||||||||||USOO5750379A United States Patent (19) 11 Patent Number: 5,750,379 Onodera et al. 45 Date of Patent: May 12, 1998

54 PROCESS FOR THE PRODUCTION OF Saito. Biochem. Physiol. Pflanzen. vol. 188, pp. 405-417. CARTHAMN 1993. (75) Inventors: Junichi Onodera; Shingo Sato, both of Saito. Plant Sci. vol. 90, pp. 1-9, 1993. Yonezawa; Toshio Kashiwagi; Isobe Kumazawa et al. Chemistry Letters, vol. 8, pp. 625-626, Tetsuhiro, both of Tokyo, all of Japan 1995. 73) Assignee: Toyo Ink Manufacturing Co., Ltd., Saito et al. J. Plant Physiol. vol. 140, pp. 121-123, 1992 Tokyo, Japan Abstract. 21 Appl. No.:711,741 Hirota et al., JP49026328, 1974-Abstract. 22 Filed: Sep. 10, 1996 Wada et al. JP61199798, 1985-Abstract. 30 Foreign Application Priority Data Mar 18, 1989 JP Japan...... 8-060482 Primary Examiner-Leon B. Lankford, Jr. Sep. 11, 1995 JP Japan ...... 7-232422 Assistant Examiner Christopher R. Tate Mar. 18, 1996 JP Japan ...... 8-060483 Attorney, Agent, or Firm-Wenderoth, Lind & Ponack (51) Int. Cl...... C12P 19/00; A61K 35/78 57 ABSTRACT

52 U.S. Cl...... 435/72; 424/195.1 Carthamin (safflower red dyestuff) which is highly useful 58) Field of Search ...... 435/72; 424/195.1 but has been limited in use due to its high price, can be 56 References Cited efficiently obtained from safflower yellow B dyestuff of which the content in safflower petals is large, and the PUBLICATIONS safflower red dyestuff therefore can be stably provided at a Saito. Z. Naturforsch, vol. 47c, pp. 205-208, 1992. low price. Saito et al. J. Plant Physiol. vol. 142, pp. 257-264, 1993 Abstract. 18 Claims, No Drawings 5,750,379 1. 2 PROCESS FOR THE PRODUCTION OF oxide together with as a receptor and an oxido CARTHAMN reductase for which works as a receptor, with saffiower yellow B. BACKGROUND OF THE INVENTION According to the present invention 3, there is further 1. Field of the Invention provided a process for the production of carthamin, which The present invention relates to a process for the produc comprises reacting a hydrolyzate of safflower yellow B with tion of carthamin, which comprises reacting a safflower an aldehyde to obtain a carthamin precursor and then yellow B dyestuff with an oxido-reductase and an aldehyde, oxidizing the carthamin precursor. or with an oxido-reductase which generates hydrogen per 10 oxide together with oxygen as a receptor and an oxido DETALED DESCRIPTION OF THE reductase for which hydrogen peroxide works as a receptor. INVENTION The carthamin produced by the above process is used as a The present inventors have made diligent studies to dyestuff for foods or in cosmetics and pharmaceutical com overcome the prior art problems and have found that positions. 15 carthamin, a red dyestuff, can be efficiently obtained by 2. Description of Related Art reacting an oxido-reductase with a safflower yellow B Safflower (Carthamus tinctorius L.) is a composite plant dyestuff in the presence of an aldehyde. It has been further and has thistle-shaped clear yellow flowers in the middle of found that a safflower yellow B dyestuff is efficiently con July. Its petals are gathered, dried or mashed and fermented verted to carthamin in the absence of an aldehyde but with to show a clear red color, and this fermented product has a combination of an oxido-reductase which generates hydro been called "Koka” (safflower) and is highly valued as gen peroxide together with oxygen as a receptor and an traditional Chinese medicine or a high-class dye. However, oxido-reductase for which hydrogen peroxide works as a the amount of water-soluble dyestuff (composed of safflower receptor. yellow A, safflower yellow B and safflower yellow C) is The process of the present invention can be carried out by overwhelmingly large, as large as about 60% by weight, 25 adding an oxido-reductase and an aldehyde, or adding an based on the dyestuffs contained in the pedals, and the oxido-reductase which generates hydrogen peroxide content of carthamin which is the useful red dyestuff is as together with oxygen as a receptor and an oxido-reductase Small as 0.4 to 0.6% by weight. In recent years, the red for which hydrogen peroxide works as a receptor, to an dyestuff of safflower is attracting attention as a dyestuff for aqueous solution containing a safflower yellow B dyestuff foods since it is excellent in heat resistance and color tone 30 and maintaining the safflower yellow B dyestuff in a buffer as compared with red beet, and studies are therefore being solution or water having a pH of 3 to 11, preferably 4 to 9, made not only on the method of efficiently extracting it but at a temperature of approximately 5° to 60° C., preferably also on the further expansion of use thereof. 20° to 40° C., for 1 minute to 1 weeks, preferably 1 minute Safflower yellow is water-soluble, and carthamin is spar to 24 hours. ingly soluble in water. On the basis of this difference, a 35 For promoting the reaction, improving the reaction effi current practice employs a method in which a red color ciency or controlling the hydrogen peroxide concentration, dyestuff is recovered from a residue remaining after the it is preferred to simultaneously add a substrate of the extraction of the yellow dyestuff with water. Since, however, oxido-reductase which generates hydrogen peroxide with the content of carthamin itself is very small, highly purified oxygen as a receptor. Further, for improving the decompo red dyestuff carthamin is expensive, which prevents the sition rate of a safflower yellow B dyestuff, the efficiency of further expansion of use thereof. the oxido-reductase in activity, the formation rate of carthamin and the formation efficiency of carthamin, it is SUMMARY OF THE INVENTION preferred to add an immobilizing adsorbent such as cellulose It is an object of the present invention to provide a process 45 or xanthone. In some cases, oxygen may be introduced into for efficiently producing a red dystuff, carthamin, from the reaction mixture for several minutes. safflower yellow B, whose contentis nearly 50% by weight, It is assumed that carthamin, a red dyestuff, is obtained based on yellow dyestuffs which are contained in a large through the following reaction scheme, in which a saflower amount in the petals of safflower. yellow B dyestuff is hydrolyzed into a hydrolyzate, which According to the present invention, there is provided a 5 can form a carthamin precursor, and safflomin A, and the process for the production of carthamin, which comprises former forms carthamin through the carthamin precursor. reacting an oxido-reductase and an aldehyde with safflower The substance called “safflower yellow B hydrolyzate” in yellow B. the reaction scheme is not only important as an intermediate According to the present invention, there is also provided for forming carthamin from the safflower yellow B dyestuff a process for the production of carthamin, which comprises 55 but also important as an intermediate for the synthesis of reacting an oxido-reductase which generates hydrogen per other dyestuff, a cosmetic material or a medicament. 5,750,379

