JP2008247837A - Allergic constitution-improving agent - Google Patents
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本発明は、食品又は医薬として有用なアレルギー体質改善剤に関する。 The present invention relates to an allergic constitution improving agent useful as a food or a medicine.
従来、アレルギー疾患の予防あるいは治療には、ステロイド剤、抗ヒスタミン剤、ケミカルメディエーター遊離抑制剤、免疫抑制剤等の薬剤が用いられてきた。また、加水分解されたグルコマンナンやガラクトマンナンが、腸内においてアレルゲンや微生物の取り込みを減少させる効果を有することが報告されている（特許文献１）。しかしながら、これらはいずれもアレルギー症状の発症を抑えるもの、あるいはアレルギー症状を緩和させるものであり、アレルギー体質を改善するものではなかった。 Conventionally, drugs such as steroids, antihistamines, chemical mediator release inhibitors and immunosuppressants have been used for the prevention or treatment of allergic diseases. In addition, it has been reported that hydrolyzed glucomannan and galactomannan have an effect of reducing uptake of allergens and microorganisms in the intestine (Patent Document 1). However, these all suppress the onset of allergic symptoms or alleviate allergic symptoms, and do not improve the allergic constitution.
これに対して、ＩｇＥ抗体の産生を抑制するような薬剤は、アレルギー体質を改善するものとして期待され、トシル酸スプラタスト、ストリクチニン等の化合物が見出されている。しかしながら、トシル酸スプラタスト等の化合物には副作用等の問題がある。一方、グルコマンナンやこれの粉砕処理物に、ＩｇＥ産生抑制作用があることも報告されているが（特許文献２）、効果が不十分であり、微細加工に特殊な装置や技術を要し、かつ作用発現までに時間を要していた。これに鑑み、本願発明者らは、水溶性食物繊維の加水分解物にＩｇＥ産生抑制作用があることを報告している（特許文献３）。 In contrast, drugs that suppress the production of IgE antibodies are expected to improve allergic constitutions, and compounds such as suplatast tosylate and strictinin have been found. However, compounds such as suplatast tosylate have problems such as side effects. On the other hand, although it is also reported that glucomannan and the pulverized product thereof have an IgE production inhibitory action (Patent Document 2), the effect is insufficient, and a special device and technology are required for fine processing, In addition, it took time to manifest the action. In view of this, the present inventors have reported that the hydrolyzate of water-soluble dietary fiber has an IgE production inhibitory effect (Patent Document 3).
一方、硫酸化多糖類には、保湿作用があること（特許文献４）、マトリックスメタロプロテアーゼ阻害物質の産生を亢進する作用があること（特許文献５）等が報告されている。
しかしながら、グルコマンナン等の中性多糖類の硫酸化物に、ＩｇＥ産生抑制作用があることはこれまでに報告されていない。
However, it has not been reported so far that sulfates of neutral polysaccharides such as glucomannan have an inhibitory effect on IgE production.
本発明は、優れたアレルギー体質改善作用を有し、且つ安全で摂取しやすい、アレルギー体質改善剤を提供することに関する。 The present invention relates to providing an allergic constitution improving agent that has an excellent allergic constitution improving action and is safe and easy to ingest.
本発明者らは、ＩｇＥ産生抑制作用を有する素材に関して検討した結果、中性多糖類を多硫酸化した場合に優れたＩｇＥ産生抑制作用が発揮され、当該硫酸化物がアレルギー体質改善のための食品又は医薬として有用であることを見出した。 As a result of examining the material having an IgE production inhibitory action, the present inventors have demonstrated an excellent IgE production inhibitory action when polysulfating neutral polysaccharides, and the sulfate is a food for improving allergic constitution. Or it discovered that it was useful as a pharmaceutical.
すなわち、本発明は以下の（１）〜（４）を提供するものである。
（１）中性多糖類の硫酸化物又はその塩を有効成分とするアレルギー体質改善剤。
（２）中性多糖類の硫酸化物又はその塩を有効成分とするＩｇＥ産生抑制剤。
（３）中性多糖類がグルコマンナン又はセルロースである前記（１）のアレルギー体質改善剤又は（２）のＩｇＥ産生抑制剤。
（４）中性多糖類の硫酸化物又はその塩を含有するアレルギー体質改善食品。
That is, the present invention provides the following (1) to (4).
(1) An allergic substance improving agent comprising a neutral polysaccharide sulfate or a salt thereof as an active ingredient.
(2) An IgE production inhibitor comprising a neutral polysaccharide sulfate or a salt thereof as an active ingredient.
(3) The allergic substance improving agent of (1) or the IgE production inhibitor of (2), wherein the neutral polysaccharide is glucomannan or cellulose.
(4) Allergic constitution improving food containing sulfate of neutral polysaccharide or salt thereof.
