KR101765988B1 - Anti-tuberculosis composition for treating or preventing tuberculosis comprising Melia azedarach L. extracts and fractions thereof - Google Patents

Abstract

In the present invention, it is traditionally used as a herbal medicine or medicinal plant in Oriental medicine and contains various physiologically active substances. Thus, the antituberculous effect on tuberculosis has not been reported in the past. In the present invention, it has been confirmed that the extracts or fractions thereof have antimicrobial activity or antibacterial activity against Mycobacterium tuberculosis, and it can be used as a composition for treatment, improvement or prevention of tuberculosis using the same.
The present invention relates to a pharmaceutical composition for treating, ameliorating or preventing tubercle bacillus containing an extract of Orthopteran or its fraction as an active ingredient, a health food composition for prevention or improvement, a feed composition for prevention or improvement, an animal pharmaceutical composition for treatment, improvement or prevention , ≪ / RTI > The pharmaceutical composition, health food composition, feed composition, animal medicine composition and quasi-drug composition according to the present invention have antimicrobial activity or antibacterial effect against Mycobacterium tuberculosis and thus can be used as an antimicrobial composition for treating, improving or preventing tuberculosis.

Description

(Anti-tuberculosis composition for treating or preventing tuberculosis containing extracts or fractions thereof)

The present invention relates to a pharmaceutical composition for treating, ameliorating or preventing tuberculosis comprising the extract of the present invention or a fraction thereof as an active ingredient, a health food composition for prevention or improvement, an animal composition for treatment, improvement or prevention, And a quasi-product composition for prevention or improvement. More specifically, Melia azedarach L. has been traditionally used as herbal medicine and oriental medicine for the treatment of diseases in oriental medicine for a long time, and is a medicinal plant containing various physiologically active substances. As the herb extract or its fraction, A pharmaceutical composition for the treatment, improvement or prevention of tuberculosis, a health food composition for prevention or improvement, a feed composition for prevention or remedy, a treatment, an improvement, a prophylactic and / or ameliorative effect of Mycobacterium tuberculosis (Mtb) Or prophylactic animal pharmaceutical compositions and quasi-drugs for prevention or improvement.

Centuries ago, infectious diseases such as measles, smallpox, cholera, pulmonary tuberculosis, and plague were high-risk infectious diseases that caused rapid deaths in all human beings, including children and adults. These infectious diseases have killed tens of millions of people over the centuries, and to date there are still some difficulties in controlling infectious diseases. Among the researches for the inhibition of infectious diseases, the discovery of penicillin has become a turning point in inhibiting or treating pathogenic microorganisms caused by antibiotics, inhibiting disease and improving health health and prolonging life span. However, direct or indirect damages caused by pathogenic microorganisms still cause various problems and diseases in economy, environment, medicine and health.

In particular, tuberculosis is an infectious disease that has taken the life of humanity the most since thousands of years ago. Tuberculosis became known to the world in 1882 when a German physician and bacteriologist Heinrich Hermann Robert Koch (1843-1910) discovered Mycobacterium tuberculosis (Mtb), a pathogen of tuberculosis, It is distributed throughout the world, including underdeveloped countries in Africa and South America, and is causing the risk of infection. Tuberculosis is a communicable disease caused by infection of Mycobacterium tuberculosis. It spreads through contact between humans and humans and is infected by cough and droplets of tuberculosis patients. According to a report by the World Health Organization (WHO), about one-third of the world's population is now infected with tuberculosis, and 1.5 million people die annually from tuberculosis. According to the Nature Journal in 2013, the rate of tuberculosis infection (Co-infection) in AIDS patients is gradually increasing in 1990.

Currently, antituberculous drugs such as Rifampicin, Isoniazid, Ethambutol, and Pyrazinamide have been used in clinical trials. However, most of them have been developed before 1970, and serious drug side effects (Rifampicin: Isoniazid: hepatitis, vasculitis, seizures, peripheral neuropathy, crustal dysfunction, hyperglycemia, renal failure, dysuria, Ethambutol: optic neuritis, diabetic nephropathy, diabetic nephropathy, , Vision impairment, hallucinations, liver disorders, digestive disorders, pyrazinamides: hepatitis, jaundice, hepatic disorders, hyperuricemia, fever) and at the same time mutagenic drug resistance. In particular, multidrug-resistant tuberculosis (MDR-TB), which is resistant to rifampicin and isoniazid, can not be treated with the first-line antituberculous drug. Secondary anti-tuberculosis drugs and combination therapy for the treatment of MDR- Has been associated with serious side effects and causes various problems in the recovery of patients' health. Such multidrug - resistant tuberculosis is causing national health problems and public health problems. Recently, new tuberculosis patients have been increasing due to latent infections and chronic infections in the younger generations of the 20s and 30s and the elderly people of the 60s and older, whose diet has been impaired by immunity despite the development of medical diagnostic technology and treatment technology.

