JPH11279076A - Neutralizer of cytotoxin - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a neutralizer of a bacterial enterotoxin produced by cholera germ, enterotoxigenic Escherichia coil, salmonella or the like by making the neutrizer include an iron-binding type lactoferrin as an active ingredient. SOLUTION: This neutralizer contains an iron-binding type lactoferrin as an active ingredient. The iron-binding type lactoferrin is obtained by subjecting a raw material of a milk of a mammal such as a cattle and a human, or a defatted milk, a whey or the like to a treatment such as an ion-exchange chromatography to provide a lactoferrin, chelating the obtained lactoferrin with an iron ion in a solution of a sodium citrate dissolving ferric chloride, dialyzing the obtained solution to desalt the solution. The iron-saturation degree of the iron-binding type lactoferrin is preferably >=20%. The daily dose of the before active ingredient is preferably 0.1-5,000 mg in a case of an adult.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a novel neutralizing agent for bacterial enterotoxin produced by cholera bacteria, toxinogenic Escherichia coli, salmonella bacteria and the like. In addition, the present invention relates to a novel medicine, food and drink having a function of neutralizing bacterial enterotoxins produced by cholera bacteria, toxogenic Escherichia coli, salmonella bacteria and the like.

[0002]

2. Description of the Related Art Food poisoning due to cholera, for example, causes severe diarrhea with abdominal pain. For this reason, patients infected with cholera may exhibit extreme dehydration due to diarrhea and die. It is known that the severe diarrhea observed in food poisoning caused by cholera is caused by a bacterial enterotoxin called cholera toxin which cholera is produced outside the cells. Cholera toxin has a molecular weight of 84,000
And consists of one A subunit and five B subunits. The B subunit of cholera toxin binds to a receptor present in small intestinal mucosal cells, and increases the amount of cyclic AMP in the cells. It is said to ooze out and cause diarrhea. Bacterial enterotoxins such as cholera toxin have also been isolated from toxinogenic Escherichia coli, Salmonella, and the like, causing food poisoning.

[0003] Incidentally, it has been confirmed that the above-mentioned receptor present in the small intestinal mucosal cells is a glycolipid.
Ganglioside GM as a receptor for cholera toxin
1 has been identified. Attempts have already been made to utilize this ganglioside GM1 as a cholera toxin neutralizer. For example, a bacterial toxin neutralizing agent comprising a ganglioside having a function of neutralizing cholera toxin, such as ganglioside GM1, immobilized on latex or the like (JP-A-56-501381)
And a heat treatment of milk fat globule coating, and a bacterial toxin neutralizing agent used without extracting ganglioside GD3, ganglioside GM3, etc. contained in milk fat globule coating (Japanese Patent Application Laid-Open No.
-72819). However, ganglioside GM1 has a problem that only a very small amount can be obtained from the brain of cattle or the like, and there is also a problem that the ganglioside content in the milk fat globule coat is not constant and the composition is not stable.

[0004] A neutralizing agent for bacterial toxins (JP-A-2-07089) has been proposed which utilizes the fact that bacterial toxins bind to sialic acid-containing glycoconjugates such as lactoferrin and exhibit neutralizing activity. Lactoferrin is a glycoprotein with a molecular weight of about 80,000 that can bind two molecules of iron into the molecule, but the tertiary structure of the molecule is different depending on whether iron is bound or not. It is known to be different.
However, regarding the neutralizing function of lactoferrin for bacterial enterotoxin, the difference in the tertiary structure, that is, the difference depending on the presence or absence of iron binding, has not been clarified at all.

[0005]

DISCLOSURE OF THE INVENTION The present inventors have been paying attention to the pharmacological action of lactoferrin and have been studying it intensively. As a result, it has been found that bacterium produced by cholera bacteria, toxogenic Escherichia coli, salmonella bacteria and the like are produced. Regarding the function of neutralizing enterotoxin, it has been found that iron-free lactoferrin has little activity, whereas iron-free lactoferrin has little activity. Then, they found that this activity depends on the iron saturation of iron-binding lactoferrin, and completed the present invention.

Accordingly, the present invention provides a novel neutralizing agent for bacterial enterotoxin produced by cholera bacteria, toxigenic Escherichia coli, Salmonella bacteria, etc., or a novel pharmaceutical agent imparting such a bacterial enterotoxin neutralizing function. And food and drink. Specifically, an object of the present invention is to provide a bacterial enterotoxin neutralizing agent containing iron-binding lactoferrin as an active ingredient. It is an object of the present invention to provide a medicine, a food and a drink having a Japanese function.

[0007]

According to the present invention, iron-binding lactoferrin is used as an active ingredient of a bacterial enterotoxin neutralizing agent, or for imparting a bacterial enterotoxin neutralizing function to medicines and foods and drinks. It is characterized by the following.

