JP2009120494A - Method for producing fucoxanthin-rich brown algae extract - Google Patents
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- 241000199919 Phaeophyceae Species 0.000 title claims abstract description 52
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Abstract
【課題】褐藻類に含まれる健康機能成分であるフコキサンチンを効率的に分離・精製し、食品（健康食品）、化粧品、医薬品、研究用試薬として利用する技術を提供すること。
【解決手段】有機溶媒を含有する溶媒で褐藻類を抽出して褐藻類エキスを得、次いで、当該エキス中のフコキサンチンを合成吸着樹脂に吸着および溶離させることを特徴とするフコキサンチン高含有褐藻類エキスの製造方法
【選択図】図１[PROBLEMS] To provide a technology for efficiently separating and purifying fucoxanthin, which is a health functional ingredient contained in brown algae, and using it as food (health food), cosmetics, pharmaceuticals, and research reagents.
A brown alga is extracted with a solvent containing an organic solvent to obtain a brown algae extract, and then fucoxanthin in the extract is adsorbed and eluted on a synthetic adsorption resin. Method for producing citrus extract [selection figure] Fig. 1
Description
本発明は、オキナワモズク等の褐藻類に含まれるフコキサンチンを高濃度で含有する褐藻類エキスを効率的に製造する方法および前記方法で得られた褐藻類エキスからフコキサンチンを精製する方法に関する。 The present invention relates to a method for efficiently producing a brown algae extract containing a high concentration of fucoxanthin contained in brown algae such as Okinawa Mozuku, and a method for purifying fucoxanthin from the brown algae extract obtained by the method.
褐藻類に特有なカロテノイドであるフコキサンチンは、抗酸化作用、脂肪燃焼作用、抗がん作用等を示し、他のカロテノイドを上回る健康機能が報告されている。 Fucoxanthin, which is a carotenoid peculiar to brown algae, exhibits an antioxidant action, a fat burning action, an anticancer action and the like, and has been reported to have a health function superior to other carotenoids.
これまでフコキサンチンの精製方法としては、海藻類のエタノール溶液を活性炭で処理するものが報告されている（特許文献１）。しかし、この方法はフコキサンチンの精製度が低く実用的なものではなかった。 So far, as a method for purifying fucoxanthin, a method of treating an ethanol solution of seaweed with activated carbon has been reported (Patent Document 1). However, this method was not practical because the purity of fucoxanthin was low.
そこで、より純度の高いフコキサンチンの取得方法が求められているが、褐藻類からフコキサンチンを得るための方法は複雑であるため、コストが高くなってしまい、その利用開発が遅れていた。例えば、現在高純度フコキサンチンが市販されているが、その価格は１ｍｇで１０万円近いものであり、商業ベースでの利用は、到底不可能であった。
従って、褐藻類に含まれるフコキサンチンを高濃度で含むエキスや、それを更に精製したフコキサンチンの高純度精製品を効率よく取得する技術を見出し、健康食品、化粧品、医薬品、研究用試薬等へのフコキサンチンの産業利用につなげることが強く求められており、本発明はこのような技術の提供をその課題とするものである。 Therefore, we have found a technology to efficiently acquire extracts containing high concentrations of fucoxanthin contained in brown algae and fucoxanthin high-purity purified products that have been further refined, to health foods, cosmetics, pharmaceuticals, research reagents, etc. Therefore, it is strongly desired to connect the fucoxanthin to industrial use, and the present invention aims to provide such a technique.
本発明者らは上記課題を解決するため鋭意研究を行った結果、褐藻類エキスを合成吸着樹脂を用いて処理することにより、低コストで、かつ、高濃度にフコキサンチンを含有する褐藻類エキスになることを見出した。また、本発明者らは前記エキスを精製することにより更に高純度のフコキサンチンが得られることを見出し、本発明を完成した。 As a result of intensive studies to solve the above-mentioned problems, the present inventors have processed brown algae extract with a synthetic adsorption resin, so that brown algae extract containing fucoxanthin at a high concentration at a low cost is obtained. I found out that Further, the present inventors have found that fucoxanthin with higher purity can be obtained by purifying the extract, and completed the present invention.
