WO2008099994A1

WO2008099994A1 - Biphenyl diol derivatives and compositions comprising the same as an active ingredient

Info

Publication number: WO2008099994A1
Application number: PCT/KR2007/003680
Authority: WO
Inventors: Deok Hoon Park; Jong Sung Lee
Original assignee: Biospectrum Inc.
Priority date: 2007-02-12
Filing date: 2007-07-31
Publication date: 2008-08-21
Also published as: KR100892596B1; KR20080075403A

Abstract

The present invention relates to a novel biphenyl diol derivative and compositions comprising the same as an active ingredient. The novel biphenyl diol derivatives of this invention have more improved hydrophilicity or hydrophobicity than original substances, honokiol and magnolol. In addition, the derivatives of this invention have significantly improved biological and physiological activity. Furthermore, the biphenyl diol derivatives of this invention can be prepared as various compositions and developed in various products due to the alteration of physical feature.

Description

BIPHENYL DIOL DERIVATIVES AND COMPOSITIONS COMPRISING THE SAME AS AN ACTIVE INGREDIENT

FIELD OF THE INVENTION The present invention relates to a novel biphenyl diol derivative, more particularly, to the novel biphenyl diol derivative and compositions comprising the same as an active ingredient having various physiological and biochemical activities.

DESCRIPTION OF THE RELATED ART In the 21^st century, a need of natural materials becomes increased in all fields.

Most of materials obtained from natural sources have a difficulty of commercialization, since the materials have worst color, perfume and stability as well as difficulty in long- term storage compared to chemically synthesized materials. Especially, some of natural materials are very slightly dissolved in water or solvents and likely to be precipitated over time, and they show lost activity even in the dissolved form, which prevents their applications to pharmaceuticals, agricultural chemicals, households and cosmetics.

Honokiol and magnolol obtained from stem of Magnolia spp. were reported to exhibit alleviation of anxiety (Yuji Maruyama et al., J. Natural Product, 61:135- 138(1998)), anti-inflammatory {Chem. Pharm. Bu., 49(6):716-720(2001)), anti-cancer effect (Hisamitsu Nagase et al., Planta Medica, 69:33-37(2006), anti-microbial effect (Beom Seok Chang et al. Planta Medica, 64: 367-369(1998)) and anti-platelet effects {Arch Pharm. Res. vol 25, No 3:325-328(2002)).

However, as described above, it has been known that honokiol and magnolol have the effects on various bacterium and fungi, but their commercialized products are not yet developed. Test results verifying effectiveness of honokiol and magnolol on bacteria and fungi (Kyu Ho Bang et al., Arch. Pharm. Res. vol 23, No. 1:46-49(2000) and Kun-Yen Ho et a\.,Phytotheraphy Research 15: 139-141(2001)) have been obtained in vitro test, using solvents or surfactants with an excellent solubility such as DMSO (dimethylsulfoxide), alcohols and Tween 80 (surfactant). Particularly, Yuji Maruyama et al. {J. Natural Product, 61; 135-138(1998)) have reported to prepare test samples by dissolving honokiol and magnolol in 0.1% Tween 80. However, where this procedure is practically performed, the samples are immediately precipitated in 0.1% of concentration. In addition, their anti-microbial activity was founded to be lost by more than 80%. The activities of honokiol and magnolol measured in the publications are suggested to be due to the residual activity of magnolol and honokiol. These findings on residual activities are also observed where using Tween 80 instead of DMSO for the field study on patch diseases in lawns. However, where DMSO solution containing magnolol or honokiol is diluted to the final concentration of 0.3%, it shows little or no activities.

This result had a similar pattern to the result described above using DMSO as solvents. The honokiol and magnolol were dissolved in DMSO, and the dissolved solution was adjusted to 0.3% of final concentration by diluting with water. Where the resultant solution was treated, the effect of honokiol and magnolol were lost completely.

Most of an agricultural chemical, households and cosmetic were used cheap solvents such as water as main solvents due to the economic feasibility and convenience in use. Therefore, if an active ingredient is better dissolved in water, it is the most easy to develop products. Furthermore, where materials with solubility in oil were dissolved by a solubilizing method, the product development was easy due to being liable to product development without difficulty. Meanwhile, in spite of not dissolving absolutely, honokiol and magnolol were able to develop product in case that the materials were dissolved and suspended in water by force by adding suitable amount of surfactant or various solvents. However, honokiol and magnolol had problem, which were not dissolved in water or oil at all and in polyol completely. Honokiol and magnolol were precipitated in emulsion or suspension using enough amount of surfactant or solvent in more than 0.1% of concentration after some time, and also precipitated in solubilized liquid form (pharmaceutical, cosmetic, food, households, sanitizer and agricultural chemical) within a short time. Namely, if the precipitation occurs, the activity is always lost. Accordingly, honokiol and magnolol is representative materials which exhibit excellent anti-microbial activity in vitro test using solvent with high solubility and have impossibility of commercialization due to poor solubility in mass production. Therefore, if it is able to prepare derivatives of honokiol and magnolol with the solubility in water or oil and the activity by improving poor solubility of these two materials, the honokiol and magnolol are likely to be developed commercially.

Throughout this application, several patents and publications are referenced and citations are provided in parentheses. The disclosure of these patents and publications is incorporated into this application in order to more fully describe this invention and the state of the art to which this invention pertains.

DETAILED DESCRIPTION OF THIS INVENTION

Honokiol and magnolol, one of biphenyl diol-based substances, have various physiological and biochemical effects; however they have a poor solubility in water and oil. Therefore, the present inventors have made intensive researches to develop novel substances with enhanced solubility as well as physiological and biochemical effects. As a result, the present inventors have found that novel derivatives prepared by organic synthetic procedures have altered physical properties and enhanced effects, allowing to provide cosmetic, food, fodder additives, fertilizer, preservative, sanitizer, agricultural chemical and pharmaceutical compositions.

Accordingly, it is an object of this invention to provide a novel biphenyl diol derivative.

It is another object of this invention to provide a skin whitening composition.

It is still another object of this invention to provide a composition for promoting collagen synthesis.

It is another object of this invention to provide a composition for improving skin wrinkles.

It is still another object of this invention to provide a composition for alleviating skin irritability.

It is another object of this invention to provide a composition for preventing hair loss or promoting hairs growth.

It is still another object of this invention to provide a composition for preventing or treating cardiovascular disorders. It is another object of this invention to provide an anti-oxidation, anti- inflammatory, alleviation of anxiety, anti-cancer, anti-allergy, anti-obesity, anti-bacterial or anti-virus composition.

It is another object of this invention to provide an agricultural chemical composition for protecting plant. It is still another object of this invention to provide a composition of fodder additives for having the activity of anti-oxidation, anti-inflammatory or anti-bacterial.

It is another object of this invention to provide a composition of fodder additives for an aquacultured fish.

It is still another object of this invention to provide a composition of fertilizer additives.

It is another object of this invention to provide a preservative composition.

It is still another object of this invention to provide a sanitizer composition.

It is another object of this invention to provide a skin whitening method.

It is still another object of this invention to provide a method for promoting collagen synthesis.

It is another object of this invention to provide a method for improving skin wrinkles.

It is still another object of this invention to provide a method for alleviating skin irritability.

It is another object of this invention to provide a method for preventing hair loss or promoting hairs growth.

It is still another object of this invention to provide a method for preventing or treating cardiovascular disorders.

It is another object of this invention to provide a method for preventing oxidation.

It is still another object of this invention to provide a method for preventing or treating inflammatory or allergy. It is another object of this invention to provide a method for preventing or treating cancers.

It is still another object of this invention to provide a method for suppressing obesity.

It is another object of this invention to provide a method for suppressing bacterial or virus proliferation.

It is still another object of this invention to provide a method for alleviating anxiety.

Other objects and advantages of the present invention will become apparent from the detailed description to follow taken in conjugation with the appended claims.

In one aspect of the present invention, there is provided a biphenyl diol derivative represented by the following formula I:

wherein Rl represents H, C₂-_I0 alkyl, C₂-_I0 alkyl carboxylic acid or its salt, or amino acid or its salt; R2 represents H, C₂-_I0 alkyl, C₂-_I0 alkyl carboxylic acid or its salt, or amino acid or its salt; R3 represents H, C₂-_I0 alkyl, C_2-I0 alkyl carboxylic acid or its salt, or amino acid or its salt; and at least one of Rl, R2 and R3 is not H.

According to a preferred embodiment, the biphenyl diol derivative is represented by the following formula II:

wherein Rl represents H, C₂-_I0 alkyl, C₂-_I0 alkyl carboxylic acid or its salt, or amino acid or its salt; R2 represents H, C₂-io alkyl, C₂-io alkyl carboxylic acid or its salt, or amino acid or its salt; and in which at least one of Rl and R2 is not H atom.

According to another preferred embodiment, the biphenyl diol derivative is represented by the following formula III:

wherein Rl represents H, C_2-I0 alkyl, C₂-_I0 alkyl carboxylic acid or its salt, or amino acid or its salt; R3 represents H, C_2-I0 alkyl, C_2-I0 alkyl carboxylic acid or its salt, or amino acid or its salt; and at least one of Rl and R3 is not H.

The term "alkyl" used as herein, refers to linear or branched chain saturated hydrocarbon group, including methyl, ethyl, n-propyl, isopropyl, isobutyl, n-butyl and t- butyl, but not limited to.

The term "alkyl carboxylic acid" used as herein, refers to a group bound carboxylic acid with a terminal of linear or branched chain saturated hydrocarbon group. For example, alkyl carboxylic acid includes acetic acid, propion acid, butyric acid, baleic acid, caroic acid, etat acid and capric acid, but not limited to. Preferably, alkyl carboxylic acid is C₂-5 alkyl carboxylic acid

Where the R group is alkyl carboxylic acid, preferably the derivatives may be in a salt type. This salt may be prepared using inorganic acids or organic acids. The inorganic acids may include hydrochloride, hydrobromide and hydroiodide. As the organic acid, acetate, adipate, alginate, aspartate, benzoate, benzensulfonate, p- toluenesulfonate, bisulfate, sulfamate, sulfate, naphthalate, butyrate, Citrate, camphorate, camphorsulfonate, cyclopenthanepropionate, digluconate, dodecylsulfate, ethanesulfonate, fumarate, glucoheptonate, glycerophosphate, hemisulfate, heptanoate, hexanoate, 2-hydroxyethansulfate, lactate, Maleate, methane sulfonate, 2-naphthalenesulfonate, nicotinate, oxalate, tosylate and undecanoate may be used. Prefeably, the salt is salt bound with inorganic acid, most preferably hydrochloric acid salt formed binding with hydrochloride.

The term "amino acid" used as herein, refers to a compound having both amino group and carboxylic group. For example, amino acid includes glysine, alanine, valine, leucine, serine, threonine, cysteine, methionine, proline, phenylalanine, tylosin, tryptophane, aspartic acid, glutamic acid, asparagine, glutamine, lysine, arginine and histidine, but not limited to. Preferably, amino acid binding to R group is glysine, alanine, serine, threonine, cysteine, methionine, tylosin, aspartic acid, glutamic acid, asparagine, glutamine, lysine and arginine, most preferably glysine.

Honokiol and magnolol as original substances of biphenyl diol derivatives of the present invention are a kind of biphenyl diol materials isolating from stem of Magnolia spp. IUPAC name of honokiol is 2-(4-hydroxy-3-prop-2-enyl-phenyl)-4-prop-2-enyl- phenol, and IUPAC name of magnolol is 2-(2-hydroxy-5-prop-2-enyl-phenyl)-4-prop- 2-enyl-phenol. It has been known that honokiol and magnolol have alleviation of anxiety, anti-inflammatory, anti-cancer, anti-bacterial and anti-platelet effect, but products are not developed due to the poor solubility in solvents.

