CN101222930B - A composition comprising an extract of tiarell polyphylla and tiarellic acid isolated therefrom having antiinflammatory, antiallergic and antiasthmatic activity - Google Patents

Abstract

The present invention relates to a composition having anti-inflammation, anti-allergic and anti-asthma effects, comprising the extract of Shuizhi and xantholic acid isolated therefrom. Extracts of twigs and twig acid isolated therefrom showed inhibitory effects on LTC ₄ release in vitro, as well as on IgE levels and cytokines (IL-4, IL-5, and IL-13) in OVA-induced asthmatic mice Inhibition of airway hyperresponsiveness and leukocyte infiltration. Therefore, they are useful as therapeutic agents or functional health foods for the treatment and prevention of inflammatory, allergic and asthmatic diseases.

Description

Composition with anti-inflammatory, anti-allergic and anti-asthmatic effects comprising twig extract and xantholic acid isolated therefrom

technical field

The present invention relates to a composition comprising Tiarella polyphylla extract and tiarellic acid isolated therefrom having anti-inflammatory, anti-allergic and anti-asthmatic effects.

Background technique

Asthma has been recognized as a complex syndrome arising in the airways manifested by various disturbances such as airflow obstruction, acute or chronic inflammation, airway hyperresponsiveness (AHR) and structural remodeling (Kumar R.K.Pharmacol. Ther., 91, pp 93-104, 2001).

Allergic inflammation occurring in the airways has been reported to play a key role in the development of asthma, and the number of patients with allergic asthma has recently increased to about 10% of the world's population. According to reports, the number of patients in the United States has reached 17 million, and the market size of allergic asthma drugs in the United States has expanded to 640 billion US dollars so far.

Asthma can be divided into two types, extrinsic asthma and intrinsic asthma. Exogenous asthma caused by exposure to antigens such as house dust mite, pollen, animal epithelium, fungi as major antigens shows a positive reaction to the antigen in skin test or bronchial stimulation test and usually occurs in young people. Intrinsic asthma due to upper respiratory infection, exercise, emotional lability, cold weather, changes in humidity occurs in adult patients.

From a pathophysiological point of view, asthma has been recognized as a chronic inflammation that occurs through a process in which inflammatory cells proliferate, proliferate, differentiate and are activated by regenerated cytokines in helper T2 immune cells, and then migrate to the airways or their adjacent tissues. Activated inflammatory cells such as neutrophils, mast cells, etc. release various inflammatory mediators, such as cytokines, chemokines, signaling molecules, adhesion molecules, and growth factors, and structural cells in the airways are involved in the development of asthma. Various stages (Elias JA et al., J Clin Invest., 111, pp 291-7, 2003). In various research and clinical studies using knockout mouse models, key observations in asthma may be grouped into several characteristic parameters such as immune response, eosinophilia, AHR and structural remodeling (Moffatt JD. Pharmacol Ther, 107, pp343 -57, 2005; Spina D et al., Trends Pharmacol Sci, 23, pp 311-5, 2002). Each parameter does not appear to be directly related to each other, however, IgE-mediated immune responses and eosinophilia are prominent symptoms in the airways of allergic asthma (Bochner B.S. et al., Annu. Rev. Immunol., 12, pp 295 -335, 1994; Bousquet J et al., N.Engl.J.Med., 323, pp1033-9, 1990), and cytokines such as IL-4, IL-5 and IL-13 produced in the allergic process in It also plays an important role in AHR development and airway remodeling (Riffo-Vasquez Y et al., Pharmacol. Ther., 94, pp 185-211, 2002). Indeed, asthma is the result of programmed inflammatory events, many of which involve specific inhibitors acting on asthmatic pathways, such as histamine H1 antagonists, plasma thromboxane antagonists, platelet activating factor antagonists, cyclooxygenase inhibitors, nitrogen monooxygenase inhibitors and prostaglandin inhibitors, and have been tried, but failed in clinical trials (Moffatt J.D., Pharmacol. Ther., 107, pp 343-57, 2005). In contrast, glucocorticoids, which suppress the origin of inflammatory cells to baseline levels by broadly inhibiting cytokine synthesis and cytokine-mediated immune cell survival, have been used for more than 30 years to control asthma in patients. Symptoms (Baatjes A.J. et al., Pharmacol, Ther., 95, pp 63-72, 2002). These reports suggest that therapeutic approaches to control asthma should focus on restoring the balance of asthma parameters rather than finding effective inhibitors of specific asthma process pathways.

Yellow water branch (Saxifrage, Saxifragaceae) is a species belonging to this genus in Korea. It grows in southwestern China, but only on the highest point of Ullung Island in Korea. Prior to this, xantholic acid was isolated using corosolic acid, tormentic acid, etc. (Park et al., Arch Pharm Res., 25, pp 57-60, 2002), and other reported its inhibitory effect on the expression of MMP-1 and type 1 procollagen in UV-irradiated skin fibroblasts (Moon et al., J Ethnopharmacol., 98, pp 185-189, 2005).

However, there is no report or disclosure in any of the above-cited documents on the inhibitory effects of twig extract and xantholic acid isolated therefrom on inflammatory, allergic and asthmatic diseases, the disclosures of which are incorporated herein by reference .

