KR101086256B1 - Preventive and curative extracts from Gentianae macrophyllae radix for respiratory organ disease - Google Patents

Abstract

The present invention relates to extracts of orthodontic extracts useful for the prevention and treatment of respiratory diseases. More specifically, the extracts of orthodontic ginseng extract on the airway contraction inhibitory activity, airway inflammation inhibitory activity, 5-lipoxygenase inhibitory activity, phosphodiesterase 4 inhibitory activity, By confirming excellent corticosteroid D4 antagonism, it is possible to prevent and treat respiratory diseases such as asthma, acute bronchitis, allergic rhinitis, acute lower respiratory tract infection (bronchitis, bronchiolitis, etc.), and acute upper respiratory tract infection (pharyngitis, tonsillitis, laryngitis). It relates to a medicament containing a useful powdered extract.

Respiratory disease, oral extract, suppress airway contraction, suppress airway inflammation, 5-lipoxygenase, phosphodiesterase 4, leukotriene D4 antagonism, asthma, bronchitis, allergic rhinitis, sore throat, tonsillitis, laryngitis

Description

Pregnancy extracts useful for the prevention and treatment of respiratory diseases {Preventive and curative extracts from Gentianae macrophyllae radix for respiratory organ disease}

The present invention relates to extracts of orthodontic extracts useful for the prevention and treatment of respiratory diseases. More specifically, the extracts of orthodontic ginseng extract on the airway contraction inhibitory activity, airway inflammation inhibitory activity, 5-lipoxygenase inhibitory activity, phosphodiesterase 4 inhibitory activity, By confirming excellent corticosteroid D4 antagonism, it is possible to prevent and treat respiratory diseases such as asthma, acute bronchitis, allergic rhinitis, acute lower respiratory tract infection (bronchitis, bronchiolitis, etc.), acute upper respiratory tract infection (sore throat, tonsillitis, laryngitis). It relates to a medicament containing a useful powdered extract.

Asthma, a representative respiratory disease in respiratory diseases, is a condition in which respiratory distress, cough, and breathing swelling (wheezing) are repeatedly seizures. About 30% of asthmatic patients within 80 years of age Asthma shows asthma symptoms at the age of 4-5 years, and the incidence rate of about 10% in Korea, and heart asthma and bronchial asthma. The former is a paroxysmal breathing difficulty seen in heart disease or high blood pressure, and is caused by hypersensitivity of the respiratory tract due to pulmonary congestion or tissue congestion and poor prognosis. The latter is a paroxysmal respiratory distress caused by narrowing of breathing due to bronchial spasm, narrowing, and mucosal swelling. In addition, there are cerebral asthma caused by disorders of the respiratory central region, and uremia asthma occurring in patients with nephropathy.

The incidence of asthma varies according to country, race, and age, but according to a 1991 UK report, about 7% of adults and 13.5% of pediatric age groups reported changes in living conditions, pollution, and stress in Korea. The trend is gradually increasing. Although asthma varies depending on the cause, in modern times when environmental pollution such as pollution is serious, the severity of asthma is maximized, such as the age of onset and the symptoms are prolonged.

The characteristic airway obstruction of asthma occurs by a three-step process. In other words, the contraction of bronchial smooth muscle, thickening of the pulmonary mucosa, and sticky mucus in the bronchus and bronchioles block the airways. Only the contraction of the double bronchial smooth muscle recovers easily.

The exogenous (allergic) asthma attack mechanism is particularly important for antibodies IgE, and some are related to IgG. IgE releases mediators (histamine, SRS-A, ECF-A, NCF, PAF, Kinin, PGs, etc.) that activate mast cells to cause hypersensitivity. Non-allergy Asthma attacks are thought to be mediated by unknown or autonomic nerves. Cholinergic stimulation in endogenous patients not only favors histamine-mediated mediators in mast cells through terminal organs, but also increases goblet cell secretion, enlarges blood vessels in the lungs, and constricts the trachea, bronchus and giant bronchioles. Bronchospasm and mucus secretion increase.

