WO2008062852A1

WO2008062852A1 - Composition neutralizing enterohemorrhagic e. coli exotoxin

Info

Publication number: WO2008062852A1
Application number: PCT/JP2007/072596
Authority: WO
Inventors: Shunsuke Imai; Koji Miyoshi
Original assignee: Shunsuke Imai; Koji Miyoshi
Priority date: 2006-11-22
Filing date: 2007-11-21
Publication date: 2008-05-29
Also published as: JPWO2008062852A1

Abstract

It is intended to provide a composition for neutralizing the exotoxin of enterohemorrhagic Escherichia coli which comprises a less expensive and easily available source as the main component. This object has been achieved by finding out that a component in a snake extract has an activity of neutralizing the exotoxin of enterohemorrhagic Escherichia coli and thus providing a composition containing the snake extract. Accordingly, a pharmaceutical composition and a food or drink composition having an activity of neutralizing the exotoxin of enterohemorrhagic Escherichia coli are provided.

Description

Specification

Composition for neutralizing enterotoxin Escherichia coli exotoxin

Technical field

[0001] The present invention relates to a composition for neutralizing bacterial exotoxins of enterohemorrhagic Escherichia coli. More specifically, the present invention relates to a composition for neutralizing enterotorrhagic Escherichia coli exotoxin containing a snake extract.

Background art

[0002] Enterohemorrhagic Escherichia coli infection is an intestinal infection caused by ingestion of food contaminated with enterohemorrhagic E. coli (EHEC) that produces verotoxin such as Ol 57. It is the subject. In addition, secondary infection from person to person is also a problem. Symptoms range from asymptomatic to mild diarrhea, severe abdominal pain, frequent watery stools, and even severe bloody stools that can cause serious complications and death. In many cases, after 3 to 5 days of incubation, bloody stools develop after frequent watery stools with severe abdominal pain (hemorrhagic colitis). The fever is mild and most is in the 37 ° C range. In the early stage of bloody stool, blood contamination is small but gradually increases. In typical cases, the stool content is low, and the blood itself is low. Severe complications such as hemolytic uremic syndrome (HUS) or encephalopathy develop in 6-7% of affected individuals within 2 weeks of the first days of onset of symptoms such as diarrhea. The mortality rate for patients with HUS is 1-5%.

[0003] The causative agent of enterohemorrhagic E. coli infection is E. coli that produces verotoxin (referred to as Verotoxin = VT or Shiga toxin = Stx). Verotoxin is given this name because it acts lethal on mouth cells, a type of cultured cells. The number of bacteria that cause humans is thought to be only about 50, and secondary infection is likely to occur because a small number of bacteria can establish infection. In addition, this bacterium exhibits strong acid resistance and survives in stomach acid.

[0004] The main virulence factors known are Intimin, which forms attaching and effacing lesions as colonization factors, and verotoxin (there are VT-1 and VT-2 due to differences in antigenicity). In Japan, the serotype of enterohemorrhagic Escherichia coli detected by patients and carriers is the largest in Ol 57 force S, followed by 026 and Olll force S. Unlike other serotypes and general Escherichia coli, O157 on the separation medium is non-degradable sorbitol and negative for β-D-glucuronidase (MUG test).

[0005] Since diarrhea due to Ol 57 infection is a bacterial infection, it is essential to use appropriate antibacterial agents. In the nationwide survey conducted in the welfare science research project, the group using antibacterial agents It was reported that those who were administered early in the study had a lower incidence of HUS

[0006] On the other hand, from the results of an in vitro test that “antibacterial agents have increased the release of oral toxins from bacteria by destroying the bacteria,” Theoretic concerns have been pointed out, “Don't destroy the bacteria and worsen the symptoms!

[0007] Because of such concerns, neutralization of bacterial exotoxin is preferable as a countermeasure against enterohemorrhagic Escherichia coli infection rather than treatment with an antibacterial agent after infection. Drugs that are effective for neutralizing bacterial exotoxins not only have the advantage of providing safer treatment, but also have the potential to exert a prophylactic effect! Development is delayed!

[0008] Examples of drugs for neutralizing bacterial exotoxins include, for example, glycosphingolipid Gb3 (Non-patent Document 1). Since the means of neutralizing bacterial exotoxin can be a preventive means, it should be a technique that can be carried out simply and inexpensively. Gb3 is, for example, the strength S contained in breast milk and milk, and it is necessary to drink about 2.4 liters of milk to take 0.5 mg of Gb3, which is an effective neutralization amount. Absent.

[0009] A preventive measure against enterohemorrhagic Escherichia coli infection becomes realistic if it is possible to ingest a drug or food or drink that neutralizes bacterial exotoxins on a daily basis. Thus, a composition for neutralizing enterotorrhagic Escherichia coli exotoxins, which is based on an inexpensive and easily available source, is desired.

[0010] Herbal medicines containing various medicinal ingredients derived from natural products are potentially useful as a supply source of substances having desired medicinal effects. However, many herbal medicines have many that are not fully understood. Therefore, in order to find a crude drug containing an active ingredient having a medicinal effect suitable for a purpose from a wide variety of crude drugs, it is generally A degree of trial and error is required.

