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Two new triterpenoid saponins from the roots of Albizia zygia (DC.) J.F. Macbr.
Phytochemistry Letters 18 (2016) 128–135
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Two new triterpenoid saponins from the roots of Albizia zygia (DC.) J.F. Macbr. Olivier Placide Notéa,b,* , Line Simoa,b , Joséphine Ngo Mbingb , Dominique Guillaumec , Sarah Ali Aouazoua , Christian Dominique Mullera , Dieudonné Emmanuel Pegnyembb , Annelise Lobsteina a Pharmacognosie et Molécules Naturelles Bioactives, Laboratoire d’Innovation Thérapeutique, UMR 7200, CNRS-Université de Strasbourg, Faculté de Pharmacie, 74 Route du Rhin, F-67401 Illkirch Cedex, France b Laboratoire de Pharmacochimie des Substances Naturelles, Département de Chimie Organique, Faculté de Sciences, Université de Yaoundé, BP 812, Yaoundé, Cameroon c Laboratoire de Chimie Thérapeutique, UMR7312, 51 Rue Cognacq-Jay, 51100 Reims, France
A R T I C L E I N F O
Article history: Received 6 July 2016 Received in revised form 12 September 2016 Accepted 20 September 2016 Available online xxx Keywords: Albizia zygia Mimosaceae Triterpenoid saponins NMR Pro-apoptotic activity Human epidermoid cancer cell A431
A B S T R A C T
As part of our search of new apoptosis-inducing triterpenoid saponins from Cameroonian Albizia genus, phytochemical investigation of the roots of Albizia zygia led to the isolation of two new oleanane-type saponins, named zygiaosides A–B (1–2), together with two known saponins, coriarioside A (3) and lebbeckoside A (4). Their structures were established on the basis of extensive 1D and 2D NMR (1H, 13C NMR, DEPT, COSY, HSQC-TOCSY, NOESY, HSQC and HMBC) and HRESIMS studies, and by chemical evidence. The apoptotic effect of saponins 1–2 was evaluated on the A431 human epidermoid cancer cell. Cytometric analyses showed that saponins 1–2 induced apoptosis of human epidermoid cancer cell (A341) in a dose-dependent manner. ã 2016 Published by Elsevier Ltd on behalf of Phytochemical Society of Europe.
Contents 1. 2. 3.
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Results and discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Experimental section . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . General experimental procedures . . . . . . . . . . . . . . . . . . . . . . . . . 3.1. 3.2. Plant material . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Extraction and isolation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.3. 3.4. Acid hydrolysis of compounds and determination of the absolute Pro-apoptotic evaluation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.5. 3.5.1. Cell lines and culture conditions . . . . . . . . . . . . . . . . . . . Pro-apoptotic evaluation . . . . . . . . . . . . . . . . . . . . . . . . . 3.5.2. 3.5.3. Statistical analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
.............................. .............................. .............................. .............................. .............................. .............................. configuration of the sugar residues .............................. .............................. .............................. .............................. .............................. ..............................
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1. Introduction
* Corresponding author at: Laboratoire de Pharmacochimie des Substances Naturelles, Département de Chimie Organique, Faculté des Sciences, Université de Yaoundé, BP 812, Yaoundé, Cameroun. E-mail addresses: [email protected], [email protected] (O.P. Noté).
Cutaneous squamous cell carcinoma represents the second most common skin cancer (Konstantopoulou et al., 2006), carrying a significant risk of metastasis and accounting for the majority of several thousand deaths attributable to non-melanoma skin cancer
http://dx.doi.org/10.1016/j.phytol.2016.09.010 1874-3900/ã 2016 Published by Elsevier Ltd on behalf of Phytochemical Society of Europe.
