WO2008023966A1

WO2008023966A1 - Novel use of panduratin a or derivatives thereof

Info

Publication number: WO2008023966A1
Application number: PCT/KR2007/004109
Authority: WO
Inventors: Jae-Kwan Hwang; Mi-Kyung Kim; Min-Soo Song; Jae-Seok Shim; Ji-Hoon Yoon
Original assignee: Industry-Academic Cooperation Foundation, Yonsei University
Priority date: 2006-08-25
Filing date: 2007-08-27
Publication date: 2008-02-28
Also published as: KR20080018855A; KR100973221B1

Abstract

The present invention relates to the novel use of a panduratin A compound, derivative thereof or an extract of Kaempferia pandurata. More particularly, it relates to the use for the antibacterial activities and acne treatment of a panduratin A compound represented by Formula 1, a panduratin A derivative represented by Formula 2 or an extract of Kaempferia pandurata. The above panduratin A compound, derivative thereof or extract of Kaempferia pandurata has excellent antibacterial activity of inhibiting the growth of bacteria selected from the group consisting of Propionibacterium acnes, Staphylococcus epidermidis, Micrococcus luteus, Staphylococcus aureus, Staphylococcus warneri, Staphylococcus haemolyticus, Staphylococcus xylosus, Staphylococcus hominis and Staphylococcus saprophyticus. Accordingly, the inventive compounds or extract may be very useful as an antibacterial composition or a composition for treating acne against the above bacteria.

Description

NOVEL USE OF PANDURATIN A OR DERIVATIVES THEREOF

Technical Field

The present invention relates to a novel use of a panduratin A compound and derivatives thereof. More particularly, it relates to a use for antibacterial activities and acne treatments of a panduratin A compound represented by Formula 1 and a panduratin A derivative represented by Formula 2.

[Formula 1]

[Formula 2]

Background Art A lot of bacteria live in the skin of human bodies. Among the bacteria, bacteria that cause skin diseases include Propionibacterium acnes, Staphylococcus epidermidis, Micrococcus luteus, Staphylococcus aureus, Staphylococcus warneri , Staphylococcus haemolyticus, Staphylococcus xylosus, Staphylococcus hominis, Staphylococcus saprophyticus, etc., and, in particular, bacteria that cause acne and inflammation include Propionibacterium acnes, Staphylococcus epidermidis, Micrococcus luteus, etc. (Raman A. et al . , Lett. Appl . Microbiol., 21: 242-245, 1995). These bacteria are also present in normal skins, and, in particular, the Propionibacterium acnes is one of major bacteria that cause acne. The bacteria enter hair follicle through a hair follicle duct in advance, and decompose sebum to produce free fatty acid while inhabiting in the deep hair follicle. At this time, secondary infection by Staphylococcus sp . causes inflammation and pus in the skin. The acne is caused by inflammatory diseases in hair sebaceous glands, and is common skin diseases that cause inflammation in the hair follicle of the facial skin of adolescence and young age groups. However, the age group suffering from the acne is broaden these days due to the air pollution, imbalance in sex hormones, drug abuses, stresses, visceral diseases, etc. When becoming adolescence, the secretion of male sex hormones are increased in both of the men and women to stimulate sebaceous glands that are close to the hair follicle, which leads to the increased in size of sebaceous glands and in amount of sebum. At the same time, a horny layer of hair pores, that is, skin pore entrances, grows thicker and more adhesive, resulting in the narrow or closed skin pores. As a result, sebum is heaped in the hair follicle, and therefore bacteria living in the hair follicle increase to cause a inflammation. Until now, methods using chemicals or antibiotics, such as benzoyl peroxide, salicylic acid, benzalkonium chloride, phenol, tetracyclin or erythromycin, have been used to suppress the pathogenic bacteria that cause acne and inflammatory skin diseases of hair sebaceous glands. The methods using antibiotics such as tetracycline are effective to treat the skin diseases, but it has been known that there are high possibility to induce side- effects such as the advent of strains having tolerance to the Propionibacterium sp . , light-sensitive reaction, etc. (Gollnick, H. et al . , Dermatology., 196: 119-125, 1998). Also, it has been reported that a salicylic acid formulation that functions mainly to remove keratin can not prescribe for suppurative dermatitis and may cause skin rubefaction and erythema, and that a benzoyl peroxide formulation that suppresses suppurative bacteria has side-effects such as allergic skin diseases and erythema, and does not fully treat an acne. In recent years, natural substances such as a tee tree oil, a royal jelly extract, a ginseng extract and the like have been also used to treat the acne, but the natural substances may lose its antibacterial activities due to the presence of other compounds that are added during formulation into skin external compositions or cosmetics since they are not composed mainly of a single compound, and they have problems of uneffective antibacterial activities to the acne- inducing bacteria due to its narrow antibacterial activity spectrum. In particular, natural substance-derived antibacterial compounds have difficulty in their formulations since a large amount of their effective components tend to be evaporated or to lost their activities due to the low heat stability in the step of heating (Higaki , S. et al . , J. Dermatology., 23: 310-314, 1996).

The present inventors have made extensive attempts to find a natural substance-derived compound having potent antibacterial activities to the skin bacteria for a long time. As a result, the present inventor found that a panduratin A compound isolated from Kaempferia pandurata, and its derivatives, or a Kaempferia pandurata extract exhibit excellent antibacterial activities to the skin bacteria, thereby completing the present invention.

Disclosure Technical Problem It is an object of the present invention to provide naturally derived compounds or an extract having excellent antibacterial activity for skin bacteria and antibacterial compositions or compositions for treating acne comprising the compounds or the extract.

Also, it is an object of the present invention to provide a method for inhibiting the growth of skin bacteria or treating acne by using the compounds or extract . Also, it is an object of the present invention to provide a novel use of the compounds or extract for preparing antibacterial agents or agents for treating acne .