OH

Safflower yellow B

OH O

Safflower yellow B hydrolyzate

5,750,379

-continued

Safflower yellow B is preferably hydrolyzed by the use of ate dehydrogenase, gluconate dehydrogenase, an reaction in the presence of an oxido-reductase, arabinosedehydrogenase, glucosedehydrogenase, galactose while safflower yellow B may also be hydrolyzed without dehydrogenase, phosphate dehydrogenase, hydrox any enzyme by maintaining it in a buffer solution having a 20 ysteroid dehydrogenase, hydroxycholanate dehydrogenase, pH of 3 to 11 at a temperature of 5° to 100° C. for 1 hour allyl , lactaldehyde reductase, ribitol to 1 week. Further, when the carthamin precursor is con dehydrogenase, fructuronate reductase, tagaturonate verted to carthamin, the reaction proceeds promptly in the reductase, hydroxypropionate dehydrogenase, tartronate presence of an oxido-reductase, while the carthamin precur semialdehyde reductase, hydroxybutylate dehydrogenase, sor may be also converted to carthamin by oxidation with 25 estradiol dehydrogenase, testosterone dehydrogenase, pyri a. doxine dehydrogenase, hydroxydecanoate dehydrogenase, The safflower used in the present invention may be fresh , gluconate dehydrogenase, octanol safflower or a dry safflower, and it includes "Mogami Koka”, dehydrogenase, aminopropanol dehydrogenase, butanediol “Chinese Koka”, “Okayama No. 1", "Israel” and "Califor dehydrogenase, lactone aldehyde reductase, lactone alde 30 hyde dehydrogenase, , isopropanol nia”. However, the safflower used in the present invention dehydrogenase, hydroxylpyruvate reductase, maleate shall not be limited in kind. The sufflower yellow B dyestuff dehydrogenase, dimethylmaleate dehydrogenase, isopropy used in the present invention may have any form such as a Imaleate dehydrogenase, ketolate reductoisomerase, purified dyestuff, a crude dyestuff extracted with an alcohol, hydroxycarboxyadipate dehydrogenase, hydroxymethylglu fresh safflower, dry safflower or crushed petals so long as 35 taryl reductase, acryl alcohol dehydrogenase, oxaloglycolate sufflower yellow B dyestuff is contained therein. reductase, , glycerol phosphate Although depending upon the amount of safflower yellow dehydrogenase, phosphoglycerate dehydrogenase, dystuff (safflower yellow B dyestuff), the amount of the diodophenylpyruvate reductase, hydroxybenzyl alcohol enzyme used in the present inventionis 1 unit to 10,000 units dehydrogenase, hydroxyfatty acid dehydrogenase, oxoacyl based on 1 g of the sufflower yellow dyestuff (sufflower ACP reductase, palmitoyl dihydroxyacetone phosphate yellow B dyestuff). reductase, dehydrosphinganine reductase, threonine The enzyme used in the present invention includes oxido dehydrogenase, oxoproline reductase, retino reductases which are classified as an enzyme for which dehydrogenase, pantoate dehydrogenase, pyridoxal CH-OH works as a donor, an enzyme which generates dehydrogenase, carnitine dehydrogenase, indole lactate hydrogen peroxide with oxygen as a receptor, and an 45 dehydrogenase, imidazole , indanol enzyme for which hydrogen peroxide works as a receptor, dehydrogenase, xylose dehydrogenase, apiose reductase, although the enzyme used in the present invention shall not ribose dehydrogenase, arabinose dehydrogenase, glucose be limited to the above classification. dehydrogenase, galactose dehydrogenase, aldose The oxido-reductase for which CH-OH works as a dehydrogenase, fucose dehydrogenase, Sorbose donor includes alcohol dehydrogenase, alcohol dehydroge 50 dehydrogenase, fructose dehydrogenase, deoxygluconate nase (used in combination with NADP+ or NAD+ as a dehydrogenase, ketodeoxygluconate dehydrogenase, ido coenzyme), , butanediol nate dehydrogenase, threonate dehydrogenase, ketoglucon dehydrogenase, acetone dehydrogenase, glycerol ate dehydrogenase, mannuronate reductase, mannose dehydrogenase, propanediol phosphate dehydrogenase, dehydrogenase, ketorhamnose reductase, deoxytalose glycerol phosphate dehydrogenase (used in combination 55 dehydrogenase, acetylglucoseamine dehydrogenase, ribitol with NAD+ as a coenzyme), xylose reductase, arabinitol phosphate dehydrogenase, mannitol dehydrogenase, , iditol dehydrogenase, galactitol phosphate dehydrogenase, hydroxyprostaglandin dehydrogenase, mannitol phosphate dehydrogenase, inositol dehydrogenase, pinitol dehydrogenase, Sequoitol dehydrogenase, glucuronate reductase, glucuronolactone dehydrogenase, perillyl alcohol dehydrogenase, hydroxys reductase, , glucose dehydrogenase, histi teroid dehydrogenase, estradiol dehydrogenase, ethiochol dinol dehydrogenase, , shikimate anolone dehydrogenase, sepiapterin reductase, ureidoglyco dehydrogenase, glyoxylate reductase, lactate late dehydrogenase, homoisocitrate dehydrogenase, , , hydroxybutylate dehydrogenase, dihydroxybutylol dehydrogenase, hydroxy dehydrogenase, hydroxyisobutylate dehydrogenase, meval butylyl CoA dehydrogenase, acetylenol pyry voyl glucose date reductase, hydroxymethylglutalyl reductase, hydroxya 65 amine reductase, erythrulose reductase, cyclopentanol cyl dehydrogenase, acetoacetyl reductase, maleate dehydrogenase, hexadecanol dehydrogenase, hydroxyhex dehydrogenase, isocitorate