JP4734648B2 - Hyaluronidase inhibitor or therapeutic agent for atopic dermatitis derived from Okinawa mozuku - Google Patents
Hyaluronidase inhibitor or therapeutic agent for atopic dermatitis derived from Okinawa mozuku Download PDFInfo
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Description
本発明は、オキナワモズクから抽出したアセチルフコイダンを有効成分とするヒアルロニダーゼ阻害剤又はアトピー性皮膚炎治療剤に関する。 The present invention relates to a hyaluronidase inhibitor or a therapeutic agent for atopic dermatitis comprising acetylfucoidan extracted from Okinawa mozuku as an active ingredient.
沖縄地方は、わが国のなかで歴史的に特異な衣食住の文化を創造してきており、この特異性が沖縄地方に住む人の長寿の大きな要因になっていると考えられている。これらのうちでも食文化について沖縄独特なものが多く見られ、特にコンブやモズクなどの海藻類を多く利用している点が特徴的である。 The Okinawa region has historically created a unique culture of food, clothing and housing in Japan, and this specificity is considered to be a major factor in the longevity of people living in the Okinawa region. Among these, there are many unique Okinawan food cultures, especially because they use a lot of seaweeds such as kombu and mozuku.
近年になってこれらの海藻類に含まれているフコイダンやその他の硫酸化多糖がさまざまな生理活性を有することがわかり、有用な生物資源として注目され、種々の研究が行われている。このフコイダンは、モズク、コンブ、ワカメ、ヒジキ等に多く含まれている硫酸化多糖類の１種であり、Ｌ−フコースに加え、Ｄ−ガラクトース、Ｄ−キシロースおよびＤ−グルクロン酸を構成糖とし、更にエステル結合した硫酸基を持つ多糖類である。
これまでフコイダンには、抗コレステロ−ル作用、抗血液凝固作用等の効果が見出されており、健康補助食品の素材としてすでに利用開発され、市場で販売されている。
In recent years, it has been found that fucoidan and other sulfated polysaccharides contained in these seaweeds have various physiological activities, attracting attention as useful biological resources, and various studies have been conducted. This fucoidan is one of the sulfated polysaccharides abundantly contained in mozuku, kombu, wakame, hijiki, etc., and in addition to L-fucose, D-galactose, D-xylose and D-glucuronic acid are used as constituent sugars. Furthermore, it is a polysaccharide having a sulfate group bonded with an ester.
Up to now, fucoidan has been found to have an anti-cholesterol action, an anticoagulant action, etc., and has already been developed and marketed as a health supplement material.
本発明者らは、海藻類に含まれる多糖類に焦点をあてて研究を行い、沖縄に自生する海藻類から機能性多糖類を分離同定し、そのゲル化機構や腫瘍細胞に対するアポトーシス誘導効果を報告している（非特許文献１参照）。また、ナガコンブからのアルギン酸について、その化学特性を報告している（非特許文献２参照）。 The present inventors focused on polysaccharides contained in seaweeds, isolated and identified functional polysaccharides from seaweeds native to Okinawa, and demonstrated their gelation mechanism and apoptosis-inducing effect on tumor cells. Has been reported (see Non-Patent Document 1). In addition, chemical properties of alginic acid from Nagacomb have been reported (see Non-Patent Document 2).
更に、本発明者らは、オキナワモズク（Cladosiphon okamuranus TOKIDA）から分子量が５００,０００で、Ｌ−フコース、Ｄ−キシロース、Ｄ−グルクロン酸、酢酸および硫酸の構成比がそれぞれ３.０〜４.０、０.１〜０.３、０.８〜１.２、０.５〜１.０、０.８〜１.２であるアセチルフコイダンを見出し、既に特許を取得しており（特許文献１参照）、このアセチルフコイダンが優れた抗腫瘍活性を有することを見出し、特許出願を行っている（特許文献２参照）。 Furthermore, the present inventors have a molecular weight of 500,000 from Okinawa mozuku (Cladosiphon okamuranus TOKIDA), and the composition ratios of L-fucose, D-xylose, D-glucuronic acid, acetic acid and sulfuric acid are 3.0 to 4. We have found acetylfucoidan which is 0, 0.1-0.3, 0.8-1.2, 0.5-1.0, 0.8-1.2 and have already obtained a patent (Patent Literature) 1), this acetylfucoidan has been found to have excellent antitumor activity, and a patent application has been filed (see Patent Document 2).
