JP5317118B2 - ポリフェノールの定量法 - Google Patents
ポリフェノールの定量法 Download PDFInfo
- Publication number
- JP5317118B2 JP5317118B2 JP2009176726A JP2009176726A JP5317118B2 JP 5317118 B2 JP5317118 B2 JP 5317118B2 JP 2009176726 A JP2009176726 A JP 2009176726A JP 2009176726 A JP2009176726 A JP 2009176726A JP 5317118 B2 JP5317118 B2 JP 5317118B2
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- JP
- Japan
- Prior art keywords
- polyphenol
- concentration
- sample
- polyphenols
- cerium
- 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.)
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- 235000013824 polyphenols Nutrition 0.000 title claims description 62
- 150000008442 polyphenolic compounds Chemical class 0.000 title claims description 61
- 238000000034 method Methods 0.000 claims description 42
- 239000013078 crystal Substances 0.000 claims description 23
- ADRVNXBAWSRFAJ-UHFFFAOYSA-N catechin Natural products OC1Cc2cc(O)cc(O)c2OC1c3ccc(O)c(O)c3 ADRVNXBAWSRFAJ-UHFFFAOYSA-N 0.000 claims description 22
- 235000005487 catechin Nutrition 0.000 claims description 22
- 150000001765 catechin Chemical class 0.000 claims description 13
- 238000006243 chemical reaction Methods 0.000 claims description 11
- 150000001785 cerium compounds Chemical class 0.000 claims description 10
- 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
- 239000000523 sample Substances 0.000 description 41
- MHAJPDPJQMAIIY-UHFFFAOYSA-N Hydrogen peroxide Chemical compound OO MHAJPDPJQMAIIY-UHFFFAOYSA-N 0.000 description 22
- 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
- CWVRJTMFETXNAD-FWCWNIRPSA-N 3-O-Caffeoylquinic acid Natural products O[C@H]1[C@@H](O)C[C@@](O)(C(O)=O)C[C@H]1OC(=O)\C=C\C1=CC=C(O)C(O)=C1 CWVRJTMFETXNAD-FWCWNIRPSA-N 0.000 description 6
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 6
- 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
- 229910052684 Cerium Inorganic materials 0.000 description 6
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- XMOCLSLCDHWDHP-UHFFFAOYSA-N L-Epigallocatechin Natural products OC1CC2=C(O)C=C(O)C=C2OC1C1=CC(O)=C(O)C(O)=C1 XMOCLSLCDHWDHP-UHFFFAOYSA-N 0.000 description 6
- 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
- 239000007853 buffer solution Substances 0.000 description 5
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 5
- WPYMKLBDIGXBTP-UHFFFAOYSA-N benzoic acid Chemical compound OC(=O)C1=CC=CC=C1 WPYMKLBDIGXBTP-UHFFFAOYSA-N 0.000 description 4
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- 238000002360 preparation method Methods 0.000 description 4
- 238000001228 spectrum Methods 0.000 description 4
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 3
- CIWBSHSKHKDKBQ-JLAZNSOCSA-N Ascorbic acid Natural products OC[C@H](O)[C@H]1OC(=O)C(O)=C1O CIWBSHSKHKDKBQ-JLAZNSOCSA-N 0.000 description 3
- 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
- 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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- 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
- 239000000203 mixture Substances 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
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- 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
- LSHVYAFMTMFKBA-TZIWHRDSSA-N (-)-epicatechin-3-O-gallate Chemical compound O([C@@H]1CC2=C(O)C=C(C=C2O[C@@H]1C=1C=C(O)C(O)=CC=1)O)C(=O)C1=CC(O)=C(O)C(O)=C1 LSHVYAFMTMFKBA-TZIWHRDSSA-N 0.000 description 2
- 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
- 241000894006 Bacteria Species 0.000 description 2
- LSHVYAFMTMFKBA-UHFFFAOYSA-N ECG Natural products C=1C=C(O)C(O)=CC=1C1OC2=CC(O)=CC(O)=C2CC1OC(=O)C1=CC(O)=C(O)C(O)=C1 LSHVYAFMTMFKBA-UHFFFAOYSA-N 0.000 description 2