According to the present invention, there is provided novel materials with an improved property by binding various chemical residues to honokiol of the following formula (IV) and magnolol of the following formula (V) by organic chemical processes. Examples demonstrate that the novel compounds of the present invention are modified into hydrophilicity and hydrophobicity according to residues bound. Furthermore, from various physiological and biochemical effects test by dissolving the novel derivatives in suitable solvents according to their physical property, it is proved in example that the derivatives of this invention have higher effects than honokiol and magnolol.

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According to more preferred embodiment, in the formula (I) to (III), Rl represents H, C_2-5 alkyl carboxylic acid, or amino acid or its salt; R2 represents H, C₂-₅ alky! carboxylic acid, or amino acid or its salt; R3 represents H, C_2-S alkyl carboxylic acid or its salt, or amino acid or its salt; and at least one of Rl, R2 and R3 is not H.

According to a preferred embodiment, only one group of Rl, R2 and R3 is H in the formula (I) to (III).

According to still more preferred embodiment, the biphenyl diol derivative of the present invention is the following compounds: 2-(4-hydroxy-3-prop-2-enylphenyl)- 4-prop-2-enylphenyl-2-aminoethanoate; 2-(4-aminoethanoyl-3-prop-2-enylphenyl)-4- prop-2-enylphenyl-2-aminoethanoate; 2-(4-aminopropanoyl-3-prop-2-enylphenyl)-4- prop-2-enylphenyl-2-aminopropanoate dihydrochloride salt; 2-(4-hydroxy-3-prop-2- enylphenyl)-4-prop-2-enylphenyl-2-aminoethanoate hydrochloride salt; 2-(4- aminoethanoyl-3-prop-2-enylphenyl)-4-prop-2-enylphenyl-2-aminoethanoate dihydrochloride salt; 3-({4-[2-(3-carboxypropanoyloxy)-5-prop-2-enylphenyl]-2-prop-2- enylphenyl}oxycarbonyl)propionic acid; 2-(2-hydroxy-5-prop-2-enylphenyl)-4-prop-2- enylphenyl-2-aminoethanoate; 2-(2-hydroxy-5-prop-2-enylphenyl)-4-prop-2- enylphenyl-2-aminoethanoate hydrochloride salt; 2-(2- aminoethanoyl -5-prop-2- enylphenyl)-4-prop-2-enylphenyl-2-aminoethanoate dihydrochloride salt; or 3-({2-[2- (3-carboxypropanoyloxy)-5-prop-2-enylphenyl]-4-prop-2- enylphenyl}oxycarbonyl)propionic acid.

Among of the biphenyl diol derivatives, 2-(4-hydroxy-3-prop-2-enylphenyl)-4- prop-2-enylphenyl-2-aminoethanoate; 2-(4-hydroxy-3-prop-2-enylphenyl)-4-prop-2- enylphenyl-2-aminoethanoate hydrochloride salt; 2-(4-aminoethanoyl-3-prop-2- enylphenyl)-4-prop-2-enylphenyl-2-aminoethanoate dihydrochloride salt; 2-(2-hydroxy- 5-prop-2-enylphenyl)-4-prop-2-enylphenyl-2-aminoethanoate; 2-(2-hydroxy-5-prop-2- enylphenyl)-4-prop-2-enylphenyl-2-aminoethanoate hydrochloride salt; 2-(2- aminoethanoyl -5-prop-2-enylphenyl)-4-prop-2-enylphenyl-2-aminoethanoate dihydrochloride salt; preferably, 2-(4-hydroxy-3-prop-2-enylphenyl)-4-prop-2- enylphenyl-2-aminoethanoate hydrochloride salt; 2-(4-aminoethanoyl-3-prop-2- enylphenyl)-4-prop-2-enylphenyl-2-aminoethanoate dihydrochloride salt; and 2-(2- hydroxy-5-prop-2-enylphenyl)-4-prop-2-enylphenyl-2-aminoethanoate hydrochloride salt; 2-(2- aminoethanoyl -5-prop-2-enylpheny!)-4-prop-2-enylphenyl-2- aminoethanoate dihydrochloride salt have the improvement of activity and hydrophilicity compared with honokiol or magnolol as starting substance.

In another aspect of the present invention, there is provided a skin whitening composition comprising the above-described biphenyl derivatives as an active ingredient. In still another aspect of the present invention, there is provided a skin whitening method, which comprises administering to a subject the above-described biphenyl derivatives as an active ingredient.

In another aspect of the present invention, there is provided a use of the above-described biphenyl derivatives for manufacturing a skin whitening composition. In still another aspect of the present invention, there is provided a composition for promoting collagen synthesis comprising the above-described biphenyl derivatives as an active ingredient.

In another aspect of the present invention, there is provided a method for promoting collagen synthesis, which comprises administering to a subject the above- described biphenyl derivatives as an active ingredient.

In still another aspect of the present invention, there is provided a use of the above-described biphenyl derivatives for manufacturing a composition for promoting collagen synthesis. In another aspect of the present invention, there is provided a composition for improving skin wrinkles comprising the above-described biphenyl derivatives as an active ingredient.

In still another aspect of the present invention, there is provided a method for improving skin wrinkles, which comprises administering to a subject the above- described biphenyl derivatives as an active ingredient.

In another aspect of the present invention, there is provided a use of the above-described biphenyl derivatives for manufacturing a composition for improving skin wrinkles. In still another aspect of the present invention, there is provided a composition for preventing hair loss or promoting hairs growth comprising the above-described biphenyl derivatives as an active ingredient.

In another aspect of the present invention, there is provided a method for preventing hair loss or promoting hairs growth, which comprises administering to a subject the above-described biphenyl derivatives as an active ingredient.

In still another aspect of the present invention, there is provided a use of the above-described biphenyl derivatives for manufacturing a composition for preventing hair loss or promoting hairs growth.

In another aspect of the present invention, there is provided a composition for preventing or treating cardiovascular disorders having anti-platelet effect comprising the above-described biphenyl derivatives as an active ingredient.

In still another aspect of the present invention, there is provided a method for preventing or treating cardiovascular disorders having anti-platelet effect, which comprises administering to a subject the above-described biphenyl derivatives as an active ingredient.

In another aspect of the present invention, there is provided a use of the above-described biphenyl derivatives for manufacturing a composition for preventing or treating cardiovascular disorders having anti-platelet effect. In still another aspect of the present invention, there is provided an anti- oxidation, anti-inflammatory, alleviation of anxiety, anti-cancer, anti-allergy, anti- obesity, anti-bacterial or anti-virus composition, which comprises the above-described biphenyl derivatives as an active ingredient. In another aspect of the present invention, there is provided a method for preventing oxidation, which comprises administering to a subject the above-described biphenyl derivatives as an active ingredient.

In still another aspect of the present invention, there is provided a method for preventing or treating inflammatory or allergy, which comprises administering to a subject the above-described biphenyl derivatives as an active ingredient.

In another aspect of the present invention, there is provided a method for preventing or treating cancers, which comprises administering to a subject the above- described biphenyl derivatives as an active ingredient.

In still another aspect of the present invention, there is provided a method for suppressing obesity, which comprises administering to a subject the above-described biphenyl derivatives as an active ingredient.

In another aspect of the present invention, there is provided a method for suppressing bacterial or virus proliferation, which comprises administering to a subject the above-described biphenyl derivatives as an active ingredient. In still another aspect of the present invention, there is provided a method for alleviating anxiety, which comprises administering to a subject the above-described biphenyl derivatives as an active ingredient.

In another aspect of the present invention, there is provided a use of the above-described biphenyl derivatives for manufacturing an anti-oxidation, anti- inflammatory, alleviation of anxiety, anti-cancer, anti-allergy, anti-obesity, anti-bacterial or anti-virus composition. It has been known that honokiol and magnolol have a potent activity of anti- oxidation and anti-inflammatory. However, the derivatives of honokiol and magnolol of this invention have the improvement of the solubility in specific solvents and exhibit strongr effects of anti-oxidation and anti-inflammatory. Namely, where the hydrophilic derivatives of this invention (e.g., 2-(4-aminoethanoyl-3-prop-2-enylphenyl)-4-prop-2- enylphenyl-2-aminoethanoate dihydrochloride salt) are dissolved in water, the derivatives have higher effects of anti-oxidation and anti-inflammatory than honokiol and magnolol, and where the hydrophobic derivatives of this invention (e.g., 3-({4-[2- (3-carboxypropanoyloxy)-5-prop-2-enylphenyl]-2-prop-2-enylphenyl}oxycarbonyl) prop ionic acid) are dissolved in organic solvents, the derivatives have higher effects of anti- oxidation and anti-inflammatory than honokiol and magnolol, which are demonstrated in example.

Furthermore, the derivatives of honokiol or magnolol used as active ingredients have more excellent whitening effect, especially the effect surpressing pigmentation by UV, compared to honkiol or magnolol. As shown in the following example clearly, the derivatives of honokiol and magnolol used as cosmetic compositions of this invention exert action on the prevention and elimination of wrinkles by more promoting collagen synthesis compared with original substances.

In another aspect of the present invention, there is provided a composition for alleviating skin irritability comprising the above-described biphenyl derivatives as an active ingredient. In the application of the biphenyl diol derivatives of this invention to skin, the derivatives show strong affinity to surface and horny layer of skin, and can improve moisture-maintaining function by being dense its structure. At the same time, the derivatives may be used as active ingredients for improving drying skin or maintaining in healthy skin condition by promoting the alteration of horny layer, and have the excellent effects of the prevention of skin aging and the contribution of wettability, softness and elasticity. The biphenyl diol derivatives of this invention contribute very effectively to the prevention of hair loss or the promotion of hairs growth. The terms "hair loss prevention" and "hairs growth promotion" used herein have the same meaning.

In addition, the biphenyl diol derivatives of this invention may be used to the prevention or treatment of cardiovascular disorders having more potent anti-platelet effect than original substances.

Furthermore, as shown in the following example clearly, the derivatives of this invention have excellent anti-oxidation, anti-inflammatory, alleviation of anxiety, anticancer, anti-allergy, anti-obesity, anti-bacterial and anti-virus effects. The biphenyl diol derivatives of this invention have more the improved antibacterial and anti-virus effects on microorganisms in food and pharmaceutical industry relative to original substances.

The derivatives of this invention have potent anti-microbial activity against pathogen inducing food poisoning, inflammatory such as impetigo and cellulites, pneumonia, gonorrhea, meningitis, anthrax, tetanus, diphtheriae, inflammatory of burn patients, cystitis, enteritis, sepsis, cholera food poisoning, peptic ulcer and leptospirosis.

In the concrete, the biphenyl diol derivatives of this invention have more the improved anti-microbial activity against pathogen in food and pharmaceutical industry included to genus and species listing below in taxology relative to original substances. The pathogen includes Staphylococcus spp., Streptococcus spp., Streptococcus pneumoniae, Clostridium tetan/, Bacillus cereus, Proteus spp., Salmonella spp., Vibriocea, rle/ocoσacter pylori and Loptospira.

The composition of the present invention may be prepared as various compositions according to objects of this invention. Preferably, the composition can be prepared as a cosmetic, pharmaceutical or food composition.

The cosmetic compositions of the present invention may contain auxiliaries as well as carrier in addition to the biphenyl diol derivatives as active ingredients. The non-limiting examples of auxiliaries include antioxidants, stabilizers, solubilizers, vitamins, colorants, odor improvers or mixtures of these ingredients.

