WO2007060811A1

WO2007060811A1 - Process for production of fucoxanthinol

Info

Publication number: WO2007060811A1
Application number: PCT/JP2006/321659
Authority: WO
Inventors: Kazuo Miyashita; Masashi Hosokawa; Tokutake Sashima; Takanori Sasaki
Original assignee: National University Corporation Hokkaido University; Marine Tec Kamaishi Inc.
Priority date: 2005-11-25
Filing date: 2006-10-30
Publication date: 2007-05-31
Also published as: JP2009033970A

Abstract

[PROBLEMS] To provide a process which can produce fucoxanthinol within a short time, at low cost and with high efficiency. [MEANS FOR SOLVING PROBLEMS] A process for production of fucoxanthinol comprises: a first step in which fucoxanthin (0.5 g) is dissolved in methanol (300 mL) and a surfactant sodium taurocholate (2.5 g) is then added and dissolved to the resulting solution; a second step in which the solution of fucoxanthin and sodium taurocholate in methanol prepared in the first step is evaporated to dryness; a third step in which the resulting dry material is dissolved in a potassium phosphate buffer (0.1 M; pH=7.0; 500 mL), a porcine pancreas-derived lipase (10 g) is added to the solution, and the resulting mixed solution is incubated at 37˚C for 2 hours; and a forth step in which fucoxanthinol thus produced is purified by column chromatography. The process can produce fucoxanthinol having high purity in a large amount, at low cost, and within a short time.

Description

Process for producing fucoxanthinol

Technical field

The present invention relates to a process for producing fucoxanthinol that enables fucoxanthin power to be obtained by enzymatic reaction.

Background art

[0002] Fucoxanthin and fucoxanthinol are the same basic skeleton as β-carotene (see Fig. 6), that is, a kind of carotenoids with conjugated double bond chains, and have various physiological functions. It has been reported. For example, in the case of fucoxanthin, the anti-obesity effect (disclosed in Non-Patent Document 1, the disclosure of which is incorporated herein by reference) and high cancer cell-inducing ability to induce apoptosis ( Non-Patent Document 2 is disclosed, and the disclosure is incorporated herein by reference thereto. In this way, fucoxanthin is highly! And has a physiological activity. Fucoxanthinol, which has a chemical structure in which the acetyl group of fucoxanthin is replaced with a hydroxyl group, has a higher physiological activity than fucoxanthin. The inventors found out.

[0003] Currently, the following three types of fucoxanthinol production methods are reported in academic papers. That is, (1) a method for extracting from marine organisms that feed on fucoxanthin (for example, sea squirts) using an organic solvent (disclosed in Non-Patent Documents 3 to 5; (2) A method for isolating and purifying fucoxanthin from a reaction product obtained by alkaline hydrolysis (disclosed in Non-Patent Documents 3 and 6) (3) a method of isolating and purifying from a reaction product obtained by hydrolysis of fucoxanthin with a hydrolase (in Non-Patent Documents 7 to 10). And the disclosure of which is incorporated herein by reference).

[0004] In the production method of (1) above, from the viewpoint of mass production of fucoxanthinol, "Securing material for extracting fucoxanthinol (marine organisms)" and " It can be said that it is inefficient because of the difficulty of isolation and purification from tenoids. For example, according to Non-Patent Document 4, the disclosure of which is incorporated herein by reference, in the case of Shirosuboya (scientific name: Didemnum moseleyi) that contains the most fucoxanthinol in the ascidian family There are seven types of carotenoids, and fucoxanthinol is present at 12.4 mg per shirosbo lOOg. In other words, in order to prepare fucoxanthinol in lg units, about 8 kg of Syringa as an extraction material is required, and there are difficulties in securing stable storage, storage and procurement of the extraction material.

[0005] Next, in the case of the production method of (2) above, the operation for obtaining fucoxanthinol is very simple as compared with the production methods of (1) and (3) above. There is a problem with the very bad point. For example, according to Non-Patent Document 6, the disclosure of which is incorporated herein by reference, fucoxanthin is added to a methanolic solution of 0.01% potassium hydroxide (potassium hydroxide and fucoxane). When xanthine molar ratio = 2.2), the component ratio of each carotenoid after 40 minutes is as follows: fucoxanthin (33%), fucoxanthinol (8%), fucoxanthin hemiketal (11%), fucoki Santinol hemiketal (3%), isofucoxanthin (17%), and isofucoxanthinol (14%) are described. According to this, the yield of fucoxanthinol is only 8%.

