New Antioxidative Phenolic Glycosides Isolated from Kokuto Non-centrifuged Cane Sugar

Biosci. Biotechnol. Biochem., 66 (1), 29 35, 2002 New Antioxidative Phenolic Glycosides Isolated from Kokuto Non-centrifuged Cane Sugar Kensaku TAKARA, Daigo MATSUI, KojiWADA, Toshio ICHIBA,* and Yoko NAKASONE Laboratory of Applied Biochemistry, Department of Bioscience and Biotechnology, Faculty of Agriculture, University of the Ryukyus, Senbaru 1, Nishihara-cho, Okinawa 903-0213, Japan *Okinawa Industrial Technology Center, Suzaki 12-2, Gushikawa-shi, Okinawa, 904-2234, Japan Received June 7, 2001; Accepted September 5, 2001 Nine compounds, 3-hydroxy-4,5-dimethoxyphenyl-b- D-glucopyranoside (1),b-D-fructfuranosyl-a-D-(6-vanilloyl)-glucopyranoside (2), b-d-fructfuranosyl-a-d-(6- syringyl)-glucopyranoside (3), 3-hydroxy-1-(4-hydroxy- 3-methoxyphenyl)-2-[4-(3-hydroxy-1-(E )-propenyl)- 2-methoxyphenoxy]propyl-b-D-glucopyranoside(4), 3- hydroxy-1-(4-hydroxy-3-methoxyphenyl)-2-[4-(3- hydroxy-1-(e )-propenyl)-2,6-dimethoxyphenoxy] propyl-b-d-glucopyranoside (5), dehydrodiconiferyl alcohol-9?-b-d-glucopyranoside (6), 4-[ethane-2-[3-(4- hydroxy-3-methoxyphenyl)-2-propen]oxy]-2,6-dimethoxyphenyl-b-d-glucopyranoside (7), 4-[ethane-2-[3-(4- hydroxy-3-methoxyphenyl)-2-propen]oxy]-2-methoxyphenyl-b-d-glucopyranoside (8), and 3-hydroxy-1-(4- hydroxy-3,5-dimethoxyphenyl)-2-[4-(3-hydroxy-1-(e )- propenyl)-2,6-dimethoxyphenoxy]propyl-b-d-glucopyranoside (9), were isolated from Kokuto noncentrifuged cane sugar. Their structures were elucidated by spectroscopic evidence, mainly based on the NMR technique. Among them, seven new glycosides were identiˆed. The 2-deoxyribose oxidation method was used to measure their antioxidative activity. All of these compounds showed antioxidative activities. Key words: cane sugar; Saccharum o cinarum L.; Kokuto; antioxidant; glycoside Kokuto has been traditionally manufactured from cane sugar in Okinawa and Amami by a unique noncentrifugal method. In our previous papers, 1,2) we have reported the isolation and identiˆcation of antioxidants from Kokuto. Phenolic compounds, which are of great interest for their radical-scavenging activities, are expected to be ešective in the prevention of many diseases and morbid states. As a continuation of our investigation of the constituents of Kokuto, we have isolated seven new phenolic glycosides (2, 3, and5 9), and two known phenolic glycosides (1 and 4). We report in this paper the structural elucidation of 1 9, on the basis of mass, UV, IR, and 1 H- and 13 C-NMR spectral data, including HMQC, HMBC and 1 H 1 H COSY experiments. The antioxidative activity of each identiˆed compound was evaluated by the deoxyribose oxidation method. Materials and Methods Material. Kokuto was produced from pressed juice of sugar cane (Saccharum o cinarum L.). Miyako Sugar Manufacturing Co. Ltd. (Tarama Plant, Okinawa, Japan) presented the sample material. Apparatus. UV spectra were recorded with a Shimadzu UV-160A spectrometer in methanol, and IRspectrawererecordedwithaBioRadFTS-3000 spectrophotometer. The spectra were measured in KBr pellets. 