There is currently no fundamental cure for asthma. Treatment involves several methods and drugs to prevent seizures and prevent complications, but it is not satisfactory. The most important thing is to find and avoid the trigger. Treatment of asthma is mainly for bronchodilators for inhalation, oral or injectable bronchodilators (sympathetic and theophylline drugs), stereoforms (for inhalation, oral, injectables, etc.), leukotrienes antagonists (montelukast, franlukast). , Ziluton and the like), and anti-allergic drugs (disodium chlorochlorate, chromoline sodium, ketotifen) are used.

Bronchitis is divided into acute and chronic, and the causes are allergic, infectious, traumatic, etc., and pathologically catarrhal, purulent, obstructive, ulcerative, and invasive. The most common cause is infection by bacteria, viruses, fungi, and the like, and people who are not normally infected are susceptible to infection when the resistance of the whole body is weakened. Allergic bronchitis is a direct reaction from inhaling allergens and partial symptoms from systemic allergic reactions. The trauma may be caused by chemical stimulation such as chlorine or sulfur dioxide, or by physical stimulation such as dust. Contaminated air in large cities is chronically irritating and inflammatory. Pathologically, acute cases include redness, swelling, and dryness in the bronchial mucosa, and mucous or purulent secretions can be seen in the bronchus. It usually recovers without causing complications, but when it is chronically developed, it causes swelling, thickening, and atrophy of the mucous membrane, which can lead to proliferation of fibers, bronchial narrowing, and emphysema. . The most prominent symptoms are cough and sputum. When the cause is caused by infection, fever and chest pain can be seen. In traumatic cases, the mucous membranes of the mouth, nose, and eyes also appear. Coughing is a type of defense in the treatment, so trying to stop it is not a good idea. You must treat the cause at the same time. In winter, increase the room temperature and use a small amount of codeine, atropine, ephedrine, and antihistamines. Steroids or theophylline may be used for inflammation, but the effect is not as expected.

Allergic rhinitis is also referred to as nasal allergy or allergic rhinitis, symptoms of sudden sneezing, watery nasal juice (clear runny nose), large amounts of nasal congestion, and heavy head and tears. The symptoms are very similar, but the antigen is not clear is called vasomotor rhinitis. In other words, when one wakes up in the morning and the body cools down temporarily, the symptoms mentioned above may occur and heal in a few hours. It is often confused with a cold, which is different from the common cold and often involves asthma or hives.

An allergic reaction of allergic rhinitis is a type of antigen-antibody hypersensitivity. Histamine is released from mast cells and basophils, and arachidonic acid is released to release prostaglandins and leukotrienes by cyclooxykenase (COX) and 5-lipooxygenase (5-LO). It produces | generates and mediates the initial reaction which appears between 2 to 90 minutes of antigen exposure, and the late reaction which appears after 4 to 8 hours. The initial response is mainly due to the mediator and the late response is mainly due to the cell infiltration. In addition, allergic and nonallergic rhinitis both act as risk factors for the development of asthma.

Treatment is desensitization (항원 感 作 분명 法) when the antigen is clear, and other drug therapy, surgical therapy, physical therapy, etc., it is difficult to recover completely.

There are many types of airway infections, such as common purulent bacteria, special bacteria (such as Mycobacterium tuberculosis, Diphtheria bacteria, Spiroherta), viruses, and fungi, and are classified into acute and chronic. When the nasal cavity, pharynx, and larynx are individually infected, they are called their respective organ names, but when their entire organs are in an infected state, they are called upper respiratory tract infections. In addition, in case of infection by special bacteria or fungi, it is often called by special names such as upper island tuberculosis, said dodiphtheria, said said candidiasis.

As described above, respiratory diseases such as asthma, allergic rhinitis, and acute bronchitis have some differences in their causes and symptoms, but they have some commonalities.

