KR20130120849A - A pharmaceutical compositon containing skullcapflavone ii or pharmaceutically acceptable salt thereof for the prevention or treatment of asthma - Google Patents

Abstract

The present invention relates to a pharmaceutical composition containing skullcapflavone II or a pharmaceutically acceptable salt thereof as an active ingredient for preventing or treating asthma. Specifically, skullcapflavone II compounds suppress body weight reduction caused by asthma, the generation of IgE, ovoalbumin-specific IgE, interleukin-4, 5, and 13, and MMP-9 in serum and a bronchoalveolar lavage fluid, alleviate airway hypersensitivity responses, and prevent collagen precipitation around bronchial tubes, inflammatory cell infiltration into bronchial tubes, and mucous secretion in bronchial tubes with low hepatotoxicity. In addition, the skullcapflavone II compounds reduce the concentration of TGF-beta 1, increase the expression of Smad7, and suppress the concentration of pSmad2/3, thereby being used as an active ingredient of a pharmaceutical composition for preventing or treating bronchial asthma. [Reference numerals] (AA) Gold E3-PR-13 (skullcapflavone ii);(BB) Document: Chem. Pharm. Bull. (1980), 28(3), 708-716;(CC) C (document) (DMSO-d_s)

Description

A pharmaceutical compositon containing Skullcapflavone II or pharmaceutically acceptable salt etc. for the prevention or treatment of asthma}

The present invention relates to a composition for the prevention or treatment of asthma containing Skullcapflavone II or a pharmaceutically acceptable salt thereof as an active ingredient.

Allergic diseases whose incidence is increasing worldwide include anaphylaxis, allergic rhinitis, asthma, atopic dermatitis, and urticaria.

Among allergic diseases, asthma is estimated to be suffering from about 3 million people in Korea, chronic inflammatory respiratory disease characterized by coughing, wheezing wheezing, and shortness of breath or chest. Patients are rapidly increasing due to deepening and westernization of eating habits. Asthma is the most common disease in developed countries such as the United States and the United Kingdom. 20-30% of the population is asthmatic.In Korea, 16% of elementary school children and 5% of adults are asthma patients and more than 4 million patients It is estimated to be seen in all age groups from infants to the elderly, and it is a common disease that affects 10% of the entire population. It has been found that atopic constitution that causes allergy in the family is fundamentally responsible for the development of the asthma, and airway hypersensitivity, eosinophilic inflammation of the airways, and hyperactivity of the Th2 immune response have been reported as the primary causes of asthma. Asthma makes it difficult to breathe, severe coughing, wheezing (wheezing), and WHO's Special Report of 2000 shows that 150 million patients worldwide suffer from asthma. 180,000 people die of bronchial asthma. In addition, the prevalence and severity of the disease continues to increase, and the health and social costs of asthma are greater than the combined costs of pulmonary tuberculosis and AIDS, according to the WHO Special Report.

In Korea, the prevalence of childhood asthma, which was only 3-4% in the early 1980s, more than doubled. According to the International Study of Asthma and Allergy in Childhood (ISAAC), published in 1998, the prevalence of childhood asthma in Korea was 13.3% for 6-7 years and 7.7% for 13-14 years. As a whole, 10 out of 100 people have asthma, and 50% of them have asthma for life. The severity of the bigger problem is that not only the prevalence of the disease but also the severity of the disease is increasing.

In general, asthma is recognized as a chronic inflammatory disease caused by inflammatory cells proliferating, differentiating and activating by interleukin-4, 5, and 13 produced by TH2 type immune cells, moving to and invading the airways and surrounding tissues (Elias JA). , et al., J. Clin . Invest. , 111, pp 291-297, 2003). In this case, a strong bronchial contraction action by inflammatory cells such as activated eosinophils, mast cells, and alveolar macrophages secretes various inflammatory mediators (cysteine leukotriene, prostaglandin, etc.) (Maggi E., Immunotechnology, 3). , pp233-244, 1998; Pawankar R., Curr. Opin.Allergy Clin.Immunol., 1, pp3-6, 2001; Barnes PJ, et al., Pharmacol Rev., 50, pp515-596, 1998).

Interleukin-4 plays the most important role in inducing homozygous for IgE in B lymphocytes (Barrett NA, Austen KF. Innate cells and T helper 2 cell immunity in airway inflammation. Immunity. 2009; 31: 425-37). Interleukin-13 can regulate the aggregation of eosinophils into the airways through various effects on epithelial and smooth muscle cells (Wills-Karp M. Interleukin-13 in asthma pathogenesis. Immunol Rev. 2004; 202: 175-90). Interleukin-5 has been associated with inducing eosinophilic inflammation and airway hypersensitivity in asthma. In addition, there is a close relationship between eosinophils, interleukin-4, 5 and 13 in the mechanisms regulating airway hypersensitivity in asthmatic lungs (Cho JY, Miller M, Baek KJ, Han JW, Nayar J, Lee SY, et. al.Inhibition of airway remodeling in IL-5-deficient mice.J Clin Invest. 2004; 113: 551-60). IgE-mediated mast cell activity contributes to airway hypersensitivity by producing many inflammatory mediators and cytokines. Therefore, the production of interleukin-4, 5, 13, and immunoglobulin E, which are involved in inflammatory cell activation, and the action of cysteine leukotriene biosynthesis secreted from inflammatory cells such as eosinophils, are major causes of inflammation and allergic reactions and asthma. As a result, many studies are being conducted to develop drugs for inhibiting their production.

TGF-β1 is a multifunctional cytokine known to be involved in the pathophysiology of asthma. Its expression is increased in human chronic lung diseases such as asthma (Chu HW, Trudeau JB, Balzar S, Wenzel SE.Peripheral blood and airway tissue expression of transforming growth factor beta by neutrophils in asthmatic subjects and normal control subjects.J Allergy Clin Immunol. 2000; 106: 1115-23), increased concentrations correlate with the degree of peribronchial fibrosis and the severity of the disease (Minshall EM, Leung DY, Martin RJ, Song YL, Cameron L, Ernst P, et al. Eosinophil-associated TGF-beta1 mRNA expression and airways fibrosis in bronchial asthma.Am J Respir Cell Mol Biol. 1997; 17: 326-33). TGF-β1 signaling pathways usually involve the activation of TGF-β1 type I and II receptors, subsequent intracellular influencers, phosphorylation of Smad2 and Smad3 and their delivery to the nucleus, which influences genes in the nucleus. You can adjust the transcription of. Another related factor, Smad7, is an intracellular inhibitor that is rapidly induced by TGF-β family members and provides a negative feedback loop (Bartram U, Speer CP. The role of transforming growth factor beta in lung development and disease. Chest. 2004; 125: 754-65). Blocking TGF-β / Smad signaling in mature T cells by expression of Smad7 enhances airway inflammation and airway response, suggesting that TGF-β regulation of T cells is important for the negative regulation of the inflammatory response. (Nakao A, Miike S, Hatano M, Okumura K, Tokuhisa T, Ra C, et al. Blockade of transforming growth factor beta / Smad signaling in T cells by overexpression of Smad7 enhances antigen-induced airway inflammation and airway reactivity.J Exp Med. 2000; 192: 151-8). In addition, TGF-β1 is a strong fibrosis factor that induces myofibroblast activity and the synthesis of extracellular matrix, and various recent studies have shown a relationship between TGF-β1 and airway remodeling, a structural change in the airways due to repeated inflammatory responses. (Bartram U, Speer CP.The role of transforming growth factor beta in lung development and disease.Chest. 2004; 125: 754-65, Kim JH, Jain D, Tliba O, Yang B, Jester WF Jr , Panettieri RA Jr., et al. TGF-beta potentiates airway smooth muscle responsiveness to bradykinin.Am J Physiol LungCell Mol Physiol 2005; 289: L511-20). Airway remodeling is characterized by bronchial epithelial thickening, hyperplasia of mucous secretory cells, and subepithelial airway fibrosis (Jim Jung-yeon, the latest findings on the proliferation and synthesis of airway smooth muscle in asthma, Korean Journal of Pediatrics Vol. 6, 2005). Therefore, inhibiting the production of TGF-β1 or blocking its bioactivity to reduce collagen production is considered one of the powerful methods for the treatment of airway remodeling (Minshall EM, Leung DY, Martin RJ, Song YL, Cameron L). , Ernst P, et al. Eosinophil-associated TGF-beta1 mRNA expression and airways fibrosis in bronchial asthma.Am J Respir Cell Mol Biol. 1997; 17: 326-33, Yamaguchi M, Niimi A, Matsumoto H, Ueda T, Takemura M, Matsuoka H, et al. Sputum levels of transforming growth factor-beta1 in asthma: relation to clinical and computed tomography findings.J Investig Allergol Clin Immunol. 2008; 18: 202-6.).

