KR20070096688A - Antibacterial composition containing the extract of fraxinus rhynchophylla or the compounds isolated therefrom - Google Patents

Abstract

An antibacterial composition containing the extract of Fraxinus rhynchophylla or compounds isolated therefrom is provided to inhibit growth of zoonoses-causing pathogenic bacteria with reduced toxicity and tolerance. An antibacterial composition for inhibiting growth of zoonoses-causing pathogenic bacteria contains the extract of Fraxinus rhynchophylla which is prepared with water, C1-C4 lower alcohol or a mixed solvent thereof, or compounds isolated from the Fraxinus rhynchophylla extract including esculetin(6,7-dihydroxy coumarin), fraxidin(6,7-dimethoxy-8-hydroxy coumarin), fraxetin(7,8-dihydroxy-6-methoxy coumarin), oleuropein, fraxidin 8-O-beta-D-glucopyranoside and escuside, wherein the zoonoses-causing pathogenic bacteria include Salmonella typhimurium.

Description

Antibacterial composition containing the extract of Fraxinus rhynchophylla or the compounds isolated therefrom

The present invention relates to an antimicrobial composition comprising an ash extract having an antimicrobial activity or a compound isolated therefrom.

It is urgent to develop new antimicrobial drugs to cope with 15% market share of medicines and 1.5 trillion won antibiotic market. In addition, in Korea, economic and cultural international exchanges are active, foreign pathogenic bacteria are more likely to be introduced, and livestock imports are increasing, which will accelerate domestic spread of new bacteria. Currently, most of the bacteria present in domestic livestock are resistant to conventional antibiotics, and the treatment is becoming increasingly difficult. These therapeutic problems will be severely transmitted to the human body through livestock products infected with these bacteria. According to recent reports, more than 50% of human infectious diseases appear to be common infectious diseases, so safe livestock supply is a prerequisite for public health. Antibiotic resistance expression of bacteria is not present in some localized antibiotics, but is expressed in almost all drugs currently in use, and the physiological and genetic characterization of the bacteria is expected to have the ability to resist any drug. Among the various bacteria resistant to antibiotics, methicillin-resistant S. aureus ( MRSA), vancomycin-resistant enterococci (VRE), vancomycin-mediated S. aureus. aureus (vancomycin intermediate S. aureus; VISA) , and Escherichia coli (E. coli) with the antibiotic resistance of at least 6 kinds of O157: H7 including a. In Korea alone, MRSA is a nosocomial infection agent in hospitals, and it is almost 80% in large general hospitals. VRE and VISA have also been rampant since the 1990s and have spread throughout the country. Escherichia coli O157: H7 is also resistant to multiple antibiotics due to multiple agent resistance and there is no complete therapeutic antibiotic yet. E. coli has already been a frequent social problem in the United States, Europe, and nearby Japan, and has occurred sporadically in humans and livestock in Korea. And since the import of cattle has been liberalized since 2001, it is certain that E. coli will prevail in Korea when livestock products or live cattle are imported from the E. coli Sangje region. These resistant bacteria do not have special antibiotic selection system in Korea, and the factor of rapid increase in resistance is much larger than in other countries. Increasing antibiotic resistance is a vicious cycle of increasing use of new expensive antibiotics, increasing treatment duration, and mortality, which in turn increases economic and social problems. Therefore, in order to solve this problem, it is urgently necessary to understand the mechanism of resistance that is occurring and to develop a new antibiotic with a structure that does not cause resistance.

In order to cope with the rapidly increasing bacterial drug resistance and effectively treat various bacteria, new drugs different from the existing antibiotics (molecular structure, efficacy of drugs, etc.) are urgently needed. In particular, due to the emergence of super bacteria that are resistant to antibiotics, the development of new antibiotics is urgently required. In addition, the condition of the new drug should be a safe drug with no side effects on human and animal cells as well as bactericidal effect. No matter how good the antibiotic is, the resistance develops over time, and natural medicines are expected to solve this problem. The reason for this is that natural products are new components that have not been known so far, and since individual actions are mixtures of various single components, it is considered that the pharmacological action of the complexes will reduce side effects such as resistance.

Many antibiotics currently used for livestock are at severe levels of resistance and some are close to 100%. In livestock, antibiotics are mainly used as feed additives or for cleaning purposes regularly between 7 and 10 days for broilers. According to the report, the antibiotic resistance rate of food poisoning bacteria such as Salmonella and enterococci isolated from cattle, pigs, and chickens from all slaughterhouses in the country was tested. As such, currently used for livestock raising, most antibiotics are resistant to bacteria, so it is urgent to develop new antibiotics that can be effectively used for livestock raising.

Recently, the research trends of medicines are getting serious side effects, toxicity, and resistance, so researches on the development of medicines from natural products are actively being carried out worldwide. On the other hand, in the East, including Korea, it has a background that has promoted the steady development of traditional medicine, which is one of natural medicines, through long periods of time. Antibiotics have been discovered since 1941 when penicillin was clinically used, and more than 5,500 species have been discovered, of which only 100 are fermented and mostly chemically synthesized. In order to be effectively used for the treatment of diseases, the therapeutic dose should not be toxic to the host and the antibacterial effect to the infectious agent should be very large, but it has poisoning or unusual side effects. to be. Therefore, there is a need for research and development of new antibiotics having low toxicity and effective against resistant bacteria, and researches for reducing the appearance and toxicity of resistant bacteria by methods such as co-administration.

The most common incidence of bacterial diseases in Korea is Salmonellosis, which is especially mediated in food for humans. More than 2,300 serotypes have been reported, and their symptoms are similar. Salmonella (Salmonella) can cause a variety of clinical symptoms and there is a variety of toxins such as endotoxins, cell toxin, enterotoxin cells exist. In particular, the pathogen can be transmitted through the intestinal epithelial cells invade host cells, and almost all mammals are susceptible to a representative strains of the most common strains of pathogens that cause food poisoning in people causing zoonotic epidemic Salmonella typhimurium (Salmonella typhimurium ) . It is spread to humans mainly through contaminated meat and usually causes symptoms such as acute enteritis, high fever and sepsis.

