Ursane Saponins from the Stems of Firmiana simplex and their Cytotoxic Activity

Three new ursane triterpene saponins, together with twelve known ursane triterpenes were isolated from the stems of Firmiana simplex. The structures of the saponines were elucidated on the basis of spectroscopic and chemical methods. The cytotoxic activity of all compounds was evaluated in vitro against lung adenocarcinoma (A549), ovarian cancer (SK-OV-3), skin melanoma (SK-MEL-2), and colon cancer (HCT-15) human cell lines, using a sulforhodamine (SRB) assay. 23-Hydroxyursolic acid showed cytotoxicity against the tested cell lines with IC50 values ranging from 11.96 to 14.11 μM.


Introduction
Firmiana simplex W. F. Wight (synonym Firmiana platanifolia Schott et Endl), family of Sterculiaceae, known as "phoenix tree", is distributed throughout Korea and China. 1,2Its seeds have been used as a folk medicine to treat symptoms of diarrhea and stomach disorders. 3Previous chemical investigations on this plant reported the isolation of quinones, 2 flavonoids, 3 and an antipsychotic neolignan. 4n the course of our continuing search for potential lead compounds from Korean traditional medicinal plants, we investigated the MeOH extract of F. simplex stems and isolated three new ursane triterpene saponins (1-3), together with twelve known ursane triterpenes (4-15) (Figure 1).All the compounds (1-15) were tested for their cytotoxic activity against the cultured human tumor cell lines lung adenocarcinoma (A549), ovarian cancer (SK-OV-3), skin melanoma (SK-MEL-2), and colon cancer (HCT-15).

General procedures
Optical rotations were obtained on a JASCO P-1020 polarimeter.Infrared (IR) spectra were recorded on a Bruker Vector 22 IR spectrophotometer.Nuclear magnetic resonance (NMR) spectra including 1 H-1 H correlation spectroscopy (COSY), distortionless enhancement by polarization transfer (DEPT), heteronuclear multiple quantum coherence (HMQC), heteronuclear multiple-bond correlation (HMBC) and nuclear Overhauser effect spectroscopy (NOESY), were recorded on a Varian UNITY INOVA 700 spectrometer operating at 700 MHz ( 1 H) and 175 MHz ( 13 C).High resolution fast atom bombardment mass spectrometry (HR-FABMS) was conducted using a JEOL JMS700 mass spectrometer.Preparative high performance liquid chromatography (HPLC) was performed using a Gilson 306 pump with a Shodex refractive index detector.Silica gel 60 (230-400 mesh, Merck) and reversed-phase (RP)-C 18 silica gel (230-400 mesh, Merck) were used for column chromatography.A Hewlett-Packard gas chromatography (GC) system 6890 Series was equipped with a 5973 mass selective detector (MSD).The system was controlled by the Enhanced Chem Station version B.01.00 program.The capillary column used for GC was an Agilent J&W HP-5MSUI (30.0 m × 0.25 mm i.d., 0.25 μm film thickness, coated with 5% diphenyl and 95% dimethylpolysiloxane).Thin-layer chromatography (TLC) was performed using Merck precoated silica gel F 254 plates and RP-18 F 254s plates.Spots were detected on TLC under ultraviolet (UV) light or by heating after spraying with 10% v/v H 2 SO 4 in EtOH.

Plant material
F. simplex stems (7.0 kg) were collected at Jecheon in Chungcheongbuk-do, Korea, in June 2012, and authenticated by one of the authors (K.R. Lee).A voucher specimen (SKKU-NPL-1209) was deposited at the herbarium of the School of Pharmacy, Sungkyunkwan University, Suwon, Korea.

Results and Discussion
The stems of F. simplex were extracted with 80% aqueous MeOH.Chemical investigation of the extract using successive column chromatography over silica gel and Sephadex LH-20, and preparative HPLC resulted in the isolation and identification of three new ursane triterpene saponins (1-3), together with twelve known ursane triterpenes (4-15).Their structures were elucidated as follows.
Compound 1 was obtained as a colorless gum, and its molecular formula C 42 H 67 O 15 was inferred from the negative HR-FABMS ion at m/z 811.4474 [M-H] -.The IR absorption bands at 3385 and 1732 cm -1 implied the presence of hydroxyl and carboxylic functionalities.The 1 H NMR spectrum (Table 1) of 1 showed the signals for an olefinic proton at d The NMR data of 1 were very similar to those of the ursane 4, 6 with the exception of an additional sugar moiety in 1.The linkage of the disaccharide moiety to the pentacyclic scaffold, and the attachment position between the two sugar units were assigned from the following HMBC correlations: d H 4.37 (d, J 8.0 Hz, H-1'') to d C 69.6 (C-6'), and d H 5.31 (d, J 8.0 Hz, H-1') to d C 178.7 (C-28) (Figure 2).
Compound 2 was obtained as a colorless gum, and its molecular formula C 42 H 67 O 16 was inferred from the negative