Introduction
Euonymus alatus Sieb. (Celastraceae) is commonly known as winged euonymus in Korea and has been widely used in traditional medicine to regulate blood circulation, relieve pain, eliminate stagnant blood and treat dysmenorrhea. The young leaves of this tree are edible kitchen herbs, and the cork cambium on the twigs which is called ‘Gui-Jun Woo’ has been traditionally used to treat cancer in Korean traditional medicine (Park et al., 2007). Biological studies have revealed that this plant possesses numerous biological effects such as anti-tumor (Lee et al., 1993), anti-inflammatory (Oh et al., 2011), anti-hyperglycemic, anti-hyperlipidemic (Park et al., 2005), nitric oxide scavenging (Jeong et al., 2004) and cytotoxic activities (Cha et al., 2003).
Recently, pharmacological studies have reported potential of E. alatus Sieb. as an anticancer agent using a variety of in vivo and in vitro models, which was also confirmed cytotoxicity in A549, SK-OV-3, SK-MEL-2, and HCT15 cancer cell lines (Kim et al., 2013). So, we investigated the active constituents of E. alatus Sieb. using bioactivityguided isolation techniques on the undetermined cancer cell lines (Jurkat T, HeLa, HL-60 and MCF-7) in previous reports.
Activity-directed isolation of the methylene chloride fraction resulted in the identification of seventeen known compounds (1 - 17) by silica gel column chromatography. In this paper, we report the isolation and structural elucidation of these compounds and their antioxidant and cytotoxic activities against Jurkat T, HeLa, HL-60 and MCF-7 cell lines.
Experimental
General – Melting points were determined on a Yanaco micro melting point apparatus and were uncorrected. Optical rotations were measured on a JASCO DIP-370 digital polarimeter. IR spectra were measured on a Mattson Polaris FT/IR-300E spectrophotometer. UV spectra were measured on a Thermo 9423AQA2200E UV spectrophotometer. NMR spectra were measured on a Varian Unity INOVA-400 spectrometer (USA), and chemical shifts are expressed as δ values using TMS as an internal standard. Low- and high-resolution EI-MS and FAB-MS data were collected on a Quattro II spectrometer. Open column chromatography was performed using silica gel (Kieselgel 60, 70 - 230 mesh and 230 - 400 mesh, Merck). TLC tests were performed on Merck precoated silica gel 60 F254 (EM 5717) and/or RP-18 F254s glass plates (0.25 mm), and spots were visualized by spraying with 10% H2SO4 and subsequent heating. All other chemicals and solvents were of analytical grade and used without further purification.
Plant material – The cork of E. alatus Sieb. was collected in July 2006 from the Palgong mountain in Gyeongsangbuk-Do, Republic of Korea. These materials were confirmed taxonomically by Professor Byung Sun Min, College of Pharmacy, Catholic University of Daegu, Korea. A voucher specimen (CUDP 200602) has been deposited at the College of Pharmacy, Catholic University of Daegu, Korea.
Extraction and isolation – The cork of Euonymus alatus Sieb. (3.5 kg) was extracted four times with MeOH under reflux for 8 hours. The MeOH extract was concentrated under reduced pressure to yield a black syrup (211.4 g). The concentrated MeOH extract was suspended in H2O (2.2 L) and partitioned successively with CH2Cl2 (5 × 2 L, 103.5 g), EtOAc (5 × 2 L, 24.6 g), n-BuOH (5 × 2 L, 28.4 g) and H2O-soluble fraction (31.5 g), respectively. The MeOH extract, CH2Cl2, EtOAc, n-BuOH, and H2O-soluble fractions were assayed for cytotoxic activities against Jurkat T, HeLa, HL-60 and MCF-7 cell lines (data not shown).