本発明者らは、海藻類の種々の医薬品への利用の可能性を調べるため、このような海藻類からの抽出物、特にフコイダンについて、その生理活性を幅広く検討した。
即ち、本発明は、ある種の海藻類の抽出物からのフコイダンを有効成分とし、その生理活性を利用する種々の治療剤を提供することをその目的とするものである。 That is, an object of the present invention is to provide various therapeutic agents using fucoidan from an extract of a certain kind of seaweed as an active ingredient and utilizing its physiological activity.
本発明者らは、種々の海藻類の抽出物の生理活性について鋭意研究を行ったところ、オキナワモズクから希塩酸等により抽出して得られるアセチルフコイダンまたはその硫酸化物が優れた生理活性を示すことを見出し、本発明を完成した。 The present inventors conducted extensive research on the physiological activity of various seaweed extracts, and found that acetylfucoidan or its sulfate obtained by extraction from Okinawa mozuku with diluted hydrochloric acid or the like exhibits excellent physiological activity. The headline and the present invention were completed.
即ち、本発明は、オキナワモズク（Cladosiphon okamuranus TOKIDA）から得られたアセチルフコイダンまたはそのアセチルフコイダンを硫酸化することにより得られる硫酸化アセチルフコイダンを有効成分とすることを特徴とする、ヒアルロニダーゼ阻害剤又はアトピー性皮膚炎治療剤を提供するものである。 That is, the present invention provides a hyaluronidase inhibitor, characterized by comprising, as an active ingredient, acetyl fucoidan obtained from Okinawa mozuku (Cladosiphon okamuranus TOKIDA) or sulfated acetyl fucoidan obtained by sulfating the acetyl fucoidan. The present invention provides a therapeutic agent for atopic dermatitis.
また、本発明は、アセチルフコイダンが、オキナワモズクから希塩酸により抽出して得られたものであることを特徴とする、ヒアルロニダーゼ阻害剤又はアトピー性皮膚炎治療剤である。 The present invention also provides a hyaluronidase inhibitor or atopic dermatitis therapeutic agent characterized in that acetyl fucoidan is obtained by extraction from Okinawa mozuku with dilute hydrochloric acid.
また、本発明は、アセチルフコイダンの硫酸化が、Ｎ,Ｎ’−ジメチルホルムアミドおよび三酸化イオウ−トリメチルアミンを用いて行われるものであることを特徴とする、ヒアルロニダーゼ阻害剤又はアトピー性皮膚炎治療剤を提供するものである。 The present invention also provides a hyaluronidase inhibitor or a therapeutic agent for atopic dermatitis, characterized in that sulfation of acetyl fucoidan is performed using N, N′-dimethylformamide and sulfur trioxide-trimethylamine. Is to provide.
更に、本発明は、上記ヒアルロニダーゼ阻害剤又はアトピー性皮膚炎治療剤を含有することを特徴とする飲食品を提供するものである。 Furthermore, this invention provides the food-drinks characterized by containing the said hyaluronidase inhibitor or the atopic dermatitis therapeutic agent.
本発明のオキナワモズクから得られたアセチルフコイダンまたはその硫酸化物を有効成分とするヒアルロニダーゼ阻害剤又はアトピー性皮膚炎治療剤は、優れたヒアルロニダーゼ阻害活性を示し、アトピー性皮膚炎の症状を和らげ、アトピー性皮膚炎に対して優れた治療効果を示す。 The hyaluronidase inhibitor or therapeutic agent for atopic dermatitis comprising acetylfucoidan obtained from Okinawa mozuku of the present invention or its sulfate as an active ingredient exhibits excellent hyaluronidase inhibitory activity, relieves the symptoms of atopic dermatitis, Excellent therapeutic effect against atopic dermatitis.
従って、本発明のオキナワモズクから得られたアセチルフコイダンを有効成分とするヒアルロニダーゼ阻害剤及びアトピー性皮膚炎治療剤は優れたヒアルロニダーゼ阻害活性を示すので、その作用を利用して抗アレルギー剤やアトピー性皮膚炎の治療等に用いることができるとともに、日常的にこれらの治療剤を含む飲食品としてこれを摂取することにより、アトピー性皮膚炎等のアレルギー性疾患の発生を抑制することもできる。 Therefore, since the hyaluronidase inhibitor and the atopic dermatitis therapeutic agent containing the acetylfucoidan obtained from the Okinawa mozuku of the present invention as an active ingredient exhibit excellent hyaluronidase inhibitory activity, anti-allergic agents and atopic properties are utilized by utilizing the action. In addition to being able to be used for the treatment of dermatitis and the like, it is possible to suppress the occurrence of allergic diseases such as atopic dermatitis by daily ingestion as foods and drinks containing these therapeutic agents.