dehydrogenase, phosphoglucon anecarbonate dehydrogenase, hydroxymalonate 5,750,379 7 8 dehydrogenase, OXopantoyl lactone reductase, OXopantoate The buffer solution used in the present invention has a reductase, hydroxymethylcholestenoate dehydrogenase, concentration of 0.001 to 1 mol. Although not specially methylenetetrahydroborate reductase, oxoadipate reductase, limited, the buffer solution includes a phosphoric acid buffer rhamnose dehydrogenase, cyclohexanediol dehydrogenase, solution, a citric acid buffer solution, an acetic acid buffer glycolate , maleate oxidase, glucose oxidase, hexose solution, a tris-hydrochloric acid buffer solution, an ammo nium acetate buffer solution, a sodium pyrophosphate buffer oxidase, cholestrol oxidase, allyl . gulono solution, a glycine-sodium buffer solution and Good's lactone oxidase, . , sor Buffer. bose oxidase, pyridoxine oxidase, alcohol oxidase, catechol It is assumed that the process of the present invention oxidase, hydroxy acid oxidase, and cho proceeds through a conversion scheme in which safflower line oxidase. However, the oxido-reductase shall not be 10 yellow B is decomposed from the safflower yellow B limited to these. dyestuff in the presence of an oxido-reductase or under some The oxido-reductase which generates hydrogen peroxide conditions, an aldehyde acts on the resultant hydrolyzate to with oxygen as a receptor is generically called “oxidase'. form a carthamin precursor, and the formation of carthamin and includes glucose oxidase, , cholesterol is promoted in the presence of the oxido-reductase or oxidase, allyl alcohol oxidase, sorbose oxidase, pyridoxine 15 through oxidation. The process of the present invention is Oxidase, alcohol oxidase, catechol oxidase, hydroxy acid also assumed to proceed as follows. The safflower yellow B oxidase, ecdysone oxidase, , amino acid dyestuff is decomposed into a hydrolyzate which forms the oxidase, amine oxidase and uric acid oxidase, although the carthamin precursor and a safflomin-A dyestuff under the above oxido-reductase shall not be limited to these. For influence of the oxido-reductase for which the oxygen works effectively feeding hydrogen peroxide, a substrate corre 20 as a receptor or some reaction conditions. The hydrolyzate sponding to the enzyme is desirably present in an amount of formed at this time forms the carthamin precursor under not more than 10 mole equivalents, preferably not more than complex activities of the oxido-reductase for which the 2 mole equivalents, based on the amount of the safflower oxygen present works as a receptor, a Substrate correspond yellow B dyestuff, although it may not be present in some ing thereto (glucose if the oxido-reductase is glucose cases. The above may be used alone or in combi 25 oxidase) and hydrogen peroxide which is consequently nation. formed. Then, the carthamin precursor is converted to Glucose oxidase is a typical example of the oxido carthamin under the complex activities of the hydrogen reductase which generates hydrogen peroxide with oxygen peroxide and the oxido-reductase for which the hydrogen as a receptor. Glucose oxidase includes those derived from 30 peroxide works as a receptor. aspergillus, penicillium amagasakiense and penicillium In both of the above two assumptions, the same result notatum, although the glucose oxidase shall not be limited to takes place. When the safflower yellow B dyestuff is hydro these. Glucose is a corresponding substrate, and glucose is lyzed in the presence of the enzyme or chemically, a desirably present in an amount of not more than 2 mole hydrolyzate which forms the carthamin precursor and the equivalents, based on the amount of the safflower yellow B 35 safflomin-A dyestuff are formed in equimolar amounts, and dyestuff, although it may not be present in some cases. the former is converted to carthamin in the presence of the Although not specially limited, the oxido-reductase for oxido-reductase or under oxidation with air through the which hydrogen peroxide works as a receptor includes carthamin precursor. That is, carthamin in a theoretical NAD+ , NADP+ peroxidase, fatty acid amount of 433 mg is obtained from 1 g of safflower yellow peroxidase, peroxidase, catalase, peroxidase, 40 B. The actual yield of carthamin is about 90% of the iodo peroxidase, glutathione peroxidase and chloride per theoretical amount. It has been found that the safflower Oxidase. yellow B dyestuff is efficiently converted to carthamin The above enzymes may be used alone or in combination. according to the process of the present invention. The above The enzyme which requires a coenzyme NAD+ or NADP+ analyses are easily confirmed by high-performance liquid may be used alone. The use of the coenzyme can improve 45 chromatography. the conversion efficiency. Carthamin obtained in the present invention is easily Catalase and peroxidase are among typical examples of recovered by allowing an immobilizing adsorbent to adsorb the oxido-reductase for which hydrogen peroxide works as it. For example, the immobilizing adsorbent includes Duo a receptor. Although not specially limited, the catalase lite S-30, ES-33 and S-37 (supplied by Diamond Shamrock includes those purified from abovine, equine or human liver. 