2013 Volume 61 Issue 6 Pages 674-677
Two new triterpenes, (1α,3β,8α,9β,10α,13α,14β)-9,10-dimethyl-25,26-dinorolean-5-en-1,3-diol (1) and (1α,3β,6β)-olean-12-en-1,3,6-triol (2) were isolated from the leaves of Aleurites fordii, together with five known triterpenes. The structures of isolates were established by one dimensional (1D)- and 2D-NMR spectroscopic data along with MS analysis. Of the isolated compounds, 1, 2 and 4 (daturadiol) displayed moderate cytotoxicities against two or more human cancer cell lines in HepG2 (hepatocellular carcinoma), SK-OV-3 (ovarian carcinoma), A-549 (lung carcinoma) and SNU-1 (gastric carcinoma).
Vernicia fordii (Hemsl.) Airy Shaw [=Aleurites fordii Hemsl.] (Euphorbiaceae), known as tung oil tree, grows in China and is cultivated in China and Korea.1) Traditionally, the fruits, leaves and roots of V. fordii have been used for the treatment of sore throat, respiratory illness, constipation and diuresis in folk medicine.2,3) Previous investigations on this plant reported the presence of various compounds such as oils, coumarins, diterpenoid esters, sterols and tannins.1,3–7) Recently, interferon (IFN)-γ inducing tigliane diterpene esters were isolated from V. fordii.7) As part of ongoing search for active constituents from this plant, two new triterpenes, (1α,3β,8α,9β,10α,13α,14β)-9,10-dimethyl-25,26-dinorolean-5-en-1,3-diol (1) and (1α,3β,6β)-olean-12-en-1,3,6-triol (2), were isolated from hexane-soluble extract of the leaves of V. fordii, along with five known triterpenes, castanopsol (3), daturadiol (4), 2,3-secofriedelane-2,3-dioic acid (5), β-sitosterol and daucosterol (see Fig. 1). All compounds were evaluated for their cytotoxicity against HepG2, SK-OV-3, A-549 and SNU-1 cell lines.
Compound 1 was obtained as white amorphous solid and the molecular formula was determined to be C30H50O2 from a molecular ion peak [M]+ at m/z 442.3824 in the high resolution-electron impact (HR-EI)-MS. The 13C- and distortionless enhancement by polarization transfer (DEPT)-NMR spectroscopic data of 1 showed 30 carbon signals, including eight methyls, nine methylenes, six methines and seven quaternary carbons, implying a triterpene skeleton. From the molecular formula and DEPT data, 1 has six degrees of unsaturation, implying this compound is composed of one double bond and five rings, suggestive of a pentacyclic triterpene. 1H-NMR spectroscopic data of 1 displayed one double bond at δH 5.70 (1H, br d, J=6.4 Hz, H-6) and two oxygenated methines at δH 3.54 (1H, t, J=2.8 Hz, H-3) and 4.03 (1H, ddd, J=10.6, 10.6, 5.3 Hz, H-1) in this structure. 1H–1H Correlated spectroscopy (COSY) correlations sequentially from H-10/H-1/H-2/H-3 and heteronuclear multiple bond connectivity (HMBC) correlations of H-3 to C-4, C-23, C24, C-5, C-1 enabled to construct planar A ring as shown in Fig. 1. A methyl group (C-25) was found on C-9, which was corroborated by the correlations from H-10 to C-25, H-25 to C-8 and C-10, H-6 to C-7 and C-8. Also, methyls (C-26 and C-27) were found on C-14 and C-13, respectively, from the observed HMBC correlations of H-8 to C-26, H-18 to C-27 and C-28. Hence, the locations of these methyls in this structure are similar to those of friedelane-type triterpenoid except C-24 is attached to C-4. The relative configuration of OH-1 and OH-3 was determined to be α-OH and β-OH, respectively, based on the coupling constants (J=10.6, 10.6, 5.3 Hz, H-1 and J=2.8 Hz, H-3). Furthermore, rotating frame Overhauser enhancement spectroscopy (ROESY) correlations between H-1 and H-25, H-25 and H-26, H-26 and both H-18 and H-28 suggested these protons are β oriented. ROESY correlations between H-10 and H-8, H-8 and H-27 assigned α orientation of these groups. Therefore, the structure of compound 1 was characterized as (1α,3β,8α,9β,10α,13α,14β)-9,10-dimethyl-25,26-dinorolean-5-en-1,3-diol.
