Title of Invention	"PHARMACEUTICAL COMPOSITION COMPRISING AN EXTRACT OF PSEUDOLYSIMACHION LONGIFOLIUM AND THE CATALPOL DERIVATIVES ISOLATED THEREFROM HAVING ANTIINFLAMMATORY, ANTIALLERGIC AND ANTIASTHMATIC ACTIVITY"
Abstract	The present invention relates to a composition comprising an extract of Pseudolysimachion genus plant, and the catalpol derivatives isolated therefrom having anti-inflammatory, antiallergic and anti-asthmatic activity. The extract of Pseudolysimachion genus plant and the catalpol derivatives isolated therefrom shows potent suppressing effect on elevated IgE, IL-4 and IL- 13 levels and eosinophilia in the plasma and BALF, and mucus overproduction in the lung tissues in an OVA-induced asthmatic mouse model. Therefore, it can be used as the therapeutics or functional health food for treating and preventing inflammatory, allergic and asthmatic disease.

Title of Invention

"PHARMACEUTICAL COMPOSITION COMPRISING AN EXTRACT OF PSEUDOLYSIMACHION LONGIFOLIUM AND THE CATALPOL DERIVATIVES ISOLATED THEREFROM HAVING ANTIINFLAMMATORY, ANTIALLERGIC AND ANTIASTHMATIC ACTIVITY"

Abstract

The present invention relates to a composition comprising an extract of Pseudolysimachion genus plant, and the catalpol derivatives isolated therefrom having anti-inflammatory, antiallergic and anti-asthmatic activity. The extract of Pseudolysimachion genus plant and the catalpol derivatives isolated therefrom shows potent suppressing effect on elevated IgE, IL-4 and IL- 13 levels and eosinophilia in the plasma and BALF, and mucus overproduction in the lung tissues in an OVA-induced asthmatic mouse model. Therefore, it can be used as the therapeutics or functional health food for treating and preventing inflammatory, allergic and asthmatic disease.