50 Chemical Co., Ltd.), Amberlite XAD-2, XAD-4, XAD-7 the liver of a sheep, a mouse, a pig or a toad, and bacteria and XAD-8 (supplied by Rohm and Hass), Diaion HP-10, such as Micrococcus lysodeikticus or Rhodopseudomonas HP-20, HP-21 and HP-40 (supplied by Mitsubishi Chemical spheroidus. Although not specially limited, the peroxidase Co., Ltd.), KS, HS, AF and L-1 (Supplied by Hokuetsu Tanso includes those derived from horseradish, a fig, a radish, a Kogyo K.K.), and polysaccharides such ascellulose, chitin, turnip, thyroid, milk, intestine or leukocyte. These enzymes 55 chitosan, starch and derivatives of these, although the adsor may be used alone or in combination. bent shall not be limited to these. The amount of the aldehyde used in the present invention Further, the co-presence of an immobilizing adsorbent in differs depending upon the amount of the safflower yellow the reaction system for the conversion from safflower yellow B dyestuff. The amount of the aldehyde is ys to 5 g B dyestuff to carthamin not only promotes the formation rate equivalents based on 1 g equivalent of the safflower yellow of carthamin but also can increase the yield of carthamin. B dyestuff. The amount of the immobilizing adsorbent is preferably 100 Although not specially limited, the aldehyde used in the mg to 1kg based on 1 kg of the dry petals of safflower. When present invention includes formaldehyde, acetoaldehyde, purified dyestuff is used, the amount of the adsorbent is butylaldehyde, crotonaldehyde, glyoxal, alkyl ester of gly calculated relative to the amount of the safflower yellow B oxylic acid, glyoxylic acid chloride, glyoxylic acid amide, 65 dyestuff and carthamin contained in the dry petals. glyoxylic acid nitrile, aldol, dimethylpropanl, benzaldehyde After the reaction, purified carthamin can be easily and its derivative and acrylaldehyde. obtained through Sephadex which is crosslinked dextran 5,750,379 9 10 (supplied by Pharmacia) or column chromatography using stirred under reaction conditions of 25°C. and pH 6.8. The silica gel, or by a method in which the carthamin is adsorbed reaction mixture showed a color change from yellow to red. on a cellulose powder and fractionated. After the reaction, a cellulose powder was added to allow the powder to stably immobilize and adsorb the red dyestuff, The present invention will be explained more in detail carthamin. Then, the pH of the reaction mixture was hereinafter. adjusted to 5-6 with citric acid to allow the cellulose powder to completely adsorb the carthamin in the reaction mixture. EXAMPLE 1. The cellulose powder was recovered by filtration and 30 Milligrams of highly purified safflower yellow B washed with water to remove water-soluble impurities, and then the carthamin was eluted from the cellulose with dyestuff, 2 mg (25 units) of and 1.5 mg 10 of glyoxylic acid were dissolved in 2 ml of a 0.5M phos ethanol. The eluate was fully dried to give a preparation of phoric acid buffer solution. The mixture was allowed to react purified carthamin (yield 7.5 mg). The analysis results of the infrared absorption spectrum, for 3 days with stirring under reaction conditions of 25°C. the ultraviolet absorption spectrum, the mass spectrometry and pH 6.8. The reaction mixture showed a color change and the thin film chromatography of the above-obtained from yellow to red. After the reaction, a cellulose powder 15 was added for to stably immobilize and adsorb carthamin as carthamin completely agreed with those of known a red dyestuff. Then, the pH of the reaction mixture was carthamin. adjusted to 5-6 with citric acid to allow the cellulose powder EXAMPLE 4 to completely adsorb the carthamin in the reaction mixture. A highly purified safflower yellow B dyestuff in an The cellulose powder was recovered by filtration and amount of 30 mg was dissolved in 2 ml of a 0.2M acetic acid washed with water to remove water-soluble impurities, and buffer solution (pH 6.8), and the mixture was allowed to then the carthamin was eluted from the cellulose with react at 25°C. for 2 days with stirring, to decompose the methanol. The eluate was fully dried to give a preparation of safflower yellow B. Then, 1.5 mg of glyoxylic acid and 0.3 purified carthamin (yield 3.0 mg). mg (30 units) of peroxidase (horseradish) were added to the The analysis results of the infrared absorption spectrum, 25 reaction mixture. The mixture was allowed to react for 1 day the ultraviolet absorption spectrum, the mass spectrometry with stirring under conditions of 25° C. and pH 6.8. The and the thin film chromatography of the above-obtained reaction mixture showed a color change from yellow to red. carthamin completely agreed with those of known After the reaction, a cellulose powder was added to allow the carthamin. powder to stably immobilize and adsorb the red dyestuff, 3) carthamin. Then, the pH of the reaction mixture was EXAMPLE 2 adjusted to 5-6 with citric acid to allow the cellulose powder A solution of 1 mg (186 units) of glucose oxidase (derived to completely adsorb the carthamin in