Therefore, the present inventors found that the extract of twig twig and the xanthiic acid isolated therefrom showed the inhibitory effect of xanthiic acid on the release of LTC ₄ in vitro, as well as the effects on IgE levels and cytokine (IL) in OVA-induced asthmatic mice. -4, IL-5 and IL-13) production, airway hyperresponsiveness and inhibition of leukocyte infiltration, which are expected to be useful in the control of asthma.

Contents of the invention

technical problem

Therefore, so far, there is still a need to find more effective non-toxic drugs for the treatment and prevention of inflammatory, allergic and asthmatic diseases.

Technical solutions

Therefore, the object of the present invention is to provide a composition for treating and preventing inflammatory, allergic and asthmatic diseases, which comprises the crude extract of Shuizhi or its organic solvent-soluble extract as an active ingredient.

The term "crude extract" disclosed herein includes extracts prepared by extracting plant material using water, C ₁ -C ₄ lower alcohols such as methanol, ethanol, preferably methanol, etc., or mixtures thereof.

The term "organic solvent-soluble extract" disclosed herein can be prepared by extracting the above-mentioned crude extract with an organic solvent such as butanol, acetone, ethyl acetate, chloroform, dichloromethane or n-hexane, preferably butanol.

The present invention provides a pharmaceutical composition, which comprises xantholic acid or a pharmaceutically acceptable salt thereof represented by the following chemical formula (I) isolated from the crude extract of Shuizhi or its organic solvent-soluble extract as an active ingredient, wherein The amounts of active ingredients described above are effective in the treatment and prevention of inflammatory, allergic and asthmatic diseases.

According to another aspect of the present invention, there is also provided the crude extract or its organic solvent soluble extract, or the xantholic acid isolated therefrom for the preparation of medicines for the treatment or prevention of inflammatory, allergic and asthmatic diseases. Drug use.

According to another aspect of the present invention, there is also provided a method for treating or preventing inflammatory, allergic and asthmatic diseases in mammals, wherein the method comprises adding a therapeutically effective amount of the crude extract of Shuizhi or its organic solvent to The soluble extract, or xantholic acid isolated therefrom, is administered to mammals suffering from inflammatory, allergic and asthmatic diseases.

The extract of the present invention isolated from Branchia chinensis and the xantholic acid isolated therefrom can be prepared according to the following preferred embodiments.

The present invention will be described in detail below.

For the present invention, for example, cut the whole plant of D. chinensis into small pieces, and the small pieces are mixed with 2 to 20 times of volume, preferably 5 to 10 times of volume of polar solvent such as water, C ₁ -C ₄ lower alcohols (such as methanol, ethanol) , butanol or a mixture thereof, preferably methanol); and heated at a temperature of 20 to 100°C, preferably 20 to 50°C, for 10 to 48 hours, preferably 20 to 30 hours, using hot water reflux extraction, cold water extraction, ultrasonic or Conventional extraction, preferably with cold water; the residue is filtered and the filtrate is dried to obtain a polar solvent soluble extract thereof.

Suspend the above crude extract prepared using the above steps in water, and then mix with 1 to 100 times the volume, preferably 1 to 5 times the volume of an organic solvent such as butanol, acetone, ethyl acetate, chloroform, methylene chloride or hexane, Preferably butanol is mixed to obtain the organic solvent soluble extract of the present invention.

The above organic solvent-soluble extract was further chromatographed on a silica gel column (70-230 mesh, 8.5×65 cm) filled with silica gel, using n-hexane: ethyl acetate (10-20% ethyl acetate, stepwise gradient) and Elution with a mixed solvent of chloroform:methanol (0-100% methanol, step gradient) yielded 9 fractions. Among these fractions, fraction 6 was subjected to repeated silica gel column chromatography (silica gel, 230-400 mesh, 6.0×60 cm, chloroform-methanol mixture, 5-50% methanol, step gradient) using a normal-phase silica gel column to obtain Xantholic acid of the present invention. Its structure was determined by NMR ( ¹ H, ¹³ C, DEPT, HMQC, HMBC), EI-MS and optical rotation using those methods reported before (Park et al., ArchPharm Res., 25, pp 57-60, 2002), Use HPLC system (Shimadzu SCL-10A with SPD-M 10Avp PDA detector, column: Phenomenex Synergi 4 μm Fusion RP-80, 4.6×150 nm, elution: ACN/0.1% TFA in distilled water (DW), 4/1 , v/v) analyzed its purity, and the purity was higher than 99.5%.

According to another aspect of the present invention, there is provided a pharmaceutical composition for treating and preventing inflammatory, allergic and asthmatic diseases, which includes the crude extract of Shuizhi prepared by the above preparation method and its organic solvent-soluble extract substances, or xantholic acid isolated therefrom as active ingredients.

According to another aspect of the present invention, there is also provided the crude extract and its organic solvent-soluble extract prepared by the above-mentioned preparation method, or the xantholic acid isolated therefrom for the treatment and prevention of inflammation and allergy Drug use for sexual and asthmatic disorders.

According to another aspect of the present invention, there is also provided a method for treating or preventing inflammatory, allergic and asthmatic diseases, wherein the method comprises administering a therapeutically effective amount of the crude extract of Shuizhi prepared by the above preparation method and An organic solvent-soluble extract thereof, or xantholic acid isolated therefrom.