Non-Patent Document 1: Shinobu WATARAI, 7 others, "Inhibition of Vero Cell Cytotoxic Activity in Escherichia coli Ol 57: H7 Lysates by Globotriao sylceramide, Gb3, from Bovine Μίΐ, Bioscience, Biotechnology, and Biochemistry, Japan, Japan Agricultural Chemistry Association, February 2001, Vol. 65, No. 2, 414—4 pp. 19

Disclosure of the invention

Problems to be solved by the invention

[0011] It is an object of the present invention to provide a composition for neutralizing enterotorrhagic Escherichia coli exotoxin, comprising as a main component an inexpensive and easily available source.

Means for solving the problem

[0012] The above-mentioned problem has been found by providing a composition containing a snake extract by finding that the component in the snake extract has a neutralizing activity of enterotorrhagic Escherichia coli exotoxin. It was resolved.

[0013] Therefore, the present invention provides the following.

(Item 1) A pharmaceutical composition for neutralizing the toxicity of enterohemorrhagic Escherichia coli exotoxin comprising a snake extract.

(Item 2) The pharmaceutical composition according to item 1, wherein the snake extract is derived from poisonous snakes. (Item 3) The pharmaceutical composition according to item 1, wherein the snake extract is a viper extract. (Item 4) The pharmaceutical composition according to item 3, wherein the beetle extract is Hampi extract. (Item 5) The pharmaceutical composition according to item 1, wherein the snake extract is a cobra extract.

(Item 6) The pharmaceutical composition according to item 5, wherein the cobra extract is a cobra tincture. (Item 7) The pharmaceutical composition according to item 1, wherein the snake extract is a hub extract. (Item 8) The pharmaceutical composition according to item 7, wherein the hub extract is hub liquor.

(Item 9) The pharmaceutical composition according to item 1, wherein the enterohemorrhagic E. coli is Ol57. (Item 10) A composition for food and drink for neutralizing the toxicity of enterotorrhagic Escherichia coli exotoxin containing a snake extract. (Item 11) The composition for eating and drinking according to Item 10, wherein the snake extract is derived from poisonous snakes. (Item 12) The composition for eating and drinking according to Item 10, wherein the snake extract is a viper extract. (Item 13) The edible composition according to Item 12, wherein the beetle extract is a Hampi extract.

(Item 14) The edible composition according to item 10, wherein the snake extract is a cobra extract

(Item 15) The composition for food and drink according to item 14, wherein the cobra extract is a cobra tincture.

(Item 16) The composition for eating and drinking according to Item 10, wherein the snake extract is a hub extract. (Item 17) The composition for eating and drinking according to item 16, wherein the hub extract is a hub liquor. (Item 18) The composition for eating and drinking according to Item 10, wherein the enterohemorrhagic Escherichia coli is 0157.

[0014] Preferred embodiments of the present invention will be described below. However, those skilled in the art can appropriately implement the embodiments of the present invention and the well-known technical skills in the field. It should be recognized that the effect is easily understood.

The invention's effect

[0015] According to the present invention, an effect is provided that a composition for neutralizing the toxicity of enterotorrhagic Escherichia coli exotoxin is provided. Furthermore, the active ingredient of the composition provided by the present invention has an effect of being inexpensive and easily available.

Brief Description of Drawings

[0016] [Fig. 1] Fig. 1 is a graph showing the effect of the herbal concentration on the survival rate when VT-1 is used.

[Fig. 2] Fig. 2 is a graph showing the effect of the crude drug concentration on the survival rate when VT-2 is used.

[Fig. 3] Fig. 3 is a graph showing cell viability of verotoxin neutralizing antibody and verotoxin when VT-1 is used.

[FIG. 4] FIG. 4 is a graph showing cell viability of verotoxin neutralizing antibody and verotoxin when VT-2 is used. [FIG. 5] FIG. 5 is a graph showing the heat treatment of a crude drug stock solution and cell viability by verotoxin when VT-1 is used.

[Fig. 6] Fig. 6 is a diagram showing the heat treatment of a crude drug stock solution and the cell viability by verotoxin when VT-2 is used.

[FIG. 7] FIG. 7 is a diagram showing the acid treatment of a crude drug stock solution and the cell viability by verotoxin when VT-1 is used.

[Fig. 8] Fig. 8 is a graph showing acid treatment of a crude drug stock solution and cell survival rate by verotoxin when VT-2 is used.

[Fig. 9] Fig. 9 shows the cell viability when VT-1 was used and when the crude drug stock solution and verotoxin solution were added in advance and each was added to the cells simultaneously. FIG.

[Fig. 10] Fig. 10 shows the viability of cells when VT-2 is used, when a pre-mixed crude drug solution and verotoxin solution is added, and when each is added to cells simultaneously. It is a figure.

[Fig. 11] Fig. 11 shows the case where VT-1 was used and the crude drug was added to the Vero cells and allowed to stand for 24 hours, after which the cell culture medium was changed and Vero toxin was added at the same time. It is a figure which shows the comparison in the case of doing.

