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Bioactive Constituents from the Leaves of Zanthoxylum schinifolium
Bioactive Constituents from the Leaves of Zanthoxylum schinifolium
Natural Product Sciences. 2015. Mar, 21(1): 1-5
Copyright © 2015, The Korean Society of Pharmacognosy
  • Received : August 18, 2014
  • Accepted : September 21, 2014
  • Published : March 31, 2015
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About the Authors
Su Yang Jeong
Phi Hung Nguyen
Bing Tian Zhao
Byung Sun Min
Eun Sook Ma
Mi Hee Woo
woomh@cu.ac.kr

Abstract
Activity-guided separation of the methylene chloride-soluble fraction of the leaves of Zanthoxylum schinifolium , resulted in the isolation of four coumarinoids ( 1 4 ), two triterpenoids ( 5 , 6 ) and three fatty acid derivatives ( 7 9 ) as active principles. Their chemical structures were identified as collinin( 1 ), 8-methoxyanisocoumarin( 2 ), 7-(6' R -hydroxy-3',7'-dimethylocta-2',7'-dienyloxy)-coumarin ( 3 ), ( E )-4-methly-6-(coumarin-7'-yloxy)hex-4-enal ( 4 ), lupeol ( 5 ), epi -lupeol ( 6 ), phytol ( 7 ), hexadec-3-enoic acid ( 8 ) and palmitic acid ( 9 ), on the basis of spectroscopic (1D, 2D and MS) data analyses and comparing with the data published in the literatures. Compounds 1 and 7 showed potent cytotoxicity against Jurkat T cells with IC 50 values of 45.58 and 47.51 μM, respectively. The others showed moderate activity with IC 50 values ranging around 80.58 to 85.83 μM, while the positive control, auraptene, possessed an IC 50 value of 55.36 μM.
Keywords
Introduction
Zanthoxylum schinifolium (Rutaceae) is an aromatic plant, whose pericarp is widely used as a pungent condiment and seasoning in Korea and in other East Asian countries. 1 The leaves, fruits, seeds and roots of Z. schinifolium have been used in the treatment of various diseases, such as inflammation, toothache, muscle pain, and ascarid. 2 - 4 Medicinal activities have also been reported from this plant, including anti-platelet aggregation, anti-oxidant, inhibition of the production of monoamine oxidase, and anti-tumor. 5 - 8 Despite many researches on Z. schinifolium , our aim is focused on the investigation into cytotoxic components from Z. schinifolium . Thus, the roots, stems, pericarps, and seeds of Z. schinifolium were extracted using MeOH, while the leaves were extracted using 80% MeOH. These extracts were examined for MTT cytotoxicity against the Jurkat T cell lines, and the result revealed that the leaves extract had a potent MTT cytotoxicity. The leaves extract was subsequently fractionated into four parts: methylene chloride, ethyl acetate, n -butanol, and water fractions. The methanol extract and fractions were examined for their cytotoxicity using an in vitro MTT assay on Jurkat T cells. Among the samples tested, the methylene chloride fraction showed the strongest inhibition against Jurkat T cell lines. Thus, assaydirected separation of this methylene chloride fraction has led to the isolation of nine active principles, including four coumarinoids ( 1 4 ), two triterpenoids ( 5 , 6 ), and three fatty acid derivatives ( 7 9 ). In this study, the isolation, structural elucidation, along with the cytotoxicity of the isolates will be discussed herein.
Experimental
General experimental procedures – Melting points were determined on a Yanaco micro melting point apparatus. Optical rotations were measured on a JASCO DIP-370 digital polarimeter (Japan). IR spectra were measured on a Mattson Polaris FT/IR-300E spectrophotometer. UV spectra were measured on a Thermo 9423 AQA2200E UV spectrophotometer. The NMR spectra were recorded on a Varian Unity INOVA-400 spectrometer (USA), and chemical shifts are expressed as δ value using TMS as an internal standard. Low- and highresolution EI-MS data were collected on a Quattro II spectrometer. Silica gel 60 (70 - 230 and 230 - 400 mesh, Merck) and Lichroprep RP-18 (40 - 63 μm, Merck) were used as stationary phases for column chromatography. For TLC and HPTLC, silica gel 60 F 254 (EM 5715, 5628) glass plates (0.25mm) were used and visualized by spraying with 10% H 2 SO 4 and subsequent heating. All other chemicals and solvents were of analytical grade and used without further purification.