Currently, various asthma treatments are commercially available, but many of them require attention due to side effects. Inhaled corticosteroids are still the most important treatments for asthma and have excellent effects, but long-term use may cause adrenal suppression, reduced bone density, growth disorders, and eye and skin complications in proportion to dose and duration of use. Known. It has also been reported that corticosteroids may rather increase the synthesis of collagen (Warshmana GS, et al., Am J Physiol 274, 499-507, 1998). Thus, in spite of years of corticosteroid treatment for chronic persistent asthma patients, asthma patients rarely normalize airway hyperresponsiveness. Long-term administration of beta-2 agonists is not known to inhibit airway remodeling (Jeffery PK, et al., Am Rev Respir Dis 145: 890-0, 1992), salmeterol and fort It has also been warned that long-acting beta-2 agonists, such as formeterol, can prevent asthma attacks and kill asthma patients. While various side effects have been reported, they are being prescribed under the judgment that the effect of relieving asthma symptoms is greater than the risk of side effects. However, as a result of measuring the growth rate of children who are sensitive to side effects, the growth rate of asthma patients who took oral leukotriene antagonist montelukast was up to 1cm per year than that of inhaled corticosteroids (Garcia Garcia ML, et al., Pediatrics 116 (2): 360-9, 2005). If asthma is not controlled during the growing season, growth of the entire body as well as the lungs can be inhibited, so it is essential to maintain normal lung function with steady treatment. However, in ongoing treatment, it is important to use safe medications and to manage the inflammation of the airways more than anything else, so careful consideration of side effects is necessary, as well as the effects of relieving asthma when choosing a treatment. Although leukotriene antagonists are known to have low frequency of side effects, they are newly used for the prevention and continuous treatment of asthma, but the effect of asthma relief is weak compared to other drugs, and only one third of the patients are remarkable. Thus, there is a need for the development of novel asthma therapies that are nontoxic, safe and capable of preventing drug resistance.

Conventionally, Patent Document 1 discloses health foods for the prevention and treatment of allergic rhinitis and colds, including Yu-Geun Skin, Dermis, Licorice, Schisandra chinensis, Ui-Jin, Eoseongcho, Astragalus, Macmun-dong, Sambaekcho, Gyeji, Golden, Vacation and Baekchul as active ingredients. The manufacturing method is disclosed.

In addition, Patent Literature 2, Chinese herbal medicine composition consisting of sujijihwang, mountain chemicals, cornus milk, baekbokyeong, mokdanpi, taxa, Schisandra chinensis, astronomical dong, mackmundong, femo, fruit fruit, pedestrians, half, citrus, gilyeong, gold, yellow lotus, licorice as an active ingredient A composition for treating asthma maintenance is disclosed.

Furthermore, Patent Literature 3 discloses a cosmetic composition containing a golden and yellow lotus extract, which has a skin moisturizing and antibacterial effect, as an active ingredient.

However, no invention has yet been reported on the use of Skullcapflavone II isolated from gold for treating asthma.

Golden (Scutellariae Radix) is the dried root of Scutellaria baicalensis GEORGE, a perennial herb that belongs to Lamiaceae. It is distributed in Korea, China, Mongolia, and eastern Siberia. It grows in the grassland of mountains, and is 20 ~ 60 cm in height. Several generations come out, grow up in hair, and have branches. Leaves are opposite to each other and have a straight edge. Flowers bloom in July-August, turn purple, and run to the side with gunshot flowers. Corollas curled at the bottom, standing straight, tubular, 1.5-2.5 cm long, lip shaped, and calyxes are bell-shaped. Two of the four surgeries are long, one pistil, and the ovary is deeply divided into four. Fruits are fruiting in September, rounded in calyx, cones are cone-shaped and fleshy yellow. In oriental medicine, the roots have been used for fever, diuresis, branches, and diarrhea. Gold has been treated as an important herb widely used in the treatment of various diseases including viral hepatitis, pancreatitis, and more recently, tumors and the like.

Therefore, the present inventors are interested in herbal medicines in consideration of in vivo side effects and safety, and while researching the development of asthma medicines, the main clinical symptoms of asthma in the asthma-induced mouse model of skullcapflavone II isolated from gold Reduction of phosphorus hypersensitivity reactions, inhibition of the production of Th2 cytokines Interleukin-4, 5, 13 and MMP-9 in the lung, inhibition of IgE and egg white albumin-specific IgE production in the lungs, low hepatotoxicity, inhibition of bronchial inflammatory cell infiltration, intrabronchial Inhibiting mucus secretion, inhibiting peribronchial collagen erosion, decreasing TGF-β1 concentration, increasing the expression of Smad7 and inhibiting the concentration of pSmad2 / 3 (phosphorylated Smad2 / 3). The present invention has been completed by revealing that it can be used for prevention or treatment.

Patent Document 1: Republic of Korea Patent Publication No. 2005-0010942 Patent Document 2: Republic of Korea Patent Publication No. 2006-0061855 Patent Document 3: Republic of Korea Patent No. 2010-0723370

An object of the present invention is to provide a new pharmaceutical composition for the prevention or treatment of asthma, which can be safely used in the treatment of asthma without side effects in vivo. Another object of the present invention to provide a composition for health food for the prevention or improvement of asthma.

In order to achieve the above object, the present invention provides a pharmaceutical composition for the prevention or treatment of asthma containing the skull cap flavone II compound represented by the following formula (1) or a pharmaceutically acceptable salt thereof as an active ingredient:

[Formula 1]

.

.

In another aspect, the present invention provides a composition for preventing or improving asthma containing a skull cap flavone II compound or a pharmaceutically acceptable salt thereof as an active ingredient.

The present invention newly suggests that a previously known skullcapflavone II compound can be used for the prevention or treatment of asthma, and the skullcapflavone II of the present invention has an excellent effect on inhibiting airway hypersensitivity in the model of asthma, and Th2 in the lung. Inhibition of the production of the cytokines interleukin-4, 5, 13 and MMP-9, Inhibition of blood IgE and egg white albumin-specific IgE production, Low hepatotoxic effect, Inhibition of inflammatory cell invasion in bronchus, Bronchial Inhibition of intramucosal secretion, inhibition of peribronchial collagen erosion, reduction of TGF-β1 concentration, and increased expression of Smad7 and inhibition of pSmad2 / 3 were identified. As a result, the skull cap flavone II of the present invention can be usefully used for the prevention or treatment of bronchial asthma, such as when the toxicity is low and steroid resistance or when long-term use of asthma treatment is required.

1 is a view showing the nuclear magnetic resonance spectroscopy results of the skull cap flavone II compound according to Preparation Example 1 of the present invention.
Figure 2 is a graph showing the weight loss inhibitory effect of Skullcapflavone II according to the present invention (type of test group: normal control group (NC), asthma induction group (OVA), dexamethasone administered asthma induction group (Dex), montelukast Asthma induction group (Monte) administered with and asthma induction group (SII10, SII30) administered with Skullcapflavone II, +: Statistical significance was recognized as compared to the normal control group (p <0.05), *: Statistical significance was recognized as compared to the asthma induction group (OVA) (p <0.05).
Figure 3 is measured by changing the concentration of methacholine Penh (Penh), the degree of airway resistance in order to determine whether airway hypersensitivity for the experimental group of the same conditions as each experimental group used in Example 1 according to the present invention A graph showing the results.
Figure 4 is a graph showing the number of inflammatory cells in bronchoalveolar lavage fluid for the experimental group under the same conditions as each experimental group used in Example 1 according to the present invention.
Figure 5 is a graph showing the concentration of IgE and egg white albumin specific IgE in serum for the experimental group of the same conditions as each experimental group used in Example 1 according to the present invention.
Figure 6 is a graph showing the activity of the liver enzymes ALT, AST, in order to measure liver toxicity for the experimental group under the same conditions as each experimental group used in Example 1 according to the present invention (**: dexamethasone is administered Statistical significance was observed compared to asthma induction group (Dex) (p <0.05).
Figure 7 shows the expression (phase) of interleukin-4, 13, MMP-9 and actin in bronchial alveolar lavage fluid (interphase) and interleukin-4, 13 and for experimental groups under the same conditions as each experimental group used in Example 1 according to the present invention. It is a graph which shows the relative density (bottom) of MMP-9.
8 shows the concentrations of interleukin-4 (upper), interleukin-5 (middle) and interleukin-13 (lower) in bronchoalveolar lavage fluid for experimental groups under the same conditions as each experimental group used in Example 1 according to the present invention. The graph shown.
9 is a graph showing inflammatory cell infiltration pictures (top) and inflammation index (bottom) around bronchioles and blood vessels for the experimental group under the same conditions as each experimental group used in Example 1 according to the present invention.
Figure 10 is a picture (top) and bronchus of differentiated goblet cells discriminated through PAS staining to measure the degree of proliferation of goblet cells in the bronchus for each experimental group of the same conditions used in Example 1 according to the present invention. It is a graph showing the percentage of goblet cells in the epithelial cells (bottom).
FIG. 11 is a cross-sectional photograph (top) and peribronchial fibrosis of a trichrome stained lung, in order to measure the effect of inhibiting peribronchial collagen erosion for the experimental group under the same conditions as each experimental group used in Example 1 according to the present invention. It is a graph (bottom) which shows the area of the stained part as a degree.
Figure 12 shows the expression (top) of TGF-β1 and actin in the lung tissue, the relative density (medium) of TGF-β1 to actin for the experimental group under the same conditions as each experimental group used in Example 1 according to the present invention and It is a graph showing the concentration (bottom) of TGF-β1 in bronchoalveolar lavage fluid.
Figure 13 shows the expression (phase) of Smad7, pSmad2 / 3 and actin in the lung tissue (Stop7), Smad7 (medium) and pSmad2 / 3 for the experimental group in the same conditions as each experimental group used in Example 1 according to the present invention It is a graph which shows the relative density of (below).

