JPH06256208A - Immunopotentiator - Google Patents
ImmunopotentiatorInfo
- Publication number
- JPH06256208A JPH06256208A JP5067479A JP6747993A JPH06256208A JP H06256208 A JPH06256208 A JP H06256208A JP 5067479 A JP5067479 A JP 5067479A JP 6747993 A JP6747993 A JP 6747993A JP H06256208 A JPH06256208 A JP H06256208A
- Authority
- JP
- Japan
- Prior art keywords
- acidic
- solution
- acidic polysaccharide
- agarase
- viscosity
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
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- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
- Medicines Containing Plant Substances (AREA)
- Polysaccharides And Polysaccharide Derivatives (AREA)
Abstract
Description
【０００１】[0001]
【産業上の利用分野】本発明は紅藻類から得られる免疫
賦活性酸性多糖を酵素処理したものを有効成分とするよ
り実用性に富んだ形態の免疫賦活剤に関する。BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a more practical form of an immunostimulant containing an enzyme-treated immunostimulatory acidic polysaccharide obtained from red algae as an active ingredient.
【０００２】[0002]
【従来の技術】海藻類は日本では古くから食用として、
また民間薬として用いられてきた。近年、その生理活性
に注目した研究開発が行われるようになってきた。例え
ば、特開昭63-316732 号公報には紅藻類由来の抗ウィル
ス剤が、また特開昭64-66126号公報には紅藻類由来の抗
腫瘍剤が、それぞれ開示されている。さらに、特開平3-
284626号公報及び特願平3-329870号（未公開）には、い
ずれも紅藻類であるアマノリ属及びオゴノリ属の海藻か
ら水性溶媒で抽出される物質を有効成分とする免疫賦活
剤がそれぞれ開示されている。オゴノリ属やアマノリ属
に属する海藻から抽出される物質は、主としてアガロー
スを基本骨格に持つ高分子の酸性多糖を主成分とするも
のであることが知られている。これらの酸性多糖は粘性
が高く、時によっては強いゲルを形成するため、これら
の免疫賦活作用を利用して、ヒト、動物等の免疫機能を
高めるために、食品や飼料に混合したり、これ自体を経
口、非経口投与に適するような剤形に製剤することは極
めて困難である。しかも製造工程での取扱いも難しく、
操作が煩雑となるなどの大きな問題点があった。2. Description of the Related Art Seaweeds have long been eaten in Japan.
It has also been used as a folk medicine. In recent years, research and development focusing on its physiological activity has been conducted. For example, JP-A-63-316732 discloses an antiviral agent derived from red algae, and JP-A-64-66126 discloses an antitumor agent derived from red algae. Furthermore, JP-A-3-
Japanese Patent Application No. 284626 and Japanese Patent Application No. 3-329870 (unpublished) each disclose an immunostimulant containing as an active ingredient a substance extracted with an aqueous solvent from seaweeds of the genus Amanori and the genus Ogonori. Has been done. It is known that substances extracted from seaweeds belonging to the genus Ogonori and the genus Anoly are mainly composed of polymeric acidic polysaccharides having agarose as a basic skeleton. These acidic polysaccharides are highly viscous and sometimes form a strong gel.Therefore, in order to enhance the immune function of humans, animals, etc. by utilizing their immunostimulatory action, they are mixed in foods or feeds, or It is extremely difficult to formulate itself into a dosage form suitable for oral and parenteral administration. Moreover, handling in the manufacturing process is difficult,
There was a big problem that the operation became complicated.
【０００３】かかる酸性多糖を加水分解する酵素とし
て、シュードモナス・アトランティカ（Pseudoomonus
atlantica)、サイトファーガ・エスピー（Cytophaga s
p.) 、ビブリオ・エスピー AP-2(Vibrio sp. AP-2)等
が、該酸性多糖中のβ-1,4結合を加水分解する能力を有
するβ−アガラーゼを生産することが報告されている
（Biochem., 105, 317-321(1967)、Chin.J.Oceanol.Lim
nol., 8 (2)135-149(1990)、Eur.J.Biochem.,133,673-6
84(1983)、Eur.J.Biochem., 187, 461-465(1990)等) 。
また、β−アガラーゼを利用して海藻類からオリゴ糖を
製造する方法が、特開平2-65788 号、特開平4-271791号
公報等において提案されている。他方、希酸による加水
分解では該酸性多糖中のα-1,3結合が比較的選択的に切
断される。しかし、酸による加水分解はこれら多糖に結
合する硫酸基をも遊離させ、構成糖の基本構造をも変化
させてしまう。As an enzyme that hydrolyzes such acidic polysaccharides, Pseudoomonus
atlantica) , Cytophaga s
p.), Vibrio sp. AP-2 ( Vibrio sp. AP-2) and the like have been reported to produce β-agarase having the ability to hydrolyze β-1,4 bond in the acidic polysaccharide. (Biochem., 105, 317-321 (1967), Chin.J.Oceanol.Lim
nol., 8 (2) 135-149 (1990), Eur.J.Biochem., 133,673-6
84 (1983), Eur. J. Biochem., 187, 461-465 (1990), etc.).
In addition, methods for producing oligosaccharides from seaweeds using β-agarase have been proposed in JP-A-2-65788 and JP-A-4-271791. On the other hand, hydrolysis with a dilute acid relatively selectively cleaves the α-1,3 bond in the acidic polysaccharide. However, acid hydrolysis also liberates the sulfate groups that bind to these polysaccharides and changes the basic structure of the constituent sugars.
【０００４】[0004]
【発明が解決しようとする課題】本発明者らは、かかる
免疫賦活活性を有する、紅藻類起源のアガロースを基本
骨格とする酸性多糖について、その粘度及びゲル形成能
を低下させてヒトや動物等への投与に適した製剤を得る
目的で鋭意研究を続けた。その結果、該酸性多糖類を加
水分解する能力を有するβ−アガラーゼを該酸性多糖に
作用させ、これを加水分解して得られる生成物が該酸性
多糖に比しはるかに低い粘性を有し、かつより優れた免
疫賦活活性を有することを見い出し、本発明を完成し
た。DISCLOSURE OF THE INVENTION The inventors of the present invention have reduced the viscosity and gel-forming ability of acidic polysaccharides having agarose derived from red algae as a basic skeleton, which have such immunostimulatory activity, thereby reducing the viscosity of human and animals. The study was continued with the aim of obtaining a formulation suitable for administration to the. As a result, β-agarase having the ability to hydrolyze the acidic polysaccharide is allowed to act on the acidic polysaccharide, and the product obtained by hydrolyzing this has a much lower viscosity than the acidic polysaccharide, Moreover, they have found that they have more excellent immunostimulatory activity and completed the present invention.
