KR20090051874A - Anti-inflammatory and anti-allergic composition containing as an active ingredient an extract of Tangerine japonica or a compound isolated therefrom - Google Patents

Abstract

The present invention Poncirus trifoliata ) The present invention relates to a composition for preventing and treating inflammatory diseases and allergic diseases containing an extract or a compound separated therefrom as an active ingredient, in detail, a tanza extract of the present invention or 21α-methylmelianodiol isolated therefrom. (21α-methylmelianodiol) or 21β-methylmelianodiol (21β-methylmelianodiol) is a pharmaceutical composition for the prophylactic treatment of inflammatory and allergic diseases because it exhibits the effect of inhibiting proliferation, cell cycle and apoptosis of interleukin-5 dependent Y16 cells and It can be usefully used for health food.

Tangerine Extract, 21α-Methylmelianodiol, 21β-Methylmelianodiol, Inflammation, Allergy

Description

Composition comprising an extract of Poncirus trifoliata and the compounds isolated there from having anti-inflammatory and anti-allergy activity

The present invention relates to a pharmaceutical composition for the prevention and treatment of inflammatory diseases and allergic diseases and health functional foods containing an extract of Tangerine or a compound isolated therefrom as an active ingredient.

Document 1 Bao S, et al., Immunology 94, pp181-188, 1998

[Document 2] Do Hae-Hyang Medicinal Dictionary Information, et al., Younglimsa, p792, 1990

Tominaga A, et al., Growth Factors 1989; 1: 135-46

Fadok VA, et al., Cell Death Differ 1998; 5: 551-62

Allergy is a type of hypersensitivity reaction due to imbalance, which is a biochemical phenomenon that shows a specific and modified response to foreign substances (antigens). Diseases include atopic dermatitis, allergic asthma, rhinitis and food allergies. 10-40% of the world's population suffers from various allergic diseases. In the normal human immune system, the Th ₁ response that regulates cellular immunity and the Th ₂ response that regulates humoral immunity are balanced, but in an allergic patient, the Th ₂ response is excessive, and the balance is broken and IgE (immunoglobulin E) It causes a variety of adverse reactions such as excessive increase of) and excessive release of histamine.

      When exposed to antigen, it is delivered to T cells and releases cytokines such as interleukin-4, 5, 6 and granulocyte-macrophage stimulator (GM-CSF). Cytokines react with B cells in lymph nodes and B cells self-denature plasma cells to secrete IgE antibodies. IgE antibodies attach to mast cells and basophils, resulting in histamine, serotonin, leukotriene B4, C4, D4, E4, platelet activator, prostaglandin D, interleukin-1, 3, 4, 5, 6, GM-CSF, tumor necrosis It releases factor-a, bradykinin and the like to mediate the inflammatory response.

Interleukin-5 is Th ₂ As a kind of cytokine, B cells and coral leukocytes are their main target cells. Interleukin-5 induces terminal differentiation of B cells into immunoglobulin-ejecting plasma cells (Bao S, et al., Immunology 94, pp181-188, 1998).

In order to treat or prevent allergies, it is necessary to suppress excessive Th ₂ responses and efficiently induce Th ₁ responses to stabilize the immune response of T cells, and to directly or indirectly act on B cells that produce excessive IgE to mediate IgE Block inflammatory reactions.

     Inflammation is an immune response of the human body in response to wounds or diseases, and oxidative stress such as ultraviolet rays, free radicals, and free radicals activate inflammatory factors, causing various diseases and aging of the skin. One of the hallmarks of inflammation is the increased oxygenation of arachidonic acid, which is metabolized by the cyclooxygenase (COX) producing prostaglandins and the 5-lipoxygenase pathway producing leukotriene. Prostaglandins and leukotrienes are mediators of inflammation. Thus, therapies designed to inhibit cyclooxygenase and / or lipoxygenase activity are of great interest. Cyclooxygenase enzymes come in two forms: cyclooxygenase-1 and cyclooxygenase-2. The latter form, cyclooxygenase-2, appears to play an important role in the progression of inflammation. Therefore, inhibiting cyclooxygenase-2 enzyme may be an effective way to reduce inflammation without the side effects associated with irreversible cyclooxygenase-1 inhibition.