The terms used in the present invention are explained.

As used herein, the term "prevention" means any action that inhibits asthma or delays progression by administration of a composition of the present invention.

As used herein, the terms "treatment" and "improvement" refer to all actions in which the symptoms of asthma are improved or beneficially altered by administration of a composition of the present invention.

The term "administering" as used herein is meant to provide any desired composition of the invention to an individual by any suitable method.

As used herein, the term "individual" refers to a mammal, including humans, monkeys, dogs, and the like, having a disease that can improve the symptoms of asthma by administering the composition of the present invention.

Hereinafter, the present invention will be described in detail.

The present invention provides a pharmaceutical composition for preventing or treating asthma, which contains a skullcapflavone II compound represented by Formula 1 or a pharmaceutically acceptable salt thereof as an active ingredient:

[Formula 1]

.

.

It is preferable to use the skull cap flavone II compound isolated from gold, but not chemically synthesized. In addition, the skull cap flavone Ⅱ may be used separately from the roots, stems, leaves, flowers, fruits and the like of gold, but preferably used separated from the roots of gold.

The present invention includes not only the compound represented by Chemical Formula 1 or a pharmaceutically acceptable salt thereof, but also all possible solvates and hydrates that can be prepared therefrom.

The compound of Formula 1 of the present invention may be used in the form of a pharmaceutically acceptable salt, and as the salt, an acid addition salt formed by a pharmaceutically acceptable free acid is useful. Acid addition salts include those derived from inorganic acids such as hydrochloric acid, nitric acid, phosphoric acid, sulfuric acid, hydrobromic acid, hydroiodic acid, nitrous acid or phosphorous acid, and aliphatic mono- and dicarboxylates, phenyl-substituted alkanoates, hydroxyalkanoates, Dioleate, aromatic acid, aliphatic and aromatic sulfonic acids. Such pharmaceutically innocuous salts include, but are not limited to, sulfate, pyrosulfate, bisulfate, sulfite, bisulfite, nitrate, phosphate, monohydrogenphosphate, dihydrogenphosphate, metaphosphate, pyrophosphate chloride, bromide, Butyrate, caprate, heptanoate, propiolate, oxalate, malonate, succinate, succinate, maleic anhydride, maleic anhydride, , Sebacate, fumarate, maleate, butyne-1,4-dioate, hexane-1,6-dioate, benzoate, chlorobenzoate, methylbenzoate, dinitrobenzoate, hydroxybenzoate, Methoxybenzoate, phthalate, terephthalate, benzene sulfonate, toluene sulfonate, chlorobenzene sulfide Propyl sulphonate, naphthalene-1-yne, xylenesulfonate, phenylsulfate, phenylbutyrate, citrate, lactate,? -Hydroxybutyrate, glycolate, maleate, Sulfonate, naphthalene-2-sulfonate or mandelate.

The acid addition salts according to the present invention are dissolved in conventional methods, for example, by dissolving the compound of the present invention in an excess of an aqueous solution of an acid, which salt is subjected to a water miscible organic solvent such as methanol, ethanol, acetone or acetonitrile. Can be prepared by precipitation.

Equivalent amounts of the compounds of formula (1) and acids or alcohols in water may be heated and then the mixture is evaporated to dryness, or the precipitated salts may be prepared by suction filtration.

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 an alkaline earth metal hydroxide solution, filtering the insoluble compound salt, and evaporating and drying the filtrate. At this time, it is preferable for the metal salt to produce sodium, potassium or calcium salt. Corresponding silver salts are also obtained by reacting alkali or alkaline earth metal salts with a suitable negative salt (eg, silver nitrate).

The skullcapflavone II compound of formula 1 according to the present invention can be synthesized by separation from natural products or by methods commonly used in the field of organic synthesis. Preferably it can be used separately from natural products. Gold may be used as the natural product, and one isolated from all parts of the roots, stems, leaves, and fruits of gold may be used. Preferably, one isolated from the golden root can be used. For example, in the case of separating the skull capflavone II of Formula 1 according to the present invention from gold can be separated by the following method.

3 kg of golden root (golden base) samples were extracted four times with methanol to obtain 250 g of total extract. This was suspended in 2 L of water and hexane (n-hexane) and ethyl acetate (ethyl acetate) were added three times, 1 L each, to obtain 13.8 g of hexane fraction and 167.0 g of ethyl acetate fraction, respectively. 160 g of ethyl acetate fraction was subjected to silica gel column chromatography (step chromatography having a concentration gradient of 30/1 to 5/1 for CHCl ₃ / MeOH), and the total fraction was divided into 10 fractions. E3-P, 7.5 g) was again subjected to silica gel column chromatography (hexane / CHCl ₃ / MeOH, 3/1 / 0.1) to obtain 14 small fractions, of which the 13th fraction (E3-PR-13) was obtained. The final fractions were purified purely (1.51 g) by recrystallization from methanol. The separated material was subjected to structural analysis using NMR (PMR, CMR, HSQC), Referring to the previous paper (Chem. Pharm. Bull (1980), 28 (3), 708-716), it was identified as Skullcapflavone II (see FIG. 1).

The therapeutically effective amount of the composition of the present invention may vary depending on several factors, such as the method of administration, the site of interest, the condition of the patient, and the like. Therefore, when used in humans, the dosage should be determined in an appropriate amount in consideration of both safety and efficiency. It is also possible to estimate the amount used in humans from an effective amount determined through animal testing. Such matters to consider in determining the effective amount may be referred to in the literature (Hardman and Limbird, eds., Goodman and Gilman's The Pharmacological Basis of Therapeutics, 10th ed. (2001), Pergamon Press; and EW Martin ed., Remington's Pharmaceutical Sciences, 18th ed. (1990), Mack Publishing Co.).

The compositions of the present invention may also include carriers, diluents, excipients or combinations of two or more commonly used in biological agents. Pharmaceutically acceptable carriers are not particularly limited so long as they are suitable for in vivo delivery of the composition, for example, compounds described in the Merck Index, 13th ed., Merck & Co. Inc., saline, sterile water, Ringer's solution, buffered saline solution, dextrose solution, maltodextrin solution, glycerol, ethanol and one or more of these components can be mixed and used, and other conventional additives such as antioxidants, buffers, bacteriostatic agents can be added as necessary. In addition, diluents, dispersants, surfactants, binders and lubricants may be additionally added to formulate into main dosage forms, pills, capsules, granules or tablets such as aqueous solutions, suspensions, emulsions and the like. Furthermore, the method described in the literature (e.g., Remington's Pharmaceutical Science (Mack Publishing Company, Easton PA, 18th, 1990)) may be suitably formulated according to each disease or component by appropriate methods in the art. have.

The composition of the present invention may further contain one or more active ingredients exhibiting the same or similar functions. The composition of the present invention may comprise 0.0001 to 10% by weight of the compound, preferably 0.001 to 1% by weight, based on the total weight of the composition.

The compositions of the present invention may be administered parenterally (eg, intravenously, subcutaneously, intraperitoneally or topically) or orally, depending on the desired method, and the dosage may be based on the weight, age, sex, health status, The range varies depending on the diet, the time of administration, the method of administration, the rate of excretion and the severity of the disease. The daily dose of the composition according to the present invention is 0.0001-10 mg / ml, preferably 0.0001-5 mg / ml, and more preferably, divided into once or several times a day.

The pharmaceutical composition containing Skullcapflavone II of Formula 1 according to the present invention can suppress weight loss caused by asthma. Referring to Example 1 of the present invention for confirming the weight loss inhibitory effect of the skull cap flavone II asthma-induced mice, the weight of the asthma induction group orally administered the skull cap flavone II of Formula 1 is 10 mg / kg It was confirmed that at the dose administered in the group 19.66 ± 0.30 g and 30 mg / kg dose to 20.14 ± 0.67 g in the group showing a weight loss inhibitory effect. This is compared with the dexamethasone (Dex) group, a synthetic steroid used mainly for the treatment of asthma and bronchial inflammation, and the montelukast (Monte) group, which is commonly used as an asthma treatment group, and showed a weight recovery effect close to the normal control group. , To the extent equivalent to inhibiting weight loss due to asthma (see Table 1 and FIG. 2).

In addition, the pharmaceutical composition according to the present invention can inhibit the airway hypersensitivity caused by asthma. Referring to Example 2 according to the present invention, in the normal control group, the pen value gradually increased with increasing methacholine concentration, but it was confirmed that the Pehn level significantly increased in the asthma induction group. On the other hand, in the dexamethasone and montelukast-administered group and the skullcapflavone II-administered group of the present invention, regardless of the methacholine concentration it can be seen that significantly reduced than asthma-induced group. This difference is more pronounced when inhaled high concentration of methacholine than low concentration of methacholine. From this, the pharmaceutical composition according to the present invention can be usefully used for the treatment of asthma by inhibiting airway hypersensitivity (see Table 2 and Figure 3).

