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Triterpenoid glycosides from the leaves of Meliosma henryi
Phytochemistry xxx (2014) xxx–xxx
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Triterpenoid glycosides from the leaves of Meliosma henryi Abdulmagid Alabdul Magid a,⇑, Hamid Morjani b, Dominique Harakat c, Claudie Madoulet d, Vincent Dumontet e, Catherine Lavaud a a
Laboratoire de Pharmacognosie, ICMR-UMR 7312, SFR CapSanté, Université de Reims Champagne-Ardenne, Bat. 18, BP 1039, 51687 Reims Cedex 2, France Laboratoire Matrice Extracellulaire Dynamique Cellulaire, ICMR-UMR 7312 CNRS, UFR de Pharmacie, SFR CapSanté, Université de Reims Champagne-Ardenne, 51 rue Cognacq-Jay, 51096 Reims Cedex, France c Service Commun d’Analyses, ICMR-UMR 7312 CNRS, Bat. 18, BP 1039, 51687 Reims Cedex 2, France d Laboratoire de Biochimie, Faculté de Pharmacie, Université de Reims Champagne-Ardenne, 51096 Reims Cedex, France e Institut de Chimie des Substances Naturelles, UPR 2301 CNRS, Avenue de la Terrasse, 91198 Gif-sur-Yvette, France b
a r t i c l e
i n f o
Article history: Received 19 June 2014 Received in revised form 24 October 2014 Available online xxxx Keywords: Meliosma henryi Diels Sabiaceae Triterpenoid glycosides Cytotoxic activity
a b s t r a c t Seven triterpenoid glycosides, named meliosmosides A–G, were isolated from the leaves of Meliosma henryi Diels (Sabiaceae). Their structures were elucidated by different spectroscopic methods including 1D and 2D NMR experiments as well as HRESIMS analysis. Isolated compounds were evaluated for their cytotoxic activity against KB cell line. Ó 2014 Elsevier Ltd. All rights reserved.
1. Introduction
2. Results and discussion
The Sabiaceae is a small family including three genera; Sabia, Ophiocaryon, and Meliosma (Wanntorp and Ronse De Craene, 2007). Meliosma with about 50–70 species has the widest distribution occurring in Asia and America, while Sabia with about 19–50 species is restricted to Asia, and Ophiocaryon with about 7 species is only found in tropical South America (Furness et al., 2007; Ronse De Craene and Wanntorp, 2008). A review of literature showed that the phytochemistry of this family has not been exhaustively studied. A short report about Meliosma pungens mentioned the presence of oleanolic acid, and nine triterpenoid glycosides were isolated from the bark of M. lanceolata (Abe et al., 1996; Rao et al., 1979). A phytochemical investigation of Meliosma henryi leaves led to the isolation of seven new triterpenoid glycosides named meliosmosides A–G. Their structures were elucidated by different spectroscopic methods including 1D NMR (1H, 13C) and 2D NMR experiments (COSY, TOCSY, ROESY, HSQC, HSQC-TOCSY and HMBC) as well as HRESIMS analysis. The antiproliferative activity of isolated compounds against KB cell line was assessed using the MTS method.
The 80% MeOH extract of the leaves of M. henryi was subjected to a Diaion HP-20 column eluted with different gradients of water and methanol mixture to give five fractions (A–E). Further purification using combination of silica gel column chromatography, RP-18 column chromatography, and semi-preparative HPLC yielded seven triterpenoid saponins as white amorphous powders (1–7). The sugar composition was determined by comparative TLC after acid hydrolysis of the saponin mixture (fraction D and E of the 80% MeOH extract) as glucose (glc), galactose (gal), arabinose (ara), apiose (api) and rhamnose (rha). The common D- configuration for the glucose, galactose, and apiose moieties and the L- configuration for the rhamnose and arabinose sugars were verified by measurement of the optical rotation of each purified sugar. The positive-ion high-resolution electrospray ionization mass spectrum (HREIMS) analysis of meliosmoside A (1) revealed a molecular formula of C45H70O16, showing an ion [M+Na]+ at m/z 889.4572 (calcd for C45H70O16Na, 889.4562). The 1H NMR spectrum of the aglycone part of 1 displayed signals assignable to five tertiary methyl groups at dH 0.83, 0.88, 0.99, 1.08, and 1.22 (3H, s), two exo-methylene protons at dH 4.64, (2H, brs), an olefinic proton at dH 5.35 (t, J = 3.8 Hz), and a proton at dH 3.17 (dd, J = 11.9, 4.4 Hz) assigned to H-3 (Table 1). The 13C NMR spectrum of the aglycone part showed 29 carbon signals of which, four typical sp2-hybridized carbon signals at dC 107.5, 124.3, 144.2, and 149.3 and one
⇑ Corresponding author. Tel.: +33 3 26 91 82 08. E-mail address: [email protected] (A. Alabdul Magid). http://dx.doi.org/10.1016/j.phytochem.2014.10.035 0031-9422/Ó 2014 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Alabdul Magid, A., et al. Triterpenoid glycosides from the leaves of Meliosma henryi. Phytochemistry (2014), http:// dx.doi.org/10.1016/j.phytochem.2014.10.035
2
1
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30
2
3
dH (J in Hz)
dC
dH (J in Hz)
1.01, 1.65, 1.73, 1.88, 3.17,
td (11.9, 3.5) m td (11.9, 3.5) m dd (11.9, 4.4)
39.8
0.99, 1.66, 1.17, 1.87, 3.17,
m dd (13.4, 3.1) brd (13.4) m dd (11.5, 4.3)
0.81, 1.44, 1.59, 1.35, 1.53,
brd (13.6) td (11.7, 3.1) m brd (11.7) m
0.82, 1.45, 1.59, 1.35, 1.54,
m dd (12.5, 3.3) m brd (12.5) td (12.5, 3.0)
1.64, t (9.8) 1.94, m 5.35, t (3.8)
1.18, 1.88, 1.87, 2.20,
brt (13.5) m m dt (13.5, 4,0)
2.76, dd (13.5, 5.0) 2.11, dd (13.5, 4.9) 2.59, t (13.5) 2.17, 2.26, 1.56, 1.94, 1.08, 0.88, 0.99, 0.83, 1.22,
brd (13.3) td (13.3, 3.9) td (13.3, 4.9) m s s s s s
4.64, brs
27.0 90.6 40.2 57.0 19.3 33.9 40.7 49.0 37.9 24.6 124.3 144.2 42.9 28.9 24.1 48.3 48.6 42.6 149.3 30.9 38.4 28.6 17.0 16.1 17.8 26.4 177.2 107.5
1.63, t (10.5) 1.95, m 5.35, t (3.5)
1.74, 1.88, 1.88, 2.21,
brt (13.1) m m td (13.1, 3.8)
2.77, dd (13.5, 4.9) 2.11, dd (13.5, 4.9) 2.59, t (13.6) 2.18, 2.27, 1.57, 1.94, 1.08, 0.88, 0.99, 0.83, 1.22,
m td (12.2, 3.6) m m s s s s s
4.65, brs
dC 39.8 28.9 90.7 40.2 57.0 19.3 33.9 40.8 49.0 37.9 24.6 124.4 144.2 42.9 27.0 24.1 48.3 48.6 42.6 149.4 30.9 38.5 28.6 17.0 16.1 18.0 26.3 177.2 107.5
4
dH (J in Hz) 1.00, 1.66, 1.17, 1.90, 3.18,
m m brd (14.1) m dd (11.5, 4.3)
0.82, 1.44, 1.60, 1.35, 1.53,
m td (11.7, 3.0) m brd (11.7) m
1.61, m 1.95, m 5.35, t (3.5)
1.73, 1.87, 1.88, 2.21,
brt (12.3) m m td (14.4, 3.2)
2.77, dd (13.4, 4.9) 2.11, dd (13.4, 5.1) 2.59, t (13.5) 2.18, 2.27, 1.59, 1.94, 1.05, 0.88, 0.99, 0.83, 1.22,
m td (13.2, 4.4) m m s s s s s
4.65, brs
dC 39.9 28.9 90.5 40.2 57.1 19.3 33.9 40.8 49.0 37.9 24.7 124.4 144.2 42.9 27.0 24.2 48.3 48.6 42.6 149.4 30.9 38.5 28.6 17.0 16.1 17.8 26.4 177.3 107.5
5
dH (J in Hz) 1.00, 1.63, 1.16, 1.86, 3.18,
m m m m dd (11.6, 4.6)
0.82, m 1.44, td (12.6, 3.2) 1.60, m 1.35, brd (12.6) 1.53,dd (12.6, 3.9) 1.61, t (10.4) 1.95, m 5.35, t (3.5)
1.73, 1.87, 1.88, 2.21,
m m m td (13.1, 3.5)
2.77, dd (13.6, 4.2) 2.11, dd (13.6, 5.0) 2.59, t (13.6) 2.20, 2.27, 1.59, 1.87, 1.08, 0.87, 0.99, 0.83, 1.22,
dd (13.2, 3.5) td (13.2, 3.5) m m s s s s s
4.65, brs
dC 39.9 28.9 90.4 40.2 57.1 19.3 33.9 40.8 49.0 37.9 24.6 124.4 144.2 42.9 27.1 24.2 48.3 48.6 42.6 149.4 30.9 38.5 28.5 17.0 16.1 17.8 26.4 177.3 107.5
6
dH (J in Hz) 1.03, 1.67, 1.17, 1.88, 3.15,
m dt (13.1, 3.3) dt (13.1, 3.3) m dd (11.6, 4.4)
0.82, 1.45, 1.60, 1.36, 1.55,
m td (12.3, 2.1) m brd (12.4) m
1.61, t (11.1) 1.95, m 5.35, t (3.7)
1.76, 1.87, 1.88, 2.21,
brt (11.1) m m td (13.8, 3.3)
2.77, dd (13.8, 5.1) 2.11, dd (13.8, 4.9) 2.59, t (13.8) 2.18, 2.26, 1.57, 1.92, 1.08, 0.88, 0.99, 0.83, 1.22,
m td (13.3, 4.1) m m s s s s s
4.65, brs
dC 39.9 28.9 90.6 40.2 57.0 19.4 33.9 40.8 49.0 37.9 24.5 124.3 144.2 42.9 27.0 24.1 48.3 48.6 42.6 149.4 30.8 38.4 28.6 17.1 16.1 18.0 26.4 177.2 107.5
7
dH (J in Hz) 0.98, 1.63, 1.69, 1.88, 3.14, – 0.81, 1.37, 1.53, 1.31, 1.47, – 1.56, – 1.86, 5.28, – – 0.98, 1.86, 1.57, 1.87, – 2.88, 0.99, 1.62, – 1.12, 1.32, 1.50, 1.69, 1.05, 0.87, 0.98, 0.83, 1.18, – 0.96, 0.99,
m m m m dd (11.3, 4.6) m m m m m m m t (3.5)
m m m m dd (12.7, 4.0) m m m m m m s s s s s s s
dC 40.1 26.44 90.6 39.8 57.1 18.9 34.7 41.0 49.0 37.9 24.2 121.8 146.3 43.2 28.6 24.2 48.0 43.0 47.7 31.5 35.1 33.9 28.3 17.1 15.8 17.8 26.4 178.1 33.8 24.2
dC 1.04, 1.64, 1.45, 1.87, 3.15, – 0.83, 1.43, 1.58, 1.35, 1.54, – 1.63, – 1.94, 5.31, – – 1.49, 1.86, 1.65, 2.00, – 2.89, 1.41, 2.18, – 1.49, 1.85, 1.63, 1.79, 1.05, 0.87, 0.98, 0.84, 1.19, –
m m m m dd (11.7, 4.4) m m m m m m brd (7.0) t (3.3)
m td (13.8, 3.3) m td (13.8, 2.6) dd (13.7, 4.0) m t (13.7) m m m m s s s s s
1.24, s
39.8 27.0 90.5 40.2 57.0 19.4 34.0 40.6 49.6 37.9 24.6 124.5 144.3 42.9 28.7 24.2 47.4 41.6 40.9 43.6 29.5 32.5 28.6 17.1 16.0 17.7 26.6 181.2 179.3 19.8
A. Alabdul Magid et al. / Phytochemistry xxx (2014) xxx–xxx
Please cite this article in press as: Alabdul Magid, A., et al. Triterpenoid glycosides from the leaves of Meliosma henryi. Phytochemistry (2014), http:// dx.doi.org/10.1016/j.phytochem.2014.10.035
Table 1 NMR spectroscopic data of the aglycone moieties for compounds 1–7 (500 MHz, CD3OD).
