JPH0820510A - Spice having antimicrobial activity and antimicrobial agent prepared by using the same - Google Patents

Abstract

PURPOSE:To provide an antimicrobial agent having a wide-ranging antimicrobial activity on methicillin-resistant Staphylococcus (MRSA) and food spoilage microorganisms. CONSTITUTION:Spices and their extracts are screened to select ones having an antimicrobial activity on MRSA. As the result of the screening, it is found that clove, allspice, sweet marjoram, cinnamon, marjoram, rosemary, nutmeg and sage are effective. In addition, those spices and their extracts have a wide- ranging antimicrobial activity also on food spoilage microorganisms. Especially, clove, cinnamon, sweet marjoram and their extracts have an excellent antimicrobial activity on food spoilage microorganisms.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a drug effective for treating and preventing intractable infectious diseases caused by methicillin-resistant Staphylococcus aureus.

[0002]

2. Description of the Related Art Staphylococcus aureu
s) is a pathogen that is widely distributed in human skin, nasal vestibule, intestinal tract, and natural environment, and is known as a pathogen for food poisoning and purulent diseases. In other words, Staphylococcus aureus has been attracting attention as a bacterium that is most familiar and highly virulent as a bacterium that causes enteritis in humans. The treatment method of the disease (staphylococcus aureus infection) due to the infection of Staphylococcus aureus is the progress of modern antibacterial therapy starting with the antibiotic penicillin,
It has not been a problem for a long time because it has been considered that it has already been clinically solved with development.

However, since the late 1980s, the therapeutic problem of Staphylococcus aureus infection has reoccurred. Methicillin-resistant Staphylococcus aureus
resistant Staphylococcus aureus: hereinafter referred to as MRSA). Methicillin is an antibiotic for penicillin-resistant staphylococci that was first developed as an antibacterial drug that has efficacy against penicillin-resistant Staphylococcus aureus. Therefore, Staphylococcus aureus that is resistant to methicillin is given the name methicillin to
I am calling.

However, the essential aspect of MRSA is that it has the property of showing resistance not only to methicillin but also to almost all β-lactam agents currently on the market. β-
Antibacterial drugs called lactams include all penicillins including resistant penicillin for Staphylococcus, all cephems called 1st, 2nd and 3rd generation, or monobactams and carbapenems. , Contains most of the clinically used antibacterial drugs. Therefore, there is almost no effective β-lactam type drug, which is one problem of the current MRSA.

Furthermore, MRSA is not only resistant to β-lactam agents but also to many aminoglycoside and macrolide antibiotics, and is being resistant to recently developed new quinolone agents. In other words, MRSA is the most embodied bacterium of today's multidrug resistant bacterium.

Further, the larger the hospital is, the larger the MRSA becomes. This is because the larger hospitals have more patients with immunodeficiency due to leukemia, cancer, etc., severe burn patients, or patients with weakened immunity such as bedridden old people and premature babies. Due to this bacterium, which is easily established in hospitals, these patients are exposed to serious infectious diseases, which poses a serious social problem.

[0007] Therefore, it is an important problem to find a countermeasure for infection by MRSA or a method for treating MRSA. Chemotherapy (disinfection), sterilization / disinfection (disinfection of hands, sterilization / disinfection of machines / equipment, disinfection of oral cavity / ear / nose / throat), etc. have been carried out as measures against MRSA infection, but they are not sufficient. Although sterilizing agents, fungicides, fibers, etc. have been developed, they are not a fundamental solution.

Recently, licorice, which is a Chinese medicine as a naturally-occurring and highly safe antibacterial substance, has been introduced, but it has not yet been put to practical use (life hygiene (Seika
tsuEisei) 37,15-19 (1993)).

Spices have long been known to have antibacterial activity, and it has been reported that they have antibacterial activity against food poisoning bacteria such as Salmonella, pathogenic Escherichia coli, Vibrio parahaemolyticus, and Bacillus cereus, and Staphylococcus aureus. (Japanese Journal of Food Industry, 29, 112-116 (1982)). But M
No effective spices against RSA are currently known. Therefore, the present inventors have completed the present invention by researching spices and their extracts having antibacterial activity against MRSA.

