Tetranortriterpenes and Limonoids from the Roots of Aphanamixis polystachya

Phytochemical investigation of the acetone extract from the roots of Aphanamixis polystachya resulted in isolation of four new tetranortriterpenes (1–4) in addition to one protolimonoid (methyl-1ξ,7R-diacetoxy-23R,25-dihydroxy-20S,24R-21,24-epoxy-3,4-seco-apotirucall-4(28),14(15)-diene-3-oate (5)), five known limonoids (rohituka 3 (6), rohituka 7 (7), nymania 1 (8), rubrin G (9), prieurianin (10)) and a steroid (2,3-dihydroxy-5-pregnan-16-one (11)). Their structures were determined by spectroscopic analyses, including 2D-NMR (COSY, HMQC, HMBC, and NOESY) and high-resolution electrospray ionization mass spectrometry (HRESIMS). Cytotoxic and anti-inflammatory activities of these compounds were evaluated. Compounds 4 and 5 showed significant inhibition against superoxide generation and elastase release by human neutrophils in response to (formyl-l-methionyl-l-leucyl-l-phenylalanine/cytochalasin B) (FMLP/CB).


Introduction
Aphanamixis polystachya (Wall.) R.N. Parker (Meliaceae) is a tropical neem tree native to Asia, especially China and India. According to previous studies, the meliaceaeous plants are famous for the limonoid-producing source and the pest control function in agriculture [1,2]. Phytochemical investigations of this plant have resulted in isolation of many limonoids with diverse skeletons [3][4][5][6][7]. Other structures such as guaiane sesquiterpenes from the seeds [8], lignans from the stem barks [9], chromone, and flavonoid glycosides from the roots [10] were also reported. In biological studies, it has been used as traditional Bangladesh medicine for the treatment of cancers, diabetes, and liver diseases [11] in addition to insecticide applications. Besides, the leaf extract showed CNS (central nervous system) depressant and analgesic activity in vivo [12], as well as antimicrobial, antioxidant, cytotoxic, and thrombolytic activities in vitro [13]. Currently, some limonoids from A. polystachya attract much attention due to their interesting anticancer [14], antifungal [15], and antifeedant activities [16,17].
Continuing our phytochemical investigation on the new anti-cancer and anti-inflammatory agents from terrestrial source, the crude extract from the roots of A. polystachya showed significant inhibition on superoxide anion generation and elastase release by human neutrophils in response to FMLP/CB at 10 µg/mL. Fractionation of the active components from the roots of this species was thus initiated. Herein, we report the isolation and structural elucidation of four new and six known triterpenoids, together with one known steroid from the roots of A. polystachya. The evaluation against human tumor cell lines and inhibitory activities on superoxide anion generation and elastase release are also discussed.

General Experimental Procedures
Optical rotations were recorded on a JASCO DIP-1000 polarimeter (JASCO, Tokyo, Japan). IR spectra were taken on a HORIBA FT-720 spectrophotometer (HORIBA, Kyoto, Japan). The 1 H-and 13 C-NMR spectra as well as 2D NMR spectra (COSY, HMQC, HMBC, and NOESY) were recorded in CDCl 3 on a Bruker AVX NMR spectrometer (Bruker, Karlsruhe, Germany) operating at 400 MHz for 1 H and 100 MHz for 13 C using the CDCl 3 solvent peak as internal standard (δ H 7.265, δ C 77.0 ppm).

Plant Material
The roots of Aphanamixis polystachya (Wall.) R.N. Parker were collected in Ping-Tong County, Taiwan, in April 2012. The plant material was identified by one of the authors (C.T.C.). A voucher specimen (code No. TP 98-1) has been deposited at the School of Pharmacy, National Taiwan University, Taipei, Taiwan.

