JP2015044755A - 抗アレルギー物質及びその製造方法 - Google Patents
抗アレルギー物質及びその製造方法 Download PDFInfo
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- JP2015044755A JP2015044755A JP2013175476A JP2013175476A JP2015044755A JP 2015044755 A JP2015044755 A JP 2015044755A JP 2013175476 A JP2013175476 A JP 2013175476A JP 2013175476 A JP2013175476 A JP 2013175476A JP 2015044755 A JP2015044755 A JP 2015044755A
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- perilla
- antiallergic
- antiallergic substance
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- histamine
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Abstract
Description
赤シソから有効成分を抽出するための溶媒としては、水、メタノールやエタノール等のアルコール、及びアセトン等が考えられるが、本発明のフラバノンをシソから抽出するための溶媒としては、抽出効率の観点から、熱水を使用することが好ましい。
本発明においては、上述の方法により得られた赤シソ熱水抽出物を逆相カラムクロマトグラフィーにより分画する。より具体的には、ポリマー系カラムを用いた逆相カラムクロマトグラフィーにおいて、メタノールで溶出することにより分画することができる。なお、メタノールで溶出後、濃縮処理を行い、その後、凍結乾燥を行う。
本発明においては、上述の方法により得られた赤シソ分画物(メタノール溶出物)を、逆相高速液体クロマトグラフィー(逆相HPLC)を使用して精製することにより、上記式(4)で表されるフラバノンを得ることができる。
(赤シソの熱水抽出)
赤ジソ粉末(三島食品/天日干し)300gを、2Lの沸騰水を使用して、1時間、攪拌抽出した。次いで、ガーゼを用いて抽出残渣を搾取した。そして、本抽出を2回繰り返した後、得られた抽出液を濾紙(アドバンテック製、商品名:FILTER PAPER 2)を使用して濾過し、濾過した抽出液に対して、エバポレーターによる濃縮処理、及び凍結乾燥処理を施すことにより、赤シソの熱水抽出物を得た。
次いで、得られた赤シソ熱水抽出物を逆相カラムクロマトグラフィーにより分画した。ポリマー系カラムとして、スチレン−ジビニルベンゼン共重合体(ST/DVD)をベースポリマーとするカラム(三菱化学製、商品名:MCLゲル CHP20P)を使用し、本担体を50%メタノールにより、1時間以上膨潤させてクロマトカラム(SIBATA製、商品名:SPCクロマトカラム)に20cm/分(141ml)充填した後、これをカラム体積の5倍量(707ml)の超純水により洗浄した。
ラット好塩基球細胞株RBL−2H3(ヒューマンサイエンス振興財団)を抗生物質(100U/ml penicillin、100μg/ml streptomycin、GIBCO製)含有MEM培地(GIBCO製)、及び10%牛胎児血清(FBS)を用いて37℃、5%CO2環境下で培養した。なお、継代の際には、pH7.2のリン酸緩衝生理食塩水(GIBCO製)を使用して、100×20mmの組織培養皿(FALCON製、商品名:353003)を洗浄後、Trypsin-EDTA(GIBCO製、商品名:25300−054)を使用して細胞を剥がし、再度、10%牛胎児血清含有MEM培地に懸濁して、新しいプレートに播種した。
対数増殖期にあるRBL−2H3細胞を24ウェルプレートに3.5×105セル/500μl/ウェル播種して、8時間、前培養した。その後、100ng/mlのIgE抗体(Monoclonal Mouse IgE anti-DNP)を加えて、16時間、感作し、IgE抗体をラット好塩基球細胞株RBL−2H3に結合させた。
次に、上述のメタノール溶出画分を、更に、メタノールで50mg/mlに調製したサンプルを準備し、このサンプルを0.20μmの親水性フィルター(アドバンテック製、商品名:DISMIC-13HP PTFE)を使用して濾過し、これを高純度シリカゲルが充填されたカラム(関東化学製、商品名:Mightysil RP−18 GP)とガードカラム(関東化学製、商品名:Mightysil RP−18 GP用ガードカラム)を使用して、逆相HPLCにより分画した。
(質量分析)
まず、HPLCにより分取した上記ピーク5のサンプルを、クロロホルム(SIGMA製、商品名05−3400)を用いて溶解し、次に、その一部をHPLC用メタノール(SIGMA製、商品名:19−2470)で十分に希釈したものをサンプルとして使用した。なお、質量測定には、質量分析計(Thermo Fisher Scientific製、商品名:LTQ Orbitrap XL)を使用し、イオン化にはESI法またはAPCI法を用いた。以上の結果を、図3に示す。
次いで、上記ピーク5のサンプルを、核磁気共鳴法(1HNMR、及び13CNMR)を用いて解析した。得られたスペクトルデータを以下に示す。
5.49(dd,1H,J=13,2.8Hz),2.85(dd,1H,J=17,3.Hz),3.10(dd,1H,J=17,13Hz),6.19(s,1H),7.26-7.48(m,1H),3.98(s,3H),3.92(s,3H).
1C NMR (125MHz, CDCl3):
80.0(d), 45.8(t), 189.2(s), 105.9(d), 152.5(s), 89.8(d), 155.0(s), 127.7(s), 149.4(s), 138.4(s), 126.3(d), 128.9(d), 128.8(d), 56.2(q), 56.4(q).
次に、8−ヒドロキシ−5,7−ジメトキシフラバノンと、シソ由来既知抗炎症成分(ロズマリン酸、ルテオリン、アピゲニン、カフェ酸)のヒスタミン遊離抑制活性を比較検討した。
BALB/cマウス(♀、5週齢:日本チャールズ・リバー製)を特定病原体不在環境下で、22℃±3℃、及び12時間ごとの明暗サイクルにて飼育した。なお、食餌はγ線照射飼料CRF−1(オリエンタル酵母製)、水は滅菌蒸留水を自由に摂取させた。
Claims (9)
- 前記溶媒が熱水であることを特徴とする請求項3に記載の抗アレルギー物質の製造方法。
- 前記カラムクロマトグラフィーが、逆相カラムクロマトグラフィーであることを特徴とする請求項3または請求項4に記載の抗アレルギー物質の製造方法。
- 前記シソが赤シソであることを特徴とする請求項2〜請求項5のいずれか1項に記載の抗アレルギー物質の製造方法。
- 請求項1に記載の抗アレルギー物質を含有することを特徴とする飲食品。
- 請求項1に記載の抗アレルギー物質を含有することを特徴とする薬品。
- 請求項1に記載の抗アレルギー物質を含有することを特徴とする化粧品。
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JP2020117456A (ja) * | 2019-01-23 | 2020-08-06 | 花王株式会社 | シソ科植物抽出物の製造方法 |
CN112569148A (zh) * | 2019-09-28 | 2021-03-30 | 广州市中通生化制品有限公司 | 一种具有舒敏防敏功效的中药组合物及其制备方法与应用 |
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JP2018002645A (ja) * | 2016-06-30 | 2018-01-11 | 国立大学法人広島大学 | 抗癌剤作用増強物質およびそれを備えた抗癌剤キット |
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JP7412888B2 (ja) | 2019-01-23 | 2024-01-15 | 花王株式会社 | シソ科植物抽出物の製造方法 |
CN112569148A (zh) * | 2019-09-28 | 2021-03-30 | 广州市中通生化制品有限公司 | 一种具有舒敏防敏功效的中药组合物及其制备方法与应用 |
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