JP6628970B2 - Method for quantifying a benzotropolone ring-containing compound - Google Patents
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Description
本発明は、液体試料に含まれるベンゾトロポロン環含有化合物を定量する方法に関する。 The present invention relates to a method for quantifying a benzotropolone ring-containing compound contained in a liquid sample.
紅茶やウーロン茶などの茶飲料には、ベンゾトロポロン環を有する様々な有色化合物が含まれている。これらの化合物は紅茶重合ポリフェノール（Black Tea Polymerized Polyphenols：BTPP）などとして知られており、茶葉の発酵過程において、茶葉に含まれるポリフェノールオキシダーゼなどの酵素作用により、カテキン又は没食子酸などのポリフェノール類が縮合して生成される。 BACKGROUND ART Tea drinks such as black tea and oolong tea contain various colored compounds having a benzotropolone ring. These compounds are known as Black Tea Polymerized Polyphenols (BTPP). During the fermentation process of tea leaves, polyphenols such as catechin or gallic acid are condensed by enzymatic action such as polyphenol oxidase contained in tea leaves. Generated.
ベンゾトロポロン環含有化合物は、様々な生理活性を有することが知られている。例えば、特許文献１ではベンゾトロポロン誘導体が抗炎症作用を有することが報告されており、特許文献２では、ベンゾトロポロン環含有化合物がリパーゼ阻害活性及びα−グルコシダーゼ阻害活性を有することが報告されている。また、ベンゾトロポロン環を含むエピテアフラガリン-3-O-ガレートやテアフラビンが中性脂肪吸収抑制作用を有することも報告されている（特許文献３及び４）。 Benzotropolone ring-containing compounds are known to have various physiological activities. For example, Patent Document 1 reports that a benzotropolone derivative has an anti-inflammatory effect, and Patent Document 2 reports that a benzotropolone ring-containing compound has a lipase inhibitory activity and an α-glucosidase inhibitory activity. . In addition, it has been reported that epitheafragalin-3-O-gallate and theaflavin containing a benzotropolone ring have a neutral fat absorption inhibitory action (Patent Documents 3 and 4).
従来知られているべンゾトロポロン環含有化合物の検出方法は、ポリフェノール類が有する２８０ｎｍの吸収極大を利用するものである（非特許文献１及び２）。しかしながら、当該方法では、ベンゾトロポロン環含有化合物と他のポリフェノール類をまとめて検出することになり、ベンゾトロポロン環含有化合物のみを定量することはできない。また、代表的なテアフラビン類の特定の化合物を分析する方法は知られているが、ベンゾトロポロン環含有化合物総量としてまとめて定量する簡便な方法は知られていない。 A conventionally known method for detecting a benzotropolone ring-containing compound utilizes the absorption maximum at 280 nm of polyphenols (Non-Patent Documents 1 and 2). However, in this method, the benzotropolone ring-containing compound and other polyphenols are collectively detected, and it is not possible to quantify only the benzotropolone ring-containing compound. In addition, a method for analyzing a specific compound of the representative theaflavins is known, but a simple method for collectively determining the total amount of the benzotropolone ring-containing compounds is not known.
本発明は、液体試料に含まれるベンゾトロポロン環含有化合物を、特定の化合物ごとに単離することなく、一群の化合物類としてまとめて特異的に定量する簡便な方法を提供することを目的とする。 An object of the present invention is to provide a simple method for collectively and specifically quantifying a benzotropolone ring-containing compound contained in a liquid sample as a group of compounds without isolation for each specific compound. .
本発明者らは、鋭意検討した結果、液体試料をカラムで処理し、カラム処理後の液体試料の３６０〜３９０ｎｍの吸光度を測定することで、液体試料に含まれるベンゾトロポロン環含有化合物を定量できることを見出した。さらに、カラム処理後の液体試料の４３７〜４６７ｎｍの吸光度も併せて測定することで、ベンゾトロポロン環含有化合物の検出特異性及び定量性を高めることができることも見出し、本発明を完成するに至った。 The present inventors have conducted intensive studies and found that a benzotropolone ring-containing compound contained in a liquid sample can be quantified by treating the liquid sample with a column and measuring the absorbance at 360 to 390 nm of the liquid sample after the column treatment. Was found. Furthermore, they have also found that the specificity and quantitativeness of the benzotropolone ring-containing compound can be enhanced by measuring the absorbance at 437 to 467 nm of the liquid sample after the column treatment, and have completed the present invention. .
即ち、本発明は、以下のものに関するが、これらに限定されない。
（１）液体試料に含まれるベンゾトロポロン環含有化合物を定量する方法であって、
液体試料をカラムで処理する工程、及び
カラム処理後の液体試料の３６０〜３９０ｎｍの吸光度を測定する工程、を含む、
前記方法。
（２）カラム処理後の液体試料の４３７〜４６７ｎｍの吸光度を測定する工程を含む、（１）に記載の方法。
（３）ベンゾトロポロン環含有化合物が茶重合ポリフェノールである、（１）又は（２）に記載の方法。
（４）茶重合ポリフェノールが紅茶重合ポリフェノール、又はウーロン茶重合ポリフェノールである、（３）に記載の方法。
（５）カラム処理工程の前に、植物原料から液体試料を抽出する工程を含む、（１）〜（４）のいずれかに記載の方法。
（６）植物原料が茶葉、紅茶葉、及びウーロン茶葉からなる群より選択される、（５）に記載の方法。
（７）液体試料をカラムで処理する工程、及びカラム処理後の液体試料の吸光度を測定する工程を、高速液体クロマトグラフィーを用いて行う、（１）〜（６）のいずれかに記載の方法。
（８）カラムが逆相系カラムである、（１）〜（７）のいずれかに記載の方法。
（９）逆相系カラムが、シリカゲル系樹脂を充填したものである、（８）に記載の方法。
（１０）逆相系カラムが、コアシェル型樹脂を充填したものである、（８）又は（９）に記載の方法。
（１１）カラム処理が、移動相に水と有機溶媒の混合溶媒を用いた、溶媒グラジエント法によるものである、（１）〜（１０）のいずれかに記載の方法。
（１２）移動相の有機溶媒が、メタノール、エタノール、及びアセトニトリルからなる群より選択される１以上を含むものである、（１１）に記載の方法。
（１３）移動相が、トリフルオロ酢酸、ギ酸、酢酸、トリクロロ酢酸、及び過塩素酸からなる群より選択される１以上を含むものである、（１１）又は（１２）に記載の方法。
（１４）液体試料のリパーゼ阻害活性を評価する方法であって、
液体試料をカラムで処理する工程、
カラム処理後の液体試料の３６０〜３９０ｎｍの吸光度を測定する工程、及び、
ベンゾトロポロン環含有化合物濃度とリパーゼ阻害活性との相関関係を利用して、液体試料のリパーゼ阻害活性を評価する工程、を含む、
前記方法。
That is, the present invention relates to the following, but is not limited thereto.