O.P. Noté et al. / Phytochemistry Letters 18 (2016) 128–135
Its leaves are used against diarrhoea (Arbonnier, 2009). In the South Western and Western regions of Cameroon, it is used for the treatment of several diseases such as fever and eczema. Remedies from root barks are also used for the treatment of venereal diseases (Laird et al., 1997). The seeds are eaten by chimpanzees and are known to be a source of gum exudate rich in mineral salts (Ushida et al., 2006). The methanolic extract of its stem bark has been reported to exhibit strong activity against Plasmodium falciparum K1 strain and Trypanosoma brucei rhodesienses (Lenta et al., 2007). Previous phytochemical studies of A. zygia stem bark reported the presence of flavonoids (Abdalla and Laatsch, 2012). In the present investigation on A. zygia roots, we report the isolation, structure characterization of two new triterpenoid saponins, named zygiaosides A–B (1–2), together with two known saponins, coriarioside A (3) and lebbeckoside A (4). The new isolated saponins (1–2) were evaluated for their pro-apoptotic effect on the A431 human epidermoid cancer cell, results are reported herein.
each year (Lewis and Weinstock, 2004). Surgical treatment for non-melanoma skin cancers is successful for the majority of patients. However, patients with advanced disease, frequently suffer chemotherapy treatment failure (Yang et al., 2013). Therefore, novel anti-skin cancer agents are sorely needed. Some natural products have been proved to be powerful anti-cancer agents. Several studies have described their mechanism of actions, through different pathways such as cell cycle arrest and/or induction of apoptosis in addition to inhibition of cell survival (Shu et al., 2010). Most cancer cells develop ways to evade apoptosis, or exhibit defective apoptosis mechanisms, thus allowing uncontrollable cell development (Erb et al., 2005). In these cases, apoptosis process becomes therefore the major target of anti-cancer chemotherapeutics. Triterpenoid saponins, commonly described in Albizia genus, are complex glycosides of acacic acid-type saponins. They have been reported to inhibit the growth of tumor cells, and thus appear as a new potential class of anticancer natural triterpenoid saponins (Lacaille-Dubois et al., 2011). In order to discover new bioactive acacic acid glycosides, we have investigated the saponins content of the roots of Albizia zygia. Albizia zygia (DC.) J.F. Macbr. (Mimosaceae) is a medium-sized (1 m of diameter) tree up to 25 m high distributed in Central and Southern Africa, occurring from Senegal to Cameroon and in Soudan (Arbonnier, 2009). In traditional medicine, its roots barks are used against cough, while its stem bark is used as a purgative, disinfectant, aphrodisiac, and to treat gastritis, toothache, conjunctivitis, as well as to fight worms and overcome female sterility.
2. Results and discussion The air-dried powdered roots of A. zygia (300 g) were extracted with aq-EtOH 70% using a Soxhlet apparatus. After evaporation of the solvent, the resulting brown residue was partitioned between water and water-saturated n-BuOH. The n-BuOH fraction was then submitted to vacuum-liquid chromatography (VLC) on reversedphase silica gel yielding three main fractions that were subjected to VLC on silica gel. Purification of the eluated subfractions by
29
11
13 14
10 9 5
4
6
8 27
7
O
21
17
MT1 O
22
O 28
16
O
OH
15
OH
O
O
OH
O HO
Fuc
18
26
1
2 3
12
30 20
19
25
129
OH
23
O
Qui I
24 OH
O
HO HO
GlcI
O
HO HO
OH
O
GlcII
Xyl
HO HO
O
O
OH
O
O
O
O
OH
HO O
O
MT2 O
Rha OH
O
GlcIII
O OH
O
HO HO
MT3
OH
O
Qui III
Qui II
OH
HO OR
HO OH
MT-Qui
R (1) (2)
MT-Qui H
Fig. 1. Structures of compounds 1–4.