1 H- and 13 C-NMR spectra were obtained at 500 MHz and 125 MHz for 1 Hand 13 C, respectively, by a Jeol A-500 spectrometer. The 1 H signals and the 13 C signals of the solvent (CD 3OD) were used as secondary references (d3.30 and 49.0 from TMS, respectively). FAB- and APCI-MS data were obtained with a Jeol JMS-700 mass spectrometer. HRMS was performed in the FAB mode, using glycerol or m-nitrobenzyl alcohol as the matrix. Column chromatography was carried out in columus of XAD-2 (5.2 i.d. 16 cm, Organo), Wakogel C-100 (2.6 i.d. 42 cm, Wako Pure Chemical), Wakogel C-200 (1.4 i.d. 50 cm, Wako Pure Chemical), Sephadex LH-20 (1.0 i.d. 45 cm, Pharmacia), LiChroprep RP-8 (25 i.d. 310 mm, Merck) and LiChroprep Si60 (10 i.d. 240 mm, Merck). HPLC was performed in Develosil ODS-5 (8.0 i.d. To whom correspondence should be addressed. E-mail: k-takara@agr.u-ryukyu.ac.jp Abbreviations: HMQC, heteronuclear multiple-quantum coherence; HMBC, heteronuclear multiple-bond correlation; 1 H 1 HCOSY, 1 H 1 H correlation spectroscopy; FAB-MS, fast atom bombardment mass spectrometry; APCI-MS, atmospheric pressure chemical ionization mass spectrometry; HRMS, high-resolution mass spectrometry; OH, hydroxyl radical; BHA, butyl hydroxyanisole; TBA, thiobarbituric acid; TCA, trichloroacetic acid

30 K. TAKARA et al. Fig. 1. Isolation of Compounds 1 9. The ˆgures in parentheses show the antioxidative activity (percentage inhibition) for 5 mg ofeachreactionmixture. 250 mm, Nomura Chemicals) and Develosil 60-5 (4.6 i.d. 250 mm, Nomura Chemicals) columns with UV detection. Isolation and identiˆcation of the active compounds from Kokuto. Kokuto (2.8 kg) was suspended in distilled water (11.2 l) and then ˆltered. The resulting ˆltrate was passed through an Amberlite XAD-2 column, and eluted with H 2O (3 l), 20z methanol, 40z methanol, 60z methanol, 80z methanol, and straight methanol (600 ml each). The resulting ˆve fractions, excepting the H 2O fraction, were evaporated to dryness. The relative weight percentages of these fractions were 24.0, 34.3, 29.0, 10.3, and 2.5z, respectively. All fractions had radical-scavenging activity by the deoxyribose oxidation method (Fig. 1). The 40z methanol eluate, the one with the greatest of yield, was dissolved in water and partitioned with n-butanol. The water-saturated n- butanol layer was then evaporated in vacuo. The residue (1.94 g) was chromatographed in a silica gel column (Wako gel C-100, CH 2Cl 2 W MeOH, 10:1, 5:1, 2:1, and 1:1). The 5:1 eluate was subjected to silica gel column chromatography (LiChroprep Si60, EtOAc W MeOH, 10:0-1:1; LiChroprep PR-8, MeOH W H 2O, 1:5 1:1), and to preparative HPLC (Develosil ODS-5, MeCN WH 2OWHCOOH, 5:95:0.1) to give 1 (9.5 mg). The 2:1 eluate from the Wako gel C-100 column was further subjected to silica gel column chromatography (Wako gel C-200, EtOAcWMeOH, 10:0 1:1; LiChroprep PR-8, MeOH WH2O, 3:7 1:1), and to preparative HPLC (Develosil ODS-5, MeOH W H O 2 WHCOOH, 15:85:0.1) to give 2 (5.5 mg), 3 (3.9 mg), and 4 (4.7 mg). The 60z methanol eluate from the XAD-2 column, the one with the second greatest yield, was also dissolved in water and partitioned with n- butanol. The water-saturated n-butanol layer was then evaporated in vacuo. The residue (2.71 g) was chromatographed in a silica gel column chromatography (Wako gel C-100, CH 2Cl 2 WMeOH, 10:1, 5:1, 2:1. 1:1, and MeOH). The 5:1 eluate was subjected to silica gel column (Wako gel C-200, EtOAcW MeOH, 20:1, 10:1, 5:1, 2:1, 1:1, and MeOH), ašording 6 fractions, A1 A6. Fraction A4 was further chromatographed in a LiChroprep RP-8 column (MeOH WH 2O, 3:7 1:1) to give fractions B1 and B2. Fraction B1 was subjected to LH-20 column chromatography (MeOH WH2O, 1:5) and then to preparative HPLC (Develosil ODS-5, MeOHWH 2OW HCOOH, 30:70:0.1) to give 5 (3.4 mg) and 6

