JP2017075104A - Allergic symptom palliative agent - Google Patents
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本発明は、ヒトなどのアレルギー症状を緩和させるための経口剤等に関するものである。 The present invention relates to an oral preparation for alleviating allergic symptoms in humans and the like.
現在では、日本人の２人に１人が何らかのアレルギー疾患に羅患していると言われており、これは従前の統計より増加している。アレルギー疾患の代表例としては、アトピー性皮膚炎、花粉症、喘息、食物アレルギーなどが挙げられ、例えば、花粉症は日本人の３分の１が羅患するいわば国民病である。 At present, it is said that one out of every two Japanese suffers from some allergic disease, which is an increase over previous statistics. Representative examples of allergic diseases include atopic dermatitis, hay fever, asthma, food allergies, etc. For example, hay fever is a national disease that affects one third of Japanese people.
花粉症は、Ｉ型アレルギーに分類され、アトピー性皮膚炎や食物アレルギーなどと同様に、体内のＩｇＥ抗体が好塩基球やマスト細胞に結合し、ここにアレルゲンが反応することによって症状が発症する。アレルゲンによる刺激がなされると、好塩基球やマスト細胞は細胞内に保持していた顆粒を細胞外に放出し、この顆粒中にはヒスタミンやロイコトリエンといったアレルギー症状を誘発する物質が含まれており、この結果、花粉症特有の目や鼻の症状が発症する。 Hay fever is categorized as type I allergy and, like atopic dermatitis and food allergy, IgE antibodies in the body bind to basophils and mast cells, and allergens react there to develop symptoms. . When stimulated by allergens, basophils and mast cells release the granules held inside the cells, and these granules contain substances that induce allergic symptoms such as histamine and leukotrienes. As a result, symptom of eyes and nose peculiar to hay fever develops.
このようなアレルギー症状を緩和するためには抗ヒスタミン薬などを用いることが多いが、副作用や継続使用による効果の大幅な低減など問題も多い。したがって、多くの人は、食経験の少ない医薬成分の服用よりはむしろ、アレルギー症状緩和効果のある食品成分の摂取により症状を和らげたいと考えている。このようなことから、食経験が豊富な食品由来成分を用いた抗アレルギー食品や抗アレルギー薬の研究・開発もいくつか進められている。 In order to alleviate such allergic symptoms, antihistamines and the like are often used, but there are also many problems such as side effects and a significant reduction in the effects of continuous use. Therefore, many people want to relieve symptoms by taking food ingredients that have an alleviating effect on allergic symptoms, rather than taking medicinal ingredients with little food experience. For this reason, several researches and developments of antiallergic foods and antiallergic drugs using ingredients derived from foods that have abundant food experience have been made.
このような食品由来成分としては、例えば、牛乳の主要タンパク質であるβ−ラクトグロブリンと柑橘フラボノイドであるノビレチンを併用したもの（特許文献１）、茶葉抽出物を利用したもの（特許文献２）、イモ焼酎粕を利用したもの（特許文献３）などが提案されているが、いまだ十分とは言えないのが現状である。 As such a food-derived component, for example, a combination of β-lactoglobulin, which is a major protein of milk, and nobiletin, which is a citrus flavonoid (Patent Document 1), a tea leaf extract (Patent Document 2), Although the thing using a potato shochu (patent document 3) etc. is proposed, it is not sufficient now.
以上のような技術背景の中、当業界においては、食経験が豊富な食品由来成分を用いた抗アレルギー食品や抗アレルギー薬、特に好塩基球やマスト細胞の脱顆粒などを効果的に抑制できるような食品由来成分を用いた剤などのさらなる開発が引き続き求められていた。 In the technical background as described above, this industry can effectively suppress anti-allergic foods and anti-allergic drugs, especially basophils and mast cell degranulation, using ingredients derived from foods with abundant dietary experience. There has been a continuing demand for further development of agents using such food-derived ingredients.
本発明は、ヒトなどのアレルギー症状を効果的に緩和させることが可能な、経口用剤として用いても安全・安心なアレルギー症状緩和剤（機能性食品、医薬品等）を提供することを目的とする。 It is an object of the present invention to provide an allergic symptom relieving agent (functional food, medicine, etc.) that can effectively relieve allergic symptoms of humans and the like and can be used safely as an oral preparation. To do.
