JP2011033347A - Assay of polyphenol - Google Patents
Assay of polyphenol Download PDFInfo
- Publication number
- JP2011033347A JP2011033347A JP2009176726A JP2009176726A JP2011033347A JP 2011033347 A JP2011033347 A JP 2011033347A JP 2009176726 A JP2009176726 A JP 2009176726A JP 2009176726 A JP2009176726 A JP 2009176726A JP 2011033347 A JP2011033347 A JP 2011033347A
- Authority
- JP
- Japan
- Prior art keywords
- polyphenol
- concentration
- sample
- cerium
- hydrogen peroxide
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
- 235000013824 polyphenols Nutrition 0.000 title claims abstract description 71
- 150000008442 polyphenolic compounds Chemical class 0.000 title claims abstract description 70
- 238000003556 assay Methods 0.000 title abstract 4
- 238000000034 method Methods 0.000 claims abstract description 44
- 239000013078 crystal Substances 0.000 claims abstract description 25
- ADRVNXBAWSRFAJ-UHFFFAOYSA-N catechin Natural products OC1Cc2cc(O)cc(O)c2OC1c3ccc(O)c(O)c3 ADRVNXBAWSRFAJ-UHFFFAOYSA-N 0.000 claims abstract description 22
- 235000005487 catechin Nutrition 0.000 claims abstract description 22
- 150000001765 catechin Chemical class 0.000 claims abstract description 13
- 238000006243 chemical reaction Methods 0.000 claims abstract description 13
- 150000001785 cerium compounds Chemical class 0.000 claims abstract description 11
- 238000011002 quantification Methods 0.000 claims description 10
- VYLVYHXQOHJDJL-UHFFFAOYSA-K cerium trichloride Chemical compound Cl[Ce](Cl)Cl VYLVYHXQOHJDJL-UHFFFAOYSA-K 0.000 claims description 5
- MHAJPDPJQMAIIY-UHFFFAOYSA-N Hydrogen peroxide Chemical compound OO MHAJPDPJQMAIIY-UHFFFAOYSA-N 0.000 abstract description 32
- 229910052684 Cerium Inorganic materials 0.000 abstract description 8
- DPUCLPLBKVSJIB-UHFFFAOYSA-N cerium;tetrahydrate Chemical compound O.O.O.O.[Ce] DPUCLPLBKVSJIB-UHFFFAOYSA-N 0.000 abstract description 4
- 210000000265 leukocyte Anatomy 0.000 abstract description 2
- 230000004807 localization Effects 0.000 abstract description 2
- 230000007935 neutral effect Effects 0.000 abstract description 2
- ZMIGMASIKSOYAM-UHFFFAOYSA-N cerium Chemical compound [Ce][Ce][Ce][Ce][Ce][Ce][Ce][Ce][Ce][Ce][Ce][Ce][Ce][Ce][Ce][Ce][Ce][Ce][Ce][Ce][Ce][Ce][Ce][Ce][Ce][Ce][Ce][Ce][Ce][Ce][Ce][Ce][Ce][Ce][Ce][Ce][Ce][Ce] ZMIGMASIKSOYAM-UHFFFAOYSA-N 0.000 abstract 2
- 230000015572 biosynthetic process Effects 0.000 abstract 1
- 239000000523 sample Substances 0.000 description 41
- WMBWREPUVVBILR-WIYYLYMNSA-N (-)-Epigallocatechin-3-o-gallate Chemical compound O([C@@H]1CC2=C(O)C=C(C=C2O[C@@H]1C=1C=C(O)C(O)=C(O)C=1)O)C(=O)C1=CC(O)=C(O)C(O)=C1 WMBWREPUVVBILR-WIYYLYMNSA-N 0.000 description 21
- 238000002835 absorbance Methods 0.000 description 20
- 239000000243 solution Substances 0.000 description 16
- 238000011088 calibration curve Methods 0.000 description 12
- JKMHFZQWWAIEOD-UHFFFAOYSA-N 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid Chemical compound OCC[NH+]1CCN(CCS([O-])(=O)=O)CC1 JKMHFZQWWAIEOD-UHFFFAOYSA-N 0.000 description 10
- 239000007995 HEPES buffer Substances 0.000 description 10
- 239000003963 antioxidant agent Substances 0.000 description 10
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- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 9
- 230000003078 antioxidant effect Effects 0.000 description 9
- 229950001002 cianidanol Drugs 0.000 description 9
- XMOCLSLCDHWDHP-IUODEOHRSA-N epi-Gallocatechin Chemical compound C1([C@H]2OC3=CC(O)=CC(O)=C3C[C@H]2O)=CC(O)=C(O)C(O)=C1 XMOCLSLCDHWDHP-IUODEOHRSA-N 0.000 description 8
- 239000000126 substance Substances 0.000 description 8
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- PZIRUHCJZBGLDY-UHFFFAOYSA-N Caffeoylquinic acid Natural products CC(CCC(=O)C(C)C1C(=O)CC2C3CC(O)C4CC(O)CCC4(C)C3CCC12C)C(=O)O PZIRUHCJZBGLDY-UHFFFAOYSA-N 0.000 description 6
