PHYTOCHEMICAL ANALYSIS Phytochem. Anal. 14, 48–53 (2003) Published online in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/pca.686 Guaiacylglycerol-7'-O-methyl 8'-vanillic Acid Ether and Related Compounds from Boreava orientalis Akiyo Sakushima,1* Kosei Ohno,1 Takashi Maoka,2 Maksut Coskun,3 Aysegul Guvenc,3 Ceyda Sibel Erdurak,3 Ayse Mine Ozkan,3 Koh-ichi Seki1 and Kazue Ohkura1 1 Faculty of Pharmaceutical Sciences, Health Sciences University of Hokkaido, Ishikari-Tobetsu, Hokkaido 061-0293, Japan Kyoto Pharmaceutical University, Misasagi, Yamashina-ku, Kyoto, Japan 3 Department of Pharmacognosy and Pharmaceutical Botany, Faculty of Pharmacy, University of Ankara, Ankara, Turkey 2 The threo and erythro forms of guaiacylglycerol-7'-O-methyl 8'-vanillic acid ethers, threo and erythro guaiacylglycerol 8'-vanillin ethers, and threo guaiacylglycerol 8'-(4-hydroxymethyl-2-methoxyphenyl) ether have been isolated from fruits of Boreava orientalis. Structural determinations were made on the basis of UV, MS, 1H- and 13C-NMR spectral data, including two-dimensional shift correlation. The relative configurations were assigned on the basis of 1H-NMR chemical shifts. Copyright # 2003 John Wiley & Sons, Ltd. Keywords: Lignan; threo and erythro guaiacylglycerol-7'-O-methyl 8'-vanillic acid ethers; threo and erythro guaiacylglycerol 8'-vanillin ethers; threo guaiacylglycerol 8'-(4-hydroxymethyl-2-methoxyphenyl) ether; guaiacylglycerol ether; Boreava orientalis; Cruciferae. INTRODUCTION The fruits of Boreava orientalis (Cruciferae), a weed which is widely distributed in Turkey, are used in traditional medicine for the treatment of coughs and skin disease (Tanker and Yenen, 1978; Tanker et al., 1983). Some of the phenolic compounds present in this plant exhibit important pharmacological and biological activities. In particular, derivatives of pyrocatechuic acid and sinapic acid have been identified as potentially useful iron-chelating agents and as strong anti-oxidative substances (Graziano et al., 1974; Maoka et al., 1996, 1997; Rice-Evans and Packer, 1998). We have previously reported the isolation and identification of the threo and erythro isomers of guaiacylglycerol 8'-vanillic acid ethers (1a and 1b) and of other compounds from the methanol extract of the fruit of B. orientalis, and we have shown that these compounds exhibit radical-scavenging activities (Sakushima et al., 1994, 1995, 1997). As a continuation of this investigation, we have succeeded in isolating guaiacylglycerol-7'-O-methyl 8'-vanillic acid ethers (2) and the related compound 4 from the same source, and here we report the structural elucidation of these isolates based on of UV, MS, 1H- and 13C-NMR spectral data, including NOE, DEPT, two-dimensional shift correlation, HMQC and HMBC analysis. injected into a Shimadzu (Kyoto, Japan) model 6AD LC system fitted with a Nucleosil 5 C18 (Nomura Chemicals, Seto City, Japan) column (250  4 mm i.d.) and equipped with a UV detector set at 280 nm. The column was eluted with water:methanol (2:3) at a flow rate of 0.5 mL/min with a pressure drop of 24 MPa/cm. EXPERIMENTAL HPLC analyses. Methanolic solutions of samples were * Correspondence to: A. Sakushima, Faculty of Pharmaceutical Sciences, Health Sciences University of Hokkaido, Ishikari-Tobetsu, Hokkaido 0610293, Japan. Email: sakushi@post.hoku-iryo-u.ac.jp Contract/grant sponsor: Ministry of Education, Science, Sports and Culture of Japan. Copyright # 2003 John Wiley & Sons, Ltd. Spectral analyses. 