Separation and Determination of Chemical Constituents in the Roots of
Rhus javanica L. var. roxburghiana
Tzong-Huei Leea( ), Jong-Liang Chioub( ), Ching-Kuo Leea( ) and Yueh-Hsiung Kuob* ( )
aGraduate Institute of Pharmacognosy Science, Taipei Medical University, Taipei, Taiwan, R.O.C. b
Department of Chemistry, National Taiwan University, Taipei, Taiwan, R.O.C.
From the roots of Rhus javanica L. var. roxburghiana, totally thirty-seven known compounds have been isolated and identified. Their structures were elucidated based on their spectral analysis as well as comparison with authentic samples. These compounds were grouped to be fifteen triterpenoids, five steroids, two lignans, two flavonoids, nine phenolics, and four other aromatic derivatives. Their cytotoxicities toward two cell lines NUGC-3 and HONE-1 were also evaluated.
Keywords: Rhus javanica L. var. roxburghiana; Anacardiaceae; Roots; Cytotoxicity; NUGC-3;
HONE-1.
INTRODUCTION
There are five species of Rhus (Anacardiaceae) found in Taiwan. R. javanica L. var. roxburghiana, a small-sized de-ciduous tree, is widely distributed in thickets and secondary forests at low altitudes throughout this island.1Its roots have been used in folk medicines as antitussives, and for the treat-ments of anasarca, jaundice, and snake bite.2The phyto-chemical studies on this plant have been reported earlier, and which have resulted in the isolation and characterization of flavonoids,3-4triterpenoids,5-7phenolics,3,8one tannin9and one aromatic alkane.5Recently, it has been shown from our preliminary pharmacological experiments that the ethyl ace-tate layer of the root extracts of this plant exhibited signifi-cant anti-proliferation activities on four cell lines including 59T (hepatoma), DLD-1 (colon cancer), HONE-1 (nasopha-ryngeal carcinoma), and SCM1 (stomach carcinoma). The roots may contain bioactive agents with anti-proliferation ac-tivities worth investigating phytochemically. Therefore, a se-ries of phytochemical examinations on the ethyl acetate layer of the root extracts of this plant was thus undertaken and has led to the isolation and characterization of thirty seven known compounds 1-37. This paper deals with the isolation and structural elucidation of these compounds as well as their cytotoxicities.
RESULTS AND DISCUSSION
The methanolic extracts of the roots of R. javanica L. var. roxburghiana were concentrated to give a residue which was subjected to partition with n-hexane and water. The aqueous layer was further partitioned with EtOAc and
n-BuOH, successively. The combined EtOAc soluble
frac-tions were then separated using Si-open column chromatog-raphy and HPLC repeatedly to yield thirty seven compo-nents: betulonic acid (1),10betulinic acid (2),11betulin (3),12 lantabetulic acid (4),133-oxoolean-18-en-28-oic acid (5),14 3b-hydroxyolean-18-en-28-oic acid (6),15 3-oxo-6 b-hy-droxyolean-18-en-28-oic acid (7),16semimoronic acid (8),13 3-O-methyl semimoronic acid (9),17 3-oxoolean-12-en-28-oic acid (10),18oleanolic acid (11),19lantanolic acid (12),20 3-oxotirucalla-7,24-dien-21-oic acid (13),21dipterocarpol (14),223 b-hydroxy-22,23,24,25,26,27-hexanordammaran-20-one (15),23b-sitosterol (16),24stigmast-4-en-3-one (17),25 stigmast-4-ene-3,6-dione (18),26stigmastane-3,6-dione (19),25 stigmast-7-en-3-ol (20),27 pinoresinol (21),28 4-oxopino-resinol (22),29 4¢,5,7-trihydroxyflavanone (23),30
trans-3,4¢,7-trihydroxyflavanone (24),31vanillin (25),32methyl ferulate (26),333,5-dihydroxytoluene (27),34 4-hydroxy-3,5-dimethoxybenzaldehyde (28),35methyl gallate (29),36 2,6-dimethoxy[1,4]benzoquinone (30),37
3,4,5-trimethoxyben-Journal of the Chinese Chemical Society, 2005, 52, 833-841 833
Dedicated to Professor Ching-Erh Lin on the Occasion of his 66thBirthday and his Retirement from National Taiwan University * Corresponding author. Tel: +886-2-23638146; E-mail: yhkuo@ntu.edu.tw
zyl alcohol (31),38gallic acid (32),39ficusol (33),40 2-hy-droxy-6-pentadec-8(Z)-enylbenzoic acid (34),41 5-formyl-mellein (35),425-hydroxymethylmellein (36),42and alkyls caffeate (37).43
When comparing the spectral data of 4 with those of 2 it was suggested 4 had the typical lupane skeleton except that the1H-NMR of 4 showed two methylene proton signals (H2-25) atd 3.71 (dd, J = 8.8, 1.6 Hz) and d 4.21 (dd, J = 8.8, 2.8
Hz) having W-shaped1H-1H correlations with those of Ha-1 and H-5, respectively. The orientation of H2-25 was assigned to beb-form by a NOESY experiment. Thus, the structure of 4 was determined to be lantabetulic acid, which was isolated originally from Lantana camara13and also found previously in R. javanica.5Compounds 8, 9 and 12 possessed spectro-scopic data closely comparable to a oleanolic acid skeleton except that a H3-25 singlet was substituted by a oxygenated methylene functionality which bridged between C-3 and C-10, the same as in 4. The1H NMR spectra also showed characteristic signals for the olefinic protons in 8 (d 5.13, s,
