KR101318896B1 - Composition for treating or preventing allergic diseases comprising 2-linoleoyl glycerol - Google Patents

Abstract

The present invention relates to a pharmaceutical composition for the prevention or treatment of allergic diseases, including 2-linoleoyl glycerol, more specifically, the residue (samsa) extract and / or 2- separated from the It relates to a pharmaceutical composition for the prevention or treatment of allergic diseases, including linoleoyl glycerol (2-linoleoyl glycerol) as an active ingredient, a food composition for improving the diseases.

Description

Composition for treating or preventing allergic diseases comprising 2-linoleoyl glycerol}

The present invention relates to a composition for the prevention or treatment of allergic diseases, including 2-linoleoyl glycerol, more specifically 2-linoleoyl glycerol (2) separated from the residue (samsam) extract (2 It relates to a pharmaceutical composition for the prevention or treatment of allergic diseases, comprising -linoleoyl glycerol) as an active ingredient, a food composition for improving the disease.

Allergic diseases are increasing day by day due to environmental pollution, stress caused by various pollution, or residential environment including changed food stocks. An allergic disease is a very difficult chronic disease caused by the body's excessively sensitive reaction to the causative agent. It is a disease caused by abnormalities in the body's immune system.

Allergic diseases are various, such as allergic rhinitis, asthma, allergic conjunctivitis, depending on the site of the immune response from the antigen coming from the outside, but the cause of the disease is common in that it is due to hypersensitivity to the causative agent from outside Because of this, drugs that can suppress an excessive immune response can be commonly used.

Asthma, a typical allergic disease, is characterized by airway hyperresponsiveness to various stimuli. Clinical symptoms such as wheezing, dyspnea, and cough caused by extensive airway narrowing are naturally or reversible by treatment. Can be improved. Most asthma are allergic, characterized by chronic airway inflammation and bronchial hyperresponsiveness (Minoguchi K and Adachi M. Pathophysiology of asthma.In: Cherniack NS, Altose MD, Homma I, editors Rehabilitation of the patient with respiratory disease.New York: McGraw-Hill, 1999, pp.97-104).

Allergic asthma patients are known to account for about 10% of the world's population (275 million, 1995), and 17 million people in the United States alone suffer from asthma, of which 5 million are reported to be adolescents. In 2000, the US market for allergic asthma drugs was about $ 6.4 billion, and the Korean market was reported to be about $ 1 billion, accounting for 20% of the Korean drug market.

Drugs currently used to treat asthma include bronchodilators for inhalation, oral or injectable bronchodilators (sympathetic and theophylline drugs), leukotriene antagonists (such as montelukast, tranlukast, and giluton), and anti-allergic drugs ( Chromoline sodium, ketotifen). Among these, airway dilators such as anticholinergic and beta-2 receptor agonists do not have an effect on inflammation that aggravates the disease, and thus only relieve symptoms. Steroids, which are known to be effective against inflammation, are problematic for long-term use due to severe side effects. Therefore, a combination of the two is often prescribed, but due to the side effects of steroids developed in the form of inhalants than the oral drug is difficult to take, there is a problem in taking compliance.

Allergic rinitis is an allergic disease that causes symptoms such as sneezing, itching, stuffy nose, runny nose, etc., caused by allergens such as pollen, dust, mold, or animal hair. When allergens come into contact with the nose, the nasal mucosa stimulates the blood vessels or secretory glands, causing a lot of runny nose.Nerve reflexes cause the nose to sneeze, sneeze, and reduce the inhalation of foreign substances from the outside. I get stuck. In addition, symptoms of allergic rhinitis are clinically indistinguishable from other chronic rhinitis symptoms in most patients, and in addition to exposure to the causative agent of allergens, various symptoms such as pollutants in the air, dust, changes in humidity or temperature, etc. Symptoms may also be caused by common stimuli, making treatment difficult.

Currently, as an allergic rhinitis treatment agent, anti-inflammatory drugs such as ibuprofen, diclofenac, naproxen, and the like, and chemicals such as tranilast and ketotifen are used. However, these chemicals are severely toxic and cause side effects such as digestive disorders, dizziness and heartburn, as well as resistance to long-term use, causing chronic rhinitis.

Allergic conjunctivitis is an inflammatory condition of the conjunctiva caused by bacteria, viruses, allergies, or environmental factors. It is the most common eye disease that causes itching, tearing, itching, red eyes, and headache. It is largely classified into infectious conjunctivitis and non-infectious conjunctivitis, and it is usually healed well in some cases, but it is fatal and may cause blindness due to tissue damage.

