JP4054170B2

JP4054170B2 - Liver function improvement preventive agent and tumor immunostimulant containing guarana extract as an active ingredient, and methods for producing them

Info

Publication number: JP4054170B2
Application number: JP2000289739A
Authority: JP
Inventors: 隆司渡辺; 康之渡辺; 重治川原
Original assignee: 三重化糧株式会社
Priority date: 2000-09-25
Filing date: 2000-09-25
Publication date: 2008-02-27
Anticipated expiration: 2020-09-25
Also published as: JP2002104981A

Description

【０００１】
【発明の属する技術分野】
本発明は、ガラナ果実の粉砕物に糖質分解酵素を作用させて得られるガラナエキスを有効成分とする肝機能障害改善予防剤及び腫瘍免疫賦活剤並びにこれらの製造方法に関する。
【０００２】
【従来の技術】
蔓状の低灌木の一種であるブラジル産ガラナ（Ｐａｕｌｌｉｎｉａｃｕｐａｎａ）の果実は、カフェインと類似の分子構造を有するグアラニンを含有しており、古来からアマゾン川流域に先住しているグアラン族が興奮性飲料として愛用していたのが始まりといわれている。このようなガラナ果実は、カテキンやタンニン（ポリフェノール）などを含有しているため、飲用による好ましい薬理活性が期待され、これまでに種々のガラナエキスが添加された飲料が提供されている。
【０００３】
ところで、従来よりガラナ果実からガラナエキスを抽出する方法にアルコール抽出法と高温水抽出法とがある。アルコール抽出法はエチルアルコールを主にした有機溶媒によりガラナ果実を抽出する方法で、ガラナ果実に含有される親油性（疎水性）組成成分を複雑な操作によることなく容易に抽出でき、また抽出・分画が効率的に行えるという長所がある反面、（１）親水性組成成分残渣が多いため収率が悪く（３０〜５０％）、（２）ガラナ果実に含有されるデンプン質がアルコールにより変性されやすい、（３）ガラナ果実に含有される親水性組成成分が十分に抽出されない、という短所があった。一方、高温水抽出法は塩類を含む高温水によりガラナ果実を抽出する方法で、ガラナ果実の組成成分にとって極めて温和な抽出法であり、また高度の技術が不要で抽出操作が簡便であるという長所がある反面、（１）親油性組成成分残渣が多いため収率が悪く（２５〜３５％）、（２）ガラナ果実に含有される親油性組成成分の抽出が困難で、抽出されるのは主として親水性組成成分であるカフェイン（グアラニン）、カテキン、タンニンなどであり、一般的に好ましい薬理活性を有するとされるフラボン系やテルペン系などの複雑な環状構造の組成成分の回収は不十分である、という短所があった。
【０００４】
また、従来より飲料として提供されているガラナエキスの薬理活性については、ガラナ果実に含有されるカテキンやタンニンなどについての研究報告がなされているにすぎず、これまでにガラナエキス固有の薬理活性についての研究報告は皆無である。
【０００５】
【発明が解決しようとする課題】
本発明は、上記従来のガラナエキスの抽出法の問題点を解決し、ガラナ果実に含有される親油性組成成分と親水性組成成分のいずれをも高収率で回収し、簡便な操作で行えるガラナエキスを有効成分とする肝機能障害改善予防剤及び腫瘍免疫賦活並びにこれらの製造方法を提供することを課題とする。
【０００６】
【課題を解決するための手段】
本発明者らは、肝機能障害・機能低下マウスをモデルにＧＯＴとＧＰＴの酵素活性値を指標としてガラナエキスの新規な薬理活性を検討すると共に、マウス・サルコーマ１８０癌細胞を移植したマウスをモデルにガラナエキスの新規な薬理活性を検討し、本発明に想到した。すなわち、本発明は、ガラナ果実の粉砕物に糖質分解酵素を作用させて得られるガラナエキスを有効成分とすることを特徴とする肝機能障害改善予防剤及び腫瘍免疫賦活剤に関する。これらの発明において、糖質分解酵素は液化酵素、セルラーゼ及び糖化酵素としてもよい。
【０００７】
上記の発明では、ガラナ果実に含まれる親油性組成成分、親水性組成成分、糖質分解酵素の作用を受けない残存高分子多糖体、アミロペクチンや繊維質などにより肝機能改善予防作用や腫瘍免疫賦活作用を発揮するものと推測される。
【０００８】
また、本発明者らは糖質分解酵素の性質に着目して、ガラナ果実に含まれる繊維質やデンプン質などを効率よく分解すると共に、残渣物を減らし高収率でガラナエキスを得ることができることに想到し本明を完成した。すなわち、本発明は、ガラナ果実の粉砕物に液化酵素、セルラーゼ及び糖化酵素を作用させ、以下の工程によりガラナエキスを得ることを特徴とする上記の肝機能障害改善予防剤又は腫瘍免疫賦活剤の製造方法に関する。
（１）ガラナ果実を粉砕して粉末化する工程
（２）高温水にガラナ果実の粉末を加え、さらに液化酵素を添加してデンプン質を液化する工程
（３）前記で得た酵素液化溶液にセルラーゼと糖化酵素を添加して糖化処理する工程
（４）前記で得た糖化処理液を濾過する工程
（５）前記で得た濾液を濃縮する工程
これらの製造方法の発明において、液化酵素は、α−アミラーゼ、β−アミラーゼ及びイソアミラーゼから選ばれる１種又は２種以上であり、糖化酵素はα−グルコシダーゼ、β−グルコシダーゼ、グルコアミラーゼ、プルラナーゼから選ばれる１種又は２種以上としてもよい。また、これらの製造方法で得られたガラナエキスに所定量のブドウ糖を添加してガラナエキス結晶粉末とする上記の肝機能障害改善予防剤又は腫瘍免疫賦活剤の製造方法に関する。
【０００９】
上記の製造方法では、主として細胞壁の構造を維持するセルロースがセルラーゼにより分解され、またガラナ果実に含まれる多糖体は糖化分解されるので、リグニン、フラボン系組成成分その他の組成成分が遊離し易くなる。
【００１０】
【発明の実施の形態】
ガラナ果実は主として種子を用いるが、果肉などを用いることもできる。予め十分に乾燥したガラナ果実をミルなどの粉砕機により粉砕し粉末化する。ガラナ果実の粉末はその重量のおよそ８〜１２倍量の高温水に加える。高温水の水温は通常８０〜９０℃であるが、使用する糖質分解酵素に応じて適宜変更してもよい。また、ガラナ果実の粉末が加えられた高温水のｐＨは通常６〜７に調整するが、使用する糖質分解酵素に応じて適宜変更してもよい。
【００１１】
次いで、上記の調整液に液化酵素を加えてガラナ果実に含まれるデンプン質を液化する。液化酵素はアミラーゼを用い、動物由来、植物由来、微生物由来を問わず用いることができる。アミラーゼには、α−アミラーゼ、β−アミラーゼ及びイソアミラーゼがあり、これらのアミラーゼを単独であるいは併用して用いることができ、特に加水分解効率の高いエンド型のα−アミラーゼが好ましく、また高温水に添加されるので耐熱性のα−アミラーゼがさらに好ましく、このようなアミラーゼは市販品を容易に入手することができる。さらに、デンプン質の液化は調整液にアミラーゼを添加後、使用する糖質分解酵素に応じ適宜変更してもよいが、通常８０〜９０℃で５０〜７０分間十分に撹拌して行う。なお、液化酵素はガラナ果実の粉末１０００ｋｇに対し約６００ｇ前後の割合で添加するのが好ましい。
【００１２】
上記の処理により得た酵素液化溶液は、使用する糖質分解酵素に応じて適宜変更してもよいが、通常ｐＨを４〜６に調整した後、セルラーゼと糖化酵素を添加し、通常４０〜５０℃で４５〜５２時間酵素処理する。添加されるセルラーゼは市販品を容易に入手でき、動物由来、植物由来及び微生物由来を問わず用いることができる。また、糖化酵素も動物由来、植物由来及び微生物由来を問わず種々の酵素を用いることができ、特にα−グルコシダーゼ、β−グルコシダーゼ、グルコアミラーゼ、プルラナーゼの中から１種又は２種以上を組み合わせて用いることが好ましく、これらの糖化酵素も市販品を用いることができる。また、ガラナエキスの甘味性を高めるためグルコースイソメラーゼなどの異性化酵素を添加してもよい。なお、セルナーゼと糖化酵素は出発原料たるガラナ果実の粉末１０００ｋｇに対して各々約６００ｇ前後の割合で添加するのが好ましい。
【００１３】
上記の処理で得た糖化処理液は、公知の操作により濾過を行い残渣と濾別する。この濾過操作は、所望により複数回繰り返される。さらに、得られた濾液は公知の操作により濃縮され、ガラナエキスを製造することができる。ガラナエキスは、白糖を加えてガラナエキスシロップに加工したり、あるいは凍結乾燥品とすることができる。また、ガラナエキスにブドウ糖を添加してガラナエキス結晶粉末に製することができ、製剤化にあたり打錠が容易となる。なお、ブドウ糖の添加量（容量）は、ガラナエキス２〜３に対して８〜７の割合が好ましい。
【００１４】
以上のようにして製造されたガラナエキスは、肝機能改善予防剤あるいは腫瘍免疫賦活剤として投与することができる。投与形態は、経口投与が一般的であるがこれに限定されない。また、剤型も用途に応じて適宜選択でき、薬学的に許容される賦形剤、結合剤、崩壊剤、軟膏基剤などを用いて散剤、顆粒剤、錠剤、カプセル剤、座剤、軟膏剤などの剤型とできる。さらに、本発明の肝機能改善予防剤及び腫瘍免疫賦活剤はそれらの薬効を失わない限り他の薬効を有する製剤（動植物由来を含む）を配合して医療用製剤として供することができ、例えば抗菌剤、制癌剤あるいはサイトカイン類を配合することができる。
【００１５】
上記製造方法で得られた肝機能改善予防剤あるいは腫瘍免疫賦活剤は、連用効果と効果の持続性に優れるので、その投与量は健常者であれば肝機能低下予防・機能促進のために１日１〜１０ｍｇ／ｋｇが好ましい。肝機能が低下している患者や悪性腫瘍の罹患者は適宜増量でき、前記の５倍量を投与するのが好ましい。
【００１６】
【実施例】
次に本発明を実施例を挙げて説明するが、本発明は以下の実施例に限定されるものではない。なお、以下の説明において特に断らない限り％は重量％を意味する。
【００１７】
実施例１（ガラナ果実からのガラナエキスの製造）
ガラナ果実（種子）を粉砕機で粉砕し粉末化した粉末１５０ｇ（水分量：７．５±０．５％）を９０℃に加温した滅菌蒸留水１５００ｍｌに加えた後（ｐＨ５．５）、炭酸カルシウムにてｐＨを６．５に調整し、得られた約１５００ｍｌの調整液にＢａｃｉｌｌｕｓｓｕｂｔｉｌｌｉｓ由来のα−アミラーゼ（アミラーゼＡＤ「アマノ」、天野製薬社製）９０ｍｇを添加し、９０℃で６０分間撹拌しながらガラナ果実粉末に含まれるデンプン質を液化した。得られた約１５００ｍｌの酵素液化溶液にシュウ酸を加えてｐＨを４．５に調整した後、Ａｓｐｅｒｇｉｌｌｕｓｎｉｇｅｒ由来のα−グルコシダーゼ（α−グルコシダーゼ「アマノ」、天野製薬社製）とＡｓｐｅｒｇｉｌｌｕｓｎｉｇｅｒ由来のセルラーゼ（セルラーゼＡ「アマノ」、天野製薬社製）を各々９０ｍｇずつ添加し、４５℃で４８時間酵素処理した（液化工程）。この酵素処理によって、植物繊維質（セルローズ）はブドウ糖にまで分解される（糖化工程）。さらに、糖化された約１５００ｍｌの糖化処理液を表面に濾過剤（パーライト）が張り付けられたヒダ付き濾紙により吸引濾過を２回繰り返した。このようにして得られた濾液を回転式真空濃縮装置を用いて濃縮し、固形分６２．７％、水分３７．３％からなる約３５０ｍｌのガラナエキスを得た。
【００１８】
上記製造方法により得られたガラナエキスの収率は約７０％で、同時に行ったアルコール抽出法の収率約４５％と高温水抽出法の収率約３０％に比べ極めて高い収率であった。また、ガラナエキスの１００ｍｌ当たりのカフェイン含有量は４．６５ｇ、タンニン含有量は１２．６ｇで、高温水抽出法で得た含有量とほぼ近似し、本発明のガラナエキスの製造方法によりガラナ果実に含まれる親水性組成成分を十分に回収できることが分かった。さらに、ガラナエキスの固形分１００ｇについて、各組成成分の含有比率を分析したところ、表１に示したようにセルラーゼとα−グルコシダーゼの分解による繊維由来ブドウ糖が最も大きな割合であった。なお、カフェインはＨＰＬＣ法、タンニンはフォーリン・デニス法、糖質はフェノール・硫酸法により分析した。
【００１９】
【表１】