[Fig. 12] Fig. 12 shows the case of using VT-2, adding crude drugs to Vero cells, letting them stand for 24 hours, then changing the cell culture medium and adding Vero toxin at the same time It is a figure which shows the comparison in the case of doing.

FIG. 11 is a photograph showing that various herbal medicines and the like contain Gb3.

BEST MODE FOR CARRYING OUT THE INVENTION

[0017] Unless otherwise stated, the terms used in the present specification are used in the meaning normally used in the art. Thus, unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In case of conflict, the present specification, including definitions, will control.

[0018] (Definition of terms) Listed below are definitions of terms particularly used in the present specification.

[0019] As used herein, the term "snake extract" refers to all or part of a snake sub-animal (dried! /, Or! Yo! /,) The extract prepared using

[0020] As used herein, the term "enterohemorrhagic E. coli" refers to E. coli that produces verotoxin (also known as exotoxin or Shiga toxin! /). A typical enterohemorrhagic enterococcus is 0157 (formally called Escherichia coli 0157: H7)

However, the present invention is not limited to this.

[0021] As used herein, the term "neutralization" refers to an activity that attenuates toxicity due to bacterial exotoxin. For example, when neutralizing activity is evaluated using cultured cells, the neutralizing activity is measured using an increase in the proportion of surviving cells as an index.

[0022] As used herein, the term "flude extracts" refers to a crude drug leachate. Typically, a flow extract is a solution prepared to contain lg of herbal medicine in 1 ml of solvent.

[0023] As used herein, the term "Tincture" refers to a solution prepared by leaching a crude drug with a mixture of ethanol or ethanol and purified water.

[0024] As used in the present specification, the term "no, npi" is a kind of Chinese herbal medicine, which means that the viper is peeled off and the internal organs are removed and dried.

(Description of Preferred Embodiment)

(Preparation method of snake extract)

Snake extract can be prepared using a variety of well-known methods. The snake used for the extraction may be either a dried snake or a non-dried snake. The solvent used for manufacturing the snake extract used in the present invention is not particularly limited. For example, one type of organic solvent such as methanol, ethanol, hexane, benzene, acetone, ethyl acetate, chlorophorem, jetinoreethenole, 1,3-butyleneglycolole, glycerin, propylene glycol, and water Or two or more types can be mixed and used. A preferred solvent is an organic solvent such as ethanol, 1,3-butylene glycol, glycerin, or propylene glycol, or water. Conditions such as extraction time, extraction temperature, and extraction method are not particularly limited. Consider the solvent's persistence, economy, and work efficiency As a matter of course, the snake is left as it is or dried, or is pulverized as it is or using 1 to 10 times the wet weight of snake ethanol or 1,3-butylene glycol, glycerin, propylene glycol at room temperature. 1 ~ 10 days of extraction power, wet weight of snake;! ~ 10 times the amount of water 80 ~; 1 ~ at 100 ° C 1 ~; It is preferable to use it. The extract can be used in the present invention as it is or after being concentrated or diluted as necessary.

[0026] (Preparation method of flow extract)

A herbal medicine extract can typically be manufactured by extracting a dried snake product with 25% to 50% ethanol (eg, 30% ethanol). Herbal medicine as a snake extract is commercially available from, for example, Mikuni Co., Ltd. (Chuo-ku, Osaka City).

[0027] (Formulation of pharmaceutical composition)

The present invention also provides a method of treating and / or preventing a disease or disorder that can be treated and / or prevented by neutralizing the action of bacterial exotoxin by administering an effective amount of the therapeutic agent to the subject. To do. By therapeutic agent is meant a composition of the invention in combination with a pharmaceutically acceptable carrier type (eg, a sterile carrier).

[0028] Therapeutic agents should be considered in terms of medical practice criteria, taking into account the individual patient's clinical condition (particularly the side effects of treatment alone), delivery site, method of administration, dosing regimen and other factors known to those skilled in the art. Prescribe and administer in a manner that complies with good medical practice (GMP). Therefore, the target “effective amount” in the present specification is determined by taking such consideration into consideration.

Yes

[0029] As a general suggestion, the total pharmaceutically effective amount of orally administered therapeutic agent per dose is preferably at least 10 mg / kg / day, more preferably about 2 Omg, based on patient weight. A dose of Gb3 in the range of / kg / day to 45 mg / kg / day S, the active ingredient of the present invention does not show toxicity, so doses exceeding 45 mg / kg / day can also be administered It is possible and, as noted above, this is left to therapeutic discretion.

[0030] Therapeutic agents can be administered orally, rectally, parenterally, intracistemally, vaginally, intraperitoneally, topically (such as by powder, ointment, gel, infusion, or transdermal patch), intraoral Alternatively, it can be administered as an oral or nasal spray. Representative routes of administration of the pharmaceutical compositions of the invention Is oral administration.

[0031] "Pharmaceutically acceptable carrier" refers to a non-toxic solid, semi-solid or liquid filler, diluent, enveloping material or any type of formulation adjuvant.