The iron-binding lactoferrin used in the present invention can be obtained by subjecting mammal milk such as cows and humans, or skim milk or whey thereof to a raw material and subjecting it to a treatment such as ion exchange chromatography. It can be prepared by chelating iron ions in lactoferrin, for example, in a solution of sodium citrate or the like in which ferric chloride is dissolved, and then dialyzing and desalting. When chelating iron with lactoferrin, lactoferrin obtained by a genetic recombination technique may be used, or commercially available lactoferrin may be used.

Milk of mammals such as cows and humans, or
As a method for obtaining lactoferrin from these skim milk and whey, a method using a monoclonal anti-lactoferrin antibody (JP-A-60-166619, JP-A-60-14
No. 5200), a method using heparin-immobilized cross-linked cellulose or chitosan as a carrier (JP-A-63-25).
No. 5299), a method using a sulfated ester of a cross-linked polysaccharide as a carrier (Japanese Patent Application Laid-Open No. 63-255300), a method using a polysaccharide affinity carrier into which a sulfone group is introduced (Japanese Patent Application Laid-Open No. 3-109400). Japanese Patent Application Laid-Open No. 5-202098) discloses a method in which a cation exchanger is used as a carrier and elution is performed by adjusting ionic strength and pH.

The iron-saturated lactoferrin used in the present invention has an iron saturation of preferably 20% or more, more preferably 50 to 100%.

[0011]

BEST MODE FOR CARRYING OUT THE INVENTION The neutralizing agent for bacterial enterotoxin of the present invention contains iron-binding lactoferrin as an active ingredient. Preferably, the bacterial enterotoxin neutralizing agent is in the form of tablets, capsules, granules, powders, powders, etc., and orally administered. These dosage forms can be produced by a conventionally known method. For example, they can be molded by mixing with a carrier or an excipient that is acceptable in the production of a pharmaceutical preparation. In addition, the pharmaceuticals and foods and beverages imparted with the bacterial enterotoxin neutralizing function of the present invention can be prepared by mixing iron-binding lactoferrin with pharmaceuticals having various dosage forms, or milk, milk drinks, coffee drinks, juices, jellies, and biscuits. , Breads, noodles, sausages and the like, or various milk powders, as well as nutritional compositions for infants, infants, low birth weight infants, and the like.
Since these medicines and foods and drinks have a bacterial enterotoxin neutralizing function, they should be used for preventing or treating food poisoning caused by infection with cholera bacteria, toxogenic Escherichia coli, salmonella bacteria, etc. Can be.

In the present invention, in order to exert a bacterial enterotoxin-neutralizing function, the amount of iron-bound lactoferrin may be adjusted in an amount of 0.1 to 5,000 mg per day for an adult.

Next, the present invention will be described in detail with reference to examples and test examples.

[0014]

Example 1 Iron-bound lactoferrin (iron saturation 100
%) To a 1% aqueous solution of commercially available lactoferrin (manufactured by Oleofina), add a 0.1 M trisodium citrate solution containing 1/5 of 0.003 M iron (III) chloride, stir for 1 hour, and add iron saturation to 100%.
% Iron-bound lactoferrin solution was prepared. The lactoferrin contained in this solution had an iron saturation of 100% due to the binding of iron ions to all lactoferrin molecules.
%. Next, the iron-bound lactoferrin solution was sealed in a dialysis tube, dialyzed against distilled water to remove excess salts, and lyophilized.
An iron-bound lactoferrin powder having an iron saturation of 100% was obtained.

Iron-free lactoferrin (iron saturation 0
%, Apolactoferrin) A 1% aqueous solution of commercially available lactoferrin (manufactured by Oleofina) was sealed in a dialysis tube, and dialyzed against a 20-fold volume of 0.1 M aqueous citric acid solution containing 0.05% EDTA at 4 ° C. for 30 hours. . Subsequently, the dialysis tube was taken out and dialyzed against distilled water for 24 hours to prepare a non-iron-bound lactoferrin (apolactoferrin) solution having an iron saturation of 0%. With respect to lactoferrin contained in this solution, it was confirmed that iron ions were not bound to all lactoferrin molecules and the iron saturation was 0%. Next, the dialyzed solution was freeze-dried to obtain an iron-free lactoferrin powder having an iron saturation of 0%.

Iron-bound lactoferrin (iron saturation 20%
The iron-binding lactoferrin (iron saturation 100%) powder obtained preparation of fine iron saturation 50%) was dissolved in water, concentration prepare 10mg / ml of iron-binding lactoferrin (iron saturation 100%) aqueous solution At the same time, the obtained powder of iron-free lactoferrin (iron saturation 0%) was dissolved in water to prepare an aqueous solution of iron-free lactoferrin (iron saturation 0%) having a concentration of 10 mg / ml.

