3310 J. Agric. Food Chem., Vol. 51, No. 11, 2003
Nakamura et al.
(2H, s, 3′, 5′-H), 6.69 (1H, d, J ) 8 Hz, 3, 5-H), 7.05 (2H, d, J ) 8
Hz, 2, 6-H), 9.10 (1H, brs, 4-OH), 12.31 (2H, brs, 2′, 6′-OH). 13C NMR
(75 MHz, (CD3)2SO): δ 18.1 (Rha-CH3), 29.3 (â), 45.7 (R), 60.3 (Glu-
6), 68.3, 69.5, 70.4, 70.5, 71.9 (Glu-4, Rha-2, 3, 4, 5), 76.6, 76.8, 77.1
(Glu-2, 3, 5), 94.8 (3′), 94.8 (5′), 97.1 (Rha-1), 100.6 (Glu-1), 105.3
(1′), 115.1 (3), 115.1 (5), 129.1 (2), 129.1 (6), 131.5 (1), 155.4 (4),
162.9 (4′), 163.7 (2′), 163.7 (6′), 205.1 (CdO). FAB-MS (matrix;
glycerol) m/z: 583 ([M + H]+). To a solution of 9b (1.2 g) in
methanol-acetonitrile (1:9, v/v, 50 mL) was added diisopropylamine
(0.26 g) and 2.0 M trimethylsilyldiazomethane solution in hexane (3
mL), and the mixture was kept at room temperature for 1 h while
stirring. Then, after 20% HCl (30 mL) added, the mixture was stirred
in a water bath (100 °C) for 30 min. The mixture was neutralized with
sodium hydroxide and then extracted with ethyl acetate. After the ethyl
acetate layer was concentrated in vacuo, the final purification was done
by absorption column chromatography with Diaion HP-20 resin to
afford the product as crystals. This product (236 mg, 41%) was identical
to 7b on the basis of the spectroscopic data as previously reported (11).
Compound 10b was also prepared by 4′-position specific methylation
of 11b according to the same procedure using trimethylsilyldiaz-
omethane. Compound 10b (39%): 1H NMR (500 MHz, (CD3)2SO):
δ 2.80 (2H, m, â), 3.30-3.70 (8H, R and sugar proton), 3.78 (3H, s,
4′-OCH3), 4.62 (1H, t, J ) 5 Hz, Glu-6-OH), 5.01 (1H, d, J ) 7 Hz,
anomeric proton), 5.05, 5.14, 5.30 (3H, d, J ) 5 Hz, Glu-2,3,4-OH),
6.13 (1H, d, J ) 2 Hz, 5′-H), 6.29 (2H, d, J ) 2 Hz, 3′-H), 6.64 (2H,
d, J ) 8 Hz, 3, 5-H), 7.03 (2H, d, J ) 8 Hz, 2, 6-H), 9.08 (1H, s,
4-OH), 13.34 (1H, s, 6′-OH). 13C NMR (75 MHz, (CD3)2SO): δ 28.8
(â), 45.1 (R), 55.6 (OCH3), 60.7 (Glu-6), 69.7 (Glu-4), 73.2 (Glu-2),
76.7 (Glu-5), 77.4 (Glu- 3), 93.5 (3′), 95.1 (5′), 100.8 (Glu-1), 106.2
(1′), 115.0 (3), 115.0 (5), 129.1 (2), 129.1 (6), 131.4 (1), 155.3 (4),
160.2 (2′), 164.9 (6′), 165.1 (4′), 205.1 (CdO). FAB-MS (matrix;
glycerol) m/z: 451 ([M + H]+). To a solution of 10b (300 mg) in
ethanol was added 20% HCl (9 mL), and the mixture was stirred in a
water bath (100 °C) for 30 min. The mixture was neutralized with
sodium hydroxide and then extracted with ethyl acetate. After the ethyl
acetate layer was concentrated in vacuo, the final purification was done
by absorption column chromatography with Diaion HP-20 resin to
afford the product as crystals. This product (163 mg, 84%) was identical
to 8b on the basis of the spectroscopic data as previously reported (11).
Evaluation of 1,1-Diphenyl-2-picrylhydrazyl (DPPH) Radical
Scavenging Activity. The DPPH radical scavenging activity was
evaluated as reported previously (14). A test compound (ethanol
solution, 2 mL) mixed with a 100 mM Tris-HCl buffer (pH 7.4, 2 mL)
was added to 0.5 mM DPPH in ethanol (1 mL), and the mixture was
shaken vigorously and kept for 20 min at room temperature in the dark.
The DPPH radical scavenging activity is expressed as the ratio of the
relative decrease in the absorbance of the test sample mixture at 517
nm to that of the 1 mM Trolox solution. The experiment was done in
triplicate. DPPH radical scavenging activity (%) ) {(ethanol alone) -
(test compound)}/{(ethanol alone) - (Trolox)} × 100.
Figure 1. Structures of flavanones (group a) and dihydrochalcones (group
b). ONeo, O-neohesperidoside; ORut, O-rutinoside; OGlu, O-glucoside.
