JPWO2015002209A1 - Antihypertensive agent, saccharification inhibitor, and method for producing the same, containing coffee bean extract as an active ingredient - Google Patents

Antihypertensive agent, saccharification inhibitor, and method for producing the same, containing coffee bean extract as an active ingredient Download PDF

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JPWO2015002209A1
JPWO2015002209A1 JP2015525245A JP2015525245A JPWO2015002209A1 JP WO2015002209 A1 JPWO2015002209 A1 JP WO2015002209A1 JP 2015525245 A JP2015525245 A JP 2015525245A JP 2015525245 A JP2015525245 A JP 2015525245A JP WO2015002209 A1 JPWO2015002209 A1 JP WO2015002209A1
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暁史 保母
暁史 保母
智博 高柳
智博 高柳
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Abstract

本発明は、温度:140〜200℃、圧力:0.15〜1.8MPa、かつ、各温度の飽和蒸気圧以上の圧力による亜臨界処理工程を経て得られるコーヒー豆の抽出液であって、アンジオテンシン変換酵素の阻害活性率が50%のとき、該コーヒー豆の抽出液の固形分濃度(IC50値)が0.16mg/mL以下であるコーヒー豆の抽出液を有効成分として含有する血圧上昇抑制剤に関する。さらに本発明は糖化抑制剤及びそれらの製造方法に関する。本発明により、コーヒー豆に由来する、より活性の高い血圧上昇抑制剤又は糖化抑制剤及びそれらの製造方法、並びに血圧上昇抑制作用及び糖化抑制作用を有するコーヒー豆の抽出液の製造方法が提供される。The present invention is a coffee bean extract obtained through a subcritical treatment step at a temperature of 140 to 200 ° C., a pressure of 0.15 to 1.8 MPa, and a pressure equal to or higher than a saturated vapor pressure at each temperature, When the inhibitory activity rate of angiotensin converting enzyme is 50%, the increase in blood pressure containing a coffee bean extract whose active ingredient is a solid content concentration (IC50 value) of 0.16 mg / mL or less as the coffee bean extract It relates to the agent. The present invention further relates to saccharification inhibitors and methods for producing them. ADVANTAGE OF THE INVENTION By this invention, the more active antihypertensive agent or saccharification inhibitor derived from coffee beans, the manufacturing method thereof, and the manufacturing method of the coffee bean extract which has an antihypertensive action and an antisaccharification action are provided. The

Description

本発明は、コーヒー豆を亜臨界処理して得られる抽出液を有効成分として含有する血圧上昇抑制剤に関する。さらに本発明は、コーヒー豆を亜臨界処理して得られる抽出液を有効成分として含有する糖化抑制剤に関する。さらに本発明は、コーヒー豆からこのような血圧上昇抑制作用又は糖化抑制作用を有するコーヒー豆の抽出液の製造方法に関する。   The present invention relates to an antihypertensive agent containing, as an active ingredient, an extract obtained by subcritical processing of coffee beans. Furthermore, the present invention relates to a saccharification inhibitor containing, as an active ingredient, an extract obtained by subcritical processing of coffee beans. Furthermore, this invention relates to the manufacturing method of the extract of coffee beans which has such a blood pressure increase inhibitory effect or saccharification inhibitory effect from coffee beans.

コーヒーは古くから世界各地で嗜好飲料として楽しまれている。一般的にコーヒーは水、熱水などによりコーヒー豆から成分を抽出した香りや味に代表される風味を楽しむ飲料として提供されている。近年、コーヒーの生理活性作用についても注目されつつあり、コーヒー中に含まれるクロロゲン酸などによる血圧降下作用について報告されている(特許文献1、特許文献2)。   Coffee has long been enjoyed as a favorite beverage around the world. Generally, coffee is provided as a beverage that enjoys flavors typified by aroma and taste extracted from coffee beans with water, hot water, and the like. In recent years, attention has also been paid to the physiologically active action of coffee, and the blood pressure lowering action caused by chlorogenic acid contained in coffee has been reported (Patent Documents 1 and 2).

特開2008−48728号公報JP 2008-48728 A 特開2007−31392号公報JP 2007-31392 A

しかしながら、コーヒーより血圧降下もしくは血圧上昇抑制作用や糖化抑制作用の生理活性を得るための熱水等の抽出方法には、ポリフェノールに代表される溶存成分濃度の抽出できる量に限界があった。そのため、血圧上昇抑制や糖化抑制の評価を行うと、抑制率が一定値以下にはならなかった。そのため、血圧上昇抑制作用や糖化抑制作用などの機能性を充分に発揮することができないことが推定された。   However, extraction methods such as hot water for obtaining the physiological activity of lowering blood pressure or suppressing blood pressure increase or saccharification from coffee have a limit in the amount of extractable dissolved component concentration typified by polyphenol. Therefore, when evaluation of suppression of blood pressure increase or suppression of glycation was performed, the suppression rate did not become a certain value or less. For this reason, it has been estimated that the functions such as the blood pressure increase inhibiting action and the glycation inhibiting action cannot be sufficiently exhibited.

そのため、コーヒー豆より血圧上昇抑制作用もしくは糖化抑制作用の生理活性を得るための熱水抽出方法以外の方法を検討することが必要であった。   Therefore, it was necessary to examine methods other than the hot water extraction method for obtaining the physiological activity of the blood pressure increase inhibitory action or the saccharification inhibitory action from the coffee beans.

本発明者らがコーヒーについて鋭意研究した結果、コーヒー生豆及び/又は焙煎コーヒー豆を亜臨界状態(各温度の飽和蒸気圧以上)で加水分解させることで得られる抽出組成液および抽出液の製造方法に特徴を見出し、ポリフェノールに代表される溶存成分共存組成液が一定量以上得られ、血圧上昇抑制作用、糖化抑制作用などの機能性が充分に発揮されることを見出した。その結果本発明のコーヒー豆の亜臨界処理により得られた抽出液は、従来の熱水抽出法により得られる抽出液よりも優れた血圧上昇抑制作用、糖化抑制作用を有することがわかった。   As a result of intensive studies on the coffee by the present inventors, an extraction composition liquid and an extraction liquid obtained by hydrolyzing green coffee beans and / or roasted coffee beans in a subcritical state (above the saturated vapor pressure at each temperature) The inventors have found characteristics of the production method, and found that a certain amount or more of a dissolved component coexisting composition liquid typified by polyphenol is obtained, and functions such as an antihypertensive action and a saccharification inhibiting action are sufficiently exhibited. As a result, it was found that the extract obtained by the subcritical processing of the coffee beans of the present invention has a blood pressure elevation inhibitory action and a saccharification inhibitory action superior to the extract obtained by the conventional hot water extraction method.

即ち、本発明の要旨は、
[1] コーヒー豆の抽出液を有効成分として含有する血圧上昇抑制剤であって、
前記コーヒー豆の抽出液は、温度:140〜200℃、圧力:0.15〜1.8MPa、かつ、各温度の飽和蒸気圧以上の圧力による亜臨界処理工程を経て得られ、
アンジオテンシン変換酵素(ACE)の阻害活性率が50%のとき、コーヒー豆の抽出液の固形分濃度(IC50値)が0.16mg/mL以下であるコーヒー豆の抽出液を有効成分として含有する血圧上昇抑制剤;
[2] コーヒー豆の抽出液を有効成分として含有する糖化抑制剤であって、
前記コーヒー豆の抽出液は、温度:140〜200℃、圧力:0.15〜1.8MPa、かつ、各温度の飽和蒸気圧以上の圧力による亜臨界処理工程を経て得られ、
糖化産物の生成阻害率が50%のとき、コーヒー抽出液の固形分濃度(IC50値)が0.06mg/mL以下であるコーヒー豆の抽出液を有効成分として含有する糖化抑制剤;
That is, the gist of the present invention is as follows.
[1] An antihypertensive agent comprising a coffee bean extract as an active ingredient,
The coffee bean extract is obtained through a subcritical processing step at a temperature of 140 to 200 ° C., a pressure of 0.15 to 1.8 MPa, and a pressure equal to or higher than a saturated vapor pressure at each temperature,
Blood pressure containing, as an active ingredient, a coffee bean extract having a solid content concentration (IC50 value) of 0.16 mg / mL or less when the angiotensin converting enzyme (ACE) inhibitory activity rate is 50% Elevation inhibitor;
[2] A saccharification inhibitor containing a coffee bean extract as an active ingredient,
The coffee bean extract is obtained through a subcritical processing step at a temperature of 140 to 200 ° C., a pressure of 0.15 to 1.8 MPa, and a pressure equal to or higher than a saturated vapor pressure at each temperature,
A saccharification inhibitor containing, as an active ingredient, a coffee bean extract having a solid content concentration (IC50 value) of 0.06 mg / mL or less when the production inhibition rate of the saccharification product is 50%;

[3] コーヒー生豆及び/又は焙煎コーヒー豆を粉砕してコーヒー豆の粉砕物を得る工程、
コーヒー豆の粉砕物に水を加えてコーヒー豆の粉砕物と水との混合物を得る工程、及び
コーヒー豆の粉砕物と水との混合物を、温度:140〜200℃、圧力:0.15〜1.8MPa、かつ、各温度の飽和蒸気圧以上の圧力の亜臨界条件で処理して、コーヒー豆の抽出液を回収する工程
を含む、血圧上昇抑制作用を有するコーヒー豆の抽出液の製造方法;並びに
[4] コーヒー生豆及び/又は焙煎コーヒー豆を粉砕してコーヒー豆の粉砕物を得る工程、
コーヒー豆の粉砕物に水を加えてコーヒー豆の粉砕物と水との混合物を得る工程、及び
コーヒー豆の粉砕物と水との混合物を、温度:140〜200℃、圧力:0.15〜1.8MPa、かつ、各温度の飽和蒸気圧以上の圧力の亜臨界条件で処理して、コーヒー豆の抽出液を回収する工程
を含む、糖化抑制作用を有するコーヒー豆の抽出液の製造方法;に関する。
[3] crushing green coffee beans and / or roasted coffee beans to obtain a coffee beans pulverized product,
A step of adding water to the ground coffee beans to obtain a mixture of the ground coffee beans and water, and a mixture of the ground coffee beans and water, temperature: 140 to 200 ° C., pressure: 0.15 to A method for producing a coffee bean extract having a blood pressure increase-inhibiting action, comprising a step of recovering the coffee bean extract by treating it under a subcritical condition of 1.8 MPa and a pressure equal to or higher than a saturated vapor pressure at each temperature. And
[4] crushing green coffee beans and / or roasted coffee beans to obtain a coffee beans pulverized product,
A step of adding water to the ground coffee beans to obtain a mixture of the ground coffee beans and water, and a mixture of the ground coffee beans and water, temperature: 140 to 200 ° C., pressure: 0.15 to A process for producing a coffee bean extract having a saccharification-inhibiting action, comprising a step of recovering the coffee bean extract by a subcritical condition of 1.8 MPa and a pressure equal to or higher than a saturated vapor pressure at each temperature; About.