(1) A method for quantifying a benzotropolone ring-containing compound contained in a liquid sample,
Treating the liquid sample with a column, and measuring the absorbance at 360 to 390 nm of the liquid sample after the column treatment,
The method.
(2) The method according to (1), comprising a step of measuring the absorbance at 437 to 467 nm of the liquid sample after the column treatment.
(3) The method according to (1) or (2), wherein the benzotropolone ring-containing compound is a tea-polymerized polyphenol.
(4) The method according to (3), wherein the tea-polymerized polyphenol is black tea-polymerized polyphenol or oolong tea-polymerized polyphenol.
(5) The method according to any one of (1) to (4), including a step of extracting a liquid sample from a plant material before the column treatment step.
(6) The method according to (5), wherein the plant material is selected from the group consisting of tea leaves, black tea leaves, and oolong tea leaves.
(7) The method according to any one of (1) to (6), wherein the step of treating the liquid sample with the column and the step of measuring the absorbance of the liquid sample after the column treatment are performed using high performance liquid chromatography. .
(8) The method according to any one of (1) to (7), wherein the column is a reversed-phase column.
(9) The method according to (8), wherein the reverse phase column is packed with a silica gel resin.
(10) The method according to (8) or (9), wherein the reversed phase column is packed with a core-shell type resin.
(11) The method according to any one of (1) to (10), wherein the column treatment is performed by a solvent gradient method using a mixed solvent of water and an organic solvent as a mobile phase.
(12) The method according to (11), wherein the organic solvent of the mobile phase contains one or more selected from the group consisting of methanol, ethanol, and acetonitrile.
(13) The method according to (11) or (12), wherein the mobile phase contains one or more selected from the group consisting of trifluoroacetic acid, formic acid, acetic acid, trichloroacetic acid, and perchloric acid.
(14) A method for evaluating a lipase inhibitory activity of a liquid sample,
Processing a liquid sample on a column,
A step of measuring the absorbance at 360 to 390 nm of the liquid sample after the column treatment, and
Utilizing the correlation between the benzotropolone ring-containing compound concentration and the lipase inhibitory activity, the step of evaluating the lipase inhibitory activity of the liquid sample,
The method.
本発明では、液体試料に含まれるベンゾトロポロン環含有化合物を、一群の化合物類として迅速かつ簡便に定量することができる。 According to the present invention, a benzotropolone ring-containing compound contained in a liquid sample can be quickly and easily quantified as a group of compounds.
１．測定対象
１−１．ベンゾトロポロン環含有化合物
本発明により定量される物質は、ベンゾトロポロン環含有化合物である。
1. Measurement target
1-1. Benzotropolone Ring-Containing Compound The substance quantified according to the present invention is a benzotropolone ring-containing compound.
本明細書においてベンゾトロポロン環含有化合物とは、式（１）に示すベンゾトロポロン環骨格を分子内に有する化合物をいう。 In this specification, a benzotropolone ring-containing compound refers to a compound having a benzotropolone ring skeleton represented by the formula (1) in a molecule.
ベンゾトロポロン環含有化合物は、分子内にベンゾトロポロン環を有するものであればよく、具体的には、テアフラビン、テアフラビン-3-O-ガレート、テアフラビン-3’-O-ガレート、テアフラビン-3,3’-O-ジガレート、テアフラバニン、テアフラバニン-3-O-ガレート、テアフラビンジガレート-トリマー1、テアフラビンジガレート-トリマー2、テアフラベートA、テアジベンゾトロポロンA、エピテアフラガリン、エピテアフラガリン-3-O-ガレート、プルプロガリン、EGCG-カテコール、3,4,6-トリヒドロキシ-1-((2R,3R)-3,5,7-トリヒドロキシクロマン-2-イル）-5H-ベンゾ［7］アヌレン-5-オン、(2R,3R)-2-(3,4-ジヒドロキシフェニル)-５,７-ジヒドロキシクロマン-3-イル2,3,4,6-テトラヒドロキシ-5-オキソ-5H-ベンゾ［7］アヌレン-8-カルボキシレートなどが挙げられるが、これらに限定されない。 The benzotropolone ring-containing compound may be any compound having a benzotropolone ring in the molecule, and specifically, theaflavin, theaflavin-3-O-gallate, theaflavin-3'-O-gallate, theaflavin-3,3 '-O-digalate, theaflavanine, theaflavanin-3-O-gallate, theaflavin digallate-trimer 1, theaflavin digallate-trimer 2, theaflavate A, theazibenzotropolone A, epitheafragalin, epitheafragalin-3- O-gallate, purprogalin, EGCG-catechol, 3,4,6-trihydroxy-1-((2R, 3R) -3,5,7-trihydroxychroman-2-yl) -5H-benzo [7] annulene -5-one, (2R, 3R) -2- (3,4-dihydroxyphenyl) -5,7-dihydroxychroman-3-yl 2,3,4,6-tetrahydroxy-5-oxo-5H-benzo [7] annulene-8-carboxylate and the like, But it is not limited to these.
ベンゾトロポロン環含有化合物としては、例えば茶重合ポリフェノール、好ましくは紅茶重合ポリフェノール、ウーロン茶重合ポリフェノールなどが挙げられる。これらのベンゾトロポロン環含有化合物は、茶葉の発酵過程において、茶葉に含まれるポリフェノールオキシダーゼなどの酵素作用により、カテキン又は没食子酸などのポリフェノール類が縮合することで生成される。 Examples of the benzotropolone ring-containing compound include, for example, tea-polymerized polyphenols, preferably black tea-polymerized polyphenols, and oolong tea-polymerized polyphenols. These benzotropolone ring-containing compounds are produced by condensing polyphenols such as catechin or gallic acid by enzymatic action such as polyphenol oxidase contained in tea leaves during the fermentation process of tea leaves.
