Furocoumarin Glycosides from the Leaves of Ficus ruficaulis Merr. var.
antaoensis
Mau-Sun Chang,†Yuh-Cheng Yang,†Yuh-Chi Kuo,‡Yueh-Hsiung Kuo,§Chen Chang,⊥Chin-Mei Chen,|and Tzong-Huei Lee*,#
Department of Medical Research, Mackay Memorial Hospital, Taipei, Taiwan 251, Department of Life Science,
Fu Jen Catholic University, Taipei, Taiwan 242, Department of Chemistry, National Taiwan University, Taipei, Taiwan 106, Department of Botany, National Museum of Natural Science, Taichung, Taiwan 404, Institute of Biochemistry,
Academia Sinica, Taipei, Taiwan 115, and Graduate Institute of Pharmacognosy Science, Taipei Medical University, Taipei, Taiwan 110, Republic of China
Received April 22, 2004
From the methanolic extract of the leaves of Ficus ruficaulis Merr. var. antaoensis, 5-O-β-D -glucopyranosyl-6-hydroxyangelicin (1), 6-O-β-D-glucopyranosyl-5-hydroxyangelicin (2), 5,6-O-β-D-diglucopyranosylangelicin (3), 8-O-β-D-glucopyranosyl-5-hydroxypsoralen (4), 5-O-β-D-glucopyranosyl-8-hydroxypsoralen (5), 3,4,5-trihydroxydehydro-R-ionol-9-O-β-D-glucopyranoside (6), rutin (7), and isoquercitrin (8) were isolated. The structures of compounds 1-4 were elucidated by the analysis of their spectroscopic data. Their in vitro antiproliferation activities were also evaluated.
Ficus ruficaulis Merr. var. antaoensis, a variable species
indigenous to Taiwan, is distributed only in the areas of Nanjenshan, Hengchun, and Lanyu Island. It is a medium-sized deciduous tree belonging to the family Moraceae.1 Since it is seldom seen in the field, the tree is rarely put into a practical use, even in folk medicines. However, it has been shown from pharmacological experiments that the water extract of the leaves of this plant exhibits antipro-liferation activity on human mononuclear cells in our previous studies.2The leaves may contain bioactive agents with immunomodulatory activities worthwhile to be inves-tigated phytochemically. Therefore, a series of phytochemi-cal examinations on a methanolic extract of the leaves of this plant were undertaken and have led to the isolation and characterization of four new furocoumarin glucosides (1-4) along with four known compounds (5-8). This paper describes the isolation and structural elucidation of the new compounds.
Results and Discussion
From the methanolic extract of fresh leaves of F.
rufi-caulis, eight major compounds, including four new
furo-coumarin glucosides, were identified. The compounds were isolated by sequential separation on Sephadex LH-20 and reversed-phase HPLC. Compound 5 was obtained as a light yellow powder whose UV, IR, MS, and1H and13C NMR data were consistent with those of 5-O-β-D -glucopyranosyl-8-hydroxypsoralen, which has been previously isolated from
Heracleum rapula Franch.3 Spectroscopic data of 3,4,5-trihydroxydehydro-R-ionol-9-O-β-D-glucopyranoside (6) were matched with those reported.4Rutin (7) was identical with the compound previously reported.5 Compound 8 was identified as isoquercitrin, and its spectroscopic data were in good agreement with published data.6
Compound 1, a light yellow amorphous powder, showed a molecular ion peak at m/z 381 in the positive FABMS
