Abstract
Three new chalcones were isolated from Desmodium oxyphyllum. Their structures were elucidated by spectroscopic methods including extensive 1D- and 2D-nuclear magnetic ressonance techniques. The compounds were evaluated for their cytotoxicity against five human tumor cell lines (NB4, A549, SHSY5Y, PC3, and MCF7). The results showed that the compounds exhibited weak cytotoxicity against some selected cell lines with IC50 values ranging from 3.6 to 8.9 μM.
chalcones; cytotoxicity; Desmodium oxyphyllum
Introduction
Desmodium oxyphyllum (Leguminosae) is a sub-shrub that grows to a height between 30 cm and 1.5 m in southern China. It has been used in folk medicine to treat rheumatic pains, leucorrhea disorder in woman, snake bites, infantile malnutrition and measles, and to reduce traumatic swelling and pain.11 Ma, X. Q.; Zheng, C. J.; Hu, C. L.; Rahman, K.; Qin, L. P.; J. Ethnopharmacol. 2011, 138, 314. Previous phytochemical studies of D. oxyphyllum revealed the presence of flavonols, isoflavones, and coumaronochromones.11 Ma, X. Q.; Zheng, C. J.; Hu, C. L.; Rahman, K.; Qin, L. P.; J. Ethnopharmacol. 2011, 138, 314.,22 Li, Y. P.; Yang, Y. C.; Li, Y. K.; Jiang, Z. Y.; Huang, X. Z.; Wang, W. G.; Gao, X. M.; Hu, Q. F.; Phytochem. Lett. 2014, 9, 41. Motivated by a search for new bioactive metabolites from local plants, our group investigated the chemical constituents of the whole plant of D. oxyphyllum growing in Puer Prefecture, which led to the isolation and characterization of three new chalcones (1-3), together with known isobavachromene (4),33 Luyengi, L.; Lee, I. S.; Mar, W.; Phytochemistry 1994, 36, 1523. artonin A (5),44 Yao, S.; Ming, Z. D.; Chin. J. Nat. Med. 2005, 3, 219. licoflavonol (6),55 Sasaki, H.; Kashiwada, Y.; Shibatav, H.; Takaishi, Y.; Planta Med. 2012, 78, 1851. leachianone G (7),66 Jung, H. A.; Jeong, D. M.; Chung, H. Y.; Lim, H. A.; Kim, J. Y.; Yoon, N. Y.; Choi, J. S.; Biol. Pharm. Bull. 2008, 31, 908. kenusanone I (8),77 Iinuma, M.; Ohyama, M.; Tanaka, T.; Mizuno, M.; Hong, S. K.; Phytochemistry 1993, 33, 1241. sophoraflavanone B (9),88 Nakahara, K.; Roy, M. K.; Ono, H.; Maeda, I.; Ohnishi- Kameyama, M.; Yoshida, M.; Trakoontivakorn, G.; J. Agric. Food Chem. 2003, 51, 6456. and naringenin (10).99 Shen, C. C.; Chang, Y. S.; Ho, L. K.; Phytochemistry 1993, 34, 843. This paper deals with the isolation and structural elucidation of these compounds, and the evaluation of the cytotoxicity of1-3 against human tumor cell lines human acute promyelocytic leukemia cells (NB4), human lung adenocarcinoma (A549), human neuroblastoma (SHSY5Y), human prostate (PC3), and human breast adenocarcinoma (MCF7).