すなわち、本発明は少なくとも有機溶媒を含有する溶媒で褐藻類を抽出して褐藻類エキスを得、次いで、当該エキスを合成吸着樹脂と接触させ、さらに樹脂に吸着したフコキサンチンを溶離させることを特徴とするフコキサンチン高含有褐藻類エキスの製造方法である。 That is, the present invention is characterized by extracting brown algae with a solvent containing at least an organic solvent to obtain a brown algae extract, then bringing the extract into contact with a synthetic adsorption resin and further eluting fucoxanthin adsorbed on the resin. This is a method for producing a fucoxanthin-rich brown algae extract.
また、本発明は前記製造方法で製造されたフコキサンチン高含有褐藻エキスと水とを混合した後、静置し、フコキサンチンを沈殿させることを特徴とするフコキサンチンの精製方法である。 In addition, the present invention is a method for purifying fucoxanthin characterized in that fucoxanthin-rich brown algae extract produced by the above production method and water are mixed and then allowed to stand to precipitate fucoxanthin.
本発明のフコキサンチン高含有海藻エキスの製造方法は、合成吸着樹脂を利用することにより、撹拌とろ過の簡単な操作でフコキサンチンの濃縮および分離を同時に行うことができる。これにより、精製操作の簡略化、処理の迅速化、溶媒使用量の削減が実現された。また、この製造方法に用いる合成吸着樹脂はシリカゲルや活性炭と異なり再生・反復使用が可能である。 The method for producing a fucoxanthin-rich seaweed extract of the present invention can simultaneously concentrate and separate fucoxanthin by a simple operation of stirring and filtration by using a synthetic adsorption resin. As a result, simplification of the purification operation, rapid processing, and reduction in the amount of solvent used were realized. The synthetic adsorption resin used in this production method can be regenerated and used repeatedly unlike silica gel or activated carbon.
従って、本発明のフコキサンチン高含有海藻エキスの製造方法は、褐藻類から高濃度のフコキサンチンを含むエキスを容易に、かつ、低コストで製造できるものである。 Therefore, the method for producing a seaweed extract with a high content of fucoxanthin of the present invention can easily produce an extract containing a high concentration of fucoxanthin from brown algae at a low cost.
また、本発明の製造方法で得られるエキスを精製することにより、簡単に高純度のフコキサンチンを得ることができる。 Moreover, highly purified fucoxanthin can be easily obtained by refine | purifying the extract obtained by the manufacturing method of this invention.
本発明のフコキサンチン高含有褐藻類エキスの製造方法（以下、単に「本発明方法」という）において、原料として用いられる褐藻類は、フコキサンチンを含有する褐藻類であれば特に制限されず、天然または養殖のいずれのものであってもよい。この褐藻類としては例えば、オキナワモズク、モズクおよびフトモズクから選ばれるモズク類、ヒジキ、ホンダワラおよびアカモクから選ばれるホンダワラ類、ワカメおよびナンブワカメから選ばれるワカメ類、マコンブおよびガゴメコンブから選ばれるコンブ類等が挙げられ、これら褐藻類の中でも特にワカメ、ヒジキ、ホンダワラおよびオキナワモズクが好ましい。これら褐藻類は、必要により、抽出処理に先立ち乾燥させ、粒度を１００μｍ以下、好ましくは粒度を１０〜５０μｍに細切または粉砕して微粉末にしておくことが好ましい。 In the method for producing a fucoxanthin-rich brown algae extract of the present invention (hereinafter simply referred to as “the method of the present invention”), the brown algae used as a raw material is not particularly limited as long as it is a brown algae containing fucoxanthin. Or any of aquaculture may be sufficient. Examples of the brown algae include mozuku selected from Okinawa mozuku, mozuku and ftmozuku, hondaki selected from hijiki, hondawara and akamoku, wakame selected from wakame and nambuwakame, kombu selected from macacombs and gagome kombu, etc. Among these brown algae, wakame, hijiki, honda walla and Okinawa mozuku are particularly preferable. If necessary, these brown algae are preferably dried prior to the extraction treatment, and finely pulverized or pulverized to a particle size of 100 μm or less, preferably 10 to 50 μm.
