KR20030035851A - Fungicide composition containing extracts derived from plant - Google Patents

Abstract

PURPOSE: Provided is a fungicide composition containing extracts derived from a plant selected from the group consisting of Foeniculum vulgare, Illicium verum Hook. fil., Asarum Sieboldii Miquel var. seoulens Nakai., Lindera erythrocarpa Makino., etc., which inhibit the growth of pathogenic fungi. CONSTITUTION: A fungicide composition characteristically comprises, as active ingredients, a compound selected from the group consisting of fenchone, eugenol, isoeugenol, methyleugenol, cinnamic alcohol, and cinnamic aldehyde which are extracted from Foeniculum vulgare, Illicium verum Hook. fil., Asarum Sieboldii Miquel var. seoulens Nakai., Lindera erythrocarpa Makino., and Eugenia caryophyllata. It has strong antifungal effects on Trichophyton and candida species, without any adverse effects.

Description

Antimicrobial composition comprising plant extract {FUNGICIDE COMPOSITION CONTAINING EXTRACTS DERIVED FROM PLANT}

[TECHNICAL FIELD OF THE INVENTION]

The present invention relates to an antimicrobial composition comprising a plant extract, and more particularly, extracts from plants selected from the group consisting of fennel, fennel, sessin, camphor and clove having antimicrobial activity against Bacillus aeruginosa (Athletes) and Candida. It relates to an antimicrobial composition comprising an extract or compound.

[Private Technology]

Skin diseases caused by fungi (fungus) are collectively called derma-tomycosis. In particular, cases caused by dermatophyte, which invade keratinous tissue such as keratin, hair, nails and toenails of the skin, are called dermato-phytosis, tinea, or superficial fungal infection. and. (Ribbon, JW (1998) Medical Mycology. In The pathogenic fungi and the pathogenic actinomyces. 2nd Ed.. WB Saunders Company. Philadelphia, London, Toronto)

Superficial fungal disease occurs in the hands and feet according to the skin invasion area, Tinea manus & Tinea pedis, Tinea faciale in the face, Tinea capitis in the head, and groin (groin). Tinea cruris, tinea ungium or Onychomy-cosis in the case of nails and toenails, and tinea corporis (Tinea corporis) are called cases (Rezabek, GH). and Friedman, AD 1992. Drugs 43 (5): 674-682 .; Kamalam, A. and Thambiah, AS 1976. Sabouraudia 14 (2): 129-148.

The main causes of superficial mycosis are microsporum, epidermo-phyton, trichophyton, candida species, and Malassezia furfur . In particular, microsporum mainly penetrates the skin and hair, epidermis (epider-mophyton) penetrates the skin and hair, and Trichophyton invades all skin, hair, and hair. (Weitzman, I. and Summerbell, RC 1995. The dermatophytes.Clinical Microbiology Reviews 8 (2): 240-259) Candida bacteria cause lesions on the skin and mucous membranes, and pneumococci cause spots (tinea versicolor). It becomes the causative agent of.

The causative agents of superficial mycosis are parasitic and multiplying keratin in the upper epithelial cells, causing superficial lesions, but sometimes inferior to the upper epithelium, or dermatophytid.

Pathogenic fungi are distributed worldwide and cause disease in animals, including humans. Pathogenic fungi do not always cause disease by contact, but infection depends on the type of fungal infection, the age of the host, the immune status, the presence of complications, the health of the skin, and the nutritional or hormonal status (Brasch, J. and Gottkehaskamp, D. 1992.The effect of selected human steroid hormones upon the growth of dermatophytes with different adaptation to man.Mycopathologia 120 (2): 87-92)

Infections of the skin and its appendages caused by the majority of the dermatophyte spp fungus are called dermatosis. These fungal groups inhabit only the epidermal keratinous layer, and some species invade and inhabit animal tissues. For convenience, depending on the host, it is referred to as enthrophillic (zoparasitic), zoophilic (zoparasitic), and geophilic (saprophysiological saprophyte) (Gupta, AK, Einarson, TR, Summerbell, RC and Shear). , NH 1998.A North American perspective of Drugs. 55 (5): 645-674)

Treatment of superficial mycosis consists in principle of the use of local and systemic antifungal agents, in some cases in parallel with the application of keratolytic agents. Treatment is required for about 2-4 weeks after the disappearance of symptoms and skin lesions. However, tinea ringworm is highly likely to fail or relapse due to carelessness, disposition of the body, and immunodeficiency.

