KR101203128B1 - Composition for Treating Atopic Dermatitis Comprising Procyanidin B3 Compound or Novel Condensed Tannin Compound As an Active Ingredient - Google Patents

Abstract

The present invention relates to a composition for the treatment, amelioration or alleviation of atopic dermatitis comprising a procyanidin B3 compound represented by Formula 1 or a condensed tannin compound represented by Formula 2 as an active ingredient. Compounds of formulas (1) and (2) of the present invention show therapeutic efficacy of atopic dermatitis in atopic dermatitis animal model NC / Nga mice, and show the number of eosinophils and the level of IgE in blood taken from NC / Nga mice. Reduce the levels of IL-4, 5 and 13, the Th2 immunoregulatory cytokines in blood and lymphocytes, and inhibit the expression of COX mRNA and protein in iNOS of cells taken from NC / Nga mouse skin. It is very likely to be developed as an immunotherapeutic agent for atopic dermatitis.

Rosa multiflora Thunberg root, extract, atopic dermatitis. Anti-atopy compounds, procyanidin B3, condensed tannin compounds, immunomodulators, NC / Nga mice, eosinophils, Th2 cytokines, iNOS, COX-2

Description

Composition for Treating Atopic Dermatitis Comprising Procyanidin B3 Compound or Novel Condensed Tannin Compound As an Active Ingredient}

The present invention relates to a composition for the treatment of atopic dermatitis comprising a procyanidin B3 compound or a novel condensed tannin compound as an active ingredient.

The term atopy is a term that has the meaning "weird" or "inappropriate" in origin. Atopic dermatitis is a relatively common chronic inflammatory skin disease that occurs most often in infancy and childhood, and improves and worsens.It can be diagnosed with three characteristics: individual or family history of atopy, severe itching, and eczema. May be exacerbated by seasonal and climate change, irritation and allergies.

The etiology of atopic dermatitis is not yet clear, but studies to date indicate a lack of functionalities due to hypersensitivity reactions with increased IgE antibodies or due to undifferentiated differentiation of T lymphocytes resulting from decreased cellular mediated immune function. Blockage of adrenergic receptors present in the skin is the cause of the onset. Therefore, atopic dermatitis is considered to be a genetic disease involving immunological abnormalities.

For the treatment and management of atopic dermatitis, it is common for most dermatologists to prescribe steroid hormones, that is, topical corticosteroids, to improve the inflammatory response along with moisturizers that keep the skin's surface moisturized. However, when the topical corticosteroids are used for a long time, there are problems that cause various skin side effects such as skin atrophy, vasodilation, pigmentation and swelling ancestors. Therefore, there is a need to develop a raw material or pharmaceuticals for treating atopic dermatitis having an anti-inflammatory effect without showing such side effects.

Rosa multiflora Thunberg is a plant belonging to the Rosaceae family and is widely distributed in East Asia such as Korea, China, and Japan. The length is about 5m and there are sparsely downward thorns on the whole. Leaves are alternate, odd one-time pinnate leaf, with thorns on petioles and veins. Small leaves 5-7, egg-shaped or upside down, 15-45 mm long, 5-28 mm butterfly. The front of the leaf is green and the back is light green or greyish green. There are sharp teeth on the edge and some hairs are not. The chin leaves are green, and are deeply divided like combs and have sharp tips. Flowers bloom in May-June, often red, but in different colors. The peduncle and the small peduncle have or have no hairs. The stamen is shorter than the petals, and the pistil is one in total and the same length as the operating table. Fruits ripen in September. This name was given to vines and rose blooms, and in the old days it was called brier or brier (food search, KFDA).

As a medicinal part of brier, youngsil (Rosae Multiflorae Fructus) is mainly used as a diuretic in folk medicine, but 'rose root', brier root, is used for diabetes, bleeding, rheumatoid arthritis and scabies. Few studies have reported using rose roots, and several reports of triterpenes and lignans have been isolated from rose roots. Korean Patent Application Publication No. 10-2002-0086326 discloses a brier extract and isolates therefrom. A cosmetic composition of antioxidant activity containing a procyanidin B3 compound as an active ingredient is disclosed. However, the therapeutic efficacy of atopic dermatitis has not yet been reported for extracts from brier and the active compounds contained in the extracts.

