Abstract:
The present invention relates to a composition comprised of a plant extract as an active component, specifically, Bamboo extract and  Scutellaria  extract, for the treatment and prevention of atopic dermatitis. The present invention is a natural ingredient can be obtained from a plant. The present invention can control immune responses by inhibiting the release of histamine and leukotrien, and thus, has effect in the treatment or prevention of allergic diseases, inflammatory diseases and skin diseases, specifically atopic dermatitis. The present invention has been proven safe and beneficial effecting the treatment of atopic dermatitis through clinical trials, and thus, can be used for the treatment and prevention of atopic dermatitis.

Description:
CROSS-REFERENCE TO RELATED APPLICATIONS 
       [0001]    This application is a continuation of U.S. application Ser. No. 13/586,741, filed Aug. 15, 2012 (now allowed); which is a continuation of U.S. application Ser. No. 12/445,159, filed Apr. 28, 2009 (now U.S. Pat. No. 8,247,007); which is a U.S. National Phase Application of International Application No. PCT/KR2007/005004, filed Oct. 12, 2007; which claims priority to Korea Application No. 10-2006-0099182, filed Oct. 12, 2006. These applications are incorporated herein by reference in their entirety. 
     
    
     TECHNICAL FIELD 
       [0002]    The present invention is a composition comprising of a plant extract as an active ingredient for treating atopic dermatitis, specifically a mixture composition comprising of Bamboo extracts and  Scutellaria  extracts. 
       BACKGROUND ART 
       [0003]    Atopic dermatitis is an allergic disease caused by a defect of a stratum corneum which is a protective wall located in the outermost part of the skin which is caused by hereditary, environmental, or immunological factors and is exacerbated in arid climates. Many people are afflicted by the atopic dermatitis, specifically 0.5-1% of the total population. In cases of minors, 5-10% of children are afflicted by the atopic dermatitis. 50% of patients can recover by their second birthday, and 25% can recover by puberty. However, 25% never recover and continue to suffer from atopic dermatitis into adulthood. 
         [0004]    The main symptoms of atopic dermatitis are severe pruritus, xeroderma, eruption or oozing of the skin, boils, scale like skin (scaly skin), etc. 
         [0005]    The pathogenesis of atopic dermatitis is not completely understood, but genetic factors are attributed to most cases of atopic dermatitis, and the pathogenesis is related to immune response. It has been shown that atopic dermatitis can be caused by a combination of dry skin, skin that is prone to itching more than the average person, infections caused by bacteria virus fungi, etc., and emotional and environmental factors. 
         [0006]    Specifically, an antibody (IgE) produced by a mast cell during the body&#39;s process of naturally eliminating a material which causes a rash to form when in contact or invading the body causes a hypersensitive reaction when this same material invades the body again producing a histamine which causes the atopic dermatitis. The mast cell is distributed widely throughout organs such as, the skin, respiratory organs, mucosa of the gastrointestinal tract, circum of lymphatic duct, brain, and is known as the cell that causes diverse inflammation and allergic reactions. The histamine released from the mast cell causes inflammation and immediate allergic reaction by inducing vasodilation, smooth muscle-contraction of the gastrointestinal and/or bronchial tract, secretion of glandular cells, exacerbation of the reactions, etc., and serves as an intermediary for diverse biological effects such as secretion of mucus and local protein. 
         [0007]    Pharmacotherapies, such as steroids, anti-histamines, antibiotics are usually prescribed for atopic dermatitis. The steroid agent (adrenal cortical hormone agent) can act as an anti-inflammatory and immuno-suppressant and has positive effect in treating the disease, but if used over a long period of time, side effects such as skin-weakening, symptom of systemic hormone, toxicity can result. Currently, uses of immune-suppression agents and novel anti-histamine agents have been studied for treating atopic dermatitis. However, anti-histamine agents cannot completely suppress the allergic reaction since other chemical transmitters in addition to the histamine can induce the allergic reaction. The mast cell releases other chemical transmitters such as leukotriene C4 and leukotriene B4 in addition to the histamine. Leukotriene C4 contracts the smooth muscle of bronchus like the histamine, and leukotriene B4 causes chronic inflammation by inducing neutrophil and eosinophil and injures neighboring cells. 
         [0008]    Thus, a novel composition for the effective treatment of atopic dermatitis without the side-effects is required. 