     Another powerful inflammatory mediator, nitric oxide (NO), is produced from L-allysine by NO synthetase (NOS) and is produced in many types of cells by stress such as ultraviolet rays, endotoxins, and cytokines. . These inflammatory stimuli increase the expression of inducible NOS in the cell, generating NO, activating the macrophages and causing an inflammatory response.

In order to treat the above inflammatory diseases, various types of therapeutic agents for inflammatory diseases have been developed. The inflammatory disease therapeutic substances reported to date are induced by tissue cells damaged by acute inflammation, cells involved in inflammation, or chemotactic factors. It is a drug that inhibits the production of eicosanoids from the cell membrane of the white blood cells. In addition, allergic agents include drugs that inhibit degranulation (histamine release) inhibition, histamine receptor blocking, leukotriene production inhibition, and leukotriene receptor blocking, which are released during antigen-antibody reactions. However, there are limited side effects from mild side effects to serious side effects such as growth inhibition and osteoporosis. Therefore, there is an urgent need for substances derived from natural products that can be applied more safely as well as efficacy and effect.

Poncirus trifoliata) is jisil (Ponciri that dried in the sun a little fruit of a deciduous shrub with trifoliate orange Fructus ) contains the main ingredients of vitamin C and flavonoid glycosides such as neohesperidin, nargin, rhoifolin and methyl-tyramine (m-methyltyramine). Et al., Younglimsa, p792, 1990).

Therefore, in the course of studying small molecules that modulate the production and intercellular activity of interleukin-5, the present inventors have found that the extract of Tangerine or the compound isolated therefrom inhibits proliferation, cell cycle change and self-killing of interleukin-5 dependent Y16 cells. The present invention was completed by confirming that there is an excellent effect on anti-inflammatory and anti-allergy through the effect.

In order to achieve the above object, the present invention is a 21α-methylmelianodiol (21α-methylmelianodiol) or 21β-methyl represented by Structural Formula (A) to Structural Formula (B) isolated from Poncirus trifoliata extract or Provided is a pharmaceutical composition for the prevention and treatment of inflammatory diseases and allergic diseases containing melianodiol (21β-methylmelianodiol) as an active ingredient.

(A)

(A)

(B)

(B)

The extract is characterized in that the crude extract or non-polar solvent soluble extract of Tangerine.

      The crude extract includes water containing purified water, a lower alcohol having 1 to 4 carbon atoms or a mixed solvent thereof, preferably a water and methanol mixed solvent, more preferably an extract available in 60 to 100% methanol.

      The nonpolar solvent soluble extract is a solvent selected from hexane, chloroform, methylene chloride, dichloromethane, ethyl acetate, glycerin, propylene glycol, butylene glycol or ether, preferably dichloromethane or ethyl acetate, more preferably soluble in dichloromethane. One extract.

Extracts as defined herein include the fruit, stem or root, preferably fruit, of a tangerine tree.

The inflammatory diseases are caused by arteriosclerosis, rheumatoid arthritis, arthritis, asthma, acute pain, chronic pain, neuropathic pain, postoperative pain, pains such as migraine and joint pain, neuropathy, nerve damage, irritable bowel syndrome, endotoxin One or more diseases selected from shock, inflammatory bowel disease, cancer, and the like.

The allergic disease includes one or more diseases selected from bronchial asthma, allergic rhinitis, allergic asthma or allergic dermatitis and the like.

       Hereinafter, a method of obtaining a tangerine extract of the present invention will be described in detail.

The crude extract of the present invention is dried and shredded fruit of P. trifoliata , water containing purified water having a volume of about 1 to 50 times, preferably about 5 to 30 times the weight, 1 to 4 carbon atoms. Extraction methods such as repeated hydrothermal extraction, reflux cooling extraction, ultrasonic extraction, etc. for 7 days at room temperature with a lower alcohol or a mixed solvent thereof, preferably a mixed solvent of water and methanol, more preferably 60 to 100% methanol. After filtering the extract extracted by the extraction method of reflux cooling extraction and concentrated under reduced pressure can be obtained crude extract of the present invention.

