Method of treating inflammation, allergy and asthma with a purified extract (ATC1) isolated from Pseudolysimachion rotundum var. subintegrum containing abundant amount of active ingredient

Abstract

The present invention relates to inventive novel industrialized method for preparing purified extract containing more abundant active ingredients such as catalpol derivatives from the extract of Pseudolysimachion rotundum var subintegrum than that prepared by the conventional preparation method disclosed in the prior art and the therapeutics or functional health food comprising the purified extract for treating and preventing inflammatory, allergic or asthmatic disease. The purified extract showed more potent anti-inflammatory, anti-allergy and anti-asthma activity than that prepared by the conventional preparation method disclosed in the prior art through various in vivo tests such as inhibition test on the reproduction of eosinophil, the release of immunoglobulin and inflammatory chemokines in plasma and bronchoalveolar fluid as well as the suppression of airway hyperresponsiveness and goblet cell hyperplasia in a OVA-sensitized/challenged mouse model.

Claims

1. A method of treatment comprising the administering of the pharmaceutical composition comprising a purified extract fractionated with butanol (ATC1) from the extract of Pseudolysimachion rotundum var subintegrum, comprising 15%-50% (w/w) verproside, 0.3%-10% (w/w) veratric acid, 0.5%-10% (w/w) catalposide, 0.3%-10% (w/w) picroside II, 0.3%-10% (w/w) isovanilloyl catalpol and 0.5%-10% (w/w) 6-O-veratroyl catalpol based on the weight of total extract (100%) of Pseudolysimachion rotundum var subintegrum to a subject in need of treatment of an ailment selected from the group consisting of an inflammatory disease, an allergy disease and an asthma disease.

2. A method of treatment comprising the administering of the pharmaceutical composition comprising a purified extract fractionated with butanol (ATC1) from the extract of Pseudolysimachion rotundum var subintegrum, comprising 12.3%-47% (w/w) catalpol derivatives selected from the group consisting of verproside, catalposide, picroside II, isovanilloyl catalpol and 6-O-veratroyl catalpol in total extract (100%) of Pseudolysimachion rotundum var subintegrum and which has a relative mixed ratio (w/w) between weight of each catalpol derivative of 15.0-18.0 (w/w) verproside, 2.10-2.60 (w/w) catalposide, 1 (w/w) picroside II, 1.00-1.30 (w/w) isovanilloyl catalpol and 2.00-2.30 (w/w) 6-O-veratroyl catalpol to a subject in need of treatment of an ailment selected from the group consisting of an inflammatory disease, an allergy disease and an asthma disease.

3. The method of claim 1, wherein the inflammatory disease is selected from the group consisting of eczema, atopic dermatitis, conjunctivitis, periodontal disease, rhinitis, otitis media, laryngopharyngitis, tonsillitis, pneumonia, gastric ulcer, gastritis, Crohn's disease, colitis, hemorrhoid, gout, ankylosing spondylitis, rheumatic fever, systemic lupus erythematosus, fibromyalgia, psoriatic arthritis, osteoarthritis, rheumatic arthritis, periarthritis of shoulder, tendinitis, tenosynovitis, peritendinitis, myositis, hepatitis, cystitis, nephritis, Sjogren's syndrome, multiple sclerosis, chronic inflammatory disease, and acute inflammatory disease; wherein the allergy disease is selected from the group consisting of allergic rhinitis, allergic dermatitis, contact dermatitis, hives, insect allergy, food allergy, drug allergy, allergic conjunctivitis, and hypersensitivity; and wherein the asthma disease is selected from the group consisting of sensitivity to dust mites, fur, dandruff, cockroach, food, drug, cough, cigarette smoke, air pollution, food additive, physical activity, exercise, weather change, yellow sand and stress.

4. The method of claim 2, wherein the inflammatory disease is selected from the group consisting of eczema, atopic dermatitis, conjunctivitis, periodontal disease, rhinitis, otitis media, laryngopharyngitis, tonsillitis, pneumonia, gastric ulcer, gastritis, Crohn's disease, colitis, hemorrhoid, gout, ankylosing spondylitis, rheumatic fever, systemic lupus erythematosus, fibromyalgia, psoriatic arthritis, osteoarthritis, rheumatic arthritis, periarthritis of shoulder, tendinitis, tenosynovitis, peritendinitis, myositis, hepatitis, cystitis, nephritis, Sjogren's syndrome, multiple sclerosis, chronic inflammatory disease, and acute inflammatory disease; wherein the allergy disease is selected from the group consisting of allergic rhinitis, allergic dermatitis, contact dermatitis, hives, insect allergy, food allergy, drug allergy, allergic conjunctivitis, and hypersensitivity; and wherein the asthma disease is selected from the group consisting of sensitivity to dust mites, fur, dandruff, cockroach, food, drug, cough, cigarette smoke, air pollution, food additive, physical activity, exercise, weather change, yellow sand and stress.

