阅读说明：本技术 治疗过敏性疾病的方法 (Method for treating allergic diseases ) 是由 吴郁彬 罗吉孟 梁惠如 林佩欣 于 2019-07-08 设计创作，主要内容包括：本发明相关于一种用于制造治疗过敏性疾病的药物的一化合物的用途,其中该化合物选自于由鱼针草内酯(ovatodiolide)、1’-乙酰氧基胡椒酚乙酸酯(1’-acetoxychavicol acetate)、花姜酮(zerumbone)及其组合组成的群组。亦提供一种用于治疗过敏性疾病的方法与组合物。(The present invention relates to the use of a compound selected from the group consisting of harringtonide (ovatodiolide), 1'-acetoxychavicol acetate (1' -acetoxychavicol acetate), zerumone (zerumbone), and combinations thereof, for the manufacture of a medicament for the treatment of allergic diseases. Also provided are methods and compositions for treating allergic diseases.)

1. A method of treating allergic disease comprising administering to a subject in need thereof a therapeutically effective amount of a compound selected from the group consisting of esparto grass lactone (ovatodiolide), 1'-acetoxychavicol acetate (1' -acetoxychavicol acetate), zerumbone, and combinations thereof.

2. The method of claim 1, wherein the compound is administered in a pharmaceutical composition.

3. The method of claim 1, wherein the compound is administered in the form of a composition comprising an herbal extract selected from the group consisting of esparto grass (anisoomeles indica), Alpinia galanga (Alpinia galanga), Zingiber zerumbet (Zingiber zerumbet), and combinations thereof.

4. The method of claim 1, wherein the allergic disease is selected from the group consisting of hay fever, food allergies, atopic dermatitis, asthma, psoriasis, allergic psoriasis, atopic dermatitis, contact dermatitis or eczema, autoimmune diseases, osteoarthritis, allergic rhinitis, seborrheic dermatitis, psoriatic arthritis, and poison ivy (poison ivy) allergy.

5. The method of claim 4, wherein the allergic disease is an allergic dermatitis.

6. The method of claim 5, wherein the allergic dermatitis is atopic dermatitis.

7. The method of claim 5, wherein the allergic dermatitis is psoriasis.

8. The method of claim 4, wherein the allergic disease is an autoimmune disease.

9. The method of claim 8, wherein the autoimmune disease is selected from the group consisting of autoimmune hepatitis, autoimmune pancreatitis, schroenger's syndrome, ulcerative colitis, Crohn's disease, reflex sympathetic dystrophy, post-myocardial infarction syndrome, rheumatoid rhinitis, multiple sclerosis, and cardiomyopathy.

10. The method of claim 8, wherein the autoimmune disease is autoimmune hepatitis.

Technical Field

The present invention relates to a method of treating allergic diseases. In particular, the present invention relates to a composition for the treatment of allergic diseases.

Background

Allergies are due to a disturbance of the body's immune system, which overreacts to allergens in the environment. The allergen and IgE antibodies that enter the body will bind to stimulate the mast cells to release substances such as histamine, which in turn generates an inflammatory reaction in the body tissue, resulting in chronic inflammation of the skin, mucosal tissue or blood vessels. In recent years, it has become a major threat to our health. Allergy is associated with a second helper T cell (Th2) among B cells and T cells. Th2 cells are characterized by production of interleukin-4, IL-4 and IL-5. IL-4 helps B cells produce sensitized antibodies to immunoglobulin E. IL-5 attracts eosinophils, which release some inflammatory mediators, resulting in many severe allergic symptoms. T helper cells of the first type are responsible for cellular immunity and suppress Th2 responses via secretion of cytokines, such as secretion of interferons: IFN-gamma, IgG2a, IL-2, IL-3, and the like.

Disclosure of Invention

The present inventors have surprisingly found that certain compounds have an anti-allergic effect.

Accordingly, in one embodiment, the present invention provides a method of treating allergic diseases comprising administering to a subject in need thereof a therapeutically effective amount of a compound selected from the group consisting of harringtonide (ovaodiolide), 1'-acetoxychavicol acetate (1' -acetoxychavicol acetate), zerumbone (zerumbone), and combinations thereof

In another embodiment, the present invention provides the use of a compound selected from the group consisting of harringtonide (ovatodiolide), 1'-acetoxychavicol acetate (1' -acetoxychavicol acetate), zerumbone (zerumbone), and combinations thereof, for the preparation of a medicament for the treatment of allergic diseases.

In another embodiment, the present invention also provides a pharmaceutical composition for treating allergic diseases, comprising a therapeutically effective amount of a compound selected from the group consisting of harringtonide (ovatodiolide), 1'-acetoxychavicol acetate (1' -acetoxychavicol acetate), zerumone (zerumone), and combinations thereof. .

In another embodiment, the present invention provides a nutraceutical composition for reducing pain caused by allergic diseases comprising a therapeutic amount of a compound selected from the group consisting of harringtonide (ovatodiolide), 1'-acetoxychavicol acetate (1' -acetoxychavicol acetate), zerumbone (zerumbone), and combinations thereof.