Experimental
General experimental procedures
UV spectra were obtained using a Shimadzu UV-2401A spectrophotometer. A Bruker Tenor 27 spectrophotometer was used for scanning infrared (IR) spectroscopy with KBr pellets. 1D- and 2D- nuclear magnetic resonance (NMR) spectra were recorded on a Bruker DRX-500 NMR spectrometer with tetramethylsilane (TMS) as internal standard. Chemical shifts (δ) are expressed in ppm with reference to the solvent signals [(CD3)2CO]. High-resolution electrospray ionisation mass spectrometry (HRESIMS) was performed on a VG Autospec-3000 spectrometer. Semipreparative high performance liquid chromatography (HPLC) was performed on a Shimadzu LC-8A preparative liquid chromatograph with Zorbax PrepHT GF (21.2 mm × 25 cm) or Venusil MP C18 (20 mm × 25 cm) columns. Column chromatography (CC) was performed using silica gel (200-300 mesh, Qing-dao Marine Chemical, Inc., Qingdao,People's Republic of China), Lichroprep RP-18 gel (40-63 μm, Merck, Darmstadt, Germany), and MCI gel (75-150 μm, Mitsubishi Chemical Corporation, Tokyo, Japan). Fractions were monitored by thin layer chromatography (TLC), and spots were visualized by heating silica gel plates sprayed with 5% H2SO4 in EtOH.
Plant material
The whole plant of D. oxyphyllum was collected in Puer Prefecture, Yunnan Province, People's Republic of China, in September 2010. The identification of the plant material was verified by Dr Yuan N. from Kunming Institute of Botany, Chinese Academy of Sciences. A voucher specimen (YNNU 2010-10-10) has been deposited in our laboratory.
Extraction and isolation
The air-dried and powdered D. oxyphyllum (1.5 kg) plant material was extracted four times with 80% aqueous ethanol (4 × 5 L) at room temperature and filtered. The filtrate was evaporated under reduced pressure, and the crude extract (140 g) was decolorized by MCI. The portion of the extract soluble in 90% methanol (50 g) was chromatographed on a silica gel column eluting with a CHCl3-MeOH gradient system (20:1, 9:1, 8:2, 7:3, 6:4, 5:5), to give six fractions (A-F). Separation of fraction C (8:2, 10.4 g) by silica gel CC, eluted with petroleum ether-acetone (9:1-1:2), yielded fractions C1-C7. Fraction C2 (8:2, 1.01 g) was subjected to silica gel CC using petroleum ether-acetone and semi-preparative HPLC (40% MeOH-H2O) to give 1 (6.6 mg), 2 (7.3 mg), 5 (1.8 mg), 7 (2.2 mg), and 10 (5.9 mg). Fraction C3 (7:3, 0.64 g) was subjected to silica gel CC using petroleum ether-acetone and semi-preparative HPLC (35% MeOH-H2O) to give 3 (6.1 mg), 4 (2.5 mg), 6 (1.2 mg), and 8 (8.2 mg). Fraction C4 (6:4, 0.28 g) was subjected to semi-preparative HPLC (32% MeOH-H2O) to give 9 (1.7 mg).
Oxyphyllumchalcone A (1)
Pale yellow gum; UV (MeOH) νmax/nm 210, 252, 362; IR (KBr) nmax/cm-1 3415, 3140, 3076, 2951, 2837, 1685, 1605, 1532, 1467, 1335, 1188, 1064, 892, 738; 1H and 13C NMR data [500 and 125 MHz, (CD3)2CO], see Table 1; positive ESIMS m/z 377 [M + Na]+; positive HRESIMS m/z, calcd. for C21H22O5Na [M + Na]+: 377.1365, found: 377.1373.
Oxyphyllumchalcone B (2)
Pale yellow gum; UV (MeOH) νmax/nm 210, 250, 360; IR (KBr) nmax/cm-13418, 3142, 3074, 2953, 2837, 1684, 1600, 1527, 1465, 1332, 1180, 1063, 899, 736; 1H and 13C NMR data [500 and 125 MHz, (CD3)2CO], see Table 1; positive ESIMS m/z 377 [M+Na]+; positive HRESIMS m/z, calcd. for C21H22O5Na [M+Na]+: 377.1365, found: 377.1358.
