Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
  • C07C51/00—Preparation of carboxylic acids or their salts, halides or anhydrides
  • C07C51/347—Preparation of carboxylic acids or their salts, halides or anhydrides by reactions not involving formation of carboxyl groups
  • C07C51/353—Preparation of carboxylic acids or their salts, halides or anhydrides by reactions not involving formation of carboxyl groups by isomerisation; by change of size of the carbon skeleton
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
  • C07C63/00—Compounds having carboxyl groups bound to a carbon atoms of six-membered aromatic rings
  • C07C63/33—Polycyclic acids
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
  • C07C63/00—Compounds having carboxyl groups bound to a carbon atoms of six-membered aromatic rings
  • C07C63/33—Polycyclic acids
  • C07C63/331—Polycyclic acids with all carboxyl groups bound to non-condensed rings

Definitions

• 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.
• 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)).
• 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.
• 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.
• Rl represents H, C 2 - I0 alkyl, C 2 - I0 alkyl carboxylic acid or its salt, or amino acid or its salt
• R2 represents H, C 2 - I0 alkyl, C 2 - 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, R2 and R3 is not H.
• 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.
• the biphenyl diol derivative is represented by the following formula II:
• Rl represents H, C 2 - I0 alkyl, C 2 - I0 alkyl carboxylic acid or its salt, or amino acid or its salt
• R2 represents H, C 2 -io alkyl, C 2 -io alkyl carboxylic acid or its salt, or amino acid or its salt
• at least one of Rl and R2 is not H atom.
• the biphenyl diol derivative is represented by the following formula III:
• Rl represents H, C 2-I0 alkyl, C 2 - 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
• at least one of Rl and R3 is not H.
• alkyl 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.
• alkyl carboxylic acid refers to a group bound carboxylic acid with a terminal of linear or branched chain saturated hydrocarbon group.
• alkyl carboxylic acid includes acetic acid, propion acid, butyric acid, baleic acid, caroic acid, etat acid and capric acid, but not limited to.
• alkyl carboxylic acid is C 2 -5 alkyl carboxylic acid
• 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-
• amino acid refers to a compound having both amino group and carboxylic group.
• 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.
• 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
• 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.
• 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.
• the derivatives of this invention have higher effects than honokiol and magnolol.
• Rl represents H, C 2-5 alkyl carboxylic acid, or amino acid or its salt
• R2 represents H, C 2 - 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.
• only one group of Rl, R2 and R3 is H in the formula (I) to (III).
• 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-( ⁇
• a skin whitening composition comprising the above-described biphenyl derivatives as an active ingredient.
• a skin whitening method which comprises administering to a subject the above-described biphenyl derivatives as an active ingredient.
• a use of the above-described biphenyl derivatives for manufacturing a skin whitening composition there is provided a use of the above-described biphenyl derivatives for manufacturing a skin whitening composition.
• a composition for promoting collagen synthesis comprising the above-described biphenyl derivatives as an active ingredient.
• a method for promoting collagen synthesis which comprises administering to a subject the above- described biphenyl derivatives as an active ingredient.
• a method for improving skin wrinkles which comprises administering to a subject the above- described biphenyl derivatives as an active ingredient.
• a use of the above-described biphenyl derivatives for manufacturing a composition for improving skin wrinkles 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.
• a composition for preventing hair loss or promoting hairs growth comprising the above-described biphenyl derivatives as an active ingredient.
• 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.
• composition for preventing or treating cardiovascular disorders having anti-platelet effect comprising the above-described biphenyl derivatives as an active ingredient.
• 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.
• a use of the above-described biphenyl derivatives for manufacturing a composition for preventing or treating cardiovascular disorders having anti-platelet effect 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.
• 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.
• a method for preventing oxidation which comprises administering to a subject the above-described biphenyl derivatives as an active ingredient.
• a method for preventing or treating inflammatory or allergy which comprises administering to a subject the above-described biphenyl derivatives as an active ingredient.
• a method for preventing or treating cancers which comprises administering to a subject the above- described biphenyl derivatives as an active ingredient.
• a method for suppressing obesity which comprises administering to a subject the above-described biphenyl derivatives as an active ingredient.
• a method for suppressing bacterial or virus proliferation which comprises administering to a subject the above-described biphenyl derivatives as an active ingredient.
• a method for alleviating anxiety which comprises administering to a subject the above-described biphenyl derivatives as an active ingredient.
• honokiol and magnolol have a potent activity of anti- oxidation and anti-inflammatory.
• 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.
• hydrophilic derivatives of this invention e.g., 2-(4-aminoethanoyl-3-prop-2-enylphenyl)-4-prop-2- enylphenyl-2-aminoethanoate dihydrochloride salt
• the derivatives have higher effects of anti-oxidation and anti-inflammatory than honokiol and magnolol
• hydrophobic derivatives of this invention e.g., 3-( ⁇ 4-[2- (3-carboxypropanoyloxy)-5-prop-2-enylphenyl]-2-prop-2-enylphenyl ⁇ oxycarbonyl) prop ionic acid
• the derivatives have higher effects of anti- oxidation and anti-inflammatory than honokiol and magnolol, which are demonstrated in example.
• 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.
• 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.
• a composition for alleviating skin irritability comprising the above-described biphenyl derivatives as an active ingredient.
• the derivatives show strong affinity to surface and horny layer of skin, and can improve moisture-maintaining function by being dense its structure.
• 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.
• 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.
• 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.
• 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/oco ⁇ acter pylori and Loptospira.
• composition of the present invention may be prepared as various compositions according to objects of this invention.
• 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.
• 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.
• 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.
• 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.
• 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.
• solvent solubilizer and emulsifier
• 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.
• 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.
• 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.
• suitable dosage unit is to administer once a day with 0.001-100 mg/kg (body weight).
• 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.
• 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).
• the food composition of this invention 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.
• an agricultural chemical composition for protecting plant comprising the above-described biphenyl derivatives as an active ingredient.
• 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.