H-19), 9 (d 5.12, s, H-19), and 12 (d 5.29, t, J = 2.8 Hz, H-12), and the location of the double bonds was deduced to be at C-18 and C-12, respectively, which was confirmed by inter-preting their HMBC spectra. Further analysis of all the 2D NMR data of 8, 9 and 12 allowed the complete assignments of their1H and13C NMR data. Accordingly, 8, 9, and 12 were assigned to be semimoronic acid, 3-O-methyl semimoronic acid, and lantanolic acid, respectively. Compounds 25, 27, 28, 29, 31, and 32 were identified as toluene derivatives with higher oxygenated on phenyl protons at C-3, -4, -5 and ben-zylic positions. The presence of 1,3,4,5-tetra-substituted aro-Constituents in the Roots of Rhus javanica J. Chin. Chem. Soc., Vol. 52, No. 4, 2005 835
matic moiety in 28, 29, 31, and 32 was supported by their1H NMR spectra, in which two phenyl protons are symmetrical. Compounds 25 and 27 possessing 1,2,5- and 1,3,5-phenyl protons, respectively, were interpreted by their coupling pat-terns. Among them, gallic acid (32) and its derivatives were the compounds most widely distributed in higher plants to be the component of hydrolysable tannins, flavonoids, or many other chemicals, and often exhibit important therapeutic ac-tivities.1H NMR spectrum of 34 showed signals for a 1,2,3-trisubstituted aromatic moiety, and an ABC system atd 6.74 (d, J = 7.2 Hz, H-5),d 6.84 (d, J = 8.0 Hz, H-3), and d 7.31 (dd, J = 8.0, 7.2 Hz, H-4) were observed. Besides, this spec-trum also displayed signals for an unsaturated long-chain alkyl functionality, one terminal methyl triplet atd 0.88 (H 3-15¢), four methylene at d 1.59 (m, H2-2¢), 2.00 (m, H2-7¢, -10¢), 2.96 (t, J = 7.6 Hz, H2-1¢), and an olefinic multiplet at d 5.33 (H-8¢, -9¢). The location of the double bond was further confirmed to be at C-8¢ by an oxidative cleavage experiment, and its configuration should be Z-form as evidenced by two signals atdc 26.9 (C-7¢) and 27.2 (C-10¢) below dc 30 in the 13
C NMR spectrum. After a complete assignment of its1H and13C NMR data, 34 was concluded to be 2-hydroxy-6-pentadec-8(Z)-enyl-benzoic acid, quite similar to the struc-ture of 6-pentadecylsalicylic acid isolated from R. javanica previously.56-Pentadecylsalicylic acid found to be an anti-thrombin agent and prolonged the clotting time in a dose-dependent manner, however, was not found in this study. Whether 34 can exert the same bioactivity or not remained to
be studied. Compound 37 were isolated as a mixture of four alkyl caffeates using HPLC with a solution of 30:70 ethyl ac-etate/hexane as eluent. The lengths of their side chains were confirmed to be C21, C23, C25, and C27by mass analysis.
All the identified compounds were grouped to be four-teen different skeletons: lupane (1, 2, 3, and 4), oleanane (5, 6, 7, 8, 9, 10, 11, and 12), tirucallane (13), dammaranoid (14 and 15), steroid (16, 17, 18, 19, and 20), lignan (21 and 22), flavonoid (23 and 24), and four aromatic alkanoid types in-cluding C6(30), C6-C1(25, 27, 28, 29, 31, and 32), phenyliso-propanoid (33), phenylphenyliso-propanoid (26 and 37), and other skel-etons (34, 35, and 36).
Compounds with higher quantities were tested for their cytotoxicity using two cancer cell lines, NUGC-3 and HONE-1. At the concentration of 20mg/mL, 29 was found to be the most cytotoxic compound, and the survival rates of NUGC-3 and HONE-1 were 13% and 24%, respectively. At the same concentration, compound 4 caused the NUGC-3 and HONE-1 cells to show 10% and 38% survival rates, respec-tively.
EXPERIMENTAL SECTION
The roots of R. javanica L. var. roxburghiana were col-lected at Kaohsiung, Taiwanin June, 2000. Melting points were collected using a Yanaco MP-53 apparatus. Optical Rotations were measured with a JASCO DIP-180 digital
polarimeter at room temperature. IR spectra were recorded on a Perkin-Elmer 983G spectrophotometer. 1H and 13C NMR spectra were recorded on Bruker AM-300 and DMX-500 instruments using tetramethylsilane (TMS) as in-ternal standard. Chemical shifts are given ind values (ppm) and coupling constants (J) are given in hertz (Hz). Elec-tron-impact mass spectrum (EI-MS) and fast atomic bombard mass spectrum (FAB-MS) were obtained onJMS-HX 110 and JEOL JMS-HX 110 mass spectrometers, respectively. For thin-layer chromatography analysis, Kieselgel 60 F254 plates (Merck, Germany) were used. HPLC was performed on GBC LC-1440 and LDC Analytical-III liquid chromatographs withLichrosorb Si-60 column (7mm, 250 ´ 10 mm, Merck, Germany).
