Two new tirucallane triterpenoids from Aphanamixis grandifolia

Two new tirucallane triterpenoids, 2 α-ethoxy-2,3-secotirucalla-2,29-epoxy-7-ene-23-oxo-3-oic acid (1) and (23E)-2 α-hydroxytirucalla-7,23,25-triene-3-one (2), along with the known 2,3-secotirucalla-2,3;2,29-diepoxy-7-ene-3,23-dione (3), were isolated from the leaves and twigs of Aphanamixis grandifolia. Their structures were elucidated by extensive NMR and MS data, and compound 3 was further confirmed by X-ray crystal diffraction analysis. Antimicrobial activities and insecticidal activities of these three compounds were also evaluated. Compound 1 showed good antimicrobial activity against Staphylococcus aureus with the MIC value of 1.56 µg/mL, while compounds 1 and 2 showed insecticidal activity at 100 ppm, with the corrected mortality 79.1% and 60.6%, respectively.


Introduction
The Meliaceae family is rich in highly complicated structural secondary metabolites with significant bioactivities, which have attracted overwhelming attention in the field of natural products. 1 The genus Aphanamixis (Meliaceae), consisting of 25 species, is distributed extensively in the tropical regions of Asia such as Southern China, India, Malaysia, and Indonesia. 2 The roots and leaves of Aphanamixis grandifolia Blume have been applied in primitive medicine for the treatment of cold and rheumatic joints pain due to arthritis as well as numbness of limbs owing to wind-cold-dampness.

Results and Discussion
Compound 1 was obtained as a colorless oil and its molecular formula was determined to be C 32 H 52 O 5 with seven degrees of unsaturation due to the positive HREIMS at m/z 516.3797 (calcd for 516.3815). The IR spectrum exhibited the existence of hydroxyl (3449 cm -1 ) and carbonyl (1755 and 1710 cm -1 ) groups. According to the 13 C NMR and DEPT spectra (Table 1), 32 carbon resonances were classified into eight methyls, ten methylenes including two oxygenated carbons (δ C 64.7 and 63.2), seven methines including an acetal carbon (δ C 99.9), and seven quaternary carbons including a carbonyl (δ C 211.2) and an olefinic carbon (δ C 144.1). Comparison of the NMR data of 1 with those of aphanamgrandin C 8 demonstrated that 1 had an additional ethoxy group [δ C 63.2 (CH 2 , 2-OCH 2 CH 3 ) and 15.1 (CH 3 , 2-OCH 2 CH 3 ), δ H 3.79, 3.47 (each 1H, m, 2-OCH 2 CH 3 ) and 1.21 (3H, t, J = 7.0 Hz, 2-OCH 2 CH 3 )], which was located at C-2 through analysis of the HMBC spectrum. As shown in Figure 2. 1a, the seven-membered ring A with a carboxyl (δ C 175.3, C-3) at C-4 (δ C 50.0) was established by the HMBC The structure of rings BD and the side chain moiety with a carbonyl (δ C 211.2) at C-23 were identical to aphanamgrandin C 8 . Then, the planar structure of compound 1 was established.

Experimental Section
General Experimental Procedures. Optical rotations were measured with a JASCO P-1020 digital polarimeter. UV spectra were determined on a Shimadzu UV-2401 PC spectrophotometer. IR spectra were detected on a Bruker Tensor-27 infrared spectrometer with a KBr disk. CD spectra were obtained on a JASCO J-810 spectro-photometer. Brucker HCT/E squire and Waters Autospec Premier P776 spectrum were respectively used for measuring ESIMS and HREIMS spectra. 1D NMR and 2D NMR spectra were recorded on a Bruker AM-400 and Bruker DRX-500 spectrometer, using TMS as an internal standard. Column chromatography was Extraction and Isolation. The leaves and twigs of A. grandifolia (9.0 kg) were percolated with 95% EtOH three times. The EtOH distillate was concentrated in vacuum to obtain a crude residue, which was partitioned with PE (petroleum ether), EtOAc, and n-BuOH, successively. The PE fraction (133.6 g) was submitted to column chromatography (CC) over silica gel (100200 mesh) using PE/acetone (100:1 → 1:1) to give five fractions 15. Fr. 2 was separated over a whole series of CC on silica gel, MCI, RP-C 18 gel, and Sephadex LH-20 to yield compounds 1 (6.0 mg), 2 (11.6 mg), and 3 (24.5 mg).    Antimicrobial Bioassays. Compounds 1−3 were evaluated their antimicrobial activities against S. aureus, P. aeruginosa, MRSA 92 # , and MRSA 98 # by the 2-fold dilution method. 12 The strains used in antimicrobial tests were obtained from the Research Center of Natural Medicine, Clinical School of Kunming General Hospital of Chengdu Military Command. The protocols of antimicrobial tests were described previously. 13 Insecticidal Bioassays 7c,7d,14 . Compounds 13 were dissolved in DMSO and then diluted with artificial seawater to the final concentrations of 100, 50, 10 ppm (mg/L), which were added to 96-well plates with each well of 1525 Artemia salina. After 48 hours of incubation at 28 C, the numbers of the dead (non-motile was considered dead) nauplii in each well were counted under a microscope. Each concentration was repeated in triplicate with toosendanin (Shanghai Standard Biotech Co. Ltd., purity  98%) as the positive control, which was treated in the same way without samples. The corrected mortality was calculated by the Abbot formula.
Corrected mortality = (the mortality of the A. salina with sample  the mortality of the A. salina of control group) / (1  the mortality of the A. salina of control group)  100%

Electronic Supplementary Material
Supplementary material is available in the online version of this article at http://dx.doi.org/ 10.1007/s13659-012-0059-3 and is accessible for authorized users.