the reaction mixture. from ) and 1.5 mg of glyoxylic acid in 2 ml The cellulose powder was recovered by filtration and of a 0.5M phosphoric acid buffer solution was added to an washed with water to remove water-soluble impurities, and aqueous solution of safflower yellow dyestuff (containing 30 35 then the carthamin was eluted from the cellulose with mg of safflower yellow B dyestuff) obtained from safflower 70%ethanol. The eluate was fully dried to give a preparation "Koka" ("California”, yellow flower variety) by 70% etha of purified carthamin (yield 4.5 mg). nol extraction. The mixture was allowed to react for 3 days The analysis results of the infrared absorption spectrum, with stirring under reaction conditions of 25° C. and pH 6.8. the ultraviolet absorption spectrum, the mass spectrometry The reaction mixture showed a color change from yellow to and the thin film chromatography of the above-obtained red. After the reaction, a cellulose powder was added to carthamin completely agreed with those of known allow the powder to stably immobilize and adsorb the red carthamin. dyestuff, carthamin. Then, the pH of the reaction mixture EXAMPLE 5 was adjusted to 5-6 with citric acid to allow the cellulose 45 Highly purified safflower yellow B dyestuff (30mg), 5 mg powder to completely adsorb the carthamin in the reaction of glucose, 1 mg (186 units) of glucose oxidase (Aspergillus mixture. The cellulose powder was recovered by filtration niger), 1.5 mg of glyoxylic acid and 0.3 mg (30 units) of and washed with water to remove water-soluble impurities, peroxidase (horseradish) were dissolved in 2 ml of a 0.5M and then the carthamin was eluted from the cellulose with phosphoric acid buffer solution. The mixture was allowed to 80% methanol. The eluate was fully dried to give a prepa 50 react for 3 days with stirring under conditions of 25°C. and ration of purified carthamin (yield 5.0 mg). pH 6.8. The reaction mixture showed a color change from The analysis results of the infrared absorption spectrum, yellow to red. After the reaction, a cellulose powder was the ultraviolet absorption spectrum, the mass spectrometry added to allow the powder to stably immobilize and adsorb and the thin film chromatography of the above-obtained the red dyestuff, carthamin. Then, the pH of the reaction carthamin completely agreed with those of known 55 mixture was adjusted to 5-6 with citric acid to allow the carthamin. cellulose powder to completely adsorb the carthamin in the reaction mixture. The cellulose powder was recovered by EXAMPLE 3 filtration and washed with water to remove water-soluble A solution of 1 mg (186 units) of glucose oxidase impurities, and then the carthamin was eluted from the (Aspergillus niger) and 0.3 mg (30 units) of peroxidase cellulose with ethanol. The eluate was fully dried to give a (horseradish) in 2 ml of a 0.5M phosphoric acid buffer preparation of purified carthamin. The yield of the carthamin solution was added to 30 mg of a highly purified safflower was 12.3 mg, or 94.6% of the theoretical value (13 mg). yellow B dyestuff. The mixture was allowed to react for 2 The analysis results of the infrared absorption spectrum, days with stirring under reaction conditions of 25°C. and pH the ultraviolet absorption spectrum, the mass spectrometry 6.8 to decompose the safflower yellow B dyestuff. Then, 2.0 65 and the thin film chromatography of the above-obtained mg of formaldehyde was added to the reaction mixture, and carthamin completely agreed with those of known the mixture was allowed to react for 1 day while it was carthamin. 5,750,379 11 12 EXAMPLE 6 of the infrared absorption spectrum, the ultraviolet absorp Highly purified safflower yellow B dyestuff (30mg), 1 mg tion spectrum, the mass spectrometry and the thin film (250 units) of alcohol dehydrogenase (Gluconobacter), 0.5 chromatography of the above-obtained preparation of puri mMNAD+, 1.5 mg of glyoxylic acid and 0.3 mg (30 units) fied carthamin completely agreed with those of known of peroxidase (horseradish) were dissolved in 2 ml of a 60 carthamin. mM sodium pyrophosphate buffer solution. The mixture was EXAMPLE 9 allowed to react for 2 days with stirring under conditions of 25° C. and pH 8.5. The reaction mixture showed a color Glucose-oxidase-producing bacteria Aspergillus niger change from yellow to red. After the reaction, a cellulose was implanted in 100 ml of an agar-free potato dextrose powder was added to allow the powder to stably immobilize 10 medium together with 3 g of a safflower yellow dyestuff and adsorb the red dyestuff, carthamin. Then, the pH of the obtained from dry Chinese "Koka” by ethanol extraction and reaction mixture was adjusted to 5-6 with citric acid to 10 g of a cellulose powder, and cultured for 3 days at 25°C. allow the cellulose powder to completely adsorb the at a pH of 5-6 with shaking the medium. The culture showed carthamin in the reaction mixture. The cellulose powder was a color change from yellow to red. After the culturing, the recovered by filtration and washed with water to remove 15 cellulose powder adsorbing carthamin was recovered by water-soluble impurities, and then