JP2004161664A - Propolis composition - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide useful applications for propolis which is safe even by long-term internal use and has excellent various biological activities. <P>SOLUTION: A composition for angiotensin II receptor antagonism, a composition for adrenaline β2 receptor activation, a female hormone-like composition, an endothelin antagonism composition, a serotonin reabsorption inhibitory composition, a serotonin release promotion composition, a serotonin receptor antagonism composition comprise each propolis, quercetin, p-coumaric acid and artepillin C and further contain each chrysin, galangin or caffeic acid phenethyl ester. The compositions for limited applications have biological activities derived from the contained components and are useful for food products, medicines, cosmetics, or the like. <P>COPYRIGHT: (C)2004,JPO

Description

【００３４】
（ｐ−クマール酸）
機種：ＬＣ−２０１０Ａ（株式会社 島津製作所）
検出器：紫外可視分光光度計 ＬＣ−２０１０Ａ（株式会社 島津製作所）
カラム：ＹＭＣ−Ｐａｃｋ ＯＤＳ−Ａ 、Ａ−３１２、Φ６ｍｍ×１５ｃｍ（株式会社 ワイエムシィ）
移動相：２．５％酢酸、メタノール及びアセトニトリルの混液（５０：８：５ｖ／ｖ／ｖ）
測定波長：３２０ｎｍ
流速：１．０ｍｌ／ｍｉｎ．
カラム温度：４０℃
【００３５】

グラジエント条件は表１に示す。
【００３６】
【表１】

【００３７】
測定波長：３３０ｎｍ
流速：１．０ｍｌ／ｍｉｎ．
カラム温度：４０℃
（クリシン及びガランギン）
機種：ＬＣ−２０１０Ａ（株式会社 島津製作所）
検出器：紫外可視分光光度計 ＬＣ−２０１０Ａ（株式会社 島津製作所）
カラム：ＹＭＣ−Ｐａｃｋ ＯＤＳ−Ａ 、Ａ−３１２、Φ６ｍｍ×１５ｃｍ（株式会社 ワイエムシィ）
移動相：５％ ギ酸、アセトニトリル及びメタノールの混液（２５：１０：１０ｖ／ｖ／ｖ）
測定波長：３６０ｎｍ
流速：１．０ｍｌ／ｍｉｎ．
カラム温度：４０℃
【００３８】
表２からもわかるように、ブラジル産プロポリスにはアルテピリンＣやｐ−クマール酸が多く含有されており、一方、中国産プロポリスにはカフェイン酸フェネチルエステルやクリシン、ガランギンが多く含有されており、ブラジル産及び中国産プロポリスの含有成分が大きく異なることがわかる。
【００３９】
【表２】

【００４０】
実施例２
アンジオテンシンＩＩ１型受容体結合試験
ヒトアンジオテンシンＩＩ１型受容体への結合試験評価は、Ｄｅｒｋ Ｊ．Ｂｅｒｇｓｍａらの方法（ＢＩＯＣＨＥＭＩＣＡＬ ＡＮＤ ＢＩＯＰＨＹＳＩＣＡＬ ＲＥＳＥＡＲＣＨ ＣＯＭＭＵＮＩＣＡＴＩＯＮＳ，１８３（３） ９８９−９９５，１９９２）を一部改変して行った。
［ＣＨＯ細胞へのヒトアンジオテンシンＩＩ１型受容体の発現］
ヒトアンジオテンシンＩＩ１型受容体プラスミド（ｐｈＡＴ_１−３）を挿入した２２５０ベースペア（ｂｐ）のエンドヌクレアーゼ（Ｅｃｏ ＲＩ）を、ｐＣＪＢ３−８クローンを産生させるために、ベクターとしてｐＣＪＢ１１ｂ（ラウス肉腫ウイルスプロモーターに対してヒトシトマガロウイルスプロモーターを置換させたｐＲＪＢ誘導体）を発現したチャイニーズハムスター卵巣細胞（以下、ＣＨＯ細胞と略す）へ再クローニングした。
次に、このＣＨＯ細胞を２４時間以内に６０〜８０％にまで集密するまで、１０−ｍｍプレート上で培養した。その後、野生型Ｔ抗原をコードするＳＶ４０の起源欠損型突然変異体ＤＮＡ２５μｇを用いて形質転換させ、３７℃で一晩インキュベーションさせた後に細胞をトリプシン−ＥＤＴＡを用いてプレート上から剥離し、そして、新鮮な完全培地を用いて６ウェルプレート（３３−ｍｍ組織培養プレート）へ移した。そして、この細胞を受容体結合試験を行う２日前より、３７℃でインキュベーションした。
【００４１】
［ヒトアンジオテンシンＩＩ１型受容体への結合試験評価］
上述の細胞を３日間、形質転換させた後、本細胞を１０ｍＭの塩化マグネシウム、０．１％グルコース及び０．２％ウシ胎児血清を含むダルベコのリン酸塩緩衝液（以下、ＤＰＢＳ^＋＋と略す）を用いて十分に洗浄する。その後、基質として４０〜５０ｐＭの［^１２５Ｉ］標識した［ｓａｒ^１，Ｉｌｅ^８］アンジオテンシンＩＩ（以下、ＳＩＡと略す）（Ｎｅｗ Ｅｎｇｌａｎｄ Ｎｕｃｌｅａｒ社製、比活性：２２００Ｃｉ／ｍｍｏｌ）を、製造例１で得られた５００μｇ／ｍｌの濃度のプロポリス組成物の存在化、非存在化で、ＤＰＢＳ^＋＋１．０ｍｌ中で２５℃で６０分間反応させた。反応後、溶媒を除去し、迅速に細胞を氷冷しておいたＤＰＢＳ^＋＋で３回洗浄し、細胞に結合した放射活性を１．０Ｎの水酸化ナトリウムを用いて溶解させた後に、γ−カウンター（８０％効力）（アロカ（株）社製）を用いて測定した。
また、ＳＩＡと１０μＭの非放射標識アンジオテンシンＩＩを用いて測定した値を、ブランク（非特異的結合）とし、データは、検量線を作成し、コンピューターソフトシステム（Ｌｕｎｄｏｎ−２ ｓｏｆｔｗａｒｅ ｐｒｏｇｒａｍ；Ｌｕｎｄｏ Ｓｏｆｔｗａｒｅ社製）を用いて算出した。なお、本測定系におけるヒトアンジオテンシンＩＩ１型受容体拮抗剤であるｓａｒａｌａｓｉｎのＩＣ_５０値は、１．９ｎＭ（１．７３×１０^−３μｇ／ｍｌ）であった。
阻害率は、８０％であった。この結果から、強力にＳＩＡを阻害する、本発明のプロポリス組成物に強いアンジオテンシンＩＩ１型受容体拮抗作用を有することがわかる。
【００４２】
実施例３
アドレナリンβ２受容体作動化作用試験
モルモット肺由来アドレナリンβ２受容体への結合試験は、ＴＯＭＭＹ ＡＢＲＡＨＡＭＳＳＯＮらの方法（Ｂｉｏｃｈｅｍ．Ｐｈａｒｍａｃ．，３７（２）２０３−２０８（１９８８））を、一部改変して行った。
３５０−５００ｇ体重のモルモット（日本クレア（株））から、肺を摘出し、膜画分を調製した。膜画分の調製は、Ｖ．Ｎｅｒｍｅらの方法（Ｂｉｏｃｈｅｍ． Ｐｈａｒｍａｃ．，３４，２９１７（１９８５））に従って行った。
すなわち、摘出した肺をホモジナイズし、遠心分離を行い、得た沈殿部分の膜画分を、氷冷しておいたトリス緩衝液（１５４ｍＭ 塩化ナトリウム、２ｍＭ 塩化マグネシウムを含む２０ｍＭ トリス−塩酸（ｐＨ７．５）から成る）を用いて、タンパク質濃度が０．２−０．６ｍｇ／ｍｌとなるように希釈し、そして、トリチウム化した放射リガンドが実験反応系中の懸濁液中に１−４ｍｇ／ｍｌとなるように上述のトリス緩衝液を用いて希釈した。ただし、タンパク質濃度の定量は、Ｌｏｗｒｙらの方法（Ｊ．Ｂｉｏｌ．Ｃｈｅｍ．，１９３，２６５（１９５１））に従って、タンパク質標準物として牛血清アルブミンを用いて測定した。
放射リガンド結合試験は、同様に、Ｖ．Ｎｅｒｍｅらの方法（Ｂｉｏｃｈｅｍ． Ｐｈａｒｍａｃ．，３４，２９１７（１９８５））に従って行った。
【００４３】
すなわち、上述にして得られた膜画分懸濁液１００μｌに１００μＭのＧＴＰ（ｇｕａｎｏｓｉｎｅ５’−ｔｒｉｐｈｏｓｐｈａｔｅ）存在化、最終容量０．２５ｍｌ中で放射リガンドと反応させた。本実験では、放射リガンドとして０．４ｎＭの［^３Ｈ］標識した（−）ＣＧＰ１２１７７（Ａｍｅｒｓｈａｍ，開発コードＮｏ．）（＋１０ｎＭ ＣＧＰ２０７１２Ａ（Ａｍｅｒｓｈａｍ，開発コードＮｏ．）を含む）を用い、反応は２２℃で２０分間行った。また、非特異的結合は５０μＭ のアロプレノロール（ａｌｐｒｅｎｏｌｏｌ）を用い、総結合の８０−９０％から成ると定義し、その非特異的結合は常に放射リガンドを加えた総量の１０％以下であった。
その後、ガラスフィルター（Ｍｏｄｅｌ ７０１、Ｓｋａｔｒｏｎ Ｉｎｃ．製）を用いて、［^３Ｈ］標識した（−）ＣＧＰ１２１７７と結合した膜標品を分離するために瀘過を行ない、２回、上述のトリス緩衝液５ｍｌにて洗浄した。このガラスフィルターをバイアルに入れ、アクアゾール（液体シンチレーション用カクテル）と混合し、液体シンチレーションカウンターにて結合［^３Ｈ］標識（−）ＣＧＰ１２１７７量を測定し、阻害率（％）を次式より算出した。
【００４４】
阻害率（％）＝１００−〔（Ｃ_１−Ｂ）／（Ｃ_０ −Ｂ）〕×１００
（式中、Ｃ_１ は、既知量の供試化合物と［^３Ｈ］標識（−）ＣＧＰ１２１７７が共存している状態での［^３Ｈ］標識（−）ＣＧＰ１２１７７の膜に対する結合量を表わし、Ｃ_０ は、供試化合物を除いた時の［^３Ｈ］標識（−）ＣＧＰ１２１７７の膜に対する結合量を表わし、Ｂは、過剰（５０×１０^−６Ｍ）のａｌｐｒｅｎｏｌｏｌ存在下での［^３Ｈ］標識（−）ＣＧＰ１２１７７の膜に対する結合量を表わす。）
なお、本測定系におけるポジティブコントロールとしてのアドレナリンβ２受容体遮断薬であるＩＣＩ１１８５５１（レファランスコンパウンド）のＩＣ_５０値は、１．９ｎＭであった。
供試化合物として、製造例１で得られたプロポリス組成物を使用した結果（濃度：５００μｇ／ｍｌ）、阻害率は４８％であった。この結果から、本発明のプロポリス組成物に強いアドレナリンβ２受容体作動化作用を有することがわかる。
【００４５】
実施例４
女性ホルモン様作用試験
ラット子宮エストロゲン受容体への結合試験評価は、Ｂ． ＫＯＦＦＭＡＮらの方法（Ｊ．Ｓｔｅｒｏｉｄ Ｂｉｏｃｈｅｍ． Ｍｏｌｅｃ． Ｂｉｏｌ．，３８（２）１３５−１３９，１９９１）を一部改変して行った。
［ｃｙｔｏｓｏｌ（サイトゾル）画分の調製］
特に記述しない限り、以下に示す全ての操作は、４℃の温度で行った。
まず、粉砕した凍結子宮組織に４倍量のリン酸緩衝液（ｐＨ８．０）を加え、ポリトロンＰＴ−１０プローブ（Ｂｒｉｎｋｍａｎ Ｉｎｓｔｒｕｍｅｎｔ Ｃｏ． Ｉｎｃ．製）を用いて、フル調節条件で、５秒間破砕し、次の破砕まで６０秒間氷中で冷却し、その後、５秒間破砕する操作を二回繰り返し行うことによりホモジナイズした。このホモジネートを１０５０００×ｇで、３０分間、遠心分離し、得られた上清をデカンテーションし、これをサイトゾル画分として使用した。サイトゾル画分のタンパク質含量は、タンパク質標準試料として牛血清アルブミンを用いて、Ｌｏｗｒｙ ｅｔ． ａｌ．の方法（Ｊ． Ｂｉｏｌ． Ｃｈｅｍ． １９３ ２６５−２７５ １９５１）により測定した。
【００４６】
［ラット子宮エストロゲン受容体への結合試験評価］
タンパク質濃度が１．２〜７．２ｍｇ／ｍｌになるように調製したサイトゾル画分をリン酸緩衝液（ｐＨ ８．０）に溶解し、１ｎＭの［^３Ｈ］で標識した１７−β−エストラジオールと製造例１で得られたプロポリス組成物５００μｇ／ｍｌとなるように加え、４０℃で２０時間反応させた。また、非特異的結合試験（ブランク）は、２００倍過剰量のジスチルベステロールの存在下で行った。
次に、遊離したステロイドや試料サンプルを、デキストラン処理した活性炭懸濁液（０．５％ノーリット Ａ、０．００５％デキストランを含むリン酸緩衝液（ｐＨ ７．４）；以下、ＤＣＣと略す）を用いて、４℃で１０分間インキュベーションすることにより除去した。その後、５０００×ｇで１０分間遠心分離することにより、ＤＣＣを沈殿させ、得られたタンパク質結合ステロイドを含む上清１００μｌに、シンチレーションカクテル溶液（Ｐａｃｋａｒｄ Ｉｎｓｔｒｕｍｅｎｔ Ｃｏ．製、Ｄｏｗｎｅｒ Ｇｒｏｖｅ、ＩＩＩ）４ｍｌを加え、シンチレーションカウンター（Ｐａｃｋａｒｄ Ｍｏｄｅｌ ２４２５、３０−４０％ ｅｆｆｉｃｉｅｎｃｙ）により測定した。抑制率は、下記式により算出した。
【００４７】
阻害率（％）＝１００−〔（Ｃ_１−Ｂ）／（Ｃ_０ −Ｂ）〕×１００
（式中、Ｃ_１ は、既知量の供試化合物と［^３Ｈ］標識１７−β−エストラジオールが共存している状態での［^３Ｈ］標識１７−β−エストラジオールのラット子宮エストロゲン受容体に対する結合量を表わし、Ｃ_０ は、供試化合物を除いた時の［^３Ｈ］標識１７−β−エストラジオールのラット子宮エストロゲン受容体に対する結合量を表わし、Ｂは、過剰の１７−β−エストラジオール（３×１０^−６Ｍ）存在下での［^３Ｈ］標識１７−β−エストラジオールのラット子宮エストロゲン受容体に対する結合量を表わす。）
結果は、阻害率８７％であった。なお、本測定系におけるポジティブコントロールとしての１７−β−エストラジオールのＩＣ_５０値は、０．８４ｎＭ（２．２９×１０^−４ｎｇ／ｍｌ）であった。この結果からわかるように、本発明のプロポリス組成物は、１７−β−エストラジオールのエストロゲン受容体結合阻害作用を拮抗的に抑制し、強い女性ホルモン（エストロゲン）様作用を有することがわかる。
【００４８】
実施例５
エンドセリン受容体結合試験
ヒトエンドセリン受容体を発現したチャイニーズハムスター卵巣細胞（以下、ＣＨＯ細胞と略す）のエンドセリン受容体への結合試験評価は、ＢＵＣＨＡＮ，Ｋ．Ｗ．らの方法（Ｂｒ． Ｊ． Ｐｈａｒｍａｃｏｌ．，１１２，１２５１−１２５７，１９９４）を一部改変し、ＣＨＯ細胞エンドセリン受容体へのエンドセリン−１の結合の阻害を測定することにより行った。
［ＣＨＯ細胞の培養］
ＣＨＯ細胞を、１０％牛胎児血清、２ｍＭ Ｌ−グルタミン、１００ｕｎｉｔｓ／ｍｌのペニシリン及び１００μｇ／ｍｌのストレプトマイシンを含むα−ＭＥＭ培地（ＧＩＢＣＯ製）を用いて３７℃、５％ＣＯ_２の条件下で培養した。細胞は、トリプシン−ＥＤＴＡを用いて１週間に２回、継代した。
【００４９】
［ＣＨＯ細胞へのヒトエンドセリン受容体の発現］
形質転換する前に、１×１０^５個のＣＨＯ細胞を１０ｃｍシャーレに蒔き、接着させるために一晩３７℃、５％ＣＯ_２の条件下で培養した。その後、ＤＮＡは、リン酸カルシウム法により形質転換させた。すなわち、０．２５Ｍ 塩酸カルシウム０．７５ｍｌにエンドセリン受容体発現プラスミド４０μｇとｐＲＳＶ−ｎｅｏ（構造的にネオマイシン耐性遺伝子を発現しているベクター）１０μｇを添加し、このＤＮＡ／塩化カルシウム混合物を等容量の２倍濃縮したＨＥＰＥＳリン酸塩緩衝液（（リン酸塩緩衝液（以下、ＰＢＳと略す）：０．２８Ｍ 塩化ナトリウム、５０ｍＭ ＨＥＰＥＳ及び２．２５ｍＭ リン酸水素二ナトリウム・２水和物から成る）（ｐＨ７．１２）とを、プラスティック攪拌棒を用いて滴下し、１０ｃｍシャーレ中のＣＨＯ細胞中へ沈殿物の形で加えた。その沈殿物を細胞上で一晩放置し、その後、ＰＢＳを用いて十分に洗浄した。そして、形質転換した細胞を、ネオマイシンのアナローグであるＧ４１８ １ｍｇ／ｍｌを用いてネオマイシン耐性発現細胞を選択する前に、標準成長培地中で４８時間培養した。
【００５０】
Ｇ４１８耐性細胞のコロニーを、選択した後２〜３週間観察し、クローンリングを用いて９６ウエルプレートのウエル中へ移した。耐性細胞の選択は完全に行ったけれども、その後、細胞を０．２５ｍｇ／ｍｌの濃度のＧ４１８で維持した。Ｇ４１８耐性クローンを増殖させ、結合試験によりエンドセリン受容体発現の有無を確認した。
継代回数の増加による不安定な受容体発現の問題を解決するために、ヒトエンドセリン受容体を形質転換した細胞を、リミッティングダイリューションクローニングを行った。すなわち、理論的密度として１ウエルあたり１細胞になるように９６ウエルプレートに移した。そして、約２週間後、単コロニー、すなわち、性質的に同じな純粋な細胞をサブクローンし、結合分析により受容体の発現を測定した。
【００５１】
［ＣＨＯ細胞からの膜の調製］
それぞれの培養フラスコから培地を除去し、ＣＨＯ細胞が密になった単層を、２５ｍｌのカルシウム及びマグネシウムを含まないＰＢＳで２回、十分に洗浄し、次いで、５ｍＭ ＥＤＴＡを含むＰＢＳ１０ｍｌを加えた。その後、３７℃で１０分間インキュベートし、細胞懸濁液を１７０×ｇ、２０℃で５分間遠心分離し、得られた沈殿を氷冷した溶解緩衝液（０．３ｍＭ ＥＤＴＡを含む１５ｍＭ トリス−塩酸緩衝液）（ｐＨ７．４）で再溶解させた。この細胞溶解液を、氷上で１０分間、ホモジナイズし、これを１７０×ｇ、４℃で１０分間遠心分離し、得られた上清を再び４００００×ｇ、４℃で１０分間遠心分離した。それぞれに、得られた沈殿物を、分析用緩衝液（５０ｍＭ トリス、バチトラシン １００μｇ／ｍｌ、１ｍＭ 塩化カルシウム及び０．１％ 牛血清アルブミンから成る）に再懸濁し、分析に用いるまで凍結保存したか、あるいは、タンパク質濃度が３０〜１００μｇ／ｍｌとなるように分析緩衝液に溶解し、直ぐに使用した。
【００５２】
［結合試験評価］
競合阻害試験は、上述にして得られたＣＨＯ細胞膜（タンパク量として、３０−１００μｇ／ｍｌ）を、［^１２５Ｉ］で放射標識したＥＴ−１（１５−２５ｐＭ）と製造例１で得られたプロポリス組成物５００μｇ／ｍｌとともに、分析用緩衝液中で２５℃で３時間、インキュベーションした。その後、３ｍｌの氷冷したＫｒｅｂｓ緩衝液で３回洗浄し、細胞を剥離し、それをガラスフィルター（Ｍｏｄｅｌ ７０１９、Ｓｋａｔｒｏｎ Ｉｎｃ．製）を用いて迅速にろ過し、フィルター上に結合した放射活性を、γ―カウンター（８０％ ｅｆｆｉｃｉｅｎｃｙ）（アロカ（株）製）を用いて測定した。
また、非特異的結合（ブランク）は、飽和濃度（０．０３ｎＭ）のエンドセリン−１の存在化での膜結合放射能として定義した。非特異的結合を全結合から差し引き、そしてその差を特異的結合と定義した。全結合は常に添加した全放射能の１５％未満であった。
結果は、阻害率９６％であった。なお、本測定系におけるポジティブコントロールとしてのエンドセリン−１のＩＣ_５０値は、０．２７ｎＭ（０．６７５×１０^−３μｇ／ｍｌ）であった。この結果からわかるように、本発明のプロポリス組成物に強いエンドセリン拮抗作用を有することがわかる。