In one embodiment of the invention, the compound may be provided by a plant or herb. For example, harringtonide (ovandiolide) and/or zerumbone (zerumbone) may be provided from the plant, esparto (Anisomeles indica); 1' -acetoxychavicol acetate from Alpinia galanga (Alpinia galanga), and zerumbone (zerumone) from Zingiber zerumbet.

In another embodiment, the present invention provides a herbal composition or pharmaceutical composition comprising an herbal extract selected from the group consisting of esparto grass (anisoomeles indica), Alpinia galanga (Alpinia galangal), Zingiber zerumbet (Zingiber zerumbet), and combinations thereof.

According to an embodiment of the invention, the allergic disease is a condition caused by an oversensitivity of the immune system to substances that are normally harmless to the environment, and the allergic disease may be selected from the group consisting of hay fever, food allergy, atopic dermatitis, asthma, psoriasis, allergic psoriasis, atopic dermatitis, contact dermatitis or eczema, autoimmune diseases, osteoarthritis, allergic rhinitis, seborrheic dermatitis, psoriatic arthritis and poison ivy (poison ivy) allergy.

In particular, the autoimmune disease is selected from the group consisting of autoimmune hepatitis, autoimmune pancreatitis, schroenger's syndrome, ulcerative colitis, Crohn's disease, reflex sympathetic dystrophy, post-myocardial infarction syndrome, rheumatoid rhinitis, multiple sclerosis, and cardiomyopathy. In one embodiment of the present invention, the autoimmune disease is autoimmune hepatitis.

It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the invention as claimed.

Drawings

The foregoing summary, as well as the following detailed description of the invention, will be better understood when read in conjunction with the appended drawings. For the purpose of illustrating the invention, there is shown in the drawings embodiments which are presently preferred.

In the drawings:

figure 1 is a schematic representation of the induction of contact dermatitis in an animal.

Figure 2 is a schematic representation of induction of psoriasis in an animal.

Figure 3 provides the results of ear swelling observations in contact dermatitis animal experiments (.: p < 0.01).

Figure 4 provides the results of mast cell infiltration of ear sections (.: p <0.01) in contact dermatitis animal experiments.

FIG. 5 provides the results of examination of skin sections of mast cell infiltration in contact dermatitis animal experiments (p < 0.05;. p < 0.01).

Figure 6 provides animal experimental results for contact dermatitis (: p < 0.05;: p < 0.01).

FIG. 7 is an analysis of serum cell dependence in an animal experiment with induced contact dermatitis in an embodiment of the present invention (p < 0.05;. p < 0.01).

FIG. 8A is a graph of desquamation levels in animal experiments with psoriasis.

FIG. 8B is a graph of the amount of redness and swelling in animal experiments with psoriasis.

FIG. 9 provides the results of real-time PCR in the experiment of animals with psoriasis (ns: p > 0.05;. p < 0.01).

FIG. 10 provides results of spleen weight in animal experiments with psoriasis.

FIG. 11 is a graph showing cytokine expression in lymphocytes in the experiment of animals with psoriasis (ns: p > 0.05;. p: p < 0.01).

Figure 12 shows the effect of AR001DS1 on GOP, GPT and body weight. Data are presented as mean ± SEM (n ═ 9). *: p <0.05 compared to Veh, as tested by Student's t. Veh, vehicle; dex, dexamethasone (dexamethasone).

Figure 13 shows the effect of AR001DS1 on liver injury. The histopathological score of necrosis is expressed as mean ± SEM (n ═ 9). ***: p <0.001 compared to Veh, as tested by Student's t. Veh, vehicle; dex, dexamethasone (dexamethasone).

FIG. 14A shows that pre-treatment with AR001DS1 or AR001DS2 inhibited PMA-induced IL-1 β expression in THP-1 cells. Q-PCR analysis (left) and immunoblotting (right) of IL-1. beta. mRNA and protein expression in THP-1 cells pretreated with AR001DS1 or AR001DS2 for 0.5 hours, followed by PMA induction for 48 hours.

FIG. 14B shows that treatment with AR001DS1 inhibited PMA-induced IL-1 β expression in THP-1 cells. Q-PCR analysis of IL-1. beta. mRNA expression in THP-1 cells stimulated with PMA for 24 hours, followed by treatment with or without AR001DS1 or AR001DS2 for an additional 24 hours.

FIG. 15 shows the results of a bio-plex assay using the human 27-plex cytokine assay to monitor the effect of AR100DS1 treatment (A-PMA) on PMA-induced cytokines; THP-1 cells were treated with AR001DS1 (10. mu.g/ml) for 0.5 h, followed by PMA (10ng/ml) for 24 h; AR001DS1 treated cells (A-PMA) were subjected to Bio-plex analysis with PMA alone and untreated THP-1 (Ctrl).

Figure 16 provides the results that AR001DS1 can inhibit mast cell degranulation in a dose-dependent manner. DNP, dinitrobenzene; DNP-BSA, dinitrobenzene-bovine serum albumin.

