Composition comprising a combined herb extract of <i>Salvia plebia </i>and red ginseng as active ingredients for preventing or treating a respiratory inflammation and the use thereof

Abstract

The present invention relates to a composition and health functional food for preventing and treating a respiratory inflammation disease using a mixed herbal mixture. Through various experiments, for example, determination of the cell number of BAL (bronchoalveolar lavage) (Experimental Example 1); Determination of CD11b+/Gr-1+ ratio in leukocyte within BAL fluid (Experimental Example 2); Determination of expressed RNA level of inflammatory cytokines in lung tissue (Experimental Example 3); Determination of expressed RNA level of inflammatory cytokines in BALF (Experimental Example 4); Lung histology (Experimental Example 5); Brief Clinical test (Experimental Example 7) etc, it has been verified that the inventive combined extract showed more potent inhibiting effect on respiratory inflammation disease than each herb extract. Therefore, the herbal extract of the present invention can be usefully used in a pharmaceutical composition, health functional food, and health supplement food for preventing and treating respiratory inflammation disease.

Claims

1. A method of treating a respiratory disease, comprising administering to the subject in need thereof a composition comprising a combined herb extract of Salvia plebeia R. Br. and red ginseng, wherein a ratio based on the dried weight of extract of Salvia plebeia R. Br. and extract of red ginseng is 2:1 (w/w), wherein the combined extract is soluble in distilled water, alcohol, methanol, ethanol, butanol, or a mixed solvent thereof, and wherein the subject is administered a dose of 1000 to 2000 mg/day of the combined extract.

2. The method of claim 1, wherein the subject is administered a dose of 500 to 1000 mg of combined extract per dose twice a day.

3. The method of claim 2, wherein the dose is administered 30 minutes after a morning meal and 30 minutes after an evening meal.

4. The method of claim 1, wherein the subject is in need of increasing a bronchodilator response and wherein administering said composition increases the bronchodilator response in said subject.

5. The method of claim 1, wherein the subject is in need of increasing a forced vital capacity and wherein administering said composition increases the forced vital capacity in said subject.

6. The method of claim 1, wherein the subject is in need of reducing one or more symptoms selected from the group consisting of reduced bronchodilator response, reduced forced vital capacity, cough and nasal congestion, and wherein administering said composition to the subject reduces said symptoms.

7. The method of claim 1, wherein the subject has suffered from one or more symptoms selected from the group consisting of cough, sputum and dyspnea for at least a month.

8. The method of claim 1, wherein the respiratory disease is a respiratory inflammation disease.

9. The method of claim 8, wherein the respiratory inflammation disease is selected from the group consisting of rhinitis, otitis media, laryngopharyngitis, tonsillitis, pneumonia, asthma, and COPD (Chronic Obstructive Pulmonary Disease).

Description

DESCRIPTION OF DRAWINGS

(1) FIG. 1 shows the histopathological analysis on broncho-alveolar tissue stained by staining agent (NC: Normal Control group; AIG: Asthma induced Group; CB3: test sample group treated with CB3 combined extract).

BEST MODE

(2) 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.

(3) 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

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

Comparative Example 1

(5) Preparation of Extract of Salvia plebeia R. Br.

(6) 1.5 kg of dried Salvia plebeia R. Br. (distributed on Buan-gun, Jeollabuk-do, Republic of Korea) was added to 22.5 L of 30% ethanol to perform extraction with reflux extraction for 4 hors at 80° C.±2° C. twice. The residue was filtered with filter paper to afford extract and the filtrated extract was concentrated under 650±30 mmHg at 52.5° C.±2.5° C. The concentrated extract was sterilized for 1 hour at 85.0° C.±2.0° C. and cooled to 55° C. The sterilized extract was dried with freeze dryer (KL-8, SeoGang Engineering Co. Ltd., inlet temp.: 190° C.±10° C., outlet temp.: 95° C.±5° C.) to afford 458 g of dried 30% ethanol extract of Salvia plebeia R. Br. (designated as “SP” hereinafter), which is used as a comparative test sample 1 in following experiment.