上記目的を達成するため、本発明者らは鋭意研究の結果、食経験が豊富な煮干し粉砕品をプロテアーゼで処理した上清液（煮干し粉砕品酵素処理液）又はその乾燥物等を有効成分とする機能性食品、医薬品等により、ヒトなどが経口用剤として安心して用いることが可能であり、且つ、その好塩基球の脱顆粒抑制やマクロファージのサイトカイン産生促進などの作用によってアレルギー症状を効果的に緩和できることを見出し、本発明を完成するに至った。 In order to achieve the above object, as a result of intensive studies, the present inventors have effectively used a supernatant liquid (boiled ground pulverized product enzyme-treated solution) obtained by treating a boiled dried ground product with abundant experience with protease or a dried product thereof. Functional foods, pharmaceuticals, etc., which are ingredients, can be used safely by humans as oral preparations, and allergic symptoms can be prevented by actions such as inhibiting basophil degranulation and promoting macrophage cytokine production. The inventors have found that it can be effectively relaxed and have completed the present invention.
すなわち、本発明の実施形態は次のとおりである。
（１）煮干し粉砕品をプロテアーゼで処理した上清液又はその乾燥物を有効成分とすることを特徴とする、アレルギー症状緩和剤。
（２）煮干し粉砕品をプロテアーゼで処理した上清から、分子量５００以下の分子を除去（主として脱塩）した処理液又はその乾燥物を有効成分とすることを特徴とする、アレルギー症状緩和剤。
（３）煮干し粉砕品をプロテアーゼで処理した上清から、分子量１４０００以上の分子を除去した処理液又はその乾燥物を有効成分とすることを特徴とする、アレルギー症状緩和剤。
（４）煮干し粉砕品をプロテアーゼで処理した上清から、分子量５００以下及び分子量１４０００以上の分子を除去した処理液又はその乾燥物を有効成分とすることを特徴とする、アレルギー症状緩和剤。
（５）上記上清液又は処理液を更に陰イオン交換カラムクロマトグラフィー処理して得た非吸着画分（液体又は乾燥物）を有効成分とすることを特徴とする、（１）〜（４）のいずれか１つに記載の剤。
（６）１００℃２０分間の加熱処理で活性が低下しないことを特徴とする、（１）〜（５）のいずれか１つに記載の剤。
（７）アレルギー症状緩和が、好塩基球の脱顆粒抑制及び／又はマクロファージのサイトカイン産生促進である、（１）〜（６）のいずれか１つに記載の剤。
That is, the embodiment of the present invention is as follows.
(1) An allergy symptom alleviating agent characterized by comprising a supernatant obtained by treating a boiled and dried product with a protease or a dried product thereof as an active ingredient.
(2) An allergy symptom alleviating agent characterized by comprising, as an active ingredient, a treatment liquid obtained by removing (mainly desalting) molecules having a molecular weight of 500 or less from a supernatant obtained by treating a boiled and dried product with a protease. .
(3) An allergy symptom alleviating agent characterized by comprising, as an active ingredient, a treatment liquid obtained by removing molecules having a molecular weight of 14,000 or more from a supernatant obtained by treating a dried and pulverized product with a protease.
(4) An allergy symptom alleviating agent characterized by comprising, as an active ingredient, a treatment liquid obtained by removing molecules having a molecular weight of 500 or less and a molecular weight of 14000 or more from a supernatant obtained by treating a dried and ground product with a protease.
(5) The non-adsorbed fraction (liquid or dried product) obtained by further subjecting the supernatant or the treatment solution to anion exchange column chromatography is used as an active ingredient, (1) to (4) ) The agent according to any one of
(6) The agent according to any one of (1) to (5), wherein the activity does not decrease by heat treatment at 100 ° C. for 20 minutes.
(7) The agent according to any one of (1) to (6), wherein the allergic symptom alleviation is suppression of degranulation of basophils and / or promotion of cytokine production of macrophages.
（８）煮干し粉砕品をプロテアーゼで処理した上清液又はその乾燥物を、１日当たり０．１〜５０ｍｇ／ｋｇ、好ましくは１．０〜３０ｍｇ／ｋｇで経口投与することを特徴とする、アレルギー症状緩和方法（ヒトに対する医療行為を除く）。 (8) A supernatant obtained by treating a dried and ground product with a protease or a dried product thereof is orally administered at 0.1 to 50 mg / kg, preferably 1.0 to 30 mg / kg per day, Allergic symptom alleviation method (except medical practice for humans).
本発明によれば、ヒトなどのアレルギー症状を緩和するための安全・安心な経口剤等の提供が可能となり、また、この剤等の経口投与により好塩基球の脱顆粒抑制やマクロファージのサイトカイン産生促進などが行われ、アレルギー症状を効果的に緩和できる。そして、この結果として、アレルギー患者などの生活の質（ＱＯＬ）をより向上させることができる。 According to the present invention, it is possible to provide a safe and reliable oral preparation for alleviating allergic symptoms in humans and the like. In addition, oral administration of this preparation or the like can suppress basophil degranulation and macrophage cytokine production. Promotion is carried out and allergic symptoms can be effectively alleviated. As a result, the quality of life (QOL) of allergic patients and the like can be further improved.