spectrum. Its strong yellow-green fluorescence under UV 365 nm excitation and the aromatic region in its1H and 13C NMR spectra were typical of furocoumarins.7The IR spectrum indicated the presence of a hydroxyl group (3377 cm-1) and a carbonyl group (1695 cm-1). Its UV absorption maxima at 218, 252, and 308 nm indicated a 5,6-dioxy-genated angelicin skeleton,8which was supported by the peak at m/z 217 for C11H5O5 in the positive FABMS spectrum. Analysis of the1H NMR spectrum for 1 showed signals characteristic of a disubstituted furan ring at δH 7.87 (H-2′, d, J ) 2.3 Hz) and 7.10 (H-3′, d, J ) 2.3 Hz) and an AX coupling system at δH6.37 (H-3, d, J ) 9.8 Hz) and 8.57 (H-4, d, J ) 9.8 Hz), in addition to an anomeric doublet at δH4.71 (H-1′′, d, J ) 7.9 Hz) and complex signals appearing at δH3.30-3.84 which corresponded to protons of the glucopyranosyl moiety. By considering the coupling constant of the anomeric proton, the sugar moiety should be the β-anomer. No aromatic proton was observed, sug-gesting that both C-5 and C-6 are substituted. This is in agreement with the placement of oxygenated functional groups at each of these carbons. The13C NMR spectrum of 1 showed 12 carbon signals at δC100-165 due to the 11 carbons of the angular furocoumarin nucleus and the anomeric carbon of the glucose, and five carbon signals at
δC60-80 due to the sugar moiety. In the HMBC spectrum, the anomeric proton at δH4.71 showed a3J correlation with the carbon signal at δC138.3 (C-5), which also connected with δH8.57 (H-4). These data unambiguously established that the glucose was attached at C-5 of the furocoumarin nucleus. Accordingly, the structure of 1 was deduced as 5-O-β-D-glucopyranosyl-6-hydroxyangelicin.
Compound 2 was also assigned a molecular formula of C17H16O10, by HRFABMS. The UV spectrum of 2 exhibited absorption maxima at 221, 252, and 313 nm, similar to that of 1, also suggesting that 2 possessed an angular furocou-marin skeleton with two oxygenated substituents at C-5 and C-6. By comparison of the13C NMR data of 2 and 1, differences involved the chemical shifts of C-5 and C-6. C-5 at δC146.8 was deduced from the HMBC spectrum due to its correlation with H-4. According to the chemical shift changes induced by the glycosylation effect,9the hydroxyl and glucopyranosyl groups located at C-5 (δC146.8) and C-6 (δC128.5), respectively, in 2 are at reverse positions
* To whom correspondence should be addressed. Tel: 886-2-27361661, ext. 6156. Fax: 886-2-23772265. E-mail: [email protected].
†Mackay Memorial Hospital. ‡Fu Jen Catholic University. §National Taiwan University.
⊥National Museum of Natural Science.
|Academia Sinica. #Taipei Medical University.
11 J. Nat. Prod. 2005, 68, 11-13
10.1021/np0401056 CCC: $30.25 © 2005 American Chemical Society and American Society of Pharmacognosy
compared to their positions in 1. Therefore, the structure of 2 was elucidated as 6-O-β-D -glucopyranosyl-5-hydroxy-angelicin.
Compound 3 also showed UV absorption bands charac-teristic of a 5,6-dioxygenated angelicin derivative similar to 1 and 2. The 1H NMR spectrum of 3 indicated the presence of an angelicin moiety, which was revealed by the following signals: δH6.40 (H-3, d, J ) 9.9 Hz), 8.30 (H-4, d, J ) 9.9 Hz), 7.23 (H-3′, d, J ) 2.0 Hz), and 8.12 (H-2′, d,
J ) 2.0 Hz). Besides these signals, two doublets at δH5.10 (H-1′′, d, J ) 7.9 Hz) and 5.39 (H-1′′′, d, J ) 7.7 Hz) were attributed to two anomeric protons. The coupling patterns of the aliphatic proton signals at δH3.07-5.55, which were assigned by the aid of the1H-1H COSY spectrum, were characteristic of a glucopyranoside. These data, coupled with the FABMS data, suggested that 3 is a diglucoside of angelicin. Two glucosyl groups in 3 were assigned at C-5 and C-6 of the angelicin moiety on the basis of the remarkable upfield shifts of the C-5 (δ 140.7) and C-6 (δ 131.3) signals, and they were also confirmed by HMBC measurements. Thus, compound 3 was characterized as 5,6-O-β-D-diglucopyranosylangelicin. Although various fu-rocoumarin glycosides have been found in plants, diglyco-sides such as 3 are rare.