Furthermore, the pharmaceutical composition according to the present invention can inhibit IgE and egg white albumin specific IgE concentrations in serum and bronchial alveolar lavage fluid correlated with the severity of asthma. Referring to Example 3 according to the present invention, it can be seen that the serum IgE antibody concentration is significantly reduced in the scullcapflavone II administration group of the present invention compared to the asthma induction group. In addition, it was confirmed that the egg white albumin-specific IgE concentration in serum was significantly decreased in the skullcapflavone II administration group of the present invention compared to the asthma induction group. From this, the pharmaceutical composition according to the present invention can be usefully used for the treatment of asthma by inhibiting the concentration of IgE and egg white albumin specific IgE (see Table 3, Table 4 and Figure 4).

In addition, the pharmaceutical composition according to the present invention can reduce the number of inflammatory cells associated with asthma in bronchoalveolar lavage fluid. Referring to Example 4 according to the present invention, it was confirmed that the total number of inflammatory cells was significantly reduced in all drug administration compared to the asthma induction group. In particular, the number of eosinophils known to play an important role in the development of asthma is as a cell that invades tissues and is mainly involved in cellular mediated immunity, spreads throughout the cells, spleen and respiratory organs, and contains inflammatory proteins. In contrast, it can be seen that all drug administration groups significantly decreased. From this, the pharmaceutical composition according to the present invention can be usefully used for treating asthma by reducing the number of inflammatory cells in the bronchus (see Table 5 and FIG. 5).

Furthermore, the pharmaceutical composition according to the present invention can minimize liver toxicity. Referring to Example 5 according to the present invention, the concentration of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in serum, which is known as an indicator of hepatocellular damage, is higher than that of asthma induction group. It was significantly increased in the administration group. On the other hand, in the montelukast-administered group and the skullcapflavone II-administered group of the present invention, significantly lower concentration values were observed than the dexamethasone-administered group. From this, the pharmaceutical composition according to the present invention can be usefully used for long-term asthma treatment due to low toxicity to the liver (see Table 6 and FIG. 6).

In addition, the pharmaceutical composition according to the present invention can inhibit the production of Th2 cytokine interleukin-4, 5, 13 and MMP-9 associated with asthma in bronchoalveolar lavage fluid. Referring to Example 6 according to the present invention, it was confirmed that the expression of interleukin 4, 5, 13 and MMP-9 is reduced in the scullcapflavone II administration group of the present invention compared to the asthma induction group. From this, the pharmaceutical composition according to the present invention can be usefully used for the treatment of asthma by inhibiting the production of Th2 cytokine interleukin-4, 5, 13 and MMP-9 (see Table 7, Table 8, Figures 7 and 8). .

Furthermore, the pharmaceutical composition according to the present invention, after antigen induction, invasion of inflammatory cells consisting of eosinophils, neutrophils and macrophages observed in the bronchus (Oh SW, et al., J, Immunol 2002; 168: 1992-2000) Can be reduced. Referring to Example 7, according to the present invention, the inflammation index around the bronchioles was found to be significantly reduced in all drug administration compared to the asthma induction group. In addition, the inflammation index around the blood vessels except for the montelukast-administered group was found to be significantly reduced compared to the asthma induction group. From this, the pharmaceutical composition according to the present invention can be usefully used for the treatment of asthma by inhibiting the infiltration of inflammatory cells (see Table 9 and Figure 9).

In addition, the pharmaceutical composition according to the present invention can inhibit the proliferation of goblet cells, which are mucin-secreting cells that cause the accumulation of mucus in the bronchus, which is a pathological feature of asthma. Referring to Example 8 according to the present invention, the proportion of goblet cells in the bronchiolepital epithelial cells was confirmed to increase in the asthma induction group compared to the normal control group. On the other hand, all drug administration groups showed a significant decrease. From this, the pharmaceutical composition according to the present invention can be usefully used for the treatment of asthma by inhibiting the proliferation of goblet cells (see Table 10 and Figure 10).

Furthermore, the pharmaceutical compositions according to the present invention can inhibit the erosion of collagen associated with subepithelial fibrotic fibrosis, a pathological feature of airway remodeling. Referring to Example 9 according to the present invention, it can be seen that the trichrome staining site significantly increased in the asthma induction group compared to the normal control group. The amount of peribronchial fibrosis decreased significantly and dose-dependently in the skullcapflavone II group, and the degree of peribronchial fibrosis also decreased significantly in the dexamethasone and montelukast groups. From this, the pharmaceutical composition according to the present invention can be usefully used for the treatment of asthma by inhibiting the erosion of collagen around the bronchus (Table 11 and Fig. 11).

In addition, the pharmaceutical composition according to the present invention may inhibit the production of TGF-β1, pSmad2 / 3 and increase the production of Smad7, which are involved in the pathophysiology of asthma. In the present invention Referring to Example 10, the TGF-β1 concentration in the lung tissue was significantly increased in the asthma induction group compared to the normal control group, and this increase in the TGF-β1 concentration was significantly increased by the administration of the scullcapflavone II of the present invention. It was confirmed that the dose-dependent decrease. Similarly, TGF-β1 concentration in bronchoalveolar lavage fluid was significantly increased in the asthma-induced group compared to the normal control group, and the increased TGF-β1 concentration was significantly and dose-dependently decreased by the administration of the scullcapflavone II of the present invention. Confirmed. Dexamethasone and montelukast administration groups were also found to reduce TGF-β1 concentrations in lung tissue and alveolar lavage fluid. The concentration of pSmad2 / 3 and Smad7 was increased in the lungs of the asthma-induced group by administration of the scullcapflavone II of the present invention, and the concentration was significantly reduced by the administration of the scullcapflavone II and dexamethasone of the present invention. In contrast, the concentration of Smad7 was found to be markedly increased in comparison with the untreated asthma induction group in the asthma induction group treated with Skullcapflavone II and dexamethasone of the present invention. No significant difference was found between the groups treated with Skullcapflavone II and dexamethasone of the present invention in terms of concentrations of pSmad2 / 3 and Smad7. From this, the pharmaceutical composition according to the present invention can be usefully used for the treatment of asthma by inhibiting the production of TGF-β1 and pSmad2 / 3 and increasing the production of Smad7 (see Table 12, Table 13, Figures 12 and 13). .

The present invention also provides a method for preventing asthma, comprising administering to a subject a pharmaceutical composition containing a pharmaceutically effective amount of a scullcapflavone II compound or a pharmaceutically acceptable salt thereof as an active ingredient.

Furthermore, the present invention provides a method for treating asthma, comprising administering to a subject a pharmaceutical composition containing a pharmaceutically effective amount of a scullcapflavone II compound or a pharmaceutically acceptable salt thereof as an active ingredient.

The pharmaceutically effective amount is 0.00001 to 10 mg / kg, preferably 0.0001 to 1 mg / kg, but is not limited thereto. The dosage may vary depending on the weight, age, sex, health condition, diet, duration of administration, method of administration, elimination rate, severity of disease, and the like of the particular patient.

The subject is a vertebrate and preferably a mammal, more preferably an experimental animal such as a rat, rabbit, guinea pig, hamster, dog, cat, and most preferably an ape-like animal such as a chimpanzee or gorilla.

The method of administration may be administered orally or parenterally, intraperitoneal, rectal, subcutaneous, intravenous, intramuscular, intrauterine epidural, intracerebroventricular or intrathoracic It can be administered by injection.

In another aspect, the present invention provides a composition for preventing or improving asthma containing a skull cap flavone II compound represented by the following formula 1 or a pharmaceutically acceptable salt thereof as an active ingredient:

[Formula 1]

.

.

The composition for health food according to the present invention may be used alone or in combination with other food or food ingredients of the skull cap compound of formula (1), may be suitably used in accordance with conventional methods.

The composition for health food according to the present invention includes components that are commonly added during food production, and include, for example, proteins, carbohydrates, fats, nutrients and seasonings.

There is no particular limitation on the kind of the food. Examples of the food to which the skull cap flavone II compound can be added include dairy products including meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gum, ice cream, various soups, beverages, Tea, drink, alcoholic beverages, vitamin complexes and the like, and includes all of the health food in the usual sense.

The composition for health drinks according to the present invention may contain various flavors or natural carbohydrates, etc. as additional ingredients, like ordinary drinks. Such natural carbohydrates are monosaccharides such as glucose and fructose, disaccharides such as maltose and sucrose, and polysaccharides such as dextrin and cyclodextrin, and sugar alcohols such as xylitol, sorbitol and erythritol. Examples of sweeteners include natural sweeteners such as tau martin and stevia extract, synthetic sweeteners such as saccharin and aspartame, and the like. The proportion of such natural carbohydrates is generally about 0.01-0.04 g, preferably about 0.02-0.03 g, per 100 ml of the composition of the present invention.