本発明のコーヒー豆の亜臨界処理による抽出液は、従来の熱水抽出法により得られる抽出液よりも優れた血圧上昇抑制作用、糖化抑制作用を有する。またその抽出方法は吸着剤処理および加熱殺菌処理や、酸、アルカリ、酵素の添加といった余分な工程を必要とせず、比較的低温で簡易的な手法により抽出液を得ることができる。   The extract obtained by subcritical processing of the coffee beans of the present invention has a blood pressure increase inhibitory action and a saccharification inhibitory action superior to those of an extract obtained by a conventional hot water extraction method. Further, the extraction method does not require extra steps such as adsorbent treatment and heat sterilization treatment, and addition of acid, alkali, and enzyme, and an extract can be obtained by a simple technique at a relatively low temperature.

図1は、ACE阻害活性測定に関するサンプル液量と阻害率の関係を示すグラフである。FIG. 1 is a graph showing the relationship between the amount of sample solution and the inhibition rate for ACE inhibitory activity measurement. 図2は、糖化産物の生成阻害率測定に関するサンプル液量と阻害率の関係を示すグラフである。FIG. 2 is a graph showing the relationship between the amount of sample liquid and the inhibition rate related to the measurement of the inhibition rate of glycation product production. 図3−1及び図3−2は、各サンプルの溶存成分濃度及びポリフェノール濃度を示すグラフである。FIGS. 3A and 3B are graphs showing the dissolved component concentration and polyphenol concentration of each sample. 図4−1及び図4−2は、アンジオテンシン変換酵素の活性を50%阻害する、各サンプルの抽出液量(μL)を示すグラフである。FIGS. 4-1 and 4-2 are graphs showing the amount of extract (μL) of each sample that inhibits the activity of angiotensin converting enzyme by 50%. 図5は、糖化産物の生成を50%阻害する、各サンプルの抽出液量(μL)を示すグラフである。FIG. 5 is a graph showing the amount of extract (μL) of each sample that inhibits the production of a saccharification product by 50%.

本発明のコーヒー豆の抽出液は、コーヒー生豆及び/又は焙煎コーヒー豆を亜臨界状態(各温度の飽和蒸気圧以上)で加水分解させることで得られる抽出液である。具体的な亜臨界状態としては、温度:140〜200℃、圧力:0.15〜1.8MPa、かつ、各温度の飽和蒸気圧以上の圧力で亜臨界処理工程を経ることである。この亜臨界処理工程を経て得られたコーヒー豆の抽出液は、血圧上昇抑制作用、糖化抑制作用を有する。本明細書において、各温度の飽和蒸気圧としては、好ましくは各温度の飽和水蒸気圧を意味する。   The coffee bean extract of the present invention is an extract obtained by hydrolyzing green coffee beans and / or roasted coffee beans in a subcritical state (above the saturated vapor pressure at each temperature). As a specific subcritical state, the temperature is 140 to 200 ° C., the pressure is 0.15 to 1.8 MPa, and the subcritical treatment step is performed at a pressure equal to or higher than the saturated vapor pressure at each temperature. The coffee bean extract obtained through this subcritical treatment step has a blood pressure increase inhibitory effect and a saccharification inhibitory effect. In the present specification, the saturated vapor pressure at each temperature preferably means the saturated water vapor pressure at each temperature.

前記コーヒー豆の抽出液は、後述する具体的な方法で抽出液を作成したとき、溶存成分濃度が30mg/mL以上であることが望ましい。さらに、具体的には、コーヒー豆と水が1:8の比率(質量比)の場合での溶存成分濃度が30mg/mL以上であることが望ましい。この溶存成分濃度は、加熱乾燥法により濃度測定を行う。加熱乾燥法とは、試料を一定条件下で乾燥し、揮発する成分を水分、残るものを乾燥物(溶存成分)とし、(乾燥物の質量)/(乾燥前の水を含んだ状態の試料の体積)として算出している。実際には、水分以外の揮発成分(アルコールなど)も除去されるが、それらは極微量で測定結果にほとんど影響を与えないと仮定して測定する。   The coffee bean extract preferably has a dissolved component concentration of 30 mg / mL or more when the extract is prepared by a specific method described below. Furthermore, specifically, it is desirable that the concentration of the dissolved component is 30 mg / mL or more when the ratio of coffee beans to water is 1: 8 (mass ratio). The dissolved component concentration is measured by a heat drying method. The heat drying method means that the sample is dried under certain conditions, the component that volatilizes is moisture, the remaining component is the dried product (dissolved component), and the sample contains (mass of dried product) / (water before drying) Volume). Actually, volatile components other than moisture (alcohol, etc.) are also removed, but they are measured on the assumption that they are extremely small and hardly affect the measurement results.

溶存成分とは、ポリフェノール、糖類、タンパク質、無機質等が混合されたものであり、また、溶存成分中には、ポリフェノール、糖類、タンパク質、無機質等以外の副生成物が得られ、これらの副生成物が血圧上昇抑制や糖化抑制に対して、異なる作用(例えば、即効性と遅効性、機能性作用と機能性補助作用)での複合的な機能性に対応することができるようになると、本発明者らは推定した。この推定により、コーヒー生豆及び/又は焙煎コーヒー豆を亜臨界状態(各温度の飽和蒸気圧以上)で加水分解させることで機能性が高い抽出液を得ることができるとも推定した。   The dissolved component is a mixture of polyphenols, saccharides, proteins, minerals, etc., and in the dissolved components, by-products other than polyphenols, saccharides, proteins, minerals, etc. are obtained, and these byproducts When a product can cope with combined functionality with different actions (for example, immediate action and delayed action, functional action and functional auxiliary action) for suppressing blood pressure rise and glycation inhibition, The inventors estimated. Based on this estimation, it was also estimated that a highly functional extract can be obtained by hydrolyzing green coffee beans and / or roasted coffee beans in a subcritical state (above the saturated vapor pressure at each temperature).

また、前述の抽出液中のポリフェノール濃度は、後述する具体的な方法で抽出液を作成したとき、6.8mg/mL以上であることが望ましい。さらに、具体的には、コーヒー豆と水が1:8の比率(質量比)の場合での抽出液中のポリフェノール濃度は、6.8mg/mL以上であることが望ましい。6.8mg/mL以上であるとき、血圧上昇抑制や糖化抑制に関する効果が得やすくなる。ポリフェノール濃度は、Folin-Ciocalteu法により測定することができる。Folin-Ciocalteu法とは、コーヒー抽出液に後述するFolin-Ciocalteu液と炭酸ナトリウム溶液とを混合させて混合液を作製し、この混合液の765nmにおける吸光度を測定し、同様に作成したクロロゲン酸水溶液による検量線からポリフェノール量をクロロゲン酸相当量(mg/mL)で算出する方法である。   Further, the polyphenol concentration in the aforementioned extract is desirably 6.8 mg / mL or more when the extract is prepared by a specific method described later. Furthermore, specifically, the polyphenol concentration in the extract when the ratio of coffee beans to water is 1: 8 (mass ratio) is desirably 6.8 mg / mL or more. When it is 6.8 mg / mL or more, it becomes easy to obtain the effect on suppression of blood pressure increase and suppression of glycation. The polyphenol concentration can be measured by the Folin-Ciocalteu method. The Folin-Ciocalteu method is a mixture of a Folin-Ciocalteu solution and a sodium carbonate solution, which will be described later, mixed with a coffee extract, and the absorbance at 765 nm of this mixture is measured. This is a method of calculating the amount of polyphenol by the amount corresponding to chlorogenic acid (mg / mL) from the calibration curve according to the above.

コーヒー抽出液は、後述する具体的な方法で抽出液を作成したとき、溶存成分濃度が30mg/mL以上であり、ポリフェノール濃度は、6.8mg/mL以上であることがより望ましい。さらに、具体的には、コーヒー豆と水が1:8の比率(質量比)の場合でのコーヒー抽出液は、溶存成分濃度が30mg/mL以上であり、ポリフェノール濃度は、6.8mg/mL以上であることがより望ましい。この2つの濃度を満たすことで、血圧上昇抑制や糖化抑制に関する効果が得やすくなる。   When the coffee extract is prepared by a specific method to be described later, the dissolved component concentration is preferably 30 mg / mL or more, and the polyphenol concentration is more preferably 6.8 mg / mL or more. More specifically, the coffee extract in the case of coffee beans and water in a ratio (mass ratio) of 1: 8 has a dissolved component concentration of 30 mg / mL or more and a polyphenol concentration of 6.8 mg / mL. The above is more desirable. By satisfying these two concentrations, it becomes easy to obtain effects related to suppression of blood pressure increase and suppression of glycation.

本願発明の一つは、アンジオテンシン変換酵素(ACE)の阻害活性率が50%のとき、コーヒー抽出液の固形分濃度(IC50値)が0.16mg/mL以下であるコーヒー豆の抽出液を有効成分として含有する血圧上昇抑制剤である。   One of the inventions of the present application is an effective use of a coffee bean extract whose solid content concentration (IC50 value) is 0.16 mg / mL or less when the inhibitory activity rate of angiotensin converting enzyme (ACE) is 50%. It is a blood pressure increase inhibitor contained as a component.

本発明における血圧上昇抑制作用は、後述する測定方法によって、血圧上昇作用を引き起こすアンジオテンシン変換酵素(ACE)の阻害活性を測定し、アンジオテンシン変換酵素(ACE)の阻害活性率が50%のときの活性阻害を示す抽出液の固形分濃度(IC50値)で規定される。   The antihypertensive effect in the present invention is determined by measuring the inhibitory activity of angiotensin converting enzyme (ACE) that causes an antihypertensive effect by the measurement method described later, and the activity when the inhibitory activity rate of angiotensin converting enzyme (ACE) is 50%. It is defined by the solid content concentration (IC50 value) of the extract showing inhibition.

ここで、アンジオテンシン変換酵素(ACE)について説明をする。アンジオテンシン変換酵素は血管内皮細胞の表面に存在し、アンジオテンシンIをアンジオテンシンIIに変換する酵素である。この作用を妨げるアンジオテンシン変換酵素阻害薬(ACE阻害薬)は昇圧抑制剤や心不全の治療薬として用いられる。ACEはangiotensin-converting enzymeの略である。アンジオテンシンは血液中に存在するペプチドの一つで、アンジオテンシンI〜IVの4種類がある。このうちアンジオテンシンII〜IVには細動脈の平滑筋を収縮させ血圧を上昇させる作用や、副腎皮質でアルドステロンの分泌を促進する作用などがある。アンジオテンシンII〜IVは特に強い血圧上昇作用をもつ。アンジオテンシンIにはそうした生理活性はない。   Here, angiotensin converting enzyme (ACE) will be described. Angiotensin converting enzyme is an enzyme that exists on the surface of vascular endothelial cells and converts angiotensin I into angiotensin II. An angiotensin converting enzyme inhibitor (ACE inhibitor) that prevents this action is used as a pressor suppressant or a therapeutic agent for heart failure. ACE is an abbreviation for angiotensin-converting enzyme. Angiotensin is one of the peptides present in blood, and there are four types of angiotensin I to IV. Among these, angiotensin II-IV has the effect | action which contracts the smooth muscle of an arteriole and raises blood pressure, and the effect | action which accelerates | stimulates the secretion of aldosterone in adrenal cortex. Angiotensin II-IV has a particularly strong blood pressure raising action. Angiotensin I has no such physiological activity.