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- CWVRJTMFETXNAD-KLZCAUPSSA-N Neochlorogenin-saeure Natural products O[C@H]1C[C@@](O)(C[C@@H](OC(=O)C=Cc2ccc(O)c(O)c2)[C@@H]1O)C(=O)O CWVRJTMFETXNAD-KLZCAUPSSA-N 0.000 description 6
- GWXLDORMOJMVQZ-UHFFFAOYSA-N cerium Chemical compound [Ce] GWXLDORMOJMVQZ-UHFFFAOYSA-N 0.000 description 6
- 235000001368 chlorogenic acid Nutrition 0.000 description 6
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- 229940074393 chlorogenic acid Drugs 0.000 description 6
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- BMRSEYFENKXDIS-KLZCAUPSSA-N cis-3-O-p-coumaroylquinic acid Natural products O[C@H]1C[C@@](O)(C[C@@H](OC(=O)C=Cc2ccc(O)cc2)[C@@H]1O)C(=O)O BMRSEYFENKXDIS-KLZCAUPSSA-N 0.000 description 6
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- KEAYESYHFKHZAL-UHFFFAOYSA-N Sodium Chemical compound [Na] KEAYESYHFKHZAL-UHFFFAOYSA-N 0.000 description 3
- 244000269722 Thea sinensis Species 0.000 description 3
- 231100000673 dose–response relationship Toxicity 0.000 description 3
- 235000012734 epicatechin Nutrition 0.000 description 3
- DZYNKLUGCOSVKS-UHFFFAOYSA-N epigallocatechin Natural products OC1Cc2cc(O)cc(O)c2OC1c3cc(O)c(O)c(O)c3 DZYNKLUGCOSVKS-UHFFFAOYSA-N 0.000 description 3
- 229930003935 flavonoid Natural products 0.000 description 3
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- 235000013305 food Nutrition 0.000 description 3
- MWDZOUNAPSSOEL-UHFFFAOYSA-N kaempferol Natural products OC1=C(C(=O)c2cc(O)cc(O)c2O1)c3ccc(O)cc3 MWDZOUNAPSSOEL-UHFFFAOYSA-N 0.000 description 3
- 238000000611 regression analysis Methods 0.000 description 3
- PPASLZSBLFJQEF-RKJRWTFHSA-M sodium ascorbate Substances [Na+].OC[C@@H](O)[C@H]1OC(=O)C(O)=C1[O-] PPASLZSBLFJQEF-RKJRWTFHSA-M 0.000 description 3
- 229960005055 sodium ascorbate Drugs 0.000 description 3
- 239000002904 solvent Substances 0.000 description 3
- PFTAWBLQPZVEMU-ZFWWWQNUSA-N (+)-epicatechin Natural products C1([C@@H]2OC3=CC(O)=CC(O)=C3C[C@@H]2O)=CC=C(O)C(O)=C1 PFTAWBLQPZVEMU-ZFWWWQNUSA-N 0.000 description 2
- PFTAWBLQPZVEMU-UKRRQHHQSA-N (-)-epicatechin Chemical compound C1([C@H]2OC3=CC(O)=CC(O)=C3C[C@H]2O)=CC=C(O)C(O)=C1 PFTAWBLQPZVEMU-UKRRQHHQSA-N 0.000 description 2
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- CIWBSHSKHKDKBQ-SZSCBOSDSA-N 2-[(1s)-1,2-dihydroxyethyl]-3,4-dihydroxy-2h-furan-5-one Chemical compound OC[C@H](O)C1OC(=O)C(O)=C1O CIWBSHSKHKDKBQ-SZSCBOSDSA-N 0.000 description 2
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- REFJWTPEDVJJIY-UHFFFAOYSA-N Quercetin Chemical compound C=1C(O)=CC(O)=C(C(C=2O)=O)C=1OC=2C1=CC=C(O)C(O)=C1 REFJWTPEDVJJIY-UHFFFAOYSA-N 0.000 description 2
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- VFLDPWHFBUODDF-FCXRPNKRSA-N curcumin Chemical compound C1=C(O)C(OC)=CC(\C=C\C(=O)CC(=O)\C=C\C=2C=C(OC)C(O)=CC=2)=C1 VFLDPWHFBUODDF-FCXRPNKRSA-N 0.000 description 2
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- Investigating Or Analysing Materials By The Use Of Chemical Reactions (AREA)
Abstract
Description
本発明は、セリウム化合物を用いたポリフェノールの定量法に関する。 The present invention relates to a method for quantifying polyphenols using a cerium compound.
ポリフェノールは、植物の果実や種子に含まれる色素、苦味成分の代表的なものとして知られ、古くから食品や化粧品に使われている。近年では、いわゆる「フレンチパラドックス」を背景とした赤ワインブームや、茶カテキン類の体脂肪蓄積抑制効果など、ポリフェノールの健康価値が注目されている（非特許文献１）。また、茶カテキン類に代表されるポリフェノールは、黄色ブドウ球菌や腸炎ビブリオ等の食中毒細菌、薬剤耐性細菌や植物病原菌に有効であることが報告されている（特許文献１−５）。 Polyphenols are known as representative pigments and bitter components contained in plant fruits and seeds, and have been used for food and cosmetics since ancient times. In recent years, the health value of polyphenols such as the red wine boom against the background of so-called “French paradox” and the body fat accumulation-inhibiting effect of tea catechins has attracted attention (Non-Patent Document 1). In addition, polyphenols typified by tea catechins have been reported to be effective against food poisoning bacteria such as Staphylococcus aureus and Vibrio parahaemolyticus, drug resistant bacteria, and plant pathogens (Patent Documents 1-5).