The cosmetic compositions of this invention may be formulated in a wide variety of form, for non-limited example, including a solution, a suspension, an emulsion, a paste, a gel, a cream, a lotion, a powder, a soap, a surfactant-containing cleanser, an oil, a powder foundation, an emulsion foundation, a wax foundation and a spray. In detail, the cosmetic composition of the present invention can be provided in a form of nutrient cream, astringent lotion, skin softener (skin lotion), lotion, essence, nutrient gel or message cream.

The cosmetically acceptable carrier contained in the present cosmetic composition, may be varied depending on the type of the formulation. For example, the formulation of pastes, creams or gels may comprise animal and vegetable fats, waxes, paraffins, starch, tragacanth, cellulose derivatives, polyethylene glycols, silicones, bentonites, silica, talc, zinc oxide or mixtures of these ingredients.

In the formulation of powder or spray, it may comprise lactose, talc, silica, aluminum hydroxide, calcium silicate, polyamide powder and mixtures of these ingredients. Spray may additionally comprise the customary propellants, for example, chlorofluorohydrocarbons, propane/butane or dimethyl ether. The formulation of solution and emulsion may comprise solvent, solubilizer and emulsifier, for example water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butyleneglycol oils, glycerol fatty esters, polyethylene glycol, fatty acid esters of sorbitan or mixtures of these ingredients. The formulation of suspension may comprise liquid diluents, for example water, ethanol or propylene glycol, suspending agents, for example ethoxylated isosteary alcohols, polyoxyethylene sorbitol esters and poly oxyethylene sorbitan esters, micocrystalline cellulose, aluminum metahydroxide, bentonite, agar and tragacanth or mixtures of these ingredients.

The formulation of cleansing compositions with surfactant may comprise aliphatic alcohol sulfate, aliphatic alcohol ether sulfate, sulfosucinnate monoester, isothinate, imidazolium derivatives, methyltaurate, sarcocinate, fatty acid amide ether sulfate, alkyl amido betain, aliphatic alcohol, fatty acid glyceride, fatty acid diethanolamide, vegetable oil, lanoline derivatives, ethoxylated glycerol fatty acid ester or mixtures of these ingredients.

The composition of this invention may be prepared as a pharmaceutical composition, and the pharmaceutically acceptable carrier as well as the active ingredient contained in the pharmaceutical composition. The pharmaceutically acceptable carrier, which is commonly used in pharmaceutical formulations, but is not limited to, includes lactose, dextrose, sucrose, sorbitol, mannitol, starch, rubber arable, potassium phosphate, arginate, gelatin, potassium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrups, methyl cellulose, methylhydroxy benzoate, propylhydroxy benzoate, talc, magnesium stearate, and mineral oils. The pharmaceutical composition according to the present invention may further include a lubricant, a humectant, a sweetener, a flavoring agent, an emulsifier, a suspending agent, and a preservative. Details of suitable pharmaceutically acceptable carriers and formulations can be found in Remington's Pharmaceutical Sciences (19th ed_v 1995). A pharmaceutical composition of this invention may be administered orally or parenterally, and the parenteral administration comprises intravenous injection, subcutaneous injection, intramuscular injection, intraperitoneal injection and dermal administration.

A suitable dosage amount of the pharmaceutical composition of the present invention may vary depending on pharmaceutical formulation methods, administration methods, the patient's age, body weight, sex, pathogenic state, diet, administration time, administration route, an excretion rate and sensitivity for a used pharmaceutical composition, and physicians of ordinary skill in the art can determine an effective amount of the pharmaceutical composition for desired treatment. According to a preferred embodiment of this invention, suitable dosage unit is to administer once a day with 0.001-100 mg/kg (body weight).

According to the conventional techniques known to those skilled in the art, the pharmaceutical composition of the present invention may be formulated with pharmaceutically acceptable carrier and/or vehicle as described above, finally providing several forms a unit dose form and a multi-dose form. Non-limiting examples of the formulations include, but not limited to, a solution, a suspension or an emulsion in oil or aqueous medium, an extract, an elixir, a powder, a granule, a tablet and a capsule, and may further comprise a dispersion agent or a stabilizer.

The composition of this invention may be prepared as a food composition. The food composition of this invention may comprise conventional additives for preparing food compositions, e.g., protein, carbohydrates, lipids, nutritive substances and flavors.

Non-limiting examples of carbohydrates described above include, but not limited to, monosaccharide {e.g., glucose and fructose); disaccharide (e.g., maltose, sucrose and oligosaccharide); and polysaccharide {e.g., dextrin and cyclodextrin); and sugar alcohol (e.g., xylitol, sorbitol and erithritol). Non-limiting examples of Flavors include, but not limited to, natural flavors [thaumatin and extract of stevia {e.g., rebaudioside A and glycyrrhizin)] and synthetic flavors {e.g., saccharin and aspartame). For example, where the food composition of this invention is provided as a drink, it may further comprise citric acid, liquid fructose, sugar, glucose, acetic acid, malic acid, fruit juice, extract of eucommia ulmoides oliv, jujube extract or extract of glycyrrhiza uralensis.

In another aspect of the present invention, there is provided an agricultural chemical composition for protecting plant comprising the above-described biphenyl derivatives as an active ingredient. Preferably, the agricultural chemical composition has a control effect of rice blast disease. The biphenyl diol derivatives of the present invention have the improved antimicrobial and anti-virus effects on agricultural microorganisms compared to original substances.

The biphenyl diol derivatives have potent sterilizing effects on pathogen or virus causing disease to plants, and are able to use as germicide for agriculture due to not having harmful effects of a medicine.

The derivatives of this invention are used for the purpose of preventing disease of plants and disease generating during storage of agricultural product, or reducing damage of disease. The plants include cereals such as rice, wheat and barley; fruit tree such as apple, pear, tangerine, grapes, banana and peach; vegetables such as coffee, tea plant, potato, red pepper, green pepper, tomato, cucumber, water melon, melon, lettuce, Chinese cabbage, celery, rape, peanut, cabbage, leek, garlic, ginger and onion; lawn, forestry and ornamental nursery tree; flowering grass such as carnation, lily, rose and chrysanthemum. In the concrete, the biphenyl diol derivatives of this invention are used for preventing disease caused by plant pathogen belonging to genus and species listed below in taxology relative to original substances.

The plant pathogen includes Alternaria alternate, Rhizoctonia so/an/, Phytophthroa capsici, Colletotrichum gloeosproioides, Alternaria brassicae, Aphanomyces raphani, Pythium ultimum, Cercosporella albomaculans, Urocystis cepulae, Puccinia all/7, Nidymella bryoriae, Colletotrichum lagenarium, Plasmodiophora brassicae, Sphaerotheca fuliginea, Pyricularia oryzae, Marssonia mali, FuMa, Podosphaera leucotricha, Venturina pyrina, Botryospheria dothidea and Diaporthe citri. In addition, the derivatives of this invention exert an excellent control effect on black mold, damping off, late blight, anthrax, brittle root rot, blast, downy mildew, sheath blight, blotch, canker and powdery mildew. Where the composition of this invention is prepared as agricultural chemical composition, the composition may be prepared as random suitable formulation such as emulsifier, liquid medicine, wettable powder, powder, particle, oil solution, aerosol, or flowables, by mixing with suitable solid carrier, liquid carrier, gas phase carrier, surfactant, dispersing agent and/or auxiliary.

The solid carrier used herein includes talc, bentonites, clay, kaolin, diatomite, vermiculite, white carbon and calcium carbonate. As the liquid carrier, alcohol such as methanol, n-hexanol and ethanol glycol; ketone such as acetone, methyl ethyl ketone and cyclohexanone; aliphatic hydrocarbons such as n-hexane and kerosene; aromatic hydrocarbons such as toluene, xylene and methylnaphthalene; ether such as diethylether, dioxane and tetrahydrofurane; ester such as ethylacetate; nitrile such as acetonitrille and isobutylnitrille; acid amide such as dimethylformamide and dimethylacetamide; plant oil dimethysulfoxide such as soybean oil and cottonseed oil; and water used. The gas phase carrier includes LPG, air, nitrogen, carbon dioxide and dimethylether.

A suitable content of biphenyl diol derivatives contained in agricultural chemical composition for protecting plant as an active ingredient may vary depending on formulation, use methods, use environment and other condition. Where agricultural chemical composition is an emulsifier, the content of biphenyl diol derivatives is 0.1 to 75 wt%, preferably 5 to 30 wt%; in case of powder, ranges from 0.3 to 25 wt%, preferably 1 to 3 wt%, in case of wettable powder, 1 to 90 wt%, preferably 5 to 50 wt%; in particle, 0.5 to 50 wt%, preferably 2 to 30 wt%.

The agricultural chemical composition of this invention is generally used as it is or after dilution. the use method of the agricultural chemical composition of this invention includes the application to plant itself (application to stem and leaf), rice seeding box (in case of rice), soil (mixing with soil or fertilizer given to growing plants), spring water and seeds. In another aspect of the present invention, there is provided a composition of fodder additives for having the activity of anti-oxidation, anti-inflammatory or antibacterial, which comprises the above-described biphenyl derivatives as an active ingredient.

According to a preferred embodiment, the composition of fodder additives is a composition for an aquacultured fish.

The following examples demonstrate that the biphenyl derivatives of the present invention may be used to fodder additives due to having more enhanced activity of anti-oxidation, anti-inflammatory or anti-bacteria.

The composition of fodder additives of this invention may be prepared adding diol derivative itself or carrier and stablilizer, as occasion demands, adding various nutrients such as vitamin, amino acid and mineral, anti-oxidation agent, antibiotics, antimicrobial agent and other additives. The formulation includes powder, granule, pellet and suspension.

Where the fodder additives are fed to land or water animals, the additives are supplied as it is or mixing with fodder.

In addition, the fodder of this invention includes powder fodder, solid fodder, moist pellet fodder, dry pellet fodder, EP (Extruder Pellet) fodder and raw food, but not limited to.

Furthermore, the composition of this invention can be prepared as a fertilizer additives, preservative or sanitizer composition.

The fertilizer additives composition of this invention are used adding to an existing fertilizer (e.g., fertilizer containing nitric acid, phosphoric acid or potassium) or mixing with germicide, insecticide, herbicide, plant growth regulator and fertilizer.

The preservative composition is used for the purpose of preserving food, cosmetic, households, living materials, construction materials or medical supplies. And the sanitizer composition is used for the sterilization of a densely populated district such as cattle pen, hospital, factory, apartment or school.

The summary of features and advantages of this invention is as follows: (i) The novel biphenyl diol derivatives of this invention have more improved physical property, especially hydrophilicity or hydrophobicity than original substances, honokiol and magnolol.

(ii) The novel biphenyl diol derivatives of this invention exhibit more improved solubility than original substances, honokiol and magnolol. (iii) The novel biphenyl diol derivatives of this invention have more excellent biological and physiological activity {e.g., skin whitening, collagen synthesis promotion, wrinkles improvement, skin irritability alleviation, hair loss prevention or hairs growth promotion, anti-platelet effect, anti-oxidation, anti-inflammatory, alleviation of anxiety, anti-cancer, anti-allergy, anti-obesity, anti-bacterial, anti-virus and plant protecting effect) than original substances.

(iv) The biphenyl diol derivatives of this invention can be prepared as various compositions and developed in various products due to the alteration of physical feature.

The present invention will now be described in further detail by examples. It would be obvious to those skilled in the art that these examples are intended to be more concretely illustrative and the scope of the present invention as set forth in the appended claims is not limited to or by the examples.

