Background Art Crocin, which is extracted from dried fruits of gardenia or from dried saffron stigma, is digentiobiose ester of crocetin.

Crocetin is obtained by hydrolyzing crocin with alkali, etc. to remove digentiobiose of crocin (c. f., for example, non-patent literature 1).

In addition to the above, there has been proposed a method for the production of crocetin, in which crocin is hydrolyzed using an enzyme (c. f., for example. Patent literature 1).

Crocin is water-soluble and used as a primary color component of gardenia yellow for coloring a wide range of foods in Japan. On the other hand, crocetin is insoluble in water and soluble in diluted alkaline water, and thus it has been limitedly used as a coloring agent for Chinese noodle (c. f., <BR> for example, patent literature 2) or soft drinks (c. f. , for example, patent literature 3), etc.

As a result of the recent progress in the studies on the functions of carotenoid pigments, there have been reported the effects of crocetin on skin as a singlet oxygen scavenger (c. f., for example, patent literature 4), collagen production

stimulants (c. f. , for example, patent literature 5), and skin<BR> immunostimulators (c. f. , for example, patent literature 6).

Moreover, physiological actions of crocetin including cholesterol lowering effect, tumor growth inhibiting effect, hepatotoxicity inhibitory effect, etc. have been reported (see, for example, non patent literature 2).

In order to utilize crocetin in skin cosmetics or pharmaceutical compositions aiming for such actions or effects, <BR> <BR> crocetin of high purity prepared not only by hydrolysis of crocin but also by the removal of impurities for purification is desired.

Although there have been disclosed methods for the preparation of crocetin in the laboratory to obtain a sample <BR> <BR> for analysis (see, for example, non patent literature 3, 4, 5, 6), manipulation and process control of those methods are complex, which means they are inappropriate for industrial production.

Therefore, an industrially advantageous method for producing crocetin of high purity in an industrial scale at low cost has not been known yet.

(Patent literature 1) Japanese Unexamined Patent Publication No. H07-018194 (Patent literature 2) Japanese Unexamined Patent Publication No. S54-064652 (Patent literature 3) Japanese Unexamined Patent Publication No. H06-248193 (Patent literature 4) Japanese Unexamined Patent Publication No. H05-320036 (Patent literature 5) Japanese Unexamined Patent Publication No. H07-285846

(Patent literature 6) Japanese Unexamined Patent Publication No. H11-246396 (Non-patent literaturel) TANIMURAAkio, et al.,"Handbook<BR> of natural coloring agents", Korin Publishing Co. , Ltd. , June 25,1979, p. 215 (Non-patent literature 2) KON Masayo,"gardenia <BR> <BR> carotenoid pigment", Bulletin of Fukuoka Women'sJunior College, 1999, No. 57, p. 31-34 (Non-patent literature 3) KAMIKURAMieko, et al.,"Journal of the Food Hygienic Society of Japan", 1985,26, No. 2, p. 151 (Non-patent literature 4) J. Agric. Food Chem., 1996,44, No. 9, p. 2613, left column, line46-61 (Non-patentliterature5) SpectrochemicaRctaPartS, 1998, 54, p. 654, left column, lines 16-24 (Non-patent literature 6) J. FoodSci. Technol. , 2001, 38, No. 4, p. 325, left column, lines 36-42 Disclosure of the Invention An object of the present invention is to provide an easily operated, industrially advantageous method for the production of crocetin.

The present inventors had conducted intensive investigations to solve the above mentioned problems and, as a result, they have found that a desired crocetin of high purity <BR> <BR> can be effectively prepared when crocetin containing impurities, which is obtained by hydrolysis of crocin existing in a plant extract, is treated with a specific organic solvent. Based on this finding, the present inventors have achieved the present invention.

Thus, the present invention relates to the following (1) to (9).

(1) A method for the purification of crocetin, which comprises the steps of (a) treating crocetin obtained by hydrolysis of crocin, which exists in a plant extract, with lower alcohol or a mixed solvent containing 50% by volume or more of lower alcohol; and (b) removing a soluble component in the solvent.

(2) A method for the purification of crocetin, which comprises the steps of (a) dissolving crocetin obtained by hydrolysis of crocin, which exists in a plant extract, into an aprotic solvent which can crystallize crocetin ; and (b) crystallizing crocetin from the solvent.