Furthermore, the composition for health food according to the present invention is a variety of nutrients, vitamins, electrolytes, flavors, coloring agents, pectic acid and salts thereof, alginic acid and salts thereof, organic acids, protective colloidal thickeners, pH regulators, stabilizers, preservatives, Glycerol, alcohols, carbonation agents used in carbonated drinks, and the like. In addition, the composition for health food of the present invention may contain a flesh for preparing 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 usually chosen in the range of 0.01 to 0.1 parts by weight per 100 parts by weight of the composition of the present invention.

As described above, the skullcapflavone II compound of the present invention according to the present invention reduces weight loss and airway hypersensitivity in an asthma-induced mouse model and produces the Th2 cytokine interleukin-4, 5, 13 and MMP-9 in the lung. Inhibitory effect, suppression of blood IgE and egg white albumin-specific IgE production, low toxicity to liver, inhibitory of bronchial inflammatory cell infiltration, inhibitory of bronchial mucus secretion, inhibition of subepithelial collagen erosion It is effective in reducing the TGF-β1 concentration, and increasing the Smad7 expression and inhibiting the pSmad2 / 3 concentration, so it can be usefully used as an active ingredient of the health food composition for the prevention or improvement of asthma.

Hereinafter, the present invention will be described in more detail with reference to the following Production Examples and Examples.

However, the following Preparation Examples and Examples are merely to illustrate the content of the present invention is not limited by the Preparation Examples and Examples.

<Preparation Example 1> Preparation of the skull Cobb flavone compound Ⅱ

3 kg of golden root (golden base) samples were extracted four times with methanol to obtain 250 g of total extract. This was suspended in 2 L of water and hexane (n-hexane) and ethyl acetate (ethyl acetate) were added three times, 1 L each, to obtain 13.8 g of hexane fraction and 167.0 g of ethyl acetate fraction, respectively. 160 g of ethyl acetate fraction was subjected to silica gel column chromatography (step chromatography having a concentration gradient of 30/1 to 5/1 for CHCl ₃ / MeOH), and the total fraction was divided into 10 fractions. E3-P, 7.5 g) was again subjected to silica gel column chromatography (hexane / CHCl ₃ / MeOH, 3/1 / 0.1) to obtain 14 small fractions, of which the 13th fraction (E3-PR-13) was obtained. The final fractions were purified purely (1.51 g) by recrystallization from methanol. The isolated material was subjected to structural analysis using NMR (PMR, CMR, HSQC), and the result of referring to the previous paper (Chem. Pharm. Bull (1980), 28 (3), 708-716) It was identified as flavone II (see FIG. 1).

Construction of Bronchial Asthma Induction Animals and Treatment of Compounds:

To prepare bronchial asthma-induced experimental animals, 6-week-old Balb / c female mice having an average body weight of about 20 g were used as experimental animals. Subjects who did not show any abnormalities on basic physical examination after one week of adaptation period were included. Intraperitoneally, 200 ul of phosphate buffer solution (pH 7.4) suspended in 2 mg of aluminum hydroxide (A8222, Sigma, St. Louis, MO) and 20 μg of egg white albumin (A5503, Sigma, St. Louis, MO) at two week intervals Was sensitized by injection. After the first intraperitoneal administration of egg white albumin (OVA), 1% egg white albumin was inhaled for 30 minutes using an ultrasonic nebulizer for 7 days from 28 to 34 days. 24 hours after the last antigen administration, airway hypersensitivity was measured, and 48 hours after the lethal dose of pentobarbital (Entoval, Hallym Pharmaceutical Co., Ltd.), the body weight was measured, and the bronchial incision was performed. Samples were collected after alveolar lavage. A group in which the egg white albumin was not administered or inhaled as a normal control group (NC), the egg white albumin was administered and inhaled in the asthma induction group (OVA), and the bronchial asthma was induced by a control group (DEXA). Montelukast (30 mg / kg, 30 mg / kg, 1 hour prior to inhalation of egg-albumin in the group administered with oral administration of dexamethasone (dexamethasone; 3 mg / kg, PO: D4902, Sigma, St. Louis, MO) and comparison group 2 (Monte) PO) orally administered group was prepared. On the other hand, the skull capflavon Ⅱ according to the present invention was prepared by the group orally administered 10 mg / kg (SII10) and 30 mg / kg (SII30), respectively, 1 hour before inhalation albumin albumin 5 rats per group Was used.

Hereinafter, in the embodiments of the present invention, the following marks are commonly used as follows.

SAL + SAL: Normal control group (NC) that was not administered or non-inhaled egg white albumin.

OVA + SAL: Asthma induction group (OVA) administered and inhaled egg white albumin.

OVA + Dex: Comparative group 1 (Dex) administered with egg white albumin and dexamethasone (Dex), a conventional asthma treatment, at a dose of 3 mg / kg.

OVA + M: Comparative group 2 (Monte) administered with egg white albumin and the conventional asthma treatment Montelukast (Monte) at a 30 mg / kg dose.

OVA + SII10: Experimental group administered with egg white albumin and Skullcapflavone II according to the present invention at a 10 mg / kg dose (SII10).

OVA + SII30: Experimental group administered with egg white albumin and the scullcapflavone II according to the present invention at a 30 mg / kg dose (SII30).

<Example 1> skull Cobb flavone Ⅱ weight loss for asthma induced inhibitory effect of the mouse

In order to determine the effect of the skull capflavon II according to the present invention on the weight loss of asthma-induced mice, the weight of each mouse prepared above was measured and the results are shown in Table 1 and FIG. 2. The statistical analysis of all measured values below calculated the mean and standard error (mean ± standard error) according to various variables, and the comparison between each group was analyzed by performing Mann-whitney U test using SPSS 10.0. It was. Statistically, it was determined that there was a significant difference when the p value was less than 0.05.

division Weight (g) Experimental group
(SⅡ10) 19.66 ± 0.30 Experimental group
(SⅡ30) 20.14 ± 0.67 Normal control
(NC) 19.83 ± 0.40 Asthma induction group
(OVA) 17.87 ± 0.23 Comparative group 1
(Dex) 18.38Ⅱ0.17 Comparative group 2
(Monte) 20.26 ± 0.51

As a result, as shown in Table 1 and Figure 2, the weight of the normal control group (NC) was 19.83 ± 0.40 g, but asthma induction group (OVA) showed a significant weight loss of 17.87 ± 0.23 g, compared to Comparative Group 1 (Dex) 18.38 ± 0.17 g silver, 20.26 ± 0.51 g of Comparative Group 2 (Monte), 19.66 ± 0.30 g, 30 mg / kg dose was administered in the group of 10 mg / kg dose of the skull capflavone II compound of the present invention In the group, 20.14 ± 0.67 g was measured. From this, it was confirmed that the body weight was recovered to a level close to the normal control group except for the comparison group 1 (Dex) of the drug administration group. Therefore, the skullcapflavone II of Formula 1 according to the present invention can be usefully used for preventing or treating asthma by inhibiting weight loss.

<Example 2> The inhibitory effect of airway hypersensitivity of asthma caused by the skull Cobb flavone Ⅱ

In order to confirm the effect of the scull capflavone II on the airway hyperresponsiveness caused by asthma, the airway resistance was measured mathematically by measuring the airway resistance using one chamber plethysmography (All Medicus, Seoul). Penh (Penh; Paen Enhanced) value that reflects the occlusion was measured and the results are shown in Table 2 and FIG. The pen value was measured for 3 minutes after inhalation of PBS using an ultrasonic nebulizer for 3 minutes after measuring the baseline value under normal respiration. Thereafter, the histamine methacholine (A2251, Sigma, St. Louis, MO), which is used in the general method of diagnosing bronchial asthma, was inhaled with increasing concentrations of 12, 25, and 50 mg / ml, and the pen level was measured. The percentage of increase in pen levels after each concentration of methacholine inhalation was expressed, and the base pen level (saline inhalation) was expressed as 100%. The calculation method of the pen value is shown in following formula (1).

Te: expiratory time (seconds)

      From intake to next intake

RT: relaxation time

     How long it takes for a tidal cycle to reach 30% of tidal volume during exhalation

PEF: Peak expiration flow

PIF: Peak inspiration flow

division Pen figures 0 mg / ml 12.5 mg / mll 25 mg / ml 50 mg / ml Experimental group
(SⅡ10) 0.5447 ± 0.07150 1.1303 ± 0.0965 1.2908 ± 0.1232 1.3499 ± 0.1549 Experimental group
(SⅡ30 0.4986 ± 0.0378 1.0248 ± 0.3163 1.0916 ± 0.2894 1.2862 ± 0.3313 Normal control
(NC) 0.4474 ± 0.0275 0.7421 ± 0.1600 0.8512 ± 0.1182 1.3062 ± 0.2379 Asthma induction group
(OVA) 0.5304 ± 0.0476 2.4787 ± 0.4835 3.8479 ± 0.7161 6.1304 ± 0.9325 Comparative group 1
(Dex) 0.4458 ± 0.0501 0.7733 ± 0.1139 1.5526 ± 0.2790 1.9719 ± 0.3172 Comparative group 2
(Monte) 0.4830 ± 0.0464 1.4258 ± 0.3044 2.1006 ± 0.4758 2.3576 ± 0.1741

As shown in Table 2 and Figure 3, in the normal control (NC) was confirmed a gentle increase in the pen level according to the increase in the concentration of methacholine, but in the asthma induction group (OVA) it was confirmed that the sharp increase in the pen level significantly. . On the other hand, the drug group of Comparative Group 1 (Dex), Comparative Group 2 (Monte), Experimental Group (SII10) and Experimental Group (SII30) was significantly reduced than asthma induction group (OVA) regardless of methacholine concentration. Was more pronounced when inhaled at higher concentrations of methacholine than at low concentrations of methacholine. From this, the skullcapflavone II of Formula 1 according to the present invention can be usefully used for preventing or treating asthma by inhibiting airway hypersensitivity.

