COMPOSITION FOR PREVENTION OR TREATMENT OF ALLERGIC DISEASE INCLUDING INOTODIOL COMPOUND AS ACTIVE INGREDIENT

Abstract

Provided is a composition for prevention or treatment of an allergic disease, the composition including an inotodiol compound and a chaga mushroom (Inonotus obliquus) extract including an inotodiol compound as an active ingredient. More particularly, it was found that when an inotodiol compound or a chaga mushroom extract containing an inotodiol compound as one of the active ingredients is administered to a mouse model in which a food allergy was induced, symptoms induced by the allergy were alleviated and cured. Thus, the inotodiol compound or the chaga mushroom extract containing an inotodiol compound as an active ingredient is expected to be useful as a composition for prevention or treatment of an allergic disease.

Claims

1-14. (canceled)

15. A method for preventing or treating an allergic disease, the method comprising administering to a subject in need thereof a therapeutically effective amount of a chaga mushroom (Inonotus obliquus) extract including an inotodiol compound represented by Formula 1 as an active ingredient. ##STR00002##

16. The method of claim 15, wherein the chaga mushroom extract is at least one selected from the group consisting of dichloromethane fractions or chloroform fractions obtained by adding dichloromethane or chloroform to an extract obtained by extracting chaga mushroom with 70 percent (%) ethanol as a solvent, respectively.

17. The method of claim 15, wherein the method suppresses degranulation of mast cells.

18. The method of claim 15, wherein the allergic disease is induced by at least one selected from the group consisting of pollen, medicines, vegetable fibers, bacteria, food, dyes, and chemicals.

19. The method of claim 18, wherein the allergic disease is induced by food.

20. The method of claim 15, wherein the allergic disease is at least one selected from the group consisting of urticaria, anaphylaxis, allergic rhinitis, bronchial asthma, and atopic dermatitis.

21. A method for preventing or treating an allergic disease, the method comprising administering to a subject in need thereof a therapeutically effective amount of an inotodiol compound represented by Formula 1 as an active ingredient. ##STR00003##

22. The method of claim 21, wherein the method suppresses degranulation of mast cells.

23. The method of claim 21, wherein the allergic disease is induced by at least one selected from the group consisting of pollen, medicines, vegetable fibers, bacteria, food, dyes, and chemicals.

24. The method of claim 23, wherein the allergic disease is induced by food.

25. The method of claim 21, wherein the allergic disease is at least one selected from the group consisting of urticaria, anaphylaxis, allergic rhinitis, bronchial asthma, and atopic dermatitis.

Description

BRIEF DESCRIPTION OF DRAWINGS

[0049] FIG. 1 shows allergy symptom improvement followed by food allergy induction and treatment with an inotodiol compound and a chaga mushroom fraction including an inotodiol compound; FIG. 1(A) shows change in rectal temperature; FIG. 1(B) shows a level of relief or treatment of anaphylaxis symptoms; and FIG. 1(C) shows diarrhea score;

[0050] FIG. 2 shows change in (A) the number of eosinophils infiltrated in small intestine; and (B) the number of mast cells in the small intestine followed by food allergy induction and treatment with an inotodiol compound and a chaga mushroom fraction including an inotodiol compound;

[0051] FIG. 3 shows change in concentration of allergy-related IgE (A); and MCPT-I (B) in a serum followed by food allergy induction and treatment with an inotodiol compound and a chaga mushroom fraction including an inotodiol compound;

[0052] FIG. 4 shows test results of CD4.sup.+T cell immunoreactivity followed by food allergy induction and treatment with an inotodiol compound and a chaga mushroom fraction including an inotodiol compound; FIG. 4(A) shows expression changes in IL-13, IL-5, and IFN-γ, which are cytokine produced by Th2 CD4.sup.+T cells; and FIG. 4(B) shows a level of activation by ovalbumin of CD4.sup.+T cells expressing DO11.00 TCR, i.e., an ovalbumin-derived peptide-specific T cell receptor and a level of the following cell division; and

[0053] FIG. 5 shows test results of immunoreactivity of mast cells followed by food allergy induction and treatment with an inotodiol compound and a chaga mushroom fraction including an inotodiol compound, and shows the change in rectal temperature in mice in which degranulation of mast cells is induced (A) and the change in MCPT-1 in blood, by a passive systemic anaphylaxis method.

BEST MODE

[0054] Hereinafter, the present disclosure will be described in more detail with reference to preferable Examples. However, the present disclosure is not limited to the Example described herein but may be embodied in other forms. Rather, the Example are provided herein such that the description presented here is thorough and complete, and the concept of the present disclosure is fully conveyed to one of ordinary skill in the art.