１−２．液体試料
本明細書において液体試料とは、カラムで処理することができる液状のものであれば特に限定されず、例えば、天然物の処理物又は抽出物、飲食物、医薬品、化粧品などが挙げられる。好ましくは茶葉抽出物であり、特に紅茶葉抽出物又はウーロン茶葉抽出物である。
1-2. Liquid sample In the present specification, a liquid sample is not particularly limited as long as it is a liquid that can be processed by a column, and includes, for example, a processed product or extract of a natural product, food and drink, a pharmaceutical product, cosmetics, and the like. . It is preferably a tea leaf extract, particularly a black tea leaf extract or an oolong tea leaf extract.
１−３．植物原料
本発明は、植物原料から液体試料を得る工程を含んでもよい。植物原料は特に限定されないが、好ましくは茶葉、より好ましくは紅茶葉、又はウーロン茶葉である。植物原料から液体試料を得る方法は特に限定されないが、例えば、植物原料を抽出処理することで液体試料を得ることができる。当該抽出工程では、植物原料として茶葉などをそのまま用いてもよいし、粉砕して用いてもよい。抽出溶媒には、水、熱水、有機溶媒（メタノール、エタノール、イソプロパノール、アセトンなど）、又はこれらの混合溶媒などを用いることができるが、熱水が好ましい。
1-3. Plant raw material The present invention may include a step of obtaining a liquid sample from a plant raw material. The plant material is not particularly limited, but is preferably tea leaves, more preferably black tea leaves, or oolong tea leaves. The method for obtaining a liquid sample from a plant material is not particularly limited. For example, a liquid sample can be obtained by extracting a plant material. In the extraction step, tea leaves or the like may be used as it is as a plant raw material, or may be used after being ground. As the extraction solvent, water, hot water, an organic solvent (methanol, ethanol, isopropanol, acetone, or the like), a mixed solvent thereof, or the like can be used, and hot water is preferable.
本明細書において紅茶葉とは、摘み取った茶葉を乾燥後に完全発酵させたものをいう。紅茶葉の産地は特に限定されないが、例えば、インド（アッサム、ダージリン、ニルギリ、ドアーズ、シッキムなど）、インドネシア（ジャワなど）、スリランカ（セイロンなど）、中国（キーマンなど）、ケニア、日本などが挙げられる。紅茶葉の品種は特に限定されないが、例えば、ケニアCTC、アッサムCTC、アッサム・ファニングス、ニルギリBOP、ジャワなどが挙げられる。また、本明細書においてウーロン茶葉とは、茶葉の発酵途中で加熱して発酵を止め、半発酵させたものをいう。ウーロン茶葉の産地は特に限定されないが、例えば、福建省武夷山、福建省安渓、広東省潮州、台湾などが挙げられる。また、ウーロン茶葉の品種も特に限定されない。 In the present specification, the term “black tea leaves” refers to tea leaves that have been completely fermented after being dried. Although the producing area of the tea leaves is not particularly limited, for example, India (Assam, Darjeeling, Nilgiri, Doors, Sikkim etc.), Indonesia (Java etc.), Sri Lanka (Ceylon etc.), China (Keyman etc.), Kenya, Japan etc. Can be The type of tea leaves is not particularly limited, but examples include Kenya CTC, Assam CTC, Assam Fannings, Nilgiri BOP, Java and the like. In the present specification, oolong tea leaves refer to tea leaves that have been heated during the fermentation to stop fermentation and have been semi-fermented. The production area of oolong tea leaves is not particularly limited, but examples include Wuyi Mountain, Fujian Province, Anxi, Fujian Province, Chaozhou, Guangdong Province and Taiwan. Also, the variety of oolong tea leaves is not particularly limited.
２．カラム
本発明において、液体試料を処理するカラムは特に限定されないが、逆相系カラムであることが好ましい。本明細書において「カラム処理」とは、液体試料をカラムに供し、べンゾトロポロン環含有化合物が溶出する保持時間において、当該化合物を含む画分を分離することをいう。べンゾトロポロン環含有化合物を含有する画分には、当該化合物以外の成分が含まれていなくても、含まれていてもよい。また、逆相系カラムの充填樹脂はシリカゲル系であることが好ましく、例えば、オクタデシル基が化学結合した充填剤（ODS）、ブチル基が化学結合した充填剤（C4）、オクチル基が化学結合した充填剤（C8）、フェニル基が化学結合した充填剤（Ph）、C30のアルキル鎖が化学結合した充填剤（C30）、オクタデシル基が合成ポリマーに結合した（ODP）、逆相系合成ポリマー担体、を用いることができるが、これらカラムに限定されるわけではない。具体的には、TSKgel ODS-80TsQA（東ソー株式会社製）等が挙げられる。また、移動相にグラジエント条件を用いる場合は、溶媒の組成変化に強いカラムを用いることが好ましい。
2. Column In the present invention, the column for treating the liquid sample is not particularly limited, but is preferably a reversed-phase column. As used herein, “column treatment” refers to applying a liquid sample to a column and separating a fraction containing the benzotropolone ring-containing compound at a retention time at which the compound is eluted. The fraction containing the benzotropolone ring-containing compound may or may not contain components other than the compound. The packing resin of the reversed-phase column is preferably silica gel. For example, a packing material in which an octadecyl group is chemically bonded (ODS), a packing material in which a butyl group is chemically bonded (C4), and a octyl group are chemically bonded. Filler (C8), Filler with Phenyl group chemically bonded (Ph), Filler with C30 alkyl chain chemically bonded (C30), Octadecyl group bonded to synthetic polymer (ODP), Reverse phase synthetic polymer carrier , But are not limited to these columns. Specifically, TSKgel ODS-80TsQA (manufactured by Tosoh Corporation) and the like can be mentioned. When gradient conditions are used for the mobile phase, it is preferable to use a column that is resistant to changes in the composition of the solvent.
本発明では、逆相系カラムの充填樹脂としてコアシェル型の樹脂を用いることができる。コアシェル型樹脂は、中心部分のノンポーラス粒子（コア）と外側部分の多孔質層（シェル）で構成される樹脂をいい、例えば、COSMOCORE 2.6 C18等が挙げられる。コアシェル型樹脂を逆相系カラムの充填樹脂として使用することで、分析時間を短縮することが可能になる。 In the present invention, a core-shell type resin can be used as the packing resin for the reversed phase column. The core-shell type resin refers to a resin composed of non-porous particles (core) in the central part and a porous layer (shell) in the outer part, such as COSMOCORE 2.6 C18. By using the core-shell type resin as the packing resin for the reversed phase column, the analysis time can be reduced.