Plant material − The leaves of Z. schinifolium were collected in Gyeongsan, Gyeongbuk, Republic of Korea in June 2004. These materials were confirmed taxonomically by Professor Chong Won Kim, Catholic University of Daegu, Gyeongsan, Republic of Korea. A voucher specimen has been deposited at the College of Pharmacy, Catholic University of Daegu.
Extraction and isolation − The leaves of Z. schinifolium (30 kg) were freeze-dried and powdered, to yield 10 kg of leaf powder. The powder was extracted with 80% MeOH, to yield 1.2 kg of extract, upon removal of the solvent. The MeOH extract was mixed in H 2 O (5 L), and the resulting H 2 O layer was partitioned with CH 2 Cl 2 (4 L × 311 g), n -BuOH (4 L × 385 g) and H 2 O (300 g). The CH 2 Cl 2 extract (311 g) was loaded on a silica gel column (9 × 75 cm, silica-gel 230~400 mesh), and eluted by a stepwise gradient of n -hexane-EtOAc (100 : 0 to 1 : 100). The eluates were combined into 23 pools (SL-MC-A~W), on the basis of silica gel TLC. SL-MC-K (3.0 g) was chromatographed on a reverse-phase column (3.5 × 15 cm, RP C-18, and eluted by a stepwise gradient of H 2 O-MeOH (40 : 60 to 0 : 100)), to yield compound 1 (500.1 mg). SL-MC-Q (89.3 mg) was chromatographed on a reverse-phase column (3.5 × 15 cm, RP C-18, and eluted by a stepwise gradient of H 2 O-MeOH (30 : 70 to 0 : 100), to yield compound 2 (4.2 mg). SL-MC-F (1.6 g) was chromatographed on a silica gel column (3.5 × 15 cm, using the n -hexane-CH 2 Cl 2 mixture as a solvent, and eluted with a stepwise gradient (100 : 1 to 40 : 1)), to yield compound 6 (400.4 mg). SL-MC-H (2.0 g) was chromatographed on a silica gel column (3.5 × 15 cm, using the n -hexane-EtOAc mixture as a solvent, and eluted with a stepwise gradient (100 : 1 to 50 : 1)), to yield compounds 5 (100.2 mg) and 7 (550.1 mg), respectively. SL-MC-I (2.8 g) was chromatographed on a silica gel column (3.5 × 15 cm, using the n -hexane-EtOAc mixture as a solvent, and eluted with a stepwise gradient (40 : 1 to 10 : 1)), to yield compounds 3 (19.4 mg), 4 (5.2 mg), and 9 (5.3 mg), respectively. SL-MC-N (1.4 g) was chromatographed on a reverse-phase column (3.5 × 15 cm, RP C-18, and eluted by a stepwise gradient of H 2 O-MeOH (40 : 60 to 0 : 100)), to yield compound 8 (42.3 mg).
Collinin (1) − White powder. IR (KBr) cm −1 : 2921 (C-H), 1704 (C=O), 1603 (C=C); 1 H NMR (CDCl 3 , 400 MHz): δ 6.24 (1H, d, J = 9.6 Hz, H-3), δ 7.61 (1H, d, J = 9.6 Hz, H-4), δ 7.13 (1H, d, J = 8.8 Hz, H-5), δ 6.86 (1H, d, J = 8.8 Hz, H-6), δ 4.68 (2H, d, J = 6.4 Hz, H-1'), δ 5.48 (1H, t, J = 6.4 Hz, H-2'), δ 2.13~2.04 (4H, m, H-4',5'), 5.07~5.04 (1H, m, H-6'), δ 1.65 (3H, s, H-8'), δ 1.74 (3H, s, H-9'), δ 1.59 (3H, s, H-10'), δ 3.97 (3H, s, H-OCH 3 ); 13 C NMR (CDCl 3 , 100 MHz): δ 160.9 (C-2), δ 113.9 (C-3), δ 143.9 (C-4), δ 113.6 (C-4a), δ 122.9 (C-5), δ 110.4 (C-6), δ 155.2 (C-7), δ 142.0 (C-8), δ 148.4 (C-8a), δ 66.5 (C-1'), δ 119.2 (C-2'), δ 136.9 (C-3'), δ 39.7(C-4'), δ 26.4 (C-5'), δ 123.9 (C-6'), δ 132.1 (C-7'), δ 25.9(C-8'), δ 16.9 (C-9'), δ 17.9 (C-10'), δ 61.6 (C-O C H 3 ); EI-MS m/z : 328 [M] + .