the carthamin was eluted filtration, washed with water to remove water-soluble impu from the cellulose with methanol. The eluate was fully dried rities and made weakly alkaline with a sodium carbonate to give a preparation of purified carthamin. The yield of the solution to elute the red dyestuff carthamin from the cellu carthamin was 11.3 mg. or 86.9% of the theoretical value (13 lose. The pH of the eluate was adjusted to 5-6 with citric mg). acid to precipitate the carthamin, and the carthamin was The analysis results of the infrared absorption spectrum, recovered by filtration, purified and dried to give a prepa the ultraviolet absorption spectrum, the mass spectrometry ration of purified carthamin. The yield of the carthamin was and the thin film chromatography of the above-obtained 240 mg. carthamin completely agreed with those of known 25 The analysis results of the infrared absorption spectrum, carthamin. the ultraviolet absorption spectrum, the mass spectrometry and the thin film chromatography of the above-obtained EXAMPLE 7 preparation of purified carthamin completely agreed with 1 gram (18,600 units) of glucose oxidase (Aspergillus those of known carthamin. niger), 1 g of glyoxylic acid and 300 mg (30,000 unit) of 30 EXAMPLE 10 peroxidase (horseradish) were dissolved in 2 liters of a 0.5M acetic acid buffer solution, and 100 g of dry safflower A highly purified safflower yellow B dyestuff (100 mg), (Chinese Koka) was added. The mixture was allowed to 50 mg of glucose, 50 units of glucose oxidase and 100 units react for 3 hours with stirring under conditions of 25°C. and of peroxidase were dissolved in 2 ml of a 0.5M citric acid pH 6.8. The reaction mixture showed a color change from 35 buffer solution, and 50 mg of a cellulose powder was added yellow to red. After the reaction, a cellulose powder was for allowing the powder to stably immobilize and adsorb added to allow the powder to stably immobilize and adsorb carthamin to be formed. The mixture was allowed to react the red dyestuff, carthamin. Then, the pH of the reaction for 15 minutes under reaction conditions of 25° C. and pH mixture was adjusted to 5-6 with citric acid to allow the 5.7. The reaction mixture showed a color change from cellulose powder to completely adsorb the carthamin in the yellow to red. After the reaction, the cellulose powder was reaction mixture. The cellulose powder was recovered by recovered by filtration and washed with water to remove filtration and washed with water to remove water-soluble water-soluble impurities, and then the carthamin was eluted impurities, and then the carthamin was eluted from the from the cellulose with methanol. The eluate was fully dried cellulose with methanol. The eluate was fully dried to give to give a purified preparation of carthamin. The yield of the a purified preparation of carthamin. The yield of the 45 was 41.8 mg. carthamin obtained by adding the oxido-reductase was 178 The analysis results of the infrared absorption spectrum, mg. The analysis results of the infrared absorption spectrum, the ultraviolet absorption spectrum, the mass spectrometry the ultraviolet absorption spectrum, the mass spectrometry and the thin film chromatography of the above-obtained and the thin film chromatography of the above-obtained preparation of purified carthamin completely agreed with preparation of purified carthamin completely agreed with 50 those of known carthamin, those of known carthamin. EXAMPLE 11 EXAMPLE 8 1.5 Grams of a safflower yellow dyestuff crude powder A safflower yellow dyestuff (30 mg) obtained from fresh (containing about 100 mg of safflower yellow B dyestuff) "Mogami Koka” (safflower) by methanol extraction, 1 mg 55 obtained from fresh safflower "Koka” (California (yellow (186 units) of glucose oxidase (Aspergillus niger), 1.5 mg of flower variety) by 70% ethanol extraction, 100 mg of glyoxylic acid and 0.3 mg (30 units) of peroxidase glucose, 25 units of glucose oxidase and 200 units of (horseradish) were dissolved in 2 ml of a 0.5M tris peroxidase were dissolved in 10 ml of a 0.5M phosphoric hydrochloric acid buffer solution. The mixture was allowed acid buffer solution, and 100 mg of a cellulose powder was to react for 3 days with stirring under conditions of 25°C. added for allowing the powder to stably immobilize and and pH 6.8. The reaction mixture showed a color change adsorb carthamin to be formed. The mixture was allowed to from yellow tored. After the reaction, the pH of the reaction react for 1 hour with stirring under reaction conditions of 25° mixture was adjusted to 5-6 with citric acid to precipitate C. and pH 6.8. The reaction mixture showed a color change carthamin in the reaction mixture, and the carthamin was from yellow to red. After the reaction, the pH of the reaction recovered by centrifugal separation, washed with 0.5% citric 65 mixture was adjusted to 5-6 with citric acid to allow the acid and dried to give a preparation of purified carthamin. cellulose powder to completely adsorb the carthamin in the The yield of the carthamin was 2.4 mg. The analysis results reaction mixture. The cellulose powder was recovered by 5,750,379 