JP2018090522A - Immunostimulator - Google Patents
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- JP2018090522A JP2018090522A JP2016234383A JP2016234383A JP2018090522A JP 2018090522 A JP2018090522 A JP 2018090522A JP 2016234383 A JP2016234383 A JP 2016234383A JP 2016234383 A JP2016234383 A JP 2016234383A JP 2018090522 A JP2018090522 A JP 2018090522A
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Abstract
Description
本発明は免疫賦活剤に関する。詳細には、フコイダンと酵母を含む免疫賦活剤に関する。 The present invention relates to an immunostimulator. In detail, it is related with the immunostimulant containing a fucoidan and yeast.
免疫機能の賦活化は、疾病の治療および予防ならびに健康増進にとり重要である。これまで様々な免疫賦活物質が見出され、合成され、研究され、利用されている。しかしながら、免疫賦活作用が優れている物質は副作用が大きい等の問題がある場合が少なくない。 Activation of immune function is important for disease treatment and prevention and health promotion. Various immunostimulatory substances have been discovered, synthesized, studied, and utilized so far. However, substances with excellent immunostimulatory effects often have problems such as large side effects.
かかる状況下において、天然由来の免疫賦活物質の探求が行われており、そのなかには海藻に含まれる多糖類であるフコイダン（特許文献１等参照）や酵母菌体（特許文献２等参照）などが含まれている。しかしながら、これらの免疫賦活物質は効果の面で満足なものとはいえない。 Under such circumstances, a search for naturally-derived immunostimulatory substances has been conducted, among which fucoidan (see Patent Document 1 and the like), which is a polysaccharide contained in seaweed, yeast cells (see Patent Document 2 and the like), and the like. include. However, these immunostimulatory substances are not satisfactory in terms of effects.
効果が優れ、しかも安全性が高い免疫賦活剤を提供することが、本発明が解決しようとする課題であった。 It was a problem to be solved by the present invention to provide an immunostimulator having excellent effects and high safety.
本発明者らは、上記課題を解決すべく鋭意研究を重ねた結果、フコイダンと酵母を共存させた場合に、免疫賦活効果が相乗的に高められること、および分子量の大きいフコイダンほど免疫賦活活性が高いことを見出し、本発明を完成するに至った。したがって、本発明は、以下のものを提供する：
（１）フコイダンまたはフコイダン含有素材および酵母を含む免疫賦活剤。
（２）フコイダンの分子量が３００ｋＤａ以上である（１）記載の剤。
（３）フコイダンがモズク由来のものである（１）または（２）記載の剤。
（４）フコイダン含有素材がモズクである（１）〜（３）のいずれかに記載の剤。
（５）酵母がSaccharomyces cerevisiaeである（１）〜（４）のいずれかに記載の剤。
（６）食品組成物である（１）〜（５）のいずれかに記載の剤。
（７）医薬組成物である（１）〜（５）のいずれかに記載の剤。
（８）分子量が３００ｋＤａ以上のモズク由来の精製フコイダンを含む免疫賦活剤。
（９）食品組成物である（８）の剤。
（１０）医薬組成物である（８）の剤。
As a result of intensive studies to solve the above problems, the present inventors have found that when fucoidan and yeast coexist, the immunostimulatory effect is synergistically increased, and fucoidan having a higher molecular weight has higher immunostimulatory activity. As a result, the present invention has been completed. Accordingly, the present invention provides the following:
(1) An immunostimulant comprising fucoidan or a fucoidan-containing material and yeast.
(2) The agent according to (1), wherein the fucoidan has a molecular weight of 300 kDa or more.
(3) The agent according to (1) or (2), wherein fucoidan is derived from mozuku.
(4) The agent according to any one of (1) to (3), wherein the fucoidan-containing material is mozuku.
(5) The agent according to any one of (1) to (4), wherein the yeast is Saccharomyces cerevisiae.
(6) The agent according to any one of (1) to (5), which is a food composition.
(7) The agent according to any one of (1) to (5), which is a pharmaceutical composition.
(8) An immunostimulant comprising a mozuku-derived purified fucoidan having a molecular weight of 300 kDa or more.
