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J. Agric. Food Chem. 2002, 50, 2142−2148
Synthesis of Theaflavin from Epicatechin and Epigallocatechin by Plant Homogenates and Role of Epicatechin Quinone in the Synthesis and Degradation of Theaflavin TAKASHI TANAKA, CHIE MINE, KYOKO INOUE, MIYUKI MATSUDA, ISAO KOUNO*
AND
School of Pharmaceutical Sciences, Nagasaki University, 1-14 Bunkyo-machi, Nagasaki 852-8521, Japan
Oxidation products of (-)-epicatechin and (-)-epigallocatechin by treatment with homogenates of 62 plants belonging to 49 families were compared. Forty-six plants were capable of synthesizing theaflavin, a black tea pigment, regardless of whether they contained catechins. Loquat, Japanese pear, and blueberry had activities higher than that of fresh tea leaves after 5 h of treatment; furthermore, these plants oxidized theaflavin to theanaphthoquinone. An additional new metabolite, dehydrotheasinensin, was generated on treatment with fresh tea leaves, eggplant, and unripened Japanese orange. Evidence for the oxidation of epigallocatechin and theaflavin by electron transfer to epicatechin quinone was demonstrated in a time course study using bananas and trapping the quinone intermediates as glutathione conjugates. KEYWORDS: Catechin; oxidation; theaflavin; dehydrotheasinensin; black tea; polyphenol
INTRODUCTION
MATERIALS AND METHODS
Enzymatic polyphenol oxidation, usually accompanied by reddish coloration, is often observed in damaged fruit or vegetables. This phenomenon is important from the viewpoints of food manufacturing and the plant defense system. However, chemical details of the reaction remain ambiguous because of the complexity of the oxidation products. In black tea manufacturing, the oxidation mechanism of four of the major catechins, (-)-epicatechin (1), (-)-epigallocatechin (2), and their 3-O-gallates, in tea leaves has been well studied, and the cooccurrence of these catechol (1 and its 3-O-gallate) and pyrogallol (2 and its 3-O-gallate) types makes the oxidation characteristic (1). Theaflavin (3) and its 3- and 3′-O-gallates, characteristic reddish orange pigments of black tea having a benzotropolone structure, are produced by oxidative condensation between 1 and 2. In the present study, we examined the production of 3 from a mixture of 1 and 2 by treatment with various plant homogenates and found that a number of plants are capable of synthesizing 3 regardless of whether the plant contains catechins. Further oxidation of 3 to theanaphthoquinone (4) (2) and generation of a new metabolite named dehydrotheasinensin (6) were also observed. Catechin oxidation by various plants and the structure determinations of the new metabolites has been investigated in this study. Evidence supporting the oxidation of 2 and 3 by epicatechin quinone (1a) is also presented.
Materials and General Procedures. UV spectra were obtained with a V-560 UV-vis spectrophotometer, and optical rotations were measured with a DIP-370 digital polarimeter (JASCO Co.). 1H and 13C NMR, 1H-1H COSY, NOESY, HSQC, and HMBC spectra were recorded in a mixture of acetone-d6 and D2O (19:1, v/v) with a Unity plus 500 spectrometer (Varian Inc.) operating at 500 MHz for 1H and at 125 MHz for 13C, respectively. Coupling constants are expressed in hertz, and chemical shifts given on a δ (parts per million) scale with tetramethylsilane as an internal standard. MS were recorded on a JMS DX-303 spectrometer (JEOL Ltd.), and glycerol or m-nitrobenzyl alcohol was used as a matrix for FAB-MS measurement. Elemental analysis was obtained with a MT-3 analyzer (Yanaco Analytical Instruments Co.). Column chromatography was done on MCI gel CHP 20P, 75-150 µm (Mitsubishi Chemical Co.), Chromatorex ODS (Fuji Silysia Chemical Ltd.), and Sephadex LH-20, 25-100 µm (Pharmacia Fine Chemical Co. Ltd.). Thin-layer chromatography (TLC) was performed on precoated Kieselgel 60 F254 plates, 0.2 mm thick (Merck), with benzene/ethyl formate/formic acid (1:7:1, v/v) or chloroform/ MeOH/H2O (14:6:1, v/v), and spots were detected by UV illumination and spraying with 2% ethanolic FeCl3 or 10% sulfuric acid reagent followed by heating. Analytical high-pressure liquid chromotography (HPLC) was performed on a Cosmosil 5C18-AR II, 250 × 4.6 mm i.d. column (Nacalai Tesque Inc.) with gradient elution from 10 to 30% (30 min) and from 30 to 75% (15 min) of CH3CN in 50 mM H3PO4 at a flow rate of 0.8 mL/min, and detection was performed with an MD910 photodiode array detector (JASCO Co.). Epicatechin and epigallocatechin were isolated from commercial green tea according to the method of Nonaka et al. (3) and recrystallized from H2O. Oxidation of Epicatechin by Banana. Banana fruit (Musa acuminata Colla cv. Giant Cavendish) purchased in a local market (100 g) was homogenized with H2O (200 mL) in a Waring blender, and the homogenate was filtered through four layers of gauze. An aqueous
* Corresponding author [telephone 81 (0)95 847 1111; fax 81 (0)95 848 4387; e-mail
[email protected]].
