JP2015182961A - Vero toxin deactivator - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a material useful for deactivating vero toxin produced by enterohemorrhagic Escherichia coli and preventing, treating or improving a symptom caused by the infection of the enterohemorrhagic Escherichia coli.SOLUTION: A vero toxin deactivator comprises theaflavin digallate as an active ingredient.

Description

  The present invention relates to an inactivating agent for verotoxin.

  Enterohemorrhagic Escherichia coli such as O-26, O-55, O-91, O-103, O-104, O-111, O-121, O-145, O-157, O-165; EHEC) is a causative bacterium for diseases such as food poisoning, hemorrhagic colitis, hemolytic uremic syndrome. Enterohemorrhagic Escherichia coli has a strong pathogenicity, often causes mass infections, and there are many deaths of infected people.

  Vero toxin (VT) is a toxin produced in enterohemorrhagic Escherichia coli and considered to cause the above-mentioned disease. Verotoxin is a toxin classified into the same Shiga toxin family as Shiga toxin, and VT1 (Stx1) having the same amino acid sequence as Shiga toxin and VT2 (Stx2) having 55% identical amino acid sequence are known. Yes. VT1 and VT2 are known to have similar biological properties but differ in their physicochemical and immunological properties.

Examples of means for preventing infectious diseases caused by enterohemorrhagic Escherichia coli include bacterial growth suppression, bacterial verotoxin production or secretion suppression, and produced verotoxin detoxification.
Vaccines and antibacterial agents are the most commonly used means for preventing bacterial infection and growth. Clinically, antibiotics such as fosfomycin have been used empirically. However, these are not always effective against enterohemorrhagic Escherichia coli in general. Because the vaccine is effective against O-157 but not against other types of enterohemorrhagic E. coli, the effect is limited by the type of bacteria, and when an antibacterial agent is used, This is because when toxins are killed, the toxins stored in the cells are released into the intestinal tract at one time, and the symptoms may become serious.

  Substances that suppress the production or secretion of verotoxin by enterohemorrhagic Escherichia coli are known. For example, pomegranate peel extract inhibited verotoxin production by enterohemorrhagic Escherichia coli (Non-patent Document 1), and epicatechin gallate (EGCg) inhibited secretion of verotoxin outside the cell ( Non-Patent Document 2), and polycatechin having a specific structure obtained from hop bract tannin has an action of inhibiting the transfer of Stx1 to the cytosol and suppressing its pathogenicity (Patent Document 1). ) Has been reported. In addition, substances that inactivate verotoxin are also known. For example, apple juice and catechin preparations have been reported to have an antitoxin action against Stx2 (Non-patent Documents 3 and 4). It has been reported that there is an effect of inactivating Stx1 (Patent Document 2).

  On the other hand, theaflavins, which are polyphenols produced from catechins, have pharmacological actions such as schulase activity inhibitory action (Patent Document 3), hyaluronidase activity inhibitory action (Patent Document 4), and lipase inhibitory action (Patent Documents 5 and 6). It has been reported that there is.

  However, it is not known at all that theaflavin digallate has an effect of inactivating verotoxin.

JP-T-2006-508924 JP 2013-136553 A JP-A-5-17352 JP-A-6-9391 JP 2010-95477 A International Publication No. 2006/004114

Supayang et al J. Health Science, 51 (5): 590-596, 2005 Konishi et al, Biochim Biophys Acta, 1472: 42-50, 1999 Okubo et al., Journal of Infectious Diseases, Volume 72, Issue 3: 211-217, March 20, 1998 Reuven et al, J. Food Science, 75 (5): 296-301, 2010

  The present invention relates to providing a verotoxin inactivating agent useful for inactivating verotoxin produced by enterohemorrhagic Escherichia coli and preventing, treating, or ameliorating symptoms caused by infection with the bacteria.

  As a result of searching for a substance that detoxifies verotoxin, the present inventors have found that theaflavin digallate has an action of reducing its cytotoxic activity against VT1 and VT2.

