KR20130096088A - Pharmaceutical composition comprising angelica dahurica bentham et hook extract for preventing or treating infectious disease of enterohamorrhagic escherichia coli - Google Patents

Abstract

PURPOSE: A pharmaceutical composition for preventing or treating enterohemorrhagic E. colibacillosis is provided to show excellent antibacterial activity against enterohemorrhagic E.coli O157:H7 using an Angelica dahurica Bentham et Hooker extract. CONSTITUTION: A pharmaceutical composition for preventing or treating enterohemorrhagic E. colibacillosis contains an Angelica dahurica Bentham et Hooker extract as an active ingredient. The extract is prepared from Angelica dahurica Bentham et Hooker using water, C1-C4 alcohol or a mixture thereof. The C1-C4 alcohol is methanol or ethanol. The extract is an ethyl acetate fraction of Angelica dahurica Bentham et Hooker. [Reference numerals] (AA) Ethyl acetate fraction of Angelica dahurica Bentham et Hook; (BB) Amphicilin

Description

Pharmaceutical composition comprising Angelica dahurica Bentham et Hook extract for preventing or treating infectious disease of Enterohamorrhagic Escherichia coli}

The present invention relates to a pharmaceutical composition for the prevention or treatment of intestinal hemorrhagic E. coli infections containing white paper extract as an active ingredient.

Escherichia coli is characterized by Enterohamorrhagic Escherichia according to virulence traits, routine epidemiology, clinical traits and pathophysiological mechanisms. coli ; EHEC), Enterotoxigenic Escherichia coli ; ETEC), Enteropathogenic Escherichia coli ; EPEC) and enteroinvasive Escherichia coli (EIEC).

Among them, E. coli was found when E. coli O157: H7 was discovered in 1982 after a hamburger food poisoning case in the United States. Intestinal hemorrhagic Escherichia coli is a causative agent of hemorrhagic colitis (HC), and it causes damage to the intestinal epithelial cells by producing verotoxin (shiga-like toxin) after mounting in the intestine. Verotoxin must have a receptor on the cell surface to enter the cell, but cows, pigs, deer, etc. do not have a receptor does not cause infection by intestinal hemorrhagic E. coli. In contrast, humans have receptors that cause intestinal hemorrhagic E. coli infections.

E. coli O157: H7 is an E. coli that can cause hemolytic uremic syndrom (HUS) in humans, especially in children and the elderly. Loss of kidney function may occur and infections of all ages have been reported. have. Intestinal hemorrhagic E. coli O157: H7 is known to cause hemorrhagic dysenteria and edema disease in weaners in young calves in livestock. In the case of calf, hemorrhagic diarrhea caused by toxins produced by these strains does not have any treatment, and when it recovers from the disease or in the case of the mother, it is asymptomatic while continuously discharging the bacteria to become the source of the disease. have. On the other hand, the fungus that causes edema of weaning pigs is a variant and is known to be independent of human disease.

As a conventional method of treating intestinal hemorrhagic E. coli infections, antibiotics, human immunoglobulin preparations, methods using bovine colostrum immunoglobulin antibodies, and the like have been used. Fosfomycin has been mainly used as an antibiotic-based method, but fosfomycin has been found to cause the release of berotoxin bound to bacterial cells, thereby killing enterohemorrgic E. coli cells. Since then, administration of fosfomycin to patients has been limited. In addition, human immunoglobulin preparations have been administered for the treatment of patients with enterohemorrhagic E. coli infections, but the effect is not clear, and it has been reported to neutralize vero toxin 1 but not vero toxin 2 to show side effects. Therefore, it can be seen that the human immunoglobulin preparations have little effect on the treatment of enterohemorrhagic E. coli infection. In addition, the method using bovine colostrum immunoglobulin antibody is expensive to obtain a large amount of serum antibodies and there is a technical problem due to the separation and storage. In the case of serum antibodies, it is not practical to have a high technology and cost according to the separation and storage of serum and immunoglobulin from blood, and colostrum is secreted only for a limited period of time, so it is impossible to produce a sufficient amount of colostrum antibody.

