Furoquinoline Alkaloids and Cytotoxic Constituents from the Leaves of Melicope semecarpifolia Original Paper
Abstract Three new furoquinoline alkaloids, melicarpine, semecarpine, and ()-8-methoxyplatydesmine, together with a known flavone, ayanin, were isolated from the leaves of Melicope semecarpifolia. The structures of the three alkaloids were determined through spectral analyses. Of the nineteen isolates obtained here and during earlier work, twelve exhibited cytotoxic activity against P-388, HT-29, or A549 cell lines in vitro. Among
Introduction
542
Furoquinoline alkaloids and their derivatives are widely distributed in plants of the family Rutaceae, including the genera Toddalia, Zanthoxylum, Dictamnus, Pelea, Stauranthus, and Melicope [1], [2], [3], [4], [5], [6], [7], [8]. Many of the isolates exhibit diverse biological activities, including antifungal [3], cytotoxic [5] and anti-platelet aggregation [8] properties. Melicope semecarpifolia (Merr.) T. Hartley [Evodia merrillii Kanehira & Sasaki ex Kanehira; Melicope confusa (Merr.) Liu] is a small-to-medium-sized evergreen trifoliated tree, distributed in lower altitude forests and thickets in Taiwan and the Philippines [9]. The roots of this plant have been used as a carminative in traditional medicine [10]. In a series of studies on the cytotoxic principles of Formosan plants, we have screened about 900 species and M. semecarpifolia was one of the active species. Previously, we have reported three new compounds, melisemine, confusadine, and melicarpinone, along with twelve known compounds, including several anti-platelet aggregation
Jih-Jung Chen1 Chang-Yih Duh2 Hung-Yi Huang1 Ih-Sheng Chen3
them, confusameline is the most cytotoxic isolate, and exhibited more potent cytotoxicity (ED50 value = 0.03 mg/mL) against the P-388 cell line than the reference compound mithramycin (ED50 value = 0.06 mg/mL). Key words Melicope semecarpifolia ´ Rutaceae ´ leaves ´ alkaloids ´ furoquinolines ´ melicarpine ´ semecarpine ´ ()-8-methoxyplatydesmine ´ cytotoxicity
agents [11] from the leaves of this plant. Continuing investigation of the minor constituents and the cytotoxic principles led to the isolation and characterization of three new alkaloids, melicarpine (1), semecarpine (2), and ()-8-methoxyplatydesmine (3), together with a known flavone, ayanin. This paper describes the structural elucidation of 1 ± 3 and the cytotoxic activity of all of the isolates.
Materials and Methods General experimental procedures All melting points were determined on a Yanaco micro-melting point apparatus and are uncorrected. IR spectra (KBr or neat) were taken on a Perkin Elmer system 2000 FT-IR spectrometer. UV spectra were obtained on a Shimadzu UV-160A spectrophotometer in EtOH. EI-mass spectra were recorded on a VG Biotech Quattro 5022 spectrometer. HR-EI, FAB, and HR-FAB-mass spectra were recorded on a JEOL JMX-HX 110 spectrometer. NMR