That is, the present invention relates to the following (1) to (2).
(1) A verotoxin inactivating agent comprising theaflavin digallate as an active ingredient.
(2) A preventive or ameliorating agent for poisoning caused by enterohemorrhagic Escherichia coli comprising theaflavin digallate as an active ingredient.

  According to the verotoxin inactivating agent and the like of the present invention, the activity of verotoxin secreted into the intestinal tract by enterohemorrhagic Escherichia coli can be reduced, food poisoning, diarrhea, hemorrhagic enteritis, hemolytic uremic syndrome, central neuropathy, Prevention, treatment or improvement of poisoning symptoms caused by verotoxins such as acute encephalopathy can be achieved.

The graph which shows VT1 inactivation effect. Error bar = standard deviation. TFDG: theaflavin digallate, EGCG: epigallocatechin gallate. The graph which shows VT2 inactivation effect. Error bar = standard deviation. TFDG: theaflavin digallate, EGCG: epigallocatechin gallate.

There are V1 and VT2 in Vero toxin (VT). VT1 and VT2 are also referred to as Stx1 and Stx2, or SLT (Shiga-like toxin) -1 and SLT-2, respectively, which are both Shiga toxin (Stx) and Shiga toxin (Stx) family. Like other toxins belonging to Stx family, it has a structure composed of A subunit and B subunit. The amino acid sequence of VT1 is registered as, for example, GenBank M 16625, and is the same as or almost the same as the amino acid sequence of Stx. On the other hand, many variants of VT2 are known, and their identity to the Stx amino acid sequence is about 55%. Both VT1 and VT2 have a lethal effect on the cell through a series of pathways beginning with binding to the cellular Gb3 receptor, whereas the amino acid sequence identity between VT1 and VT2 is 58 in the A subunit. .2% and B subunit is only 60.7%. Therefore, VT1 and VT2 share the same biological properties (toxicity to living organisms), but differ in physical and immunological properties. For example, VT1 has the same antigenicity as Stx and is neutralized by an anti-Shiga toxin antibody, but VT2 does not have the same antigenicity as VT1 or Stx (Bacterial Toxin Handbook Editor; Sakurai Jun, Takeshi Honda, Keiji Oguma Publisher: Science Forum, Inc. Publication year: 2002).
In the present invention, “verotoxin” includes both VT1 and VT2.

In the present invention, “verotoxin inactivation” is to reduce or eliminate the cytotoxic activity of verotoxin (VT1 and VT2). Verotoxin inactivation can reduce or eliminate the toxicity or adverse effects that cells receive from verotoxin.
Here, “cells” to which verotoxin can exert its toxicity include African green monkey-derived Vero cells, and intestinal cells that can be directly or indirectly affected by verotoxin in the living body, such as humans, non-human primates, mice Intestinal epithelial cells derived from rodents such as rats, or rabbits, colonic mucosal epithelial cells, renal cells, central nerve cells and the like.

  In the present invention, “theaflavin digallate” (TFDG) is a kind of red pigment component produced from catechins in the fermentation process of tea (Camellia sinensis) leaves, and the following “theaflavin 3,3′-di-” It means “theafravin 3,3′-di-O-gallate”.

  The theaflavin digallate of the present invention can be obtained by extraction and fractionation by a known method from a plant containing the same, for example, tea leaves, or synthesis using an enzymatic reaction (for example, JP 2010-35548 A). Is possible. Moreover, it is also possible to use a commercial item (Nagara Science Co., Ltd.)

As shown in the examples below, when Vero cells are cultured in the presence of Vero toxin VT1 or VT2, the cells are hardly viable due to the toxicity of VT1 or VT2, but VT1 or VT1 or When VT2 is added, the survival rate is improved. That is, theaflavin digallate has an action of reducing verototoxic activity on cells. In particular, the inactivating effect on VT1 is strong, about 5 times that of EGCg.
Therefore, infecting verotoxin produced and secreted by enterohemorrhagic Escherichia coli by administering, ingesting or contacting theaflavin digallate to the subject, or preventing or improving poisoning symptoms caused by enterohemorrhagic Escherichia coli be able to.