1H- and 13C-NMR spectra were recorded at 400 and 100 MHz, respectively, using a Jeol (Tokyo, Japan) model EX 400 spectrometer. Samples were dissolved in CD3OD. Chemical shifts are given in ppm relative to TMS as internal standard. EI/MS were measured by direct inlet using a Jeol model JMS GCMATE mass spectrometer at an electron energy of 70 eV and an ion source temperature of 200°C. Received 8 August 2001 Revised 30 January 2002 Accepted 5 February 2002 LIGNANS FROM BOREAVA ORIENTALIS Plant material. Plants of Boreava orientalis were collected near Ankara, Turkey, in 1990. A voucher specimen is lodged in the Ankara Üniversitesi Eczacilik Fakültesi herbaryumu (AEF). Extraction and Isolation. Dried fruit (1 kg) was extracted with petroleum ether (1 L  3), in order to remove the oil, and then with methanol (1 L  3). The concentrated methanolic extract was suspended in water (1 L) and extracted successively with diethyl ether (400 mL  4), chloroform (400 mL  4), ethyl acetate (400 mL  4), and butanol (400 mL  4). The chloroform extract (3.7 g) was subjected to CC over silica gel eluted with a gradient of chloroform:methanol; fractions 23 and 24 were purified by silica gel CC and preparative HPLC as described previously (Sakushima et al., 1997) to give 3a (2.2 mg) and 3b (1.9 mg), respectively. The ethyl acetate extract (1.1 g) was subjected to CC using a Sephadex LH-20 column eluted with water and methanol; fractions 15 and 16 were purified by LH-20 CC eluted with a gradient of water:methanol to give 2a (4.2 mg) and 2b (2.4 mg), respectively. The butanol extract (3 g) was subjected to LH-20 CC eluted with water and methanol; fraction 5, which eluted with 30% aqueous methanol, was re-chromatographed on LH-20 eluted with a gradient of water:methanol. The fraction which contained 4 was purified using Sephadex G-10 and preparative HPLC as described previously (Sakushima et al., 1997) to yield 2.5 mg of the compound. Base hydrolysis of 2a and 2b. A sample of the compound (0.5 mg of 2a or 0.6 mg of 2b) was stirred with 0.5 M sodium hydroxide (1 mL) at room temperature for 24 h. The reaction mixture was adjusted to pH 6.0 with dilute hydrochloric acid, extracted with ethyl acetate (3 mL), the extract washed with water (1 mL  3) and then evaporated to dryness in vacuo. The residue was analysed by HPLC. Acidic hydrolysis of 2a and 2b. A sample of the compound (0.5 mg of 2a or 0.6 mg of 2b) was refluxed with 3% hydrochloric acid (1 mL) for 2 h. The reaction mixture was extracted with ethyl acetate (3 mL), the extract washed with water (1 mL  3) and then evaporated to dryness in vacuo. The residue was analysed by HPLC. Determination of 2a and 2b. Dried fruit (100 g) was extracted with petroleum ether, in order to remove the oil, and then with ethanol. The concentrated ethanol extract was suspended in water and extracted successively with diethyl ether, chloroform, ethyl acetate, and butanol. The concentrated chloroform and ethyl acetate extracts were subjected to silica gel CC eluted with chloroform:ethanol. Fractions containing 2a and 2b were purified