For this reason, the development and dissemination of new anti-tuberculosis drugs which are more effective and have less side effects are urgently required. Currently, various TB drugs are actively developed around multinational pharmaceutical companies around the world, but effective anti-tuberculosis drugs that can replace existing therapeutic drugs have not been developed yet. In Korea, the development of therapeutic drugs for tuberculosis has not been developed. As a basic study, a small number of researchers have attempted tuberculosis research.

Tuberculosis is also a serious livestock epidemic disease in the livestock industry due to serious infectious diseases not only in humans but also in various animals including cattle, goats, pigs and birds. In particular, bovine tuberculosis can develop into a respiratory or digestive tract of bovine tuberculosis, and infect people through tuberculosis infected cattle saliva or excreta. Currently, antituberculosis drugs such as Rifampicin, Isoniazid and Ethambutol, which are used in humans, are used to treat livestock infected with bovine tuberculosis. However, infected livestock is totally slaughtered to prevent transmission to humans. For this reason, anti-tuberculosis drug plays a very important role not only in human beings but also in animal husbandry.

Most of the currently used anti-tuberculosis agents are produced through chemical synthesis, and thus exhibit many limitations such as causing the above-mentioned resistance and side effects. Therefore, recently, researches for isolating and developing new anti-tuberculosis active substances from natural products including animals, plants, microorganisms and various herbal medicines and medicinal plants are being actively carried out all over the world, and development of environmentally friendly substances is an important task . However, research and development of new antimicrobial substances against these infectious microbes are not easy. In order to develop a new antimicrobial substance, it should be considered that the antimicrobial effect is wide, it has an effect on the existing resistant strains, has an effect on latent infection and chronic infection, and should be safe even without long term administration.

It is a medicinal plant used as a herbal medicine in Oriental medicine and contains various physiologically active substances. The bark or bark of Melia azedarach Linne var. Japonica Makino, a deciduous tree belonging to the Meliaceae family, is a bark of rubella (rubella), acne, Acne, leprosy, high fever, skin eczema, diuretic, itching, itching, gynecological diseases and vaginitis. It has also been reported that pharmacological actions are effective in insecticidal action, antipyretic action, tapeworm or concretion elimination (antiparasitic effect) and trichomonas vaginitis (antigenic effect).

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Currently, Artemisinin or Artesunate is used as a therapeutic drug for malaria, which is one of the World Infectious Diseases designated by the World Health Organization (WHO). Artemisinin or Artesunate is also a physiologically active substance extracted from a medicinal plant, It is widely used worldwide. In this regard, the present inventors have sought to identify new functionalities for diseases by verifying the new efficacy and efficacy of various medicinal plants including spirulina. Under these circumstances, the present inventors have made efforts to develop antimicrobial active substances derived from natural products and medicinal plants, and have found that ghoshinia or fractions thereof inhibit the proliferation and survival of Mycobacterium tuberculosis and inhibit the growth of Mycobacterium tuberculosis, And thus the present invention has been completed.

It is an object of the present invention to provide a pharmaceutical composition for the treatment, improvement or prevention of tuberculosis comprising the extract of the present invention or a fraction thereof as an active ingredient.

Another object of the present invention is to provide a health food composition for preventing or ameliorating tuberculosis comprising the extract of the present invention or a fraction thereof as an active ingredient.

It is still another object of the present invention to provide a feed composition for preventing or ameliorating tuberculosis comprising an extract of the present invention or a fraction thereof as an active ingredient.

Another object of the present invention is to provide an animal pharmaceutical composition for treating, ameliorating or preventing tuberculosis comprising the extract of the present invention or a fraction thereof as an active ingredient.

Still another object of the present invention is to provide a quasi-drug composition for preventing or ameliorating tuberculosis comprising the extract of the present invention or a fraction thereof as an active ingredient.

In order to achieve the above object, the present invention provides a pharmaceutical composition for treating, improving or preventing tuberculosis comprising the extract of the present invention or a fraction thereof as an active ingredient. The pharmaceutical composition may preferably be achieved by having an antibacterial activity or an antibacterial effect against Mycobacterium tuberculosis.

The term " Melia azedarach L. " as used in the present invention refers to the bark or flesh of Melia azedarach L. var. Japonica Makino and is used to treat rubella and skin eczema It has been reported that pharmacologically active diuretic and antipyretic action is effective in the removal of intestinal parasites such as cones, tapeworms or horses and Trichomonas vaginitis. It has a semi-tubular or tubular shape with a grayish brown color on the outside, a longitudinally torn pattern and a horizontal shrub, and a cork layer with a reddish brown color. The folded surface is yellowish white and fibrous, and the quality is hard but easy to break. It is also known as the hard-bodied skin, the skin, the skin of the root, or the gold ring.