本発明では、アレルギー症状緩和剤等の有効成分として、煮干し粉砕品をプロテアーゼで処理した上清液又はその乾燥物等を使用する。 In the present invention, as an active ingredient such as an allergy symptom alleviating agent, a supernatant obtained by treating a boiled and ground product with a protease or a dried product thereof is used.
この煮干し粉砕品は、カタクチイワシなどの小魚を煮てから干した乾物を粉砕したものを意味し、特に、カタクチイワシの煮干しを粉砕機、ハンマーミル等で処理して細粉砕したものを用いることが好ましい。 This boiled and dried pulverized product means a product obtained by pulverizing dried fish boiled after a small fish such as anchovy, and in particular, an anchovy simmered by processing with a pulverizer, a hammer mill, etc. It is preferable.
そして、この煮干し粉砕品をプロテアーゼ処理する。使用するプロテアーゼは、微生物から得られた、作用最適ｐＨが酸性域（例えばｐＨ２〜６など）の食品用プロテアーゼが好ましく、特に、糸状菌Ａｓｐｅｒｇｉｌｌｕｓ属由来の酸性プロテアーゼを用いるのが好適である。市販品としては、デナプシン２Ｐ（Ａｓｐｅｒｇｉｌｌｕｓ ｎｉｇｅｒ由来、ナガセケムテックス株式会社製品）などが例示される。
Then, the dried pulverized product is treated with protease. As the protease to be used, a protease for food obtained from a microorganism and having an optimum pH of action in an acidic range (for example,
プロテアーゼ処理は、例えば次のようにして行う。まず、煮干し粉砕品に１〜１０倍量（好ましくは３〜５倍量）加水し、必要に応じてｐＨ２〜６に調整を行ってから酵素を０．０５〜３．０％（好ましくは０．１〜１．０％）添加して、使用酵素の至適温度である２０〜５５℃（好ましくは３０〜５０℃）で０．５〜３０時間（好ましくは１〜２０時間）反応を行う。なお、酵素反応は攪拌しながら行うのが好ましいが、酵素反応を円滑に進める手段を行うのであればこれに限定されるものではない。
For example, the protease treatment is performed as follows. First, add 1 to 10 times (preferably 3 to 5 times) amount of water to the boiled and dried product, adjust to
プロテアーゼ処理後は、必要であれば中和処理を行い、７０℃以上（好ましくは９０℃〜１００℃）の温度に２〜６０分間（好ましくは５〜３０分間）保持して酵素失活を行った後、不溶性固形分除去処理（遠心分離、フィルター濾過）を行って上清液を得る。その後、必要に応じて濃縮処理、滅菌、殺菌処理、粉末化（乾燥化）処理などを行っても良い。なお、粉末化は、スプレードライ、ドラムドライ、凍結乾燥などの定法により行うことができる。これにより、粉末剤形態の製剤などを得ることができる。また、不溶性固形分除去処理後の上清液から、さらに透析等により低分子成分や高分子成分を除去しても良い。透析を行う場合、例えば、分子量５００以下カットオフの透析膜、分子量１４０００以上カットオフの透析膜等により除去処理を行う。 After protease treatment, neutralization treatment is performed if necessary, and enzyme inactivation is performed by maintaining the temperature at 70 ° C. or higher (preferably 90 ° C. to 100 ° C.) for 2 to 60 minutes (preferably 5 to 30 minutes). After that, insoluble solid content removal treatment (centrifugation, filter filtration) is performed to obtain a supernatant. Thereafter, concentration treatment, sterilization, sterilization treatment, powderization (drying) treatment, or the like may be performed as necessary. In addition, powdering can be performed by a conventional method such as spray drying, drum drying, or freeze drying. Thereby, the formulation of a powder form etc. can be obtained. Further, low molecular components and high molecular components may be further removed from the supernatant after the insoluble solid content removal treatment by dialysis or the like. When dialysis is performed, for example, the removal treatment is performed using a dialysis membrane having a molecular weight of 500 or less and a dialysis membrane having a molecular weight of 14,000 or more and a cutoff.