The compounds of the present invention represented by formula (I) can be converted into their pharmaceutically acceptable salts and solvates by conventional methods well known in the art. As such salts, acid addition salts thereof formed from pharmaceutically acceptable free acids thereof are useful and can be prepared by conventional methods. For example, after dissolving the compound with an excess of acid solution, the salt is precipitated by a water-miscible organic solvent such as methanol, ethanol, acetone, or acetonitrile to prepare its acid addition salt, which can then further be heated to an equivalent amount of the compound and dilute the acid and water or an alcohol such as ethylene glycol monomethyl ether, followed by evaporation to dryness or filtration under reduced pressure to obtain its dry salt form.

As the free acid in the above method, an organic acid or an inorganic acid can be used. For example, organic acids such as methanesulfonic acid, p-toluenesulfonic acid, acetic acid, trifluoroacetic acid, citric acid, maleic acid, succinic acid, oxalic acid, benzoic acid, lactic acid, glycolic acid, gluconic acid, Galacturonic acid, glutamic acid, glutaric acid, glucuronic acid, aspartic acid, ascorbic acid, carbonylic acid (carbonylic acid), vanillic acid, hydroiodic acid, etc., usable inorganic acids such as hydrochloric acid, phosphoric acid , sulfuric acid, nitric acid, tartaric acid, etc.

In addition, pharmaceutically acceptable metal salt forms of the compounds of the invention can be prepared using bases. Its alkali metal or alkaline earth metal salts can be prepared by conventional methods, for example, after dissolving the compound with an excess of alkali metal hydroxide or alkaline earth metal hydroxide solution, filtering the insoluble salt and evaporating and drying the remaining filtrate to obtain its metal salts. As metal salts of the present invention, sodium, potassium or calcium salts are pharmaceutically acceptable, and the corresponding silver salts can be prepared by reacting alkali metal salts or alkaline earth metal salts with suitable silver salts such as silver nitrate.

If not expressly stated herein, pharmaceutically acceptable salts of the compounds of the present invention include all acid or base salts which may exist in the form of the compounds. For example, the pharmaceutically acceptable salts of the present invention include hydroxyl salts, such as sodium, calcium and potassium salts thereof; amino salts, such as hydrogen bromide, sulfate, hydrogen sulfate, phosphate, hydrogen phosphate, dihydrogen phosphate, Acetate, succinate, citrate, tartrate, lactate, mandelate, methanesulfonate (mesylate) and p-toluenesulfonate, etc., these can be used conventionally known in the art Method preparation.

The composition of the present invention for treating and preventing inflammatory, allergic and asthmatic diseases may comprise 0.1-50% by weight of the above-mentioned extracts or compounds based on the total weight of the composition.

The composition of the present invention may be a pharmaceutical composition containing a pharmaceutically acceptable carrier, adjuvant or diluent, such as lactose, glucose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch , Gum Arabic, Alginate, Gel, Calcium Phosphate, Calcium Silicate, Cellulose, Methylcellulose, Polyvinylpyrrolidone, Water, Methyl-Hydroxybenzoate, Propyl-Hydroxybenzoate, Talc, magnesium stearate and mineral oil. These formulations may also include fillers, anti-aggregating agents, lubricants, moisturizers, flavoring agents, emulsifiers, preservatives and the like. The compositions of the present invention can be formulated so as to provide quick, sustained or delayed release of the active ingredient after administration to the patient, using any procedure known in the art.

For example, the composition of the present invention can be dissolved in oil, propylene glycol or other solvents commonly used in the manufacture of injections. Suitable examples of the carrier include physiological saline, polyethylene glycol, ethanol, vegetable oil, isopropyl myristate, etc., but are not limited thereto. For topical administration, the extracts of the invention may be formulated in ointments and creams.

The pharmaceutical preparation containing the composition of the present invention can be prepared in any form, such as oral dosage form (powder, tablet, capsule, soft capsule, aqueous medicine, syrup, elixir, pill, face powder, granule), or topical preparation (milk ointments, ointments, lotions, gels, balms, patches, pastes, spray solutions, aerosols, etc.), or injectable preparations (solutions, suspensions, emulsions).

The pharmaceutical form of the composition of the present invention can be used in the form of its pharmaceutically acceptable salts, alone or in a suitable combination, and can also be used in combination with other pharmaceutically active compounds.

The required dose of the extract or compound of the present invention varies with the subject's condition and body weight, severity, dosage form, administration route and time, and can be selected by those skilled in the art. However, in order to obtain the desired effect, it is generally recommended to administer the extract of the present invention in an amount of 0.0001-100 mg/kg body weight/day, preferably 0.001-10 mg/kg body weight/day. This dosage may be administered alone or in several divided doses per day.

The pharmaceutical composition of the present invention can be administered to test animals such as mammals (rats, mice, domestic animals or humans) through various routes. All modes of administration are contemplated, for example, oral administration, rectal administration or administration by intravenous, intramuscular, subcutaneous, intradermal, intrathecal, dura mater or intracerebroventricular injection.

Another object of the present invention is to provide a functional health food for preventing and alleviating inflammatory, allergic and asthmatic diseases, which includes the extract of Shuizhi or a compound isolated therefrom and dietary acceptable additives.

For the development of functional health foods, examples of foods that can be added including the above-mentioned extracts or compounds of the present invention are various foods, beverages, chewing gums, vitamin complexes, health-improving foods, etc., which can be used as powders, Granules, tablets, chewable tablets, capsules or beverages, etc.