Example 1. Isolation of Inotodiol Compound

Example 1-1. Preparation of Chaga Mushroom Extraction and Content Check of Inotodiol Compound

[0055] The inotodiol compound of the present disclosure was isolated from a chaga mushroom (Inonotus obliquus) extract.

[0056] 630 grams (g) of chaga mushroom was pulverized into fine particles, 3,000 mL of 70% [v/v] ethanol was added thereto, and extraction was performed under ultrasonic agitation at 40° C. and 90 hertz (Hz) three times to obtain 70% ethanol extract (3×3.0 liters (L)). Then, 58 g of dried brown 70% ethanol extract was obtained using a rotary evaporator

[0057] 58 g of chaga mushroom 70% ethanol extract was dissolved in water, followed by addition of (dichloromethane, CH.sub.2Cl.sub.2) to obtain 6.0 g of a dichloromethane fraction. After isolating the dichloromethane fraction, ethyl acetate (EtOAc) was added to the remaining fraction to obtain 3.2 g of an ethyl acetate fraction and 48.5 g of a water fraction.

[0058] High performance liquid chromatography (HPLC) was performed to measure a content of the inotodiol compound included in the obtained chaga mushroom extract and the fractions. HPLC was performed under a condition of an acetonitrile concentration gradient (0-30 minutes; 60%.fwdarw.95% [v/v], 30-60 minutes; 95% [v/v] constantly) using LC-10AD (Shimadzu Co. Kyoto, Japan). Each of the chaga mushroom extract and the fraction were dissolved in methanol to a concentration of 2 mg/mL, followed by filtering with a 0.2 μm syringe filter. Then, 10 μL thereof was injected. A content of the inotodiol compound was measured using a column of HECTOR-M 018 (4.6×250 mm, 5 μm particle size, RStech, Korea), at a flow of 1 mL/minute, with UV detection (210 nm). As a result, it was found that about 1% of the inotodiol compound was present in the 70% ethanol extract (EtOH Ext.), 7.8% of the inotodiol compound was present in the dichloromethane fraction (CH.sub.2C1.sub.2 Frac.), 0.9 of the inotodiol compound was present in the ethyl acetate fraction (EtOAc Frac.), and 0.05% of the inotodiol compound was present in the water fraction (H.sub.2O Frac.) having the lowest inotodiol compound content.

[0059] In the case of a chloroform fraction obtained by using chloroform instead of dichloromethane, the chloroform fraction was found to contain a similar amount of the inotodiol compound as the dichloromethane fraction.

Example 1-2. Isolation of Inotodiol Compound

[0060] The inotodiol compound was isolated from the dichloromethane fraction of chaga mushroom obtained in Example 1-1.

[0061] 2.5 g of the dichloromethane fraction was subjected to fraction through silica gel column chromatography using 1 L of each of mixed solvents in which n-hexane was mixed with ethyl acetate at a ratio of 20:1, 9:1, 4:1, 2:1, and 1:2 [v/v] according to a concentration gradient dissolution condition, thereby obtaining 13 minor fractions (Frac. C1-013). Frac. C6 was mixed with Frac. C7, and the mixture was subjected to silica gel column chromatography according to a solvent condition of a solution in which n-hexane is mixed with ethyl acetate at 20:1 [v/v], thereby isolating 210 mg of the inotodiol compound.

Example 2. Confirmation of Physicochemical Structure of Inotodiol Compound

[0062] Inotodiol;

[0063] White amorphous powder;

[0064] ESI-MS: [M+ Na].sup.+ 465.37 m/z (C.sub.30H.sub.500.sub.2: 442.27);

[0065] .sup.1HNMR and .sup.13C NMR (see Table 1 for the data);

[0066] According to the ESI-MS analysis result and .sup.13C NMR data, the molecular formula was found to be C.sub.30H.sub.50O.sub.2.

[0067] In .sup.13C-NMR for inotodiol, 30 peaks of carbon were observed, and it was found that at peaks of δ.sub.C121.2, 134.0, 134.4, and 134.8, 2 double bonds were present, and at peaks of δ.sub.C73.2 and 78.7, 2 hydroxyl groups were present (see Table 2). In .sup.1H-NMR spectrum (see Table 2), at 0.67, 0.76, 0.82, 0.88, 0.93, 1.60, and 1.69 (3H, s) a singlet methyl peak was observed, and at δ.sub.H 0.88, a doublet methyl peak was observed. Also, an olefinic peak corresponding to H-24 was observed at θδ.sub.H 5.16 (1H, t, J=7.2 Hz), and a peak of oxymethine bound to oxygen was observed at δ.sub.H 3.18 (1H, m, H-3) and at δ.sub.H 3.65 (1H, H-22).

