A New Cytotoxic Pregnanone fromCalotropis gigantea
Zhu-Nian Wang
Mao-Yuan Wang
Wen-Li Mei
Zhuang Han
Hao-Fu Dai
Author to whom correspondence should be addressed; E-Mail:hfdai@yahoo.cn; Tel: +86-898-6698-8061; Fax: +86-898-6698-8061.
Received 2008 Sep 25; Revised 2008 Nov 26; Accepted 2008 Dec 3; Collection date 2008 Dec.
Licensee Molecular Diversity Preservation International, Basel, Switzerland. This article is an open-access article distributed under the terms and conditions of the Creative Commons Attribution license (http://creativecommons.org/licenses/by/3.0/).
Abstract
A new pregnanone, named calotropone (1), was isolated from the EtOH extract of the roots ofCalotropis gigantea L. together with a known cardiac glycoside. The structures were elucidated by a study of their physical and spectral data. Compounds1 and2 displayed inhibitory effects towards chronic myelogenous leukemia K562 and human gastric cancer SGC-7901 cell lines.
Keywords:Calotropis gigantea L., Calotropone, Cytotoxicity.
Introduction
The genusCalotropis (Asclepiadaceae) is comprised of about six species of shrubs distributed throughout tropical and subtropical Africa and Asia. Two of them,Calotropis gigantea L. andCalotropis procera L. occur in China, and are two sister species.C. gigantea is a high biomass, fast growing perennial shrub growing as a weed in the Hainan province of China [1]. It was used as a traditional folk medicine for the treatment of anthelmintic, carminative, cough, leprosy, and asthma by the people of the Li nationality, who are autochthonous to Hainan island in China. The chemical constituents ofC.gigantea have been extensively investigated, leading to the isolation of many cardenolides [2,3,4,5], flavonoids [6], terpenes [7,8,9,10], pregnanes [11,12] and a nonprotein amino acid [13]. During our screening for cytotoxic agents from tropical medicinal plants, the ethanol extract of the roots ofC. gigantea showed cytotoxic activity towards human chronic myelogenous leukemia (K562) and human gastric cancer (SGC-7901) cell linesin vitro by MTT method with IC50 values of 9.7μg/mL and 6.7μg/mL, respectively. Bioassay-guided fractionation led to the isolation of a new pregnanone, calotropone, together with one known cardiac glycoside gofruside (2) from the ethanol extract ofC. gigantea, their structures were elucidated using spectral means especially 1D and 2D NMR spectroscopy. Both compounds1 and2 showed significant cytotoxicity against K562 and SGC-7901 cell lines. In this paper, we describe the isolation, structural elucidation, and cytotoxicity of1 and2.
Figure 1.
Structures of Compounds1 and2.
Results and Discussion
Bioassay-guided fractionation of the ethanol extract ofC. gigantea led to the isolation of compounds1 and2. Compound1 was obtained as yellow gum. The [M+Na]+ atm/z 491.2409 (calcd 491.2404) in the high-resolution ESI-Mass spectrum corresponded to the molecular formula C28H36O6. This formula can also be validated through1H-NMR,13C-NMR and DEPT spectra. The IR spectrum of1 showed absorption bands due to a hydroxyl (3,431 cm-1) and a carbonyl (1,712 cm-1) group, whereas the UV spectrum of1 suggested the presence of a benzoyl group (absorption maxima at 241, 267, and 284 nm). The1H-NMR spectrum of1 suggested the presence of a benzoyl group [signals atδ 7.43 (2H, t,J = 7.5 Hz), 7.56 (1H, t,J = 7.5 Hz), 7.93 (2H, d,J = 7.5 Hz)]. In addition, one olefinic proton (δ 5.41, m) and three high field methyl singlet at δ 2.06, 1.41, and 0.98 were also observed. The13C NMR (DEPT) spectra of1 showed the coexistence of three methyl groups, seven methylene groups, two aliphatic sp3 methine carbons, two oxygenated sp3 methine carbons, four sp3 quaternary carbon atoms, one tri-substituted double bond, one benzoyl group, and one ketone. This observation suggested that1 was likely to be a lineolon-type compound. Comparing the13C-NMR spectral data with those of the 12-O-benzoyllineolon showed that1 had one tertiary carbon more and one quarternary carbon less than 12-O-benzoyllineolon [12]. The1H-1H COSY, HMQC, and HMBC spectra allowed the complete assignments of chemical shifts of1 (Table 1). The chemical shift of C-8 of1 was upfield shifted to δ 37.0, which suggested that C-8 was not substituted by a hydroxyl group as 12-O-benzoyllineolon. The relative stereochemistry of1 was determined by ROESY correlations (Figure 2). Based on the above evidence, the structure of compound1 was identified as 12β-O-benzoyl-3β,14β,17β-trihydroxy-pregnane-20-one, named calotropone.