Extraction and Isolation
The air dried roots of R. javanica L. var. roxburghiana (18.0 kg) were extracted with 120 L MeOH three times (seven days each time) at room temperature. The combined extracts were evaporated under vacuum to give a residue, which was suspended in 8 L water and then partitioned with EtOAc and n-BuOH, successively. The EtOAc layer was evaporated to yield residues of 500 g, which were subjected to cytotoxicity tests first. At the concentration of 50mg/mL, the cell viabilities of 59 T, DLD-1, HONE-1, and SCM1 were 19%, 56%, 16%, and 42%, respectively. Based on these find-ings, the ethyl acetate layer was then chromatographed by Si-column and HPLC, repeatedly. The eluent systems are combinations of n-hexane and EtOAc, EtOAc, EtOAc and MeOH in a step elution mode. After a series of separations, the components obtained were 1 (50 mg), 2 (1 mg), 3 (3 mg), 4 (6 mg), 5 (13 mg), 6 (2 mg), 7 (12 mg), 8 (12 mg), 9 (4 mg), 10 (2 mg), 11 (2 mg), 12 (9 mg), 13 (3 mg), 14 (14 mg), 15 (7 mg), 16 (70 mg), 17 (18 mg), 18 (9 mg), 19 (16 mg), 20 (6 mg), 21 (8 mg), 22 (3 mg), 23 (6 mg), 24 (7 mg), 25 (4 mg), 26 (14 mg), 27 (30 mg), 28 (4 mg), 29 (2.5 g), 30 (1 mg), 31 (1 mg), 32 (16 mg), 33 (2 mg), 34 (120 mg), 35 (3 mg), 36 (1 mg), and 37 (5 mg).
The physical and chemical data including major1 H-NMR interpretations of compounds 1-37 were list as follows:
Betulonic acid (1) IRnmax: 3300-2500, 1706, 1697, 1645 cm-1; mp: 243-244 °C; 1H-NMR (CDCl3)d: 0.89, 0.93, 0.95, 0.98, 1.03 (each 3H, s), 1.65 (3H, s, H-30), 2.3-2.5 (2H, m, H-2), 2.96 (1H, td, J = 10.8, 4.4 Hz, H-19), 4.57, 4.70 (each 1H, brs, H-29); EI-MS m/z (%): 454 (M+, 58), 248 (64), 219 (42), 205 (76), 189 (88), 136 (100), 121 (90). Betulinic acid (2) IRnmax: 3462, 3300-2500, 1690, 1646 cm-1; mp: 300-301 °C;1H-NMR (CDCl3)d: 0.73, 0.80, 0.91, 0.94, 0.95 (each 3H, s), 1.67 (3H, s, H-30), 2.97 (1H, td, J = 10.4, 4.8 Hz, H-19), 3.16 (1H, dd, J = 10.8, 5.4 Hz, H-3), 4.58, 4.71 (each 1H, brs, H-29); FAB-MS m/z (%): 457 (M++H, 7), 307 (25), 154 (100), 136 (60). Betulin (3) IRnmax: 3355, 1645 cm-1; mp: 252-254°C;1H-NMR (CDCl3)d: 0.74, 0.80, 0.94, 0.96, 1.00 (each 3H, s), 1.66 (3H, s, H-30), 2.35 (1H, td, J = 10.6, 5.8 Hz, H-19), 3.16 (1H, dd, J = 11.0, 5.3 Hz, H-3), 3.31, 3.78 (each 1H, d, J = 10.8 Hz), 4.56, 4.66 (each 1H, brs); EI-MS m/z (%): 442 (M+, 40), 411 (60), 203 (95), 189 (100), 95 (85). Lantabetulic acid (4) IRnmax: 3372, 3300-2500, 1692, 1644 cm-1; mp: 252-254°C;1H-NMR (CDCl3)d: 0.83, 0.93, 0.94, 1.00 (each 3H, s), 1.66 (3H, s, H-30), 2.96 (1H, td, J = 10.8, 4.8 Hz, H-19), 3.71 (1H, dd, J = 8.8, 1.6 Hz, H-25), 4.21 (1H, dd, J = 8.8, 2.8 Hz, H-25), 4.59, 4.70 (each 1H, brs); EI-MS m/z (%): 442 (M+, 58), 411 (60), 203 (95), 189 (100), 95 (85). 3-Oxoolean-18-en-28-oic acid (5) IRnmax: 3300-2500, 1700, 1654 cm-1; mp: 222°C;1 H-NMR (CDCl3)d: 0.76, 0.92, 0.95, 0.97, 0.98, 0.99, 1.05 (each 3H, s), 2.4-2.5 (2H, m, H-2), 5.14 (1H, s, H-19); EI-MS m/z (%): 454 (M+, 10), 248 (68), 235 (77), 191 (100), 190 (73). 3b-Hydroxyolean-18-en-28-oic acid (6) IR nmax: 3460, 3300-2500, 1694, 1653 cm-1; mp: 271-273°C;1H-NMR (CDCl3)d: 0.74, 0.75, 0.84, 0.94, 0.95, 0.96, 0.98 (each 3H, s), 3.18 (1H, dd, J = 11.0, 5.4 Hz, H-3), 5.16 (1H, s, H-19); EI-MS m/z (%): 456 (M+, 20), 248 (82), 203 (72), 189 (100), 163 (58).