Description

DESCRIPTION OF DRAWINGS

(1) 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;

(2) FIG. 1 shows HPLC analysis of the crude extract of Pseudolysimachion rotundum var subintegrum prepared in comparative Example 1;

(3) FIG. 2 shows HPLC analysis of the inventive purified extract (ATC1) of Pseudolysimachion rotundum var subintegrum prepared in Example 1;

(4) FIG. 3 shows HPLC analysis of the inventive purified extract (ATC2) of Pseudolysimachion rotundum var subintegrum prepared in Example 2;

(5) FIG. 4 shows the suppressive effect of the inventive purified extract on airway hyperresponsiveness in a OVA-sensitized/challenged mouse model;

(6) FIG. 5 shows the inhibitory effect of the inventive purified extract on the recruitment of inflammatory cells in bronchoalveolar lavage fluid;

(7) FIG. 6 shows the inhibitory effect of the inventive purified extract on the release of immunoglobulin (total IgE) in blood serum;

(8) FIG. 7 shows the inhibitory effect of the inventive purified extract on the release of OVA-specific IgE in blood serum;

(9) FIG. 8 represents the inhibitory effect of the inventive purified extract on the release of IL-1 beta;

(10) FIG. 9 presents the inhibitory effect of the inventive purified extract on the release of inflammatory chemokines;

(11) FIG. 10 represents the inhibitory effect of the inventive purified extract on the recruitment of inflammatory cells on lung tissue cell using by the histological examination of bronchoalveolar lavage;

(12) FIG. 11 represents the inhibitory effect of the inventive purified extract on the mucus secretion in lung tissue cell using by the histological examination of bronchoalveolar lavage;

(13) FIGS. 12 and 13 represent the inhibitory effect of the inventive purified extract on the recruitment of inflammatory cells on lung tissue cell using by the histological examination of bronchoalveolar lavage;

(14) FIGS. 14 and 15 represents the inhibitory effect of the inventive purified extract on the mucus secretion in lung tissue cell using by the histological examination of bronchoalveolar lavage.

BEST MODE FOR CARRYING OUT THE INVENTION

(15) 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.

(16) 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

(17) The following Reference Example, Examples and Experimental Examples are intended to further illustrate the present invention without limiting its scope.

Comparative Example 1

Preparation of the Crude Extract of Pseudolysimachion rotundum Var Subintegrum

(18) 1-1. Preparation of Crude Extract (ATE)

(19) 1 kg of dried Pseudolysimachion rotundum var subintegrum (cultivated at 244, Soi-myeon Eumseong-gun Chungcheongbuk-do in Korea according to GAP) cut into small pieces and mixed with 10 L of 40% ethanol. The mixture was stirred at room temperature for 24 hours and extracted with reflux extraction at 78 C. for 12 hours to collect the filtrate, three times. The extract was filtered with filter paper to remove the debris. The collected filtrate was concentrated by rotary evaporator (EYELA, N-2100, Japan) at 55-65 C. under reduced pressure and dried with freezing dryer to obtain 202 g of dried crude extract (designated as ACE hereinafter) for used as a comparative example.

(20) 1-2. Preparation of Crude Extract (ATM)

(21) 1.1 kg of dried Pseudolysimachion rotundum var subintegrum (cultivated at 244, Soi-myeon Eumseong-gun Chungcheongbuk-do in Korea according to GAP) cut into small pieces and mixed with 5 L of methanol. The mixture was stirred at room temperature for 24 hours and extracted with reflux extraction at 78 C. for 12 hours to collect the filtrate, three times. The extract was filtered with filter paper to remove the debris. The collected filtrate was concentrated by rotary evaporator (EYELA, N-2100, Japan) at 55-65 C. under reduced pressure and dried with freezing dryer to obtain 100.5 g of dried crude extract (designated as ACM hereinafter) for used as a comparative example.

(22) 1-3. Component Analysis

(23) The component analysis was performed using by HPLC (Agilent 1260 model, USA) according to the condition in Table 1 and the result was shown in FIG. 1.

(24) As can be seem in FIG. 1, it has been confirmed that each ingredient was detected at 9.548 mins (Verproside), 10.817 mins (Veratric acid), 16.728 mins (Catalposide), 20.346 min (Picroside II), 21.853 mins (Isovanilloyl catalpol), and 30.462 mins (6-O-veratrolyl catalpol) respectively.

(25) The content of each ingredient (%) in the sample was calculated based on the HPLC pattern (retention time) according to math formulae 1.
content of each ingredient=conc. of standard (mg/ml)/conc. of test sample (mg/ml)At/Aspurity of standard (%)Math formulae 1
wherein At denotes the ingredient area in test sample and As denotes that in standard provided that the sampled volume of test sample and standard is identical to each other.