Comparative Example 2

(7) Preparation of Red Ginseng Extract

(8) 2-1. Preparation of Red Ginseng

(9) 10 kg of dried 6 years-old fresh ginseng root (Panax ginseng C. A. Meyer) procured from KT&G Corp (100, Pyeongchon-dong, Seo-gu, Daejeon, Republic of Korea, cultivated from geumsangun chungcheongnamdo, South Korea) was washed with distilled water and further washed again for 30 mins using by conventional ultrasonic cleaner (Branson 5210, Emerson Electric Co. United States). The washed fresh ginseng was subjected to steaming process with vapor at about 80° C.-100° C. for 90-110 mins using by steaming apparatus (KMC-1221, Jeiotech Co. Ltd., Daejeon, Korea) and dried at about 60° C.-65° C. for 9 hours using by drying apparatus (KMC-1202D3, Jeiotech Co. Ltd., Daejeon, Korea) to afford the 1.sup.st dried ginseng (water content: 45-55%). The 1.sup.st dried ginseng was subjected to 2.sup.nd drying process in drying room for 13-17 days to make red ginseng (water content: about 14%, designated as “RG1” hereinafter).

(10) 2-2. Preparation of Red Ginseng Extract

(11) 500 g of red ginseng prepared from the above step was subjected to 3.sup.rd drying process at about 100° C.-120° C. for 15-20 mins using by far-infrared drying apparatus (Korea Energy Technicals Co. Ltd., HKD-LAB) and cut into pieces. The dried red ginseng was poured into 4-8 folds distilled water and perform reflux extraction for 8-12 hours at 85° C. The solution was filtered with filter paper and cooled to 0° C.-10° C. The residue was poured into 4-8 folds distilled water and perform reflux extraction for 8-12 hours at 85° C. and the extraction was repeated 4 times. The extract was collected, filtered with filter paper and cooled to 0° C.-10° C. The cooled extract was centrifuged for 10-20 mins, at 4° C. at the speed of 5,000-8,000 rpm to remove unnecessary debris using by centrifuge apparatus (Supra22K, Hanil Science Medicals Co. Ltd., Daejeon City) and evaporated with evaporator at 50° C.-60° C. to remove water to obtain the concentrated extract (71° brix), Finally, the concentrated red ginseng extract was dissolved in distilled water to make diluted extract (15° brix) and performed spray drying to afford 230 g of a red ginseng extract (designated as “RG” hereinafter), which was used as a comparative sample 2 in following experiments.

Example 1

(12) Preparation of Combined Formulation (CB1-CB7)

(13) The dried extract of Salvia plebeia R. Br. and red ginseng extract prepared in the above Comparative Example was thoroughly mixed with different mixed weigh ratios (See Table 1) using by mixer (Vortex genie-2, scientific industries, USA) to obtain the various kinds of invention formulation (designated as “CB1-CB7” hereinafter), which are used as a test samples in following experiment.

(14) TABLE-US-00001 TABLE 1 various kinds of invention formulation Example *weight ratio of A:B combination name Example 1-1 0.3:1.sup.  CB1 Example 1-2 1:1 CB2 Example 1-3 2:1 CB3 Example 1-4 3:1 CB4 Example 1-5 4:1 CB5 Example 1-6 5:1 CB6 Example 1-7 10:1  CB7 *A: extract of Salvia plebeia R. Br./B: red ginseng extract

Experimental Example 1

(15) Determination of the Cell Number of BAL (Bronchoalveolar Lavage).

(16) In order to confirm synergistically potent inhibiting activity on the cell number in BAL (bronchoalveolar lavage) of the inventive combinations than the comparative Examples, following test was performed by the method disclosed in the literature (Schins et al., Toxicol. Appl. Pharmacol., 195(1), pp 1-11, 2004; Smith et al., Toxicol. Sci., 93(2), pp 390-399, 2006).