Figure 17 provides results that AR001DS1 can inhibit TNF-a secretion by Dendritic Cells (DCs) in a dose-dependent manner. DMSO, DCs + 0.1% DMSO; LPS: DCs + LPS + 0.1% DMSO; and (3) Que: DCs + LPS + quercetin (quercetin); untreated: DCs + medium; AR001DS 1: DC + LPS + AR001DS 1.

Detailed Description

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

As used herein, the singular forms "a", "an" and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a sample" includes a plurality of such samples and equivalents thereof known to those skilled in the art.

The present invention provides a method of treating allergic diseases comprising administering to an individual in need thereof a therapeutically effective amount of a compound selected from the group consisting of harringtonide (ovatodiolide), 1'-acetoxychavicol acetate (1' -acetoxychavicol acetate), zerumbone (zerumone), and combinations thereof.

In the present invention, harringtonide (also referred to herein as "AR 001DS 1"), which can be isolated and purified from harringtonia barbata (Anisomeles indica) has the following structure

In the present invention, 1' -acetoxychavicol acetate, also referred to herein as "AR 001DS2," can be isolated and purified from Alpinia galanga (Alpinia galanga), having the following structure

In the present invention, zerumbone, also referred to herein as "AR 001DS 3", is isolated and purified from Zingiber zerumbet, which has the following structure

The present invention provides the use of a compound selected from the group consisting of harringtonide (ovatodiolide), 1'-acetoxychavicol acetate (1' -acetoxychavicol acetate), zerumone (zerumbone), and combinations thereof, for the preparation of a medicament for the treatment of allergic diseases.

The present invention also provides a health or pharmaceutical composition for treating allergic diseases, comprising a therapeutically effective amount of a compound selected from the group consisting of harringtonide (ovatodiolide), 1'-acetoxychavicol acetate (1' -acetoxychavicol acetate), zerumbone (zerumone), and combinations thereof.

Since the compound may be provided by a plant or herb. For example, the plant esparto grass (Anisomeles indica) may provide esparto grass lactone (ovandiolide) and/or zerumbone; 1' -acetoxychavicol acetate from Alpinia galanga (Alpinia galanga), and zerumbone (zerumone) from Zingiber zerumbet. Accordingly, the present invention provides an herbal composition or a pharmaceutical composition comprising an herbal extract selected from the group consisting of esparto grass (anisoomeles indica), Alpinia galanga (Alpinia galangal), Zingiber zerumbet (Zingiber zerumbet) and combinations thereof.

As used herein, the term "allergic disease" refers to a condition caused by the hypersensitive immune system to substances that are generally harmless in the environment, and particularly includes autoimmune diseases. In an embodiment of the invention, the allergic disease is selected from the group consisting of hay fever, food allergy, atopic dermatitis, asthma, psoriasis, allergic psoriasis, atopic dermatitis, contact dermatitis or eczema, seborrhoeic dermatitis, psoriatic arthritis, poison ivy (poison ivy) allergy, rheumatoid rhinitis, multiple sclerosis, osteoarthritis, allergic rhinitis and cardiomyopathy. Symptoms may include redness, itchy rash, sneezing, runny nose, shortness of breath, or swelling. In the present example, the allergic disease is allergic dermatitis, especially atopic eczema or psoriasis.

As used herein, the term "autoimmune disease" refers to a condition caused by an abnormal immune response to a normal body site. There are at least 80 autoimmune diseases. Examples of autoimmune diseases include, but are not limited to, autoimmune hepatitis, autoimmune pancreatitis, celiac disease, type 1 diabetes, schroenger's syndrome, ulcerative colitis, Crohn's disease, reflex sympathetic dystrophy, post-myocardial infarction syndrome, Graves ' disease, inflammatory bowel disease, multiple sclerosis, psoriasis, rheumatoid arthritis, cardiomyopathy, and systemic lupus erythematosus.

According to the present invention, an extract of esparto grass (Anisomeles indica) can be prepared by the following method: needle fish (anisoyles indica) was extracted with ethanol to obtain a crude extract, which was loaded into a silica-filled chromatography column and subjected to gradient elution, eluting: n-hexane/ethyl acetate, hexane/ethyl acetate/methanol, and methanol to obtain a separated liquid; the separated liquid was separated with a silica-filled chromatography column, using the eluent: performing gradient elution with dichloromethane, dichloromethane/methanol, and methanol to obtain a concentrate; the concentrate was recrystallized from n-hexane/ethyl acetate to give crystals. The harzilin (ovandiolide) and/or zerumbone (zerumbone) may be provided from the plant harzilin (Anisomes indica).

According to the present invention, an extract of Alpinia galanga (Alpinia galangal) can be prepared by the following method: extracting galangal (Alpinia galanga) with cyclohexane to obtain a crude extract, loading the crude extract into a silica-filled chromatography column, and performing gradient elution to obtain an elution solution: n-hexane/ethyl acetate, n-hexane/ethyl acetate/methanol, and methanol to obtain a separated substance; and the isolate was recrystallized using n-hexane/ethyl acetate to obtain crystals. 1' -acetoxychavicol acetate can be extracted from Alpinia galanga (Alpinia galanga).