A. Alabdul Magid et al. / Phytochemistry xxx (2014) xxx–xxx
carbonyl carbon signal dC 177.2. Taken together, these data were indicative of a typical 30-nor-oleana-12,20(29)-dien-28-oic acid. This assumption was confirmed by analysis of the COSY, ROESY, HSQC-Jmod and HMBC spectra which allowed the full assignment of the proton and carbon resonances of the aglycone (Table 1). The deshielding effect observed at H-18 signal (dH 2.76), due to magnetic anisotropy effect of carbonyl at C-28, is in agreement with a cis fusion between D and E rings. In the ROESY spectrum, correlations observed between H-27/H-19ax (dH 2.59, t, J = 13.5 Hz), H-27/H-16ax (dH 2.20, dt, J = 13.5–4.0 Hz), H-27/H-9, and H-19ax/H-16ax) indicated that E ring must therefore be a-oriented. The ROESY experiment revealed also several correlations, including H-3/H-5 and H-5/H-9 which indicated that the OH-3 group was in a b-orientation and H-25 with H-24 and H-26 indicating their b-orientation. The chemical shifts values for protons and carbons were in accordance with the literature data of 3bhydroxy-30-nor-oleana-12,20-(29)-dien-28-oic acid (akebonoic acid) (Hamed et al., 2011; Wang et al., 2012; Zhao et al., 2014). 1 H and 13C NMR spectra showed that akebonoic acid was common as aglycone to saponins 1–5. Full assignments of its proton and carbon resonances for each compound (2–5) were achieved by analysis of the COSY, HSQC-Jmod and HMBC spectra (Table 1). The sugar part of 1 consisted of three residues as evidenced by 1 H and 13C NMR spectra which displayed three anomeric protons at dH 4.30 (d, J = 6.7 Hz), 4.31 (d, J = 6.6 Hz), and 5.38 (d, J = 8.1 Hz), whose carbon resonances were assigned by HSQC-Jmod experiments at dC 107.1, 104.7 and 95.7, respectively. Starting from the anomeric proton at dH 5.38 (glc), the NMR signals belonging to the same system were assigned to a 6-monosubstituted D-glucopyranose (dC-6 69.2) (Gao et al., 2009) (Table 2). This assignment was further supported by the HMBC and the COSY experiments. The b-anomeric configuration of the D-glucopyranose unit was determined by the vicinal J1–2 trans coupling constant (8.1 Hz). The two remaining sugar units were identified as terminal L-arabinopyranoses (ara-I and ara-II) by their typical pattern in the COSY spectrum. Analysis of HSQC-Jmod and HMBC spectra, and comparison of the 13C NMR chemical shifts with those of related systems reported in the literature confirmed their identification as a-L-arabinopyranoses (Ishii et al., 1981) (Table 2). The L-arabinopyranose units were determined to be in an a-configuration on the basis of the J1–2 values (6.7 and 6.6 Hz) and this was confirmed by observation of ROESY correlations between H-1 and H-3 and between H-1 and H-5 for both arabinoses. The first arabinose unit (ara-I) was linked to C-3 of the aglycone as shown by the long-range correlation in the HMBC spectrum between dH 3.17 (H-3 of aglycone) and dC 107.1 (C-1 of ara-I). The HMBC spectrum showed also connectivities from H-1-ara-II (dH 4.31) to C-6-glc (dC 69.2), and from H-1-glc (dH 5.38) to C-28 of the aglycone (dC 177.2). Consequently, the structure of meliosmoside A (1) was concluded to be 3b-O-a-L-arabinopyranosyl-30-nor-oleana-12,20-(29)-dien-28-oic acid-28-Oa-L-arabinopyranosyl-(1 ? 6)-b-D-glucopyranoside. The HRESIMS of meliosmoside B (2) indicated a C50H78O20Na molecular formula, as deduced from the [M+Na]+ ion at m/z 1021.4976 (calcd for 1021.4984) in the positive ion mode. The MS2 experiment of this ion gave positive fragment at m/z 889.5 [M+Na132]+ suggesting a supplementary pentose unit compared to 1. This hypothesis was confirmed by the presence of four anomeric proton and carbon signals in the 1H and 13C NMR spectra. Severe overlap of some proton and carbon resonances requested the use of the HSQC-TOCSY experiment to map the spin systems. The detailed analysis of 1D and 2D NMR spectra led to the identification as in 1 of a 6-monosubstituted b-D-glucopyranose (glc, dC-6 69.4), a terminal a-L-arabinopyranose (ara-I), and a 4-monosubstituted a-L-arabinopyranose (ara-II, dC-4 77.4) (Table 2). The supplementary sugar residue showed NMR signals for two methines and two methylenes groups, in addition to a quaternary carbon, in
3
agreement with an apiofuranose moiety (Ishii and Yanagisawa, 1998; Eskander et al., 2006). The deshielded chemical shifts of the anomeric signals at dH 5.15 (d, J = 2.7 Hz) and dC 112.1 and the HMBC correlations observed between anomeric proton H-1 with its C-3 and C-4 indicate that this sugar residue is a b-erythroapiofuranose (Table 2) and the most commonly D- configuration for apiofuranose was assumed (Ishii and Yanagisawa, 1998). The sequence of the trisaccharide chain at C-28 was deduced from the HMBC experiment, as long-range correlations were observed between H-1-api (dH 5.15)/C-4-ara-II (dC 77.4), H-1-ara-II (dH 4.29)/C-6-glc-(dC 69.4), and H-1-glc (dH 5.38)/C-28 of akebonoic acid (dC 177.2). The glycosidic chain at C-3 was constituted by one a-L-arabinopyranose unit (ara-I) as indicated by the cross-peak between H-1-ara-I (dH 4.31) and C-3 of the akebonoic acid (dC 90.7). These findings led to the identification of meliosmoside B (2) as 3b-O-a-L-arabinopyranosyl-30-nor-oleana-12,20-(29)-dien28-oic acid-28-O-b-D-apiofuranosyl-(1 ? 4)-a-L-arabinopyranosyl-(1 ? 6)-b-D-glucopyranoside. For the three following meliosmosides C-E (3–5), the analysis of their 2D NMR spectra (COSY, HSQC-Jmod, HMBC, and ROESY) and comparison of the 1H and 13C NMR values of oligosaccharide part with those of 2, indicated that 3–5 contained the same ester trisaccharide chain b-D-apiofuranosyl-(1 ? 4)-a-L-arabinopyranosyl(1 ? 6)-b-D-glucopyranoside linked to C-28 of akebonoic acid whereas an a-L-arabinopyranosyl was linked to C-3 of the aglycone (Tables 1 and 2). Meliosmoside C (3) displayed a molecular ion peak [M+Na]+ at m/z 1153.5400 in the positive HRESIMS, in agreement with a molecular formula of C55H86O24Na (calcd for 1153.5407). The MS2 experiment of this ion gave positive fragment at 1021.6 [M+Na132]+, suggesting a supplementary pentose unit compared to 2. The analysis of the 2D NMR spectra of 3 led to the identification of a supplementary a-L-arabinopyranose unit (ara-III) in terminal position (Table 2). This arabinose was linked to C-2-ara-I as deduced from the HMBC correlation observed between H-1-ara-III (dH 4.53) and C-3-ara-I (dC 83.4). Thus, meliosmoside C (3) was concluded to be 3b-O-a-L-arabinopyranosyl(1 ? 3)-a-L-arabinopyranosyl-30-nor-oleana-12,20-(29)-dien-28-oic acid-28-Ob-D-apiofuranosyl-(1 ? 4)-a-L-arabinopyranosyl-(1 ? 6)b-D-glucopyranoside. Meliosmoside D (4) showed a molecular ion peak [M+Na]+ at m/ z 1183.5524 (calcd for C56H88O25Na, 1183.5512), in the positive HRESIMS. Analysis of the 1H and 13C NMR spectra of compound 4 revealed the presence of five sugar units as in 3. Comparison of its 1H and 13C NMR spectra with those of 3 showed the absence of the ara-III moiety. Instead, a b-D-galactopyranose unit (gal) was identified starting from the anomeric proton dH 4.53 (d, J = 7.7 Hz), characterized by the large coupling constants JH-1,H-2 and JH-2,H-3 and the small coupling constant between H-3 and H4 (JH3,H4 = 3.2 Hz) as summarized in Table 2. In the HMBC spectrum of 4, the correlation of H-1-gal with C-3-ara-I (dC 83.9) and H-1ara-I with C-3 of the aglycone (dC 90.5) indicated the linkage of a disaccharide chain b-D-galactopyranosyl-(1 ? 3)-a-L-arabinopyranoside at C-3 of akebonoic acid. The structure of meliosmoside D (4) was therefore elucidated as 3b-O-b-D-galactopyranosyl(1 ? 3)-a-L-arabinopyranosyl-30-nor-oleana-12,20-(29)-dien-28oic acid-28-O-b-D-apiofuranosyl-(1 ? 4)-a-L-arabinopyranosyl(1 ? 6)-b-D-glucopyranoside. Meliosmoside E (5) was assigned to the molecular formula C56H88O24Na, as determined from the molecular ion peak at m/z 1167.5555 [M+Na]+ in the positive ion mode HRESIMS. The 1D and 2D NMR spectra of 5 compared with those of 4, indicated that a-L-arabinopyranose (ara-I) which was C-3 monosubstituted in 4, is here C-2 monosubstituted (dC-2 76.8) in 5 (Table 2). The fifth sugar unit with its anomeric proton resonating at dH 5.13 (d, J = 1.7 Hz) was identified as a terminal a-L-rhamnopyranose (rha)
Please cite this article in press as: Alabdul Magid, A., et al. Triterpenoid glycosides from the leaves of Meliosma henryi. Phytochemistry (2014), http:// dx.doi.org/10.1016/j.phytochem.2014.10.035
4
1
Ara-I 1 2 3 4 5
2
3
4
5
6
7
dH (J in Hz)
dC
dH (J in Hz)
dC
dH (J in Hz)
dC
dH (J in Hz)
dC
dH (J in Hz)
dC
dH (J in Hz)
dC
(at C-3) 4.30, d (6.7) 3.60, dd (8.4, 6.7) 3.56, dd (8.4, 3.1) 3.84, brt (3.1) 3.53, m 3.87, dt (12.2, 3.4)
107.1 72.8 74.1 69.5 66.5
4.31, 3.57, 3.53, 3.83, 3.56, 3.86,
107.2 72.8 74.3 69.5 66.4
4.31, 3.73, 3.63, 4.00, 3.59, 3.89,
107.0 72.1 83.4 69.7 66.9
4.31, 3.73, 3.66, 4.07, 3.58, 3.88,
107.0 72.1 83.9 69.7 66.6
4.57, 3.57, 3.53, 3.82, 3.51, 3.87,
104.8 76.8 73.0 68.4 63.7
4.57, 3.80, 3.76, 3.82, 3.51, 3.87,
104.9 76.8 73.0 68.4 63.7
d (6.7) dd (8.9, 6.7) dd (8.9, 3.4) t (3.4) m dd (12.3, 3.3)
