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A study of an EtOH extract obtained from the roots of the Madagascan plant Terminalia tropophylla H. Perrier (Combretaceae) led to the isolation of the new oleanane-type triterpenoid saponin 1, the new lignan derivative 2, and the two known saponins arjunglucoside I (3) and sericoside (4). The structures of the new compounds 1 (terminaliaside A) and 2 (4′-O-cinnamoyl cleomiscosin A) were elucidated using 1D and 2D NMR experiments and mass spectrometry. Compound 1 showed antiproliferative activity against the A2780 human ovarian cancer cell line with an IC50 value of 1.2 μM.
In our continuing search for bioactive molecules from the Madagascar rainforests as part of an International Cooperative Biodiversity Group (ICBG) program, we obtained an extract of the roots of Terminalia tropophylla (Perrier) (Combretaceae). This extract, designated MG 4035, showed activity against the A2780 ovarian cancer cell line, with an IC50 value 11 μg/mL. The extract was selected for bioassay-guided fractionation based on the above activity.
Previous phytochemical studies have revealed the genus Terminalia to be a rich source of secondary metabolites, such as lignans (Valsaraj et al., 1997), flavonoids (Srivastava et al., 1999), terpenoids (Conrad et al., 1998, 2001), and tannins (Kandil and Nassar, 1998; Conrad et al., 2001). Some of these metabolites have shown a wide range of biological activities, including antimalarial (Valsaraj et al., 1997), antifungal (Valsaraj et al., 1997; Conrad et al., 1998, 2001), antibacterial (Conrad et al. 1998, 2001; Eldeen et al., 2006), and cytotoxic (Kandil and Nassar, 1998; Conrad et al., 2001) activities. The anthelmintic and haemolytic properties of terpene esters from T. macroptera have been studied (Conrad et al., 1998). Tannins from the same plant showed bacteriostatic activity, and the hydrolysable tannin isoterchebulin showed moderate nematocidal and piscicidal activity (Conrad et al., 2001). The antioxidant effects of an aqueous extract of T. chebula have also been investigated (Lee et al., 2005).
In this paper, we report the isolation, structure elucidation, and antiproliferative activities against A2780 cells of three triterpenoid saponins (1, 3 and 4) and one lignan derivative (2) obtained from the roots of Terminalia tropophylla.
Liquid-liquid partitioning of a portion of an EtOH extract (2.2 g) of the roots of Terminalia tropophylla into hexane, CH2Cl2 and aqueous MeOH fractions indicated that the aqueous MeOH fraction (1.7 g) was the most active fraction, with an IC50 value of 10 μg/mL against A2780. Purification of this fraction using a C18 open column, followed by preparative HPLC on a C18, column led to the isolation of compounds 1, 3, and 4. Compound 2 was purified from the CH2Cl2 fraction after repeated chromatography on silica gel.
Compound 1 was obtained as a white solid. Its HRFABMS (positive-ion mode) exhibited a quasimolecular ion peak at m/z 1079.5376, consistent with a molecular composition of C53H84O21Na. The aglycone of 1 was identified as an oleanane triterpenoid by analysis of its 1H and 13C NMR spectra (Table 1) and from observation of connectivities in the 1H-1H COSY, TOCSY, ROESY, HSQC, and HMBC NMR spectra. The NMR spectra indicated the presence of one trisubstituted double bond (12-position), one ketone (16-position), three oxygenated methines (3-, 21-, and 22-positions), and one oxygenated methylene (28-position) in the aglycon of 1. The signals for H3-23 (δH 1.06, s) and H3-24 (δH 0.86, s) showed 3J HMBC correlations to C-3 (δC 90.8) (Figure 1). Of the seven methyl groups in the aglycon, only H3-27 (δH 1.22, s) exhibited a 3J HMBC correlation to C-13 (δC 140.6), which confirmed the location of the double bond at the 12-position. H2-28 (δH 4.18, d, J = 9.6 Hz; 4.01, d, J = 9.6 Hz) exhibited 3J HMBC correlations to C-16 (δC 216.8) and C-22 (δC 77.1), while H3-29 (δH 0.95, s) and H3-30 (δH 0.88, s) correlated to C-21 (δC 77.7). ROESY correlations between H3-23 and H-3 (δH 3.24), and H3-24 and H3-25 (δH 1.01, s) revealed that H-3 has an α-axial orientation. H-21 (δH 3.66, d, J = 9.3 Hz) was also determined as α-axial due to its coupling constant, which was confirmed by a ROESY correlation between H3-29 and H-21 (Figure 2). In turn, H-22 (δH 3.56, dd, J = 9.3, 2.8 Hz) of 1 was assigned as β-axial due to its coupling constant and ROESY correlations to H-18 (δH 2.79, dd, J = 13.8, 3.0 Hz) and H3-30. Both H-18 and H-22 showed ROESY correlations to H2-28, which further confirmed the relative configurations of the E ring of the aglycon. The aglycon of compound 1 has not been reported before hence the 13C NMR chemical shifts of the aglycon were compared with those of similar known aglycons. The 13C NMR chemical shifts of the aglycon (A-B-C-D-E: chair-chair-boat-boat-chair) of 1 matched those of the carbons in rings A, B, C, and E of marsglobiferin (Qiu et al., 1993), and those of the carbons in ring D of 3-O-[α-L-rhamnopyranosyl-(1→2)-β-D-glucopyranosyl-(1→2)-β-D-glucuronopyranosyl]-3β,22α-dihydroxyolean-12-en-16-one (De Leo et al., 2007).