本発明では、カラム処理に用いる移動相に水又は有機溶媒を用いることができる。有機溶媒は特に限定されないが、水に可溶なものが好ましく、例えば、エタノール、メタノール、アセトニトリルなどが挙げられる。水と１種類以上の有機溶媒とを混合して用いることもでき、その混合比率も特に限定されず、例えば、移動相中の有機溶媒の比率を０〜１００％として用いることができる。移動相は酸性である方が分離能の向上及び分析対象化合物の安定性向上などのために好ましく、トリフルオロ酢酸、ギ酸、酢酸、トリクロロ酢酸、過塩素酸などを０．００１％〜５％混合して用いることができる。特に、０．０５％〜０．１％のトリフルオロ酢酸を用いることが好ましい。 In the present invention, water or an organic solvent can be used for the mobile phase used for the column treatment. The organic solvent is not particularly limited, but is preferably soluble in water, and includes, for example, ethanol, methanol, and acetonitrile. Water and one or more kinds of organic solvents may be used as a mixture, and the mixing ratio is not particularly limited. For example, the ratio of the organic solvent in the mobile phase may be 0 to 100%. The mobile phase is preferably acidic in order to improve the separation ability and the stability of the compound to be analyzed. For example, trifluoroacetic acid, formic acid, acetic acid, trichloroacetic acid, perchloric acid and the like are mixed at 0.001% to 5%. Can be used. In particular, it is preferable to use 0.05% to 0.1% of trifluoroacetic acid.
本発明では、移動相の有機溶媒比率、移動相のイオン強度、移動相のｐＨなどを時間に応じて変化させるグラジエント法を用いることができる。本発明では、移動相の有機溶媒比率を時間に応じて変化させる、溶媒グラジエント法を用いることが好ましい。本発明において溶媒グラジエント法を用いることにより、液体試料中におけるベンゾトロポロン環含有化合物を含有する画分の分離能を向上させることができる。溶媒グラジエント法では、移動相の有機溶媒比率を時間経過に応じて直線的、非直線的（凸型又は凹型）、又は段階的に増加及び／又は低下させることができる。また、移動相の有機溶媒比率を一定に保持する時間を設けることもできる。例えば、本発明のグラジエント分析の一態様では、移動相の有機溶媒比率を時間経過に応じて段階的に増加させ、その後、一定時間移動相の有機溶媒比率を一定に保持し、さらに、移動相の有機溶媒比率を分析開始時の有機溶媒比率にまで時間経過に応じて又は急速に低下させることができる。以下にグラジエント分析条件の一例を示すが、液体試料中のベンゾトロポロン環含有化合物のピークが得られる条件であれば、特に限定されない。
＜グラジエント分析条件例＞
・カラム：TSK-gel ODS-８０TsQA（4.6mmφ×150mm、東ソー株式会社）
・移動相：（Ａ）0.05%トリフルオロ酢酸／10%アセトニトリル／水、（Ｂ）0.05%トリフルオロ酢酸／80%アセトニトリル／水
・流速：１．０ｍＬ／分
・グラジエント条件：分析開始から５分後まではＢ液０％、
５分から１１分まででＢ液８％、
１１分から２１分まででＢ液１０％、
２１分から２２分まででＢ液１００％、
２２分から３０分までＢ液１００％保持、
３０分から３１分まででＢ液０％、
３１分から４２分までＡ液１００％保持
移動相の流速は特に限定されないが、例えば好ましくは０．２〜２ｍＬ／分、より好ましくは０．５〜１．５ｍＬ／分である。
In the present invention, a gradient method in which the ratio of the organic solvent in the mobile phase, the ionic strength of the mobile phase, the pH of the mobile phase, and the like are changed with time can be used. In the present invention, it is preferable to use a solvent gradient method in which the ratio of the organic solvent in the mobile phase is changed with time. By using the solvent gradient method in the present invention, the separation ability of the fraction containing the benzotropolone ring-containing compound in the liquid sample can be improved. In the solvent gradient method, the organic solvent ratio of the mobile phase can be increased and / or decreased linearly, non-linearly (convex or concave), or stepwise over time. Further, a time period for keeping the organic solvent ratio of the mobile phase constant can be provided. For example, in one embodiment of the gradient analysis of the present invention, the organic solvent ratio of the mobile phase is increased stepwise with the passage of time, and thereafter, the organic solvent ratio of the mobile phase is kept constant for a certain period of time. Can be reduced with time or rapidly to the organic solvent ratio at the start of the analysis. An example of the gradient analysis conditions is shown below, but is not particularly limited as long as the peak of the benzotropolone ring-containing compound in the liquid sample can be obtained.
<Example of gradient analysis conditions>
・ Column: TSK-gel ODS-80TsQA (4.6mmφ × 150mm, Tosoh Corporation)
-Mobile phase: (A) 0.05% trifluoroacetic acid / 10% acetonitrile / water, (B) 0.05% trifluoroacetic acid / 80% acetonitrile / water-Flow rate: 1.0 mL / min-Gradient condition: 5 minutes from the start of analysis Until later, 0% of solution B,
8% of solution B from 5 minutes to 11 minutes
10% of solution B from 11 minutes to 21 minutes
100% solution B from 21 minutes to 22 minutes
100% retention of solution B from 22 minutes to 30 minutes
0% of solution B from 30 minutes to 31 minutes
100% retention of solution A from 31 minutes to 42 minutes The flow rate of the mobile phase is not particularly limited, but is preferably, for example, 0.2 to 2 mL / min, and more preferably 0.5 to 1.5 mL / min.
３．検出
本発明において、ベンゾトロポロン環含有化合物の検出は、３６０〜３９０ｎｍ、好ましくは３６５〜３８５ｎｍ、より好ましくは３７０〜３８０ｎｍの任意の波長における吸光度を測定することで行う。ベンゾトロポロン環含有化合物を検出するための最も好ましい波長は３７５ｎｍである。
3. Detection In the present invention, the detection of the benzotropolone ring-containing compound is performed by measuring the absorbance at an arbitrary wavelength of 360 to 390 nm, preferably 365 to 385 nm, more preferably 370 to 380 nm. The most preferred wavelength for detecting a benzotropolone ring-containing compound is 375 nm.