         [0009]    Bamboo belongs to the Poaceae family. There are about 280 known species of bamboo all over the world, and about 70 species grow naturally or are cultivated in Korea. There are 11 representative kinds of Bamboo;  Phyllostachys nigra, Phyllostachys bambusoides  ( Cedrela sinesis ),  Phyllostachys edulis  ( Phyllostachys pubescen ),  Phyllostachys nigra  for.  Punctata, Sasa borealis  var.  gracilis, Arundinaria simonii, Sasa borealis  var.  chiisanensis, Sasa borealis, Sasa albo - marginata, Pseudosasa japonica,  etc. Among them,  Phyllostachys bambusoides  ( Cedrela sinesis ),  Phyllostachys nigra  and  Phyllostachys edulis  are cultivated. According to Dongeui-Bogam, Compendium of Materia Medica and the divine Farmer&#39;s Materia Medica, Bamboo is effective in treating palsy and hypertension, and was used to treat pneumonia and bronchitis to bring down fever, loosen phlegm and as a coolant. Recently, it has been reported that Bamboo has been used to treat hypertension, atherosclerosis and cardiovascular disease. Bamboo is also known to have anti-oxidant effect which is effective in the prevention of cancer and aging. Also, phytochemicals such as organic acid, dietary fiber, tannin, benzofuran within the plant are expected to contribute to preventing diseases of the circulatory system. 
         [0010]    The conventional studies for bioactive compounds focusing on antimicrobial activity have been reported mostly in Korean and Japan. Japanese researchers discovered the 2,6-dimethylbenzoquinone and benzoic acid which are antimicrobial compounds in the leaf of Bamboo, and Korean Patent No. 10-0465113 discloses the effects of bamboo extract in improving blood circulation and preventing inflammation. Japanese Patent Publication H09-278662 discloses fats and oils which have anti-allergic effect contains the Bamboo extract obtained by using the soxhlet method using ether as a solvent, and WO 2002/07745 discloses that Bamboo extract obtained by using water has antipruritic effect which is effective in the treatment of atopic dermatitis. 
         [0011]      Scutellaria  has bioactive and pharmacological properties and has been used in oriental medicine for treating fevers and allergies. It acts by dilating blood vessels and brings down blood pressure, and inhibits atherosclerosis. Bicalin contained in  Scutellaria  is a kind of flavonoid which is effective to sedate or stop bleeding by suppressing the permeability of capillaries. Also, bicalin inhibits the release of chemical transmitters by strengthening the mast cell membrane and so can do anti-allergic action. Specifically, it is known that the pharmacological properties of  Scutellaria  are improving infections caused by allergies, inhibiting increased vascular permeability and alleviating inflammatory discharge of blood and congestion by strong anti-inflammatory effect, and these pharmacological properties are derived from bicalin. Bicalin is hydrolyzed to baicalein and glucuronic acid. Baicalein acts as a diuretic and glucuronic acid acts as deintoxicant. Korean Patent Publication No. 1996-0003725 discloses a therapeutic agent comprising of the flavonoid ingredient of  Scutellaria.  Korean Patent Publication No. 1996-0040370 discloses a composition for the prevention and treatment of alcohol disorder comprising of  Scutellaria  extract and flavone glycoside. Korean Patent Publication No. 2002-0031608 discloses a  Scutellaria  extract that has positive antimicrobial effect, and the process for preparing the extract and the pharmaceutical composition of the extract. Korean Patent No. 10-0522579 discloses a mixture extract of  Scutellaria  and Omija ( Schizandra chinensia  Baillon) which has anti-stress effect. 
         [0012]    The above properties of Bamboo or  Scutellaria  have been known, but there has not been reported any therapeutic effect for atopic dermatitis using the mixture composition comprising of Bamboo extract and  Scutellaria  extract. 
         [0013]    The inventors of the present invention have studied a novel compound for the treatment of atopic dermatitis. As a result, they discovered and confirmed that the mixture composition comprising Bamboo extract and  Scutellaria  extract can strongly inhibit the release of histamine and leukotrien without any side-effects and has positive therapeutic effect on atopic dermatitis, to complete the present invention. 
       DISCLOSURE OF THE INVENTION 
       [0014]    The object of the present invention is to provide a composition comprising of a plant extract as an active ingredient which will have a positive therapeutic effect for the treatment and prevention of atopic dermatitis without any side-effects. 
         [0015]    Also, the object of the present invention is to provide a use of mixture composition of Bamboo extract and  Scutellaria  extract for the manufacture of a medicament for the treatment and prevention of atopic dermatitis. 
         [0016]    Also, the object of the present invention is to provide a method of the treatment and prevention of atopic dermatitis by administering to the subject a therapeutically effective amount of mixture composition of Bamboo extract and  Scutellaria  extract. 
     