EXAMPLES

Example 1: 2-(4-aminoethanoyl-3-prop-2-enylphenyl)-4-prop-2- enylphenyl-2-aminoethanoate di hydrochloride salt

In a 250 ml round bottom flask, Boc-Gly:(2-{[l-(l,l-dimethyl ethyloxy)ethenyl]amino}ethanoic acid) (1.74 g, 10 mmol, Sigma) was dissolved in 50 ml of anhydrous chloroform and 50 ml of dioxin and cooled to O⁰C, followed by adding DCC (l,3-diaza-l,3-dicyclohexylpropa-l,2-diene) (Sigma). 2-(4-hydroxy-3-prop-2- enylphenyl)-4-prop-2-enylphenyl-l-ol (2.93 g, 0.01 M) and TEA (triethylamine, 1.26 ml, 0.09 M) were slowly added to the reaction solution at O⁰C and stirred for 1 day at room temperature, followed by stirring for 6 hr at 4O⁰C to complete the reaction. After filtration of the solution, the filtrate was washed with 1 N HCI, 5% NaHCO₃ solution and water. Moisture was removed from the residual solution using anhydrous calcium carbonate and organic solvent was eliminated under vacuum. The residual was dissolved in ethylacetate and was subjected to bubbling with HCI for 4 hr at room temperature. Following concentration, the residual was recrystallized with ether/petroleum ether, thereby obtaining 1.51 g (yield: 43%) of 2-(4-aminoethanoyl-3- prop-2-enylphenyl)-4-prop-2-enylphenyl-2-aminoethanoate dihydrochloride salt represented by the following formula VI (the prepared sample names as "1"): NMR (δ, ppm D₂O+DMSO): 8.4 (2H,b), 7.0-7.2 (6H,m), 5.9 (2H,m), 5.1 (4H,d), 3.9 (2H,s), 3.4 (4H,q); IR (cm^"1; C=O) 1630, 1625; ¹³C (ppm; C=O) 171, 172 Formula VI

Example 2: 2-(4~aminopropanoyl-3~prop-2-enylphenyl)-4-prop-2- enylphenyl-2-aminopropanoate dihydrochloride salt

In a 250 ml of round bottom flask, Boc-Ala:(2-{[1-(1,1- dimethyl ethyloxy)etheny!]amino}propanoic acid) (1.88 g, 10 mmol, Sigma) was dissolved in 50 ml of anhydrous chloroform and 50 ml of dioxin, cooled to O⁰C, followed by adding DCC (l,3-diaza-l,3-dicyclohexylpropa-l,2-diene) (Sigma). 2-(4-hydroxy-3-prop-2- enylphenyl)-4-prop-2-enylphenyl-l-ol (2.93 g, 0.01 M) and TEA (triethylamine, 1.26 ml, 0.09 M) were slowly added to the reaction solution at O⁰C and stirred for 1 day at room temperature, followed by stirring for 6hr at 40⁰C to complete the reaction. After filtration of the solution, the filtrate was washed with 1 N HCI, 5% NaHCO₃ solution and water. Moisture was removed from the residual solution using anhydrous calcium carbonate and organic solvent was eliminated under vacuum. The residual was dissolved in ethyacetate and was subjected to bubbling with HCI for 4 hr at room temperature. Following concentration, the residual was recrystallized with ether/petroleum ether, thereby obtaining 1.68 g (yield: 45%) of 2-(4-aminopropanoyl- 3-prop-2-enylphenyl)-4-prop-2-enylphenyl-2-arninopropanoate dihydrochloride salt represented by the following formula VII: NMR(δ, ppm D₂O-I-DMSO): 8.4 (2H,b), 7.0- 7.3 (6H,m), 5.9 (2H,m), 5.1 (4H,q), 3.9 (lH,d), 3.4 (2H,d), 1.3 (3H,d); IR (cm^"1; C=O) 1630, 1625; ¹³C (ppm; C=O) 171, 172 Formula VII

Example 3: 3-({4-[2-(3-carboxypropanoyloxy)-5-prop-2-enylphenyl]-2- prop-2-enylphenyl}oxycarbonyl)propionic acid

In a 250 ml of round bottom flask, 2-(4-hydroxy-3-prop-2-enylphenyl)-4-prop- 2-enylphenyl-l-ol (2.93 g, 0.01 M) was dissolved in 200 ml of pyridine at 5O⁰C, maleicanhydride (3,4-dihydrofuran-2,5-dion) (4 g, 0.04M, Sigma) was added, and the solution was reacted for 2 days. After completion of the reaction, a solvent were removed by vacuum, and the residual was dissolved in 100 ml of 5% NaHCO₃ solution. The resultant solution was extracted twice with 50 ml of ethylacetate, the residual was eliminated, and cooled to O⁰C. 200 ml of 10% citric acid was added to thereto for acidifying and extracted twice with 100 ml of ethylacetate. The extract was washed twice with water, the moisture was removed from the extract using anhydrous calcium carbonate and organic solvent was eliminated under vacuum distillation, thereby obtaining 2.5 g (yield: 53%) of 3-({4-[2-(3-carboxypropanoyloxy)-5-prop-2- enylphenyl]-2-prop-2-enylphenyl}oxycarbonyl)propionic acid represented by the following formula VIII (the prepared sample names as "2"): NMR (δ, ppm CDCI₃): 7.0- 7.3 (6H,m), 5.9 (2H,m), 5.1 (4H,q), 4.1 (lH,md), 3.4 (4H,d), 2.9 (4H,md), 2.6 (4H,md); IR (cm^"1 ; C=O) 1765, 1770; ¹³C (ppm ; C=O) 171, 172

Formula VIII

Example 4: 2-(2- aminoethanoyl -5-prop-2-enylphenyl)-4-prop-2- enylphenyl-2-aminoethanoate dihydrochloride salt

In a 250 ml of round bottom flask, Boc-Gly:(2-{[l-(l,l-dimethyl ethyloxy)ethenyl]amino}ethanoic acid) (1.74 g, 10 mmol, Sigma) was dissolved in 50 ml of anhydrous chloroform and 50 ml of dioxin, cooled to O⁰C, followed by adding DCC (l,3-diaza-l,3-dicyclohexylpropa-l,2-diene) (Sigma). 2-(2-hydroxy-5-prop-2- enylphenyl)-4-prop-2-enylphenyl-l-ol (2.93 g, 0.01 M) and TEA (triethylamine, 1.26 ml, 0.09 M) were slowly added to the reaction solution at O⁰C and stirred for 1 day at room temperature, followed by stirring for 6hr at 4O⁰C to complete the reaction. After filtration of the solution, the filtrate was washed with 1 N HCI, 5% NaHCO₃ solution and water. Moisture was removed from the residual solution using anhydrous calcium carbonate and organic solvent was eliminated under vacuum. The residual was dissolved in ethyacetate and was subjected to bubbling with HCI for 4 hr at room temperature. Following concentration, the residual was recrystallized with ether/petroleum ether, thereby obtaining 1.40 g (yield: 40%) of 2-(2- aminoethanoyl - 5-prop-2-enylphenyl)-4-prop-2-enylphenyl-2-aminoethanoate dihydrochloride salt represented by the following formula IX (the prepared sample names as "3"): NMR(δ, ppm D₂O+DMSO): 8.4 (2H,b), 7.0-7.2 (6H,m), 5.9 (2H,m), 5.1 (4H,d), 3.9 (2H,s), 3.4 (4H,q); IR(Cm-¹; C=O) 1630, 1625; ¹³C (ppm ; C=O) 171, 172 Formula IX

Example 5: 3-(-{2-[2-(3-carboxypropanoyloxy)-5-prop-2-enylphenyl]-4- prop-2-enylphenyl}oxycarbonyl)propionic acid

In a 250 ml of round bottom flask, 2-(2-hydroxy-5-prop-2-enylphenyl)-4-prop- 2-enylphenyl-l-ol (2.93 g, 0.01 M) was dissolved in 200 ml of pyridine at 5O⁰C, maleicanhydride (3,4-dihydrofuran-2,5-dion) (4 g, 0.04M, Sigma) was added, and the solution was reacted for 2 days. After completion of the reaction, a solvent were removed by vacuum, and the residual was dissolved in 100 ml of 5% NaHCO₃ solution.

The resultant solution was extracted twice with 50 ml of ethylacetate, the residual was eliminated, and cooled to O⁰C. 200 ml of 10% citric acid was added to thereto for acidifying and extracted twice with 100 ml of ethylacetate. The extract was washed twice with water, the moisture was removed from the extract using anhydrous calcium carbonate and organic solvent was eliminated under vacuum distillation, thereby obtaining 2.36 g (yield: 53%) of 3-({2-[2-(3-carboxypropanoyloxy)-5-prop-2- enylphenyl]-4-prop-2-enylphenyl}oxycarbonyl)propioni c acid represented by the following formula X (the prepared sample names as "4"): NMR (δ, ppm CDCI₃): 7.0-7.3 (6H₇ITi), 5.9 (2H,m), 5.1 (4H,q), 4.1 (lH,md), 3.4 (4H,d), 2.9 (4H,md), 2.6 (4H,md); IR (cm-¹; C=O) 1630, 1625; ¹³C (ppm ; C=O) 171, 172 Formula X

1

Example 6: Anti-oxidation effect

Superoxide dismutase (SOD) has been known as an anti-oxidation-catalyzing enzyme which converts superoxide anion into H₂O₂ and O₂. This experiment evaluates anti-oxidation activity of sample by observing removal of superoxide anion generated by xanthine oxidase. The experiment was performed using the kit purchased from Dojindo (JP) in accordance with manufacture's protocol. A sample was used by dissolving in DMSO, Tween 80 (0.1% solution), water or refined olive oil. Where Tween 80 was used, 0.1% Tween 80 was contained in the final prepared solution and pH value of the solution was adjusted to 5.0. All solutions containing Tween 80 used in Examples were prepared with the same concentration and pH. Where the sample was dissolved in water, pH value was also adjusted to 5.0. All experiments using water as solvents were carried out at pH 5.0. In particular, several experiments using culture media or sodium acetate buffer as a solvent were performed at pH 5.0. The samples tested were analyzed to exhibit the anti-oxidation activity by observing removal of superoxide anion. The results were summarized in Table 1. Table 1

Untreatment

Solvents Honokiol Magnolol group

DMSO ++ ++ ++ +

-: no activity of anti-oxidation, +: 1-10% activity of anti-oxidation, ++: 11-20% activity of anti-oxidation, +++: 20-30% activity of anti-oxidation

As shown in Table 1, it was founded that magnolol and honokiol had a strong activity of anti-oxidation, and derivatives of the two substances, samples 1, 2, 3 and 4 exhibited stronger activity than original substances. Where Nos. 2 and 4 derivatives having low solubility in water were dissolved in water, their anti-oxidation effect was analyzed to be poor due to their insolubility. In addition, because Nos. 1 and 3 derivatives had no solubility in oil, a mixture of the derivatives and oil did not exhibit the activity. In contrast, where Nos. 1 and 3 derivatives were dissolved in water, the derivatives showed a potential anti-oxidation effect, which was not observed in original substances. The effect of Nos. 1 and 3 derivatives dissolved in water was much better than that of original substances dissolved in DMSO, demonstrating that the derivatives of this invention have improved physical properties to increase their activities. Where the derivatives of the present invention are formulated in pharmaceutical compositions, particularly for injection, the Nos. 1 and 3 derivatives are very useful due to their excellent solubility in water. This is because most of injections are a water phase and a water phase of Nos. 1 and 3 derivatives is available as injection without additional processing. Furthermore, where Nos. 2 and 4 derivatives were dissolved in oil, the derivatives exhibited stronger effect than original substances having no effect. However, the effect of Nos. 2 and 4 derivatives were lower than that of Nos. 2 and 4 derivatives dissolved in DMSO or Nos. 1 and 3 derivatives dissolved in water. This is because in case that Nos. 2 and 4 derivatives were dissolved in oil, they were separated in a reaction solution in the experiment of in vitro. It was founded that the reason of this result was the incomplete reaction. Therefore, the activity of real improved derivatives did not exert by 100%. Nevertheless, these results address that the derivatives of this invention have considerably improved effect relative to original substances.