New Antioxidative Phenolic Glycosides Isolated from Kokuto 31 Fig. 2. Chemical Structures of Compounds 1 9 Isolated from Kokuto. (4.6 mg). Fraction B2 was subjected to HPLC (Develosil 60-5, CHCl 3 W MeOH W H 2O, 8:2:1, lower layer; and Develosil ODS-5, MeOH W H2O W HCOOH, 27:73:0.1) to give 7 (11.5 mg) and 8 (2.2 mg). Fraction A5 was chromatographed in a LiChroprep RP-8 column (MeOHWH 2O, 3:7 1:1) and then subjected to preparative HPLC (MeOH W H 2O W HCOOH, 30:70:0.1) to give 9 (9.3 mg). Compound 1. FAB-MS (positive, glycerol) m W z 333 [M+H] +.UVl max (MeOH) nm: 209, 224(sh), 270. IR n max cm 1 : 3400, 1603, 1508, 1102, 1075. 1 H- NMR (CD 3OD) d:6.33(1h,d,j=2.7 Hz, H-6), 6.27 (1H, d, J=2.7 Hz, H-2), 4.78 (1H, d, J=7.6 Hz, H-1?), 3.89 (1H, dd, J=12.1,2.0Hz,H-6?a), 3.79 (3H, s, 5-OMe), 3.71 (3H, s, 4-OMe), 3.68 (1H, dd, J=12.0,5.8Hz,H-6?b), 3.46 3.34 (4H, m, H-2?, 3?, 4?, 5?). 13 C-NMR (CD 3OD) d: 155.8 (C-1),154.9 (C-5), 151.8 (C-3), 133.2 (C-4), 102.9 (C-1?), 98.7 (C-2), 94.8 (C-6), 78.2 (C-5?), 78.0 (C-3?), 74.9 (C-2?), 71.5 (C-4?), 62.6 (C-6?), 61.1 (4-OMe), 56.4 (5-OMe). Compound 2. APCI-MS (negative) m Wz 491 [M H].UVl max (MeOH) nm: 208, 220, 263, 291. IR n max cm 1 : 3430, 1700, 1604, 1516, 1288, 1117, 1053. 1 H-NMR (CD 3OD) d: 7.59(1H,dd,J=8.9, 1.9 Hz, H-6), 7.50 (1H, d, J=2.0 Hz, H-2), 6.84 (1H, d, J=8.9 Hz, H-5), 5.40 (1H, d, J=3.7 Hz, H-1?), 4.57 (1H, dd, J=12.1,2.1Hz,H-6?a), 4.43 (1H, dd, J=12.1, 4.6 Hz, H-6?b), 4.13 (1H, ddd, J=9.9, 4.6, 2.3 Hz, H-5?), 4.09 (1H, d, J=8.4 Hz, H-3!), 3.99 (1H, dd, J=8.2, 8.2 Hz, H-4!), 3.90 (3H, s, 4-OMe), 3.76 3.72 (3H, m, H-3?, 1!a, 5!), 3.65 (1H, m, H-6!a),3.61(2H,m,H-1!b, 6!b), 3.45 (1H, m, H-2?), 3.43 (1H, m, H-4?). 13 C-NMR (CD 3 OD) d: 168.1 (C-7), 152.9 (C-4), 148.8 (C-3), 125.3 (C-6), 122.4 (C-1), 115.9 (C-5), 113.7 (C-2), 105.4 (C-2!), 93.6 (C-1?), 83.8 (C-5!), 79.3 (C-3!), 75.9 (C-4!), 74.5 (C-3?), 73.2 (C-2?), 72.2 (C-5?), 71.7 (C-4?), 64.9 (C-6?), 64.1 (C-6!), 63.8 (C-1!), 56.5 (4-OMe). Compound 3. APCI-MS (negative) mwz 521 [M H].UVl max (MeOH) nm: 217, 276. IR n max cm 1 : 3430, 1700, 1620, 1516, 1337, 1117, 1053. 1 H- NMR (CD 3 OD) d: 7.36(2H,s,H-2,6),5.42(1H,d, J=3.8 Hz, H-1?), 4.61 (1H, dd, J=12.1, 2.1 Hz, H- 6?a), 4.43 (1H, dd, J=12.1, 4.7 Hz, H-6?b), 4.14 (1H, ddd, J=9.9, 4.6, 2.6 Hz, H-5?), 4.09 (1H, d, J=8.4 Hz, H-3!), 3.98 (1H, dd, J=8.1, 7.9 Hz, H-4!), 3.89 (6H, s, 3, 6-OMe), 3.74 3.72 (3H, m, H-3?,1!a, 5!), 3.66 (1H, m, H-6!a),3.62(2H,m,H- 1b!, 6!b), 3.44 (2H, m, H-2?, 5?). 13 C-NMR (CD 3OD) d: 168.1 (C-7), 148.9 (C-3, 5), 143.0 (C-4), 121.3 (C-1), 108.4 (C-2, 6), 105.5 (C-2!), 93.6 (C-1?), 83.8 (C-5!), 79.3 (C-3!), 75.9 (C-4!), 74.5 (C-3?), 73.2 (C-2?), 72.3 (C-5?), 71.8 (C-4?), 65.1 (C-6?), 64.0 (C-6!), 63.7 (C-1!), 56.9 (3, 5-OMe).