Antibiotics have been continuously used everywhere for the control of bacteria, and antibiotic-resistant bacteria have recently emerged due to the misuse and abuse of such antibiotics and antimicrobial substances, which has become a big problem in society. As awareness of these problems spreads, societal atmospheres that prevent the use of antibiotics or antimicrobial agents are being created. In addition, research on substances that can replace antibiotics has been actively conducted, and plants and probiotics (which have been used as folk remedies for a long time) ( Probiotics have been actively used to obtain antimicrobial and immune enhancing effects.

Accordingly, the present inventors completed the present invention by confirming that the ash extract of the present invention and the compound separated therefrom have an excellent antibacterial effect while identifying a substance having an antimicrobial effect from natural drug resources.

It is an object of the present invention to provide an antimicrobial composition comprising ash tree extract having a low toxicity and resistance to living organisms and having antimicrobial activity against a common infectious disease strain and a compound isolated therefrom.

In order to achieve the above object, the present invention has a low toxicity and resistance to living organisms and shows antibacterial activity ( Fragxinus) rhynchophylla ) provides an antimicrobial composition against a common infectious disease strain comprising the extract as an active ingredient.

In addition, the present invention is esculletin (esculetin; 6,7-dihydroxy coumarin), fraxidin (fraxidin; 6,7-dimethoxy-8) isolated from ash tree ( Fraxinus rhynchophylla ) exhibiting low toxicity and antimicrobial activity in vivo -hydroxy coumarin), fraxetin (7,8-dihydroxy-6-methoxy coumarin), oleuropine, fruccidin 8- O -beta-di-glucopyranoside (fraxidin 8- O- β It provides an antimicrobial composition comprising a compound selected from -D-glucopyranoside, esculin (esculetin 6-O-β-D-glucopyranoside) and escuside or a pharmaceutically acceptable salt thereof.

The present invention ash tree ( Fragxinus) rhynchophylla ) Provides antimicrobial supplements for the prevention and treatment of common infectious diseases including extracts as active ingredients.

In addition, the present invention ash tree ( Fraxinus esculetin (6,7-dihydroxy coumarin), fraxidin (6,7-dimethoxy-8-hydroxy coumarin), fraxetin (7,8-dihydroxy-6-methoxy) isolated from rhynchophylla coumarin), oleic right pane (oleuropein), proxy Dean 8- O-beta-D-gluconic nose Llano side (fraxidin 8- O -β-D- glucopyranoside), Esculin (esculin; esculetin 6-O- β-D It provides an antimicrobial adjuvant for the prevention and treatment of common infectious diseases comprising a compound selected from -glucopyranoside and escuside as an active ingredient.

The ash extract and the compound separated therefrom are characterized in that it is extracted and separated from the dermis (秦 皮, Fraxini Cortex), the stem bark of the ash.

In addition, the extract is a crude extract, a polar solvent soluble extract or a non-polar solvent soluble extract, the crude extract is extracted with water, a lower alcohol having 1 to 4 carbon atoms or a mixed solvent thereof, preferably water or methanol Wherein the polar solvent soluble extract is a solvent selected from water, methanol, butanol or a mixed solvent thereof, preferably an extract soluble in butanol, and the nonpolar solvent soluble extract is a solvent selected from hexane, chloroform, dichloromethane and ethyl acetate , Preferably extracts soluble in hexane and dichloromethane.

Animals of the acquired common infectious diseases include mammals such as pigs, cattle and goats; Fish such as carp and goldfish; And poultry such as pheasants, chickens, ducks, turkeys and the like.

In addition, the acquired common infectious disease strain is Mycobacterium tuberculosis , Mycobacterium bovis , Bacillus anthracis ), Brucella genus abortus, B. melitensis, B. suis, B. canis), Leptospira disease isolates (Leptospira canicola, L. pomona, L. icterohaemorrhagiae, L. ballum), Visionary strain (Pseudomonas mallei), the analogy that the strain (Pseudomonas Pseudomallei ), Erysipelothrix rhusiopathiae (E. insidiosa), Shigella disenteriae, S. flexneri, S. boydii, S. sonnei, Hersinia pestis, Francicella tularensis, Ersinia pseudotuberculosis, Ersinia enterrocerisis enterocolitis, Listeria monocytogenes, Pasteurella multocida, Streptobacillus moniliformis, Spirilium minor (Spirillum minus) Borrelia recurrentis, Streptococcus pyogenes, Streptococcus agalactiae, Streptococcus equimyris, Streptococcus facal S. faecalis, Clostridium tetani, Clostridium perfringens, Clostridium novi, Cl. Novyi, Clostridium septicum, Clostri Cl. Histoliticum, Clostridium sporogenes, Fusobacterium necrophorum, Salmonella typhi, Salmonella paratyphi, Salmonella gallina Salmonella gallinarium, Salmonella typhimurium, Salmonella enteritidis, Champylobacter fetus (subsp.fetus, venerealis), Champilobacter jejuni (C. jejuni, C. coli, Actinomyces bovis, Actinomyces isralii, Nocardia asterioides, Nocardia N. madurae, N. farcinica, Rabies virus, Hantan virus, Seoul virus, Pumara virus, herpes Viruses from the genus Herpesvirus simiae (herpesvirus B), Filoviridae (Marburg virus), Paramyxovirus, Newcastle virus, and Orthopoxvirus. , Pseudocowpoxvirus in Parafaxvirus, Contagious pustular dermatitis virus in Parafaxvirus, Foot-and-mouth disease virus and mouth disease virus, Vesicular stomatitis virus, Lymphocytic choriomeningitis virus of the genus Arenavirus, Lassa fever genus of the Arenavirus virus, Influenza virus of Orthomyxoviridae, Japanese B encephalitis virus of the genus Flavivirus, Aspergillus fumigata , Cryptococcus neoformans, Candida albicans, Sportoricum scheckii, Coccidioides immitis, Blastomyces dematitidis dermatitidis, Histoplasma capsulatum, Haplosporangium parvum, Haflospo Clear Kress sense (H. crescens, Trichphyton schoenleini, Richettia typhi (R. mooseri), Ricketta prowazekii, Rickettsia tsutsugamusi (R. orientali), R. akari, Coxiella burnetii, Chlamydia psittaci, Entamoeba histolytica, Toxoplasna gondii, Barantidium colli (Balantidium coli), Fasciola hepatica, Fasciola gigantica, Clonorchis sinensis, Paragonimos westermanii, Dibotherio cephalas latum (Dibothriocephalus latum), Taenia saginatus, Taenia solium, Echinococcus glanulosus, Ascaris lumbricoides, Ascaria Suum (Ascaris suum), Vibrio cholera (Vibrio cholerae), yikolrayi (Escherichia coli) and S. aureus (methicillin-resistant S. aureus; MRSA ), preferably Vibrio cholerae O1 (Vibrio cholerae O1), Vibrio cholerae O139, Shigella decenteria dysenteriae ), Shigella flexeneri , E. coli O157, Salmonella typhi, Salmonella paratypy A ( Salmonella) paratyphi A), methicillin resistance S. aureus 104 (MRSA 104), methicillin resistance S. aureus 6 (MRSA 6), E. coli O157 ( E. coli O157), methicillin resistance S. aureus ( MRSA), Salmonella Galina Leeum (Salmonella gallinarium), Salmonella typhimurium (Salmonella includes typhimurium), Salmonella Entebbe utility disk (Salmonella enteritidis), E. coli K99 (E. coli K99) and the E. coli K88 (E. coli K88).