Until now, many antifungal agents have been used to treat skin fungi, but due to the high recurrence rate and increased awareness of skin toxicity, efforts have been made to find safer and better antifungal substances in natural products.

Therefore, an object of the present invention is to provide a plant extract having antibacterial activity against fungi.

It is another object of the present invention to provide a plant extract having antibacterial activity against ringworm or Candida.

It is another object of the present invention to provide a plant-derived compound having antibacterial activity against ringworm or candida.

It is another object of the present invention to provide an antifungal drug composition that is stable to the skin and has excellent antifungal properties.

1 illustrates a process for separating eugenol from a clove methanol extract,

2 shows a process for separating eugenol and isoeugenol from clove oil.

In order to achieve the above object, the present invention provides an antimicrobial composition comprising an extract extracted from at least one plant selected from the group consisting of fennel, fennel, sessin, camphor and cloves.

The present invention also provides an antimicrobial composition comprising at least one compound selected from the group consisting of phenchon, eugenol, isoeugenol, methyl eugenol, cinnamil alcohol and cinnanamaldehyde.

Hereinafter, the present invention will be described in detail.

The inventors have confirmed that fennel, sessin, camphor and clove have antifungal activity and have completed the present invention.

Fennel ( Foeniculum vulgare ) is known to be almost the same as the main components of the scented thyme, also known as octagonal fennel ( Illicium verum ) (Kim Chang-min, Shin Mingyo, Ahn Deok-kyun, Lee Kyung-soon, etc. 1998. Chinese Medicine Dictionary, vol. ; vol. 10, 6597-6603.)

Camphor plants include Cinnamomum sieboldii , Cinnamon C. cassia , C. zeylanica , Cassia and C. laureirii . In Chinese medicine, the peeled and dried bark of the camphor plant (root, stem, and eggplant) is dried, and the bark of smaller branches is dried, cinnamon, and the very thin eggplant is dried. . Also, peeled and dried bark of the Camphor tree of Cinnamomum ( Cinnamomum ) is called cinnamon or broiler.

The clove is Eugenia caryophyllata or Syzygium aromaticum , the essential oil of the plant is commercialized as clove oil, in particular, clove bud oil (clove bud oil) only collect the buds of the clove Plant essential oil obtained by.

The plants of the present invention are prepared by a conventional extraction method, preferably by mixing plant comminutes with distilled water, alcohol or an organic solvent, fractionating and drying the solvent. The alcohol preferably has 1 to 5 carbon atoms, more preferably ethanol, methanol, propanol or butanol, and most preferably methanol. The organic solvent is preferably hexane, chloroform or ethyl acetate, most preferably hexane. Extraction part is extracted from the flowers, leaves, stems, buds, roots, resins, fruits, seeds, plants of plants, etc.Fennel extract is preferably fennel extract, Sessin extract is preferably Sessin root extract, Camphor extract And clove extract is preferably a root extract. However, it is not limited to the preferable extraction site described above.

The plants of the present invention can also be prepared with essential oils. Plant essential oils can be extracted by a conventional essential oil extraction method, or a commercial oil can be purchased and used. Representative extraction methods include distillation using water or steam and compression. Commercial essential oils include Pennyl Sweet Oil (Fennel Oil), Cinnamon Oil (Cinnamon Oil), and Clove Bud Oil (Clove Oil).

The plant extract or essential oil of the present invention showed excellent antifungal activity as a result of measuring the clear zone by applying to the fungus-induced ringworm and Candida fungi, among which the sericin extract, cloves extract, cinnamon oil and cloves oil were better. It showed high antifungal activity.

The present invention also provides plant-derived antifungal compounds. Antifungal compounds include eugenol and isoeugenol, which are clove-derived compounds, fenchon, which is a fennel-derived compound, cinnamil alcohol and cinnamic aldehyde, which are broiler-derived compounds, and methyl eugenol. The antifungal compound may be purchased by using a commercially available compound, and may be separated and purified from the plant by a known technique.

The present invention provides an antimicrobial composition comprising a plant extract or an antifungal compound. The antimicrobial composition has growth inhibitory activity against pathogenic fungi, and preferably has growth inhibitory activity against ringworm or candida.

The antifungal composition of the present invention has an antifungal effect against the pathogenic fungus described in Table 1 below. Representative pathogenic fungi include Trichophyton rubrum (KCTC 6345), Micosporum audouinii (KCTC 6346), Trichophyton ferrugineum (KCTC6351), Epidermophyton floccosum a KCTC 6586), tricot piton menta thereof blood test (Trichophyton mentagrophytes, KCTC 6077) and Candida albicans (Candida albicans, KCTC 6586).