by preparative HPLC as described previously (Sakushima et al., 1997). The retention times (R t) and 1H-NMR spectral data of the compounds thus purified agreed with those reported previously for 2a and 2b. Checking for artefact formation. A sample (0.5 mg) of guaiacylglycerol 8'-vanillic acid ether was dissolved in ethanol (3 mL), stored at room temperature for 1 week and then heated in boiling water for 3 h. The absence of guaiacylglycerol-7'-O-ethyl 8'-vanillic acid ether in the Copyright # 2003 John Wiley & Sons, Ltd. 49 treated solution was confirmed by HPLC and from 1HNMR spectral data. RESULTS AND DISCUSSION Threo guaiacylglycerol-7'-O-methyl 8'-vanillic acid ether (2a) Colourless amorphous powder; melting point, 85–88°C; HR-EI/MS m/z 378.13076 [M]‡
, corresponding to C19H22O8 (calculated 378.13140); brown colour following FeCl3 reaction; TLC [silica gel, EtOAc:MeCOEt: HCOOH:C6H6:H2O (4:3:1:1:2; upper layer); system A], R f 0.90 (R f 0.80 for 1a); HPLC [H2O:MeOH (2:3); solvent A], R t 15.7 min; UV lmax (nm) in MeOH (e), 257 (6699), 285.5 (4799); IR n (cm 1) film— 3100–3200 (OH), 1700 (—COO—), 1602 (C=C), 1558 (aromatic C=C), 1508 (aromatic C=C), 1418 (aromatic C=C), 1269 (OMe), 1220 (C—O), 1222, 1032 (C—O); EI/MS m/z (%)— 378 [M]‡
(27.2), 316 [M–C2H6O2]‡ (20.1), 211 [M–C9H11O3]‡ (6.7), 180 [M–C10H14O4]‡
(15.6), 168 [C9H11O3 ‡ H]‡
(100.0), 167 [C9H11O3)]‡ (66.0), 152 [C8H7O3 ‡ H]‡
(34.6), 137 [C7H5O3]‡
(34.4), 119 (7.7), 91 (5.4); 1H- and 13C-NMR spectra (Table 1). Erythro guaiacylglycerol-7'-O-methyl 8'-vanillic acid ether (2b) Colourless amorphous powder; HR-EI/MS m/z 378.13397 [M]‡
, corresponding to C19H22O8 (calculated 378.13140); brown colour following FeCl3 reaction; TLC [system A], R f 0.90; HPLC (solvent A), R t 15.8 min; UV, MS and IR data as for 2a; 1H- and 13C-NMR spectra (Table 1). Threo guaiacylglycerol 8'-vanillin ether (3a) Colourless amorphous powder; melting point, 67–71°C; HR-EI/MS m/z 348.1211 [M]‡
, corresponding to C18H20O7 (calculated 348.1209); brown colour following FeCl3 reaction. TLC [system A], R f 0.82. HPLC (solvent A), R t 16.1 min; UV lmax (nm) in MeOH (e)—226 (15376), 279 (8066), 304 (5100); IR n (cm 1) film— 3100–3200 (OH), 2963 (CH), 1734 (—COH—), 1559 (C=C), 1507 (aromatic C=C), 1457 (aromatic C=C), 1419 (aromatic C=C), 1270 (OMe), 1161 (C—O), 1032 (C—O); EI/MS m/z (%)—348 [M]‡
(<5%), 300 [M– (CH4O2)]‡
(18.1), 211 (12.7), 178 [M–H2O– (C8H9O3)]‡
(77.7), 153 [C8H9O3]‡ (37.7), 152 [C8H7O3 ‡ H]‡
(66.9), 151 [C8H7O3]‡ (100.0), 137 [C7H5O3]‡ (34.2), 125 (13.4), 123 [C7H7O2]‡ (26.0), 119 (13.3), 93 (21.6), 91 (14.8); 1H- and 13C-NMR spectra (Table 2). Erythro guaiacylglycerol 8'-vanillin ether (3b) Colourless amorphous powder; slightly soluble in chloroform, brown colour following FeCl3 reaction; TLC [system A], R f 0.82; HPLC (solvent A), R t 16.4 min; UV, MS and IR data as for 3a; 1H- and 13C-NMR spectra (acetone, d6) (Table 2); 1H-NMR (400 MHz, CDCl3) d 3.79–3.95 (2H, m, 9-CH2-), 3.86 (3H, s, -OMe), 3.91 (3H, s, -OMe), 4.38 (1H, m, 8'-CH-), 4.97 (1H, d, J = 5.4, 7'CH-), 6.87–7.42 (6H, Ar H), 9.85 (1H, s, CHO); literature Phytochem. Anal. 14: 48–53 (2003) 50 A. SAKUSHIMA ET AL. Table 1. 