32 K. TAKARA et al. Compound 4. APCI-MS (negative) m Wz 537 [M H].UVl max (MeOH) nm: 220(sh), 266. IR n max cm 1 : 3430, 1606, 1509, 1266, 1078, 1029. 1 H-NMR (CD 3OD) d: 7.07(1H,d,J=1.8 Hz, H-2), 7.05 (1H, d, J=1.8 Hz, H-3?), 6.95 (1H, d, J=8.2Hz, H-5), 6.89 (2H, dd, J=8.2, 1.8 Hz, H-6, 5?), 6.73 (1H, d, J=8.2 Hz, H-6?), 6.52 (1H, d, J=15.9 Hz, H-7?), 6.24 (1H, dt, J=15.9, 5.6 Hz, H-8?), 5.05 (1H, d, J=5.5 Hz, H-7), 4.56 (1H, d, J=7.6 Hz, H-1!), 4.50 (1H, dd, J=9.8, 5.5 Hz, H-8), 4.19 (2H, dd, J=5.5, 1.5 Hz, H-9?), 3.87 (3H, s, 3-OMe), 3.82 (3H, s, 2?-OMe), 3.80 (1H, br d, J=11.9Hz, H-9a), 3.70 (1H, dd, J=12.0, 2.4 Hz, H-6!a), 3.57 (1H, dd, J=12.0, 5.5 Hz, H-6!b), 3.44 (1H, dd, J=12.0, 5.2 Hz, H-9b), 3.34 (1H, m, H-5!), 3.25 (1H, dd, J=9.1, 7.6 Hz, H-4!), 3.27 (1H, dd, J=9.8, 2.1Hz, H-2!), 3.16 (1H, ddd, J=9.8, 5.5, 2.4 Hz, H-3!). 13 C-NMR (CD 3OD) d: 151.5 (C-3), 148.9 (C-2?), 148.6 (C-1?), 147.3 (C-4), 132.9 (C-1), 131.7 (C-4?), 131.4 (C-7?), 128.5 (C-8?), 121.4 (C-6), 120.7 (C-5?), 117.9 (C-5), 115.6 (C-6?), 112.6 (C-2), 111.2 (C-3?), 104.7 (C-1!), 85.1 (C-8), 81.3 (C-7), 78.0 (C-3!), 77.8 (C-5!), 75.7 (C-2!), 71.4 (C-4!), 63.8 (C-9?), 62.6 (C-6!), 61.5 (C-9), 56.5 (3 or 2?-OMe), 56.4 (3 or 2?-OMe). Compound 5. FAB-MS (positive, glycerol) mwz 569 [M+H] +. HR-FABMS: calcd. for C 27H 37O 13, 569.2234; found, 569.2250 [M+H] +. UV l max- (MeOH) nm: 218(sh), 271. IR n max cm 1 : 3420, 1590, 1506, 1226, 1123, 1078. 1 H-NMR (CD 3OD) d: 7.08 (1H, d, J=2.0 Hz, H-2), 6.92 (1H, dd, J=8.2, 1.8Hz,H-6),6.77(2H,s,H-3?, 5?), 6.75 (1H, d, J=8.1 Hz, H-5), 6.55 (1H, d, J=15.9 Hz, H-7?), 6.33 (1H, dt, J=15.7, 5.6 Hz, H-8?), 5.14 (1H, d, J=6.6 Hz, H-7), 4.59 (1H, d, J=7.6 Hz, H-1!), 4.29 (1H, dt, J=6.6, 3.6 Hz, H-8), 4.22 (2H, dd, J=5.6, 1.5 Hz, H-9?), 3.88 (6H, s, 2?-OMe, 6?-OMe), 3.85 (3H,s,3-OMe),3.74(1H,m,H-9a),3.60(2H,brdd, J=12.1, 3.7 Hz, H-9b, 6!a), 3.37 (1H, m, H-5!), 3.32 (2H, m, H-2!, 4!), 3.20 (1H, m, H-6!b), 3.18 (1H, m, H-3!). 13 C-NMR (CD 3OD) d: 154.5 (C-2?,6?), 148.5 (C-3), 147.2 (C-4), 136.1 (C-1?), 135.0 (C-4?), 131.9 (C-1), 131.3 (C-7?), 130.1 (C-8?), 121.3 (C-6), 115.6 (C-5), 112.5 (C-2), 105.2 (C-1!), 104.8 (C-3?, 5?), 87.0 (C-8), 82.2 (C-7), 78.1 (C-3!), 77.8 (C-5!), 75.6 (C-2!), 71.4 (C-4!), 63.6 (C-9?), 62.5 (C-9), 61.2 (C-6!), 56.7 (2?, 6?-OMe), 56.4 (3-OMe). Compound 6. FAB-MS (positive, glycerol) m W z 519 [M H] +. HR-FABMS: calcd. for C 26H 31O 11, 519.1866; found, 519.1876 [M H] +. UV l max (MeOH) nm: 218(sh), 278, 300(sh). IR n max cm 1 : 3425, 1610, 1520, 1076., 1030. 1 H-NMR (CD 3OD) d: 6.98 (1H, br s, H-6?), 6.96 (1H, br s, H-2?), 6.94 (1H, d, J=1.8 Hz, H-2), 6.82 (1H, dd, J=8.2, 1.8 Hz, H-6), 6.76 (1H, d, J=8.2 Hz, H-5), 6.62 (1H, d, J=16.0 Hz, H-7?), 6.23 (1H, br dt, J=16.0, 6.7 Hz, H-8?), 5.52 (1H, d, J=6.3 Hz, H-7), 4.49 (1H, ddd, J=12.5,5.9,1.4Hz,H-9?a), 4.36 (1H, d, J=7.8 Hz, H-1!), 4.30 (1H, ddd, J=12.5, 6.9, 1.4 Hz, H-9?b), 3.89 (1H, m, H-6!a), 3.87 (3H, s, 3?-OMe), 3.83 (1H, dd, J=11.0, 5.4 Hz, H-9a), 3.81 (3H, s, 3-OMe), 3.77 (1H, dd, J=11.1,7.0Hz,H-9b),3.67 (1H, dd, J=11.9, 5.4 Hz, H-6!b), 3.48 (1H, dd, J=6.4, 6.1 Hz, H-8), 3.36 3.26 (3H, m, H-3!, 4!, 5!), 3.21 (1H, dd, J=9.0, 8.2 Hz, H-2!). 13 C-NMR (CD 3OD) d: 149.4(C-4?), 149.1 (C-3), 147.6 (C-4), 145.5 (C-3?), 134.5 (C-1), 134.3 (C-7?), 132.4 (C-1?), 130.3 (C-5?), 124.2 (C-8?), 119.7 (C-6), 116.7 (C-6?), 116.2 (C-5), 112.1 (C-2?), 110.5 (C-2), 103.1 (C-1!), 89.4 (C-7), 78.1 (C-3!), 78.0 (C-5!), 75.1 (C-2!), 71.7 (C-4!), 71.0 (C-9?), 64.9 (C-9), 62.8 (C-6!), 56.7 (3?-OMe), 56.4 (3-OMe), 55.2 (C-8). Compound 7. FAB-MS (negative, glycerol) mwz 521 [M H]. HR-FABMS: calcd. for C 26 H 33 O 11, 521.2022; found, 521.2060 [M H]. UV l max (MeOH) nm: 268, 300(sh). IR n max cm 1 : 3400, 1600, 1512, 1275, 1123. 1 H-NMR (CD 3OD) d: 6.92(1H,s, H-2),6.75(1H,d,J=8.5 Hz, H-6), 6.58 (1H, d, J= 8.2Hz,H-5),6.54(2H,s,H-2?, 6?), 6.20 (1H, d, J= 16.0 Hz, H-7), 5.95 (1H, dd, J=15.8, 8.0 Hz, H-8), 4.77 (1H, d, J=7.5 Hz, H-1!), 3.83 (3H, s, 3-OMe), 3.77 (6H, s, 3?, 5?