【００５３】
実施例６
セロトニン受容体結合阻害試験
セロトニン受容体結合阻害試験は、Ｊａｎｅｔ Ｇ．Ｍｕｌｈｅｒｏｎらの方法（Ｔｈｅ Ｊｏｒｎａｌ ｏｆ ｂｉｏｌｏｇｉｃａｌ ｃｈｅｍｉｓｔｒｙ，２６９，１７，１９９４）を参考に行った。バキュロウィルスＳＦ９細胞にヒトセロトニン受容体を発現させ、これを実験に用いた。既存の方法に従って調製した膜は、湿重量に対して４０倍量の ５０ｍＭ Ｔｒｉｓ−ＨＣｌ （ｐＨ７．４） を加え、氷浴中にて Ｔｅｆｌｏｎ−ｇｌａｓｓ ｈｏｍｏｇｅｎｉｚｅｒ を用いてホモジナイズ （３分、１ ｓｔｒｏｋｅ／ 分） し、その後遠心分離（３７，０００ × ｇ、２０分、４℃）した。得られた沈渣を氷冷緩衝液［ ５０ ｍＭ Ｔｒｉｓ−ＨＣｌ （ｐＨ７．４）］で再懸濁し、ポジティブコントロールである［^３Ｈ］８−ＯＨ−ＤＰＡＴ（８−ヒドロキシ−１，２，３，４−テトラヒドロナフタレン臭化水素酸塩）と製造例１で得られたプロポリス組成物５００μｇ／ｍｌの濃度で添加した。室温で６０分間反応させた後、ガラス繊維濾紙上に速やかに低圧吸引濾過した。それから、濾紙上に残存した受容体結合放射活性を液体シンチレーションカウンターで測定した。
結果は、阻害率９０％であった。ここで示す阻害率とは、８−ＯＨ−ＤＰＡＴの作用を抑制する割合である。なお、８−ＯＨ−ＤＰＡＴのＩＣ_５０値（セロトニン受容体結合を５０％阻害する濃度）は、０．４７ ｎＭであった。このように、本発明のプロポリス組成物に、セロトニン１Ａ受容体結合阻害作用を有することがわかる。
【００５４】
実施例７
セロトニントランスポーター受容体結合阻害試験
セロトニントランスポーター受容体結合阻害試験は、Ｔａｔｓｕｍｉらの方法を参考に行った（Ｅｕｒｏｐｅａｎ Ｊｏｕｒｎａｌ ｏｆ Ｐｈａｒｍａｃｏｌｏｇｙ ３４０，２４９−２５８，１９９７）。ヒトセロトニントランスポーターｃＤＮＡをｐＲｃ／ＣＭＶベクターに発現させ、ＨＥＫ２３９細胞にトランスフェクションした。それから既存の方法に従って細胞膜を調製し、５０ ｍＭ トリス（ヒドロキシメチル）アミノメタン、１２０ ｍＭ塩化ナトリウム、５ ｍＭ 塩化カリウム、を含む緩衝液（ｐＨ７．４）で懸濁した。これに製造例１で得られたプロポリス組成物を５００μｇ／ｍｌの濃度で添加し、セロトニントランスポーター阻害剤である［^３Ｈ］イミプラミンを添加し、２２℃で３０分間インキュベートした。それから、０．２％のポリエチレンイミン浸漬のＧＦ／Ｂフィルターで濾過し、反応を停止した。フィルター上の放射活性は、液体シンチレーションカウンターによって測定した。
結果は、抑制率が９３％であった。ここで示す抑制率とは、イミプラミンのセロトニントランスポーター受容体結合阻害作用を抑制する割合である。なお、イミプラミンのＩＣ_５０値（セロトニントランスポーター受容体結合を５０％阻害する濃度）は、５．８ ｎＭであった。このように、本発明のプロポリス組成物は、イミプラミンのセロトニントランスポーター受容体結合阻害作用を競合的に抑制すること、すなわち、セロトニントランスポーター受容体結合阻害作用を有することがわかる。
【００５５】
実施例８
セロトニンの再吸収阻害試験
セロトニンの再吸収阻害試験は、Ｓ．Ｐｅｒｏｖｉｃらの方法（Ａｒｚｎｅｉｍ．−Ｆｏｒｓｃｈ．／ＤｒｕｇＲｅｓ． ４５ （ＩＩ），１１，１９９５）を参考に行った。断頭により殺した２２０−２５０ ｇのラットの脳から、シナプトソームを調製した。氷浴中にて脳皮質を湿重量に対して２０倍量の０．３２ Ｍのショ糖液で、ホモジナイズし、遠心（１０００ × ｇ、１０分、４℃）を行った。得られたシナプトソーム画分をリン酸緩衝液で懸濁して、製造例１で得られたプロポリス組成物を５００μｇ／ｍｌの濃度で加え、３０℃で５分間プレインキュベートした。リン酸緩衝液の組成は、１．２ ｍＭリン酸二水素ナトリウム、２５ ｍＭ 炭酸水素ナトリウム、１１８ ｍＭ 塩化ナトリウム、５ ｍＭ 塩化カリウム、２．５ ｍＭ 硫酸マグネシウム、５０ μＭ アスコルビン酸、１０ μＭ ＥＧＴＡ［エチレングリコールビス（２−アミノエチルエーテル）四酢酸］、１１ ｍＭ グルコースである。その後、５μＭの［^３Ｈ］−セロトニンを加え、全量１ ｍｌとし、３７℃で２０分間インキュベートを行った。反応は冷リン酸緩衝液を加え停止し、直ちにＧＦ／Ｂフィルターで濾過した。フィルター上の放射活性は、液体シンチレーションカウンターによって測定した。
結果は、阻害率が８１％であった。ここで示す阻害率とは、［１００−（サンプルを添加した時の［^３Ｈ］−セロトニン取り込み量）／（緩衝液のみのコントロールの［^３Ｈ］−セロトニン取り込み量）×１００］である。なお、本実験系でのポジティブコントロールであるイミプラミンのＩＣ_５０値（セロトニン再吸収を５０％阻害する濃度）は、０．０２４ μＭであった。このように本発明のプロポリスは、セロトニン再吸収阻害作用を有することがわかる。
【００５６】
実施例９
セロトニンの放出作用試験
セロトニンの放出作用をみる実験系は、Ｇｉａｍｂａｔｔｉｓｔａらの方法を参考に行った（Ｊｏｒｎａｌ ｏｆ Ｎｅｕｒｏｃｈｅｍｉｓｔｒｙ， ６３ （３），１１６３−１１６６，１９９４）。ラット大脳皮質より、既存の方法に従って粗シナプトソーム画分を調製し、以下の緩衝液で懸濁した。緩衝液の組成は、１．２ ｍＭ 塩化カルシウム、１．２ ｍＭ 塩化マグネシウム、１ ｍＭリン酸二水素ナトリウム、２５ ｍＭ 炭酸水素ナトリウム、１２５ ｍＭ 塩化ナトリウム、３ｍＭ 塩化カリウム、２２ ｍＭ炭酸水素ナトリウム、１０ ｍＭグルコースである。得られたシナプトソームに、０．０８ μＭ［^３Ｈ］―セロトニンを添加し、３７℃で１５分間インキュベートした。その後、製造例１で得られたプロポリス組成物を最終濃度が５００μｇ／ｍｌに成るように加え、３７℃で９５％Ｏ_２、５％ＣＯ_２条件下において潅流を行い、最後にシナプトソームの放射活性を測定した。
結果は、誘発率７４％であった。誘発率とは、［１００−（緩衝液のみのコントロールの［^３Ｈ］−セロトニン放出量）／（サンプルを添加した時の［^３Ｈ］−セロトニン放出量）×１００］である。なお、本実験系でのポジティブコントロールであるフェンフルラミンのＥＣ_５０値（セロトニン放出を５０％促進する有効濃度）は、０．０２８ μΜであった。このように、本発明のプロポリス組成物は、セロトニン放出促進作用を有することがわかる。
【００５７】
実施例１０
ブラジキニンＩＩ型受容体結合試験
モルモット回腸由来ブラジキニンＩＩ型受容体への結合試験評価は、Ｙ．−Ｊ．Ｉ．ＪＯＮＧらの方法（Ｐｒｏｃ．Ｎａｔｌ．Ａｃａｄ．Ｓｃｉ．ＵＳＡ，９０，１０９９４−１０９９８（１９９３））を一部改変して行った。
すなわち、７５−ｃｍ^２の培養フラスコにてコンフルエント（８−１２×１０^６細胞）になった細胞（ＷＩ−３８繊維芽細胞；ＡＴＣＣ ＣＣＬ ７５）を、４℃で、ダルベコのリン酸塩緩衝液（以下、ＰＢＳと略す）も用いて３回洗浄し、次ぎに、１ｍＭ トリス緩衝液（ｐＨ７．５）用いて１回洗浄し、そして、１ｍＭ トリス緩衝液（ｐＨ７．５）３ｍｌ中で１０分間、膨潤させた。その後、緩衝液Ｈ（１０ｍＭ 塩化ナトリウム、２ｍＭ ＥＧＴＡ、２ｍＭ 塩化マグネシウム及びプロテアーゼ阻害剤（１００μＭ フェニルメタンスルホニルフルオライド、１６．８μＭ ロイペプチン、４μｇ／ｍｌ キモステイン、５．８μＭ ペプステイン、６．６μＭ アンチパイン、トリプシンインヒビター ０．０８ｕｎｉｔｓ アプロチニン／ｍｌ、１０ｍＭ ベンズアミジン、０．２５ｍｇ／ｍｌ 大豆トリプシンインヒビター及び５ｍＭ ε−アミノカプロン酸）から成る）１０ｍＭ ＨＥＰＥＳ緩衝液）（ｐＨ７．５）９ｍｌを添加した。そして、細胞を直接に剥離し、Ｂ Ｄｏｕｎｃｅホモジナイザーを用いてホモジナイズした。このホモジネートを５０×ｇで５分間遠心分離し、得られた上清をさらに、４７０００×ｇで３０分間、遠心分離した。そして得られた膜画分を、上述の緩衝液 Ｈを用いて再懸濁させた。
【００５８】
５０μｌ中にタンパク質量が１５０−２００μｇに調整した膜画分と０．０３ｎＭのトリチウム標識したブラジキニン（ＤｕＰｏｎｔ社製；７０−１０７Ｃｉ／ｍｍｏｌ；１Ｃｉ＝３７ＧＢｑ、以下、［^３Ｈ］ＢＫと略す）、製造例１で得られたプロポリス組成物５００μｇ／ｍｌと結合緩衝液１００μｌに１００μＭグアノシン ５’−［γ−チオ］トリリン酸（Ｂｏｅｈｒｉｎｇｅｒ Ｍａｎｎｈｅｉｍ社製、以下、ＧＴＰ［γ−Ｓ］と略す）の存在化で２２℃で９０分間インキュベーションさせた。その後、氷冷した５０ｍＭ トリス緩衝液（ｐＨ７．１）３ｍｌを添加して反応を停止し、０．３％ ポリエチレンイミン水溶液を用いて予め処理しておいたガラスファイバーフィルター（Ｓｃｈｌｅｉｃｈｅｒ ＆ Ｓｃｈｕｅｌｌ社製 Ｎｏ．３１）を用いてろ過し、同緩衝液３ｍｌを用いて洗浄した後、シンチレーションカウンター（Ｐａｃｋａｒｄ Ｍｏｄｅｌ ２４２５、３０−４０％ ｅｆｆｉｃｉｅｎｃｙ）により測定し、下記式により阻害率（％）を算出した。
なお、１μＭ 非放射標識ブラジキニンの存在化での［^３Ｈ］ＢＫ非特異的結合は、常に全結合の２５％以下であった。
【００５９】
阻害率（％）＝１００−〔（Ｃ_１−Ｂ）／（Ｃ_０ −Ｂ）〕×１００
（式中、Ｃ_１ は、既知量の供試化合物と［^３Ｈ］標識ブラジキニンが共存している状態での［^３Ｈ］標識ブラジキニンの膜に対する結合量を表わし、Ｃ_０ は、供試化合物を除いた時の［^３Ｈ］標識ブラジキニンの膜に対する結合量を表わし、Ｂは、過剰のブラジキニン（１×１０^−６Ｍ）存在下での［^３Ｈ］標識ブラジキニンの膜に対する結合量を表わす。）
また、本反応系におけるブラジキニン受容体拮抗剤であるＤ−Ａｒｇ−［Ｈｙｐ．３，Ｄ−Ｐｈｅ７］−ブラジキニン（ＮＰＣ５６７）（シグマ社）のＩＣ_５０値（結合を５０％阻害する濃度）は、５．８ｎＭであった。
結果は、阻害率８９％であった。本発明のプロポリス組成物は、ブラジキニン受容体拮抗作用を有することがわかる。
【００６０】
実施例１１
オキシトシン受容体結合試験
オキシトシン受容体への結合試験評価は、Ｄｏｕｇｌａｓ Ｊ． Ｐｅｔｔｉｂｏｎｅらの方法（Ｅｕｒｏｐｅａｎ Ｊｏｕｒｎａｌ ｏｆ Ｐｈａｒｍａｃｏｌｏｇｙ−Ｍｏｌｅｃｕｌａｒ Ｐｈａｒｍａｃｏｌｏｇｙ Ｓｅｃｔｉｏｎ ，１８８ ， ２３５−２４２，１９９０）を一部改変して行った。
すなわち、屠殺する１８〜２４時間前に０．３ｍｇ／ｋｇの合成発情ホルモン剤、ジプロピオン酸ジエチルベスチルベステロールを静脈注射することにより処理したラットから摘出した子宮組織を、１ｍＭ ＥＤＴＡ、０．５ｍＭジチオスレイトールを含む１０ｍＭトリス緩衝液（ｐＨ７．４）を用いて、ホモジナイズし、４８０００×ｇ、４℃で３０分間、遠心分離し、得られた沈殿物を、５ｍＭ塩化マグネシウム、０．１％牛血清アルブミンを含む５０ｍＭトリス緩衝液（ｐＨ７．４）（以下、緩衝液Ａとする）で再溶解させ、４８０００×ｇ、４℃で３０分間、遠心分離し、沈殿物を得た。この得られた沈殿物を、緩衝液Ａを用いて溶解させ、分析するまで氷冷した。
【００６１】
次に、競合試験は、１０ｎＭの［^３Ｈ］標識したオキシトシン（Ｎｅｗ Ｅｎｇｌａｎｄ Ｎｕｃｌｅａｒ製、比活性；３０−６０Ｃｉ／ｍｍｏｌ）と、製造例１で得られたプロポリス組成物５００μｇ／ｍｌを、緩衝液Ａ中で、２２℃、６０分間反応させた。
その後、ガラスフィルター（Ｍｏｄｅｌ ７０１９、Ｓｋａｔｒｏｎ Ｉｎｃ．製）を用いて、［^３Ｈ］標識したオキシトシンと結合した膜標品を分離するために瀘過を行ない、２回、緩衝液Ａ ５ｍｌにて洗浄した。このガラスフィルターをバイアルに入れ、アクアゾール（液体シンチレーション用カクテル）と混合し、液体シンチレーションカウンターにて結合［^３Ｈ］標識オキシトシン量を測定した。
【００６２】
阻害率（％）＝１００−〔（Ｃ_１−Ｂ）／（Ｃ_０ −Ｂ）〕×１００
（式中、Ｃ_１ は、既知量の供試化合物と［^３Ｈ］標識オキシトシンが共存している状態での［^３Ｈ］標識オキシトシンの膜に対する結合量を表わし、Ｃ_０ は、供試化合物を除いた時の［^３Ｈ］標識オキシトシンの膜に対する結合量を表わし、Ｂは、過剰のオキシトシン（５×１０^−６Ｍ）存在下での［^３Ｈ］標識オキシトシンの膜に対する結合量を表わす。）
なお、本測定系におけるポジティブコントロールとしてのオキシトシンのＩＣ_５０値は、３．１ｎＭ（０．３２μｇ／ｍｌ）であった。
製造例１のプロポリス組成物（最終濃度：５００μｇ／ｍｌ）を用いた結果は、阻害率１０１％であった。本発明のプロポリス組成物に強いオキシトシン拮抗作用を有することがわかる。
【００６３】
実施例１２
ニコチン性アセチルコリン受容体結合阻害試験
ニコチン性アセチルコリン受容体結合阻害試験は、Ｌａｕｒｉｅ Ａ． Ｐａｂｒｅｚａらの方法（Ｍｏｌｅｃｕｌａｒ Ｐｈａｒｍａｃｏｌｏｇｙ，３９，９−１２，１９９１）を参考に行った。ラット大脳皮質を５０ ｍＭ トリス（ヒドロキシメチル）アミノメタン、１２０ ｍＭ塩化ナトリウム、５ ｍＭ 塩化カリウム、１ ｍＭ 塩化マグネシウム、２．５ ｍＭ 塩化カルシウムを含む緩衝液（ｐＨ７．０）でホモジナイズし、懸濁した。その後、４０，０００ ×ｇで１０分間（４℃）遠心分離した後、ペレットを再度、前記緩衝液でホモジナイズした。再び同条件で遠心分離を行った後、２回分の上清を４０，０００ ×ｇで１０分間（４℃）遠心分離した。それから、沈渣を前記緩衝液で懸濁して、大脳皮質の膜標品とした。この膜標品（４００−６００μｇの蛋白質含む）に、［^３Ｈ］シチシン（最終濃度１．５ ｎＭ）、製造例１で得られたプロポリス組成物（最終濃度：５００μｇ／ｍｌ）をそれぞれ加え（最終液量２５０μｌ）、４℃で７５分間インキュベーションした。反応は、すばやくブランデール社セルハーベスタを用いて、ワットマンＧＦ／Ｂフィルター（０．１％ウシ血清アルブミン含有の０．５％ポリエチレンイミン水溶液に最低３時間前処理する）上に吸引濾過し、冷却した緩衝液で３回洗浄した。フィルターをバイアル瓶に入れ、液体シンチレータを加えた後、フィルターに結合した放射能（トリチウム）を液体シンチレーションカウンターで測定を行った。また、非特異的結合は、１０μＭ（−）−ニコチンの存在下で測定した。
結果は、阻害率５３％であった。ここで示す阻害率（％）は、次式により算出した。
【００６４】
阻害率（％）＝１００−〔（Ｃ_１−Ｂ_０）／（Ｃ_０ −Ｂ_０）〕×１００
（式中、Ｃ_１ は、既知量のサンプルと［^３Ｈ］シチシンが共存している状態での［^３Ｈ］シチシンの膜画分に対する結合量を表わし、Ｃ_０ は、サンプルを除いた時の［^３Ｈ］シチシンの膜画分に対する結合量を表わし、Ｂ_０は、過剰のニコチン（１０μＭ）存在下での［^３Ｈ］シチシンの膜画分に対する結合量を表わす。）
なお、本反応系におけるポジティブコントロールとしてのニコチン性アセチルコリン受容体の作動化剤であるニコチンのＩＣ_５０値（ニコチン性アセチルコリン受容体結合を５０％阻害する濃度）は８．０ ｎＭであった。
本発明のプロポリス組成物は、ニコチン性アセチルコリン受容体に対するシチシン結合を競合的に阻害し、ニコチン性アセチルコリン受容体作動化作用を有することがわかる。
【００６５】
実施例１３
ムスカリン性アセチルコリン受容体結合試験
ムスカリン受容体に対する結合試験評価は、ＦＲＡＮＫ ＤＯＲＪＥらの方法（Ｊ． Ｐｈａｒｍａｃｏ． Ｅｘｐ． Ｔｈｅｒ．，２５６（２），７２７−７３３，１９９０）を、一部改変して行った。
［細胞の培養］
チャイニーズハムスター卵巣細胞（以下、ＣＨＯ細胞と略す）を、ＡＴＣＣ（Ａｍｅｒｉｃａｎ Ｔｙｐｅ Ｃｕｌｔｕｒｅ Ｃｏｌｌｅｃｔｉｏｎ）より購入した。導入操作を行うまでは、細胞を５％ＣＯ_２下で、１０％牛胎児血清、１００ ｕｎｉｔｓ／ｍｌ ペニシリンＧ、１００ ｕｎｉｔｓ／ｍｌストレプトマイシン及び４ ｍＭ グルタミン（Ｍ．Ａ． Ｂｉｏｐｒｏｄｕｃｔｓ社製）を含むＤＭＥＭ培地（Ｇｉｂｃｏ社製）中で単層培養した。
【００６６】
［導入操作］
ＣｈｅｎとＯｋａｙａｍａの方法（Ｃｈｅｎ， Ｃ． ＡＮＤ Ｏｋａｙａｍａ， Ｈ．：Ｍｏｌ． Ｃｅｌｌ． Ｂｉｏｌ． ７，２７４５−２７５２，１９８７）を参考に、選択マーカーとしてｐｃＤ ｎｅｏを用いる改良型カルシウムリン酸法を用いた。ＣＨＯ細胞は、ヒトムスカリンＭ１受容体の遺伝子配列を含むｐｃＤ発現ベクターが挿入されたプラスミドを用いて形質転換した。抗生物質の一種ネオマイシンのアナローグであるＧ−４１８（Ｇｉｂｃｏ社製）６００μｇ／ｍｌを用いた選択操作は、導入後７２時間目より開始し、２〜３週間、継続して行った。培地は、３日おきに交換した。クローナル細胞株は、リミッティングダイリューションクローニングを行うことにより得た。すなわち、理論的密度として１ウエルあたり１細胞になるように９６ウエルプレートに移し、約２週間後、単コロニー、すなわち、性質的に同じな純粋な細胞をサブクローンし、［^３Ｈ］ピレンゼピン結合能を測定することにより得た。
【００６７】
［膜画分の調製］
細胞を約８０％のコンフルエントにまで培養し、洗浄後、氷冷した５ｍＭ 塩化マグネシウムを含む２５ ｍＭ リン酸ナトリウムから成る結合緩衝液（ｐＨ７．４）（以下、結合緩衝液と略す）中で剥離し、Ｂｒｉｎｋｍａｎｎ Ｈｏｍｏｇｅｎｉｚｅｒ（調節位置５）を用いて３０秒間、ホモジナイズした。膜を１６，０００×ｇで１５分間遠心分離して沈殿させ、この沈殿物を同条件でもう一度ホモジナイズした。タンパク質濃度は、Ｂｒａｄｆｏｒｄの方法（Ｂｒａｄｆｏｒｄ， Ｍ． Ｍ．：Ａｎａｌ． Ｂｉｏｃｈｅｍ． ７２，２４８−２５４，１９７６）に従って、Ｂｉｏ−Ｒａｄ ｐｒｏｔｅｉｎ ａｓｓａｙ ｋｉｔ（Ｂｉｏ−Ｒａｄ社製）を用いて測定した。膜画分は、使用するまで−８０℃で凍結保存した。
【００６８】
［結合阻害試験評価］
結合緩衝液を用いて、分析は総容量１ｍｌ中で行い、膜画分は、タンパク質濃度が６μｇ／ｍｌとなるように調整した。［^３Ｈ］ピレンゼピンの飽和結合試験は、放射性リガンドの濃度（２〜１４００ ｐＭ）を８〜１０段階の濃度に分けて行った。結合阻害試験においては、［^３Ｈ］ピレンゼピン（最終濃度２ ｎＭ）と製造例１で得られたプロポリス組成物（最終濃度：５００μｇ／ｍｌ）とを同時に加えることにより行った。非特異的結合は、１μＭのアトロピンの存在化中で結合する［^３Ｈ］ピレンゼピンの差として定義した。インキュベーションは、２２℃で６０分間行い、ガラスフィルター（Ｍｏｄｅｌ ７０１９、Ｓｋａｔｒｏｎ Ｉｎｃ．製）を用いて迅速にろ過することにより反応を停止させた。フィルター上に結合した膜画分を、氷冷した結合緩衝液５ ｍｌを用いて３回洗浄し、乾燥させ、シンチレーションカクテル（Ｎｅｗ Ｅｎｇｌａｎｄ Ｎｕｃｌｅａｒ Ａｑｕａｓｏｌ）１０ｍｌ中に移し、ＬＫＢ β−カウンターを用いて放射活性を測定した。その結果、阻害率は、６９％であった。阻害率（％）は、次式により算出した。