1H- and 13C-NMR spectral dataa of the compounds 1, and 2a/2b 1ab C Benzoic acid moiety 1 124.7 2 114.2 3 148.1 4 154.0 5 117.0 6 124.3 7 167.3 56.3 3-OCH3 2ab H 7.56(d, J = 2.0 Hz) 7.21(d, J = 8.3 Hz) 7.58(dd, J = 8.3, 2.0 Hz) 3.81(s) Guaiacylglycerol moiety 1' 133.8 2' 111.6 7.10(d, J = 2.0 Hz) 3' 146.9 4' 151.0 5' 115.2 6.76(d, J = 8.0 Hz) 6' 120.5 6.90(dd, J = 8.0, 2.0 Hz) 7' 73.7 4.92(d, J = 5.4 Hz) 8' 86.9 4.47(m) 9' 62.0 a, 3.75(dd, J = 3.9, 12.2 Hz) b, 3.57(dd, J = 5.9, 12.2 Hz) 56.5 3.90(s) 3'-OCH3 7'-OCH3 a b C 124.1 114.4 148.4 154.4 115.4 124.3 167.3 56.3 130.7 112.0 147.3 150.8 116.3 121.5 84.0 85.2 62.0 56.5 57.0 2bb H 7.55(d, J = 2.0 Hz) 7.15(d, J = 8.3 Hz) 7.58(dd, J = 8.3, 2.0 Hz) 3.89(s) 7.06(d, J = 2.0 Hz) 6.80(d, J = 7.8 Hz) 6.87(dd, J = 7.8, 2.0 Hz) 4.44(d, J = 6.4 Hz) 4.55(m) a, 3.65(dd, J = 3.9, 11.7 Hz) b, 3.53(dd, J = 6.4, 11.7 Hz) 3.83(s) 3.20(s) C 124.3 114.3 148.2 153.6 115.1 124.2 167.3 56.3 H 7.49(d, J = 2.0 Hz) 7.04(d, J = 8.3 Hz) 7.54(dd, J = 8.3, 2.0 Hz) 3.82(s) 130.6 112.4 147.3 150.8 116.2 122.0 84.3 83.5 61.9 6.77(d, J = 8.3 Hz) 6.87(dd, J = 8.3, 2.0 Hz) 4.43(d, J = 5.9 Hz) 4.59(m) 3.78(dd, J = 4.9 Hz, 2H) 56.4 58.0 3.83(s) 3.20(s) 7.06(d, J = 2.0 Hz) Chemical shifts in ppm (J values in Hz). Spectra measured in acetone-d6 (35°C). 1 H-NMR (400 MHz, CDCl3–D2O; Katayama et al., 1981): d 3.80–3.95 (2H, m, 9-CH2-), 3.84 (3H, s, -OMe), 3.89 (3H, s, -OMe), 4.41 (1H, m, 8-CH-), 4.95 (1H, d, J = 5.5, 7-CH-), 6.80–7.38 (6H, Ar H), 9.85 (1H, s, CHO). Threo guaiacylglycerol 8'-(4-hydroxymethyl-2methoxyphenyl) ether (4) Colourless amorphous powder; melting point, 67–71°C; HR-EI/MS m/z 387.1058 [M ‡ Na]‡, corresponding to C18H20O8Na (calculated 387.10582); brown colour following FeCl3 reaction; TLC [system A], R f 0.83; HPLC (solvent A), R t 8.7 min; UV lmax (nm) in MeOH (e)— 250 (sh), 282 (2655), 328.5 (sh); IR n (cm 1) film— 3100–3300 (OH), 2956 (CH), 1602 (C=C), 1507 (aromatic C=C), 1471 (aromatic C=C), 1456 (aromatic C=C), 1300 (OMe), 1150 (C—O); EI/MS m/z (%)— 350 [M]‡
, 332 [M–H2O]‡, 211, 180, 168 (100), 166, 154, 153 (80.8), 151 (62.1), 137 (38.3), 124 (35.2), 109 (4.0), 107 (24.4), 93 (17.5); 1H- and 13C-NMR spectra (Table 2). Compounds 2a and 2b were obtained as pale yellow powders by preparative HPLC, and both exhibited a positive ferric chloride reaction. IR spectra suggested the presence of hydroxyl, carbonyl and aromatic rings. UV spectra showed absorption maxima at 257 and 286 nm, and shifts of the maxima upon addition of sodium ethoxide were similar to those observed for isomers of guaiacylglycerol 8'-vanillic acid ether (Sakushima et al., 1997). Neither compound could be hydrolysed with 3% hydrochloric acid or 0.5 M sodium hydroxide as has been previously noted for isomers 1a and 1b. The compounds showed molecular weights of 378 amu whilst their Copyright # 2003 John Wiley & Sons, Ltd. molecular formulae were determined to be C19H22O8 by HR-EI/MS. The EI/MS fragment ions at m/z 123, 137, 151 [M-227]‡, 167 [M-211]‡, 180, 211 and 278 [M100]‡