(9) The agent according to (8), which is a food composition.
(10) The agent according to (8), which is a pharmaceutical composition.
本発明において、フコイダンと酵母を共存させることにより相乗的に強力な免疫賦活作用が見られる。しかも分子量の大きいフコイダンほど免疫賦活活性が高い。フコイダンはモズク、ワカメ、コンブ、ヒバマタなどの食用海藻に含まれる多糖類であり、安全性が高い。酒類やパンなどの飲食物の製造に用いられる酵母についても安全性が高い。従って、本発明の免疫賦活剤は、効果が優れ、しかも安全性が高いものである。 In the present invention, a synergistic and potent immunostimulatory effect is seen by coexisting fucoidan and yeast. In addition, fucoidan having a higher molecular weight has higher immunostimulatory activity. Fucoidan is a polysaccharide contained in edible seaweeds such as mozuku, wakame, kombu, and hibamata, and is highly safe. Yeast used for the production of food and drink such as alcohol and bread is also highly safe. Therefore, the immunostimulant of the present invention has an excellent effect and high safety.
本発明者らは、フコイダンおよび酵母が相乗的に免疫賦活作用を増強すること、および分子量の大きいフコイダンほど免疫賦活活性が高いことを今回初めて見出した。したがって、本発明は、１の態様において、フコイダンおよび酵母を含む免疫賦活用の剤に関する。フコイダンは、天然界に見出される含硫多糖で、モズク、メカブなどの海藻類に多く含まれている。 The present inventors have found for the first time that fucoidan and yeast synergistically enhance the immunostimulatory action, and that fucoidan having a higher molecular weight has higher immunostimulatory activity. Therefore, in one aspect, the present invention relates to an immunostimulating agent containing fucoidan and yeast. Fucoidan is a sulfur-containing polysaccharide found in nature and is abundant in seaweeds such as mozuku and mekabu.
本発明の剤中の有効成分たるフコイダンの由来は限定されず、海藻類、とりわけ褐藻類が挙げられる。褐藻類としては、モズク（沖縄モズク、糸モズク、太モズク、石モズクなど）、メカブ（ワカメの胞子葉）、ワカメ、アラメ、ガゴメ、マコンブ、クロメ、カジメ、ミツイシコンブ、ヨレモク、ヒジキ、ホンダワラ、ヤツマタモク、アカモク、ヒバマタ、ウミトラノオなどが例示されるが、これらに限定されない。本発明の食品組成物に用いられる好ましいフコイダンはモズク由来である。特に沖縄モズクまたは糸モズク由来のフコイダンが好ましい。なお、本明細書において「モズク」という場合にはあらゆる種類のモズクを包含する。 The origin of fucoidan as an active ingredient in the agent of the present invention is not limited, and examples include seaweeds, especially brown algae. As brown algae, there are mozuku (Okinawa mozuku, thread mozuku, thick mozuku, stone mozuku, etc.), mekabu (squid foliage of wakame), wakame, alame, gagome, kombu, kurome, kajime, mitsuishi kombu, yoremoku, hijiki, honda walla, yamatamamok , Akamoku, Hibamata, Umitorano and the like, but are not limited thereto. A preferred fucoidan used in the food composition of the present invention is derived from mozuku. Fucoidan derived from Okinawa mozuku or thread mozuku is particularly preferable. In this specification, “mozuku” includes all kinds of mozuku.
フコイダンは精製品であってもよく、粗精製品、例えばモズクなどの海藻からの抽出物であってもよい。好ましくは、本発明の食品組成物中の有効成分たるフコイダンは精製品である。フコイダンの精製方法は公知であり、いずれの方法を用いてもよい。フコイダンの精製は、熱水や有機酸溶液での抽出、および限外濾過などの濾過を含むプロセスを経て行われるのが一般的である。本明細書において、精製されたフコイダンとは、少なくとも１回の抽出工程および少なくとも１回の分画工程（濾過、クロマトグラフィー等）を経て得られたフコイダンをいう。 The fucoidan may be a purified product or a crude product, for example, an extract from seaweed such as mozuku. Preferably, fucoidan as an active ingredient in the food composition of the present invention is a purified product. Methods for purifying fucoidan are known, and any method may be used. The purification of fucoidan is generally performed through a process including extraction with hot water or an organic acid solution and filtration such as ultrafiltration. In the present specification, the purified fucoidan refers to fucoidan obtained through at least one extraction step and at least one fractionation step (filtration, chromatography, etc.).