Numerous papers and patent documents are referenced and cited throughout this specification. The disclosures of the cited papers and patent documents are incorporated herein by reference in their entirety to better understand the state of the art to which the present invention pertains and the content of the present invention.

The present inventors have tried to find an active ingredient having a therapeutic effect of atopic dermatitis from natural products. As a result, a single compound procyanidin B3 and a new condensed tannin compound isolated from the root extract of Rosa multiflora Thunberg and a new condensed tannin compound were tested using Nc / Nga mouse, an animal model for atopic dermatitis, The present invention was completed by confirming that the atopic dermatitis treatment effect was excellent in the in vivo experiment.

Accordingly, an object of the present invention is to provide a composition for the treatment or improvement of atopic dermatitis comprising a procyanidin B3 compound or a novel condensed tannin compound as an active ingredient.

The objects and advantages of the present invention will become more apparent from the following detailed description of the invention, claims and drawings.

According to an aspect of the present invention, the present invention provides a pharmaceutical composition comprising (a) a pharmaceutically effective amount of a procyanidin B3 compound represented by Formula 1 or a condensed tannin compound represented by Formula 2 below; And (b) provides a pharmaceutical composition for the treatment or prevention of atopic dermatitis comprising a pharmaceutically acceptable carrier.

          [Formula 1] [Formula 2]

According to another aspect of the present invention, the present invention is (a) a cosmetically effective amount of a procyanidin B3 compound represented by the formula (1) or a condensed tannin compound represented by the formula (2); And (b) provides a cosmetic composition for improving atopic dermatitis comprising a cosmetically acceptable carrier.

According to another aspect of the invention, the present invention for improving atopic dermatitis comprising a procyanidin B3 compound represented by the formula (1) or a condensed tannin compound represented by the formula (2) as an active ingredient Provides a functional food composition.

The present inventors have tried to find an active ingredient having a therapeutic effect of atopic dermatitis from natural products. As a result, a single compound, procyanidin B3, isolated from Rosa multiflora Thunberg root extract, and a novel condensed tannin compound, were tested using Nc / Nga mouse, an animal model of atopic dermatitis, and various in vivo. The present invention was confirmed to show a very good atopic dermatitis treatment efficacy in vivo experiments.

The procyanidin B3 compound represented by the formula (1) or the condensed tannin compound represented by the formula (2), which is an active ingredient of the present invention, is a natural product, for example, from the root of Rosa multiflora Thunberg. Extracted ones may be used, but it is apparent to those skilled in the art that chemically synthesized compounds have the same effect as extracted ones.

Procyanidin B3 compounds represented by Formula 1 and condensed tannin compounds represented by Formula 2 may be isolated from Rosa multiflora Thunberg root extract.

The brier root extract in the present invention can be separated according to conventional methods known in the art for extracting extracts from natural products, i.e. using conventional solvents under the presence of conventional temperature, pressure.

As the extraction solvent for extracting the brier root extract of the present invention can be used a solvent that can be generally used in the extraction process, water, anhydrous or hydrous lower alcohol having 1 to 4 carbon atoms, acetone, ethyl acetate, butyl acetate and It is preferably extracted using a solvent selected from the group consisting of 1,3-butylene glycol.

From the Rosa multiflora Thunberg root extract, a single compound represented by the formula (1) or (2), which is an active ingredient, can be obtained by a separation and purification method of a conventional compound. For example, filtration using a silica gel or celite gel column, size-exclusion chromatography using ion column chromatography, ion-exchange chromatography, partition chromatography, affinity ( Affinity chromatography or a combination of these chromatography can be used to purify the separation.

Single compounds isolated from natural extracts are Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectroscopy (MALDI-TOF MS), Electron Ionization Mass Spectroscopy (EI MS), Chemical Ionization Mass Spectroscopy (CI), Fast Atom Bombardment Ionization (FABI) It can be finally identified by using mass spectrometry such as), infrared spectroscopy (IR Sectrum), Nuclear Magnetic Resonance (NMR), Circular Dichroism (CD) and the like.