Compound 4, with the same molecular formula as 1, also exhibited an IR absorption band at 1695 cm-1, indicating the presence of a carbonyl. However, its UV spectrum, showing absorption maxima at 220, 246.5, 272, 287, and 317 nm, quite similar to those of 5, indicated a 5,8-dioxygenated psoralen skeleton.3,10 The downfield region of the1H NMR spectrum showed two pairs of doublets at
δH6.25 and 8.23 (J ) 9.8 Hz) attributed to H-3 and H-4, respectively, while the second pair at δH7.18 and 7.93 (each 1H, d, J ) 2.0 Hz) confirmed the presence of the benzofuran moiety. The upfield region contained one sugar unit, as evident from the presence of an anomeric proton signal at
δH5.30 (H-1′′, d, J ) 7.5 Hz). From comparison of the13C NMR spectroscopic data of compounds 4 and 5, the glu-copyranosyl moiety of 4 was proposed to be located at the C-8 position. In the HMBC spectrum, the anomeric proton signal at δH5.30 (H-1′′) showed correlation with the carbon signal at δC121.9 (C-8). Therefore, the structure of 4 was elucidated as 8-O-β-D-glucopyranosyl-5-hydroxypsoralen. The inflammatory response is a nonspecific immune response triggered by pathogenic microorganism infection or tissue injury and provides early protection in restricting the tissue damage to the site of infection or tissue injury.11 Several immune cells including lymphocytes, neutrophils, monocytes, eosinophils, and basophils are involved in the inflammatory response, which in response to invasive organisms, if sufficiently intense or inappropriately pro-longed, could paradoxically aggravate the injury or even cause death. The use of anti-inflammatory medications must therefore be discreet. Blockage of the lymphocyte activation and proliferation is an anti-inflammatory mech-anism.12In this investigation the isolated pure compounds
1-5 from F. ruficaulis were tested for their
antiprolifera-tion activity on human mononuclear cells involving T lymphocytes, B lymphocytes, and macrophages isolated from peripheral blood.13 Heparinized human peripheral blood (50 mL) was obtained from healthy donors. Mono-nuclear cells were isolated from human peripheral blood by the Ficoll-Hypaque gradient density method.14 The lymphoproliferation test was performed according to the published method.15Compound 1, having an IC
50value of 33.4 µM, was moderately active in contrast to cyclosporine
A, with an IC50value of 12 nM. However, compounds 2-5 did not show any activity up to 100 µM concentration.
Experimental Section
General Experimental Procedures. Optical rotations
were measured using a Perkin-Elmer Model 341 polarimeter. UV spectra were measured in H2O on a Hitachi U-2000
spectrophotometer. The IR spectra were recorded on a Nicolet 510P FT-IR spectrometer. The NMR spectra were recorded in MeOH-d4or DMSO-d6at room temperature on a Bruker
DRX-500 SB spectrometer, and the solvent resonances were used as internal shift references. FABMS and HRFABMS were recorded on a JEOL SX-102A instrument using m-nitrobenzyl alcohol (NBA) as the matrix. Sephadex LH-20 (Pharmacia Biotech) was used for open column chromatography. TLC was performed using silica gel 60 F254 plates (200 µm, Merck).
HPLC was performed using an ODS column (Hyperprep HS C18, 10 mm i.d.× 250 mm, Thermo Electron Corp., Bellefonte, PA; detector, RI).
Plant Material. Leaves of F. ruficaulis were sampled from
the National Museum of Natural Science in Taichung in October 2003 and were identified by Dr. Chen Chang, an assistant researcher in the Department of Botany, National Museum of Natural Science. Voucher specimens (No. 20031014) have been deposited at the Graduate Institute of Pharmacog-nosy Science, Taipei Medical University, Taipei, Taiwan.