<Example 3> skull Cobb flavone serum IgE inhibitory effect of wash liquid within Ⅱ

In order to confirm the effect of the skull capflavon II according to the present invention on the concentration of IgE and egg white albumin specific IgE correlated with the severity of asthma, the immunoenzyme method was used in serum. Table 3 and serum egg albumin IgE concentrations are shown in Table 4 below. Serum and bronchial alveolar saline solution from each group was dissolved in 96-well plate (ELISA plate) in egg white albumin (OVA) in 0.1 M NaHCO ₃ buffer at pH 8.3 at 20 ug / ml and coated overnight at 4 ° C. After suppressing nonspecific reaction with PBS containing 1% bovine serum albumin, serum samples were diluted 1: 400 and reacted at room temperature for 2 hours. After washing well, anti-mouse IgE monoclonal antibody was diluted 300-fold for 2 hours, followed by HRP-conjugated goat anti-rat IgG polyclonal A conjugated with peroxidase. ) Was diluted 4000 times and reacted at room temperature for 1 hour, followed by washing. The color was reacted with a 3,3 ', 5,5'-tetramethylbenzidine substrate and the spectral absorbance was measured at 650 nm.

Total IgE Concentration in Serum
(ug / ml) Experimental group
(SⅡ10) 21.62 ± 1.13 Experimental group
(SⅡ30) 19.26 ± 0.89 Normal control
(NC) 7.46 ± 1.91 Asthma Control
(OVA) 30.18 ± 2.18 Comparative group 1
(Dex) 16.34 ± 2.29 Comparative group 2
(Monte) 21.22 ± 2.30

As shown in Table 3 and Figure 4, as a result of measuring the concentration of IgE antibody in the serum according to the present invention, the serum IgE concentration in the present invention against 30.18 ± 2.18 ug / ml in the asthma induction group (OVA) According to the present study, the dose of 10 mg / kg of Skullcapflavone II was significantly decreased to 21.62 ± 1.13 ug / ml and 19.26 ± 0.89 ug / ml in the 30 mg / kg dose group.

Egg white albumin specific IgE concentration in serum
(ng / ml) Experimental group
(SⅡ10) 196.20 ± 4.91 Experimental group
(SⅡ30) 191.60 ± 15.43 Normal control
(NC) 61.40 ± 3.28 Asthma induction group
(OVA) 438.00 ± 9.01 Comparative group 1
(DEXA) 202.20 ± 14.84 Comparative group 2
(Monte) 170.20 ± 7.69

In addition, as shown in Table 4 and Figure 4, the egg white albumin-specific IgE concentration in serum is 10 mg / kg scullcapflavone II according to the present invention compared to 438.00 ± 9.01 ng / ml in the asthma induction group (OVA) The dose was significantly decreased to 196.20 ± 4.91 ng / ml and the dose to 30 mg / kg was 191.60 ± 15.43 ng / ml. From this, the scullcapflavone II of Formula 1 according to the present invention can be usefully used for preventing or treating asthma by inhibiting IgE and egg white albumin specific IgE concentrations.

<Example 4> skull Cobb flavone Ⅱ BAL inflammatory cell inhibitory effect of

In order to confirm the effect on the number of inflammatory cells in the bronchoalveolar lavage fluid of the scull capflavone II according to the present invention, the dead cells were stained with Trypan blue immediately after recovering the bronchoalveolar lavage fluid of each mouse prepared above. Calculate the total number of cells excluding hemocytometer, smear the sample with Cytospin II, and perform Diff-Quick staining (Sysmex, Switzerland) to perform eosinophils and other inflammation. The cells were differentially calculated and the results are shown in Table 5 and FIG. 5.

division Cell count (× 10 ³ cells / mouse) Eosinophils
(eosinopill) Other inflammatory cells
(other cell) Total inflammatory cells
(total cell) Experimental group
(SⅡ10) 85.56 ± 34.71 60.76 ± 21.69 191.08 ± 65.35 Experimental group
(SⅡ30) 41.84 ± 26.98 27.16 ± 13.58 89.96 ± 44.69 Normal control
(NC) 0.00 ± 0.00 0.76 ± 0.34 20.72 ± 2.71 Asthma induction group
(OVA) 506.03 ± 132.47 157.80 ± 34.21 853.45 ± 191.26 Comparative group 1
(Dex) 1.44 ± 0.51 4.84 ± 0.77 64.36 ± 11.36 Comparative group 2
(Monte) 87.95 ± 41.17 60.20 ± 24.77 223.50 ± 85.61

As shown in Table 5 and FIG. 5, the total number of inflammatory cells was 20.72 ± 2.71 in the normal control group (NC), 853.45 ± 191.26 in the asthma induction group (OVA), 64.36 ± 11.36 in the comparison group 1 (Dex), and the comparison group. 2 (Monte) at 223.50 ± 85.61, 191.08 ± 65.35 in the group administered with the skullcapflavone II according to the present invention at a dose of 10 mg / kg, 89.96 ± 44.69 in the group administered at the 30 mg / kg dose, In contrast, all drug groups significantly decreased. In particular, the number of eosinophils was 0.00 ± 0.00 in the normal control group, 506.03 ± 132.47 in the asthma induction group (OVA), 1.44 ± 0.51 in the comparative group 1 (Dex), 87.95 ± 41.17 in the comparative group 2 (Monte), the skull of the present invention. Capflavone II was significantly reduced in all groups treated with asthma induction (OVA) compared with asthma induction group (OVA) at 85.56 ± 34.71 and 30 mg / kg in the 10 mg / kg dose group. From this, Skullcapflavone II described in Formula 1 according to the present invention can be usefully used for preventing or treating asthma by reducing the number of inflammatory cells in the bronchus.

<Example 5> toxicity between the skull Cobb flavone Ⅱ

In order to confirm the liver toxicity of Skullcapflavone II according to the present invention, using a commercial ELISA kit (Beckman Coulter, INC., Fullerton, CA, USA) and an automated analyzer (Beckman CX4, CA, USA), hepatic liver The activity of the enzymes ALT and AST was measured and the results are shown in Table 6 and FIG. 6.

Liver Enzyme Activity (IU / L) ALT AST Experimental group
(SⅡ10) 106.67 320.67 Experimental group
(SⅡ30) 46.00 136.00 Normal control
(NC) 48.67 89.33 Asthma induction group
(OVA) 92.00 262.67 Comparative group 1
(Dex) 235.33 458.67 Comparative group 2
(Monte) 72.66 195.33

As shown in Table 6 and Figure 6 ALT and AST activity was significantly increased in Comparative Group 1 (Dex) than asthma induction group (OVA). On the other hand, in the group administered with Comparative Group 2 (Monte) and Skullcapflavone II it was confirmed that the concentration value significantly lower than the Comparative Group 1 (Dex). From this, the scullcapflavone II of Formula 1 according to the present invention can be usefully used for prolonged prevention or treatment of asthma due to low liver toxicity.

< Example  6> Skull cap flavones Inhibition of Interleukin-4, 5, 13 and MMP-9 Expression in Bronchoalveolar Lavage Solution of Ⅱ

MRNA of each cytokine was used to confirm the effect on the expression of Th2 cytokine interleukin-4, 13 and MMP-9 related to asthma of the scullcapflavone II according to the present invention and the results are shown in Table 7 and FIG. It was. In order to analyze the expression level of interleukin-4, 13 and MMP-9 in the supernatant of the bronchial alveolar lavage fluid collected from each mouse prepared above, RNA was isolated from the extracted lung using Trizol (Invitrogen). Reverse transcriptase polymerase chain reaction (quantitative RT-PCR, Promega, WI) was performed. Relative densitometric value (RDV) when the expression level of the normal control group was evaluated as 1 was measured.

 As shown in Table 7 and FIG. 7, the group administered scullcapflavone II significantly inhibited the expression of interleukin-4, 13 and MMP-9 in bronchoalveolar lavage fluid as compared to the asthma induction group (OVA).