Analysis of the 1H, 13C NMR, and HSQC spectra of 1 indicated the presence of three sugar units and an additional α,β–unsaturated ester unit. The anomeric proton of one sugar, defined as the first sugar here, showed a 3J HMBC correlation to C-3 (δC 90.8). The spin system from the anomeric proton (H-1′, δH 4.43, d, J = 8.0 Hz) to the other protons of this sugar was clearly exhibited in a 1D TOCSY spectrum [H-1′ (selected): δH 4.43, d, J = 8.0 Hz; H-2′: δH 3.33, t, J = 9.6 Hz; H-3′: δH 5.02, t, J = 9.6 Hz; H-4′: δH 3.46, t, J = 9.6 Hz; H-5′: δH 3.20, m; H-6′: δH 3.65 and 3.85, m]. Based on the coupling constants between the protons in the spin system and ROESY correlations between H-1′, H-3′ and H-5′, the first sugar unit was identified as a β-glucopyranoside with a functional group at the 3′-position, since H-3′ showed a 3J HMBC correlation to the carbonyl carbon (C-A1, δC 169.4) of an α,β-unsaturated ester unit. Both H3-A4 (δH 1.98, dq, J = 7.2, 1.4 Hz) and H3-A5 (δH 1.92, br s) showed HMBC correlations to C-A2 (δC 129.5) and C-A3 (δC 138.3), and H3-A5 also correlated to C-A1. The ROESY correlation between H-A3 (δH 6.09, qq, J = 7.2, 1.7 Hz) and H3-A5 confirmed that the substituent at the 3′-position was an angeloyl group. The 13C NMR chemical shifts of C-1′~C-6′ matched well those of the 3″-O-acetylglucosyl group of 3″-O-acetylsaikosaponin d (Ebata et al., 1996), and the 13C NMR chemical shifts of the angeloyl carbons matched those of methyl angelate (Zhong et al., 1999). The H-1″ proton of the second sugar (δH 4.32, d, J = 7.7 Hz) showed a 3J HMBC correlation to C-28 (δC 75.1), establishing the connectivity from the anomeric position of the second sugar unit to the 28-position of the aglycone. The spin system from the anomeric proton to the other protons of the second sugar was also clearly exhibited in a 1D TOCSY spectrum [H-1″ (selected): δH 4.32, d, J = 7.7 Hz; H-2″: δH 3.43, t, J = 9.6 Hz; H-3″: δH 3.51, t, J = 9.6 Hz; H-4″: δH 3.29, t, J = 9.6 Hz; H-5″: δH 3.22, m; H-6″: δH 3.65 and 3.85, m] in which all the protons had similar coupling constants to those of the corresponding protons in the first sugar. The second sugar unit was thus also identified as a glucoside. The presence of a substituent at the 2″-position was indicated by a 3J HMBC correlation from the H-1 proton of the third sugar unit (δH 4.62, d, J = 8.0 Hz) to C-2″ (δC 81.5), thus establishing the connectivity from C-1 (δC 104.8) to C-2″ via an oxygen bridge. The third sugar unit was identified as a glucoside as by its 1D TOCSY data [1D TOCSY: H-1 (selected): δH 4.62, d, J = 8.0 Hz; H-2: δH 3.16, t, J = 9.6 Hz; H-3: δH 3.20, t, J = 9.6 Hz; H-4: δH 3.30, t, J = 9.6 Hz; H-5: δH 3.20, m; H-6: δH 3.65 and 3.85, m]. The 13C NMR chemical shifts of the sophorose moiety at the 28-position matched those of justicioside E (Kanchanapoom et al., 2005). Hence, the structure of compound 1 was determined as shown, and was named terminaliaside A ((3β,21β,22α)-3-O-(3′-O-angeloylpyranoglucosyl)-21,22-dihydroxy-28-O-sophorosyl-16-oxoolean-12-ene).