上記した褐藻類の抽出は、少なくとも有機溶媒を含有する溶媒（以下、「抽出溶媒」という）を用いる以外は常法に従い行うことができる。具体的に褐藻類の抽出は、褐藻類を抽出槽に入れ、褐藻類の２〜１０倍量の抽出溶媒を加え、１５〜４０℃の温度で、０.５〜２４時間行えばよい。また、抽出の際には撹拌することが好ましい。 The above-described extraction of brown algae can be performed according to a conventional method except that a solvent containing at least an organic solvent (hereinafter referred to as “extraction solvent”) is used. Specifically, the brown algae can be extracted by placing the brown algae in an extraction tank, adding 2 to 10 times the amount of the extraction solvent of the brown algae at a temperature of 15 to 40 ° C. for 0.5 to 24 hours. Moreover, it is preferable to stir in the case of extraction.
上記抽出に用いられる抽出溶媒は、有機溶媒あるいはこれと水系溶媒の混合物であり、フコキサンチンを抽出できるものであれば特に種類を制限するものではない。この抽出溶媒の例としては、メタノール、エタノール等のアルコール類、アセトン等のケトン類、クロロホルム等のアルキルハロゲン類等の有機溶媒の少なくとも１種以上または前記有機溶媒と水との混液が挙げられる。また、本発明方法で得られるフコキサンチン高含有褐藻類エキスを食品用途に用いる場合には、溶媒としてエタノールまたはエタノールと水との混液を用いることが好ましい。上記抽出後、褐藻類エキスは必要により、ろ過、遠心分離等を行い、固形分と分離しておくことが好ましい。 The extraction solvent used for the extraction is an organic solvent or a mixture of this and an aqueous solvent, and the type is not particularly limited as long as fucoxanthin can be extracted. Examples of the extraction solvent include at least one organic solvent such as alcohols such as methanol and ethanol, ketones such as acetone, and alkyl halogens such as chloroform, or a mixture of the organic solvent and water. Moreover, when using the fucoxanthin high content brown algal extract obtained by the method of this invention for a food use, it is preferable to use ethanol or a mixture of ethanol and water as a solvent. After the extraction, the brown algae extract is preferably separated from the solid content by filtration, centrifugation, etc., if necessary.
上記で得られた褐藻類エキスは、次に、合成吸着樹脂で処理し、当該エキス中のフコキサンチンを合成吸着樹脂に吸着させ、その後、当該樹脂からフコキサンチンを溶離させる。合成吸着樹脂にフコキサンチンを吸着させる方法の一例としては、前記褐藻類エキスに、水と合成吸着樹脂を添加して攪拌等により混合する方法が挙げられる（バッチ法）。この方法で、合成吸着樹脂にフコキサンチンを吸着させるには、例えば、水と合成吸着樹脂とを、褐藻類エキス１００容量部に対して、それぞれ５０〜１５０容量部および１〜３０容量部、好ましくは８０〜１２０容量部および３〜６容量部で加え、これを３０分〜２時間、好ましくは３０分〜１時間程度で攪拌すれば良い。以上のようにしてフコキサンチンを吸着させた合成吸着樹脂は、常法に従い、ろ過、遠心分離等により回収することができる。また、別の方法としては、上記の要領で褐藻類エキスに水を加え混合し、合成吸着樹脂が充填されたカラムに通液する方法が挙げられる（カラム法）。 Next, the brown algae extract obtained above is treated with a synthetic adsorption resin, fucoxanthin in the extract is adsorbed on the synthetic adsorption resin, and then fucoxanthin is eluted from the resin. As an example of a method for adsorbing fucoxanthin on a synthetic adsorption resin, there is a method in which water and a synthetic adsorption resin are added to the brown algae extract and mixed by stirring or the like (batch method). In this method, in order to adsorb fucoxanthin to the synthetic adsorption resin, for example, 50 to 150 parts by volume and 1 to 30 parts by volume, preferably 100 parts by volume of water and the synthetic adsorption resin, respectively, May be added at 80 to 120 parts by volume and 3 to 6 parts by volume, and this may be stirred for 30 minutes to 2 hours, preferably about 30 minutes to 1 hour. The synthetic adsorption resin to which fucoxanthin is adsorbed as described above can be recovered by filtration, centrifugation or the like according to a conventional method. As another method, there is a method in which water is added to and mixed with brown algae extract as described above, and the mixture is passed through a column filled with a synthetic adsorption resin (column method).