That is, theaflavin digallate can be a verotoxin inactivating agent, a preventive or ameliorating agent for poisoning caused by enterohemorrhagic Escherichia coli (hereinafter referred to as “verotoxin inactivating agent etc.”). Therefore, it can be used for prevention or amelioration of symptoms of poisoning caused by enterohemorrhagic Escherichia coli. Here, the use is for human or non-human animals (non-human primates, mice, rats and other rodents, rabbits, etc.) or specimens derived from them (intestinal epithelial cells, colonic mucosal epithelial cells, kidneys) Cells, cells such as central nerve cells, tissues, organs and the like containing these cells), and the use for humans may be a therapeutic use or a non-therapeutic use. Here, “non-therapeutic” means a concept that does not include medical practice, that is, a concept that does not include a method for operating, treating, or diagnosing a person, more specifically, a doctor or a person who has received instructions from a doctor It is a concept that does not include a method for performing surgery, treatment, or diagnosis on the subject.
Theaflavin digallate can also be used to produce verotoxin inactivating agents and the like.
Since the verotoxin inactivating agent and the like of the present invention can inactivate verotoxin that has already been secreted, it can also be used to increase the severity of symptoms caused by the massive release of verotoxin caused by bacterial destruction by antibiotics. It is applicable and advantageous compared to conventional therapeutic agents.

In the present invention, “intoxication symptoms due to enterohemorrhagic Escherichia coli” include symptoms such as food poisoning, diarrhea, hemorrhagic enteritis, hemolytic uremic syndrome, renal disorder, central nervous disorder, acute encephalopathy, etc. It is not limited.
In the present invention, “prevention” means prevention or delay of the onset of a disease or symptom in an individual or reduction of the risk of onset of a disease or symptom in an individual. “Improvement” refers to improvement of a disease or symptom, prevention or delay of deterioration of the disease or symptom, or reversal, prevention or delay of progression of the disease or symptom.

The verotoxin inactivating agent or the like of the present invention is effective for humans or animals that exerts an effect of preventing or improving verotoxin inactivation and intoxication symptoms due to enterohemorrhagic Escherichia coli when ingested or administered to animals including humans. It may be a pharmaceutical, a quasi-drug, or may be a material or raw material used in combination with the pharmaceutical, quasi-drug, food or feed.
In addition, foods formulated with theaflavin digallate as a raw material or raw material are based on the concept of inactivation of verotoxin and improvement of poisoning symptoms due to enterohemorrhagic Escherichia coli, foods that indicate that fact, functional foods, Includes food for the sick and food for specified health use. These foods are foods whose functions are permitted to be distinguished from general foods.

The dosage form of the above-mentioned pharmaceutical (including quasi-drugs) may be any of injections, suppositories, inhalants, transdermal absorption agents, various external preparations, tablets, capsules, granules, powders, syrups, etc. Good. The administration form may be any of oral administration (internal use) and parenteral administration (external use, injection, nasal, enteral), but oral administration is preferred.
In addition, the preparations of various dosage forms such as the theaflavin digallate of the present invention alone or other pharmaceutically acceptable excipients, binders, extenders, disintegrants, surfactants, lubricants. It can be prepared by appropriately combining a mixture of a powder, a dispersant, a buffer, a preservative, a flavoring agent, a fragrance, a coating agent, a carrier, a diluent, other medicinal ingredients (for example, antibiotics, antibacterial agents, etc.). . For example, oral liquid preparations can be prepared by conventional methods with the addition of flavoring agents, buffering agents, stabilizers and the like.

  As the form of the food, fruit juice drinks, carbonated drinks, tea drinks, coffee drinks, milk drinks, alcoholic drinks, soft drinks, breads, noodles, pasta, jelly-like foods, various snacks, cakes, confectionery, In addition to various foods such as ice creams, soups, dairy products, frozen foods, instant foods, other processed foods, seasonings, supplements, and other foods and nutritional foods, the same forms as the above-mentioned oral preparations (tablets, Capsules, syrups, etc.). Further, food for sick people, for example, food for elderly people who are difficult to supplement with an appropriate amount of nutrition or those in bed rest, can be in the form of nutritional compositions such as enteral nutrients.