本発明のヒアルロニダーゼ阻害剤又はアトピー性皮膚炎治療剤は、オキナワモズク由来のアセチルフコイダンまたはこのアセチルフコイダンを硫酸化することにより得られる硫酸化アセチルフコイダンを有効成分とするものである。 The hyaluronidase inhibitor or the therapeutic agent for atopic dermatitis according to the present invention comprises, as an active ingredient, acetylfucoidan derived from Okinawa mozuku or sulfated acetylfucoidan obtained by sulfating this acetylfucoidan.
原料となるオキナワモズク（Cladosiphon okamuranus TOKIDA）は、沖縄地方に生育する天然のオキナワモズクのほかに、人工的に養殖される養殖オキナワモズクを使用することができる。 The Okinawa Mozuku (Cladosiphon okamuranus TOKIDA), which is a raw material, can be used in addition to natural Okinawa Mozuku grown in the Okinawa region, as well as artificially cultivated Okinawa Mozuku.
本発明のヒアルロニダーゼ阻害剤又はアトピー性皮膚炎治療剤は、このオキナワモズクを希塩酸水溶液等の酸性溶液により抽出して得られる多糖の一種であるアセチルフコイダンまたはこのアセチルフコイダンを硫酸化することにより得られる硫酸化アセチルフコイダンを有効成分とするものである。 The hyaluronidase inhibitor or the therapeutic agent for atopic dermatitis of the present invention is obtained by sulfating acetylfucoidan, which is a kind of polysaccharide obtained by extracting this Okinawa mozuku with an acidic solution such as dilute hydrochloric acid aqueous solution, or this acetylfucoidan. Sulfated acetyl fucoidan is an active ingredient.
本発明のヒアルロニダーゼ阻害剤又はアトピー性皮膚炎治療剤の有効成分であるオキナワモズク由来のアセチルフコイダンは、酢酸基を含むフコイダンであり、概ね酢酸基を３.９〜８.０質量％含有するものである。また、このアセチルフコイダンの組成の一例を挙げれば、概ねＬ−フコース、Ｄ−キシロース、Ｄ−グルクロン酸、酢酸および硫酸の構成比がそれぞれ３.０〜４.０、０.１〜０.３、０.８〜１.２、０.５〜１.０および０.８〜１.２のものである。このオキナワモズクからアセチルフコイダンを得る方法は特には限定されないが、例えば、本発明者らの特許３３７１１２４号に記載の方法が挙げられる。また、このアセチルフコイダンは陽イオン交換樹脂等により陽イオン交換されたものであっても良い。 The acetyl fucoidan derived from Okinawa Mozuku, which is an active ingredient of the hyaluronidase inhibitor or the atopic dermatitis therapeutic agent of the present invention, is a fucoidan containing an acetate group, and generally contains 3.9 to 8.0% by mass of an acetate group. It is. Moreover, if an example of a composition of this acetyl fucoidan is given, the composition ratios of L-fucose , D-xylose, D-glucuronic acid, acetic acid and sulfuric acid are approximately 3.0 to 4.0, 0.1 to 0.3, respectively. 0.8-1.2, 0.5-1.0 and 0.8-1.2. The method for obtaining acetylfucoidan from this Okinawa mozuku is not particularly limited, and examples thereof include the method described in the inventors' patent 3371124. The acetyl fucoidan may be cation exchanged with a cation exchange resin or the like.