【００２０】
参考例１（肝機能障害・機能低下マウスの作製）
肝臓の機能障害・機能低下が惹起される肝機能障害・機能低下マウスは、Ｃｏｎｔｉらの方法（Ｊｐｎ．Ｊ．Ｐｈａｒｍａｃｏｌ．，６０：３１５，１９９２）に準じて行った。すなわち、後記実施例２の各実験群毎に３匹宛の５週齢の正常ｄｄＹ系雌マウス（平均体重：２５ｇ）を準備し、生理食塩水で６０ｍｇ／ｍｌの濃度になるように溶解させたＤ−ガラクトサミン塩酸塩（以下、Ｇａｌ−Ｎという）試薬液０．２ｍｌをマウスの腹腔内に投与（４８０ｍｇ／ｋｇ）することにより肝機能障害・機能低下マウス（以下、Ｇａｌ−Ｎ処理マウスという）を作製した。
【００２１】
参考例２（ＧＯＴ及びＧＰＴ活性値の測定法）
ＧＯＴ（ＡＳＴ）及びＧＰＴ（ＡＬＴ）の活性値の測定は、ＧＯＴあるいはＧＰＴ測定用キット試薬（アズウエル社製）をセットしたマルチ自動分析機（東芝マルチ６０Ｍ型）によるＵＶ−Ｒａｔｅ法（主波長：３４０ｎｍ／副波長：３７５ｎｍ）により行った。すなわち、血清１０μｌに酵素液（Ｒ１）３００μｌを添加後、３７℃で３分間インキュベートし、その後、基質液（Ｒ２）７５μｌを加え、１〜５分間に３４０ｎｍ付近での１分間当たりの吸光度の変化（ＮＡＤＨ→ＮＡＤ^＋への減少速度）を測定し、ＮＡＤＨ分子吸光係数からＧＯＴあるいはＧＰＴの活性値（単位：ＩＵ／ｌ）を算出した。なお、以下の各実施例における有意差検定（危険率５％以下、ｐ∠０．０５、有意差有り）はＳｔｕｄｅｎｔｔ−ｔｅｓｔ法により算出した。
【００２２】
実施例２（ガラナエキスのＧＯＴ及びＧＰＴ活性値に与える効果の検討）
（ガラナエキスの濃度効果）上記参考例１で作製されたＧａｌ−Ｎ処理マウスにガラナエキスを投与し、Ｇａｌ−Ｎ処理マウスのＧＯＴ活性値及びＧＰＴ活性値に与えるガラナエキスの濃度効果を検討した。すなわち、Ｇａｌ−Ｎ処理後、４時間目に投与量が１００ｍｇ／ｋｇ、２００ｍｇ／ｋｇ、４００ｍｇ／ｋｇ、８００ｍｇ／ｋｇとなるように実施例１で得られたガラナエキスの濃度を各々調整し、Ｇａｌ−Ｎ処理マウスに各々０．５ｍｌ経口投与した後、４８時間目にエーテル麻酔下マウスの眼球を摘出し、流血液をエチレンジアミン四酢酸ナトリウム添加小試験管に採取した。この血液を遠心分離（４℃、５０００ｒｐｍ、１０分間）後、血清を採取（２００〜３００μｌ）し、ＧＯＴ及びＧＰＴの活性値を参考例２の方法で測定した。
【００２３】
上記のようにしてＧＯＴ及びＧＰＴの活性値を測定したところ、表２に示したように、非投与対照群（正常マウス）のＧＯＴ活性値は平均９２ＩＵ／ｌであったが、Ｇａｌ−Ｎ単独投与群では、ＧＯＴ活性値は著しく上昇（平均３１３５ＩＵ／ｌ）した。このＧａｌ−Ｎ処理マウスに、ガラナエキス（１００〜８００ｍｇ／ｋｇ）を経口投与した後、４８時間目にＧＯＴ活性値を測定したところ、投与濃度に依存して酵素活性値は低下し、ガラナエキスの２００ｍｇ／ｋｇ投与で有意差が認められ（危険率５％以下）、４００ｍｇ／ｋｇ以上の投与では顕著な有意差（危険率１％以下）が認められた。また、ＧＰＴ活性値においても、表３に示したようにＧＯＴと同様にガラナエキスの投与濃度に依存して酵素活性値の低下が認められた。このように、ガラナエキスの投与濃度を高めることにより、ＧＯＴとＧＰＴの活性値が正常の酵素活性閾値に近づく傾向を示したことから、ガラナエキスには機能障害・機能低下を招いた肝機能を緩解・回復させる組成成分が含まれていることが示唆された。なお、興味あることに、Ｇａｌ−Ｎ処理によって、ＧＯＴ活性値は正常値の約３４倍まで上昇したのに対して、ＧＰＴ活性値は１４ＩＵ／ｌから２１８２ＩＵ／ｌまで上昇し、約１５６倍にまで上昇したことから、Ｄ−ガラクトサミン塩酸塩は肝由来ＧＯＴ活性値に対するよりもＧＰＴ活性値に対してより強く作用することが示唆された。
【００２４】
【表２】

