College of Pharmaceutical Sciences, Matsuyama University, 4-2 Bunkyo-cho, Matsuyama, Ehime , Japan

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1 (379) Biosci. Biotechnol. Biochem., 75 (11), , 2011 Polyphenolic Compounds in Clove and Pimento and Their Antioxidative Activities Morio YSHIMURA, Yoshiaki AMAKURA, and Takashi YSHIDA y College of Pharmaceutical Sciences, Matsuyama University, 4-2 Bunkyo-cho, Matsuyama, Ehime , Japan Received June 29, 2011; Accepted July 31, 2011; nline Publication, November 7, 2011 [doi: /bbb ] Two new polyphenolic glucosides, acetylisobiflorin (1) and (2S)-3-(4-hydroxy-3-methoxyphenyl)-propane-1,2-diol 1--(6 0 --galloyl)--d-glucoside (2), were respectively isolated from the flower buds of Syzygium aromaticum and berries of Pimenta dioica. Each structure was elucidated on the basis of spectral analyses (NMR, MS and ½Š D ) and chemical conversion. A total of twenty-seven known compounds from the plants were also characterized. The antioxidative activity of their extracts and the twenty-nine compounds, including gallo- and ellagitannins from among the isolates, was estimated by oxygen radical absorbance capacity (RAC) assay, and eugenol (3), which was the most abundant ingredient in each plant extract, showed the most potent antioxidative activity [RAC value of 39,270 mol TE (trolox equivalent)/g]. Key words: clove; pimento; polyphenol; antioxidant; oxygen radical absorbance capacity (RAC) The consumption of polyphenol-rich foods and beverages is considered to decrease the risks of cardiovascular diseases, 1,2) arterial sclerosis 3) and other diseases related to oxidative stress. 4) Clove (flower buds of Syzygium aromaticum) and pimento (berries of Pimenta dioica), which belong to the family Myrtaceae, have long been used as well-known condiments and reported to be rich in polyphenols, especially hydrolyzable tannins. 5,6) These plant sources also contain considerable amounts of eugenol (3), a volatile aromatic compound, which is known to show strong antioxidative activity. 7) Although eugenol is a hydrophobic antioxidant, hydrophilic constituents such as hydrolyzable tannins and flavonoids may make large contributions to the antioxidative activity of these plants. In our continuing study on natural polyphenolic compounds from myrtaceous plants, 8) we re-examined the correlation between the polyphenolic composition and antioxidative effect of these classical condiments, and isolated two new polyphenolic glucosides (1 and 2). Compound 1 obtained from clove was characterized as acetylisobiflorin, and compound 2 from pimento was elucidated as (2S)-3-(4-hydroxy-3-methoxyphenyl)- propane-1,2-diol 1--(6 0 --galloyl)--d-glucoside on the basis of spectral analysis and chemical conversion. Their structural elucidation and antioxidative activities evaluated by the oxygen radical absorbance capacity (RAC) are described in this paper. Materials and Methods General. ptical rotation data were recorded on a Jasco P-1030 polarimeter. NMR spectra were recorded on a Bruker AVANCE 500 instrument (500 MHz for 1 H and 126 MHz for 13 C), with chemical shifts given in (ppm) values relative to those of the solvent [Med 4 ( H 3.30, C 49.0) and acetone-d 6 ( H 2.05, C 29.8)] on a TMS scale. HR-ESI-MS spectra were obtained with a Bruker micrtf-q mass spectrometer. Normal-phase HPLC was conducted in a YMCpack SIL A-002 column (4:6 250 mm, YMC Co.) eluted with n-hexane:me: tetrahydrofuran:hc (55:33:11:1) containing oxalic acid (450 mg/l). Reversed-phase HPLC was performed with a Shimadzu LC-10Avp system, using a YMC-pack DS-AQ column (2:0 150 mm) at 40 C with flow rate of 0.25 ml/min. The analyses were conducted with solvent A, a 100 mm phosphate buffer, and solvent B, CH 3 CN, with a linear gradient of 0 50% B from 0 30 min and then 50 60% B from min, with monitoring at 280 nm. Analytical and preparative chiral HPLC were conducted in a Chiral Pak IA column (4:6 150 mm, Daicel Chemical) eluted with n-hexane:2-propanol (92:8) at 35 C and a flow rate of 1.0 ml/min. Column chromatography was carried out on Silica gel 60 (75 mm, Nacalai Tesque), Diaion HP-20, MCI-gel CHP20P (Mitsubishi Kasei Co.), Toyopearl HW-40 (coarse grade, Tosoh Co.), YMC-gel DS AQ12S50 (YMC Co.) and Sephadex LH-20 (GE Healthcare Japan). Samples and reagents. Clove (flower buds of S. aromaticum) and pimento (berries of P. dioica) were obtained from Nagaoka Perfumery Co. (saka, Japan). Tannase prepared from Aspergillus niger, osmium tetroxide (immobilized catalyst I), N-methylmorpholine N-oxide, 2,2 0 - azobis-(2-amidinopropane)-dihydrochloride (AAPH), L-cystein methyl ester hydrochloride and o-tolyl isothiocyanate were purchased from Wako Pure Chemical Industry (saka, Japan). Fluorescein sodium salt and 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (trolox) were purchased from Sigma-Aldrich. Isolation procedure for clove. Clove (dried flower buds of S. aromaticum, 570 g) was macerated with 80% Et (6 L) for 48 h at room temperature. The filtrate was concentrated in vacuo to 1 L at below 40 C and then successively extracted with n-hexane (1L 3), EtAc (1L 3) and n-bu (1L 3) to give n-hexane (100.7 g), EtAc (44.7 g), n-bu (17.5 g) and H 2 (24.4 g) extracts. The n- hexane extract (514.0 mg) was purified by preparative TLC (Silica gel F254, Merck) to yield eugenol (3; mg, 10.58% of dried material) and acetyleugenol (4; 33.5 mg, 1.15%). A precipitate deposited from the EtAc extract (15.0 g) in 30% Me was collected, and the soluble portion was fractionated by column chromatography in a Toyopearl HW-40C column (2:2 40 cm) with aqueous Me ð30%! 40%! 50%! 60%! 70%Þ!H 2 -acetone (3:7, v/v) to fractions S1 S37 (30%, S1 S8; 40%, S9 S13; 50%, S14 S26; 60%, S27 S35; 70%, S36 S37); and 70% acetone eluate, monitored by reversed-phase HPLC. The precipitate was almost all ellagic acid (6; 5.23 g, 1.36%). Fractions S19 (235.1 mg) and S35 (123.2 mg) were respectively identified as tellimagrandins I (17; 1: %) and II (18; 6: %). 3,3 0,4-Tri--methyellagic acid (7; 2.1 mg, 1: %) was obtained as crystals from S6. Fraction S5 (100.0/ mg) was purified in a Sep-pak C18 column (Waters) eluted with y To whom correspondence should be addressed. Fax: ; tyoshida@cc.matsuyama-u.ac.jp

