Composition containing monoacetyldiglyceride compound as active ingredient for preventing or treating atopic dermatitis

Abstract

The present invention relates to a pharmaceutical composition, functional health food composition, quasi-drug composition, and cosmetic composition containing a monoacetyldiacylglycerol compound as an active ingredient for preventing, treating or improving atopic dermatitis. The monoacetyldiacylglycerol compound of the present invention has an excellent effect of suppression of IL-4 and IgE secretion, thereby overcoming side effects of an atopic dermatitis treatment agent currently in use, having no toxicity, and exhibiting an excellent treatment effect, and thus can be useful as a composition for preventing, treating, and ameliorating atopic dermatitis.

Claims

1. A method of treating atopic dermatitis, comprising: administering to a patient in need thereof a composition comprising a monoacetyldiacylglycerol compound of Formula 1 as an active ingredient, Formula 1 being ##STR00006## wherein R1 and R2 are independently a fatty acid group of 14 to 20 carbon atoms.

2. The method according to claim 1, wherein R1 and R2 are independently selected from the group consisting of palmitoyl, oleoyl, linoleoyl, linolenoyl, stearoyl, myristoyl, and arachidonoyl.

3. The method according to claim 1, wherein R1 and R2(R1/R2) is selected from the group consisting of oleoyl/pahnitoyl, palmitoyl/oleoyl, palmitoyl/linoleoyl, palmitoyl/linolenoyl, pahnitoyl/arachidonoyl, palmitoyl/stearoyl, palmitoyl/paimitoyl, oleoyl/stearoyl, linoleoyl/palmitoyl, linoleoyl/stearoyl, stearoyl/linoleoyl, stearoyl/oleoyl, myristoyl/linoleoyl, myristoyl/oleoyl.

4. The method according to claim 1, wherein the monoacetyldiacylglycerol compound is a compound of Formula 2: ##STR00007##

5. The method according to claim 1, wherein the monoacetyldiacylglycerol compound of Formula 1 is separated and extracted from natural deer antler.

6. The method according to claim 1, wherein the administration of the monoacetyldiacylglycerol compound reduces the secretion of IL-4 in the patient.

7. The method according to claim 1, wherein the administration of the monoacetyldiacylglycerol compound reduces the secretion of IgE in the patient.

8. The method according to claim 1, wherein the monoacetyldiacylglycerol compound of Formula 1 is present in the composition in an amount of 0.001 to 50% by weight of the composition.

9. The method according to claim 1, wherein the composition is in the form of a health functional food composition.

10. The method according to claim 1 wherein the composition is in the form of a quasi-drug composition.

11. The method according to claim 1, wherein the composition is in the form of a cosmetic composition.

12. The method according to claim 1, wherein the patient is a non-human patient.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 is a schematic view for explaining the causes of atopic.

(2) FIG. 2 is a diagram showing the expression level of IL-4 for up to two weeks after treatment of EC-18 in mice.

(3) FIG. 3 is a diagram showing the expression level of IgE for up to two weeks after treatment of EC-18 in mice.

(4) FIG. 4 is a photograph showing the degree of atopic disease for 3 weeks in mice of the EC-18 administration group and the mice of olive oil administration group (control group).

(5) FIG. 5 is a graph showing the expression level of Th2 cytokine (IL-4) when EC-18 was administrated to normal subjects for 4 weeks.

(6) FIG. 6 is a graph showing the expression level of Th1 cytokine (IFN-γ) when EC-18 was administrated to normal subjects for 4 weeks.

(7) FIG. 7 is a photograph of the atopic symptoms change after EC-18 was administrated to a patient having strong atopic symptom.

(8) FIG. 8 is a photograph of the atopic symptoms change after EC-18 was administrated to a patient having weak atopic symptom.

DETAILED DESCRIPTION OF THE INVENTION

(9) A more detailed description of the invention will be made by reference to the attached drawings, which are intended for better understanding of the present invention and will not limit the present invention. Hereinafter, unless otherwise noted, ratios and percentages are by weight.

EXAMPLE

Induction of Atopic Dermatitis by DNCB Application

(10) DNCB (1-Chloro-2,4-dinitrobenzene, Sigma Aldrich) was diluted to 0.2% by the solvent in which olive oil and acetone were mixed in a ratio of 1:4. Diluted DNCB was applied onto back of each 7-week-old Balb/c mouse whose hairs on back were removed. During the first week application was made seven times continuously, and from the second week, application was made every two days to induce atopy.