Affiliation 1 Department of Pharmacy, Tajen Institute of Technology, Pingtung, Taiwan, Republic of China 2 Institute of Marine Resources, National Sun Yat-sen University, Kaohsiung, Taiwan, Republic of China 3 School of Pharmacy, Kaohsiung Medical University, Kaohsiung, Taiwan, Republic of China Correspondence Prof. Dr. I. S. Chen ´ School of Pharmacy ´ Kaohsiung Medical University ´ Kaohsiung ´ Taiwan ´ Republic of China ´ E-mail:
[email protected] ´ Fax: +886-7-3210683 Dr. J. J. Chen ´ Department of Pharmacy ´ Tajen Institute of Technology ´ Pingtung ´ Taiwan ´ Republic of China ´ E-mail:
[email protected] ´ Fax: +886-8-7625308 Received October 17, 2002 ´ Accepted February 15, 2003 Bibliography Planta Med 2003; 69: 542±546 ´ Georg Thieme Verlag Stuttgart ´ New York ´ ISSN 0032-0943
Original Paper 543
spectra including COSY, NOESY, HMBC and HSQC experiments were recorded on a Varian Unity 400 and Varian Inova 500 spectrometer either at 400 or 500 MHz (1H) and 100 or 125 MHz (13C), and chemical shifts are given in ppm (d) with TMS as internal standard. Silica gel (60 ± 230, 230 ± 400 mesh) (Merck) was used for CC and silica gel 60 F-254 (Merck) for TLC and preparative TLC. Optical rotations were measured using a Jasco DIP-370 polarimeter in CHCl3 or MeOH. Plant material The leaves of M. semecarpifolia were collected from Lai-I, Pingtung County, Taiwan, in April 1998 and identified by Dr. I. S. Chen. A voucher specimen (Chen 5685) was deposited in the herbarium of the School of Pharmacy, Kaohsiung Medical University, Kaohsiung, Taiwan, Republic of China. Extraction and isolation The dried leaves (11.2 kg) were extracted with cold MeOH and the extract concentrated under reduced pressure. The MeOH extract was partitioned between H2O/CHCl3 (1 : 1) to afford a CHCl3-soluble fraction. Bases in the CHCl3-soluble fraction were extracted with 2 % H2SO4. The acid-soluble part was basified with NH4OH and extracted with CHCl3 to give the basic fraction (fr. A, 32.1 g). Fr. A was washed with MeOH to yield con-
fusameline (7.5 g). The evaporated MeOH washing (19.7 g) was chromatographed over silica gel (658 g) (column 6 47 cm) eluting with CHCl3, gradually increasing the solvent strength with MeOH to obtain 13 frs: fr. A1 (2500 mL, CHCl3), fr. A2 (2500 mL, CHCl3-MeOH, 9 : 1), fr. A3 (2500 mL, CHCl3-MeOH, 4 : 1), fr. A4 (1000 mL, CHCl3-MeOH, 4 : 1), frs. A5 - A9 (each 1000 mL, CHCl3-MeOH, 1 : 1), and frs. A10 - A12 (each 1000 mL, MeOH), fr. A13 (5000 mL, MeOH). Fr. A4 (1.47 g) was rechromatographed on silica gel (48 g) eluting with CHCl3-MeOH (1 : 0 ± 1 : 1) to obtain 7 frs (each 500 mL, fr. A4 ± 1 - fr. A4 ± 7). Fr. A4 ± 3 (267 mg) was rechromatographed on silica gel (9.3 g) using CHCl3-MeOH (20 : 1) as eluent to give 8 frs (each 300 mL, fr. A4 ± 3-1 - fr. A4 ± 3-8). Fr. A4 ± 3-3 (39 mg) was further purified by preparative TLC (CHCl3-Me2CO, 5 : 1) to obtain 1 (3.7 mg) (Rf = 0.60). Fr. A5 (2.37 g) was rechromatographed on silica gel (74 g) eluting with CHCl3-Me2CO (5 : 1) to obtain 8 frs (each 800 mL, fr. A5 ± 1 - fr. A5 ± 8). Fr. A5 ± 3 (189 mg) was further separated by preparative TLC (CHCl3-Me2CO, 5 : 1) to obtain 3 (2.2 mg) (Rf = 0.29). Fr. A10 (429 mg) was rechromatographed on silica gel (14 g) eluting with CH2Cl2/MeOH (10 : 1) to obtain 9 frs (each 150 mL, fr. A10 ± 1 - fr. A10 ± 9). Fr. A10 ± 5 (68 mg) was further purified by preparative TLC (Me2CO-MeOH, 4 : 1) to afford 2 (3.4 mg) (Rf = 0.22). Fr. A10 ± 2 (57 mg) was further purified by preparative TLC (CH2Cl2-MeOH, 45 : 1) to afford 4 (8.4 mg) (Rf = 0.76). Chen J-J et al. Furoquinoline Alkaloids and ¼ Planta Med 2003; 69: 542 ± 546
Original Paper 544
Isolates Melicarpine (1): Colorless needles from MeOH, m. p. 211 ± 213 8C. UV: lmax (log e) = 207 (4.29), 242 (4.47), 287 nm (sh, 3.83). IR: nmax = 3445 (br. OH), 1652 cm±1 (C = O). FAB-MS: m/z (rel. int.) = 306 ([M + H]+, 6). EI-MS: m/z (rel. int.) = 305 (M+, 1), 290 (25), 276 (21), 275 (99.8), 274 (94), 261 (17), 260 (100), 246 (16), 231 (23), 230 (38), 221 (11), 216 (11), 202 (26), 200 (12), 174 (11), 173 (11), 172 (11), 160 (13). HR-EI-MS: C15H15O6N, found: 305.0893 [M]+, calcd.: 305.0899. 1H-NMR (CDCl3, 400 MHz): d = 3.28 (6H, s, OMe-8 2), 3.90 (3H, s, OMe-7), 4.40 (3H, s, OMe-4), 5.85 (1H, s, H-6), 7.12 (1H, d, J = 2.6 Hz, H-3), 7.69 (1H, d, J = 2.6 Hz, H-2). 13C-NMR (CDCl3, 125 MHz): d = 52.3 (OMe8 2), 56.2 (OMe-7), 60.0 (OMe-4), 96.0 (C-8), 106.5 (C-3), 107.1 (C-6), 107.9 (C-3a), 114.6 (C-4a), 143.9 (C-2), 154.9 (C-8a), 162.1 (C-4), 165.1 (C-9a), 166.9 (C-7), 183.4 (C-5). Semecarpine (2): Pale yellow needles from CHCl3-Me2CO, m. p. 145 ± 147 8C. UV: lmax (log e) = 227 (4.61), 262 (4.51), 279 (4.44), 325 (4.05), 411 nm (3.78). IR: nmax = 1445, 1500, 1596 cm±1 (aromatic ring C = C stretch). FAB-MS: m/z (rel. int.) = 284 ([M + H]+, 100); HR-FAB-MS: C17H18O3N, found: 284.1284 [M + H]+, calcd.: 284.1287. 1H-NMR (acetone-d6, 400 MHz): d = 1.48 [3H, s, OCH (Me)C(Me)2], 1.54 [3H, s, OCH(Me)C(Me)2], 1.70 [3H, d, J = 6.8 Hz, OCH(Me)C(Me)2], 4.53 (3H, s, OMe-4), 4.78 [1H, q, J = 6.8 Hz, OCH(Me)C(Me)2], 6.70 (1H, d, J = 9.4 Hz, H-6), 7.49 (1H, d, J = 2.4 Hz, H-3), 7.56 (1H, d, J = 9.4 Hz, H-5), 7.79 (1H, d, J = 2.4 Hz, H-2); (CDCl3, 400 MHz): d = 1.55 [3H, s, OCH(Me)C(Me)2], 1.60 [3H, s, OCH(Me)C(Me)2], 1.68 [3H, d, J = 6.8 Hz, OCH (Me)C(Me)2], 4.42 (3H, s, OMe-4), 4.65 [1H, q, J = 6.8 Hz, OCH (Me)C(Me)2], 6.92 (1H, d, J = 9.4 Hz, H-6), 7.09 (1H, d, J = 2.5, H-3), 7.42 (1H, d, J = 2.5 Hz, H-2), 7.64 (1H, d, J = 9.4 Hz, H-5). 13 C-NMR (acetone-d6, 125 MHz): d = 14.5 [OCH(Me)C(Me)2], 21.0 [OCH(Me)C(Me)2], 27.6 [OCH(Me)C(Me)2], 47.1 [OCH(Me)C (Me)2], 61.3 (OMe-4), 72.1 [OCH(Me)C(Me)2], 102.5 (C-3a), 106.2 (C-4a), 108.8 (C-3), 119.8 (C-8), 122.5 (C-6), 127.5 (C-4), 142.6 (C2), 143.0 (C-8a), 156.5 (C-9a), 163.5 (C-4), 170.8 (C-7). [a]D24: + 2.6 8 (c 0.14, MeOH).