本発明品の形態は、上記のような粉末剤のみならず、顆粒剤、錠剤、カプセル剤、散剤、スティック剤、液剤、ゲル剤、ペースト剤、シロップ剤などでも良く、経口投与可能な形態であれば特段の限定はない。そして、本発明品では、有効成分の効果を妨げない範囲で、賦形剤、結合剤、崩壊剤、滑沢剤、矯味矯臭剤、溶解補助剤、懸濁剤、コーティング剤などの製剤技術分野において通常使用しうる既知の補助剤の１種又は２種以上を適宜組み合わせて配合した製剤とすることもできる。そして、形態、組成等を勘案し、原料の段階から製品が完成するまでの工程で本発明の有効成分と補助剤等を適宜配合して製造すればよい。 The form of the product of the present invention may be not only a powder as described above, but also a granule, tablet, capsule, powder, stick, liquid, gel, paste, syrup, etc. If there is no special limitation. And in the product of the present invention, formulation technical fields such as excipients, binders, disintegrants, lubricants, flavoring agents, solubilizing agents, suspending agents, coating agents, etc., as long as the effects of the active ingredients are not hindered. In addition, it can also be set as the formulation which mix | blended combining suitably the 1 type (s) or 2 or more types of the known adjuvant which can be normally used. Then, considering the form, composition, etc., the active ingredient of the present invention, adjuvants and the like may be appropriately blended and manufactured in the process from the raw material stage to the completion of the product.
本発明のアレルギー症状緩和剤の用法ないし用量については、使用目的（予防、治療、又は保健の用途）、被検者の年齢、剤形等に応じて適宜定めればよく、０．１〜５０ｍｇ／ｋｇ／日、好ましくは１．０〜３０ｍｇ／ｋｇ／日の煮干し粉砕品酵素処理液又はその乾燥物等を、ヒトなどに対して例えば３０日間以上経口投与するのが好適である。しかしながら、本有効成分は安全性について全く問題がないので、上記範囲よりも多量使用しても一向にさしつかえない。 The usage or dose of the allergic symptom alleviating agent of the present invention may be appropriately determined according to the purpose of use (prevention, treatment, or health use), the age of the subject, the dosage form, etc. It is suitable to orally administer a boiled pulverized product enzyme-treated solution or a dried product thereof, for example, for 30 days or more to a human / kg / day, preferably 1.0 to 30 mg / kg / day. However, since this active ingredient has no safety problem, even if it is used in a larger amount than the above range, there is no problem.
なお、本発明は、煮干し粉砕品をプロテアーゼで処理した上清液又はその乾燥物等を有効成分とするアレルギー症状緩和剤をヒトなどに経口投与してアレルギー症状を緩和させるものであるが、これは医薬品（医薬剤）だけでなく、健康補助食品、保健機能食品、サプリメント等の、特定の機能及び形態を有し、健康維持などを目的として摂取され、有効成分の用量（有効量）や用法が規定され且つ単位包装当たりでその用量が摂取できる、単に食品としてのみ利用されるものとは明確に区別される食品組成物（医薬部外品を含む）も含まれる。 In the present invention, allergic symptom-relieving agent comprising a supernatant liquid obtained by treating a dried pulverized product with a protease or a dried product thereof as an active ingredient is orally administered to humans, etc. This has specific functions and forms, such as health supplements, health functional foods, and supplements, as well as pharmaceuticals (pharmaceuticals), and is taken for the purpose of maintaining health. Also included are food compositions (including quasi-drugs) that are clearly distinguished from those that are used only as food, where usage is prescribed and the dose can be taken per unit package.
このようにして、煮干し粉砕品をプロテアーゼで処理した上清液又はその乾燥物等を有効成分としてなるアレルギー症状緩和剤等を使用し、これをアレルギー患者などに経口投与することで、安全に且つ効果的にアレルギー症状を緩和させることが可能となる。 In this way, by using an allergic symptom-relieving agent or the like comprising an active ingredient of a supernatant or a dried product obtained by treating a boiled and dried product with protease, it can be safely administered orally to allergic patients, etc. And it becomes possible to relieve allergic symptoms effectively.
なお、本発明は、煮干し粉砕品をプロテアーゼで処理した上清液又はその乾燥物等を有効成分とするアレルギー症状緩和剤というプロセスによって特定された物の発明であるが、この有効成分中の具体的な活性成分はいまだ特定できておらず、また、この活性成分特定は著しく過大な経済的支出や時間を要するものであると推察されるため、この活性成分をその構造又は特性により直接特定することが不可能であるか、又はおよそ実際的でないという事情が存在すると認められる。 The present invention is an invention of a product specified by a process called an allergy symptom alleviating agent comprising a supernatant obtained by treating a boiled and dried product with a protease or a dried product thereof as an active ingredient. The specific active ingredient has not yet been identified, and it is surmised that the identification of this active ingredient requires significantly excessive economic expenditure and time. Therefore, this active ingredient is directly identified by its structure or characteristics. It is recognized that there are circumstances where it is impossible or nearly impractical to do.
以下、本発明の実施例について述べるが、本発明はこれらの実施例のみに限定されるものではなく、本発明の技術的思想内においてこれらの様々な変形が可能である。 Examples of the present invention will be described below, but the present invention is not limited to these examples, and various modifications can be made within the technical idea of the present invention.