【００６９】
阻害率（％）＝１００−〔（Ｃ_３−Ｂ_１）／（Ｃ_２ −Ｂ_１）〕×１００
（式中、Ｃ_３は、既知量のサンプルと［^３Ｈ］ピレンゼピンが共存している状態での［^３Ｈ］ピレンゼピンの膜画分に対する結合量を表わし、Ｃ_２は、サンプルを除いた時の［^３Ｈ］ピレンゼピンの膜画分に対する結合量を表わし、Ｂ_１は、過剰のアトロピン（１μＭ）存在下での［^３Ｈ］ピレンゼピンの膜画分に対する結合量を表わす。）
なお、本反応系におけるポジティブコントロールとしてのムスカリン１受容体の拮抗剤であるピレンゼピンのＩＣ_５０値は、１３ ｎＭ（４．５７×１０^−３μｇ／ｍｌ）であった。
本発明のプロポリス組成物は、ムスカリン１受容体に対するピレンゼピン結合を競合的に阻害し、強いムスカリン性受容体作動化作用を有することがわかる。
【００７０】
実施例１４
ドーパミン１受容体結合阻害試験
ドーパミン１受容体結合阻害試験は、Ｚｈｏｕらの方法を参考に行った（Ｎａｔｕｒｅ，３４７，７６−８０，１９９０）。
すなわち、３．０ ｋｂのＥｃｏＲｌ−Ｓａｃｌ断片をｐＢＣ１２ＢｌベクターのＨｉｎｄ ｌｌｌの Ｂａｍ Ｈ ｌサイト間にインサートし、カルシウムリン酸法によってＣＯＳ−７細胞に導入した。それから膜標品の調製は、既存の方法に従って行い、最終的にＴＥＭ緩衝液［２５ ｍＭ トリス緩衝液ｐＨ７．４、６ ｍＭ 塩化マグネシウム、１ ｍＭ エチレンジアミン四酢酸（ＥＤＴＡ）］に懸濁し、受容体膜標品とした。各試験管に、製造例１で得られた各プロポリス組成物（最終濃度：５００μｇ／ｍｌ）を加え、ドーパミン１受容体拮抗薬である［^３Ｈ］ＳＣＨ ２３３９０（アマシャム社製、最終濃度０．３ ｎＭ）、受容体膜標品（２０〜３０μｇの蛋白質を含む）および上記緩衝液を加えて反応液（総量５００ μｌ）とし、反応の開始は膜標品の添加により行った。３０℃、６０分間のインキュベーションの後、受容体に結合した標識リガンドをセルハーベスター（ブランデル社製）を用いてワットマンＧＦ／Ｂグラスファイバーフィルター上に吸引濾過して反応を停止し、直ちに、氷冷５０ ｍＭトリス−塩酸緩衝液（ｐＨ７．７）５ ｍｌで３回洗浄した。次いで、フィルター上の放射能活性を液体シンチレーションカウンターにより測定し、全結合量を求めた。また、同時に測定した１ μＭ ＳＣＨ ２３３９０存在下における結合量を非特異的結合量とし、これを全結合量から差し引くことにより特異的結合量を求めた。
結果は、阻害率７０％であった。ここで示す阻害率（％）は、次式により算出した。
【００７１】
阻害率（％）＝１００−〔（Ｃ_５−Ｂ_２）／（Ｃ_４ −Ｂ_２）〕×１００
（式中、Ｃ_５は、既知量のサンプルと［^３Ｈ］ＳＣＨ ２３３９０が共存している状態での［^３Ｈ］ＳＣＨ ２３３９０の膜画分に対する結合量を表わし、Ｃ_４は、サンプルを除いた時の［^３Ｈ］ＳＣＨ ２３３９０の膜画分に対する結合量を表わし、Ｂ_２は、過剰のＳＣＨ ２３３９０（１μＭ）存在下での［^３Ｈ］ＳＣＨ ２３３９０の膜画分に対する結合量を表わす。）
なお、本反応系におけるポジティブコントロールとしてのドーパミン１受容体の拮抗剤であるＳＣＨ ２３３９０のＩＣ_５０値（ドーパミン１受容体結合を５０％阻害する濃度）は、１．２ ｎＭであった。
本発明のプロポリス組成物に、ドーパミン１受容体作動化作用を有することがわかる。
【００７２】
実施例１５
ベンゾジアゼピン受容体結合阻害試験
ベンゾジアゼピン受容体結合阻害試験は、Ｒｏｂｅｒｔ Ｃ． Ｓｐｅｔｈらの方法を参考に行った（Ｌｉｆｅ Ｓｃｉｅｎｃｅｓ，２４，３５１−３５８，１９７９）。
すなわち、７〜８週令のウイスター系ラットの脳より調製した粗シナプトゾーム膜分画を１１８ ｍＭ塩化ナトリウム，４．８ ｍＭ塩化カリウム，１．２８ ｍＭ塩化カルシウムおよび１．２ ｍＭ硫酸マグネシウムを含む１５ ｍＭトリス−塩酸緩衝液（ｐＨ７．４）に懸濁（１ｇ脳湿重量／ ２０ｍｌ）し、受容体膜標品とした。また、標識リガンドとしては［^３Ｈ］フルニトラゼパムを用いた。各試験管に製造例１で得られたプロポリス組成物（最終濃度：５００μｇ／ｍｌ）を加え、［^３Ｈ］フルニトラゼパム（最終濃度０．４ ｎＭ）、受容体膜標品および上記緩衝液を加えて反応液（総量１ｍｌ）とし、反応の開始は膜標品の添加により行った。０℃、２０分間のインキュベーションの後、受容体に結合した標識リガンドをセルハーベスター（ブランデル社製）を用いてワットマンＧＦ／Ｂグラスファイバーフィルター上に吸引濾過して反応を停止し、直ちに、氷冷５０ ｍＭトリス−塩酸緩衝液（ｐＨ７．７）５ｍｌで３回洗浄した。次いで、フィルター上の放射能活性を液体シンチレーションカウンターにより測定し、全結合量を求めた。
また、同時に測定した１ μＭジアゼパム存在下における結合量を非特異的結合量とし、これを全結合量から差し引くことにより特異的結合量を求めた。
結果は、阻害率７８％であった。ここで示す阻害率（％）は、次式により算出した。
【００７３】
阻害率（％）＝１００−〔（Ｃ_７−Ｂ_３）／（Ｃ_６ −Ｂ_３）〕×１００
（式中、Ｃ_７は、既知量のサンプルと［^３Ｈ］フルニトラゼパムが共存している状態での［^３Ｈ］フルニトラゼパムの膜画分に対する結合量を表わし、Ｃ_６は、サンプルを除いた時の［^３Ｈ］フルニトラゼパムの膜画分に対する結合量を表わし、Ｂ_３は、過剰のジアゼパム（１μＭ）存在下での［^３Ｈ］フルニトラゼパムの膜画分に対する結合量を表わす。）
なお、本反応系におけるポジティブコントロールとしてのベンゾジアゼピン受容体の作動化剤であるジアゼパムのＩＣ_５０値（ベンゾジアゼピン受容体結合を５０％阻害する濃度）は、１０ ｎＭであった。
本発明のプロポリス組成物は、ベンゾジアゼピン受容体に対するフルニトラゼパム結合を競合的に阻害し、ベンゾジアゼピン受容体作動化作用を有することがわかる。
【００７４】
実施例１６
γ−アミノ酪酸（ＧＡＢＡ）受容体結合阻害試験
プロポリス組成物の［^３Ｈ］ＧＡＢＡのラット由来大脳皮質ＧＡＢＡ受容体への結合阻害試験は、ＡＫＩＲＡ ＴＳＵＪＩらの方法（ＡＮＴＩＭＩＣＲＯＢ． ＡＧＥＮＴＳ ＣＨＥＭＯＴＨＥＲ．，３２（２）１９０−１９４，１９８８）を、一部改変して行った。
すなわち、２３０−２７０ｋｇ体重のＳＤ系ラット（日本クレア（株））の大脳皮質を摘出し、ポリトロンホモジナイザー（Ｂｒｉｎｋｍａｎ Ｉｎｓｔｒｕｍｅｎｔ Ｃｏ． Ｉｎｃ．製）を用いて、速度５にて３０秒間、５０倍容量の１ｍＭ アスコルビン酸、１ ｍＭ テトラ酢酸ジナトリウムエチレンジアミンを含む５０ ｍＭ トリス緩衝液（ｐＨ７．４）（以下、緩衝液Ａと略す）中にてホモジナイズした。すべての操作において、組織を０℃に維持した。その後、ホモジネートを４０，０００×ｇにて１５分間遠心分離し、得られた沈殿物を５０倍容量（原重量）の緩衝液Ａに再懸濁し、上述の条件と同条件で、ポリトロンにてホモジナイズした。遠心分離及び再懸濁を、３回繰り返して行った。次いで、３７℃にて１０分間、インキュベーションを行った。このホモジネートを、４０，０００×ｇで１５分間、遠心分離し、１０倍容量（原重量）の緩衝液Ａに再懸濁させ（以下、膜画分と略す）、測定まで、−２０℃以下で保存した。
【００７５】
ラット由来大脳皮質ＧＡＢＡ受容体への結合試験を行うにあたり、上述の膜画分を３７℃にまで解氷した後、１３６ ｍＭ 塩化ナトリウム、５ ｍＭ 塩化カリウム、２ ｍＭ 硫酸マグネシウム、２ ｍＭ リン酸二水素カリウム、２ ｍＭ 塩化カルシウム及び１ ｍＭ アスコルビン酸、１ ｍＭ テトラ酢酸ジナトリウムエチレンジアミンを含む２０ ｍＭ トリス緩衝液（ｐＨ７．４）から成る２０ ｍＭ トリス−クレブス緩衝液（以下、緩衝液Ｂと略す）で２０容量（原重量）にまでメスアップした。この膜画分を、ポリトロンホモジナイザーを用いて、速度５にて３０秒間ホモジナイズし、３７℃にて１５分間インキュベーションし、２０，０００×ｇで１０分間、遠心分離した。これにより得られた沈殿物に、洗浄、再懸濁を２回以上繰り返し、最終的に得られた沈殿物を、１００倍容量（原重量）の緩衝液Ｂに再懸濁し、これを以下の試験に用いた。
【００７６】
次ぎに、製造例１で得られたプロポリス組成物５０μｌ（最終濃度：５００μｇ／ｍｌ）と、［^３Ｈ］ＧＡＢＡ（最終濃度１０ ｎＭ）を５０μｌ加え、これに上述の膜画分９００μｌを加えることにより反応を開始させた。２２℃で２０分間インキュベーションした後、ブランデル・セル・ハーベスター（Ｂｒａｎｄｅｌｌ Ｃｅｌｌ Ｈａｒｖｅｓｔｅｒ）を用い、減圧下、２×７．５ｍｌのろ過洗液を用い、ホワットマン（Ｗｈａｔｍａｎ）ＧＦ／Ｂフィルターを介するろ過により、反応を停止させた。ろ過後フィルター上に保持された放射能を、液体シンチレーションカウンター（Ｐａｃｋａｒｄ Ｍｏｄｅｌ ２４２５、３０−４０％ ｅｆｆｉｃｉｅｎｃｙ）により測定した。
また、同時に測定した１００μＭ非放射標識ＧＡＢＡ存在下における結合量を非特異的結合量とし、これを全結合量から差し引くことにより特異的結合量を求めた。結果は、阻害率９０％であった。ここで示す阻害率（％）は、次式により算出した。
【００７７】
阻害率（％）＝１００−〔（Ｃ_９−Ｂ_４）／（Ｃ_８ −Ｂ_４）〕×１００
（式中、Ｃ_９は、既知量のサンプルと［^３Ｈ］ＧＡＢＡが共存している状態での［^３Ｈ］ＧＡＢＡの膜画分に対する結合量を表わし、Ｃ_８は、サンプルを除いた時の［^３Ｈ］ＧＡＢＡの膜画分に対する結合量を表わし、Ｂ_４は、過剰のＧＡＢＡ（１００μＭ）存在下での［^３Ｈ］ＧＡＢＡの膜画分に対する結合量を表わす。）
なお、本測定系におけるポジティブコントロールとしてのＧＡＢＡのＩＣ_５０値（ＧＡＢＡ受容体結合を５０％阻害する濃度）は、１１ ｎＭ（１．１３×１０^−３μｇ／ｍｌ）であった。
本発明のプロポリス組成物が、強いＧＡＢＡ受容体作動化作用を有することがわかる。
【００７８】
実施例１７
ヒトコルチコイド受容体リガンド結合試験
ヒトコルチコイド受容体リガンド結合活性の測定は、Ａｂｂｏｔ Ｆ． Ｃｌａｒｋらの方法（Ｉｎｖｅｓｔ． Ｏｐｈｔｈａｌｍｏｌ． Ｖｉｓ． Ｓｃｉ．，３５，８０５−８１３，１９９６）を一部改変して行った。
すなわち、ヒトリンパ芽球腫細胞ＩＭ９（ＡＴＣＣ、Ｂｅｔｈｅｓｄａ、ＭＤ）を、可溶性グルココルチコイド受容体（以下、ＧＲと略す）として用いた。
上述の細胞を１０％牛胎児血清（ＨｙＣｌｏｎｅ、Ｌｏｇａｎ、ＵＴ）、１００Ｕ／ｍｌ ペミシリン、１００μｇ／ｍｌ ストレプトマイシン及び２ｍＭ Ｌ−グルタミン（Ｇｉｂｃｏ）を含むＲＰＭＩ １６４０培地（Ｇｉｂｃｏ、Ｇｒａｎｄ Ｉｓｌａｎｄ、ＮＹ）を用いて、３７℃、７％ ＣＯ_２ の条件下で１０×１０^５細胞／ｍｌになるまで培養した。その後、この細胞を、１５００ｇで１０分間遠心分離することにより、培地から剥離した。剥離した細胞を１２倍量のダルベコのリン酸塩緩衝液（以下、ＰＢＳと略す：Ｇｉｂｃｏ）を用いて洗浄した後、再び１５００ｇで１０分間遠心分離して沈殿を得、これをＰＢＳにて洗浄した。
【００７９】
洗浄した細胞に、１０ｍＭ ＴＥＳ、１０ｍＭ モリブデン酸ナトリウム、１ｍＭ ＥＤＴＡ、２０ｍＭ ２−メルカプトエタノール及び１０％ グリセロールから成るホモジネーション緩衝液（ｐＨ ７．４）（以下、ＨＢと略す）を５〜６倍量加え懸濁させ、細胞をＮ_２キャビテーター（Ｐａｒｒ Ｉｎｓｔｒｕｍｅｎｔ、Ｍｏｌｉｎｅ、ＩＬ）を用いて、０℃で、１５分間、６００〜７５０ｐｓｉの窒素圧で２回、キャビテーションすることにより破砕し、この細胞破砕物を、ニコンダイアフォトを用いたＨｏｆｆｍａｎ対比型顕微鏡（Ｇａｒｄｅｎ Ｃｉｔｙ、ＮＹ）を用いて明確化した。
その後、細胞破砕調製物を、２７０００ｇで１５分間遠心分離し、結果得られた上清を、１０３０００ｇ、４℃で６０分間遠心分離した。この得られた上清画分中のタンパク質量は、ＢＣＡ ａｓｓａｙ ｋｉｔ（Ｐｉｅｒｃｅ Ｃｈｅｍｉｃａｌ、Ｒｏｃｋｆｏｒｄ、ＩＬ）を用い、標準物質として牛血清アルブミンを用いて定量し、上清画分の標品は、ドライアイス−アセトンバス中で急冷し、−７０℃で凍結保存した。
【００８０】
競合結合試験は、ＩＭ９細胞サイトゾル１ｍｇ、１．５ｎＭの［^３Ｈ］標識したトリアムシノロン（０．０２５μＣｉ：Ａｍｅｒｓｈａｍ、Ａｒｌｉｎｇｔｏｎ、Ｈｅｉｇｈｔｓ、ＩＬ）と製造例１で得たプロポリス組成物を最終濃度が５００μｇ／ｍｌに成るように調製したものを、ＨＢ（総容量２００μｌ）中に加え、０℃で１８時間、インキュベーションさせた。木炭−デキストラン混合物（２％ 活性炭、０．５％ デキストラン、１％ ＥＤＴＡを含む１０ｍＭ トリス緩衝液（ｐＨ ７．４））１００μｌを添加することにより反応を停止させ、この混合物をさらに０℃で１０分間、インキュベーションし、８２００ｇで５分間、遠心分離した。結果として得られた上清１００μｌを、液体シンチレーションスペクトロメトリーを用いて以下の式にて放射活性を測定した。
結果は、阻害率８８％であった。なお、１０μＭ 非放射標識デキサメタソンの存在化での［^３Ｈ］トリアムシノロンの非特異的結合は、常に全結合の１５％以下であった。
【００８１】
阻害率（％）＝１００−〔（Ｃ_１−Ｂ）／（Ｃ_０ −Ｂ）〕×１００
（式中、Ｃ_１は、既知量の供試化合物と［^３Ｈ］標識トリアムシノロンが共存している状態での［^３Ｈ］標識トリアムシノロンの膜に対する結合量を表わし、Ｃ_０は、供試化合物を除いた時の［^３Ｈ］標識トリアムシノロンの膜に対する結合量を表わし、Ｂは、過剰のデキサメタソン（１０×１０^−６Ｍ）存在下での［^３Ｈ］標識トリアムシノロンの膜に対する結合量を表わす。）
なお、本測定系におけるポジティブコントロールとしてのデキサメタソンのＩＣ_５０値（結合を５０％阻害する濃度）は、２．７ｎＭであった。本発明のプロポリス組成物が強いＧＣ様作用を有することがわかる。
【００８２】
実施例１８
メラトニン受容体結合阻害
メラトニン受容体結合阻害試験は、Ｄａｒｒｙｌ Ｓ．らの方法（Ｅｕｒｏｐｅａｎ Ｊｏｕｒｎａｌ ｏｆＰｈａｒｍａｃｏｌｏｇｙ，１７５，７１−７７，１９９０）を参考に行った。
すなわち、シリアンハムスター（２〜４ヶ月齢、雄）を１６時間明期、８時間暗期という条件下で飼育し、既存の方法に従って脳からラジオレセプターアッセイ用の粗膜画分を調製した。なお懸濁には、氷冷緩衝液［５０ ｍＭ トリス（ヒドロキシメチル）アミノメタン（ｐＨ７．４）］を用いた。この膜標品（５０−１００μｇの蛋白質含む）に、［^１２５Ｉ］ヨードメラトニン（最終濃度０．１ ｎＭ）、製造例１で得られたプロポリス組成物（最終濃度：１００μｇ／ｍｌ）を加え（最終液量２００μｌ）、４℃で３０分間インキュベーションさせた後、ガラス繊維濾紙上に速やかに低圧吸引濾過した。それから、濾紙上に残存した受容体結合放射活性を液体シンチレーションカウンターで測定した。また、非特異的結合は、３０μＭメラトニンの存在下で測定した。
結果は、阻害率８９％であった。ここで示す阻害率（％）は、次式により算出した。
【００８３】
阻害率（％）＝１００−〔（Ｃ_１−Ｂ_０）／（Ｃ_０ −Ｂ_０）〕×１００
（式中、Ｃ_１ は、既知量のサンプルと［^１２５Ｉ］ヨードメラトニンが共存している状態での［^１２５Ｉ］ヨードメラトニンの膜画分に対する結合量を表わし、Ｃ_０ は、サンプルを除いた時の［^１２５Ｉ］ヨードメラトニンの膜画分に対する結合量を表わし、Ｂ_０は、過剰のメラトニン（３０μＭ）存在下での［^１２５Ｉ］ヨードメラトニンの膜画分に対する結合量を表わす。）
なお、本反応系におけるポジティブコントロールとしてのメラトニン受容体の作動化剤であるヨードメラトニンのＩＣ_５０値（メラトニン受容体結合を５０％阻害する濃度）は１８８ ｎＭであった。
本発明のプロポリス組成物は、メラトニン受容体に対するヨードメラトニン結合を競合的に阻害し、メラトニン受容体と高い親和性を有することが分かる。
【００８４】
実施例１９
カルシウムチャンネル阻害試験
カルシウムチャンネル阻害試験は、Ｈｏｗａｒｄ Ｒ． Ｌｅｅらの方法（Ｌｉｆｅ Ｓｃｉｅｎｃｅｓ，３５（７），７２１−７３２，１９８４）を一部改変して行った。
すなわち、体重２５０−３５０ｇの雄性ＳＤラットを断頭して殺した後、脳を速やかに取り出した。そして、大脳皮質を氷冷したベトリ皿上で脳の残屑から解剖した。その後、得た大脳皮質を１９倍容量のＫｒｅｂｓのリン酸緩衝液（１２０ｍＭ ＮａＣｌ、４．８ｍＭＫＣｌ、２．１ｍＭ ＭｇＳＯ_４、１．３ｍＭ ＣａＣｌ_２、２０．３ｍＭ Ｎａ_２ＨＰＯ_４、３．２ｍＭ ＨＣｌ、１０ｍＭ Ｄ−ｇｌｕｃｏｓｅ、ｐＨ ７．４）を遠心管に加え、ポリトロン（Ｂｒｉｎｋｍａｎ社製）を用いて１０秒間、３回（調節速度；４０％）、ホモジナイズした。その後、このホモジナイズした大脳皮質を２回、４８０００×ｇで１０分間、遠心分離することにより洗浄し、上述のＫｒｅｂｓのリン酸緩衝液にて再懸濁させた。
【００８５】
このようにして得られた新鮮な大脳皮質ホモジネートを湿重量で１−５ｍｇ／組織になるように調整し、その後、Ｋｒｅｂｓのリン酸緩衝液を含むチューブ中に総容量が２ｍｌとなるように０．０４ｎＭ［^３Ｈ］標識したイソプロピル ４−（２，１，３−ベンゾキサジアゾル−４−イル）−１，４−ジヒドロ−２，６−ジメチル−ピリジン−３，５−ジカルボキシル酸メチル １−メチルエチルエステル（ｉｓｏｐｒｏｐｙｌ ４−（２，１，３−ｂｅｎｚｏｘａｄｉａｚｏｌ−４−ｙｌ）−１，４−ｄｉｈｙｄｒｏ−２，６−ｄｉｍｅｔｈｙｌ−ｐｙｒｉｄｉｎｅ−３，５−ｄｉｃａｒｂｏｘｙｌｉｃ ａｃｉｄ ｍｅｔｈｙｌ １−ｍｅｔｈｙｌｅｔｈｙｌｅｓｔｅｒ、以下［^３Ｈ］（＋）ＰＮ ２００−１００と略す）（８９Ｃｉ／ｍｍｏｌ、Ｎｅｗ Ｅｎｇｌａｎｄ Ｎｕｃｌｅａｒ，Ｂｏｓｔｏｎ，Ｍａｓｓ．社製）及び製造例１で得られた最終濃度が５００μｇ／ｍｌの濃度になるように調整したプロポリス組成物を加え、ナトリウムランプを用いた暗室で、２２℃、９０分間、インキュベーションした。
【００８６】