d (7.4) dd (9.1, 7.4) dd (9.1, 3.3) m dd (12.4, 3.0) dd (12.4, 3.1)
ara-III (at ara-I C-3) 4.53, d (7.0) 3.67, dd (9.0, 7.0) 3.58, dd (9.0, 3.4) 3.82, m 3.82, m 3.88, dd (12.8, 2.4)
1 2 3 4 5
106.0 72.8 74.1 69.6 66.5
6
d (7.6) dd (9.1, 7.6) dd (9.1, 3.2) brd (3.2) dd (11.1, 1.5) dd (11.1, 2.4)
gal (at ara-I C-3) 4.53, d (7.7) 3.64, dd (9.6, 7.7) 3.58, dd (9.6, 3.2) 3.85, d (3.2) 3.57, brt (6.8)
106.1 73.0 74.6 70.3 76.7
d (6.4) dd (8.3, 6.4) dd (8.3, 3.3) m brd (11.8) dd (11.8, 6)
rha (at ara-I C-2) 5.13, d (1.7) 3.92, dd (3.4, 1.7) 3.72, dd (9.8, 3.4) 3.42, t (9.8) 3.83, m
102.0 72.1 72.1 73.9 70.2
d (6.5) dd (9.1, 6.5) dd (9.1, 3.0) m dd (11.9, 2.7) dm (11.9)
rha (at ara-I C-2) 5.13, d (1.4) 3.91, dd (3.5, 1.4) 3.72, dd (9.6, 3.5) 3.41, t (9.6) 3.83, m
102.0 72.7 72.7 73.9 70.6
rha (at ara-I C-2) 5.13, d (1.3) 3.92, dd (3.2, 1.3) 3.72, dd (9.5, 3.2) 3.42, t (9.5) 3.83, m
62.5
1.25, d (6.2)
17.8
1.25, d (6.2)
17.8
1.25, d (6.2) (at C-29) 4.91, d (8.0) 3.40, t (8.0) 3.47, dd (9.0, 8.0) 3.40, t (9.0) 3.41, m 4.26, dd (11.9, 5.3) 4.44, dd (11.9, 1.7)
(at C-28) 5.38, d (8.1) 3.37, t (8.1) 3.45, dd (9.0, 8.1) 3.44, t (9.0) 3.54, m 3.74, dd (11.4, 5.0) 4.07, dd (11.4, 2.3)
95.7 73.7 78.0 71.1 77.5 69.2
5.38, 3.35, 3.43, 3.44, 3.53, 3.79, 4.07,
d (8.1) t (8.1) dd (8.7, 8.1) t (8.7) m dd (11.5, 4.7) dd (11.5, 2.1)
95.7 73.7 78.0 71.1 77.7 69.4
5.38, 3.35, 3.44, 3.43, 3.53, 3.74, 4.07,
d (8.1) t (8.1) dd (9.4, 8.1) t (9.4) m dd (11.6, 4.6) dd (11.6, 2.2)
95.7 73.8 78.1 71.2 78.1 69.4
5.38, 3.38, 3.45, 3.43, 3.53, 3.73, 4.07,
d (8.1) t (8.1) dd (9.2, 8.1) t (9.2) m dd (12.2, 5.0) dd (12.2, 2.2)
95.7 73.8 78.1 71.2 77.7 69.4
5.38, 3.35, 3.45, 3.43, 3.53, 3.74, 4.07,
d (8.1) t (8.1) dd (8.1, 9.4) t (9.4) m dd (11.5, 4.7) dd (11.5, 2.2)
95.7 73.9 78.1 71.2 77.6 69.4
5.39, 3.36, 3.44, 3.42, 3.53, 3.76, 4.08,
d (8.1) t (8.1) t (8.1) dd (8.6, 8.1) m dd (11.7, 5.2) dd (11.7, 2.0)
95.7 73.9 78.1 71.2 77.6 69.4
ara-II 1 2 3 4 5
(at glc-C-6) 4.31, d (6.6) 3.63, dd (8.4, 6.6) 3.54, dd (8.4, 3.3) 3.83, t (3.3) 3.56, m 3.87, dd (12.2, 3.4)
104.7 72.3 74.3 69.3 66.4
4.29, 3.55, 3.59, 3.82, 3.86, 4.03,
d (6.6) m dd (8.4, 3.0) t (3.0) dd (12.3, 3.3) dd (12.3, 3.0)
104.9 72.7 74.3 77.4 66.2
4.29, 3.58, 3.61, 3.82, 3.51, 4.02,
d (6.7) dd (9.0, 6.7) dd (9.0, 3.1) m brd (12.5) dd (12.5, 3.2)
104.9 72.7 73.7 77.4 66.2
4.29, 3.58, 3.61, 3.82, 3.51, 4.02,
d (6.6) m dd (9.0, 3.2) brd (3.2) dd (11.9, 4.6) dd (11.9, 1.9)
104.9 72.7 73.7 77.4 66.2
4.29, 3.57, 3.61, 3.83, 3.51, 4.02,
d (6.6) dd (9.0, 6.6) dd (9.0, 3.4) m dd (12.2, 2.9) dd (12.2, 3.4)
104.9 72.7 74.3 77.4 66.2
4.31, 3.57, 3.61, 3.82, 3.52, 4.03,
d (6.7) dd (9.0, 6.7) dd (9.0, 3.4) m dd (12.3, 1.4) dd (12.3, 2.9)
104.8 72.7 74.3 77.4 66.2
104.8 76.8 73.1 70.2 63.8
102.0 72.1 72.1 73.9 68.4 18.0
104.4 75.3 77.6 71.1 76.0 64.0
Maltol
6 7
5
d (6.6) dd (8.4, 6.6) dd (8.4, 3.2) m dd (11.7, 2.3) dd (11.7, 5.9)
3.74, m 3.77, dd (11.5, 6.8)
glc 1 2 3 4 5 6
api 1 2 3 4
4.57, 3.80, 3.76, 3.85, 3.51, 3.87,
(at ara-II-C-4) 5.15, 4.01, – 3.78, 4.05, 3.64,
d (2.7) d (2.7) d (9.6) d (9.6) s
112.1 78.0 80.5 75.0 65.6
5.15, 4.01, – 3.78, 4.04, 3.64,
d (2.7) d (2.7) d (9.7) d (9.7) s
112.0 78.1 80.5 75.0 65.6
5.15, 4.01, – 3.78, 4.04, 3.64,
d (2.7) d (2.7) d (9.7) d (9.7) s
112.0 78.1 80.5 75.0 65.6
5.15, 4.01, – 3.79, 4.04, 3.64,
d (2.7) d (27) d (9.7) d (9.7) s
112.0 78.1 80.5 75.0 65.6
5.15, 4.01, – 3.78, 4.04, 3.64,
d (2.7) d (2.7) d (9.6) d (9.6) s
112.0 78.1 80.5 75.0 65.6
6.48, d (5.3)
164.5 143.0 176.9 117.5
8.05, d (5.3) 2.43, s
159.1 16.0
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Please cite this article in press as: Alabdul Magid, A., et al. Triterpenoid glycosides from the leaves of Meliosma henryi. Phytochemistry (2014), http:// dx.doi.org/10.1016/j.phytochem.2014.10.035
Table 2 NMR spectroscopic data of the sugar moieties for compounds 1–7 (500 MHz, CD3OD).
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with its methyl group at dH-6 1.25 (d, J = 6.2 Hz) and dC-6 17.8 (Backinowsky et al., 1980) (Table 2). In the ROESY spectrum, the rOe effects observed between H-1-rha/H-2-ara-I and between H-1-ara-I/H-3 aglycone indicated that the disaccharide a-L-rhamnopyranosyl-(1 ? 2)-a-L-arabinopyranoside was linked at C-3 of akebonoic acid. These data was further supported by the correlations observed on the HMBC spectrum of 5 between H-1-rha and C-2-ara-I (dC 76.8) and between H-1-ara-I and C-3 of the aglycone (dC 90.6). The structure of meliosmoside E (5) was therefore identified as 3b-O-a-L-rhamnopyranosyl(1 ? 2)-a-L-arabinopyranosyl-30-nor-oleana-12,20-(29)-dien-28-oic acid-28-O b-D-apiofuranosyl-(1 ? 4)-a-L-arabinopyranosyl-(1 ? 6)b-D-glucopyranoside. Meliosmoside F (6) was closely related to compound 5 for the glycosidic part. Comparison of the 13C NMR data of 6 to those of 5 and analysis of the 2D NMR spectra of 6 showed that both possessed the same two sugar chains (Table 2). HRESIMS data
5
([M+Na]+ at m/z 1183.5885) suggested that the molecular formula of 6 was C57H92O24. The difference was the absence of the exocyclic methylene group (C-29) of the aglycone. An extra geminaldimethyl moiety was detected in 6 (dH 0.96 and 0.99). The HMBC spectrum revealed proper correlations for these methyl groups: Me-30 (dH 0.99) exhibited correlations with C-20 (dC 31.5), C-21 (dC 35.1), C-19 (dC 47.7), as well as C-29 (dC 33.8). The chemical shifts values for all protons and carbons of the aglycone moiety (Table 1) were in accordance with the literature data of oleanolic acid (Agrawal and Jain, 1992). Based on the abovementioned evidence, the structure of compound 6 was assigned as 3b-O-a-L-rhamnopyranosyl-(1 ? 2)-a-L-arabinopyranosyl-oleanolic acid-28-O-b-D-apiofuranosyl-(1 ? 4)-a-L-arabinopyranosyl(1 ? 6)-b-D-glucopyranoside. Meliosmoside G (7) gave an ion peak at m/z 1057.5000 [M+Na]+ in the positive HRESIMS, from which the molecular formula C53H78O20 was deduced. Comparison of the 1H and 13C NMR data
Fig. 1. Chemical structures of saponins 1–7, isolated from Meliosma henryi.
Please cite this article in press as: Alabdul Magid, A., et al. Triterpenoid glycosides from the leaves of Meliosma henryi. Phytochemistry (2014), http:// dx.doi.org/10.1016/j.phytochem.2014.10.035
6
A. Alabdul Magid et al. / Phytochemistry xxx (2014) xxx–xxx
of the aglycone part in 7 with those in 6 revealed superimposable signals for the protons and carbons in rings A–D, but differences were observed in the ring E. In the HMBC experiment methyl protons at dH 1.24 (Me-30) exhibited 3JH-C correlations with C-19 (dC 40.9), C-20 (dC 43.6), C-21 (dC 29.5), and a carboxyl group resonating at dC 179.3 (Me-29), suggesting the replacement of the methyl group at C-29 in 6 by a carboxyl group in 7. The b-axial orientation of the methyl group (Me-30) was established by a rOe effect with H-18 observed in the ROESY spectrum; the 20-position carboxylic acid group (C-29) must therefore be a-equatorial oriented. Further analysis of the 2D NMR data was used to identify the aglycone as serratagenic acid (or 3b-hydroxyolean-12-ene-28,29-dioic acid) (Jitsuno and Mimaki, 2010; Yu et al., 1995). The 1H and 13C NMR spectra of 7 displayed three sugar anomeric proton signals at dH 4.57 (d, J = 6.6 Hz), 4.91 (d, J = 8.0 Hz), and 5.13 (d, J = 1.3 Hz) and their corresponding carbon signals at dC 104.8, 104.4, and 102.0, respectively. Assignment of the sugar signals suggested the presence of a-L-rhamnopyranosyl, a-L-arabinopyranosyl, and b-D-glucopyranosyl moieties in 7 (Table 2). The HMBC spectrum showed cross peaks between H-1-rha (d 5.13) and C-2-ara (d 76.8), and between H-1-ara (d 4.57) and C-3 of the aglycone (d 90.5) indicating a disaccharide chain ether attached to C-3 of the aglycone. Among the 58 carbons in the 13C NMR spectrum, 30 were assigned to the triterpenoid aglycone, 17 to the oligosaccharide moiety, and the remaining 6 carbons to a maltol group (3-hydroxy-2-methyl4H-pyran-4-one) by detailed analysis of 1D and 2D NMR spectra and by comparison with literature data (Kite et al., 2007; Li et al., 2000; Tada et al., 2002) (Table 2). The 1H and 13C NMR spectrum showed signals for one methyl group at dH 2.43 [3H, s, dC 16.0, (C-700 )], and a disubstitued double-bond at dH 6.48 and 8.05 [each 1H, d, J = 5.3 Hz, dC 117.5 (C-500 ), and 159.1 (C-600 )]. These two olefinic protons (H-500 and H-600 ) were correlated in the COSY spectrum. The HMBC spectrum showed correlations between H-600 with C-400 (dC 176.9) and C-200 (dC 164.5), whereas H-500 correlated with C-300 (dC 143.0) (Fig. 1) and the methyl group protons (H-700 ) exhibited correlations with C-300 . The connectivity of the maltol moiety to C-10 of the glucose moiety and the linkage of the glucose unit at C-29 of the aglycone were defined unambiguously by the HMBC correlations observed between glc-H-10 (dH 4.91) and maltol-C-300 (dC 143.0) and between glc-H-60 (dH 4.26, 4.44) and C-29 of the aglycone (dC 179.3). Based on the foregoing evidence, the structure of meliosmoside G (7) was established as maltol 300 -O{6-O-[3b-O-a-L-rhamnopyranosyl-(1 ? 2)-a-L-arabinopyranosylolean-12-ene-28,29-dioic acid-29]}-b-D-glucopyranoside.