In general, in addition to the well-known E. coli O157: H7, a variety of serotypes, such as O26 and O111, have been isolated from patients with intestinal hemorrhagic E. coli infections. E. coli has been widely distributed in livestock such as cattle. If E. coli, which produces a specific serotype of verotoxin, is used as an antigen of a prophylactic vaccine, only a limited effect of preventing the serotype can be expected. Therefore, as a study on toxins in which all serotypes are commonly produced and which are a direct cause of pathogenesis, researches on the development of mutant strains using genetic recombination technology, that is, mutations of genes that produce toxins, to prepare attenuation strains have been conducted. However, commercialization is expected to take a lot of effort and seal, such as safety issues. In this regard, Korean Patent Publication No. 10-766775 discloses isolates of domestic hemorrhagic Escherichia coli (EHEC), which produce shigatoxins, the causative agent of intestinal hemorrhagic E. coli infection, or verotoxins, the same concept. The present invention relates to a toxoid vaccine for preventing hemorrhagic E. coli infection, which is used as a vaccine strain, and contains a detoxified Shiga toxin as an active ingredient by mass production and inactivation of Shigatoxin secreted by these vaccine strains.

As described above, currently, the most effective treatment for intestinal hemorrhagic E. coli infections is temporary, severe side effects, and safety problems. Therefore, there is an urgent need for the development of an intestinal hemorrhagic E. coli infectious agent that can minimize side effects, harm to the human body and can be safely taken.

On the other hand, Angelica dahurica Bentham et Hook is the root of the perennial plant of the umbelliferae, which is known to be sweet, very warm and nonpoisonous. In Chinese medicine, herbal medicines made by drying roots are called white paper, and are distributed in Korea, Japan, northeastern China, and eastern Siberia. Since ancient times, white paper is used to treat sweating, sedation, pain, colds, headaches, and toothache, and is known to have whitening, pore contraction, and anti-inflammatory effects. In particular, liver function protection, antibacterial, antioxidant, and antimutagenic functions have been reported.

As described above, various pharmacological activities of the white paper have been reported, but the prevention or treatment effect against intestinal hemorrhagic E. coli infection is not known yet, and there is no research on this.

The inventors of the present invention, while studying the preventive or therapeutic effect of the extract of E. coli hemorrhagic E. coli, confirmed that the white paper extract exhibits excellent antibacterial activity against E. coli O157: H7, and completed the present invention.

Accordingly, the present invention is to provide a pharmaceutical composition for the prevention or treatment of intestinal hemorrhagic E. coli infections containing white paper extract as an active ingredient.

The present invention provides a pharmaceutical composition for the prevention or treatment of intestinal hemorrhagic E. coli infections containing a white paper extract as an active ingredient.

Hereinafter, the present invention will be described in detail.

The white paper extract which is an active ingredient of the composition of the present invention can be obtained by a conventional extraction method, and a commercially available one can be purchased and used. Typical extraction methods include, but are not limited to, ultrasonic extraction, filtration, and reflux extraction.

In the present invention can be obtained by extracting the white paper extract in the following manner. First, white paper is washed with water, dried and then crushed. Water, C ₁ ~ C ₄ alcohol or a mixed solvent of water and C ₁ ~ C ₄ alcohol is added to the pulverized white paper and refluxed for 1 to 5 hours, preferably 2 hours. The extract is filtered and water, C ₁ to C ₄ alcohol or a mixed solvent of water and C ₁ to C ₄ alcohol is added to the residue, and the mixture is further refluxed for 2 hours. At this time, the volume of the solvent is 1 to 10 times, preferably 2 to 5 times the weight of the pulverized white paper. The alcohol of C ₁ ~ C ₄ may be selected from methanol or ethanol, preferably ethanol. Thereafter, the white paper extract was filtered, combined with the filtrate previously obtained, concentrated under reduced pressure, and lyophilized to obtain a white paper ethanol extract in powder form. Distilled water was added to the powdered white paper ethanol extract, and the organic solvent fractions were obtained by sequentially dividing with n-hexane, methylene chloride, ethyl acetate and butanol. The antibacterial activity against E. coli O157: H7 in the white paper ethyl acetate fraction of the organic solvent fraction is the best.