Procyanidin B3 compound of formula 1 and condensed tannin compound of formula 2, which are the active ingredients of the composition of the present invention, show the efficacy of treating atopic dermatitis in animal model NC / Nga mice that induced atopic dermatitis, and increased eosinophilic leukocytes in atopic dermatitis. reduce the number of (eosinophil) and levels of immunoglobulin E (IgE), reduce the expression of interleukin-4, 5, 13, a Th2 immunoregulatory cytokine associated with lesions of atopic dermatitis, and NC / Nga mice Inhibits mRNA and protein expression of iNOS and COX-2 in skin cells.

The pharmaceutical composition for treating or preventing atopic dermatitis of the present invention includes a pharmaceutically acceptable carrier in addition to the compound of Formula 1 or Formula 2 as an active ingredient.

Pharmaceutically acceptable carriers included in the pharmaceutical compositions of the present invention are those commonly used in the preparation, such as lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia rubber, calcium phosphate, alginate, gelatin, Calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil, and the like It doesn't happen. In addition to the above components, the pharmaceutical composition of the present invention may further include a lubricant, a humectant, a sweetener, a flavoring agent, an emulsifier, a suspending agent, a preservative, and the like. Suitable pharmaceutically acceptable carriers and formulations are described in detail in Remington's Pharmaceutical Sciences (19th ed., 1995).

The appropriate dosage of the pharmaceutical composition of the present invention may vary depending on factors such as the formulation method, administration method, age, body weight, sex, pathological condition, food, administration time, administration route, excretion rate, . On the other hand, the oral dosage amount of the pharmaceutical composition of the present invention is preferably 0.001-100 mg / kg (body weight) per day.

The pharmaceutical composition of the present invention can be administered orally or parenterally, and when administered parenterally, it can be administered by local application to the skin, intravenous injection, subcutaneous injection, muscle injection, intraperitoneal injection, transdermal administration, . Given that the pharmaceutical composition of the present invention is applied for the treatment of atopic dermatitis, administration of the composition is preferably applied topically to the skin.

The concentration of the compound of Formula 1 or Formula 2, which is an active ingredient included in the composition of the present invention, may be determined in consideration of the purpose of treatment, the condition of the patient, the period of time required, and the like, and is not limited to a specific range of concentration.

The pharmaceutical composition of the present invention may be formulated into a unit dose form by formulating it using a pharmaceutically acceptable carrier and / or excipient according to a method which can be easily carried out by a person having ordinary skill in the art to which the present invention belongs. Or by intrusion into a multi-dose container. The formulations may be in the form of solutions, suspensions or emulsions in oils or aqueous media, or in the form of excipients, powders, granules, tablets or capsules, and may additionally contain dispersing or stabilizing agents.

According to a preferred embodiment of the present invention, the pharmaceutical composition of the present invention has an external dermal formulation. The external dermal formulation is not particularly limited and is preferably a powder, gel, ointment, cream, liquid or aerosol formulation.

The compound of Formula 1 or Formula 2 of the present invention can be used as an active ingredient in cosmetic compositions for improving atopic dermatitis. As used herein, the term “cosmetic effective amount” means an amount sufficient for the compounds of the invention described above to achieve atopic dermatitis improving efficacy.

The cosmetic composition of the present invention may be prepared in any form conventionally produced in the art and may be in the form of a solution, suspension, emulsion, paste, gel, cream, lotion, powder, soap, surfactant- , Oil, powder foundation, emulsion foundation, wax foundation and spray, but is not limited thereto. More specifically, it can be manufactured in the form of a soft lotion, a nutritional lotion, a nutritional cream, a massage cream, an essence, an eye cream, a cleansing cream, a cleansing foam, a cleansing water, a pack, a spray or a powder.

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

In the case where the formulation of the present invention is a powder or a spray, lactose, talc, silica, aluminum hydroxide, calcium silicate or polyamide powder may be used as a carrier component. Especially, in the case of a spray, a mixture of chlorofluorohydrocarbons, propane / Propane or dimethyl ether.

