TRITERPENOID AND STEROL COMPOUNDS ISOLATED FROM ANODENDRON PANICULATUM (ROXB.) A. DC

This phytochemical study on the aerial parts of Anodendron paniculatum led to the isolation of three triterpenes, namely ursolic acid (1), esculentic acid (2), cycloartenol (3), and one sterol: desmosterol (4). Their chemical structures were elucidated on the basis of spectroscopic analyses. The cytotoxicity of the isolated compounds against the growth of human cancer cell lines was evaluated using the sulforhodamine B bioassay. Compounds 1 exhibited a moderate cytotoxicity against the LU–1 and MKN–7 cell lines with IC50 values of 44.37 ± 5.40 and 30.89 ± 3.60 μg/mL, respectively. Meanwhile, compound 4 possessed moderate cytotoxic effects toward the LU–1, KB, Hep–G2, MKN–7 and SW–480 cell lines with IC50 values ranging from 28.11 ± 1.95 to 41.41 ± 2.31 μg/mL.


Introduction
Anodendron paniculatum (Roxb.) A. DC. is the climbing species of the Apocynaceae family and broadly distributed in Sri Lanka, India, Bangladesh, Burma, and Southeast Asia [1]. The roots of this plant have been used in traditional folk medicine as a remedy for vomiting and cough in India [2]. In addition, its latex is used to cure snake poisoning and centipede bites [3]. Several cardenolides have been isolated from A. paniculatum in the previous chemical studies [4,5]. However, the knowledge about the chemical composition of this plant is rather limited. Previously, two flavonoid glycosides (kaempferol-3-O-rutinoside and rutin), and one phenylpropanoid glycoside (sargentol) were isolated from the aerial parts of A. paniculatum by Hanh et al. [6]. In our ongoing search for bioactive components from Vietnamese natural resources, three triterpenoids, namely ursolic acid (1), esculentic acid (2), and cycloartenol (3), and one sterol: desmosterol (4) were isolated. In this paper, we present the isolation, structure elucidation, and cytotoxic activities of these compounds.

Plant material
The aerial parts of A. paniculatum (Roxb.) A. DC. were collected in the Dakrong district, Quang Tri province in June, 2014, and were identified by Dr. Nguyen The Cuong, Institute of Ecology and Biological Resources, VAST, Vietnam. A voucher specimen (AV03) was deposited at the Faculty of Pharmacy, University of Medicine and Pharmacy, Hue University.
Melting points (mp) were determined on a Buchi Melting Point B-545 apparatus (Sigma-Aldrich, Missouri, USA). Infrared spectra were recorded on an IR Prestige-21 spectrometer with KBr discs (Shimadzu, Kyoto, Japan). Electrospray ionization mass spectrometry (ESIMS) was studied on an Agilent 6310 Ion Trap and Shimadzu LCMS-IT-TOF mass spectrometers. Nuclear magnetic resonance (NMR) spectra were measured on a Bruker AM500 FT-NMR spectrometer using tetramethylsilane as internal standard.

Extraction and isolation
The dried aerial parts of A. paniculatum (2.5 kg) were extracted with MeOH (3 times, 10.0 L each) at room temperature to yield 105 g of a dark solid extract. This extract was then suspended in water and successively partitioned with chloroform (CHCl3) and ethyl acetate (EtOAc) (3 times, 2.0 L each) to obtain the CHCl3 fraction (AC, 50.7 g), the EtOAc fraction (AE, 10.2 g), respectively, and the water layer (AW, 27.5 g) after removing the solvents in vacuum.