(17) 1-1. Test Procedure

(18) 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. The mice were sensitized by INT (Intra-Nazal-Trachea) injection of 50 μL of fine dust mixture prepared by mixing alum with fine dust mixture (025 mg/ml of coal, 10 mg/ml of fly ash and 0.25 mg/ml of diesel exhaust particle) to be final concentration of 8%, at 3rd day and 6.sup.th day after the initial sensitization to prepare asthma-induced animal model.

(19) Briefly, mice were divided into four groups of which each group consists of 6 mice, i.e., (a) normal control group (NC): the groups not-treated with fine dust mixture; (b) asthma-induced group (AIG): the groups treated with fine dust mixture to induce asthma; (c) comparative groups: the groups orally treated with comparative groups prepared in Comparative Examples by dissolving in 0.5% CMC (sodium carboxymethyl cellulose, 419273, Sigma-Aldrich) every day for 10 days; and (d) test sample groups orally treated with test sample groups prepared in Examples by dissolving in 0.5% CMC (sodium carboxymethyl cellulose, 419273, Sigma-Aldrich) every day for 10 days.

(20) At 11.sup.th day after the experiment, the mice were performed to autopsy and the BAL (bronchoalveolar lavage) fluid of mice was collected.

(21) 1-2. Test Result

(22) As shown in Table 2, the total cell number in BAL (bronchoalveolar lavage) fluid in test sample group treated with inventive combinations was synergistically reduced than those in the comparative groups treated with the sole extract of Salvia plebeia R. Br. (SP) or red ginseng extract (RG), respectively.

(23) TABLE-US-00002 TABLE 2 The total cell number in BAL fluid total Cell number Inhibition percentage (×10.sup.5 cells/ml) (%)* NC 32.3 ± 6.14 AIG 117.0 ± 8.5  SP 65.0 ± 5.24 44% RG 67.3 ± 5.74 42% CB1 47.5 ± 5.36 59% CB2  40.1 ± 11.63 66% CB3 37.8 ± 2.13 68% CB4 33.3 ± 1.75 72% CB5 44.1 ± 3.28 62% CB6 49.7 ± 8.30 58% CB7 54.8 ± 7.26 53% *data based on the inhibition of AIG

Experimental Example 2

(24) Determination of CD11b+/Gr-1+ Ratio in Leukocyte within BAL Fluid.

(25) In order to confirm synergistically potent inhibiting activity on the CD11b+/Gr-1+ ratio in leukocyte within BAL fluid of the inventive combinations than the comparative Examples, following test was performed by the method disclosed in the literature (Beutner E. H., bacteriological Reviews, 25(1), pp 49-76, 1961).

(26) 2-1. Test Procedure

(27) BAL (bronchoalveolar lavage) fluid of mice collected from Experimental Example 1 was performed to specific fluorescent antibody staining method using by fluorescence-labeled CD11b antibody (553310, BD Biosciences, San Jose, Calif., USA) and Gr-1 antibody (553128, BD Biosciences, San Jose, Calif., USA). The CD11b+/Gr-1+ ratio in total leukocyte within BAL fluid was determined according to FACS method (Fluorescence-activated cell sorting, BD Biosciences, San Jose, Calif., USA).

(28) 2-2. Test Result

(29) As shown in Table 3, the CD11b+/Gr-1+ ratio in total leukocyte within BAL fluid in test sample group treated with inventive combinations was synergistically reduced than those in the comparative groups treated with the sole extract of Salvia plebeia R. Br. (SP) or red ginseng extract (RG), respectively.