[0032] The therapeutic agent of the present invention may also be appropriately administered by a sustained release system. Suitable examples of sustained-release therapeutic agents are oral, rectal, parenteral, intracistemally, vaginal, intraperitoneal, topical (powder, ointment, gel, infusion, or transdermal patch) Etc.), or by mouth or as an oral or nasal spray. “Pharmaceutically acceptable carrier” means a non-toxic solid, semi-solid or liquid filler, diluent, enveloping material or any type of treatment supplement.

An auxiliary agent. The term “parenteral” as used herein refers to modes of administration including intravenous and intramuscular, intraperitoneal, intrasternal, subcutaneous and intraarticular injections and infusions.

[0033] The therapeutic agent of the present invention is also appropriately administered by a sustained release system. Suitable examples of sustained release therapeutic agents include suitable polymer materials (eg, semipermeable polymer matrices in the form of molded articles (eg, films or microcapsules)), suitable hydrophobic materials (eg, acceptable quality oils). Or as an ion exchange resin, and poorly soluble derivatives (eg, poorly soluble salts).

[0034] The sustained-release matrix includes polylatatide (US Pat. No. 3,773,919, EP58,481), a copolymer of L-glutamic acid and γ-ethyl-L-glutamate (Sidman et al., Biopolymers 22: 547). 556 (1983)), poly (2-hydroxyethylenomethacrylate) (Langer et al., J. Biomed. Mater. Res. 15: 167—277 (1981), and: Langer, Chem. Tech. 12: 98-105 (1982)), ethylene vinylenoacetate (Langer et al., Ibid.) Or poly-D-(—) 1-hydroxybutyric acid (EP133, 988).

[0035] Sustained release therapeutic agents also include the therapeutic agents of the present invention encompassed by ribosomes (generally

Langer, Science 249: 1527—I 5d3 (1990); Treat et al., Liposomes in the Therapy of Infectious Disease and Cancer, Lopez—Berestein and v ι dler (ed.), Liss, New York, 317-327 and 353-365 (See 1989)). Ribosomes containing therapeutic agents can be prepared by methods known per se: DE3, 218, 121; Epstein et al., Proc. Natl. Acad. Sci. USA 82: 3688— 3692 ( 1985); Hwang et al., Proc. Natl. Acad. Sci. USA 77: 4030-4034 (1980); E P52, 322; EP36, 676; EP88, 046; EP143, 949; EP142, 641; 83-118008; U.S. Pat. Nos. 4,485,045 and 4,544,545 and EP 102,324. Usually, ribosomes are small (about 200-80θΑ) unilamella types, where the lipid content is adjusted at a selected rate greater than about 30 mol% cholesterol for optimal therapeutic agents.

[0036] In still further embodiments, the therapeutic agents of the invention can be delivered by a pump (Lager, supra; Sefton, CRC Crit. Ref. Biomed. Eng. 14: 201 (1987); Buchw aid et al., Surgery 88: 507 (1980); see Saudek et al., N. Engl. J. Med. 321: 574 (198 9)).

[0037] Other controlled release systems are discussed in a review by Langer (Science 249: 1527-1533 (1990)).

[0038] For parenteral administration, in one embodiment, in general, the therapeutic agent is administered to the recipient in the desired degree of purity, in a pharmaceutically acceptable carrier, ie, the dosage and concentration used. It is formulated by mixing in a unit dosage injectable form (solution, suspension or emulsion) with one that is non-toxic and compatible with the other ingredients of the formulation. For example, the formulation preferably contains oxidation, and other compounds known to be harmful to therapeutic agents!

[0039] In general, a formulation is prepared by contacting the therapeutic agent uniformly and intimately with a liquid carrier or a finely divided solid carrier or both. The product is then shaped into the desired formulation, if necessary. Preferably, the carrier is a parenteral carrier, more preferably a solution that is isotonic with the recipient's blood. Examples of such carrier vehicles include water, saline, Ringer's solution, and dextrose solution. Non-aqueous vehicles such as non-volatile oils and ethyl oleate are also useful herein, as are ribosomes.

[0040] The carrier suitably contains trace amounts of additives such as substances that enhance isotonicity and chemical stability. Such substances are not toxic to recipients at the dosages and concentrations used, such as phosphate, succinate, succinate, acetic acid and other Buffers such as organic acids or salts thereof; antioxidants such as ascorbic acid; low molecular weight (less than about 10 residues! /) Polypeptide (eg, polyarginine or tripeptide); serum anolebumin, gelatin or Proteins such as immunoglobulins; hydrophilic polymers such as polybulurpyrrolidone; amino acids such as glycine, glutamic acid, aspartic acid or arginine; monosaccharides, disaccharides, including cellulose or its derivatives, glucose, mannose or dextrin, and Other carbohydrates; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; counterions such as sodium; and / or nonionic surfactants such as polysorbates, poloxamers or PEG .

