JP2004051523A - Infection-protecting agent - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide an infection-protecting agent for inhibiting the invasion of pathogenic bacteria participating in the crises and serious states of colds or the like and for inhibiting the adhesion of the pathogenic bacteria to epithelial cells to prevent the infections of such as upper and lower respiratory tract infections. <P>SOLUTION: This infection-protecting agent comprises amylase. A bacterium adhesion inhibitor comprises amylase. A composition for oral cavities contains amylase. Thereby, the adhesion of the bacteria to the epithelial cells can be inhibited to protect the infection of the upper and lower respiratory tracts with the bacteria. <P>COPYRIGHT: (C)2004,JPO

Description

[0001]
TECHNICAL FIELD OF THE INVENTION
The present invention relates to an infection preventive agent that suppresses the invasion of pathogens involved in the onset and severity of colds and the like into the body.
[0002]
Saliva is a component that supports the environment in the human oral cavity. Saliva is secreted from the three major salivary glands (parotid, submandibular and sublingual) and minor salivary glands, with a total daily secretion of approximately 1.5 L. Its functions include chewing and swallowing due to moistening and smoothing of food, washing, antibacterial action and protection of oral mucosa. 99.5% of saliva is water, but the remaining components are extremely complex, including organic components such as proteins, lipids and carbohydrates, inorganic components such as electrolytes and salts, and cells such as epithelium and leukocytes detached from mucous membranes. It consists of components and microorganisms resident in the oral cavity.
[0003]
Among them, more than 40 kinds of protein components in saliva, including polypeptides, are known electrophoretically, and have various physiological functions. For example, it has lysozyme, lactoferrin, peroxidase, sIgA which is an antibacterial component in the oral cavity, histamine, prostaglandin, PAF which is a chemical mediator mediating inflammatory response, substance P, VIP, a mucosal defense mechanism and agglutination of bacteria. Salivary mucins (MG1 and MG2), EGF and NGF, fibronectin, which are expected to be involved in tissue repair, cystatin, a protease inhibitor that acts as a protective agent, and suppresses crystallization of calcium phosphate in the oral cavity. Statin, proline-rich protein, etc., which inhibit the formation of debris, are known, and all components protect the oral cavity, which is the entrance to the digestive tract and lower respiratory tract, from harmful stimuli such as bacterial infection and trauma, and repair tissue. Has a role to promote.
[0004]
On the other hand, it is known that upper and lower respiratory tract infections such as pneumonia and bronchitis are caused through infection of the pharynx (primary infection) by pathogens. It has been reported that Haemophilus influenzae, which is the causative bacterium, adheres to pharyngeal epithelial cells by adhering to ganglioside D2, which is a receptor component of pharyngeal epithelial cells, and proliferates.
Therefore, as a preventive measure against upper and lower respiratory tract infections, to suppress the growth of pathogenic bacteria in the oral cavity or pharynx, it is effective to inhibit the adhesion of pathogenic bacteria to epithelial cells in the oral cavity or pharynx.
[0005]
[Problems to be solved by the invention]
An object of the present invention is to provide an agent for suppressing the attachment of pathogenic bacteria to epithelial cells and preventing infectious diseases such as upper and lower respiratory tract infections.
[0006]
[Means for Solving the Problems]
The present inventors have found that amylase suppresses bacterial adhesion to epithelial cells and is useful for preventing infectious diseases via the pharynx.
[0007]
That is, the present invention provides an infection protective agent comprising an amylase and suppression of bacterial adhesion to epithelial cells.
[0008]
The present invention also provides an oral composition containing amylase.
BEST MODE FOR CARRYING OUT THE INVENTION
Amylase is a generic term for enzymes that act on starch to produce glucose, maltose, and oligosaccharides. Α-amylase that produces oligosaccharides by randomly decomposing α-1,4-glucoside bonds in starch and starch in starch β-amylase, which decomposes successively in maltose units from the non-reducing terminal of the α-1,4-glucoside-linked chain, glucoamylase, which produces glucose from the non-reducing terminal in starch, α-1,6-glucoside in starch There are branching enzymes such as pullulanase and isoamylase that degrade the binding chain.