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         [0017]      FIG. 1  is a graph showing the improvement rate of clinical trial items on antecubital space and popliteal space after administering the present extract. 
           [0018]      FIG. 2  is a digital steel photo showing the improvement effect of atopic dermatitis according to administration of the present extract by comparing photos taken before and after using the product. 
           [0019]      FIG. 3  is a graph showing measurement result of the moisture loss rate (g/m 2  h) which occurred per unit area and per unit time by using Tewameter TM300 (Courage+ Khazaka, Germany) on 10 cm lower part of popliteal fossa and antecubital fossa at the time before the product was used and after the product was used. 
       
    
    
     DETAILED DESCRIPTION OF THE INVENTION 
       [0020]    To achieve the above objectives, the present invention provides a composition for the treatment of atopic dermatitis comprising of Bamboo extract and  Scutellaria  extract as an active ingredient. 
         [0021]    Also, the present invention provides a use of mixture composition of Bamboo extract and  Scutellaria  extract for the manufacture of a medicament for the treatment and prevention of atopic dermatitis. 
         [0022]    Also, the present invention provides a method of the treatment and prevention of atopic dermatitis by administering to the subject a therapeutically effective amount of mixture composition of Bamboo extract and  Scutellaria  extract. 
         [0023]    In the composition of the present invention, Bamboo is selected from  Phyllostachys, Sasa  or  Pseudosasa,  and  Phyllostachys  is preferably selected from the group consisting of  Phyllostachys edulis, Phyllostachys nigra  var.  henonis, P. nigra, P. bambusoides, P. pubescence, P. nigra  for.  punctata  and  P. comprossa , and  Sasa  is preferably selected from the group consisting of  Sasa coreana Nakai, S. coreana, S. kurilensis, S. quelpaertensis, S. borealis, S. borealis  var . chiisanensis  and  S. borealis  var.  gracilis,  and  Pseudosasa  is preferably selected from  Pseudosasa japonica  and  Pseudosasa japonica  var.  purpurascens.    
         [0024]    In the composition of the present invention, for Bamboo and  Scutellaria  commercially purchased herbs can be used. The whole herb, branch, shell, leaf, sprout, root, endodermis, etc., can be used, preferably in the form of powder or extract. 
         [0025]    The Bamboo extract and  Scutellaria  extract of the present invention can be used by extracting Bamboo and  Scutellaria  with water, organic solvent, or mixing solvents thereof. Although all conventional solvents can be used as the above organic solvent, polar solvent such as water, C 1-4  alcohol (such as methanol, ethanol etc.), etc., or mixing solvent thereof is preferred. Preferably, water-insoluble fraction of 50-90% of ethanol extract or ethanol-soluble fraction of hot water extract can be used as the above bamboo extract. 
         [0026]    The above extraction may be carried out by conventional methods such as hot water extraction, sonication, etc., and a lyophilized product of the extract can be used for the present composition. In addition, the extract can be further purified by conventional fractionation method or chromatography, and such fractionated material or purified material is also within the scope of the present invention. 
         [0027]    In the composition of the present invention, Bamboo or  Scutellaria  can be used alone, but it is preferable to use a combined composition that Bamboo extract is additionally mixed with  Scutellaria  extract to show synergistic effect. 