Full Text	PHARMACEUTICAL COMPOSITION COMPRISING AN EXTRACT OF PSEUDOLYSIMACHION LONGIFOLIUM AND THE CATALPOL DERIVATIVES ISOLATED THEREFROM HAVING ANTIINFLAMMATORY, ANTIALLERGIC AND ANTIASTHMATIC ACTIVITY BACKGROUND OF THE INVENTION Technical Field The present invention relates to a composition comprising an extract of Pseudolysimachion longifolium and the catalpol derivatives isolated therefrom having anti-inflammatory, anti-allergic and anti-asthmatic activity. Background Art Asthma has been regarded as a complex syndrome occurring in the airways, which shows various disorders such as airflow obstruction, acute or chronic inflammation, airway hyper-responsiveness (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 critical role in asthma development and the number of patients suffering from allergic asthma has been increased to about 10% of the population in the world recently. It has been reported that the number has been reached to seventeen million in America and the market scale of the medication for allergic asthma has been enlarged to 640 billion $ in America till now. Asthma can be classified into two types, i.e., extrinsic asthma and intrinsic asthma. Extrinsic asthma caused by the exposure of antigen such as house dust mite Dermatophagoides as a main antigen, pollen, epithelium of animal, fungi etc shows positive reaction in skin test or bronchial provocation test against the antigen, and generally occurs in younger people. Intrinsic asthma caused by upper respiratory infection, exercise, emotional instability, cold weather, the change of humidity occurs in adult patients. According to the aspect of pathophysiology, asthma has been recognized as a chronic inflammation occurred by following procedure; Inflammatory cells are proliferated, differentiated, and activated caused by cytokines reproducing in T-helper 2 immune cells and is moved to air way or neighboring tissue thereof. The activated inflammatory cells such as neutrophil, mast cell etc release a variety of inflammatory mediators, such as cytokines, chemokines, signaling molecules, adhesion molecules and growth factors and the structural cells in airways are involved in various stages of asthma (Elias JA et al., J Clin Invest., Ill, pp 291-7, 2003). In numerous studies using knockout mice models and clinical research, the critical observations in asthma could fall into several characteristic parameters, such as immune responses, eosinophilia, AHR and structural remodeling (Moffatt JD. Pharmacol Ther, 107, pp 343-57, 2005; Spina D et al., Trends Pharmacol Sci, 23, pp 311-5, 2002). Each of the parameters seems not to have direct correlations with one another; however, IgE-mediated immune response 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, pp 1033-9, 1990), and the produced cytokines such as IL-4, IL-5 and IL-13 in the allergic process also play an important role in AHR development and airway remodeling (Riffo-Vasquez Y et al., Pharmacol. Ther., 94, pp 185-211, 2002). Indeed, asthma is a result of orchestrated inflammatory events, many of which involve specific inhibitors acting on the pathway of asthma, for example, histamine HI antagonists, thromboxane antagonists, platelet-activating-factor antagonists, cyclooxygenase inhibitors, nitrogen monooxygenase inhibitors and prostaglandin inhibitors, have been tried but have failed in clinical trials (Moffatt J.D., Pharmacol. Ther., 107, pp 343-57, 2005). In contrast, glucocorticoids, which suppress the progenitor levels of inflammatory cells to baseline by widespread inhibition of cytokine synthesis and cytokine mediated immune-cell survival, has been used to manage the symptoms of asthmatic patients over a period of 30 years as far (Baatjes A.J. et al., Pharmacol, Then, 95, pp 63-72, 2002). These reports suggest that the therapeutic approach for asthma management should focus on restoring the balance of asthmatic parameters rather than searching for potent inhibitors of specific pathways of the asthmatic process. Pseudolysimachion longifolium belonged to Pseudolysimachion genus, is a perennial herb distributed in Korea, China, Russia and Europe. Numerous species of same genus for example, Pseudolysimachion ovutum, Pseudolysimachion kiusianum, Pseudolysimachion kiusianum var diamanticum, Pseudolysimachion kiusianum var villosum, Pseudolysimachion dahuricum, Pseudolysimachion pyrethrinum, Pseudolysimachion linarifolium, Pseudolysimachion linarifolium var. villosulum, Pseudolysimachion rotundum var. subintegrum, Pseudolysimachion rotundum var. coreanum, Pseudolysimachion insulare, and Pseudolysimachion undulata have been reported and the plants contains mannitol, 6-hydroxyIuteolin as a main ingredient (Chung BS and Shin MK, HyangyakDaeSaJeon, Youngrimsa, pp913-914, 1998). However, there has been not reported or disclosed about the suppressive effect on inflammatory, allergic and asthmatic disease of the extract from P. longifolium and the catalpol derivatives isolated therefrom in any of above cited literatures, the disclosures of which are incorporated herein by reference. Accordingly, the present inventors have discovered that the extract of P. longifolium and the catalpol derivatives isolated therefrom show the suppressive effect on asthmatic parameters, such as IgE level, cytokine release, and eosinophilia, AR and mucus hypersecretion in OVA-sensitized/challenged mouse model and finally completed the present invention. SUMMARY OF THE INVENTION The present invention provides a pharmaceutical composition and a health food comprising an extract or catalpol derivatives isolated from P. longifolium as an active ingredient in an effective amount to treat and prevent inflammatory, allergic and asthmatic disease. The present invention also provides a use of an extract of P. longifolium and the catalpol derivatives isolated therefrom showing anti-inflammatory, anti-allergic and anti-asthmatic activity. The present invention also provides a method of treating or preventing inflammatory, allergic and asthmatic disease in a mammal comprising administering to said mammal an effective amount of an extract of P. longifolium and the catalpol derivatives isolated therefrom, together with a pharmaceutically acceptable carrier thereof. Disclosure of the invention Accordingly, it is an object of the present invention to provide a composition comprising a crude extract or organic solvent soluble extract of Pseudolysimachion genus plant, as an active ingredient for the treatment and prevention of inflammatory, allergic and asthmatic disease. The term "crude extract" disclosed herein comprises the extract prepared by extracting plant material with water, lower alcohols such as methanol, ethanol, preferably methanol and the like, or the mixtures thereof. The term "organic solvent soluble extract" disclosed herein can be prepared by extracting the above described crude extract with organic solvent, for example, butanol, acetone, ethyl acetate, chloroform or dichloromethane, preferably butanol. The term "Pseudolysimachion genus" disclosed herein comprises P. longifolium, P. ovtum, P. kiusianum, P. kiusianum var. diamanticum, P. kiusianum var. villosum, P. dahuricum, P. pyrethrinum, P, linarifolium, P. linarifolium var. villosulum, P. rotundum var. subintegrum, P. rotundum var. coreanum, P. insulare and P. undulate, The present invention provides a pharmaceutical composition comprising catalpol derivatives represented by following chemical formula (I), or a pharmaceutically acceptable salt thereof as an active ingredient in an effective amount to treat and prevent inflammatory, allergic and asthma disease. (Figure Removed) Wherein, R is independently at least one groups selected from a hydrogen atom, benzoyl or cinnamoyl group substituted with C\-Ci lower alkyl group or C\-Cj lower alkoxy group. In the above formula (I), preferably, R group comprises 3, 4-dihydroxybenzoyl, 4-hydroxy-3-methozybenzoyl, 3-hydroxy-4-methozybenzoyl, 4-hydroxybenzoyl, 3,4-dimethoxybenzoyl, 3,4-dihydroxycinnamoyl and 3-hydroxy-4-methoxycinnamoyl. The catalpol derivatives of the present invention can be isolated from P. long/folium or synthesized by general procedure well known in the art (Herbert O. house., Modern Synthetic Reactions, 2nd Ed., The Benjamin/Cummings Publishing Co., 1972). In accordance with another aspect of the present invention, there is also provided a use of crude extract or organic solvent soluble extract of Pseudolysimachion genus plant, or the catalpol derivatives isolated therefrom for manufacture of medicines employed for treating or preventing inflammatory, allergic and asthmatic disease. In accordance with another aspect of the present invention, there is also provided a method of treating or preventing inflammatory, allergic and asthmatic disease in mammals, wherein the method comprises administering a therapeutically effective amount of crude extract or organic solvent soluble extract of Pseudolysimachion genus plant, or the catalpol derivatives isolated therefrom into the mammal suffering with inflammatory, allergic and asthmatic disease. An inventive extract isolated from Pseudolysimachion genus plant, and the catalpol derivatives isolated therefrom may be prepared in accordance with the following preferred embodiment. Hereinafter, the present invention is described in detail. For the present invention, for example, the dried leave off! longifolium is cut into small pieces and the piece was mixed with 2 to 20-fold, preferably, 5 to 10-fold volume of polar solvent, for example, water, Q-C4 lower alcohol such as methanol, ethanol, butanol, or the mixtures thereof, preferably methanol; and was heated at the temperature ranging from 20 to 100°C, preferably from 20 to 50 °C, for the period ranging 10 to 48 hours, preferably 20 to 30 hours, by reflux extraction with hot water, cold water extraction, ultra-sonication or conventional extraction, preferably by cold water extraction; the residue was filtered and then the filtrate is dried to obtain polar solvent soluble extract thereof. In the above crude extract prepared by above step, is suspended in water, and then is mixed with 1 to 100-fold, preferably, 1 to 5-fold volume of organic solvent butanol, acetone, ethyl acetate, chloroform or dichloromethane, preferably butanol to obtain organic solvent soluble extract of the present invention. The above organic solvent soluble extract is further subjected to silica gel column chromatography filled with silicagel eluting with mixture solvent of chlorofornrmethanol with increasing the polarity by changing the mixed ratio (methanol 0-100%, step gradient) to obtain several fractions. Among the fractions, the 3rd fraction is further subjected to repeated silica gel column chromatography using a normal phase silica column (methanol 10-50% step gradient) to obtain the catalpol derivatives of the present invention. The structure was confirmed by NMR, EI-MS and optical rotation with those reported previously (Afifi-Yazar Ft) et al., Helv Chim Acta, 63, pp 1905-7, 1980) and the purity of the catalpol derivatives was analyzed as more than 99.5% by HPLC system. In accordance with another aspect of the present invention, there is provided a pharmaceutical composition comprising a crude extract and organic solvent soluble extract of P. longifolium or the catalpol derivatives isolated therefrom prepared by the above describe preparation method for the treatment and prevention of inflammatory, allergic and asthmatic disease as active ingredients. In accordance with another aspect of the present invention, there is also provided a use of comprising a crude extract and organic solvent soluble extract of P. longifolium or the catalpol derivatives isolated therefrom prepared by the above describe preparation method for manufacture of medicines employed for treating or preventing inflammatory, allergic and asthmatic disease. In accordance with another aspect of the present invention, there is also provided a method of treating or preventing inflammatory, allergic and asthmatic disease, wherein the method comprises administering a therapeutically effective amount of comprising a crude extract and organic solvent soluble extract of P. longifolium or the catalpol derivatives isolated therefrom prepared by the above describe preparation method. The inventive compound represented by general formula (§j) can be transformed into their pharmaceutically acceptable salt and solvates by the conventional method well known in the art. For the salts, acid-addition salt thereof formed by a pharmaceutical ly acceptable free acid thereof is useful and can be prepared by the conventional method. For example, after dissolving the compound in the excess amount of acid solution, the salts are precipitated by the water-miscible organic solvent such as methanol, ethanol, acetone or acetonitrile to prepare acid addition salt thereof and further the mixture of equivalent amount of compound and diluted acid with water or alcohol such as glycol monomethylether, can be heated and subsequently dried by evaporation or filtrated under reduced pressure to obtain dried salt form thereof. As a free acid of above-described method, organic acid or inorganic acid can be used. For example, organic acid such as methansulfonic acid, p-toluensulfonic 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, vanillic acid, hydroiodic acid and the like, and inorganic acid such as hydrochloric acid, phosphoric acid, sulfuric acid, nitric acid, tartaric acid and the like can be used herein. Further, the pharmaceutical ly acceptable metal salt form of inventive compounds may be prepared by using base. The alkali metal or alkali-earth metal salt thereof can be prepared by the conventional method, for example, after dissolving the compound in the excess amount of alkali metal hydroxide or alkali-earth metal hydroxide solution, the insoluble salts are filtered and remaining filtrate is subjected to evaporation and drying to obtain the metal salt thereof. As a metal salt of the present invention, sodium, potassium or calcium salt are pharmaceutical ly suitable and the corresponding silver salt can be prepared by reacting alkali metal salt or alkali-earth metal salt with suitable silver salt such as silver nitrate. The pharmaceutically acceptable salt of the present compound comprise all the acidic or basic salt which may be present at the compounds, if it does not indicated specifically herein. For example, the pharmaceutically acceptable salt of the present invention comprise the salt of hydroxyl group such as the sodium, calcium and potassium salt thereof; the salt of amino group such as the hydrogen bromide salt, sulfuric acid salt, hydrogen sulfuric acid salt, phosphate salt, hydrogen phosphate salt, dihydrophosphate salt, acetate salt, succinate salt, citrate salt, tartarate salt, lactate salt, mandelate salt, methanesulfonate(mesylate) salt and p-. toluenesulfonate (tosylate) salt etc, which can be prepared by the conventional method well known in the art. The inventive composition for treating and preventing inflammatory, allergic and asthmatic disease may comprises the above described extracts or compounds as 0.1 ~ 50% by weight based on the total weight of the composition. The composition according to the present invention can be provided as a pharmaceutical composition containing pharmaceutically acceptable carriers, adjuvants or diluents, e.g., lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starches, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, polyvinyl pyrrolidone, water, methylhydroxy benzoate, propylhydroxy benzoate, talc, magnesium stearate and mineral oil. The formulations may additionally include fillers, anti-agglutinating agents, lubricating agents, wetting agents, flavoring agents, emulsifiers, preservatives and the like. The compositions of the invention may be formulated so as to provide quick, sustained