WO2010041777A1

WO2010041777A1 - Anti-halitosis composition comprising panduratin derivatives

Info

Publication number: WO2010041777A1
Application number: PCT/KR2008/005859
Authority: WO
Inventors: Jae-Kwan Hwang; Sung Yong Cho; Sang Woo Cho; Nak Eon Choi; Sun-Hee Lee; Kwan Hyoung Lee; Sung Kyung Kim
Original assignee: Seoul Perfumery Co., Ltd.
Priority date: 2008-10-06
Filing date: 2008-10-06
Publication date: 2010-04-15
Also published as: JP2012504662A

Abstract

An anti-halitosis composition comprising a panduratin derivative or Boesenbergia pandurata extract as an active ingredient, and an anti-halitosis composition comprising an emulsified panduratin derivative or Boesenbergia pandurata extract as an active ingredient. The compositions may be safely applied to the buccal cavity without side effects and have high anti-halitosis effects. In particular, the anti-halitosis composition comprising the emulsified panduratin derivative or Boesenbergia pandurata extract as an active ingredient may be applied to a variety of products including liquid food products.

Description

ANTI-HALITOSIS COMPOSITION COMPRISING PANDURATIN DERIVATIVES

TECHNICAL FIELD The present invention relates to an anti-halitosis composition comprising a panduratin derivative or Boesenbergia pandurata extract as an active ingredient. The composition according to the present invention may be safely applied to the buccal cavity without side effects and has high anti-halitosis effects.

BACKGROUND ART

In general, halitosis is caused by acquired systematic disorders or volatile compounds formed from amino acids produced by breakdown of proteins or food residues in saliva by bacteria in the buccal cavity, by a decalcination enzyme or deaminase (Duarte, S., et al., Biol. Pharm, Bull., 26, 527-531 , 2003). According to research on halitosis using gas chromatography, the main substances causing halitosis may be a sulfur compounds, methyl mercaptan, hydrogen sulfide, dimethyl disulfide produced from sulfur-containing amino acids such as cystein and methionine, nitrogen compounds such as putrescine and cadaverine produced from arginine and lysine. In particular, the ratio of methylmercaptan/hydrogen sulfide of a patient having a periodontal disease is 8 times higher than that of a control without the periodontal disease, indicating that methyl mercaptan is a main ingredient of volatile sulfur compounds found in the patient having a periodontal disease. On the other hand, in a patient having a healthy buccal cavity, hydrogen sulfide produced from thiol such as cystein is a main cause of halitosis. In addition, halitosis increases if the surface of the buccal cavity is in a dry condition, since compounds such as indole, scatole, putrescine, cadaverine, butyric acid, and valeric acid contained in the saliva are easily released to the buccal cavity from the surface of mucosa with the saliva dried. Theses compounds are not easily volatilized from the saliva (Persson, S., et al., Oral Microbiol., Immunol., 5, 195-201 , 1990). In order to remove or prevent halitosis, compounds such as triclosan and sanguinarine or antibiotics such as chlorohexidine and penicillin have been used, and research on various herb medicines has been conducted. It has been reported that herb-derived ingredients for removing halitosis were obtained from Thuja orientalis leaves, lotus root, Artemisia Princeps leaves, Phellodendri cortex extract and evening primrose oil, and the like. Japanese Patent Publication No. sho 60-75418 discloses use of sage or rosemary for anti-halitosis. However, since the ingredients used in prior arts have low continuous activity for anti-halitosis, they fail to sufficiently remove halitosis or the buccal cavity may be damaged by overdose or long-term use thereof. Even though a variety of anti-halitosis compositions have been developed, there is still a need to develop an anti-halitosis composition that can be safely applied to the buccal cavity and has excellent anti-halitosis effects. While searching for a natural substance having excellent anti-halitosis effects and that can be safely applied to the buccal cavity, the present inventors have found that an extract of Boesenbergia pandurata belonging to the Zingiberaceae family or a panduratin derivative isolated therefrom has excellent anti-halitosis effects and safety in applications.

DETAILED DESCRIPTION OF THE INVENTION

TECHNICAL PROBLEM The present invention provides an anti-halitosis composition comprising a panduratin derivative or Boesenbergia pandurata extract.

The present invention also provides an anti-halitosis composition comprising a emulsified panduratin derivative or Boesenbergia pandurata extract by emulsification.

TECHNICAL SOLUTION

Hereinafter, the present invention will now be described more fully with reference to the accompanying drawings, in which exemplary embodiments of the invention are shown.

The present invention provides an anti-halitosis composition comprising: at least one panduratin derivative selected from the group consisting of panduratin derivatives represented by Formulae 1 to 3 below, or Boesenbergia pandurata extract comprising the panduratin derivative, as an active ingredient: Formula 1

Formula 2

Formula 3

The panduratin derivatives represented by Formulae 1 to 3 respectively indicate panduratin A, isopanduratin A, and 4-hydroxypanduratin A.

The panduratin derivatives of the anti-halitosis composition according to the present invention may be derived from extracts of Boesenbergia pandurata.