• 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.
• 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.
• 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.
• 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
• 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.
• 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.
• 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.
• the composition of fodder additives is a composition for an aquacultured fish.
• 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.
• 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.
• the additives are fed to land or water animals, the additives are supplied as it is or mixing with fodder.
• 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.
• 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.
• an existing fertilizer e.g., fertilizer containing nitric acid, phosphoric acid or potassium
• the preservative composition is used for the purpose of preserving food, cosmetic, households, living materials, construction materials or medical supplies.
• the sanitizer composition is used for the sterilization of a densely populated district such as cattle pen, hospital, factory, apartment or school.
• novel biphenyl diol derivatives of this invention exhibit more improved solubility than original substances, honokiol and magnolol.
• 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.
• 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.
• 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 O 0 C, followed by adding DCC (l,3-diaza-l,3-dicyclohexylpropa-l,2-diene) (Sigma).
• 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 O 0 C, followed by adding DCC (l,3-diaza-l,3-dicyclohexylpropa-l,2-diene) (Sigma).
• 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 O 0 C, followed by adding DCC (l,3-diaza-l,3-dicyclohexylpropa-l,2-diene) (Sigma).
• the resultant solution was extracted twice with 50 ml of ethylacetate, the residual was eliminated, and cooled to O 0 C. 200 ml of 10% citric acid was added to thereto for acidifying and extracted twice with 100 ml of ethylacetate.
• Superoxide dismutase has been known as an anti-oxidation-catalyzing enzyme which converts superoxide anion into H 2 O 2 and O 2 .
• 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.
• 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.
• 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.
• the derivatives were used as water phase or oil phase materials according to formulation and type of product.
• 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.
• reaction volume per well was adjusted to 500 ⁇ l, the sample compound 2, which dissolved in lipopolysaccahride (1 ⁇ g/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 ⁇ l (in EtOH, 0.1 ⁇ Ci/ml, Sigma) were added to each well, and cultivated for 10 min under the same condition.
• 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
• NF- ⁇ B luciferase reporter activity was performed.
• NF- ⁇ B promoter was reported to play an important role in the generation of cytokine related to inflammatory.
• the activity of NF- ⁇ B luciferase was measured using the following method: Human monocytic cell, 1 x 10 5 of THP-I cell (Korean Cell Line Bank) were aliquoted into each well, NF- ⁇ B luciferase reporter plasmid DNA (Stratagene) was transfected using superfect transfection reagent (In vitrogen).
• 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.
• SE lamp wavelength 290-320 nm, Toshiba
• total irradiation energy 1350 mJ/cm 2 .
• 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.
• 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 ( ⁇ L * ) between application initial point and application terminal point were obtained and then using these values, their effects of the applied samples were estimated.
• ⁇ L * L * value at 00 days after application-L * value at application initial day.
• Nos. 2 and 4 derivatives with the insolubility in water had an effect only in solubilized part by Tween 80.
• Nos. 1 and 3 derivatives exhibited considerably potent whitening activity, whereas the effect of honokiol and magnolol was inactivated completely.
• the Nos. 2 and 4 derivatives had no effect.
• 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 10 5 cell/well) and after 17 hours, the well plate was incubated in a 5% CO 2 incubator for 24 hr at 37 0 C. 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.
• a coilagenase antibody 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 ELx808TM 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.
• 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 24 0 C to 26 0 C 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
• the derivatives of the present invention showed significantly greater (3-4 folds) effects on the alleviation of wrinkles compared to the untreatment group.
• 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.
• Nos. 2 and 4 derivatives with the insolubility in water had no or little effect.
• 6 types of samples except untreatment group exhibited potent effects and significantly greater (10-12 folds) effects compared to the untreatment group.
• 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.
• 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 ⁇ g/ml of collagen was used for the platelet aggregation.
• ASA acetylsalisylic acid
• 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.
• MTT Microculture Tetrazolium
• Various cancer cells were adjusted to 4.Ox 10 4 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.
• medium A composed of RPMI 1640 medium (GIBCO) containing 10% fetal calf serum
• the plate was incubated in a carbon dioxide gas incubater for 20 hr at 37 0 C, 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 37 0 C.
• 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 37 0 C. 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% (IC 50 ) 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 37 0 C, 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 37 0 C, according to the above method.
• 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.
• 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
• mouse carcinoma cells of the colon colon-26
• DSCG sodium cromoglycate, Merck
• mice 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.
• 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.
• 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.
• Example 16 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 experiment results were as follows.
• the control group used the moxidil commercially available from Hanmi pharmaceutical. Co. Ltd. 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.
• 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.
• 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.
• 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.
• 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.
• test plants were inoculated with spore suspension of Pyricularia oryzae, and kept in a room at a temperature of 20-22 0 C, and a humidity of 100% for 48 hours. Then the test plants were transferred to a greenhouse at a temperature of 24-26 0 C. 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
• the original substances were not improved significantly relative to the untreatment group.
• 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.
• the original substances and Nos. 2 and 4 derivatives did not exhibit at all.
• 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
• the derivatives can be prepared as fertilizer additives and agricultural chemicals for applying to agricultural plants in addition to lawn.
• 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 ⁇ l 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
• 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 mx ⁇ .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.
• 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
• 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.
• 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)
• 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.
• the biphenyl diol derivatives can be prepared as various compositions and developed in various products due to the alteration of physical feature.

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