本発明の中性多糖類の硫酸化物又はその塩は、ＩｇＥ産生抑制作用を有することから、Ｉ型アレルギーを発症しやすい人の体質改善に有効である。また、原料である中性多糖類は安価で入手しやすく日常的に食されていることから、本発明品も安全性が高く、高齢者から乳幼児に至るまで、安全で摂取しやすい食品又は医薬品となり得る。 Since the neutral polysaccharide sulfate or salt thereof of the present invention has an IgE production inhibitory action, it is effective in improving the constitution of a person who is prone to develop type I allergy. In addition, since the neutral polysaccharide as a raw material is inexpensive and easily available, it is eaten on a daily basis. Therefore, the product of the present invention is also highly safe and is a safe food that is easy to consume from the elderly to infants. Can be.
本発明の中性多糖類の硫酸化物とは、中性多糖類を多硫酸化した硫酸化物を意味する。中性多糖類としては、例えばデンプン、セルロース、グアガム、アラビアガム（アラビノガラクタン）、キサンタンガム、グルコマンナン、寒天アガロース、アミロペクチン、アミロース、デキストラン、プルラン、マンナン、グルカン等のＤ−グルコース、Ｄ−ガラクトース、Ｄ−マンノース、Ｄ−キシロース等を構成単糖とする多糖類が挙げられ、このうち、ＩｇＥ産生抑制効果の点からグルコマンナン、セルロース、デンプン等が好ましく、コンニャクグルコマンナン、セルロースがより好ましい。
斯かる中性多糖類は、硫酸化されることにより水溶性が大幅に向上し、例えばセルロースのように、水に難溶性の中性多糖類であっても水溶性となり、優れたＩｇＥ産生抑制効果が得られる。
The neutral polysaccharide sulfate of the present invention means a sulfate obtained by polysulfating a neutral polysaccharide. Neutral polysaccharides include, for example, starch, cellulose, guar gum, gum arabic (arabinogalactan), xanthan gum, glucomannan, agar agarose, amylopectin, amylose, dextran, pullulan, mannan, glucan, etc. D-glucose, D-galactose , D-mannose, polysaccharides having D-xylose and the like as constituent monosaccharides are mentioned. Of these, glucomannan, cellulose, starch and the like are preferable, and konjac glucomannan and cellulose are more preferable from the viewpoint of the IgE production inhibitory effect.
Such neutral polysaccharides are significantly improved in water solubility by being sulfated. For example, neutral polysaccharides that are sparingly soluble in water, such as cellulose, become water-soluble and have excellent IgE production inhibition. An effect is obtained.
本発明の中性多糖類の硫酸化物の塩としては、ナトリウム塩、カリウム塩等のアルカリ金属塩、カルシウム、マグネシウム等のアルカリ土類金属塩、アミン塩等が挙げられる。 Examples of the neutral polysaccharide sulfate salt of the present invention include alkali metal salts such as sodium salt and potassium salt, alkaline earth metal salts such as calcium and magnesium, and amine salts.
本発明の中性多糖類の硫酸化物が含有する硫酸基の数としては、繰り返し糖単位当たり平均０．１〜３．０個、好ましくは平均０．２〜２．０個である。 The number of sulfate groups contained in the neutral polysaccharide sulfate of the present invention is 0.1 to 3.0, preferably 0.2 to 2.0 on average per repeating saccharide unit.
本発明の中性多糖類の硫酸化物又はその塩の平均分子量は、０．５万〜３００万程度であることが望ましく、好ましくは、０．５万〜１０万程度であることが望ましい。 The average molecular weight of the neutral polysaccharide sulfate or salt thereof of the present invention is preferably about 5,000 to 3,000,000, and preferably about 50,000 to 100,000.
中性多糖類に硫酸基を導入する方法としては、公知の方法、例えば中性多糖類と硫酸化剤を適当な溶媒（例えば、ピリジン、ジメチルホルムアミド、ジメチルスルホキシド等）中、加温下で反応させる方法が挙げられる。硫酸化剤としては、例えば、三酸化イオウ−ＤＭＦ錯体、三酸化硫黄−ピリジン錯体、三酸化硫黄−トリメチルアミン錯体、クロルスルホン酸、ピペリジン−Ｎ−硫酸、無水硫酸−ジメチルホルムアミド錯体等が挙げられる。中性多糖類の硫酸化率は、硫酸化剤の使用割合及び反応条件を適宜選択することにより調整することができる。一般に、多糖類１重量部に対して硫酸化剤が０．５〜３質量部となるような割合で使用するのが好ましい。 As a method for introducing a sulfate group into a neutral polysaccharide, a known method, for example, reacting a neutral polysaccharide and a sulfating agent in an appropriate solvent (eg, pyridine, dimethylformamide, dimethyl sulfoxide, etc.) under heating. The method of letting it be mentioned. Examples of the sulfating agent include sulfur trioxide-DMF complex, sulfur trioxide-pyridine complex, sulfur trioxide-trimethylamine complex, chlorosulfonic acid, piperidine-N-sulfuric acid, anhydrous sulfuric acid-dimethylformamide complex, and the like. The sulfation rate of the neutral polysaccharide can be adjusted by appropriately selecting the use ratio of the sulfating agent and the reaction conditions. In general, it is preferable to use the sulfating agent at a ratio of 0.5 to 3 parts by mass with respect to 1 part by weight of the polysaccharide.