(3) The method for the purification of crocetin according to the above (2), wherein an aprotic solvent is dimethylformamide or dimethylsulfoxide or a mixture thereof.

(4) A method for the purification of crocetin, which comprises the steps of (a) treating crocetin obtained by hydrolysis of crocin, which exists in a plant extract, with lower alcohol or a mixed solvent containing 50% by volume or more of lower alcohol to remove a soluble component in the solvent; and (b) crystallizing the above treated crocetin from an aprotic solvent which can crystallize crocetin.

(5) The method for the purification of crocetin according to the above (4), wherein an aprotic solvent is dimethylformamide or dimethylsulfoxide or a mixture thereof.

(6) A purified crocetin which is prepared by the method

described in any of the above (1) to (5).

(7) A purified crocetin, of which color value (Elotlcm) is in a range of 27,000 to 35,700.

(8) A cosmetic product or pharmaceutical composition, which comprises the purified crocetin described in the above (6) or (7).

(9) Food or health food, which comprises the purified crocetin described in the above (6) or (7).

Best Mode for Carrying Out the Invention Crocin, which is a yellow carotenoid pigment, is contained in dried fruits of gardenia (rubiaceae) (Gardenia augustaMERRIL var. grandiflora HORT., Gardenia jasminoides ELLIS) and dried saffron stigma, and the fruits of gardenia are preferably used as an industrial material to obtain crocin.

According to the present invention, crocin is extracted frompulverized, driedfruitsofgardeniawithwateroranalcohol or a mixture thereof. Although water is preferable as an extracting solvent, the extraction rate is low. Thus, usually a mixture of water and an alcohol is used for industrial purposes.

Examples of the alcohol include ethanol, methanol and the like, among which methanol is preferably used. The preferable mixing ratio of an alcohol relative to the mixture of the alcohol and water is, for example, not less than 50% by volume. After extraction, the extract is collected all together and filtered through filter paper or filter cloth optionally using a filter aid such as diatomite. The filtrate is concentrated to recover alcohol, whereby a concentrate can be obtained.

In the present invention, it is preferable to further

remove an iridoid glycoside such as geniposide, etc. from the extract of gardenia by absorbent resin treatment or membrane separation treatment, etc.

Absorbent resin treatment is carried out in such a manner that a column is filled with a porous absorbent and the concentrate which is diluted to the appropriate concentration is supplied to the column. For example, after pouring a pigment solution (i. e. the above diluted concentrate containing crocin) until columns are saturated with the pigment, impurities are washed out with water or a mixture of a dilute alcohol and water. Then, the pigments were desorbed i. e. eluted by 50 to 70 % by volume of alcohol and the eluted solution is concentrated to give a concentrate in which geniposide is significantly reduced.

Examples of the alcohol include a lower alcohol having 1 to 4 carbon atoms such as methanol, ethanol, propanol, isopropanol, <BR> <BR> n-butanol, i-butanol, sec-butanol, tert-butanol, etc. , among which ethanol is preferably used. Examples of the porous absorbentinclude Smberlite D Smberlite DiD-7 gTrademark : Organo Corporation), Diaion HP-20, HP-21, HP-40 (Trademark : Mitsubishi Chemical Corporation), and the like.

Membrane separation treatment is carried out by, for example, pouring the concentrate which is diluted to the appropriate concentration onto ultrafiltration membrane modules with a molecular weight cutoff of 2,000 to 20,000.

Impurities permeate the membrane together with water, and pigments including crocin are concentrated. These procedures are repeated, while supplying water, until the desired degree of purity is obtained. Lastly, the liquid is separated and concentrated to prepare a concentrate in which geniposide is

significantly reduced.

Concentration is carried out according to a common method by using a condensing vessel, which usually results in a concentrate having moisture content of about 10 to 80 % by weight, pref erably about 15 to 40 % by weight with the color value (Elo% lcm) of about 100 to 650, preferably about 400 to 600.

In the present invention, crocetin can be obtained by hydrolysis of the said extract, the said concentrate or the concentrate, in which geniposide is significantly reduced.

During the process, the concentrate is subjected to hydrolysis after, if desired, diluted with water to the appropriate concentration.