When the formulation of the present invention is a solution or an emulsion, a solvent, a dissolving agent or an emulsifying agent is used as a carrier component, and examples thereof include water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, , 3-butyl glycol oil, glycerol aliphatic ester, polyethylene glycol or sorbitan fatty acid esters.

In the case where the formulation of the present invention is a suspension, a carrier such as water, a liquid diluent such as ethanol or propylene glycol, a suspending agent such as ethoxylated isostearyl alcohol, polyoxyethylene sorbitol ester and polyoxyethylene sorbitan ester, Cellulose, aluminum metahydroxide, bentonite, agar or tracant, etc. may be used.

When the formulation of the present invention is a surfactant-containing cleansing, the carrier component is an aliphatic alcohol sulfate, an aliphatic alcohol ether sulfate, a sulfosuccinic acid monoester, an isethionate, an imidazolinium derivative, a methyltaurate, a sarcosinate, a fatty acid amide. Ether sulfates, alkylamidobetaines, aliphatic alcohols, fatty acid glycerides, fatty acid diethanolamides, vegetable oils, lanolin derivatives or ethoxylated glycerol fatty acid esters and the like can be used.

In addition to the active ingredient and the carrier ingredient, the ingredients included in the cosmetic composition of the present invention include ingredients conventionally used in cosmetic compositions, and include, for example, conventional auxiliaries such as antioxidants, stabilizers, solubilizers, vitamins, pigments and flavorings. It may include.

In addition, the compound of Formula 1 or Formula 2 of the present invention can be used as an active ingredient in a functional food composition for improving atopic dermatitis.

The functional food composition of the present invention includes components that are ordinarily added during the manufacture of food, and includes, for example, proteins, carbohydrates, fats, nutrients, and seasonings. For example, when prepared with a drink, flavoring agents or natural carbohydrates may be included as additional ingredients in addition to the active ingredient. For example, natural carbohydrates include monosaccharides (eg, glucose, fructose, etc.); Disaccharides (eg maltose, sucrose, etc.); oligosaccharide; Polysaccharides (eg, dextrins, cyclodextrins, etc.); And sugar alcohols (eg, xylitol, sorbitol, erythritol, and the like). As the flavoring agent, natural flavoring agents (e.g., taumartin, stevia extract, etc.) and synthetic flavoring agents (e.g., saccharin, aspartame, etc.) can be used.

The features and advantages of the present invention are summarized as follows:

(i) The procyanidin B3 compound of formula 1 or the condensed tannin compound of formula 2, which is an active ingredient of the composition of the present invention, has a therapeutic, alleviating or alleviating activity of atopic dermatitis.

(Ii) The compound of formula 1 or formula 2, which is an active ingredient of the composition of the present invention, showed significant therapeutic efficacy of atopic dermatitis in atopic dermatitis animal model NC / Nga mice.

(Iii) In addition, the compounds of Formula 1 or Formula 2 of the present invention reduced the number of eosinophilic leukocytes and the levels of IgE in blood taken from NC / Nga mice, and was a Th2 immunoregulatory cytokine in blood and lymphocytes. Levels of IL-4, 5 and 13 were reduced and inhibited the expression of COX mRNA and protein in iNOS of cells taken from NC / Nga skin.

As described in detail above, the present invention provides a composition for the treatment, amelioration or alleviation of atopic dermatitis comprising a procyanidin B3 compound represented by Formula 1 or a condensed tannin compound represented by Formula 2 as an active ingredient. It is about. Compounds of formulas (1) and (2), which are the active ingredients of the composition of the present invention, exhibit very good atopic dermatitis treatment efficacy in NC / Nga mice, which are animal models of atopic dermatitis, and the number of eosinophilic leukocytes in blood taken from NC / Nga mice. And reduced levels of IgE, decreased levels of the Th2 immunoregulatory cytokines IL-4, 5 and 13 in blood and lymphocytes, and reduced COX mRNA and protein of iNOS in cells taken from NC / Nga mouse skin. It is very likely to be developed as an immunotherapeutic agent for atopic dermatitis because it has the effect of suppressing expression.

Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these embodiments are only for describing the present invention in more detail and that the scope of the present invention is not limited by these embodiments in accordance with the gist of the present invention .

Example

Example 1: Isolation of Active Compound

   1-1. Experimental material

The root of Rosa multiflora Thunberg used in this experiment was purchased from Gyeongdong Market in March 2007 and used after botanical appraisal compared to the root of brier directly collected from Yeongcheon, Gyeongbuk.

   1-2. TLC test

Brier root extract extracted with 80% acetone was tested by Thin Layer Chromatography (TLC). Benzene: ethylformate: formic acid = 1: 7: 1 using a Silica gel 60 F ₂₅₄ plate, sprayed with 10% sulfuric acid and an analdehyde aldehyde sulfuric acid and heated to confirm the FeCl ₃ Each band was identified using the method of color development (see FIG. 2).

   1-3. Extraction and Component Separation and Structure Identification

The dried bark root material (1.7 kg) was extracted three times at room temperature with 80% acetone for 24 hours, filtered and the extract was concentrated under reduced pressure to obtain 160 g of the extract. The extract was suspended in H ₂ O and filtered under reduced pressure with Celite 545 (Duksan pure chemical, Ansan, Korea), followed by Sephadex LH-20 column chromatography (2 kg, 10 ㅧ 80 cm) for the water-soluble portion. The mobile phase was eluted with 10% of methanol from water, increasing to 100%, and divided into three sub-fractions in total. For fraction 2 (Fr. 2), MCI-gel CHP20P (600 g, 5 X 60 cm) was divided into a total of four subfractions using a water-methanol gradient system, and repeatedly MPLC ( Middle pressure liquid column chromatography was performed to obtain 150 mg of catechin- (4α → 6) -catechin (ProB-3) and 237 mg of ent-guibourtinidol- (4β → 6) -catechin (RM-1).

Example 2: Structure

   2-1. Structure Identification of catechin- (4α → 6) -catechin (Procyanidin B-3)

The property and structure data of the isolated Procyanidin B3 compound were as follows.

Brown amorphous powder

EI-MS m / z: 578.1655 [M] ⁺ (see FIG. 4)

IRυ ^KBr _max Cm ^-1 : 3350 (OH), 1600 and 1475 (aromatic C = C) cm-1 (see Figure 5)

1H-NMR (600 MHz, DMSO-d6 + D2O) and 13C-NMR (125 MHz, DMSO-d6 + D2O): data is shown in Table 1 below.

TABLE 1 ¹ H- and ¹³ C-NMR data of Procyanidin B-3 (in DMSO- d ₆ + D ₂ O)

   2-2. Structural Identification of ent-guibourtinidol- (4β → 6) -catechin (RM-1)

The properties and structural data of the newly isolated condensed tannin compound RM-1 were as follows.

Reddish brown amorphous powder,

[α] ²⁵ _D : +100 Hz (c 0.0014, MeOH)

HRFAB-MS m / z 545.1453 [M ⁻ H] ⁻ (calculated for C30H25O10,545.1448) (see FIG. 6)

IRυ ^KBr _max Cm ⁻¹ : 3350 (OH), 1600 and 1475 (aromatic C = C) cm ⁻¹ (see FIG. 7)

cd [θ] ₂₈₆ 22.25, [θ] ₂₈₁ 0, [θ] ₂₇₂ -35.18, [θ] ₂₄₈ 0, [θ] ₂₄₃ 18.19, [θ] ₂₃₉ 0 (see Fig. 8)

¹ H-NMR (600 MHz, DMSO- d ₆ + D ₂ O) and ¹³ C-NMR (125 MHz, DMSO- d ₆ + D ₂ O): data are shown in Table 2 below.

Correlation based on 2D-NMR is shown in FIG. 9.