本発明の剤に使用されるフコイダンは様々な形態のものが用いられ、固体（例えば、粉末、顆粒など）、液体（例えば、フコイダン水溶液、フコイダン懸濁液、海藻からの抽出液など）、半固体（例えば、ペーストなど）のいずれであってもよい。 The fucoidan used in the agent of the present invention may be in various forms, such as solid (eg, powder, granule, etc.), liquid (eg, fucoidan aqueous solution, fucoidan suspension, extract from seaweed, etc.), half Any of solid (for example, paste etc.) may be sufficient.
本発明の剤中のフコイダンは高分子であるほど免疫賦活作用が大きい。本発明の剤中のフコイダンの分子量は、好ましくは約２００ｋＤａ以上、より好ましくは約２５０ｋＤａ以上、さらにより好ましくは約３００ｋＤａ以上である。所望の分子量のフコイダンを得る方法も公知である。例えば適切なポアサイズのフィルターを用いてフコイダン抽出液を限外濾過することによって、所望の分子量のフコイダンを得ることができる。あるいはゲル濾過クロマトグラフィーを用いて所望の分子量のフコイダンを得ることができる。フコイダンの分子量は公知の方法、例えばゲルろ過クロマトグラフィーを用いて測定することができる。通常は、分子量測定の際に既知分子量を有するプルランをマーカーとして用いる。高分子化合物の分子量は平均分子量、数平均分子量あるいは重量平均分子量で表されることが一般的であるが、本明細書において、特に断らないかぎり、分子量というときは重量平均分子量を意味する。重量平均分子量は、公知の手段・方法により計算することができ、例えばゲルろ過クロマトグラフィーのパターンから計算することができる。 Fucoidan in the agent of the present invention has a greater immunostimulatory effect as the polymer is higher. The molecular weight of fucoidan in the agent of the present invention is preferably about 200 kDa or more, more preferably about 250 kDa or more, and even more preferably about 300 kDa or more. A method for obtaining fucoidan having a desired molecular weight is also known. For example, a fucoidan having a desired molecular weight can be obtained by ultrafiltration of the fucoidan extract using a filter having an appropriate pore size. Alternatively, fucoidan having a desired molecular weight can be obtained using gel filtration chromatography. The molecular weight of fucoidan can be measured using a known method, for example, gel filtration chromatography. Usually, pullulan having a known molecular weight is used as a marker in the molecular weight measurement. The molecular weight of a polymer compound is generally expressed by an average molecular weight, a number average molecular weight, or a weight average molecular weight. In the present specification, unless otherwise specified, the molecular weight means a weight average molecular weight. The weight average molecular weight can be calculated by a known means / method, for example, from a pattern of gel filtration chromatography.
フコイダン含有素材を本発明の剤の有効成分として用いてもよい。フコイダン含有素材は、フコイダンを含有し、ヒトに対して無毒のものであればいずれの素材であってもよい。本発明において好ましいフコイダン含有素材としては、海藻類、とくに褐藻類が挙げられる。フコイダン含有褐藻類しては、モズク（沖縄モズク、糸モズク、太モズク、石モズクなど）、メカブ（ワカメの胞子葉）、ワカメ、アラメ、ガゴメ、マコンブ、クロメ、カジメ、ミツイシコンブ、ヨレモク、ヒジキ、ホンダワラ、ヤツマタモク、アカモク、ヒバマタ、ウミトラノオなどが例示されるが、これらに限定されない。本発明の剤に用いる好ましいフコイダン含有素材はモズクである。モズクのなかでも沖縄モズクまたは糸モズクが特に好ましい。これらの海藻類をそのまま本発明の剤に使用してもよく、あるいはこれらの海藻類に切断、細断、破砕等の適当な処理を施して本発明の剤に用いてもよい。 You may use a fucoidan containing raw material as an active ingredient of the agent of this invention. The fucoidan-containing material may be any material as long as it contains fucoidan and is non-toxic to humans. Preferred fucoidan-containing materials in the present invention include seaweeds, particularly brown algae. Fucoidan-containing brown algae include mozuku (Okinawa mozuku, thread mozuku, thick mozuku, stone mozuku, etc.), mekabu (squirt of wakame), wakame, arame, gagome, macomb, kurome, kajime, mitsuishikonbu, yoremoku, hijiki, Examples include, but are not limited to, Honda Walla, Yatsumata Moku, Akamoku, Hibamata, Umitorano. A preferred fucoidan-containing material used in the agent of the present invention is mozuku. Of the mozuku, Okinawa mozuku or thread mozuku is particularly preferable. These seaweeds may be used as they are in the agent of the present invention, or these seaweeds may be used in the agent of the present invention after appropriate treatment such as cutting, shredding, crushing or the like.