[0068] The above result was compared with data reported in a paper (Du. D., et al., 2011) to verify the structure of the inotodiol compound.

TABLE-US-00001 TABLE 1 δ.sub.C (100 MHz) δ.sub.H (400 MHz) 1 35.4 2 27.6 3 78.7 3.18 (m, 1H) 4 38.7 5 50.2 0.94 (m, 1H) 6 18.9 7 26.3 8 134.4 9 134.0 10 36.8 11 20.8 12 28.9 13 44.5 14 49.2 15 30.7 16 30.7 17 47.0 1.55 (m, 1H) 18 15.5 0.67 (s, 3H) 19 18.0 0.93 (s, 3H) 20 41.5 1.97 (m, 1H) 21 12.4 0.88 (d, J = 6.5 Hz, 1H) 22 73.2 3.65 (m, 1H) 23 27.0 24 121.2 5.16 (t, J = 7.2 Hz, 1H) 25 134.8 26 25.7 1.60 (s, 3H) 27 17.9 1.69 (s, 3H) 28 27.7 0.90 (s, 3H) 29 15.2 0.76 (s, 3H) 30 24.1 0.82 (s, 3H)

<Experimental Example 1. Preparation of Allergy Mouse Model and Administration of Extract

[0069] To prepare allergy mouse models, 5-week-old male BALB/C mice were used. Food allergy models were prepared using ovalbumin of chicken as an allergy-inducing material (allergen).

[0070] For immunization of the mice, 20 μg of ovalbumin was mixed with 2 mg of alum, and the mixture was intraperitoneally injected to the mice two times every two weeks. Food allergy induction was induced by performing the second immunization injection and two weeks thereafter, 50 mg of ovalbumin was orally administered thereto five times every three days to cause an allergic response. In addition, to confirm allergy therapeutic effect, the chaga mushroom extract, the fraction, and the inotodiol compound obtained in Example 1 were orally administered to the mice every day during the administration period of ovalbumin. Here, as a positive control, a mice group was used to which dexamethasone was administered every day. 50 mg of ovalbumin alone or the chaga mushroom extract, the fraction, or the inotodiol compound was lastly administered thereto, followed by observation on symptoms caused by allergy one hour thereafter. For each experimental group, six mice were used, and each experimental group is shown in Table 2.

TABLE-US-00002 TABLE 2 Group Sample treatment Normal Not treated Allergy induction Ovalbumin (food allergy induction) Positive control 20 mg/kg of ovalbumin and dexamethasone (Dexa.) EtOH Ext. 320 mg/kg of ovalbumin and chaga mushroom 70% ethanol CH.sub.2C1.sub.2 Frac. 320 mg/kg of ovalbumin and the dichloromethane fraction of H.sub.2O Frac. 320 mg/kg of ovalbumin and the water fraction of chaga Inotodiol 20 mg/kg of ovalbumin and inotodiol compound

<Experimental Example 2. Confirmation of Improvement in Allergy Symptoms

Experimental Example 2-1. Confirmation of Change in Rectal Temperature

[0071] In order to see the change in rectal temperature followed by food allergy induction and drug treatment, mice corresponding to each group were subjected to measurement of rectal temperature. Rectal temperature was measured by inserting the tip of the sensor of a rectal thermometer into rectum. The results thereof are shown in FIG. 1(A).

[0072] As shown in FIG. 1(A), it was found that an allergy-induced group (Allergy) had a lower rectal temperature than a normal group (Normal). On the other hand, it was found that the group treated with dexamethasone (Dexa.) or the group treated with the inotodiol compound isolated from chaga mushroom (Inotodiol), the group treated with the chaga mushroom ethanol extract including an inotodiol compound (EtOH Ext.), and the group treated with the dichloromethane fraction (CH.sub.2Cl.sub.2 Frac.) of the present disclosure had a higher rectal temperature than the food allergy-induced group (Allergy). In particular, the group treated with the inotodiol compound and the group treated with the dichloromethane fraction were found to have a similar level of rectal temperature with the normal group. On the other hand, the group treated with the water fraction of chaga mushroom (H.sub.2O Frac.) was found to have a similar level of rectal temperature with the food allergy-induced group.