The aqueous solution of iron-binding lactoferrin (iron saturation 100%) and the aqueous solution of non-iron-binding lactoferrin (iron saturation 0%) are mixed at a ratio of 2: 8 to give an iron saturation of 20%. An iron-binding lactoferrin solution was prepared and freeze-dried to obtain an iron-binding lactoferrin powder having an iron saturation of 20%.

The aqueous solution of iron-binding lactoferrin (iron saturation 100%) and the aqueous solution of non-iron-binding lactoferrin (iron saturation 0%) are mixed at a ratio of 5: 5 to obtain an iron saturation of 50%. An iron-binding lactoferrin solution was prepared and freeze-dried to obtain an iron-binding lactoferrin powder having an iron saturation of 50%.

[0019]

[Test Example 1] Using iron-bound lactoferrin and iron-non-bound lactoferrin of each iron saturation prepared by the method shown in Example 1 as samples, the morphological changes of Chinese hamster ovary cells caused by toxogenic Escherichia coli enterotoxin or cholera toxin were tested. The inhibitory effect was investigated. The test was performed as follows.

(1) Determination of the concentration of toxinogenic Escherichia coli enterotoxin or cholera toxin used in measuring the activity of Chinese hamster ovary cell morphological change CFA, two types of enterotoxins having different antigenicities
/ I (LT-I) and CFA / II (LT-II) toxinogenic Escherichia coli H-10407-p strain (provided by Tokyo Metropolitan Institute of Public Health) is shake-cultured in a common CAYE medium, and then cultured. Was centrifuged, and the obtained supernatant was serially diluted from 1/10 to 1/1000. Next, each dilution was placed on ice
The sample was placed in a Lab-Tek8 chamber (manufactured by Flow Laboratories). In addition, cholera toxin CT (manufactured by Risto Biological Laboratories) is diluted in the same manner as described above.
It was placed in the ab-Tek8 chamber. Then, Dulbecco's medium containing 1% fetal calf serum was placed in each well of the Lab-Tek8 chamber.
(FCS / DMEM) 400 μl was added, and 10% FCS
/ Chinese hamster ovary K1 cultured in DMEM
5,000 cells / ml of cells (CHO-K1 cells, ATCC-CCL6)
Then, 50 μl of the cell suspension suspended in 10% FCS / DMEM was added thereto, and the cells were cultured overnight at 37 ° C. in an incubator having a 5% carbon dioxide concentration. After that, discard the medium,
The chamber was washed with phosphate buffered saline (PBS (-)), and the cells in the chamber were stained and air-dried.

According to the microscope, 200 to
Observe 400 cells, count the percentage of deformed cells,
For each toxin, the morphological change rate of CHO-K1 cells per each concentration was calculated. As a result, in cholera toxin CT, 10 μg / ml of toxinogenic Escherichia coli enterotoxin LT-
In I and LT-II, CHO-K
It was found that one cell changed its morphology by at least 70% or more. Therefore, in the following experiments, each concentration of each toxin was used.

(2) Confirmation of Inhibitory Effect on CHO-K1 Cell Morphological Change 10 μl of a culture supernatant containing cholera toxin CT at a concentration of 10 μg / ml or toxin Escherichia coli enterotoxin LT-I and LT-II was added. Lab-T on ice
ek8 chamber, and iron-bound lactoferrin (iron saturation 20%, 50% and 100%) and non-iron-bound lactoferrin (iron saturation 0%) were added to PBS (- ) Was added, and the mixture was shaken and reacted. Thereafter, methanol fixation and Giemsa staining were performed, and the morphological change rate of CHO-K1 cells was calculated.
The results are shown in FIGS.

According to this, iron-binding lactoferrin was compared to CHO-K1 in comparison with non-iron-binding lactoferrin.
It has an inhibitory effect on cell morphological changes, and has cholera toxin CT, enterotoxigenic Escherichia coli enterotoxin LT-I.
And LT-II were strongly neutralized. Further, the strength of the inhibitory effect on the CHO-K1 cell morphological change depends on the iron saturation of iron-bound lactoferrin, and it was found that the stronger the iron saturation, the stronger the effect.

(3) Confirmation of an inhibitory effect on the binding between the B subunit of cholera toxin CT and ganglioside GM1 As described above, in cholera toxin CT,
After the subunit binds to the cell via the ganglioside GM1 receptor on the cell surface, the A subunit invades the cell and develops toxicity. Therefore, the inhibitory effect on the binding between cholera toxin CT • B subunit and ganglioside GM1 was examined.