その後、膜に結合した放射活性をグラスファイバーフィルター（Ｗｈａｔｍａｎ社製、ＧＦ／Ｂ）を用いて吸引濾過してトラップし、次いで、０．９％食塩水５ｍｌを用いて３回洗浄した。そして、フィルターを乾燥させ、シンチレーション溶液中で一晩浸漬することにより、フィルター中の放射活性を溶出させた後、この溶出物中の放射活性を液体シンチレーションスペクトロメトリー（４３％ ｅｆｆｉｃｉｅｎｃｙ）を用いて測定し、以下に示す式を用いてカルシウムチャンネル阻害活性を算出した。
また、特異的結合は、１μＭのニフェジピン（ｎｉｆｅｄｉｐｉｎｅ）と置換される結合と定義し、タンパク質量は、Ｌｏｗｒｙらの方法（Ｏ．Ｈ．Ｌｏｗｒｙ， Ｎ．Ｊ．Ｒｏｓｅｎｂｒｏｕｇｈ， Ａ．Ｌ．Ｆａｒｒ ａｎｄ Ｒ．Ｊ．Ｒａｎｄａｌｌ；Ｊ． Ｂｉｏｌ． Ｃｈｅｍ． １９３， ２６５−２７５ （１９５１））により測定した。
【００８７】
阻害率（％）＝１００−〔（Ｃ_１−Ｂ）／（Ｃ_０ −Ｂ）〕×１００
（式中、Ｃ_１ は、既知量の供試化合物と［^３Ｈ］（＋）ＰＮ ２００−１００が共存している状態での［^３Ｈ］（＋）ＰＮ ２００−１００の膜画分に対する結合量を表わし、Ｃ_０ は、供試化合物を除いた時の［^３Ｈ］（＋）ＰＮ ２００−１００の膜画分に対する結合量を表わし、Ｂは、過剰のｎｉｆｅｄｉｐｉｎｅ（１×１０^−６Ｍ）存在下での［^３Ｈ］（＋）ＰＮ ２００−１００の膜画分に対する結合量を表わす。）
なお、本反応系におけるポジティブコントロールとしてのカルシウムチャンネル阻害剤であるニトレンジピン（ｎｉｔｒｅｎｄｉｐｉｎｅ、ファイザー製薬社製）のＩＣ_５０値は、１．７ｎＭであった。
用いたプロポリス組成物では、阻害率が７４％であった。本発明のプロポリス組成物は、カルシウムチャンネルに対するニトレンジピンの結合を競合的に阻害し、強いカルシウムチャンネル阻害作用を有することがわかる。
【００８８】
実施例２０
メラトニン凝集ホルモン（ＭＣＨ）受容体結合阻害試験
ＭＣＨ受容体結合阻害試験は、Ｊｏｎ Ｃｈａｍｂｅｒｓらの方法（Ｎａｔｕｒｅ， ４００， ２６１−２６５， １９９９）を参考にして行った。
すなわち、ヒトＭＣＨ受容体であるＳＬＣ１を発現させたチャイニーズハムスター卵巣細胞（ＣＨＯ細胞）（２〜４μｇ／タンパク質となるように調整）、０．１ｎＭの［^１２５Ｉ］標識した［Ｐｈｅ^１３，Ｔｙｒ^１９］−ＭＣＨ（比活性；２２００Ｃｉ／ｍｍｏｌ、Ｎｅｗ Ｅｎｇｌａｎｄ Ｎｕｃｌｅａｒ製）及び最終濃度が５００μｌ／ｍｌになるように調整した製造例１で得られたプロポリス組成物とを、２２℃で６０分間、インキュベートした。その後、グラスファイバーフィルター（Ｗｈａｔｍａｎ社製、ＧＦ／Ｃ）を用いて吸引濾過して放射活性をトラップし、次いで、０．３％脱脂粉乳５ｍｌを用いて３回洗浄した。そして、フィルターを乾燥させ、シンチレーション溶液中で一晩浸漬することにより、フィルター中の放射活性を溶出させた後、この溶出物中の放射活性を液体シンチレーションスペクトロメトリー（４３％ ｅｆｆｉｃｉｅｎｃｙ）を用いて測定し、以下に示す式を用いてＭＣＨ受容体阻害活性を算出した。
結果は、阻害率が５６％であった。また、特異的結合は、０．１μＭのＭＣＨと置換される結合と定義し、タンパク質量は、Ｌｏｗｒｙらの方法（Ｏ．Ｈ．Ｌｏｗｒｙ， Ｎ．Ｊ．Ｒｏｓｅｎｂｒｏｕｇｈ， Ａ．Ｌ．Ｆａｒｒ ａｎｄ Ｒ．Ｊ．Ｒａｎｄａｌｌ；Ｊ． Ｂｉｏｌ． Ｃｈｅｍ． １９３， ２６５−２７５ （１９５１））により測定した。
【００８９】
阻害率（％）＝１００−〔（Ｃ_１−Ｂ）／（Ｃ_０ −Ｂ）〕×１００
（式中、Ｃ_１ は、既知量の供試化合物と［^１２５Ｉ］標識した［Ｐｈｅ^１３，Ｔｙｒ^１９］−ＭＣＨが共存している状態での［^１２５Ｉ］標識した［Ｐｈｅ^１３，Ｔｙｒ^１９］−ＭＣＨの膜画分に対する結合量を表わし、Ｃ_０ は、供試化合物を除いた時の［^１２５Ｉ］標識した［Ｐｈｅ^１３，Ｔｙｒ^１９］−ＭＣＨの膜画分に対する結合量を表わし、Ｂは、過剰のＭＣＨ（１×１０^−５Ｍ）存在下での［^１２５Ｉ］標識した［Ｐｈｅ^１３，Ｔｙｒ^１９］−ＭＣＨの膜画分に対する結合量を表わす。）
なお、本反応系におけるポジティブコントロールとしてのＭＣＨ受容体作動化薬であるＭＣＨのＩＣ_５０値は、０．２５ｎＭであった。
本発明のプロポリスは、ＭＣＨ受容体に対するＭＣＨの結合を競合的に阻害し、ＭＣＨ受容体と高い親和性を有することが分かる。
【００９０】
実施例２１
アンジオテンシン変換酵素（ＡＣＥ）阻害活性試験
ＡＣＥ阻害活性試験は、Ｈｏｏｒｎ，Ｃ．Ｍ．とＲｏｔｈ，Ｒ．Ａ．の方法（Ｂｒｉｔ．Ｊ．Ｐｈａｒｍａｃｏｌ．，１１０，５９７−６０２，１９９３）を一部改変して行った。
すなわち、ガラス試験管中に、製造例１で得られたプロポリス組成物を最終濃度が５００μｇ／ｍｌとなるように１０％ジメチルスルホキシド（以下、ＤＭＳＯと略す）に溶解したものを０．０６ｍｌ加え、次いで、１００ｍＵ／ｍｌのＡＣＥ（シグマ社製）０．０３ｍｌを添加し、３７℃で５分間、プレインキュベーションした後、１００ｍＭ ホウ酸緩衝液 ｐＨ ８．３、５００ｍＭ ＮａＣｌ、６．５ｍＭ ［^３Ｈ］標識したヒプリルグリシルグリシン（ｈｙｐｐｕｒｙｌｇｌｙｃｙｌｇｌｙｃｉｎｅ、以下、［^３Ｈ］ＨＧＧと略す）０．３ｍｌを加え、３７℃で５時間、インキュベーションし、１Ｎ 塩酸０．５ｍｌを加えて反応を停止させた。その後、酢酸エチル１．５ｍｌを加えて１分間攪拌し、ＡＣＥにより遊離した［^３Ｈ］したヒプリル酸（ｈｉｐｐｕｒｉｃ ａｃｉｄ）を抽出し、この酢酸エチル層１．０ｍｌを分取して乾固させた後、液体シンチレーションを加え、放射活性を測定し、次式にて阻害率（％）を算出した。
【００９１】
阻害率（％）＝（Ａ−Ｂ）／Ａ×１００
（ここで、Ａは阻害剤を含まない場合の放射活性値を表わし、Ｂは阻害剤を添加した場合の放射活性値を表わす。）
結果は、阻害率６０％であった。なお、本反応系におけるポジティブコントロールとしてのＡＣＥ阻害剤であるカプトプリル（ｃａｐｔｏｐｒｉｌ、和光純薬製）のＩＣ_５０値は、０．００１１μＭであった。 本発明のプロポリス組成物は強いＡＣＥ阻害作用を有することがわかる。
【００９２】
実施例２２
トロンボキサンＡ２（ＴＸＡ２）合成酵素阻害活性試験
ＴＸＡ２合成酵素阻害活性試験は、ＷＡＤＥ， Ｍ． Ｌ．とＦＩＴＺＰＡＴＲＩＣＫ， Ｆ． Ａ．の方法（Ａｒｃｈ． Ｂｉｏｃｈｅｍ． Ｂｉｏｐｈｙｓ．， ３４７， １７４−１８０， １９９７）を一部改変して、ヒト血小板ミクロソームのトロンボキサン合成酵素阻害活性を調べた。
すなわち、１０ｍＭ リン酸緩衝液（ｐＨ ７．４）中に、血小板ミクロソーム（０．５ｍｇ タンパク質）、１０μＭ プロスタグランジンＨ２（以下、ＰＧＨ２と略す）、５ｍＭ エピネフリン、２μＭ ヘミン、及び前記製造例１で調製したプロポリス組成物（最終濃度：５００μｇ／ｍｌ）を含む反応液（全量 ０．２ｍｌ）を用いた。プロポリス組成物と血小板ミクロソームを２２℃で３分間プレインキュベーションした後、基質として１０μＭ ＰＧＨ２を添加して２２℃で２分間反応させた。その後、塩化スズ溶液を添加して反応を停止し、生成したトロンボキサンＢ２（以下、ＴＸＢ２と略す）の量を、ＴＸＢ２ ＥＩＡ（Ｅｎｚｙｍｅ Ｉｍｍｕｎｏ Ａｓｓａｙ）キット（アマシャム ファルマシア バイオテク社製）を用いて測定した。
結果は、阻害率９３％であった。なお、本反応系におけるポジティブコントロールとしてのＴＸＡ２合成酵素阻害剤であるｆｕｒｅｇｒｅｌａｔｅのＩＣ_５０値（酵素活性を５０％阻害する濃度）は０．２１μＭであった。本発明のプロポリス組成物は、強力なＴＸＡ２合成酵素阻害活性を有することが分かる。
【００９３】
実施例２３
一酸化窒素合成酵素阻害活性試験
プロポリス抽出物のＮＯＳ阻害活性試験は、Ｅｓｔｒａｄａらの方法（Ｂｉｏｃｈｅｍ．Ｂｉｏｐｈｙｓ．Ｒｅｓ．Ｃｏｍｍｕｎ．，１８６，４７５−４８２（１９９２））を、一部改変して行った。
マウス由来のマクロファージ様細胞ＲＡＷ２６４−７を５×１０^５／ｗｅｌｌで培養プレートに播種し、３７℃で２４時間インキュベーションした。この培養プレートに、ＬＰＳ（リポポリサッカライド；１０ｎｇ／ｍｌ）および製造例１で得られたプロポリス組成物を５００μｇ／ｍｌの濃度になるように調整して添加し、３７℃で１８時間インキュベーションした。得られた培養液（１００μｌ）とＧｒｉｅｓｓ試薬（１００μｌ）を反応させ、５４０ｎｍの吸光度を測定した。そして、プロポリス抽出物の非存在下での吸光度を１００％とし、プロポリス抽出物存在下での百分率（％）を求め、阻害率を算出した。
その結果は、阻害率９２％であった。なお、本反応系におけるポジティブコントロールとしてのＮＯＳ阻害剤であるＬ−ＮＭＭＡのＩＣ_５０値（酵素活性を５０％阻害する濃度）は、６９μＭであった。本発明のプロポリス組成物に、強いＮＯＳ阻害作用を有することがわかる。
【００９４】
実施例２４
ヒト免疫不全ウイルス−１プロテアーゼ阻害活性試験
ＨＩＶ−１プロテアーゼ阻害活性の測定は、ＴＯＴＨ， Ｍ． Ｖ．とＭＡＲＳＨＡＬＬ， Ｇ． Ｒ．の方法（Ｉｎｔ． Ｊ． Ｐｅｐｔｉｄｅ． Ｐｒｏｔｅｉｎ Ｒｅｓ．，３６，５４４−５５０，１９９０）を一部改変して行った。
すなわち、ＨＩＶ−１プロテアーゼ（遺伝子組み換えにより大腸菌に発現させたもの、バケム社製）を、２００ｍＭ 塩化ナトリウム、５ｍＭ ジチオトレイトール、１０％（ｖ／ｖ）グリセロールを含む１００ｍＭ 酢酸ナトリウム緩衝液（ｐＨ４．９）（以下、緩衝液Ａとする）し、０．０２ｍｇ／ｍｌの酵素溶液とした。また、１ｍｇ／ｍｌのＨｉｓ−Ｌｙｓ−Ａｌａ−Ａｒｇ−Ｖａｌ−Ｌｅｕ−ｐ−ｎｉｔｒｏ−Ｐｈｅ−Ｇｌｕ−Ａｌａ−Ｎｌｅ−Ｓｅｒ−ＮＨ_２（バケム社製）を蒸留水に溶解し、基質溶液とした。
０．５ｍｌ容量のプラスチックチューブに、緩衝液Ａ１００μｌ、基質溶液１０μｌ及び製造例１で得られたプロポリス組成物１０μｌ（最終濃度：５００μｇ／ｍｌ）を加え、３７℃で５分間、プレインキュベートした後、上記酵素溶液２５μｌを加え十分に攪拌した後、３７℃で４０分間、反応を行った。その後、１０％トリフルオロ酢酸１５μｌを添加して反応を停止させた。
反応停止後、酵素反応により遊離してくるｐ−ｎｉｔｒｏ−Ｐｈｅ−Ｇｌｕ−Ａｌａ−Ｎｌｅ−Ｓｅｒ−ＮＨ_２量を、下記条件下でＨＰＬＣにより定量した。
【００９５】
＜ＨＰＬＣ条件＞
カラム：ＹＭＣ−Ｐａｃｋ Ｃ８（Ａ−２０２、１５０×４．６ｍｍＩ．Ｄ．、（株）ワイエムシィ製）
溶出溶媒：０．１％トリフルオロ酢酸を含む０〜６３％のアセトニトリルの直線濃度勾配（２０分）
流速：１ｍｌ／ｍｉｎ．
検出波長：３００ｎｍ
また、阻害率（％）は、次式により算出した。
阻害率（％）＝（Ｂ−Ｃ）／Ｂ×１００
〔式中、Ｂ：阻害剤を含まない場合のｐ−ｎｉｔｒｏ−Ｐｈｅ−Ｇｌｕ−Ａｌａ−Ｎｌｅ−Ｓｅｒ−ＮＨ_２のピーク面積、Ｃ：阻害剤を添加した場合のｐ−ｎｉｔｒｏ−Ｐｈｅ−Ｇｌｕ−Ａｌａ−Ｎｌｅ−Ｓｅｒ−ＮＨ_２のピーク面積を表す。〕
上述して得た結果、阻害率は、９４％であった。なお、本測定系におけるポジティブコントロールとしてのペプステイン（ｐｅｐｓｔａｉｎ）ＡのＩＣ_５０値は、５．２μＭ（３．５７μｇ／ｍｌ）であった。本発明のプロポリス組成物に強いＨＩＶ−１プロテアーゼ阻害作用を有することがわかる。
【００９６】
以下に処方例を示す。
処方例１
［錠剤の製造］
製造例１で得られたブラジル産プロポリス及び中国産プロポリスのエタノール抽出物を重量比で１：１に混合したブレンドプロポリスエキスを用いて、常法に従って、下記の組成の錠剤を製造した。
（組 成） （配合：質量％）
ブレンドプロポリスエキス ２４
乳糖 ６３
コーンスターチ １２
グァーガム １
【００９７】
処方例２
［ジュースの製造］
製造例１で得られたブラジル産プロポリス及び中国産プロポリスのエタノール抽出物を重量比で１：１に混合したブレンドプロポリスエキスを用いて、常法に従って、下記の組成のジュースを製造した。
（組 成） （配合：質量％）
冷凍濃縮温州みかん果汁 ５．０
果糖ブドウ糖液糖 １１．０
クエン酸 ０．２
Ｌ−アスコルビン酸 ０．０２
香料 ０．２
色素 ０．１
ブレンドプロポリスエキス ０．２
水 ８３．２８
【００９８】
処方例３
［フェイスクリームの製造］
製造例１で得られたブラジル産プロポリス及び中国産プロポリスのエタノール抽出物を重量比で１：１に混合したブレンドプロポリスエキスを用いて、常法に従って、下記の組成のフェイスクリームを製造した。
（組 成） （配合：重量％）
イソステアリン酸イソプロピル ８．０
ホホバ油 ６．０
セタノール ８．０
ステアリルアルコール ２．０
ポリオキシエチレンラウリルエーテル １．５
プロピレングリコール ６．０
ソルビトール １．０
パラベン ０．４
ブレンドプロポリスエキス ０．５
ビタミンＥ ０．５
香料 ０．１
精製水 ６６．０
【００９９】
【発明の効果】
本発明で特定する成分を有するプロポリス組成物は、上記各実施例で示すような生物活性を有し、前記用途に有用である。これらの生物活性は含有されている成分に由来すると考えられる。また、このような組成物は、長期間服用しても安全である。[0001]
[Technical field to which the invention belongs]
The present invention relates to the use of a composition containing propolis.
[0002]
[Prior art]
Propolis is a substance that is produced by worker bees by collecting sap such as young shoots of plants and mixing it with their own saliva, and is a substance that contains resin components, aromatic oil, beeswax, volatile oil, pollen, minerals, etc. .
This propolis functions as a defense fence that prevents the entry of foreign enemies into the beehive, functions as a reinforcing agent when closing the nest cracks, and covers corpses such as small animals and insects to prevent corruption. It is known that it is used for this purpose (Patent Document 1).
Propolis having such various functions is recognized to have a wide range of physiological activities such as anti-inflammatory action, anti-ulcer action, antibacterial action, and antioxidant action in humans. It is widely used as a health food because it is considered useful for the treatment and prevention of diseases such as these. However, it cannot be said that the physiological activity of propolis has been sufficiently studied, and further elucidation and development of applications using the same have been desired.
[0003]
In recent years, many researchers have been researching the components of propolis by production area. For example, Brazilian propolis contains, as a main component, p-coumaric acid, artepillin C having a prenyl group, drupanin, (E) -prenyl-4- (2,3-dihydrocinnamoyloxy) ) Cinnamic acid), ((E) -3-prenyl-4- (2,3-dihydrocinnamoyloxy) cinnamic acid), (E) -3- (2,2-dimethyl-8-prenyl-2H-benzopyran-6 Cinnamic acid derivatives such as (yl) -2-propenonic acid and ((E) -3- (2,2-dimethyl-8-prenyl-2h-benzopyran-6-yl) -2-propenoic acid) . On the other hand, propolis such as Chinese, Japanese, Australian and Uruguayic acid have different components depending on the region (country), but the main components and composition show similar patterns, and as main components Many flavonoids such as chrysin, pinocembrin, galangin, pinobanksin-3-acetate, and pinobanksin are detected. Due to the difference between these main components, Brazilian propolis is qualitatively shown to be “cinnamate derivative-based type”, while other propolis such as China is “flavonoid-centered type” (non- Patent Document 1).