The above-mentioned composition of the present invention can be added in food, additive or beverage, wherein, the amount of above-mentioned extract or compound in food or beverage usually accounts for about 0.01～80w/w% of the food gross weight of health food composition, preferably 0.01 ~15w/w%, and based on 100ml health drink composition, it is 0.02~5g, preferably 0.3~1g.

As long as the health drink composition of the present invention contains the above-mentioned extracts or compounds in the indicated ratios as essential ingredients, there are no special restrictions on other liquid ingredients, which may be deodorants or natural carbohydrates, etc., such as conventional beverages. Examples of the aforementioned natural carbohydrates are monosaccharides such as glucose, fructose, etc.; disaccharides such as maltose, sucrose, etc.; conventional edible sugars such as dextrin, cyclodextrin; and sugar alcohols such as xylitol, erythritol wait. As deodorants other than the above, natural deodorants such as sweet protein, stevioside extracts such as levaudioside A, glycyrrhizin, etc., and synthetic deodorants such as saccharin, aspen Ba sweet (aspartame) and so on. The amount of the above-mentioned natural carbohydrates is usually about 1-20 g, preferably 5-12 g, based on 100 ml of the beverage composition of the present invention.

Ingredients other than the aforementioned compositions are various nutrients, vitamins, minerals or electrolytes, synthetic flavoring agents, coloring agents and improvers in cheese chocolate, etc., pectic acid and its salts, alginic acid and its salts, Organic acids, protective colloidal binders, pH controllers, stabilizers, preservatives, glycerol, alcohols, carbonating agents for carbonated drinks, etc. Other ingredients than the aforementioned may be fruit juice, juice drink or vegetable drink for preparing natural fruit juice, wherein said ingredients may be used alone or in combination. The ratio of each component is not critical, but is generally about 0 to 20 w/w% per 100 w/w% of the composition of the present invention. Examples of foods that can be added including the aforementioned extract of the present invention are various foods, beverages, chewing gums, vitamin complexes, health-improving foods, and the like.

The extract of the present invention has no toxicity and negative effect, so it can be used safely.

The following examples illustrate the present invention more specifically. However, it should be understood that the present invention is not limited to these examples in any way.

Beneficial effect

The present invention provides a pharmaceutical composition and a health food comprising an extract of twig twig or xantholic acid isolated therefrom as an active ingredient in an amount effective for treating and preventing inflammatory, allergic, and asthmatic diseases .

Description of drawings

In conjunction with the accompanying drawings, the above and other objects, features and other advantages of the present invention can be more clearly understood from the following detailed description, in the accompanying drawings:

Fig. 1 shows the effects of the extracts of Shuizhi and Dianzi acid on lung tissue cells by histological examination using bronchoalveolar lavage (A: normal control mice, B: PBS-treated mice, C: Shuizhi extract treated mice, D: xantholic acid-treated mice),

Fig. 2 shows the inhibitory effect of twig extract and twig acid on OVA-induced inflammation in lung tissue.

Detailed ways

It will be apparent to those skilled in the art that various modifications and changes can be made in the composition, use and preparation of the present invention within the spirit or scope of the present invention.

The following examples illustrate the present invention more specifically. However, it should be understood that the present invention is not limited to these examples in any way.

Example

The following reference examples, examples and experimental examples are used to further illustrate the present invention, but do not limit the scope of the present invention.

Example 1 The preparation of the crude extract of Shuizhi

It was collected in Ulleungdo, South Korea in August 2003, and its voucher specimen (PEB 3068) was collected by the Korea Research Institute of Bioscience and Biotechnology (KRIBB) in Daejeon, Korea. ) plant extract library preservation.

Cut 1.1kg of dried twigs into small pieces, mix with 5L of methanol, stir the mixture at room temperature for 24 hours, and extract three times with cold water. Filter the extract with filter paper to remove residue. The filtrate was collected, concentrated under reduced pressure using a rotary evaporator at 55-65° C., and dried with a freeze dryer to obtain 100.5 g of dried crude extract of Shuizhi chrysanthemum.

The preparation of embodiment 2 butanol soluble components

Add 1 L of distilled water to 100.5 g of the crude extract obtained in Example 1, place it in a separatory funnel, add 1 L of butanol, and shake vigorously to separate it into a butanol-soluble layer and a water-soluble layer.

The above-mentioned butanol-soluble layer was concentrated using a rotary evaporator, and dried with a freeze dryer to obtain a butanol-soluble extract. Finally, 80.0 g of butanol-soluble extract and water-soluble extract were obtained, which were used as samples in the following experiments.