10.1021/jf011301a CCC: $22.00 © 2002 American Chemical Society Published on Web 02/26/2002
Oxidation of Catechins by Various Plants solution (25 mL) of 1 (0.5 g) was mixed with the banana homogenate (100 mL) at room temperature and stirred vigorously for 5 h. Acetone (500 mL) was added and the mixture filtered. The filtrate was concentrated in vacuo to ∼100 mL and extracted three times with ethyl acetate (100 mL). The organic layer was concentrated and subjected to Sephadex LH-20 column chromatography (20 × 2.0 cm) with ethanol to give 1 (162 mg) and a fraction containing oxidation products. The products were further purified by MCI gel CHP20P column chromatography (15 × 1.5 cm) with H2O containing increasing proportions of MeOH (0-40%) to afford compound 5 (8.8 mg) as a yellow amorphous powder: [R]D -140° (c 0.3, MeOH); negative FAB-MS m/z 863 (M - H)-; UV λmaxEtOH(log ) 256sh (5.2), 282 (5.2), 384 (5.4). 1H NMR (500 MHz, in acetone-d6) δ 7.92 (1H, s, OH), 7.10 (1H, d, J ) 2.1 Hz, H-2), 6.95 (1H, d, J ) 0.5 Hz, B-2), 6.93 (1H, dd, J ) 2.1, 8.5 Hz, H-6), 6.79 (1H, d, J ) 8 Hz, H-5), 6.79 (1H, s, D-7 OH), 6.36 (1H, s, B-5), 6.21 (1H, d, J ) 2.3 Hz, A-8), 6.19 (1H, s, G-6), 6.10 (1H, s, D-6), 6.04 (1H, s, E-5), 5.99 (1H, d, J ) 2.3 Hz, A-6), 5.51 (1H, s, E-3 OH), 5.15 (1h, br s, I-2), 5.09 (1H, dt, J ) 2.5, 9.2 Hz, F-3), 4.46 (1H, br s, C-2), 4.24 (1H, br s, I-3), 4.20 (1H, d, J ) 2.8 Hz, F-2), 3.83 (1H, br s, C-3), 3.60, 3.31 (each 1H, d, J ) 5.3 and 4.4 Hz, respectively, C-3 OH and I-3 OH), 3.31 (1H, dd, J ) 4.9, 16.3 Hz, I-4), 3.21 (1H, dd, J ) 9.2, 17.6 Hz, F-4), 3.14 (1H, d, J ) 12.6 Hz, E-2), 2.82 (1H, dd, J ) 1.8, 16.3 Hz, I-4), 2.60 (1H, br d, J ) 16.0 Hz, C-4), 2.53 (1H, dd, J ) 2.5, 17.6 Hz, F-4), 2.29 (1H, dd, J ) 4.6, 16.0 Hz, C-4), 2.23 (1H, d, J ) 12.6 Hz, E-2); 13C NMR (125 MHz, in acetone-d6) δ 190.50 (E-4), 164.30, 164.23 (E-6, G-5), 163.65 (G-7), 157.62, 157.24 (A-5, A-7), 155.84 (D-5), 154.70 (A-8a), 154.67 (D-7), 154.25 (G-8a), 152.24 (D-8a), 145.35 (2C) (H-3, 4), 144.38, 143.86 (B-3, 4), 132.16, 131.95 (B-1, H-1), 122.59 (B-6), 119.53 (H6), 118.83 (B-5), 115.78 (B-2), 115.40 (2C) (H-2, H-5), 110.05 (E-5), 108.19 (D-8), 105.49 (G-8), 102.70 (G-4a), 99.93 (D-4a), 99.67 (A4a), 96.93 (D-6), 96.39 (A-8), 95.79 (A-6), 94.32 (E-3), 91.96 (G-6), 90.74 (E-1), 80.26 (F-2), 79.79 (I-2), 77.19 (C-2), 68.76 (F-3), 66.18 (I-3), 64.75 (C-3), 39.74 (E-2), 28.96 (I-4), 28.53 (C-4), 24.18 (F-4). Anal. Calcd for C45H36O18‚2.5H2O: C, 59.41%; H, 4.54%. Found: C, 59.65%; H, 4.80%. Oxidation of a Mixture of Catechins by Plant Homogenates. Fresh plant (50 g) was homogenized with H2O (100 mL) in a Waring blender and filtered through four layers of gauze. The plant homogenate (1.5 mL) was mixed with an aqueous solution (0.25 mL) of 1 (5 mg) and 2 (5 mg) and stirred vigorously for 5 h at room temperature. After addition of acetone (5 mL), the reaction mixture was filtered, and the filtrate was concentrated in vacuo. The residue was redissolved in 50% aqueous MeOH (5.0 mL), passed through a membrane filter (0.45 µm), and analyzed by HPLC with a photodiode array detector. Concurrently, a blank solution was treated in the same manner. Quantification of compounds 3, 4, and 6 in Table 1 was performed by reporting the peak area into the calibration curve of the corresponding compound (3 at 375 nm, 4 at 440 nm, and 6 at 390 nm). For the time course experiment, the scale of the reaction was twice as much and aliquots (0.1 mL) of the reaction mixture were withdrawn at the times indicated, diluted with EtOH (0.4 mL), and analyzed by HPLC. Isolation of Dehydrotheasinensin (6). Fresh tea leaves (Camellia sinensis var. assamica) (720 g) were homogenized with H2O (1800 mL) and Polyclar AT, polyvinylpolypyrrolidone (GAF Corp., Linden, NJ) (240 g), and filtered through four layers of gauze. The filtrate (1.2 L) was mixed with an aqueous solution (100 mL) containing 1 (2.0 g) and 2 (6.0 g) and stirred vigorously for 14 h at room temperature. The mixture was poured into acetone (1.5 L) and filtered through filter paper. After concentration, the filtrate was extracted five times with ethyl acetate to yield an ethyl acetate extract (3.2 g). The extract was subjected to Sephadex LH-20 column chromatography (25 × 2.5 cm) with ethanol containing increasing proportions of H2O (0-20%) and then with 50% acetone. The fractions containing polyphenols were combined and applied to an MCI gel CHP20P column (15 × 2.0 cm) with H2O containing increasing proportions of MeOH (0-80%) to yield unchanged 1 (829 mg), 6 (246.6 mg), and a mixture of theaflavin (3) and theanaphthoquinone (4). The separation of 3 (314.3 mg) and 4 (6.5 mg) was achieved by Chromatorex ODS column chromatography (040% MeOH). In this experiment, 2 was completely consumed.