[0010]

SUMMARY OF THE INVENTION The present invention has been made to solve the above problems, and its object is to have an effective antibacterial activity against MRSA and to develop resistant bacteria. The purpose is to provide an antibacterial agent that is free and highly safe.

Another object of the present invention is to provide an antibacterial agent against food poisoning which is highly safe and has a wide range of application.

[0012]

Means for Solving the Problems The spice of the present invention is MR
It is a spice having an antibacterial property against SA, and thereby the above-mentioned object is achieved.

The extract from the spices of the invention is MRSA
An extract having antibacterial properties against the above, whereby the above object is achieved.

Further, the antibacterial agent of the present invention is an antibacterial agent containing the above spice or extract having antibacterial properties against MRSA, whereby the above object can be achieved.

Furthermore, the spice or extract of the present invention has an antibacterial activity against MRSA and food poisoning bacteria, thereby achieving the above object.

The spices used in the present invention are those sold as spices, and any spices having antibacterial properties against MRSA can be used. Suitable spices include cloves, allspice, oregano, cinnamon, marjoram, rosemary, nutmeg, or sage.

The extract in the present invention may be any extract as long as it is an extract from spices and has an antibacterial property against MRSA. As spices, clove, allspice, oregano, cinnamon, marjoram, rosemary, nutmeg, or sage having the above-mentioned antibacterial activity can be preferably used, but the spices are not limited thereto. From the antibacterial activity, an extract from rosemary or sage is particularly preferable. Even if the spice has no antibacterial activity, if the extract has antibacterial activity, that spice can also be used.

The extract referred to here is an extract extracted from spices, a concentrated solution obtained by concentrating the extract, or a product obtained by drying the above extract or the concentrated solution, and further the above extract or concentrate. The product further purified from is also included.

The extract is obtained by extracting the spice with water or an organic solvent. Examples of organic solvents include hexane, acetone, dimethyl sulfoxide (hereinafter abbreviated as DMSO), alcohols such as methyl alcohol,
Examples include ethyl alcohol. Water or the above organic solvent is added in an amount of 20 to 10 times the weight of the spice, and the mixture is heated to room temperature or an appropriate temperature (for example, 25 to 50 ° C) and left for several hours to 1 day for extraction. To do. Preferably 5-
Extract at 37 ° C. using 10 volumes. The extract thus obtained can be used as it is or after further purification for the measurement of antibacterial activity.

For the purification, known methods such as extraction with a solvent, purification by an ion exchange resin or column chromatography can be used.

The antibacterial activity of the spices against MRSA was found to be hospital-derived S. aureus T581 (hereinafter SA. B strain), S. aureus T5.
82 (hereinafter SA.C strain), S.aureus T583 (hereinafter SA.D)
Strain). Trypticase Soy
It can be cultured in broth (BBS) medium at 37 ° C. for 18 to 24 hours, and the culture solution can be diluted to about 10 ⁶ cfu / ml with sterile physiological saline and used as a bacterial solution for sensitivity measurement. 0.1 ml of the obtained susceptibility measurement bacterial solution was smeared on Mueller-Hinton agar medium (BBL), the surface was dried, and then powdered spices were added.
It can be measured by applying 20 to 50 mg and culturing at 37 ° C. and measuring the formation and size of MRSA growth inhibition circles around spices. The antibacterial activity of the extract can be measured by the same method as the antibacterial activity of spices. That is, a sensitivity measuring disk (8 mm, thin: Advantech) impregnated with the extract obtained by the above method,
Instead of spices, place on an agar medium and culture, then MRSA
It can be measured by examining the formation of growth arresting circles and their size.