(26) TABLE-US-00001 TABLE 1 HPLC condition HPLC condition Pump Agilent 1260 Series, 1260 quart pump Detector Agilent 1260 Series, 1260 DAD Column Agilent Eclipse XOB C18, 4.6 50 cm, 5 m Flow rate 1.5 ml/min UV Absorbance 266 nm Mobile phase Mobile phase A: phosphate buffer (pH = 3.5) Mobile phase B: methanol Mobile phase A Mobile phase B Time (%) (%) 0~5 80 20 5~20 75 25 20~25 75 25 25~30 55 45 30~35 55 45 35~36 80 20 36~40 80 20 Injection volume 10 l

(27) At the result, it has been confirmed that the crude extract of Pseudolysimachion rotundum var subintegrum contains only 8.49% (w/w) catalposide derivatives, i.e., 5.9% (w/w) verproside, 0.21% (w/w) veratric acid, 0.82% (w/w) catalposide, 0.40% (w/w) picroside II, 0.42% (w/w) isovanillyl catalpol, and 0.74% (w/w) 6-O-veratroyl catalpol, respectively, as can be seen in Table 2.

(28) TABLE-US-00002 TABLE 2 HPLC result (crude extract: ACE) Comparative Example 1 Active ingredient Retention Time (mins) Content (w/w %) Verproside 9.548 5.90 Veratric acid 10.817 0.21 Catalposide 16.728 0.82 Picroside II 20.346 0.40 Insovanilloyl catalpol 21.853 0.42 6-O-veratroyl catalpol 30.462 0.74 Total 8.49

Example 1. Preparation of the Purified Extract (ATC1) of Pseudolysimachion rotundum Var Subintegrum

(29) The crude extract (ACE) of Pseudolysimachion rotundum var subintegrum prepared by the conventional method according to Comparative Example 1, was suspended in 2 L of distilled water and the suspension was added with 2 L of butanol to fractionate into butanol-soluble fraction and water-soluble fraction. The butanol soluble fraction was collected, concentrated under reduced pressure and dried to afford 82 g of the inventive purified extract fractionated with butanol (ATC1) used as a test example.

(30) The component analysis was performed using by HPLC (Agilent 1260 model, USA) according to the condition in Table 1 and the result was shown in FIG. 2.

(31) As can be seem in FIG. 2, it has been confirmed that each ingredient was detected at 9.545 mins (Verproside), 10.821 mins (Veratric acid), 16.727 mins (Catalposide), 20.345 min (Picroside II), 21.853 mins (Isovanilloyl catalpol), and 30.462 mins (6-O-veratroyl catalpol) respectively.

(32) The content of each ingredient (%) in the sample was calculated based on the HPLC pattern (retention time) according to math formulae 1.

(33) At the result, it has been confirmed that the inventive purified extract fractionated with butanol (ATC1) of Pseudolysimachion rotundum var subintegrum contains 25.64% (w/w) catalposide derivatives, i.e., 17.60% (w/w) verproside, 0.72% (w/w) veratric acid, 2.62% (w/w) catalposide, 1.08% (w/w) picroside II, 1.26% (w/w) isovanillyl catalpol, and 2.36% (w/w) 6-O-veratroyl catalpol, respectively, as can be seen in Table 3.

(34) TABLE-US-00003 TABLE 3 HPLC result (purified extract: ATC1) Example 1 Active ingredient Retention Time (mins) Content (w/w %) Verproside 9.545 17.60 Veratric acid 10.821 0.72 Catalposide 16.727 2.62 Picroside II 20.345 1.08 Insovanilloyl catalpol 21.853 1.26 6-O-veratroyl catalpol 30.462 2.36 Total 25.64

Example 2. Preparation of the Purified Extract (ATC2) of Pseudolysimachion rotundum Var Subintegrum

(35) The inventive purified extract fractionated with butanol (ATC1) of Pseudolysimachion rotundum var subintegrum according to Example 1, was dissolved in 75 mL of mixed solvent (distilled water:methanol=1:0.003) and 75 g of the solution was loaded on reverse phase column chromatography (C18(IV)-D-75-120 nm, AGC Si-Tech Co. Ltd., Japan, 450 g) with eluting the suspension using by eluting solvent (distilled water:methanol=90:10.fwdarw.60:40). 8.4 L of the eluted solution running at the initial eluting solvent system (distilled water:methanol=90:10) was collected and concentrated under reduced pressure. 5.6 L of the eluted solution running at the late eluting solvent system (distilled water:methanol=60:40) was collected, concentrated under reduced pressure and dried to afford 33 g of the inventive purified extract with the secondary fractionation (ATC2) used as a test example.

(36) The component analysis was performed using by HPLC (Agilent 1260 model, USA) according to the condition in Table 1 and the result was shown in FIG. 3.

(37) As can be seem in FIG. 3, it has been confirmed that each ingredient was detected at 9.525 mins (Verproside), 10.818 mins (Veratric acid), 16.721 mins (Catalposide), 20.346 min (Picroside II), 21.857 mins (Isovanilloyl catalpol), and 30.462 mins (6-O-veratroyl catalpol) respectively.

(38) The content of each ingredient (%) in the sample was calculated based on the HPLC pattern (retention time) according to math formulae 1.