The most cytotoxic methylene chloride fraction (103.5 g) was chromatographed on a silica gel column (15 × 35 cm) and was eluted with n-hexane-CH2Cl2 (100 : 0 to 0 : 100) and CH2Cl2-MeOH-H2O (100 : 0 : 0.1 to 0 : 100 : 0.1) gradient. Fractions (M1 to M27) were collected and pooled according to their similar TLC patterns. Fraction M6 (208.2 mg) was chromatographed on a normal phase column (3.5 × 15 cm) using the n-hexane-CH2Cl2 mixture as a solvent and eluted with a stepwise gradient (50 : 1 to 10 : 1) to yield compounds 2 (98.1 mg) and 3 (75.2 mg). Fraction M8 (1.25 g) was chromatographed on a normal phase column (4.5 × 15 cm) using the n-hexane-CH2Cl2 mixture as a solvent and eluted with a stepwise gradient (10 : 1 to 3 : 1) to yield compounds 4 (169.6 mg) and 7 (9.5 mg). Fraction M12 (216.0 mg) was chromatographed on a normal phase column (3.5 × 15 cm) using the nhexane-CH2Cl2 mixture as a solvent and eluted with a stepwise gradient (40 : 1 to 5 : 1) to yield compounds 13 (12.5 mg) and 5 (73.7 mg). Fraction M15 (525.3 g) was chromatographed on a normal phase column (4.5 × 15 cm) using the n-hexane-CH2Cl2 mixture as a solvent and eluted with a stepwise gradient (50 : 1 to 4 : 1) to yield compounds 1 (267.9 mg) and 14 (13.1 mg). Fraction M18 (146.2 mg) was chromatographed on a normal phase column (3.5 × 15 cm) using the n-hexane-CH2Cl2 mixture as a solvent and eluted with a stepwise gradient (40 : 1 to 3 : 1) to yield compounds 8 (7.9 mg), 11 (10.5 mg) and 15 (41.6mg). Fraction M20 (184.1mg) was chromatographed on a normal phase column (3.5 × 15 cm) using the n-hexane-CH2Cl2 mixture as a solvent and eluted with a stepwise gradient (20 : 1 to 2 : 1) to yield compounds 9 (29.9 mg) and 10 (59.2 mg). Fraction M26 (1.03 g) was chromatographed on a normal phase column (4.5 × 15 cm) using the n-hexane-CH2Cl2 mixture as a solvent and eluted with a stepwise gradient (10 : 1 to 1 : 1) to yield compounds 6 (29.7 mg), 12 (19.4 mg), 16 (21.3 mg) and 17 (75.2 mg).
β-Sitosterone (1) – White powder; m.p. 77 - 80 ℃; IR (KBr) cm−1 3019, 3959, 3938, 3874, 1663, 1614, 1467, 1378, 1232; EI-MS m/z 412 [M]+; The spectral data were identical with those reported in the literature (Gaspar et al., 1993).
Lupenone (2) – White powder; m.p. 171 - 172 ℃; IR (KBr) cm−1 3076, 1732, 1454, 1365, 1250, 1026, 978, 877; EI-MS m/z 424 [M]+; The spectral data were identical with those reported in the literature (Da et al., 1996).
Arborinone (3) – Colorless needles, m.p. 217 - 219 ℃; IR (KBr) cm−1 3400, 2930, 2980, 1701, 1655, 1270; EI-MS m/z 424 [M]+; The spectral data were identical with those reported in the literature (Akihisa et al., 1992).
β-Sitosterol (4) – White powder; m.p. 135 - 139 ℃; IR (KBr) cm−1 3420, 2935, 2864, 1457, 1375, 1052; EI-MS m/z 414 [M]+; The spectral data were identical with those reported in the literature (Su et al., 2009).
Lupeol (5) – Colorless needles; m.p. 202 - 205 ℃; IR (KBr) cm−1 3320, 2930, 1632, 1445, 1372, 1040, 880; EIMS m/z 426 [M]+; The spectral data were identical with those reported in the literature (Fuchino et al., 1995).
epi-Lupeol (6) – Colorless needles; m.p. 202 - 205 ℃; IR (KBr) cm−1 3466, 2924, 1560; EI-MS m/z 426 [M]+; The spectral data were identical with those reported in the literature (De Souza et al., 2001).
Taraxerol (7) – Colorless crystal; m.p. 280 - 282 ℃; IR (KBr) cm−1 3480, 1642, 813; EI-MS m/z 426 [M]+; The spectral data were identical with those reported in the literature (Lee et al., 1992).