Technical Solution

To achieve the above objects, the present invention provides an antibacterial composition comprising a panduratin A compound represented by Formula 1, a panduratin A derivative represented by Formula 2 or their pharmaceutically acceptable salts as an effective component, which has antibacterial activities against bacteria selected from the group consisting of Propionibacterium acnes, Staphylococcus epidermidis, Micrococcus luteus, Staphylococcus aureus, Staphylococcus warneri , Staphylococcus haemolyticus, Staphylococcus xylosus, Staphylococcus hominis and Staphylococcus saprophyticus. [Formula 1]

[Formula 2]

In another aspect, the present invention provides an antibacterial composition comprising an extract of Kaempferia pandurata as an effective component, which has antibacterial activities against bacteria selected from the group consisting of Propionibacterium acnes, Staphylococcus epidermidis, Micrococcus luteus, Staphylococcus aureus, Staphylococcus warneri, Staphylococcus haemolyticus, Staphylococcus xylosus, Staphylococcus hominis and Staphylococcus saprophyticus . In another aspect, the present invention provides a composition for treating acne comprising a panduratin A compound represented by Formula 1, a panduratin A derivative represented by Formula 2 or their pharmaceutically acceptable salts as an effective component . In another aspect, the present invention provides a composition for treating acne comprising an extract of Kaempferia pandurata as an effective component.

In another aspect, the present invention provides a method for inhibiting the growth of bacteria selected from the group consisting of Propionibacterium acnes, Staphylococcus epidermidis, Micrococcus luteus, Staphylococcus aureus, Staphylococcus warneri , Staphylococcus haemolyticus, Staphylococcus xylosus, Staphylococcus hominis and Staphylococcus saprophyticus, comprising administering an effective amount of a panduratin A compound represented by Formula 1 or a panduratin A derivative represented by Formula 2 to a subject in need thereof.

In another aspect, the present invention provides a method for inhibiting the growth of bacteria selected from the group consisting of Propionibacterium acnes, Staphylococcus epidermidis, Micrococcus luteus, Staphylococcus aureus, Staphylococcus warneri, Staphylococcus haemolyticus, Staphylococcus xylosus, Staphylococcus hominis and Staphylococcus saprophyticus, comprising administering an effective amount of an extract of Kaempferia pandurata to a subject in need thereof .

In another aspect, the present invention provides a method for treating acne, comprising administering an effective amount of a panduratin A compound represented by Formula 1 or a panduratin A derivative represented by

Formula 2 to a subject in need thereof.

In another aspect, the present invention provides a method for treating acne, comprising administering an effective amount of an extract of Kaempferia pandurata to a subject in need thereof.

In another aspect, the present invention provides a use of a panduratin A compound represented by Formula 1 or a panduratin A derivative represented by Formula 2 for preparing antibacterial agents against bacteria selected from the group consisting of Propionibacterium acnes, Staphylococcus epidermidis, Micrococcus luteus, Staphylococcus aureus, Staphylococcus warneri, Staphylococcus haemolyticus, Staphylococcus xylosus, Staphylococcus hominis and Staphylococcus saprophyticus . In another aspect, the present invention provides a use of an extract of Kaempferia pandurata for preparing antibacterial agents against bacteria selected from the group consisting of Propionibacterium acnes, Staphylococcus epidermidis, Micrococcus luteus, Staphylococcus aureus, Staphylococcus warneri, Staphylococcus haemolyticus, Staphylococcus xylosus, Staphylococcus hominis and Staphylococcus saprophyticus. In another aspect, the present invention provides a use of a panduratin A compound represented by Formula 1 or a panduratin A derivative represented by Formula 2 for preparing agents for treating acne. In still another aspect, the present invention provides a use of an extract of Kaempferia pandurata for preparing agents for treating acne.

Hereinafter, the present invention will be described in detail.

As used herein, the term "effective amount" refers to an amount showing effect of the antibacterial activity against the skin bacteria or the treatment of acne in vivo or in vitro.

As used herein, the term "subject" means mammals, particularly animals including human beings. The subject may be patients in need of treatment.

As used herein, the term "skin bacteria" means the bacteria selected from the group consisting of

Propionibacterium acnes, Staphylococcus epidermidis,

Micrococcus luteus, Staphylococcus aureus, Staphylococcus warneri, Staphylococcus haemolyticus, Staphylococcus xylosus, Staphylococcus hominis and Staphylococcus saprophyticus .

The present invention is characterized by providing a novel use of a panduratin A compound represented by Formula 1 or a panduratin A derivative represented by- Formula 2.

The present invention is characterized by providing a novel use of an extract of Kaempferia pandurata.

[Formula 1]

[Formula 2]

The compounds according to the present invention may be used in the form of a salt, and preferably a pharmaceutically acceptable salt. Preferably, the salt is the acid-addition salt formed by a pharmaceutically acceptable free acid. The free acid used in the present invention may be organic acids and inorganic acids. The organic acids include, but are not limited to, citric acid, acetic acid, lactic acid, tartar acid, maleic acid, fumaric acid, formic acid, propionic acid, oxalic acid, trifluoroacetic acid, benzoic acid, gluconic acid, methane sulfonic acid, glycolic acid, succinic acid, 4- toluene sulfonic acid, glutamic acid and aspartic acid. Also, the inorganic acids include, but are not limited to, hydrochloric acid, bromic acid, sulfuric acid and phosphoric acid.

The inventive compounds can be obtained from whole or parts of plants according to any conventional extraction and isolation method. They may be isolated and purified preferably from Kaempferia pandurata, more preferably from rhizome of Kaempferia pandurata. To obtain the desired extracts, the stems, roots or leaves are either suitably dehydrated and macerated, or simply dehydrated. The desired extracts are then purified by any purification method known to the persons skilled in the art. Moreover, the synthetic compounds or their derivatives corresponding to a panduratin A represented by Formula 1 or a panduratin A derivative represented by Formula 2 are commercially available or can be chemically synthesized by a known synthetic method. Specifically, the inventive compounds can be extracted from herb medicine resources like Kaempferia pandurata, or synthesized according to a normal chemical synthesis method. The compounds according to the present invention can be isolated and purified from water- or a C1-C6 organic solvent-extracts of rhizome of Kaempferia pandurata or from oil obtained by pressing Kaempferia pandurata .

