JP2629166B2 - Antibacterial agent effective against methicillin-resistant Staphylococcus aureus - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a medicament effective for treating and preventing intractable infections caused by methicillin-resistant Staphylococcus aureus.

[0002]

2. Description of the Related Art Staphylococcus aureus is a pathogenic bacterium that causes food poisoning and various purulent diseases. Its main habitat is skin, sebaceous glands, mucous membranes and the like of mixed animals,
It is a resident bacterium that is also detected in high concentrations from the nasal cavity.

[0003] In recent years, among these Staphylococcus aureus, bacteria having resistance to most antibiotics currently used, including β-lactam drugs, have appeared. This bacterium was transformed into methicillin-resistant Staphylococcus aureus (methicillin re-sistant Staphylococcs a).
ureus (hereinafter abbreviated as MRSA).

This Staphylococcus aureus was susceptible to penicillin G when chemotherapy with penicillin G was established, but thereafter, penicillin-resistant penicillin-resistant bacteria appeared. Furthermore, with the history of antibiotic use, resistance to most antibiotics such as tetracycline, chloramphenicol, aminoglycoside antibiotics, narrow-semi-synthetic penicillin, first generation cephem antibiotics, third generation cephem antibiotics, etc. The acquired Staphylococcus aureus has emerged.

[0005] Although MRSA is not usually a problem for healthy people, it is a problem in the surgical field as a causative bacterium of post-operative infection in post-operative patients.
It is the main causative bacterium of respiratory tract infection, biliary tract infection, and postoperative infectious enteritis. Once it develops, it is intractable and may fall from sepsis to severe multifunction dysfunction.

[0006] Hands, clothes, medical instruments and the like of healthcare workers are cited as causes of the occurrence and spread of infection, and infection in hospitals is a problem.

[0007] Since MRSA is becoming widespread in hospitals nationwide and there are few effective drugs, measures against infection and treatment methods have become important issues. As effective drugs for MRSA, minocycline, rifampicin, vancomycin, new quinolones However, since none of them show sufficient antibacterial activity, at present, treatment is generally carried out by using two or more drugs in combination.

[0008] In addition, since there is a high possibility that bacteria having resistance to these drugs will appear, it is strongly desired to develop an antibacterial agent having no sufficient bacteria and having sufficient antibacterial activity.

On the other hand, it has long been known that licorice root contains various antibacterial substances, but few reports directly mention the relationship between the antibacterial properties and components.

[0010] It is known that a crude extract of licorice root has an antibacterial activity against MRSA. Among the various components contained in the crude extract, specifically, which component is MRSA
No antibacterial activity has been known so far.

The present inventors have examined in detail the relationship between various components contained in licorice root and the antibacterial activity against MRSA. As a result, isoliquiritigenin or lycochalcone A strongly inhibits MRSA. The present invention has been completed.

[0012]

SUMMARY OF THE INVENTION Accordingly, an object of the present invention is to provide an antibacterial agent which is effective against methicillin-resistant Staphylococcus aureus without the appearance of resistant bacteria.

[0013]

SUMMARY OF THE INVENTION The above objects of the present invention are as follows.
This has been achieved by an antibacterial agent containing as an active ingredient isoliquiritigenin or lycochalcone A, which is a flavonoid component in licorice root.

The antimicrobial agent effective against methicillin-resistant Staphylococcus aureus of the present invention will be described below. Isoliquiritigenin and lycochalcone A used as an active ingredient of an antibacterial agent effective against methicillin-resistant Staphylococcus aureus in the present invention can be usually extracted from licorice root. An example of extracting A from licorice root will be described.

First, licorice root is extracted with a 70% aqueous acetone solution, and the extract is concentrated under reduced pressure. Next, the obtained concentrated extract is suspended in water and eluted with ether, ethyl acetate and n-butanol in that order to obtain a crude flavonoid fraction from the n-butanol-soluble fraction. The obtained crude flavonoid fraction is eluted with a mixed solution of chloroform and methanol (volume ratio 98: 2) using silica gel chromatography to obtain lycochalcone A and lycochalcone B.
Lycochalcone A and lycochalcone B can be separated by repeating silica gel chromatography.