Hydrolysis is carried out by the action of an acid, an alkali or an appropriate hydrolase. Examples of the acid include hydrochloric acid, sulfuric acid, phosphoric acid, etc. and examples of the alkali include sodium hydroxide, potassium hydroxide, sodium carbonate, potassium carbonate, etc. An example of the hydrolase includes-glucosidase, etc.

In industrial practice, usually an alkali is preferably used for hydrolysis. The method may follow a common method and is not particularly limited. During hydrolysis, the reaction solution may or may not be stirred, or may or may not be heated, while preferably it is stirred and heated to proper temperature to accelerate the reaction.

After hydrolysis by an alkali, an appropriate amount of an aqueous solution of an inorganic acid such as hydrochloric acid, sulfuric acid, phosphoric acid, etc. or an aqueous solution of an organic acid such as citric acid, etc. is added to the reaction solution to adjust the pH to about 4.0 or lower,

preferably about 3.0 or lower, or alternatively, the reaction solution is added to an aqueous solution of an inorganic acid such as hydrochloric acid, sulfuric acid, phosphoric acid, etc. or an aqueous solution of an organic acid such as citric acid, etc. to adjust the pH to about 4.0 or lower, preferably about 3.0 or lower, so that crocetin is precipitated. A mixture containing crocetin is filtered through filter paper or filter cloth to collect paste-like solid substance containing crocetin.

When an acid is used for hydrolysis, crocetin is precipitated upon initiation of the reaction and becomes a suspension. After the reaction, the mixture containing crocetin is filtered through filter paper or filter cloth to collect paste-like solid substance containing crocetin.

The said solid substance is washed with sufficient amount of water to remove acid, neutral salt or impurities derived from the raw materials which are attached to the surface, then remaining water is removed by evaporation using, for example, a vacuum shelf dryer at 50 °C or lower temperature preferably in the presence of nitrogen gas.

Crocetin thus obtained, however, has a relatively low purity, because it includes materials other than crocetin such as lipid and its decomposition products, polyphenols (e. g. chlorogenic acid) or saccharides (e. g. glucose, gentiobiose) which are generated by hydrolysis and not completely removed by water washing. Therefore, it is not appropriate to use such crocetin as it is in cosmetic products, skin cosmetics, foods, health foods or pharmaceutical compositions.

The present invention relates to a method for preparing crocetin of high purity by further treating the said crocetin

with a specific solvent.

According to the first method of the present invention, the said crocetin is treated with a lower alcohol. Examples of the lower alcohol include one member or a mixture of two or more members selected from the group of lower alcohols having 1 to 4 carbon atoms such as methanol, ethanol, propanol, isopropanol, n-butanol, i-butanol, sec-butanol, tert-butanol, etc. , among which methanol is preferably used. The amount of methanol is about 1 to 2000 parts by volume, preferably about 10 to 250 parts by volume, more preferably about 10 to 40 parts by volume relative to 1 part by weight of crocetin.

A mixture of the said crocetin and methanol is stirred at about 30 to 65 °C, preferably at about 45 to 50 °C for about 0.5 to 1 hour under gentle reflux. Then the mixture is cooled to room temperature and filtered through filter paper or filter cloth. The filtering method includes suction filtration, pressure filtration or centrifugal separation, etc. Crocetin remaining on filter paper or filter cloth is collected, and the filtrate containing soluble impurities in methanol is discarded.

In the method of the present invention, the said crocetin may be treated with a mixed solvent containing about 50% by volume or more, preferably about 70% by volume or more of lower alcohol.

Examples of the solvent to be mixed with the lower alcohol include solvents having high compatibility with lower alcohol such as water, saturated or unsaturated aliphatic alcohols, ketones (e. g. acetone, ethylmethylketone), ethers (e. g. diethyl ether), ethyl acetate, and the like.

The mixture of the said crocetin and the mixed solvent containing 50% by volume or more of lower alcohol is stirred

at a temperature below the boiling point of the mixed solvent for 0.5 to 1 hour preferably under gentle reflux. Then the mixture is cooled to room temperature and filtered through filter paper or filter cloth. Crocetin remaining on filter paper or filter cloth is collected, and the filtrate containing soluble impurities in the mixed solvent is discarded.