In addition, the tangja nonpolar solvent soluble extract of the present invention is a solvent selected from hexane, chloroform, methylene chloride, dichloromethane, ethyl acetate, glycerin, propylene glycol, butylene glycol or ether, preferably obtained from Dichloromethane or ethyl acetate, more preferably dichloromethane, followed by extraction fractions may be performed by silica gel column chromatography to obtain the tangia nonpolar solvent soluble extract of the present invention.

       In addition, the compounds of the present invention can be separated as follows.

The dichloromethane soluble extract of the non-polar solvent extract obtained by the above-mentioned method was separated with 11 fractions (PF1-11) through a silica gel column using an n-hexane / ethyl acetate mixed solution, and the fraction PF7 was a dichloromethane / acetone mixed solution. Separation of 6 fractions (PF71-76) through a silica gel column, fraction PF73 is separated into six fractions (PF731-736) through a silica gel column using n-hexane / ethyl acetate mixed solution in the gradient mode, Fraction PF732 can separate the fractions through a C18 reversed phase column using a methanol / water mixed solvent and finally obtain 21a-methylmelianodiol and 21β-methylmelianodiol of the present invention.

       The present invention provides a pharmaceutical composition for the prophylaxis and treatment of inflammatory diseases and allergic diseases containing an extract of the tangja tree obtained by the above method or a compound isolated therefrom as an active ingredient.

       The pharmaceutical composition for preventing and treating inflammatory diseases and allergies of the present invention comprises 0.1 to 50% by weight of the extract or a compound separated therefrom based on the total weight of the composition.

Pharmaceutical compositions comprising an extract of the present invention or a compound isolated therefrom may further comprise suitable carriers, excipients and diluents commonly used in the manufacture of pharmaceutical compositions.

Pharmaceutical dosage forms of the extracts of the present invention or compounds isolated therefrom may be used in the form of their pharmaceutically acceptable salts, and may be used alone or in combination with other pharmaceutically active compounds as well as in a suitable collection.

Pharmaceutical compositions comprising the extract according to the present invention or a compound isolated therefrom are oral formulations, external preparations, suppositories, such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, etc., according to conventional methods, respectively. It can be formulated and used in the form of sterile injectable solutions. Carriers, excipients and diluents that may be included in the composition comprising the compound include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate , Cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. When formulated, diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrating agents, and surfactants are usually used. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, and the like, and such solid preparations may contain at least one excipient such as starch, calcium carbonate, sucrose, or the like. ) Or lactose, gelatin and the like are mixed. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Oral liquid preparations include suspensions, solvents, emulsions, and syrups, and may include various excipients, such as wetting agents, sweeteners, fragrances, and preservatives, in addition to commonly used simple diluents such as water and liquid paraffin. . Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, suppositories. As the non-aqueous solvent and suspending agent, propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate and the like can be used. As the base of the suppository, witepsol, macrogol, tween 61, cacao butter, laurin butter, glycerogelatin and the like can be used.

Preferred dosages of the extracts of the present invention or compounds isolated therefrom vary depending on the condition and weight of the patient, the extent of the disease, the form of the drug, the route of administration and the duration, and may be appropriately selected by those skilled in the art. However, for the desired effect, the extract of the present invention is preferably administered at 0.0001 to 100 mg / kg, preferably 0.001 to 10 mg / kg per day. Administration may be administered once a day or may be divided several times. The dosage does not limit the scope of the invention in any aspect.

The extract of the present invention or a compound isolated therefrom can be administered to mammals such as mice, mice, livestock, humans, etc. by various routes. All modes of administration can be expected, for example by oral, rectal or intravenous, intramuscular, subcutaneous, intrauterine dural or intracerebroventricular injection.

In another aspect, the present invention provides a health functional food for the prevention and improvement of inflammatory diseases and allergic diseases comprising the tangja extract or the compound isolated therefrom as an active ingredient exhibiting a prophylactic and improvement effect of inflammatory diseases and allergic diseases.