-OMe), 3.72 (1H, m, H-6!a), 3.64 (1H, ddd, J=12.1,5.3,1.7Hz,H-6!b),3.58(2H,d, J=5.8 Hz, H-9), 3.45 (1H, m, H-2!), 3.40 3.34 (2H, m, H-4!, 5!), 3.34 (2H, m, H-8?), 3.18 (1H, m, H-3!), 2.86 (1H, dd, J=12.8, 4.6 Hz, H-7?a), 2.60 (1H, dd, J=12.7, 8.6Hz, H-7?b). 13 C-NMR (CD 3OD) d: 153.8 (C-3?, 5?), 149.0 (C-3), 147.1 (C-4), 138.6 (C-1?), 134.5 (C-4?), 132.8 (C-7), 131.3 (C-1), 129.3 (C-8), 120.5 (C-6), 116.1(C-5), 110.2 (C-2), 108.5 (C-2? or 6?), 108.4 (C-2? or 6?), 105.6 (C-1!), 78.3 (C-3!), 77.8 (C-5!), 75.7 (C-2!), 71.3 (C-4!), 66.1 (C-9), 62.5 (C-6!), 57.0 (3? or 5?-OMe), 56.9 (3? or 5?-OMe), 56.3 (3-OMe), 49.9 (C-8?), 39.2 (C-7?). Compound 8. FAB-MS (negative, glycerol) m W z 491 [M H]. HR-FABMS: calcd. for C 25H 31O 10, 491.1917; found, 491.1906 [M H]. UV l max (MeOH) nm: 270, 300(sh). IR n max cm 1 : 3430, 1628, 1514, 1072. 1 H-NMR (CD 3OD) d: 7.04(1H,d,J= 8.2 Hz, H-5?), 6.91 (1H, d, J=1.8 Hz, H-2), 6.83 (1H, d, J=1.8 Hz, H-2?), 6.75 (1H, dd, J=8.2, 1.8 Hz, H-6 or 6?), 6.74 (1H, dd, J=8.4, 2.0 Hz, H-6 or 6?), 6.68 (1H, d, J=8.1 Hz, H-5), 6.18 (1H, d, J= 16.0 Hz, H-7), 5.94 (1H, dd, J=15.9, 8.4 Hz, H-8), 4.83 (1H, d, J=7.5 Hz, H-1!), 3.86 (1H, m, H-6!a), 3.83 (3H, s, 3-OMe), 3.77 (3H, s, 3?-OMe), 3.67 (1H, dd, J=12.0, 4.9 Hz, H-6!b), 3.56 (2H, d, J=6.1Hz, H-9),3.45(2H,m,H-2!, 5!), 3.37 (2H, m, H-3!, 4!), 3.34 (2H, m. H-8?), 2.86 (1H, dd, J=13.0, 8.6 Hz, H-7?a), 2.60 (1H, m, H-7?b). 13 C-NMR

(CD 3OD) d: 150.3 (C-3?), 149.0 (C-3), 147.0 (C-4), 146.2 (C-4?), 136.7 (C-1?), 132.7 (C-7), 131.3 (C-1), 129.4 (C-8), 123.0 (C-6?), 120.5 (C-6), 117.7 (C-5?), 116.1 (C-5), 115.1 (C-2?), 110.2 (C-2), 102.9 (C-1!), 78.1 (C-3!), 77.8 (C-5!), 74.9 (C-2!), 71.3 (C-4!), 66.2 (C-9), 62.5 (C-6!), 56.7 (3?-OMe), 56.3 (3-OMe), 48.7 (C-8?), 38.6 (C-7?). Compound 9. FAB-MS (positive, m-nitrobenzyl alcohol) m W z 622 [M+Na] +,599[M+H] +.FAB-MS (glycerol, negative) m Wz 597 [M H].HR-FABMS (glycerol): calcd. for C 28H 37O 14, 597.2183; found, 597.2163 [M H]. UV l max(meoh) nm: 221(sh), 269. IR n max cm 1 : 3430, 1620, 1224, 1121. 1 H-NMR (CD 3 OD) d: 6.80(2H,s,H-2,6),6.77(2H,s,H-3?, 5?), 6.55 (1H, d, J=15.7 Hz, H-7?), 6.33 (1H, dt, J=15.9, 5.6 Hz, H-8?), 5.14 (1H, d, J=6.1 Hz, H-7), 4.57 (1H, d, J=7.6 Hz, H-1!), 4.31 (1H, dt, J=6.3, 3.6 Hz, H-8), 4.22 (2H, dd, J=5.6, 1.4 Hz, H-9?), 3.87 (6H, s, 2?, 6?-OMe), 3.84 (6H, s, 3, 5-OMe), 3.74 (1H, dd, J=12.0,2.3Hz,H-6!a), 3.61 (2H, m, H-9a, 6!b),3.37(1H,m,H-5!), 3.35 3.33 (2H, m, H-2!,4!), 3.23 (1H, dd, J=12.4, 3.9 Hz, H- 9b), 3.20 (1H, m H-3!). 13 C-NMR (CD 3OD) d: 154.5 (C-2?, 6?), 148.8 (C-3, 5), 136.2 (C-4), 136.1 (C-1?), 135.0 (C-4?), 131.3 (C-7?), 131.0 (C-1), 130.1 (C-8?), 106.0 (C-2, 6), 105.1 (C-1!), 104.8 (C-3?, 5?), 86.9 (C-8), 82.3 (C-7), 78.1 (C-3!), 77.8 (C-5!), 75.6 (C-2!), 71.4 (C-4!), 63.6 (C-9?), 62.5 (C-6!), 61.3 (C-9), 56.8 (3, 5-OMe), 56.7 (2?, 6?-OMe). 2-Deoxyribose oxidation method. 2-Deoxyribose is oxidized by OH that is formed by the Fenton reaction, and degraded to malondialdehyde. 3,4) The reaction mixture (2.0 ml) containing a test compound (0.5 mm in ˆnal concentration), 14 mm 2-deoxyribose (0.2 ml), 1.25 mm H 2O 2 (0.1 ml), a 34 mm phosphate bušer (ph 7.4, 1.6 ml), 2 mm FeCl 3 (50 ml), and 2.08 mm EDTA (50 ml) was incubated at 379C for 2 h. The extent of deoxyribose degradation was measured by the TBA method, whereby 1 ml of 1z TBA and 1 ml of 2.8z TCA were added to the mixture, which was then heated in a water bath at 1009C for 10 min. The absorbance of the resulting solution was measured spectrophotometrically at 532 nm. The control was without a test compound, and the blank was without incubation, BHA being used as a positive control. The percentage inhibition was determined as inhibition (z)=100 100(A t A 1)W(A 0 A 1), where A 0, A 1,andA t are the absorbance