The term "individual" as used herein includes a human or non-human animal, such as a companion animal (e.g., dog, cat, etc.), a farm animal (e.g., cow, sheep, pig, horse, etc.), or an experimental animal (e.g., rat, mouse, guinea pig, etc.).

The term "treating" as used herein refers to administering one or more active agents to an individual in need thereof, who has, is suffering from, or is affected by an allergic disease. The purpose is to cure, treat, alleviate, reduce, alter, correct, ameliorate, or affect a disease, a symptom of a disease, or the effects of a disease.

The term "therapeutically effective amount" as used herein refers to an amount of a compound or agent that results in a therapeutic, healing, prophylactic effect, or reduces a disease, disorder, or side effect, or reduces the rate of progression of a disease or disorder, as compared to a corresponding individual not receiving the amount. The term also includes amounts within the range that are effective to enhance normal physiological function.

For use in therapy, a therapeutically effective amount of the composition is formulated as a pharmaceutical composition for administration. Accordingly, the present invention provides a pharmaceutical composition comprising a therapeutically effective amount of active ingredient(s) and one or more pharmaceutically acceptable carriers.

The term "pharmaceutically acceptable carrier" as used herein refers to an acceptable carrier(s), diluent(s) or excipient(s) that is compatible with the other ingredients of the formulation and not injurious to the subject to which the pharmaceutical composition is administered. Any carrier, diluent or excipient commonly known or used in the art may be used in the present invention, depending on the requirements of pharmaceutical formulation.

According to the present invention, the pharmaceutical composition may be in the form of troches, pills, powders, buccal tablets, sachets, troches, elixirs, suspensions, ointments, emulsions, solutions, syrups, soft and hard gelatin capsules, suppositories, sterile injections and packaged powders. In one embodiment of the invention, the pharmaceutical composition is formulated in the form of an ointment. Such formulations may be prepared via any method known in the art of pharmacy.

According to the present invention, the pharmaceutical composition may be adapted for administration via any suitable route, including but not limited to oral, rectal, nasal, topical, vaginal or parenteral routes. In one embodiment of the invention, the pharmaceutical composition is formulated for topical administration. Such formulations may be prepared via any method known in the art of pharmacy.

According to the present invention, the methods, uses or compositions described herein may be administered to a subject in combination with at least one additional allergic agent. Exemplary allergic agents corresponding thereto include, but are not limited to, ketorolac tromethamine (ketorolac tromethamine), pemirolast potassium (pemirolast potassium), ketotifen (ketotifen), loratadine (loratadine), neodolicin sodium (neodecocimil sodium), fexofenadine (fexofenadine), loteprednol etodolate (loteprinoacetate), azelastine (azelastine), ipratropium bromide (ipratropium bromide), epinephrine, beclomethasone (beclomethasone), diphenhydramine (diphenhydramine), desloratadine (desolatatadine), loratadine (loratadine), dexamethasone (loratadine), epinastine (epinastine), and fluticasone (fluticasone).

The invention is further illustrated by the following examples, which are to be construed as merely illustrative, and not limitative of the remainder of the disclosure in any way whatsoever. Without further elaboration, it is believed that one skilled in the art can, based on the description herein, best utilize the present invention.

Preparation example

1. Animal experiment for contact dermatitis

Animal model for contact dermatitis:

after 7 to 8 weeks after BALB/c mice birth, a back shave is performed and the symptoms of contact dermatitis, such as atopic eczema, are induced using the allergy drug 1-chloro-2, 4-Dinitrobenzene (DNCB) as an irritant. As shown in FIG. 1, on days 1-3, the back was coated with 100. mu.l of 0.5% DNCB; the skin was stimulated with 100. mu.l of 1% DNCB, and the ear was stimulated with 20. mu.l of 1% DNCB on day 14. Smearing may also be performed on days 17, 21, 24, 28, 31, and 35. On the other hand, from day 14 onwards, the test ointment was administered five times a week for three weeks. On day 36, mice were sacrificed.

Grouping mice:

the mice were divided into five groups of four mice each. The names, drugs and ointments are shown in table 1:

TABLE 1

Mice were treated with a combination of harringtonine (ovatodiolide) (AR001DS1) and 1' -acetoxychavicol acetate (AR001DS2) added to the salve and a combination of harringtonine (ovatodiolide) (AR001DS1) and zerumbone (AR001DS3) added to the salve, designated as group a100 and group V2, respectively, wherein the amount of each compound was 2.5%.

Ear swelling measurement:

after the experiment, the thickness of the mouse ear was measured and recorded

Hematoxylin and Eosin (HE) staining:

the skin and ears were removed from the mice, fixed in formaldehyde and made into wax tablets. After sectioning, eosinophil (eosinophil) infiltration and skin swelling results were observed by HE staining.

Skin toluidine blue staining:

after the skin and ear of the mouse were fixed and sectioned, the infiltration of mast cells was observed by staining with toluidine blue.