Allergic conjunctivitis has been treated with antihistamine or steroid eye drops. Steroidal anti-inflammatory drugs of the glucocorticoid family, a typical treatment for conjunctivitis, cause two categories of side effects: first, sudden discontinuation after long-term use, and second, symptomatic of long-term overuse. Acute adrenal insufficiency during prolonged administration of corticosteroids causes symptoms such as systemic weakness, fever, myalgia, arthralgia, and loss of appetite, increases the risk of infections with bacteria, viruses, increases body weight, and changes body shape. Insomnia is well accompanied. In particular, eye drops or ointments of glucocorticoids are prone to side effects such as glaucoma, cataracts, or bacterial infections due to intraocular pressure increase when used over a long period of time, at the discretion of the patient. Because of these side effects, there are many problems in using glucocorticoids.

Allergic diseases are most often caused by mast cells in tissues activated by antigen-antibody reactions and basophils in blood and eosinophils (primarily histamine, leukotriene, prostaglandins, TNF-α, cytokines, etc.) do. The use of allergic drugs currently used is either symptomatic or has side effects, and therefore, the development of more fundamental and safe therapeutic drugs is urgently needed.

Prostaglandindes, eicosanoids including leukotriens, and platelet activating factor (PAF), which are key mediators for inducing allergic diseases, include phospholipase A2. , PLA2) and cyclooxygenase and lipoxygenase are produced from the precursor arachidonic acid.

Phospholipase A2 (PLA2), an arachidonic acid metabolic rate-limiting enzyme, is divided into intracellular (cPLA2) and extracellular (secretory PLA2, sPLA2), depending on the location of intracellular distribution, and isozyme of sPLA2. isozyme), and among these enzymes, sPLA2-IIA and sPLA2-V are known to be directly and indirectly involved in various inflammatory diseases and allergic reactions (Murakami M et al, Phospholipase A2 enzymes.Prostaglandins Other Lipid Mediat) , 68-69, pp. 3-58, 2002; Reid RC, Inhibitors of secretory phospholipase A2 group IIA.Curr. Med. Chem. 12, pp. 3011-3026, 2005). Phospholipase A2 acts on the sn-2 positions of phosphatidylcholine and phosphatidylethanolamine, which are cell membrane phospholipids, to release arachidonic acid. Arachidonic acid is converted to eicosanoids by cyclooxygenase. As a result, inhibition of phospholipase A2 decreases the eicosanoids, which are inflammation-inducing agents, and these mechanisms are responsible for the symptoms of asthma, arthritis, and rheumatic disease in mammals, including humans. (Pinon P. et al., Rev. Esp. Cardiol. 59, pp. 247-250, 2006; Farooqui AA et al., LA. Neuroscientist. 12, pp.245-). 260, 2006).

Cyclooxygenase is an enzyme that has an important effect on blood clotting and inflammation and is bound to cell membranes. The enzyme is an oxidation reaction of arachidonic acid and forms the main products of prostaglandin, prostacyclin and thromboxane. Tromoxane is produced by an enzyme called COX-1 present in platelets and has a function of inducing vasoconstriction and sticky properties of platelets. In contrast, an enzyme called COX-2 is expressed in blood vessels to produce prostaglandins and prostacyclins. Prostacyclin has the ability to dilate blood vessels and block platelet activity. Most drugs with cyclooxygenase inhibitory action have analgesic, anti-edema, antipyretic, anti-inflammatory and platelet anticoagulation by inhibition of prostaglandin production in topical inflammation (Learney, PM, et al. BMJ, 332). , pp. 1302-1308, 2006; Silva, PJ, et al. Theoret.Chem. Accounts, 110, pp.345-351, 2003; Vally, M et al.Curr. Pharm.Biotechnol. 7, pp.241- 246, 2006; Capone, ML et al. Expert.Opin.Drug Metab.Toxicol. 1, pp. 269-282, 2005).

Lipoxygenase is the first published enzyme in the biosynthesis of leukotriene by catalyzing the oxidation of arachidonic acid. Lipoxygenase converts arachidonic acid to hydroperoxyeicosatetraenoic acid (HPETE), which is the first step in the metabolic pathway to produce hydroxyeicosatetraenoic acid (HETE) and leukotriene. Products from lipoxygenase metabolism of arachidonic acid are involved and they are involved as regulators in various disease states. For example, aerosols of leukotriene C4 and D4 cause bronchial contraction, and leukotriene B4 and 5-HETE are potent chemotactic factors for inflammatory cells such as polymorphonuclear leukocytes. They are also found in the blood of patients with rheumatoid arthritis. Leukotriene is also involved as an important regulator in allergic rhinitis, psoriasis, adult respiratory disease symptoms, Crohn's disease, endotoxin shock and ischemia induced by myocardial injury. Thus, lipoxygenase enzymes are known to play an important role in various diseases such as inflammation, asthma, allergic arthritis, psoriasis and cancer (Radmark OJ Lipid Mediat. Cell Signal. 12, pp.171-184, 1995 Dwyer JH, et al. New Engl. J. Med. 350, pp. 4-7, 2004; Taccone-Gallucci M, et al. Kidney Int. 69, pp. 1450-1454, 2006; Manev H et al. , Med. Hypotheses 66, pp. 501-503, 2006).