Hereinafter, a method of obtaining the extract and the compound of the present invention will be described in detail.

Ash tree extract of the present invention, the dried bark of the ash tree, preferably the ash bark of the stem bark of about 1 to 20 times the dry weight, preferably about 3 to 15 times the water, C ₁ to C ₄ lower Cold extraction with an alcohol or a mixed solvent thereof, preferably water or methanol, at an extraction temperature of 20 to 100 ° C., preferably 50 to 100 ° C. for about 1 to 10 days, preferably about 2 to 5 hours, Extraction method such as hot water extraction, ultrasonic extraction, reflux cooling extraction, preferably 1 to 10 times, preferably 2 to 7 times repeatedly extracted using a reflux cooling extraction method to obtain a crude extract by concentration under reduced pressure Stage 1; A second step of suspending the crude extract in distilled water and then extracting each with hexane, dichloromethane and butanol; Dichloromethane: methanol (16: 1 (v / v)) was separated into four fractions by elution with dichloromethane solvent extract obtained in the second step as an elution solvent; A fourth step of crystallizing a third fraction of the third step fraction to obtain esculletin; A fifth step of separating the first fraction from the fraction of the third step by silica gel column chromatography to obtain proxidine; The second fraction of the fraction of the third step is separated into four primary lower fractions by Sephadex column chromatography, and the second fraction is divided into five secondary subfractions by silica gel column chromatography. A sixth step of obtaining oleuropine by performing reverse phase medium pressure column chromatography; The third fraction of the second lower fraction of the sixth step was subjected to reverse phase medium pressure column chromatography using acetonitrile: distilled water (1: 9 (v / v)) as an eluting solvent and subjected to proxidine 8- O -beta-di-glu A seventh step of obtaining copyranoside; The fourth fraction of the four first subfractions of the sixth step was chromatographed with dichloromethane: methanol (5: 1 (v / v)) as the eluent to obtain ecucin and the five bottom fractions. step; The fifth fraction of the five subfractions obtained in the eighth step was subjected to reverse phase medium pressure column chromatography using methanol: distilled water (3: 7 (v / v)) as an eluting solvent to obtain escuside. Through the manufacturing process it is possible to obtain the ash extract of the present invention and the purely separated compound therefrom.

In addition, conventional fractionation processes can also be carried out (Harborne JB, Phytochemical methods: A guide). to modern techniques of plant analysis , 3 rd Ed . , pp. 6-7, 1998).

The present invention is effective for esculletin, proxidine, paroxetine, leuuropine, proxidine 8- O -beta-di-glucopyranoside, esculin or escuside isolated from the ash tree obtained by the above method. It provides an antimicrobial composition against a common infectious disease strain comprising as a component.

The ash extract of the present invention and the compound isolated therefrom are characterized by low toxicity and resistance and antibacterial activity against common infectious disease strains.

The compounds of the present invention can be prepared with pharmaceutically acceptable salts and solvates according to methods conventional in the art.

As the pharmaceutically acceptable salt, acid addition salts formed by free acid are useful. Acid addition salts are prepared by conventional methods, for example by dissolving a compound in an excess of aqueous acid solution and precipitating the salt using a water miscible organic solvent such as methanol, ethanol, acetone or acetonitrile. Equimolar amounts of the compound and acid or alcohol (eg, glycol monomethylether) in water can be heated and the mixture can then be evaporated to dryness or the precipitated salts can be suction filtered.

In this case, organic acids and inorganic acids may be used as the free acid, and hydrochloric acid, phosphoric acid, sulfuric acid, nitric acid, tartaric acid, and the like may be used as the inorganic acid, and methanesulfonic acid, p -toluenesulfonic acid, acetic acid, trifluoroacetic acid, and citric acid may be used as the organic acid. , Maleic acid, succinic acid, oxalic acid, benzoic acid, tartaric acid, fumaric acid, manderic acid, propionic acid, citric acid, lactic acid, glycolic acid, gluconic acid acid), galacturonic acid, glutamic acid, glutaric acid, glucuronic acid, glucuronic acid, aspartic acid, ascorbic acid, carbonic acid, vanillic acid, hydroiodic acid, and the like.

Bases can also be used to make pharmaceutically acceptable metal salts. An alkali metal or alkaline earth metal salt is obtained by, for example, dissolving a compound in an excess alkali metal hydroxide or alkaline earth metal hydroxide solution, filtering the insoluble compound salt, and then evaporating and drying the filtrate. At this time, as the metal salt, it is particularly suitable to prepare sodium, potassium or calcium salt, and the corresponding silver salt is obtained by reacting an alkali metal or alkaline earth metal salt with a suitable silver salt (for example, silver nitrate).