Compound 2, obtained as white amorphous solid, showed a molecular ion peak [M]+ at m/z 458.3772 in the HR-EI-MS, corresponding to the molecular formula C30H50O3. The 1H-, 13C- and DEPT-NMR spectra of 2 were characteristic of an oleanane-type triterpene, showing eight methyls, eight methylenes, seven methines and seven quaternary carbons. The 1H- and 13C-NMR data of 2 were almost similar to those of daturadiol (3β,6β-olean-12-en-3,6-diol),8) except for the presence of one more hydroxyl group. The location of the hydroxyl group was confirmed as C-1 by HMBC correlations of δH 3.55 (H-1) to δC 73.5 (C-3), 48.6 (C-5) and 17.8 (C-25). In addition, 1H–1H COSY spectrum displayed consecutive correlations of H-1/H-2/H-3, supporting the hydroxyl group was attached to C-1. The stereochemistry of 2 was consistent with that of daturadiol based on chemical shifts, coupling constants and ROESY correlations between H-3 and H-5, H-5 and H-9, H-5 and H-6, suggesting the presence of 3β, 6β hydroxyl groups. The relative configuration of OH-1 was deduced as α orientation by the observed coupling constant (2.9 Hz) and ROESY correlations between H-1 and H-25. Taken together, the structure of 2 was determined as (1α,3β,6β)-olean-12-en-1,3,6-triol.
Compounds 3–7 were identified as the known compounds, castanopsol (3), daturadiol (4), 2,3-secofriedelane-2,3-dioic acid (5), β-sitosterol (6) and daucosterol (7). Of these known compounds, compound 5 was isolated from natural sources for the first time by comparison of their spectroscopic data with those of literature.8–11)
All the isolated compounds were evaluated for their cytotoxicity against four human cancer cell lines, HepG2 (hepatocellular carcinoma), SK-OV-3 (ovarian carcinoma), A549 (lung carcinoma) and SNU-1 (gastric carcinoma), using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) method (Table 2). Compounds 1 and 2 exhibited moderate cytotoxicity against all the cell lines with the IC50 values from 17.4 to 26.1 µm, while compound 4 was found to be cytotoxic against HepG2 and A549 cancer cell lines. Earlier report on this plant proved that tung oil containing predominantly eleostearic acid exhibited cytotoxic effects on several tumor cell lines including HepG2 and A549.12) An ingredient of tung oil, α-eleostearic acid, was evaluated for its cancer chemopreventive effect in 7,12-dimethylbenz[a]anthraxene (DMBA) and 1,2-dimethylhydrazine (DMH)-induced mammary and colon murine models and found inactive.13) The present study disclosed that new constituents in this plant, triterpenes, may be in part responsible for cytotoxic activity.
| No. | 1 | 2 | ||
|---|---|---|---|---|
| δHa) | δCb) | δHa) | δCb) | |
| 1 | 4.03 (1H, ddd, 10.6, 10.6, 5.3) | 67.6 | 3.55 (1H, t, 2.9) | 73.2 |
| 2 | 2.00, 1.90 (2H, m) | 39.0 | 2.00, 1.76 (2H, m) | 34.6 |
| 3 | 3.54 (1H, t, 2.8) | 76.5 | 3.65 (1H, dd, 12.3, 4.3) | 73.5 |
| 4 | — | 41.0 | — | 39.9 |
| 5 | — | 139.5 | 1.23 (1H, d, 2.2) | 48.6 |
| 6 | 5.70 (1H, br d, 6.4) | 125.1 | 4.64 (1H, br s) | 69.3 |
| 7 | 1.99, 1.85 (2H, m) | 23.5 | 1.77, 1.51 (2H, m) | 40.5 |
| 8 | 1.51 (1H, m) | 47.7 | — | 38.8 |
| 9 | — | 36.6 | 2.37 (1H, dd, 11.7, 6.2) | 37.8 |
| 10 | 2.12 (1H, br d, 10.2) | 57.2 | — | 40.4 |
| 11 | 1.96, 1.66 (2H, m) | 37.3 | 2.07, 1.82 (2H, m) | 23.2 |
| 12 | 1.34 (2H, m) | 30.8 | 5.23 (1H, t, 3.6) | 121.7 |
| 13 | — | 39.0 | — | 144.8 |
| 14 | — | 38.2 | — | 42.9 |
| 15 | 1.49, 1.27 (2H, m) | 32.6 | 1.83, 0.98 (2H, m) | 26.3 |
| 16 | 1.55, 1.37 (2H, m) | 36.2 | 1.98, 0.80 (2H, m) | 27.1 |
| 17 | — | 30.1 | — | 32.6 |
| 18 | 1.56 (1H, m) | 43.1 | 1.97 (1H, m) | 47.4 |
| 19 | 1.36, 1.22 (2H, m) | 35.2 | 1.67, 1.04 (2H, m) | 47.0 |
| 20 | — | 28.4 | — | 31.2 |
| 21 | 1.48, 1.25 (2H, m) | 33.2 | 1.34, 1.10 (2H, m) | 34.9 |
| 22 | 1.52, 0.91 (2H, m) | 39.1 | 1.43, 1.22 (2H, m) | 37.1 |
| 23 | 1.07 (3H, s) | 28.5 | 1.11 (3H, s) | 28.0 |
| 24 | 1.12 (3H, s) | 25.4 | 1.19 (3H, s) | 17.0 |
| 25 | 1.00 (3H, s) | 16.6 | 1.32 (3H, s) | 17.8 |
| 26 | 1.12 (3H, s) | 20.2 | 1.29 (3H, s) | 18.6 |
| 27 | 1.02 (3H, s) | 18.6 | 1.14 (3H, s) | 26.3 |
| 28 | 1.16 (3H, s) | 32.1 | 0.84 (3H, s) | 28.5 |
| 29 | 0.94 (3H, s) | 34.7 | 0.87 (3H, s) | 33.5 |
| 30 | 0.98 (3H, s) | 32.4 | 0.87 (3H, s) | 23.8 |
a) Recorded at 400 MHz. b) Recorded at 100 MHz.
