Cytotoxic and Antioxidant Compounds from the Stem Bark of Goniothalamus tapisoides Mat Salleh

Eleven compounds: goniomicin A (1), goniomicin B (2), goniomicin C (3), goniomicin D (4), tapisoidin (5), goniothalamin (6), 9-deoxygoniopypyrone (7), pterodondiol (8), liriodenine (9), benzamide (10) and cinnamic acid (11), were isolated from the stem bark of Goniothalamus tapisoides. All compounds were identified by spectroscopic analysis and, for known compounds, by comparison with published data. Goniothalamin (6) exhibited mild cytotoxic activity towards a colon cancer cell line (HT-29), with an IC50 value of 64.17 ± 5.60 µM. Goniomicin B (2) give the highest antioxidant activity in the DPPH assay among all compounds tested, with an IC50 of 0.207 µM.


Introduction
The genus Goniothalamus (Annonaceae) comprises about 160 species of shrubs and trees native to tropical and subtropical Asia [1]. Goniothalamus tapisoides Mat Salleh, locally known as "selada" or "semukau", is endemic to Borneo, especially the Southern part of Sarawak. This species is a small tree of about 5 m in height and is used by the native folks as an abortifacient and to cure poisonous animal bites such as snake, scorpion or insect bites. It is also used to relieve stomachaches [2].
In recent years, Goniothalamus species have been receiving considerable attention because of their reputation for producing styryl lactones and acetogenins which possess remarkable cytotoxic and antitumor properties against various human tumor cell lines such as A-549 (lung carcinoma), HL-60 (promyelocytic leukemia), and SGC-7901 (stomach cancer) [1,3]. Previous studies on the mechanism of action of (R)-goniothalamin have shown that it is able to induce apoptosis in MCF-7 (breast cancer) and HL-60 human cancer cells [3][4][5][6].