Kaempferia pandurata Roxb. is a plant of the zingiberaceae family, and its root has been widely used to treat a cold, enteritis, skin diseases and urethral pains. It has been reported that Kaempferia pandurata contains pinocembrin chalcone, cardamonin, pinocembrin, pinostrobin, 4-hydropanduratin A and the like, and that these components have an anticancer effect (see Trakoontivakorn, G., et al . , J. Agric. Food Chem. , 49, 3046-3050, 2001) . It is also reported that flavonoid- based dihydrochalcone compounds have an insecticidal effect (see Pandj i , C, et al . , phytochemistry, 34, 415- 419, 1993) . However, the antibacterial effects of the panduratin A compound represented by Formula 1, or its derivatives represented by Formula 2 on skin bacteria, in particular, the acne-inducing bacteria, have not been reported until now.

The inventive panduratin A represented by Formula 1 or panduratin A derivative represented by Formula 2 can be isolated and purified from water- or a C1-C6 organic solvent-extracts of Kaempferia pandurata or from oil obtained by pressing Kaempferia pandurata. Preferred examples of the extraction solvent may include water, methanol, ethanol, propanol, isopropanol, butanol, acetone, ether, benzene, chloroform, ethyl acetate, methylene chloride, hexane, cyclohexane, petroleum ether and the like, which can be used alone or a mixture thereof. The isolation and purification of the inventive compounds from an extract of Kaempferia pandurata may be performed by one or combination of, for example, column chromatography and high-performance liquid chromatography (HPLC) , packed with various synthetic resins, such as silica gel or activated alumina. However, the method for extracting and isolating the active ingredient needs not to be limited to these techniques. The inventive panduratin A represented by Formula 1 or panduratin A derivative represented by Formula 2 show an excellent antibacterial activity against acne-causing bacteria including Propionibacterium acnes and are so excellent in thermal stability as to maintain their antibacterial activity even upon heating.

The panduratin A represented by Formula 1 or the panduratin A derivative represented by Formula 2 shows an excellent antibacterial activities since they have minimal inhibitory concentrations of 2 μg/mβ and 4 βg/ml to the Propionibacterium acnes, respectively, which are very lower than the minimal inhibitory concentration (500-1000 βg/vd) of the benzoyl peroxide used as the acne-treating agent (Decker, LC. et al . , Antimicro. Agents. Chemother., 33 (3): 326-330, 1989), and are lower (about 150 times) than the minimal inhibitory concentration (200~300 /zg/mϋ.) of the benzalkonium chloride (Gloor, M. et al . , Arch. Dermatol. Res., 265 (2): 207-212, 1979) or the minimal inhibitory concentration (300-500 βg/val) of the tee tree oil component terpinen-4-ol (Raman, A. et al . , Lett. Appl. Microbiol., 21 (4): 242-245, 1995). Accordingly, the inventive panduratin A compound represented by Formula 1 or the panduratin A derivative represented by Formula 2 may be effectively used as a composition for treatment of the acne .

Also, the MIC (Minimal Inhibitory Concentration) of the inventive panduratin A represented by Formula 1 or panduratin A derivative represented by Formula 2 against the bacteria selected from the group consisting of Staphylococcus epidermidis, Micrococcus luteus, Staphylococcus aureus, Staphylococcus warneri, Staphylococcus haemolyticus, Staphylococcus xylosus, Staphylococcus hominis and Staphylococcus saprophyticus as well as the Propionibacterium acnes, was 2 ~ 4 βg/ml. So the inventive compounds show an excellent antibacterial activity. Accordingly, the inventive compounds can be used as an antibacterial composition.

The inventive extracts can be obtained by extracting with water or a C1-C6 organic solvent, according to any conventional extracting method. These organic solvents may be used in the forms of C1-C6 alcohol, acetone, ether, benzene, chloroform, ethyl acetate, methylene chloride, hexane, cyclohexane, petroleum ether or the mixture thereof. Preferably, Kaempferia pandurata is extracted by using ethanol as a solvent and then the ethanol extract is fractionated by using ethyl acetate to prepare ethyl acetate extract .

In one example of the present invention, MIC (Minimal Inhibitory Concentration) of the inventive ethanol extract and ethyl acetate extract against Propionibacterium acnes are 39 and 19.5 //g/mϋ. respectively, so the inventive ethanol extract and ethyl acetate extract show an excellent antibacterial activity. Accordingly, the inventive extracts of Kaempferia pandurata can be used in form of a composition for treating acne or an antibacterial composition.

Accordingly, the present invention can be used as an antibacterial composition or a composition for treating acne, which contains the panduratin A compound represented by Formula 1, panduratin A derivative represented by Formula 2 or a pharmaceutically acceptable salt thereof as an active ingredient. And these compositions can be used as drug formulations, not being limited to these.

Also, the inventive extract of Kaempferia pandurata can be used as an antibacterial composition or a composition for treating acne. These compositions can be used as drug formulations, not being limited thereto.

The common drug formulations may be prepared using fillers, thickeners, binders, wetting agents, disintegrants, and diluents such as surfactants, or excipients. Solid formulations for oral administration include tablets, pills, powders, granules, and capsules and are prepared by combining the inventive compounds or the extracts with at least one excipient, for example, starch, calcium carbonate, sucrose, lactose or gelatin. Also, except the simple excipient, lubricant such as magnesium stearate or talc may be used. Examples of liquid formulations for oral administration include suspensions, liquids, emulsions and syrups. The liquid formulations may comprise a simple diluent such as water, liquid paraffin, and various excipients, for example, humectants, sweet agents, aromatic agents and preservatives. Examples of pharmaceutical formulations for parenteral administration include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, ointments and creams. The nonaqueous solutions and suspensions may be prepared using propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyloleate .

Also, the parenteral administration includes subcutaneous, intravenous, intramuscular or intraperitoneal injection. For parenteral administration, the inventive panduratin A compound represented by- Formula 1, its derivative or the extract of Kaempferia pandurata may be mixed with a stabilizer or buffer in water to prepare a solution or suspension, which may then be provided as ampules or vials each containing a unit dosage form. The dosage units can contain, for example, 1, 2, 3, or 4 times of an individual dose or 1/2, 1/3 or 1/4 times of an individual dose. An individual dose preferably contains the amount of an effective drug which is given in one administration and which usually corresponds to all, 1/2, 1/3 or 1/4 times a daily dose.