TABLE 2 ¹ H- and ¹³ C-NMR data of RM-1 (in DMSO- d ₆ + D ₂ O)

Example 3: Anti-Atopic Efficacy Assessment

   3-1. Establish an Animal Model for Atopic Dermatitis

A total of 20 five-week-old female Nc / Nga mice (Vestergaard C, Yoneyama H, Murai M, Nakamura K, Tamaki K, Terashima Y, Imai T, Yoshie O, Irimura T, Mizutani H and Matsushima K. Overproduction of Th2 -specific chemokines in NC / Nga mice exhibiting atopic dermatitis-like lesions, J Clin Invest 104 (8), 1097-105; 1999) in a 12 hour light / 12 hour dark condition cycle, 22-23 ° C. Atopic dermatitis was induced by using a mite patch and DPCP at 55 ° C. and 15 ° C., and the experimental group was divided into four groups.

Group I: Negative control group (vehicle) baseline

Group II: 1% ProB-3 Compound Administration Group as Experimental Group

Group III: 1% RM-1 Compound Administration Group as Experimental Group

Group IV: positive control group treated with 1% plancol

   3-2. Preparation of Cream Samples (Ointments)

O / W cream was prepared according to the composition of Table 3 below.

TABLE 3 Composition of O / W Cream Containing ProB3 or RM-1

   3-3. Efficacy evaluation

3-3-1. SCORAD

The skin appearance of atopic dermatitis of NC / Nga mice to which the sample containing the active ingredient and the samples of the negative control group and the positive control group were applied to the skin was observed for 4 weeks (see photo of FIG. 11). In addition, the SCORAD method was used to assess the extent of erythema, hemorrhage, edema, abrasion, erosion, scaling and dryness for 24 weeks. The sum was evaluated (see FIG. 12). As a result of SCORAD evaluation, it was confirmed that atopic dermatitis improved in the group administered 1% ProB-3 and 1% RM-1.

3-3-2. Eosinophil Count

NC / Nga mouse blood was collected in a capillary tube, and 150 μl of saline was added to 30 μl of mouse whole blood, which was diluted 6-fold. Eosinophils were counted using a Sysmex XE-2100 hematology analyzer. The calculation of eosinophilic leukocytes was performed by making peripheral blood smears, staining Wright-Giemsa, and differentially calculating 200 leukocytes. Experimental results showed that after administration of 1% Pro-B3 and 1% RM-1, the number of eosinophils (Eosinophil) was significantly lower than before administration (see FIG. 13).

3-3-3. IgE level measurement

NC / Nga mouse blood was collected in a capillary tube, and serum IgE (immunoglobulin E) levels were measured in the blood of mice using an ELISA method. Experimental results showed that the levels of IgE in the 1% Pro-B3 and 1% RM-1 group was significantly reduced compared to before administration (see results in FIG. 14).

3-3-4. Th2 cytokine

Th2 cytokines (IL-4, IL-5 and IL-13) were measured by taking samples from the blood and spleen of NC / Nga mice. For blood samples, blood was drawn from the aorta to measure levels of Th2 cytokines in the serum. In the case of spleen samples, spleens were pulverized and passed through a mesh to separate the cells into single cells, and RBC was dissolved by adding RBC lysis buffer. Subsequently, the supernatant was removed by centrifugation, and then cultured in a 24 well plate by adding RPMI medium so that the lymphocyte concentration was 1 × 10 ⁶ / ml. After 3 days the levels of Th2 cytokines IL-4, IL-5 and IL-13 in cultured media were measured. As a result, it was confirmed that after administration of 1% Pro-B3 and 1% RM-1, the levels of Th2 cytokines IL-4, IL-5 and IL-13 were significantly reduced (see FIG. 16).

3-3-5. mRNA and protein expression measurements of iNOS and COX

NC / Nga mice that developed atopic dermatitis were treated with 1% Pro-B3 (Procyanidin B3 compound of Formula 1) and 1% RM-1 (new condensed tannin compound of Formula 2), and mouse skin cells were isolated to isolate iNOS and MRNA levels of COX-2 were measured using the RT-PCR method. PCR primers used for the measurement of mRNA levels are shown in the table below.

As a result, it was confirmed that mRNA expression of iNOS and COX-2 was significantly suppressed in the 1% Pro-B3 and 1% RM-1 administration groups (see FIGS. 17 and 18).

