WO2003035093A1

WO2003035093A1 - Antibacterial composition comprising plant extract

Info

Publication number: WO2003035093A1
Application number: PCT/KR2002/000826
Authority: WO
Inventors: Haeng-Byoung Lee; Soon-Il Kim; Byeong-Mook Park; Young-Jun Ahn
Original assignee: Naturobiotech Co., Ltd.
Priority date: 2001-10-24
Filing date: 2002-05-03
Publication date: 2003-05-01

Abstract

The present invention relates to an antibacterial composition comprising plant extracts, and more particularly to an extract of one or more kinds of plants selected from a group consisting of Foeniculum vulgare, Illicium verum, Asarum heterotropoides, Cinnamomum plants, and cloves, and an antibacterial compound therefrom.

Description

ANTIBACTERIAL COMPOSITION COMPRISING PLANT EXTRACT

BACKGROUND OF THE INVENTION

(a) Field of the Invention

The present invention relates to an antibacterial composition

comprising plant extracts, more particularly to an antibacterial composition

comprising an extract of one or more kinds of plants selected from a group

consisting of Foeniculum vulgare, lllicium verum, Asarum heterotropoides, Cinnamomum plants, and cloves, or a compound derived therefrom, having

antibacterial activity for Candida and five species containing Trichophyton .

(b) Description of the Related Art

Skin diseases caused by fungi are generically called dermatomycosis. Particularly, those caused by a dermatophyte that invades corneous tissues

such as keratin, hair, fingernails, toenails, etc. and are parasitic thereon are called dermatophytosis, Tinea, or superficial fungal infection (Ribbon, J. W.

(1998) Medical Mycology. In The pathogenic fungi and the pathogenic

actinomyces. 2^nd Ed. W.B Saunders Company. Philadelphia, London, Toronto.).

Superficial fungal infections are referred to differently according to the

invaded skin region, for example Tinea manus & Tinea pedis for hand and foot,

Tinea faciale for face, Tinea crusis for the groin, Tinea capitis for the head,

Tinea ungrium or Onychomy-cosis for fingernails and toenails, and Tinea

corporis for the other regions (Rezabek, G.H. and Friedman, A. D. 1992. Drugs

43(5):674-682.; Kamalam, A. and Thambiah, A.S. 1976. Sabouraudia 14(2):129-148).

Major causes for superficial fungal infection are Microsporum,

epidermophyton, Trichophyton, Candida species, and Malassezia furfur.

Microsporum mainly invades skin and hair; epidermophyton invades skin and

nails; and Trichophyton invades skin, hair, and nails (Weitzman, I. and

Summerbell, R. C. 1995. The dermatophytes. Clinical Microbiology Review

8 (2): 240-259). Candida causes disease on skin and mucous membranes, and Malassezia furfur causes tinea versicolor, which causes spots.

Superficial fungal infection-causing bacteria are parasitic on keratin of

the upper part of epithelial cells and cause superficial diseases, but sometimes they may cause inflammation below the upper part of the epithelial cells, or cause dermatophytid.

Pathogenic fungi have a worldwide distribution, and cause diseases in

animals including humans. Pathogenic fungi do not always cause disease by contact, and whether or not infection occurs therewith depends on the kinds of

infecting fungi, age of host, immune condition, existence and nonexistence of complications, health condition of skin, nutrition, or hormone conditions (Brash,

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) .

Most skin and subsidiary organ infections with Dematophyte spp fungi

are called Dermatophytosis. These fungi groups locally inhabit the epidermis

keratin layer, and some species invade and inhabit animal tissue. For convenience, they are divided into Anthrophillic (parasitic on humans),

Zoophilic (parasitic on animals) and Ziophilic (saprophyte parasitic in soil)

according to the host (Gupta, A. K. Einarson, T. R. Summerbell, R. C. and

Shear, N. H. 1998. A North American perspective of Drugs. 55(5): 645-674).

Superficial fungal infection is treated using local and general anti-

fungals in principal, and according to circumstances, a keratin-solvent is

simultaneously applied. Even after symptoms and skin disease disappear,

approximately 2 to 4 weeks of treatment is required. However, there is high

possibility of Tinea pedis treatment failing or of recurrence due to carelessness

or physical properties and immunodeficiency of the patient.