【００２５】
【表３】

【００２６】
（ガラナエキスの投与回数効果）
Ｇａｌ−Ｎ処理後、２時間毎にガラナエキス（２００ｍｇ／ｋｇ）を経口投与（計３回）した後、４８時間目に上記のガラナエキスの濃度効果の実験群と同様の方法で血清を採取し、ＧＯＴ及びＧＰＴの活性値を測定した。その結果、表４と表５に示したようにＧａｌ−Ｎ処理によって著しく上昇したＧＯＴとＧＰＴの活性値は投与回数の増加に伴い低下し、２回投与によって危険率５％以下の有意差が認められ、３回投与によっては危険率１％以下の有意差が認められ、投与回数を重ねることによりガラナエキスの肝機能改善予防効果が増強されることが示唆された。
【００２７】
【表４】

【００２８】
【表５】

【００２９】
（ガラナエキスの持続効果）
ＧＡＬ−Ｎ処理後、４時間目にガラナエキス（４００ｍｇ／ｋｇ）を経口投与した後、２４、４８及び７２時間目に上記のガラナエキスの濃度効果の実験群と同様の方法で血清を採取し、ＧＯＴとＧＰＴの活性値を測定した。その結果、表６と表７に示したように、Ｇａｌ−Ｎ＋ガラナエキス投与群での両酵素活性値は共にＧａｌ−Ｎ単独投与群における活性値よりも著しく低値であり（２４〜４８時間後の危険率５％以下、７２時間後の危険率１％以下）、しかも時間と共に徐々に減少したことから、１回の投与によって機能障害・機能低下した肝機能に対するガラナエキスの緩解・回復効果が体内でかなりの間持続することが明らかになった。
【００３０】
【表６】

【００３１】
【表７】

【００３２】
ＧＰＴはＧＯＴに比べ、正常肝細胞に限局しており、しかも肝細胞の細胞質由来のＧＰＴ活性値はＧＯＴ活性値よりも高いことが知られており、ヒトが急性肝炎に罹患した場合にはＧＰＴ活性値の方がＧＯＴ活性値よりも急激に上昇することから、マウスをＧａｌ−Ｎ処理することによりヒトにみられる急性肝炎と類似の肝機能障害が誘発されたものと考えられ、上記の各実験結果からガラナエキスはヒトの急性肝炎に対し極めて有効であろうことが示唆された。
【００３３】
参考例３（肝ホモジネートの作製）
参考例１に記載の方法でマウスをＧａｌ−Ｎ処理（４８０ｍｇ／ｋｇ）し、その６０分後にガラナエキス（４００ｍｇ／ｋｇ）を経口投与した。投与後、６、２４及び７２時間目にエーテル麻酔下のマウスを屠殺し、肝臓を摘出・細断した。次いで、肝の細断片を１５０ｍＭ塩化ナトリウム、１．０ｍＭＥＤＴＡ添加１０ｍＭトリス塩酸緩衝液に入れ、ガラスホモジナイザーにて肝ホモジネートを作製した。肝ホモジネート中の蛋白質濃度は、ローリー法にて定量し、肝ホモジネート１ｍｌ当たり１００ｍｇ蛋白量に調整した。
【００３４】
実施例３（ガラナエキスの肝脂肪過酸化抑制作用（抗酸化作用））
肝脂肪の過酸化実験法は、Ｃａｍｐｏｓらの方法（ＰｌａｎｔａＭｅｄｉｃａ，５５：４１７，１９８９）に準じて行った。すなわち、参考例３に記載の方法でガラナエキスの経口投与後（４００ｍｇ／ｋｇ）、６、２４及び７２時間目の肝ホモジネート（肝ホモジネート１に対しトリス緩衝液４を加え、終濃度を２０ｍｇ蛋白質／ｍｌに調整）にマロンアルデヒド（以下、ＭＤＡという）・チオバルビタール酸複合体を添加し、産生されるＭＤＡ濃度をイムノアッセイ自動分析機（ＢｅｈｒｉｎｇＥＬＩＳＡＰ−２型）にて５３５ｎｍで測定し、Ｅ＝１．５６×１０^５１０００ｍｌ／ｍｏｌｅ／ｃｍの条件係数を用いて計測した（単位：ｎｍｏｌｅ／ｍｇ蛋白質）。なお、本法での標識物は、ＮＡＤ^＋−ＡＴＰ補酵素を用い、ＮＡＤＰＨ−Ｆｅ^＋−ＡＤＰ産生現象を利用した測定方法である。結果は表８に示したように、非投与対照群の正常マウス肝内におけるＭＤＡ産生量は実験期間（７２時間）を通じてほぼ一定（５０〜６８ｎｍｏｌｅ／ｍｇ蛋白質：平均５６．８ｎｍｏｌｅ／ｍｇ）であったが、Ｇａｌ−Ｎ処理によって４倍量以上のＭＤＡの増加がみられ、肝脂肪の酸化が著しく亢進されていることが認められた。これに対して、ガラナエキスを経口投与することによって、ＭＤＡの産生は時間の経過と共に軽減され、投与２４時間後では危険率１％以下、７２時間後では０．１％以下の著しい有意差が認められた。このことより、肝機能の低下によって生じ易くなる脂肪（特に、中性脂肪、脂肪酸）の酸化や過酸化に対して、ガラナエキスは抑制的に作用することが明らかになった。一方、ヒトの肝機能障害・肝機能低下時における肝内での過酸化脂肪の生成・蓄積は、肝癌発症の重要な要因となることからも、ガラナエキスには発癌予防効果をも有するであろうことが十分考えられる。また、脂肪酸の酸化などを防ぐことにより加齢変化に伴う生活習慣病の発症を予防する効果が期待される。
【００３５】
【表８】