The inventive panduratin A compound represented by Formula 1, its derivative or the extract of Kaempferia pandurata can be administered in an effective dosage of 0.1-50 mg/kg, and preferably 1-10 mg/kg, 1-3 times a day.

The dose level for certain patients may vary depending on the body weight, age, sex, health condition, diet, time of administration, method of administration, excretion rate and disease severity of patients.

The inventive panduratin A compound represented by Formula 1, its derivative or the extract of Kaempferia pandurata was tested for toxicity in oral administration to rats, and as a result, it was observed that the 50% lethality (LD50) was more than 2,000 mg/kg.

Particularly, the inventive antibacterial composition or composition for treating acne can be formulated in the form of drugs for skin application, i.e., ointments and creams, and it may be properly combined by the form of drugs in the range of 0.001-10.0 wt%, and preferably 0.001-5.0 wt%, based on the total weight of a formulation. If the composition is added in an amount of less than 0.001 wt%, it will provide low antibacterial activity, and if it is added in an amount of more than 10 wt%, it will show no significant difference in antibacterial activity while increasing only their addition amount.

Also, the inventive antibacterial composition or composition for treating acne can be formulated in the form of cosmetics for acne skin. The cosmetics include, but are not limited to, cleaners, cream, lotion, essence, and cosmetic water. The inventive compositions can be formulated in various forms (e.g., cream, cosmetic water, gel, water-soluble liquid, essence, oil-in-water, water- in-oil, etc.) to be used in the cosmetics but can be easily prepared in some forms according to any of the methods known in the art. For example, in the case of preparing cleaner, which contains the inventive compositions, it can be easily prepared by adding the inventive compositions to a conventional facial cleaner base. It is possible to add keratolytic or exfoliating agent such as salicylic acid, α -hydroxy acid, β -hydroxy acid or sulfur for improving the effect of treating acne. Also, it is possible to add perfume, a pigment, bactericidal agent, an antioxidant, a preservative, moisturizer and the like, and to add thickening agents, inorganic salts or synthetic polymers for improving physical properties.

The inventive antibacterial composition or composition for treating acne may be properly added in the range of 0.001-10.0 wt%, preferably in the range of 0.001-5.0 wt%, based on the total weight of cosmetic agents, according to forms of cosmetics. If the composition is added at an amount of less than 0.001 wt%, it will show a low antibacterial activity, and if it is added at an amount of more than 10 wt%, it will show no significant difference in antibacterial activity while increasing only their addition amount.

The inventive panduratin A compound represented by Formula 1, its derivative or the extract of Kaempferia pandurata can be used in a method for inhibiting the growth of bacteria or a method for treating acne.

Specifically, the compounds can be used in a method for inhibiting the growth of bacteria or for treating acne by administering an effective amount to a subject in need thereof. The bacteria mean skin bacteria selected from the group consisting of Propionibacterium acnes, Staphylococcus epidermidis, Micrococcus luteus, Staphylococcus aureus, Staphylococcus warneri, Staphylococcus haemolyticus, Staphylococcus xylosus, Staphylococcus hominis and Staphylococcus saprophyticus .

The inventive compounds or extract can be administered in an effective dosage of 0.1-50 mg/kg, and preferably 1-10 mg/kg, 1-3 times a day. The dosage of the inventive compounds or extract may vary depending on, for example, the body weight, age, sex, health condition, diet, time of administration, method of administration, excretion rate and disease severity for a certain patient. The inventive compounds or extract can be administered by oral or parenteral ways. The parenteral administration includes subcutaneous, intravenous, intramuscular or intraperitoneal injection.

The inventive compounds or extract can be used in preparing antibacterial agents or agents for treating acne. The bacteria mean skin bacteria selected from the group consisting of Propionibacterium acnes, Staphylococcus epidermidis, Micrococcus luteus, Staphylococcus aureus, Staphylococcus warneri, Staphylococcus haemolyticus, Staphylococcus xylosus, Staphylococcus hominis and Staphylococcus saprophyticus. The antibacterial agents or agents for treating acne mean drugs or cosmetics which contain the inventive compounds or extract as an active ingredient, not being limited thereto. The inventive panduratin A compound represented by Formula 1, its derivative or the extract of Kaempferia pandurata may be used as a use for preparing agents for treating acne. The agents for treating acne mean cosmetics (e.g., cleaners, cream, lotion, essence, and cosmetic water, etc.) which contain the inventive compounds or extract as an active ingredient.

Description of Drawings FIG.l shows the ¹³C-NMR spectrum of the inventive Panduratin A compound.

FIG.2 shows the ¹H-NMR spectrum of the inventive Panduratin A compound.

FIG.3 shows the ¹H-¹H cosy spectrum of the inventive Panduratin A compound.

FIG.4 shows the ¹H-¹³C HMBC spectrum of the inventive Panduratin A compound.

FIG.5 shows the FAB Mass spectrum of the inventive Panduratin A compound. FIG.6 shows the ¹³C-NMR spectrum of the inventive Panduratin A derivative.

FIG.7 shows the ¹H-NMR spectrum of the inventive Panduratin A derivative.

FIG.8 shows the DEPT spectrum of the inventive Panduratin A derivative.

FIG.9 shows the El/MS spectrum of the inventive Panduratin A derivative. FIG.10 is a graph showing the results of viable cell counting, which indicate the antibacterial activity of the inventive panduratin A compound against Propionibacterium acnes . ♦: dimethyl sulfoxide

■ : panduratin A sample (0.0004%) A: erythromycin sample (0.0004%)

FIG.11 is a graph showing the results of viable cell counting, which indicate the antibacterial activity of the inventive panduratin A derivatives against Propionibacterium acnes .

♦: dimethyl sulfoxide

■: panduratin A derivative sample (0.0008%) A: erythromycin sample (0.0008%)

Mode for Invention

Hereinafter, the present invention will be described in detail by examples. It is to be understood, however, that these examples are for illustrative purpose only and are not construed to limit the scope of the present invention.