The health functional food may be prepared in powder, granule, tablet capsule or beverage form.

It may also be added to foods or beverages for the purpose of preventing and improving inflammatory and allergic diseases. At this time, the amount of the extract or a compound isolated therefrom in the food or beverage may be added to 0.01 to 15% by weight of the total food weight, the health beverage composition is 0.02 to 5 g, preferably 0.3 to 1 based on 100 ml It can be added at a ratio of g.

The health beverage composition of the present invention is not particularly limited to other ingredients except for containing the extract or a compound separated therefrom as an essential ingredient in the indicated proportions, and various flavors or natural carbohydrates as additional ingredients are used as in the general beverage. It may contain. Examples of the above-mentioned natural carbohydrates include monosaccharides such as glucose, fructose and the like; Disaccharides such as maltose, sucrose and the like; And conventional sugars such as polysaccharides such as dextrin, cyclodextrin, and sugar alcohols such as xylitol, sorbitol, and erythritol. As flavoring agents other than those mentioned above, natural flavoring agents (tauumatin, stevia extract (for example, rebaudioside A, glycyrrhizin, etc.) and synthetic flavoring agents (saccharin, aspartame, etc.) can be advantageously used. The proportion of said natural carbohydrates is generally about 1-20 g, preferably about 5-12 g per 100 ml of the composition of the present invention.

In addition to the above, the extract of the present invention or a compound isolated therefrom is used in various nutrients, vitamins, minerals (electrolytes), synthetic flavors and natural flavors, such as flavoring agents, coloring and neutralizing agents (such as cheese, chocolate), pectic acid and Salts thereof, alginic acid and salts thereof, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohols, carbonation agents used in carbonated drinks and the like. In addition, the extracts of the present invention may contain flesh for the production of natural fruit juices and fruit juice beverages and vegetable beverages. These components can be used independently or in combination. The proportion of such additives is not so critical but is usually selected in the range of 0 to about 20 parts by weight per 100 parts by weight of the extract of the present invention.

Tangerine extract of the present invention or a compound isolated therefrom is useful as a pharmaceutical composition and health functional food for the prevention and treatment of inflammatory and allergic diseases because it exhibits proliferation inhibition, cell cycle change and self-killing effect of interleukin-5 dependent Y16 cells. Can be used.

Hereinafter, the present invention will be described in detail by the following Examples and Experimental Examples.

However, the following Examples, Reference Examples and Experimental Examples are merely illustrative of the present invention, and the content of the present invention is not limited by the following Examples, Reference Examples and Experimental Examples.

Reference Example 1. Reagents and Instruments

The analyzer includes a polarimeter (JASCO DIP-1000 polarimeter, Japan), a spectrometer (JASCO FT-IR 300E spectrophotometer, Japan), a spectrometer (JASCO V-550 spectrophotometer, Japan), an NMR spectrometer (Bruker DMX 250 spectrometer, Germany) ) And FAB mass spectrometer (JEOL JMS-700 spectrometer, Japan) were used. Various reagents were manufactured by Sigma-Aldrich, St. Louis, USA, and other solvents were used without first-class reagent purification.

Example 1 Preparation of Tangerine Crude Extract

After drying and cutting the fruit of P. trifoliata purchased from Daegu market, 80% methanol was added to the dried fruit (10 kg) of the fruit, and extracted repeatedly at room temperature for 7 days. Concentration under gave 500 g of crude extract (hereinafter referred to as “PT”), which was used in the following experimental example.

Example 2. Preparation of Tangerine Nonpolar Solvent Soluble Extract

500 g of the PT crude extract obtained in Example 1 was extracted with dichloromethane, ethyl acetate and butanol, respectively, 160 g of dichloromethane extract (hereinafter referred to as "PT-D"), and ethyl acetate extract (hereinafter referred to as "PT-E"). 135.5 g and 44.2 g of butanol extract (hereinafter referred to as "PT-B") were obtained and used in the following experimental example.