上記吸着に用いられる合成吸着樹脂は、フコキサンチンを吸着させることが可能であれば、特に種類を制限するものではないが、例えば、スチレン−ジビニルベンゼン系合成吸着樹脂、メタクリル系合成吸着樹脂等が好ましい。これら合成吸着樹脂のうち、スチレン−ジビニルベンゼン系合成吸着樹脂としては、例えば、ダイヤイオンＨＰ２０、ＨＰ２１、セパビ−ズＳＰ７０、ＳＰ７００、ＳＰ８２５、ＳＰ８５０、ＳＰ２０７等（いずれも三菱化学社製）、アンバーライトＸＡＤ２、ＸＡＤ４、ＸＡＤ１６ＨＰ等（オルガノ社（供給元：米国ローム＆ハース社製））を使用することができる。また、メタクリル系合成吸着樹脂としてはダイヤイオンＨＰ１ＭＧ、ＨＰ２ＭＧ等（三菱化学社製）、アンバーライトＸＡＤ７ＨＰ等（オルガノ社（供給元：米国ローム＆ハース社製））を使用することができる。 The synthetic adsorption resin used for the adsorption is not particularly limited as long as fucoxanthin can be adsorbed. For example, styrene-divinylbenzene synthetic adsorption resin, methacrylic synthetic adsorption resin, etc. preferable. Among these synthetic adsorption resins, examples of the styrene-divinylbenzene synthetic adsorption resin include Diaion HP20, HP21, Sephaze SP70, SP700, SP825, SP850, SP207 (all manufactured by Mitsubishi Chemical Corporation), Amberlite, etc. XAD2, XAD4, XAD16HP, etc. (Organo Corporation (supplier: manufactured by Rohm & Haas, USA)) can be used. As the methacrylic synthetic adsorption resin, Diaion HP1MG, HP2MG, etc. (Mitsubishi Chemical Co., Ltd.), Amberlite XAD7HP, etc. (Organo Co., Ltd. (supplier: Rohm & Haas, USA)) can be used.
前記のようにして回収した、フコキサンチンを吸着させた合成吸着樹脂からフコキサンチンを溶離させるには、合成吸着樹脂に新たな溶離液を接触させればよい。具体的に合成吸着樹脂からフコキサンチンを溶離させるには、合成吸着樹脂と溶離液とを混合するバッチ法、合成吸着樹脂を充填したカラムに溶離液を通液させるカラム法等が挙げられる。この溶離液の例としては、メタノール、エタノール等のアルコール類、アセトン等のケトン類等の極性有機溶媒の少なくとも１種以上または前記極性有機溶媒と水との混液が挙げられ、好ましくは前記極性有機溶媒を８０質量％（以下、単に「％」という）以上含むものである。また、本発明方法で得られるフコキサンチン高含有褐藻類エキスを食品用途に用いる場合には、溶離液としてエタノールを用いることが好ましい。 In order to elute fucoxanthin from the synthetic adsorption resin adsorbed with fucoxanthin collected as described above, a new eluent may be brought into contact with the synthetic adsorption resin. Specifically, fucoxanthin is eluted from the synthetic adsorption resin by a batch method in which the synthetic adsorption resin and the eluent are mixed, a column method in which the eluent is passed through a column filled with the synthetic adsorption resin, and the like. Examples of the eluent include at least one or more polar organic solvents such as alcohols such as methanol and ethanol, and ketones such as acetone, or a mixture of the polar organic solvent and water, preferably the polar organic The solvent contains 80% by mass (hereinafter simply referred to as “%”) or more. Moreover, when using the fucoxanthin high content brown algae extract obtained by the method of this invention for a food use, it is preferable to use ethanol as an eluent.