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The term " Mycobacterium tuberculosis "as used in the present invention is classified as a human tubercle bacillus, a bovine type tubercle bacillus, a western type tubercle bacillus, and a bird type bacillus. It is an aerobic gram-positive bacterium in the form of a very fine rod-like bacterium with a width of about 4.0 μm and a width of about 0.3 to 0.5 μm. Mycobacterium tuberculosis is classified into four types A, I, B, and C, and its optimum growth condition is pH 6.8 ~ 7.0 and temperature 37 ℃.

The term " tuberculosis "as used herein refers to a disease that is directly infected by a fine needle or droplet from the respiratory tract from a patient with M. tuberculosis infection, and is by definition an infection caused by M. tuberculosis. Currently, tubercle bacillus is well-developed in lung tissue because it is transmitted through air. Thus, tuberculosis usually refers to pulmonary tuberculosis. However, tuberculosis can invade most tissues and organs such as pleura, lymph node, spine, brain, nerve, kidney, gastrointestinal tract, genitalia, bone and muscle as well as lung.

Tuberculosis is a major cause of tuberculosis-related organ complications such as pulmonary tuberculosis, hip tuberculosis, bone tuberculosis, pelvic tuberculosis, testicular tuberculosis, bladder tuberculosis, renal tuberculosis, tuberculosis, skin tuberculosis, tuberculosis, laryngeal tuberculosis, tuberculosis, Tuberculous tuberculosis, tuberculous peritonitis, tuberculous ovarian tuberculosis, and chronic tuberculosis. Mycobacterium tuberculosis grows slowly using the nutrients in the body of the host, and when it is caught in tuberculosis, symptoms such as lack of energy, easy fatigue, and decrease in body weight appear. In addition, symptoms vary according to the organ that is infected with Mycobacterium tuberculosis. In pulmonary tuberculosis, symptoms such as cough, sputum and chest pain occur, and urinary frequency, urination difficulty and hematuria occur in tuberculosis of urography. In osteoarthritic tuberculosis, If tuberculosis, the lymph nodes of the whole body, especially the neck or armpit, grow, causing pain and pressure. In addition, back pain in patients with spinal tuberculosis, central nervous system tuberculosis symptoms such as headache and vomiting appear. When a person's body is infected with Mycobacterium tuberculosis, the normal tissue of the body is destroyed at a very slow rate by the inflammatory reaction with immune cells, and a cheese-like pus is formed and granuloma (lumps) do.

The term " antibacterial "as used in the present invention means an effect against bacteria, more specifically, an effect of inhibiting the growth of bacteria and fungi by a drug, a physiologically active substance or a known chemical substance. For the purpose of the present invention, Inhibiting growth and inhibiting growth.

The tuberculosis that can be treated, improved or prevented by the pharmaceutical composition of the present invention is not limited thereto, but it may be preferably pulmonary tuberculosis, more preferably infectious tuberculosis caused by infection with Mycobacterium tuberculosis.

In the present invention, the extracts or fractions thereof are dissolved in 50% ethanol, and the solution is prepared by using the culture medium. Then, the extracts and fractions of the extracts and fractions of Mycobacterium tuberculosis using the Resazurin microtiter assay (REMA) The activity or antibacterial effect was measured. In one embodiment of the present invention, it has been confirmed that the extracts or fractions thereof have an antibacterial activity against Mycobacterium tuberculosis (Figs. 1 to 2).

In the present invention, the extracts or their fractions were dissolved in 50% ethanol, respectively, and the solutions were prepared by using the medium. The extracts and fractions of the extracts and fractions against the Mycobacterium tuberculosis were measured by MGIT SYSTEM. In one embodiment of the present invention, it has been confirmed that the extracts or fractions thereof of the present invention exhibit an antibacterial activity and an antibacterial effect against Mycobacterium tuberculosis (Figs. 3 to 4).

In the present invention, a solution of each extract in a 50% ethanol solution was prepared, and the solution was prepared by using the medium. The MGIT SYSTEM assay was used to determine the antituberculous synergistic effect of the extract against mycobacterium tuberculosis ) Were measured. In one embodiment of the present invention, the synergistic effect of anti-Mycobacterium tuberculosis on the extract of Mycobacterium tuberculosis was examined, and it was confirmed that the synergistic effect of anti-Mycobacterium tuberculosis was strongly induced in the mixed administration group as compared with the single extract group (Fig. 5).

In the present invention, the therapeutic, ameliorative or preventive effect of the pharmaceutical composition on tuberculosis may be achieved by having antimicrobial activity or antibacterial effect which inhibits proliferation and growth of Mycobacterium tuberculosis. In one embodiment of the present invention, by confirming that the extract or its fraction has an antimicrobial activity or antimicrobial effect inhibiting the growth and proliferation of Mycobacterium tuberculosis through the Resazurin microtiter assay and the MGIT SYSTEM assay, (Fig. 1 to Fig. 5).