アンジオテンシン変換酵素(ACE)は、体内の血圧調整メカニズムの一つであるレニン-アンジオテンシン系において、アンジオテンシンIから昇圧作用を有するアンジオテンシンIIに変換させる働きを持つ。アンジオテンシンIIにより血圧を昇圧させることで体内の血圧を調整しているのである。アンジオテンシンIからアンジオテンシンIIへの変換を阻害する割合は、アンジオテンシン変換酵素(ACE)阻害活性率と呼ばれている。   Angiotensin converting enzyme (ACE) has a function of converting angiotensin I into angiotensin II having a pressor action in the renin-angiotensin system which is one of the blood pressure regulating mechanisms in the body. The blood pressure in the body is adjusted by increasing the blood pressure with angiotensin II. The ratio of inhibiting the conversion of angiotensin I to angiotensin II is called an angiotensin converting enzyme (ACE) inhibitory activity rate.

本願発明においては、アンジオテンシンIからアンジオテンシンIIへの変換を阻害する割合が50%の時、すなわちアンジオテンシン変換酵素(ACE)の阻害活性率50%時のコーヒー抽出液中の固形分濃度(IC50値)での評価を行う。   In the present invention, when the ratio of inhibiting the conversion of angiotensin I to angiotensin II is 50%, that is, the angiotensin converting enzyme (ACE) inhibitory activity rate is 50%, the solid content concentration in the coffee extract (IC50 value) Perform an evaluation at.

本発明における血圧上昇抑制作用は、後述する測定方法で血圧上昇作用を引き起こすアンジオテンシン変換酵素(ACE)の活性阻害率を測定することにより評価されるのが一般的である。通常は一定固形分濃度下で何%のACE阻害活性率を示すかで規定され、その濃度は低い方が望ましい。通常は試料間の比較を容易にするため、アンジオテンシン変換酵素(ACE)の阻害活性率50%における阻害活性を示す抽出液の固形分濃度(IC50値)で規定される。(参考文献:“食品機能性の科学”、食品機能性の科学編集員会編、株式会社産業技術サービスセンター発行)   In general, the antihypertensive action in the present invention is evaluated by measuring the activity inhibition rate of angiotensin converting enzyme (ACE) that causes the antihypertensive action by the measurement method described later. It is usually defined by what percentage of ACE inhibitory activity is exhibited at a constant solid content concentration, and the lower the concentration is desirable. Usually, in order to facilitate comparison between samples, it is defined by the solid content concentration (IC50 value) of the extract showing inhibitory activity at an inhibitory activity rate of 50% of angiotensin converting enzyme (ACE). (Reference: “Science of Food Functionality”, edited by Science Editor of Food Functionality, published by Industrial Technology Service Center Co., Ltd.)

アンジオテンシン変換酵素(ACE)の活性阻害率の測定について、今回行った測定例に基づいて説明をする。ACE阻害活性測定は株式会社同仁化学研究所製ACE−Kit WSTのキットを使用する。まず、コーヒー豆の抽出液を用いて希釈率の異なるもしくは抽出液濃度の異なる希釈サンプルを4つもしくは5つを用意する。具体的には、予め溶存成分濃度を測定しておいたコーヒー抽出液(ここでは45.3mg/mLとする。)を0.04μL(サンプルA1)、0.12μL(サンプルB1)、0.20μL(サンプルC1)、0.28μL(サンプルD1)とサンプル液量が異なる4つのサンプルを用意する。それぞれのサンプルに水(例えば、純水)を追加して、20μLとなるように希釈サンプルを作成する。また、希釈サンプルの標準サンプルとして、コーヒー抽出液を添加しない水20μL(サンプルE1)も用意する。それぞれの希釈サンプルA1〜E1(液量:20μL)に、キットに付属しているサブストレート バッファー20μLを入れて、その後、キット内付属のEnzyme Working溶液20μLを加えて、希釈サンプルの全量を60μL(=0.06mL)としてACEの活性阻害率の測定サンプルを準備する。その後、ACEの活性阻害率の測定サンプルを温度37℃、40分培養してアンジオテンシン変換酵素を生成させる。培養後、各ウェルにキット内付属のIndicator Working溶液200μLを加え、室温で、10分間静置させる。その後、吸光プレートリーダー マルチスキャンJX(Thermo Scientific)を使用し、450nmの吸光度によりアンジオテンシン変換酵素の活性を算出する。なお、吸光度の測定値から、アンジオテンシン変換酵素活性の阻害率を以下の算出式により求める。
阻害率(%)={(サンプルE1の吸光度)−(希釈サンプルの吸光度)/(サンプルE1の吸光度)}*100
各サンプルの阻害率を下記表1に示す。
The measurement of the activity inhibition rate of angiotensin converting enzyme (ACE) will be described based on the measurement example performed this time. ACE inhibitory activity is measured using a kit of ACE-Kit WST manufactured by Dojindo Laboratories. First, four or five diluted samples having different dilution rates or different concentrations of the extract are prepared using the coffee bean extract. Specifically, 0.04 μL (sample A1), 0.12 μL (sample B1), 0.20 μL of a coffee extract (here, 45.3 mg / mL) whose dissolved component concentration was measured in advance. (Sample C1) and four samples having different sample liquid amounts from 0.28 μL (sample D1) are prepared. Water (for example, pure water) is added to each sample, and a diluted sample is prepared so as to be 20 μL. Further, 20 μL of water (sample E1) to which no coffee extract is added is also prepared as a standard sample for the diluted sample. To each diluted sample A1 to E1 (liquid volume: 20 μL), 20 μL of the substrate buffer attached to the kit is added, and then 20 μL of Enzyme Working solution included in the kit is added, and the total volume of the diluted sample is reduced to 60 μL ( = 0.06 mL), a measurement sample of the ACE activity inhibition rate is prepared. Thereafter, a sample for measuring the ACE activity inhibition rate is cultured at 37 ° C. for 40 minutes to produce angiotensin converting enzyme. After the culture, 200 μL of the Indicator Working solution included in the kit is added to each well and allowed to stand at room temperature for 10 minutes. Thereafter, the activity of angiotensin converting enzyme is calculated from the absorbance at 450 nm using an absorbance plate reader Multiscan JX (Thermo Scientific). The inhibition rate of angiotensin converting enzyme activity is determined from the measured absorbance using the following formula.
Inhibition rate (%) = {(absorbance of sample E1) − (absorbance of diluted sample) / (absorbance of sample E1)} * 100
The inhibition rate of each sample is shown in Table 1 below.

Figure 2015002209
Figure 2015002209

表1で得られた結果を元に、横軸にサンプル液量、縦軸に阻害率(%)をプロットする(図1)。そのとき、阻害率の軸の阻害率50%の点から水平方向に直線を引いて、上記プロットとの交点として交点Xが得られる。この交点Xから、垂直方向に直線を引くと、サンプル液量軸との交点T1(0.182μL(0.182×10-3mL))が得られる。Based on the results obtained in Table 1, the amount of sample solution is plotted on the horizontal axis and the inhibition rate (%) is plotted on the vertical axis (FIG. 1). At that time, a straight line is drawn in the horizontal direction from the point of the inhibition rate 50% on the inhibition rate axis, and an intersection point X is obtained as an intersection with the plot. When a straight line is drawn from this intersection X in the vertical direction, an intersection T1 (0.182 μL (0.182 × 10 −3 mL)) with the sample liquid volume axis is obtained.

交点T1で得られたサンプル液量からアンジオテンシン変換酵素の阻害活性率50%でサンプルの固形分濃度(IC50値)は、下式により求める。
固形分濃度(IC50値)[mg/mL]
=(アンジオテンシン変換酵素の阻害活性率50%における抽出液量[mL])*溶存成分濃度[mg/mL]/測定サンプル全量(0.06[mL])
=(0.182×10-3mL)*(45.3mg/mL)/(0.06mL)
=0.137mg/mL
The solid content concentration (IC50 value) of the sample with an inhibitory activity rate of 50% of the angiotensin converting enzyme is obtained from the amount of the sample solution obtained at the intersection T1 by the following equation.
Solid content concentration (IC50 value) [mg / mL]
= (Extracted solution amount [mL] at 50% inhibitory activity of angiotensin converting enzyme) * Dissolved component concentration [mg / mL] / Measurement sample total amount (0.06 [mL])
= (0.182 × 10 −3 mL) * (45.3 mg / mL) / (0.06 mL)
= 0.137 mg / mL

このサンプルの測定結果は一例であり、サンプルの抽出量、溶存成分濃度等により、希釈サンプルの液量は変えることが必要である。アンジオテンシン変換酵素の阻害活性率50%におけるサンプル液量については、上記記載に従って得られる。   The measurement result of this sample is an example, and it is necessary to change the liquid volume of the diluted sample according to the sample extraction amount, dissolved component concentration, and the like. The amount of the sample solution at an angiotensin converting enzyme inhibitory activity rate of 50% is obtained according to the above description.

コーヒー豆の抽出液の有効成分の特徴として、アンジオテンシン変換酵素(ACE)の活性阻害率が50%の活性阻害を示す抽出液の固形分濃度(IC50値)が0.16mg/mL以下であることが挙げられる。この固形分濃度(IC50値)が0.16mg/mL以下であることで血圧上昇抑制の効果を最大限に発揮する。また、固形分濃度(IC50値)が0.16mg/mLを越えると、血圧上昇抑制の効力が発揮されにくくなり、好ましくない。   As a feature of the active ingredient of the coffee bean extract, the solid concentration (IC50 value) of the extract showing an activity inhibition rate of 50% of angiotensin converting enzyme (ACE) is 0.16 mg / mL or less. Is mentioned. When the solid content concentration (IC50 value) is 0.16 mg / mL or less, the effect of suppressing blood pressure increase is exhibited to the maximum. On the other hand, if the solid content concentration (IC50 value) exceeds 0.16 mg / mL, it is difficult to exert the effect of suppressing blood pressure rise, which is not preferable.

また、アンジオテンシン変換酵素(ACE)の活性阻害率が50%のとき、活性阻害を示す抽出液の固形分濃度(IC50値)は0.001mg/mLが測定限界などの理由により下限値となる。この下限値までは、血圧上昇抑制の効力を確認することができる。   Further, when the activity inhibition rate of angiotensin converting enzyme (ACE) is 50%, the solid content concentration (IC50 value) of the extract showing activity inhibition is 0.001 mg / mL, which is the lower limit due to the measurement limit. Up to this lower limit value, the effect of suppressing blood pressure increase can be confirmed.