以上のように、合成吸着樹脂からフコキサンチンを溶離液で溶離させることによって、フコキサンチンが高濃度となったフコキサンチン高含有褐藻類エキスを得ることができる。このフコキサンチン高含有褐藻類エキスは、そのまま、あるいは濃縮・乾燥を行い溶媒を除去して、食品（健康食品）や化粧品等に利用することができる。 As described above, a fucoxanthin-rich brown algae extract having a high concentration of fucoxanthin can be obtained by eluting fucoxanthin from the synthetic adsorption resin with an eluent. This fucoxanthin-rich brown algae extract can be used for food (health foods), cosmetics, etc. as it is or after concentration and drying to remove the solvent.
また、フコキサンチン高含有褐藻類エキス中のフコキサンチンの濃度を更に高めるには、上記合成吸着樹脂によるフコキサンチンの吸着およびこれからの溶離を複数回行えばよい。 In order to further increase the concentration of fucoxanthin in the fucoxanthin-rich brown algae extract, the adsorption of fucoxanthin by the synthetic adsorption resin and the elution from this may be performed a plurality of times.
上記の合成吸着樹脂を利用した吸着および溶離操作は、１回行った場合でも、褐藻類の抽出液を、簡便・迅速に１０倍程度に濃縮する効果があり、濃縮工程を効率化することができる。 Even when the adsorption and elution operations using the above synthetic adsorption resin are performed once, there is an effect of easily and quickly concentrating the extract of brown algae about 10 times, which can make the concentration process more efficient. it can.
また、前記フコキサンチン高含有褐藻類エキスを水と混合した後、静置することによりフコキサンチンを選択的に沈殿させ、フコキサンチンを精製、取得することができる。具体的にフコキサンチンを精製するには、前記フコキサンチン高含有褐藻類エキス１００容量部に５０〜１５０容量部程度の水を加え、低温（−１０〜２０℃程度）で１晩〜１週間程度、静置し、沈殿物を形成させれば良い。そしてこの沈殿物をろ過等により回収すればよい。このようにすることにより、純度が１０〜８０％程度のフコキサンチンを得ることができる。 In addition, after the fucoxanthin-rich brown algae extract is mixed with water, the fucoxanthin can be selectively precipitated by allowing to stand to purify and obtain fucoxanthin. Specifically, to purify fucoxanthin, about 50 to 150 parts by volume of water is added to 100 parts by volume of the above-mentioned fucoxanthin-rich brown algae extract, and the temperature is low (about -10 to 20 ° C.) overnight to about 1 week. It may be left standing to form a precipitate. And what is necessary is just to collect | recover this deposit by filtration. By doing so, fucoxanthin having a purity of about 10 to 80% can be obtained.
また、このフコキサンチンは、更に、液−液分配、再結晶およびクロマトグラフィーからなる群から選ばれる精製手段の１種以上に付してもよい。これらの精製手段を行うと、フコキサンチンの純度を９５％以上にすることができる。 Further, this fucoxanthin may be further applied to one or more purification means selected from the group consisting of liquid-liquid distribution, recrystallization, and chromatography. By performing these purification means, the purity of fucoxanthin can be increased to 95% or more.
具体的に液−液分配による精製は、フコキサンチンをメタノール、エタノール、アセトンなどの極性溶媒に溶解させ、それに更にヘキサンや石油エーテル類などの非極性溶媒を加え、フコキサンチンを液−液分配することにより行われる。また、これらの操作は複数回、好ましくは２〜５回繰り返すと効果的に精製することができる。 Specifically, the purification by liquid-liquid partitioning involves dissolving fucoxanthin in a polar solvent such as methanol, ethanol or acetone, and further adding a nonpolar solvent such as hexane or petroleum ether to liquid-liquid partition the fucoxanthin. Is done. These operations can be effectively purified by repeating a plurality of times, preferably 2 to 5 times.
また、再結晶による精製は、前記のようにして得られたフコキサンチンをメタノール、エタノール、アセトンなどの極性溶媒に溶解させ、それに適量、例えば、全量の２〜５割程度の水を加え、フコキサンチンを晶析（再結晶）させることにより行われる。これらの操作を複数回、好ましくは２〜５回繰り返すと効果的に精製することができる。 For purification by recrystallization, fucoxanthin obtained as described above is dissolved in a polar solvent such as methanol, ethanol, and acetone, and an appropriate amount, for example, about 20 to 50% of the total amount of water is added thereto. It is performed by crystallizing (recrystallizing) xanthine. It can be effectively purified by repeating these operations a plurality of times, preferably 2 to 5 times.