Isolation and Chemistry
The 1 H-1 H COSY spectrum and 1 H-13 C HSQC spectrum confirmed the connectivities between C-2-C-3-C-4-C-5-C-6-C-7. The HMBC correlations indicated the linkage of the C-2 to carbonyl C-1 and C-7 to the phenyl ring C-8 (Table 1). Thus, the structure of goniomicin A (1) was elucidated as illustrated in Figure 1. The occurrence of compound 1 in Goniothalamus tapisoides is of interest since it may be a precursor of goniothalamin (6). A plausible biogenetic pathway for the formation of 6 from 1 is illustrated in Scheme 1. Compound 1 undergoes a dehydration and cyclization reaction to form 6. Scheme 1. Dehydration and cyclization of 1 to 6. The IR spectrum showed a strong absorption for a conjugated carbonyl group of an ester at 1718 cm −1 , while the UV absorption bands at 207 and 251 nm indicated the presence of a phenyl group [11]. The 1 H-NMR and 13 C-NMR spectra of 1 and 2 (Table 1) are very similar to each other, except for the existence of a three proton singlet at δ 3.72 and a methyl carbon at δ 51.6 in the 1 H-and 13 C-NMR spectra, respectively. This observation suggested the presence of an additional methoxyl group, which is attached to the C-1 carbonyl group. A noticeable difference was also observed in the coupling constant value (15.6 Hz) of protons H-2 and H-3, suggesting a trans-configuration. From the 1 H-NMR, 13 [11]. The 1 H-NMR spectrum of 3 is relatively similar to that of goniothalamin (6). However, the olefinic proton signals of the lactone ring are absent. Instead two methylene proton signals and one oxymethine proton signal appeared at δ 2.73 and δ 3.82, corresponding to H 2 -2 and H-3, respectively. Another oxymethine signal, assignable to H-5, appeared at δ 5.20 as a ddd with coupling constants of 11.0, 6.4 and 3.5 Hz respectively (Table 2). In addition, one singlet corresponding to three protons of a methoxyl group was apparent at δ 3.36. The 13 C-NMR spectrum showed the expected fourteen carbons: one methyl, two methylene, nine methines and two quaternary carbons. The oxymethine carbons, C-3 and C-5, resonated at δ 71.4 and δ 76.2, respectively. In the HMBC spectrum (Table 2), the carbonyl carbon (C-1) signal (δ 169.7) correlated with the protons signal at δ 2.73 (H 2 -2), while the carbon at δ 71.4 (C-3) correlated with the proton at δ 5.20 (H-5). Thus it can be deduced that the methoxy group is attached to C-3.
The relative stereochemistry of 3 was established by the NOESY spectrum. H-5α, which is axially oriented, correlated with H-4, therefore suggesting that H-4 adopts an α spatial orientation [12]. H-6 showed correlation to H-5β thus implying that H-6 is β oriented.With the aid of the 1 H-NMR, 13 Table 3). The downfield shift of H 2 -14 and H 2 -15 were due to the neighboring nitrogen and oxygen. The 13 C-NMR showed signals corresponding to fifteen carbons: four methylenes, nine methines, one quaternary and one carbonyl carbon. The methylene carbons C-14 and C-15 resonated downfield at δ 82.9 and δ 58.9, respectively. The carbonyl carbon (C-1) gave a peak at δ 168.9. The HMBC spectrum (Figure 1) revealed cross peaks between the methylene at δ 4.13/4.64 (H 2 -14) and C-3 (δ 54.1), C-5 (δ 77.3) and C-15 (δ 58.9). Two methylene signals of H 2 -2 (δ 2.28/2.64) and H 2 -15 (δ 3.82/4.34) were correlated to the carbonyl C-1. From a 1 H-1 H COSY experiment, the entire sequence C-2-C-3-C-4-C-5-C-6 was identified. Thorough analyses of 1D and 2D NMR data analyses allowed the full assignment of all protons and carbons as listed in Table 3.  The relative stereochemistry of 4 was established with the aid of the NOESY spectrum. H-15α which is axially oriented showed a NOESY correlation with H-3, which in turn showed correlation with H-5, therefore suggesting that both H-3 and H-5 assume an α-spatial orientation. The structure and relative stereochemistry was further confirmed using single-crystal X-ray diffraction analysis CCDC 912021 contains the supplementary crystallographic data for this paper. These data can be obtained free of charge via www.ccdc.cam.ac.uk/conts/retrieving.html (or from the CCDC, 12 Union Road, Cambridge CB2 1EZ, UK; fax: +44 1223 336033; e-mail: deposit@ccdc.cam.ac.uk). An ORTEP drawing of 4 is shown in Figure 2. (H-8, dd, J = 7.9, 1.4). Signals for three methyl groups at δ 3.96, δ 3.89 and δ 4.00 were assigned to the methoxyl groups at N-11, C-3 and C-4, respectively. The positions of the methoxyl groups were established from the NOESY and HMBC spectra. In the NOESY spectrum, the methyl protons of the methoxyl group at C-3 showed correlations with H-2. In the HMBC spectrum, the proton signal at δ 7.23 (H-2) correlated with the carbons signal at δ 59.7 (3-OCH 3 ) and δ 56.0 (4-OCH 3 ), thereby confirmed the assignments of the methoxyl groups. The 1 H-NMR spectrum of 5 also showed signals at δ 2.73 (H-9α, t, J = 13.8), δ 3.49 (H-9β, dd, J = 14.2, 6.0), δ 4.60 (H-10, dd, J = 13.8, 6.0), indicating that C-9 and C-10 are hydrogenated. To the knowledge of the authors, this is the first occurrence of a 9,10-dihydroaristolactam alkaloid. The 13 C-NMR spectrum (Table 4) showed the presence of eighteen carbons; three methyl, one methylene, six methine, seven quaternary and one carbonyl. The carbonyl carbon (C-12) resonated at δ 167.9. Thus, taking into consideration all the NMR data and analyses, the structure of tapisoidin (5) was elucidated as illustrated in Figure 1. Identification of the known compounds was done by comparison of 1 H-and 13 C-NMR data with reported values [13][14][15][16][17][18].

Bioactivity
The result of cytotoxicity tests on the hexane, CH 2 Cl 2 and MeOH crudes of G. tapisoides against lung cancer (A549), breast cancer (MCF-7) and prostate cancer (DU-145) cell lines are shown in Table 5. Hexane and CH 2 Cl 2 crude extracts exhibited cytotoxicity against the three cancer cell lines. Among the isolated compounds, compounds 1-6 were evaluated for their cytotoxicity (Table 6). Only compound 6 exhibited cytotoxic activity against all the eight cell lines. No cytotoxic activity was found for compounds 1-5, most probably due to lack of pharmacophoric groups responsible for the high antiproliferative activity [3]. Compound 6 is the most potent against the colon cancer cell line (HT-29). It exhibited an IC 50 of 64.17 ± 5.60 µM, which is comparable to that of cisplatin (77.24 ± 3.23 µM). Therefore the cytotoxicity of both hexane and dichloromethane extracts may be attributed to the presence of compound 6, which has been previously reported to exhibit cytotoxicity against various cancer cell lines [13,19]. In addition, reports have shown that the antiproliferative activity of 6 is selective for cancer cell lines with no significant cytotoxicity toward non-malignant cells [13,20].
Since free radicals are associated with DNA damage and protein modifications, including apoptotic modulators which could lead to carcinogenesis [21], we have also evaluated the antioxidant activity using a DPPH radical scavenging assay. Compound 2 gave the highest antioxidant activity, with an IC 50 of 0.207 µM, followed by compounds 4 (IC 50 = 0.252 M) and 1 (IC 50 = 0.328 M). The high antioxidant effect of compounds 2, 4 and 1 may be attributed to the presence of the hydroxyl group adjacent to the conjugated double bond that could donate electron to the DPPH free radical. Carotenes and xanthophyll which possess hydroxyl groups and conjugated double bonds have been reported to show high antioxidant activity [22,23]. Compounds 3 (IC 50 = 1.748 M), 5 (IC 50 = 0.772 M) and 6 (IC 50 = 2.024 M) which lack hydroxyl groups in their structures showed very low antioxidant activity.