従って、本発明の血圧上昇抑制剤において、ACE阻害に関するIC50値としては、0.157mg/mL以下のコーヒー豆の抽出液がより好ましく、0.150mg/mL以下のコーヒー豆の抽出液がさらに好ましい。   Therefore, in the blood pressure increase inhibitor of the present invention, the IC50 value relating to ACE inhibition is more preferably an extract of coffee beans of 0.157 mg / mL or less, and further preferably an extract of coffee beans of 0.150 mg / mL or less. .

このような優れた作用を発揮するコーヒー豆の抽出液は、後述の亜臨界処理工程を経て得ることができる。   The coffee bean extract exhibiting such excellent action can be obtained through a subcritical processing step described later.

本発明における糖化抑制作用は、後述する測定方法で老化現象を引き起こす還元糖化物質の生成を抑制する生成阻害率を測定し、糖化産物の生成阻害率が50%のときコーヒー豆の抽出液の固形分濃度(IC50値)で規定される。   The saccharification-inhibiting action in the present invention is determined by measuring the production inhibition rate that suppresses the production of a reduced saccharified substance that causes an aging phenomenon by the measurement method described later. When the production inhibition rate of the saccharification product is 50%, It is defined by the partial concentration (IC50 value).

糖化とは体内における老化メカニズムの一つであり、糖とタンパク質が結合し、糖化産物を生成する反応である。この糖化反応による糖化産物の生成を抑制する割合を糖化産物の生成阻害率と呼ぶ。本願発明においては、糖とタンパク質が結合する糖化産物の生成阻害割合が50%の時、すなわち糖化産物の生成阻害率50%時のコーヒー豆の抽出液の固形分濃度(IC50値)での評価を行う。   Saccharification is one of the aging mechanisms in the body, and is a reaction in which sugar and protein are combined to produce a saccharified product. The rate at which the production of a saccharified product due to this saccharification reaction is suppressed is referred to as the saccharified product production inhibition rate. In the present invention, when the production inhibition rate of the saccharification product in which sugar and protein bind is 50%, that is, when the production inhibition rate of the saccharification product is 50%, the evaluation is performed with the solid content concentration (IC50 value) of the coffee bean I do.

本発明における糖化抑制作用は、後述する測定方法で老化現象を引き起こす還元糖化物質の生成を抑制する生成阻害率を測定することにより評価されるのが一般的である。通常は一定固形分濃度下で何%の生成阻害率を示すかで規定され、その濃度は低い方が望ましい。通常は試料間の比較を容易にする糖化産物の生成阻害率50%での抽出液の固形分濃度(IC50値)により規定されることが薬学研究においては広く一般的に用いられている。(参考文献:M. Hori et al., Anti-Aging Medicine, 9 (2012) 135.)   The saccharification-inhibiting action in the present invention is generally evaluated by measuring the production inhibition rate that suppresses the production of a reduced saccharified substance that causes an aging phenomenon by a measurement method described later. Usually, it is defined by what percentage of production inhibition is shown at a constant solid content concentration, and the lower the concentration is desirable. It is generally and widely used in pharmaceutical research that it is defined by the solid content concentration (IC50 value) of the extract at a glycation product production inhibition rate of 50%, which facilitates comparison between samples. (Reference: M. Hori et al., Anti-Aging Medicine, 9 (2012) 135.)

糖化抑制作用を評価するための糖化産物の生成阻害率測定について、今回行った測定例に基づいて説明をする。まず、コーヒー豆の抽出液を用いて希釈率の異なるもしくは抽出液濃度の異なる希釈サンプルを4つもしくは5つを用意する。具体的には、予め溶存成分濃度を測定しておいたコーヒー豆の抽出液(ここでは27.2mg/mLとする。)を1.33μL(サンプルA2)、2.00μL(サンプルB2)、2.67μL(サンプルC2)、4.00μL(サンプルD2)、5.33μL(サンプルE2)とサンプル液量が異なる5つのサンプルを用意する。それぞれのサンプルに水(例えば、純水)を追加して、100μLとなるように希釈サンプルを作成する。また、希釈サンプルの標準サンプルとして、コーヒー抽出液を添加しない水100μL(サンプルF2)も作成する。それぞれの希釈サンプル(100μL)に、10mg/mL牛血清アルブミン(BSA)200μL、0.1Mリン酸緩衝液500μL、蒸留水100μL及び2Mフルクトース100μLを混合し、全量1mLに調整した測定サンプルを準備する。その後、Infinite F200-マイクロプレートリーダー(TECMAN)を使用し、励起波長370nm、蛍光波長440nmで還元糖化物質由来の蛍光を測定し、希釈サンプルにおける還元糖物質の蛍光強度を測定する。その後、以下の算出式により、希釈率サンプルにおける還元糖物質の生成阻害率を求める。   The production inhibition rate measurement of a saccharification product for evaluating the saccharification inhibiting action will be described based on the measurement example performed this time. First, four or five diluted samples having different dilution rates or different concentrations of the extract are prepared using the coffee bean extract. Specifically, the coffee bean extract (here 27.2 mg / mL) whose dissolved component concentration has been measured in advance is 1.33 μL (sample A2), 2.00 μL (sample B2), 2 Five samples having different sample liquid amounts are prepared: .67 μL (sample C2), 4.00 μL (sample D2), and 5.33 μL (sample E2). Water (for example, pure water) is added to each sample, and a diluted sample is prepared so as to be 100 μL. In addition, 100 μL of water (sample F2) to which no coffee extract is added is also prepared as a standard sample for the diluted sample. Each diluted sample (100 μL) is mixed with 200 μL of 10 mg / mL bovine serum albumin (BSA), 500 μL of 0.1M phosphate buffer, 100 μL of distilled water and 100 μL of 2M fructose, and a measurement sample adjusted to a total volume of 1 mL is prepared. . Thereafter, using an Infinite F200-microplate reader (TECMAN), the fluorescence derived from the reduced saccharified substance is measured at an excitation wavelength of 370 nm and a fluorescence wavelength of 440 nm, and the fluorescence intensity of the reduced sugar substance in the diluted sample is measured. Then, the production | generation inhibition rate of the reducing sugar substance in a dilution rate sample is calculated | required with the following calculation formulas.

阻害率(%)={(サンプルF2の蛍光強度)−(希釈サンプルの蛍光強度)/(サンプルF2の蛍光強度)}*100 Inhibition rate (%) = {(fluorescence intensity of sample F2) − (fluorescence intensity of diluted sample) / (fluorescence intensity of sample F2)} * 100

各希釈サンプルの阻害率を下記の表2に示す。   The inhibition rate of each diluted sample is shown in Table 2 below.

Figure 2015002209
Figure 2015002209

表2で得られた結果を元に、横軸にサンプル液量、縦軸に阻害率(%)をプロットする(図2)。そのとき、阻害率の軸から阻害率50%から水平方向に直線を引いて、検量線との交点として交点Yが得られる。この交点Yから、垂直方向に直線を引くと、サンプル液量軸との交点T2(2.64μL(2.64×10-3mL))が得られる。Based on the results obtained in Table 2, the amount of sample solution is plotted on the horizontal axis, and the inhibition rate (%) is plotted on the vertical axis (FIG. 2). At that time, a straight line is drawn in the horizontal direction from the inhibition rate 50% from the inhibition rate axis, and an intersection point Y is obtained as an intersection with the calibration curve. When a straight line is drawn from the intersection Y in the vertical direction, an intersection T2 (2.64 μL (2.64 × 10 −3 mL)) with the sample liquid volume axis is obtained.

交点T2で得られた抽出液量から糖化産物の生成阻害率50%でサンプルの固形分濃度(IC50値)は、下式により求める。
固形分濃度(IC50値)[mg/mL]
=(糖化産物の生成阻害率50%における抽出液量[mL])*溶液成分濃度[mg/mL]/測定サンプル全量(1[mL])
=(2.64×10-3mL)*(27.2mg/mL)/(1mL)
=0.0718mg/mL
From the amount of the extract obtained at the intersection T2, the solid content concentration (IC50 value) of the sample at a glycation product production inhibition rate of 50% is obtained by the following equation.
Solid content concentration (IC50 value) [mg / mL]
= (Extracted solution volume [mL] at 50% inhibition rate of glycation product production) * Solution component concentration [mg / mL] / Measurement sample total volume (1 [mL])
= (2.64 × 10 −3 mL) * (27.2 mg / mL) / (1 mL)
= 0.0718 mg / mL

このサンプルの測定結果は一例であり、サンプルの抽出量、溶存成分濃度等により、希釈サンプルの液量を変えることが必要である。糖化産物の生成阻害率50%におけるサンプル液量については、上記記載に従って得られる。   The measurement result of this sample is an example, and it is necessary to change the liquid volume of the diluted sample according to the sample extraction amount, dissolved component concentration, and the like. The amount of sample solution at a glycation product production inhibition rate of 50% is obtained according to the above description.

コーヒー豆の抽出液の有効成分の特徴として、糖化産物の生成阻害率が50%におけるコーヒー抽出液の固形分濃度(IC50値)が0.06mg/mL以下であることが挙げられる。この固形分濃度(IC50値)を0.06mg/mL以下にすることで糖化抑制の降下を最大限に発揮することができるのである。   A feature of the active ingredient of the coffee bean extract is that the solid concentration (IC50 value) of the coffee extract at a glycation product production inhibition rate of 50% is 0.06 mg / mL or less. By making this solid content concentration (IC50 value) 0.06 mg / mL or less, the decrease in saccharification inhibition can be exhibited to the maximum.

糖化産物の生成阻害率50%におけるコーヒー抽出液の固形分濃度(IC50値)は0.001mg/mLが測定限界などの理由により下限値となる。この下限値までは、糖化抑制の効力を確認することができる。   The solid content concentration (IC50 value) of the coffee extract at a glycation product production inhibition rate of 50% is 0.001 mg / mL, which is the lower limit for reasons such as the measurement limit. Up to this lower limit, the efficacy of saccharification inhibition can be confirmed.

また、固形分濃度(IC50値)が0.06mg/mLを越えると糖化抑制の効力が発揮されにくくなり、好ましくない。   On the other hand, if the solid content concentration (IC50 value) exceeds 0.06 mg / mL, the effect of inhibiting saccharification becomes difficult to be exhibited, which is not preferable.

従って、本発明の糖化抑制剤において、糖化産物の生成阻害に関するIC50値としては、0.0597mg/mL以下のコーヒー豆の抽出液がより好ましく、0.0590mg/mL以下のコーヒー豆の抽出液がさらに好ましい。   Therefore, in the saccharification inhibitor of the present invention, the IC50 value relating to the inhibition of saccharification product production is more preferably an extract of coffee beans of 0.0597 mg / mL or less, and an extract of coffee beans of 0.0590 mg / mL or less. Further preferred.