The extracts of the present invention or fractions thereof are not limited thereto, but can be preferably obtained by using an extraction method, a separation method, or a purification method derived from the use of spiral blood. The extraction method is not limited thereto. Preferably, organic solvent extraction, hot water extraction, hot water extraction, cold extraction, steam reflux extraction, reflux cooling extraction, ultrasonic extraction or freeze- It can be used properly. Further, fractions method of the extract is, or are not limited to, preferably, hexane, chloroform, acetonitrile, ethyl acetate, ether, dichloromethane, isopropanol, dichloromethane, methanol, water, distilled water, C ₁ ~ C ₆ alcohol, or their May be used.

In the present invention, the spinach extract is obtained through the step of pulverizing fine roots and the step of extracting. Wherein the pulverized material hexane (n-Hexane), chloroform (CHCl _3), ethyl acetate _{(ethyl acetate, CH 3 COOC 2} H 5), in acetonitrile (acetonitrile, CH ₃ CN), in dichloromethane (dichloromethane, CH ₂ Cl _2), ether (ethyl ether, C ₂ H ₅ OC ₂ H _5), ethyl ether (methylethyl ether, CH ₃ OCH ₂ CH _3), isopropanol (isopropanol, CH ₃ CH (OH) CH _3), water ( H ₂ O), C ₁ to C ₆ alcohols or a mixed solvent thereof. In addition, the above-mentioned extract may contain any one of the extract obtained by the extraction treatment, the diluted or concentrated liquid of the extract, the dried product obtained by drying the extract, the lyophilized product or the adjusted product or the purified product thereof.

The extraction method is not particularly limited and may be carried out by a conventional extraction method of a person skilled in the art. Preferably, extraction can be carried out at room temperature, cold temperature or warming under the condition that the active ingredient is not destroyed or minimized. More specifically, a method for obtaining an extract of Glycyrrhizae is as follows. (N-Hexane), chloroform (CHCl ₃ ), ethyl acetate, dichloromethane (CH ₂ Cl ₂ ), ether, acetonitrile and the like in a volume of about 2 to 100 times, preferably about 3 to 50 times, acetonitrile (CH ₃ CN), isopropanol _{(CH 3 CH (OH) CH} 3) or ethanol (EtOH), butanol (BuOH), methanol (MeOH) C ₁ ~ C ₆ alcohol, water (H ₂ O containing ) Or a mixed solvent thereof is used as a solvent and the extraction temperature is 20 to 100 DEG C, preferably 30 to 70 DEG C, the extraction period is about 3 to 10 days, preferably 3 to 72 hours, Extraction is carried out using extraction methods such as hot water extraction, hot water extraction, cold extraction, steam reflux extraction, reflux cooling extraction, ultrasonic extraction or freeze drying. In addition, the extract can be extracted from flowers, roots, stems, leaves, seeds, fruits or tissue cultures of plants.

According to a specific embodiment of the present invention, the extract is extracted with the solvent, and then the extract is filtered at room temperature using a vacuum pump and filter paper, and then concentrated by evaporation at 30 to 70 ° C under vacuum.

The fraction of the extract can be obtained by fractionating the above extract with a polar solvent or a non-polar solvent mixture to obtain a polar solvent fraction and a non-polar solvent fraction, respectively. The method for obtaining the fractions is not limited thereto, but can be obtained by fractionation by HPLC or column chromatography using a polar or non-polar solvent, and can also be obtained through fractionation through a solvent fraction. The solvent fractions can be fractionated into n-hexane fraction, chloroform fraction, ethyl acetate fraction, dichloromethane fraction, acetonitrile fraction, ethanol fraction, butanol fraction, methanol fraction, dichloromethanol fraction, isopropanol fraction and water fraction It is not. The solvent fraction may also be continuously treated with a polar or nonpolar solvent such as H ₂ O or distilled water, acetonitrile, dichloromethane, isopropanol, methanol, ethanol, propanol, butanol, ethyl acetate, chloroform, n- Or a mixture of two or more species as a mobile phase can be obtained by fractionation and separation through HPLC, column chromatography or phylogenetic fractions, and the active ingredient effective for the treatment, improvement or prevention of tuberculosis can be separated and used.

As used herein, the term "treatment" means any action that improves or alleviates symptoms of the disease upon ingestion or administration of the composition, and "improvement" means the onset or administration of the composition, Or mitigating the symptoms of tuberculosis by inhibiting or reducing the growth and proliferation of tuberculosis. As used herein, the term "prevention" means any action that inhibits or delays the onset of the disease by ingestion or administration of the composition.