By real-time polymerase chain reactionThe gene expression of the skin should be analyzed (PCR):

a piece of skin was taken to extract its mRNA and analyzed for gene expression of IL-4 and IL-5 via real-time PCR.

2. Animal experiment for psoriasis

Animal model for psoriasis:

the animals were 5-8 week old BALB/c mice from the center of the experimental animals. The experimental environment was maintained at 25 ℃. The light and dark periods cycle every 12 hours. The mice had free access to sufficient food and water.

Psoriasis is induced in mice by Imiquimod (IMQ). As shown in fig. 2, the day before the experiment, after the mice were dehaired with a razor and depilatory cream, the mice were divided into two groups.

The group treated with a combination of harringtonide (ovandiolide) (AR001DS1) and 1' -acetoxychavicol acetate (AR001DS2) is referred to as AR 100; the group treated with harringtonide (AR001DS1) was designated AR 111; the group treated with the combination of harringtonide (ovandiolide) (AR001DS1) and zerumbone (AR001DS3) was designated AR 112. The mice were divided into 6 groups of 8 mice each. The names, drugs and ointments are shown in table 2:

TABLE 2

Sensitized (S, IMQ/basal) mice were smeared with 62.5mg IMQ on the back of the mice. After 4 hours, the mice were treated with 50mg of AR100(IMQ/AR100), 50mg of AR111(IMQ/AR111), or 50mg of AR112(IMQ/AR 112). The medicine is applied once a day for 6 days. The body weight of the mice was recorded and photographs of the mice were taken daily. The skin was observed for redness and desquamation and scored. On the last day of the experiment, mice were sacrificed and their skin was taken for immunohistological staining and cytokine expression analysis.

3. Animal experiment of autoimmune hepatitis

Reagent

Concanavalin A (Con A) and dexamethasone (Dexamethasone, Dex) were purchased from Sigma-Aldrich (USA). Procartaplex^TMImmunoassay kits were purchased from Corning Inc (USA). GOP and GPT Fuji Dri-Chem slides were purchased from Winning Medical Inc. (Taiwan, China).

Animal(s) production

Male BALB/c mice (7-9 weeks old) were purchased from BioLASCO Taiwan co., Ltd or experimental animal center (NLAC, Taiwan). Animals were housed 5 per cage and food and water were provided ad libitum throughout the experiment. The room temperature was maintained at 23. + -. 2 ℃ with 12 hour light-dark cycling. Animals were acclimated for one week to minimize the effect of stress prior to the experiment. All experimental protocols relating to animals and their care were approved by the Institutional Animal Care and Use Committee (IACUC) in ITRI (ITRI-IACUC-2018-.

Design of experiments and hepatitis Induction

Con A was dissolved in pyrogen-free physiological saline at a concentration of 3mg/ml and injected intravenously at a dose of 15mg/kg or 20mg/kg body weight to induce hepatitis. AR100DS1 and Dex were administered orally 30 minutes before Con a treatment and 4 and 8 hours after Con a treatment. Blood and liver tissue were collected 24 hours after Con a treatment. Sera were stored at-80 ℃ until analyzed.

Liver enzyme assay

To assess the level of hepatocyte injury following Con a treatment, serum GPT and GOT levels were measured by Fuji Dri-Chem slides (Fuji, Japan).

Serum cytokine analysis

The same set of sera was pooled for cytokine assay. According to the manufacturer's instructions, via ProcartaPlex^TMThe immunoassay kit measures cytokine levels.

Histopathology

Liver tissue was fixed in 10% phosphate buffered formaldehyde, embedded in paraffin, and stained with hematoxylin and eosin (H & E) to confirm tissue damage. Tissue damage was examined with a veterinary pathology microscope from BioLASCO Taiwan co. The severity grading system criteria for all microscopic lesions were as follows: 0 ═ none; 1 ═ necrosis of individual cells; 2 ≤ 30% lobular necrosis; lobular necrosis is less than or equal to 60 percent when the ratio is 3; lobular necrosis > 60%.

Statistical analysis

Data are presented as mean ± SEM. In this study, the Student's t test was used to analyze differences between drug and vehicle treatment groups. When the p-value is less than 0.05, the difference is considered statistically significant.

4. Immune response to mast cells and dendritic cells

Beta-hexosaminidase secretion assay

RBL-2H3 cells were seeded in 24-well plates and then incubated at 37 ℃ with 5% CO₂Sensitization to Dinitrobenzene (DNP) -IgE is carried out in the atmosphere. After washing, the cells were treated with AR001DS1(12.5, 25, 50 and 100. mu.g/ml) at 37 ℃ for 30 minutes, and then stimulated with DNP-Bovine Serum Albumin (BSA) at 37 ℃ for 30 minutes to induce degranulation of the cells. The supernatant was transferred to a 96-well microplate and allowed to stand for 2 hours in 0.1M citrate buffer (pH4.5) with an equal volume of 5mM 4-nitrophenyl N-acetyl- β -aminoglycoside. The reaction was stopped by adding 200ml stop buffer (0.05M sodium carbonate, pH 10) and the OD at 405nm was measured using an ELISA disk reader.