[0006] Further, when 0.1% potassium hydroxide in methanol was added to the methanol solution (molar ratio of potassium hydroxide to fucoxanthin = 24), the component ratio of each carotenoid after 35 minutes was calculated as fucoxanthin ( 20%), fucoxanthinol (6%), fucoxanthin hemiketal (10%), fucoxanthinol hemiketal (3%), isofucoxanthin (4%), isofucoxanthinol (7%) Are listed. That is, the yield of fucoxanthinol is only 6%.

[0007] Even in Non-Patent Document 3, the disclosure of which is incorporated herein by reference, only 8 mg of fucoxanthinol is obtained from 200 mg of fucoxanthin. In other words, in the alkaline hydrolysis of fucoxanthin, the yield based on fucoxanthin as a raw material is very low, 10% or less. Therefore, compared with the production method (3) of fucoxanthinol described later, this production method (2) has a yield of fucoxanthinol of 10% or less and is suitable for a large amount of production method! You can say /!

[0008] In the production method of (3) above, fucoki is produced in a higher yield than the production methods of (1) and (2). Although Santinol can be synthesized, there are still problems with the production methods reported so far in terms of cost and yield.

[0009] For example, according to Non-Patent Document 7, the disclosure of which is incorporated herein by reference, the acetyl group of fucoxanthin is hydrolyzed using cholesterol esterase derived from microorganisms (Pseudomonas fluorescens (scientific name)). Decomposes and synthesizes fucoxanthinol.

[0010] The reaction is carried out by mixing the following three solutions A, B, and C. Solution A is a fucoxanthin solution that is 10 L of fucoxanthin in acetone prepared to a concentration of 0.33 g / L. Solution B is a cholesterol esterase solution, which is a stock solution prepared as follows from a microorganism-derived cholesterol esterase having an activity of 2300 units Zg at 37 ° C. and pH = 7.0. Dissolve lOlmg of cholesterol esterase in 10 mL of Tris-HCl buffer adjusted to pH = 7.0 and stock the resulting solution. Use 45.8 L of this stock solution as Solution B. Solution C is a buffer, which is a Tris-HCl buffer prepared to 94.2 L pH = 7.0, 0.05M (Monole / L). Mix these three solutions A, B, and C and react for 30 minutes. In general, purified cholesterol esterase is expensive. Non-patent document 7 reports that the deacetylation reaction of fucoxanthin using this enzyme has a slow reaction rate and the yield is 5% or less. And

[0011] According to Non-Patent Document 8, the disclosure of which is incorporated herein by reference, fucoxanthin is obtained by hydrolyzing the acetyl group of fucoxanthin using esterase (PLE) derived from pig liver. Synthesizes a knoll. The reaction is performed by adding the following solutions C and D to fucoxanthin. Solution C is a mixed solution of methanol and Tris-HCl buffer (pH = 7.3, 50 mM), and the ratio is methanol: Tris-HCl buffer = 2: 3 (volume ratio or OR weight ratio?). Solution D is a porcine liver-derived esterase (PLE) enzyme solution. First, prepare PLE (activity is 355 units Zmg). Dissolve PLE19mg in 100 / zL ammonium sulfate solution. The ammonium sulfate solution used here was prepared to a concentration of 3.2 M. The solutions C and D prepared in this way are reacted under the following conditions. After lmg fucoxanthin is dissolved in 6.5mL of solution C, 100mg Cover solution D of L. The mixed solution is allowed to react for 24 hours while stirring with a stirrer at 25 to 28 ° C. in a nitrogen atmosphere to carry out a hydrolysis reaction.

[0012] Under these reaction conditions, 0.33 mg (yield 45%) of fucoxanthinol was obtained. There is still room for improvement in yield. Furthermore, since the reaction takes 24 hours to complete, there is still a need to complete the reaction in a short time.