By eluting with a chloroform-methanol mixture (volume ratio: 40: 1) after the above elution, isoliquiritigenin and liquiritigenin can be obtained. Isoliquiritigenin and Liquiritigenin can be separated by repeating silica gel chromatography.

The licorice root used as a raw material may be either a raw or dried plant of licorice or a homologous plant thereof, or a residue obtained by extracting a plant body with water to extract glycyrrhizin useful as a sweetener.

Further, since the structure of the compound having antibacterial activity used in the present invention has already been elucidated, it may be produced by synthetic or plant biotechnology.

When the isoliquiritigenin or lycochalcone A of the present invention is administered as a pharmaceutical, the degree of symptoms,
Although it depends on the age, weight, etc. of the patient, it is usually sufficient for an adult to administer about 50 mg to 500 mg per day, which can be administered once or several times a day.

The method of administration may be parenteral administration by intravenous injection, subcutaneous injection, intramuscular injection, etc., oral administration by tablets, capsules, granules, fine granules, powders, etc., or suppositories, external solutions, ointments, etc. And is not particularly limited.

The compound of the present invention can be formulated into various forms according to the administration method using additives commonly used in pharmaceutical preparations, and examples thereof include emulsions, wettable powders, aqueous solutions, Dosage forms such as tablets, capsules, powders, and pills can be exemplified. In addition, as additives used for formulation, commonly used additives such as excipients, disintegrants, binders, dispersants, plasticizers, fillers, and carriers may be used.

It should be noted that, as the glycosides of isoliquiritigenin, there are isoliquiritin, apioisoliquiritin and the like. In the case of oral administration and parenteral administration, it is decomposed in vivo to form isoliquiritigenin. Therefore, these glycosides may be used.

Since the compound of the present invention is a component contained in licorice root which has been used as a traditional Chinese medicine, it is not necessary to use a high-purity preparation. In the case of oral administration, the present compound is contained in a decoction or extract. If an effective amount of the compound of the invention is contained, it can be used.

In addition to being used as a medicine, it can be used in various applications for the purpose of preventing infection, for example, disinfection of medical equipment, disinfection of hands of healthcare workers, application to clothes of healthcare workers, and clothing of patients. It can be used in addition to, for example, application to food, besides application to beds, sheets, etc.

[0025]

The present invention will be described below with reference to examples.

Embodiment 1 FIG. Preparation of isoliquiritigenin and lycochalcone A Isoliquiritigenin and lycochalcone A used for anti-MRSA activity measurement were prepared by the following method. First, licorice root was extracted with a 70% acetone aqueous solution and concentrated under reduced pressure to obtain 2 kg of a crude extract. The obtained crude extract is suspended in water,
Elution was carried out with ether, ethyl acetate and n-butanol in that order to obtain 200 g as a crude fraction from the n-butanol soluble fraction. Further, the crude fraction was packed with silica gel in a column having an inner diameter of 10 cm and a length of 100 cm, and eluted sequentially with a chloroform-methanol mixture (volume ratio: 98: 2) and a chloroform-methanol mixture (volume ratio: 40: 1). Then, the eluted fraction of a chloroform-methanol mixture (volume ratio: 98: 2) was concentrated under reduced pressure to give a concentrated extract (1200 mg), and the eluted fraction of a chloroform-methanol mixture (volume ratio: 40: 1) was concentrated under reduced pressure. Thus, 2600 mg of concentrated extract was obtained. Further, the concentrated extract was chromatographed using a silica gel column having an inner diameter of 50 mm and a length of 500 mm with a chloroform-methanol mixture (volume ratio: 98: 2) to obtain 370 mg of a crude fraction containing lycochalcone A. The product was purified by high performance liquid chromatography using a silica gel column (ODS-120T manufactured by Tosoh Corporation) to obtain 150 mg of Ricochalcon A. Similarly, the concentrated extract was chromatographed using a silica gel column with an inner diameter of 50 mm and a length of 500 mm, eluting with a mixed solution of chloroform and methanol (volume ratio: 98: 2), and subjected to crude fractionation containing isoliquiritigenin. After obtaining 750 mg / min, the product was further purified by high performance liquid chromatography using a silica gel column (ODS-120T, manufactured by Tosoh Corporation) to obtain isoliquiritigenin 120.
mg and liquiritigenin, apioisoliquiritiligenin, and formononetin, respectively.
130 mg, 60 mg and 110 mg were obtained.