（アレルギー症状緩和剤の製造）
以下の方法で、本発明品である煮干し粉砕品をプロテアーゼで処理した上清液（煮干し粉砕品酵素処理液）を有効成分とするアレルギー症状緩和剤を製造した。
(Manufacture of allergic symptom relief agents)
By the following method, an allergic symptom alleviating agent comprising as an active ingredient a supernatant liquid (boiled and dried pulverized product enzyme-treated solution) obtained by treating the dried product of the present invention with protease was prepared.
煮干し粉砕品に、その重量に対して３．７倍量の水を添加し、ｐＨ調整（ｐＨ３．３〜４．３）を行ってから、０．３％のプロテアーゼ（デナプシン２Ｐ：ナガセケムテック株式会社製品）を添加し、酵素の至適温度（４０〜５０℃）で１５〜２０時間処理した。その後、中和処理（ｐＨ５．２〜５．６に調整）を行い、加熱処理（９７〜９９℃で３０分間加熱）により酵素を失活させ、遠心分離操作により固形分を除去し、上清液を回収した。このようにして得られた煮干し粉砕品酵素処理液をアレルギー症状緩和剤とした。 After adding 3.7 times the amount of water to the dried pulverized product and adjusting the pH (pH 3.3 to 4.3), 0.3% protease (denapsin 2P: Nagase Chem) (Tech Co., Ltd. product) was added and treated at the optimum enzyme temperature (40-50 ° C.) for 15-20 hours. Thereafter, neutralization treatment (adjusted to pH 5.2 to 5.6) is performed, the enzyme is deactivated by heat treatment (heating at 97 to 99 ° C. for 30 minutes), the solid content is removed by centrifugation, and the supernatant is removed. The liquid was collected. The boiled and dried product enzyme-treated solution thus obtained was used as an allergy symptom alleviating agent.
（アレルギー症状緩和機能評価確認試験Ｉ）
実施例１で製造したアレルギー症状緩和剤の効果を機能評価確認するため、以下の試験を実施した。なお、機能評価確認試験を実施するために、煮干し粉砕品酵素処理液を分子量５００以下カットオフの透析膜で脱塩処理したものを用いた。
(Allergy symptom alleviation function evaluation confirmation test I)
In order to confirm the functional evaluation of the effect of the allergic symptom-relieving agent produced in Example 1, the following tests were conducted. In addition, in order to carry out a function evaluation confirmation test, a desalted dialysis membrane with a molecular weight of 500 or less cut off was used.
まず、好塩基球によるヒスタミンを含む顆粒の放出を指標とした、ラット好塩基球細胞株ＲＢＬ−２Ｈ３細胞（ヒューマンサイエンス研究資源バンクより分与）を用いた脱顆粒評価系を用いて確認した。脱塩処理した煮干し粉砕品酵素処理液を各種タンパク質濃度に希釈したもので常法により培養したＲＢＬ−２Ｈ３細胞を処理し、コントロール（バッファー処理）も含め、これらの細胞を溶解処理して得た細胞溶出物についてブランク（細胞培養上清）とともに活性を測定した。なお、脱顆粒抑制活性は、ヒスタミンと同様に顆粒中に含まれ、定量がヒスタミンより容易なβ−ヘキソサミニダーゼ放出率の測定・算出により評価した。また、細胞溶解処理直前の相対生細胞数も測定し、細胞の生存に対する影響も確認した。 First, it confirmed using the degranulation evaluation system using the rat basophil cell line RBL-2H3 cell (distributed from the human science research resource bank) which used the release | release of the granule containing histamine by a basophil as an parameter | index. Obtained by treating RBL-2H3 cells cultured in a conventional manner with diluted desiccated dried lysed enzyme treatment solutions to various protein concentrations and lysing these cells, including controls (buffer treatment) The cell eluate was assayed for activity along with a blank (cell culture supernatant). The degranulation inhibitory activity was evaluated by measuring and calculating the β-hexosaminidase release rate, which is contained in the granule in the same manner as histamine and is easier to quantify than histamine. In addition, the relative number of living cells immediately before the cell lysis treatment was measured, and the influence on cell survival was also confirmed.
この結果、煮干し粉砕品酵素処理液を有効成分とするアレルギー症状緩和剤は、抗原刺激によって誘発されるＲＢＬ−２Ｈ３細胞の脱顆粒を濃度依存的に抑制した（図１の左側グラフ）。また、この時、細胞の生存には影響を与えないことから（図１の右側グラフ）、これが細胞傷害性による脱顆粒の抑制ではないことが明らかになった。 As a result, the allergic symptom alleviating agent comprising the boiled and ground product enzyme-treated solution as an active ingredient suppressed the degranulation of RBL-2H3 cells induced by antigen stimulation in a concentration-dependent manner (left graph in FIG. 1). In addition, at this time, since it does not affect the survival of the cells (right graph in FIG. 1), it was revealed that this is not suppression of degranulation due to cytotoxicity.