本発明の剤の有効成分として用いられる酵母の種類は特に限定されないが、ヒトや動物に対して無害なものが好ましい。本発明の剤に用いられる好ましい酵母の例としては、酒類やパンの製造に用いられるSaccharomyces cerevisiaeが挙げられる。酵母は生菌であってもよく、死菌であってもよい。また、酵母は、生菌または死菌の破砕物であってもよく、あるいは生菌または死菌の抽出物であってもよい。公知の方法を用いて酵母の破砕物や抽出物を得ることができる。本発明の剤に用いられる酵母の形態は、粉末、顆粒などの固体、細胞懸濁液などの液体、あるいはパスタ、クリームなどの半固体であってもよい。 Although the kind of yeast used as an active ingredient of the agent of the present invention is not particularly limited, those which are harmless to humans and animals are preferable. Examples of preferable yeast used in the agent of the present invention include Saccharomyces cerevisiae used for the production of alcoholic beverages and bread. The yeast may be live or dead. The yeast may be a crushed product of live or dead bacteria, or may be an extract of live or dead bacteria. Yeast fragments and extracts can be obtained using known methods. The form of yeast used in the agent of the present invention may be a solid such as a powder or a granule, a liquid such as a cell suspension, or a semi-solid such as pasta or cream.
本発明の剤の形態や用法は特に限定されない。本発明の剤は固形、液体、半固形に成形することができる。これらの剤形の種類や製造方法は公知である。本発明の剤を味噌汁、スープ、めんつゆ、あるいは練り製品などの飲食物に添加して用いてもよい。本発明の剤を凍結乾燥等の処理をして粉末あるいは顆粒などの形態とし、適当な包装や容器に入れる等の処理加工を施して、調味料やふりかけのような形態とし、利用者が自ら任意の飲食物に添加して摂取できるようにしてもよい。あるいは本発明の剤を医薬あるいは医薬に準じる形態として使用してもよい。 The form and usage of the agent of the present invention are not particularly limited. The agent of the present invention can be formed into a solid, liquid or semi-solid. The types and production methods of these dosage forms are known. The agent of the present invention may be used by adding it to food and drink such as miso soup, soup, noodle soup, or kneaded products. The agent of the present invention is processed into a form such as powder or granule by freeze-drying, etc., and is processed into a form such as seasoning or sprinkling by processing such as putting it in an appropriate packaging or container. You may make it ingestible by adding to arbitrary food and drink. Or you may use the agent of this invention as a form according to a medicine or medicine.
本発明の剤の摂取量または投与量は特に制限はなく、例えば成人１日あたりフコイダンが約０．８ｇ（乾燥重量として）以上、酵母が約１ｇ（乾燥重量として）以上摂取されるような量であってもよい。 There are no particular restrictions on the intake or dose of the agent of the present invention, for example, an amount such that about 0.8 g (as dry weight) or more of fucoidan and about 1 g (as dry weight) of yeast per day are consumed. It may be.
本発明の剤を公知の免疫賦活剤と併用してもよい。 You may use the agent of this invention together with a well-known immunostimulant.