[0013] According to Non-Patent Document 9, the disclosure of which is incorporated herein by reference, the acetylase of fucoxanthin is hydrolyzed using a lipase derived from a microorganism (Candida cylindracea (scientific name)). Fucoxanthinol is synthesized. The reaction conditions were as follows: lmL Britton-Robinson buffer containing 5mg of lipase was added to 10µL of fucoxanthin in acetone with 26pmol fucoxanthin dissolved. C, hydrolysis is carried out by incubating for 6 hours. This document does not describe the yield of fucoxanthin to fucoxanthinol. However, a yield of about 60% is predicted from the HPLC analysis results. This method is at the laboratory level, is performed in a very small amount, and uses expensive lipase, so it is doubtful whether it is a technology capable of mass production.

[0014] According to Non-Patent Document 10, the disclosure of which is incorporated herein by reference, fucoxanthinol is obtained by hydrolyzing the acetyl group of fucoxanthin using a cholesterol esterase derived from porcine spleen. Have gained. The reaction conditions were: 5 / z mol fucoxanthin and lOOmg taurocholic acid dissolved in a small amount of dichloromethane Zmethanol (2: 1), the solvent was evaporated with argon gas, and 10mL potassium containing lOunit cholesterol esterase Fucoxanthin is hydrolyzed by dissolving in phosphate buffer and incubating at 37 ° C for 2 hours. Although the yield is completely described, it is unsuitable for mass production because it uses expensive cholesterol esterase.

[0015] Non-Patent Document 1: H. Maeda et al., Biochemical and Biophysical Research Communication, 332 (2005) 392—397.

Non-Patent Document 2: Hosokawa, BIO INDUSTRY, 21 (2004) 52—57.

Non-Patent Document 3: H. Nitsche, Biochemica et Biophysica Acta 338 (197 4) 572-576. Non-Patent Document 4: M. Ookubo and T. Matsuno, Comp. Biochem. Physiol., 81B (1985) 137—141.

Patent Document 5: 1. Konishi et al., Comparative Biochemistry and Phys iology. Part Pharmacology, toxicology & endocrinology, In p ress.

Non-Patent Document 6: J. A. Haugan et al., Acta Chemica Scandinavica 46

(1992) 614-624.

Non-Patent Document 7: P. B. Jacobs et al., Comp. Biochem. Physiol., 72 B (1982) 157-160.

Non-Patent Document 8: T. Aakermann et al., Biocatalysis and Biotransformation, 13 (1996) 157-163.

Non-Patent Document 9: T. Sugawara et al., J. Nutri. 132 (2002) 946-951 Non-Patent Document 10: A. Asai et al., Drug Metabolism and Disposition, 3 2 (2004) 205-211.

Disclosure of the invention

Problems to be solved by the invention

The present invention has been made in view of the problems of the prior art, and can produce fucoxanthinol in a short time and at a low cost with a high yield. The purpose is to provide a manufacturing method.

Means for solving the problem

[0017] As a result of intensive studies on the above problems, the present inventors have made fucoxanthinol effective at 80% or more when lipase derived from animal viscera is allowed to act on fucoxanthin at an appropriate temperature, at an appropriate concentration, and at an appropriate time. It was found that it can be obtained.

[0018] That is, the method for producing fucoxanthinol of the present invention comprises a first step of dissolving fucoxanthin and a surfactant in a first organic solvent, evaporating the first organic solvent, and A lipase is added to a second step of drying fucoxanthin and the surfactant, and a pH buffer solution in which the dried product obtained in the second step is dissolved. And a third step of incubating only for a time.

[0019] A critical difference between the present invention and the conventional method is that a lipase, particularly an animal-derived lipase is used as a hydrolase of fucoxanthin. In the case of an lipase derived from an animal, fucoxanthinol can be obtained by hydrolysis of fucoxanthin in a sufficiently high yield even with an unpurified lipase.

[0020] The fucoxanthinol obtained up to the third step may be further dissolved in a second organic solvent, and may further include a fourth step of purification by chromatography.

[0021] The fucoxanthin may be an organic compound, or an animal or plant or algae extract that has been isolated and purified, or a bacterial fungus extracted and purified. The first organic solvent is at least one selected from alcohols, ethers, ketos, halogenated compounds of aliphatic hydrocarbons, aliphatic hydrocarbons, and aromatic hydrocarbons. Also good.