or delayed release of the active ingredient after their administration to a patient by employing any of the procedures well known in the art. For example, the compositions of the present invention can be dissolved in oils, propylene glycol or other solvents that are commonly used to produce an injection. Suitable examples of the carriers include physiological saline, polyethylene glycol, ethanol, vegetable oils, isopropyl myristate, etc., but are not limited to them. For topical administration, the extract of the present invention can be formulated in the form of ointments and creams. Pharmaceutical formulations containing present composition may be prepared in any form, such as oral dosage form (powder, tablet, capsule, soft capsule, aqueous medicine, syrup, elixirs pill, powder, sachet, granule), or topical preparation (cream, ointment, lotion, gel, balm, patch, paste, spray solution, aerosol and the like), or injectable preparation (solution, suspension, emulsion). The composition of the present invention in pharmaceutical dosage forms may be used in the form of their pharmaceutically acceptable salts, and also may be used alone or in appropriate association, as well as in combination with other pharmaceutically active compounds. The desirable dose of the inventive extract or compound varies depending on the condition and the weight of the subject, severity, drug form, route and period of administration, and may be chosen by those skilled in the art. However, in order to obtain desirable effects, it is generally recommended to administer at the amount ranging from 0.0001 to lOOmg/kg, preferably, 0.001 to lOmg/kg by weight/day of the inventive extract of the present invention. The dose may be administered in single or divided into several times per day. The pharmaceutical composition of present invention can be administered to a subject animal such as mammals (rat, mouse, domestic animals or human) via various routes. All modes of administration are contemplated, for example, administration can be made orally, rectally or by intravenous, intramuscular, subcutaneous, intracutaneous, intrathecal, epidural or intracerebroventricular injection. It is the other object of the present invention to provide a functional health food comprising the extract or compounds isolated from P. longifolium together with a sitologically acceptable additive for the prevention and alleviation of inflammatory, allergic and asthmatic disease. To develop for functional health food, examples of addable food comprising the above extracts or compounds of the present invention are various food, beverage, gum. vitamin complex, health improving food and the like, and can be used as powder, granule, tablet, chewing tablet, capsule or beverage etc. The above described composition therein can be added to food, additive or beverage, wherein, the amount of the above described extract or compound in food or beverage may generally range from about 0.01 to 80w/w%, preferably 0.01 to 15w/w% of total weight of food for the health food composition and 0.02 to 5g, preferably 0.3 to Ig on the ratio of 100ml of the health beverage composition. Providing that the health beverage composition of present invention contains the above described extract or compound as an essential component in the indicated ratio, there is no particular limitation on the other liquid component, wherein the other component can be various deodorant or natural carbohydrate etc such as conventional beverage. Examples of aforementioned natural carbohydrate are monosaccharide such as glucose, fructose etc; disaccharide such as maltose, sucrose etc; conventional sugar such as dextrin, cyclodextrin; and sugar alcohol such as xylitol, and erythritol etc. As the other deodorant than aforementioned ones, natural deodorant such as taumatin, stevia extract such as levaudioside A, glycyrrhizin et al., and synthetic deodorant such as saccharin, aspartam et al., may be useful favorably. The amount of above described natural carbohydrate is generally ranges from about 1 to 20 g, preferably 5 to 12 g in the ratio of 100 mi of present beverage composition. The other components than aforementioned composition are various nutrients, a vitamin, a mineral or an electrolyte, synthetic flavoring agent, a coloring agent and improving agent in case of cheese chocolate et al., pectic acid and the salt thereof, alginic acid and the salt thereof, organic acid, protective colloidal adhesive, pH controlling agent, stabilizer, a preservative, glycerin, alcohol, carbonizing agent used in carbonate beverage et al. The other component than aforementioned ones may be fruit juice for preparing natural fruit juice, fruit juice beverage and vegetable beverage, wherein the component can be used independently or in combination. The ratio of the components is not so important but is generally range from about 0 to 20 w/w % per 100 w/w % present composition. Examples of addable food comprising aforementioned extract therein are various food, beverage, gum, vitamin complex, health improving food and the like. Inventive extract of the present invention has no toxicity and adverse effect therefore they can be used with safe. The present invention is more specifically explained by the following examples. However, it should be understood that the present invention is not limited to these examples in any manner. Brief description of Drawings The above and other objects, features and other advantages of the present invention will more clearly understood from the following detailed description taken in conjunction with the accompanying drawings, in which; Fig. 1 shows the effects of an extract of P. longifolium extract on the recruitment of inflammatory cells in bronchoalveolar lavage fluid, Fig. 2 shows the effects of the fraction 3 isolated from P. longifolium, verproside and picroside n on the recruitment of inflammatory cells in bronchoalveolar lavage fluid, Fig. 3 represents the effects of an extract of P. longifolium on lung tissue cell using by the histological examination of bronchoalveolar lavage (A: normal control mice, B: PBS-treated mice, C: P. longifolium extract-treated mice), Fig. 4 presents the effects of an extract of P. longifolium and the compounds isolated therefrom on lung tissue cell using by the histological examination of bronchoalveolar lavage (A: normal control mice, B: PBS-treated mice. C: verproside-treated mice, D: picroside n-treated mice, E: montelukast-treated mice). Best Mode for Carrying Out the Invention It will be apparent to those skilled in the art that various modifications and variations can be made in the compositions, use and preparations of the present invention without departing from the spirit or scope of the invention. The present invention is more specifically explained by the following examples. However, it should be understood that the present invention is not limited to these examples in any manner. EXAMPLES The following Reference Example, Examples and Experimental Examples are intended to further illustrate the present invention without limiting its scope. Example 1. Preparation of the crude extract of P. longifolium 7.9kg of dried P. longifolium cut into small pieces, mixed with SOL of methanol and the mixture was stirred at room temperature for 24 hours, extracted by cold water three times. The extract was filtered with filter paper to remove the debris. The filtrate was pooled and concentrated by rotary evaporator 55-65*0 under reduced pressure and dried with freezing dryer to obtain 950.5g of dried crude extract of P. longifolium. Example 2. Preparation of polar solvent and non-polar solvent soluble extract 2-1. Preparation of ethyl acetate soluble fraction 10L of distilled water was added to 425 g of the crude extract obtained in Example 1. 1OL of ethyl acetate was added thereto in separator/ funnel and shaken vigorously to divide into ethyl acetate soluble layer and water soluble layer. Above ethyl acetate soluble layer was concentrated by rotary evaporator, dried with freeze dryer to obtain ethyl acetate soluble extract. 2-2. Preparation of butanol/water soluble fraction Water soluble layer was fractionated by mixing with 10 L of butanol and finally, 144.Og of n-butanol soluble extract and water soluble extract were obtained to use as a sample in the following experiments. Example 3. Preparation of catalpol derivatives from the extract of P. longifolium 3-1. Preparation of verproside(6-O-3,4-Dihydroxybenzoyi catalpol) 144.0g of n-butanol soluble fraction was subjected to a silica gel column chromatography (70-230 mesh, 8.5X65cm) and eluted with a chloroform-methanol mixture (methanol 0-100%, step gradient) to obtain five fractions. 29. Ig of fraction 2 (between chloroform-methanol 7/3-6/4, v/v) was subjected to repeated column chromatography using a normal phase silica column chromatography (silica gel, 230-400 mesh, 6.0X60 cm, chloroform-methanol mixture, methanol 10-50% step gradient). The fractions 2-4 was performed to recrystalization in methanol to obtain 14.2g of verproside, i.e., 6-0-3,4-Dihydroxybenzoyl catalpol. The structure was confirmed by NMR ('H, 13C, DEPT, HMQC, HMBC), EI-MS and optical rotation with those reported previously (Afifi-Yazar FO et al., Helv Chim Acta, 63, pp 1905-7, 1980) and the purity of verproside was analyzed as more than 99.5% by HPLC system (Shimadzu SCL-10A with SPD-M 10A vp PDA detector, column; Phenomenex Synergi 4 urn Fusion RP-80, 4.6x150 mm, elution: MeOH/DW, 35/65, v/v, 0.8 ml/min). 6-Q-3.4-Dihvdroxvbenzoyl catalpol(verproside) 'H NMR (400 MHz, DMSO-d6) 8: 2.47(1H, dd, J=8.0, 9.2 Hz, H-9), 2.59(1H, dddd, J=\.6, 4.0, 8.0, 8.0, H-5), 3.00(1H, m, H-G4), 3.05 (1H, m, H-G2), 3.14(1H, m, H-G5), 3,18(1H, m, H-G3), 3.42, 3.71(2H, m, H-G6). 3.67(1 H, s, H-7), 3.71, 3.91(2H, d, J=13.2 Hz, each, H-10), 4.61(1H, d, J=7.6 Hz, H-G1), 4.94(1H, dd, 7=4.0, 6.0 Hz, H-4), 5.03 (1H, d, J=8.0 Hz, H-6), S.09(1H, d, .7=9.2 Hz, H-l), 6.41(1H, dd, .7=1.6. 6.0 Hz, H-3), 6.82(1H, d, .7=8.0 Hz, H-51), 7.35(1H, dd, .7=2.0, 8.0 Hz, H-6'), 7.39(1 H, d, 7=2.0 Hz, H-2'). 13C-NMR (100 MHz, DMSO- d6) 5: 93.0(C-1), 141.1(C-3), 101.8 (C-4), 35.2(C-5), 79.5(C-6), 58.2(C-7), 65.8(C-8), 41.8(C-9), 120.0 (C-l1), 116.4(C-2'), 145.1(C-3T), 150.8(C-4'), 115.4(C-5'), 122.6 (C-61), 165.6(C-7'), 97.9(C-G1), 73.4(C-G2), 76.4(C-G3), 70.3(C-G4), 77.5(C-G5), 61.4(C-G6). 3-2. Preparation of isovanillyl catalpol from the extract of P. long/folium 17.3g of fraction 3 was subjected to column chromatography using a normal phase silica column (silica gel, 230-400 mesh, 6.0x60 cm, chloroform-methanol mixture, methanol 10-50% step gradient). 8.5g of fraction 3-3 was performed to recrystalization in methanol to obtain 7.2g of isovanillyl catalpol, i.e., 6-0-3-hydroxy-4-methoxybenzoly catalpol. 6-0-3-hvdroxv-4-methoxvbenzolv catalpoKisovanillvl catalpol) 'H-NMR (400 MHz, DMSO-d6) 8: 2.47(1H, m, H-9), 2.55(1H, m H-5), 3.00(1H, m, H-G4), 3.05 (IH, m, H-G2), 3.14(1H, m, H-G5), 3.18(1H, m, H-G3), 3.43, 3.70(2H, m, H-G6), 3.70(1H, br s, H-7), 3.72, 3.92(2H, d, .7=13.2, each, H-10), 4.62(1H, d, .7=8.0 Hz, H-Gl), 4.95(1H, dd, .7=4.4, 6.0 Hz, H-4), 5.06 (IH, d, .7=8.0 Hz, H-6), 5.11(1H, d, .7=9.2 Hz, H-l), 6.42(1H, d, 7=6.0 Hz, H-3), 7.04(1H, d, .7=8.4 Hz, H-51), 7.42(1H, brs, H-2'), 7.48(1H, d, J= 8.4 Hz, H-61), 3.84(3H, s, 4'-O-CH3). UC-NMR (100 MHz, DMSO- d6) 5: 93.0(C-1), 141.0(C-3), 101,6 (C-4), 35.2(C-5), 79.7(C-6), 58.2(C-7), 65.8(C-8), 41.8(C-9), 58.4(C-10), 121.7(C-I'), 115.7(C-2'), 146.3(C-3'), 152.1(C-4'), 111.4(C-5'), 121.3 (C-61), 165.3(C-7'), 97.8(C-Gl), 73.4(C-G2), 76.4(C-G3), 70.3(C-G4), 77.4(C-G5), 61.4(C-G6), 55.7(4'-OCH3). 3-3. Preparation of picrosidell and verminoside from the extract of P. longifolium 1.5g of fraction 3-5 was subjected to reversed phase silica gel column(RP-l 8, YMC Gel ODS-A, 6.0X60 cm, methanol/water, 1/4, v/v), subjected to sepadex LH-20 column chromatography(methanol/water, 85/15, v/v) to obtain lOl.Omg of picrosidell, i.e., 6-O-4-hydroxy-3-methozybenzoyl and 30.0mg of verminoside, i.e., 6-0-3,4-dihydroxycinnamoyl catalpol. 6-0-4-hvdroxv-3-methozvPcnzovl (picrosidell) 'H-NMR (400 MHz, DMSO-d6) 6: 2.47(1 H, M, 7=8.0, 9.6 Hz, H-9), 2.58(1H, dddd, 7=1.2, 6.0, 8.0, 8.4 Hz, H-5), 3.00(1 H, m, H-G4), 3.05 (IH, m, H-G2), 3.14(1H, m, H-G5), 3.18(1H, m, H-G3), 3.42, 3.71(2H, m, H-G6), 3.67(1H, brs, H-7), 3.72, 3.92(2H, oT, 7=13.2, each, H-10), 4.62(1H, d, J=7.6 Hz, H-G1), 4.99(1H, dd, 7=4.4, 6.0 Hz, H-4), 5.06 (IH, d, .7=8.4 Hz, H-6), 5.11(1H, d, 7=9.6 Hz, H-1), 6.42(1H, dd, 7=1.2. 6.0 Hz, H-3), 6.89(1H, d, 7=8.4 Hz, H-51), 7.46(1 H, d, 7=2.0 Hz, H-21), 7.52(1 H, dd, 7=2.0, 8.4 Hz, H-6'), 3.83(3H, s, 3'-O-CH3). 13C-NMR (100 MHz, DMSO- d6) 8: 93.0(C-1), 141.1(0-3), 101.8 (0-4), 35.2(0-5), 79.7(0-6), 58.2(0-7), 65.8(0-8), 41.8(0-9), 58.5(0-10), 120.0(0-1'), 112.7(0-2'), 147.5(0-3'), 152.0(0-4'), 115.3(0-5'), 123.8 (C-61), 165.6(0-7'), 97.9(0-Gl), 73.4(C-G2), 76.4(0-03), 70.3(C-G4), 77.5(0-05), 61.4(C-G6), 55.7(3'-OCH3). 6-O-3.4-dihvdroxvcinnamoyl catalpol(verminoside) 'H-NMR (400 MHz, DMSO-d6) 5: 2.43(1H, m, H-9), 2.45(1H, m, H-5), 3.01(1H, m, H-G4), 3.05 (IH, m, H-G2), 3.14(1H, m, H-G5), 3.18(1H, m, H-G3), 3.42, 3.70(2H, m, H-G6), 3.64(1H, brs, H-7), 3.71, 3.90(2H, d, 7=13.2 Hz, each, H-10), 4.61(1H, d, 7=8.4 Hz, H-G1), 4.94(1 H, dd, 7=4.0, 5.6 Hz, H-4), 4.99 (IH, dt .7=7.2 Hz, H-6), 5.08(1 H, d, .7=9.2 Hz, H-1), 6.42(1H, d, .7=5.6 Hz, H-3), 6.77(1H, d, 7=8.0 Hz, H-5'), 7.08(1H, d, 7=1.6 Hz, H-2'), 7.05(1H, dd, .7=1.6, 8.0 Hz, H-6'). 13C-NMR (100 MHz, DMSO- d6) 8: 92.9(0-1), 141.1(0-3), 101.7 (0-4), 35.1(0-5), 79.2(0-6), 58.2(0-7), 65.7(0-8), 41.8(0-9), 58.5 (C-10), 125.4(0-1'), 115.8(0-2'), 146.0(0-3'), 148.6(0-4'), 113.3 (C-51), 121.6(0-6'), 145.6(0-7'), 115.0 (C-81), 97.9(C-G1), 73.4(C-G2), 76.4(C-G3), 70.3(C-G4), 77.5(C-G5), 61.4(0-06). 3-4. Preparation of 6-0-veratroyl catalpol from the extract of P. longifolium 6.2g of fraction 4 was subjected to column chromatography. 1.2g of fraction 4-3 was performed to recrystalization in methanol to obtain 672.6mg of 6-O-veratroyl catalpol, i.e., 6-O-3,4-Dimethoxybenzoyl. 6-O-(3.4-dimethoxybenzovl) catalpol (6-O-veratrovl catalpol) 'H-NMR (400 MHz, DMSO-d6) 8: 2.47(1 H, dd, 7=8.0, 9.6 Hz, H-9), 2.59(1H, dddd, 7=1.6, 4.8, 8.0,8.0 Hz, H-5), 3.00(1H, m, H-G4), 3.05 (IH, m, H-G2), 3.14(1H, m, H-G5), 3.18(1H, m, H-G3), 3.42, 3.71(2H, m, H-G6), 3.70(1H, brs, H-7), 3.72, 3.90(2H, d, 7=13.2 Hz, each, H-10), 4.61(1H, d, J=7.6 Hz, H-G1), 4.97(1 H, dd, .7=4.8, 6.0 Hz, H-4), 5.08 (IH, d, 7=8.8 Hz, H-6), 5.10(1H, d, 7=9.6 Hz, H-1), 6.42(1H, dd, 7=1.6. 6.0 Hz, H-3), 7.09(1H, d, 7=8.4 Hz, H-5'), 7.46(1H, d, 7=2.0 Hz, H-2'), 7.64(1 H, dd, .7=2.0, 8.4 Hz, H-61), 3.81, 3.84(6H, s each, 3', 4'-OCH3). I3C-NMR (100 MHz, DMSO- d6) 5: 92.9(C-1), 141.1(C-3), 101.8 (C-4), 35.2(C-5), 79.9(C-6), 58.2(C-7), 65.9(C-8), 41.8(C-9), 58.4 (C-10), 121.3(C-1'), 111.8(C-2'), 148.5(C-3'), 153.2(C-4'), 111.2 (C-51), 123.5(C-6'), 165.5(C-7'), 97.8(C-Gl), 73.4(C-G2), 76.4(C-G3), 70.3(C-G4), 77.5(C-G5), 61.4(C-G6), 55.6, 55.7(3', 4'-OCH3). 3-5. Preparation of minecoside from the extract of P. longifolium 261.Omg of fraction 4-4 and 288.Omg of fraction 4-5 were subjected to repeated silica gel column chromatography (chloroform-methanol mixture, methanol 10-20% step gradient) to obtain 52.5mg of minecoside, i.e., 6-0-3-hydroxy-4-methozycinnamoyl catalpol. 6-0-3-hvdroxy-4-methozvcinnamoyl catalpol(minecoside) 'H-NMR (400 MHz, DMSO-d6) 5: 2.46(1H, m, H-9), 2.48(1H, m, H-5), 3.00(1H, m, H-G4), 3.05 (1H, m, H-G2), 3.14(1H, m, H-G5), 3.18(1H, m, H-G3), 3.42, 3.70(2H, m, H-G6), 3.67(1 H, br s, H-7), 3.72, 3.91(2H, d, .7=13.2 Hz, each, H-10), 4.61(1H, d, .7=8.8 Hz, H-G1), 4.94(1H, dd, J=4.0, 6.0 Hz, H-4), 5.00 (1H, , .7=7.2 Hz, H-6), 5.09(1H, d, J=9.2 Hz, H-l), 6.42(1H, dd, .7=1,2, 5.6 Hz, H-3), 6.96(1H, d, 7=8.0 Hz, H-51), 7.13(1H, d, .7=2.0 Hz, H-2'), 7.17(1H, dd, .7=2.0, 8.0 Hz, H-61), 3.82(3H, s, -OCH3). 