Compound 2 was isolated as a colorless powder. Its positive ion HRFAB mass spectrum revealed a pseudo-molecular ion [(M+H)+] consistent with the molecular formula C29H25O9, requiring eighteen double-bond equivalents. Its 1H NMR spectrum in DMSO-d6 (Table 2) showed signals for thirteen protons between 6.30 and 8.00 ppm [δH 7.94, d, J = 9.7 Hz; 7.82, d, J = 16.2 Hz; 7.76, m (2H); 7.46, m (3H); 7.24, d, J = 1.7 Hz; 7.22, d, J = 8.0 Hz; 7.09, dd, J = 8.0, 1.7 Hz; 6.90, s; 6.85, d, J = 16.2 Hz; 6.33, d, J = 9.7 Hz;]. Signals for two oxygenated methines (δH 5.15, d, J = 8.0 Hz; 4.43, m), one oxygenated methylene (δH 3.40, m and 3.70, m), two methoxy groups (δH 3.75, s; 3.75, s), and one hydroxyl group (δH 5.33, dd, J = 5.3, 4.7 Hz) were also observed. The cis-coupling between H-3 (δH 6.33, d, J = 9.7 Hz) and H-4 (δH 7.94, d, J = 9.7 Hz) and the HMBC correlation (Figure 3) between H-4 (δH 7.94, d, J = 9.7 Hz) and C-5 indicated the existence of a 6,7,8-trisubstituted coumarin. Signals for a cinnamoyl group [H-7″: δH 7.82, d, J = 16.2 Hz; H-8″: δH 6.85, d, J = 16.2 Hz; mono-substituted benzene ring: δH 7.76, m (2H) and 7.46, m (3H)] were also observed in the 1H NMR spectrum of 2. H-7′ (δH 5.15, d, J = 8.0 Hz) in the 1,2,3-trioxygenated propanyl group exhibited 3J HMBC correlations to C-2′ and C-6′ of the 1,3,4-trisubstituted benzene ring (H-2′: δH 7.24, d, J = 1.7 Hz; H-5′: 7.22, d, J = 8.0 Hz; H-6′: δH 7.09, dd, J = 8.0, 1.7 Hz) and C-7 of the coumarin moiety, while H-8′ (δH 4.43, m) correlated to C-8 and C-1′, which indicated the existence of a 2,3-dihydro-1,4-dioxine group. The positions of the two methoxy groups were determined by the NOESY correlations between H-2′ (δH 7.24, d, J = 1.7 Hz;) and the methoxy at δH 3.75 ppm, and H-5 and the methoxy at δH 3.75 ppm (Figure 3). The 9′-OH (δH 5.33, dd, J = 5.3, 4.7 Hz) showed COSY correlation to H2-9′ (δH 3.40 & 3.70, m), therefore, the cinnamoyl group was deduced to be located at the 4′-position. The relative stereochemistries of H-7′ and H-8′ were determined as trans from the coupling constant between H-7′ and H-8′ (J = 8.0 Hz) and the NOESY correlation between H-7′ and H2-9′. The structure of the new lignan was thus assigned as 4′-O-cinnamoyl cleomiscosin A (2). A comparison of the 13C NMR data of 2 with those of cleomiscosin A (Ahmad et al., 2004) showed only fair agreement, probably in part because of the different solvents used (DMSO-d6 in the current work, and pyridine-d5 in the literature work). The 13C NMR spectrum of 2 was thus calculated using the ChemDraw software (Table 2), and the calculated spectrum was in excellent agreement with the observed spectrum except for the shift of C-3′, which was outside the standard deviation of the software (5 ppm).
Compounds 3 and 4 were determined as arjunglucoside I (Nandy et al., 1989) and sericoside (Zhou et al., 1992), respectively, by comparison of their 1H and 13C NMR and mass spectroscopic data with literature data.
Compounds 1–4 were tested in the A2780 assay. Compound 1 was the most active compound with an IC50 value of 1.2 μM, while compound 3 was weakly active with an IC50 value of 16.5 μM. Compounds 2 and 4 were both inactive with IC50 values >30 μM. It thus appears that the antiproliferative activity of olean-12-ene triterpene derivatives such as terminaliaside A (1) and gummiferaosides A-C (Cao et al., 2007a) is enhanced by substituents at the 3-, 16-, 21-, and 28-positions.
This work provides a further example of the importance of oleanane-type saponins as potential anticancer agents. Although saponins have not traditionally been regarded with favor by the pharmaceutical industry, the saponin OSW-1 is being actively investigated as a potential anticancer agent (Zhou et al. 2005), and several other saponins have shown useful activity in animal models (Bachran et al., 2008). Several of the saponins listed in this review are oleanane triterpene derivatives with sugar substitution at C-3 only (the soyasaponins), at C-3 and C-28 (gypsoside A), or at the C-3, C-21, and C-28 positions (avicin G). It is thus reasonable to infer that an oleanane-type saponin may eventually be approved for cancer chemotherapy.