[0041] The therapeutic agent is typically about 6-9 to provide Gb3 at a concentration of about 10 mg / ml to 50 mg / ml, preferably 25-40 mg / ml (assuming 60 kg body weight). Formulated in such vehicles at pH. It is understood that by using the specific excipients, carriers or stabilizers described above, salts are formed.

[0042] Any drug to be used for therapeutic administration may be in a state free of organisms other than viruses as an active ingredient, ie, in a sterile state. Aseptic conditions are easily achieved by filtration through sterile filtration membranes (eg, 0.2 micron membranes). In general, the therapeutic agent is placed in a container having a sterile access port, eg, an intravenous solution bag or vial with a stopper that can be punctured with a hypodermic needle.

[0043] The therapeutic agents are usually stored in unit dose or multi-dose containers, such as sealed ampoules or vials, as an aqueous solution or lyophilized formulation for reconstitution. As an example of a lyophilized formulation, a 10 ml vial is filled with 5 ml of sterile filtered 5% (w / v) aqueous therapeutic agent and the resulting mixture is lyophilized. The lyophilized therapeutic agent is reconstituted with injection bacteriostatic water to prepare an infusion solution.

[0044] The present invention also provides a pharmaceutical pack or kit comprising one or more containers filled with one or more components of the therapeutic agent of the present invention. A notice in the form of a government agency that regulates the manufacture, use or sale of pharmaceuticals or biological products may be attached to such containers, and this notice may be used by the government regarding the manufacture, use or sale for human administration. Represents approval by an institution. In addition, the therapeutic agent may be used in combination with other therapeutic compounds. [0045] The therapeutic agents of the present invention can be administered alone or in combination with other therapeutic agents. The combination can be administered, for example, simultaneously as a mixture; simultaneously or concurrently but separately; or over time. This represents the indication that the combined drugs are administered together as a therapeutic mixture, and also the procedure where the combined drugs are administered separately but simultaneously, for example, to the same individual through separate intravenous lines. Including. Administration “in combination” further includes separate administration of one of the compounds or agents given first, followed by the second.

[0046] In formulating the bacterial exotoxin neutralizing agent of the present invention, the above-mentioned extract may be used as it is, but this is further fractionated to further concentrate Gb3 as an active ingredient. Used as a fraction contained in

[0047] (Production of food and beverage composition)

The suitable aspect of this invention is a composition for eating and drinking. That is, the pharmaceutical composition or edible composition containing the phospholipid obtained as described above as an active ingredient is used as it is in a liquid, gel or solid food such as juice, soft drink, coffee, Black tea, Japanese tea, Oolong tea, Vegetable juice, Natural fruit juice, Milk drink, Milk, Soy milk, Sports drink, Your water drink, Nutrition drink, Coffee drink, Cocoa, Soup, Dressing, Mousse, Jelly, Yogurt, Pudding, Sprinkle , Infant formula, processed milk, sports drink, energy drink, cake mix, bread, pizza, pie, crackers, biscuits, cake, woodpecker, spaghetti, macaroni, nosta, udon, soba, ramen, candy, softkey Yanday, gum, chocolate, rice cake, potato chips, snack, a Powdered or liquid dairy products, such as chair cream, shaichi ^ noto, cream, cheese, powdered milk, condensed milk, milk drinks, buns, oysters, rice cakes, soy sauce, sauce, soy sauce, sauce, dashi-no-moto, Stew, soup, mixed seasoning, curry, mayonnaise, ketchup, retort curry, letnore stew, letnore soup, letnore soup, canned, ham, hamburger, meatballs, croquettes, dumplings, pilaf, rice balls , Frozen foods and refrigerated foods, chikuwa, rice cakes, bento rice, sushi, baby milk, baby food, baby food, sports food, nutritional supplements, supplements, health foods, etc. It is processed into pellets, tablets, granules, etc. together with excipients such as lactose and starch, fragrances and pigments, and gelatin. It can be coated and molded into capsules and used as health foods, dietary supplements, and the like. The compounding amount of the snake extract of the present invention in these foods or eating and drinking compositions is difficult to define uniformly depending on the type and condition of the food or composition, but is about 0.0; ˜50 wt%, more preferably 0.;! ˜30 wt%. If the amount is less than 0.01% by weight, the desired effect of oral intake is small. If it exceeds 50% by weight, depending on the type of food, the flavor may be impaired or the food may not be prepared. It should be noted that the extract having the activity of neutralizing bacterial exotoxin of the present invention can be used for food as it is.

[0048] References such as scientific literature, patents and patent applications cited herein are hereby incorporated by reference in their entirety to the same extent as if each was specifically described. The

[0049] As described above, the present invention has been described by showing preferred embodiments for easy understanding. Hereinafter, the present invention will be described based on examples. However, the above description and the following examples are provided for illustrative purposes only and are not provided for the purpose of limiting the present invention. Therefore, the scope of the present invention is not limited to the embodiments or examples specifically described in this specification, and is limited only by the scope of the claims.

Example

[0050] Ability to describe the present invention in more detail with reference to the following examples The present invention is not limited to the following examples.

