Two new 28-nor-oleanane-type triterpene saponins from roots of Camellia oleifera and their cytotoxic activity

Abstract Two new 28-nor-oleanane-type triterpene saponins, oleiferoside U (1), and oleiferoside V (2) were isolated from the 50% EtOH extract of the roots of Camellia oleifera C. Abel. Their structures were elucidated as camellenodiol 3β-O-β-d-galactopyranosyl-(1→2)-β-d-xylopyranosyl-(1→2)-[β-d-galactopyranosyl-(1→3)]-β-d-glucuronopyranoside and camellenodiol 3β-O-β-d-galactopyranosyl-(1→3)-β-d-xylopyranosyl-(1→2)-[β-d-galactopyranosyl-(1→3)]-β-d-glucuronopyranoside. Their chemical structures were established mainly on the basis of integrated spectroscopic techniques. In vitro, cytotoxic activities of the two new triterpene saponins were evaluated against three human tumor cell lines (A549, SMMC-7721, and MCF-7) using the MTT assay. Both of them showed a certain cytotoxic activities toward the tested cell lines and gave IC50 values in the range of 45.04–63.22 μM.


Introduction
As a part of the Theaceae family, Camellia oleifera C. Abel is mainly distributed in China, Japan, India, and other South-East Asian countries [1,2]. As an oil crop, it has been widely cultivated in many countries including China, Brazil, Philippines, India, and South Korea [3,4]. At the same time, some are used as folk medicines. Especially, the roots of C. oleifera C. are traditionally used for the treatment of common cold, bovillae, ardent fever, urinary tract infection, nephritis, edema and threatened abortion due to their antimicrobial, antioxidant, and antitumor activities [5]. The 50% EtOH extract of the roots of C. oleifera C. Abel was successively subjected to column chromatography over D101 marcroporous resin, silica gel and LX-2000 microreticular resin and semi-preparative HPLC to give two new 28-nor-oleanane-type triterpene saponins, named oleiferoside U (1) and oleiferoside V (2), and the structures of the newly characterized triterpene saponins were elucidated by means of extensive spectroscopic analyses ( Figure 1). Furthermore, cytotoxic activities of the two new triterpene saponins were evaluated against three human tumor cell lines ABSTRACT Two new 28-nor-oleanane-type triterpene saponins, oleiferoside U (1), and oleiferoside V (2) were isolated from the 50% EtOH extract of the roots of Camellia oleifera C. Abel. Their structures were elucidated as (A549, SMMC-7721, and MCF-7) using the MTT in vitro assay with IC 50 values being in the range of 45.04-63.22 μM. Herein, we report on the isolation, structural elucidation, and cytotoxity of the two new triterpene saponins.
In conclusion, two new 28-nor-oleanane-type triterpene saponins oleiferoside U (1) and oleiferoside V (2) were isolated from the 50% EtOH extract of the roots of C. oleifera C., and their structures were determined. Preliminary cytotoxicity screening revealed that both of them exhibited a certain cytotoxicity against A549, SMMC-7721, and MCF-7 cells shown in Table 2.

General experimental procedures
The specific rotation values were determined with a Perkin-Elmer model 241 polarimeter (Perkin-Elmer Inc., Waltham, MA, USA), and IR spectra were measured on a Perkin-Elmer

Plant material
The roots of Camellia oleifera C. were collected in Qichun, Hubei province of China in November 2011, and identified by Professor Xiao-ran Li at Soochow University. A voucher sample (No.11-11-06-01) was deposited in the herbarium of the College of Pharmacy, Soochow University.

Extraction and isolation
The air-dried plant materials (10 kg) were crushed with a grinder into fine debris, then extracted twice with 50% EtOH (100 L) at 80 °C under reflux. The solvent was subsequently removed under reduced pressure to give the residue (0.45 kg), which was then dissolved in distilled water and passed through a D101 macroporous resin column (i. All the semi-preparative HPLC separations were conducted at a flow rate of 2 ml/min and the detection wavelength was 203 nm. The purities of the isolated saponins were >95% by HPLC with ELSD detection.

General acid hydrolysis of compounds 1 and 2
A solution of each saponin (2 mg) was dissolved in 2 N HCl (dioxane-H 2 O, 1:1, 2.0 ml) and stirred at 80 °C for 4 h. The reaction mixture was extracted with CHCl 3 and the aqueous layer was evaporated to give a mixture of monosaccharides. Each residue was dissolved in dry pyridine (1 ml), followed by the addition of l-cysteine methyl ester hydrochloride. After heating at 60 °C for 2 h, the solvent was evaporated under N 2 , and 0.2 ml trimethylsilylimidazole was added. Then the mixture was heated at 60 °C for another 2 h and partitioned between n-hexane and water. The organic layer was investigated by GC under the following conditions: FID detector with a temperature of 280 °C; injector temperature 250 °C; initial temperature 160 °C, then raised to 280 °C at 5 °C/min, final temperature maintained for 10 min; carrier gas N 2 (2.0 kg/cm 3 ). Under these conditions, the following sugar units of each compound were identified by comparison with authentic sample. The retention times of persilylated d-glucuronic acid, d-galactose, and d-xylose were 19.770, 25.643, and 32.257 min.

Antitumoral cytotoxic bioassays
The MTT assay was performed to evaluate the viability of the cells after treated with different concentrations of the isolated saponins against A549, SMCC-7721, and MCF-7 human tumor cell lines (Cell Bank, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences). Cells were planted into 96-well flat-bottomed cultured plates at a concentration of 1 × 10 5 cells per well in fresh culture medium. After 24 h, the test solutions were applied to the cells in different final concentrations at 6.25, 12.5, 25, 50, and 100 μM. After 24 h, MTT solution was added to the wells and the plates were incubated at 37 °C for 4 h. The positive control group was treated with norcantharidin (purity higher than 99.0% as determined by HPLC; Nanjing Zelang Medical Technology Co. Ltd., Nanjing, China). After the medium was removed, each well was added 100 μl of DMSO. The amount of formazan was determined by photometry at 570 nm. The 50% inhibitory concentration (IC 50 ) was defined as the concentration that reduced the absorbance of the untreated wells by 50% of the vehicle in the MTT assay [7]. The IC 50 values (50% inhibitory concentration) of compounds 1 and 2 are shown in Table 2.