P-388 cells were cultured in Fisher's medium supplemented with 10 % heat-inactivated (56 8C for 30 min) fetal calf serum (FCS). A549 cells were cultured in Eagle's minimum essential medium (EMEM) containing Earle's salts and supplemented with 0.1 mM of non-essential amino acids and 10 % heat-inactivated FCS. HT-29 cells were maintained in Rosewell Park Memorial Institute (RPMI) 1640 medium containing 10 % heat-inactivated FCS. All cell lines were maintained in an incubator at 37 8C in humidified air containing 5 % CO2. The cytotoxic activities of compounds against P-388, A549, and HT-29 were assayed by a modification of the MTT [3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyltetrazolium bromide] colorimetric method [12]. For P-388 cells, 200 mL cultures were established at 1500 cells/well in 96-well tissue culture plates (Falcon). Compounds were dispensed to established cultures at eight concentrations in triplicate. After three days of incubation, P-388 cells were enumerated with MTT. To measure the cytotoxic activities of purified compounds against A549 and HT-29 cells, each cell line was initiated at 1000 cells/well in 96-well microtiter plates. Eight concentrations (triplicate) of test compounds encompassing a 128-fold range were added to each cell line. A549 and HT-29 cells were enumerated using MTT after exposure to test compounds for 6 days, respectively. Fifty mL of 1 mg/mL MTT were added to each well, and plates were incubated at 37 8C for a further 5 h. Formazan crystals were redissolved in DMSO (E. Merck) for 10 min with shaking, and the plate was read immediately on a microtiter plate reader (Dynatech) at a wavelength of 540 nm. The ED50 was defined as the concentration of the test compound resulting in a 50 % reduction of absorbance compared to untreated cells in the MTT assay. Above assays were repeated three times. The clinically applied anticancer agent mithramycin was used as a positive control.
Results and Discussion ()-8-Methoxyplatydesmine (3): Colorless prisms from CHCl3MeOH, m. p. 166 ± 168 8C. UV: lmax nm (log e) = 217 (4.44), 248 (4.59), 280 (3.82), 314 (3.45), 327 (3.44). IR: nmax = 3426 (OH), 1630, 1589, 1520, 1458 cm±1 (aromatic ring C = C stretch). EIMS: m/z (rel. int.) = 289 (M+, 100), 288 (36), 275 (11), 271 (13), 270 (15), 260 (18), 230 (37), 217 (11), 216 (26), 204 (16), 202 (23), 200 (12), 188 (25), 187 (23), 174 (10), 170 (12), 158 (11), 131 (10), 118 (13), 103 (11). HR-EI-MS: C16H19NO4, found: 289.1313 [M]+, calcd: 289.1314. 1H-NMR (CDCl3, 400 MHz): d = 1.26 [3H, s, C(Me)2OH], 1.42 [3H, s, C(Me)2OH], 1.95 [1H, br. s, exchangeable with D2O, C(Me)2OH], 3.60 (2H, d, J = 8.6 Hz, H-3), 4.00 (3H, s, OMe-8), 4.24 (3H, s, OMe-4), 4.63 (1H, t, J = 8.6 Hz, H-2), 6.98 (1H, dd, J = 8.0, 1.2 Hz, H-7), 7.26 (1H, dd, J = 8.4, 8.0 Hz, H6), 7.62 (1H, dd, J = 8.4, 1.2 Hz, H-5). [a]D24: 0 8 (c 0.12, CHCl3). Compounds 5 ± 19: refer to our previous paper [11]. Cytotoxicity assay P-388 cells were kindly provided by Prof. J. M. Pezzuto, University of Illinois at Chicago; A549 (Human lung adenocarcinoma) and HT-29 (human colon carcinoma) were purchased from American Type Culture Collection. Chen J-J et al. Furoquinoline Alkaloids and ¼ Planta Med 2003; 69: 542 ± 546
Melicarpine (1) was isolated as colorless needles. The EI-MS afforded a positive ion at m/z = 305 [M]+, implying a molecular formula of C15H15O6N, which was confirmed by the HR-EI-MS. The presence of a conjugated carbonyl group was revealed by an IR absorption at 1652 cm±1, along with a resonance signal in the 13 C-NMR spectrum at d = 183.4. The 1H-NMR spectrum of 1 showed four methoxy groups, a singlet at d = 4.40 (3H) was characteristic of OMe-4, a singlet at higher field d = 3.28 (6H) due to OMe-8 2, and another singlet at d = 3.90 (3H) attributed to OMe-7. In addition, a singlet signal at d = 5.85 was assigned to H-6, and a pair of ortho-coupled protons at d = 7.69 and 7.12 (each 1H, d, J = 2.6 Hz) were characteristic of H-2 and H-3 of the furan ring, respectively. On the basis of the above results and NOESY experiments (Fig. 1), the structure of 1 was elucidated as 4,7,8,8-tetramethoxy-8H-furo[2,3-b]quinolin-5-one, named melicarpine. The assignments of the 13C-NMR resonances were confirmed by HMQC and HMBC techniques which also supported the structure of 1. Semecarpine (2) was obtained as pale yellow needles. The molecular formula was established as C17H17O3N by FAB ([M +
Fig. 1 NOESY contacts (a) and HMBC connectivities (b) of compound 1.