本発明の剤の典型的な形態は食品組成物である。本発明の剤が食品組成物である場合、その形態は限定されず、味噌汁、スープ、めんつゆ、練り製品、パック詰めモズク、ふりかけ、調味料など、あらゆる飲食物の形態であってよい。本発明の食品組成物は、食品の原料にフコイダンまたはフコイダン含有素材および酵母を添加することにより、食品の製造工程においてフコイダンまたはフコイダン含有素材および酵母を添加することにより、あるいは製品である食品にフコイダンまたはフコイダン含有素材および酵母を添加することにより製造することができる。 A typical form of the agent of the present invention is a food composition. When the agent of the present invention is a food composition, its form is not limited, and may be any food or drink such as miso soup, soup, noodle soup, kneaded product, packed mozuku, sprinkle, seasoning. The food composition of the present invention comprises fucoidan or a fucoidan-containing material and yeast added to the raw material of the food, fucoidan or fucoidan-containing material and yeast added in the food production process, or fucoidan to the food product. Or it can manufacture by adding a fucoidan containing raw material and yeast.
また本発明の剤は、サプリメントとして提供されてもよい。本明細書において、サプリメントは食品組成物に含められる。サプリメントの形態としては、粉末、顆粒、錠剤、カプセル剤などの固形であってもよく、溶液、懸濁液、抽出液などの液体であってもよく、パスタ、クリームなどの半固体であってもよい。これらの形態のサプリメントの製造方法は当業者によく知られている。本発明の剤は機能性食品としても有用である。 The agent of the present invention may be provided as a supplement. As used herein, supplements are included in food compositions. The form of the supplement may be a solid such as powder, granule, tablet or capsule, may be a liquid such as a solution, a suspension or an extract, or may be a semi-solid such as pasta or cream. Also good. Methods for producing these forms of supplements are well known to those skilled in the art. The agent of the present invention is also useful as a functional food.
本発明の剤のさらなる典型的な形態は医薬組成物である。その投与方法は特に限定されず、経口投与、注射による投与、経皮投与などのいずれの方法であってもよいが、経口投与が好ましい。本発明の剤の形態も特に限定されない。経口投与の場合、粉末、顆粒、錠剤、カプセル剤、ドリンク剤などの様々な経口用剤形に処方することができる。これらの剤形の製法は公知であり、混合、溶解、粉砕、打錠、乾燥等のプロセスを適宜用いることができ、目的に応じた担体や賦形剤を使用することができる。本発明の経口投与剤に香料、甘味料、着色料などを適宜添加してもよい。本発明の経口投与剤はそのまま投与することもできるが、例えばみそ汁、スープ、麺類、その他の食品に本発明の経口投与剤を随意に添加して投与してもよい。 A further exemplary form of the agent of the present invention is a pharmaceutical composition. The administration method is not particularly limited, and any method such as oral administration, administration by injection, and transdermal administration may be used, but oral administration is preferred. The form of the agent of the present invention is not particularly limited. For oral administration, it can be formulated into various oral dosage forms such as powders, granules, tablets, capsules and drinks. Methods for producing these dosage forms are known, and processes such as mixing, dissolution, pulverization, tableting, and drying can be used as appropriate, and carriers and excipients according to the purpose can be used. A fragrance, a sweetener, a colorant and the like may be appropriately added to the oral administration agent of the present invention. The oral administration agent of the present invention can be administered as it is, but for example, the oral administration agent of the present invention may optionally be added to miso soup, soup, noodles, and other foods.
本発明の剤は獣医薬組成物であってもよい。その投与方法は特に限定されず、経口、注射、経皮等いずれの方法であってもよいが、経口投与が好ましい。経口投与剤の場合、濃縮液、粉末、顆粒、錠剤、カプセル剤、ドリンク剤、飼料などの様々な経口用剤形に処方することができる。これらの剤形の製法は公知であり、混合、溶解、粉砕、打錠、乾燥等のプロセスを適宜用いることができ、獣医薬上許容される担体や賦形剤を使用することができる。本発明の経口投与剤に香料、着色料などを適宜添加してもよい。本発明の剤を獣医役組成物として用いる場合も、その投与量には特に制限はない。 The agent of the present invention may be a veterinary pharmaceutical composition. The administration method is not particularly limited and may be any method such as oral, injection, or transdermal, but oral administration is preferred. In the case of oral administration, it can be formulated into various oral dosage forms such as concentrates, powders, granules, tablets, capsules, drinks, and feeds. Methods for producing these dosage forms are known, and processes such as mixing, dissolution, pulverization, tableting, and drying can be used as appropriate, and veterinary acceptable carriers and excipients can be used. A fragrance, a coloring agent or the like may be appropriately added to the oral administration agent of the present invention. Even when the agent of the present invention is used as a veterinary composition, the dosage is not particularly limited.