Fungi (Mold Fungus) ecology Breaking into the fur Onset Microsporum sp. M. audouinii Human friendly Hair follicle (hair external parasitic) tofu M. canis Animal friendly Hair follicle (hair external parasitic) Tofu, solo, hair, face, body M. gypseum Soil-friendly Hair follicle (hair external parasitic) tofu M. fulvum Soil-friendly Hair follicle (hair external parasitic) M. cookei Soil-friendly M. distortum Animal friendly Epidermophyton sp. E. floccosum Human friendly Complete, feet, armor Trichophyton sp. T. rubrum Human friendly Veterinary, Facial, Body, Complete, Foot, Armor T. mentagrophytes Animal friendly Hair follicle (hair external parasitic) Tofu, originality, hair, face, body, complete, foot, armor T. ferrugineum Human friendly Hair follicle (hair external parasitic) Tofu, body T. violaceum Human friendly Inner Hair (Internal Hair Parasitic) Tofu, armor T. verrucosum Animal friendly Hair follicle (hair external parasitic) Tofu, hair T. tonsurans Human friendly Inner Hair (Internal Hair Parasitic) tofu T. schoenleinii Human friendly Inner Hair (Internal Hair Parasitic) Tofu, yellow wire T. simii Animal friendly Inner Hair (Internal Hair Parasitic) tofu T. terrestre Soil-friendly T. gallinase Animal friendly T. megninii Human friendly

The antifungal composition of the present invention can be used for the purpose of inhibiting the growth of pathogenic fungi, and preferably, it can be used as an agent for treating derma-tomycosis such as athlete's foot, athlete's foot, and disinfectant.

Formulations of antifungal composition include warning agents, granules, lotions, liniments, limonades, fragrances and powders. Syrups, ophthalmic ointments, liquids, aerosols, extracts, elixiers, ointments, fluids X), emulsion, suspension, acupuncture, premise, eye drops, tablets, suppositories, injectables, spirits, kata Plasma, capsules, cream, troches, tincture, pasta or pills are preferred, but not limited to the above formulations.

The antifungal composition may further include a physiologically acceptable carrier according to the formulation and the method of use, and the content of the plant extract or the essential oil in the antifungal composition is preferably 1 to 20% by weight. The dose of the antifungal agent is preferably administered once to three times at 10 to 50 mg per day, but more preferably adjusted according to the severity of the disease and the health of the patient.

Hereinafter, examples of the present invention will be described. The following examples are only for illustrating the present invention and the present invention is not limited to the following examples.

Example 1 Preparation of Antimicrobial Plant Extracts

Fruits of fennel ( Foeniculum vulgare ), roots of Asarum heterotropoides , buds of broiler ( Cinnamomum sieboldii ) or cloves ( Eugenia caryophyllata ) and cinnamon were purchased and methanol extracts were obtained for each. Methanol extract manufacturing method was carried out in the same manner, as an example showed a fruit methanol extraction method of fennel.

300 g of Foeniculum vulgare fruit was ground finely in a blender, placed in a 500 ml Erlenmeyer flask, mixed with 200 ml of methanol, and left in a dark room at room temperature for 2 days. After 2 days, the filtrate obtained by filtration under reduced pressure was concentrated using a rotary vacuum concentrator (EYELA autojack NAJ-160, Japan). The filtered material filtered through the filter paper was again mixed with methanol to finally prepare a methanol extract.

Example 2: Preparation of Antimicrobial Plant Essential Oils

Jinhyang Co., Ltd. Fennel oil, broiler oil, cinnamon oil, clove oil were purchased.

The essential oil is prepared by steam distillation. A small amount of distilled water, fennel, broiler, and clove were each finely ground in a mixer and placed in a 3 L concentrated flask. The flask's mantle temperature is about 70 ° C. on a heating mentle, and the flask's mantle temperature is 30 to 40 ml (1/1, v / v) containing diethyl ether and hexane, respectively. It heated at 40 degreeC (about 38 degreeC) for 2 hours. The amount of sample required for distillation once was 250 g, and after extraction, a small amount of anhydrous sodium sulfate (Na ₂ SO ₄ ) was added to remove moisture contained in the extract, and then left in a desiccator. ), And concentrated.