【０００５】[0005]
【課題を解決するための手段】本発明は紅藻類に属する
海藻から酸性多糖を水性溶媒で抽出し、固液分離して得
られる抽出液に該酸性多糖を加水分解する能力を有する
β−アガラーゼを作用させて酸性多糖を低粘性化して得
られる溶液、もしくは該海藻を該β−アガラーゼを含有
する水性溶媒を接触させて酸性多糖の抽出と同時にその
低粘性化を行い、ついで固液分離して得られる溶液、ま
たは上記溶液を酸性糖を精製するための操作に付して得
られる溶液中の、主として低粘性酸性糖よりなる固形分
を有効成分として含有する免疫賦活剤に関する。以下に
本発明を詳しく説明する。The present invention is a β-agarase having the ability to hydrolyze an acidic polysaccharide from a seaweed belonging to the red algae by extracting the acidic polysaccharide with an aqueous solvent and subjecting it to solid-liquid separation. To obtain a solution obtained by lowering the viscosity of the acidic polysaccharide or by contacting the seaweed with an aqueous solvent containing the β-agarase to simultaneously reduce the viscosity of the acidic polysaccharide and then perform solid-liquid separation. The present invention relates to an immunostimulant containing as an active ingredient a solid content mainly composed of a low-viscosity acidic sugar in a solution obtained by the above, or a solution obtained by subjecting the above solution to an operation for purifying acidic sugar. The present invention will be described in detail below.
【０００６】本発明では、原材料としてアガロースを基
本骨格に持つ酸性多糖を主成分として含有する紅藻類に
属する海藻を用いる。好適に用い得る海藻としては、オ
ゴノリ属に属する海藻、例えばオゴノリ、ツルシラモ、
シラモ、オオオゴノリ、ミゾオゴノリ、カバノリ、アマ
ノリ属に属する海藻、例えばマルバアマノリ、ツクシア
マノリ、オニアマノリ、コスジノリ、ウップルイノリ、
アサクサノリ、スサビノリ、マルバチシマクロノリ、オ
オノノリ、フイリタサを挙げることができる。この他ウ
シケノリ属のウシケノリ、フノリノウシゲも本発明に使
用でき、またイトグサ属及びフノリ属に属する海藻のな
かにも本発明に使用できるものがある。In the present invention, a seaweed belonging to the group of red algae containing an acidic polysaccharide having agarose as a basic skeleton as a main component is used as a raw material. As seaweed that can be preferably used, seaweed belonging to the genus Ogonori, for example, Ogonori, Tsurusemo,
Silamo, scorpionfish, mizogonori, birch, seaweed belonging to the genus Amanori, for example, Marubaa manori, Tsukusia manori, Onia manori, Kosujinori, Upruinori,
Mention may be made of Asakusanori, Susabi Nori, Marubachishi Macronori, Onono Nori, and Firitasa. In addition to this, Ushikenori and Funorinoushige of the genus Ushikenori can also be used in the present invention, and some seaweeds belonging to the genera of Genus Rhizophora and Funori are also usable in the present invention.
【０００７】これらの海藻は最初から水性溶液による抽
出に付しても活性成分を得ることもできるが、油分、少
糖類、油溶性色素を除く意味でこれらを溶解し得る有機
溶媒でまず海藻を処理するのがよい。かかる有機溶媒と
しては例えばメタノール、エタノール、n-プロパノー
ル、イソプロパノール等の低級アルカノール、アセトン
等の低級アルカノン等を用いることができる。また、こ
れらの溶媒と少量の水（80:20v/v程度まで）との混合物
であってもよい。メタノール、エタノール及びこれらと
水との混合溶媒が好ましい。かかる有機溶媒（水との混
合溶媒を含む）の使用量は特に制限はないが、海藻（乾
燥物基準）100gに対して0.5 〜10Ｌぐらいが適当であ
る。有機溶媒抽出の温度時間は特に制限はないが、室温
以上例えば30℃〜有機溶媒の沸点で５分以上例えば15分
〜１時間が適当である。[0007] Although these seaweeds can be obtained from the beginning by extraction with an aqueous solution to obtain the active ingredient, the seaweeds are first dissolved in an organic solvent capable of dissolving them in the sense of removing oils, oligosaccharides and oil-soluble pigments. Good to handle. As such an organic solvent, for example, lower alkanol such as methanol, ethanol, n-propanol and isopropanol, lower alkanone such as acetone and the like can be used. Further, it may be a mixture of these solvents and a small amount of water (up to about 80:20 v / v). Preferred are methanol, ethanol and a mixed solvent of these and water. The amount of such an organic solvent (including a mixed solvent with water) is not particularly limited, but 0.5 to 10 L is suitable for 100 g of seaweed (dry matter standard). The temperature time for extraction with an organic solvent is not particularly limited, but is preferably room temperature or more, for example, 30 ° C. to 5 minutes or more at the boiling point of the organic solvent, for example, 15 minutes to 1 hour.
【０００８】次に有機溶媒をデカント、濾過、遠心分離
等で除去して得られる残渣を水性溶媒による抽出に付
す。水性溶媒としては水、酸性水溶液または塩基性水溶
液が用いられるが、少量の例えば10容量％以下の親水性
有機溶媒をさらに含有していてもよい。「酸性」及び
「塩基性」を与えるのは通常それぞれ酸及び塩基である
が、常用される緩衝剤であってもよい。酸としては特に
制限はないが、塩酸、硫酸リン酸等の無機酸、酢酸、プ
ロピオン酸、クエン酸等の有機酸を用いることができ
る。塩基としては特に制限はないが、アンモニア、アル
カリもしくはアルカリ土類金属の水酸化物、炭酸塩もし
くは重炭酸塩、例えば水酸化ナトリウム、水酸化カリウ
ム、炭酸ナトリウム、炭酸カリウム、重炭酸ナトリウ
ム、重炭酸カリウム等を通常使用する。塩基としてはさ
らにピリジン等の親水性有機塩基であってもよい。緩衝
剤としてはトリス系、リン酸系、クエン酸系、ホウ酸系
等の常用の緩衝剤を用いることができる。Next, the residue obtained by removing the organic solvent by decanting, filtering, centrifuging, etc. is subjected to extraction with an aqueous solvent. As the aqueous solvent, water, an acidic aqueous solution or a basic aqueous solution is used, but it may further contain a small amount of a hydrophilic organic solvent such as 10% by volume or less. It is an acid and a base which give “acidic” and “basic”, respectively, but a commonly used buffer may be used. The acid is not particularly limited, but inorganic acids such as hydrochloric acid and phosphoric acid sulfate, and organic acids such as acetic acid, propionic acid and citric acid can be used. The base is not particularly limited, but ammonia, hydroxide of alkali or alkaline earth metal, carbonate or bicarbonate such as sodium hydroxide, potassium hydroxide, sodium carbonate, potassium carbonate, sodium bicarbonate, bicarbonate. Usually potassium or the like is used. The base may be a hydrophilic organic base such as pyridine. As the buffering agent, a conventional buffering agent such as Tris-based, phosphoric acid-based, citric acid-based, boric acid-based can be used.