13C-NMR (100 MHz, DMSO- d6) 5: 93.0(C-1), 14U(C-3), 101.7 (C-4), 35.1 (C-5), 79.3(C-6), 58.2(C-7), 65.7(C-8), 41.8(C-9), 58.5 (C-10), 126.8(C-1'), 114.5(C-2'), 146.7(C-3'), 150.2(C-4'), 112.0(C-5'), 121.4(C-6'), 145.7(C-7'), 114.5 (C-81), 97.9(C-G1), 73.4(C-G2), 76.4(C-G3), 70.3(C-G4), 77.5(C-G5), 61.4(C-G6), 55.6 (4'-OCH3). 3-6. Preparation of catalpol from the extract of P. longifolium Verproside was hydrolyzed to yielded catalpol (compound 1) with 0.1N of KOH, The solution was stirred for 8 hours at room temperature and neutralized with 0.1N of HCL solution. The product was concentrated by rotary evaporator under reduced pressure, subjected to reversed phase silica gel column(RP18, methanol/water, 1/4, v/v), and yielded 54.0mg of catalpol. Catalpol 'H-NMR (400 MHz, DMSO-d6) 6: 2.12(1H, dddd, ,7=1.6, 4.0, 8.0, 8.0 Hz, H-5), 2.31(1H, d, .7=8.0, 9.6 Hz, H-9), 3.00(1H, m, H-G4), 3.05(1H, m, H-G2), 3.11(1H, m, H-G5), 3.17(1H, m, H-G3), 3,34(1H, br s, H-7), 3.40, 3.70(2H, w, H-G6), 3.63, 3.87(2H, d, 7=12.8, each, H-10), 3.76 (1H, d, .7=8.0 Hz, H-6), 4.59(1H, d, .7=8.0 Hz, H-G1), 4.90(1H, d, .7=9.6 Hz, H-l), 5.01(1H, dd, .7=4.6, 6.0 Hz, H-4), 6.36(1H, dd, J=1.6, 6.0 Hz, H-3). I3C-NMR (100 MHz, DMSO- d6) 8: 93.2(C-1), 140.2(C-3), 103.3(0-4), 37.4(0-5), 77.1(0-6), 60.7(0-7), 64.8(0-8), 42.1(0-9), 58.9(0-10), 97.8(C-G1), 73.4(C-G2), 76.4(C-G3), 70.2(C-G4), 77.4(C-G5), 61.3(C-G6). Experimental Example 1. MTT assay To investigate the cytotoxic effect of inventive extract of P. longifolmm extract and the compound isolated therefrom was determined by (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide(MTT) assay method(Wang Z et al., Biol., Pharm. Bull., 24, pp 159-162, 2001). Promyelotic HL-60 cells (HL-18103, 5X105 cells/ml) were seeded in 96-well plates under NGF-free condition. After 24 hours incubation, the cells were treated with the mixture of samples dissolved in lOpl of DMSO and lOpl of MTT solution (5mg/ml), and incubated for 4 hours under similar condition. 4 hours later, MTT was removed and lOOul of DMSO was dropped into each well to dissolve crystals. At 570nm, UV absorbance was measured by microplate reader (BIO-RAD, U.S.A.) to calculate the cell viability. As shown in Table 1, the result demonstrates that the cell viability ranges from 98% to 116% in 50uM, from 95% to 114% in lOOuM. It is confirmed that an inventive extract or compound the present invention has no cell toxicity. [Table 1] Effect of compounds isolated from P. hngifolium on HL-60 cells. (Table Removed) Experimental Example 2. Airway hyperresponsiveness (AHR) The AHR was evaluated by calculation Penh values (enhanced pause) 24 hours after the final OVA challenge. The Penh value of the OVA-treated group was significantly higher than that of the PBS control group. In the P. longifolium extract +OVA-challenged group, the Penh value was significantly reduced compared with that of the OVA-treated group at 30 mg/ml methacholine. (table 2) In the verproside+OVA-challenged group, the Penh value was significantly reduced compared with that of the OVA-treated group (P (Table Removed) Experimental Example 3. Effect of P. longifolium on OVA-induced eosinophilia in BALF 3-1. Animal sensitization and airway challenge Specific pathogen-free female BALB/c mice aged 8-10 weeks, which were routinely screened serologically for relevant respiratory pathogens, were purchased from ORIENT Co Ltd (Seoul, Korea). Following treatment: (1) sham-sensitization plus challenge with phosphate-buffered saline (PBS; ipNeb); (2) sensitization plus challenge with OVA (ovalbumin: Sigma A5503; Sigma, St. Louis, MO)(ipNeb); (3) sensitization with OVA(i.p.) plus challenge with OVA (Neb) and samples (extract of P. longifolium or montelukast) was performed to Group of mice (n=5). Briefly, mice were sensitized by intraperitoneal injection of 20#g OVA, which was emulsified with 2 mg aluminum hydroxide in 100 ul of PBS buffer (pH 7.4) on days 0 and 14. The mice were challenged through the airways with OVA(1% in PBS) for 20 min using an ultrasonic nubuilizer (NE-U12; Omron Corp., Tokyo, Japan) on days 28,29 and 30 after the initial sensitization. The mice were sacrificed 48 h after the last challenge (day 32) to determine the suppression effect of extract of P. longifolium or verproside on the airways of allergic asthma. 3-2. Sample treatment The extract of P. longifolium, and verproside were suspended in PBS and administered intragastrically using a 25-gauge stainless steel blunt feeding needle 1 h before each challenge, and control animals were exposed only on the PBS solution. As a positive control, montelukast (MSD Korea Ltd., Seoul, Korea) was treated with the same procedure in the experiment. The mice were sacrificed with an overdose of pentobarbital (Sigma P3761) 24h after the last challenge, and a tracheotomy was performed. After ice-cold 0.5ml of PBS was instilled into the lungs, bronchoalveolar lavage fluid (BALF) was obtained by aspiration three times (total 1.5ml) via tracheal cannulation (Yamazaki T, J. Jap. Bot., 43, pp 117-24, 1968) 3-3. Inflammatory cell counts in bronchoalveolar lavage fluid The total inflammatory cell number was assessed by counting cells in at least five squares of a hemocytometer after excluding dead cells confirmed by staining with trypan blue (Daigle I. et al., Swiss Med Wkly, 131, pp 231-7, 2001). lOOyl of BALF was loaded onto a slide and centrifuged (200Xg, 4°C, lOmin) to fix the cells onto the slide using a cytospine machine (Hanil Science Industrial, Korea). The cells were stained by Diff-Quick® Stain reagents (Sysmex, Cat No.38721, Switzerland) according to the manufacturer's instructions. Statistical significance was determined by Student's two-tailed Hest for independent means and the critical level for significance was set at P To evaluate the suppression of verproside on the eosinophilia in OVA-challenged mice, the recruited cells in BALF were counted 48 hours after the last challenge. OVA caused a marked influx of leucocytes into the BALF from a PBS control group. As shown in Figure 2, the total cells were counted as 40.5 ± 16.4 x 104 cells/mouse (P 80.7 ± 11.1% decrease in eosinophils (P Experimental Example 4. Lung histology To estimate the suppressive effect of verproside on the eosinophilia, lung tissues were collected 48 hours after the last challenge. The lung tissue was fixed for 24 h in 10% neutral-buffered formalin. After being embedded in paraffin, it is sliced into 4-um thickness of sections and the tissue was stained with H&E solution (hematoxylin; Sigma MHS-16 and eosin, Sigma HT110-1-32). In the OVA-challenged mice, leukocytes were found to be infiltrated into the peri-bronchiole and peri-vascular connective tissue; of these leukocytes, eosinophilia was mainly observed (Fig. 3-11, P In Periodic acid Schiff (PAS) staining, mucus overproduction in the OVA-treated mice was clearly observed as a violet color in the bronchial airways compared with the normal mice. In contrast, mucus was markedly diminished in the verproside + OVA-challenged mice (Fig. 4-A). Goblet-cell hyperplasia in the airway epithelium was quantified based on a five-point system: 0, no goblet cells; 1, 75% of the epithelium. For each mouse, five airway sections that were randomly distributed throughout the left lung were analysed, and their average scores were calculated. Quantitative analysis of mucus production was performed using an image analyzer (Leica Microsystem Imaging solution Ltd.; Cambridge, UK). As shown in Figure 4-B, the mucus area was scored as 3.60 ± 0.64 in the OVA-treated mice compare with PBS-treated mice (P These results demonstrated that verproside reduced eosinophilia and mucus hypersecretion significantly in the airway remodeling process. Experimental Example S. Measurement of IgE and cytokines Complementary capture and detection antibody pairs for mouse IgE antibodies were purchased from PharMingen (San Diego, CA), and the IgE enzyme-linked immunosorbent assay (ELISA) was performed according to the manufacturer's directions. Duplicate samples in plasma were diluted to 1:100. IgE levels in each sample were measured from optical density readings at 450 nm, and IgE concentrations were calculated from a standard curve that was generated using recombinant IgE (5-2,000 ng/ml). The amount of IL-4 and IL-13 contained in BALF was measured with a specific mouse ELISA kit (R&D Systems; Minneapolis, MN). The detection limit of the assays was 250 pg/ml. As shown in Fig. 5-A & 5-B, The IgE levels were found to be greatly increased in the OVA-treated mice: 85.6 ± 17.3 ug/ml (plasma, P 31.4 ± 14.2 yg/ml (plasma, P To determine the effect of verproside on cytokine release in the OVA-induced asthmatic mice, the levels of cytokines (IL-4 and IL-13) in BALF were measured using ELISA 48 hours after the last challenge. OVA challenge induced a significant elevation of the cytokines to 14.1 ± 6.1 pg/ml (IL-4) and 178.5 ± 96.4 pg/ml (IL-13) in the BALF compared with the control (IL-4, 0.1 ± 0,5 pg/ml; IL-13, 0.1 ± 1.0 pg/ml). In the verproside-treated group, the cytokines were significantly suppressed; 64.5 ± 27.7% decrease in IL-4 (P from a OVA-challenged group. Montelukast also showed a significant reduction in both of IL-4 (69.5 ± 22.0% decrease, P 0.05) from the control. These results demonstrate that verproside reduced the IL-4 and IL-13 concentrations in the BALF of the asthmatic model as montelukast did (Figure 5-C and 5-D). Hereinafter, the formulating methods and kinds of excipients will be described, but the present invention is not limited to them. The representative preparation examples were described as follows. Preparation of injection Dried powder of Example 1 orverproside IQQmg Sodium metabisulfite 3.0mg Methyl paraben O.Smg Propyl paraben 0.1 mg Distilled water for injection optimum amount Injection preparation was prepared by dissolving active component, controlling pH to about 7.5 and then filling all the components in 2ml ample and sterilizing by conventional injection preparation method. Preparation of powder Dried powder of Example 1 or verproside 500mg Corn Starch lOOmg Lactose lOOmg Talc lOmg Powder preparation was prepared by mixing above components and filling sealed package. Preparation of tablet Dried powder of Example 1 orverproside 200mg Corn Starch lOOmg Lactose lOOmg Magnesium stearate optimum amount Tablet preparation was prepared by mixing above components and entabletting. Preparation of capsule Dried powder of Example 1 orverproside lOOmg Lactose 50mg Corn starch 50mg Talc 2mg Magnesium stearate optimum amount Tablet preparation was prepared by mixing above components and filling gelatin capsule by conventional gelatin preparation method. Preparation of liquid Dried powder of Example 1 orverproside lOOOmg Sugar 20g Polysaccharide 20g Lemon flavor 20g Liquid preparation was prepared by dissolving active component, and then filling all the components in 1000M ample and sterilizing by conventional liquid preparation method. Preparation of health food Dried powder of Example 1 or verproside lOOOmg Vitamin mixture optimum amount Vitam i n A acetate 70 (ag Vitamin E l.Omg Vitamin Bi 0.13mg Vitamin 82 0.15mg Vitamin B6 O.Smg Vitamin B12 0.2jag Vitamin C lOmg Biotin lOj^g Amide nicotinic acid 1,7mg Folic acid 50\|ag Calcium pantothenic acid O.Smg Mineral mixture optimum amount Ferrous sulfate 1.75mg Zinc oxide 0.82mg Magnesium carbonate 25.3mg Monopotassium phosphate 15mg Dicalcium phosphate 55mg Potassium citrate 90mg Calcium carbonate lOOmg Magnesium chloride 24.8mg The above mentioned vitamin and mineral mixture may be varied in many ways. Such variations are not to be regarded as a departure from the spirit and scope of the present invention. Preparation of health beverage Dried powder of Example 1 or verproside 1 OOOmg Citric acid lOOOmg Ol igosaccharide 1 OOg Apricot concentration 2g Taurine lg Distilled water 900m£ Health beverage preparation was prepared by dissolving active component, mixing, stirred at 85 °C for 1 hour, filtered and then filling all the components in 1000in£ ample and sterilizing by conventional health beverage preparation method. The invention being thus described, it will be obvious that the same may be varied in many ways. Such variations are not to be regarded as a departure from the spirit and scope of the present invention, and all such modifications as would be obvious to one skilled in the art are intended to be included within the scope of the following claims. Industrial Applicability As described in the present invention, the extract of P. longifolium and the catalpol derivatives isolated therefrom show the suppression of elevated IgE, IL-4 and IL-13 levels and eosinophilia in plasma and BALD, and mucus overproduction in the lung tissues using by OVA-induced asthmatic mouse model. Therefore, it can be used as the therapeutics or functional health food for treating and preventing inflammatory, allergic and asthmatic disease. We Claim 1. A pharmaceutical composition comprising a crude extract or organic solvent soluble extract of Pseudolysimachion genus plant as an active ingredient and pharmaceutically acceptable carriers, adjuvants or diluents for the treatment and prevention of inflammatory, allergic and asthmatic disease, wherein the composition contains the extract in the range of from 0.1 to 50% by weight and the pharmaceutically acceptable carriers, adjuvants or diluents in the range of from 99.9 to 50% by weight based on the total weight of the composition. 2. The pharmaceutical composition as claimed in claim 1, wherein said crude extract or organic solvent soluble extract is extracted with the solvent selected from the group consisting of water, lower alcohols, acetone, ethyl acetate, chloroform, dichloromethane or mixtures thereof. 3. The pharmaceutical composition as claimed in claim 1, wherein said extract is extracted from P. longifolium, P. ovtum, P. khisianum, P. kiusianum var. diamanticum. P. kiusianum var. villosum, P. dahuricum, P. pyrethrinum, P. linarifolium, P. linarifolium var. villosulum, P. rotundum var. suhintegrum, P. roiundum var. coreanum. P. insulare or P. undulate. 4. A pharmaceutical composition comprising catalpol derivatives represented by general formula (I) or pharmaceutically acceptable salt thereof as an active ingredient and pharmaceutically acceptable carriers, adjuvants or diluents for treatment and prevention of allergic and asthmatic disease, wherein the composition contains the catalpol derivatives in the range of from 0.1 to 50% by weight and the pharmaceutically acceptable carriers, adjuvants or diluents in the range of from 99.9 to 50% by weight based on the total weight of the composition; (Formula Removed) wherein, R is independently at least one groups selected from a hydrogen atom, 3.4-dihydroxybenzoyl, 3-hydroxy-4-methoxybenzoyl, 4-hydroxyben/oyl, 3.4-dimethoxybenzoyl, and cinnamoyl group substituted with C1-C3 lower alkyl group or C1-C3 lower alkoxy group. 5. The pharmaceutical composition as claimed in claim 4, wherein said cinnamoyl group substituted with C1-C3 lower alkyl group or C1-C3 lower alkoxy group is 3.4-dihydroxycinnamoyl or 3-hydroxy-4-methoxycinnamoyl group. 6. A functional health food comprising, a extract of crude extract or organic solvent soluble extract of Pseudolysimachion genus plant as set forth in claim 1, or the catalpol derivatives as set forth in claim 4 together with a sitologically acceptable additive for the prevention and improvement of inflammatory, allergic and asthmatic disease, wherein the food contains the extract or the catalpol derivatives in the range of from 0.01 to 80% w/w and the sitologically acceptable additive in the range of from 99.99 to 20% w/w based on the total weight of the food. 7. The functional health food as claimed in claim 7, wherein said health food is provided as a form of pill, powder, granule, tablet, chewing table, capsule or beverage type.