First, all are aspects of inflammatory diseases. These respiratory diseases are caused by allergies and infections, but inflammation plays an important role in the aggravation and treatment of the disease. That is, influx and activation of leukocytes triggered by allergy, infection, etc. into the respiratory tract and various cytokines and inflammatory mediators released from leukocytes make the disease worse and difficult to treat.

Second, the contraction and relaxation of the airway is not normal, breathing is difficult. In other words, the airway is damaged, excessive response to the general stimulation (asthma) or narrowed airway (bronchitis) is difficult to breathe, so appropriate treatment is necessary.

Third, anti-inflammatory agents, airway contraction inhibitors and dilatants, airway secretion inhibitors play an important role in the main drug therapy, many drugs are commonly used. Antihistamines, anticholinergic agents, beta2 receptor agonists, steroids, leukotrienes D4 receptor antagonists, and phosphodiesterase 4 inhibitors of theophylline are widely used. Nevertheless, airway dilators such as anticholinergic agents and beta2 receptor agonists do not have an effect on the inflammation that aggravates the disease, and thus only relieve symptoms. Steroids, which are known to be effective against inflammation, are problematic for long-term use due to severe side effects and are known to be less effective for chronic bronchitis. Therefore, a combination of the two is often prescribed, due to the side effects of steroids developed in the form of inhalants than oral administration is difficult to take because there is a problem that the dosage compliance falls.

Overcoming the limitations of these current drug therapies, there is a need for the development of new therapeutic agents that can fundamentally cure the cause and effectively improve symptoms. However, as described above, respiratory diseases are involved with various leukocyte cells and various cytokines and inflammatory mediators that are released from them, so it is difficult to treat them with a single chemical, and natural extracts with various ingredients and mechanisms can be effective treatments. have.

In addition, respiratory diseases such as asthma, bronchitis, allergic rhinitis, acute lower respiratory tract infections (bronchiolitis, bronchiolitis, etc.), acute upper respiratory tract infections (sore throat, tonsillitis, laryngitis), etc., are very diverse and cause only conventional treatment. There is a problem in the treatment such as recurrence even after treatment. Therefore, prevention and treatment of respiratory diseases are emerging as one of the important tasks of the medical community, and the development of fundamental prevention and treatment is urgently needed.

On the other hand, the herbal medicine used in the present invention (Gentianae macrophyllae radix) is a perennial herb, the height is 40 ~ 60 cm, the root is firm and not elastic. It is distributed in Heilongjiang, Liaoning, North China, Henan, and Sichuan in China. It is collected in spring and autumn. Remove the foliage, beard root, black, and dry in the sun or in pairs to dry in the sun, and when it is reddish yellow or yellowish gray, spread it out and dry it again in the sun. In oriental medicine, it is effective in driving away wind, removing moisture, harmonizing blood, releasing muscles and tendons, removing heat, and diuresis. Pain caused by customs, spasms of musculature, jaundice, blood transfusion, and urine It has been used for the purpose of treating. The major components known so far are gentianine, gentianidine, gentianol, gentiopicroside, etc. [Chinese Dictionary, Book Publishing Jungdam, pp. 5171-5177; In 1998, the Korean Medicine and Herbal Medicines Standards Collection, Korea Medical Index, p. 352; 1988].

 Therefore, the present inventors have studied to develop a drug for the treatment of respiratory diseases, the results showed that the extract of the jinchuan fruit extract airway contraction inhibitory activity, airway inflammation inhibitory activity, 5-lipoxygenase inhibitory activity, phosphodiesterase 4 inhibitory activity, rukotri The present invention was completed by confirming that the endo D4 antagonist activity was excellent.

Therefore, an object of the present invention is to provide a medicament for the prevention and treatment of respiratory diseases containing the extract as an active ingredient.

The present invention is characterized by a drug for the prevention and treatment of respiratory diseases containing Gentianae macrophyllae radix extract as an active ingredient.