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Dehydrotheasinensin (6): yellow amorphous powder; [R]D +21.1° (c 0.12, MeOH); FABMS (negative mode), m/z 607 [M - H]-; UV λmaxEtOH (log ) 269 (4.11), 397 (4.59); 1H NMR (500 MHz, in acetoned6) δ 8.48 (1H, s, B-3-OH), 8.37, 8.23, 8.18, 8.09 (each 1H, s, A-ring OH), 7.036 (1H, d, J ) 1.0 Hz, E-6), 6.11 (2H, br s, B-5-OH, F-2), 6.03, 6.02, 6.00, 5.98 (each 1H, d, J ) 2.5, 2.2, 2.5, 2.2 Hz, respectively, A-6, A-8, D-6 and D-8), 4.45 (1H, ddd, J ) 1.3, 1.8, 5.0 Hz, C-3), 4.26 (1H, br s, F-3), 4.17 (1H, br s, C-2), 3.60 (1H, br s, F-3-OH), 2.97 (1H, d, J ) 11.4 Hz, B-6), 2.94 (1H, dd, J ) 4.4, 16.5 Hz, F-4), 2.85 (1H, dd, J ) 1.8, 17.6 Hz, C-4), 2.71 (1H, dd, J ) 2.5, 16.5 Hz, F-4), 2.71 (1H, dd, J ) 5.0, 17.6 Hz, C-4), 2.33 (1H, dd, J ) 1.1, 11.4 Hz, B-6); 13C NMR (125 MHz, in acetone-d6) δ 189.56 (B-4), 157.61, 157.55, 157.39, 157.05, 156.89 (A-5, A-7, D-5, D-7, F-8a), 155.54 (A8a), 152.17 (E-3), 150.95 (E-5), 139.73 (B-3), 134.25 (B-2), 130.84 (E-1), 129.40 (E-4), 112.40 (E-2), 110.21 (E-6), 100.16 (D-4a), 98.47 (A-4a), 96.51, 96.17, 95.66, 95.35 (A-6, A-8, D-6, D-8), 94.67 (B-5), 89.57 (B-1), 76.80 (F-2), 72.18 (C-2), 66.43 (F-3), 65.00 (C-3), 40.67 (B-6), 28.28 (F-4), 25.02 (C-4). Anal. Calcd for C30H24O14‚6.5H2O: C, 49.66%; H, 5.14%. Found: C, 49.85%; H, 5.04%. Oxidation in the Presence of Glutathione. A mixture of 1 (0.5 g), 2 (0.5 g), and glutathione (reduced form, 1.0 g) in H2O (25 mL) was mixed with the banana homogenate (100 mL), prepared as described above, and stirred vigorously for 3.5 h at room temperature. After addition of acetone (500 mL) and filtration, the filtrate was concentrated and applied to a Sephadex LH-20 column (25 × 3.0 cm) with H2O containing increasing proportions of MeOH to give 3 (32.1 mg), unchanged 2 (361.9 mg), and a mixture of 7, 8, and 9. The mixture of glutathione conjugates was further separated by MCI gel CHP20P chromatography (25 × 2.0 cm) with H2O/MeOH to yield 7 (64.9 mg), 8 (111.5 mg), 9 (498.9 mg), and a mixture of 7 and 8 (195.1 mg). 2′-Glutathionyl-(-)-epigallocatechin (7): white amorphous powder; [R]D -60.9° (c 0.3, MeOH); FABMS, m/z 612 [M + H]+, 634 [M + Na]+; 1H NMR (300 MHz, in acetone-d6 + D2O) δ 6.96 (1H, s, H-6′), 6.07, 5.99 (each 1H, d, J ) 2.1 Hz, H-6, H-8), 5.47 (1H, s, H-2), 4.54 (1H, dd, J ) 5.8, 6.3 Hz, Cys-R), 4.29 (1H, br s, H-3), 3.80-3.93 (3H, m, Gly-R, Glu-R), 3.15 (2H, d like, Cys-β), 2.92 (1H, dd, J ) 3.8, 16.5 Hz, H-4), 2.81 (1H, br d, J ) 16.5 Hz, H-4), 2.56 (2H, m, Glu-γ), 2.19 (2H, m, Glu-β); 13C NMR (75 MHz, in acetone-d6 + D2O) δ 174.80, 174.01, 173.42, 172.07 (COO), 157.33, 156.90, 156.87 (C5, C-7, C-8a), 148.02, 147.33 (C-3′, 5′), 134.90, 132.85 (C-1′, C-4′), 109.02, 108.15 (C-2′, C-6′), 99.67 (C-4a), 96.34, 95.64 (C-6, C-8), 77.15 (C-2), 65.70 (C-3), 54.71, 54.25 (Glu-R, Cys-R), 42.08 (Gly-R), 37.94 (Cys-β), 32.27 (Glu-γ), 29.39 (C-4), 26.87 (Glu-β). Anal. Calcd for C25H29N3O13S‚2H2O: C, 46.37%; H, 5.14%; N, 6.49%. Found: C, 46.11%; H, 5.24%; N, 6.15%. 