Phytochemistry Letters 5 (2012) 800–803
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Two new germacranolides from Elephantopus tomentosus Wen-Li Mei a,1, Bei Wang a,b,c,1, Wen-Jian Zuo a, You-Xing Zhao a, Wen-Hua Dong a, Guo-dao Liu b,*, Hao-Fu Dai a,* a Key Laboratory of Biology and Genetic Resources of Tropical Crops, Ministry of Agriculture, Institute of Tropical Bioscience and Biotechnology, Chinese Academy of Tropical Agricultural Sciences, Haikou 571101, Hainan, PR China b Tropical Crops Genetic Resources Institute, Chinese Academy of Tropical Agriculture Sciences, Danzhou 571737, Hainan, PR China c Horticultural and Garden College, Hainan University, Haikou 570228, Hainan, PR China
A R T I C L E I N F O
A B S T R A C T
Article history: Received 1 April 2012 Received in revised form 28 August 2012 Accepted 17 September 2012 Available online 2 October 2012
The bioassay-guided chemical investigation of the whole plant of Elephantopus tomentosus led to the isolation of two new germacranolides, tomenphantopin C (1) and tomenphantopin D (2), together with four known compounds (3–6). Their structures were determined by spectroscopic techniques (UV, IR, MS, 1D and 2D NMR). All compounds were evaluated for cytotoxic and antibacterial activities. ß 2012 Phytochemical Society of Europe. Published by Elsevier B.V. All rights reserved.
Keywords: Elephantopus tomentosus Tomenphantopin C Tomenphantopin D
1. Introduction Elephantopus species are known to be an abundant source of sesquiterpene lactones, which possessed significant antileishmanial (Fuchino et al., 2001; Satake et al., 2001) and cytotoxic (Kupchan et al., 1969; Lee et al., 1973, 1975, 1980; Zhang et al., 1986; But et al., 1996, 1997; Tabopda et al., 2007) activity. Elephantopus tomentosus Linn. (Asteraceae) is a traditional medicine hosted in south of China, the whole plant of which is used as diuretic, antifebrile, antiviral, and antibacterial agent, as well as in the treatment of hepatitis, bronchitis, fever, the cough associated with pneumonia, and arthralgia (Chen, 1985). Some cytotoxic germacranolides against KB cell line from the chloroform extract of E. tomentosus were reported (Hayashi et al., 1987, 1999), while both water and ethanol extracts of the plant were found to possess inhibitory effect on bacteria (Lin, 2011). The EtOAc extract of the whole plant of E. tomentosus was found to possess cytotoxic and antibacterial activities by our previous screening, which induced the investigation on its active constituents and resulted in the isolation of two new germacranolides, tomenphantopin C (1) and tomenphantopin D (2), together with four known compounds, molephantin (3), 2, 5-epoxy-2b-hydroxy-4a-methoxy-8a-(2methylpropenoyloxy)-10(14), 11(13)-germacradien-12, 6a-olide
* Corresponding authors. Tel.: +86 898 66961869; fax: +86 898 66961869. E-mail addresses:
[email protected],
[email protected] (H.-F. Dai). 1 These authors contributed equally.