Sulforhodamine B assay for evaluating cytotoxic activity
The cytotoxic activity of isolated compounds was tested using the sulforhodamine B assay against the growth of five monolayer human cancer cell lines, namely LU-1 (lung adenocarcinoma), KB (epidermoid carcinoma), HepG2 (hepatoma cancer), MKN-7 (stomach cancer), and SW-480 (colon adenocarcinoma). Stock cultures were grown in T-75 flasks containing 50 mL of Dulbecco's Modified Eagle Medium (DMEM) with 2 mM L-glutamine, 1.5 g/L sodium bicarbonate and 10 % Fetal Bovine Serum (FBS). Media were changed at 48-hour intervals. The cells were dissociated with 0.05 % Trypsin-EDTA, sub-cultured every 3-5 days with the ratio of (1:3) and incubated at 37 °C under the humidified atmosphere with 5 % carbon dioxide. Tumor cells were cultivated in the humidified atmosphere with 5 % CO2 at 37 °C for 48 hours. Cell viability was examined with the sulforhodamine B (SRB) method for cell density determination, based on the measurement of cellular protein content [7].
Viable cells were seeded in the growth medium (180 µL) into 96-well microplates (4  10 4 cells per well) and allowed to attach overnight. The tested samples were added carefully into each well of the 96-well plates and the cultivation was continued under the same conditions for another 72 h. Thereafter, the medium was removed and the remaining cell monolayers are fixed with cold 20 % (w/v) trichloroacetic acid for 1 h at 4 °C and stained using 1X SRB staining solution at room temperature for 30 min, after which the unbound dye was removed by washing repeatedly with 1 % (v/v) acetic acid. The protein-bound dye was dissolved in a 10-mM-Tris base solution for optical density determination at 515 nm on an ELISA Plate Reader (Bio-Rad). Dimethylsulfoxide (DMSO) 10 % was used as a blank sample while ellipticine was used as a positive control. The cytotoxicity was measured at doses of 100 g/mL, 20 g/mL, 4 g/mL, and 0.8 g/mL and estimated as a half maximal inhibitory concentration (IC50), which was calculated using the program TableCurve Version 4.0. All experiments were performed in triplicates. The inhibition rate (IR) of cells was calculated according to the following formula: Where, At is the average optical density value at day 3 A0 is the average optical density value at time-zero Ac is the average optical density value of the blank DMSO control sample 3 Results and discussion The 13 C-NMR and DEPT spectra showed the signals of thirty carbons belonging to seven methyl, nine methylene, seven methine, and seven quaternary carbons. Among those, the signal at δC 180.8 (C-28) was assigned to the carbonyl carbon. The signals of two olefinic carbons at δC 138.3 (C-13) and 125.6 (C-12), and one oxygenated carbon at δC 79.0 (C-3) confirmed the presence of one tri-substituted double bond and one carbinol group, respectively. On the basis of the aforementioned observations, compound 1 was determined to be 3β-hydroxy-12-ursen-28-oic acid (known as ursolic acid) (Fig. 1) [8]. . Furthermore, the signals of four tertiary methyl (δH 0.87, 1.00, 1.07, 1.14) and two secondary methyl groups (δH 0.93, 0.96) were observed ( Table 1).
The cytotoxicity of the isolated compounds against the growth of various cancer cell lines was tested using the sulforhodamine B assay, and the results are described in Table 2. The data showed that compounds 1 exhibited a moderate cytotoxicity against the LU-1 and MKN-7 cell lines with IC50 values of 44.37 ± 5.40 and 30.89 ± 3.60 μg/mL, respectively. Compound 4 possessed moderate cytotoxic effects toward the LU-1, KB, Hep-G2, MKN-7 and SW-480 cell lines with IC50 values ranging from 28.11 ± 1.95 to 41.41 ± 2.31 μg/mL. Meanwhile, compounds 2 and 3 showed no inhibition against two tested cell lines (IC50 values > 100 μg/mL).

Conclusion
Four compounds, namely ursolic acid, esculentic acid, cycloartenol, and desmosterol were isolated from the aerial parts of Anodendron paniculatum for the first time. The structures of all the isolated compounds were identified using extensive spectroscopic methods, including 1D, 2D-NMR, IR, and ESIMS. Ursolic acid and desmosterol showed a moderate inhibition against various cancer cell lines. Based on the obtained results, these two compounds were considered to be useful for developing new anticancer therapeutic agents for the human.