[0051] (Materials and Methods)

Nineteen kinds of herbal medicines (flow extract or tincture; Mikuni Co., Ltd., Chuo-ku, Osaka City) were appropriately diluted with a phosphate buffer (PBS) to obtain a sample solution. Vero cells were purchased from Dainippon Pharmaceutical. As a culture solution, a culture solution containing 5% Fetal Bovine Serum in MEMa medium (GIBCO) was used. The culture was performed at 37 ° C and 5% CO. As a Vero toxin, v

2

erotoxin― 1 (VT― 1 8 verotoxin― 2 (VT

—2) (both manufactured by NACALAI TESQUE INC, lOOmg) was used after diluting with appropriate culture medium. Anti-Verotoxin-1, 2 (manufactured by NACALAI TESQUE INC, lOOmg) was used as a Vero toxin-neutralizing antibody diluted with an appropriate culture medium. Measure absorbance using Cell Count Reagent (NACALAI TESQUE INC) as a measurement reagent. I got it.

[0052] (Test method)

Add 100 Vero cell suspension (10 ⁵ cells / ml) to each well of a 96-well flat-bottomed microplate and incubate in a 37 ° C, 5% CO incubator for 48 hours.

2

Add 10 μl of the solution (including heat-treated and acid-treated) or mouth-toxin neutralizing antibody Anti- Verotoxin 1 and 2 and Vera toxins verotoxin-1 and verotoxin-2 to each other and stir. After further incubation for 48 hours in a 37 ° C, 5% CO incubator, the reagent for measurement

2

Use Cell Count Reagent SF reagent to develop the color of the cells, measure the absorbance with UV light (450 m) using a microplate reader, and use the absorbance of cells without any addition as 100% viability. The survival rate was calculated from the absorbance of the cells. As a control, only cells cultured were used.

[0053] (Example 1: Cell viability with 20 kinds of herbal extracts or tincture and verotoxin)

The stock solutions of the following 20 crude drugs were diluted 4, 16, and 64 times with PBS. Vero toxin verotoxin-1 and verotoxin-2 stock solutions were diluted 80, 320 and 1280 times.

[0054] As herbal medicines, flow extracts (Ikariso, Yezoukogi, Ogi, Ousei, Kukosi, Koujin, Sanshu, Sanjak, Ji, Taiso, Toki, Tochuu Saw, Tochu, Dikujuyo, Carrot, Batamondo, No, Npi, and ), And tincture (strength Ikujin and Rokujiyo) were used. As a result, the other crude drugs except Hampi extract of 20 types did not show antitoxin activity against verotoxin, and the viability of Vero cells was not much different from that when only toxin was added. However, only the Hampi-flow extract showed very high antitoxin activity (suppressing the cytotoxic effect of Vero toxin on Vero cells).

[0055] Regarding the concentration of the crude drug stock solution to be added and the cell viability due to verotoxin, the crude drug A stock solution was diluted with PBS and added to the culture solution so as to be 0 to 41.7 mg / ml.

[0056] The crude drug stock solution is specified to extract lg (the active ingredient) of herbal medicine per ml (Japanese Pharmacopoeia). The sample used in the test is a Japanese product and conforms to this rule, so this concentration was used as a reference. Toxin solution was adjusted to 100 ml of lOOmg as the amount of protein.

[0057] (Example 2: Effect of crude drug concentration on survival rate when VT-1 is used) Nine toxin concentrations of VT-1 (0.4 〃g / re 0.8 〃g / L 1.6 μg / L, 3.3 μg / L, 6.5 g / L, 13. Ο (v), 26.0 μg / L, 52.1 μg / L, and 104.5 μg / L), the survival rate of Vero cells increased depending on the concentration of herbal medicine.

[0058] In the case of VT—1, in the case of toxin alone, the survival rate is low even if the toxin concentration is low (0.4 _§ / _§ ).

It was less than 20%. However, as the crude drug concentration is increased, the survival rate of Vero cells increases, and if the crude drug concentration is 20 mg / ml or higher, the survival rate is close to 50% even if the amount of toxin is 100 g / 1, or even higher. Survival rate. However, even if the concentration of herbal medicine was increased further, the survival rate was not increased. The results are shown in Figure 1.

[0059] (Example 3: Effect of crude drug concentration on survival rate when VT-2 is used)

For the nine toxin concentrations of VT-2, the survival rate of Vero cells increased depending on the concentration of the crude drug. In the case of VT-2, the overall survival rate was higher than that of VT-1. The lower the toxin concentration, the higher the survival rate than VT-1. The result is shown in figure 2

(Example 4: Cell viability with verotoxin neutralizing antibody and verotoxin)

Vero toxin-neutralizing antibody was added instead of crude drug, and the survival rate of Vero cells when reacted with toxin was measured. The toxin-neutralizing antibody 2. was 3~225 _§ / 1111. The neutralizing antibody solution was 54 mg of protein and 10 ml of culture solution. This was first diluted 2-fold (the neutralizing antibody concentration in this solution was 2.7 mg / ml). 10 μl of this was added to cell culture solution and crude drug solution 110 1 (the power of neutralizing antibody in 120 1 is 27 μg, and 225 μg in 1 ml). Figure 3 shows the results of VT-1! /, And Figure 4 shows the results of VT-2!