更に、クロマトグラフィーによる精製は、前記のようにして得られたフコキサンチンを高速液体クロマトグラフィー、中圧分取クロマトグラフィー等の各種クロマトグラフィーで処理し、フコキサンチンを分離することにより行われる。特にフコキサンチンを医薬品または研究用試薬として利用する場合、上記液−液分配および再結晶を行ったうえで、更に高速液体クロマトグラフィーで最終精製を行うことが好ましい。これを行うとフコキサンチンの純度を高められるのはもちろんのこと、シス体とトランス体の分離も可能となる。 Further, the purification by chromatography is carried out by treating the fucoxanthin obtained as described above with various chromatographies such as high performance liquid chromatography and medium pressure preparative chromatography to separate fucoxanthin. In particular, when fucoxanthin is used as a pharmaceutical or research reagent, it is preferable to perform final purification by high performance liquid chromatography after performing the liquid-liquid partition and recrystallization. When this is done, the purity of fucoxanthin can be increased, as well as separation of the cis form and the trans form.
斯くして得られるフコキサンチンは特に純度が高いので、食品（健康食品、添加物）、化粧品等だけでなく、医薬品、研究用試薬等に利用することができる。 Since the fucoxanthin thus obtained has a particularly high purity, it can be used not only for foods (health foods, additives), cosmetics, but also for pharmaceuticals, research reagents and the like.
以下に、実施例を挙げて本発明を更に説明するが、本発明はこれら実施例になんら制約されるものではない。 EXAMPLES The present invention will be further described below with reference to examples, but the present invention is not limited to these examples.
実 施 例 １
フコキサンチン高含有褐藻類エキスの製造：
（１）褐藻類エキスの製造
乾燥ワカメの５ｋｇをカッターミル ＭＫＣＭ−５（増幸産業製）および超微粒摩砕機
ＭＫＣＡ１０−１５Ｊ（増幸産業製）により粉砕し、粒度５０μｍのワカメ粉砕物とした。このワカメ粉砕物に１８Ｌのアセトン（１００％）を加え、室温で１時間撹拌して抽出を行った。その後、抽出液をろ過し、残渣について１８Ｌのアセトンで再度抽出を行った。これらの抽出液を混合し、褐藻類エキスとした。
Example 1
Production of fucoxanthin-rich brown algae extract:
(1) Production of brown algae extract 5 kg of dried seaweed was pulverized with a cutter mill MKCM-5 (manufactured by Masuko Sangyo) and an ultrafine grinding machine MKCA10-15J (manufactured by Masuko Sangyo) to obtain a crushed wakame having a particle size of 50 μm. 18 L of acetone (100%) was added to this ground seaweed and the mixture was stirred at room temperature for 1 hour for extraction. Thereafter, the extract was filtered, and the residue was extracted again with 18 L of acetone. These extracts were mixed to obtain a brown algae extract.
（２）フコキサンチンの吸着（１回目）
上記で得られた褐藻類エキスの３６Ｌに合成吸着樹脂であるダイヤイオンＨＰ２０（三菱化学製）を１.５Ｌ投入し、水３６Ｌを加えながら１時間撹拌し、ダイヤイオンＨＰ２０にフコキサンチンを吸着させた。撹拌後、ろ過により、フコキサンチンを吸着した合成吸着樹脂を回収した。
(2) Adsorption of fucoxanthin (first time)
To 36 L of the brown algae extract obtained above, 1.5 L of Diaion HP20 (Mitsubishi Chemical), which is a synthetic adsorption resin, is added and stirred for 1 hour while adding 36 L of water to adsorb fucoxanthin to Diaion HP20. It was. The synthetic adsorption resin which adsorb | sucked the fucoxanthin was collect | recovered by filtration after stirring.
（３）フコキサンチンの溶離（１回目）
上記で得られたフコキサンチンを吸着したダイヤイオンＨＰ２０の１.５Ｌをクロマト管に充填し、１００％のアセトン２Ｌを通液し、フコキサンチンをダイヤイオンＨＰ２０から溶離させ、フコキサンチンを含むアセトン溶出液を得た。
(3) Elution of fucoxanthin (first time)
Fill the chromatography tube with 1.5 L of Diaion HP20 adsorbing fucoxanthin obtained above, and pass 2 L of 100% acetone to elute fucoxanthin from Diaion HP20 and elute with acetone containing fucoxanthin. A liquid was obtained.
