WO2013141575A1 - Material for preventing or treating asthma and derivatives thereof - Google Patents

Material for preventing or treating asthma and derivatives thereof Download PDF

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Publication number
WO2013141575A1
WO2013141575A1 PCT/KR2013/002246 KR2013002246W WO2013141575A1 WO 2013141575 A1 WO2013141575 A1 WO 2013141575A1 KR 2013002246 W KR2013002246 W KR 2013002246W WO 2013141575 A1 WO2013141575 A1 WO 2013141575A1
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WIPO (PCT)
Prior art keywords
asthma
acetylsinococuline
acid
preventing
composition
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PCT/KR2013/002246
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French (fr)
Korean (ko)
Inventor
황재효
조순장
박대훈
서정욱
한상섭
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Hwang Jae Hyo
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Publication of WO2013141575A1 publication Critical patent/WO2013141575A1/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/4353—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom ortho- or peri-condensed with heterocyclic ring systems
    • A61K31/4375—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom ortho- or peri-condensed with heterocyclic ring systems the heterocyclic ring system containing a six-membered ring having nitrogen as a ring heteroatom, e.g. quinolizines, naphthyridines, berberine, vincamine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/439—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom the ring forming part of a bridged ring system, e.g. quinuclidine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system
    • A61P11/06—Antiasthmatics

Definitions

  • the present invention relates to a composition for preventing or treating asthma.
  • Asthma is the third most common disease among children with low incomes and is the third most common disease among children under 15 years of age.
  • Exogenous asthma is symptomatic when exposed to causative antigens.
  • a skin test or bronchial challenge test for causative antigens is positive and the onset age is young.
  • House dust mites are the most common cause antigens, and pollen, animal epithelium, and fungi act as causative antigens.
  • Endogenous asthma is caused or worsened by upper respiratory tract infection, exercise, emotional instability, cold climate and humidity changes, and is common in adult asthma.
  • Mixed asthma is caused by a combination of exogenous and endogenous factors, which are common in pediatric asthma.
  • Aspirin-induced asthma is a specific constitutional response to aspirin and refers to the three major symptoms of bronchial asthma, nasal polyps, and aspirin intolerance.
  • Exercise-induced asthma is associated with the development of hyperventilation due to exercise, and thus loss of heat or water in the airways.
  • Occupational asthma is caused by substances that are inhaled in the workplace, initially without symptoms, then asthmatic symptoms appear months or years later, tend to relieve on weekends or vacations, and worsen when returning to work.
  • various symptoms of respiratory distress occur, prompting rapid breathing and rapid pulse.
  • chest examination a loud wheezing can be heard during inspiration and most exhalations and an extension of the exhalation can be observed.
  • More severe asthma attacks can lead to rapid and shallow breathing due to fatigue and severe breathing difficulties, and cyanosis as the symptoms become more severe. If the patient shows conscious turbidity and helplessness, it means progression to respiratory failure due to CO 2 coma, in which case severe mucus plugging hardly hears wheezing and exacerbates gas exchange by exhaustion.
  • the most obvious signs of a severe seizure include difficulty breathing, unable to talk, and the use of cyanosis, acute veins (> 20-30 mmHg) and apnea even at rest. Severity can be most accurately assessed through arterial blood gas measurements.
  • Asthma is a respiratory disease caused by hypersensitivity to foreign bodies. It is caused by the interleukin (IL) -4 / IL-13 / Stat-6 pathway, l-selectin and intercellular adhesion molecule-1. It is known to occur.
  • IL interleukin
  • lung function eg early-phase [or acute] and late-phase bronchospasm, increased airway resistance, and bronchoconstriction
  • physiological changes eg , bronchial hypersensitivity [BHR], airway inflammation, peripheral blood eosinophilia, changes in mucus quality, increased immunoglobulin E (IgE) levels in bronchoalveolar lavage [BAL] fluid, increased expression of various T cell- and mast cell-derived cytokines, such as IL-1b, IL-5, IL-4, IL-9, IL-10, IL-13, and tumor necrosis factor-alpha (TNFa), and mediators, such as cysteinyl leukotrienes and isoprostanes, and pathological changes (eg, increased epithelial thickness, goblet cell hyperplasia, mucus plugs, epithelial cell denudation, mucus gland hyperplasia, smooth muscle hypertrophy
  • pathological changes eg, increased epithelial thickness
  • Asthma is one of the biggest problems to be solved in modern medicine, and the prevalence is increasing rapidly due to various causes, but anti-inflammatory drugs such as bronchodilators and steroids, which are being developed and used, cannot reach cure. It has only enough effect to control it. In addition, long-term use of steroids, which are the most effective drugs, can have several side effects. Therefore, there is an urgent need for the development of drugs without risks and with minimal physiological side effects.
  • the present invention is to provide a pharmaceutical composition or health food using the compound to purify the compound effective in treating asthma.
  • the present invention provides a composition for preventing or treating asthma comprising 6,7-di-O-acetylsinococuline (6,7-di-O-acetylsinococuline) or a pharmacologically acceptable salt thereof as an active ingredient.
  • the 6,7-di-O-acetylcynococulin or a pharmacologically acceptable salt thereof can inhibit inflammatory cells lymphocytes, neutrophils and eosinophils.
  • the present invention provides a pharmaceutical composition for preventing or treating asthma comprising 6,7-di-O-acetylsinococuline or a pharmacologically acceptable salt thereof as an active ingredient. .
  • the present invention provides a health food composition for preventing or improving asthma comprising 6,7-di-O-acetylsinococuline (6,7-di-O-acetylsinococuline) or a pharmacologically acceptable salt thereof as an active ingredient. do.
  • the composition containing 6,7-di-O-acetylcynococulin of the present invention is an inflammation of lymphocytes, neutrophils, or eosinophils that cause excessive immune responses and cause inflammation of mucosal tissues. It is effective in the treatment and prevention of asthma by inhibiting cellular and airway hypersensitivity, reducing the infiltration of inflammatory cells infiltrating around the bronchus, and inhibiting mucus secretion and mucosal cell thickening in the lumen, thereby preventing or treating asthma. And it can be utilized as a health food composition.
  • Figure 1 shows the HPLC data of the fraction before the sentence.
  • Figure 7 shows histopathological changes by FK-3000 in the lungs of an animal model with asthma, T1 shows 4 mg / kg of FK-3000; T2 represents 10 mg / kg FK-3000; T3 represents FK-3000 40 mg / kg.
  • FIG. 8 shows changes in bronchial epithelium, inflammatory cell infiltration and mucous secretion in the lungs according to groups.
  • FIG. 9 shows changes in capillary bronchial epithelium (Gc), inflammatory cell infiltration (arrows), and mucus secretion (M) according to the dose concentration of FK-3000.
  • the present invention is a composition for preventing or treating asthma comprising 6,7-di-O-acetylsinococuline (6,7-di-O-acetylsinococuline) or a pharmacologically acceptable salt thereof as an active ingredient and a pharmaceutical using the same
  • 6,7-di-O-acetylsinococuline 6,7-di-O-acetylsinococuline
  • a pharmacologically acceptable salt thereof as an active ingredient and a pharmaceutical using the same
  • compositions or health food compositions are provided.
  • the present invention provides a composition for preventing or treating asthma comprising 6,7-di-O-acetylsinococuline (6,7-di-O-acetylsinococuline) or a pharmacologically acceptable salt thereof as an active ingredient.
  • 6,7-di-O-acetylsinococuline (6,7-di-O-acetylsinococuline) can be represented by the following formula (1), also known as FK-3000 compounds, which are separated from natural products Physiologically, it can minimize toxicity and side effects.
  • the 6,7-di-O-acetylcynococulin is not limited to the scope of the present invention by the method of obtaining it, whether synthesized by a chemical synthesis method or separated and purified from natural products.
  • 6,7-di-O-acetylcynococulin was cooled by distillation under a reduced pressure of 70-90% aqueous ethanol ( Stephania delavayi ) to obtain an ethanol extract, suspended in distilled water and then ethyl acetate It was obtained by separating and purifying the main fraction of the fraction obtained by solvent fractionation using (ethyl acetate).
  • an acid addition salt formed by free acid may be used as the pharmaceutically acceptable salt of the 6,7-di-O-acetylcynococulin.
  • Acid addition salts can be 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 such as methanol, ethanol, acetone or acetonitrile. Equal 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.
  • acid or alcohol eg, glycol monomethylether
  • organic acids and inorganic acids may be used as the free acid
  • hydrochloric acid, phosphoric acid, sulfuric acid, nitric acid, tartaric acid, and the like may be used as the inorganic acid
  • methanesulfonic acid, p-toluenesulfonic acid, acetic acid, trifluoroacetic acid, and citric acid may be used as the organic acid.
  • Bases can also be used to make pharmaceutically acceptable metal salts.
  • An alkali metal or alkaline earth metal salt can be obtained by, for example, dissolving a compound in an excess alkali metal hydroxide or alkaline earth metal hydroxide solution, filtering the insoluble compound salt, and then evaporating and drying the filtrate.
  • the metal salt it is particularly suitable to prepare sodium, potassium or calcium salts, and the corresponding silver salt can be obtained by reacting an alkali metal or alkaline earth metal salt with a suitable silver salt (for example, silver nitrate).
  • the pharmaceutically acceptable salts of the 6,7-di-O-acetylcynococulin are acidic or basic, which may be present in the compound of 6,7-di-O-acetylcynococulin, unless otherwise indicated. Salts.
  • pharmaceutically acceptable salts include salts of sodium, calcium and potassium of the hydroxy group
  • other pharmaceutically acceptable salts of the amino group include hydrobromide, sulfate, hydrogen sulphate, phosphate, hydrogen phosphate, dihydrogen Phosphate, acetate, succinate, citrate, tartrate, lactate, mandelate, methanesulfonate (mesylate) and p-toluenesulfonate (tosylate) salts, which are known in the art. It can be prepared through.
  • Asthma is a disease that causes bronchial hyperresponsiveness and intermittent airway constriction due to inflammation of the airways, causing symptoms of respiratory distress, such as cough, aerobic wheezing, or dyspnea, and bronchial inflammation and airway hypersensitivity. May appear.
  • the 6,7-di-O-acetylcynococulin can inhibit lymphocytes, neutrophils, and eosinophils.
  • the eosinophil is one of the most frequently observed intermediate phenotypes in asthma, the number of peripheral blood and eosinophils in target tissues is closely related to the severity and exacerbation of the disease.
  • 6,7-di-O-acetylcynococurin of the present invention inhibits airway hyperresponsiveness (AHR), inflammatory cell infiltration, mucous membranes, mucosal cell hyperplasia, etc. Can be effectively prevented or treated.
  • AHR airway hyperresponsiveness
  • the present invention provides a pharmaceutical composition for preventing or treating asthma comprising 6,7-di-O-acetylsinococuline or a pharmacologically acceptable salt thereof as an active ingredient. .
  • the compound may be included in 0.0001 to 99% by weight based on the total weight of the composition, but is not limited to the scope of the present invention.
  • compositions comprising a compound of the present invention may further comprise a suitable carrier, excipient or diluent according to conventional methods.
  • Carriers, excipients and diluents that may be included in the compositions of the present invention 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.
  • compositions comprising the compounds of the present invention are each formulated in the form of oral dosage forms, external preparations, suppositories, or sterile injectable solutions, such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, etc., in accordance with conventional methods. Can be used.
  • diluents or excipients such as commonly used fillers, extenders, binders, wetting agents, disintegrating agents, surfactants, and the like.