試料中の総ポリフェノールを測定する方法としては、酒石酸鉄吸光光度法、フォーリン・デニス法及びフォーリン・チオカルト法が知られている。これらの方法は、試料中に含まれる総ポリフェノール量を発色的に定量する方法である。また、フラボノール類を測定する方法として、ＨＰＬＣによる分離分析法が知られている（特許文献６）。
しかし、比色による方法では、試料液の着色が測定の障害になる場合があり、また反応に数時間要するといった欠点を有する。
Known methods for measuring total polyphenol in a sample include iron tartrate absorptiometry, the foreign dennis method, and the foreign thiocult method. These methods are methods for quantitatively determining the total amount of polyphenol contained in a sample. Moreover, the separation analysis method by HPLC is known as a method of measuring flavonols (patent document 6).
However, the colorimetric method has the disadvantages that the coloration of the sample solution may hinder measurement, and the reaction takes several hours.
従来、過酸化水素がセリウムと反応して結晶性のセリウムペルヒドロキシド（ＣＰ）を形成することを利用して、植物や白血球における過酸化水素の局在を可視化する手法が報告されている。この手法では、電子顕微鏡により過酸化水素の発生の有無、発生部位及び発生量を定量することができる（非特許文献２−５）。
一方、カテキン類に代表されるポリフェノールは、中性からアルカリ性域で過酸化水素を発生することが報告されている（非特許文献６）。
しかしながら、これまでポリフェノールの定量にこれ自体から生じる過酸化水素を利用したものはなく、また、過酸化水素とセリウムとが反応して形成される結晶物と、ポリフェノール濃度との間にどのような関係があるのか全く知られていない。
Conventionally, there has been reported a technique for visualizing the localization of hydrogen peroxide in plants and leukocytes by utilizing the fact that hydrogen peroxide reacts with cerium to form crystalline cerium perhydroxide (CP). In this technique, the presence / absence, generation site, and generation amount of hydrogen peroxide can be quantified with an electron microscope (Non-patent Documents 2-5).
On the other hand, polyphenols represented by catechins have been reported to generate hydrogen peroxide in a neutral to alkaline region (Non-patent Document 6).
However, no hydrogen peroxide generated by itself has been used for the determination of polyphenols, and what kind of crystallinity is formed between the reaction between hydrogen peroxide and cerium and the concentration of polyphenols? It is not known at all whether there is a relationship.
本発明は、簡便な操作で、迅速に試料中のポリフェノール濃度を測定できるポリフェノールの定量法を提供することに関する。 The present invention relates to providing a polyphenol quantification method capable of quickly measuring the polyphenol concentration in a sample by a simple operation.
本発明者らは、ポリフェノール濃度と、当該ポリフェノールから発生した過酸化水素とセリウムとが反応して形成される結晶物の量との間に極めて高い相関性があることを見出した。そして、ポリフェノールを含有する試料にセリウムを作用させて生成された結晶物の量を、予め求めた結晶物の量とポリフェノールの濃度との関係に照らし合わせることで、試料中のポリフェノール濃度を定量できることを見出した。 The present inventors have found that there is a very high correlation between the concentration of polyphenol and the amount of crystals formed by the reaction of hydrogen peroxide and cerium generated from the polyphenol. And the amount of polyphenols in the sample can be quantified by comparing the amount of crystals produced by the action of cerium on the sample containing polyphenols against the relationship between the amount of crystals obtained in advance and the concentration of polyphenols. I found.
すなわち、本発明は、ポリフェノールを含有する試料にセリウム化合物を作用させ、生成した結晶物を定量することによってポリフェノールの濃度を算出することを特徴とするポリフェノールの定量法を提供するものである。 That is, the present invention provides a polyphenol quantification method characterized in that the concentration of polyphenol is calculated by allowing a cerium compound to act on a sample containing polyphenol and quantifying the produced crystal.
本発明によれば、試料中のポリフェノール濃度を簡便・迅速に算出することができる。 According to the present invention, the polyphenol concentration in a sample can be calculated easily and quickly.
本発明に用いられるポリフェノールを含有する試料としては、ポリフェノールを含有するものであれば特に制限されず、例えば茶抽出液、果汁、コーヒー抽出液、ココア飲料その他飲食品などが挙げられる。ポリフェノールを含有する試料には、ポリフェノールの他に、例えば軟化剤、乳化剤、防腐剤、香科、安定剤、着色剤、紫外線吸収剤、抗酸化剤、保湿剤、増粘剤等の各種添加剤、食品素材等が含まれていてもよい。
試料中のポリフェノールの含有量は、特に限定されるものではないが、通常、試料中に溶解した状態で０．００１質量％程度以上であるのが好適である。また、所望により、試料を緩衝液などの適当な溶媒に溶解又は分散させて用いてもよい。緩衝液としては、例えばＨＥＰＥＳ Ｂｕｆｆｅｒなどのグッドバッファーが挙げられる。
The polyphenol-containing sample used in the present invention is not particularly limited as long as it contains polyphenol, and examples thereof include tea extract, fruit juice, coffee extract, cocoa beverage, and other foods and drinks. In addition to polyphenols, samples containing polyphenols include various additives such as softeners, emulsifiers, preservatives, fragrances, stabilizers, colorants, ultraviolet absorbers, antioxidants, humectants, thickeners, etc. , Food materials and the like may be included.