【０００９】水性溶媒のpHは特に制限はないが通常１〜
13、特に２〜12が適当である。さらにpH3.5 以下、特に
pH３以下で得られた活性成分はそれより上のpHで得られ
る活性成分に比し一般に免疫賦活剤作用が強い。またpH
3.5 以下、特にpH３以下で処理する場合それより上のpH
で処理する場合に比し、一般に活性成分の収率が優れて
いる。水性溶媒の使用量は特に制限はないが通常原海藻
（乾燥物基準）100gに対して１〜10 Lぐらいが適当であ
る。水性溶媒による抽出の温度時間は特に制限はない
が、４℃〜系の沸騰温度、通常室温〜系の沸騰温度で通
常５分以上、例えば30分〜20時間が適当である。なお、
水、酸性水溶液、塩基性水溶液による抽出は２つ以上直
列に組み合わせて行ってもよい（例えば水抽出残渣を酸
性水溶液で抽出する等）。The pH of the aqueous solvent is not particularly limited, but usually 1 to
13 and especially 2 to 12 are suitable. PH 3.5 or below, especially
An active ingredient obtained at a pH of 3 or less generally has a stronger immunostimulant action than an active ingredient obtained at a pH higher than that. Also pH
PH below 3.5, especially above pH 3
Generally, the yield of the active ingredient is excellent as compared with the case of treatment with. The amount of the aqueous solvent used is not particularly limited, but is usually about 1 to 10 L per 100 g of the raw seaweed (dry matter basis). The temperature time for extraction with an aqueous solvent is not particularly limited, but it is usually 4 minutes to the boiling temperature of the system, usually room temperature to the boiling temperature of the system, and usually 5 minutes or more, for example, 30 minutes to 20 hours is suitable. In addition,
Two or more extractions with water, an acidic aqueous solution, and a basic aqueous solution may be combined in series (for example, a water extraction residue is extracted with an acidic aqueous solution).
【００１０】抽出液を残渣の海藻から通常の固液分離手
段（デカンテーション、濾過、遠心分離等）により分離
し、ついで通常この段階で酵素処理に付す。抽出を酸性
水溶液または塩基性水溶液を用いて行った場合には酵素
処理に適したpHに調整した後酵素処理に付すか、または
好ましくは一旦抽出液を中和し（中和に用いる塩基、酸
はそれぞれ塩基性水溶液及び酸性水溶液に用いる塩基、
酸でよい）、ついで脱塩処理（有機溶媒沈澱後水に溶
解、透析、限外濾過等）を行った後に酵素処理に付す。The extract is separated from the residual seaweed by a conventional solid-liquid separation means (decantation, filtration, centrifugation, etc.), and then usually subjected to an enzyme treatment at this stage. When the extraction is performed using an acidic aqueous solution or a basic aqueous solution, the pH is adjusted to a suitable pH for the enzyme treatment and then subjected to the enzyme treatment, or preferably the extract is neutralized once (a base or an acid used for neutralization is used). Is the base used for the basic aqueous solution and the acidic aqueous solution,
Acid), followed by desalting (precipitation of organic solvent, dissolution in water, dialysis, ultrafiltration, etc.), followed by enzymatic treatment.
【００１１】酵素処理に使用する酵素β−アガラーゼと
しては、前記紅藻類に含まれる酸性多糖を加水分解（β
−１，４結合の加水分解）する能力を有するものであれ
ば、起源を問わず使用することができ、例えば先に例示
したシュードモナス・アトランティカ、サイトファーガ
・エスピー、ビブリオ・エスピーＡＰ−２起源のβ−ア
ガラーゼを使用することができる。このうち、シュード
モナス属の微生物を起源とするβ−アガラーゼ製剤が市
販されている。The enzyme β-agarase used for the enzymatic treatment hydrolyzes the acidic polysaccharides contained in the red algae (β
It can be used regardless of its origin as long as it has the ability to hydrolyze -1,4 bonds). For example, Pseudomonas atlantica, Cytoferga sp., Vibrio sp. The β-agarase of origin can be used. Among these, β-agarase preparations originating from Pseudomonas microorganisms are commercially available.
【００１２】酵素処理は、多糖1g当たりβ−アガラーゼ
0.01〜50,000ユニット、より好ましくは0.1 〜1,000 ユ
ニットを、pH５〜９、温度15〜60℃で 0.5〜72時間、よ
り好ましくは５〜48時間作用させることにより行うこと
ができる。反応は静置状態で進行させることができる
が、攪拌し、さらにはpHを常に酵素反応が最も効率的に
行える値に調整しながら行うこともできる。酵素反応
後、通常酵素を加熱等により失活させる。上記酵素処理
によって酸性多糖はそのβ−１，４結合が、通常部分的
に、加水分解されて低粘性の酸性糖（酸性多糖＋酸性オ
リゴ糖）になる。該酸性糖の分子量分布は通常300 〜1,
400,000 の範囲内の範囲である。なお、酵素処理は、上
記においては水性溶媒抽出液に対して行っているが、水
性溶媒抽出と同時に、すなわちβ−アガラーゼ含有水性
溶媒を用いて抽出を行うことにより行うこともできる。
この場合には酵素反応条件は上記と同様でよいが、抽出
条件は酵素反応条件による制約を受ける。また水性溶媒
抽出液から酸性多糖を一旦後述の精製手段により精製
し、ついで水に再溶解した後、β−アガラーゼを作用さ
せてもよい。[0012] Enzymatic treatment is performed using β-agarase per 1 g of polysaccharide.
It can be carried out by operating 0.01 to 50,000 units, more preferably 0.1 to 1,000 units at pH 5 to 9 and temperature 15 to 60 ° C. for 0.5 to 72 hours, more preferably 5 to 48 hours. The reaction can be allowed to proceed in a static state, but it can also be carried out with stirring and while the pH is constantly adjusted to a value at which the enzymatic reaction can be carried out most efficiently. After the enzymatic reaction, the enzyme is usually inactivated by heating or the like. By the above-mentioned enzyme treatment, the β-1,4 bond of the acidic polysaccharide is usually partially hydrolyzed to a low-viscosity acidic sugar (acidic polysaccharide + acidic oligosaccharide). The molecular weight distribution of the acidic sugar is usually 300-1,
It is in the range of 400,000. In addition, although the enzyme treatment is performed on the aqueous solvent extract in the above, it may be performed at the same time as the aqueous solvent extraction, that is, by performing the extraction using the β-agarase-containing aqueous solvent.
In this case, the enzyme reaction conditions may be the same as above, but the extraction conditions are restricted by the enzyme reaction conditions. Alternatively, the acidic polysaccharide may be once purified from the aqueous solvent extract by the purification means described below, and then redissolved in water, and then β-agarase may be allowed to act thereon.
【００１３】酵素処理液は、そのまままたは濃縮、乾燥
して免疫賦活剤またはその有効成分として使用すること
もできるが、酸性糖を精製するための一般的処理に付し
てもよい。かかる精製処理としては除蛋白、中和、脱
塩、有機溶媒添加による沈澱、塩析、透析、限外濾過、
分配クロマトグラフィー、イオン交換樹脂処理、電気泳
動等があり、これらは単独で、または通常組み合わせて
用いられる。これらの精製処理は公知の方法（例えば特
開平3-284626号記載の方法）に準じて行うことができ
る。The enzyme-treated solution can be used as it is or after being concentrated and dried to be used as an immunostimulant or its active ingredient, but it may be subjected to a general treatment for purifying acidic sugar. Such purification treatment includes deproteinization, neutralization, desalting, precipitation by adding an organic solvent, salting out, dialysis, ultrafiltration,
There are partition chromatography, ion exchange resin treatment, electrophoresis and the like, and these are used alone or usually in combination. These purification treatments can be performed according to known methods (for example, the method described in JP-A-3-284626).