2 M. YSHIMURA et al. H 2 to yield gallic acid (5; 65.2 mg, 1: %). Fraction S2 (1.0 g) was subjected to column chromatography in a YMC-gel DS-AQ column (1:1 25 cm) eluted with H 2! aqueous Me (10%! 30%! 50%), and the H 2 and 30% Me eluates were further separately chromatographed in an LH-20 column (1:1 25 cm) eluted with Et which afforded biflorin (9; 18.9 mg, 1: %), isobiflorin (10; 33.0 mg, 2: %) and acetylisobiflorin (1; 16.2 mg, 9: %). Fractions S7 S9 (803.6 mg) were applied to a YMC-gel DS-AQ column (1:1 22 cm) eluted with H 2! aqueous Me ð10%! 20%! 30%! 40%Þ!Me, and the 30% Me eluate was obtained as eugenol-4--(6 0 -galloyl)-glucoside (8; 13.7 mg, 7: %). The 20% and 30% Me eluates (95.1 mg) were respectively chromatographed in a Toyopearl HW-40F column (1:1 6 cm) eluted with 30% Me, and a Sephadex LH-20 column (1:1 18 cm) eluted with Et, yielding quercetin 3--glucuronide methyl ester (12; 29.1 mg, 1: %) and isorhamnetin 3--glucoside (11; 8.4 mg, 4: %). Fraction S15 (272.8 mg) was chromatographically separated in a YMC-gel DS AQ column (1:1 25 cm) with H 2! aqueous Me (10%! 20%! 30%) as the eluent to yield strictinin (16; 5.8 mg, 3: %) and 1,2,3-tri--galloylglucose (13; 6.2 mg, 3: %). Fractions S24 S26 (226.1 mg) were subjected to chromatography in a YMC-gel DS AQ column (1:1 22 cm) eluted with H 2! aqueous Me ð10%! 20%! 30%! 40%Þ! Me to yield 1,2,3,6-tetra--galloylglucose (14; 24.6 mg, 1: %), and the 20% Me eluate (112.1 mg) was further chromatographed in a Sephadex LH-20 column (1:1 18 cm) eluted with Et-Me (3:2, v/v) to yield 1,3-di--galloyl-4,6- HHDP-glucose (15; 24.7 mg, 1: %). Fraction S30 (195.4 mg) was subjected to chromatography in a Sephadex LH-20 column (1:1 25 cm) eluted with Et-Me (7:3, v/v) to yield casuarictin (19; 19.7 mg, 1: %) Acetylisobiflorin (1). A pale yellow amorphous powder. ½Š 23 D þ11:5 (c 1.0, Me). 1 H-NMR (Me-d 4 ) : 6.24 (1H, s, H-6), 6.08 (1H, d, J ¼ 1:0 Hz, H-3), 4.89 (1H, d, J ¼ 10:0 Hz, Glc H-1), 4.39 (1H, dd, J ¼ 1:5, 12.0 Hz, Glc H-6), 4.21 (1H, dd, J ¼ 5:5, 12.0 Hz, Glc H-6), 4.08 (1H, br t, Glc H-2), 3.59 (1H, br t, Glc H-5), (2H, m, Glc H-3, 4), 2.40 (3H, d, J ¼ 1:0 Hz, 2-Me), 2.03 (3H, s, acetyl). 13 C-NMR (Me-d 4 ) : (C-4), (ester carbonyl), (C-2), (C-7), (C-5), (C-8a), (C-3), (C-4a), (C-8), 99.9 (C-6), 79.8 (Glc C-3), 79.6 (Glc C-5), 75.4 (Glc C-1), 72.7 (Glc C-2), 71.8 (Glc C-4), 65.3 (Glc C-6), 20.7 (acetyl methyl), 20.3 (2-Me). HR-ESI-MS: [ðm HÞ calcd. for C 18 H 19 10, ]. Isolation procedure for pimento. Pimento (dried berries of P. dioica, 570 g) was extracted with 80% Et (6 L) for 48 h at room temperature. After filtration and concentration in vacuo to 1 L, the concentrate was successively extracted with n-hexane (1L 6), EtAc (1L 6) and n-bu (1L 6) to furnish n-hexane (27.3 g), EtAc (10.8 g) and n-bu extracts (8.3 g), and a water-soluble portion (20.4 g). The n-hexane extract was analyzed by reversed-phase HPLC to detect eugenol (3) and thymol (20) which were identified by comparing with authentic samples. The EtAc extract (8.4 g) was subjected to column chromatography in a Toyopearl HW-40C column (2:2 40 cm), eluting with aqueous Me ð30%! 