【００３６】
実施例４（ガラナエキスの肝内の活性酸素抑制作用（フリーラジカル・スカベンジャー）
肝障害や肝炎等の発症に伴い増加する肝マクロファージであるクッパー細胞に起因する活性酸素（フリーラジカル酸素）は、Ｖｉｄｅｌａらの方法（Ｂｉｏｃｈｅｍ．Ｐｈａｒｍａｃｏｌ．，３４：２２０９，１９８５）に準じて測定を行った。すなわち、参考例３に記載した方法でガラナエキスを経口投与後（４００ｍｇ／ｋｇ）、６時間目の肝ホモジネート（肝ホモジネート１に対してトリス緩衝液２００を加え、終濃度０．５ｍｇ蛋白質／ｍｌに調整）をバイアル瓶（１３×３０ｍｍ）に採取し、肝ホモジネート中に発生するケミルミニセンス（化学的発光量）の１分間当たりの発光数値（単位：ｃｐｍ／ｍｇ蛋白質）を液体シンチレーション測定装置（ＢｅｃｋｍａｎｎＬＳ−３１５０Ｐ型）にて測定した。なお、本法での化学発光標識物には、イソルミノール誘導体のひとつであるＮ−（４−アミノブチル）−Ｎ−エチルイソルミノールを用いた。結果は表９に示したように、Ｇａｌ−Ｎ処理によって、正常マウス肝内に産生された活性酸素（フリーラジカル酸素）は平均１１７８ｃｐｍ／ｍｇ蛋白質から２．３倍（平均２６７５ｃｐｍ／ｍｇ蛋白質）にまで増加し、肝機能の低下が確実に惹起されていた。すなわち、Ｇａｌ−Ｎ処理によって受けた損傷あるいは破壊された肝細胞や肝由来細胞を体外へ排除するために、クッパー細胞内に存在する活性酸素による殺細胞（殺細菌）システムが異常に亢進し、その結果、クッパー細胞からの活性酸素が多量に産生されたことに起因していることが立証された。このようなマウスにガラナエキスを投与することによって、活性酸素は正常閾値（１２８０ｃｐｍ／ｍｇ蛋白質）にまで低下し、肝細胞等の損傷や障害が著しく軽減されたことを示している（危険率１％以下の有意差）。これらのことから、ガラナエキスにも脂溶性ビタミン類、セルロプラスミンあるいはスーパーオキシドデスムターゼ（ＳＯＤ）等と同様の強い活性酸素消去作用をすることが示唆された。このことにより、ガラナエキスには活性酸素に一因があるといわれる発癌や老化の予防効果が期待される。
【００３７】
【表９】

【００３８】
参考例４（ガラナエキスの低中分子組成成分画分の分離と精製）
実施例１で製造したガラナエキスを３０％ガラナエキスに調整し（４００ｍｇ／ｍｌ）、冷却高速遠心機にて１０００ｒｐｍ、６０分遠心した後、その上清液を限外濾過用メンブランを装着した限外濾過器にて吸引濾過し濾液を採取した。この濾液中に含まれる分子量５０００以下の低中分子組成成分画分を凍結乾燥し、さらに生理食塩水にて１０ｍｇ／ｍｌの濃度に調整し低中分子組成成分画分液を得た。このガラナエキスの低中分子組成成分画分液中には、カフェイン、カテキン、タンニン、リンゴ酸、α−グルコシダーゼ分解繊維質由来ブドウ糖、二糖類などが含まれており、これを以下の悪性肉腫形成・肥大抑制作用の実験に供試した。
【００３９】
実施例５（ガラナエキスの悪性肉腫形成・肥大抑制作用）
イーグルＭＥＭ培養液１ｍｌ当たり５×１０^５コのマウス・サルコーマ１８０癌細胞浮遊液０．２ｍｌ（１×１０^５コ／マウス）をマウス背部皮下に移植（各群８匹宛）する２日前、移植直後（０日）、移植後１日目、２日目、５日目及び８日目に参考例４で得られたガラナエキスの低中分子組成成分画分液０．５ｍｌ（４００ｍｇ／ｋｇ）及び実施例１で得たガラナエキス０．５ｍｌ（４００ｍｇ／ｋｇ）を各々癌細胞移植周辺背部皮下に計６回投与した。移植後２８日に腫大化した肉腫を摘出し、重量を計測した。その結果、表１０及び図１に示したように非投与対照群の移植後２８日目における肉腫の平均重量は１５．３５ｇであったのに対して、ガラナエキスの低中分子組成成分画分液を投与した場合には、肉腫の形成と肥大は多少とも抑制されていたものの、有意差は認められなかった（平均重量：１２．４４ｇ）。一方、ガラナエキスを投与した場合には、肉腫に対する著しい形成・肥大抑制効果が認められ（平均重量：９．８８ｇ）、しかも危険率１％以下の有意差であった。このように、ガラナエキスの遠心上清画分中に含まれる低〜中分子量組成成分、例えばカテキンやタンニンの抗腫瘍効果の報告があるが、低分子量から高分子量のすべての組成成分を含有するガラナエキスそのものの方がマウス・サルコーマ１８０肉腫に対して極めて強い抗腫瘍効果を示したことから、本実験で得られたガラナエキスの強力な抗腫瘍効果は、カテキンやタンニンなどによる直接的な癌細胞傷害作用のみに起因するというより、むしろ糖質分解酵素で分解されなかったガラナ果実に由来する高分子多糖体、アミロペクチンや植物繊維などによる動物体内の免疫監視機構中の非特異性免疫系や腫瘍免疫系の活性化、すなわちナチュラルキラー（ＮＫ）細胞、キラーＴリンパ球やマクロファージの活性化と免疫関連因子であるインターロイキン１２やγ−インターフェロンの産生亢進に基づいた間接的な癌細胞傷害作用との相乗効果によるものであろうことが推測された。さらに、実施例３と実施例４で示したように、ガラナエキス中には脂肪に対する抗酸化作用と活性酸素消去作用を有する組成成分が含まれていることからも、ガラナエキスのマウス・サルコーマ１８０悪性肉腫に対する抗腫瘍効果は、免疫学的実験結果からも裏付けられたものと考えられる。
【００４０】
【表１０】