(39) At the result, it has been confirmed that the inventive purified extract with the secondary fractionation (ATC2) of Pseudolysimachion rotundum var subintegrum contains 65.63% (w/w) catalposide derivatives, i.e., 43.83% (w/w) verproside, 1.80% (w/w) veratric acid, 7.07% (w/w) catalposide, 2.93% (w/w) picroside II, 3.85% (w/w) isovanillyl catalpol, and 6.15% (w/w) 6-O-veratroyl catalpol, respectively, as can be seen in Table 4.

(40) TABLE-US-00004 TABLE 4 HPLC result (purified extract: ATC2) Example 2 Active ingredient Retention Time (mins) Content (w/w %) Verproside 9.524 43.83 Veratric acid 10.818 1.80 Catalposide 16.721 7.07 Picroside II 20.346 2.93 Insovanilloyl catalpol 21.857 3.85 6-O-veratroyl catalpol 30.462 6.15 Total 65.63

Experimental Example 1. Preliminary Determination of the Total Serum IgE Level in OVA-Sensitized/Challenged Mouse Model

(41) In order to found the purified extract showing more pharmacologically potent activity than the crude extract prepared in comparative Example, following preliminary test was performed by the method disclosed in the literature (Elias, J. A. et al., J. Clin. Invest., 111, pp 297-297, 2003).

(42) 1-1. Animal Sensitization and Airway Challenge

(43) Specific pathogen-free female BALB/c mice (about 20 g), aged 6 weeks, which were routinely screened serologically for relevant respiratory pathogens, were purchased from ORIENT Co. (Seoul, Korea) and acclimated with the experimental environment for 1 week.

(44) Briefly, mice were sensitized by intraperitoneal injection of 20 g OVA (Ovalbumin; A5503, Sigma, St. Louis, Mo.), which was emulsified in 2 mg aluminum hydroxide in 200 l of PBS buffer (pH 7.4), biweekly. The mice were challenged through the airways with OVA (1% in PBS) for 30 min using an ultrasonic nebulizer (NE-U12; Omron Corp., Tokyo, Japan) from the 28th day to 34th day after the initial sensitization. 24 hrs after the antigen treatment, the airway hyperresponsiveness was determined and the mice were sacrificed 48 hrs after the last challenge. The mice were sacrificed with an overdose of pentobarbital (Entobal, Hanrim Pharm. Co. Ltd.) 24 h after the last challenge, and a tracheotomy was performed. After 1.2 ml of physiological saline solution (PBS) was instilled into the lungs, bronchoalveolar lavage fluid (BALF) was obtained by aspiration three times (total 1.5 ml) via tracheal cannulation.

(45) The groups were divided into several groups, i.e., (a) normal control group (NC): the groups treated or not-treated with OVA; (b) asthma-induced group (OVA): the groups treated with OVA to induce asthma; and (c) comparative group: the groups treated with positive control group (M30, montelukast; 30 mg/kg, PO, Sigma-Aldrich Co. Ltd., SML 0101) 1 hr prior to OVA inhalation.

(46) The test group consists of 6 mice for each group and 1 hour prior to OVA inhalation, various concentrations of the test sample, ATC1 (30 mg/kg and 100 mg/kg) and ATM (30 and 100 mg/kg) were orally administrated to the mice.

(47) As shown in Table 5, the total level of IgE in blood serum in asthma-induced group (OVA) was significantly increased whereas those in the test sample group orally administrated with various concentrations of test samples (ATC1, 30 mg/kg and 100 mg/kg) were more reduced than that in the group treated with crude extract of Pseudolysimachion rotundum var subintegrum (ATM, 30 and 100 mg/kg). (See Table 5)

(48) TABLE-US-00005 TABLE 5 The level of total IgE in blood serum total IgE (mg/ml) NC 0.57 0.02 OVA 5.23 0.34 ATC1 30 mg/kg 2.05 0.12 100 mg/kg 2.14 0.25 ATM 30 mg/kg 3.09 0.54 100 mg/kg 2.49 0.35 Monte30 1.82 0.40

Experimental Example 2. Anti-Asthamtic Effect Using by Airway Hyperresponsiveness Test in a OVA-Sensitized/Challenged Mouse Model

(49) In order to confirm the anti-asthmatic effect of test samples prepared in Examples using by airway hyperresponsiveness test in a OVA-sensitized/challenged mouse model, following test was performed by the method disclosed in the literature (Elias, J. A. et al., J. Clin. Invest., 111, pp 297-297, 2003).

(50) 1-1. Animal Sensitization and Airway Challenge

(51) Specific pathogen-free female BALB/c mice (about 20 g), aged 6 weeks, which were routinely screened serologically for relevant respiratory pathogens, were purchased from ORIENT Co. (Seoul, Korea) and acclimated with the experimental environment for 1 week.