During the treatment in Tween 80 solution, honokiol and magnolol were precipitated slightly and showed a partial effect due to the feature of honokiol and magnolol inactivated, but all of 4 type derivatives of the present invention exhibited an excellent effect. However, Nos. 2 and 4 derivatives having solubility in oil had lower effect than Nos. 1 and 3 derivatives having solubility in water, since Tween 80 used were of small quantity to completely solubilize the derivatives. Accordingly, Nos. 2 and 4 derivatives were used with not a completely solubilized condition but a partial solubilized condition in the experiment. These experiment results demonstrate that the Nos. 2 and 4 derivatives incompletely solubilized had lower effect than the derivatives fully dissolved in DMSO. However, it was founded that the activity of derivatives were remarkably improved relative to original substances. In contrast, four types of derivatives dissolved in DMSO exhibited a powerful activity. In particular, it was founded that the activity of derivatives were more improved relative to original substances.

Where an anti-oxidation agent would be used adding in pharmaceutical composition, food or fodder, this may be applied to human body or animal and administered with the various types of the formulation. An ointment type applied to skin may include emulsion of oil in water type and petrolatum ointment consisting of oil ingredient. At this time, the Nos. 1 and 3 derivatives of the water phase type were capable of applying to emulsion and the oil phase type of derivatives like Nos. 2 and 4 derivatives may be able to prepare as oil phase type of ointment such as petrolatum.

Moreover, in case of food, drug, sanitizer and fodder additives, the derivatives were used as water phase or oil phase materials according to formulation and type of product. However, insoluble materials, which were not completely solubilized to water, oil and organic solvent such as polyol, had a difficulty of product development and allowed to cause an economical loss due to the formulation development for improving insolubility and the addition of product process. Therefore, the oil and water phase of derivatives of the present invention were able to commercially use insoluble honokiol and magnolol by dramatically improving these insolubility.

Example 7: Evaluation of anti-inflammatory effect

To determine whether the derivatives of this invention had anti-inflammatory effect, the experiment of COX (cyclooxygenase) inhibitory ability was carried out using human monocytic cell, THP-I cell (Korean Cell Line Bank) according to conventional method. The COX activity was measured in accordance with Methods in Enzymo/ogy 43:9 (1994) published by F. J. Van de Ouderaaa and Muytenhek. THP-I cell line was cultivated and aliquoted into 24-well plate. The reaction volume per well was adjusted to 500 μl, the sample compound 2, which dissolved in lipopolysaccahride (1 μg/ml, Sigma) and solvents described in the following table 2 respectively, was added and followed by cultivating for 24-48 hr under the same condition. After 24-48 hr, calcium ionophore (Sigma) and [1-14C] arachidonic acid 1 μl (in EtOH, 0.1 μCi/ml, Sigma) were added to each well, and cultivated for 10 min under the same condition. Following the cultivation, citric acid was added to each well for adjusting to pH 3.5, shaked, each 500 μl of cultivation solution taken from the plate was aliquoted into micro centrifuge tube, 500 μl of ethylacetate was added to thereto, and the solution was shaking extracted for 10 min. 500 μl of ethylacetate layer was concentrated using speed vacuum dryer for 20 min, 20 μl of the concentrate was dissolved in ethylacetate, and the resultant was employed with authentic standard in TLC plate. The radioactive band was identified by authentic eicosanoid standards, the radioactivity of the identified band was measured using BAS 2000 bio-imaging analyzer (Fuji, JP). The DMSO value was considered as the control group and the inhibitory effect of each enzyme was represented using relative % against the control group. Table 2

-: no COX inhibitory ability, +: 1-10% inhibitory ability, ++: 11-20% inhibitory ability, +++: 20-30% inhibitory ability

As indicated in Table 2, it was founded that magnolol and honokiol had a potent COX inhibitory activity, i.e., anti-inflammatory effect and derivatives of the two substances, sample 1, 2, 3 and 4 exhibited stronger anti-inflammatory activity than original substances. However, where Nos. 2 and 4 derivatives having no solubility in water were used medium as solvent, the effect of anti-inflammatory showed poorly due to their insolubility in the media. In addition, because Nos. 1 and 3 derivatives had no solubility in oil, a mixture of the derivatives and oil did not exhibit the activity. Meanwhile, according to each improved feature of the derivatives, namely where Nos. 1 and 3 derivatives were dissolved in medium (water phase) and employed, the derivatives showed a powerful effect of anti-inflammatory, which did not exhibit in original substances at all. It was founded that the derivatives of this invention had the complete activity, since the derivatives were solubilized completely whereas the original substances were not dissolved in medium at all and used as the precipitated condition. In particular, these results demonstrate that the medium have no negative influence on cells compared to DMSO, since the effect of derivatives solubilized in medium was more excellent than that of them solubilized in DMSO. These results address that in the utilization of suitable solvent {e.g., DMSO) capable of solubilizing original substances, the original substances have the activity, but in the utilization of solvent capable of insolubilizing them, some activity was disappeared. Particularly, it was founded that the effect of the original substances was disappeared absolutely. Furthermore, the effect of Nos. 2 and 4 derivatives solubilized in oil were relatively stronger than original substances having no activity, but the effect of the

Nos. 2 and 4 derivatives were lower than that of Nos. 2 and 4 derivatives dissolved in DMSO or Nos. 1 and 3 derivatives dissolved in medium. This is because in case that

Nos. 2 and 4 derivatives were dissolved in oil, they were separated in a reaction solution in the experiment of in vitro. It was founded that the reason of this result was the incomplete reaction. Therefore, the activity of real improved derivatives did not exert by 100%. Nevertheless, these results address that the derivatives of this invention have a considerably improved effect relative to original substances.

During the treatment in Tween 80 solution, honokiol and magnolol were precipitated slightly and showed a partial effect due to the feature of honokiol and magnolol inactivated, but all of 4 type derivatives of the present invention exhibited an excellent effect. However, Nos. 2 and 4 derivatives having solubility in oil had a lower effect than Nos. 1 and 3 derivatives having solubility in medium or water, since Tween 80 used were of small quantity to completely solubilize the derivatives. Accordingly, Nos. 2 and 4 derivatives were used with not a completely solubilized condition but a partial solubilized condition. These experiment results demonstrate that the Nos. 2 and 4 derivatives incompletely solubilized had lower effect than the derivatives fully dissolved in DMSO.

However, it was founded that the activity of 4 types derivatives absolutely solubilized in DMSO exhibited a potent activity. In particular, it was founded that the activity of derivatives were more improved relative to original substances.

Example 8: Evaluation of Immunosuppressive effects

For examining whether the derivatives of this invention suppressed the immune response related to inflammatory, the experiment of NF-κB luciferase reporter activity was performed. NF-κB promoter was reported to play an important role in the generation of cytokine related to inflammatory. The activity of NF-κB luciferase was measured using the following method: Human monocytic cell, 1 x 10⁵ of THP-I cell (Korean Cell Line Bank) were aliquoted into each well, NF-κB luciferase reporter plasmid DNA (Stratagene) was transfected using superfect transfection reagent (In vitrogen). After 24 hr, lipopolysaccharid (100 ng/ml) was treated to activate THP-I cell and each samples was dissolved in 0.1% Tween 80 solution, medium solution or DMSO for adjusting to 0.1% of concentration. Following 24 hr, the cell was collected and the luciferase activity was measured using luminometer (Berthold Technologies GmbH&Co.KG, Germany). The results were summarized in Table 3. Table 3

-: no NF-κB promoter inhibitory ability, +: 1-10% inhibitory ability, ++: 11-20% inhibitory ability, +++: 20-30% inhibitory ability

As shown in Table 3, it was founded that magnolol and honokiol had an immunosuppressive activity and derivatives of the two substances, sample 1, 2, 3 and 4 exhibited stronger immunosuppressive activity than original substances. In the treatment of samples dissolved in DMSO, the original substances and 4 types of derivatives had the effect and the effect of the derivatives was increased. Where 0.1% of Tween 80 was used, it was founded that the original substances showed a low immunosuppressive activity. The original substances was not solubilized and dissolved completely even in 0.1% of Tween 80. Therefore, this result demonstrates that the solubilized part by 0.1% of Tween 80 contributes to some activity. In medium solution, the effect of honokiol and magnolol was inactivated completely, whereas Nos. 1 and 3 derivatives exhibited considerably potent inhibitory activity. In contrast, Nos. 2 and 4 derivatives had a poor effect due to the insolubility in medium and the dispersion. Meanwhile, where Nos. 2 and 4 derivatives were dissolved in olive oil, the derivatives showed a powerful effect. Nos. 1 and 3 derivatives did not show the effect at all, because of the insolubility in oil. In spite of the utilization of solubility in oil, the Nos. 2 and 4 derivatives had lower effect than the derivatives dissolved in DMSO, since the derivatives dissolved in oil was separated to upper part.

Example 9: Measurement of skin whitening effect Evaluation of whitening effect in animal using brown guinea pigs (tortoiseshell guinea pigs), known to increase its pigmentation upon exposure to ultraviolet light, like in humans, a whitening effect of samples used in this invention was measured.

To cause pigmentation in the brown guinea pig by ultraviolet (UV), aluminum foil with square windows of 3x3 cm² was adhered to hair-removed abdominal skin of brown guinea pig, and then UV light was irradiated thereon with a SE lamp (wavelength 290-320 nm, Toshiba) (total irradiation energy = 1350 mJ/cm²). After UV irradiation, the aluminum foil was removed and samples were applied as the following method. Increased pigmentation was observed at 2 or 3 days after UV irradiation and reached a maximum after about 2 weeks. From the maximum, samples were applied. Applications performed once or twice a day for 50 days. The samples were dissolved or diluted in a certain solvent described in the following Table 4 and applied by a swab. The control with only the solvent was applied to another site. Occurrence of cumulative irritation also was examined. In this example, water, DMSO or Tween 80 solution (containing 0.1% Tween 80) was used to be gel phase using a suitable amount of a viscosity-increasing agent due to a low viscosity of the solvents. As the viscosity-increasing agent, 0.5% of Xanthan Gum was used.

The degree of pigmentation of skin was determined using a chromameter (CR2002, MINOLTA, JP) to estimate the effects of applied samples. The results are shown in Table 4 below. L^*a^*b^* colorimetric system was used to classify color and L^* value was used as standard in the present invention. The L^* value was corrected using white board standard and was measured more than five times at one site, repeatedly. Pigmentation was evenly distributed. Skin color differences (ΔL^*) between application initial point and application terminal point were obtained and then using these values, their effects of the applied samples were estimated. These results were summarized in the following Table 4.

ΔL^* = L^* value at 00 days after application-L^* value at application initial day.

ΔL^* values were obtained both at sample application site and control application site and compared, whereby the effects of the whitening substances can be estimated. Table 4

-: no whitening effect, +: 1-10% whitening effect, ++: 11-20% whitening effect, +++: 20-30% whitening effect

As indicated in Table 4, it was founded that magnolol and honokiol had a skin whitening effect and derivatives of the two substances, sample 1, 2, 3 and 4 exhibited stronger whitening effect than original substances. In the treatment of samples dissolved in DMSO, all treatment groups had the potent effect except untreatment group. Among 6 types of samples, other samples except magnolol were a similar effect. Where Tween 80 was used, it was founded that honokiol and magnolol showed a poor activity due to the partial solubilization. In contrast, the derivatives of this invention exhibited a powerful whitening effect. In particular, Nos. 1 and 3 derivatives exhibited the most potent whitening activity due to the solubility of water. In contrast, it was founded that Nos. 2 and 4 derivatives with the insolubility in water had an effect only in solubilized part by Tween 80. Where water was used as solvent, Nos. 1 and 3 derivatives exhibited considerably potent whitening activity, whereas the effect of honokiol and magnolol was inactivated completely. However, the Nos. 2 and 4 derivatives had no effect.