13 14 filtration and washed with water to remove water-soluble and then the carthamin was eluted from the cellulose with impurities, and then the carthamin was eluted from the dimethylformamide. The eluate (150 g) was diluted to 200 cellulose with 80% methanol. The eluate was fully dried to ml with dimethylformamide, and measured for absorption give a preparation of purified carthamin. The yield of the intensity at 530 nm. Separately, the above procedures were carthamin was 41.3 mg. repeated without glucose, glucose oxidase and peroxidase, The analysis results of the infrared absorption spectrum, and the resultant eluate was measured for absorption inten the ultraviolet absorption spectrum, the mass spectrometry sity at 530 nm. The yield of the carthamin obtained by and the thin film chromatography of the above-obtained adding glucose, glucose oxidase and peroxidase was about preparation of purified carthamin completely agreed with 7 times as large as the yield of the carthamin obtained those of known carthamin. 10 without adding glucose, glucose oxidase and peroxidase. EXAMPLE 12 EXAMPLE 1.5 5 Grams of fresh petals of safflower "Mogami Koka” A highly purified safflower yellow B dyestuff (100 mg), from preferably Yamagata, Japan, 500 mg of glucose, 75 50 units of glucose oxidase and 100 units of peroxidase were 15 units of glucose oxidase and 1,500 units of peroxidase were dissolved in 2 ml of a 0.5M citric acid buffer solution, and dissolved in 100 ml of a 0.5M citric acid buffer solution, and 50 mg of a cellulose powder was added for allowing the 2 g of a cellulose powder was added for allowing the powder powder to stably immobilize and adsorb carthamin to be to stably immobilize and adsorb carthamin to be formed. formed. The mixture was allowed to react for 24 hours under The mixture was allowed to react for 18 hours with shaking reaction conditions of 25° C. and pH 5.7. The reaction 20 under reaction conditions of 25°C. and pH 5.7. The reaction mixture showed a color change fromyellow to red. After the mixture showed a color change from yellow to red. After the reaction, the cellulose powder was recovered by filtration reaction, the cellulose powder was recovered by filtration and washed with water to remove water-soluble impurities, and washed with water to remove water-soluble impurities, and then the carthamin was eluted from the cellulose with and then the carthamin was eluted from the cellulose with methanol. The eluate was fully dried to give a preparation of 25 dimethylformamide. The eluate (150 g) was diluted to 200 purified carthamin. The yield of the carthamin was 28.8 mg. ml with dimethylformamide, and measured for absorption The analysis results of the infrared absorption spectrum, intensity at 530 nm. Separately, the above procedures were the ultraviolet absorption spectrum, the mass spectrometry repeated without glucose, glucose oxidase and peroxidase, and the thin film chromatography of the above-obtained and the resultant eluate was measured for absorption inten preparation of purified carthamin completely agreed with 30 sity at 530 nm. The yield of the carthamin obtained by those of known carthamin. adding glucose, glucose oxidase and peroxidase was about 10 times as large as the yield of the carthamin obtained EXAMPLE 13 without adding glucose, glucose oxidase and peroxidase. 5 Grams of dry petals of safflower “Koka” from Shin According to the present invention, the safflower red Chan, China, 500 mg of glucose, 75 units of glucose oxidase 35 dyestuff (carthamin) which has high usefulness but has been and 1,500 units of peroxidase were dissolved in 100 ml of limited in use due to its high price can be efficiently obtained a 0.5M citric acid buffer solution, and 2 g of a cellulose from safflower yellow B dyestuff of which the content in powder was added for allowing the powder to stably immo safflower petals is large, and the safflower red dyestuff bilize and adsorb carthamin to be formed. The mixture was therefore can be stably provided at a low price. allowed to react for 18 hours with shaking under reaction 40 What is claimed is: conditions of 25° C. and pH 5.7. The reaction mixture 1. A process for the production of carthamin, which showed a color change from yellow to red. After the comprises reacting an aqueous solution of a safflower yellow reaction, the cellulose powder was recovered by filtration B dyestuff with an oxido-reductase and an aldehyde other and washed with water to remove water-soluble impurities, than saccharide for a suitable time and under suitable and then the carthamin was eluted from the cellulose with 45 conditions to obtain a carthamin, the reaction proceeding dimethylformamide. The eluate (150 g) was diluted to 200 through the formation of a hydrolyzate of the safflower ml with dimethylformamide, and measured for absorption yellow B dyestuff, and then isolating the carthamin with an intensity at 530 nm. Separately, the above procedures were immobilizing adsorbent. repeated without glucose, glucose oxidase and peroxidase, 2. A process according to claim 1, wherein the aqueous and the resultant eluate was measured for absorption inten 50 solution of a safflower yellow B dyestuff is an aqueous sity at 530 nm. The yield of the carthamin obtained by solution of an extract prepared by extracting safflower adding glucose, glucose oxidase and peroxidase was about petals. 