[0022] The surfactant includes taurocholic acid, dexoxylic acid, glycolic acid and their alkali salts or lecithins, fatty acid glycerin ester, fatty acid propylene glycol ester, fatty acid sorbitan ester, fatty acid sucrose ester, oleic acid alkali salt Fatty acid morpholine salt, calcium stearyl lactate, or protein may be at least one kind selected.

[0023] The pH of the pH buffer solution is preferably 5 or more and 9 or less. The lipase can be a lipase derived from an animal viscera. The predetermined temperature of the incubation is desirably 10 ° C or higher and 50 ° C or lower. Furthermore, the predetermined incubation time may be not less than 30 minutes and not more than 48 hours.

[0024] The second organic solvent and the developing solvent for the chromatography were selected from alcohols, ethers, ketos, halogenated aliphatic hydrocarbons, aliphatic hydrocarbons, and aromatic hydrocarbons. It can be at least one kind. The packing material used in the chromatography is normal phase silica gel, celite, alumina, calcium hydroxide, or reverse phase linear alkyl group, aromatic functional group, hydrophilic functional group, or polarity. It may be at least one selected from those in which the encapsulated functional group is chemically bonded to the silica gel. The invention's effect

[0025] A first step of dissolving fucoxanthin and a surfactant in a first organic solvent; and evaporating the first organic solvent to dry the fucoxanthin and the surfactant. This process consists of a third step in which lipase is added to the pH buffer solution in which the dried product obtained in the second step is dissolved and incubated at a predetermined temperature for a predetermined time. The fucoxanthinol is dissolved in a second organic solvent and further purified by chromatography, so that fucoxanthinol can be produced in a short time and at a low cost with a high yield. It becomes possible to do.

[0026] Further, by adding this fucoxanthinol to a food material, a functional food material having various functions can be obtained.

BEST MODE FOR CARRYING OUT THE INVENTION

Hereinafter, the power to specifically describe the embodiment of the present invention The present invention is not limited only to this embodiment. FIG. 1 is a flowchart of the method for producing fucoxanthinol of the present invention. Extraction of wakame force, isolated 'purified fucoxanthin force was also shown for a series of operations to synthesize fucoxanthinol using porcine spleen lipase

[0028] The fucoxanthin used as the raw material here is fucoxanthin extracted from seaweed. However, even fucoxanthin obtained by organic synthesis is fucoxanthin extracted from an animal containing fucoxanthin. Even if it is fucoxanthin biosynthesized by Escherichia coli or yeast, these fucosanthins may be used in combination. In other words, the fucoxanthin, which is the final product, can be obtained at a low cost and in a high yield in a short time using fucoxanthin obtained by any means.

[0029] First, as the first step, fucoxanthin and sodium taurocholate, which is a surfactant, are dissolved in methanol as the first organic solvent. Here, in the case where sodium taurocholate is difficult to dissolve, it is possible to use a soaker.

[0030] Next, as a second step, from an methanol solution as the first organic solvent of fucoxanthin and sodium taurocholate obtained in the first step, an evaporator is used to evaporate the methanol solution. Let Furthermore, dry nitrogen gas is blown onto the dried solids, and methanol Try to remove it.

[0031] Further, as a third step, the obtained dried product is dissolved in a potassium phosphate buffer solution as a pH buffer solution, and subsequently, for example, porcine spleen-derived lipase is added as a lipase. The porcine liver lipase used here does not need to be an expensive and purified product, and there is no particular problem in reaction efficiency and reaction time even if crude lipase is used. Therefore, crude lipase is sufficient to produce fucoxanthinol at low cost.

[0032] The pH of the pH buffer solution is 5 or more and 9 or less. If the pH is less than 5, the 5,6-epoxide in the fucoxanthin becomes 5,8-epoxide, which is unfavorable. On the other hand, if the pH exceeds 9, it is described in the background art Fucoxanthinol production method (2) As mentioned above, the decomposition of fucoxanthin is accelerated, which is not preferable.