  • Solid form preparations for oral administration include tablets, pills, powders, granules, capsules, and the like, which form at least one excipient such as starch, calcium carbonate, sucrose, etc. ), Lactose, gelatin and the like can be mixed.
  • lubricants such as magnesium stearate, talc can also be used.
  • Liquid preparations for oral use include suspensions, solvents, emulsions, and syrups, and 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 and suppositories.
  • 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 pharmaceutical composition of the present invention may be administered at 0.01 to 200 mg / kg per day, preferably 0.1 mg / kg to 40 mg / kg per day, but the scope of the present invention thereby Is not limited.
  • the dosage will vary depending on factors such as the formulation method, mode of administration, age, weight, sex, morbidity, food, time of administration, route of administration, rate of excretion, and reaction sensitivity of the patient, and will be appropriately selected by those skilled in the art.
  • the administration may be administered once a day, may be divided several times. The dosage does not limit the scope of the invention in any aspect.
  • the pharmaceutical dosage forms of the compounds of the present invention 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.
  • the pharmaceutical composition of the present invention can be administered to mammals such as rats, mice, livestock, humans in various ways, orally or parenterally, and in the case of parenteral administration, intravenous injection, subcutaneous injection, muscle It may be administered by infusion, intraperitoneal infusion, transdermal infusion, trachea infusion, or airway infusion, and most preferably, directly into the respiratory tract via a respiratory system or the like.
  • compositions of the present invention may be prepared in unit dosage form by formulating with a pharmaceutically acceptable carrier and / or excipient according to methods which can be easily carried out by those skilled in the art. Or may be prepared by incorporation into a multi-dose container.
  • the formulation may be in the form of powder, granules, tablets, capsules, suspensions, emulsions, syrups, oral formulations of aerosols, external preparations, suppositories, or injections, and may further include dispersants or stabilizers.
  • the present invention provides a health food composition for preventing or improving asthma comprising 6,7-di-O-acetylsinococuline (6,7-di-O-acetylsinococuline) or a pharmacologically acceptable salt thereof as an active ingredient. do.
  • Examples of the food to which the 6,7-di-O-acetylcynococulin can be added include various foods, beverages, gums, teas, vitamin complexes, and health functional foods.
  • the amount of the 6,7-di-O-acetylcynococulin in the food or beverage may be added at 0.001 to 30% by weight of the total food weight, the health beverage composition is 0.001 to 30 g, preferably based on 100 ml Preferably, it may be added in a ratio of 0.02 to 5 g, but the scope of the present invention is not limited thereto.
  • the health functional beverage composition of the present invention is not particularly limited to other ingredients except for containing 6,7-di-O-acetylcynococurine as an essential ingredient in the indicated ratios, and various flavoring or natural ingredients such as ordinary drinks Carbohydrates and the like may be included as additional components.
  • 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.
  • natural flavoring agents such as, tauumatin, stevia extract (e.g., Rebaudioside A, glycyrrhizin, etc.) and synthetic flavoring agents (saccharin, aspartame, etc.) can be advantageously used.
  • the proportion of such natural carbohydrates is generally about 1 to 20 g, preferably about 5 to 12 g per 100 ml of the composition of the present invention.
  • the composition of the present invention includes various nutrients, vitamins, minerals (electrolytes), flavors such as synthetic flavors and natural flavors, coloring and neutralizing agents (such as cheese, chocolate), pectic acid and salts thereof, alginic acid and its Salts, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohols, carbonation agents used in carbonated beverages, and the like.
  • the composition of the present invention may contain a natural fruit juice and a pulp for the production of fruit juice drinks and vegetable drinks. These components can be used independently or in combination, and the proportion of such additives is not so critical but is generally selected from the range of 0.01 to about 20 parts by weight per 100 parts by weight of the composition of the present invention.
  • the stephania delavayi used in this study was distributed from the Extract Bank of Biotechnology.
  • NMR experiments used a Varian Mecury 400 MHz NMR spectrometry. NMR solvent was used CD 3 OD. In one sentence, 6,7-di-O-acetylsinococuline (FK-3000) was purified. This material was identified as the structure of Table 1 by 1 H NMR and 2D NMR experiments as a white solid (FIGS. 2 to 6).
  • mice 72 female 5 week-old pathogenic member (SPF) BALB / c mice were purchased from Orient Bio Co., Ltd. (143-1, Sangwon-dong, Jungwon-gu, Seongnam-si, Gyeonggi-do), and the females were about 8 weeks old when the compound of the present invention was injected.
  • SPF pathogenic member
  • the breeding condition of the mouse is the temperature of the room, the humidity range is 23 ⁇ 3 °C, relative humidity 50 ⁇ 10%, the contrast cycle of the breeding room is 12 hours (08:00 lights ⁇ 20:00 lights off) fluorescent lighting
  • the number of ventilation of the breeding room was 10-20 times / hr, the illuminance of the breeding room was 150-300 Lux, the feed was sterilized by the use of radiation sterilized feed, and the negative water was passed through the UV-water sterilizer and the microfiltration device. Allowed free intake of feed and drinking water.
  • White powder of ovalbumin (A-5503) (Sigma) was used as an asthma-inducing substance and white liquid Alum (Imject alum, 77161) (PIERCE) was used as an adjuvant.
  • Bronchial contractile was used as a white powder of metacholine (A-2251) (Sigma), dexamethasone as a positive control, FK-3000 (6,7-di-O-) as a test substance. Acetylcynococulin) was used.
  • OVA was dissolved in saline to give 5% OVA solution (5 g OVA + 100 mL saline).
  • Methacholine (Methacholine, Sigma, A-2251) was prepared by dissolving in saline to 10 mg / mL and 20 mg / mL and subdivided by 1 mL to store frozen.
  • the solution prepared for OVA I.P injection was intraperitoneally administered 500 ⁇ l (20 ⁇ g as OVA) per mouse in all groups except the normal control group (Day 0).
  • the normal control group was intraperitoneally administered saline solution.
  • Asthma-induced control group was intraperitoneally administered once more in the same way a week after initial sensitization (Day 7).
  • the solution prepared for OVA inhalation was inhaled daily for 14 minutes to 18 days after initial sensitization using whole body plethysmograph and nubulizer (NE-U17, OMRON Co. Ltd, Japan) (Day 14 ⁇ 18).
  • Dose amount was calculated at 5 mL per kg based on body weight.
  • mice 24 hours after the last OVA inhalation (Day 19, 20), the mice were placed in whole body plethysmograph (BUXCO, USA) and 0.5 mL of solution of concentrations of 0, 10 and 20 mg / mL of methacholine were aerosolized. ) And inhalation for 3 minutes each, Penh was measured for 4 minutes from the end of the administration and the average value was taken as the Penh value at the corresponding dose of methacholine.
  • BUXCO whole body plethysmograph
  • Pentotalsodium (50mg / kg, I.P) was anesthetized by intraperitoneal injection, and the left lung was fixed with forceps, and then injected into the bronchus and lungs using 0.4 mL of PBS solution. The supernatant was discarded by centrifugation at 1,500 g and suspended again by adding 0.2 mL of PBS to the precipitate. The total number of leukocytes, neutrophils, eosinophils, neutrophils and lymphocytes was measured using an automated hematology analyzer (Advia 120 coulter counter, BAYER, Germany).
  • FK-3000 inhibits eosinophils in BAL fluid of asthma model animals, especially at 40 mg / kg, which is lower than the result of dexamethasone, a positive control, which is an excipient. Similar to the results of the control group. Therefore, it is thought to effectively suppress eosinophilia, which is the most representative physiological index identified during asthma.
  • the tissues in which paraffin was infiltrated using a tissue processor were cut into a thickness of 3-4 ⁇ m using a microtome. Made an intercept The slides were then immersed in xylene and ethanol, deparaffinized, and then stained with haematoxylin and eosin, covered with slides and covered with balsam to infiltrate inflammatory cells under a microscope. Mucus plugging and goblet cell proliferation were observed.
  • FK-3000 inhibited inflammatory cell infiltration, mucous plug, mucosal cell hyperplasia, etc. in asthma model animals in a concentration-dependent manner.
  • pulmonary epithelium and blood vessels did not show mucus secretion (M) and inflammatory cell infiltration (arrow), respectively.
  • Inflammatory cell infiltration was observed around blood vessels and bronchioles in primarily induced group (B) and positive control group (C).
  • Normal mucus secretions, including cell debris, have been found in capillaries of the induced groups.
  • Goblet cell (Gc) hyperplasia and mucus secretion were also seen in the induced cells and positive control groups. Goblet cell hyperplasia, mucus secretion and inflammatory cell infiltration were more noticeable in the induced group than in the positive control.
  • Figure 9 shows the changes in the capillary bronchial epithelium (bronchioles epithelium, Gc), inflammatory cell infiltration (arrows), mucus secretion (M) according to the dose concentration of FK-3000, hyperplasia of goblet cells, inflammatory cell infiltration And the degree of mucus secretion decreased with dose concentration.
  • bronchioles epithelium, Gc bronchioles epithelium
  • M mucus secretion

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Abstract

The present invention relates to a composition for preventing or treating asthma, comprising, as an active ingredient, 6,7-di-O-acetylsinococuline or pharmacological acceptable salts thereof. More particularly, the 6,7-di-O-acetylsinococuline prevents inflammatory cells, such as lymphocytes, neutrophils, eosinophils or the like, which might cause inflammation to the mucous membrane tissue of the airway, from entering the bronchoalveolar space, inhibits hypersensitivity of the airway, reduces infiltration of inflammatory cells which infiltrate around bronchial tubes, and inhibits secretion of mucus, mucosal cell thickening or the like in a lumen. Therefore, the 6,7-di-O-acetylsinococuline is effective in treating asthma, and thus can be used in a pharmaceutical composition for preventing or treating asthma and in a health food composition.

Description

천식예방 또는 치료용 물질과 그 유도체Asthma-preventing or therapeutic substances and derivatives thereof
본 발명은 천식 예방 또는 치료용 조성물에 관한 것이다.The present invention relates to a composition for preventing or treating asthma.
2008년 미국의 미국건강조사 (National Health Interview Survey)에 따르면 약 7만 명의 어린이를 포함한 2,300만 명이 천식을 갖고 있는 것으로 확인되었다. 특히 천식은 수입이 낮은 계층에서 빈발하며 15세 이하 어린이 환자의 3번째로 많이 발생하는 질병으로, 매년 사용되는 의료비는 미국에서만 2천만 불이었다. According to the 2008 US National Health Interview Survey, 23 million people, including about 70,000 children, have asthma. Asthma is the third most common disease among children with low incomes and is the third most common disease among children under 15 years of age.