Example 3 Preparation of xanthohydric acid from the extract of Shuizhi

3.29 kg of dried Cladia japonica plants were extracted twice with methanol (10 L) at room temperature to obtain 352 g of extract. The extract was suspended in 1 L of water, and the phases were separated with an equal volume of n-hexane. Then 65.1 g of n-hexane soluble fractions were subjected to silica gel column chromatography (70-230 mesh, 8.5×65 cm), and were washed successively with n-hexane-ethyl acetate mixture (10-20% ethyl acetate, stepwise gradient) and chloroform - Elution with methanol mixture (0-100% methanol, step gradient) to obtain 9 fractions (Fr.1-Fr.9). 7.4 g of component 6 (chloroform-methanol 9/1-7/3, between v/v) was subjected to column chromatography using a normal phase silica gel column (silica gel, 230-400 mesh, 6.0×60 cm, chloroform-methanol mixture, 5-50% methanol, step gradient) to obtain 400 mg xantholic acid. Its structure was determined by NMR ( ¹ H, ¹³ C, DEPT, HMQC, HMBC), EI-MS and optical rotation using those methods reported before (Park et al., ArchPharm Res., 25, pp 57-60, 2002), Use HPLC system (Shimadzu SCL-10A with SPD-M 10Avp PDA detector, column: Phenomenex Synergi 4 μm Fusion RP-80, 4.6×50 mm, elution: ACN/0.1% TFA in distilled water (DW), 4/1 , v/v) analyzed its purity, and the purity was higher than 99.5%.

Xanthoic acid

Acicular (MeOH);

mp 254-256°C;

[α] ^D ₂₃ +94 (pyrimidine, c 0.14);

IR (KBr, cm ⁻¹ ): 3491 (OH), 1689 (CO), 1645 (C=C), 1450, 1388, 1262, 1222, 1044; HRMS m/z 472.3552 (M ⁺ , for C ₃₀ H ₄₈ O ₄ calculated value: 472.3553);

EIMS (rel.int.) m/z: 472[M] ⁺ (61), 454[MH ₂ O] ⁺ (34), 436[M-2H ₂ O] ⁺ (62), 424(42), 396 (26), 205(75), 187(71), 175(87), 173(100);

¹³ C-NMR (150 MHz, pyrimidine-d ₅ ): 13.0 (C-24), 17.4 (C-25), 17.5 (C-26), 18.7 (C-6), 18.8 (C-28), 19.4 ( C-30), 21.3(C-11), 25.8(C-15), 26.7(C-12), 27.9(C-2), 30.1(C-21), 37.7(C-10), 38.2(C -7), 38.3(C-16), 39.2(C-1), 39.6(C-13), 40.4(C-22), 40.8(C-8), 42.9(C-4), 43.0(C- 17), 48.1(C-19), 49.2(C-5), 51.4(C-18), 51.6(C-9), 60.4(C-14), 68.2(C-23), 73.6(C-3 ), 110.2(C-29), 150.9(C-20), 178.3(C-27);

¹ H-NMR (600 MHz, pyrimidine-d ₅ ): 1.05, 1.71 (2H, m, per hydrogen, H-1), 1.82, 1.91 (2H, m, per hydrogen, H-2), 4.02 (1H , dd, J=4.7, 11.6Hz, H-3), 1.51 (1H, dd, J=1.5, 12.0Hz, H-5), 1.48, 1.65 (2H, m, each hydrogen, H-6), 1.87, 2.06 (2H, m, per hydrogen, H-7), 2.02 (1H, dd, J = 1.7, 12.7Hz, H-9), 1.32, 1.64 (2H, m, per hydrogen, H-11 ), 1.87, 2.60 (2H, m, per hydrogen, H-12), 1.88 (1H, m, H-13), 1.67, 2.28 (2H, m, per hydrogen, H-15), 1.70, 1.78 (2H, m, per hydrogen, H-16), 1.81 (1H, m, H-18), 2.60 (1H, m, H-19), 1.36, 1.97 (2H, m, per hydrogen, H- 21), 1.16, 1.40 (2H, m, per hydrogen, H-22), 3.57, 4.07 (2H, d, J = 10.4, per hydrogen, H-23), 1.04 (3H, s, H-24 ), 1.01 (3H, s, H-25), 1.21 (3H, s, H-26), 0.90 (1H, s, H-28). 4.76, 4.96 (2H, s, each hydrogen, H-29 ), 1.86 (3H, d, J=6.4Hz, H-30).

Experimental Example 1 Animal sensitization and airway challenge

Several groups of mice (n=5-6) were studied; they were treated as follows: (1) sham-sensitization and challenge with phosphate-buffered saline (PBS; ipNeb); (2) OVA (ovalbumin: SigmaA5503; Sensitization and excitation of Sigma, St.Louis, MO) (ipNeb); (3) Sensitization with OVA (ip) and excitation with OVA (Neb) and sample (xanthic acid or zileuton, po) . Briefly, mice were sensitized by intraperitoneal injection of 20 μg OVA emulsified with 2 mg aluminum hydroxide in 100 μl PBS buffer solution (pH 7.4) on days 0 and 11. Mice were airway challenged with OVA (1% in PBS) for 20 min using an ultrasonic nebulizer on days 19, 20, 21 and 25 after the initial sensitization. The mice were sacrificed 48 hours after the last challenge (day 27), and the inhibitory effects of the extracts of twig and twig acid on the airway of allergic asthma were determined.

Experimental example 2 MTT analysis

In order to study the cytotoxic effect of the twig extract of the present invention and the twig acid separated therefrom, bromination (3-[4,5-dimethylthiazol-2-yl]-2,5- Diphenyltetrazolium salt (MTT) analysis (Wang Z et al., Biol., Pharm. Bull., 24, pp 159-162, 2001).