(CD3OD). HRESIMS experiments were performed using a Micromass Q-TOF micro instrument (Manchester, UK). Optical rotations were determined in MeOH with a Perkin-Elmer 341 polarimeter. TLC was performed on pre-coated silica-gel 60 F254 Merck and compounds were visualized by spraying the dried plates with 50% H2SO4, followed by heating. CC was carried out on Diaion HP-20 (Sigma–Aldrich), Kieselgel 60 (63–200 mesh), or LiChroprep RP-18 (40–63 lm) Merck. HPLC was performed on a Dionex apparatus equipped with an ASI-100 autosampler, a P580 pump, a diode array detector UVD 340S and a Chromeleon software. RP-18 column (Phenomenex 250 10 mm, Luna 5 l) was used for semi preparative HPLC with binary gradient eluent (Solvent A, H2O (pH 2.4 with TFA); Solvent B, MeOH) and a flow rate 5 ml/min; the chromatogram was monitored at 205 and 210 nm. 4.2. Plant material The leaves of Meliosma henryi Diels were collected at Pà Co. Mai Châu (Hoà Binh) Vietnam in April 1996, and authenticated by one of us (Vincent Dumontet). A voucher specimen (DAI-VN 080) was deposited at the Herbarium of the Institute of Ecology and Biological Resources (NCST), Hanoi, Vietnam. 4.3. Extraction and isolation
4. Experimental
Dried and powdered leaves (800 g) were boiled under reflux in 10 l of hexane, for 4 h. After filtration, the solvent was evaporated (13 g of hexane extract) and the leaf powder dried, then macerated for 2 h with 10 l of 80% aqueous MeOH and further refluxed for 4 h. After cooling, the solution was filtered and evaporated and the residue suspended in methanol (800 ml). The solution was poured into 5 l of acetone, and the precipitate was filtered and dried over NaOH in vacuo (18.5 g). The precipitate was dissolved in H2O and chromatographed on Diaion HP-20 column chromatography CC (4.3 40 cm) eluted successively with 0, 25, 50, 75, and 100% MeOH in H2O, each 2 l to give five fractions (A: 10.5 g, B: 0.23 g, C, 1.5 g, D: 3.0 g, and E: 2.4 g, respectively). Fractions D and E (5 g) were gathered and applied to a silica gel CC (4.5 23 cm) eluted with a gradient of CHCl3–MeOH–H2O (8:2:0–6:4:0.7) to afford 80 fractions (each 250 ml). Frs [7–9] (213 mg), eluted with CHCl3–MeOH (8:2), were further separated by RP-18 CC eluted with a gradient of MeOH–H2O (from 6:4 to 10:0) to yield 23 mg of compound 7 eluted with MeOH–H2O (7:3). Frs [10–14] (268 mg), eluted with CHCl3–MeOH (8:2), were further separated by RP-18 CC eluted with a gradient of MeOH–H2O (from 6:4 to 10:0) to yield compound 1 (52 mg) eluted with MeOH–H2O (7:3). Frs [47–74] (1 g), eluted with CHCl3–MeOH–H2O (7:3:0–7:3:5), were further separated silica gel CC eluting with a gradient of CHCl3–MeOH–H2O (8:2:0–7:3:5), to afford 80 subfractions (each 50 ml). Subfractions [14–16] (120 mg) eluted with CHCl3–MeOH (7.5:2.5), were purified by semi-prep HPLC using an isocratic gradient at 65% of methanol aqueous for 30 min yielding compounds 5 (Rt 17.4 min, 52 mg), 3 (Rt 19.3 min, 21 mg), 2 (Rt 22.1 min, 14 mg), and 6 (Rt 24.0 min, 5.5 mg). Subfractions [22–33] (216 mg) eluted with CHCl3–MeOH–H2O (7:3:0), were also purified by semi-prep HPLC using an isocratic gradient at 65% of methanol aqueous for 30 min yielding compound 4 (Rt 15.8 min, 10 mg).
4.1. General experimental procedures
4.4. Acid hydrolysis
NMR spectra were acquired in CD3OD on Bruker Avance DRX III 500 instruments (1H at 500 MHz and 13C at 125 MHz). Standard pulse sequences and parameters were used to obtain 1D 1H and 13 C and 2D COSY, ROESY, TOCSY, HSQC-Jmod, HSQC-TOCSY, and HMBC spectra. Chemical shift referencing was carried out using the internal solvent resonances at dH 3.33 (CHD2OD) and dC 49
A part of the saponin mixture (400 mg of fractions D and E of Diaion HP-20 CC) was refluxed (90 °C) with 10 ml of 2 M TFA for 3 h. After extraction with ethyl acetate (3 10 ml), the aqueous layer was evaporated to furnish the monosaccharide residue (221 mg). Five sugars were identified as glucose, galactose, arabinose, apiose, and rhamnose by comparison with authentic samples
3. Bioassays In a preliminary biological screening, the MeOH extract of the leaves of M. henryi inhibit the growth of KB cell at the dose of 10 lg/mL (15% inhibition). Thus, the new saponins (1–7) isolated from M. henryi leaves were investigated for their cytotoxic activity against KB cell line. Compound 2, 3, and 6 exhibited moderate cytotoxic activity in vitro against KB cells with an IC50 of 31, 22, and 24 lM, respectively. Compounds 1, 4, 5 and 7 were inactive at the dose of 20 lM (less than 10% inhibition).
Please cite this article in press as: Alabdul Magid, A., et al. Triterpenoid glycosides from the leaves of Meliosma henryi. Phytochemistry (2014), http:// dx.doi.org/10.1016/j.phytochem.2014.10.035
A. Alabdul Magid et al. / Phytochemistry xxx (2014) xxx–xxx
on TLC in MeCOEt:iso-PrOH:Me2CO:H2O (20:10:7:6); visualization was achieved by spraying with 50% H2SO4 followed by heating. A part of the monosaccharide residue (100 mg) was subjected to a preparative TLC using the same solvent. The optical rotation of each purified sugar was measured to afford L-rhamnose {3 mg; Rf 20 0.73; [a]20 D +12, (c 0.25, H2O)}, L-arabinose {2.6 mg; Rf 0.59; [a]D 20 +43, (c 0.13, H2O)}, D-glucose {6 mg; Rf 0.48; [a]D +29.8, (c 0.5, H2O)}, D-galactose {2.6 mg; Rf 0.42; [a]20 D +45, (c 0.13, H2O)}, and 20 D-apiose {1 mg, Rf 0.58; [a]D +4.3, (c 0.08, H2O)} by comparison with authentic samples (+8, +103, +98, +79, and +5.6, respectively). 4.5. Meliosmoside A (1) 1 13 [a]20 C NMR (CD3OD) spectroD +28.3 (c 0.71, MeOH); H and scopic data, see Tables 1 and 2; HRESIMS m/z 889.4572 [M+Na]+ (calcd for C45H70O16Na, 889.4562); ESIMS/MS: 595.4 [M+Na-araglc]+.
4.6. Meliosmoside B (2) 1 13 [a]20 C NMR (CD3OD) spectroscopic D +13.6 (c 1, MeOH); H and data, see Tables 1 and 2; HRESIMS m/z: 1021.4976 [M+Na]+ (calcd for C50H78O20Na, 1021.4984); ESIMS2 (1021.5) m/z 889.5 [M+Na-api]+, 727.4 [M+Na-api-glc]+, 595.4 [M+Na-api-glc-ara]+.
7
supplemented with 10% fetal bovine serum and antibiotics. After culture, the cells were treated with saponins for 72 h. The cell cultures were then analyzed using 3-(4,5-dimethylthiazol-2-yl)-5-(3carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium inner salt (MTS) according to the manufacturer’s instructions (Promega Corporation, Charbonnières, France). Camptothecin was used as positive control. MTS is bioreduced by cells into a colored formazan product. Absorbance was analyzed at a wavelength of 540 nm with a Multiskan Ex microplate absorbance reader (Thermo Scientific, Paris, France). The results of these assays were used to obtain the dose–response curves from which IC50 values were determined. The values represent averages of three independent experiments. Acknowledgments The authors wish to thank Djoumala Abddedaim for technical assistance (ICMR CNRS UMR 7312, Reims). The authors are grateful to CNRS, Conseil Régional Champagne Ardenne, Conseil Général de la Marne, Ministry of Higher Education and Research (MESR), EUprogramme FEDER to the PlANET CPER project for financial support, and Dr. Jacqueline Eskander, Ceapro Inc. Alberta, Canada for critical reading of the manuscript.
4.7. Meliosmoside C (3)
Appendix A. Supplementary data
1 13 [a]20 C NMR (CD3OD) spectroscopic D +26.8 (c 1, MeOH); H and data, see Tables 1 and 2; HRESIMS m/z: 1153.5400 [M+Na]+ (calcd for C55H86O24Na, 1153.5407); ESIMS2 (1153.5) m/z 1021.6 [M+Na-api], 859.5[M+Na-api-glc]+, 859.5[M+Na-api-glc-ara]+.
Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.phytochem. 2014.10.035.
4.8. Meliosmoside D (4) 1 13 [a]20 C NMR (CD3OD) spectroD +18.8 (c 0.83, MeOH); H and scopic data, see Tables 1 and 2; HRESIMS m/z: 1183.5524 [M+Na]+ (calcd for C56H88O25Na, 1183.5512); ESIMS2 (1183.5) m/z 1051.5[M+Na-api], 919[M+Na-api-ara]+, 757[M+Na-api-ara-glc]+.
4.9. Meliosmoside E (5) 1 13 [a]20 C NMR (CD3OD) spectroscopic D +8.5 (c 0.33, MeOH); H and data, see Tables 1 and 2; HRESIMS m/z: 1167.5555 [M+Na]+ (calcd for C56H88O24Na, 1167.5563); ESIMS2 (1167.6) m/z 1135[M+Na-api], 727.4 [M+Na-api-glc]+, 741.4 [M+Na-api-glc-ara]+.
4.10. Meliosmoside F (6) 1 13 [a]20 C NMR (CD3OD) spectroscopic D +8.3 (c 0.46, MeOH); H and data, see Tables 1 and 2; HRESIMS m/z: 1183.5885 [M+Na]+ (calcd for C57H92O24Na, 1183.5876); ESIMS2 (1183.6) m/z 1151[M+Na-api], 757[M+Na-api-ara-glc]+.
4.11. Meliosmoside G (7) 1 13 [a]20 C NMR (CD3OD) spectroscopic D 16.8 (c 1, MeOH); H and data, see Tables 1 and 2; HRESIMS m/z: 1057.5000 [M+Na]+ (calcd for C53H78O20Na, 1057.4984); ESIMS2 (1057.5) m/z 931.5 [M+Na-maltol C6O3H6]+.