Relative densitometric value (RDV)
(NC: NC = 100%) Interleukin-4 Interleukin-13 MMP-9 Experimental group
(SⅡ10) 1.76 ± 0.02 1.42 ± 0.17 0.93 ± 0.16 Experimental group
(SⅡ30) 1.84 ± 0.21 1.34 ± 0.17 0.80 ± 0.07 Normal control
(NC) 1.00 + 0.02 1.00 ± 0.13 1 ± 0.09 Asthma induction group
(OVA) 2.19 ± 0.09 2.06 ± 0.21 1.13 ± 0.04 Comparative group 1
(Dex) 1.82 ± 0.10 1.64 ± 0.18 0.94 ± 0.07 Comparative group 2
(Monte) 1.91 ± 0.14 1.50 ± 0.13 0.93 ± 0.05

The concentrations of interleukin-4, 5, and 13 in bronchoalveolar lavage fluid were measured by enzyme immunoassay, and the results are shown in Table 8 and FIG. 8. 48 hours after the last egg albumin administration, the asthma induction group (OVA) was significantly increased compared to the normal control group (NC). Increased cytokine concentrations were significantly and dose-dependently decreased in the group administered SkullCapflavone II. In addition, Comparative Group 1 (Dex) and Comparative Group 2 (Monte) also significantly reduced the concentrations of interleukin-4, 5 and 13 in bronchoalveolar lavage fluid.

Concentration (pg / ml) Interleukin-4 Interleukin-5 Interleukin-13 Experimental group
(SⅡ10) 7.83 11.43 11.09 Experimental group
(SⅡ30) 4.37 4.12 10.45 Normal control
(NC) 4.02 0 4.53 Asthma induction group
(OVA) 27.40 56.11 29.64 Comparative group 1
(Dex) 8.79 4.39 5.30 Comparative group 2
(Monte) 2.4 10.65 14.00

From this, the scullcapflavone II of Formula 1 according to the present invention can be usefully used for preventing or treating asthma by inhibiting the production of interleukin-4, 5, 13 and MMP-9.

<Example 7> histopathological analysis by a skull Cobb flavone Ⅱ administration

In order to confirm the effect on the infiltration of inflammatory cells of scullcapflavone II according to the present invention, the lungs of each mouse prepared above were removed without performing bronchoalveolar lavage, and histopathological examination was performed. It is shown in Table 9 and FIG.

The isolated lungs without bronchoalveolar lavage in each group were subjected to hematoxylin and eosin (H & E) staining by making permanent tissue sections 4 μm thick through conventional formalin fixation and paraffin embedding. After H & E staining, the inflammation indexes of five sites were randomly measured and averaged per tissue section of each individual. Inflammation index 0 is when no inflammatory cells are found around the bronchus, Inflammation index 1 is observed when inflammatory cells are observed intermittently, Inflammation index 2 is when a thin layer of inflammatory cells is observed around most bronchus, and inflammation index 3 is mostly It was evaluated that a thick inflammatory cell layer was observed around the bronchus.

Inflammation index Around the bronchioles Around the blood vessel Experimental group
(SⅡ10) 2.00 ± 0.28 1.60 ± 0.22 Experimental group
(SⅡ30) 1.40 ± 0.22 1.00 ± 0.40 Normal control
(NC) 0.13 ± 0.11 0.00 ± 0.00 Asthma induction group
(OVA) 2.86 ± 0.13 2.71 ± 0.17 Comparative group 1
(Dex) 0.80 ± 0.18 0.40 ± 0.22 Comparative group 2
(Monte) 1.60 ± 0.36 2.00 ± 0.28

As a result, in the asthma induction group (OVA) according to the present invention, many inflammatory cells including eosinophils were infiltrated around the bronchioles, and hyperproliferated epithelial cells and thickened bronchial smooth muscle were also confirmed. On the other hand, as shown in Table 9, the drug administration group significantly reduced the infiltration of inflammatory cells and when quantified the asthma induction group (OVA) had an inflammation index around the bronchioles 2.86 ± 0.13, compared to Comparative Group 1 ( Dex) is 0.80 ± 0.18, Comparative Group 2 (Monte) is 1.60 ± 0.36, the group administered the skull capflavone II according to the invention at a dose of 10 mg / kg 2.00 ± 0.28, 30 mg / kg dose group Was 1.40 ± 0.22, which was significantly decreased compared to asthma induction group (OVA).

In addition, the inflammation index around the blood vessels in the asthma induction group (OVA) was found to be 2.71 ± 0.17, Comparative Group 1 (Dex) 0.40 ± 0.22, Comparative Group 2 (Monte) is 2.00 ± 0.28, skull according to the present invention In the group administered capcapone II at a dose of 10 mg / kg, the group administered at 1.60 ± 0.22 and the dose administered at 30 mg / kg were 1.00 ± 0.40, except for comparison group 2 (Monte). Significantly decreased compared to OVA.

From this, Skullcapflavone II described in Formula 1 according to the present invention can be usefully used for preventing or treating asthma by inhibiting infiltration of inflammatory cells.

<Example 8> skull Cobb flavone goblet cell inhibitory effect of Ⅱ

In order to confirm the effect on the number of goblet cells of the skull capflavon II according to the present invention was measured the degree of proliferation of goblet cells in each bronchus prepared above, the results are shown in Table 10 and FIG. The isolated lungs without bronchoalveolar lavage in each group were made by permanent staining and paraffin embedding to make permanent tissue sections 4 μm thick, and discriminated by Periodic acid Schiff (PAS) staining. The rate of goblet cell proliferation was evaluated by measuring the proportion of cells in bronchial epithelial cells.

PAS + cells / bronchioles (%) Experimental group
(SⅡ10) 28.47 ± 2.54 Experimental group
(SⅡ30) 26.90 ± 3.10 Normal control
(NC) 1.44 ± 0.62 Asthma induction group
(OVA) 55.02 ± 2.47 Comparative group 1
(Dex) 15.78 ± 3.27 Comparative group 2
(Monte) 30.12 ± 3.08

As shown in Table 10 and Figure 10, in the normal control group (NC), the proportion of goblet cells in bronchiolemal epithelial cells was 1.44 ± 0.62%, but increased to 55.02 ± 2.47% in the asthma induction group (OVA) 15.78 ± 3.27% in Group 1 (Dex), 30.12 ± 3.08% in Comparative Group 2 (Monte), 28.47 ± 2.54%, 30 mg / in the group administered with 10 mg / kg of Skullcapflavone II according to the present invention In the group administered with the kg dose, all were significantly decreased to 26.90 ± 3.10%.

From this, Skullcapflavone II described in Formula 1 according to the present invention can be usefully used for preventing or treating asthma by inhibiting proliferation of goblet cells.

< Example  9> Skull cap flavones Inhibitory Effect of Ⅱ Bronchial Collagen Erosion

Masson's trichrome staining was performed to confirm the effect of the scull capflavone II on the peribronchial collagen erosion. Image Pro Plus (Media Cybernetics Company, Silver Spring, MD, USA) was used to measure the length of the airway basement membrane and the area stained with collagen to quantify the stained area per unit area. Ten airways were randomized per slide and the results are shown in Table 11 and FIG. 11.

Trichrome staining site (um ² / um) Experimental group
(SⅡ10) 384.43 Experimental group
(SⅡ30) 296.26 Normal control
(NC) 211.42 Asthma induction group
(OVA) 915.69 Comparative group 1
(Dex) 319.46 Comparative group 2
(Monte) 407.63

As shown in Table 11 and FIG. 11, the trichrome staining site area was significantly increased in the asthma induction (OVA) group compared to the normal control (NC). In the group administered Skullcapflavone II according to the present invention, the degree of peribronchial fibrosis decreased significantly and dose-dependently. Comparative Group 1 (Dex) and Comparative Group 2 (Monte) also significantly reduced the degree of peribronchial fibrosis.

From this, Skullcapflavone II described in Formula 1 according to the present invention can be usefully used for preventing or treating asthma by inhibiting the erosion of collagen around the bronchus.

< Example  10> Skull cap flavones Ⅱ TGF -β1 inhibitory effect and Smad  Effect on expression

The relative density of TGF-β1 to actin, the concentration of TGF-β1, and the concentration of TGF-β1 in the lung tissues of the mice prepared above to determine the effect of Scapcapflavone II on TGF-β1 inhibition and Smad expression according to the present invention The relative density of Smad against actin was measured and the results are shown in Table 12, Table 13, FIG. 12 and FIG. Lung tissue was homogeneous in the presence of protease inhibitors capable of producing lung protein extracts. Protein concentration was determined using Bradford reagent (Bio-Rad Inc., Hercules, Calif.) And samples (30 ug of protein per lane) were analyzed by 10% SDS-PAGE. The isolated protein was transferred to a membrane of polyvinylidene difluoride (Amersham Pharmacia Biotech, Piscataway, NJ, USA) by wet transfer methtod (250 mA, 90 min). Nonspecific binding was blocked by treatment with 5% skim milk in Tris buffer saline Tween 20 (TBST; 25 mM Tris pH 7.5, 150 mM NaCl, 0.1% Tween 20) for 2 hours, pSmad2 / 3, Smad7 or TGF-β1 Incubated overnight at 4 ° C. with antibody (Cell Signaling Technology Inc., Beverly, MA, USA). Anti-rabbit horseradish peroxidase-conjugated IgG (anti-rabbit horseradish peroxidase-conjugated IgG) was used as a secondary antibody and the results were visualized by enhanced chemiluminescence assay after exposure to photographic film.