このような優れた作用を発揮するコーヒー豆の抽出液は、後述の亜臨界処理工程を経て得ることができる。また、本発明の血圧上昇抑制剤及び糖化抑制剤は、種々の動物に対して有効であり、哺乳動物(例えばヒト、サル、ウマ、ウシ、ヤギ、ヒツジ、イヌ、ネコ、ネズミ等)に対してより有効に作用する。   The coffee bean extract exhibiting such excellent action can be obtained through a subcritical processing step described later. In addition, the blood pressure elevation inhibitor and glycation inhibitor of the present invention are effective for various animals, and for mammals (eg, humans, monkeys, horses, cows, goats, sheep, dogs, cats, mice, etc.). It works more effectively.

次に、本発明の製造方法について説明する。
本発明の製造方法の一つの態様としては、
コーヒー生豆及び/又は焙煎コーヒー豆を粉砕してコーヒー豆の粉砕物を得る工程、
コーヒー豆の粉砕物に水を加えてコーヒー豆の粉砕物と水との混合物を得る工程、及び
コーヒー豆の粉砕物と水との混合物を、温度:140〜200℃、圧力:0.15〜1.8MPa、かつ、各温度の飽和蒸気圧以上の圧力の亜臨界条件で処理して、コーヒー豆の抽出液を回収する工程
を含む、血圧上昇抑制作用を有するコーヒー豆の抽出液の製造方法である。
Next, the manufacturing method of this invention is demonstrated.
As one aspect of the production method of the present invention,
Crushing green coffee beans and / or roasted coffee beans to obtain a coffee beans pulverized product,
A step of adding water to the ground coffee beans to obtain a mixture of the ground coffee beans and water, and a mixture of the ground coffee beans and water, temperature: 140 to 200 ° C., pressure: 0.15 to A method for producing a coffee bean extract having a blood pressure increase-inhibiting action, comprising a step of recovering the coffee bean extract by treating it under a subcritical condition of 1.8 MPa and a pressure equal to or higher than a saturated vapor pressure at each temperature. It is.

本発明の製造方法の別の態様としては、
コーヒー生豆及び/又は焙煎コーヒー豆を粉砕してコーヒー豆の粉砕物を得る工程、
コーヒー豆の粉砕物に水を加えてコーヒー豆の粉砕物と水との混合物を得る工程、及び
コーヒー豆の粉砕物と水との混合物を、温度:140〜200℃、圧力:0.15〜1.8MPa、かつ、各温度の飽和蒸気圧以上の圧力の亜臨界条件で処理して、コーヒー豆の抽出液を回収する工程
を含む、糖化抑制作用を有するコーヒー豆の抽出液の製造方法である。
As another aspect of the production method of the present invention,
Crushing green coffee beans and / or roasted coffee beans to obtain a coffee beans pulverized product,
A step of adding water to the ground coffee beans to obtain a mixture of the ground coffee beans and water, and a mixture of the ground coffee beans and water, temperature: 140 to 200 ° C., pressure: 0.15 to A method for producing a coffee bean extract having a saccharification-inhibiting action, comprising a step of recovering the coffee bean extract by treating it at 1.8 MPa and a subcritical condition at a pressure equal to or higher than a saturated vapor pressure at each temperature. is there.

本発明における、水による亜臨界処理、即ち加水分解反応に用いる水は、高温の水処理であれば液体状態でも気体状態でも利用することができる。温度は望ましくは100℃以上であり、望まれる反応場としては気体よりも液体状態の方が反応は進みやすいので、密閉に近い容器で強制的に液体の状態にしたいわゆる亜臨界の状態の水の使用が好ましい。   In the present invention, the water used for the subcritical treatment with water, that is, the hydrolysis reaction, can be used in a liquid state or a gas state as long as it is a high-temperature water treatment. The temperature is desirably 100 ° C. or higher, and the desired reaction field is that the reaction proceeds more easily in the liquid state than in the gas. Therefore, water in a so-called subcritical state in which the liquid state is forcibly forced in a container close to a sealed state. Is preferred.

亜臨界処理とは、所定温度及び圧力の条件下で亜臨界状態にした亜臨界流体と抽出原料とを接触させることにより、抽出原料から所定の成分を抽出するものである。例えば、水は、圧力22.12MPa、温度374.15℃まで上げると液体でも気体でもない状態を示す。この点を水の臨界点といい、臨界点より低い温度・圧力の熱水を亜臨界水という。亜臨界水は、誘電率低下とイオン積の向上により、優れた成分抽出作用と加水分解作用を有する。   The subcritical process is to extract a predetermined component from an extraction raw material by bringing a subcritical fluid brought into a subcritical state under a predetermined temperature and pressure into contact with the extraction raw material. For example, when water is raised to a pressure of 22.12 MPa and a temperature of 374.15 ° C., it shows a state where it is neither liquid nor gas. This point is called the critical point of water, and hot water at a temperature and pressure lower than the critical point is called subcritical water. Subcritical water has an excellent component extraction action and hydrolysis action due to a decrease in dielectric constant and an improvement in ionic product.

より具体的には金属やセラミックスなどの耐圧容器にコーヒー豆と抽出剤である水を入れて、密閉状態に近い状態にし、亜臨界状態(コーヒー豆の粉砕物と水との混合物を、温度:140〜200℃、圧力:0.15〜1.8MPa、かつ、各温度の飽和蒸気圧以上の圧力)を一定時間維持することによってコーヒー豆の抽出液を得、これを亜臨界処理抽出液としている。   More specifically, coffee beans and water as an extractant are placed in a pressure vessel such as metal or ceramics to bring them into a closed state, and a subcritical state (a mixture of ground coffee beans and water, temperature: 140 to 200 ° C., pressure: 0.15 to 1.8 MPa, and a pressure equal to or higher than the saturated vapor pressure at each temperature) for a certain period of time to obtain a coffee bean extract, which is used as a subcritical processing extract Yes.

亜臨界処理に用いる抽出剤は、水以外に、例えばエチレン、エタン、プロパン、二酸化炭素、メタノール、エタノール、及びそれらの混合物が挙げられる。これらの中で、安全性の観点から水を用いるのが最も好ましい。   Examples of the extraction agent used for the subcritical treatment include, in addition to water, ethylene, ethane, propane, carbon dioxide, methanol, ethanol, and mixtures thereof. Among these, it is most preferable to use water from the viewpoint of safety.

コーヒー豆の亜臨界処理を行うための温度は、140〜200℃の間で行うことが望ましく、140〜180℃の間で行うことがより望ましい。この温度範囲にすることにより、ポリフェノールに代表される溶存成分が生成しやすくなるからである。亜臨界処理の温度が140℃未満では、溶存成分を生成させることが難しいことがある。亜臨界処理の温度が200℃を越えると、コーヒー豆中の溶存成分が分解を起こしてしまい、溶存成分の生成量を減少させてしまうのである。   The temperature for carrying out the subcritical treatment of coffee beans is preferably between 140 and 200 ° C, more preferably between 140 and 180 ° C. It is because dissolved components represented by polyphenol are easily generated by adjusting to this temperature range. If the temperature of the subcritical treatment is less than 140 ° C., it may be difficult to generate dissolved components. If the temperature of the subcritical treatment exceeds 200 ° C., the dissolved components in the coffee beans are decomposed, and the amount of dissolved components generated is reduced.

コーヒー豆の亜臨界処理における処理圧力は、圧力:0.15〜1.8MPa、かつ、各温度の飽和蒸気圧以上の圧力で行うことである。また、各温度の飽和蒸気圧以上とは、その一例としては、160℃のときには、0.62MPa以上、200℃以上の時には1.55MPa以上で行うことである。この圧力にすることにより、ポリフェノールに代表される溶存成分を生成しやすくなるからである。亜臨界の処理圧力は、各温度の飽和蒸気圧未満では、溶存成分を生成させることが難しいし、処理時間を要することもある。亜臨界の処理の圧力の上限は特に上記に限定されるものではないが、高圧装置の仕様上、20〜30MPaあたりに抑えることが望ましい。   The processing pressure in the subcritical processing of coffee beans is that pressure: 0.15 to 1.8 MPa and a pressure equal to or higher than the saturated vapor pressure at each temperature. For example, “at or above the saturated vapor pressure at each temperature” means that at 160 ° C., 0.62 MPa or more, and at 200 ° C. or more, 1.55 MPa or more. This is because by using this pressure, dissolved components typified by polyphenol are easily generated. If the subcritical processing pressure is less than the saturated vapor pressure at each temperature, it is difficult to generate dissolved components, and processing time may be required. The upper limit of the subcritical processing pressure is not particularly limited to the above, but it is desirable to suppress the pressure to around 20 to 30 MPa due to the specifications of the high pressure apparatus.

コーヒーの亜臨界の処理時間は、3〜30分の間で行うことが望ましい。亜臨界の処理時間が3分未満では、ポリフェノールに代表される溶存成分を生成させることが難しい。亜臨界の処理時間が30分を越えると、コーヒー豆中のポリフェノールが分解を起こしてしまい、ポリフェノールの生成量を減少させてしまうのである。   The subcritical processing time of the coffee is preferably between 3 and 30 minutes. If the subcritical processing time is less than 3 minutes, it is difficult to produce dissolved components typified by polyphenols. If the subcritical processing time exceeds 30 minutes, the polyphenol in the coffee beans will be decomposed, and the amount of polyphenol produced will be reduced.

焙煎コーヒー豆の亜臨界の処理時間は、3〜15分の間で行うことがより望ましく、3〜9分の間で行うことがさらに望ましい。この処理時間の範囲にすることにより、ポリフェノールに代表される溶存成分を生成しやすくなるからである。亜臨界の処理時間が15分を越えると、コーヒー豆中のポリフェノールが分解を起こしてしまい、ポリフェノールの生成量を減少させてしまう傾向がある。   The subcritical processing time of roasted coffee beans is more preferably 3 to 15 minutes, and more preferably 3 to 9 minutes. It is because it becomes easy to produce | generate the dissolved component represented by polyphenol by setting it as the range of this processing time. If the subcritical processing time exceeds 15 minutes, the polyphenols in the coffee beans tend to be decomposed and the production amount of polyphenols tends to be reduced.

コーヒー生豆の亜臨界の処理時間は、10〜30分の間で行うことがより望ましい。この処理時間の範囲にすることにより、ポリフェノールに代表される溶存成分を生成しやすくなるからである。   The subcritical processing time of green coffee beans is more preferably between 10 and 30 minutes. It is because it becomes easy to produce | generate the dissolved component represented by polyphenol by setting it as the range of this processing time.

このとき、コーヒー豆の抽出液を亜臨界処理による加水分解条件の一例としては、温度:140〜200℃、圧力:0.15〜1.8MPa、かつ、各温度の飽和蒸気圧以上の圧力、処理時間は、3〜30分、より好ましくは3〜15分で行うことが望ましいのである。この条件で行うことで、ポリフェノールに代表される溶存成分を生成しやすくなるからである。   At this time, as an example of hydrolysis conditions by subcritical processing of the coffee bean extract, temperature: 140-200 ° C., pressure: 0.15-1.8 MPa, and a pressure equal to or higher than the saturated vapor pressure at each temperature, The treatment time is desirably 3 to 30 minutes, more preferably 3 to 15 minutes. It is because it becomes easy to produce | generate the dissolved component represented by polyphenol by performing on these conditions.