8-Methoxyanisocoumarin (2) − Colorless syrup. IR (KBr) cm −1 : 3400 (OH), 2960 (C-H), 1725 (C=O), 1610 (C=C); 1 H NMR (CDCl 3 , 400 MHz): δ 6.25 (1H, d, J = 9.6 Hz, H-3), δ 7.64 (1H, d, J = 9.6 Hz, H-4), δ 7.37 (1H, d, J = 8.4 Hz, H-5), δ 6.86 (1H, dd, J = 8.4, 2.4 Hz, H-6), δ 6.82 (1H, d, J = 2.4 Hz, H-8), δ 4.61 (2H, d, J = 6.4 Hz, H-1'), δ 5.49 (2H, t, J = 6.4 Hz, H-2'), δ 2.79 (2H, d, J = 6.4 Hz, H-4'), δ 5.65 (2H, m, H-5',6'), δ 1.33 (3H, s, H-8'), δ 1.75 (3H, s, H-9'), δ 1.33 (3H, s, H-10'); 13 C NMR (CDCl 3 , 100 MHz): δ 162.3 (C-2), δ 113.2 (C-3), δ 143 (C-4), δ 113.4 (C-4a), δ 128.9 (C-5), δ 112.7 (C-6), δ 161.5 (C-7), δ 101.7 (C-8), δ 156.1 (C-8a), δ 65.6 (C-1'), δ 119.5 (C-2'), δ 141.3 (C-3'), δ 42.3 (C-4'), δ 124.0 (C-5'), δ 140.7 (C-6'), δ 70.9 (C-7'), δ 30.0 (C-8'), δ 17.0 (C-9'), δ 30.0 (C-10'); FAB-MS m/z : 337 [M+ Na] + .
7-(6'R-hydroxy-3',7'-dimethylocta-2',7'-dienyloxy)-coumarin (3) − Colorless crystal. IR (KBr) cm −1 : 3329 (OH), 1703 (C=O), 1604 (C=C); 1 H NMR (CDCl 3 , 400 MHz): δ 6.23 (1H, d, J = 9.6 Hz, H-3), δ 7.63 (1H, d, J = 9.6 Hz, H-4), δ 7.36 (1H, d, J = 8.4 Hz, H-5), δ 6.84 (1H, dd, J = 8.4, 2.4 Hz, H-3), δ 6.80 (1H, d, J = 2.4 Hz, H-8), δ 4.59 (2H, d, J = 6.4 Hz, H-1'), δ 5.49 (1H, t, J = 6.4 Hz, H-2'), δ 2.13 (2H, m, H-4'), δ 1.67 (2H, m, H-5'), δ 4.05 (1H, t, J = 6.4 Hz, H-6'), δ 4.94 (1H, s, H-8'a), δ 4.84 (1H, s, H-8'b), δ 1.73 (3H, s, H-9'), δ 1.77 (3H, s, H-10'); 13 C NMR (CDCl 3 , 100 MHz): δ 162.3 (C-2), δ 113.2 (C-3), δ 143.7 (C-4), δ 113.4 (C-4a), δ 128.9 (C-5), δ 112.6 (C-6), δ 161.5 (C-7), δ 101.7 (C-8), δ 156.0 (C-8a), δ 65.6 (C-1'), δ 118.8 (C-2'), δ 142.3 (C-3'), δ 35.6 (C-4'), δ 32.9 (C-5'), δ 75.6 (C-6'), δ 147.5 (C-7'), δ 111.4 (C-8'), δ 17.0 (C-9'), δ 17.7 (C-10'); EI-MS m/z : 314 [M] + .