Such amylase is used as a desizing agent for fibers, food processing such as production of isomer sugar and prevention of aging of bread, and an enzyme for diagnosis. It is not known at all that it can be used as food.
[0009]
As the amylase in the present invention, the above-mentioned amylase can be used, and those having various origins such as human saliva, microorganisms, and plants can be used.
Among them, α-amylase is preferable from the viewpoint of the protective effect against infection, and α-amylase derived from human saliva is particularly preferable.
[0010]
Such an amylase can be obtained, for example, from Bacillus genus, Aspergillus genus microorganism, barley malt, or human saliva by extraction, separation and purification according to a conventional method. Commercially available products include Type II-A, Type VIII-A, Α-amylases such as TypeIX-A, TypeX-A, and TypeXIII-A (all manufactured by Sigma) can be used.
Alternatively, it can be obtained by extracting an amylase gene from a eukaryote as a synthetic amylase, preparing a plasmid, introducing the plasmid into Bacillus subtilis, yeast, or the like, and producing the recombinant amylase.
[0011]
Such an amylase has an effect of adhering to and trapping bacteria and suppressing the attachment of extracellular matrix to fibronectin in human epithelial cell tissues, as shown in Examples described later.
Fibronectin is a glycoprotein that exists on the surface of normal fibroblasts and endothelial cells, on the basement membrane of intestinal epithelial cells, and on the adhesion surface between various cells. (A. Ljungh et al., 1996. FEMS Immunol. Med. Microbiol. 16, 117-126), if it is possible to suppress and control bacterial adhesion to fibronectin, the upper and lower airways Infection can be effectively prevented.
[0012]
Bacteria which can be protected by amylase of the present invention, bacteria infecting attached and fixed on fibronectin, for example, Haemophilus influenzae (Haemophilus influenzae), Staphylococcus aureus (Staphylococcus aureus), Pseudomonas aeruginosa (Pseudomonas aeruginasa), pneumococcus (Streptococcus pneumoniae ), Branhamella catarrhalis, and the like. Among them, the infection protective agent and the bacterial adhesion inhibitor of the present invention are particularly effective against Haemophilus influenzae and Staphylococcus aureus, which are causative bacteria of upper and lower respiratory tract infections.
[0013]
The amylase of the present invention can be applied alone to animals including humans as a protective agent for infection or an inhibitor of bacterial adhesion to epithelial cells.
Such an infection protective agent and a bacterial adhesion inhibitor to epithelial cells can be used as various foods and drinks, pharmaceuticals and animal feeds. (Powder, granule, etc.), paste, liquid, spray or suspension, and if necessary, add various components commonly used in the production of foods and beverages such as sweeteners, sour agents, vitamins, etc. Then, it can be formulated as an oral composition such as candy, chewing gum, troche, soft drink, confectionery and the like. In particular, a form of an oral composition such as candy, chewing gum, troche and the like which stays in the oral cavity for a relatively long time is preferable because the infection protective effect is more effectively exhibited.
[0014]
In addition, when used as a pharmaceutical, it can be administered in various forms, but preferably includes oral administration by tablets, capsules, granules, powders, syrups, etc., as well as tube administration and enteral administration. it can. These preparations can be formulated by appropriately mixing excipients, binders, disintegrants, lubricants, flavoring agents, solubilizing agents, suspending agents, coating agents and the like according to a conventional method.
[0015]
The daily dose of the above-mentioned preparations or foods and drinks depends on the patient's condition, body weight, age, sex and the like, but it is usually about 50 mg to 5 g, preferably about 0.2 to 2 g per day for an adult as amylase. This is preferably applied once or twice or four times a day.