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semiprep-HPLC afforded two new triterpenoid saponins, named zygiaosides A–B (1–2), together with two known saponins, coriarioside A (3) and lebbeckoside A (4) (Fig. 1). Zygiaoside A (1) was obtained as a white, amorphous powder. Its positive HR-ESI–MS gave a pseudo-molecular ion peak at m/z 2354.1581 [M + NH4]+ with its isotopic peak at m/z 2353.1597, 2355.1637 and the corresponding doubly charged ion peaks at m/z 1185.5927, 1186.1004, and 1186.5977 [M + 2(NH4)]2+. Consequently, compound 1 was assigned a C113H178O50 molecular formula. Upon acid hydrolysis with 2.0 M HCl, 1 gave an acacic acid lactone unit, which was identified with an authentic sample, together with glucose (Glc), xylose (Xyl), fucose (Fuc), rhamnose (Rha), and quinovose (Qui), which were identified by co-TLC with authentic samples (Section 3). The absolute configuration of these sugar residues was determined to be D for Glc, Xyl, Qui, and Fuc, and L for Rha based on GC analysis of their trimethylsilyl thiazolidine derivatives (Section 3) (Chaabi et al., 2010). 1H NMR spectrum of 1 showed seven angular methyl groups as singlets at dH 0.89, 1.02, 1.08, 1.10, 1.11, 1.23, and 1.79 (each 3H, s), one olefinic proton at dH 5.67 (1H, brs), and sugar proton signals at dH 4.84-6.42. Its 13C NMR spectrum showed two olefinic carbon signals at dC 123.3 and 143.4, evidencing that 1 was an oleanane type triterpenoid saponin. 1D (1H, 13C NMR, DEPT) and 2D (COSY, HSQC and HMBC) NMR techniques permitted the unambiguous assignment of all 1H and 13 C NMR signals of the aglycone of 1 identified as acacic acid (3b, 16a, 21b-trihydroxyolean-12-ene-28-oic acid) by comparison of its 1H and 13C NMR signals with those reported in the literature (Table 1) (Cao et al., 2007; Noté et al., 2009; Liu et al., 2010). Substitutions at C-3 and C-28 of acacic acid were evidenced by the observed glycosylation-induced shifts: C-3 at dC 89.4 and C-28 at dC 175.5. Substitution at C-21 was ascertained from the acylationinduced shift observed for C-21 (dC 77.3) establishing that 1 was a 21-acyl 3, 28-bidesmosidic acacic acid derivative with sugar chains linked to C-3 and C-28 of the aglycon through ether and ester bond, respectively, and with an acyl group attached at C-21 (Fig. 2). The 1H NMR spectrum of 1 showed nine anomeric protons at dH 4.84 [d, J = 7.0 Hz, glucose (GlcI)], 4.92 [d, J = 8.1 Hz, fucose (Fuc)], 6.17 [d, J = 7.9 Hz, glucose (GlcII)], 6.43 [brs, rhamnose (Rha)], 5.39 [d, J = 8.1 Hz, glucose (GlcIII)], 5.31 [d, J = 7.2 Hz, xylose (Xyl)], 4.90 [d, J = 7.8 Hz, quinovose (QuiI)], 4.88 [d, J = 7.8 Hz, quinovose (QuiII)], and 4.97 [d, J = 8.0 Hz, quinovose (QuiIII)], which correlated with nine anomeric carbon atom resonances at dC 105.4, 105.9, 95.6, 101.5, 106.5, 106.6, 99.6, 99.6, and 97.2, respectively, in the HSQC spectrum (Tables 2 and 3). The 1H and 13C NMR data (Tables 2 and 3) of the monosaccharide residues were assigned starting, either from the readily identifiable anomeric proton of each hexosyl or pentosyl unit, or from the CH3-proton doublet of each 6deoxyhexosyl unit, by means of COSY, HSQC-TOCSY, HSQC, NOESY, and HMBC spectra obtained for this compound. Data from the above experiments indicated that these nine sugar residues were in their pyranose form. The anomeric centers of the D-glucose, Dfucose, D-quinovose, and D-xylose units in their pyranose form were each determined to have a b-configuration based on large 3JH1,H-2 values. The a-anomeric configuration of the L-rhamnose was judged by the broad singlet of its anomeric proton and the chemical shift value of C-5 (d 68.7) (Luo et al., 2008). In addition, the 1H NMR spectrum of compound 1 exhibited three olefinic proton signals at dH 6.90 (d, J = 7.3 Hz), 7.00 (d, J = 7.1 Hz), and 7.08 (d, J = 7.4 Hz), and three groups of one-substituted olefin proton signals, one group at dH 6.24 (dd, J = 10.9; 17.6 Hz), 5.27 (d, J = 10.9 Hz) and 5.47 (d, J = 17.6 Hz), another group at dH 6.27 (dd, J = 10.9; 17.6 Hz), 5.25 (d, J = 10.9 Hz) and 5.45 (d, J = 17.6 Hz), and the other group at dH 6.23 (dd, J = 11.0; 17.2 Hz), 5.29 (d, J = 11.0 Hz) and 5.40 (d, J = 17.2 Hz), indicating that compound 1 had three units of monoterpenoid moieties (Liu et al., 2010).