values for the control, blank, and the added test sample, respectively. The percentage inhibition was determined from the means of three independent experiments. Results and Discussion The 40z methanol and 60z methanol eluent fractions from the XAD-2 column were fractionated by a New Antioxidative Phenolic Glycosides Isolated from Kokuto combination of partition with n-butanol and water, and chromatography in silica gel, RP-8, LH-20 and ODS columns to yield nine phenolic compounds. Compounds 1 and 4 were identiˆed as 3-hydroxy- 4,5-dimethoxyphenyl-b-D-glucopyranoside 5) and 3-hydroxy-1-(4-hydroxy-3-methoxyphenyl)-2-[4-(3- hydroxy-1-(e )-propenyl)-2-methoxyphenoxy] propyl-b-d-glucopyranoside, 6) respectively. Compound 2 was isolated as a white amorphous powder. Its molecular formula, C 20 H 28 O 14, was deduced from an [M H] peak at 491 of negative APCI-MS, and 20 carbon signals in the 13 C-NMR spectrum. The 1 H-NMR spectrum contained the signals for three aromatic protons at d7.59 (1H, dd, J=8.9, 1.9 Hz), 7.50 (1H, d, J=2.0 Hz), and 6.84 (1H, d, J=8.9 Hz), corresponding to a typical 1,2,4- trisubstituted phenyl group. The 13 C-NMR spectrum exhibited a carbonyl group at d168.1 and a methoxyl group at d56.5. In the HMBC spectrum, the carbonyl group at d168.1 and the aromatic carbon at d148.8 showed correlation with aromatic protons at d7.59 and methoxyl protons at d3.90, respectively. In addition, the absorption at 1700 cm 1 in the IR spectrum and maximum absorption at 263 and 291 nm in the UV spectral data suggest 2 to have an vanilloyl moiety. The remaining 12 carbon signals are in good agreement with those of the sucrose. The linkage of the isovanilloyl moiety and sucrose was solved by an analysis of the HMBC spectrum. The carbonyl group at d168.1 showed correlation with the proton at d4.43, suggesting the vanilloyl moiety to be connected to C-6? of the glucoside moiety. This was further supported by the results of the 13 C-NMR analysis, which showed a downˆeld shift of the resonance of C-6 of glucose, as well as an upˆeld shift of the neighboring C-5 resonance when compared with those of sucrose. The assignment of the proton and carbon signals was achieved by a combination of the HMBC and 1 H 1 H COSY spectral data. These results enabled compound 2 to be determined as b-d-fructfuranosyl-a-d-(6-vanilloyl)-glucopyranoside. Compound 3, a white amorphous powder, showed an [M H] peak at m W z 521 in the negative APCI- MS data. The 1 H-NMR spectrum of 3 was very similar to that of 2, except for the presence of two equivalent methoxyl groups at d3.89 (6H, s) and two equivalent aromatic protons at d7.36 (2H, s). These facts suggested that the aglycone moiety of 3 was a 4- hydroxy-3,5-dimethoxybenzoic (syringyl) moiety. The remaining proton signals and carbon signals are in good agreement with those of 2. The assignment of the proton and carbon signals was achieved by comparing with those of the corresponding signals for 2, and by a combination of the HMBC and 1 H 1 H COSY spectral data. Compound 3 was therefore determined to be b-d-fructfuranosyl-a-d-(6-syringyl)- glucopyranoside. Compound 5 wasobtainedasacolorlessoil.the 33

34 K. TAKARA et al. HR positive ion FABMS data corresponded to a molecular formula of C 27H 37O 13, havingonemore methoxyl group than 4. The NMR spectra of 5 were similar to those of 4. The presence of one extra methoxyl group was conˆrmed in the 1 H-NMR spectrum, in which the methoxyl proton peak was observed as one singlet peak of 6H at d3.88. Of the aromatic proton signals, one singlet peak for two protons was observed at d6.77, suggesting the presence of a 2,6-dimethoxyhydroxyphenyl residue. The proton and carbon signals due to glucose were observed with the same chemical shifts as those in 4, and the coupling constant of a anomeric proton of d4.59 was J=7.6Hz, showing a b-linkage. The assignment of the proton and carbon signals was achieved by a combination of 1 H 1 H COSY and HMQC spectral data. The structure of 5 was determined to be 3-hydroxy-1-(4-hydroxy-3- methoxyphenyl)-2-[4-(3-hydroxy-1-(e )-propenyl)- 2,6-dimethoxyphenoxy]propyl-b-D-glucopyranoside. Compound 6, a colorless oil, showed an [M H] peak at mwz 519.1876, suggesting the molecular formula C 26H 32O 11. The 13 CNMRspectrumof6 was similar to that of dehydrodiconiferyl alcohol in the preceding paper of this series. 