5. Cytotoxicity bioassay by MTS KB cells (epithelial carcinoma) were trypsinized, harvested, and spread onto 96-well flat-bottom plates at a density of 1000 cells per well, and then incubated for 24 h in RPMI 1640 Medium
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Triterpenoid glycosides from the leaves of Meliosma henryi Abdulmagid Alabdul Magid a,⇑, Hamid Morjani b, Dominique Harakat c, Claudie Madoulet d, Vincent Dumontet e, Catherine Lavaud a a
Laboratoire de Pharmacognosie, ICMR-UMR 7312, SFR CapSanté, Université de Reims Champagne-Ardenne, Bat. 18, BP 1039, 51687 Reims Cedex 2, France Laboratoire Matrice Extracellulaire Dynamique Cellulaire, ICMR-UMR 7312 CNRS, UFR de Pharmacie, SFR CapSanté, Université de Reims Champagne-Ardenne, 51 rue Cognacq-Jay, 51096 Reims Cedex, France c Service Commun d’Analyses, ICMR-UMR 7312 CNRS, Bat. 18, BP 1039, 51687 Reims Cedex 2, France d Laboratoire de Biochimie, Faculté de Pharmacie, Université de Reims Champagne-Ardenne, 51096 Reims Cedex, France e Institut de Chimie des Substances Naturelles, UPR 2301 CNRS, Avenue de la Terrasse, 91198 Gif-sur-Yvette, France b
a r t i c l e
i n f o
Article history: Received 19 June 2014 Received in revised form 24 October 2014 Available online xxxx Keywords: Meliosma henryi Diels Sabiaceae Triterpenoid glycosides Cytotoxic activity
a b s t r a c t Seven triterpenoid glycosides, named meliosmosides A–G, were isolated from the leaves of Meliosma henryi Diels (Sabiaceae). Their structures were elucidated by different spectroscopic methods including 1D and 2D NMR experiments as well as HRESIMS analysis. Isolated compounds were evaluated for their cytotoxic activity against KB cell line. Ó 2014 Elsevier Ltd. All rights reserved.
1. Introduction
2. Results and discussion
The Sabiaceae is a small family including three genera; Sabia, Ophiocaryon, and Meliosma (Wanntorp and Ronse De Craene, 2007). Meliosma with about 50–70 species has the widest distribution occurring in Asia and America, while Sabia with about 19–50 species is restricted to Asia, and Ophiocaryon with about 7 species is only found in tropical South America (Furness et al., 2007; Ronse De Craene and Wanntorp, 2008). A review of literature showed that the phytochemistry of this family has not been exhaustively studied. A short report about Meliosma pungens mentioned the presence of oleanolic acid, and nine triterpenoid glycosides were isolated from the bark of M. lanceolata (Abe et al., 1996; Rao et al., 1979). A phytochemical investigation of Meliosma henryi leaves led to the isolation of seven new triterpenoid glycosides named meliosmosides A–G. Their structures were elucidated by different spectroscopic methods including 1D NMR (1H, 13C) and 2D NMR experiments (COSY, TOCSY, ROESY, HSQC, HSQC-TOCSY and HMBC) as well as HRESIMS analysis. The antiproliferative activity of isolated compounds against KB cell line was assessed using the MTS method.
The 80% MeOH extract of the leaves of M. henryi was subjected to a Diaion HP-20 column eluted with different gradients of water and methanol mixture to give five fractions (A–E). Further purification using combination of silica gel column chromatography, RP-18 column chromatography, and semi-preparative HPLC yielded seven triterpenoid saponins as white amorphous powders (1–7). The sugar composition was determined by comparative TLC after acid hydrolysis of the saponin mixture (fraction D and E of the 80% MeOH extract) as glucose (glc), galactose (gal), arabinose (ara), apiose (api) and rhamnose (rha). The common D- configuration for the glucose, galactose, and apiose moieties and the L- configuration for the rhamnose and arabinose sugars were verified by measurement of the optical rotation of each purified sugar. The positive-ion high-resolution electrospray ionization mass spectrum (HREIMS) analysis of meliosmoside A (1) revealed a molecular formula of C45H70O16, showing an ion [M+Na]+ at m/z 889.4572 (calcd for C45H70O16Na, 889.4562). The 1H NMR spectrum of the aglycone part of 1 displayed signals assignable to five tertiary methyl groups at dH 0.83, 0.88, 0.99, 1.08, and 1.22 (3H, s), two exo-methylene protons at dH 4.64, (2H, brs), an olefinic proton at dH 5.35 (t, J = 3.8 Hz), and a proton at dH 3.17 (dd, J = 11.9, 4.4 Hz) assigned to H-3 (Table 1). The 13C NMR spectrum of the aglycone part showed 29 carbon signals of which, four typical sp2-hybridized carbon signals at dC 107.5, 124.3, 144.2, and 149.3 and one
⇑ Corresponding author. Tel.: +33 3 26 91 82 08. E-mail address: [email protected] (A. Alabdul Magid). http://dx.doi.org/10.1016/j.phytochem.2014.10.035 0031-9422/Ó 2014 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Alabdul Magid, A., et al. Triterpenoid glycosides from the leaves of Meliosma henryi. Phytochemistry (2014), http:// dx.doi.org/10.1016/j.phytochem.2014.10.035
2
1
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30
2
3
dH (J in Hz)
dC
dH (J in Hz)
1.01, 1.65, 1.73, 1.88, 3.17,
td (11.9, 3.5) m td (11.9, 3.5) m dd (11.9, 4.4)
39.8
0.99, 1.66, 1.17, 1.87, 3.17,
m dd (13.4, 3.1) brd (13.4) m dd (11.5, 4.3)
0.81, 1.44, 1.59, 1.35, 1.53,
brd (13.6) td (11.7, 3.1) m brd (11.7) m
0.82, 1.45, 1.59, 1.35, 1.54,
m dd (12.5, 3.3) m brd (12.5) td (12.5, 3.0)
1.64, t (9.8) 1.94, m 5.35, t (3.8)
1.18, 1.88, 1.87, 2.20,
brt (13.5) m m dt (13.5, 4,0)
2.76, dd (13.5, 5.0) 2.11, dd (13.5, 4.9) 2.59, t (13.5) 2.17, 2.26, 1.56, 1.94, 1.08, 0.88, 0.99, 0.83, 1.22,
brd (13.3) td (13.3, 3.9) td (13.3, 4.9) m s s s s s
4.64, brs
27.0 90.6 40.2 57.0 19.3 33.9 40.7 49.0 37.9 24.6 124.3 144.2 42.9 28.9 24.1 48.3 48.6 42.6 149.3 30.9 38.4 28.6 17.0 16.1 17.8 26.4 177.2 107.5
1.63, t (10.5) 1.95, m 5.35, t (3.5)
1.74, 1.88, 1.88, 2.21,
brt (13.1) m m td (13.1, 3.8)
2.77, dd (13.5, 4.9) 2.11, dd (13.5, 4.9) 2.59, t (13.6) 2.18, 2.27, 1.57, 1.94, 1.08, 0.88, 0.99, 0.83, 1.22,
m td (12.2, 3.6) m m s s s s s
4.65, brs
dC 39.8 28.9 90.7 40.2 57.0 19.3 33.9 40.8 49.0 37.9 24.6 124.4 144.2 42.9 27.0 24.1 48.3 48.6 42.6 149.4 30.9 38.5 28.6 17.0 16.1 18.0 26.3 177.2 107.5
4
dH (J in Hz) 1.00, 1.66, 1.17, 1.90, 3.18,
m m brd (14.1) m dd (11.5, 4.3)
0.82, 1.44, 1.60, 1.35, 1.53,
m td (11.7, 3.0) m brd (11.7) m
1.61, m 1.95, m 5.35, t (3.5)
1.73, 1.87, 1.88, 2.21,
brt (12.3) m m td (14.4, 3.2)
2.77, dd (13.4, 4.9) 2.11, dd (13.4, 5.1) 2.59, t (13.5) 2.18, 2.27, 1.59, 1.94, 1.05, 0.88, 0.99, 0.83, 1.22,
m td (13.2, 4.4) m m s s s s s
4.65, brs
dC 39.9 28.9 90.5 40.2 57.1 19.3 33.9 40.8 49.0 37.9 24.7 124.4 144.2 42.9 27.0 24.2 48.3 48.6 42.6 149.4 30.9 38.5 28.6 17.0 16.1 17.8 26.4 177.3 107.5
5
dH (J in Hz) 1.00, 1.63, 1.16, 1.86, 3.18,
m m m m dd (11.6, 4.6)
0.82, m 1.44, td (12.6, 3.2) 1.60, m 1.35, brd (12.6) 1.53,dd (12.6, 3.9) 1.61, t (10.4) 1.95, m 5.35, t (3.5)
1.73, 1.87, 1.88, 2.21,
m m m td (13.1, 3.5)
2.77, dd (13.6, 4.2) 2.11, dd (13.6, 5.0) 2.59, t (13.6) 2.20, 2.27, 1.59, 1.87, 1.08, 0.87, 0.99, 0.83, 1.22,
dd (13.2, 3.5) td (13.2, 3.5) m m s s s s s
4.65, brs
dC 39.9 28.9 90.4 40.2 57.1 19.3 33.9 40.8 49.0 37.9 24.6 124.4 144.2 42.9 27.1 24.2 48.3 48.6 42.6 149.4 30.9 38.5 28.5 17.0 16.1 17.8 26.4 177.3 107.5
6
dH (J in Hz) 1.03, 1.67, 1.17, 1.88, 3.15,
m dt (13.1, 3.3) dt (13.1, 3.3) m dd (11.6, 4.4)
0.82, 1.45, 1.60, 1.36, 1.55,
m td (12.3, 2.1) m brd (12.4) m
1.61, t (11.1) 1.95, m 5.35, t (3.7)
1.76, 1.87, 1.88, 2.21,
brt (11.1) m m td (13.8, 3.3)
2.77, dd (13.8, 5.1) 2.11, dd (13.8, 4.9) 2.59, t (13.8) 2.18, 2.26, 1.57, 1.92, 1.08, 0.88, 0.99, 0.83, 1.22,
m td (13.3, 4.1) m m s s s s s
4.65, brs
dC 39.9 28.9 90.6 40.2 57.0 19.4 33.9 40.8 49.0 37.9 24.5 124.3 144.2 42.9 27.0 24.1 48.3 48.6 42.6 149.4 30.8 38.4 28.6 17.1 16.1 18.0 26.4 177.2 107.5
7
dH (J in Hz) 0.98, 1.63, 1.69, 1.88, 3.14, – 0.81, 1.37, 1.53, 1.31, 1.47, – 1.56, – 1.86, 5.28, – – 0.98, 1.86, 1.57, 1.87, – 2.88, 0.99, 1.62, – 1.12, 1.32, 1.50, 1.69, 1.05, 0.87, 0.98, 0.83, 1.18, – 0.96, 0.99,
m m m m dd (11.3, 4.6) m m m m m m m t (3.5)
m m m m dd (12.7, 4.0) m m m m m m s s s s s s s
dC 40.1 26.44 90.6 39.8 57.1 18.9 34.7 41.0 49.0 37.9 24.2 121.8 146.3 43.2 28.6 24.2 48.0 43.0 47.7 31.5 35.1 33.9 28.3 17.1 15.8 17.8 26.4 178.1 33.8 24.2
dC 1.04, 1.64, 1.45, 1.87, 3.15, – 0.83, 1.43, 1.58, 1.35, 1.54, – 1.63, – 1.94, 5.31, – – 1.49, 1.86, 1.65, 2.00, – 2.89, 1.41, 2.18, – 1.49, 1.85, 1.63, 1.79, 1.05, 0.87, 0.98, 0.84, 1.19, –
m m m m dd (11.7, 4.4) m m m m m m brd (7.0) t (3.3)
m td (13.8, 3.3) m td (13.8, 2.6) dd (13.7, 4.0) m t (13.7) m m m m s s s s s
1.24, s
39.8 27.0 90.5 40.2 57.0 19.4 34.0 40.6 49.6 37.9 24.6 124.5 144.3 42.9 28.7 24.2 47.4 41.6 40.9 43.6 29.5 32.5 28.6 17.1 16.0 17.7 26.6 181.2 179.3 19.8
A. Alabdul Magid et al. / Phytochemistry xxx (2014) xxx–xxx
Please cite this article in press as: Alabdul Magid, A., et al. Triterpenoid glycosides from the leaves of Meliosma henryi. Phytochemistry (2014), http:// dx.doi.org/10.1016/j.phytochem.2014.10.035
Table 1 NMR spectroscopic data of the aglycone moieties for compounds 1–7 (500 MHz, CD3OD).