France). Isosakuranetin (2a) was obtained from Apin Chemicals, Ltd.
(Abingdon, U.K.). Hesperetin (3a) and phloridzin (11b) were obtained
from Tokyo Chemical Industry Co., Ltd. (Tokyo, Japan). Hesperidin
(6a) was purchased from Alps Pharmaceutical Industry Co., Ltd. (Gifu,
Japan). The structures of flavanones and dihydrochalcones are shown
in Figure 1. All other chemicals were purchased from Wako Pure
Chemical Industries (Osaka, Japan).
Preparation of Dihydrochalcones. Isosakuranetin dihydrochalcone
(2b), hesperetin dihydrochalcone (3b), eriodictyol dihydrochalcone (4b),
and hesperidin dihydrochalcone (6b) were prepared by hydrogenation
of flavanones as previously reported (10). Briefly, 5% palladium on
carbon (1 g) was added to a flavanone solution (10 mmol) in 10%
potassium hydroxide (50 mL). The mixture was shaken with hydrogen
(3 kg/cm2) at 30 °C for 30 min. After it was filtered, the filtrate was
neutralized with hydrogen chloride and recrystallized. The final
purification was done by absorption column chromatography with
Diaion HP-20 resin to afford the corresponding dihydrochalcones as
crystals in good yields (∼80%). The corresponding products were
identical to 2b, 3b, and 4b as previously reported (11-13). Compound
6b: 1H NMR (500 MHz, CD3OD): δ 1.17 (3H, d, J ) 6 Hz, rha-
CH3), 2.76 (2H, t, J ) 7 Hz, R), 3.24 (2H, t, J ) 7 Hz, â), 3.30-3.96
(10H, d, sugar), 3.78 (3H, s, 4-OCH3), 4.80, 5.04 (2H, d, J ) 7 Hz,
anomeric proton), 6.10 (2H, s, 3′, 5′-H) 6.66 (1H, dd, J ) 2, 8 Hz,
6-H), 6.72 (1H, d, J ) 2 Hz, 2-H), 6.84 (1H, d, J ) 8 Hz, 5-H). 13C
NMR (75 MHz, CD3OD): δ 17.4 (Rha-CH3), 30.8 (â), 46.4 (R), 56.9
(OCH3), 66.8 (Glu-6), 69.6, 70.4, 71.1, 71.3, 73.2 (Glu-4, Rha-2, 3, 4,
5), 73.8, 76.0, 76.6 (Glu-2, 3, 5), 96.6 (3′), 96.6 (5′), 99.9 (Glu-1),
101.3 (Rha-1), 107.1 (1′), 113.6, 116.5 (2), 121.2 (6), 135.7 (1), 144.5
(3), 146.6 (4), 163.8 (4′), 164.1 (2′), 164.1 (6′), 208.1 (CdO). FAB-
MS (matrix; glycerol) m/z: 613 ([M + H]+).
Antioxidant Assay of Rabbit Erythrocyte Membrane Ghost
System. Commercially available rabbit blood was obtained from Japan
Biotest Institute Co., Ltd. The preparation of erythrocyte membrane
ghost and peroxidation induced by tert-butylhydroperoxide were
performed by the procedure previously reported (15). In brief, rabbit
blood (100 mL) was diluted with 300 mL of 10 mM phosphate buffer
(pH 7.4) containing 152 mM NaCl. After it was centrifuged (3500 rpm,
20 min), the blood was lysed in 300 mL of 10 mM phosphate buffer
(pH 7.4) without NaCl. The erythrocyte membrane ghosts were pelleted
by centrifugation (11 500 rpm, 40 min), and the precipitate was diluted
to give a suspension (1 mg protein/mL). The test compound dissolved
in 25 µL of dimethyl sulfoxide and 25 µL of 24 mM tert-butylhydro-
peroxide aqueous solution were added to the ghost suspension (450
µL), and the mixture thus obtained was incubated at 37 °C for 20 min.
The quantity of thiobarbituric acid-reacting substances was determined
according to the method of Osawa et al. (16). The experiment was
done in triplicate.
Preparation of 2′-O-Methyl and 4′-O-Methyl Derivatives of 1b.
2′-Methyl-phloretin (7b) and 4′-O-methyl-phloretin (8b) were prepared
by methylation and sugar hydrolysis of naringin dihydrochalcone (9b)
and asebotin (10b), respectively. Compound 9b was prepared by the
reduction of narindin (9a) according to the procedure described above
(10). Compound 9b: 1H NMR (500 MHz, (CD3)2SO): δ 1.21 (3H, d,
J ) 6 Hz, Rha-CH3), 2.81 (2H, m, R), 3.24 (2H, t, J ) 7 Hz, â), 3.25-
5.30 (sugar proton), 5.13 (2H, d, J ) 7 Hz, anomeric proton), 6.04
RESULTS AND DISCUSSION
Antioxidant Activities of Dihydrochalcones and Fla-
vanones. Antioxidant assays were performed by a DPPH