【００１４】例えば、除蛋白はトリクロロ酢酸等により
蛋白質、ポリペプチド等を沈澱させる等の手段により行
うことができる。有機溶媒による沈澱は通常活性成分を
溶解しないか少ししか溶解しない親水性有機溶媒を添加
して有効成分を沈澱させることにより行う。かかる有機
溶媒としては通常メタノール、エタノール、n-プロパノ
ール、イソプロパノール等の低級アルカノール、アセト
ン等の低級アルカノン等を用いることができる。メタノ
ール、エタノール、n-プロパノール、イソプロパノール
等の低級アルカノールが好ましい。塩析工程に用いる塩
析剤は硫安、食塩、塩化カリ、炭酸バリウム等である
が、硫安の使用が最も好ましい。また塩析工程の後処理
（脱塩）として通常透析、限外濾過、ゲル濾過、逆浸透
膜処理等のいずれか１つまたはこれらの２以上の工程を
組み合わせて行うFor example, deproteinization can be performed by means such as precipitating a protein or polypeptide with trichloroacetic acid or the like. Precipitation with an organic solvent is usually carried out by adding a hydrophilic organic solvent which does not dissolve or only slightly dissolves the active ingredient to precipitate the active ingredient. As such an organic solvent, a lower alkanol such as methanol, ethanol, n-propanol and isopropanol, a lower alkanone such as acetone and the like can be usually used. Lower alkanols such as methanol, ethanol, n-propanol and isopropanol are preferred. The salting-out agent used in the salting-out step is ammonium sulfate, sodium chloride, potassium chloride, barium carbonate, etc., but ammonium sulfate is most preferable. As the post-treatment (desalting) of the salting-out step, any one of ordinary dialysis, ultrafiltration, gel filtration, reverse osmosis treatment, etc., or a combination of two or more of these steps is performed.
【００１５】透析は通常セロファン膜、コロジオン膜な
どの半透膜を用いて行う。ゲル濾過はデキストランまた
はポリアクリルアミドゲルなどを充填したカラムを用い
て行う。セファデックス、バイオゲルの名称で販売され
ている充填剤が通常用いられている。限外濾過、逆浸圧
法はいずれも加圧下で膜を用いて分面する方法である。
前者は 0.5〜５kg/cm2、後者は20〜35kg/cm2で行うのが
通常である。分配クロマトグラフィーについては疏水性
基の結合した担体を用いた逆相分配クロマトグラフィー
を用いて低分子分画を除去する、あるいは親水性基の結
合した担体を用いた順相分配クロマトグラフィーを用い
て極性の低い画分を除去する等の処理を行う。また、上
記操作に加えて必要に応じイオン交換処理を行ってもよ
い。Dialysis is usually carried out using a semipermeable membrane such as cellophane membrane or collodion membrane. Gel filtration is performed using a column packed with dextran or polyacrylamide gel. Fillers sold under the names Sephadex and Biogel are commonly used. Both the ultrafiltration and the reverse osmosis method are methods in which the membrane is divided under pressure under a pressure.
The former is usually 0.5 to 5 kg / cm 2 , and the latter is usually 20 to 35 kg / cm 2 . For partition chromatography, remove low-molecular-weight fractions using reverse-phase partition chromatography using a hydrophobic group-bonded carrier, or use normal-phase partition chromatography using a hydrophilic group-bonded carrier. Processing such as removal of the less polar fraction is performed. In addition to the above operation, an ion exchange treatment may be carried out if necessary.
【００１６】酵素処理液からの酸性糖の精製において、
低分子量物質、特に中性オリゴ糖（糖の重合度が10以
下）を除去することにより低粘性酸性糖の免疫賦活活性
が飛躍的に増大することが判明した。中性オリゴ糖の除
去は上記精製手段により行うことができるが、具体的に
は例えば、分画分子量が5,000 〜10,000の限外濾過膜
（例えばミリポア社製ＰＴＧＣ膜、アドバンテック東洋
社製ＱＯ１００等）で排除される画分、または陰イオン
交換樹脂（例えば、東ソー社製DEAE−トヨパール650M、
ファルマシア社製Q-セファロースHP等）に保持されない
画分として中性オリゴ糖の除去を行うことができる。ま
た本発明の免疫賦活剤の有効成分である低粘性酸性糖を
上記精製手段によって分画したもの（例えば免疫賦活活
性に特に寄与する分子量分画、酸性多糖分画等）を有効
成分としてもよい。In the purification of acidic sugar from the enzyme-treated solution,
It was found that the immunostimulatory activity of low-viscosity acidic sugars is dramatically increased by removing low molecular weight substances, especially neutral oligosaccharides (the degree of polymerization of sugars is 10 or less). The removal of neutral oligosaccharides can be carried out by the above-mentioned purification means. Specifically, for example, an ultrafiltration membrane having a molecular weight cut off of 5,000 to 10,000 (eg, PTGC membrane manufactured by Millipore, QO100 manufactured by Advantech Toyo Co., Ltd.) Fraction eliminated by, or anion exchange resin (for example, Tosoh DEAE-Toyopearl 650M,
Neutral oligosaccharides can be removed as a fraction not retained by Pharmacia Q-Sepharose HP. The low-viscosity acidic sugar, which is the active ingredient of the immunostimulant of the present invention, may be fractionated by the above-mentioned purification means (for example, molecular weight fraction particularly contributing to immunostimulatory activity, acidic polysaccharide fraction, etc.) as the active ingredient. .
【００１７】以上の精製操作を経るか、また経ないで得
られる低粘性酸性糖を含有する溶液中の固形分（免疫賦
活在の有効成分）は、後記分析法によるものとして、一
般に次の物性を有する。 分子量分布 300 〜140 万の範囲内の範囲 粘度（ mPa・s ）（50℃での値) １〜40 全糖含有量(w/w%) 60〜95、好ましくは70〜95 蛋白質含量(w/w%) 20以下、好ましくは５以下 3,6-アンヒドロガラクトース含量(w/w%) 5〜33、好ましくは10〜30 硫酸エステル含量(w/w%) 3.5 〜20、好ましくは4.5 〜15The solid content in the solution containing the low-viscosity acidic sugar (immunogenic active ingredient) obtained with or without the above-mentioned purification operation is generally determined by the analytical method described below to have the following physical properties. Have. Molecular weight distribution Range within the range of 300 to 1.4 million Viscosity (mPa · s) (value at 50 ° C) 1 to 40 Total sugar content (w / w%) 60 to 95, preferably 70 to 95 Protein content (w / w%) 20 or less, preferably 5 or less 3,6-anhydrogalactose content (w / w%) 5 to 33, preferably 10 to 30 sulfate ester content (w / w%) 3.5 to 20, preferably 4.5 ~ 15
【００１８】以上の精製操作を経て得られる低粘性酸性
糖を含有する溶液はそのまま免疫賦活剤として用いるこ
ともできるが、通常、噴霧乾燥、凍結乾燥、熱風乾燥等
から適宜選ばれる方法で乾燥し、それ自体としてまたは
製薬上許容される種々の担体を混合した剤型で免疫賦活
剤として用いる。経口投与の場合には、それに適用され
る錠剤、顆粒剤、散剤、カプセル剤などは、通常それら
の組成物中に製剤上一般に使用される結合剤、滑沢剤、
賦形剤、崩壊剤、湿潤剤のような添加物を含有する。ま
た経口用液体剤として用いる場合は、内用水剤、振盪合
剤、懸濁液剤、乳剤、シロップ剤の形態であっても良
く、また使用する前に再溶解させる乾燥生成物の形態で
あってもよい。さらに、このような液体製剤は通常用い
られる添加剤、保存剤のいずれを含有していても良い。
注射用の場合には、その組成物は通常安定剤、緩衝剤、
保存剤、等張化剤などの添加剤を含有し、通常単位投与
量アンプルまたは多投与量容器の形態で提供される。な
お、上記組成物は水溶液、懸濁液、溶液、油性または水
性ビヒクル中の乳液のような形態であっても良く、一方
活性成分は使用する前に適当なビヒクルたとえば発熱物
質不含の滅菌した水で再溶解させる粉末であっても良
い。The solution containing the low-viscosity acidic sugar obtained through the above-described purification operation can be used as it is as an immunostimulant, but it is usually dried by a method appropriately selected from spray drying, freeze drying, hot air drying and the like. , As an immunostimulant, as such or in the form of a mixture of various pharmaceutically acceptable carriers. In the case of oral administration, tablets, granules, powders, capsules and the like applied to them are usually binders, lubricants, and / or lubricants commonly used in their compositions.