were due to the loss of vanillic acid, guaiacyl and guaiacylglycerol residues. The fragment ion at m/z 211 [M-167]‡ was due to the elimination of a deprotonated vanillic acid moiety (167 amu), suggesting the presence either of a vanillic acid residue on the guaiacylglycerol moiety (Houghton, 1985), or of guaiacyl methanol methyl ether moieties (167 amu). Both the ether location in guaiacylglycerol and the relative stereochemistry were determined by detailed analysis of the 1H- and 13C-NMR spectra, and from results of two-dimensional shift correlation, DEPT, HMQC, HMBC and NOE experiments. The 1H-NMR spectra of both 2a and 2b showed the presence of two sets of ABX-type signals in the aromatic region, two signals from methine protons, and two aryl methoxy signals and one alcoholic methyl ether signal in the aliphatic region (Table 1). Coupling constants and chemical shifts of the 8'-H methine proton in 2a and 2b were similar to those of 1a (Table 1). However, the signals attributed to the proton of C-7' of the glycerol core were shifted upfield by ca. 0.4 ppm when compared with 1a. The methyl group was thus determined to be at the C-7' position in the guaiacylglycerol unit. Chemical shifts for 2a and 2b in the 13C-NMR spectrum agreed with values estimated for 1a, except for a downfield shift of ca. 10 ppm of the C-7' signals of both 2a and 2b, compared with 1a, indicating etherification of the hydroxyl group at C-7 in the guaiacylglycerol moiety. Therefore, the alcoholic methyl ether was identified to be at the C-7' position of the glycerol core, and this was supported by the fragment ion of m/z 167 in the EI/MS of 2a and 2b. Phytochem. Anal. 14: 48–53 (2003) Copyright # 2003 John Wiley & Sons, Ltd. Table 2. 1H- and 13C-NMR spectral dataa of the compounds 3a/3b and 4 3ab C Guaiacylglycerol moiety 1' 133.7 2' 111.4 3' 147.1 4' 151.5 5' 115.2 6' 120.4 7' 73.7 8' 85.1 9' 61.0 3'-OCH3 b c 7.43(d, J = 2.0 Hz) 7.31(d, J = 8.3 Hz) 7.46(dd, J = 8.3, 2.0 Hz) 9.84(s) 3.93(s) 7.11(d, J = 2.0 Hz) 6.76(d, J = 8.3 Hz) 6.90(dd, J = 8.0, 2.0 Hz) 4.92(d, J = 5.9 Hz) 4.56(m) a, 3.62(dd, J = 5.9, 11.8 Hz) b, 3.52(dd, J = 3.4, 11.8 Hz) 3.81(s) C 131.6 111.3 148.0 154.9 116.1 126.2 191.2 56.2 H 7.38(d, J = 2.0 Hz) 7.20(d, J = 8.3 Hz) 7.42(dd, J = 8.3, 2.0 Hz) 9.82(s) 3.87(s) C 130.6 110.9 148.9 153.5 115.9 127.0 191.2 56.3 133.2 111.8 150.9 148.9 115.4 123.7 63.2 56.1 4c 134.0 111.7 146.8 151.6 116.1 120.7 73.9 85.2 62.2 6.73(d, J = 8.3 Hz) 6.91(dd, J = 8.3, 2.0 Hz) 4.91(d, J = 5.3 Hz) 4.62(m) a, 3.80(overlapping) 133.2 111.4 147.8 145.6 114.5 119.9 72.5 83.8 60.8 56.3 3.81(s) 56.1 7.13(d, J = 2.0 Hz) H 7.46(br-s) 6.91(d, J = 8.3 Hz) 7.40(d, J = 8.3 Hz, br-s) 4.26(overlapping) 3.68(s) 6.96(d, J = 2.0 Hz) 6.67(d, J = 8.3 Hz) 6.79(dd, J = 8.3, 2.0 Hz) 4.87(d, J = 5.3 Hz) 4.49(m) a, 3.73(dd, J = 5.9, 11.7 Hz) b, 3.51(dd, J = 5.9, 11.7 Hz) 3.79(s) Vanillyl alcohol C 133.5 111.9 147.8 145.3 115.3 120.5 64.1 56.1 Chemical shifts in ppm (J values in Hz). Spectra measured in acetone-d6 (35°C). Spectra measured in acetone-d6:D2O (1:1) (45°C). 51 Phytochem. Anal. 14: 48–53 (2003) a 56.3 H Vanillic acid C LIGNANS FROM BOREAVA ORIENTALIS Benzoic acid moiety 1 131.0 2 111.2 3 148.0 4 155.3 5 116.3 6 126.3 7 191.2 56.1 3-OCH3 3bb 52 A. SAKUSHIMA ET AL. Table 3. Comparison of the 1H-NMR spectral dataa of the Isomers of compounds 1, 2, 3 and 4 threo isomer (compound suf®x a) Compounds 1a and 1bb 2a and 2bb 3a and 3bb 4c a b c erythro isomer (compound suf®x b) 7' 8' 9' 7' 8' 9' 4.92 (5.4) 4.44 (6.4) 4.92 (5.9) 4.87 (5.3) 4.47 (m) 4.55 (m) 4.56 (m) 4.49 (m) 3.75, 3.57 (3.9, 5.9, 12.2) 3.65, 3.53 (3.9, 6.4, 11.7) 3.62, 3.52 (3.4, 5.9, 11.8) 3.73, 3.51 (5.9, 11.7) 4.90 (5.4) 4.43 (5.9) 4.90 (5.3) 4.54 (m) 4.59 (m) 4.62 (m) 3.82 (overlapping) 3.78 (4.9) 3.8 (overlapping) Chemical shifts in ppm (J values in Hz). Spectra measured in acetone-d6 (35°C). Spectra measured in acetone-d6:D2O (1:1) (45°C). Assignment of the relative configuration was based on H-NMR chemical shifts for protons of the glycerol core in guaiacylglycerol (Table 3). 