ELISA assay for TNF-alpha secretion

DCs were isolated from C57BL/6 mice and cultured at 37 ℃ for 7 days. DCs were then seeded in 24-well plates at 5% CO₂The culture was carried out overnight at 37 ℃ in the atmosphere. DCs were treated with AR001DS1(12.5, 25, 50, and 100 μ g/ml) for 1 hour, followed by stimulation with Lipopolysaccharide (LPS) for 4 hours. Supernatants were collected and quantified using an ELISA assayTNF-alpha secretion in culture supernatants.

Example 1 animal experiments for inducing atopic dermatitis

(1) Appearance observation and analysis

After sacrificing the mice at the end of the experiment, the appearance of the skin of the mice was photographed. The skin of the mice of the normal group (N) was intact; mice in sensitized group (S) had rough and inflamed skin and swollen ears. Skin remodeling phenomena are found after about 3-4 weeks after DNCB stimulation. In the SV group, the symptoms of the skin were not improved. However, an improvement effect on atopic eczema was found in each of the groups A100 and V2, in which skin redness and swelling were significantly improved. In addition, in the process of destroying the neck of the mouse, it was found that the a100 group and the V2 group had no skin crack phenomenon, in which the fragile skin caused by atopic eczema was improved. Therefore, it can be concluded that the symptoms caused by atopic eczema can be improved by the treatment with a combination of harringtonide (ovatodiolide) and 1' -acetoxychavicol acetate (a100), and a combination of harringtonide (ovatodiolide) and zerumzone (V2).

(2) Ear thickness measurement and analysis

The ear thickness of the SV group was measured to be 0.69. + -. 0.08mm, which was significantly thicker than that of the normal group (N, 0.23. + -. 0.04 mm). As shown in FIG. 3, the ear thicknesses of groups A100 and V2 were measured at 0.27 + -0.07 mm (p <0.01) and 0.31 + -0.10 mm (p <0.001), respectively. It was found that ear swelling was significantly improved in the A100 and V2 groups. In both the a100 and V2 groups, the appearance of the skin and the swelling of the ears were improved.

(3) Observation and analysis of ear and skin sections

The ears and skin of the sacrificed mice were sectioned and HE stained. The skin and ear of the sensitized group were found to have a phenomenon of thickening of the dermal layer, and many eosinophils penetrated into the skin tissue. On the other hand, in the a100 group and the V2 group, swelling of the skin and ears was reduced, and infiltration of eosinophils was reduced.

Specifically, eosinophils tend to accumulate in allergic sites, such as the asthmatic lung and the skin portion of atopic dermatitis. The group of eosinophils that accumulated in the allergic site released more inflammatory material, resulting in more severe inflammation at the infiltration site. Therefore, if eosinophil infiltration can be suppressed, the symptoms of atopic dermatitis will be significantly improved. It can be concluded that eosinophil infiltration into the skin and ears was significantly improved in groups A100 and V2.

Mouse skin and ear sections were also stained with toluidine blue and mast cells were observed. The results are shown in fig. 4 and 5. Mast cells are important immune cells that induce allergic reactions. When IgE, allergen and mast cells form cross-links, mast cells will be activated and induce the release of histamine and leukotrienes etc. resulting in allergic reactions to the tissue. When large numbers of activated mast cells penetrate into the affected area of dermatitis, severe hypersensitivity and itching can result. In the experiment, many mast cell infiltrations were found in the skin or ear of the sensitized group (as shown in fig. 4 and 5), indicating that the number of mast cells was decreased compared to the S group, resulting in a great improvement in skin allergy symptoms. Although the ear sections of the A100 group were not significantly different from the ear and skin sections of the V2 group, infiltration of mast cells was reduced.

(4) Measurement of antibodies in blood

As shown in fig. 6, 7 and 8, IgE production was inhibited in the a100 and V2 groups, whereas IgE production was inhibited more in the a100 group than in the V2 group (see fig. 8). It can be concluded that the combination of harringtonide (ovatodiolide) and 1' -acetoxychavicol acetate (A100) and the combination of harringtonide (ovatodiolide) and zerumzone (V2) inhibited IgE production and showed a significant improvement in skin allergy.

It was also found that atopic dermatitis and excessive activation of Th2 cells are closely related to each other. As shown in FIG. 7, the IL-4, IL-5 and TNF-. alpha.levels in the A100 group were significantly different from those in the control group in terms of IL-4 reduction (p < 0.05). As shown in FIG. 7, the compositions containing AR001DS1 and AR001DS2 had an effect on the levels of IL-5 and TNF- α, but showed a downward trend (see FIG. 7). In the V2 group, the combination of AR001DS1 and AR001DS3 showed the effect of lowering IL-4, IL-5 and TNF- α levels, but not significantly.

As shown in fig. 6, the composition containing AR001DS1 and AR001DS2(AR100 group) had a tendency to inhibit IgG1 and promote IgG2 a. The levels of IgG1 and IgG2a were not significantly regulated in the blood of group V2 compared to the AR100 group.