亜臨界処理を行った後、耐圧容器内の圧力を大気圧まで戻し、亜臨界処理物、容器洗浄水を回収する。この2つの回収物に蒸留水を加えて、全量を調整し、コーヒー豆:蒸留水が一定比率になるように調整液を得る。この調整液をろ過し、得られたろ過液を亜臨界処理液とすることができる。   After performing the subcritical process, the pressure in the pressure vessel is returned to atmospheric pressure, and the subcritical process product and the container washing water are recovered. Distilled water is added to the two recovered materials to adjust the total amount, and a prepared liquid is obtained so that the ratio of coffee beans: distilled water becomes a constant ratio. This adjustment liquid can be filtered and the obtained filtrate can be used as a subcritical processing liquid.

このとき、調整液のろ過は、一般的な方法により行うことができ、具体的には、ろ紙やコーヒーフィルターを用いた濾過、デカンテーション、スクリュープレス、ローラープレス、ロータリードラムスクリーン、ベルトスクリーン、振動スクリーン、多重板振動フィルター、真空脱水、加圧脱水、ベルトプレス、遠心濃縮脱水、多重円板脱水のいずれかで行うことができるのである。   At this time, filtration of the adjustment liquid can be performed by a general method. Specifically, filtration using a filter paper or a coffee filter, decantation, screw press, roller press, rotary drum screen, belt screen, vibration, It can be performed by any one of a screen, a multi-plate vibration filter, vacuum dehydration, pressure dehydration, belt press, centrifugal concentration dehydration, and multi-disc dehydration.

本発明に用いられるコーヒー豆は、生豆でもよく、焙煎した豆でも良い。
焙煎コーヒー豆は、コーヒーの生豆を適切な焙煎度まで焙煎したものである。コーヒー生豆の焙煎は、直火式、熱風式、又は半熱風式などの方法によって行うことができる。焙煎方法、温度、及び時間は、所望の焙煎度を達成するために適宜選択してよい。
The coffee beans used in the present invention may be raw beans or roasted beans.
Roasted coffee beans are roasted coffee beans to an appropriate roasting degree. The coffee beans can be roasted by a method such as a direct fire method, a hot air method, or a semi-hot air method. The roasting method, temperature, and time may be appropriately selected in order to achieve a desired roasting degree.

コーヒー生豆の種類は、特に限定されないが、アラビカ種及びロブスタ種の何れであってもよい。より詳細には、ブラジル、コロンビア、ベトナム、タンザニア及びモカから選択されるコーヒー豆であってよいがこれらに限定されない。コーヒー豆は1種を単独で用いてもよく、或いは複数種をブレンドして用いてもよい。   The type of green coffee beans is not particularly limited, and may be any of Arabica and Robusta. More specifically, the coffee beans may be selected from, but not limited to, Brazil, Colombia, Vietnam, Tanzania and Mocha. One kind of coffee beans may be used alone, or a plurality of kinds may be blended and used.

焙煎コーヒー豆の粉砕は、ロールグラインダータイプやフラットカッタータイプおよびコニカルカッタータイプなどいずれのタイプの粉砕機を用いて実施することができる。ここで、血圧上昇抑制には、生豆、焙煎した豆のどちらを用いてもよいが、生豆を用いることがより望ましい。糖化抑制には、生豆、焙煎した豆のどちらを用いてもよいが、焙煎した豆を用いることがより望ましい。   The pulverization of roasted coffee beans can be carried out using any type of pulverizer such as a roll grinder type, a flat cutter type, or a conical cutter type. Here, for suppressing blood pressure increase, either raw beans or roasted beans may be used, but it is more preferable to use raw beans. For suppressing saccharification, either raw beans or roasted beans may be used, but it is more preferable to use roasted beans.

以下、本発明を実施例に基づいて説明するが、本発明はこれらの実施例等により何ら制限されるものではない。   EXAMPLES Hereinafter, although this invention is demonstrated based on an Example, this invention is not restrict | limited at all by these Examples.

実施例1
容積2Lの耐圧容器に、焙煎され粉砕されたコーヒー豆100g、蒸留水500gを入れて、処理温度:140℃、処理圧力:0.36MPa、処理時間3分間で亜臨界処理を行った。
Example 1
100 g of roasted and crushed coffee beans and 500 g of distilled water were placed in a pressure-resistant container having a volume of 2 L, and subcritical processing was performed at a processing temperature of 140 ° C., a processing pressure of 0.36 MPa, and a processing time of 3 minutes.

亜臨界処理を行った後、耐圧容器内の圧力を大気圧まで戻し、亜臨界処理物、容器洗浄水を回収した。この回収物に蒸留水を加えて、全量を900gに調整し、コーヒー豆:蒸留水=1:8(質量比)となるように調整液を得た。この調整液をコーヒーフィルター(市販)でろ過し、得られたろ過液を亜臨界処理液とした。   After performing the subcritical treatment, the pressure in the pressure vessel was returned to atmospheric pressure, and the subcritical treatment product and the vessel washing water were collected. Distilled water was added to this recovered material to adjust the total amount to 900 g, and an adjusted liquid was obtained so that coffee beans: distilled water = 1: 8 (mass ratio). This adjustment liquid was filtered with a coffee filter (commercially available), and the obtained filtrate was used as a subcritical treatment liquid.

実施例2
亜臨界処理を処理温度:140℃、処理圧力:0.36MPa、処理時間9分で行った以外は、実施例1と同じ条件で亜臨界処理による亜臨界処理液を作製した。
Example 2
A subcritical processing solution by subcritical processing was prepared under the same conditions as in Example 1 except that the subcritical processing was performed at a processing temperature of 140 ° C., a processing pressure of 0.36 MPa, and a processing time of 9 minutes.

実施例3
亜臨界処理を処理温度:160℃、処理圧力:0.62MPa、処理時間6分で行った以外は、実施例1と同じ条件で亜臨界処理による亜臨界処理液を作製した。
Example 3
A subcritical processing solution by subcritical processing was prepared under the same conditions as in Example 1 except that the subcritical processing was performed at a processing temperature of 160 ° C., a processing pressure of 0.62 MPa, and a processing time of 6 minutes.

実施例4
亜臨界処理を処理温度:180℃、処理圧力:1.0MPa、処理時間3分で行った以外は、実施例1と同じ条件で亜臨界処理による亜臨界処理液を作製した。
Example 4
A subcritical processing solution by subcritical processing was prepared under the same conditions as in Example 1 except that the subcritical processing was performed at a processing temperature of 180 ° C., a processing pressure of 1.0 MPa, and a processing time of 3 minutes.

実施例5
亜臨界処理を処理温度:180℃、処理圧力:1.0MPa、処理時間9分で行った以外は、実施例1と同じ条件で亜臨界処理による亜臨界処理液を作製した。
Example 5
A subcritical processing solution by subcritical processing was prepared under the same conditions as in Example 1 except that the subcritical processing was performed at a processing temperature of 180 ° C., a processing pressure of 1.0 MPa, and a processing time of 9 minutes.

実施例6
容積2Lの耐圧容器に、粉砕されたコーヒー生豆100g、蒸留水500gを入れて、処理温度:165℃、処理圧力:0.70MPa、処理時間30分間で亜臨界処理を行った。
Example 6
In a pressure-resistant container having a volume of 2 L, 100 g of crushed green coffee beans and 500 g of distilled water were placed, and subcritical processing was performed at a processing temperature of 165 ° C., a processing pressure of 0.70 MPa, and a processing time of 30 minutes.

亜臨界処理を行った後、耐圧容器内の圧力を大気圧まで戻し、亜臨界処理物、容器洗浄水を回収した。この回収物に蒸留水を加えて、全量を900gに調整し、コーヒー豆:蒸留水=1:8(質量比)となるように調整液を得た。この調整液をコーヒーフィルター(市販)でろ過し、得られたろ過液を亜臨界処理液とした。   After performing the subcritical treatment, the pressure in the pressure vessel was returned to atmospheric pressure, and the subcritical treatment product and the vessel washing water were collected. Distilled water was added to this recovered material to adjust the total amount to 900 g, and an adjusted liquid was obtained so that coffee beans: distilled water = 1: 8 (mass ratio). This adjustment liquid was filtered with a coffee filter (commercially available), and the obtained filtrate was used as a subcritical treatment liquid.

実施例7
亜臨界処理を処理温度:180℃、処理圧力:1.0MPa、処理時間20分で行った以外は、実施例6と同じ条件で亜臨界処理による亜臨界処理液を作製した。
Example 7
A subcritical processing solution by subcritical processing was prepared under the same conditions as in Example 6 except that the subcritical processing was performed at a processing temperature of 180 ° C., a processing pressure of 1.0 MPa, and a processing time of 20 minutes.

実施例8
亜臨界処理を処理温度:195℃、処理圧力:1.4MPa、処理時間10分で行った以外は、実施例6と同じ条件で亜臨界処理による亜臨界処理液を作製した。
Example 8
A subcritical processing solution by subcritical processing was prepared under the same conditions as in Example 6 except that the subcritical processing was performed at a processing temperature of 195 ° C., a processing pressure of 1.4 MPa, and a processing time of 10 minutes.

実施例9
亜臨界処理を処理温度:195℃、処理圧力:1.4MPa、処理時間30分で行った以外は、実施例6と同じ条件で亜臨界処理による亜臨界処理液を作製した。
Example 9
A subcritical processing solution by subcritical processing was prepared under the same conditions as in Example 6 except that the subcritical processing was performed at a processing temperature of 195 ° C., a processing pressure of 1.4 MPa, and a processing time of 30 minutes.

比較例1 熱水抽出
焙煎され粉砕されたコーヒー豆40g、蒸留水320g(コーヒー豆:蒸留水=1:8(質量比))を用意し、市販のコーヒーメーカー(TOPVALUE社製、品番:TCO-CA6)を用いて熱水抽出を行った。コーヒーメーカーにコーヒー豆、蒸留水をセットし、95℃に加熱された蒸留水を、コーヒー用ペーパーフィルター上のコーヒー豆に滴下した。このフィルターを通過したコーヒー豆の抽出液を回収することで熱水抽出液を得た。
Comparative Example 1 Hot Water Extraction 40 g of roasted and ground coffee beans and 320 g of distilled water (coffee beans: distilled water = 1: 8 (mass ratio)) were prepared, and a commercial coffee maker (TOPVALUE, product number: TCO) was prepared. -Hot water extraction was performed using -CA6). Coffee beans and distilled water were set in a coffee maker, and distilled water heated to 95 ° C. was dropped onto the coffee beans on the paper filter for coffee. The hot water extract was obtained by recovering the coffee bean extract that passed through this filter.