The content of polyphenol in the sample is not particularly limited, but usually it is preferably about 0.001% by mass or more in a dissolved state in the sample. If desired, the sample may be dissolved or dispersed in an appropriate solvent such as a buffer solution. Examples of the buffer include good buffers such as HEPES Buffer.
本発明の測定対象であるポリフェノールは過酸化水素を発生させるものである。そのようなポリフェノールとしては、フェニルカルボン酸系、リグナン系、クルクミン系、クマリン系又はフラボノイド系のポリフェノールが挙げられ、具体的には、フェニルカルボン酸系のポリフェノールとして、クロロゲン酸、カフェ酸又はフェルラ酸が挙げられる。また、フラボノイド系として、カテキン類、アピゲニン、ルチン、ルテオリン、ケルセチン、ケンフェロール、イソラムネチン又はヘスペレチンが挙げられる。 The polyphenol to be measured according to the present invention generates hydrogen peroxide. Examples of such polyphenols include phenylcarboxylic acid-based, lignan-based, curcumin-based, coumarin-based, or flavonoid-based polyphenols. Specifically, phenylcarboxylic acid-based polyphenols include chlorogenic acid, caffeic acid, or ferulic acid. Is mentioned. Examples of flavonoids include catechins, apigenin, rutin, luteolin, quercetin, kaempferol, isorhamnetin, and hesperetin.
なかでも、フラボノイド系のポリフェノールが好ましく、特にカテキン類が好ましい。カテキン類としては、カテキン、カテキンガレート、ガロカテキン及びガロカテキンガレート等の非エピ体カテキン類並びに、エピカテキン、エピガロカテキン、エピカテキンガレート及びエピガロカテキンガレート等のエピ体カテキン類の１種以上が挙げられる。また、カテキン類は、重合体であってもよい。 Of these, flavonoid polyphenols are preferable, and catechins are particularly preferable. The catechins include one or more of non-epimeric catechins such as catechin, catechin gallate, gallocatechin and gallocatechin gallate, and epicatechins such as epicatechin, epigallocatechin, epicatechin gallate and epigallocatechin gallate. Can be mentioned. The catechins may be a polymer.
本発明において用いられるセリウム化合物は、過酸化水素と反応して結晶物を形成するものであればよく、例えば酢酸セリウム、塩化セリウム、硝酸セリウム、硫酸セリウムなどが挙げられる。なかでも、取扱性・反応性の点から塩化セリウムが好ましい。 The cerium compound used in the present invention only needs to react with hydrogen peroxide to form a crystal, and examples thereof include cerium acetate, cerium chloride, cerium nitrate, and cerium sulfate. Of these, cerium chloride is preferred from the viewpoint of handling and reactivity.
ポリフェノールを含有する試料にセリウム化合物を作用させる方法は、特に制限されないが、例えば試料に最終濃度で０．００１〜１質量％、好ましくは０．０１〜０．５質量％のセリウム化合物を添加、混合することにより行うのが好ましい。これにより、ポリフェノールから発生する過酸化水素とセリウムが反応し、セリウムの結晶物が生成される。この際、セリウム化合物は、前記と同様の緩衝液、イオン交換水などの適当な溶媒に溶解又は分散させて用いてもよい。 The method for allowing the cerium compound to act on the sample containing polyphenol is not particularly limited, but for example, 0.001-1 mass%, preferably 0.01-0.5 mass% of cerium compound is added to the sample at the final concentration, It is preferable to carry out by mixing. As a result, hydrogen peroxide generated from polyphenol reacts with cerium to produce a cerium crystal. In this case, the cerium compound may be used after being dissolved or dispersed in a suitable solvent such as the above buffer solution or ion-exchanged water.
反応温度は０〜４０℃、反応時間は１０秒〜２０分程度が好ましい。また、反応液のｐＨは６．５以上、特にｐＨ８〜１２の範囲とするのが、ポリフェノールからの過酸化水素発生が良好である点から好ましい。なお、反応液のｐＨは、水酸化ナトリウムなどにより調整することができる。 The reaction temperature is preferably 0 to 40 ° C., and the reaction time is preferably about 10 seconds to 20 minutes. In addition, the pH of the reaction solution is preferably 6.5 or more, particularly in the range of pH 8 to 12, from the viewpoint of good generation of hydrogen peroxide from polyphenol. The pH of the reaction solution can be adjusted with sodium hydroxide or the like.