The remaining solvent is removed from the crocetin purified by the above method using, for example, a vacuum shelf dryer at a temperature about 80 °C or lower, preferably about 60 °C or lower, more preferably about 50 °C or lower, preferably in the presence of nitrogen gas.

According to the second method of the present invention, the said crocetin, which is a hydrolysis product of crocin, is subjected to crystallization procedure in an aprotic solvent which can crystallize crocetin such as dimethylformamide, dimethylsulfoxide, dimethylacetamido, N-methyl-2-pyrrolidone, acetonitorile, y-butyrolactone, hexamethylphosphorictriamide, sulfolan 1, 3-dimethyl-2-imidazolidinone, N-methylcaprolactam, acetone, tetrahydrofuran, etc.

Preferably dimethylformamide, dimethylsulfoxide or a mixture thereof, more preferably dimethylformamide is used. Those aprotic solvents may be used independently or as a mixed solvent of one or more of them. The said aprotic solvents may be mixed with a nonpolar solvent or other solvents as long as the mixture can crystallize crocetin. Examples of such nonpolar solvent include benzene, toluene, hexane, etc. , and examples of such other solvents include water, methanol, ethanol, chloroform, etc.

The crystallization method may follow a common method and

is not particularly limited. That is, the amount of the solvent used may be any amount that can produce a saturated solution at a temperature at which crocetin dissolves, and for example the amount of dimethylformamide relative to 1 part by weight of crocetin is about 10 to 40 parts by volume, preferably about 15 to 20 parts by volume.

The mixed solution of the said crocetin and dimethylformamide is heated to between room temperature and 100 °C, preferably about between 50 °C and 80 °C, and stirred for about 0.5 to 1 hour to dissolve crocetin. In the above dissolution process, it is preferable to dissolve crocetin in an aprotic solvent until crocetin is saturated in the solution.

In the case where the concentration of crocetin does not reach saturation point during dissolution, the solution may be concentrated preferably under vacuum after dissolution. The solution is gradually cooled to room temperature (about 1 °C to 30 °C) or lower after impurities, if present, are removed from the solution by filtration. The solution is allowed to stand calmly preferably at the original temperature to form crystals, then the mixed solution containing formed crocetin crystals is filtered through filter paper or filter cloth.

Crocetin remaining on filter paper or filter cloth is collected, and the filtrate containing soluble impurities in dimethylformamide is discarded.

When dimethylsulf oxide is used, crystals are occasionally not precipitated depending on the amount of the solvent used even if the solution is cooled to room temperature. In such a case, crystals may be precipitated by, for example, adding the appropriate amount of water.

According to the present invention, resulting crocetin <BR> <BR> crystalsarewashedwithmethanolorethanol, preferablyethanol.

The amount of ethanol is about 5 to 80 parts by volume, preferably about 10 to 40 parts by volume relative to 1 part by weight of crocetin crystals. Solvents such as dimethylformamid adhered to the surface of crocetin crystals are removed by washing with ethanol, which makes it easier to remove solvents in the finishing operation.

In the second method, the purified crocetin obtained in the first method may be used as starting material in place of the said crocetin. Use of the purified crocetin increases the possibility of obtaining crocetin crystals of higher purity.

The remaining solvent is removed from the crocetin crystals purified by the above method using, for example, a vacuum shelf dryer at a temperature about 80 °C or lower, preferably about 60 °C or lower, more preferably about 50 °C or lower, preferably in the presence of nitrogen gas.

According to the methods of the present invention, crocetin with the purity of about 70 % by weight or more, often about 80 % by weight or more, can be obtained by the first method, and crocetin with the purity of about 90 % by weight or more, often about 95 % by weight or more, can be obtained by the second method. Meanwhile, in the present invention, the color value of the purified crocetin of high purity prepared with crystallization step is measured by the below-mentioned method to result in the value 35,700, based on which the above purity is calculated.