The white paper extract according to the present invention exhibits excellent antimicrobial activity against enterohemorrhagic E. coli O157: H7, and can be usefully used for the prevention or treatment of enterohemostatic E. coli infections.

The hemorrhagic E. coli infection is caused by E. coli O157: H7 infection, including but not limited to hemorrhagic colitis, hemorrhagic diarrhea, hemorrhagic anemia, acute renal failure, hemolytic uremic syndrome or thrombocytopenia.

The composition of the present invention may contain one or more known active ingredients having a prophylactic or therapeutic effect with enteric hemorrhagic E. coli infection together with the white paper extract.

The composition of the present invention may further comprise at least one pharmaceutically acceptable carrier in addition to the above-described effective ingredients for administration. Pharmaceutically acceptable carriers may be used in combination with saline, sterile water, Ringer's solution, buffered saline, dextrose solution, maltodextrin solution, glycerol, ethanol and one or more of these components, if necessary, as an antioxidant, buffer And other conventional additives such as bacteriostatic agents can be added. In addition, diluents, dispersants, surfactants, binders, and lubricants may be additionally added to formulate into injectable solutions, pills, capsules, granules or tablets such as aqueous solutions, suspensions, emulsions and the like. Further, it can be suitably formulated according to each disease or ingredient, using appropriate methods in the art or by the method disclosed in Remington's Pharmaceutical Science (recent edition), Mack Publishing Company, Easton PA.

The composition of the present invention can be administered orally or parenterally (eg, applied intravenously, subcutaneously, intraperitoneally or topically) according to the desired method, and the dosage is based on the weight, age, sex and health of the patient. The range varies depending on the diet, the time of administration, the method of administration, the rate of excretion and the severity of the disease. The daily dose of the white paper extract is about 100-3000 mg / kg, preferably administered once or several times a day.

The composition of the present invention may be used alone or in combination with methods using surgery, hormonal therapy, drug therapy and biological response modifiers for the prevention or treatment of enterohemorrhagic E. coli infections.

The white paper extract according to the present invention exhibits excellent antimicrobial activity against enterohemorrhagic E. coli O157: H7, and can be usefully used for the prevention or treatment of enterohemostatic E. coli infections.

1 is a diagram confirming the antimicrobial activity of the white paper extract of the present invention by the disk diffusion method.

Hereinafter, preferred embodiments of the present invention will be described in order to facilitate understanding of the present invention. However, the following examples are provided only for the purpose of easier understanding of the present invention, and the present invention is not limited by the examples.

Example  One  : Preparation of White Paper Extract

White roots were washed with water, dried and ground. 3,000 ml (5 times the content of white root) of 80% ethanol was added to 600 g of the ground white root, and refluxed for 2 hours. The extract was filtered and 1,500 ml of 80% ethanol was added to the residue, followed by further reflux extraction for 2 hours. The extract was filtered, combined with the filtrate previously obtained, concentrated under reduced pressure, and lyophilized to obtain a white paper ethanol extract in powder form. Distilled water was added to the obtained white paper ethanol extract in powder form, and the organic solvent fractions were sequentially fractionated with the same amount of n-hexane, methylene chloride, ethyl acetate and butanol (n-hexane fraction: 39.9 mg, methylene chloride fraction: 32.04 mg). , Ethyl acetate fraction: 47.54 mg, butanol fraction: 37.8 mg). Distilled water was added to each fraction to give a final concentration of 10 mg / ml, and filtered through a 0.45 µm filter to be used in the following experiment.

Experimental Example  One  : Determination of Antimicrobial Activity of White Paper Extract-Disc Diffusion Method

In order to confirm the antimicrobial activity of the white paper extract of the present invention, the following experiment was performed.

One. Intestinal hemorrhage  Escherichia coli O157 : H7 Cultivation of

E. coli O157: H7 EDL932 (E. coli 0157: H7; ATCC ^® 43894) was grown on LB (Luria-Bertani) agar plates at 37 ° C. for 17 hours. The isolated colonies were picked and inoculated in 100 ml of sterile LB medium [10 g of tryptone per liter, 5 g of yeast extract and 10 g of sodium chloride] and then incubated overnight at 37 ° C. for 17 hours with shaking at 250 rpm. Cultures were diluted 1: 100 using pre-warmed LB medium. Diluted cultures were incubated at 37 ° C. with shaking at 250 rpm until the final absorbance (OD ₆₀₀ ) reached 0.8. The culture was diluted to 1:10 using LB medium, and then cultured at 37 ° C with shaking at 250 rpm.

