JP3266719B2 - Purification method of astaxanthin - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a method for purifying astaxanthin.

[0002] Astaxanthin is a natural pigment that is often contained in crustaceans such as crabs and shrimps, and fish such as salmon and trout, and is used as a feed additive for fishes for the purpose of improving meat color. .

[0003]

2. Description of the Related Art As a method for chemically synthesizing astaxanthin, several methods are conventionally known. For example,
JP-A-59-53463 describes a method of oxidizing canthaxanthin with monoperphthalic acid, peracetic acid, and the like. US Pat. No. 5,097,063 discloses a method of oxidizing canthaxanthin. 2-phenylsulfonyl-3
-A method of oxidizing with phenyloxaziridine and the like. According to these methods, astaxanthin can be produced with a good yield. However, since the obtained astaxanthin contains unreacted canthaxanthin and adnirubin, it is necessary to separate astaxanthin from these carotenoids.

Conventionally, the following method has been known as a method for purifying astaxanthin. (1) Column chromatography using methylene chloride / diethyl ether as a developing solvent, and crystallization with a methylene chloride / methanol solvent (JP-A-59-53463). (2) Column chromatography using methylene chloride / diethyl ether as a developing solvent and crystallization with a methylene chloride / pentane solvent (US Pat. No. 5,097,063)
Specification). (3) Column chromatography using methylene chloride / ether / methanol as a developing solvent, and crystallization with a chloroform / methanol solvent, a pyridine / water solvent or a methylene chloride / methanol solvent (JP-A-55-388). (4) Crystallization with a methylene chloride / methanol solvent (JP-A-55-389, JP-A-62-294635, JP-A-1-265504, Helv. Chi)
m. Acta. 64 2405 (1981), H
elv. Chim. Acta. 64 2436
(1981), Helv. Chim. Acta.
64 2469 (1981)). (5) Crystallization with a methylene chloride / hexane solvent (JP-A-52-68157).

[0005] Astaxanthin is thus purified by column chromatography, crystallization using two or more solvents, and the like.

[0006]

Purification of astaxanthin by column chromatography requires a large amount of a solvent and a filler such as silica gel. Further, the operation is extremely complicated, such as preparation of a developing solvent, and the necessity of separating the developing solution according to the analysis value of the developing solution eluted sequentially and removing the solvent individually.

[0007] Purification of astaxanthin by crystallization involves the problem that the use of two or more solvents complicates the recovery of the solvent used.

[0008]

Means for Solving the Problems The present inventors have found that extremely high-purity astaxanthin can be obtained by crystallization of astaxanthin using chloroform as a solvent, and have reached the present invention.

That is, the present invention provides a method for purifying astaxanthin, which comprises crystallizing astaxanthin from a solution of chloroform containing astaxanthin.

The astaxanthin purification operation according to the method of the present invention is carried out by adding chloroform to astaxanthin containing carotenoids such as canthaxanthin and adnirubin and stirring the mixture, and then lowering the temperature of the solution or evaporating the solvent. .

As shown in Comparative Examples 1 and 2 below, astaxanthin and other carotenoids can be efficiently used even when methylene chloride, which is a halogen solvent similar to chloroform, or toluene, which is an aromatic solvent, is used alone. It is difficult to separate well, and the present invention using chloroform alone is an extremely effective means for purifying astaxanthin.

The amount of chloroform used is from half the weight of astaxanthin to be purified to an amount sufficient to completely dissolve the crystals, that is, about 3000 times the weight. However, in some cases, it is not necessary to use an amount of chloroform that completely dissolves the crystals.

[0013] The time for stirring chloroform in purified astaxanthin is usually in the range of 1 minute to 10 hours. This stirring is usually performed in a range from -60 ° C to the boiling point of the solution. However, in order to suppress the solubility of astaxanthin and the isomerization reaction of astaxanthin, the stirring is performed at 0 ° C.
To 55 ° C. is desired. When the precipitation of astaxanthin crystals is insufficient during the stirring, it is effective to lower the temperature from several degrees to several tens degrees centigrade during the stirring, that is, 5 minutes to 8 hours after the start of the stirring. In addition to lowering the temperature, there is another method for effectively depositing crystals by removing chloroform from the solution. In this case, chloroform can be distilled off at normal pressure, but chloroform may be distilled off under reduced pressure.

The produced astaxanthin crystals are filtered,
Alternatively, it is separated from the mother liquor by settling. The chloroform recovered from the filtrate obtained by the filtration may be used as it is in the next crystallization without any problem.