本発明において、生成した結晶物を定量する方法は、特に限定されないが、反応液の濁度を測定することにより行うのが好ましい。濁度を測定するには、例えば所定の吸収波長における試料の吸光度を測定すればよい。吸光波長は、好ましくは６００ｎｍ〜６６０ｎｍである。 In the present invention, the method for quantifying the produced crystal is not particularly limited, but it is preferably performed by measuring the turbidity of the reaction solution. In order to measure the turbidity, for example, the absorbance of the sample at a predetermined absorption wavelength may be measured. The absorption wavelength is preferably 600 nm to 660 nm.
上記により求めた結晶物の定量値を、予め既知濃度のポリフェノール試料を用いて求めた結晶物の量とポリフェノールの濃度との関係に照らし合わせることで、試料中のポリフェノール濃度を算出する。この両者の関係は、以下の方法により求めることができる。
先ず、既知濃度のポリフェノール試料に、前記と同様にセリウム化合物を添加、混合して結晶物を生成させる。ここで、ポリフェノール試料は、ポリフェノールを前記と同様の緩衝液などの適当な溶媒に溶解又は分散させて所定の濃度に希釈したものを用いることができる。
The polyphenol concentration in the sample is calculated by comparing the quantitative value of the crystal obtained as described above with the relationship between the amount of the crystal obtained in advance using a polyphenol sample having a known concentration and the concentration of polyphenol. The relationship between the two can be obtained by the following method.
First, a cerium compound is added to and mixed with a polyphenol sample having a known concentration in the same manner as described above to form a crystal. Here, the polyphenol sample may be prepared by dissolving or dispersing polyphenol in an appropriate solvent such as the above buffer solution and diluting to a predetermined concentration.
次いで、生成した結晶物を定量し、該定量値とポリフェノールの濃度をプロットする。さらに、ポリフェノール試料の濃度を変えて同様の操作を数回行い、結晶物の量とポリフェノールの濃度との関係を示すグラフを作成し、検量線とする。後記実施例に示すように、ポリフェノールの濃度に応じて生成する結晶物の量は増大する。すなわち、結晶物の量とポリフェノールの濃度とは相関関係にある。 Next, the produced crystal is quantified, and the quantitative value and the concentration of polyphenol are plotted. Further, the same operation is performed several times by changing the concentration of the polyphenol sample, and a graph showing the relationship between the amount of the crystalline substance and the concentration of the polyphenol is prepared as a calibration curve. As will be shown in the examples described later, the amount of crystallized matter generated increases with the concentration of polyphenol. That is, the amount of crystal and the concentration of polyphenol are correlated.
従って、生成した結晶物の量とポリフェノールの濃度との関係を示す検量線や関係式を予め作製しておけば、同様な方法でポリフェノールを含有する試料から生成した結晶物を定量し、その定量値を前記検量線や関係式と照らし合わせることで試料中のポリフェノールの濃度を知ることができる。また、予めポリフェノールの特定濃度に相当する箇所に目盛りや印などを付しておけば、結晶物の量がその箇所を超えるか超えないかにより、半定量的に評価することもできる。なお、反応時のｐＨや定量したいポリフェノールの種類により結晶物の量は異なるので、一つの系に対して一つの検量線が必要である。 Therefore, if a calibration curve or a relational expression showing the relationship between the amount of the produced crystal and the concentration of polyphenol is prepared in advance, the crystal produced from the sample containing polyphenol is quantified by the same method, and the quantification is performed. The concentration of polyphenol in the sample can be known by comparing the value with the calibration curve or the relational expression. In addition, if a scale or mark is attached to a location corresponding to a specific concentration of polyphenol in advance, it can be evaluated semi-quantitatively depending on whether the amount of the crystal exceeds or does not exceed the location. In addition, since the amount of the crystal varies depending on the pH during the reaction and the type of polyphenol to be quantified, one calibration curve is required for one system.
上記方法を利用して定量可能なポリフェノールの濃度は特に規定されないが１０００ｐｐｍ以下が好ましく、更に５００ｐｐｍ以下がより好ましい。 The concentration of polyphenol that can be quantified using the above method is not particularly limited, but is preferably 1000 ppm or less, and more preferably 500 ppm or less.
実施例
［試料］
EGCg；エピガロカテキンガレート(ニュートリションジャパン（株）商品名；テアビゴ)
EGC；エピガロカテキン(SIGMA社製)
ECg；エピカテキンガレート(SIGMA社製)
EC；エピカテキン(SIGMA社製)
CA；クロロゲン酸（Cayman CHEMICAL）
L(+)-アスコルビン酸ナトリウム 和光特級（和光純薬工業株式会社）
L(+)-アスコルビン酸 和光特級（和光純薬工業株式会社）
［緩衝液］
HEPES Buffer (Irvine Scientific）
Example [Sample]
EGCg: Epigallocatechin gallate (trade name of Nutrition Japan Co., Ltd .; Teabigo)
EGC: Epigallocatechin (manufactured by SIGMA)
ECg: Epicatechin gallate (SIGMA)
EC: Epicatechin (manufactured by SIGMA)
CA; chlorogenic acid (Cayman CHEMICAL)
L (+)-sodium ascorbate Wako Special Grade (Wako Pure Chemical Industries, Ltd.)
L (+)-Ascorbic acid Wako Special Grade (Wako Pure Chemical Industries, Ltd.)
