JPH021806B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to antimicrobial compositions, particularly compositions effective in inhibiting the growth of harmful microorganisms on human skin. On human skin, Propionibacterium
Microorganisms of the genus Propionibacterium, such as Propionibacterium acnes and Propionibacterium avidum, are always present, and when these microorganisms multiply for some reason, they can worsen acne and cause skin inflammation. is believed to occur. Traditionally, antibiotics such as tetracycline, erythromycin, and clindamycin have been used to prevent the growth of microorganisms that induce skin inflammation, but these antibiotics have too broad an antibacterial spectrum and are not suitable for use on the skin. It has the disadvantage of killing even the most useful microorganisms. Therefore, the present inventors searched for an antibacterial substance that exhibits an action as selective as possible against harmful microorganisms on the skin, particularly Propionibacterium bacteria, and investigated various antibacterial substances, including those that have been reported to have antibacterial activity. Screening was conducted for the plant components of the following. As a result, in the slightly acidic pH range of 5.0 to 5.5, which is the same as human skin, no substance was found that independently exhibited the desired antibacterial activity; however, quercetin and rutin, quercetin and isorhamnetin-3-O-rutinoside, Moreover, it was discovered that a mixture of these three exhibits excellent antibacterial activity against Propionibacterium bacteria in the PH region of the skin surface, leading to the completion of the present invention. That is, the present invention provides an antibacterial composition characterized by containing quercetin and rutin and/or isorhamnetin-3-O-rutinoside in an amount (weight ratio) of 1 to 10 times that amount. . Furthermore, rutin and isorhamnetin
When 3-O-rutinoside is used in combination, the total amount thereof should be 1 to 10 times that of quercetin. The above three types of compounds constituting the composition of the present invention are flavonoids each having the following molecular structure. It has been previously reported that quercetin has weak antibacterial activity in weakly alkaline or weakly acidic conditions, but the present inventors have confirmed that its activity against Propionibacterium bacteria in weakly acidic conditions is extremely weak. It is. Therefore, quercetin alone cannot be used as an antibacterial agent for skin drugs or cosmetics. Furthermore, rutin and isorhamnetin-3-O-rutinoside exhibit weak antibacterial activity in weakly alkaline conditions, but do not exhibit any activity in weakly acidic conditions. However, when these three components are used in combination in a specific ratio as mentioned above, they exhibit a strong antibacterial effect under weakly acidic conditions. This could hardly be predicted from the confirmed characteristics. In addition, as will be described later, the Sophia extract, which is known to exhibit antibacterial activity, contains rutin and trace amounts of quercetin and isorhamnetin-3-O-rutinoside; The total amount of -3-O-rutinoside is usually 20 times or more that of quercetin, which is outside the weight ratio range of 1 to 10 at which the synergistic action confirmed by the present invention appears, and its antibacterial action is weak. The antibacterial spectrum of the antibacterial composition according to the present invention shows remarkable activity against Propionibacterium acnes and Propionibacterium avidum, which are resident bacteria on the skin, under slightly acidic conditions, and moderate activity against Staphylococcus aureus. However, it only acts bacteriostatically against E. coli and Pseudomonas aeruginosa. The above activity of the antibacterial composition of the present invention has a pH of about 5.0.
It is most excellent at weak acidity of ~5.5, and its activity decreases at neutral to weak alkalinity. In the case of a product containing only isorhamnetin-3-O-rutinoside as an auxiliary ingredient, when the pH becomes about 7.5 or higher,
Most of the activity is lost. On the other hand, those containing rutin as an auxiliary component exhibit weak activity in weak alkalinity, but this is the antibacterial activity exhibited by rutin alone. The concentration of quercetin when using the composition of the present invention usually needs to be about 0.2% (wt%) or more, and if it is less than 0.2%, quercetin and/or isorhamnetin-3-O-rutinoside No matter how large the amount is used, a sufficient level of antibacterial activity cannot be obtained. The preferred concentration range of quercetin is about 0.2-0.6%, and even if the concentration is higher than 0.6%, there is almost no increase in activity. Therefore, the quercetin concentration should be approximately 0.2-0.6%, and
~10 times the concentration of rutin and/or isorhamnetin-3-O-rutinoside will coexist. For example, when the concentration of quercetin at the time of use is 0.4%, if about 0.4% or more of rutin and/or isorhamnetin-3-O-rutinoside is allowed to coexist, a weak synergistic effect will appear, and the amount of coexistence will be about 2%.
% or more, the synergistic effect becomes particularly pronounced, resulting in extremely strong antibacterial activity. Even if rutin is increased further, the effect does not change much, and rutin is about 4
% or more, the activity actually decreases. The effective concentration of isorhamnetin-3-O-rutinoside is also
It is almost the same as that of rutin. Due to the above-mentioned properties, the antibacterial activity of the composition of the present invention can be applied to human skin, which is slightly acidic. It is suitable for use in skin medicines such as skin lotions, cosmetics, and the like. The antibacterial composition of the present invention can be applied to skin products such as skin drugs and cosmetics in which quercetin, rutin and/or isorhamnetin-3-O-rutinoside is blended with other drugs or cosmetic components. It may also be an antibacterial agent consisting of quercetin, rutin and/or isorhamnetin-3-O-rutinoside for inclusion in the above skin application products. To produce the composition of the present invention, quercetin, rutin and/or isorhamnetin-3-O-rutinoside are collected from a plant extract containing some or all of them and mixed in a suitable ratio. Bye. Examples of plants that provide extracts that can be used as raw materials for the compositions of the present invention include Sophora (scientific name: Sophora
japonica), and the dry powder of this drug sold as a crude drug usually contains 0.7 to 0.8 quercetin.
%, rutin 15-25%, isorhamnetin-3-O
- Contains about 1% of rutinosides. This can be extracted with a polar solvent such as ethanol or acetone, and necessary components can be separated and purified from the resulting extract to be used in the composition of the present invention. To give an example of a purification method, first, a Sophia extract was purified by chromatography using Sephadex LH-20.
It is divided into fraction A containing quercetin and fraction B containing rutin and isorhamnetin-3-O-rutinoside. Fraction B was suspended in water, and low polar substances were extracted and removed using a chloroform-methanol mixed solvent (9:1). A portion of rutin in the aqueous phase was crystallized with 50% methanol, and the remaining liquid was is fractionated by polyamide dry column chromatography to obtain a rutin-containing fraction C and an isorhamnetin-3-O-rutinoside-containing fraction D. When fraction D is purified by silica gel chromatography, an even more purified isorhamnetin-3-O-rutinoside fraction can be obtained. The weight ratio of rutin/quercetin in the Laminaria extract is usually about 20 or more and is in a range exceeding the upper limit of the rutin/quercetin ratio of 1 to 10 in the composition of the present invention mentioned above. In other words, there is more rutin than necessary compared to quercetin, so if you try to produce the composition of the present invention only from the Sophia extract, there will be a surplus of rutin.
Excess rutin can be converted to quercetin by treatment with β-glucosidase and hydrolyzed, which can be used in the composition of the present invention. Alternatively, by treating Sophora extract with β-glucosidase,
At that time, the product obtained by hydrolyzing only a portion of rutin so that the rutin/quercetin ratio in the treated product is approximately 1 to 10 may be purified and used in the composition of the present invention. . Furthermore, it has been confirmed that kaempferol-3-O-rutinoside, which is contained in about 0.2% in Sophora oleracea, is a substance that can play the same role as isorhamnetin-3-O-rutinoside in the composition of the present invention. Therefore, when producing the composition of the present invention using Lagomorpha as a raw material, it is desirable to use this substance without removing it. The present invention will be explained below with reference to Examples. Example 1 Quercetin, rutin and isorhamnetin-3
A methanol solution of each of the -O-rutinosides was prepared, and the antibacterial activity of these or a mixture thereof was examined by the method described below. Antibacterial activity test method: Sterilized GAM bouillon medium (PH5.5, agar concentration: 1.5% for base layer, 0.75% for upper layer)
Prepare. Substrate medium 16 in a 9cm diameter shearley
Sprinkle ml of Propionibacterium on top of this.
6 ml of medium for the upper layer (temperature: 50°C) containing 10 ⁷ to 10 ⁸ bacteria of Avidum (freshly planted and precultured for 18 to 20 hours) is spread and hardened. Apply 25 μl of the test solution to a paper disc with a diameter of 8 mm.
After drying this disk with a hair dryer, place it on the above medium. After culturing at 37℃ for 18 to 20 hours with the disk placed on it, the circular area around the disk where bacterial growth was inhibited (hereinafter referred to as inhibition circle)
Measure the diameter [mm] of. Table 1 shows the measurement results of the inhibition circle diameter.

