Provided is an antibacterial agent exhibiting an excellent antibacterial activity effect and exerting a mild influence on the environment and humans. The antibacterial agent of the present invention comprises, as an effective component, a mixture of coumarin analogues extracted from a citrus fruit pericarp, particularly a mixture of coumarin analogues obtained from citrus cold press oil.

PREFFERED EMBODIMENT OF THE INVENTION

The antibacterial agent meant in the present invention is an agent comprising, as an essential component, a mixture of coumarin analogues obtained from the pericarp of citrus fruits. Although the mixture of coumarin analogues may be obtained from the above pericarp by extraction with a solvent, it may also be obtained from citrus cold press oil derived from the pericarp of citrus fruits.

The mixture of coumarin analogues meant in the present invention is obtained from the pericarp of citrus fruits and a mixture containing plural compounds selected from compounds having a coumarin skeleton and compounds having a furocoumarin skeleton. There are many known coumarin analogue mixtures. Typical examples among these mixtures include auraptene, marmin, limettin, melanzin, 5-geranoxy-7-methoxycoumarin, citropten, bergapten, bergamottin, bergaptol, epoxybergamottin, dihydroxybergamottin and 5-geranoxy-psoralen. However, these compounds are not intended to be limiting of the present invention.

The higher the content of the above coumarin analogue in the mixture of compounds obtained from the pericarp of citrus fruit, the more greatly the antibacterial ability is improved and so a higher content of the coumarin is advantageous. In the antibacterial agent of the present invention, the coumarin analogue is contained in the mixture in an amount of 40% by weight or more, more preferably 60% by weight or more and still more preferably 80% by weight or more in view of operability when it is added and compounded.

The citrus cold press oil meant in the present invention has been widely known. The citrus cold press oil is usually prepared from citrus fruits, particularly, from the pericarp thereof. The citrus cold press oil may be prepared from citrus fruits though a commercially available one may be used usually as the citrus cold press oil. As the citrus fruit, lemon, orange, lime, grapefruit, bergamot and the like are known. Among these fruits, lemon and lime are preferable in particular.

Next, explanations will be furnished as to a method for obtaining a coumarin analogue from the citrus cold press oil.

The above citrus cold press oil is separated into a high-boiling point fraction and a low-boiling point fraction by a usual method. For example, when the citrus cold press oil is fractionated by a distillation method, the citrus cold press oil is introduced into a distiller and gradually heated under reduced pressure. Then, the distillate is called a low-boiling point fraction and the residue left in the distiller is called a high-boiling point fraction. In the present invention, the high-boiling point fraction means the residue left after the citrus cold press oil is heated at 90 to 120.degree. C. under reduced pressure (e.g., about 133 kPa). This high-boiling point fraction is a mixture consisting of nonvolatile components.

Then, this high-boiling point fraction is further fractionated. As the fractionating method, various methods are known. As a typical method, a method of fractionating using a silica gel chromatographic method will be explained.

First, the above high-boiling point fraction may be pretreated in advance. For instance, it may be thickened by heating or made to have a low viscosity by adding a solvent. In this case, it is generally preferable to add the solvent in an amount of 0.1 to 30 parts by volume and preferably 0.5 to 20 parts by volume based on one part by weight of the extract.

Then, a method may be adopted in which the high-boiling point fraction is poured into, for example, a column for chromatography which is made and adjusted in advance, then an eluent constituted of a solvent is poured into the column to flow out the fraction retained temporally in the column with the solvent and the flown-out solvent is divided into several parts by a known means. In this invention, hydrocarbons such as n-pentane, n-hexane, branched hexane, benzene and toluene may be used as a non-polar solvent. Also, as a polar solvent, esters such as methyl acetate and ethyl acetate, ethers such as ethyl ether and alcohols such as methanol, ethanol and propanol can be used. However, the solvent to be used in the present invention is not limited to these solvents.

In the case of using usual silica gel chromatography, it is preferable to flow out each fraction by using n-hexane, ethyl acetate or a mixed solvent of these solvents. When the mixed solvent is used, there is no particular limitation on the proportion of each solvent. As to the elution temperature, the elution is carried out at ambient temperature. However, no particular limitation is imposed on the temperature and the elution may be carried out either at low temperatures or at high temperatures.

In the present invention, particularly the fraction is first flown out only by hexane and then a mixed solvent of hexane and ethyl acetate is used, namely the content of hexane is decreased. Also, the fraction may be flown out only by ethyl acetate.

Next, the solvent flown out by the above method using a known means is fractionated to obtain each fraction. A fraction or a combination of plural fractions containing much coumarin analogues is processed to distill the solvents thereby obtaining a concentrate and thus an antibacterial agent can be prepared. In this case, a little solvent may be left. Furthermore, a treating step may be added in which the concentrate is treated repeatedly by refining operations such as high performance liquid chromatography. The crucial point is that the antibacterial agent is made to contain a significant coumarin analogue mixture.

