Two New Lanostane Triterpenoids from the Branches and Leaves of Polyalthia oblique

Two new lanostane triterpenoids, 20-hydroxyeuphorbol-7-one (1) and 15α-hydroxyeuphorbol-7,11-dione (2), together with four known triterpenoids, euphorbol-7-one (3), friedelin (4), stigmast-4-ene-6α-ol-3-one (5), stigmasta-4-en-3,6-dione (6), were isolated from ethanol extract of the branches and leaves of Polyalthia obliqua. The structures of 1 and 2 were elucidated on the basis of extensive spectroscopic analysis and comparisons with related known compounds. Antibacterial activities of two new compounds and four known compounds were tested.


Introduction
There are about 120 species of Polyathia in the world, of which only 17 species are distributed in China and Polyalthia obliqua is one of the seven Polyalthia species growing on Hainan Island, P.R. China. The genus is known for its folk medicine applications against a number of ailments such as stomach ache, dysmenorrhea and pharynx neurosis [1]. There were a series of interesting compounds isolated from the genus Polyathia, such as daphnane diterpenoids, alkaloids and phenanthrenes [2,3]. The lanostane triterpenoid suberosol isolated from P. Suberosa showed good anti-HIV activity [4]. In

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our previous work on this genus, 10 compounds were isolated from the roots of P. obliqua like suberosol, marcanine A and so on [5]. In continuation of our research, the petroleum ether fraction of the EtOH extract of the branches and leaves of P. oblique showed antibacterial activity against the Escherichia coli, with the MIC value of 20 μg/mL. In order to study on bioactive components from the branches and leaves of P. oblique, two new lanostane triterpenoids, namely, 20-hydroxyeuphorbol-7one (1); and 15α-hydroxyeuphorbol-7,11-dione (2); together with four known triterpenoids, euphorbol-7one (3); friedelin (4); stigmast-4-ene-6α-ol-3-one (5); stigmasta-4-en-3,6-dione (6) ( Figure 1) were isolated from the EtOH extract of the branches and leaves of P. oblique, collected from Hainan province. This paper described the isolation, structural characterization and biological activities of these compounds.