40%! 50%! 60%! 80%Þ!Me! H 2 -acetone (3:7, v/v) to provide fractions P1 P37 (30%, P1 P10; 40%, P11 P13; 50%, P14 P19; 60%, P20 P26; 80%, P27 P32), and Me and 70% acetone eluates. Ellagic acid (6, mg, 2: %) was obtained from the Me eluate. A part (42.3 mg) of P7 (563.5 mg) was purified in a Sep-pak C18 cartidge, eluting with H 2 to yield gallic acid (5, 19.1 mg, 5: %). Fraction P6 (217.0 mg) was subjected to chromatography in a YMC-gel DS-AQ column (1:1 25 cm), eluting with 20% Me to yield syringic acid (21, (4.7 mg, 1: %), vanillin (23, 11.0 mg, 2: %), (4R)-, (4S)--terpineol 8--[( galloyl)-arabinosyl]-(1!6)-glucoside (28, 7.6 mg, 1: %; and 29, 10.9 mg, 2: %) and P6-1. Fraction P6-1 (21.9 mg) was further purified in a Sephadex LH-20 column (1:1 20 cm) eluted with Et, which gave (2S)-3-(4-hydroxy-3-methoxyphenyl)-propane-1,2- diol 1--(6 0 --galloyl)--d-glucoside (2, 12.5 mg, 2: %). Fraction P9 (570.2 mg) was subjected to chromatography in a YMCgel DS-AQ column (1:1 20 cm), eluting with aqueous Me (15%! 20%! 30%! 40%) to yield pimentol (22, mg, 6: %), and (4R)- and (4S)--terpineol 8--(6 0 --galloyl)- glucoside (26, 2.1 mg, 4: %; and 27, 25.4 mg, 5: %). Fraction P28 (220.0 mg) was chromatographed in a YMC-gel DS-AQ column (1:1 25 cm), using aqueous Me (10%! 15%! 20%! 25%) as the eluant, to give casuarinin (24, 16.7 mg, 3: %) and 1,2,3,6-tetra--galloylglucose (14, 16.0 mg, 3: %). Fraction P31 (186.8 mg) was subjected to chromatography in a YMC-gel DS-AQ column (1:1 25 cm) eluted with aqueous Me (10%! 15%! 20%! 25%! 30%) to yield penta--galloylglucose (25, 31.1 mg, 7: %). (2S)-3-(4-Hydroxy-3-methoxyphenyl)-propane-1,2-diol 1--(6 0 -galloyl)--d-glucoside (2). A pale yellow amorphous powder. ½Š 23 D 3:8 (c 1.0, Et). 1 H-NMR (Me-d 4 ) : 7.08 (2H, s, galloyl-h), 6.77 (1H, d, J ¼ 2:0 Hz, H-2), 6.66 (1H, d, J ¼ 8:0 Hz, H-5), 6.61 (1H, dd, J ¼ 2:0, 8.0 Hz, H-6), 4.51 (1H, dd, J ¼ 2:0, 11.5 Hz, Glc H-6), 4.38 (1H, dd, J ¼ 6:0, 11.5 Hz, Glc H-6), 4.29 (1H, d, J ¼ 7:5 Hz, Glc H-1), 3.93 (1H, ddd, J ¼ 3:0, 7.0, 10.0 Hz, H-8), 3.83 (1H, dd, J ¼ 3:0, 10.0 Hz, H-9), 3.77 (3H, s, -Me), 3.55 (1H, ddd, J ¼ 2:0, 6.0, 8.0 Hz, Glc H-5), (3H, m, H-9, Glc H-3, 4), 3.26 (1H, m, Glc H-2), 2.70 (1H, dd, J ¼ 7:0, 14.0 Hz, H-7), 2.62 (1H, dd, J ¼ 7:0, 14.0 Hz, H-7). 13 C-NMR (Me-d 4 ) : (galloyl C-7), (C-3), (2C, galloyl C-3, 5), (C-4), (galloyl C-4), (C-1), (C-6), (galloyl C-1), (C-5), (C-2), (2C, galloyl C-2, 6), (Glc C-1), 77.8 (Glc C-3), 75.5 (Glc C-5), 75.2 (Glc C-2), 74.6 (C-9), 73.0 (C-8), 71.7 (Glc C-4), 64.8 (Glc C-6), 56.4 (-Me), 40.3 (C-7). HR-ESI-MS: [ðm HÞ calcd. for C 23 H 27 13, ]. Synthesis of (RS)-2a from eugenol (3). Eugenol (3, 1.0 g) was dissolved in 75 ml of CH 3 CN-acetone-H 2 (1:1:1, v/v/v), and microencapsulated-s 4 (900 mg) and N-methylmorpholine N-oxide (1.1 g) were added to the solution. After stirring for 18 h at room temperature, the reaction mixture was extracted with EtAc (75 ml 3). The EtAc layer was evaporated in vacuo, and the product was applied to a silica gel column (1:5 18 cm) and eluted with n-hexane-etac (1:1! 