さらに、この脱顆粒抑制効果が、単に抗原と抗体の結合を阻害することに由来するものであるかを検討するため、ＩｇＥ抗体とその抗原であるジニトロフェニル（ＤＮＰ）の抗原抗体反応に及ぼすアレルギー症状緩和剤の影響を検討した。具体的には、抗原（ＤＮＰ−ＨＳＡ）、１次抗体（抗ＤＮＰ−ＩｇＥ）及び２次抗体（抗マウスＩｇＥ）を用いた系に脱塩処理した煮干し粉砕品酵素処理液を加え、コントロールとともにＥＬＩＳＡ法により比色定量を行った。 Furthermore, in order to examine whether this degranulation inhibitory effect is simply derived from inhibiting the binding between an antigen and an antibody, allergies exerted on the antigen-antibody reaction of IgE antibody and its antigen dinitrophenyl (DNP). The effects of symptom relief agents were examined. Specifically, the desalted and dried boiled product enzyme treatment solution was added to the system using the antigen (DNP-HSA), the primary antibody (anti-DNP-IgE) and the secondary antibody (anti-mouse IgE), and the control was performed. At the same time, colorimetric determination was performed by ELISA.
この結果、煮干し粉砕品酵素処理液を有効成分とするアレルギー症状緩和剤は、抗原抗体反応には何ら影響しないことが明らかになった（図２）。したがって、この脱顆粒抑制効果は、単に好塩基球細胞表面での抗体による抗原刺激を阻害するものではなく、抗原刺激が成立したあとの細胞内のシグナル伝達を阻害することで脱顆粒を抑制していることが明らかになった。 As a result, it was clarified that the allergic symptom alleviating agent containing the boiled and pulverized product enzyme-treated solution as an active ingredient has no effect on the antigen-antibody reaction (FIG. 2). Therefore, this degranulation inhibitory effect does not simply inhibit antigen stimulation by antibodies on the surface of basophil cells, but inhibits degranulation by inhibiting intracellular signal transduction after antigen stimulation is established. It became clear that.
また、Ａ２３１８７誘導性の脱顆粒について細胞内カルシウムイオン濃度の挙動も含めて同様に確認を行った結果、煮干し粉砕品酵素処理液を有効成分とするアレルギー症状緩和剤は、細胞内カルシウムイオン濃度上昇の抑制なしにＡ２３１８７誘導性の脱顆粒を抑制することが示された（図３）。 Moreover, as a result of confirming A23187-induced degranulation in the same manner including the behavior of intracellular calcium ion concentration, the allergic symptom-relieving agent containing a boiled and ground product enzyme-treated solution as an active ingredient It was shown to suppress A23187-induced degranulation without suppression of elevation (FIG. 3).
ここで、煮干し粉砕品酵素処理液中に含まれる活性物質を推察するため、まず、分子量１４０００以下カットオフの透析膜で透析したあとのサンプルを各種タンパク質濃度に希釈したものでＲＢＬ−２Ｈ３細胞を処理した際の脱顆粒抑制活性（β−ヘキソサミニダーゼ放出率）を同様に測定・算出した。この結果、透析処理により脱顆粒抑制活性は完全に消失した（図４）。このことから、煮干し粉砕品酵素処理液中に含まれる活性物質の分子量は１４０００よりも小さい分子であることが推察された。 Here, in order to infer the active substance contained in the boiled and pulverized product enzyme-treated solution, first, RBL-2H3 cells were prepared by diluting the sample after dialysis with a dialysis membrane having a molecular weight of 14,000 or less and cut off to various protein concentrations. The degranulation inhibitory activity (β-hexosaminidase release rate) at the time of treatment was similarly measured and calculated. As a result, degranulation inhibitory activity completely disappeared by dialysis treatment (FIG. 4). From this, it was speculated that the molecular weight of the active substance contained in the enzyme-treated solution of the dried and dried product was a molecule smaller than 14,000.
次にＳｕｐｅｒＱ ６５０Ｍゲル（ＴＯＳＯＨ社製品）による陰イオン交換カラムクロマトグラフィーによる精製を試みた。脱塩処理した煮干し粉砕品酵素処理液をこの陰イオン交換カラムによる分画の後、得られたフラクションの脱顆粒抑制活性（β−ヘキソサミニダーゼ活性）を同様に測定・算出した結果、活性は非吸着画分（フラクション１）に認められた（図５）。 Next, purification by anion exchange column chromatography using SuperQ 650M gel (product of Tosoh Corporation) was attempted. As a result of measuring and calculating the degranulation inhibitory activity (β-hexosaminidase activity) of the obtained fraction in the same manner after fractionating the desalted dried boiled product enzyme-treated solution with this anion exchange column, Activity was found in the non-adsorbed fraction (fraction 1) (FIG. 5).