(30) TABLE-US-00003 TABLE 3 The CD11b+/Gr-1+ ratio in total leukocyte within BAL fluid CD11b+/Gr-1+ ratio Inhibition percentage (%) (%)* NC  7.1 ± 0.2 AIG 51.2 ± 3.6 SP 35.3 ± 3.2 36% RG 40.8 ± 1.6 20% CB1 29.7 ± 1.5 42% CB2 25.1 ± 4.6 51% CB3 21.3 ± 2.7 58% CB4 19.1 ± 3.6 63% CB5 24.9 ± 1.6 51% CB6 28.4 ± 2.5 44% CB7 32.0 ± 3.6 35% *data based on the inhibition of AIG

Experimental Example 3

(31) Determination of Expressed RNA Level of Inflammatory Cytokines in Lung Tissue

(32) In order to confirm the synergistically potent inhibiting activity on the expressed RNA level of inflammatory cytokines in lung tissue of the inventive combinations than the comparative Examples, following RT-PCR (Realtime quantitative polymerase chain reaction) test was performed by the method disclosed in the literature (Adelroth E., Cancer Respir J., pp 18A-21A, 1998).

(33) 3-1. Test Procedure

(34) (1) Isolation and Extraction of RNA from Lung Tissue

(35) The lung tissue was delivered rather than BAL (bronchoalveolar lavage) fluid of mice according to the method disclosed in Experimental Example 1. The delivered lung tissue was added to 500 mL of RNAzolB (Tel-Test, Friendswood, USA) and mashed to be dissolved. 50 mL of CHCl.sub.3 was added to the suspension and stirred again for 15 seconds. the suspension was left alone in ice for 15 mins and centrifuged at the speed of 13,000 rpm. About 200 mL of collected supernatant was added to the equivalent amount of 2-propanol (19516, Sigma-Alrich, USA) and stirred gently to be left alone in ice for 15 mins. The solution was centrifuged again at the speed of 13,000 rpm, washed with 80% ethanol and dried in vaccuo for 3 mins to extract RNA. The extracted RNA was dissolved in 20 mL of distilled water treated with DEPC (Diethyl pyrocarbonate, 750023, Thermo Scientific, Massachusetts, USA), and inactivated at 75° C. to be used in cDNA (first strand complementary DNA).

(36) (2) cDNA Synthesis

(37) 2 μg of total RNA was added to 2 U/tube DNase I (AB0620, Thermo Scientific, Massachusetts, USA), reacted for 30 mins at 35° C., denatured for 10 mins and added to the reaction mixture consisting of 2.5 mL of 10 mM dNPTs mix (4030, TaKaRa Shiga, Japan), 1 mL of random sequence hexanucleotides (N8080127, Thermo Scientific, Massachusetts, USA), 1 mL of RNase inhibitor (2313A, TaKaRa Shiga, Japan), 1 mL of 100 mM DTT (4029, TaKaRa Shiga, Japan) and 4.5 mL of 5×RT buffer (M5313, Promega, Wisconsin-Madison, USA). The solution was added to 4.5 mL of M-MLV RT (M1701, Promega, Wisconsin-Madison, USA) and dissolved in distilled water treated with DEPC (Diethyl pyrocarbonate, 750023, Thermo Scientific, Massachusetts, USA) to be final volume of 20 mL. After thoroughly stirring, the solution was centrifuged for 5 seconds at the speed of 2000 rpm, reacted in heating block (Multi-block heater, TRIPUNITHURA, USA) at 37° C. for 60 mins to synthesize cDNA and then left alone for 5 mins at 95° C. to inactivate M-MLV RT. The synthesized cDNA was used in PCR method.

(38) (3) PCR

(39) The synthesized cDNA was performed to RT-PCR method according to the procedure disclosed in the literature (Galli et al., Nat. Immunol., 6(2), pp 135-142, 2005).

(40) Sper-Taqman PCR Master mix (4304437, Applied Biosystems, San Mateo, USA) as well as various primers disclosed in Table 4 (final concentration: 200 nM) were used in the RT-PCR method. RT-PCR was performed by pre-denaturing 2 mins at 50° C., for 10 mins at 94° C., and reacting for 40 cycles, i.e., 0.15 mins at 95° C. and for 1 min at 60° C. GAPDH (Glyceraldehyde-3-phosphatedehydrogenase, 4352339E, Thermo Scientific, Massachusetts, USA) was used as an internal standard.