1:3! 1:4! EtAc) to yield a racemic mixture of (RS)-2a (81.2 mg) from the n-hexane-etac (1:3) eluate. Racemate (RS)-2a (3.0 mg) was separated by preparative HPLC in a chiral column, using n-hexane-2-propanol (92:8) as the eluant, to give each enantiomer (S)-2a (t R 21.1 min, ½Š 23 D 23:4, 1.3 mg) and (R)-2a (t R 22.7 min, ½Š 23 D þ20:2, 1.4 mg). Enzymatic hydrolysis of compound 2. To a solution of compound 2 (1.0 mg) in distilled water (1 ml) was added tannase (1 mg). The solution was stored for 10 h at room temperature. After adding Me to deactivate the tannase and then centrifuging, the resulting supernatant was analyzed by chiral HPLC to detect an aglycone identical to (S)-2a. To confirm the sugar moiety, the supernatant was evaporated under an N 2 stream, and the resulting residue was heated with L-cystein methyl ester hydrochloride (0.5 mg) in pyridine at 60 C for 1 h. o-tolyl isothiocyanate (0.5 mg) was then added to the reaction mixture which was further reacted at 60 C for 1 h. The converted product was analyzed by HPLC, eluting with 25% Me in 50 mm H 3 P 4. The retention time of the product (t R 20.6 min) was identical with that prepared under the same conditions from the D-glucose standard (t R 20.7 min) [t R 18.0 min for the product from L-glucose]. Antioxidative assay. The antioxidative activity was estimated by an RAC assay. A sample solution of a 75 mm phosphate buffer (ph 7.0, 20 ml) and 94.4 nm fluorescein solution (200 ml) were pipetted into each well of a 96-well microplate and incubated for 15 min at 37 C. After adding a 31.7 mm AAPH solution (75 ml) and shaking for 15 s, the fluorescence was recorded every 2 min for 90 min at respective excitation and emission wavelengths of 485 and 535 nm. The area under the curve (AUC) of each sample was calculated relative to the initial value (the fluorescence intensity at 0 min), and the blank value was subtracted. RAC values (mmol Trolox equivalent/g) were calculated from a curve of Trolox concentration vs. AUC value. Data are expressed as the mean SD of more than two separate experiments.

3 Polyphenols in Clove and Pimento H CH 2 H 1 8 H H H H 2 C H 1 H 8 Me H H H H 2 C H 9 H H H 2 10 CH 2 (S )-2a 3: R= H 4: R= CCH 3 5: R= H 21: R= CH 3 6: R= H 7: R= CH 3 H H R' 2 C R C 13: R= R = H 14: R= H, R = G 25: R= R = G G= CH 2 H H C 20 Me H C C H H H H H H H H H 2 C 11 15: R 1 = G (β), R 2 = H, R 3 = G 16: R 1 = G (β), R 2 = R 3 = H 17: R 1 = H, R 2 = R 3 = G 18: R 1 = G (β), R 2 = R 3 = G Me H 2C 22 CCH 2 C R 3 Me H R Me R 2 H C R R 1 23 CH Me H H H H H R H H Me H R H H 3 CC CCH 2 C H C H C H H H H C CH 2 C H C 24 R C C R C H H H H 2 C 26: = 27: = H H H CH 2 2 C 28: = 29: = H H H Me H 2 C 30 Fig. 1. Structures of Isolated Compounds from Clove and Pimento. Results and Discussion An aqueous Et extract of clove (dried flower buds of