さらに、脱塩処理した煮干し粉砕品酵素処理液を１００℃で加熱処理した後の残存活性を上記と同様に測定・算出した。その結果、２０分までの加熱処理は、脱顆粒抑制活性には影響しないことが明らかになった（図６）。
これらのことから、煮干し粉砕品酵素処理液に含まれるアレルギー症状緩和効果を有する主成分は、陰イオン交換カラムの非吸着画分にある分子量が５００〜１４０００の間の物質であり、熱に安定であることが明らかになった。
Furthermore, the residual activity after heat-treating the desalted boiled and dried product enzyme-treated solution at 100 ° C. was measured and calculated in the same manner as described above. As a result, it was clarified that the heat treatment up to 20 minutes does not affect the degranulation inhibitory activity (FIG. 6).
From these things, the main component which has the allergy symptom relieving effect contained in the boiled and ground product enzyme treatment liquid is a substance having a molecular weight of 500 to 14000 in the non-adsorbed fraction of the anion exchange column, It became clear that it was stable.
（アレルギー症状緩和機能評価確認試験ＩＩ）
実施例１で製造したアレルギー症状緩和剤の効果をさらに機能評価確認するため、以下の試験を実施した。なお、実施例２と同様に、機能評価確認試験を実施するため煮干し粉砕品酵素処理液を分子量５００以下カットオフの透析膜で脱塩処理したものを用いた。
(Allergy symptom alleviation function evaluation confirmation test II)
In order to further confirm the functional evaluation of the effect of the allergic symptom alleviating agent produced in Example 1, the following tests were conducted. In addition, in the same manner as in Example 2, a boiled and pulverized product enzyme-treated solution that had been desalted with a dialysis membrane having a molecular weight of 500 or less was used in order to perform a function evaluation confirmation test.
ここでは、マウスマクロファージ細胞株Ｊ７７４．１細胞株、及びマウス腹腔から回収した初代腹腔マクロファージＰ−Ｍａｃのサイトカイン産生に及ぼす煮干し粉砕品酵素処理液の効果、及び、ＴＮＦ−α産生に及ぼす効果を検討した。 Here, the effect of the boiled and ground enzyme treatment solution on the cytokine production of the mouse macrophage cell line J774.1 cell line and the primary peritoneal macrophage P-Mac recovered from the mouse abdominal cavity, and the effect on the TNF-α production investigated.
脱塩処理した煮干し粉砕品酵素処理液を各種濃度に希釈したもので両細胞を処理したところ、Ｊ７７４．１細胞株、およびＰ−Ｍａｃの双方のＴＮＦ−α産生が、濃度依存的に促進されることが明らかになった。また、ＩＬ−６産生に及ぼす効果を検討した結果、ＩＬ−６産生も非常に強く促進されることが明らかになり、煮干し粉砕品酵素処理液はマクロファージを活性化する効果があることが明らかになった。 When both cells were treated with diluted desiccated dried and dried enzyme treatment solution to various concentrations, T77-α production of both J774.1 cell line and P-Mac was accelerated in a concentration-dependent manner. It became clear that Moreover, as a result of examining the effect on IL-6 production, it became clear that IL-6 production was also promoted very strongly, and it was found that the boiled and ground product treated with enzyme was effective in activating macrophages. Became.
さらに、受動皮膚アナフィラキシーモデルマウスに及ぼす煮干し粉砕品酵素処理液の投与効果を確認した。具体的には、受動皮膚アナフィラキシーモデルマウス（ＢＡＬＢ／ｃ（メス、７週齢）：日本エスエルシー社製品）にＩｇＥの耳介皮下投与を行い、それから２３時間後にサンプル（脱塩処理した煮干し粉砕品酵素処理液）を経口投与し、それから１時間後にアレルゲンの尾静脈注射を行い、その０．５時間後に耳介を回収してＥＬＩＳＡ法により比色定量を行った。 Furthermore, the administration effect of the boiled ground enzyme treatment solution on passive skin anaphylaxis model mice was confirmed. Specifically, IgE was subcutaneously administered to passive skin anaphylaxis model mice (BALB / c (female, 7 weeks old): product of Nippon SLC Co., Ltd.), and a sample (desalted boiled and dried after 23 hours) The pulverized product enzyme-treated solution) was orally administered, 1 hour later, the allergen was injected into the tail vein, and 0.5 hours later, the auricle was collected and colorimetrically determined by the ELISA method.