Boesenbergia pandurata is a herb belonging to the Zingiberaceae family and contains pinocembrin chalcon, cardamonin, pinocembrin, pinostribin, 4-hydroxypanduratin A, panduratin A₁ isopanduratin A, or the like. It has been reported that these compounds have anti-cancer effects (Trakoontivakorn, G., et al., J.Arig. Food

3 chem.., 49, 3046-3050, 2001), anti-inflammatory effects (Yun, J. M., et al., Planta Medica, 69, 1102-1108, 2006), anti-aging effects (Shim, J.S., et al., Planta Medica, 74, 239-244, 2008), or anti-bacterial effects (Hwang, J. K., et al., J. Antimicrob. Agents, 23, 377-381 , 2004), but their anti-halitosis effects have not been reported. The panduratin derivatives contained in the anti-halitosis composition according to the present invention may be obtained by extracting dried Boesenbergia pandurata rhizome using purified water or ethanol that are suitable for food processing and purifying the product, or by compressing Boesenbergia pandurata herb to obtain Boesenbergia pandurata oil and purifying the product therefrom. In order to obtain the panduratin derivatives or the extract containing them, a variety of solvents such as methanol, propanol, isopropanol, butanol, acetone, ether, benzene, chloroform, ethyl acetate, methylene chloride, hexane, cyclohexane, and petroleum ether may also be used for the extraction, wherein the solvents may be used alone or in a combination thereof. The panduratin derivative may be isolated from Boesenbergia pandurata and purified by column chromatography using various synthetic resins such as silica gel or activated alumina as fillers and/or high-performance liquid chromatography (HPLC). However, the method of extraction and purification of the panduratin derivatives are not limited thereto. According to an embodiment of the present invention, the amount of the panduratin derivative or Boesenbergia pandurata extract may be in the range of 0.001 to 10 wt%, and preferably 0.01 to 5 wt% based on the total weight of the anti-halitosis composition. If the amount of the panduratin derivative or Boesenbergia pandurata extract is less than 0.001 wt% in the anti-halitosis composition, anti-halitosis effects may not be sufficient. On the other hand, if the amount of the panduratin derivative or

Boesenbergia pandurata extract is greater than 10 wt%, the increase in the anti-halitosis effects is not significantly proportional to the increase in the amount.

The anti-halitosis composition comprising the panduratin derivative or Boesenbergia pandurata extract according to the present invention may be efficiently used in: oral hygiene products such as mouthwash, toothpaste, spray, and refrigerant; food products such as chewing gum, candy, jelly, and chocolate; and pharmaceuticals such as adjuvant; animal feed; or the like. The panduratin derivative or Boesenbergia pandurata extract may be a water-soluble panduratin derivative or Boesenbergia pandurata extract solubilized using an emulsifier and an emulsion adjuvant.

The panduratin derivative or Boesenbergia pandurata extract mainly comprises oil-soluble ingredients and crude panduratin derivative or Boesenbergia pandurata extract has high viscosity. Thus, it is difficult to widely apply as ingredient the panduratin derivative or Boesenbergia pandurata extract to food products, particularly, to liquid food products such as beverages. Thus, in order to use the panduratin derivative or Boesenbergia pandurata extract in various products, it is required to solubilize the panduratin derivative or Boesenbergia pandurata extract to improve water-solubility while maintaining anti-halitosis activity.

The oil-soluble ingredients such as panduratin may be solubilized by emulsifying the oil-soluble ingredients using lipophilic polysaccharides such as gum arabic, by emulsifying the oil-soluble ingredients using a hydrophilic emulsifier such as polyglycerin fatty acid ester, sugar ester, and polysorbate, by inhibiting crystallization thereof using an emulsifier and middle-chain fatty acid triglyceride, or by increasing stability of the emulsified from using polyhydric alcohol, glycerin, propylene glycol, or the like.

In order to prepare an aqueous liquid formulation with low viscosity from the oil-soluble panduratin derivative or Boesenbergia pandurata extract, an emulsion having high transparency when diluted in a water-soluble solvent such as water, acid resistance, heat resistance, and salt resistance needs to be prepared. The panduratin derivative or Boesenbergia pandurata extract is mixed with a water-soluble solution in which polyglycerin fatty acid ester, sugar ester, or the like are dissolved, and the mixture is emulsified to prepare a solubilized aqueous composition comprising the panduratin derivative or pandurata extract, polyglycerin fatty acid ester and sugar ester. The aqueous composition comprising the solubilized panduratin derivative or Boesenbergia pandurata extract is an oil-in-water emulsion retaining anti-halitosis activity of the panduratin derivative or pandurata extract, and having high heat resistance so that heat sterilization may be performed without affecting its stability. Thus, the solubilized panduratin derivative or Boesenbergia pandurata extract may be efficiently used in liquid food products, in particular, aqueous liquid food products.