(41) TABLE-US-00004 TABLE 4 The used primers in RT-PCR method Target- Sequence gene primer sequences I. D. MUC5AC Forward 5′-AGAATATCTTTCAGGACCC 1 CTGCT-3′ Reverse 5′-ACACCAGTGCTGAGCATAC 2 TTTT-3′ CCR5 Forward 5′-ATTCTCCACACCCTGTTTC 3 G-3′ Reverse 5′-AAGGTGGTCAGGAGGAGGA 4 C-3′ DAPDH-VIC Probe 5′-CATGTTCCAGTATGACTCC 5 ACTCACG-3
3-2. Test Result

(42) As shown in Table 5, the expressed RNA level of inflammatory cytokines in lung tissue such as MUCSAC, CCR5 etc in test sample group treated with inventive combinations was synergistically reduced than those in the comparative groups treated with the sole extract of Salvia plebeia R. Br. (SP) or red ginseng extract (RG), respectively.

(43) TABLE-US-00005 TABLE 5 The expressed RNA level of inflammatory cytokines in lung tissue MUC5AC CCR5 Inhibition Inhibition expressed percentage expressed percentage level (%)* level (%)* NC 1.66 ± 0.94 0.76 ± 0.19 AIG 5.29 ± 0.75 8.80 ± 1.59 SP 2.92 ± 0.84 45% 4.99 ± 1.95 43% RG 3.40 ± 0.63 36% 7.93 ± 1.65 10% CB1 2.62 ± 0.31 50% 4.69 ± 0.69 47% CB2 2.46 ± 1.20 53% 4.20 ± 0.71 52% CB3 2.22 ± 0.98 58% 4.14 ± 1.39 53% CB4 1.81 ± 0.72 66% 3.36 ± 0.90 62% CB5 2.12 ± 0.63 60% 3.98 ± 0.87 55% CB6 2.38 ± 0.28 55% 4.03 ± 1.64 54% CB7 2.65 ± 0.83 50% 4.90 ± 0.76 44% *data based on the inhibition of AIG

Experimental Example 4

(44) Determination of Expressed RNA Level of Inflammatory Cytokines in BALF

(45) In order to confirm the synergistically potent inhibiting activity on the expressed RNA level of inflammatory cytokines in BALF of the inventive combinations than the comparative Examples, following ELISA (Enzyme-Linked Immuno Sorbent Assay) test was performed by the method disclosed in the literature (Brandt E. B. et al., J. Allergy Clin. Immunol., 132(5), pp 1194-1204, 2013).

(46) 4-1. Test Procedure

(47) BAL (bronchoalveolar lavage) fluid of mice collected from Experimental Example 1 was performed to ELISA (Enzyme-Linked Immuno Sorbent Assay) test to determine the level of IL-17A, TNF-alpha, MIP2, and CXCL-1, similarly to the method disclosed in Experimental Example 3. IL-17A antibody (M1700, R&D Systems, Minneapolis, USA), TNF-alpha antibody (MTA00B, R&D Systems, Minneapolis, USA), MIP2 antibody (MM200, R&D Systems, Minneapolis, USA), and CXCL-1 antibody (MKC00B, R&D Systems, Minneapolis, USA) were diluted with buffer solution and coated with micro cell to incubate at 95° C., for 16 hours. Each well was washed with washing buffer solution three times and 100 μL of 10 fold diluted serum was inoculated thereto. After being left alone at room temperature for 1 hour, the well was washed twice and 100 μL of Avidin-HRP-conjugated antibody (DY007, R&D System, Minneapolis, USA) was treated therewith to be left alone at room temperature for 1 hour. After washing again, 100 μL of TMB substrate (DY999, R&D System, Minneapolis, USA) was inoculated thereto to be left alone in shadow for 30 mins. 50 μL of stop solution (DY994, R&D System, Minneapolis, USA) was treated therewith and then the absorbance of the solution was determined at 450 nm.