Betulinic acid (8) – White powder; m.p. 275 - 278 ℃; IR (KBr) cm−1 3060, 1630, 880; EI-MS m/z 438 [M]+; The spectral data were identical with those reported in the literature (Haque et al., 2000).
24R-Methyllophenol (9) – White powder; m.p. 140 - 146 ℃; IR (KBr) cm−1 3421, 2930, 2878, 1450; EI-MS m/z 414 [M]+; The spectral data were identical with those reported in the literature (Akihisa et al., 1981).
Germanicol (10) – White powder; m.p. 174 - 175 ℃; IR (KBr) cm−1 3600, 3030, 2940, 2850, 1630, 1450, 1360, 1040, 855; EI-MS m/z 426 [M]+; The spectral data were identical with those reported in the literature (Koul et al., 2000).
Hexatriacontane (11) – White powder; IR (KBr) cm−1 2952; EI-MS m/z 506 [M]+; The spectral data were identical with those reported in the literature (Turner et al., 1980).
Nonacosan-1-ol (12) – White powder; IR (KBr) cm−1 3429, 2967; EI-MS m/z 424 [M]+; The spectral data were identical with those reported in the literature (Kokpol et al., 1993).
Benzoic acid (13) – White powder; m.p. 122 - 122 ℃; IR (KBr) cm−1 3202, 2363, 1675, 1246; EI-MS m/z 122 [M]+; The spectral data were identical with those reported in the literature (Araora et al., 2005).
Tetradecyl(E)-ferulate (14) – White powder; m.p. 65.4 - 66.3 ℃; IR (KBr) cm−1 3429, 1696, 1618, 1583, 1499; FAB-MS m/z 413 [M + Na]+; The spectral data were identical with those reported in the literature (Das et al., 1997).
Di(2-ethylhexyl) phthalate (15) – Colorless syrup; IR (KBr) cm−1 3030, 2960, 2930, 2880, 1730, 1600, 1585, 1470, 1385, 1080; EI-MS m/z 390 [M]+; The spectral data were identical with those reported in the literature (Rao et al., 2000).
Trilinolein (16) – White powder; IR (KBr) cm−1 1732, 1452, 1435, 990; EI-MS m/z 878 [M]+; The spectral data were identical with those reported in the literature (Morelli et al., 2006).
Monopalmitin (17) – White powder; IR (KBr) cm−1 3432, 1709, 1472, 1463, 1410, 1300, 930; EI-MS m/z 330 [M]+; The spectral data were identical with those reported in the literature (Tsuzuki et al., 1995).
Cytotoxicity – Cytotoxicity was measured using a modified MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. Jurkat T cells (2.5 × 104 cells/well), HeLa cells (5.0 × 103 cells/well), HL-60 cells (1.0 × 104 cells/well) and MCF-7 cells (3.0 × 103 cells/well) were seeded on 96-well microplates and precultured for 36 h. MTT solution (1.1 mg/mL) was added to each well and incubated for an additional 4 h. The colored MTT formazan crystals were dissolved in dimethyl sulfoxide (DMSO). The optical density (OD) values of the solutions were measured at 540 nm using a plate reader. All cell lines were purchased from the Korean Cell Line Bank (Seoul, Korea).
DPPH radical scavenging activity – DPPH (2,2-diphenyl-1-picrylhydrazyl) radical scavenging activity was measured using the method described by Tagashira et al (Tagashira et al., 1998). Briefly, 10 μL of each sample dissolved in EtOH was prepared in a 96-well microplate, and then 200 μL of 100 μM methanolic DPPH solution was added. After mixing and left standing at room temperature for 10 min, the absorbance of the reaction mixture was measured at 517 nm. L-Ascorbic acid (Sigma-Aldrich; purity: ＞ 99%) was used as the positive control for DPPH radical scavenging activity.