3-Oxo-6b-hydroxyolean-18-en-28-oic acid (7)
IRnmax: 3405, 3300-2500, 1701 cm-1;1H-NMR (CDCl3) d: 0.72, 0.94, 0.97, 1.12, 1.32, 1.38, 1.42 (each 3H, s), 2.78 (1H, td, J = 15.2, 6.4 Hz, H-2), 4.44 (1H, brs, H-6), 5.14 (1H, s, H-19); EI-MS m/z (%): 470 (M+, 10), 426 (42), 235 (64), 189 (100), 163 (56). Semimoronic acid (8) IRnmax: 3412, 3300-2500, 1698 cm-1; mp: 260-261°C; 1 H-NMR (CDCl3)d: 0.72, 0.85, 0.94, 0.95, 0.97, 1.00 (each 3H, s), 3.71 (1H, dd, J = 8.4, 1.6 Hz, H-25), 4.26 (1H, dd, J = Constituents in the Roots of Rhus javanica J. Chin. Chem. Soc., Vol. 52, No. 4, 2005 837
8.4, 2.4 Hz, H-25), 5.13 (1H, s, H-19); EI-MS m/z (%): 470 (M+, 10), 424 (24), 189 (84), 163 (100), 119 (80), 105 (95).
3-O-Methyl semimoronic acid (9)
IRnmax: 3300-2500, 2929, 2861, 1700, 1606 cm-1; mp: 235-238°C;1H-NMR (CDCl3)d: 0.72, 0.94, 0.94, 0.94, 0.96 (each 3H, s), 1.16 (1H, m, Ha-2), 2.16 (1H, m, Hb-2), 3.20 (3H, s, H-31), 3.72 (1H, dd, J = 8.4, 1.6 Hz, H-25), 4.26 (1H, dd, J = 8.4, 2.4 Hz, H-25), 5.12 (1H, s, H-19); EI-MS m/z (%): 484 (M+, 20), 438 (22), 249 (28), 235 (23), 189 (82). 3-Oxoolean-12-en-28-oic acid (10) IRnmax: 3300-2500, 3082, 1697, 1387, 1367, 1271, 738 cm-1; mp: 183-185°C;1H-NMR (CDCl3)d: 0.79, 0.88, 0.91, 1.01, 1.02, 1.06, 1.12 (each 3H, s), 2.34 (1H, ddd, J = 15.6, 6.8, 3.6 Hz, Ha-2), 2.52 (1H, ddd, J = 15.6, 11.6, 7.6 Hz, Hb-2), 2.81 (1H, dd, J = 9.6, 4.0 Hz, H-18), 5.28 (1H, t, J = 4.0 Hz, H-12); FAB-MS m/z (%): 455 (M++H, 7), 409 (3), 391 (3), 248 (9), 154 (100). Oleanolic acid (11) IR nmax: 3450, 3300-2600, 3005, 1696, 1388, 1037 cm-1; mp: 273-275°C;1H-NMR (CD3OD)d: 0.76 (1H, brd, J = 9.6 Hz, H-5), 0.78, 0.82, 0.91, 0.95, 0.95, 0.98, 1.16 (each 3H, s), 2.85 (1H, dd, J = 14.0, 4.0 Hz, H-18), 3.15 (1H, dd, J = 11.2, 4.8 Hz, H-3), 5.24 (1H, t, J = 3.6 Hz. H-12); FAB-MS m/z (%): 457 (M++H, 1), 391 (3), 154 (100). Lantanolic acid (12) IRnmax: 3462, 3300-2600, 2942, 1704, 1643 cm-1; mp: 307-309°C;1H-NMR (CDCl3)d: 0.71, 0.88, 0.90, 0.95, 1.01, 1.10 (each 3H, s), 2.80 (1H, dd, J = 13.6, 4.4 Hz, H-18), 3.87 (1H, dd, J = 8.8, 1.6 Hz, H-25), 4.19 (1H, dd, J = 8.8, 2.8 Hz, H-25), 5.29 (1H, t, J = 2.8 Hz, H-12); EI-MS m/z (%): 470 (M+, 35), 424 (38), 248 (38), 241 (100), 203 (96). 3-Oxotirucalla-7,24-dien-21-oic acid (13) IRnmax: 3382, 3300-2600, 1704, 1654 cm-1; mp: 274-276 °C; 1H-NMR (CDCl3)d: 0.88, 0.98, 1.00, 1.04, 1.10 (each 3H, s), 1.57 (3H, s, H-26), 1.67 (3H, s, H-27), 2.73 (1H, td, J = 14.8, 4.8 Hz, Hb-2), 5.08 (1H, t, J = 6.8 Hz, H-24), 5.30 (1H, brs, H-7); EI-MS m/z (%): 454 (M+, 35), 439 (100), 421 (64), 297 (82). Dipterocarpol (14) IRnmax: 3482, 2954, 2875, 1705 cm-1; mp: 133-134°C; 1 H-NMR (CDCl3)d: 0.87, 0.93, 0.99, 1.02, 1.07, 1.14, 1.62, 1.67 (each 3H, s), 5.11 (1H, t, J = 7.2 Hz, H-24); EI-MS m/z (%): 424 (M+, 58), 355 (24), 205 (58), 109 (100). 