本発明は、さらなる態様において、分子量が３００ｋＤａ以上のモズク由来の精製フコイダンを含む免疫賦活用の剤を提供するものである。このような剤もまた、食品組成物、医薬組成物、獣医薬組成物などとして用いることができる。 In a further aspect, the present invention provides an agent for immunostimulation comprising purified fucoidan derived from mozuku having a molecular weight of 300 kDa or more. Such agents can also be used as food compositions, pharmaceutical compositions, veterinary pharmaceutical compositions and the like.
以下に実施例を示して本発明をより詳細かつ具体的に説明するが、実施例は本発明を限定するものと解してはならない。 The present invention will be described in more detail and specifically below with reference to examples, but the examples should not be construed as limiting the present invention.
フコイダンと酵母を同時にマクロファージに作用させてマクロファージを活性化させ、活性化の程度を調べた。マクロファージはマウス由来のＪ７７４．１細胞を用いた。フコイダンは分子量３３２ｋＤａの精製品を用いた。酵母はSaccharomyces cerevisiae NBW5株を用いた。活性化は、２０マイクロリットルのフコイダン及び酵母を懸濁した蒸留水（フコイダン濃度は２０ｍｇ／ｍｌ、酵母は１２ｍｇ／ｍｌ（酵母菌は湿重量））を１ｍｌの細胞培養液に加えることにより行った。 Fucoidan and yeast simultaneously acted on macrophages to activate macrophages, and the degree of activation was examined. As macrophages, mouse-derived J774.1 cells were used. As the fucoidan, a purified product having a molecular weight of 332 kDa was used. Saccharomyces cerevisiae NBW5 strain was used as yeast. Activation was performed by adding 20 microliters of fucoidan and distilled water in which yeast was suspended (fucoidan concentration 20 mg / ml, yeast 12 mg / ml (wet weight of yeast)) to 1 ml of cell culture. .
活性化後、ＣＯＸ２およびＧＡＰＤＨ（ローディングコントロール）に関してイムノブロットを行った結果を図１Ａに示す。さらに、ＧＡＰＤＨ（ローディングコントロール）の量により補正したＣＯＸ２タンパク質の量を図１Ｂに示す。 FIG. 1A shows the results of immunoblotting for COX2 and GAPDH (loading control) after activation. Furthermore, the amount of COX2 protein corrected by the amount of GAPDH (loading control) is shown in FIG. 1B.
図１からわかるように、フコイダンおよび酵母を加えていないサンプル（レーン１）では、ほどんど活性化が見られなかった。酵母を加えずにフコイダンの量を増やしていくと活性化が増強された。フコイダンを単独で０．１ｍｇ／ｍｌの濃度で作用させた場合（レーン３）、フコイダンおよび酵母を加えていないサンプル（レーン１）の７倍の活性化がみられた。酵母を単独で０．２４ｍｇ／ｍｌ作用させた場合（レーン４）、フコイダンおよび酵母を加えていないサンプル（レーン１）と同等の活性化しか見られなかった。酵母を０．２４ｍｇ／ｍｌとしてフコイダンの量を増加させていくと、活性化がさらに増強され、フコイダンを０．１ｍｇ／ｍｌとした場合（レーン６）、フコイダンおよび酵母を加えていないサンプル（レーン１）の２４倍の活性化が見られた。レーン６の効果は、レーン３とレーン４の効果の和よりも３．３倍多く、フコイダンと酵母との相乗効果であるといえる。 As can be seen from FIG. 1, almost no activation was observed in the sample to which fucoidan and yeast were not added (lane 1). Increasing the amount of fucoidan without adding yeast enhanced activation. When fucoidan was allowed to act alone at a concentration of 0.1 mg / ml (lane 3), 7-fold activation was observed compared to the sample without addition of fucoidan and yeast (lane 1). When yeast was allowed to act alone at 0.24 mg / ml (lane 4), only activation equivalent to the sample without addition of fucoidan and yeast (lane 1) was observed. When the amount of fucoidan was increased by setting the yeast to 0.24 mg / ml, the activation was further enhanced. When fucoidan was 0.1 mg / ml (lane 6), the sample without addition of fucoidan and yeast (lane) 24 times the activation of 1) was observed. The effect of lane 6 is 3.3 times greater than the sum of the effects of lane 3 and lane 4, and can be said to be a synergistic effect of fucoidan and yeast.