Cytotoxicity Assay
Cytotoxicity was tested against human Hep-G2 (hepatocellular carcinoma), HEp-2 (laryngeal carcinoma), A549 (lung carcinoma) and MCF-7 (breast adenocarcinoma) tumor cell lines. The assay procedure using MTT (3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyltetrazolium bromide) was carried out as previously described [18,19]. The cells were cultured in RPMI-1640 medium. After seeding of cells in a 96-well microplate for 4 h, 20 µL of sample was placed in each well and incubated at 37 • C for 3 days, and then 20 µL MTT was added for 5 h. After removing the medium and putting DMSO (200 µL/well) into the microplate with shaking for 10 min, the formazan crystals were redissolved and their absorbance was measured on a microtiter plate reader (MR 7000, Dynatech, Missouri City, TX, USA) at a wavelength of 550 nm. Mitomycin C was used as a positive control.

Anti-Inflammatory Assays, Inhibitory Effect on Superoxide Anion Generation and Elastase Release by Human Neutrophils
Neutrophils were obtained by means of dextran sedimentation and Ficoll centrifugation. Superoxide generation and elastase release were carried out according to a procedure described previously [20]. Superoxide anion production was assayed by monitoring the superoxide dismutaseinhibitable reduction of ferricytochrome c. Elastase release experiments were performed using MeO-Suc-Ala-Ala-Pro-Valp-nitroanilide as the elastase substrate. Genistein was used as a standard compound, which showed inhibition of 65.0 ± 5.7 and 51.6 ± 5.9 at 10 µg/mL, respectively, on superoxide anion generation and elastase release.
The relative configuration of 1 was determined by the analysis of NOESY correlations (Figure 3). Assuming that H-5 of 1 was α-oriented similar to that of the A-seco apotirucallane tetranortriterpenes [15,29], the NOESY correlations between H-6α/H-5/H-9/H3-18/H-16α revealed that these protons were on the α-face. The NOESY correlations of H3-19 with H-1, H-6β and H-11β, as well as Me-30 with H-7 indicating these protons were on the β-face. In addition, NOESY correlations of H-20/H-21/H-22α/H-23 suggested that these protons were all in α-face and the C-21 methoxy was β-oriented. On comparing the 1 H-and 13 C-NMR spectra of 1 with those of chisopanin C [30] and polystanins C and D [31], it was noted that the configurations of C-20, C-21 and C-23 were assigned S, S and R, respectively, the same as those of polystanin D [31], and the hydroxy at C-24 was assigned as αdisposition, the same as that of chisopanin C [30]. Therefore, structure 1 was established and a name aphataiwanin A was given. The 1D and 2D NMR spectra of 2 were similar to those of 1, suggesting that 2 was an analogue of 1. On comparing the 13 C-NMR spectra of 1 and 2, it was found that the C-21 and C-17 chemical shifts of 2 (δc 109.3, 58.1) were downfield compared to the same carbons of 1 (δ 104.6, 52.9). Thus it was suggested that the configuration of the C-21 methoxy group in 2 was different from that in 1. Also, both the COSY and HMBC correlations showed that all structural fragments were similar to those of 1, confirming that compound 2 is an epimer of 1, in which the C-21 methoxy was α-oriented.
The relative configuration of 2 was determined by NOESY experiment, in which the H3-19 and H3-30 were assigned to be β-oriented while H3-18, H-9, and H-5 were α-orientation. NOESY correlations between H-21/H-22β and H-20/H-23, and H-23/H-22β indicated that the methoxy group was α-oriented. Comparing the 13 C-NMR data with those of chisopanin C [30] assigned the configuration of C-13, C-17, C-20, C-23 and C-24 as S, S, S, R, R and R, respectively. Therefore, the structure of compound 2 was determined and it was named aphataiwanin B.