Promyelocytic HL-60 cells (HL-18103, 5×10 ⁵ cells/ml) were seeded on 96-well plates in the absence of NGF. After 24 hours of incubation, cells were treated with a sample mixture dissolved in 10 µl of DMSO and 10 µl of MTT solution (5 mg/ml), and incubated under similar conditions for 4 hours. After 4 hours, the MTT was removed and 100 μl of DMSO was dropped into each well to dissolve the crystals. Cell viability was calculated by measuring UV absorbance at 570 nm with a microwell reader (BIO-RAD, USA).

As shown in Table 1, it was confirmed that the extracts or compounds of the present invention did not show cytotoxicity.

[Table 1] Effects of Shuizhi extract and compounds isolated therefrom on HL-60 cells

sample Cell viability (%) 50μM 100μM Swig Extract 100 102 Xanthoic acid 100 102

Experimental Example 3 Preparation and activation of bone marrow-derived mast cells (BMMC)

BMMC were obtained from male Balb/c mice, and were treated with 10% fetal cells containing IL-3 (Sigma I4144, 2ng/ml) (Murakami M et al., J.Bio.Chem., 39, pp 22653-22656, 1995). 50% enriched medium supplemented with bovine serum (RPMI, containing 2mM L-glutamic acid, 25mM HEPES buffer, 2mg/ml sodium bicarbonate, 100 units/ml penicillin G, 100μg/ml streptomycin sulfate, 0.25μg/ml ml amphotericin B) for up to 4 weeks. After 3 weeks of culture, assessment by toluidine blue staining revealed greater than 98% BMMC in the cells.

BMMC were suspended in enriched medium at a cell density of 1 × ¹⁰⁶ cells/ml, then incubated at 37 °C in DMSO (DMSO final concentration less than 0.5%) with or without sample at humidity Incubate in a 5% CO ₂ incubator for 30 min. After being stimulated with stem cell factor (SCF, Sigma S9915, 100 ng/ml) for 20 min, the release of LTC ₄ in the supernatant was measured using an enzyme immunoassay kit (Cayman Chemical, Ann Arbor, MI, USA) according to the manufacturer's instructions. All experiments were performed in triplicate and the inhibition of LTC ₄ release was determined by calculating the percent reduction of LTC ₄ release relative to the control group (Lee SH et al., Biol. Pharm. Bull., 27, pp 786-788, 2004).

Experimental example 4 The effect of twig extract and twig acid on the release of LTC ₄

As described in Experimental Example 3, the inhibitory effect of the extracts of twig twig and twig acid on the release of cysteinyl leukotrienes in BMMB was determined by using the ELISA method of LTC ₄ monoclonal antibody.

The results demonstrate that the _IC50 of xantholic acid is significant, but lower than that of zileuton, which is known as a 5-lipoxygenase inhibitor (see Table 2).

[Table 2]

sample LTC ₄ Release Inhibition (IC ₅₀ ) Swig Extract 19.5μg/ml Xanthoic acid 2.49μM Ziliuton 0.11μM

Experimental Example 5 The effect of twig extract and twig acid on airway hyperresponsiveness (AHR)

AHR was measured 24 hours after the last aerosol challenge using a whole body plethysmograph (OCP3000; Allmedicus, Korea) (Hamelmann E et al., Am J Respir Crit Care Med., 156, pp 766-775, 1997). Each mouse was placed in a biometric plethysmography box and challenged with aerosolized PBS followed by increasing concentrations of aerosolized methacholine (12.5, 25 and 50 mg/ml) for 3 minutes. At each concentration, bronchoconstriction was recorded for an additional 5 min. The highest Penh value for each sample was obtained at each methacholine challenge and expressed as a percentage of the basal Penh value in response to a control (PBS) challenge.

As shown in Table 3, at any concentration of methacholine from 5 to 20 mg/ml, the Penh value of the OVA-treated group was significantly higher than that of the PBS control group (p<0.05). In the challenged group of xanthiic acid+OVA, the Penh value was significantly lower than that of the OVA-treated group (p<0.05). The positive control group of zileuton, which was developed as an anti-asthma drug, showed a decrease in AHR, but lower than that of xantholic acid (see Figure 2).

[Table 3] Effects of twig extract and twig acid on airway hyperresponsiveness (AHR)

sample Penh value Methacholine (mg/ml) 0 5 10 20 OVA-Control 0.73±0.20 1.23±0.48 2.19±0.58 2.89±0.73

Swig Extract 0.60±0.06 1.79±0.47 3.25±0.75 2.54±0.57 ^* Xanthiic acid (30mg/ml) 0.46±0.08 0.96±0.45 ^* 1.54±0.74 ^* 2.15±0.52 ^* Zileuton (30mg/ml) 0.55±0.14 1.17±0.59 1.97±0.83 2.49±0.90 ^* Significant difference from OVA-treated group, p<0.05

Experimental Example 6 The effect of xantholic acid on OVA-specific IgE

Forty-eight hours after the final challenge, mice were sacrificed with an overdose of pentobarbital (Sigma P3761) and a tracheotomy was performed. Ice-cold PBS (0.5 ml) was injected into the lungs, and bronchoalveolar lavage fluid (BALF) (1.5 ml in total) was obtained by inhaling three times through the endotracheal tube. Centrifuge the BALF and collect the supernatant, which is stored at -70 °C until use. The amount of IL-4, IL-5 and IL-13 in BALF was determined using specific mouse ELISA kits (R&D Systems; Minneapolis, MN) according to the manufacturer's instructions. The detection limit of the assay was 250 pg/ml.