Beverages generally have very low viscosity and about 1 year of the shelf life at room temperature, and most carbonated beverages and fruit juice beverages except some tea beverages have a pH of 4.0 or less. The higher the viscosity of the beverage, the more stable the emulsified ingredient in the beverage is. However, since a very small amount of precipitate or layer separation of a low-viscosity beverage is formed and easily recognized in a transparent PET bottle, high stability of the emulsion is required. In particular, in an acidic condition, solubility of most food materials decreases, and thus the food materials may be crystallized. Only a few emulsifiers are approved as a food additive and have sufficient emulsifying capacity for use in a beverage. They include gum arabic, polyglycerin fatty acid ester, hydrophilic sugar ester, polysorbate or any combination thereof.

In order to solubilize the panduratin derivative or Boesenbergia pandurata extract in the composition according to the present invention, an emulsifier may be selected from the group consisting of gum arabic, polyglycerin fatty acid ester, hydrophilic sugar ester, polysorbate and any combination thereof, and an emulsion adjuvant may be selected from the group consisting of glycerin, sorbitol, propylene glycol and any combination thereof. Preferably, the emulsifier may be a mixture of polyglycerin fatty acid ester and sugar ester, and the emulsion adjuvant may be a mixture of glycerin and propylene glycol.

The hydrophilic polyglycerin fatty acid ester used to solubilize the panduratin derivative or Boesenbergia pandurata extract herein may have a hydrophile lipophile balance (HLB) of 12 or greater, and an average degree of polymerization of glycerin of 6 or greater and preferably 10 or greater may be used. The fatty acid constituting the ester may have 8 to 18 carbon atoms, and may be lauric acid, myristic acid or palmitic acid. Preferably, the hydrophilic polyglycerin fatty acid ester may be decaglycerin monopalmitic acid ester. The amount of the hydrophilic polyglycerin fatty acid ester may be in the range of 150 to 300 parts by weight based on 100 parts by weight of the panduratin derivative or pandurata extract. In the composition according to the present invention, if the amount of the hydrophilic polyglycerin fatty acid ester, an emulsifier is less than 150 parts by weight based on 100 parts by weight of the panduratin derivative or pandurata extract, emulsifying effects are not sufficient. On the other hand, if the amount of the hydrophilic polyglycerin fatty acid ester is greater than 300 parts by weight based on 100 parts by weight of the panduratin derivative or pandurata extract, the emulsifying effects increase, but an intrinsic unpleasant smell of the emulsifier also significantly increases.

The hydrophilic sucrose fatty acid ester used to solubilize the panduratin derivative or Boesenbergia pandurata extract herein may have a HLB of 12 or greater. The fatty acid constituting the ester may have 8 to 18 carbon atoms. The amount of the hydrophilic sugar ester may be in the range of 150 to 300 parts by weight based on 100 parts by weight of the panduratin derivative or pandurata extract. In the composition according to the present invention, if the amount of the hydrophilic sugar ester constituting an emulsifier is less than 150 parts by weight based on 100 parts by weight of the panduratin derivative or pandurata extract, emulsifying effects are not sufficient. On the other hand, if the amount of the hydrophilic sugar ester is greater than 300 parts by weight based on 100 parts by weight of the panduratin derivative or pandurata extract, the emulsifying effects increase, but an unpleasant flavor of the emulsifier and manufacturing costs also increase.

The total amount of the emulsifier and the emulsion adjuvant used to solubilize the panduratin derivative or pandurata extract herein may be in the range of 150 to 400 parts by weight based on 100 parts by weight of the panduratin derivative or pandurata extract. If the amount of the emulsifier and emulsion adjuvant increases, the emulsifying and solubilizing effects increase, but unpleasant flavor and smell from the emulsifier and manufacturing costs also increase.

In the composition according to the present invention, the solubilized water-soluble panduratin derivative or Boesenbergia pandurata extract may be prepared by pre-emulsification and main-emulsification. In an embodiment of the present invention, the emulsified water-soluble panduratin derivative or Boesenbergia pandurata extract may be prepared by melting a mixture of a panduratin derivative or Boesenbergia pandurata extract, polyglycerin fatty acid ester, sugar ester, propylene glycol, and glycerin at a temperature ranging from 50 to 80D and emulsifying the melted mixture at a temperature ranging from 65 to 75D . In an embodiment of the present invention, the mixture for solubilizing the panduratin derivative or Boesenbergia pandurata extract may comprise polyglycerin fatty acid ester, sugar ester, propylene glycol, and glycerin in an amount of 150 to 400 parts by weight based on 100 parts by weight of the panduratin derivative or Boesenbergia pandurata extract. In an embodiment of the present invention, the composition comprising the water-soluble panduratin derivative or Boesenbergia pandurata extract may be prepared by pre-emulsification and main-emulsification. First, the panduratin derivative or Boesenbergia pandurata extract or a mixture of the panduratin derivative or Boesenbergia pandurata extract and ethanol or propylene glycol is heated to a temperature ranging from 50 to 8OD to increase fluidity. Then, a mixture comprising polyglycerin fatty acid ester, sugar ester, propylene glycol and water that is maintained at a temperature ranging from 50 to 8OD is added thereto while stirring at a high speed to pre-emulsify the mixture. Specifically, predetermined amounts of the panduratin derivative or pandurata extract and any other oil-soluble ingredients are maintained at a predetermined temperature while stirring at a high speed using a conventional stirrer such as a turbine stirrer or a paddle stirrer to prepare an oil phase. In the same manner, the hydrophilic polyglycerin fatty acid ester and any other water-soluble ingredients are maintained at a predetermined temperature and melted to prepare an aqueous phase. The hydrophilic polyglycerin fatty acid ester may be added to the oil phase. The temperature at which the oil phase and aqueous phase are formed may be in the range of 50 to 8OD . However, the temperature is not limited thereto. If the temperature is lower than 5OD , the pandurata derivative or panduratin extract has high viscosity and the emulsifier is not completely dissolved. On the other hand, if the temperature is higher than 8OD , granulation may not be effectively performed. Then, the oil phase is added to the aqueous phase while stirring to perform pre-emulsification. The pre-emulsified mixture is further emulsified, for example using a high-pressure emulsifying device, to regulate the particle size to a desired level.