[Buffer solution]
HEPES Buffer (Irvine Scientific)
実施例１ 検量線の作製
(１)EGCｇ、EGC、ECg、ECの４種のカテキン溶液を0.1、0.05、0.025、0.0125、0.00625、０質量％の濃度になるようにｐＨ6.0〜8.0の10mＭ HEPES Buffer(Irvine Scientific）にそれぞれ溶解して試料を調製後、96穴タイタープレート（Falcon）に0.18mLずつ分注した。分注した試料に、イオン交換水に溶解し1.0質量％に調製したCeCl3溶液を0.02mLずつ添加し、室温（25℃、以下同じ）で０、５、10、20、30、60分間静置した後、撹拌し、直ちに吸光度測定器（SPECTRA MAX190 Molecular Devices）にて吸光度（650nm）を測定した。
Example 1 Preparation of calibration curve
(1) Four kinds of catechin solutions of EGCg, EGC, ECg, and EC are added to 10 mM HEPES Buffer (Irvine Scientific) at pH 6.0 to 8.0 so that the concentration becomes 0.1, 0.05, 0.025, 0.0125, 0.00625, and 0% by mass. Each sample was prepared by dissolution and then dispensed in 0.18 mL portions into a 96-well titer plate (Falcon). To the dispensed sample, add 0.02 mL of CeCl 3 solution dissolved in ion-exchanged water and adjusted to 1.0% by mass, and let it stand for 0, 5 , 10, 20, 30 , 60 minutes at room temperature (25 ° C, the same shall apply hereinafter) After placing, the mixture was stirred and the absorbance (650 nm) was immediately measured with an absorbance meter (SPECTRA MAX190 Molecular Devices).
得られた吸光度と対応するカテキンの濃度をそれぞれプロットし、グラフを作成して検量線とした。図１−１及び図１−２は、５分間反応させた、吸光度（生成した結晶物の量）とカテキン濃度との関係を示すグラフである。図１−１及び図１−２に示すように、吸光度とカテキン濃度の関係は用量依存的に変化し相関関係があることが確認された。また、最小二乗法による回帰分析を行い、相関係数と回帰式を求めたところ、高い相関係数が得られた。反応液はｐＨ6.5以上であれば測定可能であった。また、ｐＨが高いほどグラフの傾きが大きかった。 The obtained absorbance and the corresponding catechin concentration were plotted, and a graph was prepared as a calibration curve. FIGS. 1-1 and 1-2 are graphs showing the relationship between the absorbance (the amount of the produced crystal product) and the catechin concentration after reaction for 5 minutes. As shown in FIGS. 1-1 and 1-2, it was confirmed that the relationship between the absorbance and the catechin concentration varied in a dose-dependent manner and had a correlation. Moreover, when a regression analysis by the least square method was performed and a correlation coefficient and a regression equation were obtained, a high correlation coefficient was obtained. The reaction solution was measurable at pH 6.5 or higher. Further, the slope of the graph was larger as the pH was higher.
CeCl3溶液を添加した後の吸光度を経時的に測定した結果を図２に示す（ｐＨ8.0）。添加直後から時間の経過とともに回帰式の傾きが小さくなり、さらには添加３０分後にはカテキン濃度と吸光度の相関が低下した。 FIG. 2 shows the results of measuring the absorbance over time after adding the CeCl 3 solution (pH 8.0). The slope of the regression equation decreased with the passage of time immediately after the addition, and the correlation between the catechin concentration and the absorbance decreased 30 minutes after the addition.
（２）クロロゲン酸を0.05、0.025、0.0125、0.00625、０質量％の濃度になるようにｐＨ8.0の10mＭ HEPES Buffer(Irvine Scientific）にそれぞれ溶解して試料を調製後、96穴タイタープレート（Falcon）に0.18mLずつ分注した。分注した溶液にｐＨ8.0、10mＭ HEPES Buffer(Irvine Scientific）に溶解し1.0質量％に調製したCeCl3溶液を0.02mLずつ添加し、室温で５分間静置後、撹拌し、直ちに吸光度測定器（SPECTRA MAX190 Molecular Devices）にて吸光度（650nm）を測定した。 (2) Dissolve chlorogenic acid in 10 mM HEPES Buffer (Irvine Scientific) at pH 8.0 to a concentration of 0.05, 0.025, 0.0125, 0.00625, and 0% by mass. ) 0.18 mL each. 0.02 mL of CeCl 3 solution dissolved in 1.0 mM HEPES Buffer (Irvine Scientific) and adjusted to 1.0% by mass was added to the dispensed solution, 0.02 mL each, and the mixture was allowed to stand at room temperature for 5 minutes, stirred, and immediately measured for absorbance. Absorbance (650 nm) was measured with (SPECTRA MAX190 Molecular Devices).
得られた吸光度と対応するクロロゲン酸の濃度をそれぞれプロットし、グラフを作成して検量線とした（図３）。
図３に示すように、吸光度（生成した結晶物の量）とクロロゲン酸濃度の関係は用量依存的に変化し相関関係があることが確認された。また、最小二乗法による回帰分析を行い、相関係数と回帰式を求めたところ、高い相関係数が得られた。
The obtained absorbance and the corresponding chlorogenic acid concentration were plotted, and a graph was prepared as a calibration curve (FIG. 3).