Example 3 Measurement of Growth Inhibition Activity of Mold

Trichophyton rubrum (KCTC 6345), Micosporumaudouinii (KCTC 6346), Trichophyton ferrugineum (KCTC6351), Epik from Korean Collection for Type Cultures (KCTC) the fur t flow koseom (Epidermophyton floccosum, KCTC 6586), tricot piton menta him blood test (Trichophyton mentagrophytes, KCTC 6077), Candida albicans (Candida albicans, KCTC 6586) was pre-sale 6 species. Inhibitory activity of fungi was assayed by the paper disc diffusion method.

After the inoculated strains were inoculated in Sabouraud's agar medium, each methanol extract (Example 1) was dissolved in methanol, or the essential oil (Example 2) was treated directly on a paper disc. After cultivation in the medium was inoculated with each strain and incubated at 28 ℃ for 5 days, the antibacterial activity was confirmed by looking at the size of the growth zone (clear zone) formed around the margin.

Table 2 below shows the measured inhibitory activity against virulent Bacillus spp. And Candida spp. When treated with methanol extract and the optimal concentration per room for essential oil.

Inhibitory ring, mm Treatment concentration, mg / filter fennel Fennel oil Sesin Broiler Broiler oil cloves Clove oil cinnamon Cinnamon milk 50 100 50 10 20 10 10 50 10 10 50 25 10 50 25 10 I'm ordering the Micos Forum. 11 14 13 8 16 16 28 25 30 25 45 44 28 > 50 > 50 > 50 Trichophyton Peregrineum 10 17 11 9 15 15 27 24 28 26 44 40 27 > 50 > 50 > 50 Candida albicans 9 14 11 9 20 16 37 26 30 24 48 45 37 > 50 > 50 > 50 Trichophyton Mentagrophytes 9 14 10 8 17 16 35 26 30 22 45 44 35 > 50 > 50 > 50 Epidermophyton Floco Island 10 15 10 9 18 15 30 25 22 25 49 42 30 45 44 42 Trichophyton Lurum 12 14 10 9 16 15 28 23 24 20 48 41 28 48 45 44

When the size of the growth inhibition ring by methanol extract or essential oil was 10 mm or more, it was determined to have fungal growth inhibition activity. As shown in Table 2, the antimicrobial activity of 50 mg of fennel methanol extract showed an antimicrobial activity for the medicinal extract of fennel methanol, and the fennel oil exhibited the antimicrobial activity of 100 mg and 50 mg per litter, and the sessin was more than 10 mg. When treated, it showed antimicrobial activity. In addition, broiler methanol extract and broiler oil formed very strong growth inhibitory rings when treated with 10 mg and 50 mg, respectively. Clove methanol extract and clove oil also formed very broad inhibitory rings when treated with 10 mg. This antimicrobial activity was similar in the other fungi.

Example 4: Isolation of Antifungal Compounds

(1) Eugenol Separation

Clove methanol extract (20 g) was partitioned into hexane (800 ml), chloroform (800 ml), ethyl acetate (800 ml) and water layer to give 12.6 g of hexane, 3.8 g of chloroform, 0.8 g of ethyl acetate and 2.4 g of water layer. . The antimicrobial activity of each fraction was assayed and the hexane fraction was confirmed to exhibit antimicrobial activity.

12.6 g of the hexane fraction was chromatographed on a silica gel column (Merck 230 mesh, 600 g, 5.5 x 70 cm in diameter) and stepwise gradient (hexane: ethyl acetate (5: 1-> 3: 1, v / v)) to eluate. The eluates were separated by concentration of H1 (1.4 g), H2 (27. g), H3 (0.4 g), and H4 (0.2 g) by TLC (Thin layer chromatography). As a result of assaying the antimicrobial activity of each fraction, H1 and H2 showed antimicrobial activity. Since H2 shows a much higher yield than H1, the antimicrobial active material was separated from H2.

H2 was subjected to high performance liquid chromatography (Spectra System P2000). Hexane: ethyl acetate (9: 1, v / v) solvent was flowed into the column (μPorasil, inner diameter 19 mm x length 300 mm) at a rate of 3 ml per minute, and the eluate was detected at 242 nm to obtain compound I. . Compound I was identified as eugenol of Formula 1 by analysis based on spectroscopic analysis such as EI-MS, ¹ H and ¹³ C-NMR.

[Formula 1]

(2) Separation of isoeugenol and eugenol from clove oil

Isoeugenol and eugenol were separated from 1 mg of clove oil (FIG. 2).