具体的に、オキナワモズクからアセチルフコイダンを得る方法を示せば次の通りである。まず、オキナワモズクの乾燥体を粉砕した後、塩酸等の酸を加えて攪拌する。次に、これを濾過し、ろ液を水酸化ナトリウム等のアルカリで中和した後、セライト５４５層（ナカライテスク製）等のカラムに通して、濃縮を行い、更にエタノールを添加し、沈殿させる。この沈殿物をエタノール等のアルコール脱水を数回行い、更に減圧乾燥を行い粗アセチルフコイダンを得る。この粗アセチルフコイダンを塩化カルシウム等の塩水溶液に溶解し、ろ過後、ろ液をセライト５４５層等のカラムに通して一晩透析を行い、さらに凍結乾燥を行ってアセチルフコイダンを得ることができる。また、このアセチルフコイダンをアンバーライトＩＲ−１２０（Rohm & Haas社製）等の陽イオン交換樹脂に通し、陽イオン交換を行った後、水酸化ナトリウム等のアルカリで中和、透析、凍結乾燥を行い、Ｎａ型のアセチルフコイダンを得ることができる。 Specifically, a method for obtaining acetyl fucoidan from Okinawa Mozuku is as follows. First, after pulverizing the dry body of Okinawa Mozuku, an acid such as hydrochloric acid is added and stirred. Next, this is filtered, and the filtrate is neutralized with an alkali such as sodium hydroxide, then passed through a column such as Celite 545 layer (manufactured by Nacalai Tesque) and concentrated, and further ethanol is added and precipitated. . The precipitate is subjected to alcohol dehydration such as ethanol several times and further dried under reduced pressure to obtain crude acetylfucoidan. This crude acetylfucoidan is dissolved in an aqueous salt solution such as calcium chloride, and after filtration, the filtrate is dialyzed overnight through a column such as Celite 545 layer and further freeze-dried to obtain acetylfucoidan. In addition, this acetyl fucoidan is passed through a cation exchange resin such as Amberlite IR-120 (Rohm & Haas), and after cation exchange, neutralization, dialysis and lyophilization with an alkali such as sodium hydroxide are performed. And Na-type acetylfucoidan can be obtained.
また、本発明のヒアルロニダーゼ阻害剤又はアトピー性皮膚炎治療剤のもう一つの有効成分である硫酸化アセチルフコイダンは、上記オキナワモズク由来のアセチルフコイダンを硫酸化することにより得ることができるものであり、概ね硫酸含量が１３〜３３質量％、好ましくは１５〜３３質量％含有するものである。また、この硫酸化アセチルフコイダンの組成の一例を挙げれば、概ねＬ−フコース、Ｄ−キシロース、Ｄ−グルクロン酸、酢酸および硫酸の構成比がそれぞれ３.０〜４.０、０.１〜０.３、０.８〜１.３、０.５〜１.０および０.８〜３.６である。 The sulfated acetyl fucoidan, which is another active ingredient of the hyaluronidase inhibitor or the atopic dermatitis therapeutic agent of the present invention, can be obtained by sulfating the above-mentioned Okinawa mozuku-derived acetyl fucoidan, The sulfuric acid content is generally 13 to 33% by mass, preferably 15 to 33% by mass. Moreover, if an example of a composition of this sulfated acetyl fucoidan is given, the composition ratios of L-fucose , D-xylose, D-glucuronic acid, acetic acid and sulfuric acid are approximately 3.0 to 4.0 and 0.1 to 0, respectively. .3, 0.8-1.3, 0.5-1.0 and 0.8-3.6.
この硫酸化アセチルフコイダンを得るための硫酸化の方法としては、多糖の水酸基をスルホン化可能な公知の方法であれば特に限定されないが、例えば、クロロスルホン酸−ピリジン錯体、ジシクロヘキシルカルノジイミド−硫酸、三酸化硫黄−トリメチルアミン錯体、Ｎ,Ｎ’−ジメチルホルムアミド−三酸化イオウ−トリメチルアミン等を用いる方法等が挙げられる。これらの方法の中でも、アセチルフコイダンにＮ,Ｎ’−ジメチルホルムアミド中、三酸化イオウ−トリメチルアミンを用いる方法が好ましい。 The sulfation method for obtaining this sulfated acetylfucoidan is not particularly limited as long as it is a known method capable of sulfonating a hydroxyl group of a polysaccharide. For example, chlorosulfonic acid-pyridine complex, dicyclohexylcarnodiimide-sulfuric acid, Examples thereof include a method using sulfur trioxide-trimethylamine complex, N, N′-dimethylformamide-sulfur trioxide-trimethylamine and the like. Among these methods, a method using sulfur trioxide-trimethylamine in N, N′-dimethylformamide for acetylfucoidan is preferable.