[0004]
Despite the fact that the components of Brazilian propolis and Chinese propolis are completely different in this way, most of the propolis-related products currently on the market are Brazilian propolis, and Chinese propolis is hardly marketed. Currently, Brazilian propolis is considered to be superior in terms of quality and biological activity in the market, but there is no definite basis for this. On the other hand, Kumazawa et al. Measured the polyphenol content and antioxidant activity of propolis by production area, and found that there was a general correlation between the total polyphenol content and the antioxidant activity. ) Propolis has higher antioxidant activity (Non-patent Documents 2 and 3).
[0005]
[Patent Document 1]
JP-A-9-149765
[Non-Patent Document 1]
Shigeru Tazawa et al .: Biopharmaceutical magazine, 54 (6), 164-168, 2000
[Non-Patent Document 2]
Kumazawa Shigenori, Nakayama Tsutomu: Bee Science, 22 (1), 1-8, 2001
[Non-Patent Document 3]
Shigenori Kumazawa: FOOD Style 21, 5 (10), 54-58, 2001
[0006]
[Problems to be solved by the invention]
An object of the present invention is to clarify the relationship between propolis components and their physiological activities and to provide a new use of propolis.
[0007]
[Means for Solving the Problems]
The present inventor has intensively studied to solve the above-described problems, and has focused on the content and biological activity of components contained in Brazilian propolis and Chinese propolis, and completed the present invention.
[0008]
That is, the present invention relates to an angiotensin II receptor antagonist composition containing adenolin, comprising propolis, characterized by containing quercetin, p-coumaric acid and artepilin C, and further containing chrysin, galangin or caffeic acid phenethyl ester. β2 receptor activator composition, female hormone-like composition, endothelin antagonist composition, serotonin reabsorption inhibitor composition, serotonin release promoting composition, serotonin receptor antagonist composition, serotonin transporter receptor binding inhibition Composition, bradykinin receptor antagonist composition, oxytocin antagonist composition, nicotinic acetylcholine receptor activation composition, muscarinic acetylcholine receptor activation composition, dopamine 1 receptor activation composition, Benzodiazepine receptor activation composition, γ -Aminobutyric acid receptor activation composition, glucocorticoid-like composition, melatonin receptor activation composition, melatonin receptor antagonist composition, calcium channel inhibitor composition, melatonin aggregation hormone receptor antagonist composition A composition for inhibiting angiotensin converting enzyme, a composition for inhibiting thromboxane A2 synthase, a composition for inhibiting nitric oxide synthase, and a composition for inhibiting human immunodeficiency virus protease. In the present invention, the propolis has a quercetin equivalent of 4.58 mg / ml or more, p-coumaric acid of 1.16 mg / ml or more, artepiline C of 6.73 mg / ml or more, and chrysin of 0.84 mg / ml or more. Any one of the above-mentioned compositions, propolis containing 0.53 mg / ml or more of galangin and 0.81 mg / ml or more of caffeic acid phenethyl ester, Any one of the above propolis compositions, which is a propolis containing at least two or more extracts, an oral composition or a parenteral composition containing any of the above compositions, and the above oral composition that is a food, The above-mentioned oral composition or parenteral composition which is a medicine and the above-mentioned parenteral composition which is a cosmetic.
[0009]
DETAILED DESCRIPTION OF THE INVENTION
The propolis in the present invention contains quercetin, p-coumaric acid and artepiline C, and further contains chrysin, galangin or caffeic acid phenethyl ester.
[0010]
Quercetin is a typical flavonol-type flavonoid widely present in plant foods such as lettuce, broccoli and onion, and has been shown to exhibit various physiological actions. Quercetin is preferably 4.58 mg / ml or more in terms of quercetin equivalent.
[0011]
p-coumaric acid is 3- (4-Hydroxyphenyl) -2-propenic acid (3- (4-hydroxyphenyl) -2-propionic acid), and has the molecular formula C ₉ H ₈ O ₃ , Molecular weight 164.16, melting point 210-213 ° C., pKa value 9.45 (25 ° C.). It is abundant in the peels of plums (Prunus serotina), Trifolium platens, and the like. The amount of p-coumaric acid is preferably 1.16 mg / ml or more.
[0012]
Artepilin C is 4-Hydroxy-3,5-diprenylcinnamic acid (4-hydroxy-3,5-diprenylcinnamic acid) and has the molecular formula C ₁₉ H ₂₄ O ₃ The molecular weight is 300.397, the melting point is 95 ° C., and 150 to 152 ° C., and it is abundant in Artemisia spp., Baccharis spp. The amount of artepilin C is preferably 6.73 mg / ml or more.
[0013]
Chrysin is 5,7-Dihydroxyflavone (5,7-dihydroxyflavone) and has the molecular formula C ₁₅ H ₁₀ O ₄ , Molecular weight 254.242, melting point 285-286 ° C., 290 ° C. (275 ° C.), pKa value 9.05, 7.42 (20 ° C., 50% ethanol). It is abundant in elm (Ulmus sieboldiana), Oryzylum indicum moth, Pinus spp., And Scutellaria spp. The amount of chrysin is preferably 0.84 mg / ml or more.
[0014]
Galangin is 3,5,7-Trihydroxyflavone (3,5,7-trihydroxyflavone) and has the molecular formula C ₁₅ H ₁₀ O ₅ , Molecular weight 270.241, melting point 214-215 ° C, 217-218 ° C. It is abundant in rhizomes of Galangal (Alpiniaofficinarum) used as one of the spices for curry. The amount of galangin is preferably 0.53 mg / ml or more.
[0015]
Caffeic acid phenethyl ester is 3- (3,4-dihydroxyphenyl) -caffeate (3- (3,4-dihydroxyphenyl) -caffeate) and has the molecular formula C ₁₇ H ₁₆ O ₄ The molecular weight is 284.3. The amount of caffeic acid phenethyl ester is preferably 0.81 mg / ml or more.
[0016]
The propolis in the present invention is typically obtained by mixing two or more kinds of propolis having different origins or extracts thereof. The production area of the propolis to be mixed is not particularly limited as long as a desired component can be obtained. For example, Brazilian propolis, Chinese propolis, Australian propolis, Uruguay propolis, Japanese propolis, etc. can be used. It is preferable to mix Brazilian and Chinese propolis from the viewpoints of components, various biological activities, versatility and the like. The mixing ratio of propolis or propolis extract from different production areas is not particularly limited. For example, in the case of a Chinese propolis extract and a Brazilian propolis extract, 0.1: 99.9 to 99.9: 0.1, preferably 1: 2 to 2: 1 is suitable.
[0017]
Propolis is a dried product obtained by drying propolis itself, a pulverized product thereof, a supercritical extract, a crude extract using water or an organic solvent such as alcohol, ether, and acetone, and a crude extract. All extract fractions obtained by stepwise purification by chromatography can be used. These may be used alone or in combination of two or more. For example, 3 kg of 99.5% ethanol extract is added to 1 kg each of the dried ingots of Brazilian propolis and Chinese propolis, and the extract obtained by soaking overnight at room temperature is used as it is or by mixing them. You may use, and you may use what was refine | purified combining various chromatography.
[0018]
The concentration of the propolis extract in the extracted propolis solution is not particularly limited, but is usually 15 to 70% by mass, preferably about 20 to 60% by mass. If this concentration is less than 15% by mass, it is necessary to evaporate a large amount of solution such as ethanol or water at the time of drying. If it exceeds 70% by mass, the viscosity of the solution becomes too high, and the processability may be deteriorated.
[0019]
Since the propolis composition of the present invention has an angiotensin II receptor antagonistic action, it can be used for lowering blood pressure. Because it has an adrenergic β2 receptor activating effect, it may be used for smooth muscle relaxation, glycogenolysis, promotion of glucagon secretion, vasodilation, bronchodilation, improvement of glucose and lipid metabolism, and reduction of blood pressure and bronchial asthma it can. Since it has a female hormone-like action, it can be used for anti-osteoporosis, improvement of menopause, prevention of prostate cancer, benign prostatic hyperplasia, breast cancer / uterine cancer, and improvement of post-pubertal acne. Since it has an endothelin antagonism, it can be used for the treatment of hypertension, the treatment of heart disease / cerebral circulation disease / renal disease, asthma, ulcer and diabetes.
[0020]
The propolis composition of the present invention has serotonin reabsorption inhibition, serotonin release promotion, serotonin receptor antagonism, and serotonin transporter receptor binding inhibitory action, so that it is intended for the treatment of diseases associated with serotonin such as depression. Can do. Since it has a bradykinin receptor antagonistic action, it suppresses vasodilation, suppresses increased vascular permeability, treats hypotension, treats pain, suppresses connective tissue growth, prevents hair loss, stasis, smooth muscle relaxation, rosacea It can be used for the treatment of acne, peripheral vasodilation, rhinosis, prevention of keloid formation, and anti-inflammatory analgesia, asthma and allergic rhinitis. Since it has an oxytocin antagonism, it can be used for suppression of labor, prevention of premature birth, and prevention / treatment of dysmenorrhea. Because it has nicotinic acetylcholine receptor activation, muscarinic acetylcholine receptor activation, dopamine 1 receptor activation, benzodiazepine receptor activation and γ-aminobutyric acid receptor activation action, schizophrenia, Tourette syndrome, It has uses as a treatment for central nervous system diseases such as attention deficit disorder and anxiety, prevention and improvement of brain function, and a brain metabolism activator. Since it has a glucocorticoid-like action, it can be used as an improvement in carbohydrate metabolism, an anti-stress agent, an anti-inflammatory agent, an apoptosis inducer, and an immune reaction inhibitor. Because of its melatonin receptor activating and antagonizing effects, melatonin-related diseases, ie, prevention and treatment of circadian rhythm sleep disorders, inflammatory bowel disorders, idiopathic constipation, digestive motility associated with irritable bowel syndrome It is effective in preventing and treating disorders.
[0021]
Furthermore, since the propolis composition of the present invention has a calcium channel inhibitory action, it is a cerebral infarction disease, migraine disease, epilepsy disease, psychosis disease, pain disease, blood pressure drop, angina pectoris, arrhythmia. Can be used for treatment. Since it has a melanin-concentrating hormone receptor antagonistic action, it can be used for the prevention of obesity. Since it has an angiotensin converting enzyme inhibitory action, it is effective for lowering blood pressure. Since it has a thromboxane A2 synthase inhibitory action, it can be used for the prevention and treatment of allergic diseases, bronchial asthma, allergic rhinitis, thrombus, myocardial infarction, cerebral infarction and angina. Due to its inhibitory action on nitric oxide synthase, sepsis, endotoxin shock, heart failure, shock, hypotension, rheumatic inflammation, rheumatoid arthritis, osteoarthritis, ulcerative colitis, stress gastric ulcer, Crohn's disease, autoimmunity Disease, tissue damage after organ transplantation, rejection, ischemia reperfusion injury, acute coronary microvascular embolism, shock vascular embolism (generalized intravascular coagulation syndrome (DIC), etc.), ischemic brain injury, arteriosclerosis , Pernicious anemia, Fanconi anemia, sickle cell anemia, pancreatitis, nephrotic syndrome, glomerulonephritis, insulin-dependent diabetes, hepatic porphyria, alcoholism, Parkinson's disease, chronic leukemia, acute leukemia, tumor, myeloma , Anticancer agent side effect reduction, infant and adult respiratory distress syndrome, emphysema, Alzheimer's disease, multiple sclerosis, vitamin E deficiency , Aging, sunburn, muscular dystrophy, cataract, influenza infection, malaria, AIDS, radiation injury, burn, prevention of such in vitro fertilization efficiency, it can be used in the treatment. Since it has a human immunodeficiency virus protease inhibitory effect, it can be used for prevention of human immunodeficiency virus infection, prevention and treatment of acquired immune deficiency syndrome.
[0022]
These propolis in the present invention can be produced, for example, as a tincture type propolis. For example, extraction from Brazil and China propolis bulks is performed using 70% aqueous ethanol and concentrated using a rotary evaporator to obtain an ethanol extract with a solid content of 20%. The obtained ethanol extracts are mixed and stirred so that the weight ratio becomes 1: 1, and then, using conventional means, sodium bicarbonate and soybean lecithin are added and mixed with stirring overnight. Thus, a tincture type propolis composition can be produced.
[0023]
The propolis composition (hereinafter referred to as propolis composition) for which various uses of the present invention have been specified can also be produced as an oral food, or an oral or parenteral pharmaceutical.
As an application method as a medicine, either oral administration or parenteral administration can be adopted. In administration, the active ingredient can be mixed with a solid or liquid nontoxic pharmaceutical carrier suitable for administration methods such as oral administration, rectal administration, and injection, and administered in the form of a conventional pharmaceutical preparation. Examples of such preparations include solid preparations such as tablets, granules, powders and capsules, liquid preparations such as solutions, suspensions and emulsions, and freeze-dried preparations. These preparations can be prepared by conventional means on the preparation. Examples of the non-toxic pharmaceutical carrier include glucose, lactose, sucrose, starch, mannitol, dextrin, fatty acid glyceride, polyethylene glycol, hydroxyethyl starch, ethylene glycol, polyoxyethylene sorbitan fatty acid ester, amino acid, gelatin, Examples include albumin, water, and physiological saline. Further, if necessary, conventional additives such as a stabilizer, a wetting agent, an emulsifier, a binder, and an isotonic agent can be appropriately added.
[0024]
As a food, the propolis composition of the present invention can be used as a health food or a food material having an anti-inflammatory action or an anti-oxidation action as it is, or with various nutritional components added or contained in food or drink. . For example, after adding suitable auxiliaries such as starch, lactose, maltose, vegetable oil powder, cocoa butter powder, stearic acid, etc., using conventional means, edible forms such as granules, granules, tablets It may be formed into capsules, pastes, etc. and used for food. It can also be used in various foods such as processed foods such as ham and sausage, processed fish foods such as kamaboko and chikuwa, bread, confectionery, butter, milk powder, fermented milk products, water, fruit juice, milk, You may add and use for drinks, such as a soft drink. The compounding quantity of the propolis composition of this invention is suitably set by the kind, state, etc. of the said edible composition.
[0025]
That is, when producing a powder composition of the propolis composition of the present invention used as a health food or the like, for example, ethanol extraction extracted with 70% hydrous ethanol from the original mass of Brazilian propolis and Chinese propolis. The liquid was mixed at a weight ratio of 1: 1, and then 1 part of this mixed liquid was first stirred with 0.1 to 0.5 parts, preferably 0.3 parts of starch with a mixer. Concentrate under reduced pressure to recover about 80% of the water-containing ethanol. The stirring speed is 100 to 500 r. p. m. , Preferably 150 to 300 r. p. m. Degree. Next, 0.1 to 0.5 parts, preferably 0.3 parts of starch and 0.05 parts to 0.5 parts of powdered cellulose, preferably 0.2 parts, if necessary, are added to the concentrate and mixed at high speed. Mix thoroughly for 2 to 7 hours, preferably 5 hours. The coarse block mixture thus produced is dried under reduced pressure at a temperature of 30 to 70 ° C. with a shelf dryer, pulverized with a chopper type pulverizer, etc., and sieved with a shifter to extract propolis. A powder of a product can be produced. The particle size of the propolis composition powder is not particularly limited, but about 40 mesh pass is appropriate.
[0026]
The effective dose of the propolis composition of the present invention is appropriately selected and determined according to the patient's age, weight, symptoms, patient grade, administration route, administration schedule, formulation form, strength of the inhibitory activity of the material, and the like. For example, in the case of oral administration, the dry weight is generally about 10 to 500 mg / kg body weight per day, preferably about 150 to 350 mg / kg body weight per day, and may be divided into several times a day. .
[0027]
Furthermore, cosmetics can be produced using the mixed ethanol extract of propolis obtained as described above using conventional means.
When the propolis composition of the present invention is contained and used as a cosmetic or a cosmetic material, for example, the propolis composition of the present invention is added to wheat germ oil or olive oil to provide an anti-inflammatory action or an antioxidant action. It can be used as a cosmetic material. The addition amount of the propolis composition is not particularly limited, but as an example, it is 0.1 wt% or more and 60 wt% or less, preferably 0.5 wt%, based on the weight of wheat germ oil or olive oil. % To 50% by weight is appropriate.
[0028]
Moreover, the propolis composition of the present invention can be directly used as a cosmetic ingredient to produce a cosmetic having an anti-inflammatory action or an antioxidant action. For cosmetics, fats and oils such as vegetable oils, higher fatty acids, higher alcohols, silicones, anionic surfactants, cationic surfactants, amphoteric surfactants, nonionic surfactants, preservatives, sugars, sequestering agents Further, a polymer such as a water-soluble polymer, a thickener, a liquid component, a UV absorber, a UV blocker, a moisturizer such as hyaluronic acid, a fragrance, a pH adjuster, a desiccant and the like can be contained. Vitamins, skin activators, blood circulation promoters, resident bacteria control agents, active oxygen scavengers, anti-inflammatory agents, whitening agents, bactericides, and other medicinal and physiologically active components can also be included.
[0029]
Cosmetics include skin cosmetics such as lotions, emulsions, creams, packs, makeup base lotions, makeup creams, emulsions, cream-like or ointment-type foundations, lipsticks, eye colors, and cheek colors. Preparations, body creams such as hand creams, leg creams and body lotions, bath preparations, oral cosmetics, and hair cosmetics. As a cosmetic containing the propolis composition obtained by the method of the present invention, for example, lotion, milky lotion, face cream, hand cream, lotion, essence, shampoo, rinse and the like are preferable from the functional aspect.
Such a cosmetic can be produced according to a conventional method. The addition amount of the propolis composition of the present invention in cosmetics is not particularly limited, but for example, it is suitably about 0.01% by weight to 20% by weight of the total weight of the cosmetics.
[0030]
The toxicity of the propolis composition of the present invention is low. For example, even if a mixture of Brazilian propolis and Chinese propolis extract is orally administered to rats for a long period of 1000 mg / kg daily for 100 days, no deaths are observed. Neither weight change was observed.
[0031]
【Example】
Hereinafter, the present invention will be specifically described with reference to examples, but the present invention is not limited thereto.
Production Example 1
[Production of propolis extract]
Brazilian and Chinese propolis were frozen and pulverized, and 3 kg of 80% hydrous ethanol (Wako Pure Chemical Industries) was added to 1 kg of powdered propolis powder, mixed and stirred with a stirrer for 10 hours, and then left to stand for 120 hours for extraction did. This was extracted twice and filtered with a suction filter, and concentrated using a rotary evaporator to obtain a propolis extract having a solid content of 20%.
[0032]
Example 1
[Quantitative determination of various components]
For the determination of artepilin C, p-coumaric acid, chrysin, galangin and caffeic acid phenethyl ester, a calibration curve was prepared, and based on this, high-performance liquid chromatography (hereinafter abbreviated as HPLC) was used.
That is, 2 ml of each propolis extract obtained in Production Example 1 is taken, an appropriate amount of methanol is added thereto and subjected to ultrasonic treatment, and further methanol is added to make up to 50 ml. Table 2 shows the results obtained by quantifying each component under the conditions shown below, using this solution as it is or appropriately diluted, followed by filtration using Millipore, and using this as a sample for HPLC.
However, Artepilin C as a standard substance was manufactured by Wako Pure Chemical Industries, and p-coumaric acid, chrysin, galangin, and caffeic acid phenethyl ester were manufactured by Sigma.
[0033]

[0034]
(P-coumaric acid)
Model: LC-2010A (Shimadzu Corporation)
Detector: UV-visible spectrophotometer LC-2010A (Shimadzu Corporation)
Column: YMC-Pack ODS-A, A-312, Φ6 mm × 15 cm (YMC Co., Ltd.)
Mobile phase: 2.5% acetic acid, methanol and acetonitrile mixture (50: 8: 5 v / v / v)
Measurement wavelength: 320 nm
Flow rate: 1.0 ml / min.
Column temperature: 40 ° C
[0035]

The gradient conditions are shown in Table 1.
[0036]
[Table 1]

[0037]
Measurement wavelength: 330 nm
Flow rate: 1.0 ml / min.
Column temperature: 40 ° C
(Chrysin and Galangin)
Model: LC-2010A (Shimadzu Corporation)
Detector: UV-visible spectrophotometer LC-2010A (Shimadzu Corporation)
Column: YMC-Pack ODS-A, A-312, Φ6 mm × 15 cm (YMC Co., Ltd.)
Mobile phase: 5% mixture of formic acid, acetonitrile and methanol (25:10:10 v / v / v)
Measurement wavelength: 360 nm
Flow rate: 1.0 ml / min.
Column temperature: 40 ° C
[0038]
As can be seen from Table 2, Brazilian propolis contains a lot of artepillin C and p-coumaric acid, while Chinese propolis contains a lot of caffeic acid phenethyl ester, chrysin and galangin, It can be seen that the content of Brazilian and Chinese propolis is greatly different.
[0039]
[Table 2]

[0040]
Example 2
Angiotensin II type 1 receptor binding test
Evaluation of binding studies to the human angiotensin II type 1 receptor is described by Derk J. et al. Bergsma et al. (BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, 183 (3) 989-995, 1992) was partially modified.
[Expression of human angiotensin II type 1 receptor in CHO cells]
Human angiotensin II type 1 receptor plasmid (phAT ₁ -3) inserted 2250 base pair (bp) endonuclease (Eco RI) as a vector to replace pCJB11b (Rous sarcoma virus promoter with human cytomagalovirus promoter) to produce pCJB3-8 clone PRJB derivatives) were cloned into Chinese hamster ovary cells (hereinafter abbreviated as CHO cells).
The CHO cells were then cultured on 10-mm plates until confluent to 60-80% within 24 hours. Subsequently, 25 μg of SV40 origin-deficient mutant DNA encoding wild type T antigen was transformed, incubated overnight at 37 ° C., and cells were detached from the plate with trypsin-EDTA, and Transfer to 6-well plates (33-mm tissue culture plates) using fresh complete medium. Then, the cells were incubated at 37 ° C. from 2 days before the receptor binding test.