Contains additives such as excipients, disintegrants, wetting agents. When it is used as an oral liquid preparation, it may be in the form of an internal solution, a shaking mixture, a suspension, an emulsion or a syrup, or in the form of a dry product to be redissolved before use. Good. Further, such a liquid preparation may contain any of commonly used additives and preservatives.
For injection, the composition will usually be a stabilizer, buffer,
It contains additives such as preservatives, isotonicity agents and is usually provided in the form of unit dose ampoules or multi-dose containers. It should be noted that the composition may be in the form of an aqueous solution, suspension, solution, emulsion in an oily or aqueous vehicle, while the active ingredient is sterilized in a suitable vehicle, eg, pyrogen-free, before use. The powder may be redissolved in water.
【００１９】本発明の免疫賦活剤はヒトまたは動物、例
えば免疫力が低下している人、特に高齢や疾病等により
免疫機能が低下している人に経口または非経口的に投与
される。経口投与は舌下投与を包含する。非経口投与は
注射剤たとえば皮下、筋肉、静脈注射、点滴を含む。本
発明の免疫賦活剤中の有効成分固形物の量は種々変える
ことができるが、通常５〜100%(w/w) 、特に10〜60%(w/
w)が適当である。本発明の免疫賦活剤の投与量は動物か
ヒトにより、また年齢、個人差、病状などに影響される
ので、場合によって下記範囲外の量を投与する場合も生
ずるが、一般にヒトを対象とする場合の経口投与量は活
性成分固形物量として大人１日体重１kg当たり 0.5〜1,
000mg 、好ましくは１〜300mg であり、１回または２も
しくは３回に分けて投与する。なお本免疫賦活剤活性成
分（具体的には各実施例で得られる乾燥物）の急性毒性
はいずれもＬＤ50（ICR 系マウス、経口投与）＞3g/kg
であった。また、本免疫賦活剤活性成分は多量に摂取し
ても生体に悪影響を与えない利点を有することから、そ
のまま、または種々の栄養分等を加えて、もしくは飲食
品中に含有せしめて免疫賦活作用の機能をもたせた機能
性食品、健康食品として食してもよい。すなわち、例え
ば各種ビタミン類、ミネラル類等の栄養分を加えて、例
えば栄養ドリンク、豆乳、スープ等の液状の食品や各種
形状の固形食品、さらには粉末状としてそのままあるい
は各種食品へ添加して用いることもできる。かかる機能
性食品、健康食品としての本免疫賦活剤中の有効成分の
含有量、摂取量はそれぞれ上記製薬における含有量、投
与量と同様でよい。The immunostimulant of the present invention is orally or parenterally administered to humans or animals, for example, to people with reduced immunity, particularly those with reduced immune function due to aging or disease. Oral administration includes sublingual administration. Parenteral administration includes injections such as subcutaneous, intramuscular, intravenous injection and infusion. The amount of the solid of the active ingredient in the immunostimulant of the present invention can be variously changed, but usually 5 to 100% (w / w), particularly 10 to 60% (w /
w) is suitable. The dose of the immunostimulant of the present invention is influenced by animals or humans, age, individual difference, medical condition, etc., and therefore, it may be sometimes administered in an amount outside the following range, but it is generally intended for humans. In this case, the oral dose is 0.5 to 1 per kg of adult daily body weight as the solid content of the active ingredient.
The dose is 000 mg, preferably 1 to 300 mg, which is administered once or in 2 or 3 divided doses. The acute toxicity of the active ingredient of the immunostimulant (specifically, the dried product obtained in each Example) was LD 50 (ICR mouse, oral administration)> 3 g / kg
Met. In addition, since the present immunostimulant active ingredient has an advantage that it does not adversely affect the living body even if it is ingested in a large amount, as it is, or by adding various nutrients or the like, or by including it in a food or drink, it has an immunostimulatory action. It may be consumed as a functional food or a health food having a function. That is, for example, by adding nutrients such as various vitamins and minerals, for example, liquid foods such as nutritional drinks, soy milk, soups and solid foods in various shapes, and further as powders as they are or added to various foods for use. You can also The content and intake of the active ingredient in the present immunostimulant as such functional food and health food may be the same as the content and dose in the above-mentioned pharmaceutical preparation, respectively.
【００２０】[0020]
参考例１ オゴノリ原藻50g を十分量の水で洗浄後、さらに85％
（v/v ) 温メタノールで繰り返し洗浄した。この繰り返
し洗浄は該メタノール 500mlを加え、45〜50℃で15分間
攪拌し、ヌッツェで濾過する操作を２回繰り返すことに
より行った。洗液を十分に除去した後、蒸留水 1.5リッ
トルを加え、沸騰水浴中で30分間、適宜攪拌しながら抽
出した。抽出液を遠心分離により回収し、残渣に再び蒸
留水を１リットル加え、同様に加温抽出し、抽出液を回
収した。得られた抽出液を合わせ、ヌッツェで吸引濾過
して固形成分を完全に除去した。得られた濾液に99.5％
エタノールを加え良く攪拌した後、１晩放置し沈澱を十
分に析出させた。沈澱を回収し、蒸留水に膨潤溶解後、
凍結乾燥して乾燥物 4.86gを得た（試料Ａ）。なお、
「原藻」は通常、市場にはアルカリ溶液で煮た物が塩蔵
して出回っているため、かかる処理をしていないものと
いう意味で使用した。なお、かかる市場品からも本発明
の免疫賦活剤の有効成分を得ることができる。Reference example 1 After washing 50 g of the algae algae with a sufficient amount of water, 85%
(V / v) Washed repeatedly with warm methanol. This repeated washing was performed by adding 500 ml of the methanol, stirring the mixture at 45 to 50 ° C. for 15 minutes, and filtering with a Nutze, twice. After sufficiently removing the washing liquid, 1.5 liters of distilled water was added, and the mixture was extracted in a boiling water bath for 30 minutes while appropriately stirring. The extract was recovered by centrifugation, 1 liter of distilled water was again added to the residue, and the extract was heated and extracted in the same manner to recover the extract. The obtained extracts were combined and suction-filtered with Nutze to completely remove solid components. 99.5% in the obtained filtrate
After adding ethanol and stirring well, the mixture was allowed to stand overnight to sufficiently precipitate. The precipitate is recovered, swollen and dissolved in distilled water,
Lyophilization gave 4.86 g of dried product (Sample A). In addition,
The term "raw algae" is usually used in the sense that it has not been subjected to such a treatment, as it is salted and cooked in an alkaline solution on the market. The active ingredient of the immunostimulant of the present invention can be obtained from such marketed products.