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- 239000002211 L-ascorbic acid Substances 0.000 description 2
- 235000000069 L-ascorbic acid Nutrition 0.000 description 2
- 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
- 238000010521 absorption reaction Methods 0.000 description 2
- HSJPMRKMPBAUAU-UHFFFAOYSA-N cerium(3+);trinitrate Chemical compound [Ce+3].[O-][N+]([O-])=O.[O-][N+]([O-])=O.[O-][N+]([O-])=O HSJPMRKMPBAUAU-UHFFFAOYSA-N 0.000 description 2
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- ZYGHJZDHTFUPRJ-UHFFFAOYSA-N coumarin Chemical compound C1=CC=C2OC(=O)C=CC2=C1 ZYGHJZDHTFUPRJ-UHFFFAOYSA-N 0.000 description 2
- 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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- 150000002215 flavonoids Chemical class 0.000 description 2
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- 238000012207 quantitative assay Methods 0.000 description 1
- 238000011158 quantitative evaluation Methods 0.000 description 1
- 235000005875 quercetin Nutrition 0.000 description 1
- 229960001285 quercetin Drugs 0.000 description 1
- FDRQPMVGJOQVTL-UHFFFAOYSA-N quercetin rutinoside Natural products OC1C(O)C(O)C(CO)OC1OCC1C(O)C(O)C(O)C(OC=2C(C3=C(O)C=C(O)C=C3OC=2C=2C=C(O)C(O)=CC=2)=O)O1 FDRQPMVGJOQVTL-UHFFFAOYSA-N 0.000 description 1
- 230000035484 reaction time Effects 0.000 description 1
- 230000009257 reactivity Effects 0.000 description 1
- 235000020095 red wine Nutrition 0.000 description 1
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- ALABRVAAKCSLSC-UHFFFAOYSA-N rutin Natural products CC1OC(OCC2OC(O)C(O)C(O)C2O)C(O)C(O)C1OC3=C(Oc4cc(O)cc(O)c4C3=O)c5ccc(O)c(O)c5 ALABRVAAKCSLSC-UHFFFAOYSA-N 0.000 description 1
- IKGXIBQEEMLURG-BKUODXTLSA-N rutin Chemical compound O[C@H]1[C@H](O)[C@@H](O)[C@H](C)O[C@@H]1OC[C@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](OC=2C(C3=C(O)C=C(O)C=C3OC=2C=2C=C(O)C(O)=CC=2)=O)O1 IKGXIBQEEMLURG-BKUODXTLSA-N 0.000 description 1
- 229960004555 rutoside Drugs 0.000 description 1
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- 238000000926 separation method Methods 0.000 description 1
- 239000003381 stabilizer Substances 0.000 description 1
- 125000004079 stearyl group Chemical group [H]C([*])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])[H] 0.000 description 1
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- Investigating Or Analysing Materials By The Use Of Chemical Reactions (AREA)
- Investigating Or Analyzing Non-Biological Materials By The Use Of Chemical Means (AREA)
Description
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。
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。
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。
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。
[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)
(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。
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。
(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。
実施例1の方法に従いEGCgの検量線を作製した。
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。
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。
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%
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。
(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。
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。
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。
Claims (5)
- 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ポリフェノールの定量法。
- ポリフェノールがカテキン類である請求項1記載のポリフェノールの定量法。
- セリウム化合物が塩化セリウムである請求項1又は2記載のポリフェノールの定量法。
- セリウム化合物をpH6.5以上の条件下で作用させる請求項1〜3のいずれか1項記載のポリフェノールの定量法。
- 結晶物の定量を、反応液の濁度を測定することにより行う請求項1〜4のいずれか1項記載のポリフェノールの定量法。
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