Extraction and Isolation. Fresh leaves (1 kg) were
extracted three times with 2.5 L of MeOH for 1 week. The MeOH extract was adjusted to 85% in aqueous solution for an n-hexane partition, which generated two fractions soluble in aqueous MeOH and n-hexane. Subsequently, the aqueous MeOH-soluble fraction was evaporated to dryness under vacuum (58.5 g) and further partitioned between EtOAc and H2O. The EtOAc layer was evaporated to dryness (17.7 g) and
redissolved in MeOH for chromatographic separation. The first separation step was carried out using gel filtration chroma-tography on a Sephadex LH-20 column (3× 55 cm) and eluted by MeOH with a flow rate of 13 mL/min.
Fractions collected were checked by TLC using EtOAc-HOAc-H2O (85:10:10) for development. Vanillin-sulfuric acid
(light blue spots) and a UV 365 nm lamp were used in the detection of furocoumarin glycosides. Subsequently, the frac-tions from the above separation were combined to give three subfractions, I, II, and III. Subfraction I was rechromato-graphed on a reversed-phase HPLC column with MeCN-H2O
(16:84) as eluent to yield 3 (2.3 mg) and 6 (8.0 mg). HPLC of subfraction II on the same column with MeCN-H2O (20:80)
as eluent afforded 1 (45.4 mg), 2 (12.0 mg), 4 (7.5 mg), 5 (6.5 mg), and 7 (105.0 mg). Subfraction III was purified by using the same column and eluent as used on subfraction II to yield
8 (2.5 mg).
5-O-β-D-Glucopyranosyl-6-hydroxyangelicin (1):
amor-phous light yellow powder; [R]20
D+4.1° (c 0.15, pyridine); IR
(KBr) νmax3377, 1695, 1581, 1454, 1064, 1034; UV λmax(H2O)
(log ) 218 (4.4), 252 (4.2), 308 (3.9) nm;1H and13C NMR data,
see Table 1; FABMS [M + H]+m/z 381; HRFABMS m/z [M +
H]+381.0821 (calcd for C
17H17O10381.0823).
6-O-β-D-Glucopyranosyl-5-hydroxyangelicin (2): amor-phous light yellow powder; [R]20
D-40.0° (c 0.04, pyridine); IR
(KBr) νmax3381, 1714, 1626, 1338, 1124, 1072; UV λmax(H2O)
(log ) 221 (4.0), 252 (4.0), 313 (3.6) nm;1H and13C NMR data,
see Table 1; FABMS [M + H]+m/z 381; HRFABMS m/z [M +
H]+381.0821 (calcd for C17H17O10381.0823).
5,6-O-β-D-Diglucopyranosylangelicin (3): amorphous
white powder; [R]20
D -30.0° (c 0.01, pyridine); IR (KBr) νmax
3360, 1716, 1558, 1541, 1055; UV λmax(H2O) (log ) 217 (4.4),
250 (4.1), 301 (3.9) nm;1H and13C NMR data, see Table 1;
FABMS [M + H]+m/z 543; HRFABMS m/z [M + H]+543.1368 (calcd for C23H27O15543.1351).
8-O-β-D-Glucopyranosyl-5-hydroxypsoralen (4): amor-phous light yellow powder; [R]20
D-28.8° (c 0.07, pyridine); IR
(KBr) νmax3327, 1695, 1491, 1446, 1340, 1066, 1055; UV λmax
(H2O) (log ) 220 (4.1), 246.5 (3.8), 272 (4.0), 287 (3.7), 317 (3.8)
nm;1H NMR data (DMSO-d 6) δ 8.23 (d, J ) 9.8 Hz, H-4), 7.93 (d, J ) 2.0 Hz, H-2′), 7.18 (d, J ) 2 Hz, H-3′), 6.25 (d, J ) 9.8 Hz, H-3), 5.30 (d, J ) 7.5 Hz, H-1′′), 3.52 (H-6′′b), 3.37 (H-6′′a), 3.31 (H-2′′), 3.25 (H-3′′), 3.17 (H-4′′), 3.10 (H-5′′);13C NMR data (DMSO-d6) δ 160 (s, C-2), 148.4 (s, C-7), 145.3 (d, C-2′), 143.1 (s, C-9), 142.7 (s, C-5), 140.1 (d, C-4), 121.9 (s, C-8), 113.6 (s, C-6), 110.9 (d, C-3), 104.9 (d, C-3′), 104.2 (s, C-10), 102.3 (d, C-1′′), 77.4 (d, C-5′′), 76.6 (d, C-3′′), 73.9 (d, C-2′′), 69.7 (d, C-4′′), 60.7 (t, C-6′′); FABMS [M + H]+m/z 381; HRFABMS m/z [M + H]+381.0822 (calcd for C 17H17O10381.0823).