The filtration operation can be carried out at the same temperature as the stirring temperature, that is, in the temperature range from -60.degree. C. to the boiling point of the solution. Usually, the filtration operation in the range from 30.degree. C. to -30.degree. When the crystals are fine and the filtration property is poor, the crystals can be obtained by sedimentation by a method such as centrifugation. Also in this case, it is desirable to carry out in the same temperature range as the filtration operation. Rinsing with a small amount of chloroform is effective for removing a small amount of filtrate contained in the obtained crude crystals of astaxanthin. The purity of the obtained crude crystals of astaxanthin can be further increased by repeating the crystallization operation.

The obtained crude crystals are dried at a temperature in the range of 0 ° C. to 180 ° C. under reduced pressure or by blowing an inert gas such as nitrogen, argon or helium into high purity astaxanthin crystals. it can.

The crude astaxanthin to be purified in the present invention is prepared, for example, by the method described in Reference Examples. That is, at a temperature of −5 ° C. to −30 ° C., 2 to 3 equivalents of sodium bistrimethylsilylamide or the like is added dropwise to a canthaxanthin-tetrahydrofuran solution suspension at a temperature of about 10 minutes to 4 hours. Stir. Thereafter, the mixture is cooled to -50 ° C to -80 ° C, and an oxidizing agent such as 2-phenylsulfonyl-3-phenyloxaziridine is added to the canthaxanthin in an amount of 2 to 5 equivalents. After stirring, acetic acid or hydrochloric acid, acid such as sulfuric acid is added dropwise,
Thereafter, the temperature is raised to room temperature, and the solvent is removed. An extraction solvent such as methylene chloride or chloroform and water are added to the residue and liquid separation is performed.The organic layer is concentrated to obtain crude astaxanthin, or methanol is added to the residue and crude astaxanthin precipitated is filtered. Can be obtained. The crude astaxanthin thus obtained usually contains canthaxanthin in the range of 1% to 70% and adnirubin in the range of trace amount to 30% with respect to astaxanthin. It is possible to purify with this.

[0018]

The present invention will be described below with reference to examples, but the present invention is not limited to these examples.

Example 1 In a nitrogen stream, 3.0 g of crude astaxanthin (containing 0.04 g of canthaxanthin, 0.014 g of adnirubin and 1.72 g of astaxanthin) were mixed with 15 g of chloroform.
(Wako Pure Chemical Industries, Ltd., first-class Wako product)
After stirring for 1 hour at 25 ° C, the mixture was stirred at 25 ° C for 1 hour. Thereafter, suction filtration was performed under reduced pressure using a 11G4 glass filter at the same temperature, and the obtained crude crystals were further washed with a small amount of chloroform. The crude crystals were dried at 40 ° C. under reduced pressure until the weight became constant, to obtain 1.54 g of crystals. The ones with high performance liquid chromatography, was analyzed by the method of using an internal standard, was comprised of canthaxanthin 0.007 g, adonirubin 0.007 g, astaxanthin 1.53 g (purity 99.35%). In addition, the Official Gazette (Extra 75th: June 3, 1991 P
17) Astaxanthin purity test (UV-VIS
As a result, the purity of the product was 99.5%.

The filtrate (15.67 g) contained 0.026 g of canthaxanthin, 0.006 g of adnirubin, and 0.183 g of astaxanthin. [High-performance liquid chromatography analysis conditions] Column: ZORBAX SIL 25 cm × 4 mm Solvent: hexane / ethyl acetate = 75/25 (volume ratio) , flow rate = 1.5 ml / min Detection: photodiode array detector Wavelength: 280 to 300 nm Standard substance: acetanilide

Example 2 Under a nitrogen stream, 26.2 g of crude astaxanthin (containing 8.25 g of canthaxanthin, 0.25 g of adnirubin, and 15.73 g of astaxanthin) were mixed with 25 ml of chloroform.
After adding 0 g and stirring at 40 ° C. for 1 hour, the mixture was stirred at 21 ° C. for 1 hour. Thereafter, suction filtration was performed under reduced pressure using a 17G3 glass filter, and further, washing with a small amount of chloroform gave 28.73 g as crude crystals. As a result of analyzing this product by high performance liquid chromatography, 0.554 g of canthaxanthin, 0.105 g of adnirubin,
It contained 13.09 g of astaxanthin. Further, 263 g of the filtrate contained 7.6 g of canthaxanthin, 0.14 g of adnirubin, and 2.62 g of astaxanthin.

Example 3 Chloroform 1 was added to 28.7 g of the crude crystals obtained in Example 2.
After adding 50 g and stirring at 40 ° C. for 1 hour, the mixture was stirred at 22 ° C. for 1 hour. Thereafter, suction filtration was performed under reduced pressure using a 17G3 glass filter, and further, washing with a small amount of chloroform gave 22.97 g as crude crystals. As a result of analyzing this product by high performance liquid chromatography, 0.21 g of canthaxanthin, 0.007 g of adnirubin,
It contained 10.29 g of astaxanthin. Further, 153 g of the filtrate contained 0.344 g of canthaxanthin, 0.098 g of adnirubin, and 2.76 g of astaxanthin.