Crocetin has been reported to show not only actions on skin as a singlet oxygen scavenger, collagen production stimulant

and skin immunostimulator as described above, but also physiological actions such as cholesterol lowering effect, tumor growth inhibiting effect, hepatotoxicity inhibitory effect, etc Therefore, the purified crocetin of the present invention is useful as a pharmaceutical composition or cosmetic product or skin cosmetics. Also, the crocetin of the present invention maybe used as a yellow pigment in food including confectioneries, noodles, dairy products, fish paste, pickled vegetables, vinegaredfood, Neri-yokan (thick sweet bean jelly), Mizu-yokan (soft sweet bean jelly), Uiro (sweet steamed rice paste), boiled beans, Neri-uni (paste of seasoned sea-urchin eggs), Furikake (seasoned fish and vegetable flakes), etc. or in health food, when it is made into cyclodextrin clathrates as disclosed in Japanese Unexamined Patent Publication No. H6-24819 3 or Japanese Unexamined Patent Publication No. H7-23736, spray-dried powder as disclosed in Japanese Published Patent No. S56-25097, emulsion or lysate (c. f. Non-patent literature 2).

Known pharmaceutical carriers, excipients or other additives to be used with crocetin may be chosen by those skilled in the art depending on the types and purposes of the use. More specifically, excipients, disintegrators, binding agents, surfactants, emulsifiers, plasticizers, wetting agents, lubricants, sugars, pH regulators, preservatives, flavoring agents, coloring agents or the like may be exemplified in the case of pharmaceutical compositions or cosmetic products, and various nutrients, vitamins, flavoring agents, coloring agents, antioxidants, flavoring materials for chocolate, cheese, etc., artificial sweeteners or the like may be exemplified in the case of foods or health foods.

The pharmaceutical compositions of the present invention may be administered orally in the form of syrup, powder, granule, pill, tablet, hard capsule, soft capsule or may be administered parenterally in the form of suppository, parenteral injection, infusion, etc. , depending on the method and root of administration. In the case of cosmetic product, crocetin may be appropriately added to produce a cosmetic product in the form of cream, lotion, ointment, liquid formulation, paste, etc.

When crocetin is applied to food or health food, it may be provided in the form of granule, tablet, chewing gum, candy, jelly, beverage, etc., though the form is not limited to those mentioned above.

With regard to the intake of crocetin, it is advisable to adjust the dosage of crocetin as an active ingredient in a range of about 0.1 mg to 500 mg, preferably about 1 mg to 300 mg, more preferably about 2 mg to 100 mg per a day per a human adult considering the absorption rate in the case of oral formulations, though the intake cannot be generalized because it depends on sex, age, health condition, etc. of those who take crocetin. In the case of parenteral formulations such as injections or infusions, it is advisable to adjust the dosage of crocetin in a range of about 0.1 mg to 100 mg, preferably about 0.1 mg to 50 mg, more preferably about 0.5 mg to 10 mg per a day per a human adult. In the case of skin cosmetics, it is preferable that crocetin is contained in an amount of about 0.001 to 10 % by weight relative to the whole weight of the skin cosmetics. In the case of food or health food, it is generally advisable to add crocetin in an amount of about 0.00003 to 10 % by weight, preferably about 0.01 to 5 % by weight, relative to

the whole weight of the food or health food.

Meanwhile, the above pharmaceutical compositions, skin cosmetics, cosmetic products, foods or health foods which contain crocetin may be administered, used or taken one to several times a day.

Examples The present invention will be specifically illustrated according to examples.

<Measuring method of color Value> Color value was measured by the method mentioned below according to"Food Additives Other Than Chemically Synthesized Compounds, voluntary standards (second edition), gardenia yellow Japan Food Additives Association.

The sample is precisely measured in such a way that the measured absorbance thereof falls between 0.3 and 0.7, and then dissolved in Kolthoff buffer (50mM Na2CO3-50mM Na2B407, pHlO. 0) to accurately prepare 500 ml of the solution. When the sample hardly dissolves, supersonic treatment is applied for the dissolution. The solution (10 mL) is exactly measured and Kolthoff buffer (50mM Na2CO3-50mM Na2B407, pH 10.0) is added thereto to prepare 50 mL of a test solution. Kolthoff buffer (50mM Na2CO3-50mM Na2B407, pHlO. 0) is used as a control.

Absorbance (A) that is the maximum absorbance at around 420 nm through 1 cm liquid length is measured, and the color value is calculated by the following formula.