(4), 2-deethoxy-2-methoxyphantomolin (5), and hedytriol (6). This paper deals with the isolation and structural elucidation of two new compounds, as well as the cytotoxic and antibacterial activities of these isolates. 2. Results and discussion Tomenphantopin C (1) was assigned the molecular formula of C21H28O7 based on an quasi-molecular ion peak at m/z: 415.1733 ([M+Na]+, calcd. 415.1732) in HRESIMS and 13C NMR data. The IR spectrum showed a intense absorption band at 1773 cm 1 due to a saturated g-lactone (Vichnewski et al., 1976), as evidenced by the characteristic 1H NMR signals at dH 2.74 (1H, dt, J = 9.0, 2.6 Hz, H11), 2.84 (1H, ddd, J = 10.9, 9.0, 7.7 Hz, H-7) and 4.74 (1H, dd, J = 7.7, 4.3 Hz, H-6) (Table 1). The methacrylate ester side chain was indicated by the IR signals at 1714 and 1635 cm 1 and was further supported by the 1H NMR signals at dH 6.11 (1H, m, H-19a), 5.74 (1H, m, H-19b) and 1.98 (3H, m, H-18), which were typical signals for a methacrylate group (Lee et al., 1980). The NMR spectral properties suggested that compound 1 had a similar germacrane skeleton to 5 (Banerjee et al., 1986). However, absence of the pair of doublets relative to exomethylene protons gave evidence for the saturation between C-11 and C-13, which was not usual for sesquiterpenoids of germacrane type. The methoxyl group (dH 3.30, dC 57.9) was located at C-13 by the key HMBC correlation from H (13-OMe) (dH 3.30) to C-13 (dC 70.1). In the 1H NMR spectrum, H-7 (dH 2.84) showed as a complex signal (ddd) with large coupling constants with H-6, H-8 and H-11 (J7,8 = 10.9,
1874-3900/$ – see front matter ß 2012 Phytochemical Society of Europe. Published by Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.phytol.2012.09.005
W.-L. Mei et al. / Phytochemistry Letters 5 (2012) 800–803
801
Table 1 1 H and 13C NMR data of 1 and 2 (500 and 125 MHz, respectively, CD3OD). No.
1
2
1
13
5.46 s
126.3 114.1 128.0 140.0 85.8 79.6 37.1 79.4 32.2 133.0 46.7 176.4 70.1 12.0 27.1 166.0 136.3 17.1 125.4 48.1 57.9
H
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 2-OMe 13-OMe
C
5.64 s 5.22 4.74 2.84 5.34 3.80
d (4.3) dd (4.3, 7.7) ddd (7.7, 9.0, 10.9) ddd (2.3, 4.2, 10.9) d (14.3), 2.18 dd (4.2, 14.3)
2.74 dt (2.6, 9.0) 3.75 dd (2.6, 9.2), 3.46 dd (2.6, 9.2) 1.81 m 1.76 d (1.4)
1.98 6.11 3.19 3.30
m m, 5.74 m s s
1
13
2.52 d (13.3), 2.40 d (13.3)
51.9 107.4 50.2 79.3 86.0 81.2 45.2 80.0 35.5 143.0 41.9 181.4 16.8 32.3 120.7 167.7 138.0 18.6 126.7
H
d s d s d d d d t s d s t q q s s q t q q
2.72 d (13.6), 1.90 d (13.6) 4.22 d (3.8) 4.26 dd (3.8, 7.7) 3.19 dt (7.7, 11.2) 5.15 overlapped 2. 66 d (16.1) 2.46 m 1.29 d (6.9) 1.62 s 5.32 s, 5.15 overlapped
2.00 s 6.17 s, 5.74 s
C t s t s d d d d t s d s q q t s s q t
Assignments are based on COSY, HMQC, and HMBC experiments.
J7,6 = 7.7, J7,11 = 9.0 Hz). Given that H-7 was a-oriented as in other naturally occurring germacranolides, H-6, H-8, H-11 were determined to be b-oriented, which were further confirmed by the correlations between H-6/H-8 and H-8/H-11 in the NOESY experiment. H-5 was determined to be b-oriented according to its coupling constant (4.3 Hz) with H-6 (Lee et al., 1980). Thus, the structure of 1 was elucidated as shown in Fig. 1 and named tomenphantopin C. Tomenphantopin D (2) had a molecular formula of C19H26O7 by HREIMS at m/z: 366.1687 ([M]+, calcd. 