3b-Hydroxy-22,23,24,25,26,27-hexanordammaran-20-one (15) IRnmax: 3398, 2938, 2869, 1708 cm-1; mp: 133-134°C; 1 H-NMR (CDCl3)d: 0.75, 0.83, 0.85, 0.95, 0.96 (each 3H, s), 2.11 (3H, s, H-21), 2.56 (1H, td, J = 11.2, 6.4 Hz, H-17), 3.18 (1H, dd, J = 11.2, 5.2 Hz, H-3); EI-MS m/z (%): 360 (M+, 20), 317 (48), 299 (100), 207 (48), 95 (63). b-Sitosterol (16) IRnmax: 3428, 1640 cm-1; mp: 144-146°C;1H-NMR (CDCl3)d: 0.66 (3H, s, H-18), 0.79 (3H, d, J = 6.5 Hz, H-27), 0.81 (3H, d, J = 6.5 Hz, H-26), 0.82 (3H, t, J = 7.2 Hz, H-29), 0.91 (3H, d, J = 6.4Hz, H-21), 1.00 (3H, s, H-19), 3.49 (1H, m, H-3), 5.33 (1H, d, J = 5.3 Hz, H-6); EI-MS m/z (%): 414 (M+, 92), 396 (100), 81 (78). Stigmast-4-en-3-one (17) IRnmax: 3040, 1683, 1380 cm-1; mp: 84-86°C;1H-NMR (CDCl3)d: 0.68, 1.15 (each 3H, s), 0.81, 0.83 (each 3H, d, J = 6.3 Hz), 0.86 (3H, t, J = 7.2 Hz), 0.95 (3H, d, J = 6.2 Hz), 5.69 (1H, s); EI-MS m/z (%): 412 (M+, 95), 397, 370, 289, 229, 124 (100). Stigmast-4-ene-3,6-dione (18) IRnmax: 1671, 1622 cm-1; mp: 172-173°C;1H-NMR (CDCl3)d: 0.70 (3H, s, H-18), 0.78 (3H, d, J = 6.8 Hz, H-27), 0.79 (3H, d, J = 7.0 Hz, H-26), 0.83 (3H, t, J = 7.0 Hz, H-29), 0.91 (3H, d, J = 6.5 Hz, H-21), 1.14 (3H, s, H-19), 6.15 (1H, s, H-4); EI-MS m/z (%): 426 (M+, 100), 412 (54), 398 (58), 137 (82). Stigmastane-3,6-dione (19) IRnmax: 1716, 1708, 1239 cm-1; mp: 205-206°C;1 H-NMR (CDCl3)d: 0.69 (3H, s), 0.82 (3H, d, J = 6.8 Hz), 0.84 (3H, d, J = 6.5 Hz), 0.85 (3H, t, J = 7.0 Hz), 0.93 (3H, d, J = 6.4 Hz), 0.96 (3H, s); EI-MS m/z (%): 428 (M+, 88), 413 (10), 331 (9), 287 (22), 245 (59), 231 (19), 149 (100). Stigmast-7-en-3-ol (20) IRnmax: 3422, 1450, 1377 cm-1; mp: 151-152°C;1 H-NMR (CDCl3)d: 0.51 (3H, s, H-18), 3.56 (1H, m, H-3), 5.14 (1H, brs, H-7); EI-MS m/z (%): 414 (M+,100), 396 (36), 271 (48), 255 (92). Pinoresinol (21) IRnmax: 3416, 2928, 2858, 1608 cm-1; mp: 136-137°C;
1 H-NMR (CDCl3)d: 3.10 (2H, m, H-1, H-5), 3.88 (6H, s, H3-3¢, H3-3²), 3.94 (2H, dd, J = 9.1, 3.5 Hz, Ha-4, Ha-8), 4.23 (2H, dd, J = 9.1 Hz, 6.8 Hz, Hb-4, Hb-8), 4.72 (2H, d, J = 4.2 Hz, H-2, H-6), 6.80 (2H, dd, J = 8.4, 1.6 Hz, H-6¢, H-6²), 6.87 (2H, d, J = 8.4 Hz, H-5¢, H-5²), 6.87 (2H, d, J = 1.6 Hz, H-2¢, H-2²); EI-MS m/z (%): 358 (M+, 45), 205 (20), 151 (100), 137 (55), 131 (28). 4-Oxopinoresinol (22) IRnmax: 3431, 2944, 2855, 1762, 1605 cm-1; mp: 127-128°C;1H-NMR (CDCl3)d: 3.24 (1H, m, H-1), 3.46 (1H, dd, J = 9.2, 4.0 Hz, H-5), 4.04 (1H, dd, J = 9.2, 4.4 Hz, Ha-8), 4.33 (1H, dd, J = 9.2, 6.8 Hz, Hb-8), 5.31 (1H, d, J = 4.0 Hz, H-6), 5.33 (1H, d, J = 4.0 Hz, H-2), 6.7-7.3 (6H, m, Ar-H); EI-MS m/z (%): 372 (M+, 88), 163 (24), 151 (100), 131 (55). 