【table】

[Table] Example 2 Quercetin solution with concentration of 8 mg/ml, concentration of 40 mg/ml
A rutin solution with a concentration of 20 mg/ml and an isorhamnetin-3-O-rutinoside solution with a concentration of 20 mg/ml (both methanol solutions) are prepared as test solutions. Separately, test bacteria Propionibacterium avidum are sown on GAM medium as soft agar, and on top of this, one or three of the above test solutions are added.
Place a dry 8 mm diameter paper disc with 25 μl applied. Measure the diameter [mm] of the inhibition zone after culturing for 18 to 20 hours. In the above method, the PH of the culture medium was varied in the range of 5.5 to 8.0, and the results obtained were judged according to the following criteria. Diameter of inhibition circle [mm] Antibacterial activity 0 - 0 or more and less than 12 ± 12 or more and less than 16 + 16 or more and less than 20 ++ 20 or more +++ The results are shown in Table 2.

[Table] Example 3 Add 0.6 g of quercetin, 2.0 g of rutin, and 0.4 g of isorhamnetin-3-O-rutinoside to a mixture of 70 ml of ethanol and 30 ml of 0.05M α-hydroxy-n-butyric acid buffer (PH5.0). Dissolve uniformly,
A liquid acne treatment was produced.

Claims (1)

[Claims] 1. Quercetin and 1 to 10 times its amount (weight ratio) of rutin and/or isorhamnetin-3
-An antibacterial composition characterized by containing O-rutinoside. 2. The composition according to claim 1, wherein the composition is a skin drug or cosmetic.