Results and Discussion
This study was conducted to identify the bio-active compounds in this plant using bioactivity-guided isolation techniques. The cork of E. alatus Sieb. was extracted with methanol, and the extract was concentrated and fractionated into four parts; methylene chloride, ethyl acetate, n-butanol and water fractions. The cytotoxic activities of the methanol extract and its fractions were examined by MTT assay. Among the samples tested, the methylene chloride fraction showed cytotoxic activity against cancer cell lines (data not shown).
Seventeen compounds (1 - 17) were isolated from the methylene chloride fraction of cork of E. alatus Sieb. by repetitive column chromatography on silica gel. Compounds 1 - 17 were identified as β-sitosterone (1), lupenone (2), arborinone (3), β-sitosterol (4), lupeol (5), epi-lupeol (6), taraxerol (7), betulinic acid (8), 24R-methyllophenol (9), germanicol (10), hexatriacontane (11), nonacosan-1-ol (12), benzoic acid (13), tetradecyl(E)-ferulate (14), di(2-ethylhexyl) phthalate (15), trilinolein (16) and monopalmitin (17), by spectroscopic methods and by comparing their data with the literature values. To the best of our knowledge, compounds 6, 11, 13 and 14 were isolated for the first time from this plant (Fig. 1).
Fig. 1.Structures of compounds 1 - 17 isolated from the cork of E. alatus Sieb.
Table 1.a) Positive control for cytotoxicity for Jurkat T cell-lines. b) Positive control for cytotoxicity for HeLa, HL-60 and MCF-7 cell-lines. c) Positive control for antioxidant activity d) Human T-lymphocyte cells e) Human cervical cancer cell-line. f) Human promyelocytic leukemia cells g) Human breast cancer cell-line.
The seventeen compounds isolated from E. alatus Sieb. were evaluated for their cytotoxic activity against Jurkat T, HeLa, HL-60 and MCF-7 cell lines using the MTT assay (Zhao et al., 2013). Compound 4 exhibited a moderate cytotoxicity against the HL-60 cell line (IC50: 6.22 μM). But, compound 4 was essentially weak cytotoxic against the other tested HeLa and MCF-7 cell lines. Compound 9 exhibited significant moderate cytotoxic activity against the Jurkat T, HeLa, HL-60 and MCF-7 cell lines (IC50: 63.31, 15.45, 15.14 and 21.72 μM, respectively). Compound 17 also showed moderate cytotoxic activity with an IC50 value of 70.71 μM in Jurkat T cell line (Table 1).
24R-methyllophenol (9) was previously found to have an anti-hyperglycemic effect. However, there is no report on the cytotoxic effect of compound 9 (Tanaka et al., 2006). Li and Xu (Li and Xu, 2012) had reported that monopalmitin (17) exhibited moderate molluscicidal activity against Pomacea canaliculata (Lamarck) and nematicidal activity against Meloidogyne incognita (Kofoid and White).
The protective effect of natural plants with respect to anticancer activity is assumed to be associated mainly the antioxidant activities of either individual or interacting bioactive components present in the natural plants (Balasubramanian and Ragunathan, 2012). Further, free radical reactions can produce deleterious modifications in membranes, proteins, enzymes and DNA, increasing the risk of diseases such as cancer and Alzheimer disease (Ahn et al., 2011). So, we simultaneously determined for antioxidant activity.
The radical scavenging effects of seventeen compounds from E. alatus Sieb. were evaluated by the DPPH radical scavenging assay (Table 1). The positive control, ascorbic acid, had a DPPH radical scavenging effect with an established IC50 value of 25.21 μM. Compounds 2, 3, 14 and 16 exhibited weak scavenging activities on DPPH with IC50 values of 151.76, 170.79, 137.46 and 139.37 μM, respectively. However, the other compounds had no scavenging activity compared to ascorbic acid.
In this study, the methylene chloride-soluble fraction of the cork of E. alatus Sieb. was found to exhibit significant cytotoxic activity against Jurkat T cells. Consequently, 24R-methyllophenol (9) and monopalmitin (17) showed moderate cytotoxicity. In addition, lupenone (2), arborinone (3), tetradecyl(E)-ferulate (14) and trilinolein (16) showed weak antioxidant activity.
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