(E)-4-methly-6-(coumarin-7'-yloxy)hex-4-enal (4) − Colorless crystal. IR (KBr) cm −1 : 2922 (C-H), 1727 (C=O), 1610 (C=C); 1 H NMR (CDCl 3 , 400 MHz): δ 6.25 (1H, d, J = 9.6 Hz, H-3), δ 7.64 (1H, d, J = 9.6 Hz, H-4), δ 7.37 (1H, d, J = 8.4 Hz, H-5), δ 6.84 (1H, dd, J = 8.4, 2.4 Hz, H-6), δ 6.80 (1H, d, J = 2.4 Hz, H-8), δ 4.60 (2H, d, J = 6.4 Hz, H-1'), δ 5.49 (1H, t, J = 6.4 Hz, H-2'), δ 2.43 (2H, t, J = 7.2 Hz, H-4'), δ 2.62 (2H, td, J = 7.2, 1.2 Hz, H-5'), δ 9.80 (1H, t, J = 1.2 Hz, H-6'), δ 1.78 (3H, s, H-7'), δ 6.25 (1H, d, J = 9.6 Hz, H-3), δ 7.64 (1H, d, J = 9.6 Hz, H-4), δ 7.37 (1H, d, J = 8.4 Hz, H-5), δ 6.84 (1H, dd, J = 8.4, 2.4 Hz, H-6), δ 6.80 (1H, d, J = 2.4 Hz, H-8); 13 C NMR (CDCl 3 , 100 MHz): δ 65.4 (C-1'), δ 119.6 (C-2'), δ 140.4 (C-3'), δ 31.6 (C-4'), δ 41.9 (C-5'), δ 201.8 (C-6'), δ 17.1 (C-7'), δ 162.1 (C-2), δ 113.2 (C-3), δ 143.7 (C-4), δ 113.3 (C-4a), δ 128.9 (C-5), δ 112.7 (C-6), δ 161.5 (C-7), δ 101.7 (C-8), δ 156.0 (C-8a); EI-MS m/z : 272 [M] + .
Lupeol (5) −White powder. IR (KBr) cm −1 : 3300 (OH), 2943 (C-H), 1637 (C=C); 1 H NMR (CDCl 3 , 400 MHz): δ 3.20 (1H, dd, J = 11.0, 3.8 Hz, H-3), δ 2.38 (1H, m, H-19), δ 0.97 (3H, s, H-23), δ 0.77 (3H, s, H-24), δ 0.83 (3H, s, H-25), δ 1.03 (3H, s, H-26), δ 0.95 (3H, s, H-27), δ 0.79 (3H, s, H-28), δ 4.69 (1H, d, J = 2.4 Hz, H-29a), δ 4.57 (1H, d, J = 2.4 Hz, H-29b), δ 1.68 (3H,s, H-30); 13 C NMR (CDCl 3 , 100 MHz): δ 38.9 (C-1), δ 27.7 (C-2), δ 79.2 (C-3), δ 39.2 (C-4), δ 55.5 (C-5), δ 18.5 (C-6), δ 34.5 (C-7), δ 41.0 (C-8), δ 50.6 (C-9), δ 37.4 (C-10), δ 21.1 (C-11), δ 25.3 (C-12), δ 38.3 (C-13), δ 43.0 (C-14), δ 27.7 (C-15), δ 35.8 (C-16), δ 43.2 (C-17), δ 28.2 (C-18), δ 48.5 (C-19), δ 151.1 (C-20), δ 30 (C-21), δ 40.2 (C-22), δ 28.2 (C-23), δ 16.2 (C-24), δ 16.3 (C-25), δ 15.9 (C-26), δ 14.7 (C-27), δ 18.2 (C-28), δ 19.5 (C-29), δ 109.5 (C-30); EI-MS m/z : 426 [M] + .