Hereinafter, the present invention will be described in detail.

In the present invention, asymptomatic, acute, by confirming that the extract of the jinchuan extract is excellent in airway contraction inhibitory activity, airway inflammation inhibitory activity, 5-lipoxygenase inhibitory activity, phosphodiesterase 4 inhibitory action, leukotriene D4 antagonistic action The present invention relates to a medicament containing extracts of gingival extract useful for the prevention and treatment of respiratory diseases such as bronchitis, allergic rhinitis, acute lower respiratory tract infections (bronchitis, bronchiolitis, etc.), and acute upper respiratory tract infections (sore throat, tonsillitis, laryngitis).

Briefly explaining the manufacturing process of the extract of Jinja according to the present invention,

1) After reflux extraction with 10-15 times water or alcohol solution of Chinese herbal medicine, it is filtered, and the residue is added with 7-12 times water or alcohol solution of mixed herbal medicine weight, heated, reextracted and filtered. Filtering by mixing with the filtrate of;                     

2) separating the filtrate obtained in 1) with the same amount of lower alcohol or nonpolar solvent, and then concentrating under reduced pressure at 60 to 70 ° C .; And

3) azeotropically concentrating with water of 25 to 50 times the total amount of the concentrate of 2), homogeneously suspending with the same amount of water, followed by freeze drying.

To explain this in detail, water or alcohol aqueous solution is added to the Chinese herbal medicine and reflux extraction for 2 to 5 hours, wherein the amount of water or alcohol aqueous solution is suitably used 10 to 15 times the weight of the herbal raw material. Then, the filtrate is collected by filtration, and the residue is added with an aqueous solution of water or alcohol of 7-12 times the weight of the crude ingredient, and then warmed again for 2 to 5 hours, filtered and mixed with the previous filtrate to increase the extraction efficiency. If the amount of water is too small, the stirring becomes difficult and the solubility of the extract is low, so that the extraction efficiency is reduced. If the amount is too large, the use of lower alcohols and nonpolar solvents used in the next purification step is not economical, causing problems in handling. Can be.

In the present invention, the method of re-extracting after the first extraction is adopted. Even in the case of mass production of the herbal extracts, even though the filtration is effective, the loss occurs because the water content of the herbal medicine itself is high, so the extraction efficiency is reduced only by the first extraction. This is to prevent this. In addition, as a result of verifying the extraction efficiency of each step, it was found that about 80 to 90% of the total extraction amount is extracted by the second extraction, and the third or more multistage extraction is not economical.

As described above, the extract obtained by extraction with an aqueous solution of water or alcohol over the first and second stages is filtered and concentrated, and then purified from impurities such as unnecessary proteins, polysaccharides and fatty acids contained in the filtrate. The impurities are purified by performing layer separation 2-4 times with alcohol or nonpolar solvent to obtain a solvent fraction. The lower alcohol is preferably alcohol having 1 to 6 carbon atoms, more preferably butyl alcohol, propyl alcohol or isopropyl alcohol, and the nonpolar solvent is ethyl acetate, dichloromethane, chloroform, carbon tetrachloride and methyl ethyl ketone. However, when the amount of the lower alcohol or the nonpolar solvent is less than that of the filtrate, fine particles are formed by unnecessary components such as fatty acids, and thus the layer separation is not smooth and the extraction content of the active ingredient is not efficient.

The lower alcohol or nonpolar solvent fraction obtained after layer separation is concentrated under reduced pressure at 60 to 70 ° C. to remove the solvent remaining in the sample. The concentrate thus obtained is azeotropically concentrated two to three times with water of 25 to 50 times the total amount of the concentrate, followed by the addition of an equal amount of water and suspended homogeneously. The reason for azeotropic concentration with water at the time of concentrated drying is to effectively control the content of the remaining lower alcohol in order to use the obtained herbal extract as a pharmaceutical raw material.