【００４１】
実施例６（ガラナエキスの急性毒性試験（ＬＤ_５０））
ｄｄＹ系雌及び雄マウス（５週齢）を１群５匹宛準備し、２倍段階希釈ガラナエキス０．５ｍｌを各々経口（最大濃度：８０ｍｇ／マウス）あるいは皮下（最大濃度：１２０ｍｇ／マウス）投与した後、５日間に亘り死亡匹数を記録し、これら投与法によるＬＤ_５０をベーレンス・カーバー法に従って計算した。その結果、ガラナエキスの経口投与における雌マウスに対するＬＤ_５０は２８５７ｍｇ／ｋｇ、雄マウスに対しては２５６０ｍｇ／ｋｇであった。一方、皮下投与における雌及び雄マウスに対するＬＤ_５０は、各々４２８４ｍｇ／ｋｇと３８４０ｍｇ／ｋｇであり、経口、皮下投与を問わず、雌マウスに対するガラナエキスの急性毒性は雄マウスに対するよりも低い濃度であった。
【００４２】
実施例７（錠剤）
ガラナエキス（凍結乾燥品）６０ｍｇ
デンプン４０ｍｇ
カルボキシメチルセルロースカルシウム２０ｍｇ
全量１２０ｍｇ
を混合し常法により錠剤を製した。
【００４３】
【発明の効果】
上記で詳述した本発明によれば以下の効果を奏する。すなわち、本発明の肝機能障害改善予防剤は、ガラナエキスを有効成分とし、肝機能の治療と予防に用いることでき、また、本発明の腫瘍免疫賦活剤は、ガラナエキスを有効成分とし、悪性腫瘍の治療と予防に用いることができると共に、さらに抗酸化作用と活性酸素消去能を有するので、加齢変化に伴う代謝機能の低下を予防ができ、医薬として極めて有用である。また、本発明の肝機能障害改善予防剤又は腫瘍免疫賦活剤の製造方法は、ガラナ果実に含まれる親油性組成成分と親水性組成成分のいずれも高収率で回収でき経済性に優れるばかりか、種々の薬理活性を有する組成成分を取得でき操作が簡便で有用である。
【図面の簡単な説明】
【図１】肉腫癌細胞の移植２８日目に摘出された肉腫の写真像
【符号の説明】
１：非投与対照群
２：低中分子組成成分画分液投与群
３：ガラナエキス投与群[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a preventive agent for improving liver dysfunction and a tumor immunostimulant containing guarana extract obtained by allowing a saccharide-degrading enzyme to act on a ground product of guarana fruit, and a method for producing them.
[0002]
[Prior art]
The fruit of Brazilian guarana (Paulinia cupana), a kind of vine-like low shrub, contains guanine, which has a molecular structure similar to caffeine, and is excited by the guaran people who have been indigenous to the Amazon basin since ancient times. It is said that it has been used habitually as a sex drink. Since such guarana fruit contains catechin, tannin (polyphenol), etc., it is expected to have preferable pharmacological activity by drinking, and beverages to which various guarana extracts have been added have been provided so far.
[0003]
By the way, conventionally, there are an alcohol extraction method and a high-temperature water extraction method for extracting a guarana extract from a guarana fruit. The alcohol extraction method is a method of extracting guarana fruits using an organic solvent mainly composed of ethyl alcohol. The lipophilic (hydrophobic) composition components contained in guarana fruits can be easily extracted without complicated operations. While there is an advantage that fractionation can be carried out efficiently, (1) the yield is poor because there are many hydrophilic composition residue (30-50%), and (2) the starch contained in guarana fruit is denatured by alcohol. (3) The hydrophilic composition component contained in the guarana fruit is not sufficiently extracted. On the other hand, the hot water extraction method is a method of extracting guarana fruits with salt-containing high temperature water, which is an extremely mild extraction method for the composition components of guarana fruits, and does not require advanced techniques and is easy to extract. On the other hand, (1) the yield is poor because there are many lipophilic composition component residues (25-35%), (2) it is difficult to extract the lipophilic composition components contained in guarana fruit, Caffeine (guanine), catechin, tannin, etc., which are mainly hydrophilic composition components, are not sufficiently recovered due to complex cyclic structures such as flavones and terpenes, which are generally considered to have favorable pharmacological activity. There was a disadvantage of being.
[0004]
In addition, as for the pharmacological activity of guarana extract, which has been provided as a beverage, there have been only reports on catechins and tannins contained in guarana fruit. There are no research reports.
[0005]
[Problems to be solved by the invention]
The present invention solves the problems of the conventional extraction method of guarana extract, and can recover both the lipophilic composition component and the hydrophilic composition component contained in guarana fruit in a high yield and can be carried out with a simple operation. It is an object of the present invention to provide a preventive agent for improving liver dysfunction and tumor immunostimulation comprising guarana extract as an active ingredient and a method for producing them.
[0006]
[Means for Solving the Problems]
The present inventors examined a novel pharmacological activity of guarana extract using GOT and GPT enzyme activity values as an index in mice with liver dysfunction / decreased mice, and modeled mice transplanted with mouse sarcoma 180 cancer cells. The present inventors have studied the novel pharmacological activity of guarana extract and arrived at the present invention. That is, the present invention relates to a preventive agent for improving liver dysfunction and a tumor immunity activator, characterized by comprising as an active ingredient a guarana extract obtained by allowing a saccharide-degrading enzyme to act on a ground product of guarana fruit. In these inventions, the saccharide-degrading enzyme may be a liquefying enzyme, cellulase or saccharifying enzyme.
[0007]
In the above invention, the lipophilic composition component, hydrophilic composition component, residual high molecular weight polysaccharide that is not affected by carbohydrase, amylopectin, fiber, etc., prevent liver function improvement and tumor immune activation. It is presumed to exert an effect.
[0008]
In addition, the present inventors pay attention to the properties of saccharide-degrading enzymes and can efficiently decompose fiber and starch contained in guarana fruit, reduce residue and obtain guarana extract in high yield. I came up with the idea that I could do it and completed this statement. That is, the present invention relates to the above-mentioned preventive agent for improving liver dysfunction or tumor immunity activator characterized in that a liquefied enzyme, cellulase and saccharifying enzyme are allowed to act on a ground product of guarana fruit to obtain guarana extract by the following steps. It relates to a manufacturing method.
(1) Grinding guarana fruit into powder
(2) Adding guarana fruit powder to high temperature water and adding liquefaction enzyme to liquefy starch
(3) A step of saccharification treatment by adding cellulase and saccharifying enzyme to the enzyme liquefaction solution obtained above.
(4) A step of filtering the saccharification solution obtained above
(5) Concentrating the filtrate obtained above
In the inventions of these production methods, the liquefying enzyme is one or more selected from α-amylase, β-amylase and isoamylase, and the saccharifying enzyme is selected from α-glucosidase, β-glucosidase, glucoamylase and pullulanase. It is good also as 1 type chosen or 2 or more types chosen. The present invention also relates to a method for producing the above-mentioned preventive agent for improving liver dysfunction or tumor immunostimulant by adding a predetermined amount of glucose to the guarana extract obtained by these production methods to obtain a guarana extract crystal powder.
[0009]
In the above production method, cellulose that maintains the structure of the cell wall is mainly decomposed by cellulase, and polysaccharides contained in guarana fruits are saccharified and decomposed, so that lignin, flavone-based components and other components are easily released. .
[0010]
DETAILED DESCRIPTION OF THE INVENTION
The guarana fruit mainly uses seeds, but pulp and the like can also be used. The guarana fruit, which has been sufficiently dried in advance, is pulverized by a pulverizer such as a mill. The guarana fruit powder is added to approximately 8-12 times its weight of hot water. The temperature of the high-temperature water is usually 80 to 90 ° C., but may be appropriately changed according to the saccharide-degrading enzyme used. Moreover, although the pH of the high temperature water to which the powder of guarana fruit was added is normally adjusted to 6-7, you may change suitably according to the saccharide-degrading enzyme to be used.
[0011]
Next, a liquefying enzyme is added to the adjustment liquid to liquefy the starch contained in the guarana fruit. The liquefying enzyme uses amylase and can be used regardless of animal origin, plant origin, or microorganism origin. The amylase includes α-amylase, β-amylase and isoamylase, and these amylases can be used alone or in combination. Endo-type α-amylase having particularly high hydrolysis efficiency is preferable. Therefore, a thermostable α-amylase is more preferable, and such an amylase can be easily obtained as a commercial product. Furthermore, starch liquefaction may be appropriately changed according to the saccharide-degrading enzyme to be used after amylase is added to the adjustment solution, but is usually sufficiently stirred at 80 to 90 ° C. for 50 to 70 minutes. The liquefying enzyme is preferably added at a rate of about 600 g per 1000 kg of guarana fruit powder.
[0012]
The enzyme liquefaction solution obtained by the above treatment may be appropriately changed according to the saccharide-degrading enzyme to be used. However, after adjusting the pH to 4 to 6, usually, cellulase and saccharifying enzyme are added, and usually 40 to 40. Enzyme treatment at 50 ° C. for 45-52 hours. The cellulase added can be easily obtained as a commercial product, and can be used regardless of whether it is derived from animals, plants, or microorganisms. In addition, various enzymes can be used regardless of whether they are derived from animals, plants, or microorganisms. In particular, one or two or more of α-glucosidase, β-glucosidase, glucoamylase, and pullulanase can be used in combination. These saccharifying enzymes are preferably used, and commercially available products can also be used. In addition, an isomerase such as glucose isomerase may be added to enhance the sweetness of guarana extract. Cellulase and saccharifying enzyme are preferably added in a ratio of about 600 g per 1000 kg of guarana fruit powder as a starting material.
[0013]
The saccharification solution obtained by the above treatment is filtered by a known operation and separated from the residue. This filtration operation is repeated a plurality of times as desired. Furthermore, the obtained filtrate can be concentrated by a known operation to produce a guarana extract. The guarana extract can be processed into guarana extract syrup by adding sucrose, or can be lyophilized. In addition, glucose can be added to guarana extract to produce guarana extract crystal powder, which facilitates tableting for formulation. In addition, as for the addition amount (capacity | capacitance) of glucose, the ratio of 8-7 is preferable with respect to guarana extract 2-3.
[0014]
The guarana extract produced as described above can be administered as a liver function improving preventive agent or a tumor immune activator. The administration form is generally oral administration, but is not limited thereto. In addition, the dosage form can be appropriately selected according to the use, and powders, granules, tablets, capsules, suppositories, ointments using pharmaceutically acceptable excipients, binders, disintegrants, ointment bases, etc. It can be a dosage form such as an agent. Furthermore, the liver function improving preventive agent and tumor immunostimulant of the present invention can be used as a medical preparation by blending preparations having other medicinal effects (including those derived from animals and plants) as long as their medicinal effects are not lost. An agent, an anticancer agent, or a cytokine can be added.
[0015]
The preventive agent for improving liver function or the tumor immunostimulant obtained by the above production method is excellent in continuous use effect and sustainability of the effect. 1-10 mg / kg per day is preferred. Patients with impaired liver function and patients with malignant tumors can increase the dose as appropriate, and it is preferable to administer the above five-fold dose.
[0016]
【Example】
EXAMPLES Next, the present invention will be described with reference to examples, but the present invention is not limited to the following examples. In the following description, “%” means “% by weight” unless otherwise specified.
[0017]
Example 1 (Production of guarana extract from guarana fruit)
After adding 150 g (water content: 7.5 ± 0.5%) of pulverized guarana fruit (seed) with a pulverizer to 1500 ml of sterilized distilled water heated to 90 ° C. (pH 5.5), The pH was adjusted to 6.5 with calcium carbonate, and 90 mg of α-amylase derived from Bacillus subtilis (Amylase AD “Amano”, Amano Pharmaceutical Co., Ltd.) was added to about 1500 ml of the obtained adjustment solution, and 60 ° C. at 60 ° C. The starch contained in guarana fruit powder was liquefied while stirring for a minute. After adjusting the pH to 4.5 by adding oxalic acid to about 1500 ml of the obtained enzyme liquefied solution, α-glucosidase derived from Aspergillus niger (α-glucosidase “Amano”, manufactured by Amano Pharmaceutical Co., Ltd.) and Aspergillus niger-derived 90 mg each of cellulase (Cellulase A “Amano”, Amano Pharmaceutical Co., Ltd.) was added and subjected to enzyme treatment at 45 ° C. for 48 hours (liquefaction step). By this enzyme treatment, plant fiber (cellulose) is broken down into glucose (saccharification step). Further, suction filtration of about 1500 ml of the saccharified saccharified solution was repeated twice with a filter paper with a fold having a filtering agent (perlite) attached to the surface. The filtrate thus obtained was concentrated using a rotary vacuum concentrator to obtain about 350 ml of guarana extract having a solid content of 62.7% and a water content of 37.3%.
[0018]
The yield of guarana extract obtained by the above production method was about 70%, which was extremely high compared to about 45% of the alcohol extraction method performed at the same time and about 30% of the high temperature water extraction method. . Further, the content of caffeine per 100 ml of guarana extract is 4.65 g and the content of tannin is 12.6 g, which is almost similar to the content obtained by the high-temperature water extraction method, and the guarana extract is produced by the method for producing guarana extract of the present invention. It was found that the hydrophilic composition component contained in the fruit can be sufficiently recovered. Furthermore, when the content ratio of each composition component was analyzed about 100 g of solid contents of a guarana extract, as shown in Table 1, the fiber origin glucose by decomposition | disassembly of a cellulase and alpha-glucosidase was the largest ratio. Caffeine was analyzed by the HPLC method, tannin was analyzed by the foreign dennis method, and saccharides were analyzed by the phenol / sulfuric acid method.
[0019]
[Table 1]