Plasma was obtained by cardiac puncture after tracheotomy. Complementary capture and detection antibody pairs for mouse IgE antibodies were purchased from BD OptEIA (San Diego, CA), and enzyme-linked immunosorbent assay (ELISA) was performed according to the manufacturer's instructions. The two plasma samples were diluted 1:100, the IgE level in each sample was obtained by measuring the optical density reading at 450 nm, and the OVA-specific IgE concentration was calculated from the standard curve using recombinant IgE (5-2,000 ng/ml )generate.

As shown in Table 4, the IgE levels of xantholic acid-treated mice were significantly reduced, while zileuton showed a similar inhibitory effect as xantholic acid.

[Table 4]

sample OVA-specific IgE OVA-Control 26.0±10.9 OVA+ xantholic acid (30mg/ml) (inhibition%) 13.3±5.27 ^* (48.8±20.3%) OVA+zileuton (30mg/ml) (inhibition%) 14.1±1.0 ^* (45.8±15.3%) ^* Significant difference from OVA-treated group, p<0.05

Experimental example 7 The effect of twig extract and twig acid on cytokine levels

In order to determine the effects of the extracts of Shuizhi and sizic acid on the release of cytokines in OVA-induced asthmatic mice, the cytokines (IL-4, IL-5 and IL-13) in BALF were measured by ELISA 48 hours after the last challenge s level.

As shown in Table 5, the cytokines in the xantholic acid treatment group were significantly inhibited; the IL-4, IL-5 and IL-13 were reduced by 90.5±4.0%, 54.6±23.0% and 43.7±28.2% or more (p<0.05). Zileuton also showed a greater reduction than the control group, but still much lower than xantholic acid. These results demonstrate that xantholic acid significantly reduces the concentrations of IL-4, IL-5 and IL-13 in the BALF of an asthma model.

[table 5]

sample IL-4 (pg/ml) IL-5(pg/ml) IL-13 (pg/ml) OVA-Control 356.1±14.7 180.4±17.3 145.0±10.9 OVA+White Branch Extract (30mg/ml) (inhibition%) 294.7±38.2 ^* (18.9%) 379.5±94.9 ^* (26.8%) 32.3±9.7 ^** (62.4%) OVA+ xantholic acid (30mg/ml) (inhibition%) 33.8±14.3 ^* (90.5%) 81.9±41.4 ^* (54.6%) 81.7±40.9 ^* (43.7%) OVA+zileuton (30mg/ml) (inhibition%) 289.5±59.0 (18.7%) 157.8±41.3 (12.5%) 130.1±16.8 (10.3%) ^* Significant difference from OVA-treated group, p<0.01 ^** Significant difference from OVA-treated group, p<0.05

Experimental Example 8 The effect of twig extract and twig acid on OVA-induced inflammation in lung tissue

The lung tissue was fixed with 10% neutral buffered formalin for 24 hours, and after embedding in paraffin, the tissue was cut into 4-μm thick sections, and then treated with H&E solution (hematoxylin, Sigma MHS-16 and eosin, Sigma HT110-1-32) staining. Subsequently, the stained tissue was fixed with Dako-mounting medium (Dakocytomation; Denmark Carpinteria CA) and coverslipped. The degree of cellular infiltration in the airways was scored in a double-blind experiment by two independent investigators (Myou S et al., J. Exp. Med., 198, pp 1573-1582, 2003). Peribronchiolitis and perivasculitis were evaluated using specific criteria, that is, a score of 0–3, where 0, no cells; 1, a few cells; 2, rings of cells with a depth of 1 to 5 cell layers; 3, a depth greater than Thick ring of cells with 5 cell layers. In order to evaluate the inhibitory effect of twig extract and xantholic acid on leukocyte infiltration, the degree of inflammation was scored by quantitative analysis in lung tissue 48 hours after the last challenge (see FIG. 2 ).

As shown in Fig. 1, salicylic acid had the most effective inhibitory effect on the inflammation in the lung tissue of mice induced by OVA, followed by the extract of spatula and zileuton. In H&E staining of lung tissue, leukocytes from OVA-treated mice infiltrated massively into the peribronchiolar and perivascular connective tissues of normal mice. The infiltration of eosinophil-rich leukocytes was significantly attenuated in mice treated with twig extract or twig acid compared with OVA-treated mice.

Experimental example 9 The effect of chrysanthemum extract and xantholic acid on carrageenan-induced mouse paw edema

Determination of the inhibitory effect of the twig extract and twig acid on the formation of edema in ICR mice prepared according to the above examples is as follows.

The mice were divided into three groups, each group consisted of 6 mice, that is, as a negative control group, T1 treated with solvent only, T2 treated with 50 mg/kg of the Aspirin-treated T3. One hour after the treatment, 1% carrageenan solution was injected into the ankle of the rat to induce edema, and the thickness of the ankle was measured using a Vernier caliper to determine the degree of edema. The measured thickness was calculated according to Mathematical Formula 1 below.

[mathematical formula 1]

Ankle thickness ratio (%)=[(the maximum thickness of rat paw edema)-(the thickness of rat paw edema before treatment)/(the thickness of rat paw edema before treatment)]×100

As shown in Table 6, edema reached a maximum after 4 hours of treatment. Therefore, it was confirmed that the extract of twig twig and twig acid showed an effective inhibitory effect on mouse paw edema.