Pharmaceutically acceptable salts of the compounds of the invention include salts of acidic or basic groups which may be present in the compounds of the invention, unless otherwise indicated. For example, pharmaceutically acceptable salts include sodium, calcium and potassium salts of the hydroxy group, and other pharmaceutically acceptable salts of the amino group include hydrobromide, sulfate, hydrogen sulphate, phosphate, hydrogen phosphate, dihydrogen Phosphate, acetate, succinate, citrate, tartrate, lactate, mandelate, methanesulfonate (mesylate) and p -toluenesulfonate (tosylate) salts. It can be prepared through the process.

The antimicrobial composition of the present invention comprises 0.1 to 50% by weight of the extract or compound based on the total weight of the composition.

Antimicrobial compositions comprising the extract or compound of the present invention may further comprise suitable carriers, excipients and diluents commonly used in the manufacture of pharmaceutical compositions.

Pharmaceutical dosage forms of the extracts or compounds of the present invention may be used in the form of their pharmaceutically acceptable salts, and may be used alone or in combination with other pharmaceutically active compounds, as well as in a suitable collection.

Compositions comprising extracts or compounds according to the invention, in the form of powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols and the like, external preparations, suppositories and sterile injectable solutions, respectively, according to conventional methods Can be formulated and used. Carriers, excipients and diluents that may be included in the composition comprising the compound include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate , Cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. When formulated, diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrating agents, and surfactants are usually used. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, and the like, and such solid preparations include at least one excipient such as starch, calcium carbonate and sucrose in the compound. ) Or lactose, gelatin and the like are mixed. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Oral liquid preparations include suspensions, solvents, emulsions, and syrups, and may include various excipients, such as wetting agents, sweeteners, fragrances, and preservatives, in addition to commonly used simple diluents such as water and liquid paraffin. . Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, suppositories. As the non-aqueous solvent and suspending agent, propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate and the like can be used. As the base of the suppository, witepsol, macrogol, tween 61, cacao butter, laurin butter, glycerogelatin and the like can be used.

Preferred dosages of the extracts or compounds of the present invention vary depending on the condition and weight of the patient, the extent of the disease, the form of the drug, the route of administration and the duration, and may be appropriately selected by those skilled in the art. However, for the desired effect, the extract or compound of the present invention is preferably administered at 0.0001 to 100mg / kg, preferably 0.001 to 10mg / kg per day. Administration may be administered once a day or may be divided several times. The dosage does not limit the scope of the invention in any aspect.

Extracts or compounds of the present invention can be administered by a variety of routes to mammals such as mice, mice, livestock, humans. All modes of administration can be expected, for example, by oral, rectal or intravenous, intramuscular, subcutaneous, intrauterine dural or intracerebroventricular injection.

Below, The invention is illustrated in detail by the following examples and experimental examples.

However, the following Examples and Experimental Examples are only illustrative of the present invention, the contents of the present invention is not limited by the following Examples, Reference Examples and Experimental Examples.

Example  1. Preparation of Ash Extract

1-1. Ash Tree Crude extract  Produce

Dried ash bark stem bark purchased from the University of Korea Medicine in Iksan-si was pulverized to a particle size of 30 mesh or less using a grinder to obtain dermis powder, and then dried ash bark stem obtained above. A 100% methanol solution corresponding to three times the mass of the bark powder (1 kg) was added, reflux cooled extraction at 100 ° C. for 3 hours, filtered and freeze-dried to obtain 109 g of ash crude extract.

1-2. Ash polar solvent and Nonpolar Solvent  Preparation of Soluble Extracts

The ash wood crude extract (109 g) obtained in Example 1-1 was suspended in distilled water (1 L) and placed in a separatory funnel, followed by hexane (800 mL), dichloromethane (800 mL) and butanol (800). ㎖) was added sequentially, followed by shaking and concentrating each soluble part using a reduced pressure concentrator to hexane soluble extract (20 g), dichloromethane soluble extract (10 g), butanol soluble extract (44 g) and water. Soluble extract (30 g) was obtained.

Example  2. Isolation of Compounds from Ash Trees

2-1. Esculletin  detach

Ash tree dichloromethane soluble extract (10 g) obtained in Example 1-2 was subjected to chromatography using silica gel column and dichloromethane: methanol (16: 1 (v / v)) as an elution solvent. Chromatography of the same pattern was combined, concentrated and divided into 4 fractions (A to D). The third fraction, C (2.1 g), was crystallized with methanol to obtain Compound 1 (1.34 g). It was confirmed that it is esculetin (6,7-dihydroxy coumarin) having the following physical properties (Razdan et al., Phytochemistry, 26 , pp. 2063-2069, 1987).

Light yellow needles; (-)-ESI-MS m / z 177 [M ⁻ H] ⁻

¹ H-NMR (DMSO- d ₆ , 500 MHz): 7.86 (1H, d, J = 9.6 Hz, H-4), 6.97 (1H, s, H-5), 6.74 (1H, s, H-8 ), 6.16 (1H, doublet, J = 9.6 Hz, H-3).

2-2. Proxy  detach

A (2 g), the first fraction of Example 2-1, was separated by silica gel column chromatography using dichloromethane: methanol (20: 1 (v / v)) as an eluting solvent to obtain compound 2 (259 mg). It was obtained, and the results of the instrument analysis confirmed that it is a proxidan (fraxidin; 6,7-dimethoxy-8-hydroxy coumarin) having the following physical properties (Tsukamoto et al., Chem . Pharm. Bull . , 33 , pp . 4069-4073, 1985).

Colorless powder; (-)-ESI-MS m / z 221 [M ⁻ H] ⁻

¹ H-NMR (DMSO- d ₆ , 500 MHz): 7.93 (1H, d, J = 9.6 Hz, H-4), 6.83 (1H, s, H-5), 6.36 (1H, d, J = 9.6 Hz, H-3), 3.81 (3H, s, 6-OC H ₃ ), 3.77 (3H, s, 7-OC H ₃ )

¹³ C-NMR (DMSO- d 6, 125 MHz): 160.6 (C-2), 115.1 (C-3), 145.2 (C-4), 100.8 (C-5), 149.6 (C-6), 140.6 (C-7), 138.8 (C-8), 139.0 (C-9), 114.9 (C-10), 56.5 (6-O C H ₃ ), 60.4 (7-O C H ₃ ).