Experimental Example 1

Inhibition Effect of IL-4 Expression by EC-18

(11) A mixture of EC-18 and olive oil was orally administrated to the mouse 7 times during the first week by 100 μl each time in dose of 2 mq/rat, and after then, for 11 times, was administered orally by 100 μl each time in dose of 1 mg/rat, that is the mixture was orally administrated totally 18 times. For the measurement of IL-4, blood was collected from veins under the eyes of mice treated, through ocular blood (Eye bleeding) method. Before administration, one week after the administration, 2 weeks after administration, 3 weeks after administration, a total of four times, blood samples were collected using a Pasteur pipette (Pasteur pipet, Hilgenberg). After collecting the blood of about 350 μl in the EP tube, the blood was centrifuged to separate only serum and IL-4 present in the serum was analyzed by ELISA (enzyme-linked immunosorbent assay) method. Mouse IL-4 capture antibody (BD bioscience) was diluted with 1×PBS to 2 μg/ml, dispensed to each of 96-well plate (Thermo) for mouse L-4 ELISA by 200 μl, and then coated overnight and washed by a wash buffer in which 0.05% tween 20 was diluted in 1×PBS. After washing three times, 1% BSA was dispensed to each well by 200 μl and blocking buffer was placed for 1 hour at room temperature (RT), and washing was performed three times under the same conditions. Thereafter, the serum obtained from the mice was dispensed to each well by 100 μl, and the wells were placed for reaction at room temperature for 2 hours after dispensing, and then washed five times under the same conditions. After washing, detection antibody (1:500) conjugated-HRP (1:250) diluted with 1% BSA was dispensed to each well by 100 μl and then was placed for reaction at room temperature for 1 hour. After seven-times-washing, a substrate solution of 100 μl was dispensed to each well, and the color development was observed in a state where light is blocked. When the color development is completed, the stop solution (2N H.sub.2SO.sub.4+DW) was dispensed by 100 μl, and the amount of color development at 450 nm wavelength was measured at ELISA Reader. As a result, comparing the levels of IL-4 (the average of the measured value 4 times) in one week after the administration and two weeks after the administration, the IL-4 level was reduced by approximately 50% v in the control group administered with olive oil, on the other hand, the IL-4 level was reduced by 80% in the test group the administration of EC-18. That is, it was confirmed that IL-4 produced by the atopic induction was rapidly reduced by administration of EC-18 (FIG. 2).

Experimental Example 2

IgE Expression Inhibitory Effect by EC-18

(12) Using the serum separated from the mouse blood, was measured the expression levels of IgE in the ELISA method as shown in Experimental Example 1. In IgE ELISA for the mouse, IgE capture antibody (BD bioscience) was diluted with 1×PBS to 2 μg/ml, dispensed to each of 96-well plate (Thermo) by 200 μl, coated for 12 hours, washed using a wash buffer in which 0.05% tween 20 was diluted in 1×PBS. After washing three times, 1% BSA was dispensed to each well by 200 μl and blocking buffer was placed for 1 hour at room temperature (RT) conditions, and then final washing was performed._Thereafter, the serum obtained from the mice was dispensed to each well by 100 μl, and the wells were placed for reaction at room temperature for 1 hours after dispensing, and unreacted IgE is washed under the same conditions. After washing, detection antibody (BD bioscience) was diluted with 1% BSA to be 2 μg/ml and dispensed to each well by 100 μl and then was placed for reaction at room temperature for 1 hour. After six-times-washing, Streptoavidin-HRP (Thermo) was mixed in 1% BSA in a ratio of 1:10,000 to be dispensed by 100 μl to add the substrate to the reacting IgE. After incubation for 30 minutes at room temperature, washing was performed 6 times, a substrate solution was dispensed to each well, and the color development was observed in a state where light is blocked. When the color development is completed, the stop solution (2N H.sub.2SO.sub.4+DW) was dispensed by 50 μl, and the amount of color development at 450 nm wavelength was measured. As a result, comparing the levels of IgE in one week after the administration and two weeks after the administration, in the control group administered with only olive oil, the IgE level was reduced by approximately 22%, on the other hand, in the test group the administration of EC-18, the IgE level was reduced by about 50% (FIG. 3).

Experimental Example 3

Visual Inspection on Improvement Effect of Atopic by EC-18

(13) Atopic disease models commonly used in the laboratory is a technique that induces contact Hypersensitivity (CHS) by sensitization to DNCB (1-Chloro-2,4-dinitro-benzene). This technique is a technique of inducing atopic by diluting DNCB with a solvent in which acetone and olive oil are mixed in a ratio of 4:1 to be 0.2%, and then smearing the diluted on the skin, and is for the experiment for adjusting and maintaining the expression time so that artificially severe atopy-like appearance appears could be observed two weeks after the smearing. The degree of atopic disease was evaluated by dividing the state of erythema/emorrhage, scarring/dryness, edema), excoriation/erosion which are induced on the skin, respectively into three steps (0=null, 1=mild, 2=moderate, 3=severe) and then assigning the score up to 12 from zero. It was induced in the experimental group and the control group that the time when severe atopic status ((visual inspection index of 10) is reached was two weeks after the smearing of DNCB. Further, in order to maintain this condition, after two weeks, DNCB smearing was performed every two days.