[0073] Accordingly, it was found that the inotodiol compound and the dichloromethane fraction of chaga mushroom including an inotodiol compound of the present disclosure improved a low rectal temperature, i.e., one of the symptoms caused by allergy induction.

Experimental Example 2-2. Anaphylaxis Observation

[0074] To see improvement or therapeutic effect of the inotodiol compound isolated from chaga mushroom and the dichloromethane fraction of chaga mushroom including the inotodiol compound of the present disclosure with regard to an anaphylaxis symptom caused by food allergy, responses of mice of each experimental group were observed, and the anaphylaxis symptoms were scored according to Table 3. The results thereof are shown in FIG. 1(B).

TABLE-US-00003 TABLE 3 ANAPHYLAXIS SYMPTOMS SCORE NORMAL 0 SLOW RESPONSE 1 NO RESPONSE 2 SHIVERING 3 DEATH 4

[0075] As shown in FIG. 1(B), the food allergy-induced group (Allergy) had a higher anaphylaxis score than the normal group (Normal). On the other hand, the group treated with dexamethasone (Dexa.), the group treated with the chaga mushroom ethanol extract (EtOH Ext.), the group treated with the dichloromethane fraction (CH.sub.2Cl.sub.2 Frac.), and the group treated with the inotodiol compound (Inotodiol) had a lower anaphylaxis score than the food allergy-induced group. In particular, the group treated with the dichloromethane fraction of chaga mushroom and the group treated with the inotodiol compound were found to have lower anaphylaxis scores. On the other hand, the group treated with the water fraction of chaga mushroom (H.sub.2O Frac.) was found to have a similar level of an anaphylaxis score with the food allergy-induced group.

[0076] Accordingly, it was found that the inotodiol compound and the dichloromethane fraction of chaga mushroom including an inotodiol compound of the present disclosure improved anaphylaxis symptoms caused by allergy induction.

Experimental Example 2-3. Stool Observation in Large Intestine

[0077] The change in a stool shape was observed due to food allergy upon treatment with the inotodiol compound isolated from chaga mushroom and the dichloromethane fraction of chaga mushroom including the inotodiol compound of the present disclosure. The stool shapes in the large intestines of mice of each experimental group were observed with a naked eye, and the symptoms were scored according to Table 4, and the results thereof are shown in FIG. 1(C).

TABLE-US-00004 TABLE 4 Diarrhea symptom Score Normal 0 Normal/watery stool 1 Frequent and 2 uncontrolled watery stool Severe diarrhea 3 Death 4

[0078] As shown in FIG. 1(A), it was found that a food allergy-induced group (Allergy) had a higher diarrhea symptom score than a normal group (Normal). Scores of diarrhea symptoms of the group treated with dexamethasone (Dexa.) or the group treated with the inotodiol compound isolated from chaga mushroom (Inotodiol) and the group treated with dichloromethane fraction of chaga mushroom including an inotodiol compound (CH.sub.2Cl.sub.2 Frac.) were lower than that of the food allergy-induced group. On the other hand, the group treated with the chaga mushroom ethanol extract (EtOH Ext.) and the group treated with the water fraction (H.sub.2O FraC.) had smaller diarrhea symptom relief effects than the chaga mushroom dichloromethane fraction. In the case of the water fraction had a similar level of the allergy-induced group.

[0079] Accordingly, it was found that the inotodiol compound isolated from chaga mushroom and the dichloromethane fraction of chaga mushroom including an inotodiol compound of the present disclosure improved diarrhea symptoms caused by allergy induction.

[0080] Also, although it is not shown in FIG. 1, a group treated with the chloroform fraction of chaga mushroom showed a similar rectal temperature, anaphylaxis score, and stool symptom score as the dichloromethane fraction of the present disclosure. On the other hand, when a group is treated with 20 mg/kg of cholesterol, which has a similar structure as the inotodiol compound, it was found that unlike the inotodiol compound, the group showed a similar level of rectal temperature, anaphylaxis score, and stool symptom score as the allergy-induced group.

[0081] Therefore, through the rectal temperature, the anaphylaxis symptom, and the stool observation, the inotodiol compound isolated from chaga mushroom was found to be one of the main active ingredients that relieve allergy symptoms. In addition, the dichloromethane fraction and the chloroform fraction of chaga mushroom including the inotodiol compound as an active ingredient were found to have excellent allergy symptom relief effects.