(48) 4-2. Test Result

(49) As shown in Table 6, the expressed RNA level of inflammatory cytokines in BALF such as IL-17A, TNF-alpha, MIP2, and CXCL-1 etc in test sample group treated with inventive combinations was synergistically reduced than those in the comparative groups treated with the sole extract of Salvia plebeia R. Br. (SP) or red ginseng extract (RG), respectively.

(50) Accordingly, it has been confirmed that the inventive combinations showed more potent reducing effect on the expressed RNA level of inflammatory cytokines in BALF such as IL-17A, TNF-alpha, MIP2, and CXCL-1 etc than those in the comparative groups treated with the sole extract of Salvia plebeia R. Br. (SP) or red ginseng extract (RG), respectively and therefore, they are useful in treating or preventing the asthma disease, an allergic disease or COPD in airway.

(51) TABLE-US-00006 TABLE 6 The expressed RNA level of inflammatory cytokines in BALF level (pg/mL)/inhibition percentage (%)* IL-17A TNF-alpha MIP2 CXCL-1 NC 4.4 45.6  70.7 95.8 AIG 13.2 87.2 164.5 231.1 SP 7.9/40% 47.2/46% 97.1/41% 152.5/34% RG 9.6/27% 53.2/39% 104.4/37%  162.9/30% CB1 7.8/41% 29.5/66% 77.8/53% 139.8/40% CB2 7.8/41% 30.0/66% 75.0/54% 138.6/40% CB3 7.5/43% 28.1/68% 72.9/56% 137.5/41% CB4 6.0/55% 20.3/77% 64.1/61% 130.1/44% CB5 7.1/46% 36.3/58% 79.1/52% 145.6/37% CB6 7.2/45% 46.6/47% 84.3/49% 150.9/35% CB7 7.8/41% 46.0/47% 89.5/46% 150.4/35% *data based on the inhibition of AIG

Experimental Example 5

(52) Lung Histology

(53) In order to confirm the anti-asthmatic effect of test samples prepared in Examples, following histopathological analysis on broncho-alveolar tissue was performed using by H&E staining and M-T staining method according to the method disclosed in the literature (Nandedkar S D. et al., Blood, 111(6), pp 2529-2538, 2008).

(54) 5-1. Test Procedure

(55) The lung tissue was delivered rather than BAL (bronchoalveolar lavage) fluid of mice according to the method disclosed in Experimental Example 1. The delivered lung tissue was fixed for 24 h in 10% neutral-buffered formalin solution (F8775, Sigma-Aldrich, USA), dissected and washed with running water for 8 hours. After being embedded in epoxy (A3183, Sigma-Aldrich, USA), the tissue was made into 4-μm thickness sections with microtome (leica RM2265, Wetzlar, Germany) and the section was stained with Masson-Trichrome (HT10516, Sigma-Aldrich, USA) to observe histopathological analysis on broncho-alveolar tissue using by optical microscope (Bright Microscope, Tokyo, Japan).

(56) 5-2. Test Result

(57) As shown in FIG. 1 showing the histopathological analysis on broncho-alveolar tissue, the increase of collagen fiber as well as the hypertrophy of tracheal muscle were also found in the asthma-induced group comparing with normal group and the significant decrease of collagen fiber and the thinning of tracheal muscle were found in test sample groups treated with inventive combination.

Experimental Example 6

(58) Acute Toxicity Test of Oral Administration in Rat

(59) The acute toxicity test was performed by administrating inventive extract (CB3) to 6-weeks aged SPF Sprague-Dawley rats.

(60) 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 hematological test and hematological biochemistry test was performed. The abnormal changes of abdominal organ and thoracic organ were observed after autopsy.

(61) 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.

(62) 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.

Experimental Example 7

(63) Brief Clinical Test.

(64) In order to confirm the clinical efficacy and safety of the inventive extract on respiratory disease, following brief clinical test was performed.