Full Text

PHARMACEUTICAL COMPOSITION COMPRISING AN EXTRACT
OF PSEUDOLYSIMACHION LONGIFOLIUM AND THE CATALPOL
DERIVATIVES ISOLATED THEREFROM HAVING
ANTIINFLAMMATORY, ANTIALLERGIC AND ANTIASTHMATIC ACTIVITY
BACKGROUND OF THE INVENTION
Technical Field
The present invention relates to a composition comprising an extract of Pseudolysimachion longifolium and the catalpol derivatives isolated therefrom having anti-inflammatory, anti-allergic and anti-asthmatic activity.
Background Art
Asthma has been regarded as a complex syndrome occurring in the airways, which shows various disorders such as airflow obstruction, acute or chronic inflammation, airway hyper-responsiveness (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 critical role in asthma development and the number of patients suffering from allergic asthma has been increased to about 10% of the population in the world recently. It has been reported that the number has been reached to seventeen million in America and the market scale of the medication for allergic asthma has been enlarged to 640 billion $ in America till now.
Asthma can be classified into two types, i.e., extrinsic asthma and intrinsic asthma. Extrinsic asthma caused by the exposure of antigen such as house dust mite Dermatophagoides as a main antigen, pollen, epithelium of animal, fungi etc shows positive reaction in skin test or bronchial provocation test against the antigen, and generally occurs in younger people. Intrinsic asthma caused by upper respiratory infection, exercise, emotional instability, cold weather, the change of humidity occurs in adult patients.
According to the aspect of pathophysiology, asthma has been recognized as a chronic inflammation occurred by following procedure; Inflammatory cells are proliferated, differentiated, and activated caused by cytokines reproducing in T-helper 2 immune cells and is moved to air way or neighboring tissue thereof. The activated inflammatory cells such as neutrophil, mast cell etc release a variety of inflammatory mediators, such as cytokines, chemokines, signaling molecules, adhesion molecules and growth factors and the structural cells in airways are involved in various stages of asthma (Elias JA et al., J Clin Invest., Ill, pp 291-7, 2003). In numerous studies using knockout mice models and clinical research, the critical observations in asthma could fall into several characteristic parameters, such as immune responses, eosinophilia, AHR and structural remodeling (Moffatt JD. Pharmacol Ther, 107, pp 343-57, 2005; Spina D et al., Trends Pharmacol Sci, 23, pp 311-5, 2002). Each of the parameters seems not to have direct correlations with one another; however, IgE-mediated immune response 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, pp 1033-9, 1990), and the produced cytokines such as IL-4, IL-5 and IL-13 in the allergic process also play an important role in AHR development and airway remodeling (Riffo-Vasquez Y et al., Pharmacol. Ther., 94, pp 185-211, 2002). Indeed, asthma is a result of orchestrated inflammatory events, many of which involve specific inhibitors acting on the pathway of asthma, for example, histamine HI antagonists, thromboxane antagonists, platelet-activating-factor antagonists, cyclooxygenase inhibitors, nitrogen monooxygenase inhibitors and prostaglandin inhibitors, have been tried but have failed in clinical trials (Moffatt J.D., Pharmacol. Ther., 107, pp 343-57, 2005). In contrast, glucocorticoids, which suppress the progenitor levels of inflammatory cells to baseline by widespread inhibition of cytokine synthesis and cytokine mediated immune-cell survival, has been used to manage the symptoms of asthmatic patients over a period of 30 years as far (Baatjes A.J. et al., Pharmacol, Then, 95, pp 63-72, 2002). These reports suggest that the therapeutic approach for asthma management should focus on restoring the balance of asthmatic parameters rather than searching for potent inhibitors of specific pathways of the asthmatic process.
Pseudolysimachion longifolium belonged to Pseudolysimachion genus, is a perennial herb distributed in Korea, China, Russia and Europe. Numerous species of same genus for example, Pseudolysimachion ovutum, Pseudolysimachion kiusianum, Pseudolysimachion kiusianum var diamanticum, Pseudolysimachion kiusianum var villosum, Pseudolysimachion dahuricum, Pseudolysimachion pyrethrinum, Pseudolysimachion linarifolium, Pseudolysimachion linarifolium var. villosulum, Pseudolysimachion rotundum var. subintegrum, Pseudolysimachion rotundum var. coreanum, Pseudolysimachion insulare, and Pseudolysimachion undulata have been
reported and the plants contains mannitol, 6-hydroxyIuteolin as a main ingredient (Chung BS and Shin MK, HyangyakDaeSaJeon, Youngrimsa, pp913-914, 1998).
However, there has been not reported or disclosed about the suppressive effect on inflammatory, allergic and asthmatic disease of the extract from P. longifolium and the catalpol derivatives isolated therefrom in any of above cited literatures, the disclosures of which are incorporated herein by reference.
Accordingly, the present inventors have discovered that the extract of P. longifolium and the catalpol derivatives isolated therefrom show the suppressive effect on asthmatic parameters, such as IgE level, cytokine release, and eosinophilia, AR and mucus hypersecretion in OVA-sensitized/challenged mouse model and finally completed the present invention.
SUMMARY OF THE INVENTION
The present invention provides a pharmaceutical composition and a health food comprising an extract or catalpol derivatives isolated from P. longifolium as an active ingredient in an effective amount to treat and prevent inflammatory, allergic and asthmatic disease.
The present invention also provides a use of an extract of P. longifolium and the catalpol derivatives isolated therefrom showing anti-inflammatory, anti-allergic and anti-asthmatic activity.
The present invention also provides a method of treating or preventing inflammatory, allergic and asthmatic disease in a mammal comprising administering to said mammal an effective amount of an extract of P. longifolium and the catalpol derivatives isolated therefrom, together with a pharmaceutically acceptable carrier thereof.
Disclosure of the invention
Accordingly, it is an object of the present invention to provide a composition comprising a crude extract or organic solvent soluble extract of Pseudolysimachion genus plant, as an active ingredient for the treatment and prevention of inflammatory, allergic and asthmatic disease.
The term "crude extract" disclosed herein comprises the extract prepared by extracting plant material with water, lower alcohols such as methanol, ethanol, preferably methanol and the like, or the mixtures thereof.
The term "organic solvent soluble extract" disclosed herein can be prepared by extracting the above described crude extract with organic solvent, for example, butanol, acetone, ethyl acetate, chloroform or dichloromethane, preferably butanol.
The term "Pseudolysimachion genus" disclosed herein comprises P. longifolium, P. ovtum, P. kiusianum, P. kiusianum var. diamanticum, P. kiusianum var. villosum, P. dahuricum, P. pyrethrinum, P, linarifolium, P. linarifolium var. villosulum, P. rotundum var. subintegrum, P. rotundum var. coreanum, P. insulare and P. undulate,
The present invention provides a pharmaceutical composition comprising catalpol derivatives represented by following chemical formula (I), or a pharmaceutically acceptable salt thereof as an active ingredient in an effective amount to treat and prevent inflammatory, allergic and asthma disease.
(Figure Removed)
Wherein,
R is independently at least one groups selected from a hydrogen atom, benzoyl or cinnamoyl group substituted with C\-Ci lower alkyl group or C\-Cj lower alkoxy group.
In the above formula (I), preferably, R group comprises 3, 4-dihydroxybenzoyl, 4-hydroxy-3-methozybenzoyl, 3-hydroxy-4-methozybenzoyl, 4-hydroxybenzoyl, 3,4-dimethoxybenzoyl, 3,4-dihydroxycinnamoyl and 3-hydroxy-4-methoxycinnamoyl.
The catalpol derivatives of the present invention can be isolated from P. long/folium or synthesized by general procedure well known in the art (Herbert O. house., Modern Synthetic Reactions, 2nd Ed., The Benjamin/Cummings Publishing Co., 1972).
In accordance with another aspect of the present invention, there is also provided a use of crude extract or organic solvent soluble extract of Pseudolysimachion genus plant, or the catalpol derivatives isolated therefrom for manufacture of medicines employed for treating or preventing inflammatory, allergic and asthmatic disease.
In accordance with another aspect of the present invention, there is also provided a method of treating or preventing inflammatory, allergic and asthmatic disease in mammals, wherein the method comprises administering a therapeutically effective amount of crude extract or organic solvent soluble extract of Pseudolysimachion genus plant, or the catalpol derivatives isolated therefrom into the mammal suffering with inflammatory, allergic and asthmatic disease.
An inventive extract isolated from Pseudolysimachion genus plant, and the catalpol derivatives isolated therefrom may be prepared in accordance with the following preferred embodiment.
Hereinafter, the present invention is described in detail.
For the present invention, for example, the dried leave off! longifolium is cut into small pieces and the piece was mixed with 2 to 20-fold, preferably, 5 to 10-fold volume of polar solvent, for example, water, Q-C4 lower alcohol such as methanol, ethanol, butanol, or the mixtures thereof, preferably methanol; and was heated at the temperature ranging from 20 to 100°C, preferably from 20 to 50 °C, for the period ranging 10 to 48 hours, preferably 20 to 30 hours, by reflux extraction with hot water, cold water extraction, ultra-sonication or conventional extraction, preferably by cold water extraction; the residue was filtered and then the filtrate is dried to obtain polar solvent soluble extract thereof.
In the above crude extract prepared by above step, is suspended in water, and then is mixed with 1 to 100-fold, preferably, 1 to 5-fold volume of organic solvent butanol, acetone, ethyl acetate, chloroform or dichloromethane, preferably butanol to obtain organic solvent soluble extract of the present invention.
The above organic solvent soluble extract is further subjected to silica gel column chromatography filled with silicagel eluting with mixture solvent of chlorofornrmethanol with increasing the polarity by changing the mixed ratio (methanol 0-100%, step gradient) to obtain several fractions. Among the fractions, the 3rd fraction is further subjected to repeated silica gel column chromatography using a normal phase silica column (methanol 10-50% step gradient) to obtain the catalpol derivatives of the present invention. The structure was confirmed by NMR, EI-MS and optical rotation with those reported previously (Afifi-Yazar Ft) et al., Helv Chim Acta, 63, pp 1905-7, 1980) and the purity of the catalpol derivatives was analyzed as more than 99.5% by HPLC system.
In accordance with another aspect of the present invention, there is provided a pharmaceutical composition comprising a crude extract and organic solvent soluble extract of P. longifolium or the catalpol derivatives isolated therefrom prepared by the above describe preparation method for the treatment and prevention of inflammatory, allergic and asthmatic disease as active ingredients.
In accordance with another aspect of the present invention, there is also provided a use of comprising a crude extract and organic solvent soluble extract of P. longifolium or the catalpol derivatives isolated therefrom prepared by the above describe preparation method for manufacture of medicines employed for treating or preventing inflammatory, allergic and asthmatic disease.
In accordance with another aspect of the present invention, there is also provided a method of treating or preventing inflammatory, allergic and asthmatic disease, wherein the method comprises administering a therapeutically effective amount of comprising a crude extract and organic solvent soluble extract of P. longifolium or the catalpol derivatives isolated therefrom prepared by the above describe preparation method.
The inventive compound represented by general formula (§j) can be transformed into their pharmaceutically acceptable salt and solvates by the
conventional method well known in the art. For the salts, acid-addition salt thereof formed by a pharmaceutical ly acceptable free acid thereof is useful and can be prepared by the conventional method. For example, after dissolving the compound in the excess amount of acid solution, the salts are precipitated by the water-miscible organic solvent such as methanol, ethanol, acetone or acetonitrile to prepare acid addition salt thereof and further the mixture of equivalent amount of compound and diluted acid with water or alcohol such as glycol monomethylether, can be heated and subsequently dried by evaporation or filtrated under reduced pressure to obtain dried salt form thereof.
As a free acid of above-described method, organic acid or inorganic acid can be used. For example, organic acid such as methansulfonic acid, p-toluensulfonic 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, vanillic acid, hydroiodic acid and the like, and inorganic acid such as hydrochloric acid, phosphoric acid, sulfuric acid, nitric acid, tartaric acid and the like can be used herein.
Further, the pharmaceutical ly acceptable metal salt form of inventive compounds may be prepared by using base. The alkali metal or alkali-earth metal salt thereof can be prepared by the conventional method, for example, after dissolving the compound in the excess amount of alkali metal hydroxide or alkali-earth metal hydroxide solution, the insoluble salts are filtered and remaining filtrate is subjected to evaporation and drying to obtain the metal salt thereof. As a metal salt of the present invention, sodium, potassium or calcium salt are pharmaceutical ly suitable and the corresponding silver salt can be prepared by reacting alkali metal salt or alkali-earth metal salt with suitable silver salt such as silver nitrate.
The pharmaceutically acceptable salt of the present compound comprise all the acidic or basic salt which may be present at the compounds, if it does not indicated specifically herein. For example, the pharmaceutically acceptable salt of the present invention comprise the salt of hydroxyl group such as the sodium, calcium and potassium salt thereof; the salt of amino group such as the hydrogen bromide salt, sulfuric acid salt, hydrogen sulfuric acid salt, phosphate salt, hydrogen phosphate salt, dihydrophosphate salt, acetate salt, succinate salt, citrate salt, tartarate
salt, lactate salt, mandelate salt, methanesulfonate(mesylate) salt and p-. toluenesulfonate (tosylate) salt etc, which can be prepared by the conventional method well known in the art.
The inventive composition for treating and preventing inflammatory, allergic and asthmatic disease may comprises the above described extracts or compounds as 0.1 ~ 50% by weight based on the total weight of the composition.
The composition according to the present invention can be provided as a pharmaceutical composition containing pharmaceutically acceptable carriers, adjuvants or diluents, e.g., lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starches, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, polyvinyl pyrrolidone, water, methylhydroxy benzoate, propylhydroxy benzoate, talc, magnesium stearate and mineral oil. The formulations may additionally include fillers, anti-agglutinating agents, lubricating agents, wetting agents, flavoring agents, emulsifiers, preservatives and the like. The compositions of the invention may be formulated so as to provide quick, sustained or delayed release of the active ingredient after their administration to a patient by employing any of the procedures well known in the art.
For example, the compositions of the present invention can be dissolved in oils, propylene glycol or other solvents that are commonly used to produce an injection. Suitable examples of the carriers include physiological saline, polyethylene glycol, ethanol, vegetable oils, isopropyl myristate, etc., but are not limited to them. For topical administration, the extract of the present invention can be formulated in the form of ointments and creams.
Pharmaceutical formulations containing present composition may be prepared in any form, such as oral dosage form (powder, tablet, capsule, soft capsule, aqueous medicine, syrup, elixirs pill, powder, sachet, granule), or topical preparation (cream, ointment, lotion, gel, balm, patch, paste, spray solution, aerosol and the like), or injectable preparation (solution, suspension, emulsion).
The composition of the present invention in pharmaceutical dosage forms may be used in the form of their pharmaceutically acceptable salts, and also may be used alone or in appropriate association, as well as in combination with other
pharmaceutically active compounds.
The desirable dose of the inventive extract or compound varies depending on the condition and the weight of the subject, severity, drug form, route and period of administration, and may be chosen by those skilled in the art. However, in order to obtain desirable effects, it is generally recommended to administer at the amount ranging from 0.0001 to lOOmg/kg, preferably, 0.001 to lOmg/kg by weight/day of the inventive extract of the present invention. The dose may be administered in single or divided into several times per day.
The pharmaceutical composition of present invention can be administered to a subject animal such as mammals (rat, mouse, domestic animals or human) via various routes. All modes of administration are contemplated, for example, administration can be made orally, rectally or by intravenous, intramuscular, subcutaneous, intracutaneous, intrathecal, epidural or intracerebroventricular injection.
It is the other object of the present invention to provide a functional health food comprising the extract or compounds isolated from P. longifolium together with a sitologically acceptable additive for the prevention and alleviation of inflammatory, allergic and asthmatic disease.
To develop for functional health food, examples of addable food comprising the above extracts or compounds of the present invention are various food, beverage, gum. vitamin complex, health improving food and the like, and can be used as powder, granule, tablet, chewing tablet, capsule or beverage etc.
The above described composition therein can be added to food, additive or beverage, wherein, the amount of the above described extract or compound in food or beverage may generally range from about 0.01 to 80w/w%, preferably 0.01 to 15w/w% of total weight of food for the health food composition and 0.02 to 5g, preferably 0.3 to Ig on the ratio of 100ml of the health beverage composition.
Providing that the health beverage composition of present invention contains the above described extract or compound as an essential component in the indicated ratio, there is no particular limitation on the other liquid component, wherein the other component can be various deodorant or natural carbohydrate etc such as conventional beverage. Examples of aforementioned natural carbohydrate are monosaccharide such as glucose, fructose etc; disaccharide such as maltose, sucrose etc; conventional sugar such as dextrin, cyclodextrin; and sugar alcohol such as xylitol, and erythritol etc. As the other deodorant than aforementioned ones, natural deodorant such as taumatin, stevia extract such as levaudioside A, glycyrrhizin et al., and synthetic deodorant such as saccharin, aspartam et al., may be useful favorably. The amount of above described natural carbohydrate is generally ranges from about 1 to 20 g, preferably 5 to 12 g in the ratio of 100 mi of present beverage composition.
The other components than aforementioned composition are various nutrients, a vitamin, a mineral or an electrolyte, synthetic flavoring agent, a coloring agent and improving agent in case of cheese chocolate et al., pectic acid and the salt thereof, alginic acid and the salt thereof, organic acid, protective colloidal adhesive, pH controlling agent, stabilizer, a preservative, glycerin, alcohol, carbonizing agent used in carbonate beverage et al. The other component than aforementioned ones may be fruit juice for preparing natural fruit juice, fruit juice beverage and vegetable beverage, wherein the component can be used independently or in combination. The ratio of the components is not so important but is generally range from about 0 to 20 w/w % per 100 w/w % present composition. Examples of addable food comprising aforementioned extract therein are various food, beverage, gum, vitamin complex, health improving food and the like.
Inventive extract of the present invention has no toxicity and adverse effect therefore they can be used with safe.
The present invention is more specifically explained by the following examples. However, it should be understood that the present invention is not limited to these examples in any manner.
Brief description of Drawings
The above and other objects, features and other advantages of the present invention will more clearly understood from the following detailed description taken in conjunction with the accompanying drawings, in which;
Fig. 1 shows the effects of an extract of P. longifolium extract on the recruitment of inflammatory cells in bronchoalveolar lavage fluid,
Fig. 2 shows the effects of the fraction 3 isolated from P. longifolium, verproside and picroside n on the recruitment of inflammatory cells in bronchoalveolar lavage fluid,
Fig. 3 represents the effects of an extract of P. longifolium on lung tissue cell using by the histological examination of bronchoalveolar lavage (A: normal control mice, B: PBS-treated mice, C: P. longifolium extract-treated mice),
Fig. 4 presents the effects of an extract of P. longifolium and the compounds isolated therefrom on lung tissue cell using by the histological examination of bronchoalveolar lavage (A: normal control mice, B: PBS-treated mice. C: verproside-treated mice, D: picroside n-treated mice, E: montelukast-treated mice).