As shown in FIG. 3, it was confirmed that the relationship between the absorbance (the amount of the produced crystal) and the chlorogenic acid concentration changed in a dose-dependent manner and had a correlation. Moreover, when a regression analysis by the least square method was performed and a correlation coefficient and a regression equation were obtained, a high correlation coefficient was obtained.
実施例２ 未知試料を用いたカテキン類の定量
（１）試料の調製
pH5.0の10mＭ HEPES Buffer(Irvine Scientific）に濃度未知となるようにEGCgを適宜溶解し試料（試料No.１〜No.３）を調製した。
Example 2 Determination of catechins using unknown sample (1) Sample preparation
EGCg was appropriately dissolved in 10 mM HEPES Buffer (Irvine Scientific) at pH 5.0 so that the concentration was unknown, and samples (samples No. 1 to No. 3) were prepared.
（２）検量線の作製
実施例１の方法に従いEGCgの検量線を作製した。
(2) Preparation of calibration curve According to the method of Example 1, an EGCg calibration curve was prepared.
（３）EGCgの定量
試料No.１〜No.３を96穴タイタープレート（Falcon）に0.18mLずつ分注した。分注した試料に、0.1N NaOHを0.01ｍLずつ添加しpHを8.0に調整し、更にイオン交換水に溶解し2.0質量％に調製したCeCl3溶液を0.01mLずつ添加し、室温で５分間静置後、撹拌し、直ちに吸光度測定器（SPECTRA MAX190 Molecular Devices）にて吸光度（650nm）を測定した。
(3) Quantification of EGCg Samples No. 1 to No. 3 were dispensed in 0.18 mL portions into a 96-well titer plate (Falcon). Add 0.1 mL of 0.1N NaOH to the dispensed sample to adjust the pH to 8.0, add 0.01 mL of CeCl 3 solution dissolved in ion-exchanged water and adjusted to 2.0% by mass, and let stand at room temperature for 5 minutes. After placement, the mixture was stirred, and immediately the absorbance (650 nm) was measured with an absorbance meter (SPECTRA MAX190 Molecular Devices).
（４）既存の方法による定量
試料No.１〜No.３中のEGCg濃度を既存の方法（特開2009-000098号公報参照）により以下の様に定量した。なお、EGCgの分解及び重合を考慮し、カテキン類及び没食子酸として検出されたものの合計をEGCgの値として濃度の算出を行った。
試料No.１〜No.３をそれぞれフィルター（0.45μm）で濾過し、島津製作所製、高速液体クロマトグラフ（型式SCL-10AVP）を用い、オクタデシル基導入液体クロマトグラフ用パックドカラムＬ−カラムTM ODS（4.6mmφ×250mm：財団法人 化学物質評価研究機構製）を装着し、カラム温度35℃でグラディエント法により行った。移動相Ａ液は酢酸を0.1mol/L含有の蒸留水溶液、Ｂ液は酢酸を0.1mol/L含有のアセトニトリル溶液とし、流速は1mL／分、試料注入量は10μL、UV検出器波長は280nmの条件で行った。グラディエント条件は以下の通りである。
(4) Quantification by Existing Method The EGCg concentration in samples No. 1 to No. 3 was quantified as follows by an existing method (see JP-A-2009-000098). In consideration of decomposition and polymerization of EGCg, the concentration was calculated using the total of those detected as catechins and gallic acid as the value of EGCg.
Samples No. 1 to No. 3 were filtered through a filter (0.45 μm), respectively, and a packed column L-column TM ODS for octadecyl group-introduced liquid chromatograph using a high performance liquid chromatograph (model SCL-10AVP) manufactured by Shimadzu Corporation (4.6 mmφ × 250 mm: manufactured by Chemicals Evaluation and Research Institute) was attached, and the gradient method was performed at a column temperature of 35 ° C. The mobile phase A solution is a distilled aqueous solution containing 0.1 mol / L acetic acid, the B solution is an acetonitrile solution containing 0.1 mol / L acetic acid, the flow rate is 1 mL / min, the sample injection volume is 10 μL, and the UV detector wavelength is 280 nm. Performed under conditions. The gradient conditions are as follows.
時間（分） Ａ液濃度（体積％） Ｂ液濃度
０ ９７％ ３％
５ ９７％ ３％
３７ ８０％ ２０％
４３ ８０％ ２０％
４３．５ ０％ １００％
４８．５ ０％ １００％
４９ ９７％ ３％
６０ ９７％ ３％
Time (minutes) Liquid A concentration (volume%)
5 97% 3%
37 80% 20%
43 80% 20%
43.50% 100%
48.5 0% 100%
49 97% 3%
60 97% 3%
（５）本発明方法と既存定量法の比較
ｐＨ8.0で塩化セリウムを作用させた本発明方法によるEGCgの定量結果と既存法による定量結果を比較した（図４）。
また、本発明方法によるEGCgの定量値を100とした時の既存法による定量値を表した結果を図５に示す。本発明方法と既存法の差は±10％以内であり、本発明方法によりポリフェノールの定量が可能であることが確認された。
(5) Comparison between the method of the present invention and the existing quantification method The quantification result of EGCg by the method of the present invention with cerium chloride acting at pH 8.0 was compared with the quantification result by the existing method (FIG. 4).