         [0028]    In the composition of the present invention, the synergistic effect at the time of administering the combination in comparison with administration of the extract alone was measured and confirmed by using the COLBY formula (COLBY S. R., Calculating synergistic and antagonistic response of herbicide combinations, Weeds 15, 20-22, 1967). 
         [0029]    As shown above, when the composition is used in combination with Bamboo extract and  Scutellaria  extract, their weight ratios of Bamboo: Scutellaria  could be in 1˜10:1˜10, but preferably 1˜5:1˜5, or more preferably 1˜3:1˜3. 
         [0030]    The composition of the present invention can be prepared into conventional pharmaceutical preparations according to conventional methods in the pharmaceutical field, for example, solution such as drinks, syrup, capsule, granule, tablet, powder, pill, ointment, and emulsion, skin external preparation such as gel, etc., by mixing it with a pharmaceutically acceptable carrier, excipient, etc.; and can be administered orally or parenterally. 
         [0031]    The composition of the present invention is appropriately administered depending on the extent of absorption of the active ingredients into the body; excretion rate; age, weight, sex, and condition of patient; severity of treated disease, etc. However, generally, the dosage for an adult is in solution 0.0001˜100 mg/kg, or preferably 0.001˜100 mg/kg, per day. It can be administered once a day or several times a day. The amount should not limit the scope of the present invention in any manner. 
         [0032]    Hereinafter, the present invention will be described in more detail with reference to the following examples, but the scope of the present invention should not be construed to be limited thereby in any manner. 
       EXAMPLES 
     Example 1 
     Preparation of Bamboo Extract 
     Example 1-1 
     Preparation of Bamboo Ethanol Extract 
       [0033]    Dried bamboo (20 kg) was extracted by adding 25% of ethanol (200 l) and heating the mixture at 80° C. for 6 hr. The extract was filtered and concentrated to remove the ethanol until the extract volume reached 5 l. The concentrated extract was then cooled to room temperature. The pellets were collected and dried to obtain the bamboo extract (390 g). 
       Example 1-2 
     Preparation of Bamboo Hot Water Extract 
       [0034]    Dried bamboo (20 kg) was extracted by adding water in the amount equivalent to 10 times the weight of the dried bamboo and heating the mixture at 100° C. for 4 hr. The extract was filtered and concentrated under reduced pressure. The concentrated extract was added to ethanol (10 l) and stirred at 70° C. for 2 hr, and then cooled to room temperature. The pellets were filtered and concentrated under reduced pressure to obtain the bamboo extract (350 g). 
       Example 2 
     Preparation of  Scutellaria  Extract 
       [0035]      Scutellaria  (1 Kg) was added to water (8 l) and extracted by refluxing at 80° C. for 2 hr. The extract was cooled, filtered and concentrated, to obtain the  Scutellaria  extract powder (330 g). 
       Example 3 
     Preparation of Mixture Composition 
       [0036]    The mixture composition was prepared by mixing Bamboo extract obtained from Example 1 and  Scutellaria  extract obtained from Example 2. The weight proportion of the Bamboo extract to the  Scutellaria  extract should be 1:1, 1:2, 1:3 or 2:1, 3:1. 
       EXPERIMENTS 
     Experiment 1 