2-3. Paroxetine  detach

B (1.76 g), the second fraction of Example 2-1, was subjected to Sephadex LH-20 column chromatography (elution solvent; dichloromethane: methanol (1: 1 (v / v)). 3 (203 mg) was obtained, and the result of the analysis of the instrument confirmed that the paroxetine (fraxetin; 7,8-dihydroxy-6-methoxy coumarin) having the following physical properties (Tsukamoto et al., Chem. Pharm . Bull . , 33 , pp. 4069-4073, 1985).

Pale yellow prisms; (-)-ESI-MS m / z 207 [M ⁻ H] ⁻

¹ H-NMR (DMSO- d ₆ , 500 MHz): 7.88 (1H, d, J = 9.6 Hz, H-4), 6.78 (1H, s, H-5), 6.21 (1H, d, J = 9.6 Hz, H-3), 3.81 (3H, s, 6-OC H ₃ )

¹³ C-NMR (DMSO- d ₆ , 125 MHz): 161.1 (C-2), 112.4 (C-3), 145.6 (C-4), 100.9 (C-5), 145.9 (C-6), 139.8 (C-7), 133.4 (C-8), 139.9 (C-9), 110.8 (C-10), 56.6 (6-O C H ₃ ).

2-4. Oleuropein  detach

The ash-butanol soluble extract (44 g) obtained in Example 1-2 was subjected to chromatography on a silica gel column, and chromatographed using dichloromethane: methanol (5: 1 (v / v)) as an elution solvent. The same pattern in the graph was combined, concentrated, and divided into four fractions (E-H). The second fraction, F (2.2 g), was placed on a silica gel column and dichloromethane: methanol (8: 1 (v / v). ) Was chromatographed using the elution solvent and subdivided into five secondary fractions (F1 to F5). Reverse phase medium pressure column chromatography (Column; ODS-S-50B, 120 Å, 50 µm) using F4 (820 mg), the fourth fraction of the second fraction, as an eluent with methanol: distilled water (2: 3 (v / v)). ) Was obtained to obtain compound 4 (167 mg), and the results of the instrument analysis confirmed that it is oleuropein (oleuropein) having the following physical properties (Damtoft et al., Phytochemistry , 31 , pp. 4197-4201, 1992).

Colorless powder; (-)-ESI-MS m / z 539 [M ⁻ H] ⁻

¹ H-NMR (CD ₃ OD, 500 MHz): 7.50 (1H, s, H-3), 6.68 (1H, d, J = 7.8 Hz, H-7 ”), 6.65 (1H, d, J = 1.8 Hz, H-4 ”), 6.54 (1H, dd, J = 7.8, 1.8 Hz, H-8”), 6.07 (1H, br. Q, J = 7.2 Hz, H-8), 5.91 (1H, br s, H-1), 4.80 (1H, d, J = 6.9 Hz, glc-1), 4.20 (1H, dt, J = 11.0, 6.9 Hz, Ha-1 ”), 4.09 (1H, dt, J = 11.0, 6.9 Hz, Hb-1 ”), 3.96 (1H, dd, J = 9.2, 4.4 Hz, H-5), 3.70 (3H, s, COOC H ₃ ), 2.76 (2H, t, J = 6.9 Hz, H-2 ”), 2.70 (1H, dd, J = 14.2, 4.4 Hz, Ha-6), 2.43 (1H, dd, J = 14.2, 9.2 Hz, Hb-6), 1.6 (3H, dd, J = 7.2, 1.3 Hz, H-10)

¹³ C-NMR (CD ₃ OD, 125 MHz): 93.9 (C-1), 153.8 (C-3), 108.1 (C-4), 30.5 (C-5), 39.9 (C-6), 171.9 ( C-7), 123.5 (C-8), 129.2 (C-9), 12.2 (C-10), 167.4 (C-11), 99.6 (glc-1), 73.4 (glc-2), 77.1 (glc -3), 70.1 (glc-4), 76.6 (glc-5), 61.4 (glc-6), 34.1 (C-1 ”), 65.6 (C-2”), 129.4 (C-3 ”), 115.7 (C-4 ”), 145.5 (C-5”), 143.6 (C-6 ”), 115.1 (C-7”), 120.0 (C-8 ”), 50.6 (COO C H ₃ ).

2-5. Proxidine  8- O -Beta-di- Of glucopyranoside  detach

Reverse phase medium pressure column chromatography (column; ODS-S) using F3 (344 mg) as the third fraction of the secondary fraction of Example 2-4 using acetonitrile: distilled water (1: 9 (v / v)) as the elution solvent. -50B, 120 Å, 50 μm) was obtained to give compound 5 (62 mg), and the results of the instrumental analysis showed the proxidine 8- O -beta-di-glucopyranoside (fraxidin 8- O -β having the following physical properties -D-glucopyranoside) (Tsukamoto et al., Chem . Pharm . Bull . , 33 , pp. 4069-4073, 1985).

Colorless powder; (-)-ESI-MS m / z 383 [M ⁻ H] ⁻

¹ H-NMR (DMSO- d ₆ , 500 MHz): 7.96 (1H, d, J = 9.6 Hz, H-4), 7.12 (1H, s, H-5), 6.40 (1H, d, J = 9.6 Hz, H-3), 5.19 (1H, d, J = 7.4 Hz, glc-1), 3.84 (3H, s, 6-OC H ₃ ), 3.84 (3H, s, 7-OC H ₃ )

¹³ C-NMR (DMSO- d ₆ , 125 MHz): 160.4 (C-2), 115.3 (C-3), 145.0 (C-4), 105.4 (C-5), 150.3 (C-6), 145.4 (C-7), 137.1 (C-8), 142.6 (C-9), 114.9 (C-10), 56.6 (6-O C H ₃ ), 61.4 (7-O C H ₃ ), 102.8 (glc -1), 74.6 (glc-2), 78.1 (glc-3), 70.4 (glc-4), 77.1 (glc-5), 61.3 (glc-6).

