Four new taraxastane-type triterpenoic acids from Cirsium setosum

Abstract Four new taraxastane-type triterpenoids acids 3β,22α-dihydroxy-20-taraxasten-30-oic acid (1), 3β-hydroxy-22-oxo-20-taraxasten-30-oic acid (2), 3-oxo-22α-hydroxy-20- taraxasten-30-oic acid (3), and 3β,19β-dihydroxy-20-taraxasten-30-oic acid (4) were isolated and characterized from Cirsium setosum (Willd.) MB. Their structures were determined by the combination of 1D and 2D NMR experiments (1H-1HCOSY, HSQC, HMBC and ROESY) and mass spectrometry. Compound 2 exhibited potent selective cytotoxicity against human ovarian cancer cell line A2780 with an IC50 value of 3.9 μM.


Results and discussion
The PE-soluble portion of the ethanolic extract of C. setosum was subjected to column chromatography on macroporous adsorbent resin, normal phase and reversed phase silica gels and Sephadex LH-20, successively to afford three major components which were further purified by preparative reversed phase HPLC to yield compounds 1-4.
Compound 1 was obtained as white amorphous powder. . The 13 C NMR spectrum of 1 showed 30 carbon signals, and the DEPT experiment differentiated them to be seven CH 3 , eight CH 2 , eight CH, and seven C including two oxygen-bearing carbons at δ C 78.1 and δ C 72.5, two olefinic carbons at δ C 135.5 and δ C 141.4, and one carboxyl carbonyl carbon at δ C 170.8. All above spectroscopic data suggested that 1 was an oxygenated triterpenoid bearing a carboxyl group.
Comparison of the 13 C NMR data ( Table 1) of 1 with those of the known (3β, 22α)-3,22-dihydroxytaraxast-20-en-30-al [12,13] suggested that 1 possesses the same skeletal structure with the exception that the C-20 aldehyde was replaced by a carboxyl group (Figure 1). The structure of 1 was finally confirmed by careful analysis of its 2D NMR spectroscopic data including 1 H-1 H COSY and HMBC (Figure 2), for spatial assignments of all the protons and carbons.
The relative stereochemistry of 1 was elucidated by a careful analysis of the NOESY spectrum and compared with known structure of 3β,22α-dihydroxytaraxast-20-en-30-al [12] as shown in Figure       H 3 -28/H-22 were in β-orientation, and the OH-22 possessed α-configuration, respectively. Based on the above spectral evidence, the structure of 1 was unambiguously established as 3β,22α-dihydroxytaraxast-20-en-30-oic acid. Compound 2 was obtained as a white amorphous powder. The chemical structure of 2 was very similar to compound 1, except the C-22 oxygen-bearing carbons at δ C 72.5 were replaced by a carbonyl carbon at δ C 206.1, and the proton signal of H-22 at δ H 3.90 (1H, d, J = 6.5 Hz) disappeared in 1 H NMR spectrum ( Table 1). The HREIMS at m/z 470.3359 [M] + , corresponded a formula of C 30 H 46 O 4 , indicating eight degrees of unsaturation which was in agreement with the NMR spectrum. Therefore, the structure of 2 was unambiguously established as 3β-hydroxy-22-oxo-20-taraxasten-30-oic acid, and the full assignments of 1 H and 13 C NMR data (Table 1) were achieved.
Compound 3 was obtained as a white amorphous powder. The NMR spectral data of 3 were very similar to those of compound 1 with the exception of C-3 oxygen-bearing carbon at δ C 78.1 was replaced by a carbonyl carbon at δ C 216.5, and the proton signal of H-3 at δ H    (Table 1), the structure assignment of 3 was unambiguously established as 3-oxo-22α-hydroxy-20-taraxasten-30-oic acid which is in full agreements of 1 H and 13 C NMR data shown in Table 1.
Compound 4 was obtained as a white amorphous powder. The molecular formula of C 30 H 48 O 4 , as determined by HREIMS at m/z 472.3542 [M] + with seven degrees of unsaturation was exactly the same with compound 1, and the spectroscopic data were very close to 1, indicating that compound 4 is an isomer of 1. Careful analysis of spectroscopic data of these two compounds revealed that the hydroxyl group is located at C-19 in compound 4 and at C-22 in compound 1. The key HMBC correlations from Me-28 to C-16, C-17, C-18 and C-22, H-18 to C-13, C-14, C-17, C-19, C-22, C-28 and C-29, H-21 to C-17, C-19, C-20, C-22 and C-30, Me-29 to C-18, C-19 and C-20, located the Me-29 and a hydroxyl group at C-19 ( Figure 3). The planar structure of 4 was thus established as shown in Figure 1.
The relative stereochemistry of 4 was elucidated by the analysis of the NOESY spectrum as shown in Figure 3, and compared with 1, 2, and 3 based on the same NOE correlations ( Figure 3). Accordingly, the structure of 4 was determined to be 3β,19β-dihydroxy-20taraxasten-30-oic acid.
Compounds 1-4 were evaluated in vitro for their cytotoxicities against five human cancer cell lines: colon cancer (HCT8), hepatoma (Bel7402), stomach cancer (BGC823), lung adenocarcinoma (A549), and ovarian cancer (A2780) using the MTT method with topotecan as a positive control and human epithelial WISH cell line as a normal control. In this test, IC 50 values of greater than 20 μM were defined as inactive. The results showed that 2 were highly selective and potent cytotoxicity against human cancer cell line A2780 with an IC 50 value of 3.9 μM (Table 2).

Plant material
The

In vitro cytotoxicity bioassays
Human colon cancer (HCT-8), hepatoma (Bel7402), stomach cancer (BGC-823), lung adenocarcinoma (A549), and ovarian cancer (A2780) cell lines were obtained from ATCC. Cells were maintained in RRMI1640 supplemented with 10% fetal bovine serum (FBS), 100 units/mL penicillin, and 100 μg/mL streptomycin. Cultures were incubated at 37 °C in a humidified 5% CO 2 atmosphere. All the cells were seeded in 96-well microtiter plates at 1200 cells/well. After 24 h, the test compound was added to the cells. After 96 h of drug treatment, cell viability was determined by measuring the metabolic conversion of MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) into purple formazan crystals by active cells. MTT assay results were read using a MK 3 Wellscan (Labsystem Drogon) plate reader at 570 nm. All compounds were tested in five concentrations and were dissolved in DMSO with a final DMSO concentration of 0.1% in each well. Each concentration of the compounds was tested in three parallel wells. IC 50 values were calculated using Microsoft Excel software.

Disclosure statement
No potential conflict of interest was reported by the authors.