The extract thus obtained is lyophilized to obtain a powdery final extract, which is expected to be very useful as a prophylactic and therapeutic agent for respiratory diseases because of its excellent airway contraction inhibitory activity and airway inflammation inhibitory effect.

Tablets, capsules, injections and the like are prepared by formulating a conventional Chinese method by containing the extract of jinjagyo, among them, a combination of lactose, microcrystalline cellulose, magnesium stearate, and the like, which is used as a base when preparing tablets. The use of X in a ratio of 2: 1 can produce tablets that are active in the prevention and treatment of respiratory diseases.

In the manufacture of such pharmaceuticals, herbal extracts may be used as such, but they may also be mixed with pharmaceutically acceptable carriers, excipients, diluents, etc., to prepare powders, granules, capsules or injections. Is possible. In addition, the extract of the jinchuan according to the present invention has been used for food and medicinal use since ancient times, and there is no particular restriction on its dosage. Rate, the severity of the disease, and the like. In general, it is preferable to administer about 0.1-10 mg / kg body weight extract per 1 kg. Therefore, the extract containing the active ingredient of the present invention is to be prepared in consideration of the effective amount range, and the unit dosage form formulated in this way according to the judgment of experts and the needs of the individual to monitor or observe the administration of the drug as needed Specialized medications can be used or administered at regular intervals.

Hereinafter, the present invention will be described in more detail based on the following examples, but the present invention is not limited thereto.

Preparation Example 1 Preparation of Orthodontic Extract

After mixing 250 g of finely chopped fine powder about 3.0 cm, 2 L of water was added thereto, followed by hot boiling extraction for 5 hours while stirring well. The filtrate was collected and collected, and the residue was extracted with 2 liters of water and re-heated for 3 hours. The filtrates were combined and concentrated to 1 liter. The same amount of saturated n-butyl alcohol was added thereto, and the layers were separated three times. Then, only n-butyl alcohol was collected and concentrated under reduced pressure until the herbal extract was dried at 65 ° C. After most of n-butyl alcohol and water were evaporated, 0.1 L of water was added to the azeotropic condensate, and the resultant was repeated twice. Finally, the same amount of distilled water was added, suspended, and lyophilized to obtain a powdered powdered extract.

Preparation Example 2 Preparation of Orthodontic Extract

The mixture was washed with water to remove impurities, and 2 liters of 50% (v / v) ethanol aqueous solution was added to 250 g of the well-dried oak, followed by extraction under reflux for 6 hours with stirring. The filtrate was collected and the residue was added to 1.5 L of 30% (v / v) alcohol aqueous solution and re-extracted for 3 hours, and the filtrates were combined and concentrated to 1 L. The same amount of saturated n-butyl alcohol was added thereto, and the layers were separated three times. Then, only n-butyl alcohol was collected and concentrated under reduced pressure until the herbal extract was dried at 65 ° C. Most of n-butyl alcohol and water were evaporated, 0.3 L of water was added, the azeotropic concentration was repeated, and the resultant was repeated twice.

Example 1: Airway Shrinkage Inhibition Test in vitro )

In order to evaluate the airway contraction inhibitory activity of the extracts prepared in Preparation Examples 1 and 2 were carried out by the following method using the extraction bronchus, the results are as shown in Table 1 below.

Experimental Method

Hartely male guinea pigs (400-450 g, SLC, Japan) were sensitized by intravenous injection of anti-ovalbumin anti-serum at a volume of 1.5 ml / kg. 48 hours after sensitization, guinea pigs were bled and bronchus was removed. After removing the other tissues attached to the bronchus from the liquid Krebs-Heseleit, the cartilage was incised in a ring shape to include 2-3 cartilage. The cartilage part of the ring was cut while preserving the bronchial muscles, the threads were connected to both sides, and suspended in an organ bath. After stabilization for a certain time, 10 μg / ml of carbachol was added to induce maximum contraction. The bronchus was washed and stabilized with Krebs-Heseleit fluid. One minute after the test to be added 2 μmol indomethacin (indomethacin) was added. After 5 minutes, 10 μg / ml of ovalbumin was added to cause contraction. Shrinkage was calculated by comparing the contraction with carbacol and egg albumin. Bronchoconstriction was measured using a physiological activity meter (MP150, BioPAC system) connected to a force transducer (FT4, BioPAC system).