具体的にアセチルフコイダンに三酸化イオウ−トリメチルアミンを作用させて硫酸化アセチルフコイダンを得る方法の例を示せば次の通りである。まず、アセチルフコイダンをＮ,Ｎ’−ジメチルホルムアミド等の溶媒に分散させ、これに三酸化イオウ−トリメチルアミンを加えて静置する。静置後、酢酸ナトリウム等の酢酸塩を加えてエタノールで沈殿させる。この沈殿物をエタノールで洗浄後、蒸留水に溶解し、アンバーライトＩＲ−１２０（Rohm & Haas社製）等の陽イオン交換樹脂を充填したカラムを通して脱塩する。最後に水酸化ナトリウム等のアルカリ溶液で中和を行い、透析後、凍結乾燥することにより硫酸化アセチルフコイダンを得ることができる。 Specifically, an example of a method of obtaining sulfated acetyl fucoidan by allowing sulfur trioxide-trimethylamine to act on acetyl fucoidan is as follows. First, acetyl fucoidan is dispersed in a solvent such as N, N'-dimethylformamide, and sulfur trioxide-trimethylamine is added thereto and allowed to stand. After standing, an acetate such as sodium acetate is added and precipitated with ethanol. This precipitate is washed with ethanol, dissolved in distilled water, and desalted through a column filled with a cation exchange resin such as Amberlite IR-120 (Rohm & Haas). Finally, it is neutralized with an alkali solution such as sodium hydroxide, and after dialysis, freeze-dried to obtain sulfated acetylfucoidan.
本発明のヒアルロニダーゼ阻害剤又はアトピー性皮膚炎治療剤は、オキナワモズクから得られるアセチルフコイダンまたはその硫酸化物をそのままあるいは公知の適当な医薬担体と組み合わせ、製剤化することにより製造することができる。本発明のヒアルロニダーゼ阻害剤又はアトピー性皮膚炎治療剤におけるオキナワモズクから得られるフコイダンまたはその硫酸化物の含有量は、ヒアルロニダーゼ阻害活性やアレルギー抑制効果が発揮される量であれば特に制限されないが、例えばオキナワモズクから得られるフコイダンまたはその硫酸化物が１３〜３３質量％、好ましくは１５〜３３質量％である。 The hyaluronidase inhibitor or the therapeutic agent for atopic dermatitis of the present invention can be produced by formulating acetylfucoidan obtained from Okinawa mozuku or a sulfate thereof as it is or in combination with a known appropriate pharmaceutical carrier. The content of fucoidan obtained from Okinawa mozuku in the hyaluronidase inhibitor or the atopic dermatitis therapeutic agent of the present invention or the sulfate thereof is not particularly limited as long as the hyaluronidase inhibitory activity and the allergy suppressing effect are exhibited. Fucoidan obtained from Okinawa Mozuku or its sulfate is 13 to 33% by mass, preferably 15 to 33% by mass.
以上のように、オキナワモズクから得られるアセチルフコイダンまたはその硫酸化物は、ヒアルロニダーゼ阻害活性を有するものであり、このような硫酸化多糖が食物因子としてアレルギー抑制作用を有する。従って、本発明のヒアルロニダーゼ阻害剤又はアトピー性皮膚炎治療剤は、通常の飲食品に含有せしめ、日常的に摂取することができる。このような飲食品の製造方法は、本発明のヒアルロニダーゼ阻害剤又はアトピー性皮膚炎治療剤を飲食品の原料素材等に有効量添加すればよく、それ以外の点は、通常の飲食品の製造方法に準じればよい。 As described above, acetylfucoidan obtained from Okinawa mozuku or its sulfate has hyaluronidase inhibitory activity, and such sulfated polysaccharide has an allergy suppressing action as a food factor. Therefore, the hyaluronidase inhibitor or the atopic dermatitis therapeutic agent of the present invention can be contained in ordinary foods and drinks and taken daily. Such a method for producing a food or drink may be prepared by adding an effective amount of the hyaluronidase inhibitor or the atopic dermatitis therapeutic agent of the present invention to a raw material for food or drink, etc. What is necessary is just to follow the method.
これら飲食品の例としては茶飲料、栄養補助飲料等の飲料、カプセル状、錠剤状、粉末状等の栄養補助食品等の食品が挙げられる。 Examples of these foods and drinks include beverages such as tea beverages and nutritional supplements, and foods such as dietary supplements such as capsules, tablets, and powders.