Straw (Sasam) ( Adenophora triphylla var. japonica Hara, Adenophora polyantha Nakai, Adenophora triphylla var. hirsuta Nakai, Adenophora lilifolia Ledeb., Adenophora stricta Miq., Adenophora taquetii Lev., Adenophora palustris Kom.) Is a perennial herb that belongs to the bellflower family, which grows anywhere in the country and grows mostly in the sunny foothills. The height is about 40-120cm, and there are fine hairs on the whole plant. Leaves are coarse, round, or regenerated along the stem, long oval or round ellipse, 4-8 cm long, 0.5-4 cm wide, with jagged edges. The main ingredient of the root is saponin such as deodeok and bellflower. However, there are no teachings or disclosures about the effects of allergic extracts or compounds isolated therefrom on allergic diseases such as asthma and conjunctivitis.

The present inventors have found that 2-linoleoyl glycerol compounds isolated from stalk extracts are various cytokines and related proteins, interleukin-4 (IL-4), interleukin-10 (IL-10), and TNF-. Inhibits the activity of α, cyclooxygenase-2 (COX-2) and lipoxygenase to effectively prevent and treat allergic diseases including rheumatoid arthritis, other types of arthritis, asthma, rhinitis, conjunctivitis, etc. Confirmed and completed the present invention.

An object of the present invention to provide a pharmaceutical composition for the prevention or treatment of allergic diseases, or a food composition for improving the diseases comprising 2-linoleoyl glycerol represented by the formula (1) as an active ingredient It is.

Another object of the present invention is a pharmaceutical composition for the prophylaxis or treatment of allergic diseases, including as the active ingredient (2-samole) extract and 2-linoleoyl glycerol isolated therefrom, or It is to provide a food composition for improvement.

As one embodiment, the present invention provides a pharmaceutical composition for the prevention or treatment of allergic diseases comprising 2-linoleoyl glycerol represented by the following formula (1) as an active ingredient.

In another aspect, the present invention provides a method for treating an allergic disease comprising 2-linoleoyl glycerol represented by Formula 1 as an active ingredient.

[Formula 1]

As another aspect, the present invention provides a pharmaceutical composition for the prevention or treatment of allergic diseases, including the residue (samsam) extract and 2-linoleoyl glycerol.

In yet another aspect, the present invention provides a method for treating an allergic disease comprising the above residue (sasam) extract and 2-linoleoyl glycerol.

The 2-linoleoyl glycerol of the present invention is preferably obtained by extracting, separating and purifying from a residue (sasam ginseng), but is not limited thereto. That is, the compound may be obtained by extraction, separation and purification from other raw materials, may be prepared by a known synthesis method, or may be used as a commercial reagent.

In another aspect, the present invention provides a method for separating and purifying 2-linoleoyl glycerol from a residue (sasam). The method of separation and purification is as follows.

First, the residue (samsam) is extracted with water, C ₁ -C ₄ alcohol or a mixed solvent thereof to obtain a residue (samsam) extract. The residue (sasam) extract is extracted with water, n-hexane (n-hexane), methylene chloride (methylene chloride), ethyl acetate (ethyl acetate), or n-butanol (n-butanol) To obtain. Finally, the residue (four ginseng) fraction is subjected to column chromatography to obtain 2-linoleoyl glycerol.

The stem (sasam) extract of the present invention may be preferably a root (samsam) root extract, but is not limited thereto.

In the present invention, the residue (sasam) extract may be extracted with C ₁ to C ₄ lower alcohols such as water, methanol, ethanol, butanol or a mixed solvent thereof. Preferably the residue (sasam) extract is extracted with ethanol, most preferably the residue (sasam) extract is 10 to 100% ethanol extract.

In addition, the extract of the present invention may include any one or more of the extract obtained by the extraction treatment, the dilution or concentrate of the extract, the dried product obtained by drying the extract, and these modifiers or purified products.

The composition comprising the residue (sasam) extract of the present invention and / or 2-linoleoyl glycerol may be a pharmaceutical composition for the prevention or treatment of allergic diseases.