ベンゾトロポロン環含有化合物を含む一般的なポリフェノール類は、２８０ｎｍ付近の波長域に吸収極大を有する（図１）。そのため、ベンゾトロポロン環含有化合物を２８０ｎｍ付近の波長で検出した場合には、カテキン類やタンニン類などの他のポリフェノール類を含むピークとして検出される。一方で、ベンゾトロポロン環含有化合物以外の他のポリフェノール類は、一般的には３６０〜３９０ｎｍ付近に吸収極大を有しない（図１）。従って、本発明では、液体試料に含まれるベンゾトロポロン環含有化合物の濃度を特異的に測定することができる。 General polyphenols containing a benzotropolone ring-containing compound have an absorption maximum in a wavelength region around 280 nm (FIG. 1). Therefore, when a benzotropolone ring-containing compound is detected at a wavelength around 280 nm, it is detected as a peak containing other polyphenols such as catechins and tannins. On the other hand, other polyphenols other than the benzotropolone ring-containing compound generally do not have an absorption maximum around 360 to 390 nm (FIG. 1). Therefore, in the present invention, the concentration of the benzotropolone ring-containing compound contained in the liquid sample can be specifically measured.
ベンゾトロポロン環含有化合物は、４３７〜４６７ｎｍの波長域にも吸収極大を有する。そのため、前記３６０〜３９０ｎｍの任意の波長における吸光度測定に加えて、さらに４３７〜４６７ｎｍ、好ましくは４４２〜４６２ｎｍ、より好ましくは４４７〜４５７ｎｍ、最も好ましくは４５２ｎｍの吸光度測定を行うことにより、ベンゾトロポロン環含有化合物をより特異的に検出及び定量することができる。これにより、ベンゾトロポロン環含有化合物の検出特異性及び定量性を高めることができる。 The benzotropolone ring-containing compound also has an absorption maximum in the wavelength range of 437 to 467 nm. Therefore, in addition to the absorbance measurement at an arbitrary wavelength of 360 to 390 nm, the benzotropolone ring is further measured by measuring the absorbance at 437 to 467 nm, preferably 442 to 462 nm, more preferably 447 to 457 nm, and most preferably 452 nm. The contained compound can be detected and quantified more specifically. Thereby, the detection specificity and quantitativeness of the benzotropolone ring-containing compound can be improved.
４．高速液体クロマトグラフィー（ＨＰＬＣ）
本発明では、液体試料をカラムで処理する工程、及びカラム処理後の液体試料の吸光度を測定する工程を、高速液体クロマトグラフィー（ＨＰＬＣ）を用いて行うことができる。本発明で使用されるＨＰＬＣ機器は、特に限定されないが、例えば、化学計測機器として市販されている通常のＨＰＬＣ装置などを用いることができる。また、検出器として紫外可視分光検出器を有するものが好ましい。さらに、溶媒グラジエントシステムを有するものがより好ましい。尚、測定対象、カラム・樹脂の種類、移動相の組成・流速、及び検出条件などの分析条件は前記と同様である。
4. High performance liquid chromatography (HPLC)
In the present invention, the step of treating the liquid sample with the column and the step of measuring the absorbance of the liquid sample after the column treatment can be performed using high performance liquid chromatography (HPLC). The HPLC instrument used in the present invention is not particularly limited, and for example, an ordinary HPLC instrument commercially available as a chemical measuring instrument can be used. Further, a detector having an ultraviolet-visible spectral detector is preferable as the detector. Further, those having a solvent gradient system are more preferred. The analysis conditions such as the measurement target, the type of column / resin, the composition / flow rate of the mobile phase, and the detection conditions are the same as described above.
５．液体試料のリパーゼ阻害活性を評価する方法
本発明の一態様は、液体試料をカラムで処理する工程、カラム処理後の液体試料の３６０〜３９０ｎｍの吸光度を測定する工程、及び、ベンゾトロポロン環含有化合物濃度とリパーゼ阻害活性との相関関係を利用して、液体試料のリパーゼ阻害活性を評価する工程、を含む、液体試料のリパーゼ阻害活性を評価する方法である。ベンゾトロポロン環含有化合物がリパーゼ阻害活性を有することは、ＷＯ２０１０／１３４５９５号公報に開示されている。さらに、ベンゾトロポロン環含有化合物濃度とリパーゼ阻害活性には相関関係が認められる（実施例３）。従って、本発明により、液体試料中のベンゾトロポロン環含有化合物の濃度を測定することで、液体試料のリパーゼ阻害活性の多寡を評価することができる。また、本発明は４３７〜４６７ｎｍの吸光度を測定する工程や、植物原料から液体試料を得る工程をさらに含んでもよい。尚、測定対象、カラム・樹脂の種類、移動相の組成・流速、及び検出条件などの分析条件は前記と同様である。
5. Method for evaluating lipase inhibitory activity of liquid sample One embodiment of the present invention includes a step of treating the liquid sample with a column, a step of measuring the absorbance at 360 to 390 nm of the liquid sample after the column treatment, and a compound containing a benzotropolone ring. A method for evaluating the lipase inhibitory activity of a liquid sample, comprising the step of evaluating the lipase inhibitory activity of a liquid sample using a correlation between the concentration and the lipase inhibitory activity. The fact that a benzotropolone ring-containing compound has a lipase inhibitory activity is disclosed in WO2010 / 134595. Furthermore, there is a correlation between the concentration of the benzotropolone ring-containing compound and the lipase inhibitory activity (Example 3). Therefore, according to the present invention, the amount of the lipase inhibitory activity of the liquid sample can be evaluated by measuring the concentration of the benzotropolone ring-containing compound in the liquid sample. Further, the present invention may further include a step of measuring the absorbance at 437 to 467 nm and a step of obtaining a liquid sample from a plant material. The analysis conditions such as the measurement target, the type of column / resin, the composition / flow rate of the mobile phase, and the detection conditions are the same as described above.