得られた中性多糖類の硫酸化物又はその塩の精製は、例えば、透析による脱塩、ゲル濾過クロマトグラフィーなどの分子サイズによる分画、有機溶媒を添加して沈殿させることによる回収操作、凍結乾燥による回収操作等を用いて行えばよい。 Purification of the obtained neutral polysaccharide sulfate or a salt thereof includes, for example, desalting by dialysis, fractionation by molecular size such as gel filtration chromatography, recovery operation by adding an organic solvent, and freezing. What is necessary is just to perform using collection | recovery operation etc. by drying.
斯くして得られた中性多糖類の硫酸化物又はその塩は、水溶性が高く、後記実施例に示すようにＩｇＥ産生抑制作用を有することから、ＩｇＥ産生抑制剤として、またアトピー性皮膚炎、気管支喘息、アレルギー性鼻炎等のＩ型アレルギーを発症しやすい人の当該アレルギー体質を改善するためのアレルギー体質改善剤として、食品、医薬品等の形態で使用できる。 Since the neutral polysaccharide sulfate or salt thereof thus obtained has high water solubility and has an IgE production inhibitory action as shown in the Examples below, it is also useful as an IgE production inhibitor and atopic dermatitis. In addition, it can be used in the form of food, medicine, etc. as an allergic constitution improving agent for improving the allergic constitution of a person who easily develops type I allergy such as bronchial asthma and allergic rhinitis.
本発明のアレルギー体質改善剤、ＩｇＥ産生抑制剤を医薬品として用いる場合、経口投与剤、非経口投与剤のいずれの製剤にもすることができ、例えば錠剤、顆粒剤、カプセル剤、粉末剤等の固形製剤、シロップ剤、エリキシル剤等の液状製剤の他、注射剤、坐剤、吸入剤（スプレー）、点眼、外用剤とすることができる。 When the allergic substance improving agent or IgE production inhibitor of the present invention is used as a pharmaceutical product, it can be made into an oral or parenteral preparation, such as a tablet, granule, capsule, powder, etc. In addition to liquid preparations such as solid preparations, syrups and elixirs, injections, suppositories, inhalants (sprays), eye drops, and external preparations can be used.
斯かる製剤は、中性多糖類の硫酸化物又はその塩を常法に従って薬学的に許容される担体と共に種々の剤型とすればよい。例えば、経口用固形製剤を調製する場合には、中性多糖類の硫酸化物又はその塩に賦形剤、必要に応じて結合剤、崩壊剤、滑沢剤、着色剤、矯味剤、矯臭剤等を加えた後、常法により錠剤、被覆錠剤、顆粒剤、散剤、カプセル剤等を製造することができる。また、注射剤とする場合は、担体として、例えば水、エチルアルコール、マクロゴール、プロピレングリコール等の各種希釈剤、ｐＨ調整剤及び緩衝剤、安定化剤、更に溶解補助剤、無痛化剤、局所麻酔剤等を適宜添加し、常法により皮下、筋肉内、静脈内用注射剤とすればよい。 Such a preparation may be prepared in various dosage forms with a neutral polysaccharide sulfate or a salt thereof together with a pharmaceutically acceptable carrier according to a conventional method. For example, when preparing an oral solid preparation, an excipient, a binder, a disintegrating agent, a lubricant, a coloring agent, a corrigent, a corrigent, if necessary, to a neutral polysaccharide sulfate or a salt thereof. Etc., tablets, coated tablets, granules, powders, capsules and the like can be produced by conventional methods. In addition, in the case of an injection, as a carrier, for example, various diluents such as water, ethyl alcohol, macrogol, propylene glycol, pH adjusters and buffers, stabilizers, further solubilizers, soothing agents, topical agents An anesthetic agent or the like may be added as appropriate, and an injection for subcutaneous, intramuscular or intravenous injection may be obtained by a conventional method.
本発明のアレルギー体質改善剤、ＩｇＥ産生抑制剤を健康食品、保健機能食品等の食品とする場合には、ビスケット類、チョコレート類、キャンデー類、チューインガム類、スナック菓子類、油菓類、アイスクリーム類、ゼリー菓子等の菓子、パン類、めん類、豆腐等の大豆加工品、ヨーグルト、バター等の乳製品、ソース、ドレッシング、マヨネーズ、ふりかけ等の調味料、発酵乳、果汁飲料、スポーツドリンク、スープ等の飲料の形態とすることができる。
尚、斯かる食品には、さらに、一般にアレルギーに効果があるとされる、茶、紫蘇、甜茶、月見草、タンポポ、柿葉、よもぎ、柑橘類等を配合しても良い。
When the allergic substance improving agent and IgE production inhibitor of the present invention are used as foods such as health foods and health functional foods, biscuits, chocolates, candy, chewing gums, snacks, oils, ice creams, Confectionery such as jelly confectionery, processed soybean products such as breads, noodles, and tofu, dairy products such as yogurt and butter, seasonings such as sauces, dressing, mayonnaise, sprinkles, fermented milk, fruit juice drinks, sports drinks, soups, etc. It can be in the form of a beverage.