In these examples and test examples, percentages are by weight unless otherwise specified.

Example 1 Separation and Structure Determination of Panduratin A Compound and Its Derivative

<1-1> Separation and Structure Determination of Panduratin A Compound from Kaempferia pandurata

400 ml of ethanol(100 volume %) was added to 100 g (dry weight) of the dried and ground rhizome of Kaempferia pandurata, and kept at a room temperature for two days, and the resulting solution was then filtered using Whatman filter paper No. 2. After the procedures were repeated twice, the ethanol filtrate was concentrated under a vacuum, freeze-dried, and then extracted with a mixed solvent of n- hexane: chloroformrethylacetate (15 :5 :2) by using a silica- gel column chromatography. Then, the remaining solvent was completely removed from the resulting ethanol filtrate, using a rotary vacuum evaporator system, to yield a crude extract of Kaempferia pandurata.

The crude extract of Kaempferia pandurata was separated on a TLC plate using a mixed solvent system of n-hexane : chloroform: ethylacetate (15 : 5 : 2) , and the separated components were determined using a TLC plate analysis. A single panduratin A compound was separated from the compounds in the extract having antibacterial activities as determined by the TLC plate analysis, the panduratin A compound having a Rf value of 0.2 determined by adjusting a ratio of a solvent-spreading distance to a compounds-spreading distance to a Rf value of 0.2 using a mixed solvent system n- hexane : chloroform: ethylacetate (15 : 5 : 2) , and having a strong absorption band for ultraviolet rays (254, 365 ran, VL-6-LC, Vilber lourmat) .

In order to determine a structure of the separated single compound, its ¹H-NMR spectrum and ¹³C-NMR spectrum were analyzed in wavelengths of 500 MHz and 125 MHz (solvent: DMSO), respectively, and shown in FIGs. 1 and 2. In order to determine a ¹H-¹H correlation and a ¹H-¹³C correlation on the basis of the results of the ¹³C-NMR spectrum and ¹H-NMR spectrum, its ¹H-¹H COSY spectrum and ¹H-¹³C COSY spectrum were analyzed, and the results are shown in FIGs . 3 and 4.

Also, the results of FAS/MS are shown in FIG. 5 to analyze a molecular weight of the separated single compound. The inventive compound was determined to have a molecular weight of 406 since the [M+H⁺] was observed at m/z 407 in the FAB/MS, and its chemical formula was C₂₆H₃₀O₄.

By comparing the above-mentioned results of ¹H-NMR, ¹³C-NMR, ¹H-¹H COSY, ¹H-¹³C HMBC and EI/MS to the previously published research report (Woo, W. S. et al . , Phytochemistry, 26: 1542-1543, 1987), it was confirmed that the separated single compound in Example 1-1 is (2 , 6-dihydroxy-4-methoxylphenyl) - [3^' -methyl-2^' -(3^" - methylbut-2^" enyl) -6^' -phenylcyclohex-3^' -enyl] methanone, which is the panduratin A compound represented by Formula 1. [Formula 1]

<l-2> Separation and Structure Determination of

Panduratin A Derivative from Kaempferia pandurata

The crude extract of Kaempferia pandurata extracted in Example 1-1 was eluted in a mixed solvent of methanol and water(9:l) using RP-18 gel 6OF₂₅₄ (Merck), and then re-eluted in a mixed solvent of chloroform and methanol (10 : 0.2) using silica-gel column chromatography, and finally eluted in a mixed solvent of n-hexane and ethylacetate (10 : 3) . A single panduratin A derivative was separated from the compounds in the extract having antibacterial activities as determined by the TLC plate analysis, the panduratin A derivative having a Rf value of 0.2 determined by adjusting a ratio of a solvent-spreading distance to a compounds-spreading distance to a Rf value of 0.2 using a mixed solvent system of n-hexane : chloroform: ethylacetate (15 : 5 : 1.5) , and having a strong absorption band for ultraviolet rays (254, 365 nm, VL-6-LC, Vilber lourmat) .

In order to determine a structure of the separated single derivative compound, its ¹³C-NMR spectrum and ¹H-

NMR spectrum were analyzed in wavelengths of 500 MHz and

125 MHz (solvent: DMSO), respectively, and shown in FIGs.

6 and 7. In order to determine a ¹H-¹H correlation and a

¹H-¹³C correlation on the basis of the results of the ¹³C- NMR spectrum and ¹H-NMR spectrum, its DEPT spectrum was analyzed, and the results are shown in FIG. 8.

Also, EI-MASS was analyzed to determine a molecular weight of the separated single derivative compound, and the results are shown in FIG. 9. The inventive derivative compound was determined to have a molecular weight of 406 since the [M⁺] was observed at m/z 406 in the FAB/MS, and its chemical formula was C₂₆H₃₀O₄.

By comparing the above-mentioned results of ¹³C-NMR, ¹H-NMR, DEPT and El/MS to the previously published research report (Woo, W. S. et al . , Phytochemistry, 26: 1542-1543, 1987), it was confirmed that the separated single derivative compound in Example 1-2 is (2-methoxy- 4, 6-dihydroxyphenyl) - [3 ' -methyl-2 ' - (3 " -methylbut-2 " - enyl) -6 ' -phenylcyclohex-3 ' -enyl] methanone, which is the panduratin A derivative represented by Formula 2. [Formula 2]

Example 2 Evaluation of Antibacterial Activity of Panduratin A Compound and Its Derivative on Bacteria

<2-l> Antibacterial activity of Panduratin A Compound The panduratin A compound prepared in Example 1-1 was dissolved in dimethyl sulfoxide (DMSO) at a concentration of 0.0004%. Propionibacterium acnes was diluted with the prepared Panduratin A sample solution to a concentration of 2 X 10⁵ CFU/m-β, added each test tube at an amount of 100 μJt , and then incubated at 37^°C for 4 hours, 8 hours, 12 hours, 16 hours, 20 hours, 24 hours and 28 hours, respectively. Then, the sample solutions were measured at an absorbance of 625 nm (wavelength) to count viable cells of Propionibacterium acnes, the representative acne- inducing bacterium.