2.5 times as large as the yield of the carthamin obtained 3. A process according to claim 1, wherein the oxido without adding glucose, glucose oxidase and peroxidase. reductase is added to the aqueous solution of a safflower 55 yellow B dyestuff to hydrolyze the safflower yellow B EXAMPLE 14 dyestuff and the aldehyde other than saccharide is added to 5 Grams of dry petals of safflower "Koka” from Un-nan, a resultant hydrolyzate. China, 500 mg of glucose, 75 units of glucose oxidase and 4. A process according to claim 1, wherein the oxido 1,500 units of peroxidase were dissolved in 100 ml of a reductase and the aldehyde other than saccharide are added 0.5M citric acid buffer solution, and 2 g of a cellulose to a hydrolyzate of safflower yellow B dyestuff. powder was added for allowing the powder to stably immo 5. A process according to claim 1, wherein the aldehyde bilize and adsorb carthamin to be formed. The mixture was other than saccharide is selected from acetaldehyde, glyoxy allowed to react for 18 hours with shaking under reaction lic acid or an alkyl ester of glyoxylic acid. conditions of 25° C. and pH 5.7. The reaction mixture 6. A process according to claim 1, wherein the oxido showed a color change from yellow to red. After the 65 reductase is at least one member selected from the group reaction, the cellulose powder was recovered by filtration consisting of an enzyme for which CH-OH works as a and washed with water to remove water-soluble impurities, donor, an enzyme which generates hydrogen peroxide with 5,750,379 15 16 oxygen as a receptor and an enzyme for which hydrogen 12. A process according to claim 10, wherein the oxido peroxide works as a receptor. reductase which generates hydrogen peroxide with oxygen 7. A process according to claim 1, wherein the oxido as a receptor is an oxido-reductase for which saccharides are reductase which generates hydrogen peroxide with oxygen substrates. as a receptor is an oxido-reductase for which saccharides are 13. A process according to claim 12, wherein saccharide substrates. is further added. 8. A process according to claim 7, wherein saccharide is 14. A process according to claim 10, wherein the oxido further added. reductase for which hydrogen peroxide works as a receptor 9. A process according to claim 1, wherein the oxido is peroxidase. reductase is a combination of an alcohol dehydrogenase with 15. A process according to claim 10, wherein the oxido nicotinamide-adenine dinucleotide (NAD+) or reductase which generates hydrogen peroxide with oxygen nicotinamide-adenine dinucleotide phosphate (NADP+) as a as a receptor is glucose oxidase. coenzyme thereof. 16. A process according to claim 15, wherein glucose is 10. A process for the production of carthamin, which further added. comprises reacting an oxido-reductase which generates 15 hydrogen peroxide with oxygen as a receptor and an oxido 17. A process for the production of carthamin, which reductase for which hydrogen peroxide works as a receptor comprises reacting a hydrolyzate of a safflower yellow B with an aqueous solution of a safflower yellow B dyestuff for dyestuff with an aldehyde other than saccharide for a suit a suitable time and under suitable conditions to obtain a able time and under suitable conditions to obtain a carthamin through a hydrolyzate of the safflower yellow B carthamin, and then isolating the carthamin with an immo dyestuff, and then isolating the carthamin with an immobi bilizing adsorbent. lizing adsorbent. 18. A process according to claim 17, wherein the aldehyde 11. A process according to claim 10, wherein the aqueous other than saccharide is selected from acetaldehyde, glyoxy solution of a safflower yellow B dyestuff is an aqueous lic acid or an alkyl ester of glyoxylic acid. solution of an extract prepared by extracting safflower 25 petals.

UNITED STATES PATENT AND TRADEMARK OFFICE CERTIFICATE OF CORRECTION PATENT NO. : 5,750,379 P age l of 2 DATED : May 12, 1998 INVENTOR(S) : Onodera et al. it is certified that error appears in the above-identified patent and that said letters Patent is hereby corrected as shown below: In columns 3 and 4 of the above-identified patent, the chemical formulas designated as "Safflower yellow B hydrolyzate" should be corrected to read as follows: 9HoH do O OH O O

OH O Safflower yellow B hydrolyzate

In columns 3 and 4 of the above-identified patent, the chemical formulas designated as "Precursor" should be corrected to read as follows:

UNITED STATES PATENT ANDTRADEMARK OFFICE CERTFCATE OF CORRECTION

PATENT NO. : 5,750,379 Page 2 of 2 DATED : May 12, 1998 INVENTOR(S) : Onodera et al. it is certified that error appears in the above-identified patent and that said Letters Patent is hereby corrected as shown below: OH O HO

too O O

Precursor

Signed and Sealed this Fourth Day of May, 1999 3.72% Q. TODD DICKINSON Attesting Officer Acting Commission ('r of Pate' its and Trutenarks