Embodiment 2 FIG. Measurement of Anti-MRSA Activity by Disk Method First, two strains derived from hospital patients were used as methicillin-resistant Staphylococcus aureus, and two strains derived from food poisoning were used as methicillin-resistant Staphylococcus aureus. The culture of these methicillin-resistant Staphylococcus aureus is based on Trypicase Soy Broth (B
BL) at 37 ° C. for 1 day, and dilute the culture solution to 10 ⁶ cfU / ml with sterile physiological saline to obtain a bacterial solution for inoculation. The diameter of the culture solution contains 20 ml of Mueller-Hinton agar medium (BBL).
0.05 ml of a Staphylococcus aureus suspension (bacterial solution for inoculation described above) was spread on agar in a 90 mm petri dish. In addition, a 1% concentration DMSO dilution of isoliquiritigenin and lycochalcone A
After 20 μl of the disc was infiltrated into a sensitivity measurement disc (8 mm thick Advantech), the disc was placed on the agar medium described above and cultured to examine the inhibition circle.

As subjects, liquiritigenin, apioisoliquiritigenin, and formononetin were similarly prepared, and the formation of an inhibition circle was examined. Table 1 shows the results.

[0029]

[Table 1]

Embodiment 3 FIG. Measurement of anti-MRSA activity (minimum inhibitory concentration by agar plate dilution method) Staphylococcus aureus (MRSA) used was derived from food poisoning.
3 strains, 3 strains from diarrhea or healthy carriers, 3 from hospital patients
A total of 49 strains of 9 strains were used. Staphylococcus aureus (MR
SA) used two strains derived from food poisoning. Culture is Trypicase
One day at 37 ° C. by Soy Broth (BBL). The culture solution was diluted to 10 ⁶ cfU / ml with sterile physiological saline to obtain a bacterial solution for inoculation. Dilutions were made by diluting Isoliquiritigenin and Lycochalcone A with DMSO, and 2 ml of each diluted solution and 18 ml of Mueller-Hinton agar medium (sterilized with autograve and cooled to about 50 ° C.) 90mm in diameter
In a Petri dish, allowed to stand at room temperature, solidified, and dried. The DMSO dilution was prepared so that it was finally contained in the agar medium at 1%, 0.1%, and 0.01%. Then, 5 μl of the above-described bacterial solution for inoculation was streaked on the agar medium, and cultured. (Minimum inhibitory concentration) was determined.
Table 2 shows the results.

[0031]

[Table 2]

[0032]

As described above, isoliquiritigenin,
Since all of Ricochalcon A show strong antibacterial activity against methicillin-resistant Staphylococcus aureus, an antibacterial agent containing flavonoid component isoliquiritigenin or lycochalcone A as an active ingredient in licorice root according to the present invention is MRS.
It was found that it can be effectively used for infections caused by A and infection prevention.

Claims (2)

(57) [Claims] 1. An antibacterial agent effective against methicillin-resistant Staphylococcus aureus, comprising isoliqueuritigenin, a flavonoid component in licorice root , as an active ingredient. 2. An antibacterial agent which is effective against methicillin-resistant Staphylococcus aureus, comprising ricochalcon A, a flavonoid component in licorice root , as an active ingredient.