JP2996838B2 - Antibacterial agent - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to an antibacterial agent, and more particularly to a highly safe and excellent antibacterial agent containing a natural organic compound as an active ingredient.

[0002]

2. Description of the Related Art Heretofore, there has been no known example in which an antibacterial substance which is derived from a natural product, particularly a citrus peel, has excellent antibacterial activity and has high safety, has been isolated and purified, and the structure has been successfully determined.

[0003] On the other hand, psoralen, a compound having a coumarin bone nucleus, ie, flo [3,2]
-G] coumarin, also known as 7H-furo [3,2-g] [1]
Benzopyran-7-one) and its derivatives are used in microorganisms (for example, caries, periodontal pathogens, and fungi parasitic on plants).
As for its excellent antibacterial activity, no other reports have been made, not only patents.

[0004]

At present, chemically synthesized products are unavoidably used as antibacterial agents, but they have a problem in terms of safety to the human body and the environment. Therefore, the development of an excellent antibacterial agent having high safety is expected in the art.

[0005]

SUMMARY OF THE INVENTION The present invention has been made to meet the needs of the industry described above, and has focused on natural products from the viewpoint of safety.

Accordingly, the present inventors have conducted intensive studies for antibacterial substances derived from natural products, and have confirmed that citrus peels such as lemon and grapefruit contain antibacterial substances. In addition to successfully separating and purifying antibacterial substances, three purified substances (LE-
I, LE-II, LE-III) were successfully determined for their structures, and furthermore, the excellent antibacterial properties of these purified substances were confirmed, and the present invention was finally completed.

That is, the present invention relates to LE-I, LE-II,
And / or an antimicrobial agent containing LE-III as an active ingredient. These LE substances are natural substances derived from citrus peel and are coumarin derivatives, and as a result of structure determination, LE-I is 8-geranoxysoralen (8-ger
anoxypsoralen), LE-II is 5-geranoxypsoralen (5-geranoxypsoralen)
n), LE-III is 5-geranoxy-7-methoxycoumarin (5-geranoxy-7-methyoxyc)
oumarin).

[0008] These LE substances exhibit excellent antibacterial activity and are extremely useful as antibacterial agents. Hereinafter, examples of the production method, structure determination, and antibacterial property of the LE substance will be described.

[0009]

Example 1 The skin of lemon pomace ground with a mixer was extracted by leaving it in methanol at 37 ° C. for 3 days. This was concentrated with an evaporator, and the concentrate was diluted with water and subjected to silica gel chromatography.
The active substance was adsorbed on the resin. This was eluted with 60% methanol to elute impurities and then with 80% methanol to obtain a sample containing the active substance. Preparative HPLC
And three fractions showing activity were obtained. This 3
The solubility was lowered by gradually adding water to the methanol solution of the fraction, and crystallization was performed by repeating heating and cooling. This crystallization (recrystallization) was repeated to obtain three purified substances (LE-I, II, III).

Each of these LE substances has ¹ H
NMR and ¹³ C NMR analyzes were performed to obtain the results shown in Tables 1, 2, and 3.

[0011]

[Table 1]

[0012]

[Table 2]

[0013]

[Table 3]

From these results, the structure of the LE substance was determined, and LE-I was determined to be 8-geranoxysporale.
n (the chemical structural formula is shown in Chemical formula 4), and LE-II
Was identified as 5-geranoxysporalen (chemical structure is shown in Chemical Formula 5), and LE-III was 5
-Geranoxy-7-methoxycoumar
in (chemical structural formula is shown in Chemical formula 6).

[0015]

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[0016]

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[0017]

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[0018]

Example 2 The antibacterial activity of the LE substance against caries and periodontal disease bacteria was measured and confirmed as follows.

For tooth decay, S
treptococcus mutans ATCC
7270 was used. For periodontal disease, the bacteria responsible for gingivitis and alveolar pyorrhea were used. That is, as the causative bacteria of gingivitis, Prevotellaintermedia and A
ctinomyces viscosus NIAH
Using 1010, as the causative bacteria of alveolar pyorrhea, Porp
hydromonasgingivalis 381 was used. And S. mutans and A.M. visc
As a growth medium for P. osus , a brain heart infusion (BHI) medium was used . inte
rmedia and P. A gum (GAM) medium was used as a growth medium for gingivalis .