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The pharmaceutical composition of the present invention may include, but is not limited to, a pharmaceutically acceptable carrier.

Examples of carriers, excipients and diluents that can be included in the pharmaceutical composition of the present invention include dextrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, calcium silicate, cellulose, methylcellulose, microcrystalline cellulose, poly Vinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, and mineral oil. Compositions comprising a pharmaceutically acceptable carrier can be of various oral or parenteral formulations. In the case of formulation, it can be prepared using diluents or excipients such as fillers, extenders, binders, humectants, disintegrants, surfactants and the like which are usually used. Solid formulations for oral administration may include tablet pills, powders, granules, capsules, and the like, which may contain one or more excipients, such as starch, calcium carbonate, sucrose, or lactose lactose, gelatin, and the like. In addition to simple excipients, lubricants such as magnesium stearate, talc, and the like may also be used. Liquid preparations for oral administration include suspensions, solutions, emulsions, syrups and the like. Various excipients such as wetting agents, sweeteners, fragrances, preservatives and the like may be included in addition to water and liquid paraffin, which are simple diluents commonly used. have. Formulations for parenteral administration may include sterile aqueous solutions, non-aqueous solutions, suspensions, amino acids, emulsions, lyophilized formulations, and suppositories. Propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like can be used as the non-aqueous solvent and suspension agent. Examples of the suppository base include witepsol, macrogol, tween 61, cacao paper, laurin, glycerogelatin and the like.

In addition, the pharmaceutical composition of the present invention may be in the form of tablets, pills, powders, granules, capsules, sustained release formulations, suspensions, suspensions, solutions, emulsions, injections, And may have any one form selected from the group consisting of a patch, a film, a coating agent, a patch, an inhalant, a syrup, an emulsion, a gel, an ointment, an eyedrop, a sterilized aqueous solution, a nonaqueous agent, And the form of the formulations which can be produced by the above examples is not limited.

The dosage form of the pharmaceutical composition of the present invention is not particularly limited and may be varied depending on the degree of absorption, body weight, age, sex, health condition, diet, administration time, administration method, excretion rate, have. The pharmaceutical composition of the present invention is prepared in consideration of an effective dose range, and the formulated unit dosage form can be prepared by using a specialized dosage regimen according to the judgment of the expert and the needs of the individual, Lt; / RTI >

In another aspect, the present invention provides a health food composition for preventing or ameliorating tuberculosis comprising an extract of Orthopteris extract or a fraction thereof as an active ingredient. The health food composition may preferably be obtained by having an antibacterial activity or antibacterial effect against Mycobacterium tuberculosis, as described above.

When the composition of the present invention is used as an additive for food, the composition may be added as it is or may be used together with other health food or ingredients thereof, and may be suitably used according to a conventional method. The mixing amount of the active ingredient can be suitably determined according to the purpose of use, and the amount of the food composition of the present invention is not particularly limited.

The food of the present invention is not particularly limited and examples of the health food to which the composition can be added include meat, sausage, bread, chocolate, candy, snack, confectionery, pizza, ramen, other noodles, gum, Dairy products, various soups, beverages, tea, juices, drinks, alcoholic beverages and vitamin complexes, and can include both normal and healthy functional foods and foods used as feed for animals can do.

In addition, when the health food composition of the present invention is used in the form of a drink, it may contain various sweeteners, flavors, or natural carbohydrates as an additional ingredient such as ordinary beverages. In addition to the above, the health food composition of the present invention may contain various nutrients, vitamins, electrolytes, flavors, colorants, pectic acid and salts thereof, alginic acid and salts thereof, organic acids, protective colloid thickening agents, pH adjusting agents, stabilizers, Alcohols, carbonating agents used in carbonated drinks, and the like. In addition, it may contain flesh for the production of natural fruit drinks, fruit juice drinks and vegetable drinks. The composition of the present invention can be prepared in the form of tea, juice, and drink, and then consumed for drinking, granulated, tableted, encapsulated, and powdered, but the form that can be manufactured by the above example is not limited.

In another aspect, the present invention provides a feed composition for preventing or ameliorating tuberculosis comprising an extract of Spinach or a fraction thereof as an active ingredient. The feed composition may preferably be obtained by having an antibacterial activity or an antibacterial effect against Mycobacterium tuberculosis, as described above.

When the composition of the present invention is used as a feed additive, the composition may be added as it is or may be mixed with other feed ingredients and used appropriately according to a conventional method. The amount of the active ingredient to be mixed may be appropriately determined according to the purpose of use, and the amount of the feed composition of the present invention is not particularly limited.