（４）フコキサンチンの吸着（２回目）
上記で得られたアセトン溶出液２ＬにダイヤイオンＨＰ２０を５００ｍＬ投入し、水１.８Ｌを加えながら３０分間撹拌し、ダイヤイオンＨＰ２０にフコキサンチンを吸着させた。撹拌後、ろ過によりフコキサンチンを吸着したダイヤイオンＨＰ２０を回収した。
(4) Adsorption of fucoxanthin (second time)
500 mL of Diaion HP20 was added to 2 L of the acetone eluate obtained above, and the mixture was stirred for 30 minutes while adding 1.8 L of water to adsorb fucoxanthin to Diaion HP20. After stirring, Diaion HP20 adsorbing fucoxanthin was collected by filtration.
（５）フコキサンチンの溶離（２回目）
上記で得られたフコキサンチンを吸着したダイヤイオンＨＰ２０の５００ｍＬをクロマト管に充填し、７０％アセトン水溶液２Ｌを通液し、フコキサンチンをダイヤイオンＨＰ２０から溶離させ、フコキサンチン高含有褐藻類エキス（フコキサンチンを０.１％程度含む）を得た。
(5) Elution of fucoxanthin (second time)
500 mL of Diaion HP20 adsorbing fucoxanthin obtained above was filled in a chromatographic tube, and 2 L of 70% acetone aqueous solution was passed through to elute fucoxanthin from Diaion HP20, and fucoxanthin-rich brown algae extract ( Containing about 0.1% fucoxanthin).
実 施 例 ２
フコキサンチンの粗精製：
上記実施例１で得られたフコキサンチン高含有褐藻類エキス（７０％アセトン溶出液）２Ｌに水１.９Ｌを加え、４℃で１８時間静置し、フコキサンチンを沈殿させた。この沈殿したフコキサンチンをろ過と遠心分離により回収した。回収したフコキサンチン沈殿物を適量のアセトンに再溶解し、適量の水を加えて再結晶させた（同操作を３回繰り返した）。乾燥ワカメ５ｋｇからフコキサンチンの粗精製物を１.３２ｇ（収率０.０２６％：純度約８０％）得た。
Example 2
Crude purification of fucoxanthin:
1.9 L of water was added to 2 L of the fucoxanthin-rich brown algae extract (70% acetone eluate) obtained in Example 1, and the mixture was allowed to stand at 4 ° C. for 18 hours to precipitate fucoxanthin. The precipitated fucoxanthin was recovered by filtration and centrifugation. The recovered fucoxanthin precipitate was redissolved in an appropriate amount of acetone and recrystallized by adding an appropriate amount of water (the same operation was repeated three times). From 5 kg of dried wakame, 1.32 g (yield 0.026%: purity about 80%) of a crude purified product of fucoxanthin was obtained.
実 施 例 ３
フコキサンチン高含有褐藻類エキスの製造およびフコキサンチンの粗精製：
乾燥ワカメの粒度を１００μｍとする以外は実施例１と同様にしてフコキサンチン高含有褐藻類エキス（フコキサンチンを０.１％程度含む）を得、更に前記エキスからフコキサンチンを実施例２と同様にして精製した。その結果、乾燥ワカメ５ｋｇからフコキサンチンの粗精製物を０.２７ｇ（収率０.００５４％：純度約８０％）得た。
Example 3
Production of fucoxanthin-rich brown algae extract and crude purification of fucoxanthin:
A fucoxanthin-rich brown algae extract (containing about 0.1% fucoxanthin) is obtained in the same manner as in Example 1 except that the particle size of the dried wakame is 100 μm, and fucoxanthin is further extracted from the extract as in Example 2. And purified. As a result, 0.27 g (yield 0.0054%: purity about 80%) of a crude purified product of fucoxanthin was obtained from 5 kg of dried wakame.