Clove oil is analyzed by high-performance liquid chromatography (analytical HPLC, μPorasil: 10 μm in diameter, 3.9 mm in diameter x 300 mm in length), 2 ml per minute, absorbance 242 nm, solvent condition hexane: ethyl acetate 9: 1 (v / v). Eugenol and eugenol were separated. Under the same conditions, compounds H1, H2, H3 were purified using a preparative column (μPorasil, resin diameter 10 μm, internal diameter 19 mm x length 300 mm). The antimicrobial activity of three compounds (H1, H2, H3) was assayed, and the antimicrobial active compounds H2 and H3 were identified.

Isoeugenol and eugenol were purchased from Sigma, and the standards of isoeugenol and eugenol and EI-MS were compared with H2 and H3. As a result, H2 was identified as eugenol and H3 was identified as isoeugenol. Isoeugenol is represented by the formula (2).

[Formula 2]

Example 5: Antifungal Assay of Compounds

The antifungal activity of eugenol and isoeugenol was assayed, and the antifungal activity of cinnamon-derived fenchon-derived broiler-derived cinnamic alcohol and cinnamic aldehyde was also tested.

Growth inhibition ring size, mm Treatment concentration, mg / filter Penchon Eugenol Isoeugenol Cinnamic Alcohol Cinnamic aldehyde Methyl Eugenol Clotrimazole 50 10 10 10 10 10 5 2.5 1.25 5 2.5 1.25 I'm ordering the Micos Forum. 16 9 31 25 28 48 32 30 15 35 23 21 Trichophyton Peregrineum 12 11 31 25 28 38 25 26 15 33 30 23 Candida Albican 13 10 30 24 28 44 25 23 13 25 22 22 Trichophyton Mentagrophytes 15 9 28 23 27 40 23 25 14 36 18 19 Epidermophyton Floco Island 14 9 25 24 25 35 30 28 14 27 24 18 Trichophyton Lurum 13 10 26 22 29 40 31 27 16 28 23 18

In Table 3, clotrimizol is a positive control. Pencoin showed strong inhibitory activity at 50 mg treatment, Eugenol and Isoeugenol showed very strong inhibitory activity at 10 mg treatment. It showed strong inhibitory activity. Cinnamic aldehyde and methyl eugenol showed the strongest antifungal activity. The size of the inhibitory ring of each compound was slightly different depending on the treatment concentration and strain, but showed similar pattern in overall growth inhibition activity.

Example 6: Clinical Trials

(1) Clinical trial on fennel oil

Fennel oil was dissolved in ethanol to prepare a fennel oil composition at 5%, and then clinical trials were conducted in five adult men infected with foot fungus by athlete's foot.

The fennel oil composition was sprayed into the foot once every three seconds (at a level of 0.15 g of the fennel oil component) to investigate the progress of athlete's foot over time.

For athletes with severe athlete's foot, the following pattern appeared according to usage time.

(1) Treatment Day 1: The oozing from the epithelial cells or tissues cracked and crushed like eczema disappeared.

(2) Day 2 of treatment: The cracked site gradually healed.

(3) Day 3 of treatment: keratin was formed at the athlete's foot, and pain at the athlete's foot disappeared.

(4) Day 4 treatment: The formation of keratin was very prominent, showing complete healing.

In case of athlete's foot, it was possible to cure athlete's foot by inducing enough keratin formation even twice a day.

In addition, no lesions and side effects were observed on the skin of the patient during the application of the fennel oil.

Therefore, the fennel oil composition causes keratinization of the athlete's foot-forming tissues, thereby bringing about changes in the growth conditions of ringworm, and treating athlete's foot.

The present invention isolated antibacterial extracts from fennel, fennel, sessin, camphor and clove, and identified the antimicrobial compounds Fenchon, Eugenol, Isoeugenol, Methyl Eugenol, Cinnamil Alcohol and Cinnanamaldehyde. Since the antimicrobial extract and the antimicrobial compound have very strong antimicrobial activity against ringworm and Candida, it is highly applicable as a natural antimicrobial agent without toxicity.

Claims (3)

An antimicrobial composition comprising a compound selected from the group consisting of phenchon, eugenol, isoeugenol, methyl eugenol, cinnamil alcohol and cinnanamaldehyde. The antimicrobial composition of claim 1, wherein the antimicrobial composition has antimicrobial activity against ringworm and Candida. The antimicrobial composition of claim 1 wherein the antimicrobial composition is used for treating athlete's foot.