The spice or extract of the present invention is MR
Not only the antibacterial activity against SA but also the antibacterial activity against food poisoning bacteria, spices having such activity include clove, cinnamon or oregano.
The antibacterial activity of these spices or extracts is
The antibacterial activity against A can be measured in exactly the same manner. In addition, the extract MIC (minimun inhibitory concentrati
on) can be determined by serially diluting each extract and measuring the lowest concentration that can form an inhibition circle. Examples of food poisoning bacteria for which the spice or extract of the present invention is effective include Salmonella, pathogenic Escherichia coli, Bacillus cereus, or Campylobacter.

Bacteria that can be used to measure the antibacterial activity against food poisoning bacteria include the following strains: (1) Staphylococcus aureus: Staphylococcus aureus (S. aur) as methicillin-sensitive Staphylococcus aureus (MSSA)
eus) 209P (hereinafter SA.A strain) and egg-derived S. aureus
OCIS 501 (hereinafter SA.E strain), S.aure derived from food poisoning cases
us OCIS 41 shares, OCIS 49 shares, OCIS 62 shares, OCIS 67 shares, OC
IS 91 strain, OCIS 95 strain, hospital-derived S. aureus T581 (hereinafter SA. B strain), S. aureus T582 (hereinafter SA. C strain), Sa as methicillin-resistant Staphylococcus aureus (MRSA)
ureus T583 (hereinafter SA.D strain), S.au derived from food poisoning cases
reus OCIS 100 and OCIS 104, T655, T67 from hospital
2, 01, and 03, (2) Escherichia coli: Escherichia coli NIHJ JC2 (hereinafter, E
C. A strain), E. coli 41F (haemorrhagic Escherichia coli, hereinafter, EC.
B strain), E. coli H10407 (toxigenic E. coli, hereinafter, E
C. C strain) and E. coli B / M702 (toxogenic E. coli, hereinafter EC.D strain), sewage-derived E.colin OCIE 1501 (hereinafter EC.E strain), (3) Salmonella: food-derived Salmonella Enteritid
is 910225NIPT4 (hereinafter Sal.A strain) and 922701 PT4
(Hereinafter referred to as Sal. B strain), S. Enteritidis 90 derived from food poisoning
9342PT34 (hereinafter Sal. C strain), S. Typhimurium 8701
NM (hereinafter, Sal. D strain) and 8842MS (hereinafter, Sal. D strain).
E strain), (4) spore bacterium (Cereus bacterium): Bacillus cereus strain isolated from food poisoning and food poisoning cases (hereinafter, BC.A strain,
BC. B strain, BC. C strain, BC. Strain D, BC. E stock),
And (5) Campylobacter jejuni CIP 7
02 (hereinafter, CJ strain) and C. coli CIF 7080 (hereinafter, C
C stock).

The above strain is stored in the Osaka City Institute of Environmental Science.

The above-mentioned strain is a suitable medium such as Tryptica.
It can be used as a bacterial solution for susceptibility measurement by culturing in se Soy broth (BBS) medium at 37 ° C. for 18-24 hours, diluting the culture solution to about 10 ⁶ cfu / ml with sterile physiological saline. When using Campylobacter, blood agar (Brucella agar
(BBL) with 7% horse defibrinated blood) under aerobic condition,
Incubate at 37 ° C for 2 days and add about 10 ⁸ cfu / m in sterile saline.
A suspension in 1 may be used as well.

Using these strains, the spice of the present invention,
Or that the extract has antibacterial activity against food poisoning bacteria,
It can be measured in the same manner as the MRSA described above.

The spice or extract confirmed to have antibacterial activity by the above method can be used as an antibacterial agent against MRSA or food poisoning. Examples of the form of the antibacterial agent include parenteral administration forms such as intravenous injection, subcutaneous injection and intramuscular injection, oral administration forms such as tablets, capsules, granules, fine granules and powders, suppositories, external preparations and ointments. There are forms of local administration such as, but not limited to. Additives used for formulation include excipients, disintegrants, dispersants, plasticizers, fillers and the like. The dosage form includes, for example, emulsion, wettable powder,
Aqueous solutions, tablets, capsules, pills, pills and the like can be mentioned.