In view of the above, the combination of AR001DS1 and AR001DS2 had an effect on the level of IL-4 and IgE, indirectly showing that a100 decreased the activity of Th2 cells. In both groups A100 and V2, a reduced expression of TNF-. alpha.can be found (inflammatory marker).

In conclusion, the compositions of AR001DS1 and AR001DS2 and the compositions of AR001DS1 and AR001DS3 all provided the effect of improving the symptoms of atopic dermatitis in mice. Although not particularly good for reducing Th2 immune cells, it was found that in the AR100 group treated with the composition of AR001DS1 and AR002DS2, the levels of IgE and IL-4 in the blood were reduced. In the AR100 and/or V2 groups, decreased infiltration of eosinophils and mast cells on the skin was found, showing an improvement in skin swelling and inflammation.

Example 2 animal experiments on psoriasis

(1) Appearance observation and analysis

The appearance of the skin of the mice was observed and found to be effective in reducing desquamation and redness of the area affected by psoriasis in the AR100 and AR112 groups, wherein the composition of AR0001DS1 and AR0002DS3 had a significant effect in reducing the symptoms of psoriasis.

(2) Scoring of redness and swelling

Mice were scored for the degree of redness based on daily observations and recordings. The degree of desquamation of the skin is shown in FIG. 8A, and redness and swelling is shown in FIG. 8B. The data in fig. 8A and 8B are based on the Psoriasis Area and Severity Index (PASI) with psoriasis skin desquamation rate, redness and desquamation being scored as 0 points (no), 1 points (mild), 2 points (moderate), 3 points (severe) and 4 points (very severe) and recorded daily during the course of the experiment. The results show that the AR100 group, the AR111 group and the AR112 group can effectively relieve the symptoms of desquamation and red swelling of psoriasis skin, wherein the AR112 group has the best curative effect.

(3) Measurement of antibodies in blood

Th1 cells (IFN-. gamma.), Th2 cells (IL-4), Th17 cells (IL-17A, IL-17F, IL-22), Th17 cells (IL-17A, IL-17F, IL-22) and inflammatory cytokines (TNF-. alpha., IL-6) were analyzed by real-time PCR to compare the effects of IMQ/basal, IMQ/AR100, IMQ/AR111 and IMQ/AR112 on the reduction of psoriasis. As shown in FIG. 9, mRNA expression for TNF- α, IL-6, IL-17, and IL-22 was decreased in the IMQ/AR100, IMQ/AR111, and IMQ/AR112 groups. It can be concluded that in the IMQ/AR112 group, compositions containing AR001DS1 and AR001DS3 can ameliorate psoriasis.

(4) Measurement of splenomegaly

In IMQ-induced psoriasis animals, splenomegaly, lymphoid organ swelling or lymphoid tissue swelling is induced. Spleen size and weight were considered as indicators of inflammation in the experiment. As shown in figure 10, the efficacy of inflammation reduction in IMQ-induced psoriasis animals was determined according to mouse spleen size and spleen weight as inflammatory indices. In the IMQ/AR100, IMQ/AR111, and IMQ/AR112 groups, the degree of splenomegaly in the IMQ-induced pattern was significantly reduced.

(5) Measurement of cytokine expression in lymphocytes

The expression of cytokines in Th1 cells (IFN-. gamma.), Th2 cells (IL-1) among the lymphocytes of the IMQ/AR100, IMQ/AR111 and IMQ/AR112 groups was analyzed by flow cytometry. As shown in FIG. 11, it was found that in the IMQ/AR100, IMQ/AR111 and IMQ/AR112 groups, the performance of Th17 cells was decreased, wherein the effects of the composition containing AR001DS1 and AR001DS2, and the composition containing AR001DS1 and AR001DS3, provided statistically significant effects.

Example 3 animal experiment with autoimmune hepatitis

Autoimmune hepatitis (AIH) is a complex disease characterized by a propensity for inflammation, necrosis and cirrhosis of the liver cells. Lectin concanavalin a (con a) -induced acute hepatitis in BALB/c mice is a common animal model of AIH that we performed to evaluate the anti-hepatitis effect of AR001DS 1. Dexamethasone (Dexamethasone, Dex), a steroid-based AIH standard treatment, was used as a positive control.

To establish Con A-induced acute hepatitis in mice, we stimulated the mice with 15 and 20mg/kg Con A. The 15mg/kg Con A-treated group showed significantly increased levels of GOT and GPT compared to the sham control group (GOT: 1450. + -.433 vs. 140. + -.23U/L; GPT: 1437. + -.398 vs. 76. + -.7U/L). Serum GOT levels were significantly elevated (2249 + -549 vs 140 + -23U/L) and serum GPT levels were elevated but not significant (2030 + -833 vs 76 + -7U/L) for the 20mg/kg Con A treated group. In nature and originNo difference was observed between mice (GOT: 140. + -. 23 vs 135. + -.20U/L; GPT: 76. + -.7 vs 83. + -.2U/L). Dex served as a positive control in this study. The results show that Dex decreases Con A induced elevation in GOT (15mg/kg Con A: 809 + -339 vs. 1450 + -433U/L; 20mg/kg Con A: 1261 + -282 vs. 2249 + -549U/L). However, Dex only mitigated GPT levels induced by 15mg/kg Con A (898 + -515 vs 1437 + -398U/L), whereas the 20mg/kg Con A group did not (1940 + -403 vs 1437 + -398U/L). In addition, all mice treated with Con a had reduced body weight.