The antibacterial agent thus obtained exhibits excellent antibacterial ability against various bacteria. Examples of these bacteria may include dental caries causal bacteria, periodontosis bacteria, acid-thermophilic bacteria, acne bacteria, hircismus bacteria, dandruff bacteria, skin normal bacteria, abscess bacteria and food poisoning bacteria.

As specific examples of these bacteria, the following bacteria may be mentioned.

Dental caries causal bacteria; Streptococcus mutans, Actinomyces naeslundii, Actionmyces viscosus;

Periodontosis bacteria; Fusobacterium nuclestum, Prevotella intermedia, Porphyromonas gingivalis;

Calcification bacteria; Corynebacterium matruchotti;

Pathogenic bacteria in a pharynx zone; Streptococcus pyogenes, Haemophilus influenzae;

Acid-thermophilic bacteria; Alicyclobacillus acidocaldarius, A. acidoterrestris, A. cycloheptanicus;

Flatsour bacteria; Bacillus coagulans;

Lactic acid bacteria; Sporolactobacillus inulinus;

Butyric acid bacteria: Clostridium pasteurianum, Clostridium butyricum;

Eumycetes; Byssochlamys uflva, Neosartorya fischeri;

Acne bacteria; Propionibacterium acnes;

Hircismus bacteria; Corynebacterium xerosis;

Dandruff bacteria; Malassezia furfur;

Skin normal bacteria; Staphylococcus aureus, Staphylococcus epidermides, Corynebacterium minutissimum;

Abscess bacteria; Bacteriodes fragilis;

Food poisoning bacteria; Vibrio parahaemolyticus, Campylobacter jejuni;

Putrefying bacteria: Bacillus subtilis; and etc.

The antibacterial agent thus obtained may be added directly to foods and the like. Also, it is possible to subject the antibacterial agent to use by dissolving or dispersing it in a proper liquid carrier or by mixing it with or adsorbing it onto a proper powder carrier. As the case may be, emulsifiers, dispersants, suspending agents, spreaders, penetrants, wetting agents or stabilizers may be added whereby the antibacterial agent is made into preparations such as emulsions, water-dispersible powders, powders or tablets. The antibacterial agent is preferably used for foods, cosmetics and antibacterial preparations. Also, the antibacterial agent of the present invention may be used in combination with other known antibacterial agents or known compounds considered to have an antibacterial activity.

As examples of the materials to which the antibacterial agent of the present invention may be added and compounded, foods, fragrant products, fundamental cosmetics, hair cosmetics, toiletry products, bath agents, body care products, detergent/finishing agents, flavorous deodorants and drugs are given, however the present invention is not limited to these materials.

Examples of the above foods may include drinks such as a non-fruit juice drink, fruit juice-containing drink, lactic acid beverage and powdery drink, frozen sweets such as an ice cream, sherbet and ice sweet, deserts such as pudding, jelly, bavaroi and yoghurt, sweets such as a gum and candy and marine products made with boiled fish paste.

Examples of the fragrant products may include perfumes, toilet water, cologne and shower cologne.

Examples of the above fundamental cosmetics may include skin cream, cleansing cream, skin lotion, after-shave lotion, foundation, lipstick and talcum powder.

Examples of the above hair cosmetics may include shampoo agents such as a shampoo, rinse, conditioner, rinse-in-shampoo and treatment, hair dressing agents such as a pomade, hair tonic, hair liquid and hair jell, hair restorer, hair dying agent and cold wave agent.

Examples of the above toiletry products may include a toilet soap, bath soap and transparent soap.

Examples of the above bath agents may include a powdery bathing agent, solid bathing agent, solid foam bathing agent, bath oil and bubble bath.

Examples of the above detergents may include a powdery detergent for clothes, liquid detergent for clothes, softening and finishing agent, kitchen detergent, lavatory detergent, bath detergent, glass cleaner and mould-removing agent.

Examples of the above air care deodorants may include a gel-like air care deodorant, liquid air care deodorants, impregnated type air sol air care deodorant and mist type air care deodorant.

Examples of the above drugs may include a tablet, liquid drug, capsule type drug and granular drug.

Generally, the amount of the above antibacterial agent to be added to and compounded in a material is preferably 0.1 .mu.g/ml to 50% by weight based on the material though it largely differs depending on the type of material and the type of bacterium. When the amount is less than 0.1 .mu.g/ml, only insufficient antibacterial ability is obtained. Even if the antibacterial agent is added in an amount exceeding 50% by weight, this is economically disadvantageous though sufficient antibacterial ability is obtained.
 

Claim 1 of 7 Claims

1. A method of inhibiting the growth of bacteria comprising contacting the bacteria with an antibacterial agent comprising a mixture of nonvolatile compounds obtained by fractionation of the portion of a citrus cold press oil remaining after citrus cold press oil is heated at 90 to 120.degree. C. under reduced pressure, said mixture of nonvolatile compounds containing 40% by weight or more of coumarin analogues.

____________________________________________
If you want to learn more about this patent, please go directly to the U.S. Patent and Trademark Office Web site to access the full patent.