Monosaccharide Composition Analysis of 1-5.
Com-pounds 1-5 (100 µg) were treated with 0.5 M methanolic HCl at 80 °C for 16 h to give a methyl glycoside derivative. Free hydroxyl groups were trimethylsilylated using the Sylon HTP trimethylsilylation reagent (Supelco, Bellefonte, PA) for 20 min at room temperature. After removal of the excess reagent and organic solvent by condensation, the resulting volatile deriva-tives were dissolved in n-hexane, and this solution was used for GC/MS analysis. GC/MS analysis of the trimethylsilylated derivatives was performed on a fused silica capillary column using a temperature gradient of 60-140 °C at 25 °C/min, increased to 250 °C at 5 °C/min, and then increased to 300 °C at 10 °C/min. Several authentic monosaccharides including arabinose, rhamnose, fructose, xylose, mannose, galactose, and glucose were used as standards, and arabitol was the internal standard. When compared with the trimethylsilylated deriva-tives of the standards, the monosaccharide composition of 1-5 was confirmed to be glucose. The retention time of the trimethylsilyl methyl glucose was found to be 16.22 and 16.55 min.
Acid Hydrolysis. Compound 1 (5 mg) was hydrolyzed by
1 M H2SO4 (2 mL) at room temperature overnight. The
reaction mixture was then partitioned with EtOAc (2× 2 mL). The lower layer was neutralized using 1 M Ba(OH)2(2 mL)
and filtered with glass wool, and the filtrate was evaporated to giveD-glucose: [R]25D+42.7° (c 0.1, H2O).
Acknowledgment. This research was supported by the
grant from Mackay Memorial Hospital (93MMH-TMU-16). We are grateful to Ms. H. Wang, Ms. L. Huang, and Ms. S.-Y. Sun for the NMR data acquisition and HR/LRFABMS measurement in the Instrumentation Center of Taipei Medical University and Instrumentation Center of the College of Science, National Taiwan University.
References and Notes
(1) Huang, T. C., Ed. Flora of Taiwan; National Science Council of the Republic of China: Taipei, 1996; Vol. 2, p 174.
(2) Unpublished data.
(3) Niu, X.-M.; Li, S.-H.; Jiang, B.; Zhao, Q.-S.; Sun, H.-D. J. Asian Nat. Prod. Res. 2002, 4, 33-41.
(4) Rastrelli, L.; Aquino, R.; Abdo, S.; Proto, M.; De Simone, F.; De Tommasi, N. J. Agric. Food Chem. 1998, 46, 1797-1804.
(5) Hasan, A.; Ahmed, I.; Jay, M.; Voirin, B. Phytochemistry 1995, 39, 1211-1213.
(6) Barakat, H. H.; el-Mousallamy, A. M.; Souleman, A. M.; Awadalla, S. Phytochemistry 1991, 30, 3777-3779.
(7) Ahluwalia, V. K.; Boyd, D. R.; Jain, A. K.; Khanduri, C. H.; Sharma, D. Phytochemistry 1988, 27, 1181-1183.
(8) Dreyer, D. L. J. Org. Chem. 1970, 35, 2294-2297. (9) Agrawal, P. K. Phytochemistry 1992, 31, 3307-3330.