The main emulsification to solubilize the panduratin derivative or Boesenbergia pandurata extract may be performed at a temperature ranging from 65 to 75D to melt the panduratin derivative or pandurata extract and the emulsifier. If the temperature is lower than 65D , the panduratin derivative of the emulsifier may not sufficiently melt. On the other hand, if the temperature is higher than 75D , the panduratin derivative or pandurata extract may be partially degraded by the heat, since reduction of the particle diameter by the homogenization decreases as the temperature increases.

The water-soluble composition containing the panduratin derivative may be obtained by mechanical emulsification using an emulsifying device that is commonly used in the art. The mechanical emulsification may be performed using a high-speed stirring emulsifying device such as homo mixer, a high-pressure homogenizer, a colloid mill, or an ultrasonic mixer.

As the particle size thereof decreases, the surface area of the solubilized emulsion increases, and thus dispersion stability and bioavailability increase, but the amount of emulsifier required also increases. The bioavailability used herein refers to the degree of exhibiting desired activity, i.e., anti-halitosis activity, obtained by directly contacting the panduratin derivative or Boesenbergia pandurata extract, as an active ingredient of the composition according to the present invention, to a target material. In an embodiment of the present invention, a median diameter of the emulsified particles in the water-soluble composition containing the panduratin derivative may be regulated to 200 nm or less by the emulsification. As the diameter of the emulsified particles increases, dispersion stability of the water-soluble composition decreases and bioavailability decreases, and thus the desired effects of the present invention and may not be obtained. In addition, as the median diameter of the emulsified particles decrease, storage stability, bioavailability, and transparency of the water-soluble composition are improved, but the amount of the emulsifier may increase, leading to high manufacturing cost.

According to an embodiment of the present invention, the mixture of the pre-emulsified panduratin derivative or Boesenbergia pandurata extract and the emulsifier and emulsion adjuvant is homogenized at 400 Kg/cm² or greater in the main emulsification. Homogenization may be suitably used for emulsification and repeated, if needed, since distribution of the particle diameters may become narrow and uniform, improving storage stability if the homogenization is repeated even at the same pressure. The panduratin derivative or Boesenbergia pandurata extract exists in a paste having very high viscosity, tends to be crystallized, and is water-insoluble at room temperature. Thus, the panduratin derivative or Boesenbergia pandurata extract as it is may not be applied to liquid food products such as yogurt, fruit juice beverages, and coffee beverages. However, the emulsified water-soluble panduratin derivative or Boesenbergia pandurata extract may be applied to a variety of products due to excellent processibility.

The water-soluble composition comprising emulsified panduratin derivative or Boesenbergia pandurata extract according to an embodiment of the present invention may be applied to food products comprising functional food products, pharmaceuticals, quasi drugs, cosmetics, and animal feed. In particular, the water-soluble composition comprising emulsified panduratin derivative or Boesenbergia pandurata extract according to an embodiment of the present invention has excellent bioavailability improved by micronization of particles, and thus activity of the panduratin derivative or pandurata extract may be more efficiently used. The anti-halitosis composition comprising the panduratin derivative or Boesenbergia pandurata extract according to the present invention or the panduratin derivative or Boesenbergia pandurata extract solubilized by emulsification, as an active ingredient, may be formulated in oral hygiene products such as mouthwash, toothpastes, sprays, and adjuvants, and refrigerant or food products such as spices, chewing gum, candy, jelly, and chocolate. However, the formulation is not limited thereto. In particular, the composition comprising the solubilized panduratin derivative or Boesenbergia pandurata extract, as an active ingredient, may be administered as it is or in a form of capsule. The composition may be applied to a variety of liquid products comprising beverages such as milk beverages, refreshing drinks, nutritional supplement drinks, and cosmetic drinks, pet food, or the like.

Hereinafter, one or more embodiments will be described in detail with reference to the following examples. However, these examples are not intended to limit the purpose and scope of the invention.