Experimental Example 3. Observation on Changes in Cells in Small Intestine Due to Food Allergy

Experimental Example 3-1. Observation on Eosinophil in Small Intestine

[0082] Eosinophil is known to play an important role in food allergy. That is, eosinophil induces allergic inflammation in in small intestines. By observing eosinophil changes in small intestines, allergy prevention and therapeutic effects of the inotodiol compound and the dichloromethane fraction of chaga mushroom including the inotodiol compound of the present disclosure were confirmed.

[0083] The mice of each experimental group in Experimental Example 1 were anesthetized and cut the abdomen. Then, the small intestine was removed, fixed with formalin, treated with paraffin, permeated with paraffin, and microsectioned using microtome, thereby obtaining tissue sections. The obtained small intestine sections were stained with hematoxylin and eosin (H&E) to observe eosinophil cells. The results thereof are shown in FIG. 2(A).

[0084] According to the results of measurement of the number of stained eosinophil cells by tissue staining as shown in FIG. 2(A), the food allergy-induced group (Allergy) showed increased infiltration of eosinophils in the small intestine, as compared with the normal group, and the group treated with dexamethasone (Dexa.), the group treated with the inotodiol compound isolated from chaga mushroom (inotodiol), and the dichloromethane fraction including an inotodiol compound isolated from chaga mushroom (CH.sub.2Cl.sub.2Fr.sub.ac.) showed decreased infiltration of eosinophils. However, the water fraction of chaga mushroom (H.sub.2O Frac.) had little decreasing effects.

[0085] Accordingly, it was found that the inotodiol compound isolated from chaga mushroom and the dichloromethane fraction of chaga mushroom including an inotodiol compound of the present disclosure prevent or treat allergy by alleviating inflammation caused by the allergy.

Experimental Example 3-2. Observation on Mast Cells in Small Intestine

[0086] By observing mast cells which are known as inducing an allergic response, allergy prevention and therapeutic effects of the inotodiol compound and dichloromethane fraction of chaga mushroom including the inotodiol compound fraction of the present disclosure were confirmed.

[0087] To observe mast cells, the tissue sections of small intestines obtained in Experimental Example 3-1 were stained with toluidine. Then, the mast cells were observed with a microscope, and the number of mast cells were measured, which are shown in FIG. 2(B).

[0088] According to the results of measurement of the number of stained mast cells by toluidine staining as shown in FIG. 2(B), the food allergy-induced group (Allergy) showed increased number of mast cells in the small intestine, as compared with the normal group, and the group treated with dexamethasone (Dexa.), the group treated with the inotodiol compound isolated from chaga mushroom (Inotodiol), and the dichloromethane fraction including an inotodiol compound isolated from chaga mushroom (CH.sub.2Cl.sub.2Fr.sub.ac.) showed decreased number of mast cells. However, the water fraction (H.sub.2O Frac.) had little decreasing effects.

[0089] Also, although it is not shown in FIG. 2, a group treated with the chloroform fraction showed a similar level of decrease in the number of eosinophils and mast cells as the dichloromethane fraction of chaga mushroom of the present disclosure. On the other hand, when a group is treated with 20 mg/kg of cholesterol, which has a similar structure as the inotodiol compound, it was found that unlike the inotodiol compound, the group showed little decrease in the number of eosinophils and mast cells.

[0090] Accordingly; it was found that the inotodiol compound isolated from chaga mushroom and the dichloromethane fraction of chaga mushroom including an inotodiol compound of the present disclosure prevent or treat allergy.

Experimental Example 4. Confirmation of Expression of IgE and MCPT-1 Due to Allergy

[0091] An allergic response is due to expression of IgE responsive to allergen and by combination of allergen and IgE, mast cells secrete granular materials associated with allergic inflammation such as histamine, serine protease, or proteoglycan. Therefore, to confirm allergy prevention and therapeutic effect of the inotodiol compound and the dichloromethane fraction of chaga mushroom including the inotodiol compound of the present disclosure, a concentration of each of IgE and a mast cell protease-1 (MCPT-I), i.e., a mast cell-specific serine protease, in serum of a mouse was measured.

[0092] The serum was obtained from the blood of a mouse one day after administration of 50 mg of ovalbumin alone or the chaga mushroom extract, the fraction, or the inotodiol compound in Experimental Example 1. The obtained serum was diluted 100 times and used.

