the nanoemulsion prepared only with cypress oil (FIG. 4).

In addition, the results of analyzing the concentrations of IgE, TNF-α and IL-4 by separating the serum from the atopic induced group and the atopic induced + miR-126 / cypress oil nanoemulsion group after 2 weeks in the atopic induced animal model. miR-126/cypress oil nanoemulsion effectively inhibited the production of IgE (FIG. 5). In addition, TNF-α was also effectively inhibited by miR-126/cypress oil nanoemulsion treatment (FIG. 6). In addition, the concentration of IL-4 inflammatory cytokine, which was increased by atopy induction, also showed a statistically significant decrease according to miR-126/cypress oil nanoemulsion treatment (FIG. 7). However, as a result of IgE, TNF-α and IL-4 experimental analysis, no significant decrease was seen in the miR-control group/cypress oil nanoemulsion treatment group compared to the atopic group.

Claims (5)

A pharmaceutical composition for preventing or treating atopic dermatitis, comprising a nanoemulsion composed of micro RNA-126 represented by the nucleotide sequence of SEQ ID NO: 1 and cypress essential oil. The pharmaceutical composition for preventing or treating atopic dermatitis according to claim 1, wherein the nanoemulsion composed of the micro RNA (micro RNA)-126 and cypress essential oil inhibits the expression of IgE. The pharmaceutical composition for preventing or treating atopic dermatitis according to claim 1, wherein the nanoemulsion composed of micro RNA-126 and cypress essential oil inhibits the expression of IL-4. The pharmaceutical composition for preventing or treating atopic dermatitis according to claim 1, wherein the nanoemulsion composed of the micro RNA-126 and cypress essential oil inhibits the expression of TNF-a. A cosmetic composition for preventing or improving atopic dermatitis, comprising a nanoemulsion composed of microRNA-126 represented by the nucleotide sequence of SEQ ID NO: 1 and cypress essential oil.

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