[0061] When the VT-1 antibody was added to the cells together with the toxin, the survival rate increased as the antibody concentration increased.

[0062] When the antibody concentration was 225 ag / mL, the effect of the toxin was almost invisible regardless of the concentration of the toxin. Even when VT-2 antibody is added, the survival rate increases as the antibody concentration increases, but it does not increase as markedly as VT-1, and at 225 g / mL, it is about 60 to 75% of the case when no toxin is added. Stayed alive. From the above results, it was confirmed that the survival rate of Vero cells was due to the action of porcine toxin.

[0063] (Example 5: Heat treatment of crude drug stock solution and cell viability by verotoxin) The effect on Vero cells when the crude drug stock solution was heat-treated (in a boiling water bath at approximately 100 ° C for 3 minutes) was examined.

[0064] With regard to VT-1, although a slight difference was observed when the crude drug concentration was high, the survival rate was almost the same at other crude drug concentrations. For VT-2, the survival rate was almost the same even when the conditions of crude drug concentration and toxin concentration were changed. From these results, it was estimated that the anti-verotoxin active substance in herbal medicine was not inactivated by heating (about 98 ° C in a boiling water bath). Figure 5 shows the results for VT-1, and Figure 6 shows the results for VT-2.

(Example 6: Acid treatment of crude drug stock solution and cell viability by verotoxin)

The effect of Vero cells when the crude drug stock solution was acid-treated (pH adjusted to 4 with acetate buffer) was examined. VT-1 showed almost the same survival rate even when the crude drug concentration and toxin concentration were changed. For VT-2, the survival rate was similar to that of VT-1, except that the cell survival rate was higher. The acid treatment was performed with acetic acid acidity (pH 4). Figure 7 shows the results for VT-1, and Figure 8 shows the results for VT-2.

[0066] (Example 7: Cell viability when a mixture of a crude drug stock solution and a verotoxin solution is added in advance and when each is added to cells simultaneously)

For the purpose of investigating whether or not herbal toxin reacts with the herbal medicine (Chinese medicine) components in vitro, the crude drug and Vero toxin solution may be added to the cell culture medium for 2 hours before mixing them, The survival rate of Vero cells when added to the mouth cells was examined. The overall survival rate for VT-1 was slightly higher when the crude drug and toxin were mixed in advance. A similar trend was observed for VT 2, but when the toxin was diluted with the culture solution, the toxicity decreased slightly over time. 1, VT-2 seems to be almost! /. Figure 9 shows the results for VT-1! And Figure 10 shows the results for VT-2.

[0067] (Example 8: Herbal medicine was added to Vero cells and allowed to stand for 24 hours, after which the cell culture medium was removed.

Instead, add verotoxin. (Comparison with the case where it was added to the control at the same time as before)

Add herbal medicines to the Vero cells and let them stand for 24 hours. Mouth toxin was added. This was compared with the control when it was added simultaneously as before. As a result, compared to Example 7, VT-1 had a survival rate reduced by about 10% when the crude drug was added 24 hours before the toxin concentration at any crude drug concentration. In VT-2, when the crude drug concentration is lower, the survival rate of the one added 24 hours before is 5 to; the 10% decreased potency The herbal drug concentration is 20.8 mg / ml, and almost the same survival rate at any toxin concentration showed that.

Figure 11 shows the results of VT-1! /, And Figure 12 shows the results of VT-2!

(Summary)

(1) Of the 20 crude drugs, only Hampi extract showed anti-toxin activity.

(2) The added crude drug concentration is proportional to the cell viability (the higher the herbal concentration, the higher the survival rate).

(3) The addition of verotoxin neutralizing antibody increased cell viability (confirmed that the causative agent causing cell damage was mouth toxin).

(4) The crude drug solution does not lose its efficacy even when heated in a boiling water bath! / (The substance with anti-verotoxin activity in the crude drug solution is heat resistant).

(5) Anti-verotoxin active substances in herbal medicine solutions did not lose their potency even with acetic acid pH 4.

((66)) Herbal medicine solution and venom toxin solution may be pre-mixed 22 hours in advance. For each degree of disability, the difference in power is unacceptable ((slightly depending on the pre-mixing) The survival rate increased and rose))). .

((77)) Add the herbal medicine solution to the fine cell vesicles and cultivate it for 2244 hours, then add the toxin toxin solution And, when the herbal medicine and the venom solution are added at the same time, the survival rate is about 1100 %% lower than the case. I'm sorry. .

As a result, the result was obtained. .

((Execution example 99 :: snake extraction extract contains GGbb33))

((11 .. Single isolation of GGbb33 as positive control)

GGbb33 is used as a cell tubule for preparation and preparation. EECCAACCCC standard quasi-microcellular vesicle, which is a small cell vesicle, DDAAIIDDII ((EECC AACCCC NNoo .. 8855001111443377)) 22 ~ 99 XX 1100 ⁵⁵ cell cysts // mmll, 1100 %% Add sushi fetal blood serum was added * In gnoretamine). The cultured cells were extracted with a black mouth form: methanol mixture (1: 1) and used as a Gb3 positive control.