[0041]
[Evaluation of binding test to human angiotensin II type 1 receptor]
After the above cells were transformed for 3 days, the cells were treated with Dulbecco's phosphate buffer (hereinafter referred to as DPBS) containing 10 mM magnesium chloride, 0.1% glucose and 0.2% fetal calf serum. ⁺⁺ Wash thoroughly with abbreviated). Thereafter, 40-50 pM [ ¹²⁵ I] labeled [sar ¹ , Ile ⁸ Angiotensin II (hereinafter abbreviated as SIA) (manufactured by New England Nuclear, specific activity: 2200 Ci / mmol) was obtained in the presence or absence of the propolis composition at a concentration of 500 μg / ml obtained in Production Example 1. , DPBS ⁺⁺ The reaction was allowed to proceed for 60 minutes at 25 ° C. in 1.0 ml. After the reaction, the solvent was removed and the cells were quickly cooled with ice in DPBS ⁺⁺ Then, the radioactivity bound to the cells was dissolved using 1.0 N sodium hydroxide, and then measured using a γ-counter (80% efficacy) (manufactured by Aloka Co., Ltd.).
In addition, a value measured using SIA and 10 μM non-radiolabeled angiotensin II was used as a blank (non-specific binding), and a data was prepared from a calibration curve, and a computer software system (Lundon-2 software program; Product). In addition, the IC of saralasin which is a human angiotensin II type 1 receptor antagonist in this measurement system ₅₀ The value is 1.9 nM (1.73 × 10 ^-3 μg / ml).
The inhibition rate was 80%. This result shows that the propolis composition of the present invention, which strongly inhibits SIA, has a strong angiotensin II type 1 receptor antagonistic action.
[0042]
Example 3
Adrenergic β2 receptor activation test
The test for binding to the guinea pig lung-derived adrenergic β2 receptor was performed by partially modifying the method of TOMMY ABRAHAMSSON et al. (Biochem. Pharmac., 37 (2) 203-208 (1988)).
A lung was extracted from a guinea pig having a body weight of 350 to 500 g (Nippon Claire Co., Ltd.), and a membrane fraction was prepared. The preparation of the membrane fraction is described in V. Nerme et al. (Biochem. Pharmac., 34, 2917 (1985)).
That is, the extracted lung was homogenized, centrifuged, and the obtained membrane fraction of the precipitated portion was cooled with ice-cooled Tris buffer (154 mM sodium chloride, 2 mM magnesium chloride-containing 20 mM Tris-hydrochloric acid (pH 7. 5) is used to dilute the protein concentration to 0.2-0.6 mg / ml, and the tritiated radioligand is 1-4 mg / ml in suspension in the experimental reaction system. It diluted using the above-mentioned Tris buffer so that it might become ml. However, quantification of the protein concentration was measured using bovine serum albumin as a protein standard according to the method of Lowry et al. (J. Biol. Chem., 193, 265 (1951)).
Radioligand binding studies are similarly described in V.C. Nerme et al. (Biochem. Pharmac., 34, 2917 (1985)).
[0043]
That is, 100 μl of the membrane fraction suspension obtained above was allowed to react with radioligand in the presence of 100 μM GTP (guanosine 5′-triphosphate) and a final volume of 0.25 ml. In this experiment, 0.4 nM [ ³ H] labeled (−) CGP12177 (Amersham, development code No.) (including +10 nM CGP20712A (Amersham, development code No.)), and the reaction was performed at 22 ° C. for 20 minutes. Nonspecific binding was defined as using 50 μM alloprenolol and consisting of 80-90% of the total binding, which was always less than 10% of the total plus radioligand.
Then, using a glass filter (Model 701, manufactured by Skatron Inc.), [ ³ H] Filtration was performed to separate the membrane preparation bound to the labeled (−) CGP12177 and washed twice with 5 ml of the above-described Tris buffer. Place this glass filter in a vial, mix with aquasol (cocktail for liquid scintillation), and combine with a liquid scintillation counter [ ³ H] The amount of labeled (-) CGP12177 was measured, and the inhibition rate (%) was calculated from the following formula.
[0044]
Inhibition rate (%) = 100 − [(C ₁ -B) / (C ₀ -B)] × 100
(Where C ₁ Is a known amount of the test compound and [ ³ [H] [(-) CGP12177 co-existing [ ³ H] represents the amount of label (−) CGP12177 bound to the membrane, ₀ Is the [ ³ H] represents the amount of labeled (−) CGP12177 bound to the membrane, and B represents an excess (50 × 10 ^-6 M) in the presence of alprenolol [ ³ H] represents the amount of labeled (−) CGP12177 bound to the membrane. )
In addition, IC of ICI118551 (reference compound) which is an adrenergic β2 receptor blocker as a positive control in this measurement system ₅₀ The value was 1.9 nM.
As a result of using the propolis composition obtained in Production Example 1 as a test compound (concentration: 500 μg / ml), the inhibition rate was 48%. From this result, it can be seen that the propolis composition of the present invention has a strong adrenergic β2 receptor activating effect.
[0045]
Example 4
Female hormone-like action test
Evaluation of binding studies to the rat uterine estrogen receptor is as follows. This method was carried out by partially modifying the method of KOFFMAN et al. (J. Steroid Biochem. Molec. Biol., 38 (2) 135-139, 1991).
[Preparation of Cytosol Fraction]
Unless otherwise stated, all operations shown below were performed at a temperature of 4 ° C.
First, 4 times the amount of phosphate buffer (pH 8.0) is added to the crushed frozen uterine tissue, and then crushed for 5 seconds under full control conditions using a Polytron PT-10 probe (manufactured by Brinkman Instrument Co. Inc.). Then, the mixture was cooled in ice for 60 seconds until the next crushing, and then homogenized by repeating the crushing operation for 5 seconds twice. This homogenate was centrifuged at 105000 × g for 30 minutes, and the resulting supernatant was decanted and used as the cytosolic fraction. The protein content of the cytosolic fraction was determined using Lowry et. Al. Using bovine serum albumin as a protein standard. al. (J. Biol. Chem. 193 265-275 1951).
[0046]
[Evaluation of binding test to rat uterine estrogen receptor]
The cytosol fraction prepared so that the protein concentration is 1.2 to 7.2 mg / ml is dissolved in a phosphate buffer (pH 8.0), and 1 nM [ ³ H] -labeled 17-β-estradiol and the propolis composition obtained in Production Example 1 were added at 500 μg / ml and reacted at 40 ° C. for 20 hours. The non-specific binding test (blank) was performed in the presence of a 200-fold excess of distilbestrol.
Next, the released steroid or sample sample was subjected to dextran-treated activated carbon suspension (0.5% Norrit A, phosphate buffer containing 0.005% dextran (pH 7.4); hereinafter abbreviated as DCC). Was removed by incubation at 4 ° C. for 10 minutes. Then, DCC is precipitated by centrifugation at 5000 × g for 10 minutes, and 4 ml of scintillation cocktail solution (Packard Instrument Co., Downer Grove, III) is added to 100 μl of the supernatant containing the protein-bound steroid obtained. , And measured with a scintillation counter (Packard Model 2425, 30-40% efficiency). The inhibition rate was calculated by the following formula.
[0047]
Inhibition rate (%) = 100 − [(C ₁ -B) / (C ₀ -B)] × 100
(Where C ₁ Is a known amount of the test compound and [ ³ H] in the presence of labeled 17-β-estradiol [ ³ H] represents the amount of 17-β-estradiol labeled to the rat uterine estrogen receptor, C ₀ Is the [ ³ H] represents the amount of labeled 17-β-estradiol bound to the rat uterine estrogen receptor, B represents an excess of 17-β-estradiol (3 × 10 ^-6 M) In the presence of [ ³ H] represents the binding amount of labeled 17-β-estradiol to rat uterine estrogen receptor. )
As a result, the inhibition rate was 87%. In addition, IC of 17-β-estradiol as a positive control in this measurement system ₅₀ The value is 0.84 nM (2.29 × 10 ^-4 ng / ml). As can be seen from the results, the propolis composition of the present invention antagonistically suppresses the estrogen receptor binding inhibitory action of 17-β-estradiol and has a strong female hormone (estrogen) -like action.
[0048]
Example 5
Endothelin receptor binding test
The binding test evaluation of Chinese hamster ovary cells expressing human endothelin receptor (hereinafter abbreviated as CHO cells) to endothelin receptor is described in BUCHAN, K. et al. W. The method (Br. J. Pharmacol., 112, 1251-1257, 1994) was partially modified to measure inhibition of endothelin-1 binding to the CHO cell endothelin receptor.
[Culture of CHO cells]
CHO cells were cultured at 37 ° C., 5% CO using α-MEM medium (GIBCO) containing 10% fetal bovine serum, 2 mM L-glutamine, 100 units / ml penicillin and 100 μg / ml streptomycin. ₂ The culture was performed under the following conditions. Cells were passaged twice a week using trypsin-EDTA.
[0049]
[Expression of human endothelin receptor in CHO cells]
1 × 10 before transformation ⁵ Seed CHO cells in a 10 cm dish and allowed to attach overnight at 37 ° C., 5% CO ₂ The culture was performed under the following conditions. Thereafter, the DNA was transformed by the calcium phosphate method. Specifically, 40 μg of endothelin receptor expression plasmid and 10 μg of pRSV-neo (a vector that structurally expresses a neomycin resistance gene) were added to 0.75 ml of 0.25 M calcium chloride, and this DNA / calcium chloride mixture was added to an equal volume. 2 times concentrated HEPES phosphate buffer (phosphate buffer (hereinafter abbreviated as PBS): 0.28 M sodium chloride, 50 mM HEPES and 2.25 mM disodium hydrogen phosphate dihydrate) (PH 7.12) was added dropwise using a plastic stir bar and added in the form of a precipitate into CHO cells in a 10 cm dish, which was left on the cells overnight, and then PBS was used. The transformed cells were washed with 1 mg / ml of neomycin analog G418. Before selecting a neomycin resistance-expressing cells had were cultured for 48 hours in a standard growth medium.
[0050]
Colonies of G418 resistant cells were observed 2-3 weeks after selection and transferred into the wells of a 96 well plate using cloning. Although the selection of resistant cells was complete, cells were subsequently maintained at a concentration of 418 mg / ml G418. G418 resistant clones were grown and the presence or absence of endothelin receptor expression was confirmed by a binding test.
In order to solve the problem of unstable receptor expression due to an increase in the number of passages, limiting dilution cloning was performed on cells transformed with the human endothelin receptor. That is, the cells were transferred to a 96-well plate so that the theoretical density was 1 cell per well. Then, after about 2 weeks, a single colony, ie, a pure cell having the same properties, was subcloned, and the expression of the receptor was measured by binding analysis.
[0051]
[Preparation of membranes from CHO cells]
The culture medium was removed from each culture flask, and the monolayer enriched with CHO cells was washed thoroughly twice with 25 ml of PBS without calcium and magnesium, and then 10 ml of PBS containing 5 mM EDTA was added. Thereafter, the mixture was incubated at 37 ° C. for 10 minutes, the cell suspension was centrifuged at 170 × g and 20 ° C. for 5 minutes, and the resulting precipitate was cooled with ice (15 mM Tris-HCl containing 0.3 mM EDTA). Buffer) (pH 7.4). The cell lysate was homogenized on ice for 10 minutes, centrifuged at 170 × g and 4 ° C. for 10 minutes, and the resulting supernatant was again centrifuged at 40000 × g and 4 ° C. for 10 minutes. In each case, the resulting precipitate was resuspended in analytical buffer (consisting of 50 mM Tris, bacitracin 100 μg / ml, 1 mM calcium chloride and 0.1% bovine serum albumin) and stored frozen until use for analysis. Alternatively, it was dissolved in an analysis buffer so that the protein concentration was 30 to 100 μg / ml and used immediately.
[0052]
[Bonding test evaluation]
The competitive inhibition test was performed using the CHO cell membrane obtained as described above (30-100 μg / ml as the protein amount), [ ¹²⁵ I] radiolabeled with ET-1 (15-25 pM) and 500 μg / ml of the propolis composition obtained in Preparation Example 1 were incubated in analytical buffer at 25 ° C. for 3 hours. Thereafter, the cells were washed three times with 3 ml of ice-cold Krebs buffer, the cells were detached, and the cells were rapidly filtered using a glass filter (Model 7019, manufactured by Skatron Inc.) to remove the radioactivity bound on the filter. , And measured using a γ-counter (80% efficiency) (manufactured by Aroca).
Nonspecific binding (blank) was defined as membrane bound radioactivity in the presence of saturating concentration (0.03 nM) of endothelin-1. Nonspecific binding was subtracted from total binding and the difference was defined as specific binding. Total binding was always less than 15% of the total radioactivity added.
As a result, the inhibition rate was 96%. In addition, endothelin-1 IC as a positive control in this measurement system ₅₀ The value is 0.27 nM (0.675 × 10 ^-3 μg / ml). As can be seen from the results, the propolis composition of the present invention has a strong endothelin antagonistic action.
[0053]
Example 6
Serotonin receptor binding inhibition test
The serotonin receptor binding inhibition test is described in Janet G. et al. The method was carried out with reference to the method of Mulheron et al. (The Journal of biochemical 269, 17, 1994). The human serotonin receptor was expressed in baculovirus SF9 cells and used for experiments. The membrane prepared according to the existing method was added with 50 mM Tris-HCl (pH 7.4) in an amount 40 times the wet weight, and homogenized (3 minutes, 1 stroke / 1) using a Teflon-glass homogenizer in an ice bath. And then centrifuged (37,000 × g, 20 minutes, 4 ° C.). The obtained precipitate was resuspended in an ice-cold buffer [50 mM Tris-HCl (pH 7.4)], which is a positive control [ ³ H] 8-OH-DPAT (8-hydroxy-1,2,3,4-tetrahydronaphthalene hydrobromide) and the propolis composition obtained in Preparation Example 1 were added at a concentration of 500 μg / ml. After reacting for 60 minutes at room temperature, low-pressure suction filtration was quickly performed on glass fiber filter paper. The receptor-bound radioactivity remaining on the filter paper was then measured with a liquid scintillation counter.
As a result, the inhibition rate was 90%. The inhibition rate shown here is a rate at which the action of 8-OH-DPAT is suppressed. 8-OH-DPAT IC ₅₀ The value (concentration that inhibits serotonin receptor binding by 50%) was 0.47 nM. Thus, it can be seen that the propolis composition of the present invention has a serotonin 1A receptor binding inhibitory action.
[0054]
Example 7
Serotonin transporter receptor binding inhibition test
The serotonin transporter receptor binding inhibition test was conducted with reference to the method of Tatsumi et al. (European Journal of Pharmacology 340, 249-258, 1997). Human serotonin transporter cDNA was expressed in pRc / CMV vector and transfected into HEK239 cells. Then, a cell membrane was prepared according to an existing method and suspended in a buffer solution (pH 7.4) containing 50 mM tris (hydroxymethyl) aminomethane, 120 mM sodium chloride, 5 mM potassium chloride. To this, the propolis composition obtained in Production Example 1 was added at a concentration of 500 μg / ml, which is a serotonin transporter inhibitor [ ³ H] imipramine was added and incubated at 22 ° C. for 30 minutes. Then, the reaction was stopped by filtering with a GF / B filter immersed in 0.2% polyethyleneimine. Radioactivity on the filter was measured with a liquid scintillation counter.
As a result, the inhibition rate was 93%. The suppression rate shown here is the rate of suppressing the serotonin transporter receptor binding inhibitory action of imipramine. Imipramine IC ₅₀ The value (concentration inhibiting 50% of serotonin transporter receptor binding) was 5.8 nM. Thus, it can be seen that the propolis composition of the present invention competitively suppresses the serotonin transporter receptor binding inhibitory action of imipramine, that is, has a serotonin transporter receptor binding inhibitory action.
[0055]
Example 8
Serotonin reabsorption inhibition test
The serotonin reabsorption inhibition test Perovic et al. (Arzneim.-Forsch./DrugRes. 45 (II), 11, 1995) was used as a reference. Synaptosomes were prepared from 220-250 g rat brains killed by decapitation. The brain cortex was homogenized with 0.32 M sucrose solution in an amount of 20 times the wet weight in an ice bath, and centrifuged (1000 × g, 10 minutes, 4 ° C.). The obtained synaptosome fraction was suspended in a phosphate buffer, the propolis composition obtained in Production Example 1 was added at a concentration of 500 μg / ml, and preincubated at 30 ° C. for 5 minutes. The composition of the phosphate buffer was 1.2 mM sodium dihydrogen phosphate, 25 mM sodium bicarbonate, 118 mM sodium chloride, 5 mM potassium chloride, 2.5 mM magnesium sulfate, 50 μM ascorbic acid, 10 μM EGTA [ Ethylene glycol bis (2-aminoethyl ether) tetraacetic acid], 11 mM glucose. Then 5 μM [ ³ H] -serotonin was added to make a total volume of 1 ml and incubated at 37 ° C. for 20 minutes. The reaction was stopped by adding cold phosphate buffer and immediately filtered through a GF / B filter. Radioactivity on the filter was measured with a liquid scintillation counter.
As a result, the inhibition rate was 81%. The inhibition rate shown here is [100- (when the sample is added [ ³ H] -serotonin uptake) / (buffer only control [ ³ H] -serotonin uptake) × 100]. IC of imipramine, which is a positive control in this experimental system ₅₀ The value (concentration that inhibits serotonin reabsorption by 50%) was 0.024 μM. Thus, it turns out that the propolis of this invention has a serotonin reabsorption inhibitory effect.
[0056]
Example 9
Serotonin release test
The experimental system for observing the serotonin release action was performed with reference to the method of Giambattista et al. (Jornal of Neurochemistry, 63 (3), 1163-1166, 1994). A crude synaptosome fraction was prepared from rat cerebral cortex according to an existing method and suspended in the following buffer solution. The composition of the buffer was 1.2 mM calcium chloride, 1.2 mM magnesium chloride, 1 mM sodium dihydrogen phosphate, 25 mM sodium bicarbonate, 125 mM sodium chloride, 3 mM potassium chloride, 22 mM sodium bicarbonate, 10 mM glucose. To the obtained synaptosome, 0.08 μM [ ³ H] -serotonin was added and incubated at 37 ° C. for 15 minutes. Thereafter, the propolis composition obtained in Production Example 1 was added to a final concentration of 500 μg / ml, and 95% O at 37 ° C. ₂ 5% CO ₂ Perfusion was performed under conditions, and finally the radioactivity of synaptosomes was measured.
The result was an induction rate of 74%. The induction rate is [100- (control of buffer only [ ³ H] -serotonin release amount) / (when the sample is added [ ³ H] -serotonin release amount) × 100]. In addition, EC of fenfluramine as a positive control in this experimental system ₅₀ The value (effective concentration that promotes serotonin release by 50%) was 0.028 μM. Thus, it can be seen that the propolis composition of the present invention has a serotonin release promoting action.
[0057]
Example 10
Bradykinin type II receptor binding test
Evaluation of binding studies on the guinea pig ileum-derived bradykinin type II receptor has -J. I. JONG et al. (Proc. Natl. Acad. Sci. USA, 90, 10994-10998 (1993)) was partially modified.
That is, 75-cm ² Confluent (8-12 × 10 ⁶ Cells) (WI-38 fibroblasts; ATCC CCL 75) at 4 ° C. using 3 times Dulbecco's phosphate buffer (hereinafter abbreviated as PBS), and then 1 mM. Washed once with Tris buffer (pH 7.5) and swelled in 3 ml of 1 mM Tris buffer (pH 7.5) for 10 minutes. Thereafter, buffer H (10 mM sodium chloride, 2 mM EGTA, 2 mM magnesium chloride and protease inhibitors (100 μM phenylmethanesulfonyl fluoride, 16.8 μM leupeptin, 4 μg / ml chymostine, 5.8 μM pepsin, 6.6 μM antipine, 9 ml of 10 mM HEPES buffer (pH 7.5) consisting of trypsin inhibitor 0.08 units aprotinin / ml, 10 mM benzamidine, 0.25 mg / ml soybean trypsin inhibitor and 5 mM ε-aminocaproic acid) was added. Cells were then detached directly and homogenized using a B Dounce homogenizer. The homogenate was centrifuged at 50 × g for 5 minutes, and the resulting supernatant was further centrifuged at 47000 × g for 30 minutes. The obtained membrane fraction was resuspended using the above-mentioned buffer H.
[0058]
50 μl of membrane fraction adjusted to 150-200 μg of protein and 0.03 nM tritium-labeled bradykinin (manufactured by DuPont; 70-107 Ci / mmol; 1 Ci = 37 GBq, hereinafter [ ³ H] BK), 100 μl of the propolis composition obtained in Production Example 1 and 100 μl of the binding buffer, 100 μM guanosine 5 ′-[γ-thio] triphosphate (manufactured by Boehringer Mannheim, hereinafter referred to as GTP [γ- In the presence of S] for 90 minutes at 22 ° C. Thereafter, 3 ml of ice-cooled 50 mM Tris buffer (pH 7.1) was added to stop the reaction, and a glass fiber filter (No. manufactured by Schleicher & Schuell, Inc.) previously treated with 0.3% polyethyleneimine aqueous solution .31) and after washing with 3 ml of the same buffer, measurement was performed with a scintillation counter (Packard Model 2425, 30-40% efficiency), and the inhibition rate (%) was calculated according to the following formula.
In the presence of 1 μM non-radiolabeled bradykinin [ ³ H] BK non-specific binding was always less than 25% of total binding.
[0059]
Inhibition rate (%) = 100 − [(C ₁ -B) / (C ₀ -B)] × 100
(Where C ₁ Is a known amount of the test compound and [ ³ H] in the presence of labeled bradykinin [ ³ H] represents the amount of labeled bradykinin bound to the membrane, C ₀ Is the [ ³ H] represents the amount of labeled bradykinin bound to the membrane, B represents excess bradykinin (1 × 10 ^-6 M) In the presence of [ ³ H] represents the amount of labeled bradykinin bound to the membrane. )
Further, D-Arg- [Hyp., Which is a bradykinin receptor antagonist in this reaction system. 3, D-Phe7] -bradykinin (NPC567) (Sigma) IC ₅₀ The value (concentration that inhibits binding by 50%) was 5.8 nM.