()-8-Methoxyplatydesmine (3) was isolated as colorless prisms. The HR-EI mass spectrum gave an [M ]+ ion peak at m/z = 289.1313, consistent with a molecular formula of C16H19O4N. The presence of a 4-oxygenated 2,3-dihydrofuroquinoline skeleton was indicated by the UV spectrum showing absorptions at 217, 248, 280, 314, and 327 nm [14]. The presence of a hydroxy group was revealed by IR absorption at 3426 cm±1. The 1H-NMR spectrum of 3 was similar to that of 7,8-dimethoxyplatydesmine [14] except that the OMe-7 of 7,8-dimethoxyplatydesmine was replaced by a H-7 in 2. Analysis of the 1H-NMR spectrum of 3 revealed three, mutually coupling, aromatic protons at d = 6.98, 7.26, and 7.62, which were assigned to H-7, H-6, and H-5, respectively. Two singlets at d = 4.24 and 4.00 (each 3H) were attributed to OMe-4 and OMe-8, respectively. The presence of a side chain ±C(Me)2OH group was clearly demonstrated from two methyl singlets resonating at d = 1.26 and 1.42, and a broad signal at d = 1.95 (exchangeable with D2O) attributed to a hydroxy pro-
The known flavone, ayanin (4) was readily identified by comparison of physical and spectroscopic data (UV, IR, 1H-NMR, and mass spectrometry data) with those of an authentic sample [7]. The cytotoxic effects of the isolates were tested in vitro against P388, A549, and HT-29 cell lines. The cytotoxicity data are shown in Table 1, and the clinically applied anticancer agent, mithramycin, was used as reference compound. Compounds 1, 4, 5 ± 8, 10 ± 12, 14, 15, and 17 were cytotoxic (ED50 value < 4 mg/mL) against the P-388, A549, or HT-29 cell line. Among them, confusameline
Fig. 3 NOESY contacts of compound 3.
545 Table 1
Cytotoxic effects of compounds isolated from Melicope semecarpifolia against P-388, HT-29, and A549 cell lines ED50 [mg mL±1]
Compound
P-388
A549
HT-29
0.07
0.08
Mithramycin*
0.06
Melicarpine (1)
8.1
11.2
2.5
Semecarpine (2)
28.1
> 50
29.3
()-8-Methoxyplatydesmine (3)
27.5
> 50
> 50
Ayanin (4)
0.22
11.2
6.9
Skimmianine (5)
2.5
7.2
0.12
Confusameline (6) Kokusaginine (7) Haplopine (8) Confusadine (9)
0.03
2.3
0.24
16.8
1.4
7.6
13.1
3.3
12.9
18.6
4.3
12.0
Dutadrupine (10)
0.09
(S)-(±)-7,8-Dimethoxyplatydesmine (11)
7.5
28.3
1.9
(S)-(+)-Isoplatydesmine (12)
4.1
9.8
1.5 30.5
Melicarpinone (13)
0.11
0.13
20.6
> 50
Z-Dimethyl rhoifolinate (14)
8.0
16.2
3.4
E-Dimethyl rhoifolinate (15)
8.1
13.5
3.6
13.2
46.0
4.0
9.2
42.1
Edulinine (18)
27.1
43.6
25.5
trans-Methyl p-coumarate (19)
10.5
8.9
15.1
Melisemine (16) Glycocitridine (17)
Fig. 2 NOESY contacts (a) and HMBC connectivities (b) of compound 2.
Original Paper
H]+, m/z = 284) and HR-FAB-mass spectrometry. The 1H-NMR spectrum of 2 was similar to that of dutadrupine [13], also isolated in this study, except that the ±OC(CH3)2CH = CH- group (C-7, C-8) of dutadrupine was replaced by an ±OCH(CH3)C(CH3)2- group in 2. The presence of an ±OCH(CH3)C(CH3)2- group was demonstrated from two singlet signals at d = 1.48, 1.54 (each 3H), an oxymethine proton at d = 4..78 (1H, q, J = 6.8 Hz), and a neighboring methyl group at 1.70 (3H, d, J = 6.8 Hz). In addition, the 1H-NMR spectrum exhibited a singlet signal at d = 4.43, typical of OMe-4, and two pairs of AB-doublets. One of the doublets at d = 7.79 and 7.49 (J = 2.4 Hz) was characteristic of H-2 and H-3 of the furan ring, while the other at d = 7.56 and 8.31 (J = 9.4 Hz) was attributed to H-5 and H-6. On the basis of the above results and NOESY experiments (Fig. 2), the structure of 2 was elucidated as 6-methoxy-1,1,2-trimethyl-1,2-dihydro-3,9-dioxa-10-azadicyclopenta[a,g]naphthalene, named semecarpine. The assignments of the 13C-NMR resonances were confirmed by the HSQC and HMBC techniques which also supported the structure of 2.