[0093] The IgE concentration in the serum was measured using an enzyme-linked immunosorbent assay (ELISA) kit. 100 μL of a carbonate coating buffer (pH 9.5) containing 2 μg/mL of ovalbumin was added to each well of a 96-well plate, and then subjected to reaction at a temperature of 4° C. overnight for coating. The plate was washed three times with a washing buffer (0.05% tween 20 in PBS), a phosphate buffered saline (PBS) containing 1% bovine serum albumin (BSA) was added to 200 μL per well, followed by treatment at room temperature for 1 hour and blocking. Thereafter, three times of washing with a washing buffer were carried out. After washing, 100 μL of serum was added to each well, reacted at room temperature for 2 hours, and washed with washing buffer. 100 μL of 1 μg/mL of biotin-conjugated anti-mouse IgE was added to each well and reacted for one hour, followed by washing with washing buffer and treatment with streptavidin-HRP to which a horseradish peroxidase (HRP) is conjugated for 30 minutes. The plate was washed five times with washing buffer, and 100 μL of tetramethylenbenzidine was treated to induce color development. After color development, 2N sulfuric acid (H.sub.2SO.sub.4) was added to each well to stop the color reaction. The absorbance at 450 nm was measured and the concentration of IgE in the serum was analyzed. The results thereof are shown in FIG. 3(A).

[0094] A concentration of a mast cell protease-1 (MCPT-1), i.e., a serine protease was measured using a mMCP-1 ELISA kit (eBioscience) according to the protocol provided by the manufacturer. The results thereof are shown in FIG. 3(B).

[0095] As shown in FIG. 3, the food allergy-induced group (Allergy) had increased concentrations of egg white protein-specific IgE (FIG. 3(A)) and MCPT-1 (FIG. 3(B)) in serum, as compared with the normal group (Normal). The group treated with the ethanol extract of chaga mushroom (EtOH Ext) and the group treated with the dichloromethane traction (CH.sub.2Cl.sub.2 Frac.) had significantly decreased concentrations of IgE and MCPT-1. The group treated with the water fraction of chaga mushroom (H.sub.2O Frac.) did not have substantial change in concentrations of IgE and MCPT-1. The group treated with dexamethasone (Dexa.) and the group treated with the inotodiol compound (inotodiol) had no substantial change in the concentration of IgE, however, the concentration of MCPT-1 of these groups decreased significantly.

[0096] Also, although it is not shown in FIG. 3, a group treated with the chloroform fraction showed a similar level of decrease in the concentration of IgE and MCPT-1 in serum as the dichloromethane fraction of chaga mushroom of the present disclosure.

[0097] This result shows that effects of activity regulation/inhibition of the mast cells are more directly related to prevention or treatment of allergy symptoms than the reducing effects of allergen specific IgE expression.

Experimental Example 5. Confirmation of Mast Cell-Specific Inhibition Effects

Experimental Example 5-1. Confirmation of CD4.SUP.+ T Cell Activity Regulation Effects

[0098] It is known that cytokines produced by T helper 2 (Th2) CD4.sup.+ T cells promote production of IgE and promote differentiation of mast cells and eosinophils, thereby deepening allergic symptoms. It has also been reported that plant extracts, which have been reported to have anti-allergic effects, have effects of inhibiting the activation of CD4.sup.+ T cells and cytokine production of Th2 CD4.sup.+ T cells.

[0099] In order to examine the effect of an inotodiol compound or a chaga mushroom extract containing an inotodiol compound on CD4.sup.+ T cell activity regulation, analysis of expression level of cytokine produced by Th2 CD4.sup.+ T cell and adoptive transfer experiments using CD4.sup.+ TCR transgenic T cells that express DO11.00 TCR, i.e., the ovalbumin-derived peptide specific T cell receptor (TCR), were performed.

[0100] For the expression of cytokine, a sample was treated in the same manner as in Experimental Example 1, and the cells were isolated from the mesenteric lymph node (mLN) of mice of each group after one day. At this time, a group treated with 10 mg/kg of dexamethasone, a group treated with 20 mg/kg of imatinib (Gleevec), which is used as a mast cell hyperthermia therapeutic agent, and a group treated with 20 mg/kg of cholesterol that is similar in structure to the inotodiol compound were added. The isolated cells were dispensed into wells of a 96-well plate at a rate of 1×10.sup.6 cells/well. RPMI complete medium (containing 10% fetal bovine serum (FBS), penicillin, streptomycin, and glutamine) containing ovalbumin or RPMI complete medium containing 100 μg/mL of ovalbumin was inoculated and culture for three days under a condition of 37° C. and 5% CO.sub.2. After three days, the cell culture solution was collected, followed by analysis using an ELISA kit according to the protocol provided by the manufacturer to analyze amount of IL-5, IL-13, and IFN-γ corresponding to each cytokine secreted as a cell culture solution. The results thereof are shown in FIG. 4 (A).