epi-Lupeol (6) − White powder. IR (KBr) cm −1 : 3666 (OH), 2924 (C-H), 1560 (C=C); 1 H NMR (CDCl 3 , 400 MHz): δ 3.38 (1H, m, H-3), δ 2.38 (1H, m, H-19), δ 0.96 (3H, s, H-23), δ 0.84 (3H, s, H-24), δ 0.83 (3H, s, H-25), δ 1.03 (3H, s, H-26), δ 0.93 (3H, s, H-27), δ 0.79 (3H, s, H-28), δ 4.69 (1H, d, J = 2.4 Hz, H-29a), δ 4.56 (1H, d, J = 2.4 Hz, H-29b), δ 1.68 (3H, s, H-30); 13 C NMR (CDCl 3 , 100 MHz): δ 38.9 (C-1), δ 27.7 (C-2), δ 79.2 (C-3), δ 39.2 (C-4), δ 55.5 (C-5), δ 18.5 (C-6), δ 34.5 (C-7), δ 41.0 (C-8), δ 50.6 (C-9), δ 37.4 (C-10), δ 21.1 (C-11), δ 25.3 (C-12), δ 38.3 (C-13), δ 43.0 (C-14), δ 27.7 (C-15), δ 35.8 (C-16), δ 43.2 (C-17), δ 48.2 (C-18), δ 48.5 (C-19), δ 151.1 (C-20), δ 30.1 (C-21), δ 40.2 (C-22), δ 28.1 (C-23), δ 16.2 (C-24), δ 16.4 (C-25), δ 15.9 (C-26), δ 14.7 (C-27), δ 18.2 (C-28), δ 19.5 (C-29), δ 109.4 (C-30); EI-MS m/z : 426 [M] + .
Phytol (7) − Colorless crystal. IR (KBr) cm −1 : 3666 (OH), 2987 (C-H), 1653 (C=C); 1 H NMR (CDCl 3 , 400 MHz): δ 4.15 (2H, d, J = 6.8 Hz, H-1), δ 5.41 (2H, t, J = 6.8 Hz, H-2), δ 1.99 (1H, t, J = 8.0 Hz, H-4), δ 0.86 (3H, d, J = 6.4 Hz, H-16), δ 0.85 (3H, d, J = 6.4 Hz, H-17), δ 0.87 (3H, d, J = 6.4 Hz, H-18), δ 0.88 (3H, d, J = 6.4 Hz, H-19), δ 1.67 (3H, s, H-20); 13 C NMR (CDCl 3 , 100 MHz): δ 59.6 (C-1), δ 123.3 (C-2), δ 140.4 (C-3), δ 40.1 (C-4), δ 25.3 (C-5), δ 36.8 (C-6), δ 33.0 (C-7), δ 37.6 (C-8), δ 24.7 (C-9), δ 37.5 (C-10), δ 32.9 (C-11), δ 37.5 (C-12), δ 25.0 (C-13), δ 39.8 (C-14), δ 28.2 (C-15), δ 22.8 (C-16), δ 22.9 (C-17), δ 20.0 (C-18), δ 19.9 (C-19), δ 16.4 (C-20); EI-MS m/z : 296 [M] + .
Hexadec-3-enoic acid (8) −White powder. IR (KBr) cm −1 : 2919 (C-H), 1693 (C=O), 1054 (C-O); 1 H NMR (CDCl 3 , 400 MHz): δ 2.94 (2H, d, J = 5.6 Hz, H-2), δ 5.51 (1H, td, J = 12.8, 5.6 Hz, H-3), δ 5.49 (1H, td, J = 12.8, 5.6 Hz, H-4), δ 1.99 (2H, dd, J = 12.8, 5.6 Hz, H-5), δ 1.33~1.19 (20H, m, H-6~15), δ 0.86 (3H, t, J = 6.4 Hz, H-16); 13 C NMR (CDCl 3 , 100 MHz): δ 175.0 (C-1), δ 37.7 (C-2), δ 134.2 (C-3), δ 122.3 (C-4), δ 32.4 (C-5), δ 31.9 (C-6), δ 29.6~29.5 (C-7~14), δ 22.6 (C-15), δ 13.3 (C-16); EI-MS m/z : 264 [M] + .
Palmitic acid (9) − White powder. IR (KBr) cm −1 : 2935 (C-H), 1699 (C=O), 1054 (C-O); 1 H NMR (CDCl 3 , 400 MHz): δ 2.36 (2H, t, J = 6.86 Hz, H-2), δ 1.63 (2H, m, H-3), δ 1.31~1.26 (24H, m, H-4~15), δ 0.89 (3H, t, J = 6.4 Hz, H-16); 13 C NMR (CDCl 3 , 100 MHz): δ 179.9 (C-1), δ 34.2 (C-2), δ 24.9 (C-3), δ 29.9~29.3 (C-4~13), δ 32.1 (C-14), δ 22.9 (C-15), δ 14.3 (C-16); EI-MS m/z : 256 [M] + .