S. aromaticum) was partitioned by solvent extraction into n-hexane-, EtAc- and n-bu-soluble portions. Preparative TLC of the n-hexane extract yielded eugenol (3) and acetyleugenol (4). The EtAc extract was subjected to a combination of column chromatography over Diaion HP-20, Toyopearl HW40, YMC-gel D- SAQ and Sephadex LH-20 to furnish compound 1 and 15 known compounds which were characterized by their NMR data or a direct HPLC comparison with authentic samples as gallic acid (5), ellagic acid (6), 3,3 0,4-tri-methylellagic acid (7), eugenol 4--(6 0 --galloyl)- glucoside (8), 5) biflorin (9), 9) isobiflorin (10), 5) isorhamnetin 3--glucoside (11), 10) quercetin 3--glucuronide methyl ester (12), 11) 1,2,3-tri--galloylglucose (13), 12) 1,2,3,6-tetra--galloylglucose (14), 12) 1,3-di--

4 M. YSHIMURA et al. galloyl-4,6-hhdp-glucose (15), 13) strictinin (16), 14) tellimagrandins I (17) 12) and II (18), 12) and casuarictin (19) 14) (Fig. 1). Compound 1, as a pale yellow amorphous powder, was assigned a molecular formula of C 18 H by high-resolution (HR)-ESI-MS data [m=z (calcd. for )]. The 1 H- and 13 C-NMR spectra of 1 were similar to those of isobiflorin (10), except for the presence of an acetyl group ( H 2.03; C 20.7 and 172.9), indicating 1 to be an acetyl derivative of 10. The acetyl group in 1 was located at -6 of the glucose moiety on the basis of the lower field shifts of glucose H-6 proton signals ( 4.21 and 4.39) than those of 10. This assignment was also verified by two- and threebond correlations between the acetyl group and glucose H-6 protons through an ester carbonyl carbon in the (S )-2a (R )-2a Fig. 2. Me H CH 2 H H H H H H 2 C H 1 2 HMBC Correlations of Compounds 1 and 2 (HMBC: H!C). (RS )-2a Hydrolyzate from min Fig. 3. Chiral HPLC Comparison of (RS)-2a with the Enzymatic Hydrolyzate from 2. S. aromaticum_80% Et ext. n-hexane ext. EtAc ext. n-bu ext. H 2 ext. P. dioica_80% Et ext. n-hexane ext. EtAc ext. n-bu ext. H 2 ext. L-ascorbic acid 6 --acetylisobiflorin (1) (2S)-3-(4-hydroxy-3-methoxyphenyl)-propane- eugenol (3) 1,2-diol 1--(6'--galloyl)-β-D-glucoside (2) acetyleugenol (4) gallic acid (5) ellagic acid (6) 3,3,4-tri--methylellagic acid (7) eugenol 4--(6 --galloyl)-glucoside (8) biflorin (9) isobiflorin (10) isorhamnetin 3--glucoside (11) quercetin 3--glucuronide 6 --methylester (12) 1,2,3-tri--galloylglucose (13) 1,2,3,6-tetra--galloylglucose (14) 1,3-di--galloyl-4,6-HHDP-glucose (15) strictinin (16) tellimagrandin I (17) tellimagrandin II (18) casuarictin (19) thymol (20) syringic acid (21) pimentol (22) vanillin (23) casuarinin (24) penta--galloylglucose (25) (4R)-α-terpineol 8--(6 --galloyl)-glucoside (26) (4S)-α-terpineol 8--(6 --galloyl)-glucoside (27) (R)-α-terpineol 8--[(2''--galloyl)-arabinosyl]- (1 6)-glucoside (28) (S)-α-terpineol 8--[(2''--galloyl)-arabinosyl]- (1 6)-glucoside (29) Fig ,000 10,000 15,000 20,000 25,000 30,000 35,000 40,000 µmol TE/g RAC Values of the Extracts and Ingredients of Clove and Pimento. Data are expressed as the mean SD of more than two separate experiments.