ADVANTAGEOUS EFFECTS

The anti-halitosis composition according to the present invention may be safely applied to the buccal cavity without side effects and has excellent anti-halitosis effects. In particular, the anti-halitosis composition comprising the solubilized panduratin derivative or Boesenbergia pandurata extract may be applied to a variety of products such as food comprising liquid food, pharmaceuticals, cosmetics, and animal feed based on its improved processing properties.

DESCRIPTION OF THE DRAWINGS The above and other features and advantages of the present invention will become more apparent by describing in detail exemplary embodiments thereof with reference to the attached drawings in which:

FIG. 1 shows an EI/MS spectrum of panduratin A; FIG. 2 shows an EI/MS spectrum of isopanduratin A; and FIG. 3 shows an EI/MS spectrum of 4-hydroxypanduratin A.

MODE OF THE INVENTION

Example 1. Preparation of Boesenbergia pandurata extract comprising panduratin Dried Boesenbergia pandurata was pulverized using a mixer, and 10O g of the pulverized Boesenbergia pandurata was added to 500 ml_ of ethanol, and then the mixture was stirred at 5OD for 30 minutes to perform extraction. The extract was filtered using Whatman No. 2 filter paper, and the filtered extract was concentrated using a rotational vacuum concentrator to remove the solvent. Then, the resultant was lyophilized to remove moisture to obtain Boesenbergia pandurata extract.

Example 2. Isolation and identification of panduratin A

(1) Isolation of panduratin A

The concentrated Boesenbergia pandurata extract obtained according to Example 1 was mixed with ethyl acetate to extract ingredients soluble in ethyl acetate. The ethyl acetate was removed under reduced pressure to concentrate the ingredients soluble in ethyl acetate. Then, the concentrated ingredients were loaded onto a column filled with silica gel (6x15cm) and eluted using a solvent system comprising hexane, chloroform, and ethyl acetate in a ratio of 15:5:1.5 (v/v/v). The collection obtained from the elution was divided into 6 fractions according to the eluting order, and each fraction was concentrated and dried. No. 3 fraction (Fraction 3) among the 6 fractions was further separated by thin layer chromatography (TLC, silica gel 60F254, Merck) with running solvent of hexane, ethyl acetate, and methanol in a ratio of 18:2:1 (v/v/v), collecting 3 fractions according to the eluting order, and each fraction was concentrated and dried. No. 2 fraction among the 3 fractions (Fraction 3-2) was further separated by recycling HPLC (column: W-252, 20.0 mm IDx 500 mm L), collecting 2 fractions according to the eluting order, and each fraction was concentrated and dried. Finally, No. 2 fraction among the 2 fractions (Fraction 3-2-2) was concentrated and dried to isolate a pure single ingredient having anti-halitosis activity. (2) Identification of panduratin A

In order to identify the structure of the single active ingredient isolated in operation (1) described above, ¹H-NMR spectrum and ¹³C-NMR spectrum were obtained respectively at 500 MHz and 125 MHz (solvent: CDCI₃). In order to identify the ¹H-¹H and ¹H-¹³C relationships based on the results of ¹³C-NMR spectrum and ¹H-NMR spectrum, ¹H-¹H COSY spectrum and ¹H-¹³C HSQC spectrum were obtained and the signal of each carbon was identified using wavelengths from carbon resonance. The results are shown in Table 1 below.

In addition, an EI/MS spectrum obtained for mass analysis of the isolated single active ingredient is shown in FIG. 1. Since [M+H+] was observed in m/z 407 in the EI/MS spectrum, it was identified that the molecular weight was 406, and the molecular formula was C₂₆H₃₀O₄.

By comparing the results of ¹H-NMR, ¹³C-NMR, ¹H-¹H COSY, ¹H-¹³C HSQC and EI/MS spectra with the results of research reported by Woo, W.S. et al., Phytochemistry, 26: 1542-1543, 1987, it was identified that the single ingredient isolated in operation (1) was

(2,6-dihydroxy-4-methoxyphenyl)-[3'-methyl-2'-(3"-methylbut-2"-enyl)-5'-phenylcyclohex- 3'-enyl]methanone that is panduratin A represented by Formula 1 below.

Table 1

SH SC

1 .

2 C H-3

3 CH H-3 H-5

4 - C

CH,

5 ΓH H-5 H-3

- C . H-5

I' CH H-I

2' CH H-2

C -

CH₁ H-2

4 CH H-4

5' H-6

6' CH H-6 r CH₂

T CH

3" - C -

CH,

CH, r" C . -

2" CH

CH

4 CH

CH

6' CH

C-O -

Formula 1

Example 3. Isolation and identification of isopanduratin A (1) Isolation of isopanduratin A