ton. The mass fragment at m/z = 230 [M - 59]+ also supported the ±C(Me)2OH substitution on C-2 [14]. In addition, an AB2 system at d = 3.60 (d, J = 8.6 Hz, H-3) and 4.63 (t, J = 8.6 Hz, H-2) could be assigned to three protons of an a-substituted dihydrofuran. The above assignments were further confirmed by the NOESY and 1H-1H COSY experiments (Fig. 3). On the basis of the above results, and without optical activity, [a]D24: 0 8, the structure of 3 was elucidated as ()-8- methoxyplatydesmine. This is the first report of the occurrence of 3 in a natural source, although it was synthesized by Barr et al. [15], [16].
0.52
* Mithramycin was used as a positive control.
Chen J-J et al. Furoquinoline Alkaloids and ¼ Planta Med 2003; 69: 542 ± 546
(6) and dutadrupine (10) showed more potent cytotoxic activities (ED50 value < 2.5 mg/mL) than the other furoquinoline alkaloids against P-388, A549, and HT-29 cell lines.
Original Paper 546
From the results of the cytotoxic tests, a number of conclusions regarding these isolates could be made: (a) Three 2-quinolone alkaloids, melisemine (16), glycocitridine (17), and edulinine (18) were generally inactive except that 17 exhibited effective cytotoxicity (ED50 value = 0.52 mg/mL) against HT-29. (b) The furoquinoline alkaloids (5 ± 10) exhibited more potent cytotoxic activity than the dihydrofuroquinoline alkaloids (3, 11) against the A549 cell line. (c) The pyranofuroquinoline alkaloid, dutadrupine (10) showed strong cytotoxicity (ED50 value < 0.13 mg/mL) against P-388, A549, and HT-29 cell lines, but the difuroquinoline alkaloid, semecarpine (2) was inactive. (d) Among the test systems, the flavone, ayanin (4) showed selective cytotoxicity (ED50 value = 0.22 mg/mL) against P-388 cell line. (e) The major isolates, 4-methoxyfuroquinolines (5 ± 8) exhibited effective cytotoxicities, and the intensity of activity was in the order 6 (with 7-hydroxy) > 5 (with 7,8-dimethoxy) > 8 (with 7-hydroxy-8-methoxy) > 7 (with 6,7-dimethoxy) against P-388 and A549 cell lines; 5 (with 7,8-dimethoxy) > 6 (with 7-hydroxy) > 7 (with 6,7-dimethoxy) > 8 (with 7-hydroxy-8-methoxy) against HT-29 cell line. (f) Two secofuroquinoline alkaloids, Z-dimethyl rhoifolinate (14) and E-dimethyl rhoifolinate (15) showed selective cytotoxicity (ED50 value < 4 mg/mL) only against the HT-29 cell line. (g) (S)(±)-7,8-Dimethoxyplatydesmine (11) exhibited more potent cytotoxic activity than ()-8-methoxyplatydesmine (3) against the P-388, A549, and HT-29 cell lines. This revealed that the 7,8-dimethoxy groups probably play an important role in cytotoxicity as in the case of skimmianine (5) and glycocitridine (17). Finally, confusameline (6) is the most cytotoxic isolate, and exhibited more potent cytotoxicity (ED50 value = 0.03 mg/mL) against the P-388 cell line than mithramycin (ED50 value = 0.06 mg/mL).
Acknowledgements This work was kindly supported by grants from the National Science Council of the Republic of China (NSC 90-2320-B-127-001) and the Tajen Institute of Technology (Tajen-89 008).
Chen J-J et al. Furoquinoline Alkaloids and ¼ Planta Med 2003; 69: 542 ± 546
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