As a result, the inhibition rate was 89%. It can be seen that the propolis composition of the present invention has a bradykinin receptor antagonistic action.
[0060]
Example 11
Oxytocin receptor binding test
The binding test evaluation to the oxytocin receptor is described in Douglas J. et al. The method of Pettibone et al. (European Journal of Pharmacology-Molecular Pharmacology Section, 188, 235-242, 1990) was partially modified.
That is, uterine tissues excised from rats treated by intravenous injection of 0.3 mg / kg of a synthetic estrus hormone agent, diethylvestilbesterol dipropionate, 18-24 hours before sacrifice were treated with 1 mM EDTA, 0. Homogenized using 10 mM Tris buffer (pH 7.4) containing 5 mM dithiothreitol, centrifuged at 48000 × g for 30 minutes at 4 ° C., and the resulting precipitate was treated with 5 mM magnesium chloride, 0.1 Redissolved with 50 mM Tris buffer (pH 7.4) (hereinafter referred to as Buffer A) containing% bovine serum albumin, and centrifuged at 48000 × g for 30 minutes at 4 ° C. to obtain a precipitate. The resulting precipitate was dissolved using buffer A and ice-cooled until analysis.
[0061]
Next, the competition test is 10 nM [ ³ H] labeled oxytocin (manufactured by New England Nuclear, specific activity; 30-60 Ci / mmol) and 500 μg / ml of the propolis composition obtained in Production Example 1 were reacted in buffer A at 22 ° C. for 60 minutes. It was.
Then, using a glass filter (Model 7019, manufactured by Skatron Inc.), [ ³ H] Filtration was performed to separate the membrane preparation bound to the labeled oxytocin, and the membrane preparation was washed twice with 5 ml of buffer A. Place this glass filter in a vial, mix with aquasol (cocktail for liquid scintillation), and combine with a liquid scintillation counter [ ³ H] The amount of labeled oxytocin was measured.
[0062]
Inhibition rate (%) = 100 − [(C ₁ -B) / (C ₀ -B)] × 100
(Where C ₁ Is a known amount of the test compound and [ ³ H] in the presence of labeled oxytocin [ ³ H] represents the amount of labeled oxytocin bound to the membrane, and C ₀ Is the [ ³ H] represents the amount of labeled oxytocin bound to the membrane, and B represents excess oxytocin (5 × 10 5 ^-6 M) In the presence of [ ³ H] represents the amount of labeled oxytocin bound to the membrane. )
IC of oxytocin as a positive control in this measurement system ₅₀ The value was 3.1 nM (0.32 μg / ml).
As a result of using the propolis composition of Production Example 1 (final concentration: 500 μg / ml), the inhibition rate was 101%. It can be seen that the propolis composition of the present invention has a strong oxytocin antagonistic action.
[0063]
Example 12
Nicotinic acetylcholine receptor binding inhibition test
The nicotinic acetylcholine receptor binding inhibition test was performed according to Laurie A. et al. The method was carried out with reference to the method of Pabreza et al. (Molecular Pharmacology, 39, 9-12, 1991). Rat cerebral cortex is homogenized with a buffer solution (pH 7.0) containing 50 mM tris (hydroxymethyl) aminomethane, 120 mM sodium chloride, 5 mM potassium chloride, 1 mM magnesium chloride, 2.5 mM calcium chloride, and suspended. did. Then, after centrifugation at 40,000 × g for 10 minutes (4 ° C.), the pellet was again homogenized with the buffer solution. Centrifugation was performed again under the same conditions, and then the supernatant for two times was centrifuged at 40,000 × g for 10 minutes (4 ° C.). Then, the sediment was suspended in the buffer solution to obtain a cerebral cortex membrane preparation. To this membrane preparation (containing 400-600 μg protein), [ ³ H] cytisine (final concentration 1.5 nM) and the propolis composition obtained in Production Example 1 (final concentration: 500 μg / ml) were added (final solution volume 250 μl), respectively, and incubated at 4 ° C. for 75 minutes. The reaction was quickly filtered by suction on a Whatman GF / B filter (pretreated with a 0.5% polyethyleneimine aqueous solution containing 0.1% bovine serum albumin for at least 3 hours) using a brand harvester cell harvester and cooled. Wash three times with the buffer solution. The filter was placed in a vial, a liquid scintillator was added, and the radioactivity (tritium) bound to the filter was measured with a liquid scintillation counter. Nonspecific binding was measured in the presence of 10 μM (−)-nicotine.
As a result, the inhibition rate was 53%. The inhibition rate (%) shown here was calculated by the following formula.
[0064]
Inhibition rate (%) = 100 − [(C ₁ -B ₀ ) / (C ₀ -B ₀ )] X 100
(Where C ₁ Is a known amount of sample and [ ³ [H] in the coexistence of cytisine [ ³ H] represents the amount of cytosine bound to the membrane fraction, C ₀ Is the [ ³ H] represents the amount of cytisine bound to the membrane fraction, B ₀ In the presence of excess nicotine (10 μM) [ ³ H] represents the amount of cytosine bound to the membrane fraction. )
IC of nicotine, which is an agonist of nicotinic acetylcholine receptor, as a positive control in this reaction system ₅₀ The value (concentration that inhibits nicotinic acetylcholine receptor binding by 50%) was 8.0 nM.
It can be seen that the propolis composition of the present invention competitively inhibits cytisine binding to the nicotinic acetylcholine receptor and has a nicotinic acetylcholine receptor activating effect.
[0065]
Example 13
Muscarinic acetylcholine receptor binding test
The binding test for the muscarinic receptor was evaluated by partially modifying the method of FRANK DORJE et al. (J. Pharmaco. Exp. Ther., 256 (2), 727-733, 1990).
[Cell culture]
Chinese hamster ovary cells (hereinafter abbreviated as CHO cells) were purchased from ATCC (American Type Culture Collection). Until the introduction procedure, the cells are 5% CO ₂ Under the above, monolayer culture was carried out in a DMEM medium (manufactured by Gibco) containing 10% fetal bovine serum, 100 units / ml penicillin G, 100 units / ml streptomycin and 4 mM glutamine (manufactured by MA Bioproducts).
[0066]
[Installation operation]
With reference to the method of Chen and Okayama (Chen, C. AND Okayama, H .: Mol. Cell. Biol. 7, 2745-2752, 1987), an improved calcium phosphate method using pcD neo as a selection marker was used. . CHO cells were transformed with a plasmid into which a pcD expression vector containing the human muscarinic M1 receptor gene sequence was inserted. The selection operation using 600 μg / ml of G-418 (manufactured by Gibco), which is an analog of the antibiotic neomycin, was started 72 hours after the introduction and continued for 2 to 3 weeks. The medium was changed every 3 days. The clonal cell line was obtained by performing limiting dilution cloning. That is, a theoretical density of 1 cell per well is transferred to a 96-well plate, and after about 2 weeks, a single colony, i.e. a purely qualitative pure cell, is subcloned and [ ³ H] It was obtained by measuring pirenzepine binding ability.
[0067]
[Preparation of membrane fraction]
Cells are cultured to about 80% confluence, washed, and then detached in binding buffer (pH 7.4) (hereinafter abbreviated as binding buffer) consisting of 25 mM sodium phosphate containing 5 mM magnesium chloride cooled on ice. And homogenized for 30 seconds using a Brinkmann Homogenizer (adjustment position 5). The membrane was precipitated by centrifugation at 16,000 × g for 15 minutes, and this precipitate was homogenized once again under the same conditions. The protein concentration was measured using a Bio-Rad protein assay kit (Bio-Rad) according to the method of Bradford (Bradford, MM: Anal. Biochem. 72, 248-254, 1976). Membrane fractions were stored frozen at −80 ° C. until use.
[0068]
[Evaluation of binding inhibition test]
The analysis was performed using binding buffer in a total volume of 1 ml and the membrane fraction was adjusted to a protein concentration of 6 μg / ml. [ ³ The saturation binding test of H] pirenzepine was performed by dividing the concentration of the radioligand (2-1400 pM) into 8-10 concentrations. In the binding inhibition test, [ ³ H] pirenzepine (final concentration 2 nM) and the propolis composition obtained in Production Example 1 (final concentration: 500 μg / ml) were added simultaneously. Non-specific binding binds in the presence of 1 μM atropine [ ³ H] pirenzepine difference. Incubation was performed at 22 ° C. for 60 minutes, and the reaction was stopped by rapid filtration using a glass filter (Model 7019, manufactured by Skatron Inc.). The membrane fraction bound on the filter was washed 3 times with 5 ml of ice-cold binding buffer, dried, transferred into 10 ml of scintillation cocktail (New England Nuclear Aquasol) and radiated using an LKB β-counter. Activity was measured. As a result, the inhibition rate was 69%. The inhibition rate (%) was calculated by the following formula.
[0069]
Inhibition rate (%) = 100 − [(C ₃ -B ₁ ) / (C ₂ -B ₁ )] X 100
(Where C ₃ Is a known amount of sample and [ ³ [H] When pirenzepine coexists [ ³ H] represents the amount of pirenzepine bound to the membrane fraction, C ₂ Is the [ ³ H] represents the amount of pirenzepine bound to the membrane fraction, B ₁ In the presence of excess atropine (1 μM) [ ³ H] represents the amount of pirenzepine bound to the membrane fraction. )
In addition, IC of pirenzepine which is an antagonist of muscarinic 1 receptor as a positive control in this reaction system ₅₀ The value is 13 nM (4.57 × 10 ^-3 μg / ml).
It can be seen that the propolis composition of the present invention competitively inhibits pirenzepine binding to the muscarinic 1 receptor and has a strong muscarinic receptor activating effect.
[0070]
Example 14
Dopamine 1 receptor binding inhibition test
The dopamine 1 receptor binding inhibition test was performed with reference to the method of Zhou et al. (Nature, 347, 76-80, 1990).
That is, a 3.0 kb EcoRl-Sacl fragment was inserted between the BamHl sites of Hindll of the pBC12Bl vector and introduced into COS-7 cells by the calcium phosphate method. The membrane preparation is then prepared according to existing methods and finally suspended in TEM buffer [25 mM Tris buffer pH 7.4, 6 mM magnesium chloride, 1 mM ethylenediaminetetraacetic acid (EDTA)] A membrane sample was used. Each propolis composition obtained in Production Example 1 (final concentration: 500 μg / ml) is added to each test tube, which is a dopamine 1 receptor antagonist [ ³ H] SCH 23390 (manufactured by Amersham, final concentration 0.3 nM), receptor membrane preparation (containing 20-30 μg of protein) and the above buffer solution were added to form a reaction solution (total amount 500 μl), and the reaction started Was performed by adding a membrane preparation. After incubation at 30 ° C. for 60 minutes, the labeled ligand bound to the receptor was suction-filtered onto a Whatman GF / B glass fiber filter using a cell harvester (Brandell) to immediately stop the reaction. The plate was washed 3 times with 5 ml of 50 mM Tris-HCl buffer (pH 7.7). Next, the radioactivity on the filter was measured with a liquid scintillation counter to determine the total amount of binding. In addition, the amount of binding in the presence of 1 μM SCH 23390 measured at the same time was defined as the amount of non-specific binding, and the amount of specific binding was determined by subtracting this from the total amount of binding.
As a result, the inhibition rate was 70%. The inhibition rate (%) shown here was calculated by the following formula.
[0071]
Inhibition rate (%) = 100 − [(C ₅ -B ₂ ) / (C ₄ -B ₂ )] X 100
(Where C ₅ Is a known amount of sample and [ ³ [H] SCH 23390 co-existing [ ³ H] represents the amount of SCH 23390 bound to the membrane fraction, C ₄ Is the [ ³ H] represents the amount of SCH 23390 bound to the membrane fraction; ₂ In the presence of excess SCH 23390 (1 μM) [ ³ H] represents the amount of SCH 23390 bound to the membrane fraction. )
In addition, IC of SCH 23390 which is an antagonist of dopamine 1 receptor as a positive control in this reaction system ₅₀ The value (concentration inhibiting 50% of dopamine 1 receptor binding) was 1.2 nM.
It can be seen that the propolis composition of the present invention has a dopamine 1 receptor activating effect.
[0072]
Example 15
Benzodiazepine receptor binding inhibition test
The benzodiazepine receptor binding inhibition test was performed by Robert C. et al. It was carried out with reference to the method of Speth et al. (Life Sciences, 24, 351-358, 1979).
That is, a crude synaptosomal membrane fraction prepared from 7-8 week old Wistar rat brain was prepared with 118 mM sodium chloride, 4.8 mM potassium chloride, 1.28 mM calcium chloride and 1.2 mM magnesium sulfate. Suspended in 1 mM Tris-HCl buffer (pH 7.4) (1 g brain wet weight / 20 ml) to prepare a receptor membrane preparation. As a labeled ligand, [ ³ H] flunitrazepam was used. The propolis composition obtained in Production Example 1 (final concentration: 500 μg / ml) was added to each test tube, and [ ³ H] flunitrazepam (final concentration 0.4 nM), a receptor membrane sample and the above buffer solution were added to obtain a reaction solution (total volume 1 ml), and the reaction was started by adding the membrane sample. After incubation at 0 ° C. for 20 minutes, the labeled ligand bound to the receptor was suction filtered onto a Whatman GF / B glass fiber filter using a cell harvester (manufactured by Brandel) to stop the reaction. The plate was washed 3 times with 5 ml of 50 mM Tris-HCl buffer (pH 7.7). Next, the radioactivity on the filter was measured with a liquid scintillation counter to determine the total amount of binding.
In addition, the amount of binding in the presence of 1 μM diazepam measured at the same time was defined as the nonspecific binding amount, and this was subtracted from the total binding amount to determine the specific binding amount.
As a result, the inhibition rate was 78%. The inhibition rate (%) shown here was calculated by the following formula.
[0073]
Inhibition rate (%) = 100 − [(C ₇ -B ₃ ) / (C ₆ -B ₃ )] X 100
(Where C ₇ Is a known amount of sample and [ ³ H] [With flunitrazepam coexisting [ ³ H] represents the amount of flunitrazepam bound to the membrane fraction, C ₆ Is the [ ³ H] represents the amount of flunitrazepam bound to the membrane fraction; ₃ In the presence of excess diazepam (1 μM) [ ³ H] represents the amount of flunitrazepam bound to the membrane fraction. )
In addition, IC of diazepam, which is a benzodiazepine receptor agonist, as a positive control in this reaction system. ₅₀ The value (concentration that inhibits benzodiazepine receptor binding by 50%) was 10 nM.
It can be seen that the propolis composition of the present invention competitively inhibits flunitrazepam binding to the benzodiazepine receptor and has a benzodiazepine receptor activating effect.
[0074]
Example 16
γ-aminobutyric acid (GABA) receptor binding inhibition test
Propolis composition [ ³ H] GABA binding inhibition test to rat-derived cerebral cortex GABA receptor was performed by partially modifying the method of AKIRA TSUJI et al. (ANTIMICROB. AGENTS CHEMOTHER., 32 (2) 190-194, 1988).
That is, the cerebral cortex of 230-270 kg body weight SD rats (CLEA Japan, Inc.) was removed, and a polytron homogenizer (Brinkman Instrument Co. Inc.) was used for 30 seconds at a speed of 5 for a 50-fold capacity. Homogenization was performed in 50 mM Tris buffer (pH 7.4) (hereinafter abbreviated as Buffer A) containing 1 mM ascorbic acid and 1 mM disodium tetraacetate ethylenediamine. In all operations, the tissue was maintained at 0 ° C. Thereafter, the homogenate is centrifuged at 40,000 × g for 15 minutes, and the resulting precipitate is resuspended in 50 times volume (original weight) of buffer A. Under the same conditions as described above, polytron is used. Homogenized. Centrifugation and resuspension were repeated three times. Then, incubation was performed at 37 ° C. for 10 minutes. This homogenate is centrifuged at 40,000 × g for 15 minutes, resuspended in 10 volumes (original weight) of buffer A (hereinafter abbreviated as membrane fraction), and measured at −20 ° C. or lower until measurement. Saved with.
[0075]
In conducting the binding test to the rat-derived cerebral cortex GABA receptor, the above membrane fraction was thawed to 37 ° C., and then 136 mM sodium chloride, 5 mM potassium chloride, 2 mM magnesium sulfate, 2 mM diphosphate 20 mM Tris-Krebs buffer (hereinafter abbreviated as Buffer B) consisting of 20 mM Tris buffer (pH 7.4) containing potassium hydrogen, 2 mM calcium chloride and 1 mM ascorbic acid, 1 mM disodium tetraacetate ethylenediamine The volume was increased to 20 volumes (original weight). This membrane fraction was homogenized for 30 seconds at speed 5 using a Polytron homogenizer, incubated at 37 ° C. for 15 minutes, and centrifuged at 20,000 × g for 10 minutes. The precipitate thus obtained is washed and resuspended twice or more, and the finally obtained precipitate is resuspended in 100 times the volume (original weight) of buffer B. Used for testing.
[0076]
Next, 50 μl (final concentration: 500 μg / ml) of the propolis composition obtained in Production Example 1, and [ ³ The reaction was initiated by adding 50 μl of H] GABA (final concentration 10 nM) and adding 900 μl of the membrane fraction described above. After incubation for 20 minutes at 22 ° C., by filtration through a Whatman GF / B filter using a Brandell Cell Harvester, using 2 × 7.5 ml of filtered wash under reduced pressure. The reaction was stopped. The radioactivity retained on the filter after filtration was measured with a liquid scintillation counter (Packard Model 2425, 30-40% efficiency).
Further, the amount of specific binding was determined by subtracting the amount of non-specific binding from the amount of binding in the presence of 100 μM non-radiolabeled GABA measured simultaneously. As a result, the inhibition rate was 90%. The inhibition rate (%) shown here was calculated by the following formula.
[0077]
Inhibition rate (%) = 100 − [(C ₉ -B ₄ ) / (C ₈ -B ₄ )] X 100
(Where C ₉ Is a known amount of sample and [ ³ [H] When GABA coexists ³ H] represents the amount of GABA bound to the membrane fraction, C ₈ Is the [ ³ H] represents the amount of GABA bound to the membrane fraction, B ₄ In the presence of excess GABA (100 μM) ³ H] represents the amount of GABA bound to the membrane fraction. )
In addition, IC of GABA as a positive control in this measurement system ₅₀ The value (concentration that inhibits GABA receptor binding by 50%) was 11 nM (1.13 × 10 6 ^-3 μg / ml).
It can be seen that the propolis composition of the present invention has a strong GABA receptor activating effect.
[0078]
Example 17
Human corticoid receptor ligand binding test
The measurement of human corticoid receptor ligand binding activity was determined by Abbott F. et al. The method of Clark et al. (Invest. Ophthalmol. Vis. Sci., 35, 805-813, 1996) was partially modified.
That is, human lymphoblastoma cell IM9 (ATCC, Bethesda, MD) was used as a soluble glucocorticoid receptor (hereinafter abbreviated as GR).
The above cells were cultured in RPMI 1640 medium (Gibco, Grand Island, NY) containing 10% fetal bovine serum (HyClone, Logan, UT), 100 U / ml pemicillin, 100 μg / ml streptomycin and 2 mM L-glutamine (Gibco). , 37 ° C, 7% CO ₂ 10 × 10 under the conditions of ⁵ Incubate until cells / ml. The cells were then detached from the culture medium by centrifuging at 1500 g for 10 minutes. The detached cells were washed with 12-fold amount of Dulbecco's phosphate buffer (hereinafter abbreviated as PBS: Gibco), and then centrifuged again at 1500 g for 10 minutes to obtain a precipitate, which was washed with PBS. did.
[0079]
5-6 times volume of homogenization buffer (pH 7.4) (hereinafter abbreviated as HB) composed of 10 mM TES, 10 mM sodium molybdate, 1 mM EDTA, 20 mM 2-mercaptoethanol and 10% glycerol to the washed cells. Add and suspend, cells are N ₂ Using a cavitation machine (Parr Instrument, Moline, IL), the cells were disrupted by cavitation twice at 0 ° C. for 15 minutes at a nitrogen pressure of 600 to 750 psi. Clarified using a Hoffman contrast microscope (Garden City, NY).
The cell disruption preparation was then centrifuged at 27000 g for 15 minutes and the resulting supernatant was centrifuged at 103,000 g for 60 minutes at 4 ° C. The amount of protein in the obtained supernatant fraction was quantified using BCA assay kit (Pierce Chemical, Rockford, IL) and bovine serum albumin as a standard substance. It was rapidly cooled in an ice-acetone bath and stored frozen at -70 ° C.
[0080]
Competitive binding studies consisted of IM9 cell cytosol 1 mg, 1.5 nM [ ³ H] labeled triamcinolone (0.025 μCi: Amersham, Arlington, Heights, IL) and the propolis composition obtained in Production Example 1 were prepared to a final concentration of 500 μg / ml, and HB (total volume 200 μl) And incubated for 18 hours at 0 ° C. The reaction was stopped by adding 100 μl of charcoal-dextran mixture (10 mM Tris buffer (pH 7.4) containing 2% activated carbon, 0.5% dextran, 1% EDTA) and Incubated for 5 minutes and centrifuged at 8200 g for 5 minutes. The radioactivity of 100 μl of the resulting supernatant was measured by the following formula using liquid scintillation spectrometry.
As a result, the inhibition rate was 88%. In the presence of 10 μM non-radiolabeled dexamethasone [ ³ Nonspecific binding of H] triamcinolone was always less than 15% of total binding.
[0081]
Inhibition rate (%) = 100 − [(C ₁ -B) / (C ₀ -B)] × 100
(Where C ₁ Is a known amount of the test compound and [ ³ H] in the presence of labeled triamcinolone [ ³ H] represents the amount of labeled triamcinolone bound to the membrane, C ₀ Is the [ ³ H] represents the amount of triamcinolone bound to the membrane, and B represents an excess of dexamethasone (10 × 10 ^-6 M) In the presence of [ ³ H] represents the amount of labeled triamcinolone bound to the membrane. )
Dexamethasone IC as a positive control in this measurement system ₅₀ The value (concentration inhibiting 50% binding) was 2.7 nM. It can be seen that the propolis composition of the present invention has a strong GC-like action.