General
Melting points were determined by a Fargo MD-1D melting point apparatus. The specific rotations were measured on a Jasco P-1020 polarimeter. UV spectra were recorded on a Shimadzu 1650 PC ultraviolet-visible spectrometer, and IR spectra on a Perkin Elmer Spectrum 400 FT-IR/FT-FIR spectrometer. 1D and 2D NMR spectra were measured with a JEOL ECA 400 spectrometer (400 MHz). The mass spectra were obtained on an Agilent Technologies 6530 Accurate-Mass-Q-TOF LC/MS. Column chromatography was performed on silica gel Merck 60 (230-400 mesh). X-ray data collection was obtained from a Bruker APEX2 unit; cell refinement: SMART; data reduction: SAINT; program(s) used to solve the structure: SHELXTL; program(s) used to refine structure: SHELXTL.

Plant Material
The stem bark of G. tapisoides was collected from Sarawak and identified by Prof. Fasihuddin bin Ahmad, Faculty of Resource Science and Technology, Universiti Malaysia Sarawak. A voucher specimen (HUMS 000108) is deposited at the Herbarium of Universiti Malaysia Sarawak, Kota Samarahan, Sarawak, Malaysia.

Cytotoxicity Assay
Cytotoxicity of the compounds were evaluated against eight types of cancer cell lines; lung (A549), prostate (DU-145), skin (SK-MEL-5), pancreatic (BxPC-3), liver (Hep G2), colon (HT-29), breast cancer (MCF-7), and (MDA-MB-231). Cell lines were cultured in Dulbecco's Modified Eagle medium (DMEM) with 10% foetal bovine serum. Cells were plated into 96-well microplates and 24 hour later, 100 µL of samples and cisplatin standard were introduced in triplicate. Cells were further incubated for 48 h and cell viability was determined using the MTS reagent (Promega Corp., Madison, WI, USA). Microplates were returned to the incubator for 1 h and absorbance was read on a microplate reader at 490 nm (Infinite 200, Tecan, Männedorf, Swizerland). The concentration required causing 50% cell death (IC 50 ) by the samples and drug standards were determined using dose-response curves in Prism 5.02 software [24].

Antioxidant Assay
The DPPH antioxidant assay was determined as described by Shimada et al. [25]. Briefly, 0.1 mM DPPH (1 mL) dissolved in ethanol was added to an ethanol solution (3 mL) of the tested compound at different concentrations (0, 50, 100, 150, 200 µg/mL). An equal volume of ethanol was added in the control test. The mixture was shaken vigorously and allowed to stand at room temperature for 30 min. Then the absorbance at 517 nm was measured with a UV-VIS spectrophotometer. The percentage of scavenging of DPPH was calculated using the following equation: DPPH scavenging effect (%) = ° ° 100 where A o is the absorbance of the control reaction and A1 is the absorbance in the presence of the sample.

Conclusions
The CH 2 Cl 2 extract of the stem bark of G. tapisoides yielded eleven compounds, of which five are new; goniomicins A-D (compounds 1-4), and tapisoidin (5). The latter, tapisoidin (5), represents the first report of the occurence of a 9,10-dihydroaristolactam alkaloid. From this study, the compound responsible for the cytotoxicity of the extracts is goniothalamin (6), which showed the highest potency against the colon cancer cell line (HT-29) withan IC 50 of 64.17 ± 5.60 µM. Goniomicin A (1), goniomicin B (2) and goniomicin D (4) displayed high antioxidant activity, which may be due to the presence of conjugated hydroxyl groups that could donate electrons to scavenge radicals [22,23].