Five New Taraxerene-Type Triterpenes from the Branch Barks of Davidia involucrata

Five new taraxerene-type triterpenes, 2-nor-D-friedoolean-14-en-28-ol (1), 2-nor-d-friedoolean-14-en-3α,28-diol (2), 6α-hydroxy-2-nor-D-friedoolean-14-en-3,21-dione (3), 6α,11α,29-trihydroxy-D-friedoolean-14-en-3,16,21-trione (4), and 6α,23,29-trihydroxy-D-friedoolean-14-en-3,16,21-trione (5), were isolated from the MeOH extract of the branch barks of Davidia involucrata, together with five known compounds. Their structures were elucidated by means of various spectroscopic analyses. Five of the identified compounds showed moderate cytotoxicities against the cell proliferation of SGC-7901, MCF-7, and BEL-7404.


Introduction
Davidia involucrata Baill., an ornamental tree known as the Chinese dove tree or handkerchief tree, is the only species in genus Davidia. D. involucrata is a relic deciduous tree species of the Tertiary OPEN ACCESS period with important ecological, scientific and horticultural values [1][2][3]. An initial and the only report of study on chemical components of D. involucrata besides our program appeared in 1989, which revealed the presence of sterols, tannins and triterpenes [4]. The branch barks of D. involucrata have been intensively studied in our previous work and were found containing diverse constituents including flavonoids, alkaloids, lignans, and phenols, among which were three alkaloids including vicosamide, strictosidinic acid and puimiloside, which were proposed to be the intermediate precursors between strictosamide and camptothecin [5][6][7][8][9][10][11][12]. Moreover, we have recently reported the identification of two novel 2-nor-ursane triterpenes having a unique five-membered A-ring from the water insoluble fraction of the branch barks of D. involucrata [13].
Compound 5, obtained as a white amorphous powder, was assigned to have a molecular formula of C30H44O6 by HR-TOF-MS at m/z = 523.3030 [M + Na] + (calcd for C30H44O6Na, 523.3036), which was an isomer of 4. The NMR data of 5 were comparable to those of 4. Observed in the 1 H-NMR spectra was the absence of proton signal of an oxymethine at H-11 (δH 4.28, t, J = 7.0 Hz, 1H), with the appearance of an additional oxygenated methylene proton resonances at δH 3.58 (d, J = 10.2 Hz, 1H) and 3.80 (d, J =10.2 Hz, 1H) in the 1 H-NMR spectra of 5. Correspondingly, an oxymethine carbon at δC 65.9 (C-11) was absent with the presence of an oxygenated secondary carbon at δC 72.5 in the 13 C-NMR spectra of 5. The HMBC correlations from H-23a (δH 3.58, d, J = 10.2 Hz, 1H) and H-23b (δH 3.80, d, J = 10.2 Hz, 1H) to C-3 (δC 220.9), C-4 (δC 54.3) and C-5 (δC 54.2) indicated that a hydroxyl group was attached to C-23, which was also confirmed by the key ROESY correlations (Figure 3) of H-23/H-5, as well as the downfield shifted carbon resonance of C-4, as compared with those of compound 4. Analysis of the ROESY spectrum suggested the relative configuration of the remainder of the molecule of 5 was identical with that of 4. Compound 5 (davinvolunone C) was therefore determined as 6α,23,29-trihydroxy-D-friedoolean-14-en-3,16,21-trione.
The novel compounds, namely davinvolunols A-B (1-2) and davinvolunones A-C (3)(4)(5), are all taraxerene-type triterpenoids with a D-friedoolean-14-en skeleton. To the best of our knowledge, compounds 1-3, as well as previously reported two ursane triterpenes, davinvolunic acid A and B from the same plant [13], are the first reported 2-nor pentacyclic triterpenoids with 29 carbons and a five-membered A-ring. The results provided important, evolutionary and chemotaxonomic knowledge of monotypic genus Davidia.

General
Optical rotations were measured with a Jasco DIP-180 digital polarimeter. IR spectra were recorded with a Perkin-Elmer 1750 FT-IR spectrometer in KBr discs. High-resolution mass spectra were recorded on an IonSpec 4.7 Tesla FTMS instrument. The NMR spectra were obtained by using a Bruker AV-400 or a DRX-500 spectrometer. Semipreparative HPLC was performed with an Elite P230 pump equipped with a Schambeck SFD GmbH RI2000 detector and a YMC-Pack SIL column (250 × 10 mm, 5 µm). Sephadex LH-20 (25-100 µm, Pharmacia Fine Chemicals, Uppsala, Sweden), Silica gel (200-300 mesh) and Silica gel H (Qingdao Oceanic Chemical Co., Qingdao, China) were used for column chromatography. Thin-layer chromatography was performed on TLC plates (H, Qingdao Oceanic Chemical Co.), with compounds visualized by spraying with 5% (v/v) H2SO4 in alcohol solution, followed by heating.

Plant Material
The

Extraction and Isolation
The air-dried branch barks of the plant (10 kg) were extracted with MeOH (2 × 10 L) at room temperature to give 600 g of crude extract, which was solubilized in water (1 L) and then filtered. The water-insoluble fraction (175 g) was separated on a silica gel column (200-300 mesh, 80 × 5 cm, i.d.) that was eluted with a gradient of Petroleum ether/Acetone (from 10:1 to 10:5, v/v) to afford seven fractions 1-7.

Cytotoxicity Assay
The in vitro cytotoxic activity was determined by the MTT colorimetric method as described previously in our paper [13]. Three tumor cell lines, SGC-7901 cells (human gastric adenocarcinoma), MCF-7 cells (human breast cancer) and BEL-7404 (human hepatocellular carcinoma), provided by Department of Hepatobiliary Surgery, Affiliated Union Hospital, Fujian Medical University, were cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum and 100 IU·mL −1 of penicillin-streptomycin at 37 °C in humidified atmosphere with 5% CO2. For the cytotoxicity tests, cells in exponential growth stage were harvested from culture by trypsin digestion and centrifuging at 180× g for 3 min, and then resuspended in fresh medium at a cell density of 5 × 104 per mL. The cell suspension was dispensed into a 96-well microplate at 100 μL and incubated for 24 h, and then treated with compounds at various concentrations. After 48 h of treatment, 50 μL solution of 1 mg·mL −1 MTT was added to each well, and the cells were further incubated for 4 h. Finally, supernatants were removed and the formazan crystals were dissolved by adding 100 μL DMSO and the optical density was recorded at 570 nm. All drug doses were tested with doxorubicin as positive control in triplicate.