Best Mode for Carrying Out the Invention
It will be apparent to those skilled in the art that various modifications and variations can be made in the compositions, use and preparations of the present invention without departing from the spirit or scope of the invention.
The present invention is more specifically explained by the following examples. However, it should be understood that the present invention is not limited to these examples in any manner.
EXAMPLES
The following Reference Example, Examples and Experimental Examples are intended to further illustrate the present invention without limiting its scope.
Example 1. Preparation of the crude extract of P. longifolium
7.9kg of dried P. longifolium cut into small pieces, mixed with SOL of methanol and the mixture was stirred at room temperature for 24 hours, extracted by cold water three times. The extract was filtered with filter paper to remove the debris. The filtrate was pooled and concentrated by rotary evaporator 55-65*0 under reduced pressure and dried with freezing dryer to obtain 950.5g of dried crude extract of P. longifolium.
Example 2. Preparation of polar solvent and non-polar solvent soluble extract
2-1. Preparation of ethyl acetate soluble fraction
10L of distilled water was added to 425 g of the crude extract obtained in Example 1. 1OL of ethyl acetate was added thereto in separator/ funnel and shaken vigorously to divide into ethyl acetate soluble layer and water soluble layer.
Above ethyl acetate soluble layer was concentrated by rotary evaporator, dried with freeze dryer to obtain ethyl acetate soluble extract.
2-2. Preparation of butanol/water soluble fraction
Water soluble layer was fractionated by mixing with 10 L of butanol and finally, 144.Og of n-butanol soluble extract and water soluble extract were obtained to use as a sample in the following experiments.
Example 3. Preparation of catalpol derivatives from the extract of P. longifolium 3-1. Preparation of verproside(6-O-3,4-Dihydroxybenzoyi catalpol)
144.0g of n-butanol soluble fraction was subjected to a silica gel column chromatography (70-230 mesh, 8.5X65cm) and eluted with a chloroform-methanol mixture (methanol 0-100%, step gradient) to obtain five fractions. 29. Ig of fraction 2 (between chloroform-methanol 7/3-6/4, v/v) was subjected to repeated column chromatography using a normal phase silica column chromatography (silica gel, 230-400 mesh, 6.0X60 cm, chloroform-methanol mixture, methanol 10-50% step gradient). The fractions 2-4 was performed to recrystalization in methanol to obtain 14.2g of verproside, i.e., 6-0-3,4-Dihydroxybenzoyl catalpol. The structure was confirmed by NMR ('H, 13C, DEPT, HMQC, HMBC), EI-MS and optical rotation with those reported previously (Afifi-Yazar FO et al., Helv Chim Acta, 63, pp 1905-7, 1980) and the purity of verproside was analyzed as more than 99.5% by HPLC system (Shimadzu SCL-10A with SPD-M 10A vp PDA detector, column; Phenomenex Synergi 4 urn Fusion RP-80, 4.6x150 mm, elution: MeOH/DW, 35/65, v/v, 0.8 ml/min).
6-Q-3.4-Dihvdroxvbenzoyl catalpol(verproside)
'H NMR (400 MHz, DMSO-d6) 8: 2.47(1H, dd, J=8.0, 9.2 Hz, H-9), 2.59(1H, dddd, J=\.6, 4.0, 8.0, 8.0, H-5), 3.00(1H, m, H-G4), 3.05 (1H, m, H-G2), 3.14(1H, m, H-G5), 3,18(1H, m, H-G3), 3.42, 3.71(2H, m, H-G6). 3.67(1 H, s, H-7), 3.71, 3.91(2H, d, J=13.2 Hz, each, H-10), 4.61(1H, d, J=7.6 Hz, H-G1), 4.94(1H, dd, 7=4.0, 6.0 Hz, H-4), 5.03 (1H, d, J=8.0 Hz, H-6), S.09(1H, d, .7=9.2 Hz, H-l),
6.41(1H, dd, .7=1.6. 6.0 Hz, H-3), 6.82(1H, d, .7=8.0 Hz, H-51), 7.35(1H, dd, .7=2.0, 8.0 Hz, H-6'), 7.39(1 H, d, 7=2.0 Hz, H-2').
13C-NMR (100 MHz, DMSO- d6) 5: 93.0(C-1), 141.1(C-3), 101.8 (C-4), 35.2(C-5), 79.5(C-6), 58.2(C-7), 65.8(C-8), 41.8(C-9), 120.0 (C-l1), 116.4(C-2'), 145.1(C-3T), 150.8(C-4'), 115.4(C-5'), 122.6 (C-61), 165.6(C-7'), 97.9(C-G1), 73.4(C-G2), 76.4(C-G3), 70.3(C-G4), 77.5(C-G5), 61.4(C-G6).
3-2. Preparation of isovanillyl catalpol from the extract of P. long/folium
17.3g of fraction 3 was subjected to column chromatography using a normal
phase silica column (silica gel, 230-400 mesh, 6.0x60 cm, chloroform-methanol
mixture, methanol 10-50% step gradient). 8.5g of fraction 3-3 was performed to recrystalization in methanol to obtain 7.2g of isovanillyl catalpol, i.e., 6-0-3-hydroxy-4-methoxybenzoly catalpol.
6-0-3-hvdroxv-4-methoxvbenzolv catalpoKisovanillvl catalpol)
'H-NMR (400 MHz, DMSO-d6) 8: 2.47(1H, m, H-9), 2.55(1H, m H-5), 3.00(1H, m, H-G4), 3.05 (IH, m, H-G2), 3.14(1H, m, H-G5), 3.18(1H, m, H-G3), 3.43, 3.70(2H, m, H-G6), 3.70(1H, br s, H-7), 3.72, 3.92(2H, d, .7=13.2, each, H-10), 4.62(1H, d, .7=8.0 Hz, H-Gl), 4.95(1H, dd, .7=4.4, 6.0 Hz, H-4), 5.06 (IH, d, .7=8.0 Hz, H-6), 5.11(1H, d, .7=9.2 Hz, H-l), 6.42(1H, d, 7=6.0 Hz, H-3), 7.04(1H, d, .7=8.4 Hz, H-51), 7.42(1H, brs, H-2'), 7.48(1H, d, J= 8.4 Hz, H-61), 3.84(3H, s, 4'-O-CH3).
UC-NMR (100 MHz, DMSO- d6) 5: 93.0(C-1), 141.0(C-3), 101,6 (C-4), 35.2(C-5), 79.7(C-6), 58.2(C-7), 65.8(C-8), 41.8(C-9), 58.4(C-10), 121.7(C-I'), 115.7(C-2'), 146.3(C-3'), 152.1(C-4'), 111.4(C-5'), 121.3 (C-61), 165.3(C-7'), 97.8(C-Gl), 73.4(C-G2), 76.4(C-G3), 70.3(C-G4), 77.4(C-G5), 61.4(C-G6), 55.7(4'-OCH3).
3-3. Preparation of picrosidell and verminoside from the extract of P. longifolium
1.5g of fraction 3-5 was subjected to reversed phase silica gel column(RP-l 8, YMC Gel ODS-A, 6.0X60 cm, methanol/water, 1/4, v/v), subjected to sepadex LH-20 column chromatography(methanol/water, 85/15, v/v) to obtain lOl.Omg of picrosidell, i.e., 6-O-4-hydroxy-3-methozybenzoyl and 30.0mg of verminoside, i.e., 6-0-3,4-dihydroxycinnamoyl catalpol.
6-0-4-hvdroxv-3-methozvPcnzovl (picrosidell)
'H-NMR (400 MHz, DMSO-d6) 6: 2.47(1 H, M, 7=8.0, 9.6 Hz, H-9), 2.58(1H, dddd, 7=1.2, 6.0, 8.0, 8.4 Hz, H-5), 3.00(1 H, m, H-G4), 3.05 (IH, m, H-G2), 3.14(1H, m, H-G5), 3.18(1H, m, H-G3), 3.42, 3.71(2H, m, H-G6), 3.67(1H, brs, H-7), 3.72, 3.92(2H, oT, 7=13.2, each, H-10), 4.62(1H, d, J=7.6 Hz, H-G1), 4.99(1H, dd, 7=4.4, 6.0 Hz, H-4), 5.06 (IH, d, .7=8.4 Hz, H-6), 5.11(1H, d, 7=9.6 Hz, H-1), 6.42(1H, dd, 7=1.2. 6.0 Hz, H-3), 6.89(1H, d, 7=8.4 Hz, H-51), 7.46(1 H, d, 7=2.0 Hz, H-21), 7.52(1 H, dd, 7=2.0, 8.4 Hz, H-6'), 3.83(3H, s, 3'-O-CH3).
13C-NMR (100 MHz, DMSO- d6) 8: 93.0(C-1), 141.1(0-3), 101.8 (0-4), 35.2(0-5), 79.7(0-6), 58.2(0-7), 65.8(0-8), 41.8(0-9), 58.5(0-10), 120.0(0-1'), 112.7(0-2'), 147.5(0-3'), 152.0(0-4'), 115.3(0-5'), 123.8 (C-61), 165.6(0-7'), 97.9(0-Gl), 73.4(C-G2), 76.4(0-03), 70.3(C-G4), 77.5(0-05), 61.4(C-G6), 55.7(3'-OCH3).
6-O-3.4-dihvdroxvcinnamoyl catalpol(verminoside)
'H-NMR (400 MHz, DMSO-d6) 5: 2.43(1H, m, H-9), 2.45(1H, m, H-5), 3.01(1H, m, H-G4), 3.05 (IH, m, H-G2), 3.14(1H, m, H-G5), 3.18(1H, m, H-G3), 3.42, 3.70(2H, m, H-G6), 3.64(1H, brs, H-7), 3.71, 3.90(2H, d, 7=13.2 Hz, each, H-10), 4.61(1H, d, 7=8.4 Hz, H-G1), 4.94(1 H, dd, 7=4.0, 5.6 Hz, H-4), 4.99 (IH, dt .7=7.2 Hz, H-6), 5.08(1 H, d, .7=9.2 Hz, H-1), 6.42(1H, d, .7=5.6 Hz, H-3), 6.77(1H, d, 7=8.0 Hz, H-5'), 7.08(1H, d, 7=1.6 Hz, H-2'), 7.05(1H, dd, .7=1.6, 8.0 Hz, H-6').
13C-NMR (100 MHz, DMSO- d6) 8: 92.9(0-1), 141.1(0-3), 101.7 (0-4), 35.1(0-5), 79.2(0-6), 58.2(0-7), 65.7(0-8), 41.8(0-9), 58.5 (C-10), 125.4(0-1'), 115.8(0-2'), 146.0(0-3'), 148.6(0-4'), 113.3 (C-51), 121.6(0-6'), 145.6(0-7'), 115.0 (C-81), 97.9(C-G1), 73.4(C-G2), 76.4(C-G3), 70.3(C-G4), 77.5(C-G5), 61.4(0-06).
3-4. Preparation of 6-0-veratroyl catalpol from the extract of P. longifolium
6.2g of fraction 4 was subjected to column chromatography. 1.2g of fraction 4-3 was performed to recrystalization in methanol to obtain 672.6mg of 6-O-veratroyl catalpol, i.e., 6-O-3,4-Dimethoxybenzoyl.
6-O-(3.4-dimethoxybenzovl) catalpol (6-O-veratrovl catalpol)
'H-NMR (400 MHz, DMSO-d6) 8: 2.47(1 H, dd, 7=8.0, 9.6 Hz, H-9), 2.59(1H, dddd, 7=1.6, 4.8, 8.0,8.0 Hz, H-5), 3.00(1H, m, H-G4), 3.05 (IH, m, H-G2), 3.14(1H, m, H-G5), 3.18(1H, m, H-G3), 3.42, 3.71(2H, m, H-G6), 3.70(1H, brs, H-7), 3.72, 3.90(2H, d, 7=13.2 Hz, each, H-10), 4.61(1H, d, J=7.6 Hz, H-G1), 4.97(1 H, dd, .7=4.8, 6.0 Hz, H-4), 5.08 (IH, d, 7=8.8 Hz, H-6), 5.10(1H, d, 7=9.6 Hz, H-1), 6.42(1H, dd, 7=1.6. 6.0 Hz, H-3), 7.09(1H, d, 7=8.4 Hz, H-5'), 7.46(1H, d, 7=2.0 Hz,
H-2'), 7.64(1 H, dd, .7=2.0, 8.4 Hz, H-61), 3.81, 3.84(6H, s each, 3', 4'-OCH3).
I3C-NMR (100 MHz, DMSO- d6) 5: 92.9(C-1), 141.1(C-3), 101.8 (C-4), 35.2(C-5), 79.9(C-6), 58.2(C-7), 65.9(C-8), 41.8(C-9), 58.4 (C-10), 121.3(C-1'), 111.8(C-2'), 148.5(C-3'), 153.2(C-4'), 111.2 (C-51), 123.5(C-6'), 165.5(C-7'), 97.8(C-Gl), 73.4(C-G2), 76.4(C-G3), 70.3(C-G4), 77.5(C-G5), 61.4(C-G6), 55.6, 55.7(3', 4'-OCH3).
3-5. Preparation of minecoside from the extract of P. longifolium
261.Omg of fraction 4-4 and 288.Omg of fraction 4-5 were subjected to repeated silica gel column chromatography (chloroform-methanol mixture, methanol 10-20% step gradient) to obtain 52.5mg of minecoside, i.e., 6-0-3-hydroxy-4-methozycinnamoyl catalpol.
6-0-3-hvdroxy-4-methozvcinnamoyl catalpol(minecoside)
'H-NMR (400 MHz, DMSO-d6) 5: 2.46(1H, m, H-9), 2.48(1H, m, H-5), 3.00(1H, m, H-G4), 3.05 (1H, m, H-G2), 3.14(1H, m, H-G5), 3.18(1H, m, H-G3), 3.42, 3.70(2H, m, H-G6), 3.67(1 H, br s, H-7), 3.72, 3.91(2H, d, .7=13.2 Hz, each, H-10), 4.61(1H, d, .7=8.8 Hz, H-G1), 4.94(1H, dd, J=4.0, 6.0 Hz, H-4), 5.00 (1H, , .7=7.2 Hz, H-6), 5.09(1H, d, J=9.2 Hz, H-l), 6.42(1H, dd, .7=1,2, 5.6 Hz, H-3), 6.96(1H, d, 7=8.0 Hz, H-51), 7.13(1H, d, .7=2.0 Hz, H-2'), 7.17(1H, dd, .7=2.0, 8.0 Hz, H-61), 3.82(3H, s, -OCH3).
13C-NMR (100 MHz, DMSO- d6) 5: 93.0(C-1), 14U(C-3), 101.7 (C-4), 35.1 (C-5), 79.3(C-6), 58.2(C-7), 65.7(C-8), 41.8(C-9), 58.5 (C-10), 126.8(C-1'), 114.5(C-2'), 146.7(C-3'), 150.2(C-4'), 112.0(C-5'), 121.4(C-6'), 145.7(C-7'), 114.5 (C-81), 97.9(C-G1), 73.4(C-G2), 76.4(C-G3), 70.3(C-G4), 77.5(C-G5), 61.4(C-G6), 55.6 (4'-OCH3).
3-6. Preparation of catalpol from the extract of P. longifolium
Verproside was hydrolyzed to yielded catalpol (compound 1) with 0.1N of KOH, The solution was stirred for 8 hours at room temperature and neutralized with 0.1N of HCL solution. The product was concentrated by rotary evaporator under reduced pressure, subjected to reversed phase silica gel column(RP18, methanol/water, 1/4, v/v), and yielded 54.0mg of catalpol.
Catalpol
'H-NMR (400 MHz, DMSO-d6) 6: 2.12(1H, dddd, ,7=1.6, 4.0, 8.0, 8.0 Hz, H-5), 2.31(1H, d, .7=8.0, 9.6 Hz, H-9), 3.00(1H, m, H-G4), 3.05(1H, m, H-G2), 3.11(1H, m, H-G5), 3.17(1H, m, H-G3), 3,34(1H, br s, H-7), 3.40, 3.70(2H, w, H-G6), 3.63, 3.87(2H, d, 7=12.8, each, H-10), 3.76 (1H, d, .7=8.0 Hz, H-6), 4.59(1H, d, .7=8.0 Hz, H-G1), 4.90(1H, d, .7=9.6 Hz, H-l), 5.01(1H, dd, .7=4.6, 6.0 Hz, H-4), 6.36(1H, dd, J=1.6, 6.0 Hz, H-3).
I3C-NMR (100 MHz, DMSO- d6) 8: 93.2(C-1), 140.2(C-3), 103.3(0-4), 37.4(0-5), 77.1(0-6), 60.7(0-7), 64.8(0-8), 42.1(0-9), 58.9(0-10), 97.8(C-G1), 73.4(C-G2), 76.4(C-G3), 70.2(C-G4), 77.4(C-G5), 61.3(C-G6).
Experimental Example 1. MTT assay
To investigate the cytotoxic effect of inventive extract of P. longifolmm extract and the compound isolated therefrom was determined by (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide(MTT) assay method(Wang Z et al., Biol., Pharm. Bull., 24, pp 159-162, 2001).
Promyelotic HL-60 cells (HL-18103, 5X105 cells/ml) were seeded in 96-well plates under NGF-free condition. After 24 hours incubation, the cells were treated with the mixture of samples dissolved in lOpl of DMSO and lOpl of MTT solution (5mg/ml), and incubated for 4 hours under similar condition. 4 hours later, MTT was removed and lOOul of DMSO was dropped into each well to dissolve crystals. At 570nm, UV absorbance was measured by microplate reader (BIO-RAD, U.S.A.) to calculate the cell viability.
As shown in Table 1, the result demonstrates that the cell viability ranges from 98% to 116% in 50uM, from 95% to 114% in lOOuM. It is confirmed that an inventive extract or compound the present invention has no cell toxicity.
[Table 1] Effect of compounds isolated from P. hngifolium on HL-60 cells.
(Table Removed)
Experimental Example 2. Airway hyperresponsiveness (AHR)
The AHR was evaluated by calculation Penh values (enhanced pause) 24 hours
after the final OVA challenge. The Penh value of the OVA-treated group was significantly higher than that of the PBS control group. In the P. longifolium extract +OVA-challenged group, the Penh value was significantly reduced compared with that of the OVA-treated group at 30 mg/ml methacholine. (table 2) In the verproside+OVA-challenged group, the Penh value was significantly reduced compared with that of the OVA-treated group (P (Table Removed)
Experimental Example 3. Effect of P. longifolium on OVA-induced eosinophilia in BALF
3-1. Animal sensitization and airway challenge
Specific pathogen-free female BALB/c mice aged 8-10 weeks, which were routinely screened serologically for relevant respiratory pathogens, were purchased from ORIENT Co Ltd (Seoul, Korea).
Following treatment: (1) sham-sensitization plus challenge with phosphate-buffered saline (PBS; ipNeb); (2) sensitization plus challenge with OVA (ovalbumin: Sigma A5503; Sigma, St. Louis, MO)(ipNeb); (3) sensitization with OVA(i.p.) plus challenge with OVA (Neb) and samples (extract of P. longifolium or montelukast) was performed to Group of mice (n=5). Briefly, mice were sensitized by intraperitoneal injection of 20#g OVA, which was emulsified with 2 mg aluminum hydroxide in 100 ul of PBS buffer (pH 7.4) on days 0 and 14. The mice were challenged through the airways with OVA(1% in PBS) for 20 min using an ultrasonic nubuilizer (NE-U12; Omron Corp., Tokyo, Japan) on days 28,29 and 30 after the initial sensitization. The mice were sacrificed 48 h after the last challenge (day 32) to determine the suppression effect of extract of P. longifolium or verproside on the
airways of allergic asthma.
3-2. Sample treatment
The extract of P. longifolium, and verproside were suspended in PBS and administered intragastrically using a 25-gauge stainless steel blunt feeding needle 1 h before each challenge, and control animals were exposed only on the PBS solution. As a positive control, montelukast (MSD Korea Ltd., Seoul, Korea) was treated with the same procedure in the experiment.
The mice were sacrificed with an overdose of pentobarbital (Sigma P3761) 24h after the last challenge, and a tracheotomy was performed. After ice-cold 0.5ml of PBS was instilled into the lungs, bronchoalveolar lavage fluid (BALF) was obtained by aspiration three times (total 1.5ml) via tracheal cannulation (Yamazaki T, J. Jap. Bot., 43, pp 117-24, 1968)
3-3. Inflammatory cell counts in bronchoalveolar lavage fluid
The total inflammatory cell number was assessed by counting cells in at least five squares of a hemocytometer after excluding dead cells confirmed by staining with trypan blue (Daigle I. et al., Swiss Med Wkly, 131, pp 231-7, 2001). lOOyl of BALF was loaded onto a slide and centrifuged (200Xg, 4°C, lOmin) to fix the cells onto the slide using a cytospine machine (Hanil Science Industrial, Korea). The cells were stained by Diff-Quick® Stain reagents (Sysmex, Cat No.38721, Switzerland) according to the manufacturer's instructions. Statistical significance was determined by Student's two-tailed Hest for independent means and the critical level for significance was set at P To evaluate the suppression of verproside on the eosinophilia in OVA-challenged mice, the recruited cells in BALF were counted 48 hours after the last challenge. OVA caused a marked influx of leucocytes into the BALF from a PBS control group. As shown in Figure 2, the total cells were counted as 40.5 ± 16.4 x 104 cells/mouse (P 80.7 ± 11.1% decrease in eosinophils (P Experimental Example 4. Lung histology
To estimate the suppressive effect of verproside on the eosinophilia, lung tissues were collected 48 hours after the last challenge. The lung tissue was fixed for 24 h in 10% neutral-buffered formalin. After being embedded in paraffin, it is sliced into 4-um thickness of sections and the tissue was stained with H&E solution (hematoxylin; Sigma MHS-16 and eosin, Sigma HT110-1-32). In the OVA-challenged mice, leukocytes were found to be infiltrated into the peri-bronchiole and peri-vascular connective tissue; of these leukocytes, eosinophilia was mainly observed (Fig. 3-11, P In Periodic acid Schiff (PAS) staining, mucus overproduction in the OVA-treated mice was clearly observed as a violet color in the bronchial airways compared with the normal mice. In contrast, mucus was markedly diminished in the verproside + OVA-challenged mice (Fig. 4-A). Goblet-cell hyperplasia in the airway epithelium was quantified based on a five-point system: 0, no goblet cells; 1, 75% of the epithelium. For each mouse, five airway sections that were randomly distributed throughout the left lung were analysed, and their average scores were calculated. Quantitative analysis of mucus production was performed using an image analyzer (Leica Microsystem Imaging solution Ltd.; Cambridge, UK). As shown in Figure 4-B, the mucus area was scored as 3.60 ± 0.64 in the OVA-treated mice compare with PBS-treated mice (P These results demonstrated that verproside reduced eosinophilia and mucus hypersecretion significantly in the airway remodeling process.
Experimental Example S. Measurement of IgE and cytokines
Complementary capture and detection antibody pairs for mouse IgE antibodies were purchased from PharMingen (San Diego, CA), and the IgE enzyme-linked immunosorbent assay (ELISA) was performed according to the manufacturer's directions. Duplicate samples in plasma were diluted to 1:100. IgE levels in each sample were measured from optical density readings at 450 nm, and IgE concentrations were calculated from a standard curve that was generated using recombinant IgE (5-2,000 ng/ml). The amount of IL-4 and IL-13 contained in BALF was measured with a specific mouse ELISA kit (R&D Systems; Minneapolis, MN). The detection limit of the assays was 250 pg/ml.
As shown in Fig. 5-A & 5-B, The IgE levels were found to be greatly increased in the OVA-treated mice: 85.6 ± 17.3 ug/ml (plasma, P 31.4 ± 14.2 yg/ml (plasma, P To determine the effect of verproside on cytokine release in the OVA-induced asthmatic mice, the levels of cytokines (IL-4 and IL-13) in BALF were measured using ELISA 48 hours after the last challenge. OVA challenge induced a significant elevation of the cytokines to 14.1 ± 6.1 pg/ml (IL-4) and 178.5 ± 96.4 pg/ml (IL-13) in the BALF compared with the control (IL-4, 0.1 ± 0,5 pg/ml; IL-13, 0.1 ± 1.0 pg/ml). In the verproside-treated group, the cytokines were significantly suppressed;
64.5 ± 27.7% decrease in IL-4 (P from a OVA-challenged group. Montelukast also showed a significant reduction in
both of IL-4 (69.5 ± 22.0% decrease, P 0.05) from the control. These results demonstrate that verproside reduced the IL-4
and IL-13 concentrations in the BALF of the asthmatic model as montelukast did
(Figure 5-C and 5-D).
Hereinafter, the formulating methods and kinds of excipients will be described, but the present invention is not limited to them. The representative preparation examples were described as follows.
Preparation of injection
Dried powder of Example 1 orverproside IQQmg
Sodium metabisulfite 3.0mg
Methyl paraben O.Smg
Propyl paraben 0.1 mg
Distilled water for injection optimum amount
Injection preparation was prepared by dissolving active component, controlling pH to
about 7.5 and then filling all the components in 2ml ample and sterilizing by
conventional injection preparation method.
Preparation of powder
Dried powder of Example 1 or verproside 500mg
Corn Starch lOOmg
Lactose lOOmg
Talc lOmg
Powder preparation was prepared by mixing above components and filling sealed
package.
Preparation of tablet
Dried powder of Example 1 orverproside 200mg
Corn Starch lOOmg
Lactose lOOmg
Magnesium stearate optimum amount
Tablet preparation was prepared by mixing above components and entabletting.
Preparation of capsule
Dried powder of Example 1 orverproside lOOmg
Lactose 50mg
Corn starch 50mg
Talc 2mg
Magnesium stearate optimum amount
Tablet preparation was prepared by mixing above components and filling gelatin
capsule by conventional gelatin preparation method.
Preparation of liquid
Dried powder of Example 1 orverproside lOOOmg
Sugar 20g
Polysaccharide 20g
Lemon flavor 20g
Liquid preparation was prepared by dissolving active component, and then filling all
the components in 1000M ample and sterilizing by conventional liquid preparation
method.
Preparation of health food
Dried powder of Example 1 or verproside lOOOmg
Vitamin mixture optimum amount
Vitam i n A acetate 70 (ag
Vitamin E l.Omg
Vitamin Bi 0.13mg
Vitamin 82 0.15mg
Vitamin B6 O.Smg
Vitamin B12 0.2jag
Vitamin C lOmg
Biotin lOj^g
Amide nicotinic acid 1,7mg
Folic acid 50|ag
Calcium pantothenic acid O.Smg
Mineral mixture optimum amount
Ferrous sulfate 1.75mg
Zinc oxide 0.82mg
Magnesium carbonate 25.3mg
Monopotassium phosphate 15mg
Dicalcium phosphate 55mg
Potassium citrate 90mg
Calcium carbonate lOOmg
Magnesium chloride 24.8mg
The above mentioned vitamin and mineral mixture may be varied in many ways. Such
variations are not to be regarded as a departure from the spirit and scope of the present
invention.
Preparation of health beverage
Dried powder of Example 1 or verproside 1 OOOmg
Citric acid lOOOmg
Ol igosaccharide 1 OOg
Apricot concentration 2g
Taurine lg
Distilled water 900m£
Health beverage preparation was prepared by dissolving active component, mixing, stirred at 85 °C for 1 hour, filtered and then filling all the components in 1000in£ ample and sterilizing by conventional health beverage preparation method.
The invention being thus described, it will be obvious that the same may be varied in many ways. Such variations are not to be regarded as a departure from the spirit and scope of the present invention, and all such modifications as would be obvious to one skilled in the art are intended to be included within the scope of the following claims.
Industrial Applicability
As described in the present invention, the extract of P. longifolium and the catalpol derivatives isolated therefrom show the suppression of elevated IgE, IL-4 and IL-13 levels and eosinophilia in plasma and BALD, and mucus overproduction in the lung tissues using by OVA-induced asthmatic mouse model. Therefore, it can be used as the therapeutics or functional health food for treating and preventing inflammatory, allergic and asthmatic disease.