Compound 3, [α] 25 D −60 (MeOH), was obtained as an amorphous solid and found to possess the molecular formula C36H56O10, (one more oxygen atom than 1) as inferred from its HRESIMS (m/z  The 1D and 2D NMR spectra of 2 were similar to those of 1, suggesting that 2 was an analogue of 1. On comparing the 13 C-NMR spectra of 1 and 2, it was found that the C-21 and C-17 chemical shifts of 2 (δc 109.3, 58.1) were downfield compared to the same carbons of 1 (δ 104.6, 52.9). Thus it was suggested that the configuration of the C-21 methoxy group in 2 was different from that in 1. Also, both the COSY and HMBC correlations showed that all structural fragments were similar to those of 1, confirming that compound 2 is an epimer of 1, in which the C-21 methoxy was α-oriented.
The relative configuration of 2 was determined by NOESY experiment, in which the H 3 -19 and H 3 -30 were assigned to be β-oriented while H 3 -18, H-9, and H-5 were α-orientation. NOESY correlations between H-21/H-22β and H-20/H-23, and H-23/H-22β indicated that the methoxy group was α-oriented. Comparing the 13 C-NMR data with those of chisopanin C [30] assigned the configuration of C-13, C-17, C-20, C-23 and C-24 as S, S, S, R, R and R, respectively. Therefore, the structure of compound 2 was determined and it was named aphataiwanin B.
Compound 3, [α] 25 D −60 (MeOH), was obtained as an amorphous solid and found to possess the molecular formula C 36 H 56 O 10 , (one more oxygen atom than 1) as inferred from its HRESIMS (m/z 671.3785 [M + Na] + ). The similar 1 H-and 13 C-NMR spectroscopic data of 3 and 1 suggested that they are close analogues. However, the 1 H-NMR spectrum of 3 exhibited eight methyl singlets (δ H 0.95, 1.00, 1.14, 1.26, 1.27, 1.78, 1.96 and 2.04) instead of seven in 1 and only three olefinic protons (δ H 4.84, 5.02 and 5.27) instead of five in 1, implying that a double bond was missing in 3. In the 13 C-NMR spectrum, compound 3 was found possessing an additional methyl (δ C 26.5) and lacking an olefinic methylene, in comparison to 1. The additional methyl group was assigned at C-27 by observation of the HMBC correlations from H 3 -27 (δ H 1.26) and H 3 -26 (δ H 1.27) to C-25 (δ C 73.1) and C-24 (δ C 76.8). The relative configuration of 3 was determined on the basis of the NOESY experiment and comparing the J values of 3 with those of 1. The result was identical to 1, suggesting the same configuration. On the basis of above interpretations, the structure of 3 was categorized into the group of ring A-seco apotirucallol and a name aphataiwanin C was given.
Compound 4 was isolated as an amorphous solid, [α] 25 D −170 (MeOH). It had the same molecular formula C 36 H 56 O 10 as 3, as derived from HRESIMS at m/z 671.3763 ([M + Na] + ). The IR spectrum revealed that 4 contained a hydroxyl (3491 cm −1 ), ester (1741 cm −1 ), and double bond (1642 cm −1 ) functionalities. The 1 H-and 13 C-NMR spectra of 4 (Tables 1 and 2) were similar to those of 3 except for the hemiacetal carbon shifted downfield to δ C 109.7 (C-21) in 3 and the oxygenated carbon C-23 shifted upfield to δ C 76.9. The 1 H-1 H COSY and HMBC of 4 revealed cross peaks similar to those of 3,

Conclusions
In the present study, 10 tetranortriterpenoids, including four new compounds and a steroid, were isolated; compounds 1-5 belong to a group of protolimonoids, ring A-seco apotirucallol, while 6-10 can be classified as rings A,B-seco prieurianins. Among these secondary metabolites, liaphataiwanins A (1) and D (4), 5, and prieurianin (10) were found to have mild cytotoxicities against cancer cells and moderate to potent anti-inflammatory activities. Compound 5, which has the moiety of six-member ether ring, showed the best biological function. The chemical constituents and the evaluation of cytotoxicities against human cancer cells and anti-inflammatory activities reported herein may provide beneficial information for further phytotherapy research.