  Examples of the feed include feed for small animals used for rabbits, rats, mice, etc., feed for pet foods used for dogs, cats, and the like, and can be prepared in the same form as the above foods.

  Various forms of food or feed are theaflavin digallate alone or other food or feed materials, solvents, softeners, oils, emulsifiers, preservatives, fragrances, stabilizers, colorants, antioxidants, It can be prepared by appropriately combining humectants, thickeners, active ingredients other than theaflavin digallate.

  The content of theaflavin digallate in the pharmaceutical, food or feed is generally preferably 0.0001% by mass or more, more preferably 0.001% by mass or more, in view of inactivation of verotoxin, and Preferably it is 5 mass% or less, More preferably, it is 1 mass% or less, More preferably, it is 0.1 mass% or less, Most preferably, it is 0.01 mass% or less. Moreover, it is 0.0001-5 mass%, More preferably, it is 0.001-1 mass%, More preferably, it is 0.001-0.1 mass%, More preferably, it is 0.001-0.01 mass%.

The subject in the case of administering or ingesting the verotoxin inactivating agent or the like of the present invention to a human is not particularly limited as long as it is a human who needs or desires it, but for example, due to infection with enterohemorrhagic Escherichia coli , Patients suffering from symptoms such as food poisoning, diarrhea, hemorrhagic enteritis, hemolytic uremic syndrome, renal disorder, central nervous disorder, acute encephalopathy, and humans suspected of such.
The dosage or ingestion amount when the verotoxin inactivating agent or the like of the present invention is administered or ingested to humans varies depending on the dosage form or use, but is 0.1 μg or more per day for adults as theaflavin digallate / 50 kg body weight, preferably 1 μg or more / 50 kg body weight, more preferably 10 μg or more / 50 kg body weight, more preferably 100 μg or more / 50 kg body weight, more preferably 1 mg or more / 50 kg body weight, more preferably 10 mg or more / 50 kg body weight, More preferably 100 mg / 50 kg body weight, still more preferably 1,000 mg / 50 kg body weight, and preferably 30,000 mg or less / 50 kg body weight, more preferably 20,000 mg or less / 50 kg body weight, more preferably 10,000 mg. Below / 50 kg body weight, more preferably 5,000 0 mg / 50 kg, more preferably from 1,000 mg / 50 kg. Also, 0.1 μg to 30,000 mg / 50 kg body weight, preferably 1 μg to 20,000 mg / 50 kg body weight, preferably 10 μg to 10,000 mg / 50 kg body weight, more preferably 100 μg to 5,000 mg / 50 kg body weight, more preferably Examples include 1 mg to 5,000 mg / 50 kg body weight, more preferably 10 mg to 5,000 mg / 50 kg body weight, more preferably 100 mg to 5,000 mg / 50 kg body weight, and still more preferably 1,000 mg to 5,000 mg / 50 kg body weight.

With respect to the above-described embodiment, the following aspects are further disclosed in the present invention.
<1> Verotoxin inactivating agent containing theaflavin digallate as an active ingredient.
<2> A preventive or ameliorating agent for poisoning caused by enterohemorrhagic Escherichia coli comprising theaflavin digallate as an active ingredient.
<3> Use of theaflavin digallate for producing verotoxin inactivating agent.
<4> Use of theaflavin digallate for producing a preventive or ameliorating agent for intoxication caused by enterohemorrhagic Escherichia coli.
<5> Theaflavin digallate for use in inactivating verotoxin.
<6> Theaflavin digallate for use in the prevention or improvement of intoxication caused by enterohemorrhagic Escherichia coli.
<7> A method for inactivating verotoxin, comprising administering or ingesting theaflavin digallate in an effective amount to a subject in need thereof.
<8> A method for preventing or ameliorating intoxication symptoms caused by enterohemorrhagic Escherichia coli, wherein theaflavin digallate is administered or ingested in an effective amount to a subject in need thereof.
<9> In the above <5> to <6> and <7> to <8>, the use and method are non-therapeutic.
<10> The poisoning symptom in the above <2>, <4>, <6> and <8> is preferably food poisoning.