Although various anti-fungals have been used to treat dermatomycosis,

there is an effort to discover safer and superior anti-fungals from natural

substances due to the high relapse rate and the increase in understanding of

skin toxicity.

SUMMARY OF THE INVENTION

Accordingly, it is an object of the present invention to provide a plant

extract having antibacterial activity for fungi.

It is another object of the present invention to provide a plant extract

having antibacterial activity for Trichophyton and Candida.

It is another object of the present invention to provide a plant-derived

compound having antibacterial activity for Trichophyton and Candida.

It is another object of the present invention to provide an anti-fungal composition that is safe to skin and has superior anti-fungal activity.

In order to achieve these objects, the present invention provides an

antibacterial composition comprising an extract of one or more kinds of plants

selected from a group consisting of Foeniculum vulgare, lllicium verum,

Asarum heterotropoides, Cinnamomum plants, and cloves.

The present invention also provides an antibacterial composition

comprising a compound selected from a group consisting of fenchone, eugenol,

isoeugenol, methyleugenol, cinnamyl alcohol, cinnamic aldehyde, and a

mixture thereof.

BRIEF DESCRIPTION OF THE DRAWINGS

Fig. 1 shows a process for separating eugenol from a methanol extract

of cloves.

Fig. 2 shows a process for separating eugenol and isoeugenol from

clove oil.

DETAILED DESCRIPTION AND THE PREFERRED EMBODIMENTS

The present inventors discovered that Foeniculum vulgare, Asarum

heterotropoides, Cinnamomum sp. plants, and cloves have anti-fungal

activities, and completed the present invention.

Foeniculum vulgare is known to have almost the same major

ingredients as lllicium verum (Changmin Kim, Minkyo Shin, Dukkyun Ann,

Kyungsun Lee, etc. 1998. Medicine Dictionary, vol. 9, Jungdam, 5813-5816;

vol. 10, 6597-6603). Cinnamomum sp. plants include Cinnamomum sieboldii, C. cassia, C.

zeylanica, a cinnamon tree, and C. laureihi. In herbal medicine, pellicles

(roots, trunks, branches) of Lauraceae plants are called C. sieboldii for those

stripped thickly from branches and dried, C. cassia for those stripped from

smaller bark and dried, and Cinnamomi Ramulus for those stripped from very

slim branches and dried. Those stripped from the bark of Cinnamomum

belonging to the Lauraceae family, and when dried are called C. cassia or

Cinnamomum sieboldii.

The scientifical name of cloves is Eugenia caryophyllata or Syzygium aromaticum, and the essential oil therefrom is commonly used as clove bud or leaf oil. In particular, clove bud oil is a plant oil obtained only from the buds of

cloves.

The plant extract of the present invention is prepared by a common extraction method, preferably by mixing the pulverized plant with distilled water, alcohol, or organic solvent; fractioning the mixture; and removing the solvent.

The alcohol is preferably C1-5; more preferably ethanol, methanol, propanol, or

butanol; and most preferably methanol. The organic solvent is preferably

hexane, chloroform, or ethyl acetate, and most preferably hexane. The extraction region is the flower, branch, trunk, bud, root, bark, fruit, seed, plant

body, etc., and Foeniculum vulgare extract is preferably from its fruit, Asarum

heterotropoides extract is preferably from its root, and Cinnamomum sp. plants

and clove extracts are preferably from their root. However, the extraction

region is not limited thereto. In addition, the plants of the present invention can be made into

essential oil. Plant essential oil is extracted by common essential oil

extraction methods, or marketed essential oil can be used. Representative

extraction methods include distillation and expression using water or vapor.

Marketed essential oil includes fennel sweet oil (Foeniculum vulgare oil),

Cinnamon oil (C. cassia oil), and clove bud oil (clove oil).

The plant extracts and essential oils of the present invention were

applied to Candida or Trichophyton, which cause athlete's foot. As a result,

all of them showed excellent anti-fungal activity, and Asarum heterotropoides extract, clove extract, C. cassia extract, and clove oil showed the best anti-

fungal activities.

In addition, the present invention provides a plant-derived anti-fungal compound. The anti-fungal compound includes clove-derived compounds

eugenol and isoeugenol, a Foeniculum vulgare-όerived compound (+)- fenchone, and Cinnamomum sieboldii-derweό compounds cinnamyl alcohol,

cinnamic aldehyde, and methyl eugenol. The anti-fungal compounds can be purchased as marketed compounds, or they can be separated and purified

from the plants.