4¢,5,7-Trihydroxyflavanone (23) IRnmax: 3384, 1631, 1605, 1462 cm-1;1H-NMR (CDCl3) d: 2.72 (1H, dd, J = 17.1, 3.0 Hz, Ha-3), 3.19 (1H, dd, J = 17.1, 12.9 Hz, Hb-3), 5.44 (1H, dd, J = 12.9, 3.0 Hz, H-2), 5.92 (1H, d, J = 2.2 Hz, H-6), 5.94 (1H, d, J = 2.2 Hz, H-8), 6.90 (1H, d, J = 8.6 Hz, H-3¢), 7.39 (1H, d, J = 8.6 Hz, H-2¢), 12.18 (1H, s, OH-5); EI-MS m/z (%): 272 (M+, 100), 179 (24), 153 (80), 120 (48). trans-3,4¢,7-Trihydroxyflavanone (24) IRnmax: 3271, 1669, 1610, 1608 cm-1;1H-NMR (CDCl3) d: 4.41 (1H, d, J = 3.2 Hz, OH-3), 4.56 (1H, dd, J = 12.0, 3.2 Hz, H-3), 5.04 (1H, d, J = 12.0 Hz, H-2), 6.39 (1H, d, J = 2.4 Hz, H-8), 6.61 (1H, dd, J = 8.4, 2.4 Hz, H-6), 6.87 (1H, d, J = 8.8 Hz, H-2¢), 7.42 (1H, d, J = 8.8 Hz, H-3¢), 7.72 (1H, d, J = 8.4 Hz, H-5); EI-MS m/z (%): 272 (M+, 20), 243 (75), 149 (32), 137 (100), 107 (45). Vanillin (25) IRnmax: 3217, 1668, 1590, 1510 cm-1; mp: 82-83°C; 1H-NMR (CDCl3)d: 3.95 (3H, s, -OCH3), 6.20 (1H, s, -OH), 6.99 (1H, d, J = 2.0 Hz, H-5), 7.38 (1H, d, J = 2.0 Hz, H-2), 7.40 (1H, dd, J = 8.0, 2.0 Hz, H-6), 9.81 (1H, s, -CHO); EI-MS m/z (%): 152 (M+, 100), 123 (30), 109 (16). Methyl ferulate (26) IRnmax: 3399, 1706, 1637, 1594, 1516 cm-1; mp: 62-63 °C;1H-NMR (CDCl3)d: 3.78 (3H, s, -COOCH3), 3.90 (3H, s, -OCH3), 5.89 (1H, s, -OH), 6.91 (1H, d, J = 8.1 Hz, H-5), 7.00 (1H, d, J = 1.9 Hz, H-2), 7.07 (1H, dd, J = 8.1, 1.9 Hz, H-6), 6.28 (1H, d, J = 15.9 Hz, H-7), 7.60 (1H, d, J = 15.9 Hz, H-8); EI-MS m/z (%): 208 (M+, 100), 177 (68), 145 (36). 3,5-Dihydroxytoluene (27) IRnmax: 3356, 1604, 1480 cm-1; mp: 108-110°C;1 H-NMR (CDCl3)d: 2.21 (3H, s, -CH3), 6.15 (1H, t, J = 2.4 Hz, H-4), 6.22 (2H, d, J = 2.4 Hz, H-2, H-6); EI-MS m/z (%): 124 (M+, 100), 123 (78), 95 (20). 4-Hydroxy-3,5-dimethoxybenzaldehyde (28) IRnmax: 3415, 1684, 1609, 1514 cm-1; mp: 114-116°C; 1 H-NMR (CDCl3)d: 3.96 (6H, s, -OCH3), 6.05 (1H, s, -OH), 7.14 (2H, s, H-2, H-6), 9.80 (1H, s, -CHO); FAB-MS m/z (%): 183 (M++1, 15), 154 (100), 136 (66). Methyl gallate (29) IRnmax: 3442, 1700, 1614, 1540 cm-1; mp: 240-242°C; 1 H-NMR (CD3COCD3)d: 3.78 (3H, s, -OCH3), 7.10 (2H, s, H-2, H-6); EI-MS m/z (%): 184 (M+, 52), 153 (100), 125 (26). 2,6-Dimethoxy[1,4]benzoquinone (30) IRnmax: 3415, 1697, 1646, 1596, 1447 cm-1; mp: 255-258°C;1H-NMR (CDCl3)d: 3.80 (6H, s, -OCH3), 5.84 (2H, s, H-2, H-6); FAB-MS m/z (%): 169 (M++1, 15), 154 (100), 136 (69). 3,4,5-Trimethoxybenzyl alcohol (31) IRnmax: 3400, 1610, 1505 cm-1; mp: 36-38°C;1H-NMR (CDCl3)d: 3.82 (3H, s, -OCH3), 3.85 (6H, s, -OCH3), 4.62 (2H, s, -CH2OH), 6.59 (2H, s, H-2, H-6); FAB-MS m/z (%): 198 (M+, 20), 154 (100), 137 (60), 136 (68). Gallic acid (32) IRnmax: 3364, 3300-2600, 1682, 1616, 1455 cm-1; mp: 258-260°C;1H-NMR (CD3OD)d: 7.06 (2H, s, H-2, H-6); EI-MS m/z (%): 170 (M+, 100), 153 (66). Ficusol (33) IRnmax: 3419, 1731, 1602, 1518 cm-1;1H-NMR (CDCl3) d: 3.69 (3H, s, -COOCH3), 3.72 (1H, dd, J = 10.5, 5.3 Hz, H-2), 3.80 (1H, dd, J = 12.9, 5.3 Hz, Hb-3), 3.87 (3H, s, -OCH3), 4.09 (1H, dd, J = 12.9, 10.5 Hz, Ha-3), 5.50 (1H, s, -OH), 6.72 (1H, dd, J = 8.6, 1.9 Hz, H-6¢), 6.76 (1H, d, J = 1.9 Hz, H-2¢), 6.85 (1H, d, J = 8.6 Hz, H-5¢); EI-MS m/z (%): 226 (M+, 100), 196 (96), 195 (94), 164 (60).