     Measurement of Inhibition Activity of Releasing Histamine and Leukotrien from the Mast Cell According to the Examples 
       [0037]    The release of histamine and leukotrien from the mast cell is one of the major causes for the allergic reaction. The effect of the mixture composition of the Bamboo extract and the  Scutellaria  extract in inhibiting the release of histamine and leukotrien from the mast cell was measured. 
       Experiment 1-1 
     Isolation of the Mast Cell from Liver 
       [0038]    Lung tissue (3 g/1 pig) was isolated from eight female guinea pigs (200 g) and fat tissue, bronchus and blood were removed from the lung tissue. The isolated lung tissue was treated with enzyme (5 mg/ml of collagenase, 1.8 unit/27 μl of elastase) by using Tyrode TGCM buffer containing Ca 2+ , Mg 2+  and 0.1% of gelatin at 3 times for 15, 15, 25 mins. The each enzyme treated lung tissue was filtered by nylon mesh and metal mesh (100 μm), and then centrifuged (called ‘monodispersed mast cell’). The pellets was suspended with TG buffer (16 ml) containing 0.1% of gelatin, but no Ca 2+  and Mg 2+ , and centrifuged by loading to rough Percoll (1.041 mg/ml density) at 1,400 rpm for 25 mins, to obtain the pellets. The pellets were re-suspended with TG buffer (8 ml) and centrifuged by loading to discontinuous Percoll (1.06-1.10 mg/ml density) at 1,400 rpm for 25 mins, to isolate several cell layers. Among the several cell layers, the third and fourth layers were washed twice with TGCM buffer since the mast cell exists in third and fourth layers. The whole cell and mast cell were stained with trypan blue and alcian blue. The purity of the mast cell was measured by calculating the number of cells, to obtain about 80-90% of the mast cell. 
       Experiment 1-2  
     Inhibition of Releasing Histamine from the Mast Cell 
       [0039]    The mast cell (4105 cells) was treated with guinea pig IgG1 antibody (anti-OVA 1 ml/106 cells) at 37° C. for 45 mins, and washed with TGCM buffer to remove anti-OVA antibodies which are not bound to the membrane of the mast cell. The mast cell was suspended with TGCM buffer (1 ml) and pre-treated with each reagents (30 μg concentration). The mast cell was reacted by sensitizing using ovalbumin (1.0 μg/μl) for 10 mins, cooled in ice, and centrifuged, to measure histamine from the supernant. 
         [0040]    The amount of histamine in each sample was measured by modifying the method of Siraganian and using automated continuous-flow extraction and a flourometic analyzer (Astoria analyzer series 300, Astoria-pacific international, Oragon, USA). 1N-hydrochloric acid, 0.73M phosphoric acid, 5N sodium hydroxide, 1N sodium hydroxide, saline diluents and sampler wash, o-phthaladehyde solution was prepared and connected to a tube linked to the analyzer. The storage solution of histamine was diluted to 20 ng, 10 ng, 5 ng, 3 ng and 1 ng, and the concentration-dependent result of standard curve was obtained. Then, each sample was diluted with 2% of perchloric acid and the amount of histamine was measured. The result showed that the Bamboo extract and the  Scutellaria  extract showed inhibition activity, respectively, and the mixture composition of the Bamboo extract and the  Scutellaria  extract also showed high inhibition activity. The synergistic effect at the time of administering the combination in comparison with administration of the extract alone was measured and confirmed by using the COLBY formula (COLBY S. R., Calculating synergistic and antagonistic response of herbicide combinations, Weeds 15, 20-22, 1967) (Table 1). 
         [0000]    
       