1) The remaining six carbon signals were in good agreement with those of the b-glucoside. In the HMBC spectrum, the carbon signal at d71.0 showed correlation with the anomeric proton at d4.36, suggesting the glucose moiety to be connected to the propenyl moiety. The assignment of the proton and carbon signals was achieved by a combination of 1 H 1 H COSY and HMQC spectral data. The structure of 6 was thus determined to be 3-[2,3-dihydro-3-(hydroxymethyl)-7-methoxy-2-(4- hydroxy-3-methoxyphenyl)-5-benzofuranyl]-2- propenyl-b-d-glucoside. Compound 7 was isolated as a white amorphous powder. Its molecular formula was established as C 26H 34O 11 by the HR negative ion FABMS data which showed a molecular ion peak at m W z 521.2060 [M H].The 1 H-NMR spectrum of 7 exhibited the signals for an (E )-coniferyl alcohol moiety and 4- hydroxy-3,5-dimethoxyphenyl ethanol moiety. The 13 C-NMR data showed the signals due to a glucopyranose moiety. In the 1 H-NMR spectrum, the coupling constant of an anomeric proton of d4.77 was J=7.5 Hz, showing a b-linkage. Additionally, the HMBC spectrum showed the correlation of C-8? at d49.9 and C-4? at d134.5 with H-9 at d3.58 and H-1! at d4.77, respectively. Thus, the (E )-coniferyl alcohol moiety and 4-hydroxy-3,5-dimethoxyphenyl ethanol moiety must have been attached to C-9 and C-8? by an -O-linkage, and the glucopyranose moiety must also have been attached to C-4?. Therefore, the structure of 7 was determined to be 4-[ethane-2-[3- (4-hydroxy-3-methoxyphenyl)-2-propen]oxy]-2,6- dimethoxyphenyl-b-d-glucopyranoside. Compound 8 was isolated as a white amorphous powder. Its molecular formula was established as C 25H 32O 10 by the HR negative ion FABMS data which showed a molecular ion peak at m W z 491.1906 [M H].The 1 H-NMR spectrum of 8 was similar to that of 7, showing the absence of one methoxyl group and the presence of a 1?,2?,4?-trisubstituted aromatic ring at d7.04 (1H, d, J=8.2Hz, H-5?), d6.83 (1H, d, J=1.8Hz, H-2?), and d6.74 (1H, dd, J=8.4, 2.0 Hz, H-6?). Compared with the NMR spectra of 7 and 8, compound 8 was determined to be 4-[ethane-2-[3- (4-hydroxy-3-methoxyphenyl)-2-propen]oxy]-2- methoxyphenyl-b-d-glucopyranoside. Compound 9 was isolated as a colorless oil. Its molecular formula was established as C 28H 38O 14 by negative the HR-FABMS data which showed a molecular ion peak at m W z 597.2163 [M H].The UV and IR spectra of 9 were similar to those of 4 and 5. The NMR spectra of 9 were very similar to those of 5, except for the presence of two equivalent methoxyl groups at d3.85 (6H, s) and two equivalent aromatic protons at d6.80 (2H, s). Therefore, the structure of 9 was determined to be 3-hydroxy-1-(4-hydroxy-3,5- dimethoxyphenyl)-2-[4-(3-hydroxy-1-(e )-propenyl)- 2,6-dimethoxyphenoxy]propyl-b-D-glucopyranoside. The 20z methanol fraction from the XAD-2 column had comparatively high antioxidative activity, even though it was di cult to isolate for complex component. A more detailed report of this fraction will be presented later. On the other hand, the 80z methanol and methanol eluent fractions from the XAD-2 column, which had comparatively high antioxidative activity too, were combined and fractionated by the method already described. Many phenolic compounds that were reported in previous papers 1,2) were also isolated. Phenolic compounds are widely distributed in nature, and many have been isolated from cane-related substances. The free aglycones of 3 and 6 have been reported in our previous paper, 1) and some aromatic glycosides, for example tachioside and arbutin, have been isolated from Kokuto 7) and cane molasses. 