Oxyphyllumchalcone C (3)
Pale yellow gum; UV (MeOH) νmax/nm 210, 252, 364; IR (KBr) nmax/cm-1 3408, 3140, 3085, 2962, 2835, 1680, 1602, 1531, 1468, 1335, 1182, 1065, 898, 734; 1H and 13C NMR data (500 and 125 MHz, (CD3)2CO), see Table 1; positive ESIMS m/z 393 [M+Na]+; positive HRESIMS m/z, calcd. for C21H22O6Na [M+Na]+: 393.1314, found: 393.1322.
Cytotoxicity assay
Colorimetric assays were performed to evaluate cytotoxicity. Human acute promyelocytic leukemia cells (NB4), human lung adenocarcinoma (A549), human neuroblastoma (SHSY5Y), human prostate (PC3), and human breast adenocarcinoma (MCF7) tumor cell lines were purchased from the American Type Culture Collection (ATCC). All cells were cultured in Roswell Park Memorial Institute-1640 (RPMI-1640) or Dulbecco's modification of Eagle's medium (DMEM) medium (Hyclone, Logan, UT) supplemented with 10% fetal bovine serum (Hyclone) at 37 ºC in a humidified atmosphere with 5% CO2. Cell viability was assessed by conducting colorimetric measurements of the amount of insoluble formazan formed in living cells based on the reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) (Sigma, St. Louis, MO). Briefly, 100 μL of suspended adherent cells were seeded into each well of a 96-well cell culture plate and allowed to adhere for 12 h before drug addition. In addition, suspended cells were seeded just before drug addition, with an initial density of 1 × 105 cells mL-1 in 100 μL of medium. Each tumor cell line was exposed to each test compound at various concentrations in triplicate for 48 h; Paclitaxel (Sigma, purity > 95%) was used as a positive control. After the incubation, MTT (100 μg) was added to each well, and the incubation was continued for 4 h at 37 ºC. The cells were lysed with 100 μL of 20% SDS-50% DMF after removal of 100 μL of the medium. The optical density of the lysate was measured at 595 nm in a 96-well microtiter plate reader (Bio-Rad 680). The IC50 value of each compound was calculated by Reed and Muench's method.1010 Reed, L. J.; Muench, H.; Am. J. Hyg. 1938, 27, 493.
Results and Discussion
Whole plants of D. oxyphyllum were extracted with 80% aqueous ethanol. The extract was subjected repeatedly to column chromatography on silica gel, RP-18, and semi-preparative RP-HPLC separation to afford compounds 1-3. The 1H and 13C NMR data of the compounds 1-3 are listed in Table 1.
Compound 1 gave a quasi-molecular ion by HRESIMS at m/z 377.1373 [M + Na]+ corresponding to the molecular formula C21H22O5. The 1H and 13C NMR signals of 1 (Table 1) were assigned by the DEPT, HSQC and HMBC spectra. The 1H, 13C, and DEPT NMR spectra showed signals for 21 carbons and 24 hydrogen atoms. The carbon signals [δC 126.9 s, 130.2 s, 144.0 s, 148.0 s, 113.8 d, 120.0 d, 114.2 s, 164.5 s, 102.9 d, 168.5 s, 107.6 d, 133.1 d, 143.2 d, 119.2 d], the characteristic protons signals for a 1,2,3,4-tetrasubstituted aromatic ring at δH 6.80 (d, 1H, J 8.5 Hz, Ph-H), 7.42 (d, 1H, J 8.5 Hz, Ph-H), and a 1,2,4-trisubstituted aromatic ring at δH 6.37 (d, 1H, J 2.2 Hz, Ph-H), 6.45 (dd, 1H, J 8.8 Hz, 2.2, Ph-H), and 8.08 (d, 1H, J 8.8 Hz, Ph-H), as well as two olefinic protons at δH 7.65 (d, 1H, J 15.1 Hz, CH) and 8.18 (d, 1H, J 15.1 Hz, CH), indicated the NMR pattern of a chalcone. The 1H NMR spectrum (Table 1) revealed a triplet at δH 5.14 (t, 1H, J 6.8 Hz, CH), a doublet at δH 3.60 (d, 2H, J 6.8 Hz, CH2), a singlet at δH 1.83 (s, 3H, CH3), and a singlet at δH 1.63 (s, 3H, CH3), which established the presence of a prenyl group. HMBC correlations (Figure 2) of H-1" (δH 3.60) with C-1, C-2, C-3, and those of H-2" (δH5.14) with C-2, together with the 1H-1H COSY correlations H-1"/H-2", confirmed the prenyl group at C-2 in 1. The connectivity of C-1' and the carbonyl carbon was established by the HMBC corrections from H-6' (δH8.08) to C-1' and the carbonyl carbon, and from H-3' (δH6.37) to C-1'. The HMBC correlations of three hydroxy protons at δH 12.51, 12.83 and 13.17, with C-4 (δC 148.0 s)/C-3 (δC 144.0 s)/C-5 (δC 113.0 d), C-4 (δC 148.0 s)/C-3 (δC 144.0 s)/C-1 (δC 126.1 s), and C-2' (δC 164.5 s), respectively, placed the phenolic OH groups at C-4, C-3, and C-2', respectively. A methoxy group was located at C-4' from HMBC and ROESY correlations of its signal at δH 3.80 with C-4' (δC 168.5 s), H-3' (δH 6.37, d, 1H, J 2.2 Hz, Ph-H) and H-5' (δH 6.45, dd,1H, J8.8 Hz, 2.2, Ph-H). Doublets atH-5, H-6, H-3', H-6' as well as a doublet of doublets at H-5' indicated no substituent at C-5, C-6, C-3', C-5' and C-6'. Thus, the structure of 1 was established as shown, and named as oxyphyllumchalcone A.
Compound 2, obtained as a pale yellow gum, showed a quasi-molecular ion at m/z 377.1358 [M + Na]+ in the HRESIMS, corresponding to the molecular formula C21H22O5. The 13C NMR and DEPT spectra of compound 2 were similar to those of 1, except that C-4 was downfield shifted (δδC +2.2 ppm), and C-4' was upfield shifted (δδC -2.4 ppm). Detailed comparison of the NMR spectra of 1 and 2 showed, as major difference, the position of the methoxy group, which was placed at C-4 in 2, as indicated by the HMBC correlation of the methoxy protons at δH 3.80 with C-4 (δC 150.2). Accordingly, the structure of oxyphyllumchalcone B (2) was determined as shown.
Compound 3, obtained as a pale yellow gum, had the molecular formulas C21H22O6 as determined by positive HRESIMS at m/z 393.1322 [M + Na]+. Comparison of 13C NMR data between 3 and 1 suggested that 3 was similar to 1 except for an additional hydroxy group at C-4" in 3. This was further confirmed by an oxygenated signal of C-4" (δC 68.9 t, δH 3.96 s, 2H, CH2), the lack of the one singlet signal of the methyl group, and the HMBC correlations from H2-4" to C-2", C-3", and C-5". The ROESY correlations of H-1" with H-5", and of H-2" with H-4" indicated an E-configuration for the C-2", C-3" double bond [Figure S17, in the Supplementary Information (SI) section].1111 Nguyen, N. T.; Mai, H. K. N.; Nguyen, H. X.; Ngan, K. N. B.; Mai, T. T. N.; J. Nat. Prod. 2012, 75, 1951.,1212 Chung, M. I.; Lai, M. H.; Yen, M. H.; Wu, R. R.; Lin, C. N.; Phytochemistry 1997, 44, 943. Accordingly, the structure of oxyphyllumchalcone C (3) was determined as shown.