We Claim
1. A pharmaceutical composition comprising a crude extract or organic solvent soluble
extract of Pseudolysimachion genus plant as an active ingredient and
pharmaceutically acceptable carriers, adjuvants or diluents for the treatment and
prevention of inflammatory, allergic and asthmatic disease,
wherein the composition contains the extract in the range of from 0.1 to 50% by weight and the pharmaceutically acceptable carriers, adjuvants or diluents in the range of from 99.9 to 50% by weight based on the total weight of the composition.
2. The pharmaceutical composition as claimed in claim 1, wherein said crude extract or organic solvent soluble extract is extracted with the solvent selected from the group consisting of water, lower alcohols, acetone, ethyl acetate, chloroform, dichloromethane or mixtures thereof.
3. The pharmaceutical composition as claimed in claim 1, wherein said extract is extracted from P. longifolium, P. ovtum, P. khisianum, P. kiusianum var. diamanticum. P. kiusianum var. villosum, P. dahuricum, P. pyrethrinum, P. linarifolium, P. linarifolium var. villosulum, P. rotundum var. suhintegrum, P. roiundum var. coreanum. P. insulare or P. undulate.
4. A pharmaceutical composition comprising catalpol derivatives represented by general formula (I) or pharmaceutically acceptable salt thereof as an active ingredient and pharmaceutically acceptable carriers, adjuvants or diluents for treatment and prevention of allergic and asthmatic disease,
wherein the composition contains the catalpol derivatives in the range of from 0.1 to 50% by weight and the pharmaceutically acceptable carriers, adjuvants or diluents in the range of from 99.9 to 50% by weight based on the total weight of the composition;