以下、実施例に基づいて本発明を説明するが、本発明はこれらの実施例に限定されるものではない。
実施例１：ＨＰＬＣ分析条件の確立
紅茶重合ポリフェノールのＨＰＬＣ条件を以下のように確立した。
＜条件＞
・カラム：TSK-gel ODS-８０TsQA（4.6mmφ×150mm、東ソー株式会社）
・移動相：（Ａ）0.05%トリフルオロ酢酸／10%アセトニトリル／水、（Ｂ）0.05%トリフルオロ酢酸／80%アセトニトリル／水
・流速：１．０ｍＬ／分
・カラム温度：４０℃
・グラジエント条件：
分析開始から５分後まではＢ液０％、
５分から１１分まででＢ液８％、
１１分から２１分まででＢ液１０％、
２１分から２２分まででＢ液１００％、
２２分から３０分までＢ液１００％保持、
３０分から３１分まででＢ液０％、
３１分から４２分までＡ液１００％保持
・検出：２８０ｎｍ、３７５ｎｍ
・注入量：１０μＬ
・標準物質：テアフラビン-3,3’-ジガレート（紅茶重合ポリフェノール（BTPP）測定のための標準物質）及びウーロンホモビスフラバンB（ウーロン茶重合ポリフェノール（OTPP）測定のための標準物質）
本ＨＰＬＣ分析では標準物質として、テアフラビン-3,3’-ジガレート及びウーロンホモビスフラバンB（ともに長良サイエンス社製）を用い、62.5、125、250、及び500μg/mL溶液を用いて検量線を作製した。作成したテアフラビン-3,3’-ジガレートの検量線を図１に示す。
Hereinafter, the present invention will be described based on examples, but the present invention is not limited to these examples.
Example 1: Establishment of HPLC analysis conditions HPLC conditions for black tea polymerized polyphenols were established as follows.
<Condition>
・ Column: TSK-gel ODS-80TsQA (4.6mmφ × 150mm, Tosoh Corporation)
-Mobile phase: (A) 0.05% trifluoroacetic acid / 10% acetonitrile / water, (B) 0.05% trifluoroacetic acid / 80% acetonitrile / water-Flow rate: 1.0 mL / min-Column temperature: 40 ° C
・ Gradient conditions:
0% solution B until 5 minutes after the start of analysis
8% of solution B from 5 minutes to 11 minutes
10% of solution B from 11 minutes to 21 minutes
100% solution B from 21 minutes to 22 minutes
100% retention of solution B from 22 minutes to 30 minutes
0% of solution B from 30 minutes to 31 minutes
100% retention and detection of solution A from 31 minutes to 42 minutes: 280 nm, 375 nm
・ Injection volume: 10 μL
-Standard substances: theaflavin-3,3'-digalate (standard substance for measuring black tea polymerized polyphenol (BTPP)) and oolong homobisflavan B (standard substance for measuring oolong tea polymerized polyphenol (OTPP))
In this HPLC analysis, a calibration curve was prepared using 62.5, 125, 250, and 500 μg / mL solutions using theaflavin-3,3'-digalate and oolong homobisflavan B (both manufactured by Nagara Science) as standard substances. did. FIG. 1 shows a calibration curve of the prepared theaflavin-3,3′-digalate.
また、テアフラビン-3,3’-ジガレートとウーロンホモビスフラバンBの吸収スペクトルを図２に示す。その結果、分子内にベンゾトロポロン環を有するテアフラビン-3,3’-ジガレートは、２８０ｎｍ付近、３７５ｎｍ付近、及び４５２ｎｍ付近に吸収極大が認められた。一方で、分子内にベンゾトロポロン環を有しないウーロンホモビスフラバンBは、２８０ｎｍ付近にのみ吸収極大が認められた。従って、テアフラビン-3,3’-ジガレートにおいて認められた３７５ｎｍ付近、及び４５２ｎｍ付近の吸収極大は、ベンゾトロポロン環に起因するものであることが明らかとなった。 FIG. 2 shows the absorption spectra of theaflavin-3,3'-digalate and oolong homobisflavan B. As a result, theaflavin-3,3'-digalate having a benzotropolone ring in the molecule showed absorption maxima near 280 nm, 375 nm, and 452 nm. On the other hand, oolong homobisflavan B having no benzotropolone ring in the molecule had an absorption maximum only at around 280 nm. Therefore, it was revealed that the absorption maxima around 375 nm and around 452 nm observed in theaflavin-3,3'-digalate are caused by the benzotropolone ring.
実施例２：ベンゾトロポロン環含有化合物のＨＰＬＣ分析
実施例１のＨＰＬＣ条件に従い、分子内にベンゾトロポロン環を有するテアフラビン-3,3’-ジガレートの250μg/mL溶液と、分子内にベンゾトロポロン環を有しないウーロンホモビスフラバンB（OHBF-B）の250μg/mL溶液の分析を行った。その結果、図３に示すように、２８０ｎｍの波長で検出した場合には、テアフラビン-3,3’-ジガレート及びウーロンホモビスフラバンB共に保持時間（R.T.）24分付近に単一ピークが認められ、分子内のベンゾトロポロン環の有無に関わらずテアフラビン-3,3’-ジガレート及びウーロンホモビスフラバンBが検出されることが示された。一方で、３７５ｎｍの波長で検出した場合には、ウーロンホモビスフラバンBのピークは検出されなかったが、テアフラビン-3,3’-ジガレートは保持時間24分付近に単一ピークとして検出された。従って、３７５ｎｍの検出波長を用いることで、ベンゾトロポロン環含有化合物を特異的に検出できることが示された。
Example 2 HPLC Analysis of Benzotropolone Ring-Containing Compound According to the HPLC conditions of Example 1, a 250 μg / mL solution of theaflavin-3,3′-digalate having a benzotropolone ring in the molecule and a benzotropolone ring in the molecule were used. Analysis of a 250 μg / mL solution of oolong homobisflavan B (OHBF-B), which does not have OH, was performed. As a result, as shown in FIG. 3, when detected at a wavelength of 280 nm, a single peak was observed near the retention time (RT) of 24 minutes for both theaflavin-3,3'-digalate and oolong homobisflavan B. It was shown that theaflavin-3,3'-digalate and oolong homobisflavan B were detected regardless of the presence or absence of a benzotropolone ring in the molecule. On the other hand, when detected at a wavelength of 375 nm, the peak of oolong homobisflavan B was not detected, but theaflavin-3,3'-digalate was detected as a single peak around a retention time of 24 minutes. Therefore, it was shown that the benzotropolone ring-containing compound can be specifically detected by using the detection wavelength of 375 nm.
さらに、テアフラビン-3,3’-ジガレートの250μg/mL溶液に、ウーロンホモビスフラバンBを31.25μg/mL、62.5μg/mL、125μg/mL、及び250μg/mLとなるように添加した混合溶液の分析を行った。結果を表１に示す。 Further, to a 250 μg / mL solution of theaflavin-3,3′-digalate, oolong homobisflavan B was added at 31.25 μg / mL, 62.5 μg / mL, 125 μg / mL, and a mixed solution of 250 μg / mL. Analysis was carried out. Table 1 shows the results.