Table 1 1 H NMR (600 MHz) and 13C NMR (150 MHz) data of the aglycone part of compounds 1–2 in pyridine-d5 (d in ppm, J in Hz).a N C
1a 1b 2a 2b 3 4 5 6a 6b 7a 7b 8 9 10 11a 11b 12 13 14 15a 15b 16 17 18 19a 19b 20 21 22a 22b 23 24 25 26 27 28 29 30
1
2
dC
dH (J in Hz)
dC
dH (J in Hz)
39.0
1.61 nd 1.92 nd 3.18, dd (10.7, 4.1) – 0.63, d (12.6) 1.51 1.30 1.63 1.55 – 1.75 – 2.47 2.03 5.67, brs – – 2.24, d (4.4) 2.08, nd 5.67 – 3.50 3.00 1.51 – 5.37 2.24 2.09 1.23, s 1.11, s 0.89, s 1.10, s 1.79, s – 1.02, s 1.08, s
38.7
1.62 1.03 1.91 2.41 3.17 dd (10.5, 4.0) – 0.62, d (12.6) 1.51 1.31 1.62 1.56 – 1.75 – 2.44 2.03 5.66, brs – – 2.24, nd 2.09, nd 5.66 – 3.50 3.02 1.50 – 5.39 2.23 2.08 1.22, s 1.10, s 0.88, s 1.11, s 1.79, s – 1.08, s 1.09, s
27.0 89.4 39.4 56.1 18.7 33.7 40.0 47.2 37.2 24.0 123.3 143.4 42.1 36.2 75.8 52.6 41.3 48.3 35.4 77.3 36.7 28.5 17.1 16.0 17.6 27.5 175.5 29.5 19.5
26.7 89.1 39.4 55.8 18.3 33.6 40.1 46.9 37.2 23.8 123.0 143.3 42.2 35.9
52.6 40.9 48.2 35.4 77.2 36.4 28.2 16.8 15.7 17.3 27.1 174.4 29.2 19.2
Assignments were based on the HMBC, HSQC, COSY, HSQC-TOCSY, NOESY, and DEPT experiments. nd, not determinated. a Overlapped proton NMR signals are reported without designated multiplicity.
Extensive analysis of 1D and 2D NMR spectra of 1, revealed that the sugar moiety at C-28 was identical to that of lebbeckosides A–B (Noté et al., 2015), and the monoterpene-quinovosyl moiety at C21 was identical to that of gummiferaoside C (Cao et al., 2007), coriarioside A (Noté et al., 2009), coriariosides C-D (Noté et al., 2010), and lebbeckoside A (Noté et al., 2015). Hence, the units attached at C-28 and C-21 of the aglycon of 1 were established as b-D-xylopyranosyl-(1 ! 4)-[b-D-glucopyranosyl-(1 ! 3)]-a-Lrhamnopyranosyl-(1 ! 2)-b-D-glucopyranoside, and {(2E,6S)-6-O{4-O-[(2E,6S)-2,6-dimethyl-6-O-(b-D-quinovopyranosyl) octa-2,7dienoyl]-4-O-[(2E,6S)-2,6-dimethyl-6-O-(b-D-quinovopyranosyl) octa-2,7-dienoyl]-b-D-quinovopyranosyl}-2,6-dimethylocta-2,7dienoyl}, respectively. In the same way, extensive analysis of 1D and 2D NMR spectra of 1, revealed that the sugar moiety at C-3 was identical to that of coriarioside E (Noté et al., 2010). Hence, the unit attached at C-3 of the aglycone was established as b-D-fucopyranosyl-(1 ! 6)-b-Dglucopyranoside. Consequently, the structure of zygiaoside A (1) was elucidated as 3-O-[b-D- fucopyranosyl-(1 ! 6)-b-D-glucopyranosyl]-21-O-{(2E,6S)-6-O-{4-O-[(2E,6S)-2,6-dimethyl-6-O-(b-Dquinovopyranosyl)octa-2,7-dienoyl]-4-O-[(2E,6S)-2,6-dimethyl6-O-(b-D-quinovopyranosyl)octa-2,7-dienoyl]-b-D-quinovopyranosyl}-2,6-dimethylocta-2,7-dienoyl}acacic acid 28-O-b-D-xylopyranosyl-(1 ! 4)-[b-D-glucopyranosyl-(1 ! 3)]-a-L-
O.P. Noté et al. / Phytochemistry Letters 18 (2016) 128–135
131
O O O
O
OH
OH
O
O
HO OH
HO HO
OH
O
O
O OH
O
OH
HO O
O
HO HO
O O
HO HO
O
O
O
OH O
OH HO HO
O OH
O
OH
O HO O
OH O
O OH
O
HMBC
HO OH
NOESY Fig. 2. Key HMBC and NOESY correlations of compound 1.