Example 3 Separation of 21α-Methylmelianodiol and 21β-Methylmelianodiol from Tangerine Extracts

160 g of Tangerine dichloromethane / water (1: 1, v / v) soluble extract (PT-D) obtained in Example 2 was added with n-hexane / ethyl acetate (10: 0, 9: 1, 8: 2 , 7: 3, 6: 4, 5: 5, 4: 6, 3: 7, 2: 8, 1: 9, 0:10, 4 L each) using a mixed solution of silica gel column (12 x 80 cm) Eleven fractions (PF1-11) were separated. Fraction PF7 (5.9 g) was separated into silica gel column (6 ×) using dichloromethane / acetone (9: 1, 8: 2, 7: 3, 6: 4, 5: 5, 3: 7, 4 L each). Six fractions (PF71-76) were separated. Fraction PF73 consists of six fractions through a silica gel column (3.5 x 50 cm) using n-hexane / ethyl acetate (6: 4, 5: 5, 4: 6, 3: 7, 3 L each) in gradient mode. (PF731-736) was separated. Fraction PF732 separates the fractions through a C18 reversed phase column (4 x 50 cm) using methanol / water (4: 6, 5: 5, 6: 4, 7: 3, 3 L each) and then finally 120 mg of 21α-methylmelianodiol (hereinafter referred to as “21α-MMD”) and 21β-methylmelianodiol (hereinafter referred to as “21β-MMD”) each having the following physical properties using high performance liquid chromatography And 20 mg were obtained and used in the following experimental example.

21α-MMD:

¹ H-NMR (250 MHz, CDCl ₃ ) δ: 5.27 (1H, brd, J = 2.8 Hz, H-7), 4.75 (1H, d, J = 2.8 Hz, H-21), 4.20 (1H, m , H-23), 3.31 (3H, s, 21-OCH ₃ ), 3.20 (1H, d, J = 9.3 Hz, H-24), 2.74 (1H, dt, J = 14.5, 5.5 Hz, H-2a ), 2.28 (1H, m, H-9), 2.23 (1H, m, H-2b), 1.98 (1H, m, H-17), 1.97 (1H, m, H-1a), 1.90 (1H, m, H-16a), 1.44 (1H, m, H-1b), 1.33 (1H, m, H-16b), 1.27, 1.23, 1.08, 1.01, 0.99, 0.98, 0.82 (3H, s, 7 ×, respectively) CH ₃ );

¹³ C-NMR (63 MHz, CDCl ₃ ) δ: 216.9 (C-3), 145.5 (C-8), 118.1 (C-7), 108.9 (C-21), 76.70 (C-23), 75.32 ( C-24), 73.03 (C-25), 55.61 (C21-OCH ₃ ), 52.29 (C-5), 50.91 (C-14), 50.24 (C-17), 48.23 (C-9), 47.83 ( C-4), 47.65 (C-20), 43.58 (C-13), 38.47 (C-1), 35.04 (C-2), 34.87 (C-10), 34.29 (C-15), 33.74 (C -22), 31.45 (C-12), 27.36 (C-30), 27.24 (C-16), 26.42 (C-27), 26.33 (C-26), 24.44 (C-29), 24.30 (C- 6), 22.56 (C-18), 21.53 (C-28), 17.72 (C-11), 12.70 (C-19).

21β-MMD:

¹ H-NMR (250 MHz, CDCl ₃ ) δ: 5.28 (1H, brd, J = 2.8 Hz, H-7), 4.71 (1H, brs, H-21), 4.40 (1H, m, H-23) , 3.31 (3H, s, 21-OCH ₃ ), 3.15 (1H, d, J = 9.3 Hz, H-24), 2.75 (1H, dt, J = 14.5, 5.5 Hz, H-2a), 2.28 (1H , m, H-9), 2.23 (1H, m, H-2b), 1.98 (1H, m, H-17), 1.97 (1H, m, H-1a), 1.90 (1H, m, H-16a ), 1.44 (1H, m, H-1b), 1.33 (1H, m, H-16b), 1.24, 1.22, 1.09, 1.02, 1.00, 0.98, 0.81 (3H, s, 7 × CH ₃ , respectively);