The MRSA of the present invention, or the spice or extract having antibacterial activity against food poisoning bacteria, is highly safe.
It is also possible to use as a disinfectant / bactericidal agent for preventive purposes, fibers having a bactericidal effect, indoor cleaning, cleaning of dishes, etc., or directly added to food.

The spices and the extracts from the spices having the antibacterial activity against MRSA and the antibacterial bacteria of the present invention will be described below with reference to Examples.

[0030]

【Example】

(Example 1) Primary screening First, 28 types of spices were used to perform a primary screening for the presence or absence of antibacterial activity against food poisoning bacteria including MRSA.

1) Spices used Red pepper, ginger, mustard, onion, garlic, clove, allspice, cinnamon, nutmeg, mace, cumin, metch, coriander, cardamom, dill, fennel, anise, star anise, turmeric, oregano, Sage, celery, thyme, marjoram,
Basil, laurel, rosemary, peppermint.

2) Strains used (1) Staphylococcus aureus: As methicillin-sensitive Staphylococcus aureus (MSSA), SA. A strain, SA. E shares, S.au
reus OCIS 41 shares, OCIS 49 shares, OCIS 62 shares, OCIS 67 shares, O
CIS 91 strain, and OCIS 95 strain, SA as methicillin-resistant Staphylococcus aureus (MRSA). B strain, SA. C strain, S
A. D strain, S. aureus OCIS 100, OCIS 104, T655, T67
2, 01, 03, (2) E. coli: EC. A strain, EC. B strain, EC. C stock,
EC. Strain D, and EC. E. strain, (3) Salmonella: Sal. A strain, Sal. B shares, S
al. C strain, Sal. Strain D, and Sal. E. strain, (4) spore bacterium (bacillus cereus): BC. A strain, BC. B
Strain, BC. C strain, BC. Strain D, BC. E strain, and (5) Campylobacter: CJ strain and CC strain.

3) Culture method and preparation of inoculum solution Trypticase Soy broth (BBS) was inoculated with the above strains,
It was cultured at 37 ° C for one day. The culture solution was diluted with sterile saline to 10 ⁶
It was diluted to cfu / ml to prepare a bacterial solution for inoculation.

In the case of Campylobacter, a bacterium that had been cultured in a blood agar medium at 37 ° C. for 2 days under microaerobic conditions was suspended in sterile physiological saline at 10 ⁸ cfu / ml and used in the same manner.

4) Primary screening for antibacterial activity of spices 90 ml in diameter containing 20 ml of Mueller-Hinton agar medium (BBL)
0.1 ml of the inoculum of each bacterium was smeared on the agar in the Petri dish, the surface was dried, 20-50 mg of each spice was added, and the plate was incubated at 37 ° C for one day. After the culture, those in which an inhibition circle was found around the spice were regarded as primary screening positive. The results are shown in Table 1.

[0036]

[Table 1]

The spices shown in Table 1 had antibacterial activity against all the strains used in the left column.

Example 2 Preparation of Spice Extract and Measurement of Antibacterial Activity (Secondary Screening) 28 kinds of spices were added to hexane, acetone, DMSO, and sterilized water at 10 w / v% of each spice. In this way, the mixture was immersed for 1 day, and each supernatant was used as an extract. However, the immersion in sterilized water was treated at 37 ° C. for 4 to 5 hours and then filtered through a membrane filter (Advantech) to obtain an extract. 20 μl of each spice extract was soaked in a disc for sensitivity measurement (8 mm, thin: Advantech), placed on each agar in the same manner as in the case of the above-mentioned primary screening, culturing, and formation of the inhibition circle. Examined.
Table 2 shows the results.

[0039]

[Table 2]

Antibacterial activity of the spice extract was not detected by the secondary screening against Escherichia coli, Salmonella, and Campylobacter, but the activity is shown in Table 3 using the concentrate described later. Therefore, it is likely that the concentration of the extract was low.

Staphylococcus aureus (MSSA and MRS
A), and for all the above used strains of Bacillus cereus, the spices listed in Table 2 hexane, acetone, D
All solvent extracts of MSO and water had antibacterial activity.