As shown in the graph of FIG. 10, it was revealed that most of all bacteria were dead in the measured time when 4 //g/ml of the panduratin A compound was added. The panduratin A compound has substantially the same effect as erythromycin used recently as an anti-acne antibiotic, as shown in FIG. 10. When considering that oral drugs are generally administered once every 8 hours, and ointments or application drugs are applied to the skin every 2-4 hours, these results indicate that drugs or acne treating agents for application comprising panduratin A compound has a very effective antibacterial activity to Propionibacterium acnes, the representative acne-inducing bacterium.

MBC (Minimal Bactericidal Concentration) of the panduratin A compound on Staphylococcus epidermidis, Micrococcus luteus, Staphylococcus aureus, Staphylococcus warneri, Staphylococcus haemolyticus, Staphylococcus xylosus, Staphylococcus hominis and Staphylococcus saprophyticus was measured in the same manner as described above. The results are listed in the following Table 1.

[Table 1]

MBC (Minimal Bactericidal Concentration) of panduratin A against skin bacteria.

As a result, it was revealed that the panduratin A may be used as an effective antibacterial composition since the panduratin A shows a very effective antibacterial activity against the bacteria, as well as the acne-inducing bacterium Propionibacterium acnes.

<2-2> Antibacterial Activity of Panduratin A Derivative

The panduratin A derivative prepared in Example 1-2 was dissolved in dimethyl sulfoxide (DMSO) at a concentration of 0.0008%, and the antibacterial activities of the panduratin A derivative were then measured in the same manner as described in Example 2-1.

As shown in the graph of FIG. 11, it was revealed that most of all bacteria were dead in the measured time when 8 μg/ml of the panduratin A derivative was added. Therefore, it was revealed that drugs or acne treating agents for application comprising panduratin A derivative has a very effective antibacterial activity to Propionibacterium acnes, the representative acne-inducing bacterium.

MBC of the panduratin A derivative on Staphylococcus epidermidis, Micrococcus luteus, Staphylococcus aureus, Staphylococcus warneri, Staphylococcus haemolyticus, Staphylococcus xylosus, Staphylococcus hominis and Staphylococcus saprophyticus was measured in the same manner as described above. The results are listed in the following Table 2. [Table 2]

MBC of panduratin A derivative against skin bacteria

As a result, it was revealed that the panduratin A derivative may also be used as an effective antibacterial composition since the panduratin A shows a very effective antibacterial activity against the bacteria, as well as the acne-inducing bacterium Propionibacterium acnes.

<2-3> Measurement of Minimal Inhibitory

Concentration (MIC) of Panduratin A Compound

The panduratin A compound prepared in Example 1-1 was dissolved in 0.5% methanol, and the blood serum plate medium was put into each well of 96-well plate in an amount of 100 fd , and then 100 μi of the panduratin A compound was put into only the first well at a concentration of 0.1%, and sequentially diluted 2 times. 100 μJt of Propionibacterium acnes (2 XlO⁵ CFU/ml) was added to each well and cultured at 37 ^°C for more than 24 hours. Then, their minimal inhibitory concentrations that do not show turbidity were measured. Also, the minimal inhibitory concentration of the panduratin A compound on Staphylococcus epidermidis, Micrococcus luteus, Staphylococcus aureus, Staphylococcus warneri , Staphylococcus haemolyticus, Staphylococcus xylosus, Staphylococcus hominis and Staphylococcus saprophyticus was measured in the same manner as described above . The results are listed in the following Table 3

[Table 3]

MIC (Minimal Inhibitory Concentration) of panduratin A against skin bacteria.

<2-4> Measurement of Minimal Inhibitory

Concentration (MIC) of Panduratin A Derivative A minimal inhibitory concentration of the panduratin A derivative prepared in Example 1-2 was measured in the same manner as described in Example 2-3. The results are listed in the following Table 4

[Table 4]

MIC of panduratin A derivative against skin bacteria.

Example 3

Effect on Treatment of Acne using Panduratin A Compound and Its Derivative

Clinical tests for prevention and treatment of acne were carried out using the panduratin A prepared in Example 1-1. 30 men and women of mid teenagers to mid thirties suffering from the acne were selected as test subjects, and 600 mg of the panduratin A was administered to the test subjects 2 times a day for 4 weeks, respectively. The results are listed in the following

Table 5. The lesion counts in the test subjects were significantly reduced after 4 weeks, compared to the lesion counts before the clinical test, and this result was statistically significant (p-value <0.05). [Table 5]

Counting of Acne Lesions In Orally Administering Panduratin A

Meanwhile, clinical test for the prevention and treatment of the acne were carried out in the same manner as described above, using the panduratin A derivative prepared in Example 1-1. The results are listed in the following Table 6. The lesion counts in the test subjects were significantly reduced after 4 weeks, compared to the lesion counts before the clinical test, and this result was statistically significant (p-value <0.05)

[Table 6]

Counting of Acne Lesions In Orally Administering

Panduratin A Derivative

Example 4

Heat Stability of Panduratin A Compound and Its

Derivative

<4-l> Heat Stability of Panduratin A Compound

The panduratin A compound prepared in Example 1-1 was dissolved in dimethyl sulfoxide (DMSO) to prepare a test sample having a concentration of 0.1%, and then the test sample was heat-treated at 60 "C , 70 ^°C , 80 ^°C , 90 ^°C , 100 ^°C and 121 ^°C for 30 minutes, respectively, and each of the test samples was measured for antibacterial activities using a well diffusion analysis. The results are listed in the following Table 7.

[Table 7]

Measurement of Antibacterial Activity according to

Temperature of Panduratin A Compound

As seen in the results of Table 7, it was revealed that the antibacterial activities of the panduratin A compound are not reduced when the panduratin A compound are heat-treated in the temperature range of 60 to 121 ^°C .

Therefore, it was confirmed that the panduratin A compound of the present invention is stable during the high temperature treatment process.