(14) FIG. 4 is a photograph showing the degree of atopic disease for 3 weeks in mice of the EC-18 administration group (test group) and the mice of olive oil administration group (control group). As shown in FIG. 4, in the control group administered with olive oil, visual inspection index was changed to 3 points after one week, 10 points after two weeks and 6 points after three weeks. In contrast, in the test group the administration of EC-18, visual inspection index was changed to 1 point after one week, 8 points after two weeks and 3 points after three weeks. This proves the therapeutic efficacy on the lesions of atopic dermatitis is also superior in a group of administration of EC-18. That is, after two weeks, when the visual inspection index in the control group was 10, the visual inspection index in the test group (EC-18 administration group) stayed in 8, which indicates that the EC-18 inhibits the production of atopic. In addition, from the visual inspection index after three weeks, the test group (EC-18 continuously-fed group) showed rapid atopic treatment and recovery than the control group (In test group the visual inspection index was reduced from 8 to 3, and in the control group the visual inspection index was reduced from 10 to 6).

Experimental Example 4

IL-4 and IFN-γ Expression after EC-18 Administration

(15) EC-18 was administered to the normal healthy people (36 people) four weeks. EC-18 Lymphocytes separated from the blood before administration and after administration were incubated with ConA (concanavalin A). In order to measure the cytokine (IFN-γ and IL-4) which is separated from the lymphocyte (T cell) activated by Con A, culture medium was incubated for 42 hours, and were stored frozen. Purified antibody (capture antibody) of cytokine to be measured was dispensed to well (Nunc-immuno module, polysorp) by 0.2 μg/well, and left overnight at 4° C. to coat antigen. After well washing, blocking buffer (1% bovine serum albumin) treatment was performed for about 1 hour to inhibit the nonspecific binding. After washing the well, a cell culture solution was placed into each well and the antibody was combined with coating antigen for 2 hours. Antibody to which a peroxidase or biotin is bound was added, and the well was left at room temperature for 1 hour and thereafter washed. TMB (3,3′,5,5′-Tetramethyl benzidine) substrate was directly added to antibody to which peroxidase is bound, and SAv-HRP (Streptavidin-Horseradish Peroxidase) was added to antibody to which biotin is bound, to react for 30 minutes, and then washed and TMB substrate was added. After approximately 10-30 minutes, stop solution was added into the well and absorbance at 450-570 nm was measured with Automatic Microplate reader (Molecular Devices, CA, USA). In the same manner, absorbance according to cytokine solution concentration was measured to plot standard curve, and the amount of cytokine contained in the sample was calculated by substituting the absorbance of the sample group. The change amount of cytokine calculated in this manner and significance (P<0.05) were shown in flowing Table 1 and FIGS. 5-6.

(16) FIG. 5 is a graph showing the expression level of Th2 cytokine (IL-4) when EC-18 was administrated to normal subjects for 4 weeks. As shown in FIG. 5, EC-18 administration to normal subjects for 4 weeks reduced the expression levels of Th2 cytokine (IL-4). FIG. 6 is a graph showing the expression level of Th1 cytokine (IFN-γ) when EC-18 was administrated to normal subjects for 4 weeks. As shown in FIG. 6, even though EC-18 was administrated to normal subjects for 4 weeks, the change did not occur uniformly in expression levels of cytokine Th1 (IFN-γ).

(17) TABLE-US-00001 TABLE 1 Before EC-18 After EC-18 administration administration (N = 36) (N = 36) P-value IL-4 (pg/mL) 246.6 ± 186.9 96.1 ± 64.2 <0.001 IFN-γ (pg/mL) 686.4 ± 162.2 611.9 ± 209.7 0.473

Experimental Example 5

Observation of Atopic Symptom Alleviation in Patients with Atopic

(18) To patients with severe atopic symptoms, the EC-18 was administrated every day at a dose of 500 mg/day for 2 weeks and atopic symptoms change which was taken were shown in FIG. 7. To a patient having a mild skin rash, the EC-18 was administrated every day at a dose of 500 mg/day for 2 days and atopic symptoms change which was taken were shown in FIG. 8. It was confirmed that as shown in FIGS. 7 and 8, when the EC-1 was administered to patients with atopic, the atopic symptoms are relaxed.

(19) From the above description, a person skilled in the art will appreciate that the invention may be embodied in other specific forms without changing the technical spirit or essential characteristics. In this regard, the examples described above are intended to be illustrative in all respects and it should be understood as not limiting. The scope of the invention should be understood to include all ranges of the above detailed description and the appended claims, rather than the ranges of the specific examples, as well as all such modifications derived from those equivalents.

INDUSTRIAL APPLICABILITY

(20) Monoacetyldiacylglycerol compound of the present invention can be used for the preparation of a pharmaceutical composition, functional health food composition, quasi-drug composition, and cosmetic composition for preventing, treating or improving atopic dermatitis.