さまざまな分子量のフコイダン（６０ｋＤａ、１３５ｋＤａ、３３２ｋＤａ（３３２ｋＤａのみ２サンプル））を用いて、マクロファージの活性化を行った。実験は実施例１と同様にして行った。 Macrophage activation was performed using fucoidans of various molecular weights (60 kDa, 135 kDa, 332 kDa (only 2 samples of 332 kDa)). The experiment was performed in the same manner as in Example 1.
活性化後、ＣＯＸ２およびＧＡＰＤＨ（ローディングコントロール）に関してイムノブロットを行った結果を図２Ａに示す。さらに、ＧＡＰＤＨ（ローディングコントロール）の量により補正したＣＯＸ２タンパク質の量を図２Ｂに示す。 FIG. 2A shows the result of immunoblotting for COX2 and GAPDH (loading control) after activation. Furthermore, the amount of COX2 protein corrected by the amount of GAPDH (loading control) is shown in FIG. 2B.
レーン１〜４は、フコイダンのみでマクロファージを活性化した結果である。分子量の大きなフコイダンのほうがマクロファージの活性化が強く起こり、分子量３３２ｋＤａのフコイダンの活性化作用が最大であった。 Lanes 1 to 4 are results of activating macrophages with fucoidan alone. Macrophage activation occurred more strongly with fucoidan having a higher molecular weight, and the activation effect of fucoidan with a molecular weight of 332 kDa was the largest.
レーン５〜８は、フコイダン（終濃度４００μｇ／ｍｌと酵母（終濃度２４０μｇ／ｍｌ）を同時に作用させた結果である。フコイダン単独で作用させた場合よりも、活性化が増強された。フコイダンの分子量を増加させていくと、活性化も増強された。最も強い活性化が観察されたのは分子量３３２ｋＤａのフコイダンであった。６０ｋＤａ、１３５ｋＤａのフコイダンは、酵母と共存させていても（レーン５、レーン６）、３３２ｋＤａのフコイダンを単独で作用させた場合（レーン４）と同程度の活性化しか観察されなかった。このことから、酵母とフコイダンを共存させてマクロファージを活性化させる場合、フコイダンの分子量が大きいほど活性化が増強され、分子量約３０万以上のフコイダンが好ましいことがわかった。 Lanes 5 to 8 show the results of simultaneous action of fucoidan (final concentration of 400 μg / ml and yeast (final concentration of 240 μg / ml)), and activation was enhanced as compared to the case of fucoidan alone. As the molecular weight was increased, the activation was also enhanced, and the strongest activation was observed for fucoidan with a molecular weight of 332 kDa, although fucoidan with a molecular weight of 60 kDa and 135 kDa coexisted with yeast (lane 5). Lane 6) When the 332 kDa fucoidan was allowed to act alone (lane 4), only the same level of activation was observed, indicating that when activating macrophages in the presence of yeast and fucoidan, fucoidan It was found that the higher the molecular weight, the higher the activation and the more preferable fucoidan having a molecular weight of about 300,000 or more.
本発明の免疫賦活剤は、効果が優れ、しかも安全性が高いものであるから、食品や医薬品の分野において利用可能である。 Since the immunostimulant of the present invention has excellent effects and high safety, it can be used in the fields of foods and pharmaceuticals.
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