¹³ C-NMR (63 MHz, CDCl ₃ ) δ: 216.8 (C-3), 145.6 (C-8), 118.1 (C-7), 104.8 (C-21), 78.84 (C-23), 76.50 ( C-24), 72.88 (C-25), 55.16 (C21-OCH ₃ ), 52.38 (C-5), 50.75 (C-14), 48.31 (C-9), 47.84 (C-4), 46.27 ( C-20), 44.97 (C-17), 43.48 (C-13), 38.51 (C-1), 35.07 (C-2), 34.89 (C-10), 34.17 (C-15), 31.56 (C -22), 31.06 (C-12), 27.41 (C-30), 27.30 (C-16), 26.38 (C-27), 26.30 (C-26), 24.47 (C-29), 24.34 (C- 6), 23.25 (C-18), 21.52 (C-28), 17.80 (C-11), 12.71 (C-19).

Experimental Example 1. Measurement of cell proliferation inhibitory effect

The inhibitory effect of interleukin-5 dependent Y16 cell proliferation of the PT extract obtained in Examples 1 to 3 or a compound isolated therefrom was measured by the following method using the method described in the literature (Tominaga A, et al., Growth Factors 1989; 1: 135-46).

After inoculating Y16 cells (1 × 10 ⁴ cells / well) in 96 wells, the samples obtained in Examples 1 to 3, interleukin-5 (3 U / mL) and 10% Fetal bovine serum (FBS) together Incubate for 48 hours. Cells in each well were diluted with 2- (4-oodophenyl) -3- (4-nitrophenyl) -5- (2,4-disulfenphenyl) -2 H -tetrazolium (WST-1) Lab, Kumamoto, Japan) 20 uL was incubated for 2 hours and the absorbance was measured at 450 nm. The standard solution (100 mg / mL or 100 mM) of the test sample was dissolved in 100% dimethyl sulfoxide (DMSO) and used after diluting in the medium at a concentration of less than 0.05% DMSO. Tyropostin AG-490 (Sigma-Aldrich, USA) was used as a positive control.

As a result of the experiment, when the samples of Examples 2 to 3 were treated at a concentration of 50 ug / mL, it was confirmed that the proliferation of interleukin-5-dependent Y16 cells was significantly reduced compared to the control group treated with interleukin-5 only ( See Table 1).

sample Proliferation inhibition rate (%) PT-D (Example 2) 94 PT-E (Example 2) 18 PT-B (Example 2) 5

When 21α-MMD, a compound obtained in Example 3, was treated at concentrations of 20 uM, 30 uM and 40 uM, it was confirmed that the proliferation of Y16 cells was inhibited at 67%, 96% and 98%, respectively. (See Figure 1A). In addition, when 21β-MMD was treated at concentrations of 10 uM, 20 uM, 30 uM and 40 uM, it was confirmed that the proliferation of Y16 cells was inhibited in a concentration-dependent manner at 24%, 77%, 95% and 99%, respectively ( See FIG. 1A). On the other hand, since both 21α-MMD and 21β-MMD had no effect on FBS-dependent RAW 264.7 macrophages (FIG. 1B), it was confirmed that they have a specific effect on the interleukin-5 dependent response (FIG. 1B). 1).

Experimental Example 2. Flow Cytometry

Cell cycle analysis was performed by flow cytometry as follows.

Y16 cells ( ⁵ × 10 ⁵ cells / well) were seeded and attached to 6-well plates, then interleukin-5 (10 U / mL), 10% FBS and the sample obtained in Example 3 were incubated together for 12 hours. After fixing the cells with 70% ethanol, propidium iodide (20 ug / mL) was treated for 30 minutes. The proportion of cells present at different stages of the cell cycle was measured 10,000 cells per sample via FACS caliber (BD Bioscience, USA) and analyzed using Modpit software.