[0020]
Reference Example 1 (Preparation of mice with liver dysfunction / decreased function)
Liver dysfunction / hypofunction mice that cause liver dysfunction / hypofunction were performed according to the method of Conti et al. (Jpn. J. Pharmacol., 60: 315, 1992). That is, 5 week-old normal ddY female mice (average body weight: 25 g) addressed to 3 animals were prepared for each experimental group of Example 2 described later, and dissolved in physiological saline to a concentration of 60 mg / ml. D-galactosamine hydrochloride (hereinafter referred to as Gal-N) reagent solution (0.2 ml) was intraperitoneally administered (480 mg / kg) to the mice with liver dysfunction / hypofunction (hereinafter referred to as Gal-N-treated mice). ) Was produced.
[0021]
Reference Example 2 (Method for measuring GOT and GPT activity values)
The activity values of GOT (AST) and GPT (ALT) are measured by a UV-Rate method (main wavelength: main wavelength: using a multi-automatic analyzer (Toshiba Multi 60M type) in which GOT or a kit reagent for GPT measurement (manufactured by Aswell) is set. 340 nm / subwavelength: 375 nm). That is, after adding 300 μl of enzyme solution (R1) to 10 μl of serum and incubating at 37 ° C. for 3 minutes, then adding 75 μl of substrate solution (R2), and the change in absorbance per minute at around 340 nm for 1 to 5 minutes (NADH → NAD ⁺ The rate of GOT or GPT activity (unit: IU / l) was calculated from the NADH molecular extinction coefficient. In addition, the significant difference test (risk rate 5% or less, p∠0.05, with significant difference) in each of the following examples was calculated by the Student t-test method.
[0022]
Example 2 (Examination of the effect of guarana extract on GOT and GPT activity values)
(Concentration effect of guarana extract) Galana extract was administered to the Gal-N-treated mice prepared in Reference Example 1 above, and the concentration effect of guarana extract on GOT activity value and GPT activity value of Gal-N-treated mice was examined. . That is, the concentration of guarana extract obtained in Example 1 was adjusted so that the dose was 100 mg / kg, 200 mg / kg, 400 mg / kg, 800 mg / kg at 4 hours after Gal-N treatment, After 0.5 ml was orally administered to each Gal-N-treated mouse, the eyeball of the mouse under ether anesthesia was removed 48 hours later, and blood was collected in a small test tube containing sodium ethylenediaminetetraacetate. After the blood was centrifuged (4 ° C., 5000 rpm, 10 minutes), serum was collected (200 to 300 μl), and the activity values of GOT and GPT were measured by the method of Reference Example 2.
[0023]
When the activity values of GOT and GPT were measured as described above, as shown in Table 2, the GOT activity value of the non-administered control group (normal mice) averaged 92 IU / l, but Gal-N alone In the administration group, the GOT activity value significantly increased (average of 3135 IU / l). After Galana extract (100-800 mg / kg) was orally administered to this Gal-N-treated mouse, the GOT activity value was measured at 48 hours. The enzyme activity value decreased depending on the administration concentration. A significant difference was observed at 200 mg / kg (risk rate of 5% or less), and a significant difference (risk rate of 1% or less) was observed at doses of 400 mg / kg or more. Also, in the GPT activity value, as shown in Table 3, a decrease in the enzyme activity value was recognized depending on the administration concentration of guarana extract as in GOT. In this way, by increasing the administration concentration of guarana extract, the activity values of GOT and GPT tended to approach the normal enzyme activity threshold. It was suggested that the composition component to relieve and recover was included. Interestingly, the Gal-N treatment increased the GOT activity value to about 34 times the normal value, while the GPT activity value increased from 14 IU / l to 2182 IU / l, about 156 times. From the above, it was suggested that D-galactosamine hydrochloride acts more strongly on the GPT activity value than on the liver-derived GOT activity value.
[0024]
[Table 2]