比較例2 熱水抽出
粉砕されたコーヒー生豆20g、蒸留水160g(コーヒー生豆:蒸留水=1:8(質量比))を用意し、市販のコーヒーメーカー(TOPVALUE社製、品番:TCO-CA6)を用いて熱水抽出を行った。コーヒーメーカーにコーヒー豆、蒸留水をセットし、95℃に加熱された蒸留水を、コーヒー用ペーパーフィルター上のコーヒー豆に滴下した。このフィルターを通過したコーヒー豆の抽出液を回収することで熱水抽出液を得た。
Comparative Example 2 Extraction with hot water 20 g of ground coffee beans and 160 g of distilled water (raw coffee beans: distilled water = 1: 8 (mass ratio)) were prepared, and a commercial coffee maker (manufactured by TOPVALUE, product number: TCO- Hot water extraction was performed using CA6). Coffee beans and distilled water were set in a coffee maker, and distilled water heated to 95 ° C. was dropped onto the coffee beans on the paper filter for coffee. The hot water extract was obtained by recovering the coffee bean extract that passed through this filter.

物性値特定結果
実施例1〜9および比較例1、2で得られた、亜臨界処理液、熱水抽出液の2種類の抽出液の物性値を測定した。測定した物性値は、ポリフェノール濃度、溶存成分濃度であった。
Physical property value identification results The physical property values of two types of extraction liquids obtained in Examples 1 to 9 and Comparative Examples 1 and 2 were measured. The measured physical property values were polyphenol concentration and dissolved component concentration.

ポリフェノール濃度は、Folin-Ciocalteu法により測定した。
蒸留水により2倍に希釈したFolin-Ciocalteu液(MP Biomedicals, Inc.)1mL、各抽出液1mL、0.4M炭酸ナトリウム水溶液5mLを混合した。混合液を50℃で5分間保持した後、30分間、常温の水で冷却した。その後、各混合液の765nmにおける吸光度を測定した。このとき、クロロゲン酸水溶液により検量線を作成し、ポリフェノール量をクロロゲン酸相当量(mg/mL)で算出した。その結果を図3−1及び図3−2に示す。
The polyphenol concentration was measured by the Folin-Ciocalteu method.
1 mL of Folin-Ciocalteu solution (MP Biomedicals, Inc.) diluted twice with distilled water, 1 mL of each extract, and 5 mL of 0.4 M aqueous sodium carbonate solution were mixed. The mixture was held at 50 ° C. for 5 minutes and then cooled with room temperature water for 30 minutes. Then, the light absorbency in 765 nm of each liquid mixture was measured. At this time, a calibration curve was prepared using a chlorogenic acid aqueous solution, and the amount of polyphenol was calculated as the amount corresponding to chlorogenic acid (mg / mL). The results are shown in FIGS. 3-1 and 3-2.

溶存成分濃度は加熱乾燥法により測定した。
各抽出液10mLをセラミックス容器に分注し、乾燥機中105℃で一晩乾燥させ、抽出液中の水分を蒸発させて容器の乾燥前後の重量差より各抽出液の溶存成分濃度(mg/mL)を算出した。その結果を図3−1及び図3−2に示す。
The dissolved component concentration was measured by a heat drying method.
Dispense 10 mL of each extract into a ceramic container, dry in a dryer at 105 ° C. overnight, evaporate the water in the extract, and determine the concentration of dissolved components (mg / mg) from the weight difference before and after drying the container. mL) was calculated. The results are shown in FIGS. 3-1 and 3-2.

図3−1及び図3−2から、熱水抽出処理よりも、亜臨界処理の方が、コーヒー豆からの溶存成分やポリフェノールをより多く抽出できることが分かった。   FIG. 3-1 and FIG. 3-2 show that the subcritical process can extract more dissolved components and polyphenols from coffee beans than the hot water extraction process.

ACE阻害活性試験
亜臨界処理液、熱水抽出液の2種類の抽出液のACE阻害活性試験を行った。
ACE inhibitory activity test The ACE inhibitory activity test of two types of extracts, a subcritical processing solution and a hot water extract, was performed.

1.サンプル準備
亜臨界処理液、熱水抽出液の2種類の抽出液を用意した。その用意した抽出液を表3に示す。
1. Sample preparation Two types of extracts were prepared: a subcritical processing solution and a hot water extract. The prepared extract is shown in Table 3.

Figure 2015002209
Figure 2015002209

2.ACE阻害活性測定
ACE阻害活性測定は株式会社同仁化学研究所製ACE−Kit WSTを使用して実施した。
上記の1で準備した亜臨界処理液、熱水抽出液のそれぞれ試験サンプルの所定量(以下、抽出液量と呼ぶ)を採取し、蒸留水により適宜希釈した。希釈率は測定した阻害率の値が50%を挟むように設定し、各サンプルにつき4点もしくは5点の希釈溶液を作製した。マイクロプレートの各ウェルに各希釈サンプル溶液20μL、キット内付属のサブストレート バッファー20μLを入れて、その後、キット内付属のEnzyme Working溶液20μLを加えて全量を60μL(=0.06mL)とし、温度37℃、40分培養してアンジオテンシン変換酵素を生成させた。培養後、各ウェルにキット内付属のIndicator Working溶液200μLを加え、室温で、10分間静置させた。その後、吸光プレートリーダー マルチスキャンJX(Thermo Scientific)を使用し、450nmの吸光度でアンジオテンシン変換酵素の阻害活性を測定し、各希釈率における阻害率を求めた。また、得られた阻害率の挙動から、アンジオテンシン変換酵素の阻害活性率50%における抽出液量を求めた。また、下記の式を用いて、得られた抽出液量からアンジオテンシン変換酵素の阻害活性率50%である各サンプルの固形分濃度(IC50値)を求めた。
2. Measurement of ACE inhibitory activity ACE inhibitory activity was measured using ACE-Kit WST manufactured by Dojindo Laboratories.
Predetermined amounts (hereinafter referred to as extract amounts) of test samples of the subcritical treatment liquid and hot water extract prepared in 1 above were collected and appropriately diluted with distilled water. The dilution rate was set so that the measured inhibition rate was 50%, and 4 or 5 dilution solutions were prepared for each sample. 20 μL of each diluted sample solution and 20 μL of substrate buffer included in the kit are added to each well of the microplate, and then 20 μL of Enzyme Working solution included in the kit is added to make a total volume of 60 μL (= 0.06 mL). An angiotensin converting enzyme was produced by incubation at 40 ° C. for 40 minutes. After culturing, 200 μL of the Indicator Working solution included in the kit was added to each well and allowed to stand at room temperature for 10 minutes. Thereafter, the inhibitory activity of angiotensin converting enzyme was measured at an absorbance of 450 nm using an absorbance plate reader Multiscan JX (Thermo Scientific), and the inhibition rate at each dilution rate was determined. Further, from the behavior of the obtained inhibition rate, the amount of the extract at 50% inhibition rate of the angiotensin converting enzyme was determined. Moreover, the solid content concentration (IC50 value) of each sample having an inhibitory activity rate of 50% of angiotensin converting enzyme was determined from the amount of the obtained extract using the following formula.

IC50[mg/mL]=(アンジオテンシン変換酵素の阻害活性率50%における抽出液量[mL])*溶存成分濃度[mg/mL]/0.06[mL] IC50 [mg / mL] = (Amount of extract (mL) at 50% inhibitory activity of angiotensin converting enzyme) * Dissolved component concentration [mg / mL] /0.06 [mL]

3.評価結果
上記の試験結果を図4−1、図4−2及び表4にまとめた。
3. Evaluation Results The above test results are summarized in FIG. 4-1, FIG. 4-2, and Table 4.

Figure 2015002209
Figure 2015002209

参考として、日本国特許第3149199号公報の段落0018〜0019に開示の、アンジオテンシン変換酵素阻害活性を有するペプチドによる同様のACE阻害試験の結果を表4に記載した。該公報の段落0020〜0021においては、自然発症高血圧ラットにこのペプチドを経口投与した結果、100mgの経口投与において十分な血圧上昇抑制効果を示したことが記載されている。実施例1〜5のIC50値はこの参考の値よりも十分に低いので、このペプチドよりもさらに十分な血圧上昇抑制効果が期待できる。   For reference, the results of a similar ACE inhibition test with peptides having angiotensin converting enzyme inhibitory activity disclosed in paragraphs 0018 to 0019 of Japanese Patent No. 3149199 are shown in Table 4. In paragraphs 0020 to 0021 of the publication, it is described that as a result of oral administration of this peptide to spontaneously hypertensive rats, 100 mg of oral administration showed a sufficient antihypertensive effect. Since the IC50 values of Examples 1 to 5 are sufficiently lower than this reference value, it is possible to expect a more sufficient blood pressure increase suppressing effect than this peptide.

以上の結果から、表4より、実施例1〜5においては、比較例1に比べて優れた血圧上昇抑制作用を有することがわかる。表4より、実施例6〜9においては比較例2に比べて優れた血圧上昇抑制作用を有することがわかる。   From the above results, it can be seen from Table 4 that Examples 1 to 5 have an excellent blood pressure increase suppressing effect as compared with Comparative Example 1. From Table 4, it can be seen that Examples 6 to 9 have superior blood pressure elevation inhibiting action as compared with Comparative Example 2.

糖化抑制試験
亜臨界処理液、熱水抽出液の2種類の抽出液の糖化抑制試験を行った。
糖化抑制試験は、生体タンパクのアミノ基や脂質とグルコースなどの還元糖化物質の生成を測定するものである。皮膚における還元糖化物質の形成や蓄積は、皮膚の弾力性や柔軟性の低下および黄色化を招きやすくするのであり、結果としての老化の原因とされている。それ故に、糖化抑制作用は、抗老化剤という位置付けになり、還元糖化物質(糖化産物)の生成を抑制することができれば、糖化抑制作用に優れるという指標となるのである。
Saccharification inhibition test A saccharification inhibition test was conducted on two types of extracts, a subcritical processing solution and a hot water extract.
The saccharification inhibition test measures the production of reducing saccharified substances such as amino groups and lipids and glucose of biological proteins. Formation and accumulation of a reduced saccharified substance in the skin easily causes a decrease in elasticity and flexibility of the skin and yellowing, which is a cause of aging as a result. Therefore, the saccharification inhibitory action is positioned as an anti-aging agent, and if the production of a reduced saccharified substance (saccharification product) can be suppressed, it becomes an index that the saccharification inhibitory action is excellent.

1.サンプル準備
亜臨界処理液、熱水抽出液の2種類の抽出液を用意した。その用意した抽出液を表5に示す。
1. Sample preparation Two types of extracts were prepared: a subcritical processing solution and a hot water extract. The prepared extract is shown in Table 5.