366.1679) and 13C NMR data. IR spectrum revealed the presence of hydroxyls (3446 cm 1), g-lactone (1754 cm 1), unsaturated ester carbonyl group (1716 cm 1), methyls (2359 cm 1), and double bond (1636, 1456 cm 1). The 13C and 1H NMR spectral properties of 2 were similar to those of 4 (Satake et al., 2001). The obvious differences were the loss of the methoxy signal, and a saturated a-methyl glactone in place of the well-defined a-methylene g-lactone ring. The assignments of H-11, H-9, H-8, H-7 and H-6 protons and one olefinic methylene (H-15), as well as the methyl at C-13, were made by 1H–1H COSY experiment. In addition, the chemical shifts of correspondent carbon atoms were also assigned unambiguously by a combination of 1H and 13C NMR, HSQC, HMBC, NOESY and 1 H–1H COSY data. The similar relative configurations to compound 1 at H-6, 7, 8, 11 were confirmed by the correlations between H-7/ H-13, H-11/H-8 and H-8/H-6 in the NOESY experiment. The borientation of H-5 was also deduced by its coupling constant with
H-6 (3.8 Hz). Moreover, the key NOE correlation of H-5/H-14 indicated the b-orientation of the methyl group of C-14. Therefore, the C-8 substituent and the C-4 hydroxyl group were proposed to be a-oriented. Based on the above analysis, the structure of 2 was elucidated as shown in Fig. 1 and named tomenphantopin D. Four known compounds, molephantin (3) (Lee et al., 1973), 2,5-epoxy-2b-hydroxy-4a-methoxy-8a-(2-methylpropenoyloxy)10(14),11(13)-germacradien-12,6a-olide (4) (Satake et al., 2001), 2-deethoxy-2-methoxyphantomolin (5) (Banerjee et al., 1986), and hedytriol (6) (Zhu et al., 2007), were identified by comparison of spectroscopic data with those of literatures. Compounds 1–6 were evaluated for their cytotoxic activities against human myeloid leukemia cell line (K562) and human hepatoma cell line (SMMC-7721) by MTT method. The results showed that compound 3 possessed the strong activities against K562 and SMMC-7721with the IC50 value of 7.9 mM and 5.8 mM, respectively. Compounds 2 and 4 also had potent activities against K562 and SMMC-7721. Compound 5 had activity against SMMC-7721, while compounds 1 and 6 were completely inactive (Table 2). These compounds were tested for in vitro antibacterial activities against Staphylococcus aureus (S. aureus) strain by the filter paper disc agar diffusion method. Compounds 2–5 showed antibacterial activities against S. aureus with MICs of 3.4, 0.57, 6.6, and 0.86 mM, respectively. Compounds 1 and 6 were completely inactive (Table 2).
Fig. 1. Structures of 1 and 2.
W.-L. Mei et al. / Phytochemistry Letters 5 (2012) 800–803
802 Table 2 Cytotoxic activities and antibacterial activities of 2–5. Compounds
2 3 4 5 Mitomycin C Kanamycin sulfate
Cytotoxic activities with IC50 values (mM)
Diameter of inhibition zone (mm)
K562
SMMC-7721
S. aureus
44.8 7.9 32.8 – 21.2
11.2 5.8 20.9 44.2 7.5
24.7 20.6 13.0 26.4 26.8
3. Experimental 3.1. General experimental procedures The NMR spectra were recorded on a Bruker Avance DRX-500 spectrometer, using TMS as an internal standard. The HRESIMS spectra and HREIMS spectra were measured with an API QSTAR Pulsar mass spectrometer. The IR spectra were obtained on a Nicolet 380 FT-IR instrument from KBr pellets. The UV spectra were measured on a Shimadzu UV-2550 spectrometer. Melting points were measured on a Beijing Taike X-5 stage apparatus and were uncorrected. Optical rotation was recorded using a Rudolph Autopol III polarimeter (USA). Column chromatography was performed with silica gel (Marine Chemical Industry Factory, Qingdao, China), RP-18 (Merck, Germany), and Sephadex LH-20 (Merck, Germany). TLC was preformed with silica gel GF254 (Marine Chemical Industry Factory, Qingdao, China). HPLC separations were using a Dionex P680 pump with a Summit P680 detetor (l = 254 nm) and a YMC C18 reversed phase column (5 mm, 10 mm 250 mm). 