As a result of the experiment, it was confirmed that the interleukin-5-dependent Y16 cells showed a cell ratio of 27% in the G1 stage, 60% in the S stage, and 13% in the G2 / M stage (see FIG. 2A). The compounds obtained in Example 3, 21α-MMD and 21β-MMD, had no effect when treated at a concentration of 10 uM (see FIGS. 2B and 2D) and 30 μM of 21α-methylmelianodiol and 21β-MMD. When treated with the concentration of G1 cells were confirmed to increase to 49% and 75%, respectively, compared to the 27% control group (see Fig. 2C and 2E). On the other hand, Tyrofostine AG-490 as a positive control group was found to have no effect on the cell cycle when treated at a concentration of 30 uM (see Figure 2F).

Experimental Example 3. Confirmation of the effect of inhibiting self-killing

Apoptosis of the compound obtained in Example 3 was measured by the following method using the method described in the literature via dual staining of FITC-labeled Annexin V and propidyl iodide (Fadok VA, et al. Cell Death Differ 1998; 5: 551-62).

After Y16 cells ( ⁵ × 10 ⁵ cells / well) were inoculated into 12-well plates and attached, the interleukin-5 (30 U / mL) and the sample obtained in Example 3 without FBS were incubated for 24 hours. Cells were reacted for 10 minutes in the dark with FITC-labeled Annexin V (0.5 ug / mL) and Propidyl Iodide (2 ug / mL), and then measured by flow cytometry.

As a result of the experiment, when treated with interleukin-5 alone for 24 hours, it was confirmed that 96% or more of Y16 cells were alive (see FIG. 3A), whereas apoptosis was treated when 21α-MMD was treated at a concentration of 40 uM (see FIG. 3A). Early apoptosis: 3.7%; and late apoptosis / cell necrosis: 58.2%), and apoptosis was reduced to 69.8% (initial apoptosis: 4.0%; and 21β-MMD at a concentration of 40 uM); and Terminal apoptosis / cell necrosis: 65.8%) was confirmed to increase significantly (see Figures 3C and 3E). As a positive control group, tyrofostine AG-490 was found to increase to 51.9% and 76.2%, respectively, when apoptosis was treated at concentration-dependent concentrations of 10 uM and 40 uM (see FIG. 3F).

Hereinafter, an example of the preparation of a pharmaceutical composition containing an extract of the present invention or a compound separated therefrom will be described, but the present invention is not intended to be limited thereto but only to be described in detail.

Formulation Example 1 Preparation of Powder

PT extract 20 mg

Lactose 100 mg

Talc 10 mg

The above ingredients are mixed and filled in an airtight cloth to prepare a powder.

Formulation Example 2 Preparation of Tablet

21α-MMD or 21β-MMD 10 mg

Corn starch 100 mg

Lactose 100 mg

2 mg magnesium stearate

After mixing the above components and tableting according to the manufacturing method of the tablet of the bankbook to prepare a tablet.

Formulation Example 3 Preparation of Capsule

PT extract 10 mg

3 mg of crystalline cellulose

Lactose 14.8 mg

Magnesium Stearate 0.2 mg

According to a conventional capsule preparation method, the above ingredients are mixed and filled into gelatin capsules to prepare capsules.

Formulation Example 4 Preparation of Injection

21α-MMD or 21β-MMD 10 mg

Mannitol 180 mg

Sterile distilled water for injection 2974 mg

Na ₂ HPO ₄ , 12H ₂ O 26 mg

According to the conventional method for preparing an injection, the amount of the above ingredient is prepared per ampoule (2 ml).

Formulation Example 5 Preparation of Liquid

PT extract 20 mg

10 g of isomerized sugar

5 g of mannitol

Purified water

According to the conventional method for preparing a liquid solution, each component is added to the purified water to dissolve it, and lemon flavor is added to the mixture, the above ingredients are mixed, and then purified water is added to adjust the total amount to 100 ml, and then sterilized by filling into a brown bottle. do.