In the present invention, the allergic disease may be a respiratory disease, an ocular disease, a nasal disease, a gastrointestinal disease, a lung disease or an inflammatory disease. Specifically, bronchial asthma, allergic asthma, allergic conjunctivitis, allergic disease Rhinitis, gastrointestinal allergy, or arthritis, and may include, but are not limited to, colds, sneezing, angioedema, eosinophilia, abdominal pain, vomiting, diarrhea symptoms.

In the present invention, "prophylaxis or treatment of a disease" includes the prevention, complete or partial treatment of the disease. It also includes reducing symptoms, improving symptoms, alleviating symptoms of pain, and reducing the incidence of disease.

Key mediators for inducing allergic diseases are interleukin, prostaglandins, leukotrienes and the like, which can be produced by phospholipase A2, cyclooxygenase-2, lipoxygenase and the like. In the specific example of the present invention, when 2-linoleoyl glycerol was treated, it was confirmed that the expression or activity of the cytokine or related protein which causes allergic diseases was suppressed. Specifically, it was confirmed that 2-linoleoyl glycerol inhibits the degranulation of inflammatory chemical mediators in adipocytes in a concentration-dependent manner (FIG. 2). As the concentration of 2-linoleyl glycerol increases, the concentration of interleukin- 4 (IL-4), interleukin-10 (IL-10), TNF-α, it was confirmed that the expression of cyclooxygenase-2 (COX-2) is inhibited (Fig. 3). It was also confirmed that 2-linoleyl glycerol inhibits the expression of proteins involved in signaling processes by COX-2 and cytokines (p-AKT, p-ERK, p-JNK, p-p38). (FIG. 4), As the concentration of 2-linoleoyl glycerol increased, it was confirmed that the activity inhibition rate of lipoxygenase increased proportionally (FIG. 5).

Therefore, it was confirmed that the present invention can be used for the prevention or treatment of allergic diseases by blocking or inhibiting the pro-inflammatory mechanism of the 2-linoleoyl glycerol of the present invention.

In the present invention, the 2-linoleoyl glycerol may also be used in the form of a pharmaceutically acceptable salt, and includes all salts, hydrates and solvates prepared by conventional methods.

As salts are acid addition salts formed with pharmaceutically acceptable free acids. Acid addition salts are prepared by conventional methods, for example by dissolving a compound in an excess of aqueous acid solution and precipitating the salt using a water miscible organic solvent (eg, methanol, ethanol, acetone or acetonitrile). Equivalent molar amounts of the compound and acid or alcohol (eg, glycol monomethylether) in water can be heated and the mixture can then be evaporated to dryness or the precipitated salts can be suction filtered.

In this case, inorganic acids and organic acids may be used as the free acid, and hydrochloric acid, bromic acid, phosphoric acid, sulfuric acid, nitric acid, tartaric acid, etc. may be used as the inorganic acid, and citric acid, acetic acid, lactic acid, tartaric acid ( tartaric acid), maleic acid, fumaric acid, formic acid, propionic acid, oxalic acid, trifluoroacetic acid, benzoic acid, gluconic acid, methanesulfonic acid, glycolic acid, succinic acid, 4-toluenesulfonic acid, galluxuronic acid, m Main acid, glutamic acid or aspartic acid, etc. can be used, but it is not limited to these.

In addition, bases can be used to make pharmaceutically acceptable metal salts. The alkali metal or alkaline earth metal salt is obtained, for example, by dissolving the compound in an excess amount of an alkali metal hydroxide or alkaline earth metal hydroxide solution, filtering the non-soluble compound salt, and evaporating and drying the filtrate. At this time, as the metal salt, it is preferable to produce sodium, potassium or calcium salt particularly, but not limited thereto. Corresponding silver salts can also be obtained by reacting alkali or alkaline earth metal salts with a suitable silver salt (eg, silver nitrate).

The composition of the present invention may further comprise a pharmaceutically acceptable carrier. The composition comprising a pharmaceutically acceptable carrier can be of various oral or parenteral formulations. In the case of formulation, a diluent or excipient such as a filler, an extender, a binder, a wetting agent, a disintegrant, or a surfactant is usually used.

Solid preparations for oral administration include tablets, troches, lozenges, aqueous or oily suspensions, prepared powders or granules, emulsions, hard or soft capsules, syrups or elixirs, and the like. Binders such as lactose, saccharose, sorbitol, mannitol, starch, amylopectin, cellulose or gelatin for preparation in formulations such as tablets and capsules; Excipients such as dicalcium phosphate; Disintegrants such as corn starch or sweet potato starch; Lubricants such as magnesium stearate, calcium stearate, sodium stearyl fumarate or polyethylene glycol wax. In the case of capsule formulations, in addition to the substances mentioned above, they contain a liquid carrier such as fatty oil.