2-6. Esculin  detach

The fourth fraction of Example 2-4, H (940 mg), was placed on a silica gel column, and chromatographed using dichloromethane: methanol (5: 1 (v / v)) as an eluting solvent. mg) and five subfractions (H1 ~ H5) were obtained, and the results of the instrument analysis revealed that Compound 6 is esculin (escuin; esculetin 6-O-β-D-glucopyranoside) having the following physical properties (Vol. et al. , Kor. J. Pharmacogn., 27 , pp.347-349, 1996).

Colorless powder; (-)-ESI-MS m / z 341 [M ⁻ H] ⁻

¹ H-NMR (CD ₃ OD, 500 MHz): 7.83 (1H, d, J = 9.1 Hz, H-4), 7.42 (1H, s, H-5), 6.80 (1H, s, H-8) , 6.21 (1H, d, J = 9.3 Hz, H-3), 4.84 (1H, d, J = 7.3 Hz, glc-1)

¹³ C-NMR (CD ₃ OD, 125 MHz): 162.4 (C-2), 111.5 (C-3), 144.7 (C-4), 115.3 (C-5), 143.1 (C-6), 152.0 ( C-7), 103.2 (C-8), 151.3 (C-9), 111.8 (C-10), 102.9 (glc-1), 73.5 (glc-2), 77.2 (glc-3), 70.0 (glc -4), 76.3 (glc-5), 61.2 (glc-6).

2-7. Escusid  detach

Reverse phase medium pressure column chromatography (Column; ODS-S-) using H5 (190 mg) as the fifth fraction of the lower fraction of Example 2-6 using methanol: distilled water (3: 7 (v / v)) as the eluting solvent. 50B, 120 microliters, 50 micrometers) was obtained, and compound 7 (86 mg) was obtained, and the instrument analysis confirmed that it was an escuside which has the following physical property (Iossifova et al., Fitoterapia , 73 , pp.386- 389, 2002).

Colorless powder; (-)-ESI-MS m / z 725 [M ⁻ H] ⁻

¹ H-NMR (DMSO- d ₆ , 500 MHz): 7.80 (1H, d, J = 9.6 Hz, H-4 ”), 7.53 (1H, s, H-3), 7.26 (1H, s, H- 5 ”), 6.76 (1H, s, H-8”), 6.13 (1H, d, J = 9.6 Hz, H-3 ”), 5.95 (1H, dd, J = 6.4, 0.9 Hz, H-8) , 5.84 (1H, s, H-1), 4.82 (1H, d, J = 7.5 Hz, H-1), 4.63 (1H, d, J = 7.8 Hz, H-1 '), 4.35 (1H, dd , J = 11.9, 2.1 Hz, Ha-6), 4.07 (1H, dd, J = 11.9, 6.4 Hz, Hb-6), 4.01 (1H, dd, J = 9.2, 4.1 Hz, H-5), 3.64 (1H, dd, J = 11.7, 1.2 Hz, Ha-6 '), 3.62 (3H, s, COOC H ₃ ), 3.55 (1H, dd, J = 9.1, 7.5 Hz, H-2), 3.44 (1H , dd, J = 11.7, 6.2 Hz, Hb-6 '), 2.69 (1H, dd, J = 14.6, 4.1 Hz, Ha-6), 2.39 (1H, dd, J = 14.6, 9.2 Hz, Hb-6 ), 1.64 (3H, dd, J = 6.4, 0.9 Hz, H-10)

¹³ C-NMR (DMSO- d ₆ , 125 MHz): 93.5 (C-1), 154.0 (C-3), 108.3 (C-4), 30.6 (C-5), 40.1 (C-6), 171.1 (C-7), 123.7 (C-8), 129.8 (C-9), 13.6 (C-10), 166.8 (C-11), 99.5 (C-1 '), 74.4 (C-2'), 77.9 (C-3 '), 70.4 (C-4'), 77.1 (C-5 '), 61.6 (C-6'), 161.1 (C-2 ”), 111.7 (C-3”), 144.9 ( C-4? ”), 115.2 (C-5”), 143.3 (C-6 ”), 154.8 (C-7”), 103.9 (C-8 ”), 151.4 (C-9”), 110.5 (C -10 ”), 102.5 (C-1), 73.7 (C-2), 76.3 (C-3), 70.3 (C-4), 73.8 (C-5), 64.2 (C-6), 51.8 (COO C H ₃ ).

Experimental Example  1. Antibacterial test

1-1. Disclosure strains and medium and strain culture

Strains used for the determination of antimicrobial activity are isolated outdoor strains and ATCC standard strains in humans or animals, and the strain list is shown in Table 1. Strains were inoculated in TSB (Tryptic Soy broth; Difco, USA) medium and incubated for 24 hours in a 37 ° C thermostat and used for experiments. It was.

List of strains for antimicrobial activity test Name origin Vibrio cholerae O1 JOL375 Vibrio cholerae O139 JOL376 Shigrlla dysenteriae JOL377 Shigella flexeneri JOL378 E. coli O157 ATCC 43890 Salmonella typhi JOL380 Salmonella paratyphi A JOL381 MRSA 104 Person isolate MRSA 6 Animal (chicken) isolate E. coli O157 Animal isolate MRSA ATCC 700698 Shigella dysenteriae ATCC 49557 Salmonella gallinarium Water-based standard strain E. coli O157 ATCC 35150 Salmonella typhimurium Water-based Standard Strains (Sal-13) Salmonella enteritidis Water-based standard strain (Sal-36) E. coli K99 Water-based Standard Strains (E-125) E. coli K88 Water-based Standard Strain (E-126)

1-2.  Check disk ( Bauer - Kirby test )

Bacterial culture was inoculated on the Muller-Hinton agar medium, and the antimicrobial test sample solution was injected onto the disk to produce an antibiotic concentration gradient on the medium. The inhibitory ring appears in size depending on the solubility of the antimicrobial substance and the degree of sensitivity of the bacteria, and the sensitivity of each antimicrobial substance is sensitive (S), intermeditely sensitive (I), and resistant ( resistant, R).

A. Determination of Antimicrobial Activity of Ash Extract

After the ash extract was treated with 1 mg / ml concentration, the antimicrobial activity was examined by the disk diffusion method, and as shown in Table 2, susceptibility to all bacteria except E. coli O157 (ATCC 43890) was shown.