Results and Discussion
The air-dried branches and leaves of P. oblique were extracted three times with 80% EtOH at room temperature. Repeated chromatography on a petroleum ether fraction from EtOH extract afforded two new compounds 1-2 and four known compounds 3-6.
Compound 1 was obtained as a white amorphous powder, and its HRESIMS in the positive mode revealed a peak at m/z: 471.3826 [M+H] + indicative of the molecular formula of C 31 H 50 O 3 , indicating seven degrees of unsaturation. Its IR spectrum exhibited the presence of hydroxyl (3,439 cm −1 ) and carbonyl groups (1,642 cm −1 ).
In the 1 H-NMR spectrum, six methyl singlet signals at δ H 0.83 (3H, s), 0.88 (3H, s), 0.94 (3H, s), 1.00 (3H, s), 1.16 (3H, s) and 1.28 (3H, s), two methyl doublet signals at δ H 1.01 (3H, d, J = 6.6 Hz) and 1.03 (3H, d, J = 6.6 Hz), one oxymethine signal at δ H 3.26 (1H, dd, J = 11.6, 3.2 Hz) and two olefinic proton signals at δ H 4.67 (1H, br s) and 4.73 (1H, br s), and a series of overlapped signals were observed. The 13 C-NMR and DEPT spectra of 1 displayed 31 carbon signals which were assigned to eight methyls, ten methylenes, four methines, and nine quaternary carbons (including one carbonyl group at δ C 199.2). The two olefinic carbon signals at δ C 106.5 (CH 2 ) and 156.3 (C) were characteristic of the exomethylene moiety. The other two olefinic carbon signals appearing at δ C 138.5 (C) and 164.8 (C) corresponded to one double bond. The 13 C-NMR chemical shifts at δ C 75.3 (C) and 78.1 (CH) confirmed the presence of two oxygenated carbons. The aforementioned data indicated that 1 was most likely a lanostane triterpenoid with a C9 side-chain moiety at C-17 containing a CH 2 substituent at C-24 [6]. Its 1 H-and 13 C-NMR spectra were very similar to those of the reported compound euphorbol-7-one (euphorbol-7-one is a lanostane triterpenoid, which has been isolated from the Euphorbia sapinii [7]), except for the disappearance of a methine signal at δ H 1.43 (1H, m) in the 1 H-NMR spectrum for 1. Furthermore, in the 13 C-NMR spectrum, the C-20 signal moved downfield significantly [δ C 75.3 (C) in 1 vs. 36.4 (CH) in euphorbol-7-one], indicating a hydroxyl group was located at C-20. The location of the hydroxyl group at C-20 was further confirmed by the HMBC correlations from H-17, C-21, C-22 to C-20. The planar structure of 1 was further determined by the 2D NMR spectra including 1 H-1 H COSY and HMBC experiments ( Figure 2). The relative configuration of 1 was determined by extensive analysis of the coupling constants of 1 H-NMR (Table 1)    Its IR spectrum showed an absorption at 3441 cm −1 for hydroxyl groups and 1669 cm −1 for carbonyl. The general features of its NMR spectroscopic data (Table 1) were markedly similar to those of (3β)-3hydroxy-24-methylenelanost-8-ene-7,11-dione (a lanostane triterpenoid isolated from the Euphorbia humifusa [8]). Detailed comparison of NMR data of these two compounds suggested that they had the same lanostane triterpenoid skeleton. The only significant difference in the 1 H-NMR spectrum was the presence of a methine signal at δ H 4.34 (1H, dd, J = 14.4, 7.2 Hz) in 2 instead of a methylene signal at δ H 2.13-2.16 (2H, m) in (3β)-3-hydroxy-24-methylenelanost-8-ene-7,11-dione, and in the 13 C-NMR spectrum a methine group at δ C 72.3 (CH) for C-15 was observed in 2, instead of a methylene group at δ C 32.2 for C-15 in (3β)-3-hydroxy-24-methylenelanost-8-ene-7,11-dione. These results suggested that there was a hydroxyl group at C-15. The location of the hydroxyl group at C-15 was confirmed by the HMBC correlation of H-15 to C-30. The gross structure of 2 was further confirmed by 1 H-1 H COSY and HMBC spectra ( Figure 2).
All compounds were examined for antibacterial activity against six terrestrial pathogenic bacteria, including Staphylococcus aureus, Micrococcus luteus, Micrococcus tetragenus, Staphylococcus albus, Bacillus cereus and Escherichia coli by the microplate assay method. The results demonstrated that compounds 4 and 5 showed antibacterial activities against E. coli and M. tetragenus with the same MIC values of 5 μg/mL. Ciprofloxacin was used as a positive control with the same MIC values of 0.313 μg/mL to against E. coli and M. tetragenus.

General Information
IR spectra were recorded on a Thermo Nicolet 6700 (using KBr disks) spectrophotometer (Thermo Scientific, Madison, WI, USA). NMR spectra were measured on a Bruker AV-400 instrument with TMS as the internal standard. HRESIMS spectra were made on the Bruker Daltonics Apex-Ultra 7.0 T (Bruker Corporation, Billerica, MA, USA). Silica gel used for column chromatography (CC) was supplied by Qingdao Marine Chemical Factory (Qingdao, China). Sephadex LH-20 was used (Pharmacia Co., Ltd., Sandwich, UK). TLC and preparative TLC were purchased from Qingdao Chemical Industry Institute (Qingdao, China). All solvents were purchased from Tianjin Fuchen Chemical Reagent Factory (Tianjin, China). The optical density was measured by an enzyme-labeled detector (Elx800, BioTek Instruments, lnc., Winooski, VT, USA).

Plant Material
The branches and leaves of Polyalthia oblique were collected in Changjiang County, Hainan Province, China, in June 2012 the fruiting season and authenticated by associate Prof. Qiong-Xin Zhong, College of Life Science, Hainan Normal University. They were not separated. The averaged diameter of the collected branches were about 2 cm. A voucher specimen was deposited in the Key Laboratory of Tropical Medicinal Plant Chemistry of Ministry of Education, Hainan Normal University.