Cytotoxic Chalcones from Desmodium oxyphyllum

Three new chalcones were isolated from Desmodium oxyphyllum. Their structures were elucidated by spectroscopic methods including extensive 1Dand 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.


Introduction
Desmodium oxyphyllum (Leguminosae) is a subshrub 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. 1 Previous phytochemical studies of D. oxyphyllum revealed the presence of flavonols, isoflavones, and coumaronochromones. 1,2Motivated 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), 3 artonin A (5), 4 licoflavonol (6), 5 leachianone G (7), 6 kenusanone I (8), 7 sophoraflavanone B (9), 8 and naringenin (10). 9This paper deals with the isolation and structural elucidation of these compounds, and the evaluation of the cytotoxicity of 1-3 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 [(CD 3 )

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.

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 °C in a humidified atmosphere with 5% CO 2 .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 × 10 5 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 °C.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 IC 50 value of each compound was calculated by Reed and Muench's method. 10

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 semipreparative RP-HPLC separation to afford compounds 1-3.The 1 H and 13 C NMR data of the compounds 1-3 are listed in Table 1.
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 C 21 H 22 O 5 .The 13 C NMR and DEPT spectra of compound 2 were similar to those of 1, except that C-4 was downfield shifted (Δδ C +2.2 ppm), and C-4' was upfield shifted (Δδ C -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 δ H 3.80 with C-4 (δ C 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 C 21 H 22 O 6 as determined by positive HRESIMS at m/z 393.1322 [M + Na] + .Comparison of 13 C 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" (δ C 68.9 t, δ H 3.96 s, 2H, CH 2 ), the lack of the one singlet signal of the methyl group, and the HMBC correlations from H 2 -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]. 11,12Accordingly, the structure of oxyphyllumchalcone C (3) was determined as shown.
Since some previously reported flavonoids from Desmodium plants exhibited cytotoxicity, 13,14 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. 15Paclitaxel 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.