(Example 3) Measurement of MIC (minimum inhibitory concentration) of spice extract by disk method Spice extract concentrate (spice) which may have antibacterial activity by primary screening and secondary screening Was extracted with hexane, and hexane was removed under vacuum). D for each extract concentrate
In addition to MSO, a 10% DMSO solution was prepared, and this was used as a stock solution to prepare 2-fold serial dilutions with DMSO. Each of the diluted solutions was absorbed in the above-mentioned disc, and subsequently placed on agar smeared with the strain in the same manner as in the primary screening, and after the culture, formation of an inhibition circle around the disc was examined. For each bacterium, the highest dilution factor, that is, the lowest concentration in the dilution series in which the inhibition circle was observed was defined as the MIC by the disc method. The antibacterial activity against food poisoning bacteria is shown in Table 3, and Staphylococcus aureus (M
The antibacterial activity against RSA and MSSA) is shown in Table 4.

[0043]

[Table 3]

[0044]

[Table 4]

As a result of the above, allspice, cinnamon,
Cloves, nutmeg, marjoram, oregano, rosemary and sage show antibacterial properties against MRSA. In particular, among these spices, the extract concentrate of rosemary and sage shows high antibacterial activity with high MRSA activity. Also, cloves,
Since cinnamon and oregano also have antibacterial activity against food poisoning bacteria, they can be effectively used for prevention of food poisoning.

[0046]

INDUSTRIAL APPLICABILITY The MRSA of the present invention, or the spice or extract having antibacterial activity against food poisoning bacteria is highly safe, and therefore, in addition to its use as a drug, disinfection for the purpose of infection prevention and prevention -It can be used as a bactericidal agent, a fiber having a sterilizing effect, indoor cleaning, washing of dishes, etc., or can be directly added to food.

 ─────────────────────────────────────────────────── ─── Continuation of the front page (72) Inventor Yoji Ishii, 419 Nishitsutsumi, Higashi-Osaka-shi, Osaka (72) Inventor Junko Odate, 4-2-21 Nakaishikiri-cho, Higashi-Osaka, Osaka

Claims (12)

[Claims] 1. Methicillin-resistant Staphylococcus aureus (MRS)
A spice having antibacterial properties against A). 2. The spice is clove, allspice, oregano, cinnamon, marjoram, rosemary,
The spice according to claim 1, which is selected from the group consisting of nutmeg and sage. 3. An extract from spices, which has antibacterial activity against methicillin-resistant Staphylococcus aureus (MRSA). 4. The spice is clove, allspice, oregano, cinnamon, marjoram, rosemary,
Selected from the group consisting of nutmeg, and sage,
The extract according to claim 3. 5. Methicillin-resistant Staphylococcus aureus (MRS)
An antibacterial agent comprising a spice having an antibacterial property against A) or an extract from the spice, the extract having an antibacterial activity against MRSA. 6. The spice is clove, allspice, oregano, cinnamon, marjoram, rosemary,
The antibacterial agent according to claim 5, which is selected from the group consisting of nutmeg and sage. 7. The antibacterial agent according to claim 6, wherein the extract is an extract from rosemary or sage. 8. A methicillin-resistant Staphylococcus aureus (MRS)
Spices having antibacterial properties against A) and antibacterial activities against food poisoning bacteria. 9. The spice according to claim 8, which is selected from the group consisting of cloves, cinnamon or oregano. 10. A methicillin-resistant Staphylococcus aureus (MR)
SA) and spices having antibacterial properties against food poisoning bacteria,
Alternatively, an antibacterial agent containing an extract from spices, which has an antibacterial activity against MRSA and food poisoning bacteria. 11. The antibacterial agent according to claim 10, wherein the spice is selected from the group consisting of cloves, cinnamon or oregano. 12. The antibacterial agent according to claim 10, wherein the food poisoning bacterium is Salmonella, Escherichia coli, Bacillus cereus, or Campylobacter.