Such foods may further contain tea, shiso, strawberry tea, evening primrose, dandelion, persimmon leaves, wormwood, citrus fruits and the like, which are generally considered to be effective for allergies.
上記アレルギー体質改善剤又はＩｇＥ産生抑制剤の１日当たりの投与量は、患者の症状、体重、年齢、性別等によって異なり一概には決定できないが、中性多糖類の硫酸化物又はその塩として通常成人１日当たり約３０ｍｇ〜３０ｇ、好ましくは約１００ｍｇ〜３ｇとすれば良く、これを１日１回又は２〜４回程度に分けて投与するのが好ましい。
以下、実施例により本発明を更に詳細に説明するが、本発明はこれらに限定されるものではない。
The daily dose of the allergic substance improving agent or IgE production inhibitor varies depending on the patient's symptoms, body weight, age, sex, etc., and cannot be determined unconditionally, but is usually an adult as a neutral polysaccharide sulfate or salt thereof About 30 mg to 30 g per day, preferably about 100 mg to 3 g may be used, and this is preferably administered once a day or divided into about 2 to 4 times a day.
EXAMPLES Hereinafter, although an Example demonstrates this invention further in detail, this invention is not limited to these.
実施例１：硫酸化コンニャクグルコマンナンのＩｇＥ産生抑制効果
（１）コンニャクグルコマンナン加水分解物の調製
コンニャクグルコマンナン（清水化学（株）製）２００ｍｇを蒸留水１７．５ｍＬに懸濁し、室温で１時間振盪してゲルを調製した。これに塩酸を終濃度０．２５Ｎとなるように加えて７５℃の水浴中で１時間振盪した。室温に戻し水酸化ナトリウムを添加して塩酸を中和し、さらに０．５Ｍリン酸緩衝液（ｐＨ６．５）を０．５ｍＬ添加して溶液のｐＨを６．５に調整した。得られた加水分解物を遠心分離（３０００ｒｐｍ、１０ｍｉｎ）して不溶物を除き、ＳｅｐｈａｃｒｙｌＳ−３００ (ＧＥヘルスケアサイエンス社)カラム（２．５×７０ｃｍ）にかけて純水で分画溶出した。得られた画分の全糖をフェノール硫酸法によって測定したところ、溶出液量１７９〜２６０ｍＬの位置にコンニャクグルコマンナンの加水分解物が溶出されることが分かった（図１）。この画分を減圧濃縮器によって濃縮し、この加水分解物の中性糖としての濃度をフェノール硫酸法によって測定した。
Example 1: Effect of inhibiting sulfated konjac glucomannan on IgE production (1) Preparation of hydrolyzate of konjac glucomannan 200 mg of konjac glucomannan (manufactured by Shimizu Chemical Co., Ltd.) was suspended in 17.5 mL of distilled water and 1 at room temperature. The gel was prepared by shaking for hours. Hydrochloric acid was added to this to a final concentration of 0.25 N, and the mixture was shaken in a 75 ° C. water bath for 1 hour. After returning to room temperature, sodium hydroxide was added to neutralize hydrochloric acid, and 0.5 mL of 0.5 M phosphate buffer (pH 6.5) was further added to adjust the pH of the solution to 6.5. The obtained hydrolyzate was centrifuged (3000 rpm, 10 min) to remove insoluble matters, and fractionated and eluted with pure water over a Sephacryl S-300 (GE Healthcare Science) column (2.5 × 70 cm). When the total sugar of the obtained fraction was measured by the phenol-sulfuric acid method, it was found that the hydrolyzate of konjac glucomannan was eluted at a position of 179 to 260 mL of the eluate (FIG. 1). This fraction was concentrated by a vacuum concentrator, and the concentration of the hydrolyzate as a neutral sugar was measured by the phenol-sulfuric acid method.