Optical rotations were recorded on a JASCO P-2000 polarimeter. IR and UV spectra were measured on MIDAC M-series FTIR and Shimadzu UV-1201 spectrophotometers, respectively. NMR spectra were obtained on a JEOL Eclipse 500 for 1H, 13C, HMQC, and HMBC and an INOVA 400 spectrometer for TOCSY, COSY, and ROESY. Chemical shifts are given in δ (ppm), and coupling constants are reported in Hz. Mass spectra were obtained on a JEOL JMS-HX-110 instrument, in the positive-ion mode. HPLC was performed on a Shimadzu LC-10AT instrument with a semi-preparative C18 Varian Dynamax column (5 μm, 250 × 10 mm) and a preparative C18 Varian Dynamax column (8 μm, 250 × 21.4 mm).
Roots of Terminalia tropophylla H. Perrier (Combretaceae) were collected on October 8th, 2006 by Richard Randrianaivo et al. at an elevation of 52 m in the Lamiranta forest, Andavakoera, 15 km from National Route 6, Madagascar. The sample was collected from a dry degraded forest. The collection coordinates were 13°05′09″S 049°13′35″E, and the voucher number was RIR 1397. The sample was a large tree 25 m high, diameter at breast height 85 cm, with white flowers. It was abundant along a river. Voucher specimens have been deposited at herbaria of the Centre National d’ Application des Recherches Pharmaceutiques, Madagascar (CNARP); the Parc Botanique et Zoologique de Tsimbazaza, Madagascar (TAN); the Missouri Botanical Garden, St. Louis, Missouri (MO); and the Muséum National d’Histoires Naturelles, Paris, France (P).
Dried roots of Terminalia tropophylla (250 g) were ground in a hammer mill, then extracted with EtOH by percolation for 24 h at rt to give the crude extract MG 4035 (8.5 g), of which 2.8 g was made available to Virginia Polytechnic Institute and State University. Extract MG 4035 (2.2 g; IC50 11 μg/mL, A2780) was suspended in aqueous MeOH (MeOH-H2O, 9:1, 100 mL) and extracted with n-hexane (3 × 100 mL portions). The aqueous layer was then diluted to 70% MeOH with H2O and extracted with CH2Cl2 (3 × 100 mL portions). The aqueous MeOH extract (1.7 g) was active with IC50 value of 10 μg/mL against A2780, while both the hexane and CH2Cl2 extracts were inactive. The aqueous MeOH extract was filtered through a C18 open column and then subjected to HPLC separation on a C18 column (28% MeCN/H2O, 10 mL/min) to give 11 fractions. Fractions 6 and 10 yielded compounds 3 (tR 14.2 min, 3 mg) and 4 (tR 23.6 min, 5 mg). Compound 1 (tR 18 min, 0.9 mg) was obtained by HPLC of fraction 11 using C18 HPLC (33% MeCN/H2O, 2 mL/min). Compound 2 (0.5 mg) was obtained from the CH2Cl2 extract after repeated chromatography on silica gel.
White solid; [α]25D −6.1 (c 0.18, MeOH); UV (MeOH) λmax (log ε) 208 (4.2) nm; IR (film) νmax 3366, 1698, 1025 cm−1; 1H NMR (500 MHz, CD3OD), see Table 1; 13C NMR (125 MHz, CD3OD), see Table 1; HRFABMS m/z 1079.5376 (calcd for C53H84O21Na, 1079.5403).
This project was supported by the Fogarty International Center, the National Cancer Institute, the National Science Foundation, the National Heart, Lung and Blood Institute, the National Institute of Mental Health, the Office of Dietary Supplements, and the Office of the Director of NIH, under Cooperative Agreement U01 TW000313 with the International Cooperative Biodiversity Groups, and this support is gratefully acknowledged. We thank Mr. B. Bebout for obtaining the mass spectra. Field work essential for this project was conducted under a collaborative agreement between the Missouri Botanical Garden and the Parc Botanique et Zoologique de Tsimbazaza and a multilateral agreement between the ICBG partners, including the Centre National d’Applications des Recherches Pharmaceutiques. We gratefully acknowledge courtesies extended by the Government of Madagascar (Ministère des Eaux et Forêts).
Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.phytochem. 2009.XX.XXX.
†Biodiversity Conservation and Drug Discovery in Madagascar, Part 39. For Part 38, see Cao et al., 2009.
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