Meanwhile, Pro-B3 and RM-1 were administered to NC / Nga mice that developed atopic dermatitis, and proteins were separated from mouse skin, and the expression levels of iNOS and COS-2 proteins were measured by western blotting. . As a result, it was confirmed that iNOS and COX-2 protein expression was significantly suppressed in the administration group of 1% Pro-B3 and 1% RM-1 (see FIGS. 19 and 20).

3-3-6. Summary of In Vivo Experiments

To evaluate the anti-atopic effect of Pro-B3 and RM-1 compounds isolated from the roots of Rosa multiflora Thunberg, Nc / Nga mice were developed using DPCP and mite patches to induce atopic dermatitis. In vivo tests were performed.

Experimental results show that ProB3 and RM-1 compounds have anti-atopic efficacy by inhibiting the number of Eosinophil, IgE levels, Th2 cytokines, iNOS and COX-2 proteins as well as in clinical observations. Proven. These compounds are very likely to be developed as preventive and therapeutic agents for atopy.

Having described the specific part of the present invention in detail, it is apparent to those skilled in the art that the specific technology is merely a preferred embodiment, and the scope of the present invention is not limited thereto. Accordingly, the actual scope of the present invention will be defined by the appended claims and their equivalents.

1 is a photograph showing the root portion and the flower portion of the brier ( Rosa multiflora Thunberg).

Figure 2 is a photograph showing the pattern tested by TLC (Thin Layer Chromatography) of Rosa multiflora Thunberg root extract. TLC was performed on benzene, ethylformate and formate = 1: 7: 1 (volume ratio) as mobile phase using pre-coated silica gel 60 F254 plates. In Fig. 2, A is a spot detected by spraying 10% sulfuric acid, B is a spot detected by spraying anisealdehyde-sulfuric acid, C is heated by spraying anisealdehyde-sulfuric acid After detecting with FeCl ₃ solution. 1: Extracting the brier roots purchased from Gyeongdong Market with 80% acetone, dissolving them in water and filtering them; 2: Extracting the brier roots collected from Yeongcheon, Gyeongbuk with 80% acetone, 3: Isolated from the brier roots ent-guibourtinidol- (4β → 6) -catechin (RM-1), 4: catechin- (4α → 6) -catechin (ProB-3) isolated from the roots of the brier.

Figure 3 is a schematic diagram showing a process of obtaining the extracts, fractions from the root of Rosa multiflora Thunberg, and again separating the compounds of Formula 1 and Formula 2.

Figure 4 shows the EI-MS spectrum of the procyanidin B3 compound of formula (1).

Figure 5 shows the results of the FT-IR spectrum of the procyanidin B3 compound of formula (I).

Figure 6 shows the EI-MS spectrum of the condensed tannin compound of formula (2).

Figure 7 shows the FT-IR spectrum of the condensed tannin compound of formula (2).

Figure 8 shows the CD spectrum results of the condensed tannin compound of formula (2).

9 shows the major correlations of COSY (H-H) and CMBC (C-> H) of the condensed tannin compound of Formula 2. FIG.

10A shows the chemical structure of the procyanidin B3 compound of formula 1 (ProB-3), and FIG. 10B shows the chemical structure of the condensed tannin compound of formula 2 (RM-1).

11 is a photograph showing the appearance of atopic dermatitis after administration of the compound of Formula 1 or Formula 2 of the present invention to NC / Nga mouse, an atopic dermatitis induced animal model. Negative controls were measured by baseline and positive controls were administered PLANCOL.

12 is a result of measuring the degree of improvement of atopic dermatitis after administration of the compound of Formula 1 or Formula 2 of the present invention to NC / Nga mouse, an atopic dermatitis induced animal model by the SCORAD method.

Figure 13 shows the results of measuring the number of eosinophilic leukocytes (Eosinophil) before and after administration of the compound of Formula 1 or Formula 2 of the present invention to NC / Nga mouse, an atopic dermatitis induced animal model. Negative controls were measured by baseline and positive controls were administered PLANCOL.