<4-2> Heat Stability of Panduratin A Derivative The panduratin A derivative prepared in Example 1-2 was measured for antibacterial activities in the same manner as described in Example 4-1. The results are listed in the following Table 8

[Table 8]

Measurement of Antibacterial Activity according to

Temperature of Panduratin A Derivative

As seen in the results of Table 8, the antibacterial activities of the panduratin A derivative are not reduced when the panduratin A derivative are heat-treated in the temperature range of 60 to 121 ^°C . Therefore, it was revealed that the panduratin A derivative according to the present invention is stable during the high temperature treatment process

Example 5

Evaluation of Antibacterial activity of Kaempferia pandurata Extract on Bacteria An antibacterial activity against the acne- inducing bacterium Propionibacterium acnes of the 100% ethanol crude extract of Kaempferia pandurata, which is prepared in the procedure of separating the panduratin A or its derivative prepared in Example 1-1 or 1-2, was measured in the same manner as described in Example 1-1. As a result, it was shown that most of all acne-inducing bacteria were dead when 78 //g/ml of the extract was added. Also, a minimal inhibitory concentration of the extract on the acne-inducing bacterium Propionibacterium acnes, Staphylococcus epidermidis and Micrococcus luteus was measured in the same manner as described in Example 2-3. As a result, it was revealed that their minimal inhibitory concentrations were 39 βg/naϋ, 156 βg/ml and 39 respectively.

Also, an antibacterial activity against the acne- inducing bacterium Propionibacterium acnes of the ethylacetate crude extract of Kaempferia pandurata, which is prepared in the procedure of separating the panduratin A or its derivative prepared in Example 1-1 or 1-2, was measured in the same manner as described in Example 2-1. As a result, it was shown that most of all acne-inducing bacteria were dead when 39 /zg/ml of the extract was added. Also, a minimal inhibitory concentration of the extract on the acne-inducing bacterium Propionibacterium acnes was measured in the same manner as described in Example 2-3. As a result, it was revealed that its minimal inhibitory concentration was 19.5 βg/mt.

Accordingly, it was revealed that the Kaempferia pandurata extract may be used for a very effective antibacterial composition since a relatively low amount of the extract has an excellent antibacterial activity against the bacteria, and, in particular, that the Kaempferia pandurata extract may be used for a composition for treating acne since it has an excellent antibacterial activity against the acne-inducing bacterium Propionibacterium acnes .

Preparative examples 1-4 Preparation of Lotion Containing Panduratin A Compound

Lotions having the following composition were prepared using the panduratin A compound according to the present invention. The panduratin A compound separated in Example 1-1 was dissolved in a certain amount of water to concentrations of 1.0, 0.1, 0.01 and 0.001%, and mixed with an aqueous phosphate solution. Ethanol , glycerine and Propylene glycol was added to the mixture and a perfume and a preservative were added to the mixture while mixing the mixture. The resulting mixture was adjusted with purified water to a certain weight and mixed homogeneously.

[Table 9]

Propy ene Propy ene Propy ene Propy ene

Experimental example 1

Measurement of Antibacterial activity of Lotion

Containing Panduratin A Compound

1 ml. of a solution containing the acne-inducing bacterium such as Propionibacterium acnes was added to each test tube including 4 vnl of the lotion, which is prepared in preparation example 1-4, containing different concentrations of panduratin A. Each of the test tubes was incubated for 24 hours while stirring, and its antibacterial activity was then measured by counting its viable cells. The results are listed in the following Table 10.

[Table 10]

As seen from the results of Table 10, the medicinal lotions according to the present invention have very high antibacterial activities against the acne-inducing bacterium such as Propionibacterium acnes, when compared to the panduratin A compound-free lotions.

Preparative examples 5-8

Preparation of Lotion containing Panduratin A

Derivative

Lotions having the following compositions were prepared in the same manner as described in Preparative example 1-4, using the panduratin A derivative of the present invention represented by Formula 2

[Table 11]

Experimental example 2

Measurement of Antibacterial Activity of Lotion containing Panduratin A Derivative

Antibacterial activities of the prepared lotions containing the panduratin A derivative were measured in the same manner as described in Experimental example 1.

The results are listed in the following Table 12

[Table 12]

As seen from the results of Table 12, the medicinal lotions according to the present invention have very high antibacterial activities against the acne- inducing bacterium such as Propionibacterium acnes, when compared to the panduratin A derivative-free lotions.

Preparative examples 9-12

Preparation of Cream containing Panduratin

Compound

Creams having the following compositions were prepared using the panduratin A compound of the present invention. First, compounds represented by " B" were dissolved respectively at 75-80 ^"C , and, cetyl alcohol and a preservative among compounds represented by " c" were dissolved at the same temperature as described above. The compound C was emulsified in the compound B, and the panduratin A compound represented by " A" was then added to the mixture at the concentrations of 3.0, 0.1, 0.01, 0.001%, and mixed. Finally, a perfume was then added to the resulting mixture, and quantified with purified water to prepare creams .

[Table 13]

Experimental example 3

Measurement of Antibacterial Activity of Cream containing Panduratin A Compound

1 ml of the solution containing the acne-inducing bacterium such as Propionibacterium acnes was added to test tubes containing 4 mi of the prepared panduratin A compound-containing creams with each concentrations, and the test tubes were incubated for 24 hours while stirring. The antibacterial activities of the creams were measured by counting viable cells in the test tubes. The results are listed in the following Table 14

[Table 14]

As seen from the results of Table 14, the creams according to the present invention have very high antibacterial activities against the acne-inducing bacterium such as Propionibacterium acnes, when compared to the panduratin A-free creams.

Preparative example 13-16

Preparation of Cream containing Panduratin A

Derivative Creams having the following compositions were prepared in the same manner as described in Preparative example 9-12, using the panduratin A derivative of the present invention represented by Formula 2.

[Table 15]

Experimental example 4

Measurement of Antibacterial Activity of Cream containing Panduratin A Derivative

Antibacterial activities of the prepared lotions containing the panduratin A derivative were measured in the same manner as described in Experimental example 3.

The results are listed in the following Table 16

[Table 16]

Preparat ve . -

Preparat ve . lO \

As seen from the results of Table 16, the creams according to the present invention have very high antibacterial activities against the acne-inducing bacterium such as Propionibacterium acnes, when compared to the panduratin A derivative-free creams.