In petrolatum, even hardly dissolved in oil, showed the feature better dispersed relatively. The effect of the original substances having low solubility in oil and high dispersion had no difference from that of Nos. 1 and 3 derivatives having low solubility in oil. In contrast, Nos. 2 and 4 derivatives dissolved in oil absolutely had a strong effect. Where water was used as solvent, the samples with insolubility in water had no effect, but where the samples with insolubility in oil were dispersed in petrolatum, the effect exhibited some extent. This is because some ingredients are absorbed by petrolatum whereas substances are not absorbed by hydrogel at all.

Example 10: Measurement of an improving effect of skin wrinkles

Test of wrinkle improvement effect may be generally measured through collagen biosynthesis ability, collagenase degradation inhibitory ability and clinical test to human. Human fibroblasts (commercially available from pacific) were seeded into a 6-well plate (2 x 10⁵ cell/well) and after 17 hours, the well plate was incubated in a 5% CO₂ incubator for 24 hr at 37⁰C. Then, the medium was removed, a dissolving samples were added to a suitable solution to adjust to 0.1% of concentration and incubated again for 24 hr. Following 24 hr, the cell medium was collected and samples were taken.

The extent of collagen synthesis was determined by measuring the amount of procollagen type I C-peptide (PICP) using Procollagen Type I C-peptide EIA kit (MKlOl, Takara, Kyoto, Japan). The method was performed in accordance with manufacturer's protocol.

As a method of measuring the activity of coilagenase, an enzyme that decomposes collagen, a coilagenase antibody was used. The experiment was performed in accordance with manufacturer's protocol. A Type 1 coilagenase assay kit (Amersham Biosciences, RPN2629) was used, and the absorbance was measured using an ELISA reader (Bio-Tek ELx808™ Series Ultra Microplate Reader, U. K). The measured average values were represented as mean ± standard deviation. A T-test with SPSS/PC+ was conducted to determine significance, and the result is shown in Table 5.

The effects of skin wrinkle improvement of compositions comprising 6 types materials used in this invention were measured through a clinical test. A cream was prepared as oil in water type of general cream according to conventional methods. The effects of wrinkle alleviation were evaluated by measuring the changes in the elasticity of the skin. Measurements were conducted on 30 healthy female test subjects (aged 25 to 35) in a stable environment of temperature ranging from 24⁰C to 26⁰C and humidity ranging from 38% to 40%. After samples, nutrient creams were applied to the facial skin of test subjects twice a day for 3 months, the elasticity was measured using a Cutometer SEM 474 (Courage+Khazaka, Cologne, D. E). Relative grades were set forth for skin elasticity within a range from zero for no elasticity to 5 for the highest elasticity measured, and the results are shown Table 5, below. Table 5

As shown in Table 5, the derivatives of the present invention showed significantly greater (3-4 folds) effects on the alleviation of wrinkles compared to the untreatment group. In case that a buffer solution was used as solvent, the Nos. 1 and 3 derivatives showed significantly greater (about 20 folds) effects on the increase ratio in collagen synthesis than the untreatment group, whereas original substances and untreatment group had no effects. In addition, Nos. 2 and 4 derivatives with the insolubility in water had no or little effect. Where DMSO was used as solvent, 6 types of samples except untreatment group exhibited potent effects and significantly greater (10-12 folds) effects compared to the untreatment group. In Tween 80, Nos. 1 and 3 derivatives with high solubility in water showed the greatest activity, the original substances, No. 2 and 4 derivatives exhibited a similar activity. Test of collagenase inhibitory activity also showed a similar tendency to collagen biosynthesis ability.

These results demonstrate that Nos. 1 and 3 derivatives dissolved in DMSO and Tween 80 had lower effects than Nos. 1 and 3 derivatives dissolved in medium, since DMSO and Tween 80 caused a poor cytotoxicity.

Example 11: Effect of alleviation of anxiety

The anti-anxiety effect was measured orally administering samples to mouse in accordance with the experimental method described in J Natural Product, 61:135- 138(1998). Saiboku-to used in the paper was used as control group. Table 6

An anti-anxiety effect on Saiboku-to was measured using Tween 80 as solvent, the measurement value was given a definition as 100, and the anti-anxiety to other samples was represented by a relative value against Saiboku-to. As indicated in Table 6, where water was used as solvent, honokiol and magnolol showed significantly lower effects on the anxiety alleviation compared to the control and experimental group, due to the insolubility in water. In contrast, it was founded that the Nos. 1 and 3 derivatives with the solubility in water showed greater (about 3 folds) effects than the control group. Furthermore, where the Nos. 2 and 4 derivatives with the solubility in oil were dissolved in water, the derivatives exhibited a similar effect to original substances. In addition, despite Saiboku-to was given a boil with water, the effect showed greater (approximately 6-7 folds) relative to the original substances. This was because several ingredients in addition to 2 types of original substances were extracted by boiling Saiboku-to. Meanwhile, in case that oil was used as solvent, the orally administering (a diet intake with dispersion condition by mixing) result of the control group, which was mixed the extract obtained by boiling the Saiboku-to and water, demonstrates that the effect was significantly decreased. Moreover, Nos. 2 and 4 derivatives showed significantly greater effects than Nos. 1 and 3 derivatives dissolved in water. This result address that oil phase of Nos. 2 and 4 derivatives were better absorbed than water phase of derivatives through small-intestine mucosa.

Example 12: Anti-platelet effect

The anti-platelet effect was examined according to well-known methods published in Arch Pharm Res. VoI 25, No. 3: 325-328(2002). The blood from mouse heart was collected using a syringe contained 0.1 ml of 2.2% Sodium citrate. Only platelet was obtained by centrifuging at 200 g for 10 min. The platelet was diluted with saline solution to be 400-450 x 1,000,000 /ml. Platelet aggregation was measured by using a platelet aggregometer (Model 500VS, Chrono-Log Corp. U.S.A). In this example, 2-5 μg/ml of collagen was used for the platelet aggregation. Table 7

ASA: acetylsalisylic acid

The experiment results demonstrate that honokiol and magnolol showed greater (about 2 folds) effects on the anti-platelet effect compared to ASA. Meanwhile, Nos. 2 and 4 derivatives with the insolubility in water had no effect. In contrast, Nos. 1 and 3 derivatives with the solubility in water exhibited significantly greater (more than 4 folds) effects on the anti-platelet effect compared to ASA. These results address that the derivatives of this invention was prepared as pharmaceutical compositions for preventing or treating cardiovascular disorders due to the anti-platelet effect of the derivatives of this invention.

Example 13: Anti-cancer effect

Cytotoxity of acetylgliotoxin against various cancer cells, judgement of apotosis and anti-tumor activity of acetylgliotoxin against various cancer cells was measured by the MTT (Microculture Tetrazolium) method. Various cancer cells were adjusted to 4.Ox 10⁴ cells/ml by using a medium (hereinafter referred go as medium A) composed of RPMI 1640 medium (GIBCO) containing 10% fetal calf serum, and each 0.1 ml of the cancer cells was pipetted into each well of a 96 well microtiter plate. The plate was incubated in a carbon dioxide gas incubater for 20 hr at 37⁰C, and each 0.025 ml of samples (test compound) suitably diluted with the medium A was added. Then, incubation was further carried out in the carbon dioxide gas incubater for 72 hr at 37⁰C. Each 0.01 ml of the MTT solution adjusted to 5 mg/ml by using Dulbecco's PBS (-) (Nissui) was added thereto, and incubated in the carbon dioxide gas incubator for 3 hr at 37⁰C. Subsequently, each 0.05 ml of 0.01 N HCI/20% SDS was added. Produced crystals were dissolved, and absorbance at 570 nm was measured by a microplate reader. By comparing the absorbance of the cells treated with the sample in the known concentration with that of non-treated cells, the sample concentration which inhibits multiplication of cells at the rate of 50% (IC₅₀) was calculated. The results are shown in Table 8. Apotosis was determined by incubating the plate in the carbon dioxide gas incubator for 20 hr at 37⁰C, adding each 0.025 ml of samples (test compound) suitably diluted with the medium A, and incubating in the carbon dioxide gas incubater for 72 hr at 37⁰C, according to the above method. Then, nucleus- staining fluorescent dye DAPI (4,6-diaminodino-2-phenylindole)(Sigma) was added to each well so as to become 0.5 ng/ml, and after leaving for 10 min at room temperature, and followed by confirming agglomeration of chromosomes by using a fluorescence microscope.

As Cells used in the measurement of cytotoxity, human leukemia cell (K562), human carcinoma cells of the colon (HCT-15, HCT-116, SW946), human leukemia cell (U937), human lung cancer cells (LX-I), mouse leukemia cell (P388) and mouse carcinoma cells of the colon (colon-26) were commercially available from Korean Cell Line Bank & Seoul National University College of Medicine. Table 8

Type of Cells

Honokiol Magnolol

HCT-15

300 403 (human carcinoma of the colon )

HCT-116

1200 1408 (human carcinoma of the colon )

SW948

1278 1457 (human carcinoma of the colon )

LX-I (human lung cancer )

U937 (human leukemia ) 300 431

P388 (mouse leukemia ) 680 892 colon-26

1200 1230 (mouse carcinoma of the colon)

As shown in Table 8, honokiol and magnolol had an anti-tumor activity against various cancers, but did not have against human lung cancer. In contrast, Nos. 1 and 3 derivatives with the improvement of solubility showed the anti-cancer effect even at lower IC₅₀ concentration (about 10-15 folds) compared to original substances. In particular, all 4 types of derivatives of this invention had the effect even against human lung cancer cell line. Furthermore, even the Nos. 2 and 4 derivatives with the property improvement showed greater effects on the anti-cancer effect compared to original substances. Therefore, this result demonstrates that the derivatives of the present invention exhibited the improved anti-cancer effect relative to the original substances.

Example 14: Anti-allergy effect

Where 0.50 mg/g body weight of the 6 type samples only were administered to each 15 animals of ICR mouse, death of the mouse was not observed at all, but 100% of death rate was shown in the group administered only mast cell stimulator (Compound 48/80; N-methyl-p-methoxyphenylethylamine) (Sigma).

In addition, 1 hr before mast cell stimulator was administered to 15 animals of ICR mouse, a systemic anaphylactic shock was induced by administering the samples dissolved in various solvents as described in the following Table 9. This result demonstrates that death rate showed dose-dependent tendency, in case that the doses was 0.50 mg/g body weight, death rate exhibited 0%.

As a comparative experiment, DSCG (disodium cromoglycate, Merck) was used and mouse were sensitized by intraperitoneal injection with 8 mg/kg body weight of stimulator. The DSCG was introduced as a potent anti-allergic drug which stabilized the mast cell membrane and suppressed the extrication of chemical-mediated material. Table 9

As indicated in Table 9, where mast cell stimulator was treated, all mice were dead. In contrast, where 0.5 mg/g body weight of DSCG was administered, the anti- allergy effect was to be about 20%. Furthermore, in case that 1.0 mg/g body weight of DSCG was treated, the death rate exhibited 0%. Where the original substances dissolved in water were orally administered, the effect did not show at all due to the insolubility of water. However, in the form of solution in Tween 80 or liposome, the original substances indicated approximately 10% of effects, since some of the substances were dissolved and they were prepared as forms capable of absorbing. Where the Nos. 1 and 3 derivatives were in the form of solution in water or Tween 80, and liposome, the effect showed 100 % of survival rate. In contrast, in the form of solution in water, the Nos. 2 and 4 derivatives exhibited about 10% of anti-allergy effect. In the form of solution in Tween 80, they showed 25-30% of survival rates. However, 100% of survival rates were indicated in liposome form.