         
               
             
               
               
             
           
               
                 TABLE 1 
               
             
             
               
                   
               
               
                 The inhibition activity of releasing histamine 
               
               
                 from the mast cell per each extract. 
               
             
          
           
               
                 Sample 
                 Inhibition activity(%) 
               
               
                   
               
               
                 Control 
                 32.5 ± 0.25 
               
               
                 Bamboo extract 
                 22.4 ± 0.09 (31.1%) 
               
               
                   Scutellaria  extract 
                 26.4 ± 0.11 (18.8%) 
               
               
                 Mixture composition (Bamboo: Scutellaria  = 1:1) 
                 10.1 ± 0.25 (70.5%) 
               
               
                 Mixture composition (Bamboo: Scutellaria  = 1:2) 
                 15.4 ± 0.46 (52.6%) 
               
               
                 Mixture composition (Bamboo: Scutellaria  = 1:3) 
                 19.7 ± 0.52 (39.4%) 
               
               
                 Mixture composition (Bamboo: Scutellaria  = 2:1) 
                  9.3 ± 0.32 (71.3%) 
               
               
                 Mixture composition (Bamboo: Scutellaria  = 3:1) 
                 13.2 ± 0.11 (59.4%) 
               
               
                   
               
             
          
         
       
     
       Experiment 1-3 
     Inhibition of Releasing Leukotrien from the Mast Cell 
       [0041]    The amount of leukotrien in each sample was measured by using the method of Aharoney et al. (Biochem. Biophys. Res. Commun., p 574-579, 1983). The leukotrien antibody was suspended with 5 mM MES buffer containing 0.1% of gelatin, and to each tube the supernant of the cell (100 μl) which was treated with a reagent (30 μg) was added. The leukotrien antibody and [ 3 H] leukotrien D4 (LTD 4 ) were added to the supernant and was allowed to react at 4° C. for 2 hr. The reaction was stopped by using dextran coated charcoal and the inhibition activity was measured by using liquid scintillation spectrometry. The results showed that the Bamboo extract and the  Scutellaria  extract showed inhibition activity, respectively, and the mixture composition of the Bamboo extract and the  Scutellaria  extract also showed high inhibition activity. The synergistic effect at the time of administering the combination in comparison with administration of the extract alone was measured and confirmed by using the COLBY formula (COLBY S. R., Calculating synergistic and antagonistic response of herbicide combinations, Weeds 15, 20-22, 1967) (Table 2). 
         [0000]    
       
         
               
             
               
               
               
             
           
               
                 TABLE 2 
               
             
             
               
                   
               
               
                 The inhibition activity of releasing leukotrien 
               
               
                 from the mast cell per each extract. 
               
             
          
           
               
                   
                 Sample 
                 Inhibition activity(%) 
               
               
                   
                   
               
               
                   
                 Control 
                 679.0 ± 54.19 
               
               
                   
                 Bamboo extract 
                 449.0 ± 40.47 (33.8%) 
               
               
                   
                   Scutellaria  extract 
                 569.4 ± 32.89 (16.1%) 
               
               
                   
                 Mixture composition 
                 149.5 ± 8.26 (78.0%) 
               
               
                   
                 (Bamboo:  Scutellaria  = 1:1) 
               
               
                   
                 Mixture composition 
                 282.1 ± 47.55 (58.5%) 
               
               
                   
                 (Bamboo:  Scutellaria  = 1:2) 
               
               
                   
                 Mixture composition 
                 350.1 ± 33.1 (48.4%) 
               
               
                   
                 (Bamboo:  Scutellaria  = 1:3) 
               
               
                   
                 Mixture composition 
                 147.5 ± 11.92 (78.3%) 
               
               
                   
                 (Bamboo:  Scutellaria  = 2:1) 
               
               
                   
                 Mixture composition 
                 322.9 ± 33.65 (52.4%) 
               
               
                   
                 (Bamboo:  Scutellaria  = 3:1) 
               
               
                   
                   
               
             
          
         
       
     
       Experiment 2 
     Clinical Trials 
       [0042]    20 patients suffering from severe atopic dermatitis were tested by using the mixture composition of Bamboo extract and  Scutellaria  extract selected from Experiment 1 for 4 weeks. The present composition was spread onto the popliteal fossa and the antecubital fossa, and the results were investigated. 
         [0043]    In the clinical trial, the effects before and after using the product were estimated by using the Local SCORAD index. The results were estimated by rating the degress of 6 intensity items, erythema, edem/population, oozing/crusting, excoriation, lichenification, dryness on a scale of 4 (0=absent, 1=mild, 2=moderate, 3=severe) for the right and left side of popliteal fossa and antecubital fossa which were then used to show improvement rate. 
         [0044]    The results showed that there was improvement effect after using the product, specifically there was more than 50% improvement in erythema, oozing/crusting and excoriation ( FIG. 1 ). The result was photographed by using a Digital Still Camera (DSC-575, Sony) at the time before the product was used and after the product was used ( FIG. 2 ). Also, the moisture loss (g/m 2 ·h) due to evaporation which occurred per unit area and per unit time was estimated by using Tewameter TM300 (Courage+ Khazaka, Germany) on 10 cm lower part of popliteal fossa and antecubital fossa at the time before the product was used and after the product was used. The moisture loss on trandermal was reduced each time, specifically the improvement on the antecubital fossa was better than on the popliteal fossa ( FIG. 3 ). 
       INDUSTRIAL APPLICABILITY 
       [0045]    The present invention is a natural ingredient obtained from a plant, and can control immune responses by inhibiting the release of histamine and leukotrien. It has been confirmed that the present invention is safe and is beneficial to the treatment of atopic dermatitis, and thus, the composition can be used for the treatment and prevention of atopic dermatitis.