2′-Glutathionyl-(-)-epicatechin (8): white amorphous powder; [R]D -30.3° (c 0.4, MeOH); FABMS, m/z 596 [M + H]+, 618 [M + Na]+, 634 [M + K]+; 1H NMR (300 MHz, in acetone-d6 + D2O) δ 7.19, 6.95 (each 1H, d, J ) 8.2 Hz, H-5′, H-6′), 6.07, 5.97 (each 1H, d, J ) 2.2 Hz, H-6, H-8), 5.48 (1H, s, H-2), 4.56 (1H, dd, J ) 5.0, 8.0 Hz, Cys-R), 4.28 (1H, br s, H-3), 3.80-3.95 (3H, m, Gly-R, Glu-R), 3.24 (2H, br s, Cys-β), 2.92 (1H, dd, J ) 3.3, 17.3 Hz, H-4), 2.80 (1H, br d, J ) 17.3 Hz, H-4), 2.55 (2H, m, Glu-γ), 2.21 (2H, m, Glu-β); 13C NMR (75 MHz, in acetone-d6 + D2O) δ 174.63, 173.47, 172.84, 171.89 (COO), 157.34, 156.97(2C) (C-5, C-7, C-8a), 146.99, 145.12 (C-3′,4′), 134.20 (C-1′), 120.48, 118.00, 116.41 (C-2′, C-5′, C-6′), 99.61 (C-4a), 96.24, 95.49 (C-6, C-8), 77.22 (C-2), 65.51 (C-3), 54.60, 54.28 (GluR, Cys-R), 41.67 (Gly-R), 37.06 (Cys-β), 32.23 (Glu-γ), 29.28 (C-4), 26.81 (Glu-β). Anal. Calcd for C25H29N3O12S‚1.5H2O: C, 48.23%; H, 5.18%; N, 6.75%. Found: C, 48.43%; H, 5.19%; N, 6.66%. 5′-Glutathionyl-(-)-epicatechin (9): white amorphous powder; [R]D -66.5° (c 0.3, MeOH), FABMS, m/z 596 [M + H]+, 618 [M + Na]+, 634 [M + K]+; 1H NMR (300 MHz, in acetone-d6 + D2O) δ 7.12, 7.03 (each 1H, br s, H-2′, H-6′), 6.06, 5.99 (each 1H, br s, H-6, H-8), 4.88 (1H, s, H-2), 4.53 (1H, br t, J ) 7.4 Hz, Cys-R), 4.30 (1H, br s, H-3), 3.86 (overlapped with HOD signal, m, Gly-R, Glu-R), 3.28 (2H, br s, Cys-β), 2.87, 2.76 (each 1H, br d, J ) 14.3 Hz, H-4), 2.53 (2H, m, Glu-γ), 2.18 (2H, m, Glu-β); 13C NMR (75 MHz, in acetone-d6 + D2O) δ 174.61, 173.81, 173.16, 172.17 (COO), 157.19, 157.01, 156.39 (C-5, C-7, C-8a), 145.41 (2C) (C-3′,4′), 131.98 (C-1′), 123.62, 119.81, 115.18 (C-2′, C-5′, C-6′), 99.51 (C-4a), 96.33, 94.20 (C-6, C-8), 78.64
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Table 1. Formation of Theaflavin (3), Theanaphthoquinone (4), and Dehydrotheasinensin (6) from 1 and 2 (5 h of Reaction unless Otherwise Noted) family name Asteridae
Asteraceae
part
3a
4a
Artemisia princeps (wormwood) Arctium lappa (edible burdock) Petasites japonicus (Japanese butterbur) Viburnum odoratissimum (sweet viburnum) Gardenia jasminoides (cape jasmine) Ligustrum japonicum (Japanese privet) Mentha spicata (spearmint) Perilla frutescens (perilla) Clerodendrum trichotomum Borago officinalis (common borage) Ipomoea batatas (sweet potato) Capsicum annuum var. grossum (bell pepper) Solanum melongena (eggplant) Solanum tuberosum (potato) Nerium oleander var. indicum (oleander) Daucus carota (carrot) Citrus paradisi (grapefruit) Citrus unshiu (Satsuma mandarin)
5.4 3.1 b 27.0 3.7 b b b 0.5 2.5 1.0 0.7 24.9 6.9 14.5 2.0 d 2.8 d 1.0 7.7 2.9 18.2 1.9 d 0.7 55.7 15.9 22.3 17.4 55.6 6.9 10.8
14.2