Experimental example 5

Skin Application of Cream Containing Panduratin A Compound or its Derivatives

Clinical tests for prevention and treatment of acne were carried out using the panduratin A-containing cream prepared in Preparative example 9. 30 men and women of mid teenagers to mid thirties suffering from the acne were selected as test subjects, and a suitable amount of the creams were applied to skins of the test subjects 2 times a day for 4 weeks, respectively. The results are listed in the following Table 17. The lesion counts in the test subjects were significantly reduced after 4 weeks, compared to the lesion counts before the clinical test, and this result was statistically significant (p- value <0.05) . [Table 17]

Counting of Acne Lesions in Skin Application of Panduratin A

Meanwhile, clinical tests for the prevention and treatment of the acne were carried out in the same manner as described above, using the panduratin A derivative- containing cream prepared in Preparative example 13. The results are listed in the following Table 18. The lesion

counts in the test subjects were significantly reduced

after 4 weeks, compared to the lesion counts before the

clinical test, and this result was statistically

significant (p-value <0.05]

[Table 18]

Counting of Acne Lesions in Skin Application

Industrial Applicability As described above, a panduratin A compound represented by Formula 1, a panduratin A derivative represented by Formula 2 or an extract of Kaempferia pandurata has excellent antibacterial activity of inhibiting the growth of bacteria selected from the group consisting of Propionibacterium acnes, Staphylococcus epidermidis, Micrococcus luteus, Staphylococcus aureus, Staphylococcus warneri , Staphylococcus haemolyticus, Staphylococcus xylosus, Staphylococcus hominis and Staphylococcus saprophyticus .

Accordingly, the inventive compounds or extract may be very useful as a composition for treating acne or an antibacterial composition against the above bacteria.

Claims

Claims :

1. An antibacterial composition comprising one selected from the group consisting of a panduratin A compound represented by Formula 1, a panduratin A derivative represented by Formula 2 and their pharmaceutically acceptable salts as an effective component : [Formula 1]

[Formula 2]

2. The antibacterial composition of Claim 1, wherein the composition has antibacterial activities against bacteria selected from the group consisting of Propionibacterium acnes, Staphylococcus epidermidis, Micrococcus luteus, Staphylococcus aureus, Staphylococcus warneri, Staphylococcus haemolyticus, Staphylococcus xylosus, Staphylococcus hominis and Staphylococcus saprophyticus .

3. An antibacterial composition comprising an extract of Kaempferia pandurata as an effective component .

4. The antibacterial composition of Claim 3, wherein the extract of Kaempferia pandurata is extracted with water or Cl ~ C6 organic solvent .

5. The antibacterial composition of Claim 3 or 4 , wherein the composition has antibacterial activities against one or more bacteria selected from the group consisting of Propionibacterium acnes, Staphylococcus epidermidis , Micrococcus luteus, Staphylococcus aureus, Staphylococcus warneri, Staphylococcus haemolyticus, Staphylococcus xylosus, Staphylococcus hominis and Staphylococcus saprophyticus.

6. A composition for treating acne comprising one selected from the group consisting of a panduratin A compound represented by Formula 1, a panduratin A derivative represented by Formula 2 and their pharmaceutically acceptable salts as an effective component .

7. A composition for treating acne comprising an extract of Kaempferia pandurata as an effective component .

8. The composition for treating acne of Claim 7, wherein the extract of Kaempferia pandurata is extracted with water or Cl ~ Cβ organic solvent.

9. A method for inhibiting the growth of one or more bacteria selected from the group consisting of Propionibacterium acnes, Staphylococcus epidermidis, Micrococcus luteus, Staphylococcus aureus, Staphylococcus warneri, Staphylococcus haemolyticus, Staphylococcus xylosus, Staphylococcus hominis and Staphylococcus saprophyticus, the method comprising administering to a subject in need thereof an effective amount of panduratin A compounds represented by Formula 1 or panduratin A derivatives represented by Formula 2.

10. A method for inhibiting the growth of one or more bacteria selected from the group consisting of Propionibacterium acnes, Staphylococcus epidermidis, Micrococcus luteus, Staphylococcus aureus, Staphylococcus warneri, Staphylococcus haemolyticus, Staphylococcus xylosus, Staphylococcus hominis and Staphylococcus saprophyticus, the method comprising administering to a subject in need thereof an effective amount of an extract of Kaempferia pandurata.

11. The method for inhibiting the growth of bacteria of Claim 10, wherein the extract of Kaempferia pandurata is extracted with water or Cl ~ C6 organic solvent .

12. A method for treating acne, comprising administering to a subject in need thereof an effective amount of a panduratin A compound represented by Formula 1 or a panduratin A derivative represented by Formula 2.

13. A method for treating acne, comprising administering to a subject in need thereof an effective amount of an extract of Kaempferia pandurata .

14. The method for treating acne of Claim 13, wherein the extract of Kaempferia pandurata is extracted with water or Cl ~ C6 organic solvent.

15. Use of a panduratin A compound represented by- Formula 1 or a panduratin A derivative represented by Formula 2 for the manufacture of antibacterial agents against one or more bacteria selected from the group consisting of Propionibacterium acnes, Staphylococcus epidermidis, Micrococcus luteus, Staphylococcus aureus, Staphylococcus warneri, Staphylococcus haemolyticus, Staphylococcus xylosus, Staphylococcus hominis and Staphylococcus saprophyticus .

16. Use of an extract of Kaempferia pandurata for the manufacture of antibacterial agents against one or more bacteria selected from the group consisting of Propionibacterium acnes, Staphylococcus epidermidis, Micrococcus luteus, Staphylococcus aureus, Staphylococcus warneri, Staphylococcus haemolyticus, Staphylococcus xylosus, Staphylococcus hominis and Staphylococcus saprophyticus .

17. Use of a panduratin A compound represented by Formula 1 or a panduratin A derivative represented by

Formula 2 for the manufacture of agents for treating acne .

18. Use of an extract of Kaempferia pandurata for the manufacture of agents for treating acne.

19. The use of an extract of Kaempferia pandurata of Claim 18, wherein the extract of Kaempferia pandurata is extracted with water or Cl ~ C6 organic solvent.