Since some previously reported flavonoids from Desmodium plants exhibited cytotoxicity,1313 Gan, N.; Yang, X.; Li, T. H.; He, P.; Zhongguo Zhongyao Zazhi 2008, 33, 2077.,1414 Gan, N.; Li, T. H.; Yang, X.; He, P.; Zhongcaoyao 2009, 40, 852. we tested compounds 1-3 for cytotoxicity against five human tumor cell lines (NB4, A549, SHSY5Y, PC3, and MCF7) using the MTT method as reported previously.1515 Mosmann, T.; J. Immunol. Methods 1983, 65, 55. Paclitaxel was used as the positive control. The results are depicted in Table 2.
Conclusions
Three new chalcones were isolated from D. oxyphyllum. The structures of 1-3 were elucidated by spectroscopic methods including extensive 1D- and 2D-NMR techniques. These three compounds presented a weak cytotoxic activity against tumor cell lines NB4, A549, SHSY5Y, PC3, and MCF7.
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Supplementary InformationSupplementary information is available free of charge at http://jbcs.sbq.org.br as a PDF file.
Acknowledgments
This project was supported financially by the Excellent Scientific and Technological Team of Yunnan High School (2010CI08), the Yunnan Minzu University Green Chemistry and Functional Materials Research for Provincial Innovation Team (2011HC008), and Open Research Fund Program of Key Laboratory of Ethnic Medicine Resource Chemistry (Yunnan Minzu University) (2010XY08).
References
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1Ma, X. Q.; Zheng, C. J.; Hu, C. L.; Rahman, K.; Qin, L. P.; J. Ethnopharmacol. 2011, 138, 314.
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2Li, Y. P.; Yang, Y. C.; Li, Y. K.; Jiang, Z. Y.; Huang, X. Z.; Wang, W. G.; Gao, X. M.; Hu, Q. F.; Phytochem. Lett. 2014, 9, 41.
-
3Luyengi, L.; Lee, I. S.; Mar, W.; Phytochemistry 1994, 36, 1523.
-
4Yao, S.; Ming, Z. D.; Chin. J. Nat. Med. 2005, 3, 219.
-
5Sasaki, H.; Kashiwada, Y.; Shibatav, H.; Takaishi, Y.; Planta Med. 2012, 78, 1851.
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6Jung, H. A.; Jeong, D. M.; Chung, H. Y.; Lim, H. A.; Kim, J. Y.; Yoon, N. Y.; Choi, J. S.; Biol. Pharm. Bull. 2008, 31, 908.
-
7Iinuma, M.; Ohyama, M.; Tanaka, T.; Mizuno, M.; Hong, S. K.; Phytochemistry 1993, 33, 1241.
-
8Nakahara, K.; Roy, M. K.; Ono, H.; Maeda, I.; Ohnishi- Kameyama, M.; Yoshida, M.; Trakoontivakorn, G.; J. Agric. Food Chem. 2003, 51, 6456.
-
9Shen, C. C.; Chang, Y. S.; Ho, L. K.; Phytochemistry 1993, 34, 843.
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10Reed, L. J.; Muench, H.; Am. J. Hyg. 1938, 27, 493.
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11Nguyen, N. T.; Mai, H. K. N.; Nguyen, H. X.; Ngan, K. N. B.; Mai, T. T. N.; J. Nat. Prod. 2012, 75, 1951.
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12Chung, M. I.; Lai, M. H.; Yen, M. H.; Wu, R. R.; Lin, C. N.; Phytochemistry 1997, 44, 943.
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13Gan, N.; Yang, X.; Li, T. H.; He, P.; Zhongguo Zhongyao Zazhi 2008, 33, 2077.
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14Gan, N.; Li, T. H.; Yang, X.; He, P.; Zhongcaoyao 2009, 40, 852.
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15Mosmann, T.; J. Immunol. Methods 1983, 65, 55.
Data availability
Publication Dates
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Publication in this collection
Apr 2015
History
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Received
18 Sept 2014 -
Accepted
12 Feb 2015