That is, an ethanol solution (1 μg / ml) of ganglioside GM1 was placed on a 96-well flat bottom plastic plate.
After adding μl, the ethanol was evaporated to dryness, and ganglioside GM1 was adsorbed to the plate. Next, a PBS solution of 1% bovine serum albumin (BSA / P) was placed on a 96-well flat-bottomed plastic plate on which the ganglioside GM1 was adsorbed.
BS) and leave for 1 hour, then 0.05% Tween 20
Was washed three times with PBS containing PBS (PBS Tween 20).

On the other hand, iron-binding lactoferrin (iron saturation
20%, 50% and 100%) and iron-free lactoferrin
(0% iron saturation) in 1% BSA / PBS solution, 25 μl of each sample and 1% BSA / PB of peroxidase-labeled cholera toxin CT / B subunit
An S solution (1 μg / ml) was mixed in advance with 25 μl and incubated at room temperature for 30 minutes to prepare a mixture.

Then, this mixed solution was added to a 96-well flat-bottomed plastic plate on which the above-mentioned ganglioside GM1 was adsorbed, left for 30 minutes, washed six times with PBS Tween 20, and washed with 2,2'-azinobis (3-ethyl Benzthiazoline-6 sulfonic acid) -2 ammonium salt (ABTS)
Add 100 μl of 0.2 M citrate buffer (pH 4.0) containing 0.006% hydrogen peroxide dissolved to a concentration of 0.3 mg / ml, and add
One minute later, the binding rate between cholera toxin CT • B subunit and ganglioside GM1 was calculated by measuring the absorbance at 405 nm. FIG. 4 shows the results.

According to the results, it was found that iron-bound lactoferrin strongly inhibited the binding between cholera toxin CT / B subunit and ganglioside GM1, as compared with iron-free lactoferrin. Further, it was found that this effect depends on the iron saturation of the iron-bound lactoferrin, and the higher the iron saturation, the stronger the effect.

[0029]

Example 2 After mixing the raw materials according to the formulation shown in Table 1, the mixture was tableted with a tableting machine to produce a tablet having a bacterial enterotoxin neutralizing function.

[0030]

[Table 1] 結合 Iron-bound lactoferrin (iron saturation 100%) 10.0 (weight %) Reduced maltose 52.0 Sugar 15.0 Lactose 18.7 Citric acid 2.0 Flavor 1.0 Lubricant 1.3 ────────────────────────────────

[0031]

Example 3 Raw materials were mixed according to the formulation shown in Table 2, filled in a container, and then sterilized by heating to produce a beverage having a bacterial enterotoxin neutralizing function.

[0032]

[Table 2] 結合 Iron-bound lactoferrin (iron saturation 100%) 2.0 (weight %) Fructose dextrose liquid sugar 8.0 citric acid 0.6 apple juice 10.0 water 79.4 ────────────────────────────────

[0033]

Industrial Applicability The neutralizing agent for bacterial enterotoxin of the present invention and the pharmaceuticals and foods and beverages imparted with the neutralizing function of bacterial enterotoxin exhibit an excellent neutralizing effect on bacterial enterotoxin. In addition, since iron-bound lactoferrin with an increased iron saturation is used, it can be supplied in large quantities at a relatively low cost, and has the features of extremely high safety.

[Brief description of the drawings]

FIG. 1 shows the inhibitory effects of iron-bound lactoferrin and non-iron-bound lactoferrin on cholera toxin CT-induced CHO-K1 cell shape change in Test Example 1.

FIG. 2 shows the inhibitory effects of iron-bound lactoferrin and non-iron-bound lactoferrin on CHO-K1 cell morphological changes caused by toxin Escherichia coli enterotoxin LT-I in Test Example 1.

FIG. 3 shows the inhibitory effects of iron-binding lactoferrin and non-iron-binding lactoferrin on CHO-K1 cell shape change by toxin Escherichia coli enterotoxin LT-II in Test Example 1.

FIG. 4 shows the inhibitory effects of iron-bound lactoferrin and non-iron-bound lactoferrin on the binding between cholera toxin CT / B subunit and ganglioside GM1 in Test Example 1.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁶ Identification code FI A23L 2/38 A61K 37/14 AGZ

Claims (4)

[Claims] 1. A bacterial enterotoxin neutralizing agent comprising iron-binding lactoferrin as an active ingredient. 2. The iron-bound lactoferrin having an iron saturation of 20.
The neutralizing agent for bacterial enterotoxin according to claim 1, which is 100% to 100%. 3. A medicament or food or drink having an iron-binding lactoferrin and imparting a bacterial enterotoxin neutralizing function. 4. An iron-bound lactoferrin having an iron saturation of 20.
The medicament or food or drink according to claim 3, wherein the amount is from 100% to 100%.