The present invention also provides an antibacterial composition

comprising plant extracts or anti-fungal compounds. The antibacterial

composition has growth-inhibition activity for pathogenic fungi, and it preferably

has growth-inhibition activity for Trichophyton and Candida.

The anti-fungal composition of the present invention has antibacterial effects for pathogenic fungi as shown in Table 1. Representative pathogenic

fungi include Trichophyton rubrum, KCTC 6345; Microsporum audouinii, KCTC

6346; Trichophyton ferrugineum, KCTC 6351; Epidermophyton floccosum,

KCTC 6586; Trichophyton mentagrophytes, KCTC 6077; and Candida albicans,

KCTC 7728.

Table 1

The anti-fungal composition of the present invention can be used for

inhibiting growth of pathogenic fungi, and preferably it is used as a cleaner, a treating agent for dermatomycosis such as athlete's foot, a disinfectant, etc.

The preparations of the anti-fungal composition, although not limited

thereto, are preferably plasters, granules, lotions, liniments, limonades,

aromatic waters, powders, syrups, eye ointments, liquids, solutions, aerosols,

extracts, elixirs, ointments, fluid extracts, emulsions, suspensions, infusions,

decoctions, ophthalmic solutions, tablets, suppositories, injections, spirits,

cataplasma, capsules, creams, troches, tinctures, pastes, or pills.

The anti-fungal composition may further comprise pharmaceutically acceptable vehicles according to its preparation and use method, and the

contents of plant extracts or essential oils in the composition are preferably 1 to 20 wt%. A dose of the composition is preferably 10 to 50 mg and the frequency is preferably one to three times per day, but it is preferably controlled according to the degree of disease and health condition of the patient.

The present invention will now be explained in more detail with reference to the following Examples. However, these are to illustrate the

present invention and the present invention is not limited to them.

Example 1.

Preparation of antibacterial plant extract

' Fruits of Foeniculum vulgare, roots of Asarum heterotropoides, buds of

Cinnamomum sieboldii or Eugenia caryophyllata, and C. cassia were

purchased, and methanol extracts were obtained from each. All of the

methanol extracts were prepared by the same method, and methanol

extraction method of Foeniculum vulgare fruit is hereinafter explained as an example.

300 g of Foeniculum vulgare fruit were finely pulverized with a mixer

and introduced into a 500 ml Erlenmeyer flask, 200 ml of methanol were mixed

therewith, and the mixture was left for 2 days. It was then filtered under

reduced pressure, and the filtrate was concentrated with a rotary vacuum condenser (EYELA autojack NAJ-160, Japan). The pulverized substance was

filtered through a filtering paper and mixed with methanol again to finally prepare a methanol extract.

Example 2. Preparation of antibacterial plant essential oil

Foeniculum vulgare oil, Cinnamomum sieboldii oil, C. cassia oil, and clove oil were purchased from Jin-a Perfume Inc.

The essential oils were prepared by steam distillation, as follows.. A

small amount of distilled water was added to each of the Foeniculum vulgare, Cinnamomum sieboldii, and cloves, and they were finely pulverized with a

mixer and introduced into 3L concentration flasks, respectively. The

temperature of the flask was set to approximately 70 ^°C on a heating mantle,

and flasks containing 40 ml each (1/1 , v/v) of diethyl ether and hexane were set

to 30 to 40 ^°C (approximately 38 ^°C), and they were all heated for 2 hours.

The amount of sample required for one distillation was 250 g, and after

extraction, a small amount of sodium hydroxide anhydride (Na₂SO₄) was added

to the flasks and they were left in a desiccator to remove moisture from the

extracts, and then the extracts were passed through a Toyo No. 2 filter to concentrate them.

Example 3.