In addition, to formulate into a parenteral formulation, the compound of Formula 1 is prepared as a solution or suspension by mixing a stabilizer, a buffer, a sterile aqueous solution, a non-aqueous solvent, a suspension, an emulsion, a lyophilized agent and a suppository, It is formulated in unit dosage forms of ampoules or vials. As the non-aqueous solvent and suspending agent, vegetable oils such as propylene glycol, polyethylene glycol and olive oil, injectable esters 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.

On the other hand, the composition of the present invention can be administered via any general route as long as it can reach the target tissue. The composition of the present invention may be administered intraperitoneally, intravenously, intramuscularly, subcutaneously, intradermally, orally, intranasally, intrapulmonarily, or rectally, though it is not intended to be limited thereto. The composition may also be administered by any device capable of transferring the active agent to the target cell.

The 2-linoleoyl glycerol of the present invention is administered in a pharmaceutically effective amount. The "pharmaceutically effective amount" means an amount sufficient to treat a disease at a reasonable benefit / risk ratio applicable to medical treatment, and the effective dose level is sex, age, health condition, diet, time of administration, method of administration, It may be appropriately changed depending on the activity of the drug, the sensitivity to the drug, the duration of treatment and the severity of the disease. The administration may be carried out once a day or divided into several doses. Thus, the dosage amounts are not intended to limit the scope of the invention in any manner.

On the other hand, as another aspect of the present invention, the present invention provides a food composition for improving allergic diseases comprising 2-linoleoyl glycerol represented by the formula (1) as an active ingredient.

The composition of the present invention can be used in a variety of drugs, food and beverages for the improvement of allergic diseases. There is no particular limitation on the kind of food to which the compound of the present invention can be added, and for example, dairy, meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gums, and ice cream. Products, various soups, beverages, teas, drinks, alcoholic beverages and vitamin complexes, etc., and can be used in the form of powders, granules, tablets, capsules, pills, liquids or beverages.

In general, in the production of food or beverage, the composition of the present invention is added in an amount of 0.01 to 10 parts by weight, preferably 0.05 to 1 part by weight based on the raw materials. However, in the case of long-term ingestion intended for health and hygiene purposes or for the purpose of controlling health, the amount can also be used in the above-mentioned range.

The food composition of the present invention may contain various proteins, fats, nutrients, flavoring agents, flavoring agents or natural carbohydrates, and the like as additional ingredients, as in general beverages. Examples of the carbohydrates described above include monosaccharides (eg, glucose, fructose, etc.), disaccharides (eg, maltose, sucrose, oligosaccharides, etc.) and polysaccharides (eg, dextrins, cyclodextrins). And other sugars such as xylitol, sorbitol, and erythritol. As flavoring agents, natural flavoring agents (e.g., stevia extracts such as taumartin, rebaudioside A, glycyrrhizin, etc.) and synthetic flavoring agents (e.g., saccharin, aspartame, etc.) can be used.

In addition to the above, the composition of the present invention may further contain various nutrients, vitamins, electrolytes, flavors, colorants, pectic acids and salts thereof, alginic acid and its salts, organic acids, protective colloid thickeners, pH adjusting agents, stabilizers, preservatives, A carbonating agent used in a carbonated beverage, and the like. In addition, the composition of the present invention may contain flesh for the production of natural fruit juice, fruit juice beverage and vegetable beverage. These components may be used independently or in combination. The proportion of such additives is not critical, but is generally selected in the range of 0.01 to 0.1 parts by weight per 100 parts by weight of the composition of the present invention.

2-linoleoyl glycerol of the present invention is to inhibit the degranulation reaction and allergen-induced cytokine interleukin-4 (IL-4), interleukin-10 (IL-10) of inflammatory chemical mediators in adipocytes , The effect of inhibiting TNF-α activity, the gene expression inhibitory effect of cyclooxygenase-2 (COX-2), an enzyme that synthesizes inflammatory products, and the effect of inhibiting the activity of lipoxygenase. It can be usefully used in a prophylactic or therapeutic composition, or a food composition for improving the diseases.

1 shows a process of obtaining a fraction from the residue (sasam) extract and separating 2-linoleoyl glycerol from the fraction.
Figure 2 shows the degranulation inhibitory effect of inflammatory chemical mediators in adipocytes by 2-linoleoyl glycerol.
Figure 3 shows the effect of inhibiting the expression of interleukin-4 (IL-4), interleukin-10 (IL-10), TNF-α, cyclooxygenase-2 (COX-2) by 2-linoleoyl glycerol will be.
Figure 4 shows the effect of concentration-dependent decrease in the activity of MAP kinase that plays an important role in the production of cytokines in the body by 2-linoleoyl glycerol.
Figure 5 shows the effect of inhibiting lipoxygenase activity by 2-linoleoyl glycerol.