次に、本発明を実施例によって更に詳しく説明するが、本発明はこれらの実施例に限定されるものではない。また、実施例中の「％」及び「部」は、いずれも特に注記しない限り質量基準である。 EXAMPLES Next, although an Example demonstrates this invention further in detail, this invention is not limited to these Examples. Further, “%” and “part” in the examples are based on mass unless otherwise noted.
（１）アセチルフコイダンの製造：
沖縄県知念漁業共同組合で収穫され、冷凍保存されていた養殖オキナワモズクを解凍して用いた。この藻体１００ｇをミキサーで５分間粉砕した。この粉砕物に全量が６００ｍＬとなるように０.０５Ｍの塩酸を加え、室温で４時間撹拌した。次にこれを８０００ｒｐｍで遠心分離し、上清を吸引ろ過し、ろ液を０.０５Ｍの水酸化ナトリウムで中和した。次いで、セライト５４５層に通して、濃縮を行い、さらに２倍量のエタノールを添加し、沈殿させた。沈殿物をエタノール脱水を数回行い、更に減圧乾燥を行い粗アセチルフコイダンを得た。この粗アセチルフコイダンを０.１Ｍの塩化カルシウム水溶液に溶解し、吸引ろ過後、ろ液をセライト５４５層に通して一晩透析、凍結乾燥を行って精製アセチルフコイダンを得た。
(1) Production of acetyl fucoidan :
The farmed Okinawa Mozuku harvested by the Chinen Fishery Cooperative Association in Okinawa and stored frozen was thawed and used. 100 g of this algal body was pulverized with a mixer for 5 minutes. To this pulverized product, 0.05 M hydrochloric acid was added so that the total amount became 600 mL, and the mixture was stirred at room temperature for 4 hours. Next, this was centrifuged at 8000 rpm, the supernatant was suction filtered, and the filtrate was neutralized with 0.05 M sodium hydroxide. Subsequently, the mixture was passed through Celite 545 layer, concentrated, and further doubled amount of ethanol was added for precipitation. The precipitate was dehydrated with ethanol several times and further dried under reduced pressure to obtain crude acetylfucoidan. This crude acetylfucoidan was dissolved in a 0.1 M calcium chloride aqueous solution, and after suction filtration, the filtrate was passed through Celite 545 layer, dialyzed overnight and freeze-dried to obtain purified acetylfucoidan.
この精製アセチルフコイダンを陽イオン交換樹脂（アンバーライトＩＲ−１２０）に通し、陽イオン交換を行った後、トリエチルアミンによりｐＨ５．５〜６．０に調整し、凍結乾燥を行い、ＴＥＡ型の精製アセチルフコイダン（以下、単に「アセチルフコイダン」または「ネイティブアセチルフコイダン」という）を得た。 This purified acetyl fucoidan was passed through a cation exchange resin (Amberlite IR-120), and after cation exchange, the pH was adjusted to 5.5 to 6.0 with triethylamine, freeze-dried, and TEA-type purified acetyl. Fucoidan (hereinafter simply referred to as “acetyl fucoidan” or “native acetyl fucoidan”) was obtained.
上記で得られたアセチルフコイダンの組成はＬ−フコース、Ｄ−キシロース、Ｄ−グルクロン酸、酢酸の構成比がそれぞれ４.０、０.２、１.１および０.８であった。また、バリウム沈殿法によりアセチルフコイダンの硫酸含量を測定したところ１３.２質量％であった。 The composition of acetylfucoidan obtained above was such that the composition ratios of L-fucose , D-xylose, D-glucuronic acid, and acetic acid were 4.0, 0.2, 1.1, and 0.8, respectively. The sulfuric acid content of acetylfucoidan was measured by the barium precipitation method and found to be 13.2% by mass.
（２）硫酸化アセチルフコイダンの製造：
上記（１）で製造したＴＥＡ型アセチルフコイダン２００ｍｇをＮ,Ｎ’−ジメチルホルムアミド２０ｍｌに溶解させ、硫酸基供与体である三酸化イオウピリジン錯体（４．５ｇ／４０ｍＬＤＭＦ）を２０ｍＬ加えて、５０℃で６時間反応させた。反応後、等量の冷水を加えて反応をとめて中和し、陽イオン交換樹脂を通して陽イオン交換を行い、透析、凍結乾燥を行って、硫酸化アセチルフコイダンを得た。
(2) Production of sulfated acetylfucoidan :
200 mg of TEA-type acetylfucoidan produced in (1) above was dissolved in 20 ml of N, N′-dimethylformamide, 20 ml of sulfur trioxide pyridine complex (4.5 g / 40 ml DMF) as a sulfate group donor was added, and 50 ° C. For 6 hours. After the reaction, the reaction was neutralized by adding an equal amount of cold water, cation exchange was performed through a cation exchange resin, dialysis and lyophilization were carried out to obtain sulfated acetylfucoidan.