| Compounds | IC50 (µm)a) | |||
|---|---|---|---|---|
| HepG2 | SK-OV-3 | A-549 | SNU-1 | |
| 1 | 21.06±0.14 | 24.46±0.21 | 20.83±0.02 | 17.36±0.11 |
| 2 | 22.78±0.09 | 24.86±0.21 | 24.27±0.21 | 26.12±1.35 |
| 3 | >40 | >40 | >40 | >40 |
| 4 | 24.74±0.18 | >40 | 26.91±0.14 | >40 |
| 5 | >40 | >40 | >40 | >40 |
| 6 | >40 | >40 | >40 | >40 |
| 7 | >40 | >40 | >40 | >40 |
| Controlb) | 26.42±0.35 | 48.92±2.09 | 6.43±0.08 | 4.32±0.18 |
a) The data represent the mean±S.D. b) Adriamycin is used for positive control.
Optical rotation was measured with a Jasco P2000 polarimeter (Jasco Corporation, Japan), and FT-IR spectra using a Jasco FT/IR-4200 (Jasco Corporation). NMR spectra were recorded on a Varian UNITY 400 (Varian, Inc., Palo Alto, CA, U.S.A.) with the tetramethylsilane as an internal standard. HR-EI-MS (JEOL, Tokyo, Japan) was performed with on a JEOL JMS AX505W spectrometer. Silica gel (230–400 mesh, SiliCycle Inc., Quebec, Canada), RP-C18 (Cosmosil 75C18-PREP, Kyoto, Japan) Sephadex LH-20 (25–100 µm, Sigma-Aldrich, Steinheim, Germany) were used for column chromatography. TLC was performed on precoated Kiesel-gel 60 F254 (0.25 mm, Merck, Darmstadt, Germany) and Kiesel-gel 60 RP-18F254s (0.25 mm, Merck, Steinheim, Germany). MTT assay was measured in Microplate reader Benchmark (Bio-RAD, Hercules, CA, U.S.A.).
Plant MaterialThe leaves of V. fordii were collected in October 2009 from Jeju island, Republic of Korea, and identified by Dr. Chan-Soo Kim. A voucher specimen (KRIBB0000463) has been deposited at the herbarium of Korea Research Institute of Bioscience and Biotechnology, Daejeon, Republic of Korea.