Hereinafter, the present invention will be described in detail with reference to Examples and Experimental Examples. However, the following Examples and Experimental Examples are merely illustrative of the present invention, and the present invention is not limited to the following Examples and Experimental Examples.

Example  1: stalk Four A) extract manufacturer

Dry Dried Powder Straw (Sasam) ( Adenophorae ) Radix ) 80 kg of ethanol was added to 1.5 kg and cold extraction was performed twice for 24 hours. The extract was filtered and concentrated with a rotary vacuum concentrator to obtain an ethanol extract.

Example  2: Derived from Zansam (Sasam) Extract Solvent fraction  detach

As shown in FIG. 1, the residue (sasam) ethanol extract prepared in Example 1 was suspended in distilled water, followed by solvent fractionation with dichloromethane, ethyl acetate, butanol extract (16 g), ethyl acetate extract (1 g) and butanol extract (9 g) were obtained. The obtained dichloromethane fractionation layer was evaporated under reduced pressure to obtain 16 g of an extract, and the extract was subjected to silica gel column chromatography. The eluate was eluted stepwise by changing the composition of hexane-acetone (1: 0, 90: 1, 70: 1, 50: 1, 30: 1, 10: 1, 5: 1, 3: 1, 2: 1). A total of 12 subfractions (G22JC-72-1 ~ 12) were divided into silica gel column chromatograph (dichloromethane-acetone = 90: 1, 70: 1, 50: 1, 30: 1, 10: 1, 5: 1, 3: 1, 2: 1), divided into nine subfractions (G22JC-75-1 ~ 9), and G22JC-75-5 and 6 The fractions were combined and prep. Compound 1 was isolated by HPLC (80% acetonitrile; 20 ml / min; ODS column). Purely separated compounds were subjected to UV, NMR, mass spectrometry to determine their structure.

Example  3: Physicochemical Characterization of Compounds Obtained from Zansam Extract

In Example 2, the purely separated compound 1 was subjected to physicochemical and spectroscopic analysis. ESI-MS (electrospray ionization mass spectrometry, Fisons VG Quattro 400 mass spectrometer, USA) was used to measure the molecular weight of the compound, and nuclear magnetic resonance (NMR, Bruker Co., 100 MHz, 400 MHz) was used to characterize the structure. Used. The compound was dissolved in CDCl ₃ in NMR Merck Co., and placed in an NMR tube. Hydrogen and carbon nuclear magnetic resonances were measured, and the peaks of the solvents were measured as internal standards or based on peaks of tetramethylsilane (TMS). .

The compound 1 was isolated into a colorless oil component, and the molecular weight was confirmed as the molecular weight of 354 amu from the molecular weight m / z 377 [M + Na] ⁺ from ESI-MS, it can be seen that the molecular formula C ₂₁ H ₃₈ O ₄ The degree of unsaturation was found to be 3. ^The olefinic group signal was confirmed from δ _H 5.31-5.41 (4H, m) and δ _C 130.2, 130.0, 128.1, 127.9 by ¹ H-NMR and ¹³ C-NMR, and δ _H 2.77 (2H, brt, Ｊ = 6.5 Hz In 1), one methylene signal between two double bonds was identified, and at δ _H 2.38 (1H, t, Ｊ = 7.5 Hz), each methylene signal connected to an ester group was identified. In addition, at δ _H 2.05 (4H, dd, Ｊ = 13.5, 6.5 Hz), two allylic methylene signals and the methylene signal at the β position of the ester bond at δ _H 1.64 (2H, m) and δ _H 1.27-1.37 (14H) , brs) was identified seven methylene signals, δ _H 0.89 (3H, t, Ｊ = 7.5 Hz) was confirmed that the signal due to the primary methyl group. The partial structure of linoleic acid was determined from the above instrumental analysis data.

In addition, the remaining signals δ _H 4.93 (1H, qui, Ｊ = 5.0 Hz, H-2), 3.83 (4H, d, Ｊ = 5.0 Hz, H-1, H-3) and δ _C 75.0 (C-2 ), 62.6 (C-1, C-3) As shown in the formula (2), Compound 1 is symmetrical C-1 and C-3, and was assumed to be a glycerol compound with linoleic acid in the C-2 position Correlation between H-2 (δ _H 4.93) and δ _C 174.1 (C-1 ') from HMBC confirmed that linoleic acid binds to glycerol position 2. The structure of Compound 1 was determined by 2-linoleoyl glycerol (2-linoleoylglycerol) in comparison with the results of the above instrumental analysis and literature, and represented by the following formula (1).