[0101] Experiments to measure a degree of ovalbumin-derived peptide specific T cell immune response from an animal experiment were performed as follows. Cells were isolated from the lymph nodes of transgenic mice expressing DO11.10TCR. The isolated cells were fluorescence-stained with carboxyfluorescein succinimidyl ester (CFSE), and 2×10.sup.6 cells were injected intravenously into BALB/C mice. Cells were injected intraperitoneally with ovalbumin (50 μg) and alum to induce division of CD4.sup.+ T cells expressing DOII.00 TCR. Oral administration of 20 mg/kg of the inotodiol compound, 320 mg/kg of the chaga mushroom ethanol extract, 10 mg/kg of dexamethasone, 10 mg/kg of imatinib, or 20 mg/kg of the cholesterol was performed once a day for four days starting from one day after ovalbumin injection. After five days of ovalbumin injection, the cells were isolated from BALB/C lymph nodes, stained with anti-CD4 antibody and anti-DO11.10 antibody, and measured CFSF fluorescence intensity of the CD4.sup.+ T cell expressing DOII.00 TCR using flow cytometry to determine the cell division degree. Then, the number of cells was measured. The results thereof are shown in FIG. 4(B), respectively. Here, the CFSF fluorescence intensity decreased as the cell division increases.

[0102] As shown in FIG. 4(A), no substantial expression of IL-5, IL-13, and IFN-γ in all groups was observed in the case of cells cultured in RPMI complete medium without ovalbumin (No OVA). However, in the case of cells cultured in RPMI complete medium including ovalbumin (OVA), the amount of each of IL-5, IL-13, and IFN-γ was increased in the allergy-induced group (Allergy). However, the amount of cytokine of the group treated with the chaga mushroom ethanol extract (EtOH Ext.) was similar to that of the normal group (Normal). However, the amount of cytokines of the group treated with the inotodiol compound (Inotodiol) was not substantially different from the expression amount of IL-5, IL-13, and IFN-γ.

[0103] In addition, as in the analysis of CD4.sup.+ T cell expressing DO11.00 TCR, i.e., an ovalbumin-derived peptide specific TCR shown in FIG. 4(B), the group treated with ovalbumin (Allergy) had a significantly decreased CFSE fluorescence intensity of CD4.sup.+T cell expressing DOII.00 TOR of and a significantly increased number of CD4.sup.+T cells, as compared with the ovalbumin-unimmune group (Normal). However, the group treated with chaga mushroom ethanol extract (EtOH Ext.) and the group treated with imatinib (Imat.) had no difference in the CFSE fluorescence intensity and the number of cells, as compared with the unimmune group. The group treated with the inotodiol compound (Inotodiol) had a similar level of decrease in CFSE fluorescence intensity of CD4.sup.+T cell expressing DOII.00 TCR, as compared with the group treated with ovalbumin, and significantly increased number of CD4.sup.+T cells.

[0104] Also, although it is not shown in FIG. 4, a group treated with the dichloromethane fraction of chaga mushroom and a group treated with the chloroform fraction of chaga mushroom according to the present disclosure were found to inhibit cytokine expression due to Th2 CD4.sup.+T cells so does the chaga mushroom extract.

[0105] Accordingly, from the foregoing results, it was found that the dichloromethane fraction of chaga mushroom and the chloroform fraction of chaga mushroom according to the present disclosure have significant Th2 CD4.sup.+T cells (induced by allergen) activity regulating ability, while the inotodiol compound does not.

Experimental Example 5-2. Confirmation of Mast Cell Immunoreactivity Regulation Effects

[0106] In Experimental Example 4, it was found that MCPT-1 expression reduction of an inotodiol compound or a chaga mushroom extract containing an inotodiol compound is related to immunoreactivity inhibition of mast cells due to an inotodiol compound.

[0107] In order to confirm degranulation inhibition effects of mast cells of the inotodiol compound or the chaga mushroom extract containing the inotodiol compound of the present disclosure, a passive systemic anaphylaxis experiment was performed.