Further, FIG. 5 shows the result of the quantitative value obtained by the existing method when the quantitative value of EGCg obtained by the method of the present invention is set to 100. The difference between the method of the present invention and the existing method is within ± 10%, and it was confirmed that polyphenols can be quantified by the method of the present invention.
実施例３ 抗酸化剤存在下でのカテキン類の定量
（１）試料の調製
抗酸化剤として0.05質量％のL(+)-アスコルビン酸ナトリウムを添加したpH8.0の10mＭ HEPES Buffer(Irvine Scientific）及び0.05質量％のL(+)-アスコルビン酸を添加したpH5.0の10mＭ HEPES Buffer(Irvine Scientific）に、それぞれEGCgを0.1、0.05、0.025、0.0125質量％の濃度になるように溶解し試料を調製した。コントロールとして、L(+)-アスコルビン酸ナトリウム未添加のpH8.0の10mＭ HEPES Buffer(Irvine Scientific）及びL(+)-アスコルビン酸未添加のpH5.0の10mＭ HEPES Buffer(Irvine Scientific）に、同様にEGCgを溶解した試料を調製した。
Example 3 Quantitative determination of catechins in the presence of an antioxidant (1)
（２）EGCgの定量
試料0.18mLを96穴タイタープレート（Falcon）に分注した。分注したpH8.0の試料に対しては、イオン交換水に溶解した１質量％ CeCl3溶液を0.02mLずつ添加した。分注したpH5.0の試料に対しては、0.1N NaOHを0.01mL添加しpH8.0に調整し、イオン交換水に溶解した２質量％ CeCl3溶液を0.01mLずつ添加した。室温で５分間静置後、撹拌し、直ちに吸光度測定器（SPECTRA MAX190 Molecular Devices）にて吸光度（650nm）を測定した。
(2) Quantification of EGCg 0.18 mL of the sample was dispensed into a 96-well titer plate (Falcon). To the dispensed pH 8.0 sample, 0.02 mL of 1 mass% CeCl 3 solution dissolved in ion-exchanged water was added. To the dispensed pH 5.0 sample, 0.01 mL of 0.1N NaOH was added to adjust the pH to 8.0, and 0.01 mL each of a 2 mass% CeCl 3 solution dissolved in ion-exchanged water was added. The mixture was allowed to stand at room temperature for 5 minutes and then stirred, and the absorbance (650 nm) was immediately measured with an absorbance meter (SPECTRA MAX190 Molecular Devices).
得られた吸光度と対応するEGCgの濃度をそれぞれプロットし、グラフを作成して検量線とした（図６の▲）。一方、コントロール溶液も同様にして吸光度を測定し、その測定値と対応するEGCg濃度をそれぞれプロットし、グラフを作成して検量線とした（図６の◆）。
その結果、図６(a)、(b)に示すように、抗酸化剤を添加した試料においては吸光度が抗酸化剤未添加試料より低くなるものの吸光度とカテキン濃度の関係は用量依存的に変化し相関関係があることが確認された。また、最小二乗法による回帰分析を行い、相関係数と回帰式を求めたところ、高い相関係数が得られた。この結果から、抗酸化剤を含む試料中のポリフェノール濃度の測定は、試料と同濃度の抗酸化剤を含む緩衝液などで濃度既知のポリフェノール試料を調製し、その試料を用いて検量線を作製することで測定可能であることが示された。なお、試料中の抗酸化剤濃度は、常法に従って、HPLC法、滴定法、比色法等により測定できる。
The obtained absorbance and the corresponding EGCg concentration were respectively plotted, and a graph was prepared as a calibration curve (▲ in FIG. 6). On the other hand, the absorbance of the control solution was measured in the same manner, the measured value and the corresponding EGCg concentration were plotted, and a graph was created to make a calibration curve (♦ in FIG. 6).
As a result, as shown in FIGS. 6 (a) and 6 (b), the absorbance and catechin concentration change in a dose-dependent manner, although the absorbance is lower in the sample to which the antioxidant is added than in the sample to which the antioxidant is not added. It was confirmed that there was a correlation. Moreover, when a regression analysis by the least square method was performed and a correlation coefficient and a regression equation were obtained, a high correlation coefficient was obtained. From this result, the polyphenol concentration in a sample containing an antioxidant is measured by preparing a polyphenol sample with a known concentration in a buffer solution containing an antioxidant at the same concentration as the sample, and creating a calibration curve using that sample. It was shown that it can be measured. In addition, the antioxidant concentration in a sample can be measured by an HPLC method, a titration method, a colorimetric method or the like according to a conventional method.
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| CN114965907A (en) * | 2022-04-02 | 2022-08-30 | 浙江大学 | Rapid tea grade detection method based on turbidity difference |
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Free format text: JAPANESE INTERMEDIATE CODE: R250 |
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Free format text: JAPANESE INTERMEDIATE CODE: R250 |
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Free format text: JAPANESE INTERMEDIATE CODE: R250 |