There is no particular limitation on the kind of the feed of the present invention, and the composition may be supplemented with various auxiliaries such as amino acids, inorganic salts, vitamins, antioxidants, microbial preparations and the like and pulverized or crushed wheat, vegetable protein feeds such as barley, corn, Can be used in combination with nutritional supplements, growth promoters, digestion-absorption promoters, and disease prevention agents in addition to the main components such as animal protein feeds, animal fat and vegetable fat. The dosage form, method and formulation of the feed can be varied, and the form that can be produced by the example is not limited.

The feed composition of the present invention can be applied to a large number of animal diets, i.e. feeds, including mammals, poultry, fish and crustaceans. Commercial livestock can be used for zoo animals such as mammals, elephants, camels, giraffes, monkeys, pets such as cows, pigs, horses, deer and goats; pets such as dogs and cats; , Pigeons, and commercially available fish and crustaceans such as carp, carp, trout, mullet, and shrimp.

In another aspect, the present invention relates to an animal pharmaceutical composition for treating, ameliorating or preventing tuberculosis comprising an extract of Orthopteris or a fraction thereof as an active ingredient. That is, the composition of the present invention can be used as an animal pharmaceutical composition for the purpose of treatment, improvement or prevention of tuberculosis. The composition may preferably be achieved by having an antibacterial activity or an antibacterial effect against Mycobacterium tuberculosis, as described above.

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When the composition of the present invention is used as an animal pharmaceutical composition, the composition may be used as it is or may be used together with other pharmaceutical or quasi-drug components, and may be suitably used according to a conventional method, but is not limited thereto. The amount of the active ingredient to be mixed can be suitably determined according to the intended use (prevention, health, improvement or therapeutic treatment).

In another aspect, the present invention relates to a quasi-drug composition for preventing or ameliorating tuberculosis comprising an extract of Spinach or a fraction thereof as an active ingredient. That is, the composition of the present invention can be added to a quasi-drug composition for preventing or improving tuberculosis. The composition may preferably be achieved by having an antibacterial activity or an antibacterial effect against Mycobacterium tuberculosis, as described above.

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When the composition of the present invention is used as a quasi-drug additive, the composition may be added as it is or may be used together with other quasi-drugs or quasi-drugs, and may be suitably used according to a conventional method, but is not limited thereto. The amount of the active ingredient to be mixed can be suitably determined according to the intended use (prevention, health, or improvement). There is no limitation on the kind of the quasi-drug composition of the present invention, but it is preferable to use a disinfectant such as disinfectant, shower foam, gagrin, wet tissue, detergent soap, spray, mask pack, hand wash, ointment, , Sanitary gloves, sanitary bags, filter fillers or masks, and the forms which can be produced by the above examples are not limited.

The pharmaceutical composition, health food composition, feed composition, animal medicine composition or quasi-drug composition according to the present invention have antimicrobial activity or antibacterial effect against Mycobacterium tuberculosis, so that they can be effectively used for treatment, improvement or prevention of tuberculosis. . ≪ / RTI >

FIG. 1 shows the result of measuring antimicrobial activity of each of the extracts against the Mycobacterium tuberculosis by applying the extract to the Resazurin microtiter assay. "+" Indicates the antibacterial activity or antibacterial effect of the test substance against Mycobacterium tuberculosis.
FIG. 2 shows the result of measuring the antimicrobial activity of each fraction of the fraction against the Mycobacterium tuberculosis by applying the respective fractions of the extract to the concentration by a concentration-dependent method (Resazurin microtiter assay). "+" Indicates the antibacterial activity or antibacterial effect of the test substance against Mycobacterium tuberculosis.
FIG. 3 shows the result of measuring the antimicrobial effect of each extract according to each concentration of Mycobacterium tuberculosis by applying the MGIT SYSTEM measurement method after preparing the extract of Goryeo crestaceae according to the concentration. In FIG. 3, NO.sub.1 is extracted with hexane extract, NO.sub.2 is extracted with chloroform, NO.sub.3 is extracted with ethyl acetate, NO.sub.4 is extracted with ethanol, NO.sub.5 is extracted with butanol, and NO.sub.6 is extracted with methanol The extract, NO 7, represents a water extract of spiny blooms.
FIG. 4 shows the result of measuring the antimicrobial effect of each fraction of the above-mentioned fractions against the Mycobacterium tuberculosis by applying the MGIT SYSTEM measurement method after each fractions of the extracts of Glycyrrhiza extract were prepared for each concentration. In Fig. 4, NO1 represents fractions of hexane, NO2, fractions of ethyl acetate, NO3, butanol and NO5, respectively.
FIG. 5 shows the result of measuring the antimicrobial synergistic effect of the extracts against mycobacterium tuberculosis by the mixed administration of the extracts to the Mycobacterium tuberculosis, . In FIG. 5, NO 1 represents the mixed administration group of 800 μg / ml of hexane extract and 400 μg / ml of high-purity ethyl acetate, NO 2 represents the mixed administration group of 800 μg / ml of the high-purity chloroform extract and 400 μg / , NO 3 was a mixed administration group of 800 ㎍ / ㎖ of water extract of Goryeong blood and 400 ㎍ / ㎖ of high-purity ethyl acetate, NO 4 was a mixed administration group of 300 ㎍ / ㎖ of high-purity ethanol and 400 ㎍ / Is a mixed administration group of 300 μg / ml of butanol and 400 μg / ml of high-purity ethyl acetate, and NO 6 is a mixed administration group of 300 μg / ml of high-purity methanol and 400 μg / ml of high-purity ethyl acetate.