この結果、煮干し粉砕品酵素処理液の経口投与により受動皮膚アナフィラキシー応答を抑制できることが明らかとなった（図７）。 As a result, it was clarified that passive skin anaphylactic response can be suppressed by oral administration of the boiled and dried product enzyme-treated solution (FIG. 7).
（アレルギー症状緩和機能評価確認試験ＩＩＩ）
実施例１で製造したアレルギー症状緩和剤の効果をヒトで確認するため、以下の試験を実施した。
(Allergy symptom alleviation function evaluation confirmation test III)
In order to confirm the effect of the allergic symptom alleviating agent produced in Example 1 in humans, the following test was performed.
実施例１で得られた煮干し粉砕品酵素処理液の乾燥物を有効成分とするアレルギー症状緩和剤を用意し、春季にアレルギー症状を感じる年齢３０〜６０の男女４名（各２名）を被験者として、当該時期に有効成分を０．１ｇ／日の量で３０日間食事と供に経口投与を行った。 Prepare allergic symptom alleviation agent which uses dry substance of boiled and dried product enzyme treatment liquid obtained in Example 1 as active ingredient, and 4 men and women (2 each) of age 30-60 who feel allergic symptoms in spring As a test subject, the active ingredient was orally administered at a dose of 0.1 g / day with meals for 30 days.
その結果、アレルギー症状が緩和したと感じた人が３名、感じられなかった人が１名であった。この結果から、煮干し粉砕品酵素処理液の経口投与により、アレルギー症状を一定程度緩和できると推察された。 As a result, there were 3 people who felt that allergic symptoms were alleviated and 1 person who did not feel it. From this result, it was speculated that allergic symptoms could be alleviated to some extent by oral administration of the boiled and dried product enzyme treatment solution.
以上より、煮干し粉砕品酵素処理液が好塩基球に脱顆粒を抑制し、また、マクロファージのサイトカイン産生を促進し、ヒトなどのアレルギー症状を緩和させることができるということが示された。なお、煮干し粉砕品酵素処理液を所定量毎日食べ続けても、副作用等は全くなく、安全に継続摂取出来ることも確認された。 From the above, it was shown that the boiled and ground product enzyme-treated solution suppresses degranulation in basophils, promotes cytokine production of macrophages, and can alleviate allergic symptoms such as humans. In addition, it was confirmed that even if the predetermined amount of the boiled and ground product enzyme-treated solution was continuously eaten every day, there was no side effect at all and it could be safely and continuously ingested.
本発明を要約すれば、以下の通りである。 The present invention is summarized as follows.
本発明は、ヒトなどのアレルギー症状を効果的に緩和させることが可能な、経口用剤として用いても安全、安心な機能性食品、医薬品等を提供することを目的とする。 It is an object of the present invention to provide functional foods, pharmaceuticals, etc. that are safe and reliable even when used as oral preparations that can effectively alleviate allergic symptoms of humans and the like.
そして、食経験が豊富な煮干し粉砕品をプロテアーゼで処理した上清液又はその乾燥物等を有効成分とする機能性食品、医薬品等により、ヒトなどが経口用剤として安心して用いることが可能であり、且つ、好塩基球の脱顆粒抑制やマクロファージのサイトカイン産生促進などの作用によってアレルギー症状を効果的に緩和することができる。 And, it is possible for humans to use it as an oral preparation with peace of mind by using functional foods and pharmaceuticals, etc., whose active ingredients are supernatant liquids obtained by treating protease-dried boiled and dried products with abundant dietary experience. Moreover, allergic symptoms can be effectively alleviated by actions such as suppression of degranulation of basophils and promotion of cytokine production by macrophages.
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| KR900001722B1 (en) * | 1987-07-06 | 1990-03-19 | 주식회사 농심 | Process for extracting from small sardine |
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| JP2016189767A (en) * | 2015-03-30 | 2016-11-10 | 真実 大西 | Natural healing (dried small sardine) supplementary food (t.690) of osteoporosis and pollen allergic symptom, natural healing (snapping turtle egg) supplementary food (t.691) of osteoporosis and pollen allergic symptom, and natural healing (crab, lobster, shark, eel and shell bone) supplementary food (t.692) of osteoporosis and pollen allergic symptom |
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| KR900001722B1 (en) * | 1987-07-06 | 1990-03-19 | 주식회사 농심 | Process for extracting from small sardine |
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| JP2016189767A (en) * | 2015-03-30 | 2016-11-10 | 真実 大西 | Natural healing (dried small sardine) supplementary food (t.690) of osteoporosis and pollen allergic symptom, natural healing (snapping turtle egg) supplementary food (t.691) of osteoporosis and pollen allergic symptom, and natural healing (crab, lobster, shark, eel and shell bone) supplementary food (t.692) of osteoporosis and pollen allergic symptom |
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