(52) Briefly, mice were sensitized by intraperitoneal injection of 20 g OVA (Ovalbumin; A5503, Sigma, St. Louis, Mo.), which was emulsified in 2 mg aluminum hydroxide in 200 l of PBS buffer (pH 7.4), biweekly. The mice were challenged through the airways with OVA (1% in PBS) for 30 min using an ultrasonic nebulizer (NE-U12; Omron Corp., Tokyo, Japan) from the 28th day to 34th day after the initial sensitization. 24 hrs after the antigen treatment, the airway hyperresponsiveness was determined and the mice were sacrificed 48 hrs after the last challenge. The mice were sacrificed with an overdose of pentobarbital (Entobal, Hanrim Pharm. Co. Ltd.) 24 h after the last challenge, and a tracheotomy was performed. After 1.2 ml of physiological saline solution (PBS) was instilled into the lungs, bronchoalveolar lavage fluid (BALF) was obtained by aspiration three times (total 1.5 ml) via tracheal cannulation.

(53) Group of mice (n=6) were studied; they received the following treatment: (1) The non-treatment group with OVA as a normal control group (NC); (2) The control group treated and inhaled with OVA as an asthma induced group (OVA); (3) The positive control group treated with known asthma therapeutics (Montelukast; 30 mg/kg, PO, Sigma-Aldrich Korea, SML-0101, M30) 1 hour prior to OVA inhalation; (4) The test sample group orally administrated with various concentrations of test samples, i.e., 5 mg/kg, 10 mg/kg, 25 mg/kg and 50 mg/kg of purified extract (ATC2) 1 hour prior to OVA inhalation.

(54) 1-2. Evaluation of Airway Hyperresponsiveness

(55) In order to evaluate the airway hyperresponsiveness of the mice, the airway resistance was determined using by apparatus (One chamber whole body plethymography, OCP3000, All Medicus, Seoul. Korea) and the determined value was statistically calculated by Pehn value (Enhance Pause) reflecting on the degree of airway obstruction. The Penh value was determined for 3 mins by the process of determining the basal value at the eupnea phase and determining the Penh value after inhaling PBS with Ultrasonic nebulizer (NE-U12, IMRON Corp., Tokyo, JAPAN) for 3 mins.

(56) Thereafter, various concentrations of methacholine (A2251, Sigma, St. Louis, Mo.), 12, 25 and 50 mg/ml, were inhaled with increasing concentration to determine the Pehn values. The increase of Penh value was expressed as percentage (%) after the methacholine inhalation and the Penh value of basal line was set to 100%. The value of Pehn was calculated according to math formulae 2 and the result was shown in FIG. 4.
Pehn=(Te/RT1)PEF/PIFMath formulae 2

(57) Te: Expiration Time (The period from a inhalation to the next inhalation);

(58) RT: Relaxation Time (The period that the exhaled volume is reached to the extent to 30% of one expiration volume during expiration)

(59) PEF: Peak Expiration Flow

(60) PIF: Peak Inspiration Flow

(61) At the result, it has been confirmed that the Penh value in the control group treated and inhaled with OVA as an asthma induced group (OVA) was sharply increased while that in the non-treatment group with OVA as a normal control group (NC) had been gradually increased with increasing the concentration of methancholine.

(62) In a while, the Penh value in the positive control group treated with Montelukast (MO) as well as the test sample group orally administrated with various concentrations of test samples (ATC-10, ATC-25, and ATC-50) were significantly reduced regardless of the concentration of methacholine. (See Table 6)

(63) TABLE-US-00006 TABLE 6 Penh Value Methacholine (Conc. mg/Ml) 0 12.5 25 50 NC 0.37 0.03 0.39 0.04 0.69 0.14 1.07 0.18 OVA 0.85 0.10 2.79 0.25 5.99 0.92 7.24 0.74 ATC2 5 0.52 0.05 1.69 0.27 2.68 0.52 5.19 0.74 (mg/kg) 10 0.63 0.06 1.54 0.18 2.01 0.52 3.08 0.40 25 0.64 0.08 1.31 0.17 1.64 0.31 2.43 0.33 50 0.51 0.06 1.02 0.17 1.36 0.09/ 1.82 0.28 M30 0.49 0.04 1.54 0.20 1.98 0.37 2.51 0.36

(64) It has been confirmed that those change in Penh value has been prominent in case of higher-dose methacholine treatment group rather than in lower-dose methacholine treatment group and the Penh value in the test sample for the same concentration of methacholine, has been remarkably decreased in a dose dependent manner.

(65) Accordingly, it has been confirmed that the inventive purified extract effectively suppressed the airway hyperresponsiveness and therefore, they are useful in treating or preventing the asthma disease, an allergic disease in airway.

Experimental Example 3. Effect on the Level of Eosinophil and Inflammatory Cells in BALF

(66) In order to confirm the inhibition effect of test samples prepared in Examples on the level of eosinophil and inflammatory cells in bronchoalveolar fluid (BALF), following test was performed by the method disclosed in the literature (Chen M. et al., Immunolgy, pp 376-384, 2011).

(67) The bronchoalveolar lavage fluid (BALF) prepared in Experimental Example 1 was recovered to determine the level of inflammatory cells.