表１に示すように、２８０ｎｍで測定した場合には、ウーロンホモビスフラバンBの添加量の増加に伴い検量線から求められるウーロン茶重合ポリフェノール（OTPP）の濃度が実際の含有量よりも増大することが示された。これに対して、３７５ｎｍで定量した場合には、ウーロンホモビスフラバンBの添加量を増加させても、検量線から求められる紅茶重合ポリフェノール（BTPP）濃度は実際の含有量250μg/mLにきわめて近い値であることが示された。従って、３７５ｎｍの波長を用いて測定することで、混在するウーロン茶重合ポリフェノールの影響を受けることなく、紅茶重合ポリフェノールのようなベンゾトロポロン環を含有する化合物の濃度を正確に測定できることが明らかとなった。 As shown in Table 1, when measured at 280 nm, the concentration of oolong tea-polymerized polyphenol (OTPP) obtained from the calibration curve increases from the actual content as the added amount of oolong homobisflavan B increases. It has been shown. On the other hand, when quantified at 375 nm, even if the added amount of oolong homobisflavan B is increased, the black tea polymerized polyphenol (BTPP) concentration obtained from the calibration curve is very close to the actual content of 250 μg / mL. Value. Therefore, it was clarified that by measuring using a wavelength of 375 nm, the concentration of a compound containing a benzotropolone ring such as black tea polymerization polyphenol can be accurately measured without being affected by the mixed oolong tea polymerization polyphenol. .
実施例３：紅茶重合ポリフェノール濃度とリパーゼ阻害活性との相関関係
（１）紅茶葉抽出物中の紅茶重合ポリフェノール濃度の測定
紅茶葉16gを800mLの熱水（90℃）で5分ごとに攪拌しながら20分間抽出した。紅茶葉は、ケニアCTC、アッサムCTC、アッサム・ファニングス、ニルギリBOP、及びジャワの5種類の品種を用いた。東洋濾紙No.2で濾過後、50%アセトニトリルで２倍希釈し、さらにミリポアフィルター（0.45μm、マイレクスLH-4）で濾過したものを、実施例１のＨＰＬＣ条件で分析した。
（２）リパーゼ阻害活性測定
特許文献２に記載の通り、ベンゾトロポロン環含有化合物はリパーゼ阻害活性を示すことが知られている。そして、この変化は濃度依存的であると考えられている。そこで、異なる濃度の紅茶重合ポリフェノールを含有する紅茶抽出物を用いて、リパーゼ阻害活性力価（1/IC50）の違いを評価した。
Example 3: Correlation between black tea-polymerized polyphenol concentration and lipase inhibitory activity (1) Measurement of black tea-polymerized polyphenol concentration in black tea leaf extract 16 g of black tea leaves were stirred every 5 minutes with 800 mL of hot water (90 ° C). While extracting for 20 minutes. For tea leaves, five varieties of Kenya CTC, Assam CTC, Assam Fannings, Nilgiri BOP, and Java were used. After filtration through Toyo Filter Paper No. 2, the suspension was diluted 2-fold with 50% acetonitrile, and further filtered through a Millipore filter (0.45 μm, Milex LH-4), and analyzed under the HPLC conditions of Example 1.
(2) Measurement of lipase inhibitory activity As described in Patent Document 2, it is known that a benzotropolone ring-containing compound exhibits lipase inhibitory activity. This change is considered to be concentration-dependent. Thus, differences in lipase inhibitory activity titers (1 / IC50) were evaluated using black tea extracts containing different concentrations of black polymerized polyphenols.
リパーゼ活性は、基質として蛍光性の４−メチルウンベリフェロンのオレイン酸エステル（4-MUO）を使用し、反応によって生成した４−メチルウンベリフェロンの蛍光を測定することにより測定した。測定にあたり、緩衝液は150mM NaCl、1.36mM CaCl2を含む13mM Tris-HCl（pH8.0）を用いた。基質である4-MUOは0.1MのDMSO溶液とした後に上記緩衝液で1000倍希釈したものを用いた。また、リパーゼはブタ膵リパーゼ（シグマ社製）を同様に上記緩衝液を用いて400U/mL溶液として調製したものを酵素測定に供した。 Lipase activity was measured by using the fluorescent oleic acid ester of 4-methylumbelliferone (4-MUO) as a substrate and measuring the fluorescence of 4-methylumbelliferone produced by the reaction. In the measurement, 13 mM Tris-HCl (pH 8.0) containing 150 mM NaCl and 1.36 mM CaCl 2 was used as a buffer. As the substrate, 4-MUO was used as a 0.1 M DMSO solution, which was then diluted 1000-fold with the above buffer solution. In addition, lipase prepared as a 400 U / mL solution of porcine pancreatic lipase (manufactured by Sigma) in the same manner as above using the above buffer solution was subjected to enzyme measurement.
酵素反応は、25℃条件下において、96穴マイクロプレートに50μLの4-MUO緩衝液、25μLの蒸留水（あるいは試料水溶液）を添加し混合した後に、25μLのリパーゼ緩衝液を添加することにより開始させた。30分間反応を行った後に、100μLの0.1Mクエン酸緩衝液（pH4.2）を添加して反応を停止させ、反応によって生成した４−メチルウンベリフェロンの蛍光（励起波長３５５ｎｍ、蛍光波長４６０ｎｍ）を蛍光プレートリーダー（Labsystem社製Fluoroskan Asent CF）を用いて測定した。 The enzyme reaction is started by adding 50 μL of 4-MUO buffer and 25 μL of distilled water (or sample aqueous solution) to a 96-well microplate at 25 ° C., mixing, and then adding 25 μL of lipase buffer. I let it. After performing the reaction for 30 minutes, the reaction was stopped by adding 100 μL of 0.1 M citrate buffer (pH 4.2), and the fluorescence (excitation wavelength: 355 nm, fluorescence wavelength: 460 nm) of 4-methylumbelliferone produced by the reaction was added. ) Was measured using a fluorescent plate reader (Fluoroskan Asent CF manufactured by Labsystem).