천식을 원인에 따라 분류하면 크게 외인성, 내인성, 혼합형, 아스피린 유발성, 운동 유발성, 직업성 천식으로 분류할 수 있다. 외인성 천식은 원인항원에 노출되었을 때 증상이 나타나는데 원인 항원에 대한 피부시험이나 기관지 유발시험이 양성 반응을 보이며 발병 연령이 젊다. 집먼지 진드기가 가장 많은 원인 항원이며, 그밖에 꽃가루, 동물의 상피, 곰팡이 등이 원인항원으로 작용한다. 내인성 천식은 상기도 감염, 운동, 정서불안, 한랭 기후 및 습도의 변화 등에 의해 유발되거나 악화되고, 성인형 천식에서 흔히 볼 수 있다. 혼합형 천식은 외인성 및 내인성 요인이 혼합되어 유발되는데 소아형 천식에 많다. 아스피린 유발성 천식은 아스피린에 대한 특이체질 반응으로서, 기관지천식, 비용종, 아스피린 불내성의 3대 증상이 있는 경우를 말한다. 운동 유발성 천식은 운동으로 인한 과호흡, 이에 따른 기도의 열 또는 수분의 손실이 발병에 관여한다. 직업성 천식은 작업장에서 흡입되는 물질에 의해 발생하는데 초기에는 증상 없이 지내다가 수개월 혹은 수년 후에 천식 증상이 나타나며 주말이나 휴가 시엔 완화되고 직장에 복귀하면 악화되는 경향이 있다.If you classify asthma according to the cause, it can be classified into exogenous, endogenous, mixed, aspirin-induced, exercise-induced, occupational asthma. Exogenous asthma is symptomatic when exposed to causative antigens. A skin test or bronchial challenge test for causative antigens is positive and the onset age is young. House dust mites are the most common cause antigens, and pollen, animal epithelium, and fungi act as causative antigens. Endogenous asthma is caused or worsened by upper respiratory tract infection, exercise, emotional instability, cold climate and humidity changes, and is common in adult asthma. Mixed asthma is caused by a combination of exogenous and endogenous factors, which are common in pediatric asthma. Aspirin-induced asthma is a specific constitutional response to aspirin and refers to the three major symptoms of bronchial asthma, nasal polyps, and aspirin intolerance. Exercise-induced asthma is associated with the development of hyperventilation due to exercise, and thus loss of heat or water in the airways. Occupational asthma is caused by substances that are inhaled in the workplace, initially without symptoms, then asthmatic symptoms appear months or years later, tend to relieve on weekends or vacations, and worsen when returning to work.
천식발작은 천명, 기침, 호흡곤란 등이 갑자기 나타나는 경우 및 점진적으로 발병하는 경우가 있는데 대부분 처음에 기침, 가슴 부위의 답답함이나 압박감 등을 느끼게 되고 천명음이 들리기도 한다. 급성 발작 동안에는 다양한 호흡곤란 증세를 나타내게 되며 빠른 호흡과 빠른 맥박을 호소한다. 흉부 진찰시에 흡기와 대부분의 호기 동안에 고조의 천명음이 들리고 호기의 연장을 관찰할 수 있다. 천식발작이 더 심해지면 피로와 심한 호흡곤란으로 인해 빠르고 얕은 호흡 양상을 보이고 더욱 심해지면 청색증이 나타나게 된다. 환자가 의식 혼탁과 무력감을 보이면 CO2 혼수에 의한 호흡부전증으로 진행되는 것을 의미하는데, 이 경우 심한 점액 폐색 (plugging)으로 인해서 천명음이 거의 들리지 않게 되고 탈진에 의해 가스 교환이 더 악화된다. 중증 발작의 가장 확실한 징후들로는 안정시에도 호흡이 곤란하고 대화를 할 수 없으며 청색증, 기이맥 (>20-30 mmHg) 및 무호흡근의 사용 등이 있다. 중증도는 동맥혈 가스 측정을 통해서 가장 정확하게 평가할 수 있다.Asthma attacks occur suddenly and wheezing, coughing, shortness of breath, etc., and most often develop cough, chest tightness or pressure, and wheezing can be heard. During acute seizures, various symptoms of respiratory distress occur, prompting rapid breathing and rapid pulse. During chest examination, a loud wheezing can be heard during inspiration and most exhalations and an extension of the exhalation can be observed. More severe asthma attacks can lead to rapid and shallow breathing due to fatigue and severe breathing difficulties, and cyanosis as the symptoms become more severe. If the patient shows conscious turbidity and helplessness, it means progression to respiratory failure due to CO 2 coma, in which case severe mucus plugging hardly hears wheezing and exacerbates gas exchange by exhaustion. The most obvious signs of a severe seizure include difficulty breathing, unable to talk, and the use of cyanosis, acute veins (> 20-30 mmHg) and apnea even at rest. Severity can be most accurately assessed through arterial blood gas measurements.
천식은 외래물질 (foreign body)에 의한 과민증상으로 야기되는 호흡기 질환으로, 인터루킨 (IL)-4/IL-13/Stat-6 경로, l-셀렉틴 (selectin)과 세포간 부착분자-1 등에 의해 발생된다고 알려져 있다. 많은 인자를 통해 천식의 정도를 평가할 수 있는데, 예를 들면 폐의 기능에 대한 평가(e.g., early-phase [or acute] and late-phase bronchospasm, increased airway resistance, and bronchoconstriction), 생리학적인 변화 (e.g., bronchial hypersensitivity [BHR], airway inflammation, peripheral blood eosinophilia, changes in mucus quality, increased immunoglobulin E (IgE) levels in bronchoalveolar lavage [BAL] fluid, increased expression of various T cell- and mast cell-derived cytokines, such as IL-1b, IL-5, IL-4, IL-9, IL-10, IL-13, and tumor necrosis factor-alpha (TNFa), and mediators, such as cysteinyl leukotrienes and isoprostanes), 그리고 병리조직학적인 변화 (e.g., increased epithelial thickness, goblet cell hyperplasia, mucus plugs, epithelial cell denudation, mucus gland hyperplasia, smooth muscle hypertrophy and hyperplasia, thickening of the lamina reticularis, accumulation of subepithelial extracellular matrix, increased numbers of submucosal myofibroblasts, increased vascularization, and neurite outgrowth) 등이 그것이다.Asthma is a respiratory disease caused by hypersensitivity to foreign bodies. It is caused by the interleukin (IL) -4 / IL-13 / Stat-6 pathway, l-selectin and intercellular adhesion molecule-1. It is known to occur. Many factors can be used to assess the extent of asthma, for example, evaluation of lung function (eg early-phase [or acute] and late-phase bronchospasm, increased airway resistance, and bronchoconstriction), physiological changes (eg , bronchial hypersensitivity [BHR], airway inflammation, peripheral blood eosinophilia, changes in mucus quality, increased immunoglobulin E (IgE) levels in bronchoalveolar lavage [BAL] fluid, increased expression of various T cell- and mast cell-derived cytokines, such as IL-1b, IL-5, IL-4, IL-9, IL-10, IL-13, and tumor necrosis factor-alpha (TNFa), and mediators, such as cysteinyl leukotrienes and isoprostanes, and pathological changes (eg, increased epithelial thickness, goblet cell hyperplasia, mucus plugs, epithelial cell denudation, mucus gland hyperplasia, smooth muscle hypertrophy and hyperplasia, thickening of the lamina reticularis, accumulation of subepithelial extracellular matrix, increased numbers of subm ucosal myofibroblasts, increased vascularization, and neurite outgrowth.
이와 같이, 천식은 현대 의학이 안고 있는 해결해야할 가장 큰 문제 중의 하나이고 여러 원인들에 의해서 유병율이 급속도로 증가하고 있으나 현재 개발되어 사용중인 기관지 확장제와 스테로이드 등의 항염증제는 완치에 도달할 수 없고 증상을 조절할 수 있을 정도의 효과만을 지니고 있다. 뿐만 아니라 가장 효과적인 약제인 스테로이드를 장기간 사용시 여러 부작용이 수반될 수 있다. 따라서 위험이 없고 생리적으로 부작용이 최소화된 약물의 개발이 절실히 요망된다. Asthma is one of the biggest problems to be solved in modern medicine, and the prevalence is increasing rapidly due to various causes, but anti-inflammatory drugs such as bronchodilators and steroids, which are being developed and used, cannot reach cure. It has only enough effect to control it. In addition, long-term use of steroids, which are the most effective drugs, can have several side effects. Therefore, there is an urgent need for the development of drugs without risks and with minimal physiological side effects.
본 발명은 천식 치료에 효과적인 화합물을 정제하고 이를 이용한 약학 조성물 또는 건강식품을 제공하고자 한다.The present invention is to provide a pharmaceutical composition or health food using the compound to purify the compound effective in treating asthma.
본 발명은 6,7-디-O-아세틸시노코큘린 (6,7-di-O-acetylsinococuline) 또는 이의 약리학적으로 허용가능한 염을 유효성분으로 포함하는 천식 예방 또는 치료용 조성물을 제공한다.The present invention provides a composition for preventing or treating asthma comprising 6,7-di-O-acetylsinococuline (6,7-di-O-acetylsinococuline) or a pharmacologically acceptable salt thereof as an active ingredient.
상기 6,7-디-O-아세틸시노코큘린 또는 이의 약리학적으로 허용가능한 염은 염증 세포인 림프구 (lymphocytes), 중성구 (neutrophils) 및 호산구 (eosinophils)를 억제할 수 있다.The 6,7-di-O-acetylcynococulin or a pharmacologically acceptable salt thereof can inhibit inflammatory cells lymphocytes, neutrophils and eosinophils.
본 발명은 6,7-디-O-아세틸시노코큘린 (6,7-di-O-acetylsinococuline) 또는 이의 약리학적으로 허용가능한 염을 유효성분으로 포함하는 천식 예방 또는 치료용 약학 조성물을 제공한다.The present invention provides a pharmaceutical composition for preventing or treating asthma comprising 6,7-di-O-acetylsinococuline or a pharmacologically acceptable salt thereof as an active ingredient. .
본 발명은 6,7-디-O-아세틸시노코큘린 (6,7-di-O-acetylsinococuline) 또는 이의 약리학적으로 허용가능한 염을 유효성분으로 포함하는 천식 예방 또는 개선용 건강식품 조성물을 제공한다.The present invention provides a health food composition for preventing or improving asthma comprising 6,7-di-O-acetylsinococuline (6,7-di-O-acetylsinococuline) or a pharmacologically acceptable salt thereof as an active ingredient. do.
본 발명의 6,7-디-O-아세틸시노코큘린을 함유하는 조성물은 과도한 면역반응을 일으키고, 점막조직에 염증을 일으키는 림프구 (lymphocytes), 중성구 (neutrophils), 또는 호산구 (eosinophils) 등의 염증 세포 및 기도 과민성을 억제하고, 기관지 주위에 침윤하는 염증세포의 침윤을 감소시키며, 내강 내에 점액 분비 및 점막세포 비후 등을 억제함으로써 천식의 치료 및 예방에 효과적이므로, 이를 천식 예방 또는 치료용 약학 조성물 및 건강식품 조성물로 활용할 수 있다.The composition containing 6,7-di-O-acetylcynococulin of the present invention is an inflammation of lymphocytes, neutrophils, or eosinophils that cause excessive immune responses and cause inflammation of mucosal tissues. It is effective in the treatment and prevention of asthma by inhibiting cellular and airway hypersensitivity, reducing the infiltration of inflammatory cells infiltrating around the bronchus, and inhibiting mucus secretion and mucosal cell thickening in the lumen, thereby preventing or treating asthma. And it can be utilized as a health food composition.
도 1은 일문전 분획물의 HPLC 데이터를 나타낸 것이다.Figure 1 shows the HPLC data of the fraction before the sentence.
도 2는 1H NMR 데이터를 나타낸 것이다.2 shows 1 H NMR data.
도 3은 13C NMR 데이터를 나타낸 것이다.3 shows 13 C NMR data.
도 4은 2D gCOSY NMR 데이터를 나타낸 것이다. 4 shows 2D gCOSY NMR data.
도 5는 2D gHSQC NMR 데이터를 나타낸 것이다.5 shows 2D gHSQC NMR data.
도 6는 2D gHMBC NMR 데이터를 나타내 것이다.6 will show 2D gHMBC NMR data.