[0033] After the lipase is dissolved, it is incubated at a temperature of 10 ° C to 50 ° C for 30 minutes to 48 hours.

If the incubation temperature is less than 10 ° C, the enzyme reaction activity decreases.In addition, the yield is low and the reaction time becomes longer, which is not preferable. On the other hand, if it exceeds 50 ° C, the enzyme reaction activity decreases. In addition, low yield and longer reaction time and decomposition of the raw material fucoxanthin and the synthetic fucoxanthinol are preferable.

Further, if the incubation time is less than 30 minutes, the amount of unreacted fucoxanthinol increases, which is unfavorable. On the other hand, if it exceeds 48 hours, the produced fucoxanthinol is decomposed, which is not preferable.

[0034] Following the above, as a fourth step, unpurified fucoxanthinol obtained in the first step to the third step is dissolved in a second organic solvent and subjected to column chromatography for purification by chromatography. . Since the second organic solvent and the developing solvent for the chromatography are desired to have a boiling point of 100 ° C. or lower when the alcohol is removed later, alcohols having 1 to 4 carbon atoms are particularly preferable. .

[0035] [Example]

Next, an embodiment of the present invention will be described.

As a first step, 0.5 g of fucoxanthin was dissolved in 300 mL of methanol, and then 2.5 g of sodium taurocholate as a surfactant was added to this solution and dissolved. Next As a second step, the methanol solution of fucoxanthin and sodium taurocholate obtained in the first step was evaporated to dryness with an evaporator, and further, dry nitrogen gas was blown onto the dried product to remove the methanol.

[0036] Further, as the third step, the obtained dried product was dissolved in 500 mL of a potassium phosphate buffer solution of 0.1 M, pH = 7.0, followed by addition of 10 g of porcine spleen-derived crude lipase. Although crude lipase was used, there was no problem in reaction efficiency or reaction time. After the lipase was dissolved, it was incubated at 37 ° C for 2 hours.

[0037] Following the above, 300 mL of methanol was added to the solution after the enzyme reaction, and the mixture was stirred and allowed to stand for 10 minutes. To this solution, 500 mL of jetyl ether was added to carry out a liquid separation operation. The gel ether phase was recovered, and the water phase was repeated until the carotenoid color disappeared. The recovered jetyl ether phase was dried with an evaporator to obtain unpurified fucoxanthinol.

[0038] Further, as the fourth step, the obtained unpurified fucoxanthinol is subjected to column chromatography (filler: silica gel, developing solvent: 50% acetone Z hexane (volume ratio or weight ratio?)). Provided. Figure 2 shows the development of column chromatography. First, sodium taurocholate elutes, followed by unreacted fucoxanthin followed by fucoxanthinol. Under the reaction conditions shown in Fig. 2 (the amount of lipase is 10 times the amount of raw fucoxanthin), the unreacted fucoxanthin appears. The reaction conditions in the flowchart of Fig. 1 (the amount of lipase is the amount of raw fucoxanthin) In the 20 times amount), no fucoxanthin band was visually confirmed in the column chromatogram.

[0039] The experiment was conducted twice under the reaction conditions shown in FIG. 2 and the reaction conditions shown in the flowchart of FIG. Under the reaction conditions shown in Fig. 2, the raw materials were 518.9 g (l time) and 620. lmg (2nd time), respectively, and fucoxanthinol was 272.8 g (l time: yield 56. 1%), 360. 6 mg ( Second time: yield 62.2%), unreacted fucoxanthin 223.5 mg (first time), 168.9 mg (second time) were obtained. On the other hand, under the reaction conditions in the flowchart of FIG. 1, the raw materials 496.7 7 mg (l time), 531.7 7 mg (second time), and the like, fucoxanthino monool 434.4 mg (l time: yield 93.4%), 412 2 mg (2nd: yield 82.8%) was obtained. Therefore, by setting the amount of fucoxanthin and lipase at an appropriate ratio, the yield of fucoxanthinol can be increased. It becomes possible.