A. Alabdul Magid et al. / Phytochemistry xxx (2014) xxx–xxx
carbonyl carbon signal dC 177.2. Taken together, these data were indicative of a typical 30-nor-oleana-12,20(29)-dien-28-oic acid. This assumption was confirmed by analysis of the COSY, ROESY, HSQC-Jmod and HMBC spectra which allowed the full assignment of the proton and carbon resonances of the aglycone (Table 1). The deshielding effect observed at H-18 signal (dH 2.76), due to magnetic anisotropy effect of carbonyl at C-28, is in agreement with a cis fusion between D and E rings. In the ROESY spectrum, correlations observed between H-27/H-19ax (dH 2.59, t, J = 13.5 Hz), H-27/H-16ax (dH 2.20, dt, J = 13.5–4.0 Hz), H-27/H-9, and H-19ax/H-16ax) indicated that E ring must therefore be a-oriented. The ROESY experiment revealed also several correlations, including H-3/H-5 and H-5/H-9 which indicated that the OH-3 group was in a b-orientation and H-25 with H-24 and H-26 indicating their b-orientation. The chemical shifts values for protons and carbons were in accordance with the literature data of 3bhydroxy-30-nor-oleana-12,20-(29)-dien-28-oic acid (akebonoic acid) (Hamed et al., 2011; Wang et al., 2012; Zhao et al., 2014). 1 H and 13C NMR spectra showed that akebonoic acid was common as aglycone to saponins 1–5. Full assignments of its proton and carbon resonances for each compound (2–5) were achieved by analysis of the COSY, HSQC-Jmod and HMBC spectra (Table 1). The sugar part of 1 consisted of three residues as evidenced by 1 H and 13C NMR spectra which displayed three anomeric protons at dH 4.30 (d, J = 6.7 Hz), 4.31 (d, J = 6.6 Hz), and 5.38 (d, J = 8.1 Hz), whose carbon resonances were assigned by HSQC-Jmod experiments at dC 107.1, 104.7 and 95.7, respectively. Starting from the anomeric proton at dH 5.38 (glc), the NMR signals belonging to the same system were assigned to a 6-monosubstituted D-glucopyranose (dC-6 69.2) (Gao et al., 2009) (Table 2). This assignment was further supported by the HMBC and the COSY experiments. The b-anomeric configuration of the D-glucopyranose unit was determined by the vicinal J1–2 trans coupling constant (8.1 Hz). The two remaining sugar units were identified as terminal L-arabinopyranoses (ara-I and ara-II) by their typical pattern in the COSY spectrum. Analysis of HSQC-Jmod and HMBC spectra, and comparison of the 13C NMR chemical shifts with those of related systems reported in the literature confirmed their identification as a-L-arabinopyranoses (Ishii et al., 1981) (Table 2). The L-arabinopyranose units were determined to be in an a-configuration on the basis of the J1–2 values (6.7 and 6.6 Hz) and this was confirmed by observation of ROESY correlations between H-1 and H-3 and between H-1 and H-5 for both arabinoses. The first arabinose unit (ara-I) was linked to C-3 of the aglycone as shown by the long-range correlation in the HMBC spectrum between dH 3.17 (H-3 of aglycone) and dC 107.1 (C-1 of ara-I). The HMBC spectrum showed also connectivities from H-1-ara-II (dH 4.31) to C-6-glc (dC 69.2), and from H-1-glc (dH 5.38) to C-28 of the aglycone (dC 177.2). Consequently, the structure of meliosmoside A (1) was concluded to be 3b-O-a-L-arabinopyranosyl-30-nor-oleana-12,20-(29)-dien-28-oic acid-28-Oa-L-arabinopyranosyl-(1 ? 6)-b-D-glucopyranoside. The HRESIMS of meliosmoside B (2) indicated a C50H78O20Na molecular formula, as deduced from the [M+Na]+ ion at m/z 1021.4976 (calcd for 1021.4984) in the positive ion mode. The MS2 experiment of this ion gave positive fragment at m/z 889.5 [M+Na132]+ suggesting a supplementary pentose unit compared to 1. This hypothesis was confirmed by the presence of four anomeric proton and carbon signals in the 1H and 13C NMR spectra. Severe overlap of some proton and carbon resonances requested the use of the HSQC-TOCSY experiment to map the spin systems. The detailed analysis of 1D and 2D NMR spectra led to the identification as in 1 of a 6-monosubstituted b-D-glucopyranose (glc, dC-6 69.4), a terminal a-L-arabinopyranose (ara-I), and a 4-monosubstituted a-L-arabinopyranose (ara-II, dC-4 77.4) (Table 2). The supplementary sugar residue showed NMR signals for two methines and two methylenes groups, in addition to a quaternary carbon, in
3
agreement with an apiofuranose moiety (Ishii and Yanagisawa, 1998; Eskander et al., 2006). The deshielded chemical shifts of the anomeric signals at dH 5.15 (d, J = 2.7 Hz) and dC 112.1 and the HMBC correlations observed between anomeric proton H-1 with its C-3 and C-4 indicate that this sugar residue is a b-erythroapiofuranose (Table 2) and the most commonly D- configuration for apiofuranose was assumed (Ishii and Yanagisawa, 1998). The sequence of the trisaccharide chain at C-28 was deduced from the HMBC experiment, as long-range correlations were observed between H-1-api (dH 5.15)/C-4-ara-II (dC 77.4), H-1-ara-II (dH 4.29)/C-6-glc-(dC 69.4), and H-1-glc (dH 5.38)/C-28 of akebonoic acid (dC 177.2). The glycosidic chain at C-3 was constituted by one a-L-arabinopyranose unit (ara-I) as indicated by the cross-peak between H-1-ara-I (dH 4.31) and C-3 of the akebonoic acid (dC 90.7). These findings led to the identification of meliosmoside B (2) as 3b-O-a-L-arabinopyranosyl-30-nor-oleana-12,20-(29)-dien28-oic acid-28-O-b-D-apiofuranosyl-(1 ? 4)-a-L-arabinopyranosyl-(1 ? 6)-b-D-glucopyranoside. For the three following meliosmosides C-E (3–5), the analysis of their 2D NMR spectra (COSY, HSQC-Jmod, HMBC, and ROESY) and comparison of the 1H and 13C NMR values of oligosaccharide part with those of 2, indicated that 3–5 contained the same ester trisaccharide chain b-D-apiofuranosyl-(1 ? 4)-a-L-arabinopyranosyl(1 ? 6)-b-D-glucopyranoside linked to C-28 of akebonoic acid whereas an a-L-arabinopyranosyl was linked to C-3 of the aglycone (Tables 1 and 2). Meliosmoside C (3) displayed a molecular ion peak [M+Na]+ at m/z 1153.5400 in the positive HRESIMS, in agreement with a molecular formula of C55H86O24Na (calcd for 1153.5407). The MS2 experiment of this ion gave positive fragment at 1021.6 [M+Na132]+, suggesting a supplementary pentose unit compared to 2. The analysis of the 2D NMR spectra of 3 led to the identification of a supplementary a-L-arabinopyranose unit (ara-III) in terminal position (Table 2). This arabinose was linked to C-2-ara-I as deduced from the HMBC correlation observed between H-1-ara-III (dH 4.53) and C-3-ara-I (dC 83.4). Thus, meliosmoside C (3) was concluded to be 3b-O-a-L-arabinopyranosyl(1 ? 3)-a-L-arabinopyranosyl-30-nor-oleana-12,20-(29)-dien-28-oic acid-28-Ob-D-apiofuranosyl-(1 ? 4)-a-L-arabinopyranosyl-(1 ? 6)b-D-glucopyranoside. Meliosmoside D (4) showed a molecular ion peak [M+Na]+ at m/ z 1183.5524 (calcd for C56H88O25Na, 1183.5512), in the positive HRESIMS. Analysis of the 1H and 13C NMR spectra of compound 4 revealed the presence of five sugar units as in 3. Comparison of its 1H and 13C NMR spectra with those of 3 showed the absence of the ara-III moiety. Instead, a b-D-galactopyranose unit (gal) was identified starting from the anomeric proton dH 4.53 (d, J = 7.7 Hz), characterized by the large coupling constants JH-1,H-2 and JH-2,H-3 and the small coupling constant between H-3 and H4 (JH3,H4 = 3.2 Hz) as summarized in Table 2. In the HMBC spectrum of 4, the correlation of H-1-gal with C-3-ara-I (dC 83.9) and H-1ara-I with C-3 of the aglycone (dC 90.5) indicated the linkage of a disaccharide chain b-D-galactopyranosyl-(1 ? 3)-a-L-arabinopyranoside at C-3 of akebonoic acid. The structure of meliosmoside D (4) was therefore elucidated as 3b-O-b-D-galactopyranosyl(1 ? 3)-a-L-arabinopyranosyl-30-nor-oleana-12,20-(29)-dien-28oic acid-28-O-b-D-apiofuranosyl-(1 ? 4)-a-L-arabinopyranosyl(1 ? 6)-b-D-glucopyranoside. Meliosmoside E (5) was assigned to the molecular formula C56H88O24Na, as determined from the molecular ion peak at m/z 1167.5555 [M+Na]+ in the positive ion mode HRESIMS. The 1D and 2D NMR spectra of 5 compared with those of 4, indicated that a-L-arabinopyranose (ara-I) which was C-3 monosubstituted in 4, is here C-2 monosubstituted (dC-2 76.8) in 5 (Table 2). The fifth sugar unit with its anomeric proton resonating at dH 5.13 (d, J = 1.7 Hz) was identified as a terminal a-L-rhamnopyranose (rha)
Please cite this article in press as: Alabdul Magid, A., et al. Triterpenoid glycosides from the leaves of Meliosma henryi. Phytochemistry (2014), http:// dx.doi.org/10.1016/j.phytochem.2014.10.035
4
1
Ara-I 1 2 3 4 5
2
3
4
5
6
7
dH (J in Hz)
dC
dH (J in Hz)
dC
dH (J in Hz)
dC
dH (J in Hz)
dC
dH (J in Hz)
dC
dH (J in Hz)
dC
(at C-3) 4.30, d (6.7) 3.60, dd (8.4, 6.7) 3.56, dd (8.4, 3.1) 3.84, brt (3.1) 3.53, m 3.87, dt (12.2, 3.4)
107.1 72.8 74.1 69.5 66.5
4.31, 3.57, 3.53, 3.83, 3.56, 3.86,
107.2 72.8 74.3 69.5 66.4
4.31, 3.73, 3.63, 4.00, 3.59, 3.89,
107.0 72.1 83.4 69.7 66.9
4.31, 3.73, 3.66, 4.07, 3.58, 3.88,
107.0 72.1 83.9 69.7 66.6
4.57, 3.57, 3.53, 3.82, 3.51, 3.87,
104.8 76.8 73.0 68.4 63.7
4.57, 3.80, 3.76, 3.82, 3.51, 3.87,
104.9 76.8 73.0 68.4 63.7
d (6.7) dd (8.9, 6.7) dd (8.9, 3.4) t (3.4) m dd (12.3, 3.3)