(65) 7-1. Selection of Volunteers

(66) 30 volunteers (aged 19-70 years old) having suffered with consistent respiratory disease such as cough, sputum, dyspnea etc for more than 1 month were divided into three groups, i.e., (a) low-dosage test group consisting of 10 volunteers (orally taking 2 capsules containing 250 mg of CB3 extract/capsule, 30 mins after morning meal and evening meal, twice a day for 12 weeks); (b) high-dosage test group consisting of 10 volunteers (orally taking 2 capsules containing 500 mg of CB3 extract/capsule, 30 mins after morning meal and evening meal, twice a day for 12 weeks); and (c) placebo group consisting of 10 volunteers (orally taking 2 capsules containing 0 mg/capsule, 30 mins after morning meal and evening meal, twice a day).

(67) 7-2. Test Procedure

(68) The brief clinical test was performed by randomized, double-blind, parallel, placebo-controlled manner in Clinical Trial Center for Functional Foods of Chon buk National University Hospital (division of Pulmonology, M. D., PARK, S. J.) and the volunteers had visited three times, i.e., (a) at 1.sup.st day (the volunteers were randomly divided into three groups to provide three kinds of test capsules); (b) at 43.sup.rd day (the volunteers were invited to perform pre-determined efficacy tests and to provide additional test capsules); and (c) at 85.sup.th day (the volunteers were invited to perform pre-determined efficacy tests and to end the brief clinical test).

(69) The pre-determined efficacy tests in the experiment consist of (1) 1.sup.st efficacy evaluation using by SGRQ (Saint George's respiratory questionnaire) and CAT (COPD assessment test) to compare the efficacy in the test sample groups with placebo group) and (2) 2.sup.nd efficacy evaluation were using by pulmonary function test, infection frequency, infection period, syndrome etc.

(70) 7-3. Test Result

(71) As shown in Table 7 and Table 8, the test sample groups taking high- and low-dosage test groups showed significant treating activity comparing with placebo group.

(72) Accordingly, it has been confirmed that the inventive combinations showed potent treating activity of respiratory disease such as COPD, asthma etc in brief clinical test and therefore, they are useful in treating or preventing the asthma disease, an allergic disease or COPD in airway.

(73) TABLE-US-00007 TABLE 7 The 1.sup.st evaluation test result significant contents between each group (P-value)* Analysis P vs L P vs H 1 CAT score — — 2 SGRQ score — — 3 CAT 10< CAT score — — SGRQ score — — 4 CAT 10> CAT score — — SGRQ score Impact 0.010 6 weeks — 0.041 12 weeks — total 0.040 12 weeks — P: placebo group, L; Low-dosage test group, H: high-dosage test group

(74) TABLE-US-00008 TABLE 8 The 2.sup.nd evaluation test result significant contents between each group (P-value)* Analysis** P vs L*** P vs H 1 PFT PF FVC <.001 12 Ws FVC 0.021  6 Ws (L) (%) FVC 0.002 12 Ws 0.013 12 Ws (%) FVC1 0.047  6 Ws (%) BR FVC 0.026 12 Ws (L) FVC 0.017 12 Ws (%) 2 CAT PF FVC 0.012 12 Ws FVC 0.021 12 Ws 10< (L) (L) FVC 0.009 12 Ws FVC 0.018 12 Ws (%) (%) FVC1 0.034 12 Ws (L) FVC1 0.039 12 Ws (%) BR FVC 0.015 12 Ws — (L) FVC 0.013 12 Ws (%) 3 CAT PF FVC 0.033 12 Ws — 10> (L) BR 4 cytokine — — 5 hs-CRP, — — ESR, CBC 6 mRNA — — 7 IF, IP, SY NC PD 0.045 NC PD 0.014 Cough PD 0.046 Cough PD 0.027 *P: placebo group, L; Low-dosage test group, H: high-dosage test group **PFT: Pulmonary Function Test, PF: Pulmonary Function, BR: Bronchodilator response, hs-CRP: High sensitive C-reactive protein, ESR: Erythrocyte sedimentation rate, CBC: Complete blood count, IF: infection frequency, IP: infection period, SY: syndrome **FVC (L): Forced vital capacity (liter), FVC (%): Forced vital capacity, Ws: weeks, NC: nasal Congestion, PD: Period

MODE FOR INVENTION

(75) 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.