The concentrated Boesenbergia pandurata extract obtained according to Example 1 was mixed with ethyl acetate to extract ingredients soluble in ethyl acetate. The ethyl acetate was removed under reduced pressure to concentrate the ingredients soluble in ethyl acetate. Then, the concentrated ingredients were loaded onto a column filled with silica gel (6x15cm) and eluted using a solvent system comprising hexane, chloroform, and ethyl acetate in a ratio of 15:5:1.5 (v/v/v). The collection obtained from elution was divided into 6 fractions according to the eluting order, and each fraction was concentrated and dried. No. 4 fraction (Fraction 4) among the 6 fractions was further separated by reversed phase-18 (Rp-18, LiChropep, 25-40 m) column chromatography using a solvent system comprising methanol and water in a ratio of 9:1 (v/v). Then, the collection obtained was divided into 2 fractions according to the eluting order. No. 2 fraction among the 2 fractions (Fraction 4-2) was concentrated and dried, and further separated by reversed phase-18 column chromatography using a solvent system comprising chloroform and methanol in a ratio of 10:0.2 (v/v). Then, the collection obtained was divided into 2 fractions according to the eluting order, and each fraction was concentrated and dried. No. 2 fraction among the 2 fractions (Fraction 4-2-2) was further separated by reversed phase-18 column chromatography using a solvent system comprising hexane and ethyl acetate in a ratio of 10:3 (v/v). The collection obtained was divided into 2 fractions, and each fraction was concentrated and dried. Finally, No. 2 fraction among the 2 fractions (Fraction 4-2-2-2) was concentrated and dried to isolate a pure single ingredient having anti-halitosis activity. (2) Identification of isopanduratin A In order to identify the structure of the single active ingredient isolated in operation (1) described above, ¹H-NMR spectrum and ¹³C-NIvIR spectrum were obtained respectively at 500 MHz and 125 MHz (solvent: CDCI₃). In order to identify the ¹H-¹H and ¹H-¹³C relationships based on the results of ¹³C-NMR spectrum and ¹H-NMR spectrum, ¹H-¹H COSY spectrum and ¹H-¹³C HSQC spectrum were obtained and the signal of each carbon was identified using wavelengths from carbon resonance. The results are shown in Table 2 below.

In addition, an EI/MS spectrum obtained for mass analysis of the isolated single active ingredient is shown in FIG. 2. Since [M+H+] was observed in m/z 407 in the EI/MS spectrum, it was identified that the molecular weight was 406, and the molecular formula was C₂₆H₃₀O₄.

(4,6-dihydroxy-2-methoxylphenyl)-[3^l-methyl-2'-(3"-methylbut-2"-enyl)-6¹-phenylcyclohex -3'-enyl]methanone that is isopanduratin A represented by Formula 2 below.

Table 2 δH 6C

10664 C

16281

1 89 (s) C H-I H-5

591 (br s) CH -

CH, -

5 5 89 (br s) CH H-5 H-3

6 C . H-5

1' 449(dd) CH

2' 249(br ό) CH

3¹ C

1 79 (br s)

4' 542 (s) CH H-4

5¹ (a) 240 (br d) at

(b) 202 (m)

6' 342 (br ddd) CH r (a) 223 (m) CH,

(b) 2 10 (m)

2 485 (0 CH

C -

1 50 (s) CH,

C .

720(10) CH

7 l8 (m) CH

7 10 (m) CH

7 l8 (m) CH

720 (m) CH

C-O -

Formula 2

Example 4. Isolation and identification of 4-hydroxypanduratin A (1 ) Isolation of 4-hydroxypanduratin A

The concentrated Boesenbergia pandurata extract obtained according to Example 1 was mixed with ethyl acetate to extract ingredients soluble in ethyl acetate. The ethyl acetate was removed under reduced pressure to concentrate the ingredients soluble in ethyl acetate. Then, the concentrated ingredients were loaded onto a column filled with silica gel (6x15cm) and eluted using a solvent system comprising hexane, chloroform, and ethyl acetate in a ratio of 15:5:1.5 (v/v/v). The collection obtained from the elution was divided into 6 fractions according to the eluting order, and each fraction was concentrated and dried. No. 6 fraction (Fraction 6) among the 6 fractions was further separated by the column chromatography using a solvent system comprising methylenechloride and methanol in a ratio of 19:1 (v/v). The collection obtained was divided into 3 fractions according to the eluting order. No. 2 fraction (Fraction 6-2) among the 3 fractions was further separated by the column chromatography eluted using a solvent system comprising chloroform and methanol in a ratio of 20:1 (v/v). The fraction obtained was divided into 2 fractions according to the eluting order. Finally, No. 2 fraction (Fraction 6-2-2) among the 2 fractions was separated by recycling HPLC (column: W-252, 20.0 mm IDx 500 mm L) to isolate a pure single ingredient having anti-halitosis activity.

(2) Identification of 4-hydroxypanduratin A

In order to identify the structure of the single active ingredient isolated in operation (1) described above, ¹H-NMR spectrum and ¹³C-NMR spectrum were obtained respectively at 500 MHz and 125 MHz (solvent: methanol). In order to identify the ¹H-¹H and ¹H-¹³C relationships based on the results of ¹³C-NMR spectrum and ¹H-NMR spectrum, ¹H-¹H COSY spectrum and ¹H-¹³C HSQC spectrum were obtained and the signal of each carbon was identified using wavelength from carbon resonance. The results are shown in Table 3 below.