【００２１】実施例１ 参考例１で得られた乾燥物（試料Ａ）1gを蒸留水に溶解
し（0.1 ％）、シュードモナス属由来β−アガラーゼ
（シグマ社製）1,000 ユニットを加えて30℃に保温し適
宜攪拌しつつ、48時間反応させた。反応液を沸騰水溶中
で15分間加熱して酵素を失活させた後凍結乾燥し、酵素
処理物を得た（試料Ｂ）。 実施例２ 実施例１と同様に操作して得た酵素反応液を熱失活させ
た後、公称分画分子量5,000 の限外濾過膜（日本ミリポ
ア・リミテッド製）で10倍に限外濾過濃縮した。処理
後、蒸留水を加えて10倍に希釈して上記膜処理を再度行
い、得られた濃縮液を凍結乾燥して乾燥物を得た（試料
Ｃ）。Example 1 1 g of the dried product (Sample A) obtained in Reference Example 1 was dissolved in distilled water (0.1%), and 1,000 units of Pseudomonas β-agarase (manufactured by Sigma) were added to 30 ° C. The reaction was carried out for 48 hours while maintaining the temperature and stirring appropriately. The reaction solution was heated in boiling water for 15 minutes to inactivate the enzyme and then freeze-dried to obtain an enzyme-treated product (Sample B). Example 2 The enzyme reaction solution obtained by the same procedure as in Example 1 was heat-inactivated, and then concentrated 10 times by ultrafiltration with an ultrafiltration membrane (manufactured by Japan Millipore Limited) having a nominal molecular weight cutoff of 5,000. did. After the treatment, distilled water was added to dilute the mixture 10-fold, the membrane treatment was performed again, and the obtained concentrated liquid was freeze-dried to obtain a dried product (Sample C).
【００２２】実施例３ オゴノリ原藻50g を十分量の水で洗浄後、さらに85％(v
/v）温メタノールで参考例１と同様にして繰り返し洗浄
した。洗液を十分に除去した後、蒸留水１リットルを加
え、沸騰水浴中で30分間、攪拌抽出した。抽出液を30℃
まで冷却し、シュードモナス属由来のβ−アガラーゼ
（シグマ社製） 5,000ユニットを加え、30℃に保温し適
宜攪拌しながら24時間反応させた。反応終了後熱失活を
施し、ヌッツェで吸引濾過して固形分を除去した。濾液
にトリクロロ酢酸を終濃度が10％となるように加え、氷
水中で30分保持した後、セライトでプリコートしたヌッ
ツェで吸引濾過し、濾液を回収した。濾液を2N水酸化ナ
トリウムで中和後、セファデックスG-25を充填したカラ
ムで脱塩し、凍結乾燥して乾燥物6.37g を得た（試料
Ｄ）。Example 3 After washing 50 g of the algae, which is a proto-algae, with 85% (v
/ v) Repeated washing with warm methanol in the same manner as in Reference Example 1. After sufficiently removing the washing liquid, 1 liter of distilled water was added, and the mixture was extracted by stirring in a boiling water bath for 30 minutes. Extract at 30 ℃
After cooling to room temperature, 5,000 units of Pseudomonas-derived β-agarase (manufactured by Sigma) were added, and the mixture was kept at 30 ° C. and reacted for 24 hours while appropriately stirring. After completion of the reaction, heat deactivation was carried out, and suction filtration was carried out using Nutze to remove solids. Trichloroacetic acid was added to the filtrate so that the final concentration was 10%, and the mixture was kept in ice water for 30 minutes and then suction-filtered with Nutze precoated with Celite to collect the filtrate. The filtrate was neutralized with 2N sodium hydroxide, desalted with a column packed with Sephadex G-25, and freeze-dried to obtain 6.37 g of a dried product (Sample D).
【００２３】参考例２ スサビノリ原藻50g を粉砕し、85％(v/v) 温メタノール
で参考例１と同様にして繰り返し洗浄した。洗液を十分
に除去した後、蒸留水0.75リットルを加え、沸騰水浴中
で30分間、攪拌抽出し、遠心分離により沈澱を回収し
た。沈澱は 0.5リットルの蒸留水で洗浄した後、１リッ
トルの蒸留水を加え、濃塩酸でpHを２に調整し、室温で
１晩放置した。抽出液をヌッツェで吸引濾過して固形分
を除去し、得られた濾液を2N水酸化ナトリウムで中和
後、４倍量の99.5％エタノールを加え良く攪拌し、１晩
放置し沈澱を十分に析出させた。沈澱を回収し、蒸留水
に膨潤溶解後、凍結乾燥して乾燥物4.1gを得た（試料
Ｅ）。Reference Example 2 50 g of Susabinori original algae was crushed and repeatedly washed with 85% (v / v) warm methanol in the same manner as in Reference Example 1. After sufficiently removing the washing solution, 0.75 liter of distilled water was added, and the mixture was extracted by stirring in a boiling water bath for 30 minutes, and the precipitate was recovered by centrifugation. The precipitate was washed with 0.5 liter of distilled water, 1 liter of distilled water was added, the pH was adjusted to 2 with concentrated hydrochloric acid, and the mixture was allowed to stand at room temperature overnight. The extract is suction-filtered with Nutze to remove solids, the resulting filtrate is neutralized with 2N sodium hydroxide, 4 volumes of 99.5% ethanol are added, and the mixture is stirred well and left overnight to allow sufficient precipitation. It was deposited. The precipitate was recovered, swollen and dissolved in distilled water, and freeze-dried to obtain 4.1 g of a dried product (Sample E).