Hereinafter, the present invention will be described in more detail with reference to examples. However, these examples are for illustrative purposes only, and the scope of the present invention is not limited to these examples.

: material

Middlebrook 7H9 Broth (Difco), Middlebrook OADC Enrichment supplement solution, MGIT 7ml broth Tube and MGIT Supplement (OADC) were purchased from Becton Dickinson (USA). Rifampicin, Isoniazid, Tween 80, Glycerol, DMSO, n-Hexane, chloroform (CHCl ₃ ), ethyl acetate (C ₄ H ₈ O ₂ ), ether, acetonitrile (CH ₃ CN), isopropanol _{(CH 3 CH (OH) CH} 3), dichloromethane (CH ₂ Cl _2), ethanol (EtOH), butanol (BuOH), methanol (MeOH) and Resazurin sodium salt powder is Deoksan Pharmaceutical Co., and Sigma-Aldrich Co. Ltd. (Sigma-Aldrich Co. LLC, St. Louis, Mo., USA).

: Preparation of Extracts and Fractions

Goryeon Pyo was purchased from Kyunghee University Oriental Medical University Oriental Hospital. (500 g) of each of the powdered powders was dissolved in a mixture of n-hexane, chloroform (CHCl ₃ ), ethyl acetate (C ₄ H ₈ O ₂ ), ethanol (EtOH), butanol (BuOH) And 5 L of distilled water (H ₂ O), followed by shaking at room temperature. After the extraction, the extract was filtered using a filter paper and a vacuum pump, and then evaporated and concentrated under vacuum at 30 to 70 ° C to obtain respective extracts. The extracts were stored frozen at -80 ° C after lyophilization and dissolved in the test.

The extracts were filtered using a filter paper and a vacuum pump, and then evaporated and concentrated under vacuum at 30 to 55 ° C to obtain respective extracts. To the extract (100 g), 3 L of distilled water and 3 L of hexane, chloroform, ethyl acetate or butanol were mixed and fractionated by the system. 3 L of hexane or chloroform was mixed with 3 L of ethanol or methanol, and each fraction was fractionated. Then, each fraction layer was filtered using a filter paper and a vacuum pump, and then evaporated and concentrated under a vacuum of 30 to 70 캜 to obtain respective fractions Respectively. The obtained fractions were stored frozen at -80 ° C after lyophilization and dissolved in the experiment.

: Culture of Mycobacterium tuberculosis

Middlebrook 7H9 medium containing 0.05% Tween 80 or 0.2% glycerol and 10% OADC Enrichment was used as a liquid medium for culturing Mycobacterium tuberculosis (H37Rv, ATCC 27294). The medium was incubated at 37 ° C. with shaking after inoculation with Mycobacterium tuberculosis. The culture of M. tuberculosis was continued until the absorbance reached 0.09 (McFarland turbidity standard 0.5) at 600 nm. After incubation, the cells were subcultured and stored at -80 ℃ for freezing.

: Measurement of antimicrobial activity by Resazurin microtiter assay (REMA)

In order to confirm the antibacterial activities of Mycobacterium tuberculosis against the extract of Mycobacterium tuberculosis or its fractions, each of the above test substances was dissolved in 50% ethanol and prepared by using the culture medium for each concentration. Rifampicin and Isoniazid were used as drug control drugs. They were dissolved in 50% DMSO (Dimethyl Sulfoxide) and H ₂ O and then used. After culturing, the Mycobacterium tuberculosis was diluted with Middlebrook 7H9 medium to a concentration of 2 × 10 ⁶ CFU / ml. Each of the test substances prepared above was diluted to 25 μg / ml, 50 μg / ml, 100 μg / / Ml and 400 μg / ml, respectively, and 200 μl of the solution was added to a 96-well plate. Rifampicin and Isoniazid were added to the culture medium of Mycobacterium tuberculosis at a concentration of 10 μg / ml, and the 96 well plate was incubated at 37 ° C for 5 days Lt; / RTI > Five days after the incubation, 30 ㎕ of coloring solution (0.02% Resazurin solution) was added to each well of a 96-well plate and cultured at 37 캜 for 36 hours. After 36 hours, the antimicrobial activity of each test substance was compared with the negative control, the positive control and the drug control. Bifidobacterium tuberculosis and Isoniazid were administered to patients with tuberculosis as a negative control group. Blank Control Group, which was not inoculated with Mycobacterium tuberculosis as a negative control group, Untreated Positive Growth Control Group as a positive control group, and Rifampicin and Isoniazid, Group (Drug-Treated Group) were cultured together to measure the antibacterial activity of test substances against Mycobacterium tuberculosis.