試料の阻害活性は、対照（蒸留水）の活性に対して50%阻害を与える試料量IC50（μM）として求めた。また、IC50値の逆数をμgあたりの相対活性値（リパーゼ阻害活性力価（units））として算出し、試料溶液中のリパーゼ阻害活性を比較した。
（３）紅茶重合ポリフェノール濃度とリパーゼ阻害活性との相関関係
上記（１）及び（２）の方法で、5種類の紅茶葉抽出物の紅茶重合ポリフェノール濃度及びリパーゼ阻害活性を測定した。結果を表２に示す。さらに、横軸に紅茶重合ポリフェノール濃度、及び縦軸にリパーゼ阻害活性力価をプロットして作成した相関図を図４に示す。その結果、紅茶重合ポリフェノール濃度が高い品種（ケニアCTC、アッサムCTC、及びアッサム・ファニング）は、リパーゼ阻害活性力価（1/IC50）も高いことが明らかとなり、両者の間には相関関係が認められた。
The inhibitory activity of the sample was determined as a sample amount IC50 (μM) that gives 50% inhibition to the activity of the control (distilled water). The reciprocal of the IC50 value was calculated as a relative activity value per μg (lipase inhibitory activity titer (units)), and the lipase inhibitory activities in the sample solutions were compared.
(3) Correlation between black tea-polymerized polyphenol concentration and lipase inhibitory activity The black tea-polymerized polyphenol concentrations and lipase inhibitory activities of the five tea leaf extracts were measured by the methods (1) and (2) above. Table 2 shows the results. Further, FIG. 4 shows a correlation diagram created by plotting the black tea polymerization polyphenol concentration on the horizontal axis and the lipase inhibitory activity titer on the vertical axis. The results revealed that varieties with high black tea-polymerized polyphenol concentrations (Kenya CTC, Assam CTC, and Assam Fanning) also had higher lipase inhibitory activity titers (1 / IC50), indicating a correlation between the two. Was done.
実施例４：改良ＨＰＬＣ法
紅茶重合ポリフェノールの分析時間を短縮するため、コアシェルタイプの充填樹脂を用いたＨＰＬＣ法を確立した。ＨＰＬＣ条件を以下に示す。
＜条件＞
・カラム：COSMOCORE 2.6 C18 3mm×75mm
・移動相：（Ａ）0.05%トリフルオロ酢酸／水、（Ｂ）0.05%トリフルオロ酢酸／90%アセトニトリル／水
・流速：０．８ｍＬ／分
・グラジエント条件：
分析開始から２．５分後まではＢ液１１．１％
２．５分から５．５分まででＢ液１７．３％
５．５分から８分まででＢ液１８．１％
８分から８．１分まででＢ液９０％
８．１分から１４．６分までＢ液９０％保持
１４．６分から１５分まででＢ液１１．１％
１５分から２１分まででＢ液１１．１％保持
・検出：３７５ｎｍ
・注入量：５μＬ
コアシェルタイプの樹脂を充填したカラムを用いることで、分析時間を約半分に短縮できることが明らかとなった。
Example 4: Improved HPLC method In order to shorten the analysis time of black tea polymerized polyphenol, an HPLC method using a core-shell type packed resin was established. The HPLC conditions are shown below.
<Condition>
・ Column: COSMOCORE 2.6 C18 3mm × 75mm
-Mobile phase: (A) 0.05% trifluoroacetic acid / water, (B) 0.05% trifluoroacetic acid / 90% acetonitrile / water-Flow rate: 0.8 mL / min-Gradient conditions:
Until 2.5 minutes after the start of analysis, 11.1% of solution B
17.3% of solution B from 2.5 minutes to 5.5 minutes
18.1% of solution B from 5.5 minutes to 8 minutes
90% liquid B in 8 minutes to 8.1 minutes
90% retention of solution B from 8.1 minutes to 14.6 minutes
11.1% from 14.6 minutes to 15 minutes
Retention and detection of 11.1% of solution B from 15 minutes to 21 minutes: 375 nm
・ Injection volume: 5 μL
It became clear that the analysis time can be reduced to about half by using a column packed with a core-shell type resin.
本発明では、液体試料をカラムで処理し、カラム処理後の液体試料の３６０〜３９０ｎｍの吸光度を測定することで、液体試料に含まれるベンゾトロポロン環含有化合物を正確に定量できる。また、本発明では液体試料に含まれるベンゾトロポロン環含有化合物を、一連の化合物群として迅速かつ簡便に定量することができる。よって、本発明の方法によれば、ベンゾトロポロン環含有化合物に由来する生理活性の高い原材料や組成物を選抜することができる。 In the present invention, a benzotropolone ring-containing compound contained in a liquid sample can be accurately quantified by treating the liquid sample with a column and measuring the absorbance at 360 to 390 nm of the liquid sample after the column treatment. Further, in the present invention, a benzotropolone ring-containing compound contained in a liquid sample can be quickly and easily quantified as a series of compound groups. Therefore, according to the method of the present invention, a highly bioactive raw material or composition derived from a benzotropolone ring-containing compound can be selected.
Claims (11)
液体試料を高速液体クロマトグラフィーにて逆相系カラムで処理する工程、及び、
カラム処理後の液体試料を高速液体クロマトグラフィーの検出器で測定する工程であって、測定する吸光度が３７０〜３８０ｎｍおよび４４７〜４５７ｎｍである工程、を含む、
前記方法。 A method for quantifying a benzotropolone ring-containing compound contained in a liquid sample,
A step of treating the liquid sample with a reversed-phase column by high-performance liquid chromatography, and
A step of measuring the liquid sample after the column treatment with a detector of high performance liquid chromatography, wherein the absorbance to be measured is 370 to 380 nm and 447 to 457 nm.
The method.
液体試料を高速液体クロマトグラフィーにて逆相系カラムで処理する工程、
カラム処理後の液体試料を高速液体クロマトグラフィーの検出器で測定する工程であって、測定する吸光度が３７０〜３８０ｎｍおよび４４７〜４５７ｎｍである工程、及び、
ベンゾトロポロン環含有化合物濃度とリパーゼ阻害活性との相関関係を利用して、液体試料のリパーゼ阻害活性を評価する工程、を含む、
前記方法。 A method for evaluating a lipase inhibitory activity of a liquid sample,
Processing a liquid sample on a reversed-phase column by high-performance liquid chromatography,
A step of measuring the liquid sample after the column treatment with a detector for high performance liquid chromatography, wherein the absorbance to be measured is 370 to 380 nm and 447 to 457 nm; and
Utilizing the correlation between the benzotropolone ring-containing compound concentration and the lipase inhibitory activity, the step of evaluating the lipase inhibitory activity of the liquid sample,
The method.
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