8,9) On the other hand, many avonoids have been reported as raw sugar pigments. 10) In this paper, however, isolated glycoside compounds 2, 3, and5 9 have not been previously reported. Kokuto is manufactured from sugar cane juice by concentrating under heating and variation of the ph value. It is not therefore clear whether the compounds isolated in this study were natural or artifacts. The hydroxy radical-scavenging activities of the isolated compounds were measured by the deoxyribose oxidation method and compared to the synthetic antioxidant, BHA. As shown in Fig. 3, all compounds showed radical-scavenging activity. The scavenging activity order of the test compounds was 2À3À4, 8, BHAÀ5, 7, 9À6À1. Each test compound had a phenolic hydroxyl group in its structure,

New Antioxidative Phenolic Glycosides Isolated from Kokuto References 35 Fig. 3. Hydroxyl Radical-scavenging EŠects by the Deoxyribose Oxidation Method. Each value is the mean±standard deviation (SD), n=3. and phenolic antioxidants have been recognized to function as electron or hydrogen donors. Thus, the hydroxy radical-scavenging activity of these compounds may be mostly related to the phenolic hydroxyl group. Furthermore, the antioxidative potency depended on the stability of the radical formed via increased electron delocalization. 11) Compounds 2 and 3, with relatively high activity, may involve the carboxyl adjacent to the phenolic group participating in stabilizing the radical by resonance. On the other hand, compound 6, which is a glycoside of a compound found in our previous work, 1,2) was less e cient than the corresponding aglycon. We suggest that the presence of the glucosyl group in compound 6 did not improve its e ciency. The hydroxy radical-scavenging activity of these compounds will be further examined by various assays. 1) Takara, K., Kinjyo, A., Matsui, D., Wada, K., Nakasone, Y., and Yogi, S., Antioxidative phenolic compounds from the non-sugar fraction in Kokuto, non-centrifuged cane sugar. Nippon N šogeikagaku Kaishi (in Japanese), 74, 885 890 (2000). 2) Nakasone, Y., Takara, K., Wada, K., Tanaka, J., and Yogi, S., Antioxidative compounds isolated from Kokuto, non-centrifuged cane sugar. Biosci. Biotechnol. Biochem., 60, 1714 1716 (1996). 3) Gutteridge, J. M. C., Reactivity of hydroxyl and hydroxyl-like radicals discriminated by release of thiobarbituric acid-reactive material from deoxy sugars, nucleosides and benzoate. Biochem. J., 224, 761 767 (1984). 4) Gutteridge, J. M. C., Ferrous-salt-promoted damage to deoxyribose and benzoate. Biochem. J., 243, 709 714 (1987). 5) Li,Y.C.andKuo,Y.H.,Amonoterpenoidandtwo simple phenols from heartwood of Ficus microcarpa. Phytochem., 49, 2417 2419 (1998). 6) Kraus, R. and Spiteller, G., Lignan glucosides from roots of Urtica dioica. Liebigs Ann. Chem., 1205 1213 (1990). 7) Matsuura, Y., Kimura, Y., and Okuda, H., EŠect of aromatic glucosides isolated from black sugar on intestinal absorption of glucose. Wakan-Yaku, 7, 168 172 (1990). 8) Palla, G., Isolation and identiˆcation of phenolic glucosides in liquid sugars from cane molasses. J. Agric. Food Chem., 30, 764 766 (1982). 9) Palla, G., Characterization of the main secondary components of the liquid sugars from cane molasses. J. Agric. Food Chem., 31, 545 548 (1983). 10) Patron, N. H., Smith, P., and Mabry, T. J., Identiˆcation of avonoid compounds in HPLC separation of sugar cane colorants. Int. Sugar J., 87, 213 215 (1985). 11) Cuvelier, M. E., Richard, H., and Berset, C., Comparison of the antioxidative activity of some acid-phenols: structure-activity relationship. Biosci. Biotechnol. Biochem., 56, 324 325 (1992).