Next, histopathological analysis of liver tissues was performed. Both the 15 and 20mg/kg Con A groups induced significant hepatic necrosis compared to the sham control group (score: 15mg/kg Con A, 2.2 + -0.2 vs. 0 + -0; 20mg/kg Con A, 2.2 + -0.7 vs. 0 + -0, p < 0.05). Dex showed mild tissue damage remission (score 1.6. + -. 0.6 vs. 2.2. + -. 0.2) following 15mg/kg Con A induction, but caused more severe lesions in the 20mg/kg Con A stimulated group (score 2.6. + -. 0.2 vs. 2.2. + -. 0.2)). Based on these results, we chose 15mg/kg Con A for further study.

Finally, 50mg/kg of AR100DS1 significantly decreased GPT levels due to Con A increase (109. + -.25 vs 368. + -.107U/L, p <0.05) and slightly improved GOT rise (261. + -.45 vs 410. + -.56U/L) (FIG. 12). In addition, histopathological analysis showed that AR100DS1 improved hepatic necrosis (score 0.2 ± 0.2 vs. 1.4 ± 0.2, p <0.05) (fig. 13).

Example 4 antagonism of the inflammatory Signaling by AR-100

Since AR001DS1 shows therapeutic effects on animal models of gouty arthritis, in which IL-1 β released by macrophages plays a key inflammatory role, AR001DS1 was tested for IL-1 β induction in phorbol-12-myristic-13-acetate (PMA) induced-activated THP-1 cells. Since AR100 consists of AR001DS1 and AR001DS2, THP-1 cells were pretreated with two components independently, followed by PMA activation. Q-PCR and immunoblotting showed that pre-treatment of THP-1 cells with AR001DS1 or AR001DS2 blocked the expression of IL-1 β (FIG. 14A). Interestingly, treatment of THP-1 cells with AR100DS1 after activation of THP-1 cells with PMA still attenuated IL-1 β expression, whereas AR001DS2 did not inhibit PMA-induced IL-1 β (FIG. 14B). These data show that both AR001DS1 and AR001DS2 inhibit the initiation of macrophage inflammatory signals; in addition, AR001DS1 is able to block inflammatory signals in activated macrophages.

Example 5 Effect of inflammatory cytokines

In addition to IL-1 β, several cytokines are involved in the inflammatory response. To identify the cytokine network affected by AR001DS1, we performed a Bio-plex assay to monitor the effect of AR001DS1 treatment on human 27 cytokine expression in PMA treated THP-1 cells. PMA was observed to induce IL-1 β, and AR001DS1 treatment attenuated its performance. In addition, cytokines such as Macrophage Inflammatory Proteins (MIP) - α and- β are highly expressed during PMA-induced macrophage differentiation, and AR001DS1 treatment blocks their expression. As shown in FIG. 15, AR001DS1 treatment also inhibited the induction of inflammatory cytokines such as IL-8, TNF- α, and RANTES. These data support that AR001DS1 can block the expression of inflammatory cytokines in macrophages.

Example 6.

Considerable evidence supports the role of Dendritic Cells (DCs) in the pathogenesis of allergic diseases. Acting with DCs, mast cells are one of the first immune cells to interact with allergens and other environmentally derived substances. Therefore, in this study, we solved the question of whether AR001DS1 inhibits the immune response of mast cells and dendritic cells by using RBL-2H3(RBL) cells (mast cell line) and mouse-derived DCs.

As shown in FIG. 16, stimulation with antigen [ DNP-Bovine Serum Albumin (BSA) ] caused significant release of β -hexosaminidase from RBL cells sensitized with anti-Dinitrophenyl (DNP) IgE. In contrast, in cells treated with AR001DS1, secretion of β -hexosaminidase was significantly inhibited in a dose-dependent manner.

Having observed that AR001DS1 can inhibit the secretion of β -hexosaminidase, we began to determine whether AR001DS1 inhibits the regulatory exocytosis of other secretory particulate matter (e.g., TNF- α) in DCs. As shown in FIG. 17, Lipopolysaccharide (LPS) stimulation induces significant secretion of TNF- α, whereas quercetin (quercetin) is one of the most well-known phytochemicals with antioxidant, antiproliferative and anti-inflammatory properties, and is effective in reducing TNF- α secretion in LPS-stimulated DCs. Surprisingly, the inhibitory effect of AR001DS1 on TNF- α secretion in LPS-stimulated DCs was superior to that of quercetin.

While the invention has been disclosed in terms of preferred embodiments, it is not intended to be limited thereto. Modifications and decorations may be effected by one of ordinary skill in the art without departing from the spirit and scope of the invention. Accordingly, the scope of the invention should be determined from the following claims.