The measurement of the growth inhibitory activity of the LE substance against these fungi was carried out by inoculating 10 ⁵ cells / ml of the bacteria into ⁵ ml of a broth medium containing these three samples.
Culture was performed at 37 ° C. for 2 days. However, P. ging
Ivalis was cultured in an anaerobic jar (manufactured by BBL) because the present bacterium is an obligate anaerobic bacterium. Thereafter, 0.1 ml of the culture solution was added to the agar medium, and this was added at 37 ° C. for 2 hours.
After culturing for days, the presence or absence of colonies was observed.

The antibacterial property was judged to be sterile when there was no colony. Then, the correlation between the sample concentration and the antibacterial effect was examined, and the strength of the activity was indicated by the minimum inhibitory concentration (MIC) at which the same bacteria was sterilized at the minimum concentration. Also, LE-I, II, III
Was dissolved in methanol, and it was confirmed that the added amount of methanol did not affect the growth of the bacteria. The results are shown in Table 4 below.

[0022]

[Table 4]

[0023]

Example 3 The activity of the LE substance to inhibit the growth of mold during fruit storage was measured and confirmed as follows.

The test mold was isolated from the lemon fruit that had developed mold. And Penicill of green mold disease
ium digitatum , Penicci of blue mold
A mixed spore of lium italicum was obtained.
As a growth medium, a potato dextrose agar medium was used.

To measure the growth inhibitory activity of the LE substance against these molds, 5 ml of an agar medium containing these three types of samples was prepared in a petri dish, and a hole having a diameter of 2 mm was formed at the center of the petri dish. There, 10 ⁵ spores / ml of inoculum to 25 ° C. The mold spores in equal amounts (5 [mu] l), 3 to 7
Cultured for days. The diameter of the mycelia of the grown mold was measured, and the degree of proliferation was observed. LE-I, II, and III were dissolved in methanol, and it was confirmed that the added amount of methanol had no effect on mold growth. The results are shown in Table 5 below.

[0026]

[Table 5]

[0027]

The antibacterial agent of the present invention is extracted from citrus peel, has extremely high safety, and is effective for use as a fungicide for fruits and vegetables. In addition, it has a high activity of inhibiting the growth of dental caries and periodontal disease bacteria, and thus can be appropriately added to, for example, toothpastes, mouthwashes, various foods, and the like.

Further, since the structure of the antibacterial agent has been determined according to the present invention, it is expected that a novel antibacterial agent can be created by producing derivatives of these compounds.

────────────────────────────────────────────────── ─── Continued on the front page (51) Int.Cl. ⁷ Identification code FI A61K 35/78 A61K 35/78 K // C07D 311/18 C07D 311/18 493/04 106 493/04 106C (56) References JP-A-5-2508 (JP, A) Agric. Food Chem. , 40 [7] (1992) pp. 1217-1221 Photochem. Photobiol. , 55 [4] (1992) pp. 529-532 J.C. Agric. Food Chem. , 35 [4] (1987) pp. 509-511J. Agric. Food Chem. , 27 [6] (1979) pp. 1334− 1337 (58) Field surveyed (Int.Cl. ⁷ , DB name) C07D 493/04 C07D 311/00-311/96 A61K 31/00-31/80 A61K 7/16-7/30 A01N 43 / 90 A61K 35/78 CA (STN) REGISTRY (STN)

Claims (6)

(57) [Claims] The present invention relates to 8-geranoxypsoralen represented by the following chemical formula (1).
An antibacterial agent comprising EI) as an active ingredient. Embedded image 2. The antibacterial agent according to claim 1, wherein LE-I is derived from a natural product such as citrus. 3. The LE-I according to claim 1, which has an antibacterial property, further comprising 5-geranoxypsoralen represented by the following chemical formulas (2) and (3).
en, LE-II) and / or 5-geranoxy-7-
Methoxygmarin (5-geranoxy-7-met)
3. Antibacterial agent according to claim 1 or 2, wherein hoxycoumarin (LE-III) is mixed. Embedded image Embedded image 4. The antibacterial agent according to claim 1, wherein the antibacterial agent is an antibacterial agent against caries and periodontal pathogens.
The antibacterial agent according to item. 5. The antibacterial agent according to claim 1, wherein the antibacterial agent is a fungicide. 6. The antibacterial agent according to claim 1, wherein the antibacterial agent is a fungicide for storing and transporting fruits and vegetables.