1) Appearance: Colorless oil

2) Molecular formula: C ₂₁ H ₃₈ O ₄

3) Molecular Weight: 354.52

4) ESI-MS m / z: 377 [M + Na] ⁺

5) ¹ H-NMR (CDCl ₃ , 400 MHz) δ: 5.31-5.38 (4H, m, H-9 ', 10', 12 ', 13'), 4.93 (1H, qui, Ｊ = 5.0 Hz, H-2), 3.84 (4H, d, Ｊ = 5.0 Hz, H-1, H-3), 2.77 (2H, brt, Ｊ = 6.5 Hz, H-11 '), 2.38 (1H, t, Ｊ = 7.5 Hz, H-2'), 2.05 (4H , dd, Ｊ = 13.5, 6.5 Hz, H-8 ', 14'), 1.64 (2H, m, H-3 '), 1.27-1.37 (14H, brs, H-4', 5 ', 6', 7 ', 15', 16 ', 17'), 0.89 (3H, t, Ｊ = 7.5 Hz, H-18 ')

6) ¹³ C-NMR (CDCl ₃ , 100 MHz) δ: 174.1, 130.3, 130.0, 128.1, 127.9, 75.0, 62.6, 34.3, 31.5, 29.7, 29.6, 29.4, 29.2, 29.1, 29.08, 27.2, 27.19, 25.6, 24.9, 22.6, 14.1

Experimental Example  1: beta- Hexosaminidays (β- hexosaminidase ) analysis

In order to measure the degranulation inhibitory effect in mast cells generated upon inflammation by 2-linoleoyl glycerol prepared in Example 2, beta-hexosaminidase analysis was performed as follows.

Hexosaminidase is an enzyme that hydrolyzes N-acetyl-D-hexosamin in N-acetyl-β-D-hexosaminid, secreted by mast cells after antigen stimulation, Depending on the amount of enzyme can be measured the degree of degranulation. Degranulation degree is measured using the RBL-2H3 cell line, which contains receptors of IgE antibodies on the surface, and when these receptors are complexed with each other by antigen-stimulated beta hexosa It secretes an enzyme called β-hexosaminidase. When the secretion of this enzyme increases rapidly, an allergic reaction occurs, and during the allergic reaction, the influx of calcium ions increases, thereby activating serine / tyrosine kinase (Syk), a type of tyrosine kinase, resulting in mitogen-activated protein kinase ( Kinase activity such as mitogen activated protein kinase (MAPK) is cascaded.

RBL-2H3 cell line, a rat basophlic leukemia cell line, was seeded at 2 × 10 ⁵ / well in a 24 well plate at MEM medium containing 15% FBS, 100 unit / ml penicillin and streptomycin, and at 37 ° C and 5% CO _2. After 24 hours incubation.

After removal of the supernatant, MEM medium containing 25 ng / ml anti-DNP IgE was added for sensitization, followed by 4 hours of incubation, washing twice with 500 μl of PIPES buffer, and containing 0.4 mM MgCl ₂ , 5.6 mM glucose and 0.1% BSA. 180 μl of PIPES buffer was added and incubated for 10 minutes. After pre-culture, the sample prepared in Example 2 was stimulated by reacting for 20 minutes at 50 ng / ml DNP-BSA for 20 minutes at a concentration of 25, 50, 100 μg / ml. After completion of the reaction by standing in ice for 10 minutes, the supernatant was put into a 96 well plate of 25 µl each, and 25 µl of 1 mM P-nitrophenyl-N-acetyl-β-D-glucosaminid was reacted at 37 ° C for 1 hour. I was. A stop solution (0.1 M NaHCO ₃ , 0.1 M Na ₂ CO ₃ ) was added to terminate the reaction, and then the absorbance was measured with an ELISA reader at 405 nm wavelength to confirm the amount of secreted beta-hexasaminides. Ketotifen (100 μg / ml) was used as a positive control, and the results are shown in FIG. 2.

As a result, as shown in Figure 2, it was confirmed that 2-linoleoyl glycerol inhibits the degranulation reaction in a concentration-dependent manner, EC ₅₀ , a concentration required to represent 50% of the degranulation maximum response was 196.85 uM (Fig. 2). Reference).

Experimental Example  2: RT - PCR  Measure

Expression of interleukin-4 (IL-4), interleukin-10 (IL-10), TNF-α, cyclooxygenase-2 (COX-2) by 2-linoleoyl glycerol prepared in Example 2 In order to measure the inhibitory effect, experiments were carried out as follows.