2-Hydroxy-6-pentadec-8(Z)-enylbenzoic acid (34)
IRnmax: 3436, 2925, 2850, 1648, 1604 cm-1; mp: 136-137°C;1H-NMR (CDCl3)d: 0.88 (3H, t, H-15¢), 1.59 (2H, m, H-2¢), 2.00 (4H, m, H-7¢, H-10¢), 2.96 (2H, t, J = 7.6 Hz, H-1¢), 5.33 (2H, m, H-8¢, H-9¢), 6.74 (1H, d, J = 7.2 Hz, H-5), Constituents in the Roots of Rhus javanica J. Chin. Chem. Soc., Vol. 52, No. 4, 2005 839
6.84 (1H, d, J = 8.0 Hz, H-3), 7.31 (1H, dd, J = 8.0, 7.2 Hz, H-4), 8.34 (1H, brs, COOH-1), 11.01 (1H, brs, OH-2); EI-MS
m/z (%): 364 (M+, 8), 302 (12), 149 (16), 108 (100). 5-Formylmellein (35) IRnmax: 3121, 2710, 1673, 1578, 1476 cm-1; mp: 127-129°C;1H-NMR (CDCl3)d: 1.58 (3H, d, J = 6.4 Hz), 3.05 (1H, dd, J = 18.0, 11.6 Hz, Ha-4), 3.95 (1H, dd, J = 18.0, 3.2 Hz, Hb-4), 4.72 (1H, m, H-3), 7.05 (1H, d, J = 8.8 Hz, H-7), 7.92 (1H, d, J = 8.8 Hz, H-6), 10.01 (1H, s, CHO-5), 11.94 (1H, brs, OH-8); EI-MS m/z (%): 206 (M+, 96), 191 (100), 163 (75), 136 (68). 5-Hydroxymethylmellein (36) IRnmax: 3415, 1675, 1615, 1480 cm-1; mp: 111-112°C; 1 H-NMR (CDCl3)d: 1.54 (3H, d, J = 6.4 Hz), 2.86 (1H, dd, J = 17.2, 9.0 Hz, Ha-4), 3.17 (1H, dd, J = 17.2, 3.2 Hz, Hb-4), 4.60 (2H, s, CH2OH-5), 4.70 (1H, m, Ha-3), 6.87 (1H, d, J = 8.4 Hz, H-7), 7.43 (1H, d, J = 8.4 Hz, H-6), 11.16 (1H, s, OH-8); EI-MS m/z (%): 208 (M+, 100), 190 (28), 178 (40), 161 (39). Alkyls caffeate (37) IRnmax: 3420, 1690, 1602, 1464 cm-1;1H-NMR (CDCl3) d: 0.85 (3H, t, J = 6.8 Hz), 1.65 (2H, m, H-2¢), 4.16 (2H, t, J = 6.6 Hz, H-1¢), 6.24 (1H, d, J = 16.0 Hz, H-8), 6.84 (1H, d, J = 8.2 Hz, H-5), 6.98 (1H, dd, J = 8.2, 2.0 Hz, H-6), 7.07 (1H, d, J = 2.0 Hz, H-2), 7.55 (1H, d, J = 16.0 Hz, H-7); EI-MS m/z (%): 572 (M+, 4), 544 (M+, 14), 516 (M+, 60), 488 (M+, 10), 180 (100), 163 (64). ACKNOWLEDGMENT
This research was supported by a grant from the Na-tional Science Council of the Republic of China (NSC 91-2323-B-002-002). We also thank the Biotechnology and Pharmaceutical Research Division, National Health Re-search Institutes for cytotoxic tests.
Received February 21, 2005.
REFERENCES
1. Huang, T. S., Ed. Flora of Taiwan. Vol. 3; Editorial Commit-tee of the Flora of Taiwan, 2ndEd., Taipei, Taiwan, 1993; p
585.
2. Kao, M. T., Ed. Popular Herbal Remedies of Taiwan (2); SMC Publishing Inc., Taipei, Taiwan, 1988; p 91.
3. Parveen, N.; Khan, N. U. D. J. Indian Chem. Soc. 1988, 65, 737-738.
4. Taniguchi, S.; Yazaki, K.; Ryoko, Y. U.; Kawakami, K. Y.; Ito, H.; Hatano, T.; Yoshida, T. Phytochemistry 2000, 53, 357-364.
5. Kuo, S. C.; Teng, C. M.; Lee, L. G.; Chiu, T. H.; Wu, T. S.; Huang, S. C.; Wu, J. B.; Shieh, T. Y.; Chang, R. J.; Chou, T. C. Planta Med. 1991, 57, 247-249.
6. Parveen, N.; Singh, M. P.; Khan, N. U.; Achari, B.; Logani, M. K. Phytochemistry 1991, 30, 2415-2416.
7. Sung, C. K.; Akiyama, T.; Sankawa, U.; Iitaka, Y.; Han, D. S.
J. Chem. Soc. Chem. Commun. 1980, 19, 909-910.
8. El, S.; El, A. Planta Med. 1966, 14, 171-176.
9. Takechi, M.; Tanaka, Y.; Takehara, M.; Nonaka, G. I.; Nishioka, I. Phytochemistry 1985, 24, 2245-2250.
10. Wang, H. C.; Fujimoto, Y. Phytochemistry 1993, 33, 151-154.
11. Barba, B.; Diaz, J. G.; Herz, W. Phytochemistry 1992, 31, 4374-4375.
12. Yadav, R. D.; Kataky, J. C. S.; Mathur, R. K. Indian J. Chem.
Sect. B 1998, 37, 1214-1216.