As a result, it was confirmed through the experiment of antimicrobial activity by the liquid culture method, that the goryeum extract or its fractions showed very high activity of Mycobacterium tuberculosis in Mycobacterium tuberculosis (Fig. 1 to Fig. 2). These results indicate that the ginseng extract or fractions thereof can be used as an antimicrobial composition against M. tuberculosis.

: Measurement of antimicrobial effect by MGIT SYSTEM measurement method

In order to confirm the antimicrobial effect of the extracts or their fractions against Mycobacterium tuberculosis, each of the test substances was dissolved in 50% ethanol, prepared by the concentration using the medium, and applied to the MGIT SYSTEM measurement method. The effect was measured. The procedure of the MGIT SYSTEM measurement method is as follows. First, 0.8 ml of MGIT Supplement solution (OADC, Becton Dickinson, USA), 0.1 ml of Mycobacterium tuberculosis solution and test substance solution were added to the MGIT 7 ml broth tube (Becton Dickinson, USA) and the final volume was measured using Middlebrook 7H9 medium After mixing, the tube is mounted on a BACTEC MGIT System (Becton Dickinson, USA) and cultured. Mycobacterium tuberculosis was cultured in Middlebrook 7H9 medium and added to the tube adjusted to 1,200,000 CFU / ml based on a total volume of 8 ml. (Untreated Positive Growth Control) as a positive control group and Drug-Treated Control group (Rifampicin and Isoniazid) administered at a concentration of 10 ㎍ / ㎖ to Mycobacterium tuberculosis as a drug control group. The antimicrobial effect was compared and measured for 3-4 weeks. The BACTEC MGIT SYSTEM is equipped with a fluorescence measurement sensor that automatically measures the proliferation and growth of Mycobacterium tuberculosis in the incubator, and the sensitivity of Mycobacterium tuberculosis or Mycobacterium tuberculosis is automatically detected and measured according to the proliferation and growth of Mycobacterium tuberculosis.

As a result, it was confirmed through the MGIT SYSTEM measurement method that the test substance extract or its fraction had an antimicrobial activity or antimicrobial activity which is highly concentrated in a concentration-dependent manner to the Mycobacterium tuberculosis (FIGS. 3 to 4). In addition, in order to confirm the synergistic effect of the extract against Mycobacterium tuberculosis extracts, the extracts of Glycyrrhiza epidermidis were prepared according to their concentrations and the results were mixed, and it was confirmed that the synergistic effect of anti-Mycobacterium tuberculosis was induced in the mixed treatment group compared to the single extract group 5). These results indicate that the ginseng extract or its fractions can be used as an antimicrobial composition against M. tuberculosis.

As described above, the measurement results of the extract and the fractions thereof according to the present invention are shown in FIGS. 1 to 5.

In Figs. 1 and 2, "+" indicates an antibacterial activity or an antibacterial effect of a test substance against Mycobacterium tuberculosis.

Claims (8)

A pharmaceutical composition for treating, ameliorating or preventing tuberculosis, which comprises an extract of Alaska pollack or a fraction thereof as an active ingredient.
The pharmaceutical composition according to claim 1, wherein the extract is extracted with a solvent selected from the group consisting of hexane, chloroform, ethyl acetate, water (H ₂ O), C ₁ to C ₆ alcohols or mixed solvents thereof.
The method according to claim 1, wherein the fraction is selected from the group consisting of hexane, chloroform, ethyl acetate, dichloromethane, ether, acetonitrile, isopropanol, water, C ₁ to C ₆ alcohols or mixtures thereof ≪ / RTI > by fractionation with two or more solvents.
The pharmaceutical composition according to claim 1, wherein the composition induces an antimicrobial activity against mycobacterium tuberculosis or an inhibitory effect against the growth of Mycobacterium tuberculosis.
A health food composition for preventing or ameliorating tuberculosis comprising an extract of Oriental peel or a fraction thereof as an active ingredient.
A feed composition for prevention or improvement of tuberculosis comprising extracts or fractions thereof as an active ingredient.
Animal pharmaceutical composition for treating, ameliorating or preventing tuberculosis comprising extracts or fractions thereof as an active ingredient.
A quasi-drug composition for preventing or ameliorating tuberculosis comprising extracts of Glycyrrhizae or fractions thereof as an active ingredient.

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