[0040] When the obtained column chromatography purified fucoxanthinol was analyzed by high performance liquid chromatography (HPLC), more than 80% of all-trans fucoxanthinol was obtained as shown in FIG. Only all trans isomers were isolated by HPLC 'purification and NMR analysis was performed. FIG. 4 is a one-dimensional proton NMR of all-transfucoxanthinol after HPLC purification, and FIG. 5 is a one-dimensional carbon NMR of all-transfucoxanthinol after HPLC purification. This confirmed that the obtained hydrolyzate of fucoxanthin was fucoxanthinol. Therefore, the fucoxanthinol production method of the present invention makes it possible to produce high-purity fucoxanthinol in large quantities, at low cost and in a short period of time.

Industrial applicability

[0041] By adding fucoxanthinol produced by the method for producing fucoxanthinol of the present invention to a food material or the like, it can be used as a functional food material. However, industrial application fields are not limited to this.

Brief Description of Drawings

FIG. 1 is a flowchart showing an embodiment of the method for producing fucoxanthinol of the present invention.

FIG. 2 is a photograph showing the development of column chromatography in an example of the method for producing fucoxanthinol of the present invention.

FIG. 3 is a HPLC analysis chart of each component after the column in the example of the method for producing fucoxanthinol of the present invention.

FIG. 4 is a one-dimensional proton NMR of all transfucoxanthinol after HPLC purification in an example of the method for producing fucoxanthinol of the present invention.

FIG. 5 is a one-dimensional carbon NMR analysis of all transfucoxanthinol after HPLC purification in an example of the method for producing fucoxanthinol of the present invention.

FIG. 6 is a chemical structural formula of fucoxanthinol and fucoxanthin.

Claims

The scope of the claims

[1] A first step of dissolving fucoxanthin and a surfactant in a first organic solvent, and evaporating the first organic solvent to dry the fucoxanthin and the surfactant. And a third step of adding lipase to the pH buffer solution in which the dried product obtained in the second step is dissolved and incubating at a predetermined temperature for a predetermined time. A method for producing a knoll.

[2] The method for producing fucoxanthinol according to claim 1, further comprising a fourth step in which fucoxanthinol obtained up to the third step is dissolved in a second organic solvent and purified by chromatography. .

[3] The fucoxanthinol according to claim 1, wherein the fucoxanthin is extracted from an organic compound, animal or plant or algal force, isolated and purified, or extracted and purified by bacterial force. Manufacturing method.

[4] The first organic solvent is at least one selected from alcohols, ethers, ketos, halogenated compounds of aliphatic hydrocarbons, aliphatic hydrocarbons, and aromatic hydrocarbons. The method for producing fucoxanthinol according to claim 1.

[5] The surfactant includes taurocholic acid, dexoxylic acid, glycolic acid and their alkali salts or lecithins, fatty acid glycerin ester, fatty acid propylene glycol ester, fatty acid sorbitan ester, fatty acid sucrose ester, and oleic acid alkali salt. 2. The method for producing fucoxanthinol according to claim 1, wherein the fatty acid morpholine salt, calcium stearyl lactate, or protein is at least one selected.

6. The process for producing fucoxanthinol according to claim 1, wherein the pH of the pH buffer solution is 5 or more and 9 or less.

7. The method for producing fucoxanthinol according to claim 1, wherein the lipase is a lipase derived from an internal organ of an animal.

8. The method for producing fucoxanthinol according to claim 1, wherein the predetermined temperature of the incubation is 10 ° C or higher and 50 ° C or lower.

[9] The method for producing fucoxanthinol according to claim 1, wherein the predetermined time for the incubation is 30 minutes or more and 48 hours or less.

[10] The second organic solvent and the developing solvent for the chromatography were selected from alcohols, ethers, ketos, aliphatic hydrocarbon halogen compounds, aliphatic hydrocarbons, and aromatic hydrocarbons. The method for producing fucoxanthinol according to claim 2, which is at least one kind.

11. The method for producing fucoxanthinol according to claim 2, wherein the filler used in the chromatography is at least one selected from normal phase silica gel, celite, alumina, and calcium hydroxide calcium hydroxide.

[12] The packing used in the chromatography is at least one selected from the group consisting of a reverse-phase linear alkyl group, an aromatic functional group, a hydrophilic functional group, or a polar inclusion functional group chemically bonded to a silica gel. The method for producing fucoxanthinol according to claim 2.