도 7는 천식이 있는 동물모델의 폐에서 FK-3000에 의한 조직병리학적 변화를 나타낸 것으로, T1은 FK-3000 4 mg/kg를 나타낸 것이며; T2는 FK-3000 10 mg/kg을 나타낸 것이고; T3는 FK-3000 40 mg/kg을 나타낸 것이다.Figure 7 shows histopathological changes by FK-3000 in the lungs of an animal model with asthma, T1 shows 4 mg / kg of FK-3000; T2 represents 10 mg / kg FK-3000; T3 represents FK-3000 40 mg / kg.
도 8은 그룹에 따라 폐 내에 있는 상피조직 (bronchial epithelium), 염증세포 침윤 (inflammatory cell infiltration) 및 점액 분비 (mucous secretion)의 변화를 나타낸 것이다.FIG. 8 shows changes in bronchial epithelium, inflammatory cell infiltration and mucous secretion in the lungs according to groups.
도 9은 FK-3000의 복용량 농도에 따른, 모세기관지 상피세포 (bronchioles epithelium, Gc), 염증세포 침윤 (arrows), 점액 분비 (M)의 변화를 나타낸 것이다.FIG. 9 shows changes in capillary bronchial epithelium (Gc), inflammatory cell infiltration (arrows), and mucus secretion (M) according to the dose concentration of FK-3000.
본 발명은 6,7-디-O-아세틸시노코큘린 (6,7-di-O-acetylsinococuline) 또는 이의 약리학적으로 허용가능한 염을 유효성분으로 포함하는 천식 예방 또는 치료용 조성물 및 이를 이용한 약학 조성물 또는 건강식품 조성물을 제공한다.The present invention is a composition for preventing or treating asthma comprising 6,7-di-O-acetylsinococuline (6,7-di-O-acetylsinococuline) or a pharmacologically acceptable salt thereof as an active ingredient and a pharmaceutical using the same Provided are compositions or health food compositions.
이하, 본 발명을 상세히 설명한다.Hereinafter, the present invention will be described in detail.
본 발명은 6,7-디-O-아세틸시노코큘린 (6,7-di-O-acetylsinococuline) 또는 이의 약리학적으로 허용가능한 염을 유효성분으로 포함하는 천식 예방 또는 치료용 조성물을 제공한다. The present invention provides a composition for preventing or treating asthma comprising 6,7-di-O-acetylsinococuline (6,7-di-O-acetylsinococuline) or a pharmacologically acceptable salt thereof as an active ingredient.
6,7-디-O-아세틸시노코큘린 (6,7-di-O-acetylsinococuline)은 하기의 화학식 1로 표시될 수 있으며, FK-3000 화합물로도 알려져 있는데, 상기 화합물은 천연물에서 분리한 것으로 생리적으로 독성 및 부작용을 최소화할 수 있다.6,7-di-O-acetylsinococuline (6,7-di-O-acetylsinococuline) can be represented by the following formula (1), also known as FK-3000 compounds, which are separated from natural products Physiologically, it can minimize toxicity and side effects.
[화학식 1][Formula 1]
Figure PCTKR2013002246-appb-I000001
Figure PCTKR2013002246-appb-I000001
상기 6,7-디-O-아세틸시노코큘린은 화학적 합성방법에 의해 합성된 것이든 천연물로부터 분리, 정제된 것이든 그 수득방법에 의해 본 발명의 범위가 한정되는 것은 아니다.The 6,7-di-O-acetylcynococulin is not limited to the scope of the present invention by the method of obtaining it, whether synthesized by a chemical synthesis method or separated and purified from natural products.
일례로, 6,7-디-O-아세틸시노코큘린은 일문전 (Stephania delavayi)을 70 내지 90% 수용성 에탄올로 냉침시킨후 감압증류하여 에탄올 추출물을 얻은 후, 이를 증류수에 현탁시킨 후 에틸 아세테이트 (ethyl acetate)를 이용하여 용매분획하여 얻은 분획물 중 주요 분획을 분리, 정제함으로 수득하였다.For example, 6,7-di-O-acetylcynococulin was cooled by distillation under a reduced pressure of 70-90% aqueous ethanol ( Stephania delavayi ) to obtain an ethanol extract, suspended in distilled water and then ethyl acetate It was obtained by separating and purifying the main fraction of the fraction obtained by solvent fractionation using (ethyl acetate).
상기 6,7-디-O-아세틸시노코큘린의 약학적으로 허용가능한 염으로는 유리산 (free acid)에 의해 형성된 산 부가염을 사용할 수 있다. 산 부가염은 통상의 방법, 일례로 화합물을 과량의 산 수용액에 용해시키고, 이 염을 수혼화성 유기 용매, 예를 들면 메탄올, 에탄올, 아세톤 또는 아세토니트릴을 사용하여 침전시켜서 제조할 수 있다. 동몰량의 화합물 및 물 중의 산 또는 알코올 (예, 글리콜 모노메틸에테르)을 가열하고 이어서 상기 혼합물을 증발시켜서 건조시키거나, 또는 석출된 염을 흡인 여과시킬 수 있다.As the pharmaceutically acceptable salt of the 6,7-di-O-acetylcynococulin, an acid addition salt formed by free acid may be used. Acid addition salts can be 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 such as methanol, ethanol, acetone or acetonitrile. Equal 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.
이때, 유리산으로는 유기산과 무기산을 사용할 수 있으며, 무기산으로는 염산, 인산, 황산, 질산, 주석산 등을 사용할 수 있고 유기산으로는 메탄술폰산, p-톨루엔술폰산, 아세트산, 트리플루오로아세트산, 시트르산, 말레인산 (maleic acid), 숙신산, 옥살산, 벤조산, 타르타르산, 푸마르산, 만데르산, 프로피온산 (propionic acid), 구연산 (citric acid), 젖산 (lactic acid), 글리콜산 (glycollic acid), 글루콘산 (gluconic acid), 갈락투론산, 글루탐산, 글루타르산 (glutaric acid), 글루쿠론산 (glucuronic acid), 아스파르트산, 아스코르브산, 카본산, 바닐릭산, 히드로아이오딕산 등을 사용할 수 있다.In this case, organic acids and inorganic acids may be used as the free acid, and hydrochloric acid, phosphoric acid, sulfuric acid, nitric acid, tartaric acid, and the like may be used as the inorganic acid, and methanesulfonic acid, p-toluenesulfonic acid, acetic acid, trifluoroacetic acid, and citric acid may be used as the organic acid. , Maleic acid, succinic acid, oxalic acid, benzoic acid, tartaric acid, fumaric acid, manderic acid, propionic acid, citric acid, lactic acid, glycolic acid, gluconic acid acid), galacturonic acid, glutamic acid, glutaric acid, glucuronic acid, aspartic acid, ascorbic acid, carbonic acid, vanillic acid, hydroiodic acid, and the like.
또한, 염기를 사용하여 약학적으로 허용가능한 금속염을 만들 수 있다. 알칼리 금속 또는 알칼리토 금속염은, 일례로 화합물을 과량의 알칼리 금속 수산화물 또는 알칼리토 금속 수산화물 용액 중에 용해하고, 비용해 화합물염을 여과한 후 여액을 증발, 건조시켜 얻을 수 있다. 이때, 금속염으로서는 특히 나트륨, 칼륨 또는 칼슘염을 제조하는 것이 제약상 적합하며, 또한 이에 대응하는 은염은 알칼리 금속 또는 알칼리토 금속염을 적당한 은염 (일례로, 질산은)과 반응시켜 얻을 수 있다.Bases can also be used to make pharmaceutically acceptable metal salts. An alkali metal or alkaline earth metal salt can be obtained by, for example, dissolving a compound in an excess alkali metal hydroxide or alkaline earth metal hydroxide solution, filtering the insoluble compound salt, and then evaporating and drying the filtrate. At this time, as the metal salt, it is particularly suitable to prepare sodium, potassium or calcium salts, and the corresponding silver salt can be obtained by reacting an alkali metal or alkaline earth metal salt with a suitable silver salt (for example, silver nitrate).
상기 6,7-디-O-아세틸시노코큘린의 약학적으로 허용가능한 염은, 달리 지시되지 않는 한, 6,7-디-O-아세틸시노코큘린의 화합물에 존재할 수 있는 산성 또는 염기성의 염을 포함할 수 있다. 일례로, 약학적으로 허용가능한 염으로는 히드록시기의 나트륨, 칼슘 및 칼륨의 염이 포함되며, 아미노기의 기타 약학적으로 허용가능한 염으로는 히드로브로마이드, 황산염, 수소 황산염, 인산염, 수소 인산염, 이수소 인산염, 아세테이트, 숙시네이트, 시트레이트, 타르트레이트, 락테이트, 만델레이트, 메탄설포네이트 (메실레이트) 및 p-톨루엔설포네이트 (토실레이트) 염이 있으며, 당업계에서 알려진 염의 제조방법이나 제조과정을 통하여 제조될 수 있다.The pharmaceutically acceptable salts of the 6,7-di-O-acetylcynococulin are acidic or basic, which may be present in the compound of 6,7-di-O-acetylcynococulin, unless otherwise indicated. Salts. In one example, pharmaceutically acceptable salts include salts of sodium, calcium and potassium of the hydroxy group, and other pharmaceutically acceptable salts of the amino group include hydrobromide, sulfate, hydrogen sulphate, phosphate, hydrogen phosphate, dihydrogen Phosphate, acetate, succinate, citrate, tartrate, lactate, mandelate, methanesulfonate (mesylate) and p-toluenesulfonate (tosylate) salts, which are known in the art. It can be prepared through.
천식은 기도의 염증으로 인해서 기관지가 과민해지고 간헐적인 기도 수축이 발생하여 호흡곤란 증상을 유발하는 질병을 의미하는데, 그 대표적인 증상으로는 기침, 호기성 천명음, 또는 호흡곤란이 있으며, 기관지 염증 및 기도 과민성을 나타날 수 있다.Asthma is a disease that causes bronchial hyperresponsiveness and intermittent airway constriction due to inflammation of the airways, causing symptoms of respiratory distress, such as cough, aerobic wheezing, or dyspnea, and bronchial inflammation and airway hypersensitivity. May appear.
상기 6,7-디-O-아세틸시노코큘린은 림프구 (lymphocytes), 중성구 (neutrophils), 및 호산구 (eosinophils)를 억제할 수 있다. 특히, 상기 호산구는 천식에서 가장 빈번하게 관찰되는 중간 표현형 중 하나로, 말초혈액 및 목표 조직 내의 호산백혈구의 개수는 질환의 심각성 및 악화와 밀접하게 연관되어 있다.The 6,7-di-O-acetylcynococulin can inhibit lymphocytes, neutrophils, and eosinophils. In particular, the eosinophil is one of the most frequently observed intermediate phenotypes in asthma, the number of peripheral blood and eosinophils in target tissues is closely related to the severity and exacerbation of the disease.
본 발명의 6,7-디-O-아세틸시노코큘린은 기도 과반응성 (airway hyperresponsiveness, AHR), 염증세포 침윤 (inflammatory cell infiltration), 점막, 점막세포 비후 (mucosal cell hyperplasia) 등을 억제함으로 천식을 효과적으로 예방 또는 치료할 수 있다.6,7-di-O-acetylcynococurin of the present invention inhibits airway hyperresponsiveness (AHR), inflammatory cell infiltration, mucous membranes, mucosal cell hyperplasia, etc. Can be effectively prevented or treated.
본 발명은 6,7-디-O-아세틸시노코큘린 (6,7-di-O-acetylsinococuline) 또는 이의 약리학적으로 허용가능한 염을 유효성분으로 포함하는 천식 예방 또는 치료용 약학 조성물을 제공한다.The present invention provides a pharmaceutical composition for preventing or treating asthma comprising 6,7-di-O-acetylsinococuline or a pharmacologically acceptable salt thereof as an active ingredient. .