Extraction and IsolationAir-dried leaves of V. fordii (2.9 kg) were pulverized and extracted with MeOH at room temperature three times to obtain 449.6 g of solid extract. The MeOH extract was suspended in H2O and then partitioned with n-hexane, EtOAc, and n-BuOH successively, to give the residue of 96.3 g, 53.9 g and 85.9 g respectively. Of these fractions, n-hexane-soluble extract was found to be cytotoxic against HepG2 cancer cell line with IC50 20.35 µg/mL. The hexane extract was then subjected to column chromatography (CC) over silica gel (230–400 mesh) eluted with hexane–EtOAc (50 : 1–1 : 5), and then with CHCl3–MeOH (10 : 1–1 : 1) to give 38 fractions (LH1–LH38). Combined fractions LH19 and LH20 were subjected to silica gel with hexane–EtOAc (10 : 1), then purified by recrystallization from CHCl3–MeOH to yield compound 6 (313 mg). Fraction LH22 was chromatographed over silica gel with hexane–EtOAc (10 : 1), obtaining subfractions LH22-1 to LH22-4. Subfraction LH22-3 was then subjected to RP18 silica gel (75 µm) eluted with MeOH–H2O (80 : 20, 90 : 10, 95 : 5, 100 : 0) and further purified by silica gel with CHCl3–acetone (200 : 1) to give compound 4 (12.0 mg). Fraction LH28 was subjected to Sephadex LH20 eluted with hexane–CH2Cl2–MeOH (10 : 10 : 1) to give subfractions LH28S1 to LH28S15. LH28S4 was further fractionated by medium pressure liquid chromatography (MPLC) column of RP-C18 (75 µm) with gradient MeOH–H2O (80 : 20 to 95 : 5), then purified by Sephadex LH20 with MeOH–H2O (80 : 20) to yield compound 1 (27.0 mg). LH28S5 was chromatographed over RP18 silica gel with MeOH–H2O (85 : 15) and further purified by silica gel with CHCl3–acetone–MeOH (20 : 10 : 1), affording compound 3 (1.9 mg). Compound 2 (17 mg) was obtained from the purification of LH28S10 by silica CC with CHCl3–acetone (10 : 1). LH34-35 were fractionated over silica gel CC with CHCl3–MeOH (50 : 1), affording subfractions LH3435-1 to LH3435-11. Among them, LH3435-10 was purified by RP18 CC with MeOH–H2O (85 : 15), yielding compound 7 (6.2 mg). Fraction LH3435-7 was further fractionated using Sephadex LH20 CC eluted with hexane–CH2Cl2–MeOH (10 : 10 : 1) and provided subfractions LH3435-7-1 to LH3435-7-8. Compound 5 (6 mg) was then obtained from the recrystallization of LH3435-7-7 with CHCl3–MeOH.
(1α,3β,8α,9β,10α,13α,14β)-9,10-Dimethyl-25,26-dinorolean-5-en-1,3-diol (1): White amorphous solid; [α]D20 +27.5° (c=0.12, CHCl3); IR (film) νmax cm−1: 3392, 2945, 2869, 1461, 1386, 759; 1H- and 13C-NMR data, see Table 1; HR-EI-MS m/z 442.3824 [M]+ (Calcd for C30H50O2: 442.3811).
(1α,3β,6β)-Olean-12-en-1,3,6-triol (2): White amorphous solid; [α]D20 +27.0° (c=0.21, CHCl3); IR (film) νmax cm−1: 3618, 3436, 2948, 2914, 2855, 1462, 1383, 1362, 759; 1H- and 13C-NMR data, see Table 1; HR-EI-MS m/z 458.3772 [M]+ (Calcd for C30H50O3: 458.3760).
Cell Lines and Cell CultureHepG2 (human liver hepatocellular carcinoma) cells were maintained in Dulbecco’s modified Eagle’s medium in the presence of 10% (v/v) fetal bovine serum (FBS) and 1% (w/v) penicillin–streptomycin. SK-OV-3 (ovary adenocarcinoma) were cultured in McCoy’s 5A medium with supplement of 10% (v/v) FBS and 1% (w/v) penicillin/streptomycin. A-549 (human lung carcinoma), and SNU-1 (human gastric carcinoma) cells were cultured in RPMI-1640 with 10% (v/v) FBS and 1% (w/v) penicillin–streptomycin. Cells were cultured at 37°C in a humidified atmosphere containing 5% CO2. All cells were obtained from the American Type Culture Collection (ATCC, Manassas, U.S.A.).
Cytotoxicity AssayCell viability was measured using MTT assay. Briefly, HepG2 (3×103), SK-OV-3, and A-549 cells (1×104) were seeded in each 96-well plates (0.1 mL). The SNU-1 cells (2×104) were seeded in each 96-well plates (0.2 mL). Test samples were dissolved in small amount of DMSO and diluted in the appropriate culture medium (final concentration of DMSO <0.5%). After incubation with compounds for 24 h, 10 µL of MTT solution (5 mg/mL in PBS) was added to each well and re-incubated for 4 h at 37°C. The formazan crystals produced were dissolved in 100 µL of DMSO. Absorption was measured by an enzyme-linked immunosorbant assay reader (ELISA, Bio-Rad) at 570 nm. Cytotoxicity was expressed as the concentration of compound inhibiting cell growth by 50% (IC50) and the given values were calculated from the mean of three different experiments.
This work was supported from KRIBB Research Initiative Program, and a Grant of the NRF (2011-0014225, Y.-W. Chin) funded by the government of Korea (MEST). We thank Dr. Dong-Ho Choung for NMR spectra data.