これらの結果より、抽出に用いるワカメの粒度が細かい方がフコキサンチンを効率よく抽出できることが判明した。 From these results, it was found that fucoxanthin can be efficiently extracted when the grain size of the seaweed used for extraction is finer.
実 施 例 ４
フコキサンチンの精製：
（１）液−液分配による精製
上記実施例１および２を繰り返して得たフコキサンチンの粗精製物３ｇをメタノール３５０ｍＬで溶解し、分液漏斗に入れた。さらに、これにヘキサン３００ｍＬと少量の水を加え、振とう、静置して分液した。その後、フコキサンチンを含むメタノール層（下層）とヘキサン層を分離し、更に、メタノール層に新たにヘキサンを加え、分配操作を繰り返し、３回の液−液分配を行った。
Example 4
Purification of fucoxanthin:
(1) Purification by liquid-liquid partition 3 g of a crude purified product of fucoxanthin obtained by repeating Examples 1 and 2 was dissolved in 350 mL of methanol and placed in a separatory funnel. Further, 300 mL of hexane and a small amount of water were added thereto, and the mixture was shaken and allowed to stand for separation. Thereafter, a methanol layer (lower layer) containing fucoxanthin and a hexane layer were separated, and hexane was newly added to the methanol layer, and the distribution operation was repeated, and liquid-liquid distribution was performed three times.
（２）再結晶による精製
上記液−液分配後のメタノール層に、少量ずつ水を加えていき、フコキサンチンを析出させ、ろ過により回収した。回収したフコキサンチンをアセトンに再溶解し、少量ずつ水を加えていき、再結晶させた。再結晶操作を３回ほど繰り返し、フコキサンチン精製物２．５７ｇ（収率８６％、純度９８％）を得た。なお、フコキサンチン精製物の純度は高速液体クロマトグラフィー（ＨＰＬＣ）、核磁気共鳴（ＮＭＲ）および質量分析（ＬＣ−ＡＰＣＩ−ＭＳ）により確認した（図１〜３）。
(2) Purification by recrystallization Water was added little by little to the methanol layer after the above liquid-liquid distribution to precipitate fucoxanthin and recovered by filtration. The recovered fucoxanthin was redissolved in acetone, and water was added little by little to recrystallize. The recrystallization operation was repeated about 3 times to obtain 2.57 g (yield 86%, purity 98%) of a fucoxanthin purified product. The purity of the fucoxanthin purified product was confirmed by high performance liquid chromatography (HPLC), nuclear magnetic resonance (NMR) and mass spectrometry (LC-APCI-MS) (FIGS. 1 to 3).
（３）ＨＰＬＣによる精製
上記再結晶後のフコキサンチン精製物をアセトンに溶解し、これを以下の条件のＨＰＬＣで精製した。これによりフコキサンチンのトランス体が３５分付近に溶出され、シス体と良好に分離できた。
＜ＨＰＬＣ条件＞
分取カラム：Ｃｏｓｍｏｓｉｌ（登録商標）ＯＤＳ ２０×２５０ｍｍ
（ナカライテスク製）
移動層：メタノール／水＝９０／１０
流速：５ｍＬ／ｍｉｎ
(3) Purification by HPLC The purified product of fucoxanthin after the recrystallization was dissolved in acetone and purified by HPLC under the following conditions. As a result, the trans form of fucoxanthin was eluted at around 35 minutes, and was successfully separated from the cis form.
<HPLC conditions>
Preparative column: Cosmosil (registered trademark) ODS 20 x 250 mm
(Nacalai Tesque)
Moving bed: methanol / water = 90/10
Flow rate: 5 mL / min
本発明方法によれば、抗酸化作用、脂肪燃焼作用、抗がん作用などを有するフコキサンチンを高濃度で含有するエキスやフコキサンチンの高純度精製品を褐藻類から低コストで得ることが可能となる。 According to the method of the present invention, it is possible to obtain an extract containing fucoxanthin having a high concentration, such as an antioxidant action, fat burning action, and anticancer action, and a highly purified product of fucoxanthin from brown algae at a low cost. It becomes.
Claims (7)
The method for purifying fucoxanthin according to claim 6, wherein the method further comprises one or more purification means selected from the group consisting of liquid-liquid distribution, recrystallization, and chromatography.
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