Table 1.
The NMR data of compound1.
| Position | δC | δH | HMBC |
|---|---|---|---|
| 1 | 37.0 | 1.75 (1H, m, H-1a), 1.13 (1H, m, H-1b) | C-2, 3, 5, 10 |
| 2 | 31.4 | 1.81, 1.46 (each 1H, m) | C-3, 4, 10 |
| 3 | 71.4 | 3.53 (1H, m) | C-1, 2, 5 |
| 4 | 41.9 | 2.33 (1H, dd, 12.8, 3.6 Hz), 2.25 (1H, m, overlapped) | C-2, 6, 5, 10 |
| 5 | 139.5 | ||
| 6 | 121.1 | 5.41 (1H, m) | C-4, 5, 7, 10 |
| 7 | 26.0 | 2.20, 1.91 (each 1H, m) | C-5, 6, 9, 14 |
| 8 | 37.0 | 1.80 (1H, m) | C-7, 10, 14 |
| 9 | 42.6 | 1.32 (1H, m) | C-1, 5, 11, 12, 14 |
| 10 | 36.7 | ||
| 11 | 26.5 | 2.06, 1.45 (each 1H, m, overlapped) | C-8, 10, 13 |
| 12 | 73.1 | 4.80 (1H, dd, 11.3, 4.5 Hz) | C-9, 14, 17, 19, 7' |
| 13 | 57.5 | ||
| 14 | 88.5 | ||
| 15 | 31.7 | 2.12 (1H, m, H-15a), 1.92 (1H, m, H-15b) | C-8, 13, 17 |
| 16 | 31.8 | 2.90 (1H, m, H-16a), 1.88 (1H, m, H-16b) | C-13, 14, 20 |
| 17 | 91.2 | ||
| 18 | 7.7 | 1.41 (3H, s) | C-12, 13, 14, 17 |
| 19 | 19.4 | 0.98 (3H, s) | C-1, 5, 9, 10 |
| 20 | 209.3 | ||
| 21 | 27.4 | 2.06 (3H, overlapped) | C-17, 20 |
| 1' | 129.9 | ||
| 2' | 128.4 | 7.93 (1H, d, 7.5 Hz) | C-1', 3', 4', 6' |
| 3' | 129.5 | 7.43 (1H, t, 7.5 Hz) | C-1', 2', 4', 5' |
| 4' | 133.2 | 7.56 (1H, t, 7.5 Hz) | C-2', 3', 5', 6' |
| 5' | 129.5 | 7.43 (1H, t, 7.5 Hz) | C-1', 3', 4', 6' |
| 6' | 128.4 | 7.93 (1H, d, 7.5 Hz) | C-1', 2', 4', 5', 7' |
| 7' | 165.3 |
The data were measured in CDCl3 with reference to TMS.
Figure 2.
Key HMBC and ROESY correlations of compound1.
Compounds1 and2 were evaluated for their cytotoxic activity against K562 and SGC-7901 cell lines using the MTT method [14], and both of them showed significant cytotoxicity against the two cell lines (Table 2).
Table 2.
IC50 values for inhibition of human cell lines of compounds1 and2.
| Compounds (IC50,μg/mL) | |||
|---|---|---|---|
| Cell lines | 1 | 2 | Mitomycin C* |
| K562 | 9.2 | 4.7 | 7.1 |
| SGC-7901 | 91.3 | 14.1 | 8.8 |
*Mitomycin C (MMC) was used as a positive control.
Conclusions
AlthoughC. gigantea was used as a very famous traditional folk medicine by many cultures, and it has been the subject of extensive phytochemical and bioactive investigations, its chemical components and bioactivities have not been completely investigated yet. Up to now, seven oxypregnane-oligoglycosides, calotroposides A−G have been isolated from the roots ofC. gigantea [5,11]. In our present study a new pregnanone was isolated and identified from the genus ofCalotropis, this is the first steroidal aglycone isolated from this genus. Meanwhile, the cytotoxicity against K562 and SGC-7901 cell lines of compounds1 and2 was evaluated for the first time, this is also the first report about the cytotoxicity of the pregnanone from this genus.
Experimental
General
Melting points were obtained on Beijing Taike X-5 stage apparatus uncorrected. The NMR spectra were recorded on Bruker AV-400 spectrometer, using TMS as an internal standard. The FAB-MS spectra were measured with a VG Autospec-3000 mass spectrometer, and the HRESI-MS spectra were measured with an API QSTAR Pulsar mass spectrometer. The IR spectra were obtained on a Nicolet 380 FT-IR instrument, as KBr pellets. The UV spectra were measured on a Beckman DU800 spectrometer. Optical rotation was recorded using Rudolph Autopol III polarimeter (U.S.A). Column chromatography was performed with silica gel (Marine Chemical Industry Factory, Qingdao, P.R. China), and Macroporous resin D101 (Shandong Lukang Pharmaceutical Co., Ltd.). TLC was preformed with silica gel GF254 (Marine Chemical Industry Factory, Qingdao, China), and developed by spraying with 10% H2SO4 followed by heating.