MTT Assay − The cytotoxic activity of each compound on Jurkat T cells was analyzed by 3-(4,5-dimethylthiazole-2-yl)-2,5-biphenyl tetrazolium bromide (MTT) assay (Park , 2000) . The Jurkat T cells (2.5 × 10 4 cells/well) were seeded on a 96-well microplate, and were precultured for 36 h. 50 μL of the MTT solution (1.1 mg/mL) was added to each well, and incubated for an additional 4 h. The colored formazan crystal produced from the MTT was dissolved in dimethyl sulfoxide (DMSO). A plate reader was used to measure the optical density (OD) values of the solutions at 540 nm.
Results and Discussion
The nine compounds ( 1 9 ) ( Fig. 1 ) were isolated from the methylene chloride fraction of the leaves of Z. schinifolium , by repetitive column chromatography, using silica gel and RP-C18. Compound 1 was obtained as white powder with a molecular weight of m/z 328 [M] + , based on EI-MS data. Compound 1 exhibited UV absorption bands at 202 and 323 nm. The IR spectrum of 1 showed absorption bands at 1701 cm −1 (C=O), 2921 (CH) and 1603 (C=C). The 1 H NMR spectrum of 1 showed the presence of a 7,8-disubstituted coumarin from the characteristic doublets of H-3 (δ H 6.24, J = 9.6 Hz) and H-4 (δ H 7.61, J = 9.6 Hz), H-5 (δ H 7.13, 1H, d, J = 8.8 Hz) and H-6 (δ H 6.86, 1H, d, J = 8.8 Hz). The terpenyl side-chain exhibited signals due to two olefinic protons at δ H 5.48 (1H, t, J = 6.4 Hz, H-2') and 5.05 (1H, m, H-6'), three methylenic protons at δ H 2.13~2.04 (4H, m, H-4',5') and δ H 4.68 (2H, d, J = 6.4 Hz, H 2 -1'), and three vinylic methyl protons at δ H 1.65 (3H, s, H-8'), δ H 1.74 (3H, s, H-9'), δ H 1.59 (3H, s, H-10'). The 13 C NMR spectrum of 1 displayed all the 20 carbons including nine carbons of the coumarin skeletons at δ H 160.9 (C-2), 113.9 (C-3), 143.9 (C-4), 113.6 (C-4a), 122.9 (C-5), 110.4 (C-6), 155.2 (C-7) and 142.0 (C-8), 148.4 (C-8a), seven carbons of the terpenyl moiety [δ 66.5 (C-1'), 119.2 (C-2'), 136.9 (C-3'), 39.7 (C-4'), 26.4 (C-5'), 123.9 (C-6'), 132.1 (C-7'), 25.9 (C-8'), 16.9 (C-9'), 17.9 (C-10')] and an additional methoxy unit at δ C 61.6 (C-O C H 3 ) and δ H 3.97 (3H, s, H-OCH 3 ). Compound 1 was finally identified as collinin by comparison of the physical and spectroscopic data with those in the literature. 9
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Chemical structures of compounds 19 isolated from the leaves of Z. schinifolium.
Compound 2 was isolated as colorless syrup, its IR spectrum displayed absorption bands at 3400 cm −1 (OH), 2960 (C-H), 1725 (C=O), 1610 (C=C). The molecular weight of compound 2 was obtained from FAB-MS with m/z 337 [M + Na] + . The 1 H and 13 C NMR spectra of compound 2 were identical with those of compound 1 except only for the signal assignable for C-5' [δ H 5.65 (1H, m) and δ C 70.9] and H-4' [δ H 2.79 (2H, d, J = 6.4 Hz, H-4') and δ C 42.3], respectively. Compound 2 was therefore elucidated as 8-methoxyanisocoumarin, it was isolated from this plant previously by Tsai et al . 10 Compound 3 was obtained as colorless crystal. The IR (KBr) displayed absorption bands at 3329 cm −1 (OH), 1703 (C=O), 1604 (C=C). The 1 H and 13 C NMR spectra of 3 showed the presence of a 7-substituted coumarin from the characteristic doublets of H-3 (δ H 6.23, 1H, d, J = 9.6 Hz) and H-4 (δ H 7.61, 1H, d, J = 9.6 Hz), a pair of ortho-coupled protons of H-5 (δ H 7.36, 1H, d, J = 8.4 Hz) and H-6 (δ H 6.86, 1H, d, J = 8.8 Hz) which was meta-coupled with H-8 (δ H 6.80, 1H, d, J = 2.4 Hz). The terpenyl side-chain exhibited signals due to one olefinic proton at δ H 5.49 (1H, t, J = 6.4 Hz, H-2'), three methylenic protons at δ H 4.59 (2H, d, J = 6.4 Hz, H-1'), δ H 2.13 (2H, m, H-4'), δ H 1.67 (2H, m, H-5'), and two vinylic methyl protons at δ H 1.73 (3H, s, H-9'), δ H 1.77 (3H, s, H-10'). In addition, the 1 H and 13 C NMR spectra of 3 possessed an oxygenated olefinic proton at δ H 4.05 (1H, t, J = 6.4 Hz, H-6') and δ C 75.6 (C-6') due to the attachment of a hydroxyl group at C-6, an germinal methylene protons at δ H 4.94 (1H, s, H-8'a), δ H 4.84 (1H, s, H-8'b) with corresponding carbons at δ C 147.5 (C-7') and 111.4 (C-8') were further supported for this observation. Detailed comparison of the 1 H and 13 C NMR spectra of compound 3 with those published in the literature, 11 led to the structural identification of compound 3 to be 7-(6' R -hydroxy-3',7'-dimethylocta-2',7'-dienyloxy)-coumarin.