상기 화합물은 전체 조성물 총 중량에 대하여 0.0001∼99 중량%로 포함될 수 있으나, 이에 의해 본 발명의 범위가 한정되는 것은 아니다. The compound may be included in 0.0001 to 99% by weight based on the total weight of the composition, but is not limited to the scope of the present invention.
본 발명의 화합물을 포함하는 조성물은 통상의 방법에 따른 적절한 담체, 부형제 또는 희석제를 더 포함할 수 있다.Compositions comprising a compound of the present invention may further comprise a suitable carrier, excipient or diluent according to conventional methods.
본 발명의 조성물에 포함될 수 있는 담체, 부형제 및 희석제로는 락토즈, 덱스트로즈, 수크로스, 솔비톨, 만니톨, 자일리톨, 에리스리톨, 말티톨, 전분, 아카시아 고무, 알지네이트, 젤라틴, 칼슘 포스페이트, 칼슘 실리케이트, 셀룰로즈, 메틸 셀룰로즈, 미정질 셀룰로스, 폴리비닐 피롤리돈, 물, 메틸히드록시벤조에이트, 프로필히드록시벤조에이트, 탈크, 마그네슘 스테아레이트 및 광물유를 들 수 있다.Carriers, excipients and diluents that may be included in the compositions of the present invention 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.
본 발명의 화합물을 포함하는 조성물은, 각각 통상의 방법에 따라 산제, 과립제, 정제, 캡슐제, 현탁액, 에멀젼, 시럽, 에어로졸 등의 경구형 제형, 외용제, 좌제 또는 멸균 주사용액의 형태로 제형화하여 사용될 수 있다. The compositions comprising the compounds of the present invention are each formulated in the form of oral dosage forms, external preparations, suppositories, or sterile injectable solutions, such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, etc., in accordance with conventional methods. Can be used.
상세하게는, 제제화할 경우에는 보통 사용하는 충진제, 증량제, 결합제, 습윤제, 붕해제, 계면활성제 등의 희석제 또는 부형제를 사용하여 조제될 수 있다. 경구투여를 위한 고형제제에는 정제, 환제, 산제, 과립제, 캡슐제 등이 포함되며, 이러한 고형제제는 상기 화합물에 적어도 하나 이상의 부형제 예를 들면, 전분, 칼슘카보네이트 (calcium carbonate), 수크로스 (sucrose), 락토오스 (lactose), 젤라틴 등을 섞어 조제될 수 있다. 또한, 단순한 부형제 이외에 마그네슘 스테아레이트, 탈크 같은 윤활제들도 사용될 수 있다. 경구를 위한 액상 제제로는 현탁제, 내용액제, 유제, 시럽제 등이 해당되는 데, 흔히 사용되는 단순 희석제인 물, 리퀴드 파라핀 이외에 여러 가지 부형제, 예를 들면 습윤제, 감미제, 방향제, 보존제 등이 포함될 수 있다. 비경구 투여를 위한 제제에는 멸균된 수용액, 비수성용제, 현탁제, 유제, 동결건조 제제 및 좌제가 포함된다. 비수성용제, 현탁제로는 프로필렌글리콜 (propylene glycol), 폴리에틸렌 글리콜, 올리브 오일과 같은 식물성 기름, 에틸올레이트와 같은 주사 가능한 에스테르 등이 사용될 수 있다. 좌제의 기제로는 위텝솔(witepsol), 마크로골, 트윈(tween) 61, 카카오지, 라우린지, 글리세로젤라틴 등이 사용될 수 있다.Specifically, when formulated, it may be prepared using diluents or excipients such as commonly used fillers, extenders, binders, wetting agents, disintegrating agents, surfactants, and the like. Solid form preparations for oral administration include tablets, pills, powders, granules, capsules, and the like, which form at least one excipient such as starch, calcium carbonate, sucrose, etc. ), Lactose, gelatin and the like can be mixed. In addition to simple excipients, lubricants such as magnesium stearate, talc can also be used. Liquid preparations for oral use include suspensions, solvents, emulsions, and syrups, and include various excipients such as wetting agents, sweeteners, fragrances, and preservatives, in addition to commonly used simple diluents such as water and liquid paraffin. Can be. Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations and 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.
본 발명의 약학적 조성물의 바람직한 투여량은 1일 0.01 내지 200mg/kg으로 투여할 수 있으며, 바람직하게는 1일 0.1 mg/kg ~ 40 mg/kg으로 투여할 수 있으나, 이에 의해 본 발명의 범위가 한정되는 것은 아니다. 상기 투여량은 제제화 방법, 투여 방식, 환자의 연령, 체중, 성, 병적 상태, 음식, 투여 시간, 투여 경로, 배설 속도 및 반응 감응성과 같은 요인들에 의해 다양하며, 당업자에 의해 적절하게 선택될 수 있으며, 상기 투여는 하루에 한번 투여할 수도 있고, 수회 나누어 투여할 수도 있다. 상기 투여량은 어떠한 면으로든 본 발명의 범위를 한정하는 것은 아니다.Preferred dosages of the pharmaceutical composition of the present invention may be administered at 0.01 to 200 mg / kg per day, preferably 0.1 mg / kg to 40 mg / kg per day, but the scope of the present invention thereby Is not limited. The dosage will vary depending on factors such as the formulation method, mode of administration, age, weight, sex, morbidity, food, time of administration, route of administration, rate of excretion, and reaction sensitivity of the patient, and will be appropriately selected by those skilled in the art. In addition, the administration may be administered once a day, may be divided several times. The dosage does not limit the scope of the invention in any aspect.
또한, 본 발명의 화합물의 약학적 투여 형태는 이들의 약학적 허용가능한 염의 형태로도 사용될 수 있고, 또한 단독으로 또는 타 약학적 활성 화합물과 결합뿐만 아니라 적당한 집합으로 사용될 수 있다. 본 발명의 약학적 조성물은 쥐, 생쥐, 가축, 인간 등의 포유동물에 다양한 방식으로 투여될 수 있는데, 경구 또는 비경구로 투여할 수 있고, 비경구 투여인 경우에는 정맥내 주입, 피하 주입, 근육 주입, 복강 주입, 경피 주입, 기관 (trachea) 주입, 또는 기도 (airway) 주입 등으로 투여할 수 있으며, 가장 바람직하게는 호흡기 등을 통하여 기도에 직접 투여할 수 있다.In addition, the pharmaceutical dosage forms of the compounds of the present invention 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. The pharmaceutical composition of the present invention can be administered to mammals such as rats, mice, livestock, humans in various ways, orally or parenterally, and in the case of parenteral administration, intravenous injection, subcutaneous injection, muscle It may be administered by infusion, intraperitoneal infusion, transdermal infusion, trachea infusion, or airway infusion, and most preferably, directly into the respiratory tract via a respiratory system or the like.
본 발명의 약학적 조성물은 당해 발명이 속하는 기술분야에서 통상의 지식을 가진 자가 용이하게 실시할 수 있는 방법에 따라, 약제학적으로 허용되는 담체 및/또는 부형제를 이용하여 제제화함으로써 단위 용량 형태로 제조되거나 또는 다용량 용기 내에 내입시켜 제조될 수 있다. 이때 제형은 산제, 과립제, 정제, 캡슐제, 현탁액, 에멀젼, 시럽, 에어로졸의 경구적 제형, 외용제, 좌제, 또는 주사제 형태일 수도 있으며, 분산제 또는 안정화제를 추가적으로 포함할 수 있다.The pharmaceutical compositions of the present invention may be prepared in unit dosage form by formulating with a pharmaceutically acceptable carrier and / or excipient according to methods which can be easily carried out by those skilled in the art. Or may be prepared by incorporation into a multi-dose container. In this case, the formulation may be in the form of powder, granules, tablets, capsules, suspensions, emulsions, syrups, oral formulations of aerosols, external preparations, suppositories, or injections, and may further include dispersants or stabilizers.
본 발명은 6,7-디-O-아세틸시노코큘린 (6,7-di-O-acetylsinococuline) 또는 이의 약리학적으로 허용가능한 염을 유효성분으로 포함하는 천식 예방 또는 개선용 건강식품 조성물을 제공한다.The present invention provides a health food composition for preventing or improving asthma comprising 6,7-di-O-acetylsinococuline (6,7-di-O-acetylsinococuline) or a pharmacologically acceptable salt thereof as an active ingredient. do.
상기 6,7-디-O-아세틸시노코큘린을 첨가할 수 있는 식품으로는, 예를 들어, 각종 식품류, 음료, 껌, 차, 비타민 복합제, 건강 기능성 식품류 등이 있다.Examples of the food to which the 6,7-di-O-acetylcynococulin can be added include various foods, beverages, gums, teas, vitamin complexes, and health functional foods.
이때, 식품 또는 음료 중의 상기 6,7-디-O-아세틸시노코큘린의 양은 전체 식품 중량의 0.001 내지 30 중량 %로 가할 수 있으며, 건강 음료 조성물은 100 ㎖를 기준으로 0.001 내지 30 g, 바람직하게는 0.02 내지 5g의 비율로 가할 수 있으나, 이에 의해 본 발명의 범위가 한정되는 것은 아니다. At this time, the amount of the 6,7-di-O-acetylcynococulin in the food or beverage may be added at 0.001 to 30% by weight of the total food weight, the health beverage composition is 0.001 to 30 g, preferably based on 100 ml Preferably, it may be added in a ratio of 0.02 to 5 g, but the scope of the present invention is not limited thereto.
본 발명의 건강 기능성 음료 조성물은 지시된 비율로 필수 성분으로서 상기 6,7-디-O-아세틸시노코큘린을 함유하는 외에는 다른 성분에는 특별한 제한이 없으며 통상의 음료와 같이 여러 가지 향미제 또는 천연 탄수화물 등을 추가 성분으로서 함유할 수 있다. 상술한 천연 탄수화물의 예는 모노사카라이드, 예를 들어, 포도당, 과당 등; 디사카라이드, 예를 들어 말토스, 슈크로스 등; 및 폴리사카라이드, 예를 들어 덱스트린, 시클로덱스트린 등과 같은 통상적인 당, 및 자일리톨, 소르비톨, 에리트리톨 등의 당알콜이다. 상술한 것 이외의 향미제로서 천연 향미제 (타우마틴, 스테비아 추출물 (예를 들어 레바우디오시드 A, 글리시르히진 등)) 및 합성 향미제 (사카린, 아스파르탐 등)를 유리하게 사용할 수 있다. 상기 천연 탄수화물의 비율은 본 발명의 조성물 100 ㎖당 일반적으로 약 1 내지 20 g, 바람직하게는 약 5 내지 12 g이다.The health functional beverage composition of the present invention is not particularly limited to other ingredients except for containing 6,7-di-O-acetylcynococurine as an essential ingredient in the indicated ratios, and various flavoring or natural ingredients such as ordinary drinks Carbohydrates and the like may be included as additional components. 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 (e.g., Rebaudioside A, glycyrrhizin, etc.) and synthetic flavoring agents (saccharin, aspartame, etc.) can be advantageously used. have. The proportion of such natural carbohydrates is generally about 1 to 20 g, preferably about 5 to 12 g per 100 ml of the composition of the present invention.