division density Bronchoconstriction inhibition rate (%) Preparation Example 1 0.25 mg / ml 72 0.5 mg / ml 76 Production Example 2 0.25 mg / ml 75 0.5 mg / ml 82 Positive control group Montelukast 10 μM 24

As shown in Table 1, the extract prepared as in the present invention can be seen that the degree of inhibition of airway contraction of the extracted bronchus.

Example 2: Airway Shrinkage Inhibition Test in vivo )

In order to evaluate the airway contraction inhibitory activity of the extracts prepared in Preparation Examples 1 and 2 were carried out by the following method using the airway palpation reaction induced when the antigen was exposed to the sensitized guinea pig, and the results are shown in the following table. As shown in 2.

Experimental Method

Anti-ovalbumin antiserum was intravenously injected into a vein of Hartley male guinea pigs (400-450 g, SLC, Japan) at a volume of 1.5 ml / kg. The drug was administered orally after 48 hours. After 30 minutes, pyrilamine maleate (10 mg / kg), indomethacin (10 mg / kg), and propranolol (Propranolol, 0.1 mg / kg) were pretreated subcutaneously. Then, the various airway indicators of the guinea pigs were measured in a double chamber plethysmograph box equipped with a plethysmometer (HSE, German) and the basic airway resistance value was measured. After 30 minutes of pretreatment, 1% ovalbumin was aerosolized using high pressure compressed air and sprayed for 2 minutes. After 4 hours the drug was orally administered again. After 24 hours, the waste broth was washed with 1.5 ml of phosphate buffer solution to collect the washing solution. The white blood cell count and eosinophil count in the wash solution were counted.                     

division density Bronchoconstriction inhibition rate (%) Preparation Example 1 200 mg / ml 46 400 mg / ml 53 Production Example 2 200 mg / ml 54 400 mg / ml 62 positivity
Control Franlukast 100 mg / kg 27

As shown in Table 2, it can be seen that the extract prepared as in the present invention has a large degree of suppressing airway contraction of the sensitized guinea pig.

Example 3: Airway Inflammation Inhibition Test

In order to evaluate the airway inflammation inhibitory activity of the extracts prepared by Preparation Examples 1 and 2, leukocyte and eosinophil apoptosis into the bronchiole induced by exposure of the antigen to sensitized mice was performed by the following method. The results are shown in Table 3 below.

Experimental Method

BALB / c female mice (6 weeks old, SLC, Japan) were sensitized by intraperitoneal administration of 0.2 ml of a mixture of 10 μg of egg albumin and alum on days 0, 7, and 14. After 8 and 10 days of final sensitization, 0.7% egg albumin was aerosolized using high-pressure compressed air and sprayed for 50 minutes to induce airway inflammation. After 24 hours of induction of inflammation, the waste bronchus was washed with 1.5 ml of phosphate buffer solution to collect the wash solution. The white blood cell count and eosinophil count in the wash solution were counted. Orthodontic extract was administered orally 7 times on the final sensitized 7-10 days.                     

division density Airway inflammation inhibition rate (%) Preparation Example 1 400 mg / ml 26 800 mg / ml 35 Production Example 2 400 mg / ml 31 800 mg / ml 40 Control group Rolipram 10 mg / ml 35

As shown in Table 3 it can be seen that the extract prepared as in the present invention has an inhibitory effect on airway inflammation. The effect of the positive control of rolifram shows a saturation of the drug at low doses already without dose dependence, but the pharmaceutical composition of the present invention has sufficient dose-dependent efficacy.