1.1 33.2 d d 1.4 c d 0.7 d 0.4 16.8 23.6 29.7e 14.7e d b 4.0 1.8 d 2.8 d 2.1 21.5 b 2.9 0.4 0.3 2.4 0.1 0.6 6.3 d 2.8
Vitaceae Euphorbiaceae Punicaceae Fabaceae Rosaceae
Vitis vinifera (grape) Phyllanthus urinaria Punica granatum (pomegranate) Phaseolus vulgaris (common bean) Eriobotrya japonica (loquat)
Hydrangeaceae Pittosporaceae
Malus pumila (apple) Prunus mume (Japanese apricot) Pyrus pyrifolia (Japanese pear) Hydrangea macrophylla (hydrrangea) Pitttosporum tobira (tobira)
leaves root stem leaves leaves leaves leaves leaves leaves leaves root fruit fruit rhizome leaves root fruit fruit (unripe) fruit (ripe) fruit fruit peel fruit leaves fruit fruit, seed fruit (unripe) fruit (ripe) fruit fruit fruit leaves leaves
Diospyros kaki (persimmon) Vaccinium myrtillus (blueberry) Brassica oleracea var. capitata (cabbage) Eutrema wasabi (wasabi) Cucurbita moschata (pumpkin) Momordica charantia (balsam pear) Carica papaya (papaya) Abelmoschus esculentus (okura) Eleaocarpus sylvestris Actinidia chinensis (kiwi fruit) Camellia japonica (common camellia) Camellia sasanqua (sasanqua) Camellia sinensis (tea) (5 h of reaction) (8 h of reaction) Polygonum cuspidatum (Japanese knotweed) Achyranthes fauriei Spinacia oleracea (spinach) Quercus dentata (Daimio oak) Myrica rubra (Chinese strawberry tree) Ficus carica (common fig) Distylium racemosum Nelumbo nucifera (Indian lotus) Houttuynia cordata (Houttuynia herb) Persea americana (avocado) Dioscorea opposita (Chinese yam) Allium fistulosum (Welsh onion) Zingiber mioga (Mioga ginger) Musa acuminata (banana) Ananas comosus (pineapple) Zea mays (maize) Colocasia esculenta (taro) Ginkgo biloba (ginkgo) Lentinus edodes (shiitake mashuroom)
fruit fruit leaves root fruit fruit fruit fruit leaves fruit leaves leaves leaves leaves leaves leaves leaves leaves fruit fruit leaves rhizome leaves fruit root leaves seedling fruit fruit seed rhizome fruit fruit body
Caprifoliaceae Rubiaceae Oleaceae Lamiaceae Verbenaceae Boraginaceae Convolvulaceae Solanaceae
Rosidae
plant name
Apocynaceae Apiaceae Rutaceae Anacardiaceae Sapindaceae
Mangifera indica (mango) Litchi chinensis (litchi)
6a 1.1 0.1
25.5 1.7 3.0 0.2 0.5 0.8 0.5 0.3 2.8
7.4
0.7 1.5
5.2 1.8 8.6 19.9
0.8
26.4 1.7 1.7
Dilleniidae Ebenaceae Ericaceae Brassicaeae Cucurbitaceae Caricaceae Malvaceae Elaeocarpaceae Actinidiaceae Theaceae
Caryophyllidae Hamamelidae
Magniliidae Liliidae Zingiberidae Commelinidae Arecidae Gymnospermae Basidiomycetes
Polygonaceae Amaranthaceae Chenopodiaceae Fagaceae Myricaceae Moraceae Hamamelidaceae Nymphaeaceae Saururaceae Lauraceae Dioscoreaceae Liliaceae Zingiberaceae Musaceae Blomeliaceae Poaceae Araceae Ginkgoaceae Tricholomataceae
2.3 2.1
7.5 2.3 2.5 2.0
3.5 0.6
0.4 9.9 7.2
1.4 1.1
15.8 0.7 2.4
a Yield (mol %) from 2 (16.3 mmol). b Catechins and theaflavin completely disappeared, and large broad peaks due to polymeric products were observed. c Yields were