上記で得られた硫酸化アセチルフコイダンの組成は、Ｌ−フコース、Ｄ−キシロース、Ｄ−グルクロン酸および酢酸の構成比がそれぞれ４.０、０.２、１.１および０.８であった。また、この硫酸化アセチルフコイダンの硫酸含量をバリウム沈殿法により測定したところ、３２.２質量％であった。 The composition of sulfated acetyl fucoidan obtained above was such that the composition ratios of L-fucose , D-xylose, D-glucuronic acid and acetic acid were 4.0, 0.2, 1.1 and 0.8, respectively. . Further, the sulfuric acid content of this sulfated acetylfucoidan was measured by a barium precipitation method and found to be 32.2% by mass.
（３）脱硫酸化アセチルフコイダンの製造：
上記（１）で製造したＴＥＡ型アセチルフコイダンを陽イオン交換樹脂を通して陽イオン交換を行い、ピリジンによりｐＨ７．０に調整してから濃縮し、２４時間透析を行い、凍結乾燥してピリジン型フコイダン（Ｐｙ型フコイダン）とした。Ｐｙ型フコイダンを、メタノールを含むジメチルスルフォキシドに溶解させ、８０℃で４時間反応させ、その後３０時間透析を行い、陽イオン交換樹脂を通して陽イオン交換を行い、中和、濃縮、凍結乾燥を行って、脱硫酸化アセチルフコイダンを得た。
(3) Production of desulfated acetylfucoidan :
The TEA-type acetylfucoidan produced in (1) above is subjected to cation exchange through a cation exchange resin, adjusted to pH 7.0 with pyridine, concentrated, dialyzed for 24 hours, freeze-dried, and pyridine-type fucoidan ( Py type fucoidan). Py-type fucoidan is dissolved in dimethyl sulfoxide containing methanol and reacted at 80 ° C. for 4 hours, followed by dialysis for 30 hours, cation exchange through a cation exchange resin, neutralization, concentration, and freeze-drying. To obtain a desulfated acetylfucoidan.
上記で得られた脱硫酸化アセチルフコイダンの組成は、Ｌ−フコース、Ｄ−キシロース、Ｄ−グルクロン酸および酢酸の構成比がそれぞれ４．０、０．３、１．５および０．５であった。また、この脱硫酸化アセチルフコイダンの硫酸含量は０．０６質量％であった。 The composition of the desulfated acetylfucoidan obtained above was such that the composition ratios of L-fucose , D-xylose, D-glucuronic acid and acetic acid were 4.0, 0.3, 1.5 and 0.5, respectively. . The sulfuric acid content of this desulfated acetylfucoidan was 0.06% by mass.
以上の３種類のアセチルフコイダンの組成及び硫酸含量をまとめると、次の表１に示すとおりである。 The composition and sulfuric acid content of the above three types of acetylfucoidan are summarized as shown in Table 1 below.
（４）ヒアルロニダーゼ阻害活性の評価
上記（１）〜（３）で得られた３種類のアセチルフコイダンについて、ヒアルロニダーゼ阻害活性を測定した。
ヒアルロニダーゼ０．１ｍＬ（３ｍｇ／ｍＬ）に、濃度の異なるネイティブアセチルフコイダン、硫酸化アセチルフコイダン、および脱硫酸化アセチルフコイダンをそれぞれ０．１ｍＬ加え、室温で２０分間インキュベートした。ヒアルロニダーゼは牛の精巣から分離したもの（シグマ社）を使用した。次に、この溶液にヒアルロニダーゼ活性化因子として１．５ＭＮａＣｌ０．１ｍＬ、及び基質であるヒトのヘソ由来のヒアルロン酸（MP Biomedicals）０．７ｍＬを加えて、３７℃で４０分間反応させ、ヒアルロニダーゼを失活させるために１００度に加熱した後、Mogan-Elson法により定量した。
(4) Evaluation of hyaluronidase inhibitory activity Hyaluronidase inhibitory activity was measured for the three types of acetylfucoidan obtained in (1) to (3) above.