[0016]
【The invention's effect】
ADVANTAGE OF THE INVENTION According to this invention, adhesion of bacteria to an epithelial cell can be suppressed and the upper and lower respiratory tract infection by the said bacteria can be prevented. The amylase of the present invention is extremely safe and harmless because it is a component of saliva and the like.
[0017]
【Example】
Example 1 Adhesion of RI-Labeled Bacteria to Salivary Amylase (1) Preparation of RI-Labeled Bacteria The Staphylococcus aureus was prepared by cryopreserving a purchased strain Staphylococcus aureus (IFO13276) by brain heart infusion ( (BHI) Agar medium was streaked, incubated at 37 ° C. in humidified air of 95% air and 5% carbon dioxide, and cultured overnight. The grown colonies were cultured in a BHI liquid medium (10 mL), and 1 mL of exponentially growing cells (2 to 3 at OD 600 nm) were used to label cell-labeled [ ³⁵ S] amino acid @ 70% [ ³⁵ S] methionine, 30% [ ³⁵ S] The cells were cultured for 18 hours in a BHI liquid medium (10 mL) supplemented with 5.3 MBq of cysteine II (manufactured by Amersham Pharmacia), and labeled. The labeled cells were collected by centrifugal washing (2800 rpm, 5 min, 20 ° C.) three times with a KCl buffer ( ^* KCl). The labeled cells were prepared at 5 × 10 ⁸ cells / mL (0.5 at OD600 nm) with ^** 1% BSA-KCl and used for the adhesion experiment.
^* KCl: 50 mM KCl, 1 mM K ₂ HPO ₄ KH ₂ PO ₄ (pH 6.0), 1 mM CaCl ₂ , 0.1 mM MgCl ₂
^** 1% BSA-KCl: ^* KCl solution of 1% bovine albumin final concentration
(2) Preparation of sample As a saliva sample, 5 mL of human whole saliva was collected, the supernatant (U) centrifuged at 2800 rpm, 10 min, and 4 ° C, and Type IX-A (manufactured by Sigma) and Type XIII-A (manufactured by Sigma) as salivary amylase. A solution (IX-A, XIII-A) was prepared by dissolving Sigma) in a 1 mM CaCl ₂ aqueous solution to a protein concentration of 0.05 to 0.5 mg / mL.
[0019]
(3) SDS-PAGE
20 μL of 2-mercaptoethanol was added to 980 μL of Laemmli Sample Buffer (manufactured by Bio-Rad) to prepare a sample buffer. Sample: Mix sample buffer at 1: 2, boil for 5 minutes at 100 ° C., apply 30 μL to ready-gel 10%, 7 comb (manufactured by Bio-Rad), and apply electricity at 20 mA constant current per gel for 90 minutes. Electrophoresis was performed.
[0020]
(4) The gel after SDS-PAGE was placed in a Western blot blotting buffer, shaken for 10 to 30 minutes, and a Polyvinylidene difluoride (PVDF) membrane was immersed in methanol for 30 seconds, immersed in a blotting buffer and shaken for 10 minutes or more. Filter paper No. 526 (8 sheets + one sheet obtained by cutting the size of the gel) (manufactured by Advantech) was immersed in the blotting buffer. Milli-blot (made by Millipore) is placed on the electrode plate so that air bubbles do not enter the four filter papers, then a hollow filter paper is set, a PVDF membrane is placed in the cut-out holes, and the blotting buffer is sufficiently filled. The gels that had been equilibrated were overlaid. The remaining four filter papers are overlapped in the same manner as the first four papers, a small amount of blotting buffer is applied to the top filter paper, the upper electrode plate is attached, and the lower +, upper-, constant current 2.3 mA / cm ² And transferred for 60 minutes. After the transfer, the mixture was gently shaken with 1% BSA-KCl for 1 hour or more.
[0021]
(5) Adhesion experiment The prepared [ ³⁵ S] -labeled bacterial cell solution and the transferred PVDF membrane were subjected to a gentle shaking reaction for 60 minutes. After the attachment reaction, the plate was gently shaken and washed 5 times with 1% BSA-KCl for 5 minutes. After air drying, a BAS-MS2040 imaging plate (manufactured by Fuji Film Co., Ltd.) was brought into close contact with the membrane on the adhesion reaction side, and after 3 to 4 days, analysis was performed with a BAS2500 image reader.