【００２４】実施例４ 参考例２で得られた乾燥物（試料Ｅ）1gを蒸留水に溶解
し（0.1 ％）、シュードモナス属由来β−アガラーゼ
（シグマ社製）2,000 ユニットを加えて30℃に保温し適
宜攪拌しつつ、48時間反応させた。反応液は、前記と同
様に加熱失活と凍結乾燥を行い、酵素処理物を得た（試
料Ｆ）。 実施例５ 実施例４と同様に操作して得た酵素反応液を熱失活させ
た後、DEAE−トヨパール650Mカラム（1.6 × 20cm)にの
せ、蒸留水 200ミリリットルで洗浄後、保持された画分
を0.5M塩化ナトリウムで溶出した。溶出液は濃縮後、セ
ファデックスG-25を充填したカラムで脱塩し、濃縮後、
凍結乾燥を行って乾燥物0.6gを得た（試料Ｇ）。 実施例６ 実施例４と同様に操作して得た酵素反応液を熱失活させ
た後、公称分画分子量5000の限外濾過膜（日本ミリポア
・リミテッド製）で10倍に限外濾過濃縮した。処理後、
蒸留水を加えて10倍に希釈して上記濃縮操作を再度行
い、得られた濃縮液を凍結乾燥して乾燥物を得た( 試料
Ｈ）。Example 4 1 g of the dried product (Sample E) obtained in Reference Example 2 was dissolved in distilled water (0.1%), 2,000 units of Pseudomonas β-agarase (manufactured by Sigma) were added, and the mixture was heated to 30 ° C. The reaction was carried out for 48 hours while maintaining the temperature and stirring appropriately. The reaction liquid was inactivated by heating and freeze-dried in the same manner as described above to obtain an enzyme-treated product (Sample F). Example 5 The enzyme reaction mixture obtained by the same procedure as in Example 4 was heat-inactivated, placed on a DEAE-Toyopearl 650M column (1.6 × 20 cm), washed with 200 ml of distilled water, and then retained. Minutes were eluted with 0.5M sodium chloride. The eluate was concentrated, desalted with a column packed with Sephadex G-25, and concentrated,
Lyophilization was performed to obtain 0.6 g of dried product (Sample G). Example 6 After the enzyme reaction solution obtained by the same operation as in Example 4 was heat-inactivated, it was concentrated 10 times by ultrafiltration with an ultrafiltration membrane (manufactured by Japan Millipore Limited) having a nominal molecular weight cutoff of 5000. did. After treatment,
Distilled water was added to the mixture to dilute it 10-fold, the above concentration operation was repeated, and the obtained concentrated liquid was freeze-dried to obtain a dried product (Sample H).
【００２５】参考例１〜２及び実施例１〜６で得られた
乾燥物の各種分析結果を表１に示した。なお、分析項目
中の全糖はフェノール硫酸法〔Dubois M. et al.: Ana
l. Chem.,28, 350-356(1956) 〕を用い、ガラクタンと
して算出した。蛋白質はローリー法〔Lowry O.H.et a
l.: J.Biol. Chem.,193 , 265-275(1951) 〕を用いて、
3,6-アンヒドロガラクトースはW. Yapheらの方法〔Yaph
e W. et al.: Anal. Biochem.,13, 143-148(1965) 〕
で、それぞれ測定した。硫酸エステルは酸加水分解後、
遊離した硫酸イオンをイオンクロマトグラフィー・シス
テムにて測定した。分子量はＧＰＣによりプルランを基
準として測定した。粘度は 1.0％の水溶液について、表
１のものでは50℃、表２のものでは25℃で、それぞれＢ
型粘度計を用いて測定した。Table 1 shows various analytical results of the dried products obtained in Reference Examples 1 and 2 and Examples 1 to 6. The total sugars in the analysis items were measured by the phenol-sulfuric acid method [Dubois M. et al .: Ana.
l. Chem., 28 , 350-356 (1956)], and calculated as galactan. Protein is a Lowry method
l .: J. Biol. Chem., 193 , 265-275 (1951)],
3,6-anhydrogalactose was prepared by the method of W. Yaphe et al. [Yaph
e W. et al .: Anal. Biochem., 13 , 143-148 (1965)).
Then, each was measured. After acid hydrolysis of the sulfuric acid ester,
The liberated sulfate ion was measured by an ion chromatography system. The molecular weight was measured by GPC with reference to pullulan. Aqueous solutions with a viscosity of 1.0% are 50 ° C for Table 1 and 25 ° C for Table 2, respectively.
It measured using the type viscometer.
【００２６】[0026]
【表１】 [Table 1]
【００２７】[0027]
【表２】 [Table 2]
【００２８】実施例７（酵素分解試料のマクロファージ
活性化作用） 免疫賦活作用の指標としてマクロファージ活性化作用を
調べた。常法によりプロテオースペプトンで誘導したマ
ウスの腹腔細胞を採取し、４×105/ウェルとなるよう96
穴プレートに分注し、１時間培養してマクロファージを
付着させた。浮遊細胞を除去した後試料を加え10％牛胎
児血清、ぺニシリン、ストレプトマイシンを含む RPMI-
1640培地中で72時間培養し、培養上清中の残存グルコー
ス及び分泌された亜硝酸イオン濃度を測定し、マクロフ
ァージ活性化の指標とした。すなわち、グルコース消費
量と亜硝酸イオン産生量は、いずれも値の大きい程マク
ロファージ活性化作用が大きいことを示す。対照として
マクロファージ活性化物質であるリポポリサッカライド
（ＬＰＳ）及びラミナリンを用いた。結果を表３に示し
た。Example 7 (Macrophage activating action of enzyme-decomposed sample) The macrophage activating action was examined as an index of immunostimulating action. Peritoneal cells of mice induced by proteose peptone were collected by a standard method, and 96 cells were collected at 4 × 10 5 / well.
It was dispensed into a well plate and cultured for 1 hour to attach macrophages. RPMI- containing 10% fetal bovine serum, penicillin, and streptomycin after removing floating cells
After culturing in 1640 medium for 72 hours, the residual glucose and secreted nitrite ion concentration in the culture supernatant were measured and used as an index for macrophage activation. That is, the larger the glucose consumption amount and the nitrite ion production amount, the greater the macrophage activating effect. As controls, macrophage activators, lipopolysaccharide (LPS) and laminarin were used. The results are shown in Table 3.
【００２９】[0029]
【表３】 [Table 3]
【００３０】表３から明らかなように、酸性多糖を酵素
処理することにより、マクロファージの亜硝酸産生能が
著しく上昇し、グルコース消費量は殆どそのまま維持さ
れた。このことは、酵素処理により免疫能が増強される
ことを如実に示すものである。As is clear from Table 3, the enzymatic treatment of acidic polysaccharide markedly increased the nitrite-producing ability of macrophages, and the glucose consumption was maintained almost as it was. This clearly shows that the enzyme treatment enhances the immunity.
【００３１】実施例８ 注射剤 試料Ｃ ６００ｇ ポリエキシエチレン硬化ヒマシ油 ５００ｇ 局方蒸留水 １０リットル 上記成分を用い常法により注射剤を調製し、１アンプル
に５ミリリットルずつ充填した。 実施例９ カプセル剤 試料Ｄ ２００ｇ コーンスターチ １５０ｇ タルク ８０ｇ ステアリン酸マグネシウム ３０ｇ 上記成分を十分混和し、60メッシュの金網を通過させて
粒度を調整した後、1,000 個のゼラチンカプセルに充填
した。Example 8 Injection Sample 600 g Polyethylene ethylene hydrogenated castor oil 500 g Pharmacopoeial distilled water 10 liters An injection was prepared by the conventional method using the above components, and each ampoule was filled with 5 ml. Example 9 Capsule Sample D 200 g Corn starch 150 g Talc 80 g Magnesium stearate 30 g The above ingredients were thoroughly mixed, passed through a 60 mesh wire mesh to adjust the particle size, and then filled into 1,000 gelatin capsules.