Measurement of fungi growth-inhibition activity

The six species of Trichophyton rubrum, KCTC 6345; Microsporum

audouinii, KCTC 6346; Trichophyton ferrugineum, KCTC 6351 ;

Epidermophyton floccosum, KCTC 6586; Trichophyton mentagrophytes, KCTC

6077; and Candida albicans, KCTC 7728 were obtained from the Korean Collection for Type Cultures, KCTC. Growth-inhibition activities were

examined by a paper disc diffusion method. The strains were inoculated on a Sabouraud's agar medium, and each

methanol extract (from Example 1) was dissolved in methanol and applied to a paper disc or each essential oil (from Example 2) was directly applied to a paper disc. The paper discs were mounted on each of the mediums

inoculated with the various fungi, the fungi were aerobically cultured for 5 days

at 28 ^°C , and antibacterial activities were confirmed by the size of a clear zone

formed around the paper discs.

Table 2 shows growth-inhibition activities of the 5 species of bacteria causing Trichophytia and 1 species of Candida caused by the methanol

extracts and essential oils, when each paper disc was applied with an

appropriate concentration of methanol extract or essential oil.

Table 2.

extract or the essential oil was 10 mm or more, fungi growth-inhibition activity was judged to exist. As shown in Table 2, Foeniculum vulgare methanol

extract showed antibacterial activity for Microsporum audouinii when 50 mg thereof were applied, Foeniculum vulgare oil showed antibacterial activity when 100 mg and 50 mg were applied, and Asarum heterotropoides showed activity

when 10 mg or more were applied. In addition, Cinnamomum sieboldii methanol extract and oil formed very strong clear zones when 10 mg and 50

mg respectively were applied, and clove methanol extract and oil also formed a

very broad clear zone when 10 mg were applied. They showed similar

aspects of antibacterial activities in other fungi.

Example 4.

Separation of anti-fungal compound

(1) Separation of eugenol

Clove methanol extract (20 g) was fractionated with hexane (800 ml),

chloroform (800 ml), ethylacetate (800 ml), and a water layer to obtain 12.6 g of hexane, 3.8 g of chloroform, 0.8 g of ethylacetate, and 2.4 g of the water layer.

Antibacterial activity of each fraction was examined, and it was confirmed that

the hexane fraction showed antibacterial activity.

A chromatographic analysis with a silica gel column (Merck 230 mesh,

600g, diameter 5.5 x 70 g) and a stepwise gradient with hexane-ethyl acetate

(hexane: ethylacetate = 5:1 ->3:1 , v/v) were performed on 12.6 g of the hexane

fraction to obtain effluents. TLC (Thin Layer Chromatography) was performed

on the effluents, and if migration of spot is same, they were gathered and

concentrated to separate them into H1 (1 .4 g), H2 (27 g), H3 (0.4 g), and H4

(0.2 g) fractions. Antibacterial activity of each fraction was examined, and

results showed antibacterial activities in the H1 and H2 fractions. Since H2

showed a larger yield than H1 , the antibacterial substance was isolated in H2.

High-speed liquid chromatography (Spectra System P2000) was

performed on H2. Hexane : ethyl acetate (9:1 . v/v) solvent was dripped into a

column (μ Porasil, inner diameter 19mm X length 300 mm) at a rate of 3 ml

per minute, and effluent was detected at 242 nm to obtain a compound I. The

compound I was identified as eugenol of Chemical Formula 1 as a result of an

analysis on the basis of a spectroscopic analysis such as EI-MS, ¹H and ¹³C-

NMR, etc.

(Chemical Formula 1 )

(2) Separation of isoeugenol and eugenol from clove oil

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

Analytical HPLC (μ Porasil: diameter 10 μm, inner diameter 3.9 mm X

length 300 m) was performed on clove oil at a rate of 2 ml per minute,

absorbance of 242 nm, solvent condition of hexane : ethylacetate 9:1 (v/v) to

separate isoeugenol and eugenol. Under the same conditions, compounds

H1 , H2, and H3 were purified using a fractioning column (μ Porasil, resin

diameter 10 μm, inner diameter 19 mm X length 300 mm). These three kinds

of compounds (H1 , H2, H3) were examined for their antibacterial activities, and

H2 and H3 were confirmed to have antibacterial activities.

Isoeugenol and eugenol were purchased from Sigma, and EI-MS of H2

and H3 were compared with those of the standard isoeugenol and eugenol

products. As a result, H2 was identified as eugenol, and H3 as isoeugenol.

Isoeugenol is represented by the Chemical Formula 2.

(Chemical Formula 2)

Example 5.