(10) Thastrup, O.; Lemmich, J. Phytochemistry 1983, 22, 2035-2037. (11) Janeway, C. A.; Travers, P.; Hunt, S.; Walport, M. Immunobiology:
The Immune System in Health and Disease; Garland Publishing Inc.: New York, 1997.
(12) Adams, D. O.; Hamilton, T. A. Annu. Rev. Immunol. 1984, 2, 283-318.
(13) Lin, C.; Kuo, Y. C.; Chou, C. J. J. Nat. Prod. 1999, 62, 405-408. (14) Kuo Y. C.; Weng S. C.; Chou C. J.; Chang T. T.; Tsai W. J. Br. J.
Pharmacol. 2003, 140, 895-906.
(15) Kuo Y. C.; Yang N. S.; Chou C. J.; Lin L. C.; Tsai W. J. Mol. Pharmacol. 2000, 58, 1057-1066.
NP0401056
Table 1. 1H and13C NMR Spectroscopic Data (CD
3OD, 500 MHz) for Compounds 1-3 [δ in ppm, mult. (J in Hz)]
1 2 3a position 13Cb 1Hc HMBC (HfC) 13Cb 1Hc HMBC (HfC) 13Cb 1Hc HMBC (HfC) 2 163.1 s 163.2 s 159.7 s 3 114.2 d 6.37 d (9.8) 2, 10 112.4 d 6.30 d (9.7) 2, 10 113.3 d 6.40 d (9.9) 2, 10 4 143.1 d 8.57 d (9.8) 2, 5, 9 142.4 d 8.28 d (9.7) 2, 5, 9 141.6 d 8.30 d (9.9) 2, 5, 9 5 138.3 s 146.8 s 140.7 s 6 135.2 s 128.5 s 131.3 s 7 148.2 s 151.6 s 148.8 s 8 116.2 s 110.7 s 114.2 s 9 141.6 s 145.7 s 142.6 s 10 111.4 s 107.7 s 110.9 s 2′ 148.0 d 7.87 d (2.3) 3′, 7, 8 146.1 d 7.74 d (2.0) 3′, 7, 8 147.4 d 8.12 d (2.0) 3′, 7, 8 3′ 104.7 d 7.10 d (2.3) 2′, 7, 8 104.6 d 7.03 d (2.0) 2′, 7, 8 103.7 d 7.23 d (2.0) 2′, 7, 8 1′′ 108.3 d 4.71 d (7.9) 5 106.9 d 4.96 d (7.9) 6 104.0 d 5.10 d (7.9) 5 2′′ 75.4 d 3.59 t (7.9) 75.3 d 3.57 t (8.0) 3′′ 73.9 or 74.1 d 3.38 3′′ 77.9 d 3.45 4′′ 77.8 d 3.44 4′′ 76.3 d 3.25 4′′ 70.9 d 3.47 3′′ 70.9 d 3.48 3′′ 69.7 or 69.9 d 3.15 5′′ 78.6 d 3.30 78.5 d 3.30 77.2 or 77.5 d 3.07 or 3.13 6′′a 62.2 t 3.78 dd (5.0, 12.0) 62.1 t 3.73 dd (4.5, 12.0) 60.7 t 3.38 6′′b 62.2 t 3.84 dd (2.3, 12.0) 62.1 t 3.80 dd (2.3, 12.0) 60.7 t 3.55 1′′′ 102.5 d 5.39 d (7.7) 6 2′′′ 73.9 or 74.1 d 3.38 3′′′ 76.3 d 3.25 4′′′ 69.7 or 69.9 d 3.15 5′′′ 77.2 or 77.5 d 3.07 or 3.13 6′′′a 60.7 t 3.38 6′′′b 60.7 t 3.55 aMeasured in DMSO-d
6.bMultiplicities were obtained from DEPT experiments.cSignals without multiplicity were picked up from
1H-1H COSY or HMQC spectra.