The 1 H and 13 C NMR spectra of compound 4 revealed an identical structure with compound 4 , except for the disappearances of the oxygenated olefinic proton at δ H 4.05 (1H, t, J = 6.4 Hz, H-6'), the germinal methylene protons at δ H 4.94 (1H, s, H-8'a), δ H 4.84 (1H, s, H-8'b) and the vinylic methyl protons at δ H 1.77 (3H, s, H-10'). Instead of that an aldehyde group was observed in compound 4 at δ H 9.80 (1H, t, J = 1.2 Hz, H-6') and the corresponding carbon at δ C 201.8 (C-6'). Compound 4 was thus identified as ( E )-4-methly-6-(coumarin-7'-yloxy)hex-4-enal. 12 Compounds 5 and 6 were isolated and identified to be lupeol 13 and epi -lupeol, 14 respectively by comparing the physicochemical and spectroscopic data with the published literatures. Three fatty acid derivatives were also characterized as phytol ( 7 ), 15 ( 8 ) hexadec-3-enoic acid, 16 and ( 9 ) palmitic acid, 17 respectively. To the best of our knowledge, compounds 4 , 6 and 8 were isolated from this plant for the first time.
The nine compounds isolated from Z. schinifolium were tested for their cytotoxic activity against the Jurkat T cells, 18 and the result are presented in Table 1 . Among those, compounds 1 and 7 showed potential growth inhibition effects with IC 50 values of 45.58 and 47.51 μM, respectively. While, auraptene, used as the positive control, possessed weaker activity with IC 50 value of 55.36 μM. Compounds 3 , 4 and 9 showed moderate activity with IC 50 values ranging around 80.58 to 85.83 μM. The other compounds 8 and 9 were weak (IC 50 value 96.87 μM) or no active (IC 50 > 100 μM), respectively. The cytotoxicities of compounds 5 and 6 were not able to investigate due to their insolubility in DMSO.
Cytotoxic activity of isolated compounds1−9from the leaves ofZ. schinifoliumagainst Jurkat T cells
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aThe inhibitory effects are represented as the molar concentration (μM) giving 50% inhibition (IC50) relative to the vehicle control. These data represent the average values of three repeated experiments. bThe compound used as positive control. cThe compounds were not tested.
Compound 1 , collinin, was previously found to be an inhibitor of hepatitis B virus (HBV) DNA replication in a HBV-transfected cell line. 19 Koheno et al. had suggested that certain phenyloxycoumarins, such as auraptene and collinin, could serve as an effective agent against colitisrelated colon cancer development in rodents. 20 Costa et al. reported an anticonvulsant effect of phytol in a pilocarpine model in mice. 21 In this study, we found that collinin ( 1 ) and phytol ( 7 ) possessed potential cytotoxicity against Jurkat T cells, suggesting that Z. schinifolium and its active principles may be useful for the development of agent in combat with cancer.
Acknowledgements
This research was supported by research grants from Catholic University of Daegu in 2014. We are grateful to Korea Basic Science Institute (KBSI) for mass spectral measurements.
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