[0108] the inotodiol compound (20 mg/kg), the chaga mushroom ethanol extract (320 mg/kg), cholesterol (20 mg/kg) similar in structure to the inotodiol compound, dexamethasone (10 mg/kg) as a positive control, and imatinib (20 mg/kg) were oral-administered to 5-week old male BALB/C mice of Experimental Example 1 once a day for 3 days. On the third day after oral administration, 3 μg of anti-dinitrophenol (DNP)-IgE was intravenously injected, and after 24 hours, mast cell degranulation was induced by intravenous injection of 80 μg of DNP-conjugated bovine serum albumin (BSA). The rectal temperature of mice induced mast cell degranulation was measured, and the amount of MCPT-1 in the blood was analyzed. The results thereof are shown in FIG. 5.

[0109] As shown in FIG. 5, upon measurement of the rectal temperature of mice (FIG. 5(A)), when the mast cell degranulation was induced (DNP-BSA treated) the temperature of the rectum was lowered, whereas the inotodiol compound (Inotodiol) and the chaga mushroom ethanol extract (EtOH Ext.) did not decrease the rectal temperature.

[0110] In addition, the amount of MCPT-1 secreted during mast cell degranulation was analyzed (FIG. 5(B)), and when the mast cell degranulation was induced (DNP-BSA treated), the concentration of MCPT-1 in the blood of the mouse increased. When treated with dexamethasone (Dexa.), imatinib (Imat.), the inotodiol compound (Inotodiol), and the chaga mushroom ethanol extract (EtOH Ext.), a level of increase in concentration of MCPT-1 was significantly decreased. When cholesterol that is similar in structure to the inotodiol compound (Chol.) is treated, unlike the inotodiol compound, MCPT-1 did not decrease.

[0111] Although it is not shown in FIG. 5, the dichloromethane fraction of chaga mushroom and the chloroform fraction of chaga mushroom according to the present disclosure inotodiol compound also reduced the amount of MCPT-1 to a similar degree.

[0112] Accordingly, it was found that the dichloromethane fraction of chaga mushroom and the chloroform fraction of chaga mushroom according to the present disclosure inhibited mast cell degranulation.

[0113] Therefore, from the foregoing results, it was found that the chaga mushroom dichloromethane fraction and chloroform fraction according to the present disclosure regulate CD4.sup.+T cell activity and inhibit mast cell degranulation, thus having multiple immune inhibition. On the other hand, unlike dexamethasone or the dichloromethane fraction and the chloroform fraction having multiple immune inhibition, the inotodiol compound according to the present disclosure selectively inhibit mast cell degranulation, thus providing a relatively selective and safe treatment of an allergic disease.

Preparation Example 1. Pharmaceutical Preparation

Preparation Example 1-1. Preparation of Tablet

[0114] 200 g of the dichloromethane fraction of chaga mushroom according to the present disclosure was mixed with 175.9 g of lactose, 180 g of potato starch, and 32 g of colloidal silicic acid. A 10% gelatin solution was added to this mixture which was then pulverized and passed through a 14-mesh sieve. The result was dried, and 160 g of potato starch, 50 g of talc, and 5 g of magnesium stearate were added thereto to prepare a mixture which was then made into tablets.

Preparation Example 1-2. Preparation of Injectable Solution

[0115] 1 g of the inotodiol compound of the present disclosure, 0.6 g of sodium chloride, and 0.1 g of ascorbic add were dissolved in distilled water such that the solution was 100 mL. The solution was added to a bottle and heated for 30 minutes at 20° C. for sterilization.

Preparation Example 2. Food Preparation

Preparation Example 2-1. Preparation of Cooking Seasonings

[0116] The inotodiol compound of the present disclosure was added to a cooking seasoning at 1 wt % to prepare a cooking seasoning for health promotion.

Preparation Example 2-2. Preparation of Flour Food

[0117] The inotodiol compound of the present disclosure was added to wheat flour at 0.1 wt %, and bread, cake, cookies, crackers, and noodles were prepared by using this mixture to prepare food for health promotion.

Preparation Example 2-3. Preparation of Soups and Gravies

[0118] The inotodiol compound of the present disclosure was added to soups and gravies at 0.1 wt % to prepare soups and gravies for health promotion.

Preparation Example 2-4. Preparation of Dairy Products

[0119] The inotodiol compound of the present disclosure was added to milk at 0.1 wt %, and this milk was used to prepare various dairy products such as butter and ice cream.

Preparation Example 2-5. Preparation of Vegetable Juice

[0120] 0.5 g of the inotodiol compound of the present disclosure was added to 1,000 mL of tomato juice or carrot juice to prepare vegetable juice for health promotion.

Preparation Example 2-6. Preparation of Fruit Juice

[0121] 1 g of the inotodiol compound of the present disclosure was added to 1,000 mL of apple juice or grape juice to prepare fruit juice for health promotion.