[0070] (Detection of Gb3 by antigen-antibody reaction)

100 L each of the sample and the positive control solution was adsorbed on a polyvinylidene fluoride (PVD F) membrane (Immobilon P, manufactured by Millipore) by suction using a dot plate. Samples are “Blank” (PBS only), “Burkitt Lymphoma” (ECACC standard cells, DAIDI, 5 × 10 ⁷ cells), “Hampi” (25% viper extract 501, Mikuni Co., Ltd.) Chuo-ku), “Hub” (Hob liquor with november 501, Mikuni Co., Ltd., Chuo-ku, Osaka), “Cobra” (15% tincture of Cobra), “Milk” (milk 100 1) .

[0071] The membrane was immersed in a blocking solution (0.4% Block Ace) at room temperature for 1 hour. Next! / ヽ mount the primary antibody solution (rat anti-human CD77 100T antibody (Serotec) diluted 50-fold or 100-fold with PBS ace PBS solution 50-fold or 100-fold) on the membrane and apply parafilm on the surface. And allowed to react at 4 ° C overnight. Wash the membrane 4-5 times with 0.5% Triton X-100 phosphate buffer; then with phosphate buffer;! Next! /, In the membrane, the secondary antibody solution (anti-rat IgG; Ameri

can Resarch Product, Inc) was mounted and allowed to react at room temperature for 2 hours. The membrane was washed as before and stained with a staining reagent (Immostine HRP-1000 manufactured by Konica Minolta MG). The results are shown in FIG.

[0072] As shown in the results, the snake extract contained Gb3. Its content was greater than that of milk, which is conventionally known to contain Gb3. Therefore, it is considered that at least part of the exotoxin neutralizing activity by the snake extract is attributable to Gb3.

[0073] Example; Summarizing the results of! ~ 8 and the result of Example 9, 2.4 liters is required when ingesting the necessary amount of fungal exotoxin neutralizer in milk, On the other hand, Hampi needs 10-20 ml, and Hubbard needs 20-30 ml. Therefore, milk needs 120 times the amount of Hampi and 80 times the amount of hub liquor. Converting this to Gb3 results in 0.5 mg for milk and 13-26 _§ Gb3 for S. This result suggests that unidentified substances with neutralizing exotoxin activity are contained in snake extracts such as potatoes and numpies. Composition of the present invention containing bi extract Can not be predicted from the prior art! / And has a remarkable effect.

[0074] As described above, the power of the present invention, which has been exemplified using the preferred embodiment of the present invention, and the present invention should not be construed as being limited to this embodiment. It is understood that the scope of the present invention should be construed only by the claims. It is understood that those skilled in the art can implement an equivalent range based on the description of the present invention and the common general technical knowledge from the description of specific preferred embodiments of the present invention. Patents, patent applications and literature references cited in this specification should be incorporated by reference as if the contents themselves were specifically described in the present specification. Is understood.

Industrial applicability

[0075] According to the present invention, there is provided a composition for neutralizing enterotorrhagic Escherichia coli exotoxins that can be easily and inexpensively prepared.

Claims

The scope of the claims

[I] A pharmaceutical composition for neutralizing the toxicity of enterotorrhagic Escherichia coli exotoxin comprising a snake extract.

[2] The pharmaceutical composition according to claim 1, wherein the snake extract is derived from a poisonous snake.

[3] The pharmaceutical composition according to claim 1, wherein the snake extract is a viper extract.

[4] The pharmaceutical composition according to claim 3, wherein the beetle extract is a Hampi extract.

5. The pharmaceutical composition according to claim 1, wherein the snake extract is a cobra extract.

6. The pharmaceutical composition according to claim 5, wherein the cobra extract is a cobra tincture.

7. The pharmaceutical composition according to claim 1, wherein the snake extract is a hub extract.

8. The pharmaceutical composition according to claim 7, wherein the hub extract is hub liquor.

[9] The pharmaceutical composition according to claim 1, wherein the enterohemorrhagic E. coli is Ol 57.

[10] A food and drink composition for neutralizing the toxicity of enterotorrhagic Escherichia coli exotoxin, comprising a snake extract.

[I I] The food and drink composition according to claim 10, wherein the snake extract is derived from poisonous snakes.

[12] The composition for eating and drinking according to claim 10, wherein the snake extract is a viper extract.

[13] The composition for eating and drinking according to claim 12, wherein the beetle extract is a Hampi extract.

14. The composition for eating and drinking according to claim 10, wherein the snake extract is a cobra extract.

[15] The dietary composition according to claim 14, wherein the cobra extract is a cobra tincture.

16. The food / drink composition according to claim 10, wherein the snake extract is a hub extract.

17. The composition for eating and drinking according to claim 16, wherein the hub extract is hub liquor.

18. The composition for eating and drinking according to claim 10, wherein the enterohemorrhagic Escherichia coli is Ol 57.