d (7.4) dd (9.1, 7.4) dd (9.1, 3.3) m dd (12.4, 3.0) dd (12.4, 3.1)
ara-III (at ara-I C-3) 4.53, d (7.0) 3.67, dd (9.0, 7.0) 3.58, dd (9.0, 3.4) 3.82, m 3.82, m 3.88, dd (12.8, 2.4)
1 2 3 4 5
106.0 72.8 74.1 69.6 66.5
6
d (7.6) dd (9.1, 7.6) dd (9.1, 3.2) brd (3.2) dd (11.1, 1.5) dd (11.1, 2.4)
gal (at ara-I C-3) 4.53, d (7.7) 3.64, dd (9.6, 7.7) 3.58, dd (9.6, 3.2) 3.85, d (3.2) 3.57, brt (6.8)
106.1 73.0 74.6 70.3 76.7
d (6.4) dd (8.3, 6.4) dd (8.3, 3.3) m brd (11.8) dd (11.8, 6)
rha (at ara-I C-2) 5.13, d (1.7) 3.92, dd (3.4, 1.7) 3.72, dd (9.8, 3.4) 3.42, t (9.8) 3.83, m
102.0 72.1 72.1 73.9 70.2
d (6.5) dd (9.1, 6.5) dd (9.1, 3.0) m dd (11.9, 2.7) dm (11.9)
rha (at ara-I C-2) 5.13, d (1.4) 3.91, dd (3.5, 1.4) 3.72, dd (9.6, 3.5) 3.41, t (9.6) 3.83, m
102.0 72.7 72.7 73.9 70.6
rha (at ara-I C-2) 5.13, d (1.3) 3.92, dd (3.2, 1.3) 3.72, dd (9.5, 3.2) 3.42, t (9.5) 3.83, m
62.5
1.25, d (6.2)
17.8
1.25, d (6.2)
17.8
1.25, d (6.2) (at C-29) 4.91, d (8.0) 3.40, t (8.0) 3.47, dd (9.0, 8.0) 3.40, t (9.0) 3.41, m 4.26, dd (11.9, 5.3) 4.44, dd (11.9, 1.7)
(at C-28) 5.38, d (8.1) 3.37, t (8.1) 3.45, dd (9.0, 8.1) 3.44, t (9.0) 3.54, m 3.74, dd (11.4, 5.0) 4.07, dd (11.4, 2.3)
95.7 73.7 78.0 71.1 77.5 69.2
5.38, 3.35, 3.43, 3.44, 3.53, 3.79, 4.07,
d (8.1) t (8.1) dd (8.7, 8.1) t (8.7) m dd (11.5, 4.7) dd (11.5, 2.1)
95.7 73.7 78.0 71.1 77.7 69.4
5.38, 3.35, 3.44, 3.43, 3.53, 3.74, 4.07,
d (8.1) t (8.1) dd (9.4, 8.1) t (9.4) m dd (11.6, 4.6) dd (11.6, 2.2)
95.7 73.8 78.1 71.2 78.1 69.4
5.38, 3.38, 3.45, 3.43, 3.53, 3.73, 4.07,
d (8.1) t (8.1) dd (9.2, 8.1) t (9.2) m dd (12.2, 5.0) dd (12.2, 2.2)
95.7 73.8 78.1 71.2 77.7 69.4
5.38, 3.35, 3.45, 3.43, 3.53, 3.74, 4.07,
d (8.1) t (8.1) dd (8.1, 9.4) t (9.4) m dd (11.5, 4.7) dd (11.5, 2.2)
95.7 73.9 78.1 71.2 77.6 69.4
5.39, 3.36, 3.44, 3.42, 3.53, 3.76, 4.08,
d (8.1) t (8.1) t (8.1) dd (8.6, 8.1) m dd (11.7, 5.2) dd (11.7, 2.0)
95.7 73.9 78.1 71.2 77.6 69.4
ara-II 1 2 3 4 5
(at glc-C-6) 4.31, d (6.6) 3.63, dd (8.4, 6.6) 3.54, dd (8.4, 3.3) 3.83, t (3.3) 3.56, m 3.87, dd (12.2, 3.4)
104.7 72.3 74.3 69.3 66.4
4.29, 3.55, 3.59, 3.82, 3.86, 4.03,
d (6.6) m dd (8.4, 3.0) t (3.0) dd (12.3, 3.3) dd (12.3, 3.0)
104.9 72.7 74.3 77.4 66.2
4.29, 3.58, 3.61, 3.82, 3.51, 4.02,
d (6.7) dd (9.0, 6.7) dd (9.0, 3.1) m brd (12.5) dd (12.5, 3.2)
104.9 72.7 73.7 77.4 66.2
4.29, 3.58, 3.61, 3.82, 3.51, 4.02,
d (6.6) m dd (9.0, 3.2) brd (3.2) dd (11.9, 4.6) dd (11.9, 1.9)
104.9 72.7 73.7 77.4 66.2
4.29, 3.57, 3.61, 3.83, 3.51, 4.02,
d (6.6) dd (9.0, 6.6) dd (9.0, 3.4) m dd (12.2, 2.9) dd (12.2, 3.4)
104.9 72.7 74.3 77.4 66.2
4.31, 3.57, 3.61, 3.82, 3.52, 4.03,
d (6.7) dd (9.0, 6.7) dd (9.0, 3.4) m dd (12.3, 1.4) dd (12.3, 2.9)
104.8 72.7 74.3 77.4 66.2
104.8 76.8 73.1 70.2 63.8
102.0 72.1 72.1 73.9 68.4 18.0
104.4 75.3 77.6 71.1 76.0 64.0
Maltol
6 7
5
d (6.6) dd (8.4, 6.6) dd (8.4, 3.2) m dd (11.7, 2.3) dd (11.7, 5.9)
3.74, m 3.77, dd (11.5, 6.8)
glc 1 2 3 4 5 6
api 1 2 3 4
4.57, 3.80, 3.76, 3.85, 3.51, 3.87,
(at ara-II-C-4) 5.15, 4.01, – 3.78, 4.05, 3.64,
d (2.7) d (2.7) d (9.6) d (9.6) s
112.1 78.0 80.5 75.0 65.6
5.15, 4.01, – 3.78, 4.04, 3.64,
d (2.7) d (2.7) d (9.7) d (9.7) s
112.0 78.1 80.5 75.0 65.6
5.15, 4.01, – 3.78, 4.04, 3.64,
d (2.7) d (2.7) d (9.7) d (9.7) s
112.0 78.1 80.5 75.0 65.6
5.15, 4.01, – 3.79, 4.04, 3.64,
d (2.7) d (27) d (9.7) d (9.7) s
112.0 78.1 80.5 75.0 65.6
5.15, 4.01, – 3.78, 4.04, 3.64,
d (2.7) d (2.7) d (9.6) d (9.6) s
112.0 78.1 80.5 75.0 65.6
6.48, d (5.3)
164.5 143.0 176.9 117.5
8.05, d (5.3) 2.43, s
159.1 16.0
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Please cite this article in press as: Alabdul Magid, A., et al. Triterpenoid glycosides from the leaves of Meliosma henryi. Phytochemistry (2014), http:// dx.doi.org/10.1016/j.phytochem.2014.10.035
Table 2 NMR spectroscopic data of the sugar moieties for compounds 1–7 (500 MHz, CD3OD).
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with its methyl group at dH-6 1.25 (d, J = 6.2 Hz) and dC-6 17.8 (Backinowsky et al., 1980) (Table 2). In the ROESY spectrum, the rOe effects observed between H-1-rha/H-2-ara-I and between H-1-ara-I/H-3 aglycone indicated that the disaccharide a-L-rhamnopyranosyl-(1 ? 2)-a-L-arabinopyranoside was linked at C-3 of akebonoic acid. These data was further supported by the correlations observed on the HMBC spectrum of 5 between H-1-rha and C-2-ara-I (dC 76.8) and between H-1-ara-I and C-3 of the aglycone (dC 90.6). The structure of meliosmoside E (5) was therefore identified as 3b-O-a-L-rhamnopyranosyl(1 ? 2)-a-L-arabinopyranosyl-30-nor-oleana-12,20-(29)-dien-28-oic acid-28-O b-D-apiofuranosyl-(1 ? 4)-a-L-arabinopyranosyl-(1 ? 6)b-D-glucopyranoside. Meliosmoside F (6) was closely related to compound 5 for the glycosidic part. Comparison of the 13C NMR data of 6 to those of 5 and analysis of the 2D NMR spectra of 6 showed that both possessed the same two sugar chains (Table 2). HRESIMS data
5
([M+Na]+ at m/z 1183.5885) suggested that the molecular formula of 6 was C57H92O24. The difference was the absence of the exocyclic methylene group (C-29) of the aglycone. An extra geminaldimethyl moiety was detected in 6 (dH 0.96 and 0.99). The HMBC spectrum revealed proper correlations for these methyl groups: Me-30 (dH 0.99) exhibited correlations with C-20 (dC 31.5), C-21 (dC 35.1), C-19 (dC 47.7), as well as C-29 (dC 33.8). The chemical shifts values for all protons and carbons of the aglycone moiety (Table 1) were in accordance with the literature data of oleanolic acid (Agrawal and Jain, 1992). Based on the abovementioned evidence, the structure of compound 6 was assigned as 3b-O-a-L-rhamnopyranosyl-(1 ? 2)-a-L-arabinopyranosyl-oleanolic acid-28-O-b-D-apiofuranosyl-(1 ? 4)-a-L-arabinopyranosyl(1 ? 6)-b-D-glucopyranoside. Meliosmoside G (7) gave an ion peak at m/z 1057.5000 [M+Na]+ in the positive HRESIMS, from which the molecular formula C53H78O20 was deduced. Comparison of the 1H and 13C NMR data
Fig. 1. Chemical structures of saponins 1–7, isolated from Meliosma henryi.
Please cite this article in press as: Alabdul Magid, A., et al. Triterpenoid glycosides from the leaves of Meliosma henryi. Phytochemistry (2014), http:// dx.doi.org/10.1016/j.phytochem.2014.10.035
6
A. Alabdul Magid et al. / Phytochemistry xxx (2014) xxx–xxx
of the aglycone part in 7 with those in 6 revealed superimposable signals for the protons and carbons in rings A–D, but differences were observed in the ring E. In the HMBC experiment methyl protons at dH 1.24 (Me-30) exhibited 3JH-C correlations with C-19 (dC 40.9), C-20 (dC 43.6), C-21 (dC 29.5), and a carboxyl group resonating at dC 179.3 (Me-29), suggesting the replacement of the methyl group at C-29 in 6 by a carboxyl group in 7. The b-axial orientation of the methyl group (Me-30) was established by a rOe effect with H-18 observed in the ROESY spectrum; the 20-position carboxylic acid group (C-29) must therefore be a-equatorial oriented. Further analysis of the 2D NMR data was used to identify the aglycone as serratagenic acid (or 3b-hydroxyolean-12-ene-28,29-dioic acid) (Jitsuno and Mimaki, 2010; Yu et al., 1995). The 1H and 13C NMR spectra of 7 displayed three sugar anomeric proton signals at dH 4.57 (d, J = 6.6 Hz), 4.91 (d, J = 8.0 Hz), and 5.13 (d, J = 1.3 Hz) and their corresponding carbon signals at dC 104.8, 104.4, and 102.0, respectively. Assignment of the sugar signals suggested the presence of a-L-rhamnopyranosyl, a-L-arabinopyranosyl, and b-D-glucopyranosyl moieties in 7 (Table 2). The HMBC spectrum showed cross peaks between H-1-rha (d 5.13) and C-2-ara (d 76.8), and between H-1-ara (d 4.57) and C-3 of the aglycone (d 90.5) indicating a disaccharide chain ether attached to C-3 of the aglycone. Among the 58 carbons in the 13C NMR spectrum, 30 were assigned to the triterpenoid aglycone, 17 to the oligosaccharide moiety, and the remaining 6 carbons to a maltol group (3-hydroxy-2-methyl4H-pyran-4-one) by detailed analysis of 1D and 2D NMR spectra and by comparison with literature data (Kite et al., 2007; Li et al., 2000; Tada et al., 2002) (Table 2). The 1H and 13C NMR spectrum showed signals for one methyl group at dH 2.43 [3H, s, dC 16.0, (C-700 )], and a disubstitued double-bond at dH 6.48 and 8.05 [each 1H, d, J = 5.3 Hz, dC 117.5 (C-500 ), and 159.1 (C-600 )]. These two olefinic protons (H-500 and H-600 ) were correlated in the COSY spectrum. The HMBC spectrum showed correlations between H-600 with C-400 (dC 176.9) and C-200 (dC 164.5), whereas H-500 correlated with C-300 (dC 143.0) (Fig. 1) and the methyl group protons (H-700 ) exhibited correlations with C-300 . The connectivity of the maltol moiety to C-10 of the glucose moiety and the linkage of the glucose unit at C-29 of the aglycone were defined unambiguously by the HMBC correlations observed between glc-H-10 (dH 4.91) and maltol-C-300 (dC 143.0) and between glc-H-60 (dH 4.26, 4.44) and C-29 of the aglycone (dC 179.3). Based on the foregoing evidence, the structure of meliosmoside G (7) was established as maltol 300 -O{6-O-[3b-O-a-L-rhamnopyranosyl-(1 ? 2)-a-L-arabinopyranosylolean-12-ene-28,29-dioic acid-29]}-b-D-glucopyranoside.