Figure 14 shows the level of IgE (immunoglobulin E) in serum before and after administration of the compound of Formula 1 or Formula 2 of the present invention to NC / Nga mice, atopic dermatitis induced animal model using ELISA (Enzyme-Linked Immunosorbent Assay) Show the measurement results. Negative controls were measured by baseline and positive controls were administered PLANCOL.

Figure 15 shows the levels of Th2 cytokines IL (interleukin) -4, IL-5 and IL-13 in the blood after administration of a compound of Formula 1 or Formula 2 of the present invention to NC / Nga mice that are animal models of atopic dermatitis Show measured results. Negative controls were determined by Baseline (Blank) and positive controls were administered PLANCOL. (student t-test; * p <0.05, ** p <0.01, *** p <0.001; compare baseline (blank) with each experimental group, n = 5).

Figure 16 shows the Th2 cytokines IL (interleukin) -4, IL-5 and IL-13 in lymphocytes after administration of the compound of Formula 1 or Formula 2 of the present invention to NC / Nga mice, which are animal models of atopic dermatitis Shows the result of measuring the level of. Negative controls were determined by Baseline (Blank) and positive controls were administered PLANCOL. (student t-test; * p <0.05, ** p <0.01, *** p <0.001; compare baseline (blank) with each experimental group, n = 5).

Figure 17 shows the results of measuring the expression level of the mRNA of COX-2 mRNA in skin cells after administration of the compound of Formula 1 or Formula 2 of the present invention to NC / Nga mouse, an atopic dermatitis induced animal model. Negative controls were determined by Baseline (Blank) and positive controls were administered PLANCOL. (student t-test; * p <0.05, ** p <0.01, *** p <0.001; compare baseline (blank) with each experimental group, n = 5).

FIG. 18 shows the results of RT-PCR measurement of mRNA expression of iNOS in skin cells after administration of a compound of Formula 1 or Formula 2 to a NC / Nga mouse, an atopic dermatitis-induced animal model. Negative controls were determined by Baseline (Blank) and positive controls were administered PLANCOL. (student t-test; * p <0.05, ** p <0.01, *** p <0.001; compare baseline (blank) with each experimental group, n = 5).

19 is a result of measuring the degree of protein expression of COX-2 in skin cells after administration of the compound of Formula 1 or Formula 2 to NC / Nga mouse, an atopic dermatitis-induced animal model by Western blotting Shows. Negative controls were determined by Baseline (Blank) and positive controls were administered PLANCOL. (student t-test; * p <0.05, ** p <0.01, *** p <0.001; compare baseline (blank) with each experimental group, n = 5).

Figure 20 shows the result of Western blotting the protein expression level of iNOS in skin cells after administration of the compound of Formula 1 or Formula 2 of the present invention to NC / Nga mouse, atopic dermatitis induced animal model . Negative controls were determined by Baseline (Blank) and positive controls were administered PLANCOL. (student t-test; * p <0.05, ** p <0.01, *** p <0.001; compare baseline (blank) with each experimental group, n = 5).

Claims (9)

(a) a pharmaceutically effective amount of a compound represented by the following formula (2); And (b) a pharmaceutical composition for treating or preventing atopic dermatitis, comprising a pharmaceutically acceptable carrier.

     [Formula 2] The pharmaceutical composition of claim 1, wherein the composition has an external preparation for skin. The pharmaceutical composition according to claim 2, wherein the external application formulation is a powder, gel, ointment, cream, liquid or aerosol formulation. (a) a cosmetically effective amount of a compound represented by the following formula (2); And (b) a cosmetic composition for improving atopic dermatitis comprising a cosmetically acceptable carrier.

    [Formula 2] The group of claim 4 wherein the composition consists of a solution, suspension, emulsion, paste, gel, cream, lotion, powder, soap, surfactant-containing cleansing, oil, powder foundation, emulsion foundation, wax foundation and spray Cosmetic composition, characterized in that it has a formulation selected from. Functional food composition for improving atopic dermatitis comprising a compound represented by the formula (2) as an active ingredient.

     [Formula 2] delete delete delete

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