Plant material
The roots ofCalotropis gigantea used in this research were collected from Eman Village of Danzhou County, Hainan Province, P. R. China, in December 2006, and authenticated by Prof. Zhu-Nian Wang of the Institute of Tropical Crops Genetic Resources, Chinese Academy of Tropical Agricultural Sciences. The voucher specimen (No 20061201) was deposited at the Institute of Tropical Crops Genetic Resources, Chinese Academy of Tropical Agricultural Sciences.
Extraction and isolation
The roots (26.7 kg) ofCalotropis gigantea were extracted three times with 95% ethanol at room temperature. Following filtration, the combined ethanol extract was evaporated to dryness under reduced pressure to give a crude extract. The crude ethanol extract was suspended in water (6.0 L) and successively partitioned with petroleum ether to give Petro-soluble fraction (236.1 g) and an aqueous residue. Then the aqueous residue was concentrated and applied to a D-101 resin column, eluting with H2O and MeOH, successively, the MeOH eluent was collected and evaporated under reduced pressure to afford the “MeOH fraction” (yield 256.1 g). The MeOH fraction was subjected to vacuum liquid chromatography (VLC) over silica gel, eluting with gradient elution CHCl3-MeOH (100:0, 50:1, 25:1, 10:1, 5:1, 2:1, MeOH) to afford seven fractions (Fr.1−Fr.7). Fr.1 (56.3 g) was subjected to further column chromatography over silica gel, with petroleum ether-acetone (6:4) as eluent, to afford compound1 (32 mg). Fr.4 (20.3 g) was subjected to column chromatography over silica gel, eluting with gradient elution CHCl3-MeOH to afford2 (35 mg).
Calotropone (1): Yellow gum, [α]
−89.7° (c 0.26, MeOH); HR-ESI-MS:m/z [M+Na]+ 491.2409( calcd. For C28H36O6Na, 491.2404); IRν
(cm-1): 3431, 2918, 2849, 1712, 1629, 1463, 1275, 1110; UVλmax nm (CHCl3): 241, 267, 284;1H-NMR (400 MHz, CDCl3),13C-NMR (100 MHz, CDCl3 ):Table 1.
Gofruside (2) [15,16]: C29H40O9, Colorless needles; m.p. 165 − 167 °C; IR (KBr) λmax (cm-1): 3437, 2937, 1738; FAB-MS (neg.)m/z 535 [M−H]−;1H-NMR (400Hz, CD3OD):δ 5.88 (1H,brs, H-22), 5.02, 4.89 (each 1H, d,J = 18.3 Hz, H-21), 4.70 (1H, d,J = 7.9 Hz, H-1'), 3.69 (1H, m, H-3), 3.65 (1H, m, H-5'), 3.29 (1H, brs, H-3'), 3.23 (1H, dd,J = 2.5, 7.7 Hz, H-2'), 3.14 (1H, dd,J = 1.8, 9.4 Hz, H-4'), 1.20 (3H, d,J = 6.0 Hz, H-6'), 0.91 (3H, s, H-18);13C-NMR (100 MHz, CD3OD):δ 32.1 (C-1), 30.2 (C-2), 78.7 (C-3), 37.0 (C-4), 45.1 (C-5), 30.0 (C-6), 28.6 (C-7), 43.8 (C-8), 51.9 (C-9), 52.8 (C-10), 22.9 (C-11), 40.6 (C-12), 50.9 (C-13), 86.1 (C-14), 32.8 (C-15), 27.9 (C-16), 52.0 (C-17), 16.3 (C-18), 210.4 (C-19), 178.3 (C-20), 75.3 (C-21), 117.9 (C-22), 177.2 (C-23), 99.9 (C-1'), 72.4 (C-2'), 72.9 (C-3'), 74.3 (C-4'), 70.5 (C-5'), 18.2 (C-6').
Cytotoxicity bioassay
Compounds1 and2 were examined for their cytotoxic activity against chronic myelogenous leukemia K562 and human gastric cancer SGC-7901 cell lines. Cancer cells were incubated for 3 days at 37 °C in the presence of various concentrations of compounds from DMSO-diluted stock solutions. The growth inhibitory property was determined using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetra-zolium bromide (MTT) assay as described by Mosmann [14].
Acknowledgements
This research was financially supported by National Basic Research Program of China (2007CB116306) and National Programs for Science and Technology development of China (2007B127B00).
Footnotes
Sample Availability: Samples of the compounds are available from the co-author Hao-fu Dai (hfdai@yahoo.cn).
References and Notes
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