상기 외에 본 발명의 조성물은 여러 가지 영양제, 비타민, 광물 (전해질), 합성 풍미제 및 천연 풍미제 등의 풍미제, 착색제 및 중진제 (치즈, 초콜릿 등), 펙트산 및 그의 염, 알긴산 및 그의 염, 유기산, 보호성 콜로이드 증점제, pH 조절제, 안정화제, 방부제, 글리세린, 알콜, 탄산 음료에 사용되는 탄산화제 등을 함유할 수 있다. 그밖에 본 발명의 조성물은 천연 과일 쥬스 및 과일 쥬스 음료 및 야채 음료의 제조를 위한 과육을 함유할 수 있다. 이러한 성분은 독립적으로 또는 조합하여 사용할 수 있으며, 이러한 첨가제의 비율은 그렇게 중요하진 않지만 본 발명의 조성물 100 중량부 당 0.01 내지 약 20 중량부의 범위에서 선택되는 것이 일반적이다.In addition to the above, the composition of the present invention includes various nutrients, vitamins, minerals (electrolytes), flavors such as synthetic flavors and natural flavors, coloring and neutralizing agents (such as cheese, chocolate), pectic acid and salts thereof, alginic acid and its Salts, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohols, carbonation agents used in carbonated beverages, and the like. In addition, the composition of the present invention may contain a natural fruit juice and a pulp for the production of fruit juice drinks and vegetable drinks. These components can be used independently or in combination, and the proportion of such additives is not so critical but is generally selected from the range of 0.01 to about 20 parts by weight per 100 parts by weight of the composition of the present invention.
이하, 본 발명을 하기의 실시예 및 실험예에 의하여 더욱 상세하게 설명한다. 단, 하기 실시예 및 실험예는 본 발명을 예시하는 것으로 본 발명의 내용이 하기 실시예 및 실험예에 의해 본 발명의 범위가 제한되는 것은 아니다.Hereinafter, the present invention will be described in more detail by the following Examples and Experimental Examples. However, the following Examples and Experimental Examples illustrate the present invention, and the scope of the present invention is not limited by the following Examples and Experimental Examples.
<실시예><Example>
실시예 1; 실험재료 및 방법Example 1; Experimental Materials and Methods
본 연구에서 사용된 일문전 (Stephania delavayi)은 생명공학연구원 추출물은행에서 분양받아 사용하였다.The stephania delavayi used in this study was distributed from the Extract Bank of Biotechnology.
1-1. 추출 및 정제 1-1. Extraction and Purification
잘 건조된 일문전 (50g)을 90% 수용성 에탄올로 7일간 냉침시킨 후 감압증류하여 에탄올 추출물(6g)을 얻었다. 이를 증류수에 현탁시킨후 에틸 아세테이트 (ethyl acetate)(500 mL x 10)를 이용하여 용매분획하여 EtOAc 분획물 3.2g을 얻었다. EtOAc 분획물 3g을 실리카겔 MPLC (MC:MeOH = 98:2(60min)~(240min) 80:20,~(120min) 20:50 ~(40min) 1:99, 40mL/min)를 실시하여 12개의 분획으로 나누고, 이 중에서 주요 분획 (major fraction)인 3번을 C-18 HPLC하여 순수한 물질을 정제하였다 (도 1).The well dried Munmunjeon (50g) was cooled for 7 days with 90% aqueous ethanol and distilled under reduced pressure to obtain an ethanol extract (6g). This was suspended in distilled water and solvent fractionated using ethyl acetate (500 mL x 10) to obtain 3.2 g of EtOAc fraction. 3 g of EtOAc fraction was subjected to silica gel MPLC (MC: MeOH = 98: 2 (60min) ~ (240min) 80: 20, ~ (120min) 20: 50 ~ (40min) 1:99, 40mL / min) The purified material was purified by performing C-18 HPLC on the major fraction (major fraction) 3 (FIG. 1).
1-2. 구조분석1-2. Structural analysis
NMR 실험은 Varian Mecury 400 MHz NMR 스펙트로메트리를 이용하였다. NMR 용매는 CD3OD를 사용하였다. 일문전에서 주요 화합물인 순수물질 6,7-디-O-아세틸시노코큘린 (6,7-di-O-acetylsinococuline; FK-3000)을 정제하였다. 이 물질은 흰색 고체로 1H NMR 및 2D NMR 실험을 통하여 표 1의 구조로 확인되었다 (도 2 내지 도 6).NMR experiments used a Varian Mecury 400 MHz NMR spectrometry. NMR solvent was used CD 3 OD. In one sentence, 6,7-di-O-acetylsinococuline (FK-3000) was purified. This material was identified as the structure of Table 1 by 1 H NMR and 2D NMR experiments as a white solid (FIGS. 2 to 6).
표 1
position δc(mult.) δH(int., mult., J in Hz) HMBC (H→C)
1 119.8 (d) 6.66 (1H, d, 8.6) 3, 11 or 12, 10
2 111.8 (d) 6.89 (1H, d, 8.6) 4, 11
3 148.4 (s)
3-OMe 56.8 (q) 3.86 (3H, s) 3
4 145.9 (s)
5 33.9 (t) 3.02 (1H, dd, 14.0, 3.2)2.37 (1H, t, 13.6) 14, 7, 6, 1312, 7, 13, 15
6 69.4 (d) 5.16 (1H, dt, 13.6, 3.6)
6'-OCOCH3 b 172.08 (s)
6''-OCOCH3 c 20.96 (q) 1.99 (3H, s) 6'
7 64.5 (d) 5.98 (1H, d, 3.6) 7', 8, 14, 6, 5
7'-OCOCH3 b 172.10 (s)
7'-OCOCH3 c 20.79 (q) 2.05 (3H, s) 7'
8 146.7 (s)
8-OMe 57.2 (q) 3.62 (3H, s) 8
9 47.9 (d) 4.98 (1H, d, 5.6) 8, 11, 14, 16, 13, 10
10 33.6 (t) 3.31 (1H, dd, 18.4, 5.2) 11 or 12, 1, 9
11d 127.7 (s) 3.13 (1H, d, 18.4) 11 or 12, 14, 9
12d 127.7 (s)
13 38.4 (s)
14 118.7 (s)
15 34.2 (t) 2.21 (1H, dd, 13.6, 4.0)2.11 (1H, td, 13.6, 4.6) 12, 14, 1312, 16, 13
16 40.7 (t) 3.18 (1H, dd, 12.8, 4.6)2.86 (1H, td, 12.8, 4.0) 1315
Table 1
position δ c (mult.) δ H (int., mult., J in Hz) HMBC (H → C)
One 119.8 (d) 6.66 (1 H, d, 8.6) 3, 11 or 12, 10
2 111.8 (d) 6.89 (1H, d, 8.6) 4, 11
3 148.4 (s)
3-OMe 56.8 (q) 3.86 (3H, s) 3
4 145.9 (s)
5 33.9 (t) 3.02 (1H, dd, 14.0, 3.2) 2.37 (1H, t, 13.6) 14, 7, 6, 1312, 7, 13, 15
6 69.4 (d) 5.16 (1H, dt, 13.6, 3.6)
6'-OCOCH 3 b 172.08 (s)
6 ''-OCOCH 3 c 20.96 (q) 1.99 (3H, s) 6 '
7 64.5 (d) 5.98 (1H, d, 3.6) 7 ', 8, 14, 6, 5
7'-OCOCH 3 b 172.10 (s)
7'-OCOCH 3 c 20.79 (q) 2.05 (3H, s) 7 '
8 146.7 (s)
8-OMe 57.2 (q) 3.62 (3H, s) 8
9 47.9 (d) 4.98 (1 H, d, 5.6) 8, 11, 14, 16, 13, 10
10 33.6 (t) 3.31 (1H, doublet of 18.4, 5.2) 11 or 12, 1, 9
11 d 127.7 (s) 3.13 (1H, d, 18.4) 11 or 12, 14, 9
12 d 127.7 (s)
13 38.4 (s)
14 118.7 (s)
15 34.2 (t) 2.21 (1H, dd, 13.6, 4.0) 2.11 (1H, td, 13.6, 4.6) 12, 14, 1312, 16, 13
16 40.7 (t) 3.18 (1H, dd, 12.8, 4.6) 2.86 (1H, td, 12.8, 4.0) 1315
a 1H and 13C NMR were recorded at 400 MHz and 100 MHz, respectively. a 1 H and 13 C NMR were recorded at 400 MHz and 100 MHz, respectively.
b, c, d Assignments may be interchangeable within the same column. b, c, d Assignments may be interchangeable within the same column.
실시예 2;Example 2; 천식효능 실험방법Asthma test method
2-1. 시험재료2-1. Test material
(주) 오리엔트 바이오 (경기도 성남시 중원구 상대원동 143-1)로부터 암컷 5주령의 정병원체부재(SPF) BALB/c 마우스 72마리를 구입하였으며, 본 발명의 화합물 주입시 암컷은 약 8주령이었다.72 female 5 week-old pathogenic member (SPF) BALB / c mice were purchased from Orient Bio Co., Ltd. (143-1, Sangwon-dong, Jungwon-gu, Seongnam-si, Gyeonggi-do), and the females were about 8 weeks old when the compound of the present invention was injected.
상기 마우스의 사육조건은 사육실의 온, 습도 범위는 온도 23 ±3℃, 상대습도 50 ±10%, 사육실의 명암 사이클 (cycle)은 형광등조명 12 시간 (08:00 점등~20:00 소등)이며, 사육실의 환기횟수는 10~20 회/hr이고, 사육실의 조도는 150~300 Lux이였고 사료는 방사선 멸균된 사료를 사용하고 음수는 자외선 유수살균장치와 미세여과장치를 통과시켜 소독하였으며, 마우스는 사료와 음수를 자유섭취하게 하였다. The breeding condition of the mouse is the temperature of the room, the humidity range is 23 ± 3 ℃, relative humidity 50 ± 10%, the contrast cycle of the breeding room is 12 hours (08:00 lights ~ 20:00 lights off) fluorescent lighting The number of ventilation of the breeding room was 10-20 times / hr, the illuminance of the breeding room was 150-300 Lux, the feed was sterilized by the use of radiation sterilized feed, and the negative water was passed through the UV-water sterilizer and the microfiltration device. Allowed free intake of feed and drinking water.
2-2. 시험물질 및 대조물질2-2. Test substance and control substance
천식유발물질로는 백색분말의 오브알부민 (Ovalbumin, A-5503) (Sigma)을 사용하였고, Adjuvant로는 백색액상의 Alum (Imject alum, 77161) (PIERCE)을 사용하였다. 기관지 수축물질로는 백색분말의 메타콜린 (Metacholine, A-2251) (Sigma)를 사용하였고, 양성대조물질로 덱사메타손 (Dexamethasone)을, 시험물질로는 FK-3000 (6,7-디-O-아세틸시노코큘린)을 사용하였다.White powder of ovalbumin (A-5503) (Sigma) was used as an asthma-inducing substance and white liquid Alum (Imject alum, 77161) (PIERCE) was used as an adjuvant. Bronchial contractile was used as a white powder of metacholine (A-2251) (Sigma), dexamethasone as a positive control, FK-3000 (6,7-di-O-) as a test substance. Acetylcynococulin) was used.
2-3. 시험물질의 조제  2-3. Preparation of Test Substance
(1) OVA I.P (복강)주사용(1) OVA I.P (abdominal cavity) injection
OVA 20 ㎍과 알루미늄 하이드록사이드 하이드레이트 (aluminum hydroxide hydrate, alum; 40 mg/mL) 1 mg을 혼합하여 500 ㎕의 생리식염수에 현탁하였다. [2 mg OVA + 100 mg alum (2.5 mL) + 47.5 mL Saline]20 μg of OVA and 1 mg of aluminum hydroxide hydrate (alum; 40 mg / mL) were mixed and suspended in 500 μl of saline. [2 mg OVA + 100 mg alum (2.5 mL) + 47.5 mL Saline]
(2) OVA 흡입투여(2) OVA inhalation administration
5% OVA용액이 되도록 OVA를 생리식염수에 녹였다 (5 g OVA+100 mL saline).OVA was dissolved in saline to give 5% OVA solution (5 g OVA + 100 mL saline).