【００３２】[0032]
【発明の効果】本発明の免疫賦活剤の有効成分である低
粘性酸性糖は原料酸性多糖に比べ、低粘度で取扱い易く
各種の剤型に製剤化が可能であり、かつ免疫賦活作用が
より優れている。EFFECT OF THE INVENTION The low-viscosity acidic sugar, which is the active ingredient of the immunostimulant of the present invention, has a low viscosity, is easy to handle, can be formulated into various dosage forms, and has a higher immunostimulatory action than the raw material acidic polysaccharide. Are better.
───────────────────────────────────────────────────── フロントページの続き (72)発明者 山本 綽 愛知県安城市昭和町19−10 新日本化学工 業（株）内 (72)発明者 伊藤 昌雄 愛知県安城市昭和町19−10 新日本化学工 業（株）内 (72)発明者 村松 五月 愛知県安城市昭和町19−10 新日本化学工 業（株）内 (72)発明者 野村 和代 愛知県安城市昭和町19−10 新日本化学工 業（株）内 ─────────────────────────────────────────────────── ─── Continuation of the front page (72) Inventor Ryo Yamamoto 19-10 Showa-cho, Anjo City, Aichi Prefecture Shin-Nippon Chemical Industry Co., Ltd. (72) Inventor Masao Ito 19-10 Showa-cho, Anjo City, Aichi Prefecture Shin-Nihon In Chemical Industry Co., Ltd. (72) Inventor May Muramatsu 19-10 Showa-cho, Anjo City, Aichi Prefecture (72) In Shin-Nippon Chemical Industry Co., Ltd. (72) Inventor Kazuyo Nomura 19-10 Showa-cho, Anjo City, Aichi Prefecture Inside Shin Nippon Chemical Industry Co., Ltd.
Claims (3)
溶媒で抽出し、固液分離して得られる抽出液に該酸性多
糖を加水分解する能力を有するβ−アガラーゼを作用さ
せて酸性多糖を低粘性化して得られる溶液、もしくは該
海藻を該β−アガラーゼを含有する水性溶媒を接触させ
て酸性多糖の抽出と同時にその低粘性化を行い、ついで
固液分離して得られる溶液、または上記溶液を酸性糖を
精製するための操作に付して得られる溶液中の、主とし
て低粘性酸性糖よりなる固形分を有効成分として含有す
る免疫賦活剤。1. An acidic polysaccharide is extracted from a seaweed belonging to red algae by extracting β-agarase having an ability to hydrolyze the acidic polysaccharide with an extract obtained by solid-liquid separation of the acidic polysaccharide extracted with an aqueous solvent. A solution obtained by lowering the viscosity, or a solution obtained by contacting the seaweed with an aqueous solvent containing the β-agarase to simultaneously reduce the viscosity of the acidic polysaccharide, and then performing solid-liquid separation, or An immunostimulant containing as an active ingredient a solid content mainly composed of low-viscosity acidic sugar in a solution obtained by subjecting the solution to an operation for purifying acidic sugar.
中に含まれる中性オリゴ糖の除去を含む請求項１記載の
免疫賦活剤。2. The immunostimulant according to claim 1, wherein the purification operation includes removal of neutral oligosaccharide contained in the solution after the action of β-agarase.
ある請求項１または２記載の免疫賦活剤。3. The immunostimulant according to claim 1, wherein the red alga belongs to the genus Ogonori or the genus Amanori.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP06747993A JP3568214B2 (en) | 1993-03-03 | 1993-03-03 | Immunostimulants |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP06747993A JP3568214B2 (en) | 1993-03-03 | 1993-03-03 | Immunostimulants |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JPH06256208A true JPH06256208A (en) | 1994-09-13 |
| JP3568214B2 JP3568214B2 (en) | 2004-09-22 |
Family
ID=13346153
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP06747993A Expired - Lifetime JP3568214B2 (en) | 1993-03-03 | 1993-03-03 | Immunostimulants |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JP3568214B2 (en) |
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| CN1102151C (en) * | 1997-12-25 | 2003-02-26 | 中国科学院水生生物研究所 | Method for extracting and separating filiform blue-green algae water-soluble polyose and ecto-polyose |
| CN1108310C (en) * | 1999-11-30 | 2003-05-14 | 中国科学院南海海洋研究所 | Algae polysaccharide and its preparation and use |
| JP2005145885A (en) * | 2003-11-14 | 2005-06-09 | Japan Science & Technology Agency | Immunologic mechanism activator comprising alginic acid oligomer |
| WO2005039597A3 (en) * | 2003-10-24 | 2005-07-14 | Nutricia Nv | Immunemodulating oligosaccharides |
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|---|---|---|---|---|
| CN1102151C (en) * | 1997-12-25 | 2003-02-26 | 中国科学院水生生物研究所 | Method for extracting and separating filiform blue-green algae water-soluble polyose and ecto-polyose |
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| US8557794B2 (en) | 2003-10-24 | 2013-10-15 | N.V. Nutricia | Immunemodulating oligosaccharides |
| WO2005039597A3 (en) * | 2003-10-24 | 2005-07-14 | Nutricia Nv | Immunemodulating oligosaccharides |
| EP1721612A3 (en) * | 2003-10-24 | 2007-05-09 | N.V. Nutricia | Immunemodulating oligosaccharides |
| EP2123282A3 (en) * | 2003-10-24 | 2010-01-20 | N.V. Nutricia | Immunemodulating oligosaccharides |
| EP2305269A3 (en) * | 2003-10-24 | 2011-07-13 | N.V. Nutricia | Immunemodulating oligosaccharides |
| JP2005145885A (en) * | 2003-11-14 | 2005-06-09 | Japan Science & Technology Agency | Immunologic mechanism activator comprising alginic acid oligomer |
| JP2009508941A (en) * | 2005-09-23 | 2009-03-05 | プキョン ナショナル ユニヴァーシティ インダストリー−アカデミック コーオペレーション ファンデーション | Capsochiffon-Fluvesson sugar hot water extract and anticancer agent based on this |
| JP2012224554A (en) * | 2011-04-15 | 2012-11-15 | Nikken Sohonsha Corp | Antiviral agent and method for producing the same |
| CN102334702A (en) * | 2011-07-12 | 2012-02-01 | 集美大学 | Production method for laver protein and polysaccharide nutrient powder |
| JP2015151344A (en) * | 2014-02-12 | 2015-08-24 | 株式会社アンチエイジング・プロ | Method for producing dioscorea japonica extract |
| JP2016065037A (en) * | 2014-09-17 | 2016-04-28 | 株式会社日健総本社 | Production method of antiviral drug and antiviral drug obtained by method thereof |
| CN104403015A (en) * | 2014-11-14 | 2015-03-11 | 青岛聚大洋藻业集团有限公司 | Processing method for red alga polysaccharide |
| JP2018168263A (en) * | 2017-03-29 | 2018-11-01 | 日油株式会社 | Vegetable proteoglycan and use thereof |
| US20220295826A1 (en) * | 2019-07-30 | 2022-09-22 | Umaro Foods, Inc. | Plant-based food products |
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