(Formula Removed)
wherein,
R is independently at least one groups selected from a hydrogen atom, 3.4-dihydroxybenzoyl, 3-hydroxy-4-methoxybenzoyl, 4-hydroxyben/oyl, 3.4-dimethoxybenzoyl, and cinnamoyl group substituted with C1-C3 lower alkyl group or C1-C3 lower alkoxy group.
5. The pharmaceutical composition as claimed in claim 4, wherein said cinnamoyl group substituted with C1-C3 lower alkyl group or C1-C3 lower alkoxy group is 3.4-dihydroxycinnamoyl or 3-hydroxy-4-methoxycinnamoyl group.
6. A functional health food comprising, a extract of crude extract or organic solvent soluble extract of Pseudolysimachion genus plant as set forth in claim 1, or the catalpol derivatives as set forth in claim 4 together with a sitologically acceptable additive for the prevention and improvement of inflammatory, allergic and asthmatic disease,
wherein the food contains the extract or the catalpol derivatives in the range of from 0.01 to 80% w/w and the sitologically acceptable additive in the range of from 99.99 to 20% w/w based on the total weight of the food.
7. The functional health food as claimed in claim 7, wherein said health food is provided as a form of pill, powder, granule, tablet, chewing table, capsule or beverage type.

Documents:

9757-DELNP-2007-Abstract-(25-01-2012).pdf

9757-delnp-2007-abstract.pdf

9757-DELNP-2007-Assignment-(05-05-2009).pdf

9757-DELNP-2007-Claims-(25-01-2012).pdf

9757-delnp-2007-claims.pdf

9757-DELNP-2007-Correspondence Others-(25-01-2012).pdf

9757-DELNP-2007-Correspondence Others-(31-01-2012).pdf

9757-DELNP-2007-Correspondence-Others-(05-05-2009).pdf

9757-delnp-2007-correspondence-others-1.pdf

9757-delnp-2007-correspondence-others.pdf

9757-delnp-2007-description (complete).pdf

9757-DELNP-2007-Drawings-(25-01-2012).pdf

9757-delnp-2007-drawings.pdf

9757-DELNP-2007-Form-1-(25-01-2012).pdf

9757-delnp-2007-form-1.pdf

9757-delnp-2007-form-18.pdf

9757-DELNP-2007-Form-2-(25-01-2012).pdf

9757-delnp-2007-form-2.pdf

9757-DELNP-2007-Form-3-(25-01-2012).pdf

9757-delnp-2007-form-3.pdf

9757-DELNP-2007-Form-5-(25-01-2012).pdf

9757-delnp-2007-form-5.pdf

9757-delnp-2007-form-6-(05-05-2009).pdf

9757-DELNP-2007-GPA-(25-01-2012).pdf

9757-delnp-2007-pct-210.pdf

9757-delnp-2007-pct-237.pdf

9757-delnp-2007-pct-304.pdf

9757-DELNP-2007-Petition-137-(25-01-2012).pdf

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Patent Number

254672

Indian Patent Application Number

9757/DELNP/2007

PG Journal Number

49/2012

Publication Date

07-Dec-2012

Grant Date

04-Dec-2012

Date of Filing

17-Dec-2007

Name of Patentee

KOREA RSEARCH INSTIUTE OF BIOSCIENCE AND BIOTECHNOLOGY

Applicant Address

51, EOEUN-DONG, USEONG-GU, DAEJEON 305-806, KOREA

Inventors:

#	Inventor's Name	Inventor's Address
1	LEE, HYEONG KYU	111-101, HANBIT APT., 99, EOEUN-DONG, YUSEONG-GU, DAEJEON 305-755, KOREA.
2	OH, SEI RYANG	210-1703, SINSEON MAEUL APT., GWANJEO-DONG, SEO-GU, DAEJEON 302-243, KOREA
3	LEE, SANG KU	504-101, YEOLMAE MAEUL APT., JIJOK-DONG, YUSEONG-GU, DAEJEON 305-330, KOREA
4	KWON, OK KYOUNG	308-704, CHUNGSOL APT., SONGGANG-DONG, YUSEONG-GU, DAEJEON 305-503,KOREA
5	JOUNG, HYOUK	404, 804, EXPO APT., JEONMIN-DONG, YUSEONG-GU, DAEJEON 305-762, KOREA
6	QUAN, GUI HUA	52, EOEUN-DONG, YUSEONG-GU, DAEJEON 305-333, KOREA.
7	KIM, MI JIN	201HO, 161-6, SINSEONG-DONG, YUSEONG-GU, DAEJEON 305-345, KOREA.
8	PARK, BO YOUNG	105-101, NARAE APT., JEONMIN-DONG, YUSEONG-GU, DAEJEON 305-390, KOREA
9	AHN, KYUNG SEOP	802-201, YEOLMAE MAEUL APT., NOEUN-DONG, YUSEONG-SU, DAEJEON 305-325, KOREA
10	LEE, JOONG KU	109-603, EXPO APT., JEONMIN-DONG, YUSEONG-GU, DAEJEON 305-761, KOREA
11	KIM, DOO YOUNG	126-208, HANBIT APT., 99, EOEUN-DONG, YUSEONG-GU, DAEJEON 305-333, KOREA.

PCT International Classification Number

A61K 31/352

PCT International Application Number

PCT/KR2006/002092

PCT International Filing date

2006-05-30

PCT Conventions:

#	PCT Application Number	Date of Convention	Priority Country
1	10-2005-0045755	2006-05-30	Republic of Korea
2	10-2005-0045756	2006-05-30	Republic of Korea
3	10-2006-0048319	2006-05-29	Republic of Korea
4	10-2006-0048104	2006-05-29	Republic of Korea