(3) 메타콜린 (Methacholine) 흡입투여용(3) Methacholine for inhalation administration
메타콜린 (Methacholine, Sigma, A-2251)을 10 mg/mL 및 20 mg/mL이 되도록 생리식염수에 녹여 조제하고 1 mL 씩 소분하여 냉동보관하였다. Methacholine (Methacholine, Sigma, A-2251) was prepared by dissolving in saline to 10 mg / mL and 20 mg / mL and subdivided by 1 mL to store frozen.
(4) 시험물질과 양성대조물질 경구투여용(4) Oral administration of test substance and positive control substance
양성대조물질을 투여용량에 맞게 칭량하여 멸균증류수에 현탁하여 조제하였다. FK-3000은 100% 에탄올 용액에 FK-3000을 가하여 녹인 후 10% 에탄올 용액이 되도록 멸균증류수를 가하고 현탁하여 조제하였다. Positive controls were weighed to the dosage and suspended in sterile distilled water. FK-3000 was dissolved by adding FK-3000 to 100% ethanol solution and then prepared by adding sterile distilled water and suspending it to 10% ethanol solution.
2-4. 투여방법2-4. Dosing method
(1) OVA초기 감작(1) OVA initial sensitization
OVA I.P 주사용으로 조제한 용액을 정상대조군 이외의 모든 군의 마우스 한 마리 당 500 ㎕ (OVA로서 20 ㎍)을 복강투여 하였다 (Day 0). 정상대조군은 생리식염수를 복강 투여하였다. 천식유발 대조군는 초기 감작(복강투여) 후 일주일에 같은 방법으로 한 번 더 복강투여 하였다 (Day 7). The solution prepared for OVA I.P injection was intraperitoneally administered 500 μl (20 μg as OVA) per mouse in all groups except the normal control group (Day 0). The normal control group was intraperitoneally administered saline solution. Asthma-induced control group was intraperitoneally administered once more in the same way a week after initial sensitization (Day 7).
(2) OVA 흡입투여(2) OVA inhalation administration
OVA 흡입투여 (inhalation)용으로 조제한 용액을 whole body plethysmograph과 nubulizer (NE-U17, OMRON Co. Ltd, Japan)를 이용하여 초기감작 후 14일부터 18일까지 매일 30분씩 흡입 투여하였다 (Day 14 ~ 18).The solution prepared for OVA inhalation was inhaled daily for 14 minutes to 18 days after initial sensitization using whole body plethysmograph and nubulizer (NE-U17, OMRON Co. Ltd, Japan) (Day 14 ~ 18).
(3) 시험물질과 양성대조물질 투여(3) Administration of test substance and positive control substance
초기감작 후 14일부터 18일까지 5일간 OVA 흡입투여하기 1시간 전에 매일 경구 투여하였다. 체중을 기준으로 kg당 5 mL로 하여 투여액량을 계산하였다.Daily oral administration was performed 1 hour prior to inhalation of OVA for 5 days from 14 to 18 days after initial sensitization. Dose amount was calculated at 5 mL per kg based on body weight.
2-5. 시험군의 구성, 투여액량 및 투여량2-5. Composition, Dose and Dose of Test Group
표 2
군 성별 동물수(마리) 동물번호 투여량(mg/kg) 투여액량(mL/kg)
V.Control F 8 1~8 0 5
천식유발대조군 F 8 9~16 0 5
P.Control (Dex.) F 8 17~24 1 5
FK-3000 F 8 49~56 4 5
FK-3000 F 8 57~64 10 5
FK-3000 F 8 65~72 40 5
TABLE 2
group gender Animal count (mari) Animal Number Dose (mg / kg) Dosage amount (mL / kg)
V.Control F 8 1-8 0 5
Asthma-Induced Control F 8 9-16 0 5
P.Control (Dex.) F 8 17-24 One 5
FK-3000 F 8 49-56 4 5
FK-3000 F 8 57-64 10 5
FK-3000 F 8 65-72 40 5
2-6. 시험방법 및 시험항목2-6. Test Method and Test Items
(1) 메타콜린 (Methacholine)에 대한 과민반응(Airway hyper-responsiveness) 조사 (1) Investigation of airway hyper-responsiveness to methacholine
마지막 OVA 흡입투여 후 24시간 뒤 (Day 19, 20)에 마우스를 whole body plethysmograph (BUXCO, USA)에 넣고 메타콜린 (methacholine) 0, 10 및 20 mg/mL 농도의 용액을 0.5 mL 취해 에어로졸 (aerosol)에 넣고 각각 3분간 흡입투여하고 투여종료시점에서부터 4분간의 Penh를 측정하여 평균값을 해당 메타콜린 (methacholine) 투여용량에서의 Penh 값으로 하였다.24 hours after the last OVA inhalation (Day 19, 20), the mice were placed in whole body plethysmograph (BUXCO, USA) and 0.5 mL of solution of concentrations of 0, 10 and 20 mg / mL of methacholine were aerosolized. ) And inhalation for 3 minutes each, Penh was measured for 4 minutes from the end of the administration and the average value was taken as the Penh value at the corresponding dose of methacholine.
표 3에서 나타난 바와 같이, FK-3000은 OVA로 유도된 천식모델 마우스의 AHR을 농도의존적으로 억제하였다. As shown in Table 3, FK-3000 concentration-dependently inhibited AHR of OVA-induced asthma model mice.
표 3
그룹 복용량(Dose)(mg/kg) 루트(Route) 메타콜린(Methacholine)
0 mg/ml 10 mg/ml 20 mg/ml
V.C 0 P.O 1.41 ± 0.23 2.13 ± 0.68 9.24 ± 5.28
I.C 0 P.O 1.50 ± 0.11 9.00 ± 4.21 17.12 ± 5.43
Dexa. 1 P.O 1.66 ± 0.24 3.24 ± 1.00 10.90 ± 8.81
FK-3000 4 P.O 1.56 ± 0.21 5.39 ± 3.06 15.17 ± 4.80
FK-3000 10 P.O 1.84 ± 0.58 5.15 ± 3.06 13.31 ± 3.35
FK-3000 40 P.O 2.41 ± 0.98 6.75 ± 5.77 11.75 ± 7.76
TABLE 3
group Dose (mg / kg) Route Methacholine
0 mg / ml 10 mg / ml 20 mg / ml
VC 0 PO 1.41 ± 0.23 2.13 ± 0.68 9.24 ± 5.28
IC 0 PO 1.50 ± 0.11 9.00 ± 4.21 17.12 ± 5.43
Dexa. One PO 1.66 ± 0.24 3.24 ± 1.00 10.90 ± 8.81
FK-3000 4 PO 1.56 ± 0.21 5.39 ± 3.06 15.17 ± 4.80
FK-3000 10 PO 1.84 ± 0.58 5.15 ± 3.06 13.31 ± 3.35
FK-3000 40 PO 2.41 ± 0.98 6.75 ± 5.77 11.75 ± 7.76
(2) 기관지 및 폐 세척액 (Bronchoalveolar lavage fluid; BAL fluid) 에서의 세포 수 및 면역세포 분포조사 (Day 20, 21)(2) Survey of cell number and immune cell distribution in bronchoalveolar lavage fluid (BAL fluid) (Day 20, 21)
펜토탈소디움 (50mg/kg, I.P)을 복강 주사하여 마취한 후 좌측 폐를 집게로 고정한 상태에서 PBS 용액 0.4 mL을 이용하여 기관지와 폐에 주입 후 빼어내어 세척액을 취하며 이를 세 번 반복하였다. 이를 1,500 g에서 원심분리하여 상층액을 버리고 침전물에 PBS 0.2 mL을 가하여 다시 현탁하였다. 이를 혈액학자동분석기 (Advia 120 coulter counter, BAYER, Germany)를 사용하여 전체 백혈구 세포, 호중구, 호산구, 중성구 및 림프구 세포의 수를 측정하였다.Pentotalsodium (50mg / kg, I.P) was anesthetized by intraperitoneal injection, and the left lung was fixed with forceps, and then injected into the bronchus and lungs using 0.4 mL of PBS solution. The supernatant was discarded by centrifugation at 1,500 g and suspended again by adding 0.2 mL of PBS to the precipitate. The total number of leukocytes, neutrophils, eosinophils, neutrophils and lymphocytes was measured using an automated hematology analyzer (Advia 120 coulter counter, BAYER, Germany).
하기 표 4 내지 6에서 나타난 바와 같이, FK-3000은 천식모델동물의 BAL fluid 내 호산구 (eosinophil)을 억제하며 특히 40 mg/kg의 경우 양성대조군인 덱사메타손 (Dexamethasone)의 결과보다도 낮으며, 이는 부형제 대조군의 결과와 유사하다. 따라서 천식발생시 확인되는 가장 대표적인 생리학적인 지표인 에오시노필리아 (eosinophilia)를 효과적으로 억제하는 것으로 판단된다.As shown in Tables 4 to 6 below, FK-3000 inhibits eosinophils in BAL fluid of asthma model animals, especially at 40 mg / kg, which is lower than the result of dexamethasone, a positive control, which is an excipient. Similar to the results of the control group. Therefore, it is thought to effectively suppress eosinophilia, which is the most representative physiological index identified during asthma.
(표 5 및 표 6에서 NEU는 neutrophi을, LYM는 lymphocyte를, MON는 monocyte를, EOS은 eosinophil을, BAS은 basophil을, LUC은 large unstained cell를 나타낸 것이다.)(Tables 5 and 6 show NEU for neutrophi, LYM for lymphocyte, MON for monocyte, EOS for eosinophil, BAS for basophil, and LUC for large unstained cells.)