（２）コンニャクグルコマンナンの硫酸化
コンニャクグルコマンナン（清水化学（株）製）１００ｍｇ分（１０ｍＬ）に対して１６ｍＬのエタノールを添加し沈殿を得た。これをエタノールで４回、ジメチルホルムアミド（ＤＭＦ）で４回洗浄した。得られた沈渣にＤＭＦ８００μＬを添加した後、ＤＭＦ１．２ｍＬ中に溶解した三酸化イオウ−ＤＭＦ錯体（ＤＭＦ−ＳＯ3）（Ｓｉｇｍａ）１９２ｍｇを加え４℃で１晩撹拌して硫酸化した。これに、ＮａＣｌ飽和アセトン６ｍＬを加えて生じた沈殿を回収し、１０ｍＭリン酸緩衝液２ｍＬに溶かした後、ｐＨを６．５に調整した。図２にＳｅｐｈａｃｒｙｌＳ−３００カラム（２．５×７０ｃｍ）で分析した場合のクロマトグラムを示す。溶出位置（１４３ｍＬ〜２３３ｍＬ）を回収し、凍結乾燥しコンニャクグルコマンナンの硫酸化物２８ｍｇを得た。このようにして得られた硫酸化コンニャクグルコマンナンの硫酸基の測定は、硫酸カリウム（Ｋ2ＳＯ4）を標準として、Ｄｏｄｇｓｏｎ−Ｐｒｉｃｅ比濁法によって行った。フェノール硫酸法による全糖１ｍｇ当たり０．８ｍｇの硫酸基が結合していると見積もられた。
(2) Sulfation of konjac glucomannan 16 mL of ethanol was added to 100 mg (10 mL) of konjac glucomannan (manufactured by Shimizu Chemical Co., Ltd.) to obtain a precipitate. This was washed 4 times with ethanol and 4 times with dimethylformamide (DMF). After adding 800 μL of DMF to the resulting precipitate, 192 mg of sulfur trioxide-DMF complex (DMF-SO 3 ) (Sigma) dissolved in 1.2 mL of DMF was added and the mixture was sulfated by stirring overnight at 4 ° C. To this was added 6 mL of NaCl-saturated acetone, and the resulting precipitate was recovered, dissolved in 2 mL of 10 mM phosphate buffer, and adjusted to pH 6.5. FIG. 2 shows a chromatogram when analyzed with a Sephacryl S-300 column (2.5 × 70 cm). The elution position (143 mL to 233 mL) was collected and freeze-dried to obtain 28 mg of konjac glucomannan sulfate. The sulfate group of the sulfated konjac glucomannan thus obtained was measured by the Dodgson-Price turbidimetric method using potassium sulfate (K 2 SO 4 ) as a standard. It was estimated that 0.8 mg of sulfate groups were bound per 1 mg of total sugar by the phenol-sulfuric acid method.
（３）硫酸化コンニャクグルコマンナンのインビトロ抗体産生系におけるＩｇＥ産生抑制効果
Ｂａｌｂ／ｃマウス（８週齢、♂）の脾臓をＩＳＣＯＶ培地中でほぐして細胞懸濁液を調製し、さらにＬｙｍｐｈｏｌｉｔｅ−Ｍ（ＣｅｄａｒＬａｎｅ Ｌａｂｏｒａｔｏｒｉｅｓ社）を用いた比重遠心分離法によりリンパ球分画を回収した。調製したリンパ球をＩＬ−４（Ｒ＆Ｄ社、最終濃度１００ｎｇ／ｍＬ）、抗ＣＤ−４０抗体（Ｓｅｒｏｔｅｃ社、最終濃度２００ｎｇ／ｍＬ）、及び２−メルカプトエタノール（最終濃度５０ｎＭ）を含むＩＳＣＯＶ培地で２×１０6細胞／ｍＬの濃度になるように調整し、９６−ウェルマイクロプレートの各ウェルに１９０μＬずつ分注した。これにＰＢＳ（−）１０μＬ及び硫酸化コンニャクグルコマンナン（１．５ｍｇ／ｍＬ、３．０ｍｇ／ｍＬ） １０μＬを添加し、炭酸ガス培養器中で７日間培養した。ウェルの培養上清を回収し、培養液中に産生された抗体濃度を測定した。ＩｇＥ濃度の測定は、Ｍｏｕｓｅ ＩｇＥ Ｑｕａｎｔｉｔａｔｉｖｅ ＥＬＩＳＡキット（Ｂｅｔｈｙｌ社）を用い、製品付属の取扱説明書の方法に従って行った。
(3) Suppressive effect of sulfated konjac glucomannan on IgE production in in vitro antibody production system The cell spleen of Balb / c mice (8 weeks old, sputum) is loosened in ISCOV medium to prepare a cell suspension, and Lympholite-M Lymphocyte fractions were collected by specific gravity centrifugation using (Cedar Lane Laboratories). Prepared lymphocytes in ISCOV medium containing IL-4 (R & D, final concentration 100 ng / mL), anti-CD-40 antibody (Serotec, final concentration 200 ng / mL), and 2-mercaptoethanol (final concentration 50 nM). The concentration was adjusted to 2 × 10 6 cells / mL, and 190 μL was dispensed into each well of a 96-well microplate. To this, 10 μL of PBS (−) and 10 μL of sulfated konjac glucomannan (1.5 mg / mL, 3.0 mg / mL) were added and cultured for 7 days in a carbon dioxide incubator. The culture supernatant of the well was collected, and the concentration of the antibody produced in the culture solution was measured. The IgE concentration was measured using a Mouse IgE Quantitative ELISA kit (Bethyl) according to the method in the instruction manual attached to the product.
図３に示すとおり、この条件でのインビトロ 抗体産生系におけるＩｇＥの産生は、（１）に記載した方法で調製したコンニャクグルコマンナン加水分解物を最終濃度で７５、１５０μｇ／ｍＬの濃度で添加した培養では、濃度依存的に抑制された。次いで、上記（１）で調製した硫酸化コンニャクグルコマンナンを同じ濃度で添加した場合には、さらにＩｇＥ産生を強く抑制した。 As shown in FIG. 3, the production of IgE in the in vitro antibody production system under these conditions was performed by adding konjac glucomannan hydrolyzate prepared by the method described in (1) at a final concentration of 75 and 150 μg / mL. In culture, it was suppressed in a concentration-dependent manner. Next, when the sulfated konjac glucomannan prepared in (1) above was added at the same concentration, IgE production was further strongly suppressed.