3.2. Plant material The whole plant of E. tomentosus Linn. was collected in Changjiang County of Hainan Province, China (October 2010), and authenticated by Associate Professor Zheng-Fu Dai of the Institute of Tropical Bioscience and Biotechnology, Chinese Academy of Tropical Agricultural Science, where a voucher specimen (No. ET201010) was deposited. 3.3. Extraction and isolation The air-dried and crushed whole plant (19.4 kg) of E. tomentosus was extracted three times with 95% EtOH at room temperature. The extract was evaporated under reduced pressure to dryness. Then, the residue was suspended in H2O and partitioned with petroleum ether, EtOAc, and then n-BuOH. The EtOAc fraction (81.3 g) was subjected to a silicon gel dry-column chromatography (2.5 kg, 200–300 mesh) eluted with CHCl3–MeOH (9:1, 3 L) to give five corresponding fractions, Fr.1–Fr.5. Fr.1 (31.4 g) was further separated on a silica gel CC (4 cm 98 cm, 350 g) eluting with PEEtOAc gradient (1:0, 50:1, 30:1, 15:1, 10:1, 8:1, 5:1, 2:1, 0:1, each 1 L) to yield nine fractions (Fr.1-1–Fr.1-9). Fr.1–7 (4.4 g) was further separated on a silica gel CC (3 cm 65 cm, 64 g) with PE-EtOAc (5:1, 3.5 L) to yield 5 fractions (Fr.1-7-1–Fr.1-7-5). Compound 3 (15.3 mg) was isolated from Fr.1-7-3 (1.8 g) by using a silica gel CC (3 cm 65 cm, 64 g) with CHCl3–MeOH (100:1, 2.5 L) as eluent repeatedly, then was further purified on Sephadex LH-20 (column: 100 cm 3 cm) in CHCl3–MeOH (1:1, v/v). Fr.1-74 (160.0 mg) was separated on a silica gel CC (2 cm 45 cm, 25 g) with PE–EtOAc (4:1, v/v), then on RP-18 with 65% MeOH–H2O to yield 2 (5.7 mg). Fr.1-7-5 (245.2 mg) was purified on Sephadex LH20 (column: 100 cm 3 cm) in EtOH and was further separated by
semi-preparative HPLC using MeOH–H2O (78:22, v/v) at 1.0 mL/min to yield 1 (5.3 mg, tR = 25.5 min) and 4 (25.0 mg, tR = 40.5 min). Fr.2 (7.6 g) was chromatographed on silica gel CC (4 cm 98 cm, 350 g) eluting with a gradient of CHCl3–MeOH (1:0, 50:1, 30:1, 20:1, 15:1, 10:1, 8:1, 5:1, 2:1, 0:1, each 1 L) to yield 10 fractions (Fr.2-1–Fr.2-10). Fr.2-2 (1.7 g) was chromatographed on silica gel CC (3 cm 65 cm, 64 g) with a PE–acetone gradient (9:1, 8:1, 7:1, each 800 mL) to yield 10 fractions (Fr.2-2-1–Fr.2-2-10), and then Fr.2-2-2 (83.7 mg) by silica gel CC (2 cm 45 cm, 25 g) eluting with PE-acetone (16:1, 1.5 L) obtained 5 (11.5 mg). Fr.2-3 (1.4 g) was chromatographed on silica gel CC (3 cm 65 cm, 64 g) with CHCl3–MeOH gradient (40:1, 30:1, 20:1, 10:1, each 800 mL) to yield eight fractions (Fr.2-3-1–Fr.2-3-8), and then Fr.2-2-5 (536.4 mg) was further purified by Sephadex LH-20 (column: 100 cm 3 cm) in EtOH and was further separated on RP-18 with 60% MeOH–H2O to afford compound 6 (8.3 mg). 3.3.1. Tomenphantopin C Colourless syrup (CH3OH); [a]D22 + 7.7 (c 0.02, MeOH); UV (MeOH) lmax (log e) 213 (4.21) nm; IR (KBr) 2360, 1773, 1714, 1669, 1652, 1635 cm 1; 1H and 13C NMR see Table 1; HRESIMS m/z 415.1733 [M+Na]+ (Calcd. 415.1732 for C21H28O7Na). 3.3.2. Tomenphantopin D Colourless crystal (CH3OH), mp 168–171 8C; [a]D22 40.7 (c 0.02, MeOH); UV (MeOH) lmax (log e): 210 (4.17) nm; IR (KBr) 3446, 2974, 2359, 1754, 1716, 1636, 1456 cm 1; 1H and 13C NMR see Table 1; HREIMS m/z 366.1687 [M]+ (Calcd. for C19H26O7, 366.1679). 3.4. Bioassay of cytotoxic activity Human myeloid leukemia cell line (K562) and human hepatoma cell line (SMMC-7721) were obtained from the Cell Bank of Type Culture Collection of the Chinese Academy of Sciences, Shanghai Institute of Cell Biology. Cells were cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum, 100 IU/mL penicillin, and 100 mg/mL streptomycin at 37 8C and 5% CO2 with 90% humidity. The logarithmic phase cells were used for experiments. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenytetrazolium bromide (MTT) assay was used to determine the growth inhibition of K562 and SMMC-7721 cells. The logarithmic phase cells (90 mL) were seeded onto 96-well plates at the concentration of 5 104 cell/mL. After 24 h, different concentrations of the sample (0.1, 0.4, 1.6, 6.3, 25, 100 mg/mL), dissolved in DMSO, were added at 10 mL per well, respectively, and each concentration had 3 replicate wells. Control cells were treated with DMSO alone and positive controls with mitomycin C. The cells were grown for 72 h and then observed with a XS-212 biological microscope. MTT was dissolved at 5 mg/mL in PBS and used essentially as previously described (Mosmann, 1983). Briefly, 15 mL of MTT solution were added to each well and the cells were further incubated at 37 8C for 4 h. Then the supernatant was removed, and 100 mL DMSO was added into each well. In the end, the absorbance (A value) at a wavelength of 490 nm was measured with a MK3 Microtiter plate reader. The inhibition rates (IR%) were calculated using OD mean values from IR% = (ODcontrol ODsample)/ODcontrol. The IC50 value, which is defined as the concentration of sample needed to reduce 50% of absorbance relative to the vehicle-treated control, was determined using the Bliss method. 3.5. Antibacterial activity All compounds were tested for in vitro antibacterial activities against S. aureus (CMCC(B) 26003, obtained from National
W.-L. Mei et al. / Phytochemistry Letters 5 (2012) 800–803
Institutes for Food and Drug Control) strain by the filter paper disc agar diffusion method (Xu et al., 2003). The media nutrient agar was used to culture the bacteria. The sterile agar media was poured into Petri-plates to a uniform depth of 5 mm and was allowed to solidify. The bacterial suspensions were streaked over the surface of media using a sterile cotton swab. 50 mL (20 mg/mL) of the compounds were impregnated on sterile filter paper discs of 6 mm size, respectively. These discs were then aseptically applied to the surface of the agar plates at well-spaced intervals. Control discs impregnated with 50 mL of CH3OH and 50 mL of kanamycin sulfate (0.64 mg/mL) were also used alongside the test discs in the experiment. The plates were incubated at 36 8C for 24 h. The MICs was performed by dilution of four compounds. Experiments were done in triplicate, and the results were presented as mean values of the three measurements. Acknowledgment This research was financially supported by National Nonprofit Institute Research Grant of CATAS-TCGRI (No. PZS083) and CATASITBB (No. ZD-0741). References Banerjee, S., Schmeda-Hirschmann, G., Castro, V., Schuster, A., Jakupovic, J., Bohlmann, F., 1986. Further sesquiterpene lactones from Elephantopus mollis and Centratherum punctatum. Planta Med. 52, 29–32. But, P.P.H., Hon, P.M., Cao, H., Che, C.T., 1996. A new sesquiterpene lactone from Elephantopus molis. Planta Med. 62, 474–476. But, P.P.H., Hon, P.M., Cao, H., Chan, T.W.D., Wu, B.M., Mark, T.C.W., Che, C.T., 1997. Sesquiterpene lactones from Elephantopus elatus. Phytochemistry 44, 113–116. Chen, Y.L., 1985. Flora Republicae Popularis Sinicae, vol. 74. Science Press, Beijing, p. 43. Fuchino, H., Koide, T., Takahashi, M., Sekita, S., Satake, M., 2001. New sesquiterpene lactones from Elephantopus mollis and their leishmanicidal activities. Planta Med. 67, 647–653.
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