Figure 2015002209
Figure 2015002209

2.糖化抑制測定
上記の1で準備した亜臨界処理液、熱水抽出液のそれぞれ試験サンプルを蒸留水により段階希釈した。希釈率は測定した阻害率の値が50%を超過、もしくは下回るように約50倍〜200倍に設定し、各サンプルにつき4点もしくは5点の希釈溶液を作製した。これらの各希釈サンプル溶液100μLと、10mg/mL牛血清アルブミン(BSA)200μL、0.1Mリン酸緩衝液500μL、蒸留水100μL及び2Mフルクトース100μLを混合し、全量1mLに調整した。その後、温度:60℃、40時間インキュベートして還元糖化物質を生成させた。その後、Infinite F200-マイクロプレートリーダー(TECMAN)を使用し、励起波長370nm、蛍光波長440nmで還元糖化物質由来の蛍光を測定し、各希釈率における還元糖物質の生成阻害率を求めた。また得られた阻害率の挙動から、糖化産物の生成阻害率が50%のときの抽出液量を求めた。また、下記の式を用いて、得られた抽出液量から糖化産物の生成阻害率50%である各サンプルの固形分濃度(IC50値)を求めた。また、比較対照として、糖化抑制作用を有するアミノグアニジンを用いた。アミノグアンジも、試験サンプルと同様の手順により、糖化産物の生成阻害率50%である固形分濃度(IC50値)を求めた。
2. Saccharification suppression measurement Each test sample of the subcritical processing solution and hot water extract prepared in 1 above was serially diluted with distilled water. The dilution rate was set to about 50 to 200 times so that the measured inhibition rate value exceeded or below 50%, and a diluted solution of 4 or 5 points was prepared for each sample. 100 μL of each of these diluted sample solutions was mixed with 200 μL of 10 mg / mL bovine serum albumin (BSA), 500 μL of 0.1 M phosphate buffer, 100 μL of distilled water, and 100 μL of 2M fructose, and adjusted to a total volume of 1 mL. Thereafter, the resultant was incubated at a temperature of 60 ° C. for 40 hours to produce a reduced saccharified substance. Thereafter, using an Infinite F200-microplate reader (TECMAN), the fluorescence derived from the reduced saccharified substance was measured at an excitation wavelength of 370 nm and a fluorescence wavelength of 440 nm, and the production inhibition rate of the reduced sugar substance at each dilution rate was determined. Further, from the behavior of the inhibition rate obtained, the amount of the extract was obtained when the production inhibition rate of the saccharification product was 50%. Moreover, the solid content concentration (IC50 value) of each sample having a production inhibition rate of saccharification product of 50% was determined from the amount of the obtained extract using the following formula. As a comparative control, aminoguanidine having a saccharification inhibiting action was used. For aminoguandi, the solid content concentration (IC50 value) with an inhibition rate of glycation product production of 50% was determined by the same procedure as the test sample.

IC50[mg/mL]=(糖化産物の生成阻害率50%における抽出液量[mL])*溶存成分濃度[mg/mL]/1[mL] IC50 [mg / mL] = (Extracted solution amount [mL] at 50% inhibition rate of glycation product production) * Dissolved component concentration [mg / mL] / 1 [mL]

3.評価結果
上記の糖化抑制試験の結果を図5及び表6に示した。
3. Evaluation Results The results of the saccharification inhibition test are shown in FIG.

Figure 2015002209
Figure 2015002209

以上の結果から実施例1〜5においては、比較例1に比べて優れた糖化抑制作用を有することがわかる。また対照として用いたアミノグアニジンの値と比べても、比較例1はアミノグアニジンと同等の活性を示しているのに対し、実施例1〜5においてはアミノグアニジンを上回る活性を示していることが分かる。   From the above results, it can be seen that Examples 1 to 5 have an excellent saccharification inhibiting action as compared with Comparative Example 1. Further, compared with the value of aminoguanidine used as a control, Comparative Example 1 shows an activity equivalent to that of aminoguanidine, whereas Examples 1 to 5 show an activity exceeding that of aminoguanidine. I understand.

本発明のコーヒー豆の抽出液は、血圧上昇抑制効果、糖化抑制効果が高いので、医薬品原料に用いたり、化粧料の配合成分やサプリメント等の健康食品に用いたりすることができる。   Since the coffee bean extract of the present invention has a high blood pressure rise inhibitory effect and a high saccharification inhibitory effect, it can be used as a raw material for pharmaceuticals or health foods such as cosmetic ingredients and supplements.

Claims (14)

コーヒー豆の抽出液を有効成分として含有する血圧上昇抑制剤であって、
前記コーヒー豆の抽出液は、温度:140〜200℃、圧力:0.15〜1.8MPa、かつ、各温度の飽和蒸気圧以上の圧力による亜臨界処理工程を経て得られ、
アンジオテンシン変換酵素(ACE)の阻害活性率が50%のとき、コーヒー豆の抽出液の固形分濃度(IC50値)が0.16mg/mL以下であるコーヒー豆の抽出液を有効成分として含有する血圧上昇抑制剤。
An antihypertensive agent containing a coffee bean extract as an active ingredient,
The coffee bean extract is obtained through a subcritical processing step at a temperature of 140 to 200 ° C., a pressure of 0.15 to 1.8 MPa, and a pressure equal to or higher than a saturated vapor pressure at each temperature,
Blood pressure containing, as an active ingredient, a coffee bean extract having a solid content concentration (IC50 value) of 0.16 mg / mL or less when the angiotensin converting enzyme (ACE) inhibitory activity rate is 50% An increase inhibitor.
前記コーヒー豆がコーヒー生豆及び/又は焙煎コーヒー豆である、請求項1に記載の血圧上昇抑制剤。   The blood pressure elevation inhibitor according to claim 1, wherein the coffee beans are green coffee beans and / or roasted coffee beans. 前記亜臨界処理の処理時間が3〜30分間である、請求項1又は2に記載の血圧上昇抑制剤。   The blood pressure increase inhibitor according to claim 1 or 2, wherein a treatment time of the subcritical treatment is 3 to 30 minutes. 前記亜臨界処理の処理時間が3〜15分間である、請求項1〜3のいずれか1項に記載の血圧上昇抑制剤。   The blood pressure increase inhibitor according to any one of claims 1 to 3, wherein a treatment time of the subcritical treatment is 3 to 15 minutes. コーヒー豆の抽出液を有効成分として含有する糖化抑制剤であって、
前記コーヒー豆の抽出液は、温度:140〜200℃、圧力:0.15〜1.8MPa、かつ、各温度の飽和蒸気圧以上の圧力による亜臨界処理工程を経て得られ、
糖化産物の生成阻害率が50%のとき、コーヒー抽出液の固形分濃度(IC50値)が0.06mg/mL以下であるコーヒー豆の抽出液を有効成分として含有する糖化抑制剤。
A saccharification inhibitor containing a coffee bean extract as an active ingredient,
The coffee bean extract is obtained through a subcritical processing step at a temperature of 140 to 200 ° C., a pressure of 0.15 to 1.8 MPa, and a pressure equal to or higher than a saturated vapor pressure at each temperature,
A saccharification inhibitor comprising, as an active ingredient, a coffee bean extract having a solid content concentration (IC50 value) of 0.06 mg / mL or less when the production inhibition rate of a saccharification product is 50%.
前記コーヒー豆がコーヒー生豆及び/又は焙煎コーヒー豆のいずれかである、請求項5に記載の糖化抑制剤。   The saccharification inhibitor according to claim 5, wherein the coffee beans are green coffee beans and / or roasted coffee beans. 前記亜臨界処理の処理時間が3〜30分間である、請求項5又は6に記載の糖化抑制剤。   The saccharification inhibitor according to claim 5 or 6, wherein a treatment time of the subcritical treatment is 3 to 30 minutes. 前記亜臨界処理の処理時間が3〜15分間である、請求項5〜7のいずれか1項に記載の糖化抑制剤。   The saccharification inhibitor according to any one of claims 5 to 7, wherein a treatment time of the subcritical treatment is 3 to 15 minutes. コーヒー生豆及び/又は焙煎コーヒー豆を粉砕してコーヒー豆の粉砕物を得る工程、
コーヒー豆の粉砕物に水を加えてコーヒー豆の粉砕物と水との混合物を得る工程、及び
コーヒー豆の粉砕物と水との混合物を、温度:140〜200℃、圧力:0.15〜1.8MPa、かつ、各温度の飽和蒸気圧以上の圧力の亜臨界条件で処理して、コーヒー豆の抽出液を回収する工程
を含む、血圧上昇抑制作用を有するコーヒー豆の抽出液の製造方法。
Crushing green coffee beans and / or roasted coffee beans to obtain a coffee beans pulverized product,
A step of adding water to the ground coffee beans to obtain a mixture of the ground coffee beans and water, and a mixture of the ground coffee beans and water, temperature: 140 to 200 ° C., pressure: 0.15 to A method for producing a coffee bean extract having a blood pressure increase-inhibiting action, comprising a step of recovering the coffee bean extract by treating it under a subcritical condition of 1.8 MPa and a pressure equal to or higher than a saturated vapor pressure at each temperature. .
前記亜臨界処理の処理時間が3〜30分間である、請求項9に記載の血圧上昇抑制作用を有するコーヒー豆の抽出液の製造方法。   The method for producing a coffee bean extract having a blood pressure increase-suppressing effect according to claim 9, wherein the treatment time of the subcritical treatment is 3 to 30 minutes. 前記亜臨界処理の処理時間が3〜15分間である、請求項9又は10に記載の血圧上昇抑制作用を有するコーヒー豆の抽出液の製造方法。   The manufacturing method of the coffee bean extract which has the blood pressure rise inhibitory effect of Claim 9 or 10 whose processing time of the said subcritical process is 3 to 15 minutes. コーヒー生豆及び/又は焙煎コーヒー豆を粉砕してコーヒー豆の粉砕物を得る工程、
コーヒー豆の粉砕物に水を加えてコーヒー豆の粉砕物と水との混合物を得る工程、及び
コーヒー豆の粉砕物と水との混合物を、温度:140〜200℃、圧力:0.15〜1.8MPa、かつ、各温度の飽和蒸気圧以上の圧力の亜臨界条件で処理して、コーヒー豆の抽出液を回収する工程
を含む、糖化抑制作用を有するコーヒー豆の抽出液の製造方法。
Crushing green coffee beans and / or roasted coffee beans to obtain a coffee beans pulverized product,
A step of adding water to the ground coffee beans to obtain a mixture of the ground coffee beans and water, and a mixture of the ground coffee beans and water, temperature: 140 to 200 ° C., pressure: 0.15 to A method for producing a coffee bean extract having a saccharification-inhibiting action, comprising a step of recovering the coffee bean extract by a subcritical condition of 1.8 MPa and a pressure equal to or higher than a saturated vapor pressure at each temperature.
前記亜臨界処理の処理時間が3〜30分間である、請求項12に記載の糖化抑制作用を有するコーヒー豆の抽出液の製造方法。   The method for producing an extract of coffee beans having a saccharification-inhibiting action according to claim 12, wherein the subcritical processing time is 3 to 30 minutes. 前記亜臨界処理の処理時間が3〜15分間である、請求項12又は13に記載の糖化抑制作用を有するコーヒー豆の抽出液の製造方法。   The method for producing a coffee bean extract having a saccharification-inhibiting action according to claim 12 or 13, wherein the treatment time of the subcritical treatment is 3 to 15 minutes.
JP2015525245A 2013-07-03 2014-07-01 Antihypertensive agent, saccharification inhibitor, and method for producing the same, containing coffee bean extract as an active ingredient Pending JPWO2015002209A1 (en)

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