Three New Ursane-Type Triterpenoids from the Stems of Saprosma merrillii

Three new ursane-type triterpenoids, 3α,6α,30-trihydroxy-ursan-28-oic acid (1), 3α,30-dihydroxy-6-oxo-ursan-28-oic acid (2) and 3α,6α,7α,30-tetrahydroxy-ursan-28-oic acid (3), together with one known triterpenoid, betulinic acid (4), one known anthraquinone, 1,7-dihydroxy-2-methylanthraquinone (5), four known phenols, 1,3,5-trimethoxybenzene (6), p-hydroxybenzoic acid (7), syringic acid (8), isovanillin (9), two steroids, sitosterol (10) and daucosterol (11), were isolated from the ethanol extract of the stems of S. merrillii. Their structures were elucidated on the basis of physical and spectral techniques, besides comparison with literature data. Compounds 1–3 showed inhibitory activities against the A549, HEPG2, and B16F10 cell lines.


Introduction
The genus Saprosma belongs to the family Rubiaceous which has about 50 species found around the world, and has been used as a traditional medicine for the treatment of fever and lumbocrural pain [1][2][3][4][5][6].
S. merrillii is an endemic plant in Hainan Island whose chemical constituents have not been investigated previously. As part of our continuing study on bioactive components from tropical medicinal plants from Hainan, the stems of S. merrillii were investigated. The EtOAc soluble fraction of the EtOH extract of S. merrillii, which showed cytotoxic activity against the A549 cell line, with an IC 50 value of 65.66 μg/mL, was further purified by column chromatography to afford three new ursane-type triterpenoids 1-3 ( Figure 1) and eight known compounds 4-11. Here, we wish to report on the isolation and structural elucidation of these compounds. Compounds 1-3 were evaluated for their cytotoxicity against the A549, MDA-MB-231, HEPG2 and B16F10 tumor cell lines.

Results and Discussion
The air-dried stems of S. merrillii were extracted three times with 80% EtOH at room temperature. The EtOH extract of the plant was suspended in water and then partitioned successively with petroleum ether and EtOAc. Column chromatography (CC) of the EtOAc-soluble fraction yielded three new compounds 1-3 and eight known compounds 4-11.

General
Optical rotations were measured with PolAAr 3005 polarimeter (Optical Activity Limited, Cambridgeshire, UK). IR spectra were recorded on a Thermo Nicolet 6700 (using KBr disks) spectrophotometer (Thermo Scientific, Madison, WI, USA). NMR spectra were acquired on a Bruker AV (400 MHz for 1H-NMR, ppm relative to TMS) spectrometer (Bruker, Hesse-Darmstadt, Germany). ESIMS spectra were recorded on an Agilent 1200 series HPLC (Agilent Technologies, Waldbroon, Germany) interfaced to an Bruker Esquire 6000 Ion Trap mass spectrometer equipped with an electrospray ionization source, and HRESIMS spectra were made on the Bruker Daltonics Apex-Ultra 7.0 T (Bruker Corporation, Billerica, MA, USA), respectively. Column chromatography of silica gel (200-300 mesh), all solvents for column chromatography were of analytical grade (Xilong Chemical Reagents Company, Ltd., Guangdong, China). Spots of compounds on TLC were visualized by spraying using 10% H 2 SO 4 in EtOH (v/v) followed by heating.

Cytotoxicity Assays
Test compounds 1-3 were dissolved in DMSO (final concentration, 0.1%). The cytotoxicity of compounds 1-3 against A549, MDA-MB-231, HEPG2 and B16F10 was determined by standard MTS assay [16]. Cells dispersed evenly in medium and were seeded in a 96-well plate with a density of 1 × 10 4 cells/well. Next day, cells were treated with various concentrations of samples (0-100 μM) for 48 h or treated with indicated concentrations for 48 h with six replicates of each treatment. After incubation, each well was added 20 μL of MTS [3-(4,5-dimethylthiazol-2-yl)-5-(3-car-boxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium] reagent and incubated for 3 h. Cell viability was determined by measuring the optical density at 490 nm using a Biotek microplate reader (Biotek, Winooski, VT, USA). Untreated cells in medium were used as control. Corresponding groups without cells were used as blanks. All experiments were carried out with four replicates.