実施例２：硫酸化セルロースのＩｇＥ産生抑制効果
（１）硫酸化セルロースの調製
濾紙粉末（東洋濾紙（株）製；濾紙粉末Ｃ）１００ｍｇを１０ｍＬのエタノールで１回、１０ｍＬのＤＭＦで４回洗浄し沈渣を回収した。これにＤＭＦ８００μＬを添加した後、ＤＭＦ１．２ｍＬ中に溶解した三酸化イオウ−ＤＭＦ（ＤＭＦ−ＳＯ3）（Ｓｉｇｍａ）１９２ｍｇを加え４℃で１晩撹拌して硫酸化した。これに、ＮａＣｌ飽和アセトン６ｍＬを加えて生じた沈殿を回収し、１０ｍＭリン酸緩衝液２ｍＬに溶かした後、ｐＨを６．５に調整した。図４にＳｅｐｈａｃｒｙｌＳ−３００カラム（２．５×１００ｃｍ）で分析した場合のクロマトグラムを示す。溶出位置（３１３ｍＬ〜４２１ｍＬ）を回収し、凍結乾燥してセルロースの硫酸化物８６ｍｇを得た。このようにして得られた硫酸化セルロースの硫酸基の測定は、硫酸カリウム（Ｋ2ＳＯ4）を標準として、Ｄｏｄｇｓｏｎ−Ｐｒｉｃｅ比濁法によって行った。フェノール硫酸法による全糖１ｍｇ当たり０．２６ｍｇの硫酸基が結合していると見積もられた。
Example 2: IgE production inhibitory effect of sulfated cellulose (1) Preparation of sulfated cellulose 100 mg of filter paper powder (manufactured by Toyo Filter Paper Co., Ltd .; filter paper powder C) was washed once with 10 mL of ethanol and four times with 10 mL of DMF. The sediment was collected. After adding 800 μL of DMF, 192 mg of sulfur trioxide-DMF (DMF-SO 3 ) (Sigma) dissolved in 1.2 mL of DMF was added, and the mixture was stirred at 4 ° C. overnight to be sulfated. To this was added 6 mL of NaCl-saturated acetone, and the resulting precipitate was recovered, dissolved in 2 mL of 10 mM phosphate buffer, and adjusted to pH 6.5. FIG. 4 shows a chromatogram when analyzed with a Sephacryl S-300 column (2.5 × 100 cm). The elution position (313 mL to 421 mL) was collected and freeze-dried to obtain 86 mg of cellulose sulfate. The sulfate group of the sulfated cellulose thus obtained was measured by the Dodgson-Price turbidimetric method using potassium sulfate (K 2 SO 4 ) as a standard. It was estimated that 0.26 mg of sulfate groups were bound per 1 mg of total sugar by the phenol-sulfuric acid method.
（２）硫酸化セルロースのインビトロ 抗体産生系におけるＩｇＥ産生抑制効果
Ｂａｌｂ／ｃマウス（８週齢、♂）の脾臓をＩＳＣＯＶ培地中でほぐして細胞懸濁液を調製し、さらにＬｙｍｐｈｏｌｉｔｅ−Ｍ（ＣｅｄａｒＬａｎｅ Ｌａｂｏｒａｔｏｒｉｅｓ社）を用いた比重遠心分離法によりリンパ球分画を回収した。調製したリンパ球をＩＬ−４（Ｒ＆Ｄ社、最終濃度１００ｎｇ／ｍＬ）、抗ＣＤ−４０抗体（Ｓｅｒｏｔｅｃ社、最終濃度２００ｎｇ／ｍＬ）、及び２−メルカプトエタノール（最終濃度５０ｎＭ）を含むＩＳＣＯＶ培地で２×１０6細胞／ｍＬの濃度になるように調整し、９６−ウェルマイクロプレートの各ウェルに１９５μＬずつ分注した。これにＰＢＳ（−）１０μＬ及び硫酸化セルロース（０．４ｍｇ／ｍＬ、４．０ｍｇ/ｍＬ） ５μＬを添加し、炭酸ガス培養器中で７日間培養した。培養後、ウェルの培養上清を回収し産生された抗体濃度を測定した。ＩｇＥ濃度の測定は、Ｍｏｕｓｅ ＩｇＥ Ｑｕａｎｔｉｔａｔｉｖｅ ＥＬＩＳＡキット（Ｂｅｔｈｙｌ社）を用い、製品付属の取扱説明書の方法に従って行った。上記（１）で調製した硫酸化セルロースを最終濃度で１０及び１００μｇ／ｍＬの濃度で添加した培養では、培地中に産生されたＩｇＥはコントロールに比べ有意に低下していた（図５）。元々のセルロース粉末は水溶性ではなく、ＩｇＥ産生抑制効果も認められない。このことは、セルロースを硫酸化することによって、原料のセルロースには存在しないＩｇＥ産生抑制効果を付与することができることを示している。
図６には、実施例１及び実施例２と同様の方法で調製した硫酸化コンニャクグルコマンナン、硫酸化セルロースを用いて、同様にインビトロ抗体産生系におけるＩｇＥ産生抑制効果を調べた結果を示す。ここでは、インビトロ抗体産生系に添加したコンニャクグルコマンナン加水分解物、硫酸化コンニャクグルコマンナン、硫酸化セルロースの最終濃度は、それぞれ１０μｇ／ｍＬ及び１００μｇ／ｍＬとした。図５に示す結果同様に、これらの硫酸化物はインビトロでＩｇＥ産生を抑制することが確認できた。
(2) In vitro effect of sulfated cellulose IgE production inhibitory effect in antibody production system A spleen of a Balb / c mouse (8 weeks old, sputum) is loosened in an ISCOV medium to prepare a cell suspension, and Lympholite-M (Cedar Lane) The lymphocyte fraction was collected by specific gravity centrifugation using Laboratories). Prepared lymphocytes in ISCOV medium containing IL-4 (R & D, final concentration 100 ng / mL), anti-CD-40 antibody (Serotec, final concentration 200 ng / mL), and 2-mercaptoethanol (final concentration 50 nM). The concentration was adjusted to 2 × 10 6 cells / mL, and 195 μL was dispensed into each well of a 96-well microplate. To this, 10 μL of PBS (−) and 5 μL of sulfated cellulose (0.4 mg / mL, 4.0 mg / mL) were added and cultured in a carbon dioxide incubator for 7 days. After culturing, the culture supernatant of the well was collected and the concentration of the produced antibody was measured. The IgE concentration was measured using a Mouse IgE Quantitative ELISA kit (Bethyl) according to the method in the instruction manual attached to the product. In the culture in which the sulfated cellulose prepared in the above (1) was added at final concentrations of 10 and 100 μg / mL, IgE produced in the medium was significantly reduced compared to the control (FIG. 5). The original cellulose powder is not water-soluble and has no IgE production inhibitory effect. This indicates that the effect of suppressing IgE production that does not exist in the raw material cellulose can be imparted by sulfating cellulose.
FIG. 6 shows the results of examining the IgE production inhibitory effect in an in vitro antibody production system using sulfated konjac glucomannan and sulfated cellulose prepared by the same method as in Example 1 and Example 2. Here, the final concentrations of konjac glucomannan hydrolyzate, sulfated konjac glucomannan, and sulfated cellulose added to the in vitro antibody production system were 10 μg / mL and 100 μg / mL, respectively. Similar to the results shown in FIG. 5, it was confirmed that these sulfates inhibit IgE production in vitro.
実施例３ ケラチノサイト抽出物によるＩｇＥ産生系に対する硫酸化コンニャクグルコマンナンの効果
アトピー性皮膚炎に伴い肌のかゆみがおこり、それを掻くことにより症状が悪化し、またかゆみが増大するという悪循環が見られる。この現象から、皮膚を掻くことによって、破壊された角化細胞（ケラチノサイト）が症状の悪化の原因であることが考えられる。ケラチノサイト（ＰＡＭ−２１２細胞）の抽出物をＢａｌｂ/ｃマウスに投与することによって、血中ＩｇＥ産生が刺激されることが明らかにされている。また、Ｂａｌｂ/ｃマウスの脾臓細胞を用いたインビトロＩｇＥ産生系にＰＡＭ−２１２細胞抽出物を添加することによって顕著にＩｇＥ産生が亢進することが明らかにされている（Yamamoto T, Kaneko S et al, (2002) Increase in serum IgE levels following injection of syngeneic keratinocyte extracts in BALB/c mice. Arch Dermatol Res 294: 117-23、森本謙一、他．マウス角化細胞株由来IgE産生増強因子のIgEクラススイッチに及ぼす影響．アレルギー51(9・10), 992(抄), 2002）。そこで、ケラチノサイト抽出物によるインビトロＩｇＥ産生系を用いて、ＩｇＥ産生亢進に対する硫酸化コンニャクグルコマンナンのＩｇＥ産生抑制効果を調べた。
Example 3 Effect of sulfated konjac glucomannan on IgE production system by keratinocyte extract Itching with skin caused by atopic dermatitis, vigorous symptom is worsened by symptom worsening and itching . From this phenomenon, it is considered that the keratinocytes destroyed by scratching the skin are the cause of the worsening of symptoms. It has been shown that administration of an extract of keratinocytes (PAM-212 cells) to Balb / c mice stimulates blood IgE production. It has also been clarified that IgE production is significantly enhanced by adding PAM-212 cell extract to an in vitro IgE production system using spleen cells of Balb / c mice (Yamamoto T, Kaneko S et al , (2002) Increase in serum IgE levels following injection of syngeneic keratinocyte extracts in BALB / c mice. Arch Dermatol Res 294: 117-23, Kenichi Morimoto, et al.IgE class switch of IgE production enhancing factor derived from mouse keratinocyte cell line Effects on allergies 51 (9 ・ 10), 992 (abstract), 2002). Therefore, the in vitro IgE production system using keratinocyte extract was used to examine the IgE production inhibitory effect of sulfated konjac glucomannan on the IgE production enhancement.