Examination of Antifungal activities of compounds

Antifungal activities of eugenol and isoeugenol were examined, and

those of Foeniculum vulgare oil-derived Fenchone, Cinnamomum sieboldii-

derived cinnamyl alcohol, and cinnamic aldehyde were examined by the same

method.

Table 3.

In Table 3, clotrimazole is a positive control. For Microsporum

audouinii, fenchone showed very strong inhibition activity when 50 mg per

paper disc were applied, eugenol and isoeugenol showed very strong activities

when 10 mg were applied, and cinnamyl alcohol showed strong activity when

25 mg or more were applied. Cinnamic aldehyde and methyleugenol showed

the strongest antifungal activities. The diameter of the clear zone from each

compound differed slightly according to application concentration and strains, but overall growth-inhibition activities thereof showed similar results.

Example 6.

Clinical Test

(1 ) Clinical test for Foeniculum vulgare oil

Foeniculum vulgare oil was dissolved in ethanol to prepare a 5%

Foeniculum vulgare oil composition, and a clinical test was performed with 5

adult men whose feet were infected with Trichophyton.

The Foeniculum vulgare oil composition was sprayed on the subjects'

feet once every day for 3 seconds (0.15 g of Foeniculum vulgare oil applied) to

examine the progress of Trichophyton over time.

Subjects having serious athlete's foot showed the following effects

according to use time.

(1 ) 1 day of treatment: Sore oozing from splitted or pressed skin such

as eczema disappeared.

(2) 2 days: Split region was slowly healed.

(3) 3 days: Keratin formed around athlete's foot region, and pain

around athlete's foot region disappeared.

(4) 4 days: Keratin formation was very conspicuous, and athlete's foot

seemed to be completely healed.

For those not having serious athlete's foot, treatment twice per day

could cause sufficient keratin formation and athlete's foot could be completely

healed.

In addition, during application of Foeniculum vulgare, particular symptom and side effects were not observed on the skin of the subjects.

Accordingly, Foeniculum vulgare oil composition causes keratinization

of athlete's foot tissues to change growth conditions of Trichophyton, thereby

treating athlete's foot.

According to the present invention, antibacterial extracts were

separated from Foeniculum vulgare, lllicium verum, Asarum heterotropoides,

Cinnamomum sp. plants, and cloves, and the antibacterial compounds

fenchone, eugenol, isoeugenol, methyleugenol, cinnamyl alcohol, and cinnamic

aldehyde were identified. The antibacterial extracts and compounds can be used as natural antibacterial compounds without toxicity because they have

very strong antibacterial activities for Trichophyton and Candida.

Claims

WHAT IS CLAIMED IS:

1. An antibacterial composition comprising an extract of one or

more kinds of plants selected from a group consisting of Foeniculum vulgare,

lllicium verum, Asarum heterotropoides, Cinnamomum sp. plants, and cloves.

2. The antibacterial composition according to Claim 1 , wherein

the Cinnamomum sp. plant is one or more kinds selected from a group consisting of Cinnamomum sieboldii, C. cassia, C. zeylanica, a cinnamon tree, and C. laureirii.

3. The antibacterial composition according to Claim 1 , wherein

the extract is prepared using at least one solvent selected from a group consisting of an organic solvent, distilled water, and alcohol.

4. The antibacterial composition according to Claim 1 , wherein

the extract is at least one selected from a group consisting of Foeniculum vulgare oil, lllicium verum oil, Asarum heterotropoides oil, Cinnamomum

sieboldii oil, C. cassia oil, clove oil, and clove bud oil.

5. The antibacterial composition according to Claim 1 , wherein

the composition has antibacterial activity for Trichophyton or Candida.

6. The antibacterial composition according to Claim 1 , wherein

the composition has antibacterial activity for one or more kinds of fungi

selected from a group consisting of Trichophyton rubrum, Microsporum

audouinii, Trichophyton ferrugineum, Epidermophyton floccosum, Trichophyton

mentagrophytes, and Candida albicans.

7. An antibacterial composition comprising a compound selected from fenchone, eugenol, isoeugenol, methyleugenol, cinnamyl alcohol,

cinnamic aldehyde, and a mixture thereof.

8. The antibacterial composition according to Claim 7, wherein

the composition has antibacterial activity for Trichophyton and Candida.

9. The antibacterial composition according to Claim 7 for treating

athlete's foot.