5 HMBC spectrum (Fig. 2). ther HMBC correlations, including those supporting the C-C linkage of glucose, were also consistent with assumed structure 1. Compound 1 was chemically confirmed by its methanolysis yielding 10 which was identified by an HPLC comparison with isobiflorin (10, t R : 23.3 min) and biflorin (9, t R : 24.2 min). The structure of compound 1 was assigned as 6 0 -acetylisobiflorin from these results. A concentrated aqueous Et extract of pimento (berries of P. dioica) was successively partitioned with n-hexane, EtAc and n-bu. The EtAc extract was fractionated by column chromatography over Diaion HP-20 and subsequent repeated column chromatography over Toyopearl HW40, YMC-gel DSAQ and Sephadex LH-20 to yield compound 2 and 14 known compounds [eugenol (3), thymol (20), syringic acid (21), gallic acid (5), ellagic acid (6), pimentol (22), 15) vanillin (23), casuarinin (24), 14) 1,2,3,6-tetra--galloylglucose (14), penta--galloylglucose (25), 12) (4R)-, (4S)--terpineol 8--(6 0 --galloyl)-glucoside (26), (27), 15) (4R)-, (4S)--terpineol 8--[( galloyl)-arabinosyl]-(1!6)-glucoside (28), and (29) 11) (Fig. 1). Isolation of the hydrolyzable tannins, casuarinin (24), 1,2,3,6-tetra--galloylglucose (14) and penta--galloylglucose (25), from the P. dioica berry was the first example. The known compounds were identified by their NMR data or a direct comparison with authentic samples by HPLC. Compound 2 was obtained as a pale yellow amorphous powder and possessed the molecular formula of C 23 H (calcd. for ) as determined by HR- ESI-MS data [m=z ðm HÞ ]. The 1 H-NMR spectrum of 2 showed an aromatic 2H-singlet ( 7.08) assignable to a galloyl group, and three aromatic protons [ 6.77 (d, J ¼ 2:0 Hz), 6.66 (d, J ¼ 8:0 Hz) and 6.61 (dd, J ¼ 2:0, 8.0 Hz)] attributable to a 1,3,4-trisubstituted phenyl group. Sugar protons, a methoxy 3H-singlet and successively coupled 5 spin signals were observed in the aliphatic region of the 1 H- 1 H CSY spectrum. The 5 spin system was assigned to a methylene ( 2.70, 2.62), oxygenated methine ( 3.93) and oxygenated methylene ( 3.83, 3.37) by 13 C-NMR and HSQC measurements. These NMR and 1 H- 1 H CSY data allowed the assignment of a 6--acylated glucose as the sugar unit by the close similarity of their chemical shifts to those of 8 and 22. The location of each unit was determined by HMBC correlations as shown in Fig. 2, leading to structure 2 for this compound. Although the glucoside (30) isomeric to 2 has previously been reported as one of the constituents of the same plant by Kikuzaki et al., 15) compound 2 was clearly different from 30 in the glucosyl position, as revealed by the HMBC correlation between the terminal methylol carbon and anomeric proton (and vice versa). To determine the absolute configuration at C-8, we first synthesized a racemic aglycone (2a) from eugenol by s 4 oxidation according to the method reported by Kikuzaki et al. 16) Each enantiomer of 2a was separated by preparative HPLC in a chiral column, and each absolute configuration was assigned by comparing their ½Š D values [(R)-: þ20:2, t R 22.7 min; (S)-: 23:4, t R 21.1 min] with data previously reported [(R)-: þ18, (S)-: 23 ] in the literature. 16) The aglycone obtained Polyphenols in Clove and Pimento ABS Fig. 5. HPLC Analyses (280 nm) of the Polyphenolic Constituents in Clove (A) and Their n-hexane (B), EtAc (C), n-bu (D) and H 2 (E) Extracts. from compound 2 by enzymatic hydrolysis was identified as (S)-2a by directly comparing the retention time (t R 21.1 min) with that of an authentic synthesized sample by chiral HPLC (Fig. 3). A sugar component was additionally identified as D-glucose according to the conversion method reported by Tanaka et al., 17) enabling the structure of compound 2 to be established as (2S)- 3-(4-hydroxy-3-methoxyphenyl)-propane-1,2-diol 1-- (6 0 --galloyl)--d-glucoside. The antioxidative activities of the extracts and compounds isolated from the respective plant materials were estimated by an assay for oxygen radical absorbance capacity (RAC) (Fig. 4). 18,19) The n-hexane extracts of clove and pimento respectively showed high RAC values [25,975 and 19,458 mmol TE (= trolox equivalent)/g], their activities being mostly attributable to the major constituent, eugenol (3), which had the most potent RAC value (39,271 mmol TE/g). The EtAc extracts containing galloylated compounds, including hydrolyzable tannins, also exhibited high RAC values (>9;000 mmol TE/g). Significant antioxidative activity of the well-known condiments was evaluated in this study by the RAC method applicable to both lipophilic and hydrophilic compounds on the basis of detailed analyses of their constituents, including new polyphenol glucosides 1 and 2. Eugenol (3) was confirmed as the major antioxidant in both materials in spite of the simple phenol (Figs. 5 and 6). Its potency, which is accounted for by the formation of a stable quinone radical, 20) is noteworthy in its superiority to polyphenolic compounds with a catechol or galloyl moiety when they are compared in weight (g) order under the same assay estimating the durable radical scavenging ability. The flavonol glycosides (11 min

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