To 0.1 mL of hyaluronidase (3 mg / mL), 0.1 mL each of native acetyl fucoidan, sulfated acetyl fucoidan and desulfated acetyl fucoidan having different concentrations was added and incubated at room temperature for 20 minutes. Hyaluronidase used was isolated from the testes of the cow (Sigma). Next, 0.1 mL of 1.5 M NaCl as a hyaluronidase activator and 0.7 mL of hyaluronic acid (MP Biomedicals) as a substrate are added to this solution and reacted at 37 ° C. for 40 minutes to lose hyaluronidase. After heating to 100 degrees for activation, the amount was determined by the Mogan-Elson method.
各試料について、ヒアルロニダーゼを失活させた溶液に、ｐ−ジメチルアミノベンズアルデヒドを溶解させた濃硫酸を１０倍希釈したものを加え、３７℃で２０分間インキュベートした後、この溶液を波長５８５ｎｍで吸光度を測定して、次式により阻害率を求めた。 For each sample, 10-fold diluted concentrated sulfuric acid in which p-dimethylaminobenzaldehyde was dissolved was added to a solution in which hyaluronidase was inactivated, and incubated at 37 ° C. for 20 minutes. Then, the absorbance of this solution was measured at a wavelength of 585 nm. The inhibition rate was determined by the following equation.
各試料についての種々の希釈濃度での阻害率を図１に示す。比較のために既にヒアルロニダーゼ阻害活性が見出されているヘパリン（Celsus Laboratory）を陽性対照として同様にして阻害試験を行った。その結果も併せて図１に示す。
この結果から、ネイティブアセチルフコイダンは阻害活性を示し、２５μｇ／ｍＬで阻害率は約８０％であった。硫酸化アセチルフコイダンは更に高い活性を示し、２５μｇ／ｍＬでほぼ１００％の阻害率であり、その半分の濃度である１２．５μｇ／ｍＬでも約８０％の阻害率であり、ヘパリンよりも高い活性が認められた。一方、脱硫酸化アセチルフコイダンはほとんど阻害活性が認められなかった。
The inhibition rates at various dilution concentrations for each sample are shown in FIG. For comparison, a heparin (Celsus Laboratory), for which hyaluronidase inhibitory activity has already been found, was used as a positive control to carry out an inhibition test in the same manner. The results are also shown in FIG.
From this result, native acetylfucoidan showed inhibitory activity, and the inhibition rate was about 80% at 25 μg / mL. Sulfated acetylfucoidan shows higher activity, almost 100% inhibition at 25 μg / mL, and about 80% inhibition at 12.5 μg / mL, which is half that concentration, higher activity than heparin Was recognized. On the other hand, desulfated acetylfucoidan showed almost no inhibitory activity.
次に、図１の結果から５０％阻害濃度（ＩＣ５０）を求め、その結果を図２に示す。ネイティブアセチルフコイダンのＩＣ５０は２５．６μｇ／ｍＬ、硫酸化アセチルフコイダンのＩＣ５０は３．６μｇ／ｍＬ、ヘパリンのＩＣ５０は８．９μｇ／ｍＬであった。脱硫酸化アセチルフコイダンのＩＣ５０は算出することができなかった。 Next, a 50% inhibitory concentration (IC 50 ) was determined from the results of FIG. 1, and the results are shown in FIG. IC 50 of native acetylfucoidan is 25.6μg / mL, IC 50 of sulfation acetylfucoidan is 3.6 g / mL, IC 50 of heparin was 8.9μg / mL. The IC 50 for desulfated acetylfucoidan could not be calculated.
本発明は、優れたヒアルロニダーゼ阻害活性を有するヒアルロニダーゼ阻害剤又はアトピー性皮膚炎治療剤として有用である。 更に、本発明はヒアルロニダーゼ阻害剤又はアトピー性皮膚炎治療剤であるとともに、これを各種飲食品に配合することにより優れた健康食品とすることができ、日常の飲食品として摂取することによって種々のアトピー性皮膚炎を防止することが期待できる。 The present invention is useful as a hyaluronidase inhibitor or an atopic dermatitis therapeutic agent having excellent hyaluronidase inhibitory activity. Furthermore, the present invention is a hyaluronidase inhibitor or an atopic dermatitis therapeutic agent, and can be made into an excellent health food by blending it into various foods and drinks. It can be expected to prevent atopic dermatitis.
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