Strain Diameter of clean area when processing ash extract from ash tree (mm) Vibrio cholerae O1 JOL375 21 Vibrio cholerae O139 JOL376 11 Shigrlla dysenteriae JOL377 10 Shigella flexeneri JOL378 25 E. coli O157 ATCC 43890 - Salmonella typhi JOL380 7 Salmonella paratyphi A JOL381 7 MRSA 104 Person Isolation 12 MRSA 6 animal (chicken) isolate 8 E. coli O157 animal isolate 8

B. Antimicrobial Activity of Soluble Extracts from Ash and Polar Solvents

Hexane, dichloromethane, butanol and water soluble extracts of the ash tree obtained in Example 1-2 were treated with 1 mg / ml concentration, respectively, and the antimicrobial activity was examined by the disk diffusion method, as shown in Table 3 below. Soluble extract and butanol soluble extract were susceptible to all strains, and dichloromethane soluble extract showed the strongest antibacterial activity.

Strain Ash diameter (mm) for clean and polar solvents Ash Hexane Soluble Extract Ash Dichloromethane Soluble Extract Ash Butanol Soluble Extract Ash Tree Soluble Extract Vibrio cholerae O1 JOL375 12 25 20 - Vibrio cholerae O139 JOL376 - 20 9 - Shigrlla dysenteriae JOL377 - 8 8 - Shigella flexeneri JOL378 - 16 10 - E. coli O157 ATCC 43890 - 8 8 - Salmonella typhi JOL380 - 8 8 - Salmonella paratyphi A JOL381 - 9 8 - MRSA 104 Person Separation 12 12 9 - MRSA 6 animal (chicken) isolate - 9 8 - E. coli O157 animal isolate - 8 8 -

1-3. Minimum inhibitory concentration ( Minimum inhibitory concentration , MIC ) Measure

Muller-Hinton agar medium was used to determine the minimum inhibitory concentration for 18 test strains of the 7 compounds (Compounds 1 to 7) obtained in Example 2. The bacteria enriched in TSB were re-sterilized using the same medium, and the absorbance was measured at 590 nm to calculate CFU (dog / ml), and then diluted to 1 × 10 ⁵ cells / ml using 0.85% saline.

The measured concentrate was dissolved in Muller-Hinton broth at 55-60 ° C., incubated with diluted bacteria, and incubated in a 37 ° C. thermostat for 24 hours. The cultured bacteria were diluted 100-fold with sterile distilled water, and then 100 μl diluted solution was added to Muller-Hinton agar medium and incubated at 37 ° C for 18 hours, followed by visual observation. Determined by the inhibitory concentration is shown in Table 4. As a result, the seven compounds obtained in Example 2 (Compounds 1-7) showed excellent antimicrobial activity with a minimum inhibitory concentration of 50 to 500 ㎍ / ㎖, respectively for all 18 species causing the common infectious disease.

Name origin Antimicrobial Activity (Minimum Growth Concentration: μg / ml) Compound 1 Compound 2 Compound 3 Compound 4 Compound 5 Compound 6 Compound 7 P / S ^{a )} K ^{b )} Vibrio cholerae O1 JOL375 NT ^{c )} NT NT 500 500 500 500 NA ^{d )} 10 Vibrio cholerae O139 JOL376 200 500 50 500 500 500 500 NA 10 Shigrlla dysenteriae JOL377 200 500 200 NT 500 500 500 NA 10 Shigella flexeneri JOL378 200 200 200 500 500 500 500 NA 10 E. coli O157 ATCC 43890 500 500 500 500 500 500 500 NA 10 Salmonella typhi JOL380 100 200 100 500 500 500 500 NA 10 Salmonella paratyphi A JOL381 200 500 200 500 500 500 500 NA 10 MRSA 104 Person isolate 500 500 200 500 500 500 500 NA 10 MRSA 6 Animal (chicken) isolate 500 500 500 500 500 500 500 NA 10 E. coli O157 Animal isolate 500 200 200 500 500 500 500 NA 10 MRSA ATCC 700698 500 500 500 500 500 500 500 One NA Shigella dysenteriae ATCC 49557 500 500 500 500 500 500 500 NA 10 Salmonella gallinarium Water-based standard strain 200 200 200 500 500 500 500 NA 10 E. coli O157 ATCC 35150 500 500 500 500 500 500 500 NA 10 Salmonella  typhimurium Water-based Standard Strains (Sal-13) 200 200 200 500 500 500 500 NA 10 Salmonella  enteritidis Water-based standard strain (Sal-36) 200 500 200 500 500 500 500 NA 10 E. coli K99 Water-based Standard Strains (E-125) 200 500 200 500 500 500 500 NA 10 E. coli K88 Water-based Standard Strain (E-126) 200 200 200 500 500 500 500 NA 10

^a) : positive control drug (penicillin / streptomycin equivalent mixture), ^b) : positive control drug (kanamycin), ^c) : no antimicrobial test, ^d) : no antimicrobial activity

Examples of the preparation of a pharmaceutical composition comprising the extract or compound of the present invention will be described, but the present invention is not intended to limit the present invention, but is only intended to be described in detail.

Formulation example  One. Powder  Produce

Ash Tree Extract 20 mg

Lactose 100 mg

Talc 10 mg

The above ingredients are mixed and filled in an airtight cloth to prepare a powder.

Formulation example  2. Preparation of Tablets

Esculletin 10 mg

Corn starch 100 mg

Lactose 100 mg

2 mg magnesium stearate

After mixing the above components, tablets are prepared by tableting according to a conventional method for preparing tablets.

Formulation example  3. Manufacture of capsule

Proxidine 10 mg

3 mg of crystalline cellulose

Lactose 14.8 mg

Magnesium Stearate 0.2 mg

According to a conventional capsule preparation method, the above ingredients are mixed and filled into gelatin capsules to prepare capsules.

Formulation example  4. Preparation of Injectables

Olerofane 10 mg

Mannitol 180 mg

Sterile distilled water for injection 2974 mg

Na ₂ HPO ₄ 12H ₂ O 26 mg

According to the conventional method for preparing an injection, the amount of the above ingredient is prepared per ampoule (2 ml).