13. Bagchi, A.; Sahai, M.; Sinha, S. C.; Ray, A. B.; Oshima, Y.; Hinkino, H. J. Chem. Res. Synop. 1985, 398-401.
14. Ahsan, M.; Armstrong, J. A.; Gray, A. I.; Waterman, P. G.
Phytochemistry 1995, 38, 1275-1278.
15. Ye, Y.; Kinoshita, K.; Koyama, K.; Takahashi, K.; Kondo, N.; Yuasa, H. J. Nat. Prod. 1998, 61, 456-460.
16. Gonzalez, A. G.; Amaro, J.; Fraga, B. M.; Luis, J. G.
Phyto-chemistry 1983, 22, 1828-1830.
17. Zhou, J.; Pan, D.; Li, Z. Shanghai Yike Daxue Xuebao 1986,
13, 340-345.
18. Sung, T. V.; Peter-Katalinic, J.; Adam, G. Phytochemistry 1991, 30, 3717-3720.
19. Okada, Y.; Shibata, S.; Javellana, A. M. J.; Kamo, O. Chem.
Pharm. Bull. 1988, 36, 1264-1269.
20. Barre, J. T.; Bowden, B. F.; Coll, J. C.; Jesus, J. D.; Fuente, V. E. D. Phytochemistry 1997, 45, 321-324.
21. Ma, C.; Nakamura, N.; Hattori, M.; Kakuda, H.; Qiao, J.; Yu, H. J. Nat. Prod. 2000, 63, 238-242.
22. Tori, M.; Matsuda, R.; Sono, M. U.; Asakawa, Y. Magn.
Reson. Chem. 1988, 26, 581-590.
23. Tanaka, R.; Matsuda, M.; Matsunaga, S. Phytochemistry 1987, 26, 3365-3366.
24. Jain, T. C.; Banks, C. M. Can. J. Chem. 1968, 46, 2325-2357.
25. Gaspar, E. M. M.; Neves, H. J. C. Phytochemistry 1993, 34, 523-528.
26. Wu, T. S.; Li, C. Y.; Leu, Y. L.; Hu, C. Q. Phytochemistry 1999, 50, 509-512.
27. Seo, S.; Uomori, A.; Yoshimura, Y.; Sankawa, U.; Ebizuka, Y. J. Chem. Soc. Chem. Commun. 1987, 1876-1878. 28. Greca, M. D.; Ferrara, M.; Fiorentino, A.; Monaco, P.;
Previtera, L. Phytochemistry 1998, 49, 1299-1304. 29. Quideau, S.; Ralph, J. J. Chem. Soc. Perkin Trans 1 1993, 6,
653-660.
30. Barros, D. A. D.; Alvarenga, M. A. D.; Gottlieb, O. R.; Gottlieb, H. E. Phytochemistry 1982, 21, 2107-2110. 31. Oyamada, B. Bull. Chem. Soc. Jpn. 1966, 39, 507-509. 32. Seca, A. M. L.; Silva, A. M. S.; Silvestre, A. J. D.; Cavaleiro,
J. A. S.; Domingues, F. M. J.; Pascoal-Neto, C.
Phyto-chemistry 2001, 56, 759-768.
33. Wallace, G.; Fry, S. C. Phytochemistry 1995, 39, 1293-1300. 34. Garg, S. N.; Misra, L. N.; Agarwal, S. K. Phytochemistry
1989, 28, 1771-1772.
35. Gutierrez, A. B.; Herz, W. Phytochemistry 1988, 27,
3871-3874.
36. Hattori, M.; Shu, Y. Z.; Tomimori, T.; Kobashi, K.; Namba, T. Phytochemistry 1989, 28, 1289-1290.
37. Otsuka, H.; Takeuchi, M.; Inoshiri, S.; Sato, T.; Yamasaki, K.
Phytochemistry 1989, 28, 883-886.
38. Olszewski, J. D.; Marshalla, M.; Sabat, M.; Sundberg, R. J.
J. Org. Chem. 1994, 59, 4285-4296.
39. Gu, J. Q.; Park, E. J.; Luyengi, L.; Hawthorne, M. E.; Mehta, R. G.; Farnsworth, N. R.; Pezzuto, J. M.; Kinghorn, A. D.
Phytochemistry 2001, 58, 121-128.
40. Li, Y. C.; Kuo, Y. H. Phytochemistry 1998, 49, 2417-2421. 41. Satoh, M.; Takeuchi, N.; Fujita, T.; Yamazaki, K.; Tobinaga,
S. Chem. Pharm. Bull. 1998, 46, 1501-1505.
42. Anderson, J. R.; Edwards, R. L.; Whalley, A. J. S. J. Chem.
Soc. Perkin Trans 1 1983, 2185-2192.
43. Tsai, I. L.; Lin, W. Y.; Teng, C. M.; Ishikawa, T.; Doong, S. L.; Huang, M. W.; Chen, Y. C.; Chen, I. S. Planta Med. 2000,
66, 618-623.