Example 15: Anti-obesity effect

An obesity inhibition test was performed by the following method using well- known animals. Table 10 shows the results.

Method for determining obesity-inhibitory activity was as follows:

Gj : ICR male mice (obtained from Charles River Japan Ltd) aged 7 weeks were preliminarily fed for 1 week, then classified into groups each having 7 animals and subjected to the test. The animals were fed in a thermo-hygrostat at a temperature of 23±1°C, and a humidity of 55±5% under illumination for 12 hours per day. They were fed with a feed Labo MR (manufactured by Nippon Nosan) and allowed to take water ad libitum. The test samples were in the form of solution in water and liposome in 5% lecithin to become 0.1%. The concentration of each sample solution was regulated so that 0.1 ml of the solution was given per 10 g body weight of animal. The doses employed were 1.5 g/kg and 1 g/kg. To a control group, water and 5% lecithin emulsion were administered. After fasting the mice, the sample was administered once by force on the next day. During the test period over 2 weeks, the body weight and general conditions were monitored.

The experiment results were as follows:

1) Body weight: the body weight gain was inhibited in the 4 types derivatives of the 6 types test samples. The original substances had no effect in the form of solution in water and liposome. In contrast, Nos. 1 and 3 derivatives showed the obesity- inhibitory activity in the form of solution in water and liposome. In addition, Nos. 2 and 4 derivatives did not exhibit the activity in the form of solution in water, but had a potent activity in liposome form. Table 10

2) General conditions: each test group showed no abnormality compared with the control group, which means that the derivatives of present invention were free from any problem in safety.

Example 16: Effect of hair loss prevention and hairs growth promotion For measuring the effect of hair loss prevention and hairs growth promotion, the test samples were prepared in the form of hydrogel base containing viscosity- increasing agent and preservative, and liposome in 3% lecithin derived from a bean. Each 3 cc of liquids for external use for promoting hairs growth prepared were applied to the hair loss sites of 10 baldness patients twice a day for 3 months. The result indicated that the liquids containing the derivatives of this invention showed an excellent effect such as the generation of the root of hair from 8 baldness patients. The experiment results were as follows. The control group used the moxidil commercially available from Hanmi pharmaceutical. Co. Ltd. Table 11

As indicated in Table 11, honokiol and magnolol had no effect of hair loss prevention and hairs growth promotion in the form of hydrogel, due to the insolubility.

In addition, Nos. 2 and 4 derivatives not dissolved in water had on effect. In contrast, all 4 type derivatives in liposome form showed a great effect on hair loss prevention and hairs growth promotion since the water and oil phase of derivatives were contained in liposome. Therefore, the derivatives of the present invention are able to develop a product.

Example 17: Anti-bacterial and anti-virus effects to agricultural microorganisms

Test of anti-bacteria and anti-virus against various microorganism in the agricultural field was performed using honokiol, magnolol and their derivatives prepared in Example 1 to 5. The measurement method of anti-bacterial activity was performed by paper disk-agar diffusion method. Bacteria and virus used in this test were about main diseases, but did not limit to. As the method determining the concentration of anti-bacterial materials of solutions, this method is that a number of disks each impregnated with a known concentration of antibiotic are placed on an agar surface previously seeded with an organism to be tested and after incubation, the diameter of the growth inhibition halo surrounding the disk was measured. Among agar diffusion methods, Kirby-Bauer method was available generally. The anti-bacterial experiment results by this method revealed that the 4 type derivatives of this invention had a great anti-bacterial effect. The experiment was performed limiting to 1000 ppm (0.1%) of the anti-bacterial test concentration. All sample materials was dissolved in DMSO and tested.

Table 12a

Table 12b

Table 12c Table 12c

Table 12d

Table 12e

Table 12f

+: weak (the diameter of the growth inhibition halo: less than 2 mm), ++: strong (the diameter of the growth inhibition halo: less than 2-5 mm), +++: very strong (the diameter of the growth inhibition halo: not less than 5 mm)

As indicated in Table 12, the derivatives (Nos. 1 to 4 materials) exhibited greater anti-microbial activity for virus and bacteria than the original substances. Therefore, the results revealed that the anti-microbial activity of the derivatives was more improved relative to that of the original substances. From allowing the derivatives of this invention to show the activity for all microorganisms tested, it was also founded that the derivatives would show the activity for most of microorganisms not tested.

Example 18: Field trial for rice blast disease

Each 10 parts of the 6 type derivatives were thoroughly mixed with 4 parts of sodium dodecylbenzene sulfonate, 2 parts of polyvinyl alcohol and 84 parts of clay, and the mixture was finely pulverized three times with a hammer mill. The pulverized mixture was homogenized to obtain a wettable powder. Each 10 ml of 0.1% or 0.3% diluent solution containing the wettable powder as active ingredient was uniformly sprayed on rice seedlings (variety: Norin 20) in 3 to 4-leaf stage in each pot with a spray gun. After the natural drying, the test plants were inoculated with spore suspension of Pyricularia oryzae, and kept in a room at a temperature of 20-22⁰C, and a humidity of 100% for 48 hours. Then the test plants were transferred to a greenhouse at a temperature of 24-26⁰C. After 3 days, the numbers of the lesions formed on the upper two leaves were counted. The counting was carried out three times and the mean value was summarized in Table 13. Table 13

As shown in Table 13, in the numbers of the lesions, the original substances were not improved significantly relative to the untreatment group. In contrast, the Nos. 1 and 3 derivatives showed the control effect close to 100%, so that the derivatives were available as an agricultural chemical against the rice blast disease. Where water was used as solvents, the original substances and Nos. 2 and 4 derivatives did not exhibit at all. In contrast, in case of solubilizing using surfactants by force, the original substances still did not showed the effect whereas the Nos. 2 and 4 derivatives indicated the potential control effect, so the derivatives may be able to use as agricultural chemicals.

Example 19: Field trial in lawn Three green and field of J golf course located at Young In Si, Gyeong Gi-Do were divided into each one pyeong, the divided sites were classified with untreatment, honokiol treatment, magnolol treatment and derivatives treatment group and an attack rate of a disease was examined. Untreatment group was treated with water containing 1% Tween 80, and all of honokiol, magnolol and derivatives were used dissolving in 1% Tween 80. The concentration of all samples used was adjusted to 0.5% of final concentration. Main lawn pathogen, 4 type bacteria was treated each at the divided trial field and allowed the bacteria to grow for 1 month. After growth, sample solution containing sample for untreatment group was sprayed in the same field for 3 months with interval of 3 weeks. The final diseased area was measured and the attack rate of a disease was calculated as percentage to one pyeong of treatment group. Table 14

As shown in Table 14, where honokiol and magnolol was used in the condition of suspension containing 1% Tween 80, the materials showed about 10% of the control effect compared to the untreatment group. In contrast, Nos. 1 and 3 derivatives with the solubility in water exhibited 100% of the control effect. This result revealed that the anti-microbial activity of honokiol and magnolol was improved. However, Nos. 2 and 4 derivatives with the solubility in oil indicated approximately 30% of the control effect. It was founded that the solubility improvement of honokiol and magnolol results in the improvement of the anti-microbial activity. Where the material property of honokiol and magnolol was altered to be dissolved in solvents such as water and oil absolutely or partially, their effects can be improved. Therefore, the derivatives can be prepared as fertilizer additives and agricultural chemicals for applying to agricultural plants in addition to lawn.

Example 20: Field trial in other plants

The field trial was performed according to conventional methods by seasons. Whether the derivatives of the present invention really had a control effect was examined using representative test bacteria on each plant. The field trial was carried out in accordance with Examples 18 and 19. Table 15

As shown in Table 15, honokiol and magnolol showed about 10% of the control effect in all plants tested, 100% of the attack of a disease occurred in untreatment group. In contrast, Nos. 1 and 3 derivatives exhibited the control effect close to 100%. Meanwhile, Nos. 2 and 4 derivatives indicated more enhanced control effect than original substances, but had the poorer effect than Nos. 1 and 3 derivatives. Nos. 2 and 4 derivatives showed about 30% of the control effect compared to untreatment group on the average.

Example 21: Anti-bacterial and anti-virus effect to microorganisms in a foods and pharmaceutical industry

An anti-microbial test was performed using the derivatives obtained in Examples 1 to 6 for various microorganisms which had the problem in the pharmaceutical industry. The anti-microbial test was used a paper disc method of conventional anti-microbial test methods. Namely, After cultivating of the test strains at suitable temperature, each 100 μl of the cultivated strains were inoculated on an agar medium. The paper discs (diameter: 8 mm) were mounted on each of the mediums inoculated with the various fungi. Each 6 type samples were dissolved in water, applied to a paper disc to contain 0.3% and cultivated for 24 hr at suitable temperature. The fungi were cultured for 48 hr. Then, antibacterial activities were confirmed by the size of a clear zone formed around the paper discs. The results were summarized in Table 16. Table 16a

Table 16b

Table 16c

-: no effect, +: weak (the diameter of the growth inhibition halo: less than 2 mm), ++: strong (the diameter of the growth inhibition halo: less than 2-5 mm), +++: very strong (the diameter of the growth inhibition halo: not less than 5 mm)

The result revealed that magnolol and honokiol identical to untreatment group treated only water, had no the antimicrobial activity in condition of insolubility in water. In contrast, Nos. 1 and 3 derivatives exhibited the strong antimicrobial activity for all bacterial strains tested. Meanwhile, Nos. 2 and 4 derivatives exhibited the partial antimicrobial activity due to the feature dispersed better. Therefore, all 4 types of the derivatives of the present invention showed improved antimicrobial effect relative to the original substances.

Example 22: Effect of fodder additives

Because the compounds of this invention had a excellent anti-inflammatory and anti-microbial activity, the compounds were added to fodder as an alternative of the antibiotics and carried out an experiment. Existing fodder and fodder contained 0.3 wt% of 6 types comparative materials were feed to nursery pigs for 50 days and the effect of an increased body weight was measured. This result was summarized in Table 17. Table 17

As indicated in Table 17, untreatment group, which was fed the mixed feed not containing samples, showed 35 kg of the increased body weight. In contrast, the fodder containing the 4 type derivatives of this invention indicated more than 40 kg of the increased body weight. This result demonstrates that the derivatives of this invention are available as fodder additives. The original substances had lower effect than the derivatives, but the substances showed higher effect compared to untreatment group.

Example 23: Effect of fodder additives for an aquacultured fish on growth promotion An outdoor test pond was divided along a stream by a board, and two net crawls (5 mm meshes), 1 mxθ.8 m (depth), in each division were settled at intervals of about 0.5 meters. Spring water (14.5°C-15.0°C) was flowed into this pond at the ratio of 1/300, and the depth of water was controlled at about 0.6 meters. The steelhead trout of the same venter, at about 7 months after hatching, were divided at random into 6 groups (30 fishes per group) and cultivated preliminarily with the same feed as used in the field studies for two weeks, then field studies were carried out. The entire test method was performed according to Korean. Pat. Reg. Pub. No. 90- 003014. After feeding for 2 months, the addition effect was estimated on the average. The feed were prepared by mixing a conventional fish fodder with 6 types derivatives used in test in an amount of 0.1%, and used. All samples were dissolved in alcohol for feeding, water or 0.1% of Tween 80, and the dissolved samples were added to the fodder. Table 18

Mean body weight (g)

Solvents Untreatment

Honokiol Magnolol group

Alcohol 59 63.1 62.4 69 67.6 65.4 67.2

Tween 80(0.1%) 61.2 63 62.7 67 68 68 66

Water 60 62.8 62.1 68.7 62.1 68 62

Olive oil 61.1 62.1 63.1 63 68.2 61.8 66.7

As shown in Table 18, the effect of honokiol and magnolol on weight gain was exhibited in 4 type solvents. Where Nos. 1 and 3 derivatives were dissolved in water and the dissolved derivatives were added to fodder, the weigh gain effect was higher than the 2 type original substances. In contrast, Where Nos. 2 and 4 derivatives were dissolved in olive oil and the dissolved derivatives were added to fodder, the derivatives showed better effect due to the feature of solubility in oil. Where Nos. 2 and 4 derivatives were dissolved in water, the derivatives showed a similar effect to original substances due to the feature of insolubility in water. Therefore, it was founded that the 4 type derivatives exhibited the effect according to physical and chemical solubility feature of the materials. Especially, despite the original substances were not dissolved in water and oil, the substances showed higher weight gain effect than untreatment group. This was because when other materials were absorbed, the fodder as pellet type of solid phase was absorbed together in the dispersion form by repetition mixing. It was also founded that the derivatives with the insolubility in water and oil had a similar effect to the original substances.