For actin
Relative Density of TGF-β1 Concentration of TGF-β1
(pg / ml) Experimental group
(SⅡ10) 5.87 176.56 Experimental group
(SⅡ30) 3.06 140.18 Normal control
(NC) 1.3364 106.06 Asthma induction group
(OVA) 15.91 312.19 Comparative group 1
(Dex) 4.73 138.19 Comparative group 2
(Monte) 2.16 127.19

As shown in Table 12 and FIG. 12, TGF-β1 protein in lung tissue was significantly increased in the asthma induction group (OVA) compared to the normal control (NC) at 48 hours of the last egg white albumin administration. The increased concentration of TGF-β1 was significantly and dose-dependently reduced by the administration of Skullcapflavone II according to the present invention. Similarly, enzyme immunoassay revealed that TGF-β1 concentration in bronchoalveolar lavage fluid increased significantly in the asthma induction group (OVA) compared to the normal control (NC) at 48 hours of the last egg white albumin administration. However, the increased concentration of TGF-β1 was significantly and dose-dependently decreased by the administration of Skullcapflavone II according to the present invention. Comparative Group 1 (Dex) and Comparative Group 2 (Monte) administration groups also decreased the concentration of TGF-β1 in the lung tissue and alveolar lavage fluid.

For actin
Relative Density of Smad7 For actin
relative density of pSmad2 / 3 Experimental group
(SⅡ10) 0.4199 0.351 Experimental group
(SⅡ30) 0.6213 0.2715 Normal control
(NC) 0. 0.6367 0.1645 Asthma induction group
(OVA) 0.2617 0.7256 Comparative group 1
(Dex) 0.4325 0.3348 Comparative group 2
(Monte) 0.6298 0.2786

As shown in Table 13 and Figure 13, the analysis of the concentration of pSmad2 / 3 and Smad7 in the lungs of the asthma-induced group by the skull capflavone II administration according to the present invention, the increase of pSmad2 / 3 7 days after egg white albumin administration Was observed and this concentration was significantly reduced by administration of Skullcapflavone II and dexamethasone. In contrast, the concentration of Smad7 was compared with the untreated asthma induction group (OVA) in the asthma induction group treated with skullcapflavone II and dexamethasone (experimental group (SII10), experimental group (SII30) and comparative group 1 (Dex)). Markedly increased. No significant difference was found between the groups that received scullcapflavone II and dexamethasone in terms of concentrations of pSmad2 / 3 and Smad7.

From this, the scullcapflavone II of Formula 1 according to the present invention can be usefully used for preventing or treating asthma by inhibiting the production of TGF-β1 and pSmad2 / 3 and increasing the production of Smad7.

< Formulation example  1> Preparation of Pharmaceutical Formulations

<1-1> Sanje  Produce

2 mg of Skullcapflavone II compound of the present invention

Lactose 1 g

The above components were mixed and packed in airtight bags to prepare powders.

<1-2> Preparation of tablets

100 mg of Skullcapflavone II compound of the present invention

Corn starch 100 mg

100 mg of milk

2 mg of magnesium stearate

After mixing the above components, tablets were prepared by tableting according to a conventional method for producing tablets.

&Lt; 1-3 > Preparation of capsules

100 mg of Skullcapflavone II compound of the present invention

Corn starch 100 mg

100 mg of milk

2 mg of magnesium stearate

After mixing the above components, the capsules were filled in gelatin capsules according to the conventional preparation method of capsules.

&Lt; 1-4 >

1 mg of Skullcapflavone II compound of the present invention

1.5 g lactose

1 g of glycerin

Xylitol 0.5 g

After mixing the above components, they were prepared so as to be 4 g per one ring according to a conventional method.

<1-5> Preparation of granules

150 mg of Skullcapflavone II compound of the present invention

Soybean extract 50 mg

200 mg of glucose

600 mg of starch

After mixing the above components, 100 mg of 30% ethanol was added and the mixture was dried at 60 캜 to form granules, which were then filled in a capsule.

< Manufacturing example  2> Manufacturing of food

Foods containing the skullcapflavone II compound of the present invention were prepared as follows.

<2-1> Production of flour food

0.5-5.0 parts by weight of the skullcapflavone II compound of the present invention was added to flour, and bread, cake, cookies, crackers and noodles were prepared using this mixture.

<2-2> soup  And juicy ( gravies Manufacturing

0.1-5.0 parts by weight of the skullcapflavone II compound of the present invention was added to soups and broths to prepare meat products for health promotion, soups of noodles and broths.

<2-3> Ground Beef  Produce

10 parts by weight of the skull capflavon II compound of the present invention was added to the ground beef to prepare a ground beef for health promotion.

<2-4> Dairy products ( dairy products Manufacturing

5-10 parts by weight of the skullcapflavone II compound of the present invention was added to milk, and various dairy products such as butter and ice cream were prepared using the milk.

<2-5> Solar  Produce

Brown rice, barley, glutinous rice, and yulmu were dried by a known method and dried, and the mixture was granulated to a powder having a particle size of 60 mesh.

Black soybeans, black sesame seeds, and perilla seeds were steamed and dried by a conventional method, and then they were prepared into powder having a particle size of 60 mesh by a pulverizer.

The scull capflavone II compound of the present invention was concentrated under reduced pressure in a vacuum concentrator, and the dried product obtained by drying with a spray and a hot air dryer was pulverized with a particle size of 60 mesh to obtain a dry powder.

The grains, seeds and skullcapflavone II compounds prepared above were blended in the following ratios.

(30 parts by weight of brown rice, 15 parts by weight of yulmu, 20 parts by weight of barley)

Seeds (7 parts by weight of perilla, 8 parts by weight of black beans, 7 parts by weight of black sesame seeds)

Skull cap flavone II compound (3 weight part),

(0.5 part by weight),

(0.5 parts by weight)

< Manufacturing example  3> Manufacturing of beverage

<3-1> Health drink  Produce

Instant sterilization by homogeneously mixing 5 mg of Skullcapflavone II of the present invention with subsidiary materials such as liquid fructose (0.5%), oligosaccharide (2%), sugar (2%), salt (0.5%) and water (75%). After it was prepared by packaging in a small packaging container such as glass bottles, plastic bottles.

<3-2> Preparation of vegetable juice

Vegetable juice was prepared by adding 5 mg of the skullcapflavone II compound of the present invention to 1,000 ml of tomato or carrot juice.

<3-3> Production of fruit juice

1 mg of the skullcapflavone II compound of the present invention was added to 1,000 ml of apple or grape juice to prepare a fruit juice.

Claims (14)

A pharmaceutical composition for preventing or treating asthma, which contains a skullcapflavone II compound represented by the following Chemical Formula 1 or a pharmaceutically acceptable salt thereof as an active ingredient:
[Chemical Formula 1]

.
The pharmaceutical composition for preventing or treating asthma according to claim 1, wherein the Skullcapflavone II compound of Formula 1 is isolated from Scutellariae Radix.
The pharmaceutical composition for preventing or treating asthma according to claim 1, wherein the Skullcapflavone II compound of Formula 1 is isolated from the root of gold.
The pharmaceutical composition for preventing or treating asthma according to claim 1, wherein the Skullcapflavone II compound of Formula 1 inhibits weight loss.
The pharmaceutical composition for preventing or treating asthma according to claim 1, wherein the Skullcapflavone II compound of Formula 1 inhibits airway hypersensitivity.
The pharmaceutical composition for preventing or treating asthma according to claim 1, wherein the Skullcapflavone II compound of Formula 1 reduces the concentration of IgE in serum.
The pharmaceutical composition for preventing or treating asthma according to claim 1, wherein the Skullcapflavone II compound of Formula 1 reduces the number of inflammatory cells in the bronchus.
The pharmaceutical composition for preventing or treating asthma according to claim 1, wherein the Skullcapflavone II compound of Formula 1 has low toxicity to the liver.
The pharmaceutical composition for preventing or treating asthma according to claim 1, wherein the scullcapflavone II compound of formula 1 inhibits the expression of interleukin-4, 5, 13 and MMP-9 in the bronchus.
The pharmaceutical composition for preventing or treating asthma according to claim 1, wherein the Skullcapflavone II compound of Formula 1 inhibits invasion of inflammatory cells in the bronchus.
The pharmaceutical composition for preventing or treating asthma according to claim 1, wherein the Skullcapflavone II compound of Formula 1 inhibits the proliferation of goblet cells in the bronchus.
The pharmaceutical composition for preventing or treating asthma according to claim 1, wherein the Skullcapflavone II compound of Formula 1 inhibits erosion of collagen around the bronchus.
The method of claim 1, wherein the skullcapflavone II compound of Formula 1 inhibits the expression of TGF-β1 and pSmad2 / 3 in lung tissue and bronchus or increases the expression of Smad7 pharmaceutical for the treatment of asthma Composition.
A composition for preventing or improving asthma containing a skull cap flavone II compound represented by the following formula (1) or a pharmaceutically acceptable salt thereof as an active ingredient:
[Chemical Formula 1]

.

Images