2. Determination of Antimicrobial Activity of White Paper Extract by Disk Diffusion Method

200 μl of E. coli 0157: H7 incubated in LB medium was plated on LB agar plates and a sterile 6 mm filter paper disc was placed firmly on the plate. The 10 mg / ml white paper ethanol extract and white paper extract fraction (n-hexane fraction, methylene chloride fraction, ethyl acetate fraction, butanol fraction) prepared in Example 1 were treated with a paper disk, followed by 17 hours at 37 ° C. Incubated. Then, the diameter of the clear zone around the paper disc was measured. 50 mg / ml of ampicillin was used as a positive control, and DMSO was used as a negative control.

The results are shown in Table 1 and Fig.

White paper extract Diameter of restraining zone (mm) White Paper Ethanol Extract 7.2 ± 0.1 White paper n-hexane fraction 7.2 ± 0.3 White paper methylene chloride fraction 7.4 ± 0.2 White Paper Ethyl Acetate Fraction 8.9 ± 0.2 White Butanol Fraction 7.3 ± 0.1 Positive control group (ampicillin) 9.9 ± 0.1 Voice Control Group (DMSO) 6.0 ± 0.0

As shown in Table 1 and Figure 1, it was confirmed that the white paper extract of the present invention showed excellent antimicrobial activity against enterohemorrhagic E. coli O157: H7, in particular, the antibacterial activity against enterohemostatic Escherichia coli O157: H7 in the ethyl acetate fraction of the white paper. It was confirmed that this appeared to be the best.

Examples of formulations for the composition of the present invention are illustrated below.

Formulation example  One  : Sanje  Produce

White Paper Extract 200mg

Lactose 100mg

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

Formulation example  2  : Preparation of Tablet

White Paper Extract 200mg

Corn Starch 100mg

Lactose 100mg

2 mg magnesium stearate

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

Formulation example  3  : Preparation of Capsule

White Paper Extract 200mg

Corn Starch 100mg

Lactose 100mg

2 mg magnesium stearate

After mixing the above components, the capsule was prepared by filling in gelatin capsules according to the conventional method for producing a capsule.

Formulation example  4  : Preparation of Injection

White Paper Extract 200mg

Mannitol 100mg

Na ₂ HPO ₄ 12H ₂ O 2mg

Sterile sterilized water for injection

Injectables were prepared by mixing the above ingredients per ampoule (2 ml) according to the usual method for preparing injectables.

Claims (7)

A pharmaceutical composition for preventing or treating intestinal hemorrhagic E. coli infections containing white paper extract as an active ingredient. The method of claim 1, wherein the white paper extract is obtained by extracting white paper with water, C ₁ ~ C ₄ alcohol or a mixed solvent of water and C ₁ ~ C ₄ alcohol prevention or treatment of intestinal hemorrhagic E. coli infection Pharmaceutical composition for. According to claim 2, wherein the C ₁ ~ C ₄ alcohol is methanol or ethanol, characterized in that the pharmaceutical composition for the prevention or treatment of hemorrhagic E. coli infection. The pharmaceutical composition of claim 1, wherein the white paper extract is white paper ethyl acetate fraction. [Claim 2] The pharmaceutical composition of claim 1, wherein the white paper extract has antibacterial activity against E. coli O157: H7. The pharmaceutical composition for preventing or treating intestinal hemorrhagic E. coli infection according to claim 1, wherein the intestinal hemorrhagic E. coli infection is caused by infection with enterohemorrhagic E. coli O157: H7. The method of claim 6, wherein the hemorrhagic E. coli infection caused by the infection with enterohemorrhagic E. coli O157: H7 is selected from the group consisting of hemorrhagic colitis, hemorrhagic diarrhea, hemorrhagic anemia, acute renal failure, hemolytic uremic syndrome, and thrombotic thrombocytopenia Pharmaceutical composition for the prevention or treatment of hemorrhagic E. coli infection, characterized in that the species or more.

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