1H-NMR spectral data of the glycerol cores of both 2a and 2b were similar to those of the threo and erythro isomers of guaiacylglycerol 8'vanillic acid ether reported previously (Sakushima et al., 1997). Downfield shifts of 8'- and 9'-H were attributed to the erythro isomer, and upfield shifts were attributed to the threo isomer. Therefore, 2a and 2b were determined to be threo and erythro guaiacylglycerol-7'-O-methyl 8'vanillic acid ethers, respectively. Both compounds are presumed to be naturally-occurring and not artefacts, since they were isolated from neutral solution (Brunow and Lundguist, 1991), and a compound related to 2 has been isolated from Myristica fragrans (Hattori et al., 1986). Furthermore, 2 could be identified by HPLC and 1 H-NMR in fruit extracts prepared with ethanol instead of methanol. Compounds 3a and 3b were obtained as pale yellow powders, and both exhibited a positive ferric chloride reaction. IR spectra showed the presence of hydroxyl, carbonyl and aromatic rings, as for 2a and 2b, whilst the UV spectra showed absorption maxima at 227, 279 and 304 nm. The 1H-NMR spectra of 3a and 3b were characterised by the presence of aldehyde protons at 9.82 and 9.83 ppm and signals of six aromatic protons and four aliphatic protons due to guaiacylglycerol and vanillin. The chemical shifts for the guaiacylglycerol moieties in the 13C-NMR spectra of both 3a and 3b agreed with those of isomers of guaiacylglycerol 8'vanillic acid ether, and the chemical shifts for the other moiety agreed with those of vanillin (Table 2). The compounds showed molecular weights of 348 amu whilst their molecular formulae were determined to be C18H20O7 by HR-EI/MS. The EI/MS fragment ions at m/z 123, 137, 151 [M-227]‡, 167 [M-211]‡, 180, 211 and 278 [M-100]‡
were due to the loss of vanillin, guaiacyl and guaiacylglycerol residues. Compounds 3a and 3b 1 were, therefore, identified as threo and erythro guaiacylglycerol 8'-vanillin ethers. These compounds have also been identified as products of the biodegradation of lignin (Katayama et al., 1980, 1981), and the diastereomeric compounds have been isolated from Larix leptolepis (Miki et al., 1980). Compound 4 was obtained as a pale yellow powder, and exhibited a positive ferric chloride reaction. The molecular formula was determined to be C18H22O7 by HR-EI/MS. The IR, UV, 1H- and 13C-NMR spectra were similar to those of 3a; the 1H-NMR spectrum of 4 exhibited a resonance of a hydroxymethyl group at 4.26 ppm, which was identified by a C-H COSY shift correlation. The 1H-NMR spectral data of the glycerol core in the guaiacylglycerol moiety of 4 resembled that of 3a rather than 3b (Table 3). In the 13C-NMR spectrum, chemical shifts of the guaiacylglycerol moiety of 4 agreed with those of guaiacylglycerol 8'-vanillic acid ether, and chemical shifts for the other moiety matched those of vanillyl alcohol (Table 2). Since the EI/MS fragmentation pattern was similar