Color Value (Elotlcm) = (A x 250)-amount of sample collected (g) Reference Example 1

A mixed solution (300 ml) of methanol and water (1: 1 by volume) was added to 150 g of pulverized, dried fruits of gardenia, and the mixture was stirred at room temperature for 3 hours, followed by suction filtration. To the extraction residue was added 300 ml of a mixed solution of methanol and water (1: 1 by volume). The mixture was stirred at room temperature for 30 minutes and subjected to suction filtration, and these procedures were repeated once again to collect the total volume of about 900 ml of the extract as filtrate. The extract was concentrated using a rotary evaporator under vacuum at 60 °C to give about 50 g of a concentrate containing crocin (color value (E1o% lcm) =573).

The resulting concentrate was mixed with 40 % by weight of sodium hydroxide solution (17 g), and the mixed solution was subjected to hydrolysis reaction at 50 °C for 3.5 hours while stirring. After the reaction, the reaction mixture was added to 4 % by weight of aqueous phosphoric acid solution (420 ml) to make it acidic and allowed to stand at room temperature for about 3 hours.

The precipitated deposits were collected by centrifugation at 10, 000 x g for 10 minutes, and twice subjected to washing with 100 ml of water and the centrifugation. The resulting paste-like solid substance was vacuum-dried at 50 °C for 8 hours to give about 1.2 g of crocetin. The color value (Eloalcm) of the crocetin was about 12,500.

Example 1 Methanol (250 ml) was added to 1 g of crocetin obtained in the same manner as described in Reference Example 1, and the

mixture was stirred at 50 °C for 30 minutes. Then the mixture was centrifuged at 10, 000 x g for 10 minutes to collect deposits and the resulting paste-like solid substance was vacuum-dried at 50 °C for 8 hours to give about 0.32 g of purified crocetin.

The color value (Elo% lcm) of the purified crocetin was about 30,100.

Example 2 A mixture (250 ml) of methanol and water (4: 1 by volume) was added to 1 g of crocetin obtained in the same manner as described in Reference Example 1, and the mixture was stirred at 50 °C for 30 minutes. Then the mixture was centrifuged at 10, 000 x g for 10 minutes to collect solid substance and the resulting paste-like solid substance was vacuum-dried at 50 °C for 8 hours to give about 0. 39 g of purified crocetin. The color value (E1otlcm) of the purified crocetin was about 27,000.

Example 3 Dimethylformamide (18 ml) was added to 1 g of crocetin obtained in the same manner as described in Reference Example 1, then dissolved at 80 °C. The insoluble substances were filtered through quantitative filter paper (No. 5C, Advantec <BR> <BR> Toyo Kaisha, Ltd. ) and the filtrate was allowed to stand at 10 °Cfor3days. Next, the mother liquor containing formed crocetin crystals was passed through a sintered glass filter No. 3, and the crystals were vacuum-dried at 50 °C after washed with 20 ml of methanol to give about 0.16 g of purified crocetin. The color value (E1oælCm) of the purified crocetin was about 34,200.

Example 4 Dimethylformamide (6. 5 ml) was added to 0. 15 g of purified crocetin obtained in Example 1, then dissolved at 80 °C while stirring. The insoluble substances were filtered through quantitative filter paper No. 5C, and the filtrate was allowed to stand at 10 °C for 3 days. Next, the mother liquor containing formed crocetin crystals was passed through a sintered glass filter No. 3, and the crystals were vacuum-dried at 50 °C after washed with 10 ml of methanol to give about 0.09 g of purified crocetin. The color value (E1°glcm) of the purified crocetin was about 35,700.

Test Example 1 Melting point determination and elemental analysis of the crystals obtained in Example 4 were carried out by an external institution. The results are shown below.

Melting point: 285 °C to 286 °C Elemental analysis: Carbon 72. 60 %, Hydrogen 7. 51 m, Oxygen 19. 89 % The above results show that the data of said crystals are identical with those described in literatures.

Industrial Applicability According to the present invention, crocetin of high purity is easily prepared in an industrial manner, and it is preferably applied to cosmetic products, skin cosmetics, pharmaceutical compositions and the like. The unpleasant taste and off-flavor of the crocetin are significantly decreased because of the purification, which makes it possible for such crocetin to be used in a broader range of food or health food.