In addition, an EI/MS spectrum obtained for mass analysis of the isolated single active ingredient is shown in FIG. 3. Since [M+H+] was observed in m/z 393 in the EI/MS spectrum, it was identified that the molecular weight was 392, and the molecular formula was C25H28O4.

(2,4,6-trihydroxypheny-[3'-methyl-2'-(3"-methylbut-2"-enyl)-6^l-phenylcyclohex-3^l-enyl]me thanone that is 4-hydroxypanduratin A represented by Formula 3 below.

Table 3 δH

- 10664 C -

16281 C

55 76 C H-3 H-5

3 591 (br s) 9089 CH

16740 CH, -

589 (t>r s) 9672 CH - H-5 H-3

162 16 C - . H-5 r 449 (dd) CH

249 (brd)

.

1 79 (br s) 2291 CH,

4' 542 (s) 12098 CH

5' (a) 240 (br d) CH₂

(b) 202 (m)

142 (brddd) CH

1 ' (a) 223 (m) CH₂

2 CH

3' C -

CH₁

CH,

1"' C .

2" 720 (m) CH

7 18 (m) CH

7 10 (m) CH

7 l8 (m) CH

720 (m) CH

- -

Formula 3

Examples 5 - 8. Preparation of water-soluble composition comprising panduratin derivative-containing extract Water-soluble compositions were prepared using the extract prepared according to Example 1 and ingredients listed in Table 4 below, and anti-halitosis effects were measured according to Experimental Example 1 below.

Experimental Example 1. Measurement of anti-halitosis effects 40 men and women were subjected to the experiments. 5 subjects were tested for each mouthwash or beverage comprising the composition prepared according to Examples. The anti-halitosis effects were measured using a Halimeter. After the subjects ate the same meal, halitosis was measured using a Halimeter to obtain an initial halitosis. Then, the mouth of each of the subjects was washed with a mouthwash or beverage for 3 minutes, and then halitosis was measured again using the Halimeter. The method of using the Halimeter is as follows.

The subjects breathed only with their nose while their mouth was closed for 2 minutes before the measurement. Then, the halitosis was measured for about 15 seconds while a straw was inserted into the mouth by about 1.5 cm. In this regard, the straw should not contact the tongue, the lips should be open by about 3 to 4 mm, and breathing should be performed only with the nose. The unit of the Halimeter is ppb. Average halitosis values for each subject were calculated to compare halitosis inhibition rates among the subjects. The halitosis inhibition rate was calculated using Equation 1 below.

Equation 1 Halitosis inhibition rate (%) = (initially measured value - value measured after washing mouth)÷(initially measured value)* 100 Table 4

®: very stable o: stable

When gum arabic was used as the emulsifier according to Example 5, the composition was stably maintained in a suspension state for 3 months or more due to strong hydrophilicity of gum arabic despite large particle sizes. However, anti-halitosis activity of the composition was far less than compositions having small particles. The anti-halitosis activity of the composition according to Example 5 was about 1/5 of that of the composition having particles where diameter is 150 nm prepared according to Example 8. If the particle diameter is 150 nm or less, anti-halitosis activity of the panduratin derivative was sufficient. The anti-halitosis activities of the compositions according to Examples 6 and 7 were 50% or more lower than that of the composition according to Example 8, and had lower stability at high temperature. On the other hand, the solubilized composition according to Example 8 showed stability in emulsion state and activity without precipitation and separation after long-term storage and centrifugation at 3,750 rpm for 4 hours.

Examples 9 - 12. Preparation of mouthwash comprising pandurata crude extract

Mouthwashes were prepared using a crude extract obtained according to Example 1 and ingredients listed in Table 5 below. Table 5

Measuring anti-halitosis effects of mouthwash comprising panduratin crude extract

Anti-halitosis effects were measured in the same manner as in Experimental Example 1 , except that mouthwashes comprising the panduratin crude extracts prepared according to Examples 9 to 12 were used. The results are shown in Table 6 below.

Table 6

Examples 13 - 16. Preparation of mouthwash comprising panduratin A

Mouthwashes were prepared using panduratin A obtained according to Example 2 and ingredients listed in Table 7 below. Table 7

Measuring anti-halitosis effects of mouthwash comprising panduratin A Anti-halitosis effects were measured in the same manner as in Experimental Example 1 , except that mouthwashes comprising panduratin A prepared according to Examples 13 to 16 were used. The results are shown in Table 8 below. Table 8

Examples 17 - 20. Preparation of mouthwash comprising isopanduratin A

Mouthwashes were prepared using isopanduratin A obtained according to Example 3 and ingredients listed in Table 9 below. Table 9

Measuring anti-halitosis effects of mouthwash comprising isopanduratin A Anti-halitosis effects were measured in the same manner as in Experimental Example 1 , except that mouthwashes comprising isopanduratin A prepared according to Examples 17 to 20 were used. The results are shown in Table 10 below. Table 10

Examples 21 - 24. Preparation of mouthwash comprising hydroxypanduratin A Mouthwashes were prepared using hydroxypanduratin A obtained according to