표 4
그룹 복용량(Dose)(mg/kg) 루트(Route) WBC (×106 cells)
mean S.D
V.C 0 P.O 0.124 ± 0.05
I.C 0 P.O 1.398 ± 0.30
Dexa. 1 P.O 0.405 ± 0.17
FK-3000 4 P.O 1.084 ± 0.37
FK-3000 10 P.O 0.938 ± 0.35
FK-3000 40 P.O 0.194 ± 0.07
Table 4
group Dose (mg / kg) Route WBC (× 10 6 cells)
mean SD
VC 0 PO 0.124 ± 0.05
IC 0 PO 1.398 ± 0.30
Dexa. One PO 0.405 ± 0.17
FK-3000 4 PO 1.084 ± 0.37
FK-3000 10 PO 0.938 ± 0.35
FK-3000 40 PO 0.194 ± 0.07
표 5
그룹 복용량(mg/kg) 루트(Route) Percentage of differential WBC (%)
NEU LYM EOS MON BAS LUC
V.C 0 P.O 22.46±4.62 26.90±11.16 7.14±11.29 0.00±0.00 5.05±1.09 38.45±17.37
I.C 0 P.O 3.20±1.51 7.44±3.02 81.17±3.95 0.27±0.18 0.45±0.29 7.47±2.18
Dexa. 1 P.O 4.77±1.90 8.64±4.17 64.06±13.60 0.28±0.54 1.59±0.68 20.66±8.84
FK-3000 4 P.O 3.46±1.35 16.88±9.58 70.90±8.90 0.39±0.33 0.58±0.32 7.79±3.28
FK-3000 10 P.O 3.75±1.37 9.62±6.42 65.92±15.71 0.40±0.28 0.66±0.45 19.64±15.20
FK-3000 40 P.O 23.77±5.93 36.95±10.88 2.41±2.00 1.05±1.57 3.07±0.84 32.75±10.11
Table 5
group Dose (mg / kg) Route Percentage of differential WBC (%)
NEU LYM EOS MON BAS LUC
VC 0 PO 22.46 ± 4.62 26.90 ± 11.16 7.14 ± 11.29 0.00 ± 0.00 5.05 ± 1.09 38.45 ± 17.37
IC 0 PO 3.20 ± 1.51 7.44 ± 3.02 81.17 ± 3.95 0.27 ± 0.18 0.45 ± 0.29 7.47 ± 2.18
Dexa. One PO 4.77 ± 1.90 8.64 ± 4.17 64.06 ± 13.60 0.28 ± 0.54 1.59 ± 0.68 20.66 ± 8.84
FK-3000 4 PO 3.46 ± 1.35 16.88 ± 9.58 70.90 ± 8.90 0.39 ± 0.33 0.58 ± 0.32 7.79 ± 3.28
FK-3000 10 PO 3.75 ± 1.37 9.62 ± 6.42 65.92 ± 15.71 0.40 ± 0.28 0.66 ± 0.45 19.64 ± 15.20
FK-3000 40 PO 23.77 ± 5.93 36.95 ± 10.88 2.41 ± 2.00 1.05 ± 1.57 3.07 ± 0.84 32.75 ± 10.11
표 6
그룹 복용량(mg/kg) 루트(Route) WBC (×106 cells)
NEU LYM MON EOS BAS LUC
V.C 0 P.O 0.0269 ±0.01 0.0313 ±0.01 0.0000 ±0.00 0.0075 ±0.01 0.0063 ±0.00 0.0525 ±0.04
I.C 0 P.O 0.0419 ±0.01 0.1056 ±0.05 0.0038 ±0.00 1.1388 ±0.27 0.0063 ±0.00 0.1013 ±0.02
Dexa. 1 P.O 0.0169 ±0.00 0.0313 ±0.01 0.0013 ±0.00 0.2725 ±0.15 0.0056 ±0.00 0.0775 ±0.03
FK-3000 4 P.O 0.0369 ±0.02 0.1850 ±0.15 0.0038 ±0.00 0.7669 ±0.27 0.0063 ±0.00 0.0856 ±0.04
FK-3000 10 P.O 0.0344 ±0.01 0.0881 ±0.06 0.0038 ±0.00 0.6431 ±0.35 0.0056 ±0.00 0.1625 ±0.11
FK-3000 40 P.O 0.0438 ±0.01 0.0763 ±0.04 0.0019 ±0.00 0.0050 ±0.00 0.0056 ±0.00 0.0619 ±0.02
Table 6
group Dose (mg / kg) Route WBC (× 10 6 cells)
NEU LYM MON EOS BAS LUC
VC 0 PO 0.0269 ± 0.01 0.0313 ± 0.01 0.0000 ± 0.00 0.0075 ± 0.01 0.0063 ± 0.00 0.0525 ± 0.04
IC 0 PO 0.0419 ± 0.01 0.1056 ± 0.05 0.0038 ± 0.00 1.1388 ± 0.27 0.0063 ± 0.00 0.1013 ± 0.02
Dexa. One PO 0.0169 ± 0.00 0.0313 ± 0.01 0.0013 ± 0.00 0.2725 ± 0.15 0.0056 ± 0.00 0.0775 ± 0.03
FK-3000 4 PO 0.0369 ± 0.02 0.1850 ± 0.15 0.0038 ± 0.00 0.7669 ± 0.27 0.0063 ± 0.00 0.0856 ± 0.04
FK-3000 10 PO 0.0344 ± 0.01 0.0881 ± 0.06 0.0038 ± 0.00 0.6431 ± 0.35 0.0056 ± 0.00 0.1625 ± 0.11
FK-3000 40 PO 0.0438 ± 0.01 0.0763 ± 0.04 0.0019 ± 0.00 0.0050 ± 0.00 0.0056 ± 0.00 0.0619 ± 0.02
(3) 폐 및 비장 조직의 관찰(3) observation of lung and spleen tissue
비장과 집게로 고정한 좌측 폐를 떼어내어 10% 포르말린용액에 하루 동안 고정 후, 조직 프로세서 (tissue processor)를 이용하여 파라핀 침투시킨 조직을 마이크로톰 (microtome)을 이용하여 3-4 ㎛의 두께로 잘라 조직 절편을 만들었다. 만들어진 슬라이드를 자일렌 (Xylene)과 에탄올 (ethanol)에 차례로 담가서 탈 파라핀 시킨 후, 헤마톡실린 (Haematoxylin)과 에오신 (Eosin) 염색하고 발삼 (balsam)으로 커버슬라이드를 덮어 현미경으로 염증세포의 침윤, 점액 마개형성 및 고블릿 (goblet) 세포증식 정도를 관찰하였다.After removing the left lung fixed with the spleen and forceps and fixing it in 10% formalin solution for one day, the tissues in which paraffin was infiltrated using a tissue processor were cut into a thickness of 3-4 μm using a microtome. Made an intercept The slides were then immersed in xylene and ethanol, deparaffinized, and then stained with haematoxylin and eosin, covered with slides and covered with balsam to infiltrate inflammatory cells under a microscope. Mucus plugging and goblet cell proliferation were observed.
도 7 내지 도 9에서 나타난 바와 같이, FK-3000은 천식모델동물에서 농도의존적으로 염증세포 침윤 (inflammatory cell infiltration), 점막 (mucous plug), 점막세포 비후 (mucosal cell hyperplasia) 등을 억제하였다. As shown in FIGS. 7 to 9, FK-3000 inhibited inflammatory cell infiltration, mucous plug, mucosal cell hyperplasia, etc. in asthma model animals in a concentration-dependent manner.
도 8에서 나타난 바와 같이, vehicle 대조군 그룹 (A)에서 폐내 상피 및 혈관 (V)은 점액 분비 (M) 및 염증세포 침윤 (arrow)을 각각 보이지 않았다. 염증세포 침윤은 일차적으로 유도된 그룹 (B) 및 양성 대조군 그룹 (C)에서 혈관 및 모세기관지 (bronchioles) 주위에서 관찰되어 졌다. 세포 잔해를 포함한 일반적인 점액 분비는 유도된 그룹의 모세기관지에서 발견되어 졌다. 고블릿 세포 (Goblet cell, Gc) 과다형성 및 점액 분비는 또한 유도된 세포 및 양성 대조군 그룹에서 보여졌다. 고블릿 세포 과다형성, 점액 분비 및 염증세포 침윤은 양성 대조군보다 유도된 그룹에서 더욱 눈에 띄었다.As shown in FIG. 8, in the vehicle control group (A), pulmonary epithelium and blood vessels (V) did not show mucus secretion (M) and inflammatory cell infiltration (arrow), respectively. Inflammatory cell infiltration was observed around blood vessels and bronchioles in primarily induced group (B) and positive control group (C). Normal mucus secretions, including cell debris, have been found in capillaries of the induced groups. Goblet cell (Gc) hyperplasia and mucus secretion were also seen in the induced cells and positive control groups. Goblet cell hyperplasia, mucus secretion and inflammatory cell infiltration were more noticeable in the induced group than in the positive control.
도 9는 FK-3000의 복용량 농도에 따른, 모세기관지 상피세포 (bronchioles epithelium, Gc), 염증세포 침윤 (arrows), 점액 분비 (M)의 변화를 나타낸 것으로, 고블릿 세포의 과다형성, 염증세포 침윤 및 점액 분비의 정도는 복용량 농도에 따라 감소되었다. 유도된 그룹 (A)와 비교시, 모든 처리된 그룹은 더 낮은 고블릿 세포 과다형성, 감소된 염증세포 침윤 및 감소된 점액 분비를 보였다. 특히, 고용량 복용 그룹 (D)의 변화는 저용량 (B) 및 평균 (C) 복용량 그룹의 변화에 비해서 더욱 눈에 띄는 변화를 보였다. Figure 9 shows the changes in the capillary bronchial epithelium (bronchioles epithelium, Gc), inflammatory cell infiltration (arrows), mucus secretion (M) according to the dose concentration of FK-3000, hyperplasia of goblet cells, inflammatory cell infiltration And the degree of mucus secretion decreased with dose concentration. Compared to induced group (A), all treated groups showed lower goblet cell hyperplasia, reduced inflammatory cell infiltration and decreased mucus secretion. In particular, changes in the high dose group (D) showed more noticeable changes than changes in the low dose (B) and average (C) dose groups.
(4) 실험이 종료되면, 잔여동물을 포함한 생존동물은 CO2 가스로 안락사시키며, 사망동물에 대해서는 따로 부검을 실시하지 않고 사체처리하였다. (4) At the end of the experiment, surviving animals including residual animals were euthanized with CO 2 gas, and dead animals were carcassed without additional necropsy.
2-7. 통계학적 방법 2-7. Statistical method
얻어진 수치 자료에 대하여서는 평균과 표준편차를 표시하였다. 얻어진 자료에 대한 통계분석은 다중비교검정법을 실시하였다. 이러한 분석은 통계프로그램인 SAS (Version 9.2, Cary, NC, USA)를 이용하여 실시하며, 검정의 위험율은 5% 및 1%로 정하였다.For the numerical data obtained, the mean and standard deviation are indicated. Statistical analysis on the obtained data was conducted by multiple comparison test. This analysis was conducted using the statistical program SAS (Version 9.2, Cary, NC, USA), and the risks of the tests were set at 5% and 1%.

Claims (4)

  1. 6,7-디-O-아세틸시노코큘린 (6,7-di-O-acetylsinococuline) 또는 이의 약리학적으로 허용가능한 염을 유효성분으로 포함하는 천식 예방 또는 치료용 조성물.6,7-di-O-acetylsinococulin (6,7-di-O-acetylsinococuline) or a pharmacologically acceptable salt thereof as an active ingredient comprising a composition for preventing or treating asthma.
  2. 제 1항에 있어서, 상기 6,7-디-O-아세틸시노코큘린 또는 이의 약리학적으로 허용가능한 염은 염증 세포인 림프구 (lymphocytes), 중성구 (neutrophils) 및 호산구 (eosinophils)를 억제하는, 천식 예방 또는 치료용 조성물.The asthma according to claim 1, wherein the 6,7-di-O-acetylcynococulin or a pharmacologically acceptable salt thereof is asthmatic, which inhibits lymphocytes, neutrophils and eosinophils, which are inflammatory cells. Prophylactic or therapeutic composition.
  3. 6,7-디-O-아세틸시노코큘린 (6,7-di-O-acetylsinococuline) 또는 이의 약리학적으로 허용가능한 염을 유효성분으로 포함하는 천식 예방 또는 치료용 약학 조성물.A 6,7-di-O-acetylsinococuline (6,7-di-O-acetylsinococuline) or a pharmacologically acceptable salt thereof as an active ingredient comprising a pharmaceutical composition for preventing or treating asthma.
  4. 6,7-디-O-아세틸시노코큘린 (6,7-di-O-acetylsinococuline) 또는 이의 약리학적으로 허용가능한 염을 유효성분으로 포함하는 천식 예방 또는 개선용 건강식품 조성물.6,7-di-O-acetylsinococuline (6,7-di-O-acetylsinococuline) or a pharmacologically acceptable salt thereof as an active ingredient comprising a health food composition for preventing or improving asthma.
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