Chemical Constituents of Excoecaria acerifolia and Their Bioactivities

A new kaurane diterpenoid, 3α,18-dihydroxy-3β,20-epoxykaur-15-ene (1), was isolated from the aerial parts of Excoecaria acerifolia (Euphorbiaceae) together with 16 known compounds. Their structures were identified by extensive spectral analysis, especially 2D NMR techniques. Antiangiogenic effects of compounds 1-6 and 9-17 were evaluated using a zebrafish model, with compound 9 being active in this bioassay. At the same time, compounds 4, 6, 10, 11 showed activity in inhibiting the growth of A549 lung cancer cells, and the compound 10 also showed apoptosis-inducing effects on A549 lung cancer cells.


Results and Discussion
Compound 1 was isolated as white crystals from methanol. Its HRESIMS indicated the molecular formula C 20 H 30 O 3 , as evidenced by the pseudo-molecular ion peak at m/z 341.2099 [M+Na] + (calcd. 341.2092), corresponding to six degrees of unsaturation. IR absorption bands at 3,381, 2,919, 1,646 and 1,058 cm −1 implied the presence of hydroxyl, methylene, olefinic and ether groups, respectively. The 1 H-NMR spectra exhibited two methyl groups as singlets (δ H 1.68, 1.15). The 13 C-NMR and DEPT spectra of 1 in CDCl 3 showed 20 carbons with 28 directly attached protons for the diterpene nucleus ( Table 1). The 1 H-and 13 C-NMR spectra also showed the presence of a double bond, four tertiary carbons, and three oxygenated carbons in this molecule. Further analysis of 2D NMR spectra ( 1 H-1 H COSY, HMQC, ROESY and HMBC) lead to the construction of a structural formula for 1 based on an anthracene skeleton with an epoxy ring formed between C-3 and C-20, cis-olefins at C-8 and C-13, and a hydroxyl methyl groups at C-18 to complete a kaurane skeleton, which was also supported by the characteristic peak of two angular methyl groups at δ H 1.68 (3H, s) and 1.15 (3H, s) [20][21]. The comparison of the 1D NMR data of 1 with those of the known compound excoecarin D [22], which was determined by X-ray diffraction, showed the 1 H-and 13 C-NMR data of these two compounds were structurally similar. The differences can be rationalized to the position change of methyl-17 from C-13 in excoecarin D to C-16 in 1, which lead to the observation of only one olefinic proton (δ H 5.04) in 1 and one methine at C-13 in 1 other than a quaternary carbon in excoecarin D. This deduction was established by the HMBC correlations of the methyl group (δ H 1.68, C-17) with δ C 42.1 (C-13), 133.3 (C-15) and 142.4 (C-16).
The relative configurations of 1 were determined on the basis of ROESY experiments. The olefinic proton at C-15 correlated with the methylene protons at C-20 and the protons at CH 3 -17. ROESY correlations of H-5 with the protons of the oxygenated methylene (C-18) and H 2 -20 with H-19 indicated C-18 were -orientated. ROESY correlations of H-9 with H 2 -14 showed both H-9 and C-14 are on the same face of the molecule with -orientation. Therefore, the structure of 1 was established as 3α, 18-dihydroxy-3β,20-epoxykaur-15-ene.

Biological Acticity
The antiangiogenic activities of compounds 1-6 and 9-17 were evaluated using a zebrafish model, in terms of the inhibition on the growth of intersegmental vessels, with PTK787 as positive control (IC 50 0.15 g/mL) [23]. The results showed that intersegmental vessels of embryos treated with compound 9 was significantly less than that of the control (0.1% DMSO in sterile salt water). The inhibition ratio of compound 9 was 64.9% at a concentration of 50 g/mL ( Table 2). The antiproliferative activities of compounds 1-6 and 9-17 were evaluated using A549 lung cancer cells by MTT assay [24]. The results indicated that compounds 4, 6, 10 and 11 showed a certain extent antiproliferative activities (Table 3). From the photos of acridine orange staining, compound 10 showed obvious effect of inducing apoptosis of A549 lung cancer cells (Figure 3). Other tested compounds didn't show any obvious antiangiogenic and antiproliferative bioactivities.

General
1D and 2D NMR experiments were performed on a Bruker AM-400 or DRX-500 spectrometer. Chemical shifts (δ) are expressed in ppm with reference to the solvent signals. Mass spectra were recorded on a VG Autospec-300 spectrometer under 70 eV. Optical rotation was measured with a Horiba SEPA-300 polarimeter. A Bio-Rad FTS-135 spectrophotometer was used for scanning IR spectroscopy of compounds with KBr pellets. Column chromatography was performed on silica gel (200-300 mesh, Qingdao Marine Chemical Inc., Qingdao, People's Republic of China), silica gel H (10-40 m, Qingdao Marine Chemical Inc.) and MCI gel CHP20P (75-150 m, Mitsubishi Chemical Corporation, Tokyo, Japan). Fractions were monitored by TLC and spots were visualized by heating plates spraying with 15% H 2 SO 4 in EtOH.

Plant Material
The aerial parts of E. acerifolia were collected from Deqin, Yunnan Province, People's Republic of China. The plant material was identified by Dr. Yuanwen Duan. A voucher specimen (No.Yangyp 080724) was deposited at Kunming Institute of Botany, Chinese Academy of Sciences.

Antiangiogenesis and antiproliferative assays
3.4.1. Antiangiogenesis [23] Stock solutions (10 mg/mL) of all samples were prepared by dissolving the test compounds in 100% DMSO. These solutions were diluted in sterile salt water (5 mM NaCl, 0.17 mM KCl, 0.4 mM CaCl 2 , 0.16 mM MgSO 4 ) to obtain solutions with the test compounds dissolved in 0.1% DMSO. These solutions were aliquoted into 96-well plates, and embryos at 24 hpf (hours post fertilization) were also transferred randomly into the above wells. After 24h of treatment, the intersegmental vessels of embryos were visualized with green fluorescent protein labeling and endogenous alkaline phosphatase staining. The antiangiogenic activities of compounds were calculated from the inhibition ratio of angiogenesis. PTK787 was used as the positive control.

Conclusions
Our current research led to the isolation of a new kaurane diterpenoid (1), together with 16 known compounds 2-17 from the aerial parts of Excoecaria acerifolia. Their structures were identified by extensive spectral evidence, including HSQC, HMBC, 1 H-1 H COSY, ROESY experiments. Most of isolated constituents are phenolic compounds, which hints that the major chemical constituents in the aerial parts of Excoecaria acerifolia may be phenolics. The antiangiogenic and antiproliferative activities of part of isolates were evaluated and some of them are bioactive.