RBL-2H3 cells were incubated in 6 well plates with 1X10 ⁶ / well and then IgE was reacted for 4 hours. Thereafter, after 2-linoleoyl glycerol treatment, DNP-BSA was added and reacted at 37 ° C. for 20 minutes. After washing twice with PBS, Trizol (Invitrogen) was added to lyse the cells, chloroform (sigma) was added and centrifuged at 13000 rpm for 15 minutes to obtain a supernatant. After mixing the supernatant and isopropanol (isopropanol) was reacted to precipitate RNA and washed once with ethanol to separate the RNA.

Using extracted RNA, cDNA was synthesized by reverse transcriptase chain polymerization using Superscript III (Invitrogen), a reverse transcriptase enzyme. The primer sequences of the interleukin-4 (IL-4), interleukin-10 (IL-10), TNF-α, cyclooxygenase-2 (COX-2), and the control GAPDH are shown in Table 1. PCR conditions were amplified DNA by 35 cycles of 94 ℃ 45 seconds, 55 ℃ 45 seconds, 72 ℃ 1 minutes 30 seconds. The inhibition of expression of each gene was compared on an agarose gel, and the results are shown in FIG. 3.

As shown in FIG. 3, as the concentration of 2-linoleoyl glycerol increased, interleukin-4 (IL-4), interleukin-10 (IL-10), TNF-α, and cyclooxygenase were concentration-dependently. Gene expression of -2 (COX-2) was confirmed to be inhibited (see Figure 3).

Experimental Example  3: Western blotting  Measure

Expression of proteins (p-AKT, p-ERK, p-JNK, p-p38) involved in the signaling process by COX-2 and cytokines by 2-linoleoyl glycerol prepared in Example 2 In order to measure the inhibitory effect, experiments were performed as follows.

RBL-2H3 cells were sensitized with IgE, and the cells stimulated with DNP-BSA were washed with PBS, followed by lysis buffer (50 mM Tris-HCl, pH 8.0 with 150 mM sodium chloride, 1% NP40, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate). The protein was extracted by centrifugation at 13000 rpm for 20 minutes, and the protein was denatured at 100 ° C. for 5 minutes. Quantified protein was transferred to nitrocellulose membrane after electrophoresis using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The membrane was blocked with TBS-T buffer containing 0.1% Tween 20 and 5% skim dried milk. In addition, the protein of the membrane is reacted with a secondary antibody (1: 10000) complexed with a primary antibody (1: 1000) and horseradish peroxidase (horseradish peroxidase) and identified the desired protein with ECL western detection reagent. The results are shown in FIG. 4.

As shown in FIG. 4, as the concentration of 2-linoleoyl glycerol increases, the expression of p-AKT, p-ERK, p-JNK, and p-p38 proteins that affect cytokine production in a concentration-dependent manner is suppressed. It could be confirmed (see Fig. 4).

Experimental Example  4: Of lipoxygenase  Determination of activity inhibitory effect

In order to determine the effect of inhibiting the activity of lipoxygenase (lipoxygenase) by the 2-linoleoyl glycerol prepared in Example 2, the experiment was carried out as follows.

To 2 ml of 0.1 M Tris buffer (pH 8.5), add 20 ul of 2-linoleoyl glycerol at each concentration, mix 20 ul of substrate linoleic acid (final concentration 110 uM), and mix for 5 minutes at room temperature. Pre-reacted. After adding 20 ul of soybean lipoxygenase (Type V, 500 UNIT / final concentration) to the reaction solution, the reaction was carried out for 5 minutes and the absorbance was measured at 234 nm.

As a result, as shown in Figure 5, it was confirmed that as the concentration of 2-linoleoyl glycerol increases, the activity inhibition rate of lipoxygenase increases proportionally.

Claims (10)

Extracting the residue (sasam) with water, C ₁ -C ₄ alcohol or a mixed solvent thereof to obtain a residue (sasam) extract;
Fractionating the extract with water, n-hexane, methylene chloride, ethyl acetate or n-butanol to obtain a fraction; And
Performing column chromatography of the fractions to obtain 2-linoleoyl glycerol represented by the following formula 1, for the prevention or treatment of allergic diseases of 2-linoleoyl glycerol represented by the following formula (1) Manufacturing Method:
[Formula 1]

According to claim 1, wherein the residue (sasam) extract is a method of extracting the residue (sasam) with ethanol.
The method of claim 1, wherein the fraction is a methylene chloride fraction.
The method of claim 1, wherein the column chromatography is performed with a mixed solvent of hexane and acetone.
The method of claim 1, wherein the allergic disease is a respiratory disease, eye diseases, nasal diseases, gastrointestinal diseases, lung diseases, or inflammatory diseases.
The method of claim 5, wherein the allergic disease is bronchial asthma, allergic asthma, allergic conjunctivitis, allergic rhinitis, gastrointestinal allergy, or arthritis.



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