In conclusion, it was confirmed that the 4 type derivatives of this invention had effects of body weight gain by adding them to fodder after dissolving in accordance with the physical and chemical feature.

Example 24: Effect of irritability alleviation

After skin was stimulated with SDS (0.01%) and skin tissue was damaged, a cream containing each 0.1% of magnolol, honokiol, No. 1 sample, No. 2 sample, No. 3 sample and No. 4 sample was applied to upper forearms of 30 healthy adults, to determine whether skin tissue was improved or not.

Finn chamber patch method (Epitest Ltd, Finland) was used. In this respect, 15 μl of the skin external applications were dropped into the chamber, and then a patch was put into the chamber. The reactivity of skin was scored using the following formula 1. The results are shown in Table 19 below. Formula 1

The mean reactivity = [[{(the number of subjects reacting x reactivity)/the total number of subjects} x maximum reactivity (at of 4)] x 100] ÷ the number of tests (9 times)

In Table 19, the reactivity is indicated as follows: ±: 1, +: 2 and ++: 4.

Table 19

Reactivity Mean

Test materials

1 week 2 week 3 week Reactivity

Control group

0.12 0.15 0.15 0.14 (Squalene)

As indicated in Table 19, where SDS (0.01%) was applied to skin, it was founded that skin severely was damaged from 4.33 of mean reactivity value. In contrast, where each test group 1, 2, 3, 4, 5 and 6 was applied to skin, it was founded that the test groups showed an alleviation effect to skin irritability. In particular, Nos. 1 and 3 samples had better alleviation effects than Nos. 2 and 4 samples. In addition, all No. 1, 2, 3 and 4 samples exhibited higher alleviation effects compared to magnolol and honokiol. These results demonstrate that all No. 1, 2, 3 and 4 samples have an alleviation effect to skin irritability.

As described hereinabove, the present invention provides novel biphenyl diol derivatives and various compositions comprising them as an active ingredient. The novel biphenyl diol derivatives showed more improved physical property, especially hydrophile or lipophilic property than honokiol and magnolol, and had also much more improved biological and physiological activity than the original substances. Furthermore, the biphenyl diol derivatives can be prepared as various compositions and developed in various products due to the alteration of physical feature. Having described a preferred embodiment of the present invention, it is to be understood that variants and modifications thereof falling within the spirit of the invention may become apparent to those skilled in this art, and the scope of this invention is to be determined by appended claims and their equivalents.

Claims

What is claimed is:

1. A biphenyl diol derivative represented by the following formula I:

wherein Rl represents H, C₂-io alkyl, C₂-_I0 alkyl carboxylic acid or its salt, or amino acid or its salt; R2 represents H, C₂-_I0 alkyl, C₂-_I0 alkyl carboxylic acid or its salt, or amino acid or its salt; R3 represents H, C₂. io alkyl, C₂-io alkyl carboxylic acid or its salt, or amino acid or its salt; and at least one of Rl, R2 and R3 is not H.

2. The biphenyl diol derivative according to claim 1, wherein the biphenyl diol derivative is represented by the following formula II:

wherein Rl represents H, C₂-₁₀ alkyl, C₂-_I0 alkyl carboxylic acid or its salt, or amino acid or its salt; R2 represents H, C₂-_I0 alkyl, C₂-_I0 alkyl carboxylic acid or its salt, or amino acid or its salt; and at least one of Rl and R2 is not H.

3. The biphenyl diol derivative according to claim 1, wherein the biphenyl diol derivative is represented by the following formula III:

wherein Rl represents H, C_2-10 alkyl, C₂-_I0 alkyl carboxylic acid or its salt, or amino acid or its salt; R3 represents H, C_2-I0 alkyl, C_2-I0 alkyl carboxylic acid or its salt, or amino acid or its salt; and at least one of Rl and R3 is not H.

4. The biphenyl diol derivative according to any one of claims 1 to 3, wherein the biphenyl diol derivative is the following compounds: 2-(4-hydroxy-3-prop-2- enylphenyl)-4-prop-2-enylphenyl-2-aminoethanoate; 2-(4-hydroxy-3-prop-2- enylphenyl)-4-prop-2-enylphenyl-2-aminoethanoate hydrochloride salt; 2-(4- aminoethanoyl-3-prop-2-enylphenyl)-4-prop-2-enylphenyl-2-aminoethanoate dihydrochloride salt; 2-(4-aminopropanoyl-3-prop-2-enylphenyl)-4-prop-2-enylphenyl- 2-aminopropanoate dihydrochloride salt; 3-({4-[2-(3-carboxypropanoyloxy)-5-prop-2- enylphenyl]-2-prop-2-enylphenyl}oxycarbonyl)propionic acid; 2-(2-hydroxy-5-prop-2- enylphenyl)-4-prop-2-enylphenyl-2-aminoethanoate; 2-(2-hydroxy-5-prop-2- enylphenyl)-4-prop-2-enylphenyl-2-aminoethanoate hydrochloride salt; 2-(2- aminoethanoyl -5-prop-2-enylphenyl)-4-prop-2-enylphenyl-2-aminoethanoate dihydrochloride salt; or 3-({2-[2-(3-carboxypropanoyloxy)-5-prop-2-enylphenyl]-4- prop-2-enylphenyl}oxycarbonyl)propionic acid.

5. A skin whitening composition comprising the biphenyl derivatives of any one of claims 1 to 4 as an active ingredient.

6. A composition for promoting collagen synthesis comprising the biphenyl derivatives of any one of claims 1 to 4 as an active ingredient.

7. A composition for improving skin wrinkles comprising the biphenyl derivatives of any one of claims 1 to 4 as an active ingredient.

8. A composition for alleviating skin irritability comprising the biphenyl derivatives of any one of claims 1 to 4 as an active ingredient.

9. A composition for preventing hair loss or promoting hair growth comprising the biphenyl derivatives of any one of claims 1 to 4 as an active ingredient.

10. A composition for preventing or treating cardiovascular disorders having antiplatelet effect, which comprises the biphenyl derivatives of any one of claims 1 to 4 as an active ingredient.

11. An anti-oxidation, anti-inflammatory, alleviation of anxiety, anti-cancer, anti- allergy, anti-obesity, anti-bacterial or anti-virus composition, which comprises the biphenyl derivatives of any one of claims 1 to 4 as an active ingredient.

12. An agricultural chemical composition for protecting plant comprising the biphenyl derivatives of any one of claims 1 to 4 as an active ingredient.

13. The agricultural chemical composition according to claim 12, wherein the composition has a control effect of rice blast disease.

14. The composition according to any one of claims 5 to 9 and 11, wherein the composition is a cosmetic composition.

15. The composition according to any one of claims 5 to 11, wherein the composition is a food composition.

16. The composition according to any one of claims 9 to 11, wherein the composition is a pharmaceutical composition.

17. A composition of fodder additives for having the activity of anti-oxidation, anti- inflammatory or anti-bacterial, which comprises the biphenyl derivatives of any one of claims 1 to 4 as an active ingredient.

18. The composition of fodder additives for an aquacultured fish according to claim 17, wherein the composition comprises the biphenyl derivatives as an active ingredient.

19. A composition of fertilizer additives comprising the biphenyl derivatives of any one of claims 1 to 4 as an active ingredient.

20. A preservative composition comprising the biphenyl derivatives of any one of claims 1 to 4 as an active ingredient.

21. A sanitizer composition comprising the biphenyl derivatives of any one of claims 1 to 4 as an active ingredient.

22. A skin whitening method, which comprises administering to a subject the biphenyl derivatives of any one of claims 1 to 4 as an active ingredient.

23. A method for promoting collagen synthesis, which comprises administering to a subject the biphenyl derivatives of any one of claims 1 to 4 as an active ingredient.

24. A method for improving skin wrinkles, which comprises administering to a subject the biphenyl derivatives of any one of claims 1 to 4 as an active ingredient.

25. A method for alleviating skin irritability, which comprises administering to a subject the biphenyl derivatives of any one of claims 1 to 4 as an active ingredient.

26. A method for preventing hair loss or promoting hairs growth, which comprises administering to a subject the biphenyl derivatives of any one of claims 1 to 4 as an active ingredient.

27. A method for preventing or treating cardiovascular disorders having anti-platelet effect, which comprises administering to a subject the biphenyl derivatives of any one of claims 1 to 4 as an active ingredient.

28. A method for preventing oxidation, which comprises administering to a subject the biphenyl derivatives of any one of claims 1 to 4 as an active ingredient.

29. A method for preventing or treating inflammatory or allergy, which comprises administering to a subject the biphenyl derivatives of any one of claims 1 to 4 as an active ingredient.

30. A method for preventing or treating cancers, which comprises administering to a subject the biphenyl derivatives of any one of claims 1 to 4 as an active ingredient.

31. A method for suppressing obesity, which comprises administering to a subject the biphenyl derivatives of any one of claims 1 to 4 as an active ingredient.

32. A method for suppressing bacterial or virus proliferation, which comprises administering to a subject the biphenyl derivatives of any one of claims 1 to 4 as an active ingredient.

33. A method for alleviating anxiety, which comprises administering to a subject the biphenyl derivatives of any one of claims 1 to 4 as an active ingredient.

34. The method according to any one of claims 22 to 26 and 28 to 33, wherein the composition is a cosmetic composition.

35. The method according to any one of claims 22 to 33, wherein the composition is a food composition.

36. The method according to any one of claims 26 to 33, wherein the composition is a pharmaceutical composition.

37. A use of the biphenyl derivatives of any one of claims 1 to 4 for manufacturing a skin whitening composition.

38. A use of the biphenyl derivatives of any one of claims 1 to 4 for manufacturing a composition for promoting collagen synthesis.

39. A use of the biphenyl derivatives of any one of claims 1 to 4 for manufacturing a composition for improving skin wrinkles.

40. A use of the biphenyl derivatives of any one of claims 1 to 4 for manufacturing a composition for alleviating skin irritability.

41. A use of the biphenyl derivatives of any one of claims 1 to 4 for manufacturing a composition for preventing hair loss or promoting hairs growth.

42. A use of the biphenyl derivatives of any one of claims 1 to 4 for manufacturing a composition for preventing or treating cardiovascular disorders having anti-platelet effect.

43. A use of the biphenyl derivatives of any one of claims 1 to 4 for manufacturing an anti-oxidation, anti-inflammatory, alleviation of anxiety, anti-cancer, anti-allergy, anti-obesity, anti-bacterial or anti-virus composition.

44. The use of the biphenyl derivatives according to any one of claims 37 to 41 and 43, wherein the composition is a cosmetic composition.

45. The use of the biphenyl derivatives according to any one of claims 37 to 43, wherein the composition is a food composition.

46. The use of the biphenyl derivatives according to any one of claims 41 to 43, wherein the composition is a pharmaceutical composition.