(68) The total inflammatory cell number was assessed by the counting of cells in at least five squares of a hemocytometer after excluding dead cells by staining with trypan blue (Daigle I. et al., Swiss Med Wkly, 131, pp 231-7, 2001). 1000 of BALF was loaded onto a slide and centrifuged (200g, 4 C., 10 min) to fix the cells onto the slide using a Cellspin machine (Cyto12.5+clip5, 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 t-test for independent means and the critical level for significance was set at P<0.05.

(69) As shown in FIG. 5, the total number of eosinophil and inflammatory cells in the control group treated and inhaled with OVA as an asthma induced group (OVA) was significantly increased comparing with those in the non-treatment group with OVA as a normal control group (NC).

(70) The total number of eosinophil and inflammatory cells in the positive control group treated with Montelukast (MO) as well as the test sample group orally administrated with various concentrations of test samples (ATC-5, ATC-10, ATC-25, and ATC-50) were significantly reduced. (See Table 7)

(71) TABLE-US-00007 TABLE 7 total number of eosinophil and inflammatory cells in BALF No. of inflammatory cells (10.sup.3 cells/mouse) No. of eosinolphil No. of inflammatory cells NC 0.00 0.00 8.28 1.46 OVA 135.44 4.54 260.48 10.39 ATC2 5 72.23 9.45 158.2 15.38 (mg/kg) 10 55.40 3.46 131.67 9.03 25 40.8 2.34 98.6 4.57 50 36.57 4.02 88.1 7.04 M30 52.03 4.06 106.67 6.48

Experimental Example 4. Effect on the Level of IgE and OVA-Specific IgE in Blood Serum

(72) In order to confirm the inhibition effect of test samples prepared in Examples on the level of IgE and OVA-specific IgE in blood serum, following test was performed by the method disclosed in the literature (Kay, A. B., The New England Journal of Medicine, 344, pp 30-37, 2001).

(73) The blood serum and bronchoalveolar lavage fluid (BALF) prepared in Experimental Example 1 was recovered to determine the level of IgE and OVA-specific IgE in blood serum.

(74) The blood serum and bronchoalveolar lavage fluid (BALF) was added to 96-well plates (ELISA plate) and coated with 0.1M NaHCO.sub.3 buffer solution (pH 8.3) containing 20 g/ml of OVA (Sigma, MO, USA) at 4 C. overnight. After inhibiting nonspecific reaction using by PBS containing 1% bovine serum albumin, the serum for testing was diluted to 1:400 and reacted together for 2 hours at room temperature. After washing, the serum was reacted with diluted (300) anti-mouse IgE monoclonal antibody (MCA419, Serotec, Oxford, UK) for 2 hours and with diluted (4000) HRP-conjugated goat anti-rat IgG polyclonal A (STAR110P, Serotec, UK) for 1 hours at room temperature. After washing, the solution was stained with 3,3, 5,5-tetramethylbenzidine (52-00-02, KPL) substrate and the reaction was stopped by 2N H.sub.2SO.sub.4 to determine the absorbance using by spectroscopy (Versamax, Molecular Devices, US) at 450 nm.

(75) As shown in FIG. 6 and FIG. 7, the level of IgE and OVA-specific IgE in blood serum in the control group treated and inhaled with OVA as an asthma induced group (OVA) was significantly increased whereas those in the positive control group treated with Montelukast (MO) as well as the test sample group orally administrated with various concentrations of test samples (ATC-5, ATC-10, ATC-25, and ATC-50) were significantly reduced. (See Table 8)

(76) TABLE-US-00008 TABLE 8 The level of IgE and OVA-specific IgE in blood serum Concentration (g/ml) level of IgE in serum level of OVA-specific IgE NC 0.92 0.17 0.05 0.00 OVA 7.68 0.42 0.17 0.02 ATC2 5 6.51 0.72 0.13 0.01 (mg/kg) 10 4.97 0.91 0.10 0.01 25 4.56 0.73 0.09 0.02 50 4.01 0.67 0.08 0.01 M30 4.76 0.73 0.09 0.02

(77) Accordingly, it has been confirmed that the inventive purified extract effectively inhibited the level of IgE and OVA-specific IgE in blood serum and therefore, they are useful in treating or preventing the allergic disease and asthma disease.

Experimental Example 5. Effect on the Level of Inflammatory Cytokines in BALF

(78) In order to confirm the inhibition effect of test samples prepared in Examples on the level of Th2 cytokines (IL-4, IL-5 and IL-13) and IL-1 in bronchoalveolar lavage fluid (BALF), following test was performed by the sandwich enzyme immunosorbent assay method disclosed in the literature (Renz H. et al., J. Exp. Med., 1777, pp 1175-1180, 1993).

(79) The blood serum and bronchoalveolar lavage fluid (BALF) was added to 96-well plates (ELISA plate) coated with cytokine antibody to induce antigen-antibody reaction for 2 hours at room temperature. The level of Th2 cytokines (IL-4, IL-5 and IL-13) and IL-1 in bronchoalveolar lavage fluid (BALF) was determined using by ELISA kit (Biosource Int. CA, USA) specifically reacting with each cytokine according to the manufacture's manual.