[0025]
[Table 3]

[0026]
(Effect of the frequency of guarana extract administration)
After treatment with Gal-N, galana extract (200 mg / kg) was orally administered every 2 hours (3 times in total), and then serum was collected at 48 hours in the same manner as in the experimental group on the concentration effect of the galana extract. Then, the activity values of GOT and GPT were measured. As a result, as shown in Tables 4 and 5, the activity values of GOT and GPT that were remarkably increased by the treatment with Gal-N decreased as the number of administrations increased, and there was a significant difference of 5% or less of the risk rate between the two administrations. A significant difference with a risk rate of 1% or less was observed after 3 administrations, suggesting that the effect of preventing gallana extract from improving liver function was enhanced by repeated administration.
[0027]
[Table 4]

[0028]
[Table 5]

[0029]
(Sustained effect of guarana extract)
After GAL-N treatment, galana extract (400 mg / kg) was orally administered at 4 hours, and then at 24, 48 and 72 hours, serum was collected in the same manner as in the experimental group on the concentration effect of guarana extract. The activity values of GOT and GPT were measured. As a result, as shown in Tables 6 and 7, both enzyme activity values in the Gal-N + Galana extract administration group were significantly lower than the activity values in the Gal-N single administration group (24 to 48 hours). (Risk rate 5% or less after 72 hours, risk rate 1% or less after 72 hours), and gradually decreased with time. Relief / recovery effect of guarana extract on liver function that was impaired or decreased in function after one administration. Was found to persist in the body for quite some time.
[0030]
[Table 6]

[0031]
[Table 7]

[0032]
GPT is limited to normal hepatocytes compared to GOT, and the GPT activity value derived from the cytoplasm of hepatocytes is known to be higher than the GOT activity value. When humans suffer from acute hepatitis, GPT Since the activity value rises more rapidly than the GOT activity value, it is considered that treatment of mice with Gal-N induced liver dysfunction similar to acute hepatitis seen in humans. Experimental results suggested that guarana extract would be extremely effective against human acute hepatitis.
[0033]
Reference Example 3 (Production of liver homogenate)
Mice were treated with Gal-N (480 mg / kg) by the method described in Reference Example 1, and guarana extract (400 mg / kg) was orally administered 60 minutes later. After administration, the mice under ether anesthesia were sacrificed at 6, 24 and 72 hours, and the liver was excised and shredded. Next, the liver fine fragment was placed in a 10 mM Tris-HCl buffer containing 150 mM sodium chloride and 1.0 mM EDTA, and a liver homogenate was prepared using a glass homogenizer. The protein concentration in the liver homogenate was quantified by the Raleigh method and adjusted to an amount of 100 mg protein per 1 ml of the liver homogenate.
[0034]
Example 3 (Inhibition of liver fat peroxidation (antioxidation) of guarana extract)
The liver fat peroxidation experiment was performed according to the method of Campos et al. (Planta Medica, 55: 417, 1989). That is, after oral administration of guarana extract by the method described in Reference Example 3 (400 mg / kg), liver homogenate at 6, 24 and 72 hours (Tris buffer 4 was added to liver homogenate 1 to a final concentration of 20 mg protein) Malone aldehyde (hereinafter referred to as MDA) / thiobarbital acid complex is added to the solution, and the produced MDA concentration is measured at 535 nm with an immunoassay automatic analyzer (Behring ELISA P-2 type). = 1.56 × 10 ⁵ It was measured using a condition coefficient of 1000 ml / mole / cm (unit: nmole / mg protein). The label in this law is NAD ⁺ -Using ATP coenzyme, NADPH-Fe ⁺ -It is a measurement method using the ADP production phenomenon. As shown in Table 8, the amount of MDA produced in the normal mouse liver of the non-administered control group was almost constant (50 to 68 nmole / mg protein: average 56.8 nmole / mg) throughout the experimental period (72 hours). However, Gal-N treatment showed an increase in MDA of 4 times or more, indicating that liver fat oxidation was significantly enhanced. On the other hand, by orally administering guarana extract, the production of MDA is reduced with the passage of time, and there is a significant difference of a risk rate of 1% or less after 24 hours and 0.1% or less after 72 hours. Admitted. From this, it became clear that guarana extract acts suppressively on the oxidation and peroxidation of fats (especially neutral fats and fatty acids) that are likely to occur due to a decrease in liver function. On the other hand, galana extract also has a carcinogenesis-preventing effect because the production and accumulation of peroxidized fat in the liver during human liver dysfunction and liver function decline is an important factor in the development of liver cancer. Deafness is considered enough. Moreover, the effect which prevents the onset of the lifestyle-related disease accompanying an aging change by preventing the oxidation of a fatty acid etc. is anticipated.
[0035]
[Table 8]

[0036]
Example 4 (Inhibition of active oxygen in the liver by guarana extract (free radical scavenger)
Reactive oxygen (free radical oxygen) caused by Kupffer cells, which are hepatic macrophages that increase with the onset of liver damage or hepatitis, is measured according to the method of Videla et al. (Biochem. Pharmacol., 34: 2209, 1985). went. That is, after oral administration of guarana extract by the method described in Reference Example 3 (400 mg / kg), liver homogenate at 6 hours (Tris buffer 200 was added to liver homogenate 1 to a final concentration of 0.5 mg protein / ml). ) Is collected in a vial (13 × 30 mm), and the luminescence value (unit: cpm / mg protein) per minute of the chemilminisense (chemiluminescence) generated in the liver homogenate is measured by liquid scintillation measurement (Beckmann LS-3150P type). Note that N- (4-aminobutyl) -N-ethylisoluminol, which is one of isoluminol derivatives, was used as the chemiluminescent label in this method. As shown in Table 9, the active oxygen (free radical oxygen) produced in normal mouse liver by the treatment with Gal-N increased from an average of 1178 cpm / mg protein to 2.3 times (average of 2675 cpm / mg protein). The decrease in liver function was surely caused. That is, in order to eliminate the damaged or destroyed hepatocytes or liver-derived cells that have been treated by Gal-N treatment outside the body, the cell killing (bactericidal) system by active oxygen present in Kupffer cells is abnormally enhanced, As a result, it was proved that it was caused by the production of a large amount of active oxygen from Kupffer cells. By administering guarana extract to such mice, the active oxygen decreased to the normal threshold (1280 cpm / mg protein), indicating that damage and damage to hepatocytes and the like were remarkably reduced (risk rate 1). % Or less significant difference). These results suggest that guarana extract also has a strong active oxygen scavenging action similar to fat-soluble vitamins, ceruloplasmin or superoxide desmutase (SOD). As a result, guarana extract is expected to be effective in preventing carcinogenesis and aging, which are said to be due to active oxygen.
[0037]
[Table 9]