Example 4: 5-lipoxygenase (5-LO) inhibitory action

In order to evaluate the activity inhibitory activity against rucotriene producing enzyme, which is the main pathogenesis of asthma, of the extracts prepared by Preparation Examples 1 and 2, the cells were carried out by the following method, and the results are shown in Table 4 below. As shown.

Experimental Method

Human peripheral blood mononulear leukocytes (PBML) were stabilized at 37 ° C. on Hank's balanced salt solution (HBSS), and then reacted for 15 minutes with the extract of jinchuan. Arachidonic acid (arachidonic acid) was added thereto as a substrate to produce rucotriene B4 (LTB4) for 15 minutes. The amount of LTB4 produced was measured using an Enzyme immunoassay (EIS) kit.                     

division density 5-lipoxygenase inhibition rate (%) Preparation Example 1 0.03 mg / ml 75 0.1 mg / ml 82 Production Example 2 0.03 mg / ml 77 0.1 mg / ml 85 * Positive control NDGA (nordihydroguaiatinic acid) IC ₅₀ = 87.4 nM

As shown in Table 4, the extract prepared according to the present invention can be seen that the degree of 5-lipoxygenase inhibitory action is large.

Example 5: Phosphodiesterase 4 (PDE4) Inhibitory Activity

In order to evaluate the inhibitory activity against phosphodiesterase 4, which plays an important role in the development of asthma of the extracts prepared by Preparation Examples 1-2, cells were used in the following method, and the results are shown in the following table. As shown in 5.

Experimental Method

Human U937 cells were stabilized at 25 ° C. on a mixed solution of 50 mM Tris-HCl and 5 mM MgCl ₂ (pH 7.5). Adenosine extract and 1.01 μM ([ ³ H] cAMP + cAMP) mixed solution were added together and reacted for 20 minutes to determine the amount of adenosine produced by measuring the amount of [ ³ H] adenosine.

division density % Phosphodiesterase 4 inhibition Preparation Example 1 0.03 mg / ml 65 0.1 mg / ml 74 Production Example 2 0.03 mg / ml 70 0.1 mg / ml 81 Positive control IBMX (3-isobutyl-1-methylxanthine) IC ₅₀ = 20.7 μM

As shown in Table 5, the extract prepared according to the present invention can be seen that the degree of phosphodiesterase 4 inhibitory action is large.

Example 6: Lukotriene D4 Receptor (Leukotriene D4, LTD4) Antagonism

In order to evaluate the inhibitory activity against LTD4 receptor, which is the main pathogenesis mechanism of asthma, of the extracts prepared by Preparation Examples 1 and 2, cells were used in the following method, and the results are shown in Table 6 below.

Experimental Method

LTD4 receptors isolated from lung tissue of Duncan Hartely series guinea pigs were 25 ° C. in 50 mM Tris-HCl buffer (5 mM CaCl ₂ , 5 mM MgCl ₂ , 100 μg / ml bacitracin, 1 mM benzamidine, 0.1 mM phenylmethylsulfonyl fluoride). Stabilized. The mixture was added with 0.2 nM [ ³ H] LTD4 and reacted. Binding rate was analyzed by radioligand binding analysis, and nonspecific binding was determined using 0.1 μM LTD4. Specific binding rate 85%, Kd 0.2 nM, Bmax 0.24 pmol / mg protein.

division density LTD4 receptor inhibition rate (%) Preparation Example 1 0.03 mg / ml 41 0.1 mg / ml 58 Production Example 2 0.03 mg / ml 49 0.1 mg / ml 63 Positive control LTD4 IC ₅₀ = 1.12 nM

As shown in Table 6, the extract prepared according to the present invention can be seen that the degree of LTD4 receptor antagonism is large.

Example 7: Oral Administration of Rats Acute Toxicity

Acute toxicity test was performed using 6-week-old SPF SD rats as follows.