(80) As shown in FIG. 8 and FIG. 9, 48 hours after the OVA-treatment, the level of Th2 cytokines (IL-4, IL-5 and IL-13) and IL-1 in the control group treated and inhaled with OVA as an asthma induced group (OVA) was significantly increased comparing with those in the non-treatment group with OVA as a normal control group (NC).

(81) The increased level of Th2 cytokines (IL-4, IL-5 and IL-13) and IL-1 in the positive control group treated with Montelukast (MO, 30 mg/kg) as well as the test sample group orally administrated with various concentrations of test samples (ATC-10, ATC-25, and ATC-50) were significantly reduced. (See Table 9)

(82) TABLE-US-00009 TABLE 9 The level of Th2 cytokines (IL-4, IL-5 and IL-13) and IL-1 conc. (pg/ml) IL-1 IL-4 IL-5 IL-13 NC 94.12 65.24 20.94 1.76 57.51 3.15 22.25 3.04 OVA 281.78 26.15 48.76 6.96 109.48 2.87 44.76 4.85 ATC2 5 243.93 27.58 35.27 5.26 90.77 12.78 34.29 7.55 (mg/kg) 10 226.33 14.21 35.18 4.45 79.26 9.60 29.50 2.76 25 190.30 17.82 29.94 2.32 73.74 9.54 27.27 4.42 50 170.70 25.43 25.26 5.55 57.92 19.99 23.53 4.10 M30 187.03 47.17 32.60 4.53 69.74 7.17 25.93 4.13

(83) Accordingly, it has been confirmed that the inventive purified extract effectively inhibited the level of level of Th2 cytokines (IL-4, IL-5 and IL-13) and IL-1 in BALF and therefore, they are useful in treating or preventing the allergic disease and asthma disease.

Experimental Example 6. Lung Histology

(84) In order to confirm the anti-asthmatic effect of test samples prepared in Examples, following histopathological analysis on broncho-alveolar tissue was performed by the method disclosed in the literature (Kwak Y G. et al., J. Clin. Invest., 111, pp 1083-1092, 2003).

(85) The delivered lung tissues of BALB/c mice which had not perform broncho-alveolar lavage was fixed for 24 h in 10% neutral-buffered formalin. After being embedded in paraffin, then made into 4-m thickness sections, the tissue was stained with H&E solution (hematoxylin; Sigma MHS-16 and eosin, Sigma HT110-I-32) and the inflammation score of five regions in each section chosen in a randomized manner, was determined. (Inflammation score 0: inflamed cells is not found in bronchial surrounding, Inflammation score 1: inflamed cells is sporadically found in bronchial surrounding, Inflammation score 2: thin inflamed cell layer is found in most of bronchial surrounding, Inflammation score 3: thick inflamed cell layer is found in most of bronchial surrounding).

(86) As shown in FIG. 10, many inflammatory cells including eosinophills were found in bronchiolar surroundings and hyperplasia of epithelial cells as well as hypertrophy of tracheal muscle were also found in the control group treated and inhaled with OVA as an asthma induced group (OVA) whereas the invasion of the inflamed cells was significantly reduced in the positive control group treated with Montelukast (MO, 30 mg/kg) as well as the test sample group orally administrated with various concentrations of test samples (ATC-10, ATC-25, and ATC-50). (See Table 10)

(87) TABLE-US-00010 TABLE 10 Inflammation score and the ratio of goblet cell in bronchiolar epithelial cell histopathological analysis Inflammation score PAS + cells/bronchiole (%) NC 0.06 0.05 2.19 0.54 OVA 2.11 0.07 52.75 1.42 ATC2 5 1.75 0.17 48.07 1.15 (mg/kg) 10 1.33 0.14 44.59 1.60 25 1.17 0.11 38.61 1.74 50 1.08 0.18 35.71 1.14 M30 1.25 0.13 39.21 2.34

Experimental Example 7. Evaluation of Goblet Cell Plasia

(88) In order to confirm the anti-asthmatic effect of test samples prepared in Examples, following goblet cell plasia analysis on broncho-alveolar tissue was performed by the method disclosed in the literature (Lee K S. et al., FASEB J., 20, pp 455-465, 2006).

(89) The delivered lung tissues of BALB/c mice which had not perform broncho-alveolar lavage was fixed for 24 h in 10% neutral-buffered formalin. After being embedded in paraffin, then made into 4-m thickness sections, the tissue was stained with Periodic acid Schiff (PAS stain kit, T-K7308, IMEB, CA, USA) to determine the ratio of goblet cell in bronchiolar epithelial cell

(90) As shown in FIG. 11, the ratio of goblet cell in bronchiolar epithelial cell was significantly increased in the control group treated and inhaled with OVA as an asthma induced group (OVA) comparing with normal control group (Nc) whereas the ratio of goblet cell in bronchiolar epithelial cell was significantly reduced in the positive control group treated with Montelukast (MO, 30 mg/kg) as well as the test sample group orally administrated with various concentrations of test samples (ATC-10, ATC-25, and ATC-50). (See Table 9)

Experimental Example 8. Acute Toxicity Test of Oral Administration in Rat

(91) The acute toxicity test was performed by administrating inventive extract to 6-weeks aged SPF Sprague-Dawley rats.