[0022]
As a result, it was confirmed that [ ³⁵ S] -labeled bacterial cells adhered to human salivary amylase (FIG. 1).
[0023]
Example 2 Trap Effect on Fluorescently Labeled Bacteria (1) Preparation of Bacteria:
Staphylococcus aureus was prepared by streaking a frozen strain of Staphylococcus aureus (IFO13276) on brain heart infusion agar, incubating at 37 ° C. in humidified air of 95% air and 5% carbon dioxide at 1 ° C. An overnight culture was performed. The grown colonies are cultured in a brain heart infusion liquid medium, and 6 mL of exponentially growing bacterial cells (2 to 3 at OD 600 nm) are diluted with phosphate buffer (PBS: KH ₂ PO ₄ 0.144 g / N, NaCl 9.0 g / N). L, Na ₂ HPO _{4 (} 0.795 g / L) and centrifugal washing (10000 rpm, 3 min, 4 ° C.) three times to collect the cells. Alexa Fluor 488C ₅ maleimide, sodium salt (manufactured by Molecular Probe) 1 mg / PBS 5 mL solution 1 mL was added to the cells, and the mixture was stirred and stored at 4 ° C. overnight under light shielding. The reaction cells washed by centrifugation three times with PBS (10000rpm, 3min, 4 ℃ ) to obtain a fluorescence-labeled bacterial cells by, prepared in 10 ⁸ / mL.
[0024]
Preparation of Haemophilus influenzae was performed by streaking a cryopreserved isolate of Nagasaki University Tropical Medicine Institute, Haemophilus influenzae (H95-135), on a chocolate agar medium at 37 ° C in 95% air and 5% carbon dioxide in humid air. And cultured overnight. The cells grown on four chocolate agar media were scraped off, washed three times with PBS (10000 rpm, 3 min, 4 ° C.) and collected. To the cells, 1 mL of Alexa Fluor 488C ₅ maleimide, sodium salt (Molecular Probe) 1 mg / PBS 5 mL solution was added, and the mixture was stirred and stored at room temperature for 3 hours under light shielding. The reaction cells washed by centrifugation three times with PBS (10000rpm, 3min, 4 ℃ ) to obtain a fluorescence-labeled bacterial cells by, prepared in 10 ⁸ / mL.
[0025]
(2) Trap test A 0.5% aqueous solution of human salivary amylase TypeIX-A (manufactured by Sigma) and a fluorescently labeled cell were mixed and reacted at a ratio of 1: 1. After the mixing reaction for 1 hour, the reaction solution was brought into contact with a human fibronectin-coated 24-well plate, which is a human epithelial cell model. After the contact for 1 hour, the plate was washed five times with PBS, and the adherence rate of the pathogenic bacteria was measured by measuring the fluorescence. As a control, the adhesion rate was calculated assuming that a sample reacted with PBS instead of an aqueous 0.5% amylase solution was 100%.
[0026]
As a result, when S. aureus was trapped with amylase, the adherence rate of S. aureus was 42% (FIG. 2a), and when T. influenzae was trapped, the adherence rate of H. influenzae was 12% (FIG. 2b). Was found to inhibit the adhesion of Staphylococcus aureus and Haemophilus influenzae.
[0027]
[Brief description of the drawings]
FIG. 1 is a graph showing the action of [ ³⁵ S] -labeled cells on salivary amylase.
FIG. 2 (a) is a diagram showing the effect of inhibiting the adhesion of Staphylococcus aureus (trap test).
(B): It is a figure which shows the adhesion suppression effect (trap test) of H. influenzae.

Claims (3)

Infection protective agent consisting of amylase. An inhibitor of bacterial adhesion to epithelial cells consisting of amylase. An oral composition containing amylase.

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