(76) Preparation of Injection

(77) CB1 extract: 100 mg

(78) Sodium metabisulfite: 3.0 mg

(79) Methyl paraben: 0.8 mg

(80) Propyl paraben: 0.1 mg

(81) Distilled water for injection: optimum amount

(82) 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.

(83) Preparation of Powder

(84) CB2 extract: 500 mg

(85) Corn Starch: 100 mg

(86) Lactose: 100 mg

(87) Talc: 10 mg

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

(89) Preparation of Tablet

(90) CB3 extract 200 mg

(91) Corn Starch 100 mg

(92) Lactose 100 mg

(93) Magnesium stearate optimum amount

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

(95) Preparation of Capsule

(96) CB4 extract: 100 mg

(97) Lactose: 50 mg

(98) Corn starch: 50 mg

(99) Talc: 2 mg

(100) Magnesium stearate optimum amount

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

(102) Preparation of Liquid

(103) CB5 extract: 1000 mg

(104) Sugar: 20 g

(105) Polysaccharide: 20 g

(106) Lemon flavor: 20 g

(107) 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.

(108) Preparation of Health Food

(109) CB6 extract: 1000 mg

(110) Vitamin mixture: optimum amount

(111) Vitamin A acetate: 70 g

(112) Vitamin E: 1.0 mg

(113) Vitamin B.sub.10: 13 mg

(114) Vitamin B.sub.2: 0.15 mg

(115) Vitamin B6: 0.5 mg

(116) Vitamin B1: 20.2 g

(117) Vitamin C: 10 mg

(118) Biotin: 10 g

(119) Amide nicotinic acid: 1.7 mg

(120) Folic acid: 50 g

(121) Calcium pantothenic acid: 0.5 mg

(122) Mineral mixture: optimum amount

(123) Ferrous sulfate: 1.75 mg

(124) Zinc oxide: 0.82 mg

(125) Magnesium carbonate: 25.3 mg

(126) Monopotassium phosphate: 15 mg

(127) Dicalcium phosphate: 55 mg

(128) Potassium citrate: 90 mg

(129) Calcium carbonate: 100 mg

(130) Magnesium chloride: 24.8 mg

(131) 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.

(132) Preparation of Health Beverage

(133) CB1 extract: 1000 mg

(134) Citric acid: 1000 mg

(135) Oligosaccharide: 100 g

(136) Apricot concentration: 2 g

(137) Taurine: g

(138) Distilled water: 900 ml

(139) 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.

(140) 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

(141) As described in the present invention, the present invention provides a combined herb extract of Salvia plebeia R. Br. and red ginseng, as an active ingredient for preventing and treating a respiratory inflammation disease and the combined herb composition shows more potent treating effect on a respiratory inflammation than the individual herb composition, which is confirmed by various experiments, for example, determination of the cell number of BAL (bronchoalveolar lavage) (Experimental Example 1); Determination of CD11b+/Gr-1+ ratio in leukocyte within BAL fluid (Experimental Example 2); Determination of expressed RNA level of inflammatory cytokines in lung tissue (Experimental Example 3); Determination of expressed RNA level of inflammatory cytokines in BALF (Experimental Example 4); Lung histology (Experimental Example 5); Brief Clinical test (Experimental Example 7) etc, it has been verified that the inventive combined extract showed more potent inhibiting effect on respiratory inflammation disease than each herb extract. Therefore, the herbal extract of the present invention can be usefully used in a pharmaceutical composition, health functional food, and health supplement food for preventing and treating respiratory inflammation disease.