EXAMPLES Hereinafter, an Example is shown and this invention is demonstrated more concretely.
Example 1 Verotoxin inactivating action (1) Test substance TFDG: Theaflavin 3,3′-di-o-gallate (Nagara Science Co., Ltd.)
EGCG: Epigallocatechin gallate (Mitsui Norin Co., Ltd.)

(2) Preparation of toxin solution and measurement of titer As enterohaemorrhagic Escherichia coli (EHEC) producing verotoxin, Escherichia coli O-157: H-7 No. 33 strain (VT1 +) was used as the VT2 producing strain. coli O-157 No. 148 strains (VT2 +) were used respectively.
No. above. No. 33 strain and No. The 148 strain was cultured overnight at 37 ° C. in 5 ml of LB medium (Becton & Dickinson). The culture broth was diluted 1000 times and 10 μl was inoculated into 5 ml (40 pieces) of fresh LB medium and further cultured overnight at 37 ° C. with shaking. 5 ml of the culture solution of each strain was put into a 15 ml centrifuge tube, polymixin B was added to the culture solution so as to be 5000 U / 1 ml, and incubated at 37 ° C. for 1 hour, and the culture solution was collected. The collected culture solution was put into a 50 ml centrifuge tube in 25 ml portions, centrifuged (3000 × g, 15 minutes), and the supernatant was once collected in a beaker and then sterilized by filtration (0.2 μm) to obtain a toxin solution. .
The titer of verotoxin in the toxin solution was calculated by examining the productivity of VT1 and VT2 using VTEC-RPLA “Seiken” (Denka Seiken) according to the attached protocol. As a result of measurement, no. The toxin solution (VT1) obtained from the 33 strain had a titer of 256 and No. The toxin solution (VT2) obtained from strain 148 had a titer of 32768. In the following verotoxin inactivation test, a toxin solution prepared by stepwise diluting VT1 and VT2 2-fold with PBS and preparing a final dilution factor of 1 to 128 times was used.

(3) Verotoxin inactivation test 100 μL of 5% FBS-added MEM-E medium in which Vero cells are suspended to a concentration of 2.0 × 10 ⁴ cells / well is added to each 96-well cell culture plate [Corning]. The wells were seeded and cultured for 24 hours in a 37 ° C. CO ₂ incubator (5% CO ₂ ). Toxin samples were mixed with TFDG or EGCG and incubated for 1 hour in a CO ₂ incubator (5% CO ₂ ) at 37 ° C. 100 μL each was added to the Vero cells after culturing, and the CO ₂ incubator at 37 ° C was added. The cells were cultured for 24 hours in (5% CO ₂ ). After the culture, the medium was removed, 100 μL of fresh 5% FBS-added MEM-E medium was added, 10 μL of MTT solution was added, and the mixture was further incubated in a CO ₂ incubator (5% CO ₂ ) at 37 ° C. for 4 hours. Thereafter, the medium was removed, and 100 μL of DMSO was added to dissolve the dye, and the absorbance at 595 nm was measured. A Microplate Reader Model 680 was used for measuring the absorbance. Wells to which only VT-free samples were added were prepared as negative controls for toxicity tests, and wells to which only VT-containing samples were added were prepared as positive controls.

(4) Results The results are shown in FIGS. Cells to which VT1 toxin solution mixed with TFDG and VT2 toxin solution mixed with TFDG were added had improved survival compared to cells to which TFDG-untreated VT1 or VT2 toxin solution was added (FIG. 1 and 2). Moreover, the inhibitory activity of TFDG on VT1 was about 5 times stronger than EGCG.

Claims (2)

  Verotoxin inactivating agent containing theaflavin digallate as an active ingredient.   A preventive or ameliorating agent for poisoning caused by enterohemorrhagic Escherichia coli comprising theaflavin digallate as an active ingredient.