Example 4 31.58 g of crude astaxanthin (1.69 g of canthaxanthin, 3.44 g of adnirubin,
5000 g of chloroform was added to 125.13 g of astaxanthin), and the mixture was stirred at 50 ° C. for 30 minutes. After cooling to 25 ° C and stirring for 2 hours, the mixture was cooled to -20 ° C and left overnight. This was filtered through a 26G4 glass filter and washed with a small amount of chloroform to obtain 195.81 g of crude crystals. The product was analyzed by high performance liquid chromatography.
96 g and astaxanthin 95.81 g.
1.66 g of canthaxanthin was added to 1060 g of the filtrate,
It contained 1.4 g of adnirubin and 29.1 g of astaxanthin.

Example 5 The same operation as in Example 4 was carried out by using 2,500 g of chloroform on 195.81 g of the crude crystal obtained in Example 4, to obtain 179.64 g of crude crystals. This was dried under reduced pressure to obtain 72.5 g of astaxanthin crystals. As a result of analysis by high performance liquid chromatography, it was 0.5 g of adnirubin and 71.85 g of astaxanthin (purity: 99.1%). As a result of an astaxanthin purity test (method using a UV-VIS spectrometer) according to the official gazette (extra issue No. 75: P3 on June 3, 1991), the purity of this product was 9
It was 9.0%.

The filtrate (2520 g) contained 1.5 g of adnirubin and 22.1 g of astaxanthin.

Comparative Example 1 Crystallization with Methylene Chloride In a nitrogen stream, 3.08 g of crude astaxanthin (containing 0.2 g of canthaxanthin, 0.05 g of adnirubin, and 2.07 g of astaxanthin) were mixed with 84 g of methylene chloride at 40 ° C Stir for 30 minutes. Then gradually -20
C. and left overnight. This was subjected to suction filtration under reduced pressure using a 11G4 glass filter, and the obtained crude crystals were further washed with a small amount of methylene chloride. The crude crystal was dried under reduced pressure until the weight became constant.
86 g were obtained. The product was analyzed by high performance liquid chromatography, and as a result, it was 0.147 g of canthaxanthin, 0.03 g of adnirubin, and 1.68 g of astaxanthin (purity: 90.32%). 79 g of the filtrate contained 0.05 g of canthaxanthin, 0.02 g of adnirubin, and 0.39 g of astaxanthin.

[0027]

Reference Example Synthesis of astaxanthin from canthaxanthin using 2-phenylsulfonyl-3-phenyloxaziridine as an oxidizing agent Under a nitrogen atmosphere, 26.2 g (97%) of canthaxanthin was placed in a 10- liter stainless steel reaction vessel. Purity: 4
5mmol) in 1350 ml of tetrahydrofuran. Here, 93.5 g of a solution of sodium bistrimethylsilylamide in tetrahydrofuran at -18 ° C.
(1.07 mol / kg concentration: 100 mmol) was added dropwise, and the mixture was stirred at the same temperature for 2 hours. After cooling to -70 ° C., 2 - full Enirusuruhoni Le - 3-phenyl-oxaziridine 28.0 g (98% purity: 105 mmol) in tetrahydrofuran 270ml solution was slowly dropwise.
After stirring at the same temperature for 2 hours, 6.0 g of acetic acid (100 mm
ol) in 30 ml of tetrahydrofuran is added dropwise.
Stirred for 0 minutes. After the temperature of the reaction solution was raised to room temperature, the solvent was removed under reduced pressure. Methanol 120 is added to the obtained solid.
After adding 0 ml, stirring for a while and filtering, 53.84 g of crude astaxanthin was obtained. The product was analyzed by high performance liquid chromatography. As a result, 2.54 g of canthaxanthin, 0.29 g of adnirubin, 21.49 g of astaxanthin (yield from canthaxanthin: 80
%).

[0029]

According to the present invention, extremely high-purity astaxanthin can be obtained by a simple purification operation in which astaxanthin is crystallized using chloroform as a solvent, and the solvent chloroform can be easily recovered. It is.

──────────────────────────────────────────────────続 き Continuation of front page (56) References JP-A-55-388 (JP, A) UK Patent Application Publication No. 2212806 (GB, A) (58) Fields investigated (Int. Cl. ⁷ , DB name) C07C 403/00-403/24

Claims (1)

(57) [Claims] 1. A method for purifying astaxanthin, comprising crystallization of astaxanthin from a chloroform solution containing astaxanthin.