[0038]
Reference Example 4 (Separation and purification of fractions of low and medium molecular components of guarana extract)
The guarana extract produced in Example 1 was adjusted to 30% guarana extract (400 mg / ml), centrifuged at 1000 rpm for 60 minutes in a cooling high-speed centrifuge, and then the supernatant was subjected to ultrafiltration membrane attachment. The filtrate was collected by suction filtration with an external filter. The low and medium molecular composition component fraction having a molecular weight of 5000 or less contained in the filtrate was freeze-dried and further adjusted to a concentration of 10 mg / ml with physiological saline to obtain a low and medium molecular composition component fraction. This guarana extract low and medium molecular composition fraction fraction contains caffeine, catechin, tannin, malic acid, α-glucosidase-degrading fiber-derived glucose, disaccharides, etc. It was used for the experiment of the formation / hypertrophy suppression action.
[0039]
Example 5 (Inhibitory action of guarana extract on malignant sarcoma formation / hypertrophy)
5 x 10 per ml of Eagle MEM broth ⁵ 0.2 ml of 1 × 10 mouse sarcoma 180 cancer cell suspension ⁵ 2 days before transplantation (six mice per group) to the back of the mouse (8 mice per group), immediately after transplantation (0 day), 1 day, 2 days, 5 days and 8 days after transplantation in Reference Example 4 0.5 ml (400 mg / kg) of the obtained low-molecular-weight composition fraction solution of guarana extract and 0.5 ml (400 mg / kg) of guarana extract obtained in Example 1 were each subcutaneously implanted in the back of the periphery of the cancer cell transplantation. Administered once. On the 28th day after transplantation, the sarcoma that had enlarged was removed and weighed. As a result, as shown in Table 10 and FIG. 1, the average weight of sarcoma on the 28th day after transplantation in the non-administration control group was 15.35 g, whereas the fraction of low and medium molecular components of guarana extract When the solution was administered, sarcoma formation and hypertrophy were somewhat suppressed, but no significant difference was observed (average weight: 12.44 g). On the other hand, when guarana extract was administered, a remarkable formation / hypertrophy inhibitory effect on sarcoma was observed (average weight: 9.88 g), and the difference was significant with a risk rate of 1% or less. Thus, there are reports of anti-tumor effects of low to medium molecular weight components, such as catechin and tannin, contained in the centrifugal supernatant fraction of guarana extract, but it contains all low molecular weight to high molecular weight components. Since guarana extract itself showed a much stronger antitumor effect against mouse sarcoma 180 sarcoma, galana extract obtained in this experiment has a strong antitumor effect due to direct cancer caused by catechin, tannin, etc. Rather than only due to cytotoxic action, non-specific immune system in the immune surveillance mechanism in the animal body, such as macromolecular polysaccharides derived from guarana fruits that were not degraded by carbohydrases, amylopectin and plant fibers Activation of tumor immune system, ie, activation of natural killer (NK) cells, killer T lymphocytes and macrophages and immune-related factors Roikin 12 and it would be of γ- interferon by synergy with indirect cancer cytotoxicity based on enhanced production was inferred. Furthermore, as shown in Example 3 and Example 4, the guarana extract contains a composition component having an antioxidant action against fat and an active oxygen scavenging action. The antitumor effect against malignant sarcoma is thought to be supported by immunological experimental results.
[0040]
[Table 10]

[0041]
Example 6 (Acute toxicity test of guarana extract (LD ₅₀ ))
ddY female and male mice (5 weeks old) were prepared for 5 mice per group, and 0.5 ml of 2-fold diluted guarana extract was orally (maximum concentration: 80 mg / mouse) or subcutaneous (maximum concentration: 120 mg / mouse). After administration, the number of dead animals was recorded over 5 days. ₅₀ Was calculated according to the Behrens-Kerber method. As a result, LD for female mice in oral administration of guarana extract ₅₀ Was 2857 mg / kg and 2560 mg / kg for male mice. On the other hand, LD for female and male mice in subcutaneous administration ₅₀ Were 4284 mg / kg and 3840 mg / kg, respectively, and the acute toxicity of guarana extract to female mice was lower than that for male mice, both oral and subcutaneous administration.
[0042]
Example 7 (tablet)
Guarana extract (freeze-dried product) 60mg
Starch 40mg
Carboxymethylcellulose calcium 20mg
Total amount 120mg
Were mixed to prepare tablets.
[0043]
【The invention's effect】
According to the present invention described in detail above, the following effects are obtained. That is, the preventive agent for improving liver dysfunction according to the present invention comprises guarana extract as an active ingredient and can be used for the treatment and prevention of liver function. The tumor immunostimulant according to the present invention comprises guarana extract as an active ingredient and is malignant. It can be used for the treatment and prevention of tumors, and further has an antioxidative action and an active oxygen scavenging ability. Therefore, it is possible to prevent a decrease in metabolic function accompanying an aging change and is extremely useful as a medicine. In addition, the method for producing a preventive agent for improving liver dysfunction or a tumor immunostimulant of the present invention is not only excellent in economic efficiency, but can recover both the lipophilic composition component and the hydrophilic composition component contained in guarana fruit in high yield. Therefore, composition components having various pharmacological activities can be obtained, and the operation is simple and useful.
[Brief description of the drawings]
1 is a photograph of a sarcoma removed on the 28th day after transplantation of sarcoma cancer cells.
[Explanation of symbols]
1: Non-administered control group
2: Low and medium molecular composition component fraction administration group
3: Guarana extract administration group

Claims

A preventive agent for improving liver dysfunction, comprising a guarana extract obtained by allowing a saccharide-degrading enzyme to act on a ground product of guarana fruit as an active ingredient.

The agent for improving and preventing liver dysfunction according to claim 1, wherein the saccharide-degrading enzyme comprises liquefaction enzyme, cellulase and saccharification enzyme.

A tumor immunostimulant comprising a guarana extract obtained by allowing a saccharide-degrading enzyme to act on a ground product of guarana fruit as an active ingredient.

The tumor immunity activator according to claim 3, wherein the saccharide-degrading enzyme comprises liquefaction enzyme, cellulase and saccharification enzyme.

The method for producing a preventive agent for improving liver dysfunction according to claim 1 or 2 , wherein a liquefied enzyme, cellulase and saccharifying enzyme are allowed to act on the ground product of guarana fruit to obtain guarana extract by the following steps. .
(1) A step of pulverizing guarana fruit to pulverize (2) A step of adding guarana fruit powder to high-temperature water and further adding a liquefied enzyme to liquefy starch. (3) In the enzyme liquefied solution obtained above Step of adding saccharification enzyme and cellulase (4) Step of filtering the saccharification solution obtained above (5) Step of concentrating the filtrate obtained above

The liquefying enzyme is one or more selected from α-amylase, β-amylase and isoamylase, and the saccharifying enzyme is one or more selected from α-glucosidase, β-glucosidase, glucoamylase and pullulanase. The method for producing a preventive agent for improving liver dysfunction according to claim 5 , wherein

A method for producing a preventive agent for improving liver dysfunction, comprising adding a predetermined amount of glucose to a guarana extract obtained by the production method according to claim 5 or 6 to obtain a guarana extract crystal powder.

The method for producing a tumor immunostimulant according to claim 3 or 4 , wherein a liquefied enzyme, cellulase and saccharifying enzyme are allowed to act on the ground product of guarana fruit to obtain guarana extract by the following steps.
(1) A step of pulverizing guarana fruit to pulverize (2) A step of adding guarana fruit powder to high-temperature water and further adding a liquefied enzyme to liquefy starch. (3) In the enzyme liquefied solution obtained above Step of adding saccharification enzyme and cellulase (4) Step of filtering the saccharification solution obtained above (5) Step of concentrating the filtrate obtained above

The liquefying enzyme is one or more selected from α-amylase, β-amylase and isoamylase, and the saccharifying enzyme is one or more selected from α-glucosidase, β-glucosidase, glucoamylase and pullulanase. The method for producing a tumor immunostimulant according to claim 8 , wherein:

A method for producing a tumor immunostimulant comprising adding a predetermined amount of glucose to a guarana extract obtained by the production method according to claim 8 or 9 , thereby obtaining a guarana extract crystal powder.