(CD3OD). HRESIMS experiments were performed using a Micromass Q-TOF micro instrument (Manchester, UK). Optical rotations were determined in MeOH with a Perkin-Elmer 341 polarimeter. TLC was performed on pre-coated silica-gel 60 F254 Merck and compounds were visualized by spraying the dried plates with 50% H2SO4, followed by heating. CC was carried out on Diaion HP-20 (Sigma–Aldrich), Kieselgel 60 (63–200 mesh), or LiChroprep RP-18 (40–63 lm) Merck. HPLC was performed on a Dionex apparatus equipped with an ASI-100 autosampler, a P580 pump, a diode array detector UVD 340S and a Chromeleon software. RP-18 column (Phenomenex 250 10 mm, Luna 5 l) was used for semi preparative HPLC with binary gradient eluent (Solvent A, H2O (pH 2.4 with TFA); Solvent B, MeOH) and a flow rate 5 ml/min; the chromatogram was monitored at 205 and 210 nm. 4.2. Plant material The leaves of Meliosma henryi Diels were collected at Pà Co. Mai Châu (Hoà Binh) Vietnam in April 1996, and authenticated by one of us (Vincent Dumontet). A voucher specimen (DAI-VN 080) was deposited at the Herbarium of the Institute of Ecology and Biological Resources (NCST), Hanoi, Vietnam. 4.3. Extraction and isolation
4. Experimental
Dried and powdered leaves (800 g) were boiled under reflux in 10 l of hexane, for 4 h. After filtration, the solvent was evaporated (13 g of hexane extract) and the leaf powder dried, then macerated for 2 h with 10 l of 80% aqueous MeOH and further refluxed for 4 h. After cooling, the solution was filtered and evaporated and the residue suspended in methanol (800 ml). The solution was poured into 5 l of acetone, and the precipitate was filtered and dried over NaOH in vacuo (18.5 g). The precipitate was dissolved in H2O and chromatographed on Diaion HP-20 column chromatography CC (4.3 40 cm) eluted successively with 0, 25, 50, 75, and 100% MeOH in H2O, each 2 l to give five fractions (A: 10.5 g, B: 0.23 g, C, 1.5 g, D: 3.0 g, and E: 2.4 g, respectively). Fractions D and E (5 g) were gathered and applied to a silica gel CC (4.5 23 cm) eluted with a gradient of CHCl3–MeOH–H2O (8:2:0–6:4:0.7) to afford 80 fractions (each 250 ml). Frs [7–9] (213 mg), eluted with CHCl3–MeOH (8:2), were further separated by RP-18 CC eluted with a gradient of MeOH–H2O (from 6:4 to 10:0) to yield 23 mg of compound 7 eluted with MeOH–H2O (7:3). Frs [10–14] (268 mg), eluted with CHCl3–MeOH (8:2), were further separated by RP-18 CC eluted with a gradient of MeOH–H2O (from 6:4 to 10:0) to yield compound 1 (52 mg) eluted with MeOH–H2O (7:3). Frs [47–74] (1 g), eluted with CHCl3–MeOH–H2O (7:3:0–7:3:5), were further separated silica gel CC eluting with a gradient of CHCl3–MeOH–H2O (8:2:0–7:3:5), to afford 80 subfractions (each 50 ml). Subfractions [14–16] (120 mg) eluted with CHCl3–MeOH (7.5:2.5), were purified by semi-prep HPLC using an isocratic gradient at 65% of methanol aqueous for 30 min yielding compounds 5 (Rt 17.4 min, 52 mg), 3 (Rt 19.3 min, 21 mg), 2 (Rt 22.1 min, 14 mg), and 6 (Rt 24.0 min, 5.5 mg). Subfractions [22–33] (216 mg) eluted with CHCl3–MeOH–H2O (7:3:0), were also purified by semi-prep HPLC using an isocratic gradient at 65% of methanol aqueous for 30 min yielding compound 4 (Rt 15.8 min, 10 mg).
4.1. General experimental procedures
4.4. Acid hydrolysis
NMR spectra were acquired in CD3OD on Bruker Avance DRX III 500 instruments (1H at 500 MHz and 13C at 125 MHz). Standard pulse sequences and parameters were used to obtain 1D 1H and 13 C and 2D COSY, ROESY, TOCSY, HSQC-Jmod, HSQC-TOCSY, and HMBC spectra. Chemical shift referencing was carried out using the internal solvent resonances at dH 3.33 (CHD2OD) and dC 49
A part of the saponin mixture (400 mg of fractions D and E of Diaion HP-20 CC) was refluxed (90 °C) with 10 ml of 2 M TFA for 3 h. After extraction with ethyl acetate (3 10 ml), the aqueous layer was evaporated to furnish the monosaccharide residue (221 mg). Five sugars were identified as glucose, galactose, arabinose, apiose, and rhamnose by comparison with authentic samples
3. Bioassays In a preliminary biological screening, the MeOH extract of the leaves of M. henryi inhibit the growth of KB cell at the dose of 10 lg/mL (15% inhibition). Thus, the new saponins (1–7) isolated from M. henryi leaves were investigated for their cytotoxic activity against KB cell line. Compound 2, 3, and 6 exhibited moderate cytotoxic activity in vitro against KB cells with an IC50 of 31, 22, and 24 lM, respectively. Compounds 1, 4, 5 and 7 were inactive at the dose of 20 lM (less than 10% inhibition).
Please cite this article in press as: Alabdul Magid, A., et al. Triterpenoid glycosides from the leaves of Meliosma henryi. Phytochemistry (2014), http:// dx.doi.org/10.1016/j.phytochem.2014.10.035
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on TLC in MeCOEt:iso-PrOH:Me2CO:H2O (20:10:7:6); visualization was achieved by spraying with 50% H2SO4 followed by heating. A part of the monosaccharide residue (100 mg) was subjected to a preparative TLC using the same solvent. The optical rotation of each purified sugar was measured to afford L-rhamnose {3 mg; Rf 20 0.73; [a]20 D +12, (c 0.25, H2O)}, L-arabinose {2.6 mg; Rf 0.59; [a]D 20 +43, (c 0.13, H2O)}, D-glucose {6 mg; Rf 0.48; [a]D +29.8, (c 0.5, H2O)}, D-galactose {2.6 mg; Rf 0.42; [a]20 D +45, (c 0.13, H2O)}, and 20 D-apiose {1 mg, Rf 0.58; [a]D +4.3, (c 0.08, H2O)} by comparison with authentic samples (+8, +103, +98, +79, and +5.6, respectively). 4.5. Meliosmoside A (1) 1 13 [a]20 C NMR (CD3OD) spectroD +28.3 (c 0.71, MeOH); H and scopic data, see Tables 1 and 2; HRESIMS m/z 889.4572 [M+Na]+ (calcd for C45H70O16Na, 889.4562); ESIMS/MS: 595.4 [M+Na-araglc]+.
4.6. Meliosmoside B (2) 1 13 [a]20 C NMR (CD3OD) spectroscopic D +13.6 (c 1, MeOH); H and data, see Tables 1 and 2; HRESIMS m/z: 1021.4976 [M+Na]+ (calcd for C50H78O20Na, 1021.4984); ESIMS2 (1021.5) m/z 889.5 [M+Na-api]+, 727.4 [M+Na-api-glc]+, 595.4 [M+Na-api-glc-ara]+.
7
supplemented with 10% fetal bovine serum and antibiotics. After culture, the cells were treated with saponins for 72 h. The cell cultures were then analyzed using 3-(4,5-dimethylthiazol-2-yl)-5-(3carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium inner salt (MTS) according to the manufacturer’s instructions (Promega Corporation, Charbonnières, France). Camptothecin was used as positive control. MTS is bioreduced by cells into a colored formazan product. Absorbance was analyzed at a wavelength of 540 nm with a Multiskan Ex microplate absorbance reader (Thermo Scientific, Paris, France). The results of these assays were used to obtain the dose–response curves from which IC50 values were determined. The values represent averages of three independent experiments. Acknowledgments The authors wish to thank Djoumala Abddedaim for technical assistance (ICMR CNRS UMR 7312, Reims). The authors are grateful to CNRS, Conseil Régional Champagne Ardenne, Conseil Général de la Marne, Ministry of Higher Education and Research (MESR), EUprogramme FEDER to the PlANET CPER project for financial support, and Dr. Jacqueline Eskander, Ceapro Inc. Alberta, Canada for critical reading of the manuscript.
4.7. Meliosmoside C (3)
Appendix A. Supplementary data
1 13 [a]20 C NMR (CD3OD) spectroscopic D +26.8 (c 1, MeOH); H and data, see Tables 1 and 2; HRESIMS m/z: 1153.5400 [M+Na]+ (calcd for C55H86O24Na, 1153.5407); ESIMS2 (1153.5) m/z 1021.6 [M+Na-api], 859.5[M+Na-api-glc]+, 859.5[M+Na-api-glc-ara]+.
Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.phytochem. 2014.10.035.
4.8. Meliosmoside D (4) 1 13 [a]20 C NMR (CD3OD) spectroD +18.8 (c 0.83, MeOH); H and scopic data, see Tables 1 and 2; HRESIMS m/z: 1183.5524 [M+Na]+ (calcd for C56H88O25Na, 1183.5512); ESIMS2 (1183.5) m/z 1051.5[M+Na-api], 919[M+Na-api-ara]+, 757[M+Na-api-ara-glc]+.
4.9. Meliosmoside E (5) 1 13 [a]20 C NMR (CD3OD) spectroscopic D +8.5 (c 0.33, MeOH); H and data, see Tables 1 and 2; HRESIMS m/z: 1167.5555 [M+Na]+ (calcd for C56H88O24Na, 1167.5563); ESIMS2 (1167.6) m/z 1135[M+Na-api], 727.4 [M+Na-api-glc]+, 741.4 [M+Na-api-glc-ara]+.
4.10. Meliosmoside F (6) 1 13 [a]20 C NMR (CD3OD) spectroscopic D +8.3 (c 0.46, MeOH); H and data, see Tables 1 and 2; HRESIMS m/z: 1183.5885 [M+Na]+ (calcd for C57H92O24Na, 1183.5876); ESIMS2 (1183.6) m/z 1151[M+Na-api], 757[M+Na-api-ara-glc]+.
4.11. Meliosmoside G (7) 1 13 [a]20 C NMR (CD3OD) spectroscopic D 16.8 (c 1, MeOH); H and data, see Tables 1 and 2; HRESIMS m/z: 1057.5000 [M+Na]+ (calcd for C53H78O20Na, 1057.4984); ESIMS2 (1057.5) m/z 931.5 [M+Na-maltol C6O3H6]+.
5. Cytotoxicity bioassay by MTS KB cells (epithelial carcinoma) were trypsinized, harvested, and spread onto 96-well flat-bottom plates at a density of 1000 cells per well, and then incubated for 24 h in RPMI 1640 Medium
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