to that of 3a, compound 4 was finally identified as threo guaiacylglycerol 8'-(4-hydroxymethyl-2-methoxyphenyl) ether, a new natural product. However, similar to the case for 3a and 3b, compound 4 has been previously identified as a breakdown product of lignin (Katayama et al., 1981; Shen and Heiningen, 1992). Acknowledgements The authors wish to thank Dr Kazuyuki Kamata (Faculty of Pharmaceutical Sciences, Health Sciences University of Hokkaido, Japan) for support and encouragement in preparing this manuscript, and Miss Yoko Akashima (Central Analytical Laboratory, Health Sciences University of Hokkaido, Japan) for recording MS and NMR spectra. This work was supported in part by a grant-in-aid for High Technology Research from the Ministry of Education, Science, Sports and Culture of Japan. REFERENCES Brunow G and Lundguist K. 1991. On the acid-catalysed alkylation of lignins. Holzforsch 45: 37±40. Graziano J, Grady RW and Cerami A. 1974. The identi®cation of 2,3-dihydroxybenzoic acid as a potentially useful ironchelating drug. J Pharm Exp Therapeut 190: 570±575. Copyright # 2003 John Wiley & Sons, Ltd. Hattori M, Hada S, Watahiki A, Ihara H, Shu Y-Z, Kakiuchi N, Mizuno T and Namba T. 1986. Studies on dental caries prevention by traditional medicines. X. Antibacterial action of phenolic compounds from mace against Streptcoccus mutans. Chem Pharm Bull 34: 3885±3893. Phytochem. Anal. 14: 48–53 (2003) LIGNANS FROM BOREAVA ORIENTALIS Houghton PJ. 1985. Lignans and neolignans from Buddleja davidii. Phytochemistry 24: 819±826. Katayama T, Nakatsubo F and Higuchi T. 1980. Initial reactions in the fungal degradation of guaiacylglycerolb-coniferyl ether, a lignan substructure model. Arch Microbiol 126: 127±132. Katayama T, Nakatsubo F and Higuchi T. 1981. Syntheses of arylglycerol-b-aryl ethers. Mokuzai Gakkaishi 27: 223± 230. Maoka T, Sakushima A, Coskun M and Ito H. 1996. Antioxidative activity of phenolic compounds of Boreava orientalis. J Jap Oil Chem Soc 45: 671±673. Maoka T, Ito H, Sakushima A, Ohno K, Coskun M and Nishibe S. 1997. Comparison of anti-oxidative activity of phenolic compounds in Boreava orientalis and their relative compounds. J Jap Oil Chem Soc 46: 1399±1402. Miki K, Takehara T, Sasaya T and Sakakibara A. 1980. Lignans of Larix leptolepis. Phytochemistry 19: 449±453. Rice-Evans CA and Packer L. 1998. The Flavonoids in Health and Disease. Marcel Dekker: New York; 35±59. Copyright # 2003 John Wiley & Sons, Ltd. 53 Sakushima A, Coskun M, Tanker M and Tanker N. 1994. A sinapic acid ester from Boreava oreientalis. Phytochemistry 35: 1481±1484. Sakushima A, Coskun M and Maoka T. 1995. Hydroxybenzoic acids from Boreava orientalis. Phytochemistry 40: 257± 261. Sakushima A, Coskun M, Maoka T and Nishibe S. 1997. Separation of guaiacylglycerol-8'-vanillic acid ether isomers from Boreava orientalis. Nat Prod Lett 11: 31±36. Shen X and Heiningen AV. 1992. Synthesis of b-O-4 lignin model dimmers and their chlorinated derivatives. Can J Chem 70: 1754±1761. Tanker M and Yenen M. 1978. Boreava orientalis. J Fac Pharm Ankara Univ 8: 1. Tanker M, Ertan M, Coskun M, Sariseker N and Yurdesin T. 1983. The investigation of possibilities in Turkey with regards to the antibiotic industry. Turkish J Med Sci 7: 99± 108. Phytochem. Anal. 14: 48–53 (2003)