Two animals per group were dissolved orally administered with the extracts prepared in Preparation Examples 1 and 2 in a single oral dose at a dose of 2 g / kg / ml. After administration of the test substance, mortality, clinical symptoms, and changes in body weight were observed. Hematological and hematological examinations were performed. Necropsy was performed to observe abdominal and thoracic organ abnormalities. As a result, no significant clinical symptoms or dead animals were noted in all animals treated with the test substance, and no toxic changes were observed in weight changes, blood tests, blood biochemical tests, and autopsy findings. Therefore, the extract of the extract according to the present invention does not show a toxic change up to 2,000 mg / kg in all rats, and the minimum lethal dose (LD ₅₀ ) of oral administration was determined to be a safe substance of 2,000 mg / kg or more.

Formulation Example 1 Preparation of Tablet

Tablets for oral administration were prepared using the wet granulation method and the dry granulation method with the following composition by using the extract of Orthodontic ginseng.

[Furtherance]                     

200 mg of orthogyo extract, 10 mg of hard silicic anhydride, 2 mg of magnesium stearate, 50 mg of microcrystalline cellulose, 25 mg of sodium starch glycolate, 113 mg of corn starch, proper amount of ethanol anhydride.

Formulation Example 2: Preparation of Ointment

Using oleaginous extract of the present invention, an ointment was prepared in the following composition.

[Furtherance]

5 g of jinkyo extract, 20 g of cetyl palmitate, 40 g of cetanol, 40 g of stearyl alcohol, 80 g of myristan isopropyl, 20 g of monostearic acid sorbitan, 60 g of polysorbate, 1 g of paraoxybenzoic acid profile, para 1 g of methyl oxyanate, phosphoric acid and purified water

Formulation Example 3 Preparation of Injection

Injection was prepared in the following composition using the extract of the jinchuan of the present invention.

[Furtherance]

100 mg of jinkyo extract, mannitol 180 mg, sodium dihydrogen phosphate 25 mg, water for injection 2974 mg

Formulation Example 4 Preparation of Transdermal

Transdermal preparations were prepared by the following method using the extract of Orthodontic extract of the present invention.

[Composition 1]

0.4 g of jinchuan extract, 1.3 g sodium polyacrylate, 3.6 g glycerin, 0.04 g aluminum hydroxide, 0.2 g methyl paraben, 14 g water.

[Composition 2]

0.8 g of cotyledon extract, propylene glycol 1.6 g, 0.8 g liquid paraffin, 0.4 g isopropyl myristate, 16.4 g gel bar 1430.

As described above, the extracts according to the present invention have a good effect in various respiratory disease models, unlike the existing synthetic medicines are effective only in some limited respiratory disease models. In other words, respiratory diseases such as asthma, acute bronchitis and allergy due to excellent airway contraction inhibitory activity, airway inflammation inhibitory activity, 5-lipoxygenase inhibitory activity, phosphodiesterase 4 inhibitory activity, leukotriene D4 antagonistic action It is very useful for the prevention and treatment of rhinitis, acute lower respiratory tract infections (bronchitis, bronchiolitis, etc.), acute upper respiratory tract infections (pharyngitis, tonsillitis, laryngitis).

Claims (4)

Among asthma, acute bronchitis, allergic rhinitis, acute lower respiratory tract infection, and acute upper respiratory tract infection, which contain the extract of Gentianae Radix as an active ingredient, wherein the active ingredient does not contain the extracts of ginkgo biloba and wild rump. Agents for the prevention and treatment of selected respiratory diseases. The pharmaceutical preparation according to claim 1, wherein the powdered extract is extracted with water or an aqueous alcohol solution and then purified with a saturated lower alcohol or a non-polar solvent. 3. The drug according to claim 2, wherein the lower alcohol is an alcohol having 1 to 6 carbon atoms and the nonpolar solvent is ethyl acetate, dichloromethane, chloroform, carbon tetrachloride or methyl ethyl ketone. delete