Example 4 and ingredients listed in Table 11 below. Table 11

Measuring anti-halitosis effects of mouthwash comprising hvdroxypanduratin A Anti-halitosis effects were measured in the same manner as in Experimental Example 1 , except that mouthwashes comprising hydroxypanduratin A prepared according to Examples 21 to 24 were used. The results are shown in Table 12 below. Table 12

Examples 25 - 28. Preparation of mouthwash comprising water-soluble crude extract Mouthwashes were prepared using water-soluble crude extracts obtained according to Examples 5 and 8 and ingredients listed in Table 13 below. Table 13

Measuring anti-halitosis effects of mouthwash comprising water-soluble crude extract

Anti-halitosis effects were measured in the same manner as in Experimental Example 1 , except that mouthwashes comprising water-soluble crude extracts prepared according to Examples 25 to 28 were used. The results are shown in Table 14 below.

Table 14

The anti-halitosis effects of the mouthwashes comprising water-soluble crude extracts of pandurata prepared according to Examples 25 to 28 were about 7 times higher than those of the mouthwashes comprising pandurata extract prepared according to Examples 9 to 12. Since the water-soluble crude extracts of pandurata according to Examples 25 to 28 were obtained from the composition comprising 15 wt% of pandurata crude extract as described in Example 8, it was identified that anti-halitosis effects were improved in the solubilized pandurata extract or panduratin derivative, when comparing anti-halitosis effects based on the amount of the pandurata crude extract used. That is, the solubilized pandurata extract or panduratin derivative is sufficiently dispersed in the mouthwashes and efficiently used, and thus anti-halitosis effects thereof were significantly improved compared non-solubilized pandurata extract or panduratin derivatives.

Examples 29 - 32. Preparation of beverage comprising water-soluble crude extract Beverages were prepared using water-soluble crude extract obtained according to Example 8 and ingredients listed in Table 15 below. Table 15

Measuring anti-halitosis effects of beverage comprising water-soluble crude extract

Anti-halitosis effects were measured in the same manner as in Experimental Example 1 , except that beverages comprising water-soluble crude extracts prepared according to Examples 29 to 32 were used. The results are shown in Table 16 below.

Table 16

While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it will be understood by those of ordinary skill in the art that various changes in form and details may be made therein without departing from the spirit and scope of the present invention as defined by the following claims.

Claims

1. An anti-halitosis composition comprising as an active ingredient: at least one panduratin derivative selected from the group consisting of panduratin derivatives represented by Formulae 1 to 3 below, or Boesenbergia pandurata extract comprising the at least one panduratin derivative:

Formula 1

Formula 2

Formula 3

2. The anti-halitosis composition of claim 1 , wherein the panduratin derivatives are extracted from Boesenbergia pandurata.

3. The anti-halitosis composition of claim 1 , wherein the amount of the panduratin derivatives or Boesenbergia pandurata extracts are in the range of 0.001 to 10 wt% based on the total weight of the anti-halitosis composition.

4. The anti-halitosis composition of any one of claims 1 to 3, wherein the panduratin derivatives or Boesenbergia pandurata extracts are emulsified water-soluble panduratin derivative or Boesenbergia pandurata extract with an emulsifier and an emulsion adjuvant.

5. The anti-halitosis composition of claim 4, wherein the emulsifier is selected from the group consisting of gum arabic, polyglycerin fatty acid ester, sugar ester, polysorbate, and any combination thereof, and the emulsion adjuvant is selected from the group consisting of glycerin, sorbitol, propylene glycol, and any combination thereof.

6. The anti-halitosis composition of claim 5, wherein the emulsifier comprises a mixture of polyglycerin fatty acid ester and sugar ester, and the emulsion adjuvant comprises a mixture of glycerin and propylene glycol.

7. The anti-halitosis composition of claim 4, wherein the emulsified water-soluble panduratin derivative or Boesenbergia pandurata extract is prepared by melting a mixture comprising a panduratin derivative or Boesenbergia pandurata extract, polyglycerin fatty acid ester, sugar ester, propylene glycol, and glycerin at a temperature ranging from 50 to 80"C and emulsifying the melted mixture at a temperature ranging from 65 to 75 "C.

8. The anti-halitosis composition of claim 7, wherein the mixture comprising a panduratin derivative or Boesenbergia pandurata extract, polyglycerin fatty acid ester, sugar ester, propylene glycol, and glycerin comprises 150 to 400 parts by weight of a mixture comprising polyglycerin fatty acid ester, sugar ester, propylene glycol, and glycerin based on 100 parts by weight of the panduratin derivative or Boesenbergia pandurata extract.

9. The anti-halitosis composition of any one of claims 1 to 3, wherein the anti-halitosis composition is formulated in a form selected from the group consisting of mouthwash, food, pharmaceuticals, and animal feed.

10. The anti-halitosis composition of claim 4, wherein the anti-halitosis composition is formulated in a form selected from the group consisting of beverage, milk, fermented milk, and liquid mouthwash.