(92) 250 mg/kg, 500 mg/kg, 1000 mg/kg, 5000 mg/kg of inventive extract was orally administrated to each group consisting of 2 rats and the symptoms of rats were observed for 14 days. After administrating the extract or compounds, all the clinical changes i.e., mortality, clinical signs, body weight changes was observed and blood test such as haematological test and hematological biochemistry test was performed. The abnormal changes of abdominal organ and thoracic organ were observed after autopsy.

(93) There did not show any changes in mortality, clinical signs, body weight changes and gross findings in any group or either gender. Furthermore, there showed any toxicity in test group treated with 5000 mg/kg of inventive extract or compounds.

(94) Accordingly, it has been confirmed that the inventive extract prepared in the present invention was potent and safe substance showing LD.sub.50 (more than 5000 mg/kg) in oral administration.

MODE FOR INVENTION

(95) 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.

(96) Preparation of Injection

(97) ATC1 extract 100 mg

(98) Sodium metabisulfite 3.0 mg

(99) Methyl paraben 0.8 mg

(100) Propyl paraben 0.1 mg

(101) Distilled water for injection optimum amount

(102) Injection preparation was prepared by dissolving active component, controlling pH to about 7.5 and then filling all the components in 2 Ml ample and sterilizing by conventional injection preparation method.

(103) Preparation of Powder

(104) ATC2 extract 500 mg

(105) Corn Starch 100 mg

(106) Lactose 100 mg

(107) Talc 10 mg

(108) Powder preparation was prepared by mixing above components and filling sealed package.

(109) Preparation of Tablet

(110) ATC1 extract 200 mg

(111) Corn Starch 100 mg

(112) Lactose 100 mg

(113) Magnesium stearate optimum amount

(114) Tablet preparation was prepared by mixing above components and entabletting.

(115) Preparation of Capsule

(116) ATC2 extract 100 mg

(117) Lactose 50 mg

(118) Corn starch 50 mg

(119) Talc 2 mg

(120) Magnesium stearate optimum amount

(121) Tablet preparation was prepared by mixing above components and filling gelatin capsule by conventional gelatin preparation method.

(122) Preparation of Liquid

(123) ATC1 extract 1000 mg

(124) Sugar 20 g

(125) Polysaccharide 20 g

(126) Lemon flavor 20 g

(127) Liquid preparation was prepared by dissolving active component, and then filling all the components in 1000 Ml ample and sterilizing by conventional liquid preparation method.

(128) Preparation of Health Food

(129) ATC2 extract 1000 mg

(130) Vitamin mixture optimum amount

(131) Vitamin A acetate 70 g

(132) Vitamin E 1.0 mg

(133) Vitamin B.sub.10. 13 mg

(134) Vitamin B.sub.2 0.15 mg

(135) Vitamin B6 0.5 mg

(136) Vitamin B1 20.2 g

(137) Vitamin C 10 mg

(138) Biotin 10 g

(139) Amide nicotinic acid 1.7 mg

(140) Folic acid 50 g

(141) Calcium pantothenic acid 0.5 mg

(142) Mineral mixture optimum amount

(143) Ferrous sulfate 1.75 mg

(144) Zinc oxide 0.82 mg

(145) Magnesium carbonate 25.3 mg

(146) Monopotassium phosphate 15 mg

(147) Dicalcium phosphate 55 mg

(148) Potassium citrate 90 mg

(149) Calcium carbonate 100 mg

(150) Magnesium chloride 24.8 mg

(151) 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.

(152) Preparation of Health Beverage

(153) ATC1 extract 1000 mg

(154) Citric acid 1000 mg

(155) Oligosaccharide 100 g

(156) Apricot concentration 2 g

(157) Taurine 1 g

(158) Distilled water 900 Ml

(159) 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 1000 Ml ample and sterilizing by conventional health beverage preparation method.

(160) 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

(161) As described in the present invention, the present invention provides inventive novel industrialized method for preparing purified extract containing more abundant active ingredients such as catalpol derivatives from the extract of Pseudolysimachion rotundum var subintegrum and the purified extract showed more potent anti-inflammatory, anti-allergy and anti-asthma activity than that prepared by the conventional preparation method disclosed in the prior art through various in vivo tests such as inhibition test on the reproduction of eosinophil, the release of immunoglobulin and inflammatory chemokines in plasma and bronchoalveolar fluid as well as the suppression of airway hyperresponsiveness and goblet cell hyperplasia in a OVA-sensitized/challenged mouse model. Therefore, it can be used as the therapeutics or functional health food for treating and preventing inflammatory, allergic or asthmatic disease.