[0082]
Example 18
Melatonin receptor binding inhibition
The melatonin receptor binding inhibition test was performed by Darryl S. et al. The method (European Journal of Pharmacology, 175, 71-77, 1990) was used as a reference.
That is, Syrian hamsters (2-4 months old, male) were bred under conditions of 16 hours light period and 8 hours dark period, and a crude membrane fraction for radioreceptor assay was prepared from the brain according to existing methods. For suspension, ice-cold buffer [50 mM Tris (hydroxymethyl) aminomethane (pH 7.4)] was used. To this membrane preparation (containing 50-100 μg protein), [ ¹²⁵ I] Iodomelatonin (final concentration 0.1 nM) and the propolis composition obtained in Production Example 1 (final concentration: 100 μg / ml) were added (final solution volume 200 μl), and the mixture was incubated at 4 ° C. for 30 minutes. Low-pressure suction filtration was quickly performed on glass fiber filter paper. The receptor-bound radioactivity remaining on the filter paper was then measured with a liquid scintillation counter. Nonspecific binding was also measured in the presence of 30 μM melatonin.
As a result, the inhibition rate was 89%. The inhibition rate (%) shown here was calculated by the following formula.
[0083]
Inhibition rate (%) = 100 − [(C ₁ -B ₀ ) / (C ₀ -B ₀ )] X 100
(Where C ₁ Is a known amount of sample and [ ¹²⁵ I] In the coexistence of iodomelatonin [ ¹²⁵ I] represents the amount of iodomelatonin bound to the membrane fraction, C ₀ Is the [ ¹²⁵ I] represents the amount of iodomelatonin bound to the membrane fraction, B ₀ In the presence of excess melatonin (30 μM) [ ¹²⁵ I] represents the amount of iodomelatonin bound to the membrane fraction. )
In addition, IC of iodomelatonin which is a melatonin receptor agonist as a positive control in this reaction system. ₅₀ The value (concentration that inhibits melatonin receptor binding by 50%) was 188 nM.
It can be seen that the propolis composition of the present invention competitively inhibits iodomelatonin binding to the melatonin receptor and has high affinity with the melatonin receptor.
[0084]
Example 19
Calcium channel inhibition test
The calcium channel inhibition test was performed by Howard R. et al. The method of Lee et al. (Life Sciences, 35 (7), 721-732, 1984) was partially modified.
Specifically, male SD rats weighing 250-350 g were decapitated and killed, and then the brain was quickly removed. The cerebral cortex was then dissected from brain debris on an ice-cold Betri dish. Thereafter, the obtained cerebral cortex was added to 19 volumes of Krebs phosphate buffer (120 mM NaCl, 4.8 mM KCl, 2.1 mM MgSO ₄ 1.3 mM CaCl ₂ 20.3 mM Na ₂ HPO ₄ 3.2 mM HCl, 10 mM D-glucose, pH 7.4) was added to the centrifuge tube, and homogenized with Polytron (manufactured by Brinkman) for 3 seconds (regulation rate; 40%). The homogenized cerebral cortex was then washed twice by centrifugation at 48000 × g for 10 minutes and resuspended in the above-mentioned Krebs phosphate buffer.
[0085]
The fresh cerebral cortex homogenate thus obtained was adjusted to a wet weight of 1-5 mg / tissue, and then adjusted to a total volume of 2 ml in a tube containing Krebs phosphate buffer. .04nM [ ³ H] -labeled isopropyl methyl 4- (2,1,3-benzoxadiazol-4-yl) -1,4-dihydro-2,6-dimethyl-pyridine-3,5-dicarboxylate 1-methylethyl Ester (isopropyl 4- (2,1,3-benzoxazol-4-yl) -1,4-dihydro-2,6-dimethyl-pyridine-3,5-dicarboxylic acid methyl 1-methylester, hereinafter [ ³ H] (+) PN 200-100) (89 Ci / mmol, New England Nuclear, Boston, Mass.) And the final concentration obtained in Production Example 1 was adjusted to a concentration of 500 μg / ml. Propolis composition was added and incubated for 90 minutes at 22 ° C. in a dark room with a sodium lamp.
[0086]
Thereafter, the radioactivity bound to the membrane was trapped by suction filtration using a glass fiber filter (manufactured by Whatman, GF / B), and then washed three times with 5 ml of 0.9% saline. The filter is dried and immersed in a scintillation solution overnight to elute the radioactivity in the filter, and then the radioactivity in the eluate is measured using liquid scintillation spectrometry (43% efficiency). And the calcium channel inhibitory activity was computed using the formula shown below.
Also, specific binding is defined as binding displaced by 1 μM nifedipine, and the amount of protein is determined by the method of Lowry et al. (OH Lowry, NJ Rosenbrough, AL Farr and R). J. Randall; J. Biol. Chem. 193, 265-275 (1951)).
[0087]
Inhibition rate (%) = 100 − [(C ₁ -B) / (C ₀ -B)] × 100
(Where C ₁ Is a known amount of the test compound and [ ³ [H] (+) PN 200-100 co-existing [ ³ H] (+) represents the amount of binding of PN 200-100 to the membrane fraction, C ₀ Is the [ ³ H] (+) PN 200-100 represents the amount of binding to the membrane fraction, B is the excess nifedine (1 × 10 ^-6 M) In the presence of [ ³ H] (+) represents the amount of binding of PN 200-100 to the membrane fraction. )
IC of nitrendipine (manufactured by Pfizer Pharmaceutical Co., Ltd.), a calcium channel inhibitor as a positive control in this reaction system. ₅₀ The value was 1.7 nM.
In the propolis composition used, the inhibition rate was 74%. It can be seen that the propolis composition of the present invention competitively inhibits the binding of nitrendipine to calcium channels and has a strong calcium channel inhibitory action.
[0088]
Example 20
Melatonin-concentrating hormone (MCH) receptor binding inhibition test
The MCH receptor binding inhibition test was performed with reference to the method of Jon Chambers et al. (Nature, 400, 261-265, 1999).
That is, Chinese hamster ovary cells (CHO cells) expressing SLC1 which is a human MCH receptor (adjusted to 2 to 4 μg / protein), 0.1 nM [ ¹²⁵ I] labeled [Phe ¹³ , Tyr ¹⁹ ] -MCH (specific activity; 2200 Ci / mmol, manufactured by New England Nuclear) and the propolis composition obtained in Preparation Example 1 adjusted to a final concentration of 500 μl / ml were incubated at 22 ° C. for 60 minutes. . Thereafter, the radioactivity was trapped by suction filtration using a glass fiber filter (manufactured by Whatman, GF / C), and then washed three times with 5 ml of 0.3% nonfat dry milk. The filter is dried and immersed in a scintillation solution overnight to elute the radioactivity in the filter, and then the radioactivity in the eluate is measured using liquid scintillation spectrometry (43% efficiency). The MCH receptor inhibitory activity was calculated using the following formula.
As a result, the inhibition rate was 56%. Also, specific binding is defined as binding displaced by 0.1 μM MCH, and the amount of protein is determined by the method of Lowry et al. (OH Lowry, NJ Rosenbrough, AL Farr and R. R.). J. Randall; J. Biol. Chem. 193, 265-275 (1951)).
[0089]
Inhibition rate (%) = 100 − [(C ₁ -B) / (C ₀ -B)] × 100
(Where C ₁ Is a known amount of the test compound and [ ¹²⁵ I] labeled [Phe ¹³ , Tyr ¹⁹ ]-When MCH coexists ¹²⁵ I] labeled [Phe ¹³ , Tyr ¹⁹ ] Represents the amount of MCH bound to the membrane fraction, C ₀ Is the [ ¹²⁵ I] labeled [Phe ¹³ , Tyr ¹⁹ ] Represents the amount of MCH bound to the membrane fraction, and B represents excess MCH (1 × 10 ^-5 M) In the presence of [ ¹²⁵ I] labeled [Phe ¹³ , Tyr ¹⁹ ] Represents the amount of MCH bound to the membrane fraction. )
In addition, IC of MCH, which is an MCH receptor agonist, as a positive control in this reaction system ₅₀ The value was 0.25 nM.
It can be seen that the propolis of the present invention competitively inhibits the binding of MCH to the MCH receptor and has a high affinity for the MCH receptor.
[0090]
Example 21
Angiotensin converting enzyme (ACE) inhibitory activity test
The ACE inhibitory activity test was performed by Hoorn, C .; M.M. And Roth, R.A. A. (Brit. J. Pharmacol., 110, 597-602, 1993) was partially modified.
That is, 0.06 ml of a 10% dimethyl sulfoxide (hereinafter abbreviated as DMSO) obtained by dissolving the propolis composition obtained in Production Example 1 in a glass test tube so as to have a final concentration of 500 μg / ml was added, Next, 0.03 ml of 100 mU / ml ACE (manufactured by Sigma) was added, pre-incubated at 37 ° C. for 5 minutes, and then 100 mM borate buffer pH 8.3, 500 mM NaCl, 6.5 mM [ ³ H] labeled hyplyglycylglycine (hereinafter referred to as [hyppurylglycylglycine] ³ H] HGG) was added (0.3 ml), incubated at 37 ° C. for 5 hours, and 0.5 ml of 1N hydrochloric acid was added to stop the reaction. Thereafter, 1.5 ml of ethyl acetate was added, stirred for 1 minute, and released by ACE [ ³ H] hippuric acid was extracted, 1.0 ml of this ethyl acetate layer was separated and dried, liquid scintillation was added, radioactivity was measured, and the inhibition rate (% ) Was calculated.
[0091]
Inhibition rate (%) = (A−B) / A × 100
(Here, A represents the radioactivity value when no inhibitor is contained, and B represents the radioactivity value when an inhibitor is added.)
As a result, the inhibition rate was 60%. IC of captopril (captopril, manufactured by Wako Pure Chemical Industries, Ltd.), an ACE inhibitor as a positive control in this reaction system. ₅₀ The value was 0.0011 μM. It can be seen that the propolis composition of the present invention has a strong ACE inhibitory action.
[0092]
Example 22
Thromboxane A2 (TXA2) synthase inhibitory activity test
The TXA2 synthase inhibitory activity test is described in WADE, M. et al. L. And FITZPATRICK, F.M. A. (Arch. Biochem. Biophys., 347, 174-180, 1997) was partially modified to examine thromboxane synthase inhibitory activity of human platelet microsomes.
That is, in 10 mM phosphate buffer (pH 7.4), platelet microsomes (0.5 mg protein), 10 μM prostaglandin H2 (hereinafter abbreviated as PGH2), 5 mM epinephrine, 2 μM hemin, and Production Example 1 A reaction solution (total amount 0.2 ml) containing the prepared propolis composition (final concentration: 500 μg / ml) was used. The propolis composition and platelet microsomes were preincubated at 22 ° C. for 3 minutes, 10 μM PGH2 was added as a substrate and reacted at 22 ° C. for 2 minutes. Thereafter, the reaction was stopped by adding a tin chloride solution, and the amount of thromboxane B2 (hereinafter abbreviated as TXB2) was measured using a TXB2 EIA (Enzyme Immuno Assay) kit (manufactured by Amersham Pharmacia Biotech). .
As a result, the inhibition rate was 93%. In addition, as a positive control in this reaction system, the IC of furegrelate which is a TXA2 synthase inhibitor ₅₀ The value (concentration inhibiting 50% of enzyme activity) was 0.21 μM. It can be seen that the propolis composition of the present invention has potent TXA2 synthase inhibitory activity.
[0093]
Example 23
Nitric oxide synthase inhibitory activity test
The NOS inhibitory activity test of the propolis extract was performed by partially modifying the method of Estrada et al. (Biochem. Biophys. Res. Commun., 186, 475-482 (1992)).
The mouse-derived macrophage-like cells RAW264-7 were 5 × 10 ⁵ / Well on a culture plate and incubated at 37 ° C. for 24 hours. To this culture plate, LPS (lipopolysaccharide; 10 ng / ml) and the propolis composition obtained in Production Example 1 were added to a concentration of 500 μg / ml and incubated at 37 ° C. for 18 hours. The obtained culture solution (100 μl) was reacted with Griess reagent (100 μl), and the absorbance at 540 nm was measured. Then, the absorbance in the absence of the propolis extract was taken as 100%, the percentage (%) in the presence of the propolis extract was determined, and the inhibition rate was calculated.
As a result, the inhibition rate was 92%. In addition, IC of L-NMMA which is a NOS inhibitor as a positive control in this reaction system ₅₀ The value (concentration that inhibits enzyme activity by 50%) was 69 μM. It can be seen that the propolis composition of the present invention has a strong NOS inhibitory action.
[0094]
Example 24
Human immunodeficiency virus-1 protease inhibitory activity test
The measurement of HIV-1 protease inhibitory activity was measured by TOTH, M. et al. V. And MARSHALL, G.M. R. (Int. J. Peptide. Protein Res., 36, 544-550, 1990) was partially modified.
Specifically, HIV-1 protease (expressed in E. coli by gene recombination, manufactured by Bachem) was added to a 100 mM sodium acetate buffer (pH 4.) containing 200 mM sodium chloride, 5 mM dithiothreitol, 10% (v / v) glycerol. 9) (hereinafter referred to as Buffer A) to obtain a 0.02 mg / ml enzyme solution. In addition, 1 mg / ml His-Lys-Ala-Arg-Val-Leu-p-nitro-Phe-Glu-Ala-Nle-Ser-NH ₂ (Bakem Co., Ltd.) was dissolved in distilled water to obtain a substrate solution.
After adding 100 μl of buffer A, 10 μl of the substrate solution and 10 μl of the propolis composition obtained in Preparation Example 1 (final concentration: 500 μg / ml) to a 0.5 ml plastic tube, preincubating at 37 ° C. for 5 minutes, After adding 25 μl of the enzyme solution and stirring sufficiently, the reaction was carried out at 37 ° C. for 40 minutes. Thereafter, 15 μl of 10% trifluoroacetic acid was added to stop the reaction.
P-nitro-Phe-Glu-Ala-Nle-Ser-NH released by enzymatic reaction after stopping the reaction ₂ The amount was quantified by HPLC under the following conditions.
[0095]
<HPLC conditions>
Column: YMC-Pack C8 (A-202, 150 × 4.6 mm ID, manufactured by YMC Co., Ltd.)
Elution solvent: linear concentration gradient of 0 to 63% acetonitrile containing 0.1% trifluoroacetic acid (20 minutes)
Flow rate: 1 ml / min.
Detection wavelength: 300 nm
Further, the inhibition rate (%) was calculated by the following formula.
Inhibition rate (%) = (B−C) / B × 100
[In the formula, B: p-nitro-Phe-Glu-Ala-Nle-Ser-NH without an inhibitor ₂ Peak area, C: p-nitro-Phe-Glu-Ala-Nle-Ser-NH with inhibitor added ₂ Represents the peak area. ]
As a result, the inhibition rate was 94%. In addition, the IC of pepstein A as a positive control in this measurement system ₅₀ The value was 5.2 μM (3.57 μg / ml). It can be seen that the propolis composition of the present invention has a strong HIV-1 protease inhibitory action.
[0096]
A prescription example is shown below.
Formulation Example 1
[Manufacture of tablets]
Using a blended propolis extract obtained by mixing ethanol extract of Brazilian propolis and Chinese propolis obtained in Production Example 1 at a weight ratio of 1: 1, tablets having the following composition were produced according to a conventional method.
(Composition) (Composition: Mass%)
Blend propolis extract 24
Lactose 63
Cornstarch 12
Guar gum 1
[0097]
Formulation Example 2
[Manufacture of juice]
Using a blended propolis extract obtained by mixing ethanol extract of Brazilian propolis and Chinese propolis obtained in Production Example 1 at a weight ratio of 1: 1, juice having the following composition was produced according to a conventional method.
(Composition) (Composition: Mass%)
Frozen and concentrated Wenzhou orange juice 5.0
Fructose glucose liquid sugar 11.0
Citric acid 0.2
L-ascorbic acid 0.02
Fragrance 0.2
Dye 0.1
Blend propolis extract 0.2
Water 83.28
[0098]
Formulation Example 3
[Manufacture of face cream]
Using a blended propolis extract obtained by mixing ethanol extract of Brazilian propolis and Chinese propolis obtained in Production Example 1 at a weight ratio of 1: 1, a face cream having the following composition was produced.
(Composition) (Composition: Weight%)
Isopropyl isostearate 8.0
Jojoba oil 6.0
Cetanol 8.0
Stearyl alcohol 2.0
Polyoxyethylene lauryl ether 1.5
Propylene glycol 6.0
Sorbitol 1.0
Paraben 0.4
Blend propolis extract 0.5
Vitamin E 0.5
Fragrance 0.1
Purified water 66.0
[0099]
【The invention's effect】
The propolis composition having the components specified in the present invention has biological activity as shown in each of the above examples, and is useful for the above-mentioned use. These biological activities are thought to be derived from the contained components. Moreover, such a composition is safe even if taken for a long time.

Claims (30)

An angiotensin II receptor antagonist composition containing propolis, containing quercetin, p-coumaric acid and artepilin C, and further containing chrysin, galangin or caffeic acid phenethyl ester. A composition for activating adrenergic β2 receptor comprising propolis, comprising quercetin, p-coumaric acid and artepilin C and further containing chrysin, galangin or caffeic acid phenethyl ester. A female hormone-like composition containing propolis, which contains quercetin, p-coumaric acid and artepilin C, and further contains chrysin, galangin or caffeic acid phenethyl ester. An endothelin antagonistic composition comprising propolis, containing quercetin, p-coumaric acid and artepilin C, and further containing chrysin, galangin or caffeic acid phenethyl ester. A composition for inhibiting serotonin reabsorption, comprising propolis, containing quercetin, p-coumaric acid and artepilin C, and further containing chrysin, galangin or caffeic acid phenethyl ester. A composition for promoting serotonin release, comprising propolis, characterized by containing quercetin, p-coumaric acid and artepiline C, and further containing chrysin, galangin or caffeic acid phenethyl ester. A composition for serotonin receptor antagonism comprising propolis, which contains quercetin, p-coumaric acid and artepilin C, and further contains chrysin, galangin or caffeic acid phenethyl ester. A composition for inhibiting serotonin transporter receptor binding, comprising propolis, comprising quercetin, p-coumaric acid and artepilin C, and further containing chrysin, galangin or caffeic acid phenethyl ester. A bradykinin receptor antagonist composition comprising propolis, containing quercetin, p-coumaric acid and artepilin C, and further containing chrysin, galangin or caffeic acid phenethyl ester. An oxytocin antagonist composition comprising propolis, containing quercetin, p-coumaric acid and artepilin C, and further containing chrysin, galangin or caffeic acid phenethyl ester. A composition for activating a nicotinic acetylcholine receptor comprising propolis, containing quercetin, p-coumaric acid and artepilin C, and further containing chrysin, galangin or caffeic acid phenethyl ester. A composition for activating a muscarinic acetylcholine receptor comprising propolis, comprising quercetin, p-coumaric acid and artepilin C, and further containing chrysin, galangin or caffeic acid phenethyl ester. A composition for activating dopamine 1 receptor, comprising propolis, containing quercetin, p-coumaric acid and artepiline C, and further containing chrysin, galangin or caffeic acid phenethyl ester. A composition for activating a benzodiazepine receptor comprising propolis, which contains quercetin, p-coumaric acid and artepilin C, and further contains chrysin, galangin or caffeic acid phenethyl ester. A composition for activating γ-aminobutyric acid receptor comprising propolis, which contains quercetin, p-coumaric acid and artepiline C, and further contains chrysin, galangin or caffeic acid phenethyl ester. A glucocorticoid-like active composition containing propolis, which contains quercetin, p-coumaric acid and artepilin C, and further contains chrysin, galangin or caffeic acid phenethyl ester. A composition for activating melatonin receptor comprising propolis, containing quercetin, p-coumaric acid and artepilin C, and further containing chrysin, galangin or caffeic acid phenethyl ester. A melatonin receptor antagonist composition containing propolis, containing quercetin, p-coumaric acid and artepilin C, and further containing chrysin, galangin or caffeic acid phenethyl ester. A composition for inhibiting calcium channels, comprising propolis, containing quercetin, p-coumaric acid and artepilin C, and further containing chrysin, galangin or caffeic acid phenethyl ester. A composition for antagonizing melatonin-aggregating hormone receptor comprising propolis, containing quercetin, p-coumaric acid and artepilin C, and further containing chrysin, galangin or caffeic acid phenethyl ester. A composition for inhibiting angiotensin converting enzyme, comprising propolis, which contains quercetin, p-coumaric acid and artepiline C, and further contains chrysin, galangin or caffeic acid phenethyl ester. A composition for inhibiting thromboxane A2 synthase containing propolis, which contains quercetin, p-coumaric acid and artepiline C, and further contains chrysin, galangin or caffeic acid phenethyl ester. A composition for inhibiting nitric oxide synthase containing propolis, which contains quercetin, p-coumaric acid, and artepilin C, and further contains chrysin, galangin, or caffeic acid phenethyl ester. A composition for inhibiting human immunodeficiency virus protease, comprising propolis, containing quercetin, p-coumaric acid and artepilin C, and further containing chrysin, galangin or caffeic acid phenethyl ester. Propolis has a quercetin equivalent of 4.58 mg / ml or more, p-coumaric acid of 1.16 mg / ml or more, artepilin C of 6.73 mg / ml or more, chrysin of 0.84 mg / ml or more, and galangin of 0.53 mg The composition according to any one of claims 1 to 24, which is a propolis containing at least 0.81 mg / ml of caffeic acid phenethyl ester / ml or more. The propolis composition according to any one of claims 1 to 25, wherein the propolis is a propolis containing at least two kinds of propolis having different origins or extracts thereof. An oral composition or a parenteral composition comprising the composition according to any one of claims 1 to 26. The oral composition according to claim 27, wherein the composition is a food. The oral composition or parenteral composition according to claim 27, which is a pharmaceutical. The parenteral composition according to claim 27, which is a cosmetic.