[Table 6]

Hour T1 T2 T3 Ankle Thickness Ratio (%) Ankle Thickness Ratio (%) inhibition(%) Ankle Thickness Ratio (%) inhibition(%) 0 100 - - - - 1 119.2±15.6 125.1±1.5 -5.0±1.3 139.3±40.6 -16.8±34.0 2 158.0±9.0 146.4±19.4 7.3±12.3 170.4±34.5 -7.8±21.8 3 194.9±12.4 166.8±18.8 14.4±9.7 180.6±28.6 7.3±14.7 4 205.9±19.0 180.6±26.9 12.3±13.1 196.9±15.4 4.3±7.5 5 201.6±5.1 193.4±22.4 4.1±11.1 198.5±12.6 1.5±6.3 T1: Control T2: 50mg/kg Shuizhi extract T3: 50mg/kg aspirin

Hereinafter, the formulation method and various excipients will be described, but the present invention is not limited thereto. Representative preparations are as follows.

Preparation of Injections

The dry powder of embodiment 1 or xantholic acid 100mg

Sodium metabisulfite 3.0mg

Methylparaben 0.8mg

Propylparaben 0.1mg

Distilled water for injection Appropriate amount

Injection forms are prepared by dissolving the active ingredient, controlling the pH to about 7.5, and then filling all components into 2 ml ampoules and sterilizing by conventional injection preparation methods.

Preparation of powder

The dry powder of embodiment 1 or xantholic acid 500mg

cornstarch 100mg

Lactose 100mg

Talc powder 10mg

Powders are prepared by mixing the above components and filling into sealed packages.

Tablet preparation

The dry powder of embodiment 1 or xanthohydric acid 200mg

cornstarch 100mg

Lactose 100mg

Magnesium Stearate Appropriate amount

Tablets are prepared by mixing the above ingredients and compressing.

Preparation of capsules

The dry powder of Example 1 or xantholic acid 100mg

Lactose 50mg

cornstarch 50mg

Talc powder 2mg

Magnesium Stearate Appropriate amount

Capsules are prepared by mixing the above ingredients and filling gel capsules by conventional gel preparation methods.

Preparation of Liquids

The dry powder of Example 1 or xantholic acid 1000mg

sugar 20g

Polysaccharide 20g

Lemon Spice 20g

Liquid dosage forms are prepared by dissolving the active ingredient, then filling all components into 1000 ml ampoules and sterilizing using conventional liquid preparation methods.

Preparation of healthy food

The dry powder of Example 1 or xantholic acid 1000mg

Vitamin Mixture Appropriate amount

Vitamin A acetate 70μg

Vitamin E 1.0mg

Vitamin _B1 0.13mg

Vitamin _B2 0.15mg

Vitamin _B6 0.5mg

Vitamin _B12 0.2μg

Vitamin C 10mg

Biotin 10μg

Niacinamide 1.7mg

Folic acid 50μg

Calcium pantothenate 0.5mg

Mixture of minerals Appropriate amount

Ferrous Sulfate 1.75mg

Zinc Oxide 0.82mg

Magnesium Carbonate 25.3mg

Potassium dihydrogen phosphate 15mg

Calcium hydrogen phosphate 55mg

Potassium citrate 90mg

Calcium Carbonate 100mg

Magnesium chloride 24.8mg

The mixture of vitamins and minerals described above can be varied in many ways. Such changes are not considered to depart from the spirit and scope of the invention.

Preparation of healthy drinks

The dry powder of embodiment 1 or xantholic acid 1000mg

Citric acid 1000mg

Oligosaccharides 100g

Apricot Concentrate 2g

Taurine 1g

Distilled water 900ml

Health drinks were prepared by dissolving the active ingredients, mixing, stirring at 85°C for 1 hour, filtering, then filling all components into 1000ml ampoules and sterilizing using conventional health drink preparation methods.

Having thus described the invention, it will, however, be obvious that the invention may be varied in many ways. Such changes are not to be regarded as a departure from the spirit and scope of the invention, and all changes obvious to one skilled in the art are intended to be included within the scope of the appended claims.

Industrial Applicability

As described in the present invention, twig extract and xantholic acid isolated therefrom showed inhibitory effects on LTC ₄ release in vitro, as well as effects on IgE levels and cytokines (IL-4, IL-4) in OVA-induced asthmatic mice. 5 and IL-13), airway hyperresponsiveness and inhibition of leukocyte infiltration. Therefore, they are useful as therapeutic agents or functional health foods for the treatment and prevention of inflammatory, allergic and asthmatic diseases.

Claims (3)

1. The use of the crude methanol extract or the butanol-soluble extract of Shuizhi for the preparation of a medicament for treating or preventing asthma. 2. The use of the crude methanol extract or the butanol-soluble extract of Shuizhi for the preparation of functional health food for preventing and improving asthma, the functional health food containing the crude extract of Shuizhi Methanol extract or butanol-soluble extract of Sclera chinensis and dietary acceptable additives. 3. The use of the crude methanol extract or the butanol-soluble extract of Shuizhi as claimed in claim 2, wherein the health food can be in the form of pills, powders, granules, tablets, capsules or beverages.

Images