Formulation Example 6 Preparation of Health Functional Food

21α-MMD or 21β-MMD 1000 mg

Vitamin mixture proper amount

70 μg of Vitamin A Acetate

Vitamin E 1.0 mg

Vitamin B1 0.13 mg

Vitamin B2 0.15 mg

Vitamin B6 0.5 mg

0.2 μg of vitamin B12

Vitamin C 10 mg

10 μg biotin

Nicotinamide 1.7 mg

50 μg folic acid

Calcium Pantothenate 0.5 mg

Mineral mixture

Ferrous Sulfate 1.75 mg

Zinc Oxide 0.82 mg

Magnesium carbonate 25.3 mg

15 mg potassium monophosphate

Dicalcium Phosphate 55 mg

Potassium Citrate 90 mg

Calcium Carbonate 100 mg

Magnesium chloride 24.8 mg

Although the composition ratio of the above-mentioned vitamin and mineral mixtures is mixed with a component suitable for a health food in a preferred embodiment, the compounding ratio may be arbitrarily modified, and the above ingredients are mixed according to a conventional health food manufacturing method. The granules may be prepared and used for preparing a health food composition according to a conventional method.

Formulation Example 7 Preparation of Healthy Drink

PT extract 1000 mg

Citric acid 1000 mg

100 g oligosaccharides

Plum concentrate 2 g

1 g of taurine

900 ml of purified water whole

After mixing the above components according to the conventional healthy beverage production method, and stirred and heated at 85 ℃ for about 1 hour, the resulting solution is filtered and obtained in a sterilized 2 L container, sealed sterilization and then refrigerated and stored Used to prepare the healthy beverage composition of the invention.

Although the composition ratio is a composition that is relatively suitable for the preferred beverage in a preferred embodiment, the compounding ratio may be arbitrarily modified according to regional and ethnic preferences such as demand hierarchy, demand country, and usage.

1 is a diagram showing the effect of inhibiting interleukin-5-dependent Y16 cells in accordance with the concentration of the extract of Tangerine and the compound isolated therefrom,

2 is a diagram illustrating the cell cycle of Y16 cells according to the concentration of 21α-methylmelianodiol and 21β-methylmelianodiol,

3 is a diagram illustrating the apoptosis of Y16 cells according to the concentration of 21α-methylmelianodiol and 21β-methylmelianodiol.

Claims (9)

Tangerine Extract ( Poncirus) trifoliata ) or inflammation containing 21α-methylmelianodiol or 21β-methylmelianodiol represented by the following structural formulas (A) to (B) as an active ingredient Pharmaceutical compositions for the prevention and treatment of diseases and allergic diseases;

(A)

(B) The pharmaceutical composition according to claim 1, wherein the extract is a crude extract or a nonpolar solvent soluble extract. The pharmaceutical composition according to claim 2, wherein the crude extract comprises an extract available in water including purified water, a lower alcohol having 1 to 4 carbon atoms, or a mixed solvent thereof. 3. The pharmaceutical composition according to claim 2, wherein the nonpolar solvent soluble extract comprises an extract soluble in a solvent selected from hexane, chloroform, methylene chloride, dichloromethane, ethyl acetate, glycerin, propylene glycol, butylene glycol or ether. Composition. The pharmaceutical composition according to claim 1, wherein the extract comprises fruits, stems, or roots of tangerine trees. The method of claim 1, wherein the inflammatory disease is arteriosclerosis, rheumatoid arthritis, arthritis, asthma, acute pain, chronic pain, neuropathic pain, postoperative pain, pains such as migraine and joint pain, neuropathy, nerve damage, irritable bowel A pharmaceutical composition comprising one or more diseases selected from syndromes, shock by endotoxins, inflammatory bowel disease, cancer and the like. The pharmaceutical composition of claim 1, wherein the allergic disease comprises one or more diseases selected from bronchial asthma, allergic rhinitis, allergic asthma, allergic dermatitis, and the like. Tangerine Extract ( Poncirus) trifoliata ) or 21α-methylmelianodiol (21α-methylmelianodiol) or 21β-methylmelianodiol represented by Structural Formulas (A) to (B) separated from the above as an active ingredient Functional foods for the prevention and improvement of inflammatory diseases and allergic diseases. The dietary supplement of claim 8 in the form of a powder, granule, tablet, capsule or beverage.

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