（１）硫酸化コンニャクグルコマンナンのインビトロ 抗体産生系におけるＩｇＥ産生抑制効果
Ｂａｌｂ／ｃマウス（８週齢、♂）の脾臓をＩＳＣＯＶ培地中でほぐして細胞懸濁液を調製し、さらにＬｙｍｐｈｏｌｉｔｅ−Ｍ（ＣｅｄａｒＬａｎｅ Ｌａｂｏｒａｔｏｒｉｅｓ社）を用いた比重遠心分離法によりリンパ球分画を回収した。調製したリンパ球をＩＬ−４（Ｒ＆Ｄ社、最終濃度１００ｎｇ／ｍＬ）、抗ＣＤ−４０抗体（Ｓｅｒｏｔｅｃ社、最終濃度２００ｎｇ／ｍＬ）、及び２−メルカプトエタノール（最終濃度５０ｎＭ）を含むＩＳＣＯＶ培地で２×１０6細胞／ｍＬの濃度になるように調整し、９６−ウェルマイクロプレートの各ウェルに１８０μＬずつ分注した。これにケラチノサイト抽出物１０μＬ及びコンニャクグルコマンナン加水分解物１０μＬを添加し、炭酸ガス培養器中で7日間培養した。硫酸化コンニャクグルコマンナンは実施例１（２）と同様にして調製した。培養後、各ウェルの培養上清を回収し、産生されたＩｇＥ抗体の濃度を測定した。ケラチノサイト抽出物の添加によりインビトロＩｇＥ産生量は増加した。これにコンニャクグルコマンナン加水分解物を最終濃度で７５、１５０μｇ／ｍＬの濃度で添加した培養では、有意なＩｇＥ産生抑制が認められなかったのに対し、硫酸化コンニャクグルコマンナンでは同濃度において強いＩｇＥ産生抑制効果を示した（図７）。
(1) In vitro effect of sulfated konjac glucomannan IgE production inhibitory effect in antibody production system Balb / c mice (8 weeks old, sputum) were loosened in ISCOV medium to prepare a cell suspension, and Lympholite-M Lymphocyte fractions were collected by specific gravity centrifugation using (Cedar Lane Laboratories). Prepared lymphocytes in ISCOV medium containing IL-4 (R & D, final concentration 100 ng / mL), anti-CD-40 antibody (Serotec, final concentration 200 ng / mL), and 2-mercaptoethanol (final concentration 50 nM). The concentration was adjusted to 2 × 10 6 cells / mL, and 180 μL was dispensed into each well of a 96-well microplate. To this, 10 μL of keratinocyte extract and 10 μL of konjac glucomannan hydrolyzate were added and cultured in a carbon dioxide incubator for 7 days. Sulfated konjac glucomannan was prepared in the same manner as in Example 1 (2). After the culture, the culture supernatant of each well was collected, and the concentration of the produced IgE antibody was measured. In vitro IgE production increased with the addition of keratinocyte extract. In the culture in which konjac glucomannan hydrolyzate was added at a final concentration of 75 or 150 μg / mL, no significant suppression of IgE production was observed, whereas sulfated konjac glucomannan showed strong IgE at the same concentration. The production inhibitory effect was shown (FIG. 7).
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| WO2013051146A1 (en) * | 2011-10-07 | 2013-04-11 | 株式会社 荻野商店 | Method for producing depolymerized konjak glucomannan and depolymerized konjak glucomannan obtained thereby |
| JPWO2013051146A1 (en) * | 2011-10-07 | 2015-03-30 | 株式会社荻野商店 | Method for producing low molecular weight konjac glucomannan and low molecular weight konjac glucomannan obtained by this method |
| JP2015218137A (en) * | 2014-05-16 | 2015-12-07 | 株式会社Cac | IgE inhibitor |
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| JPH08253502A (en) * | 1994-10-13 | 1996-10-01 | Cv Therapeutics Inc | Production of very high-molecular-weight sulfated polysaccharide |
| JP2004099664A (en) * | 2002-09-05 | 2004-04-02 | Shoji Takigami | Sulfated konjak mannan as new functional material and method for synthesizing the same |
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| JPH08253502A (en) * | 1994-10-13 | 1996-10-01 | Cv Therapeutics Inc | Production of very high-molecular-weight sulfated polysaccharide |
| JP2004099664A (en) * | 2002-09-05 | 2004-04-02 | Shoji Takigami | Sulfated konjak mannan as new functional material and method for synthesizing the same |
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| WO2013051146A1 (en) * | 2011-10-07 | 2013-04-11 | 株式会社 荻野商店 | Method for producing depolymerized konjak glucomannan and depolymerized konjak glucomannan obtained thereby |
| JPWO2013051146A1 (en) * | 2011-10-07 | 2015-03-30 | 株式会社荻野商店 | Method for producing low molecular weight konjac glucomannan and low molecular weight konjac glucomannan obtained by this method |
| JP2015218137A (en) * | 2014-05-16 | 2015-12-07 | 株式会社Cac | IgE inhibitor |
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