Formulation example  5. Liquid  Produce

Proxidine 8- O -beta-di-glucopyranoside 20 mg

10 g of isomerized sugar

5 g of mannitol

Purified water

According to the conventional method of preparing a liquid solution, each component is added and dissolved in purified water, lemon flavor is added to the mixture, and then the above ingredients are mixed, purified water is added to adjust the total amount to 100 ml, and then filled in a brown bottle. The solution is prepared by sterilization.

As mentioned above, the ash tree extract of the present invention and the compound isolated from the ash tree can be used as an antimicrobial composition by showing an excellent antimicrobial effect against the common common infectious disease strains.

Claims (10)

Ash Tree ( Fracxinus) rhynchophylla ) Antimicrobial composition against a common infectious disease strain comprising an extract as an active ingredient. The antimicrobial composition of claim 1, wherein the extract is a crude extract, a polar solvent soluble extract, or a nonpolar solvent soluble extract. The antimicrobial composition of claim 2, wherein the crude extract is an extract available in a solvent selected from water, lower alcohols having 1 to 4 carbon atoms, or a mixed solvent thereof. The antimicrobial composition according to claim 2, wherein the polar solvent soluble extract is an extract soluble in a solvent selected from water, methanol, butanol or a mixed solvent thereof. The antimicrobial composition according to claim 2, wherein the nonpolar solvent soluble extract is an extract soluble in a solvent selected from hexane, chloroform and dichloromethane. Esculetin (6,7-dihydroxy coumarin), Fraxidin (6,7-dimethoxy-8-hydroxy coumarin), Fraxetin (7,8-dihydroxy-6-methoxy coumarin), Oleruro Payne (oleuropein), proxy Dean 8- O-beta-D-glucoside pyrazol furnace side (fraxidin 8- O -β-D- glucopyranoside), Esculin (esculin; esculetin 6-O- β-D-glucopyranoside) and eseukusi Antimicrobial composition against a common infectious disease strain comprising a compound selected from escuside as an active ingredient. The method of claim 6, wherein the compound is Fraxinus rhynchophylla ) antimicrobial composition characterized in that it is separated from. The antimicrobial composition of claim 1 or 6, wherein the extract or compound comprises 0.1 to 50% by weight of the compound, based on the total weight of the composition. The method according to claim 1 or 6, wherein the acquired common infectious disease strains are puppet bacteria (Mycobacterium tuberculosis), Bovine bacterium (Mycobacterium bovis), Bacillus Anthrasis (Bacillus anthracis), Brucella genus strain (Brucella abortus , B. melitensis, B. suis , B. canis), Leptospirosis strains (Leptospira canicola , L. pomona , L. icterohaemorrhagiae , L. ballum), Visitor strain (Pseudomonas mallei), Ubiquitous strain (Pseudomonas Pseudomallei), Erysipelotrix rhusiopathiae (E. insidiosa), Shigella disenteriae, S. flexneri, S. boydii, Siegel S. sonnei, Hersinia pestis, Francicella tularensis, Ersinia pseudotuberculosis, Ersinia enterocolitis ), Listeria monocytogenes, Pasteurella multocida, Streptobacillus moniliformis, Spirilium minor (Spirillum minus) Recurentis (Brrelia recurrentis), Streptococcus pyogenes, Streptococcus agalactiae, S. equimilis, Streptococcus faecalis ecalis, Clostridium tetani, Clostridium perfringens, Clostridium novi, Cl. novyi, Clostridium septicum, Clostridium histories Cl. Histoliticum, Clostridium sporogenes, Fusobacterium necrophorum, Salmonella typhi, Salmonella paratyphi, Salmonella gallinarium ), Salmonella typhimurium, Salmonella enteritidis, Champylobacter fetus (subsp.fetus, venerealis), Champilobacter jejuni (C. jejuni, C. coli, Actinomyces bovis, Actinomyces isralii, Nocardia asterioides, Nocardia N. madurae, N. farcinica, Rabies virus, Hantan virus, Seoul virus, Pumara virus, herpes Viruses from the genus Herpesvirus simiae (herpesvirus B), Filoviridae (Marburg virus), Paramyxovirus, Newcastle virus, and Orthopoxvirus. , Pseudocowpoxvirus in Parafaxvirus, Contagious pustular dermatitis virus in Parafaxvirus, Foot-and-mouth disease virus and mouth disease virus, Vesicular stomatitis virus, Lymphocytic choriomeningitis virus of the genus Arenavirus, Lassa fever genus of the Arenavirus virus, Influenza virus of Orthomyxoviridae, Japanese B encephalitis virus of the genus Flavivirus, Aspergillus fumigata , Cryptococcus neoformans, Candida albicans, Sportoricum scheckii, Coccidioides immitis, Blastomyces dematitidis dermatitidis, Histoplasma capsulatum, Haplosporangium parvum, Haflospo Clear Kress sense (H. crescens, Trichphyton schoenleini, Richettia typhi (R. mooseri), Ricketta prowazekii, Rickettsia tsutsugamusi (R. orientali), R. akari, Coxiella burnetii, Chlamydia psittaci, Entamoeba histolytica, Toxoplasna gondii, Barantidium colli (Balantidium coli), Fasciola hepatica, Fasciola gigantica, Clonorchis sinensis, Paragonimos westermanii, Dibotherio cephalas latum (Dibothriocephalus latum), Taenia saginatus, Taenia solium, Echinococcus glanulosus, Ascaris lumbricoides, Ascaria Suum (Ascaris suum), Vibrio cholera (Vibrio cholerae), yikolrayi (Escherichia coli) and S. aureus (methicillin-resistantS. aureus ; MRSA) antibacterial composition characterized in that the strain selected from. The extract of claim 1 or the esculetin (6,7-dihydroxy coumarin), frixidan (6,7-dimethoxy-8-hydroxy coumarin), fraxetin (fraxetin; 7,8-dihydroxy-) 6-methoxy coumarin), oleic right pane (oleuropein), proxy Dean 8- O-beta-D-gluconic nose Llano side (fraxidin 8- O -β-D- glucopyranoside), Esculin (esculin; esculetin 6-O- An antimicrobial adjuvant for the prevention and treatment of common infectious diseases comprising a compound selected from β-D-glucopyranoside and escuside as an active ingredient.