rhamnopyranosyl-(1 ! 2)-b-D-glucopyranosyl ester, a new acacic acid derivative. Zygiaoside B (2), obtained as a white amorphous powder, exhibited in its HRESIMS spectrum (positive-ion mode) a pseudomolecular ion peak at m/z 2042.0073 [M +NH4]+ with its corresponding doubly charged ion peak at m/z 1029.5181 [M + 2 (NH4)]2+. Consequently, compound 2 was assigned a C97H154O44 molecular formula. As already observed for 1, acid hydrolysis of 2 also afforded an acacic acid lactone, identified by co-TLC with authentic sample, together with D-glucose, D-xylose, D-fucose, Lrhamnose, and D-quinovose units which were identified by GC analysis of their trimethylsilyl thiazolidine derivatives (Section 3). Comparison of 1D and 2D NMR data of 2 with those of 1 indicated that 2 had acacic acid as aglycon while the major difference regarding the aglycone moiety was the absence of one monoterpene-quinovosyl (MT-Qui) unit in 2, as already suggested by its
mass spectrum, which displayed 312 mass unit less than that of 1. The observation of glycosylation- and acylation-induced shifts at dC 89.1 (C-3 of Agly), dC 77.2 (C-21 of Agly), and dC 174.4 (C-28 of Agly) in the 13C NMR spectrum of 2 suggested that it should be also a 21-acyl 3,28-bidesmosidic acacic acid derivative with sugar chains linked to C-3 and C-28 through an ether and ester bond, respectively, and with an acyl group attached at C-21. The 1H NMR spectrum of 2 showed eight anomeric protons signals at dH 4.84 [d, J = 7.4 Hz, glucose (GlcI)], 4.91 [d, J = 7.7 Hz, fucose (Fuc)], 6.17 [d, J = 7.9 Hz, glucose (GlcII)], 6.43 [brs, rhamnose (Rha)], 5.34 [d, J = 7.8 Hz, glucose (GlcIII)], 5.31 [d, J = 7.0 Hz, xylose (Xyl)], 4.89 [d, J = 7.6 Hz, quinovose (QuiI)], and 4.91 [d, J = 7.7 Hz, quinovose (QuiII)], which gave correlations with eight anomeric carbon resonances at dC 105.1, 105.6, 95.2, 101.2, 106.2, 106.2, 99.3, and 99.3, respectively in the HSQC spectrum (Tables 2 and 3). Comparison of their NMR spectra revealed that the units attached
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Table 2 1 H NMR (600 MHz) and 13C NMR (150 MHz) data of the sugar moieties attached at C-3 and C-28 of compounds 1–2 in pyridine-d5 (d in ppm, J in Hz).a Position
1
dC 3-O-sugars GlcI 1 2 3 4 5 6a 6b Fuc 1 2 3 4 5 6 28-O-sugars GlcII 1 2 3 4 5 6a 6b
105.4 75.8 78.7 71.7 76.3 70.4
105.9 71.9 75.8 72.4 71.4 17.6
2
dH (J in Hz)
4.84, d (7.0) 4.03 4.13 4.18 4.05 4.90 4.27
4.92, d (8.1) 4.06 4.01 4.08 3.82 1.54, d (6.4)
dC
105.1 75.6 78.6 71.5 76.4 70.1
105.6 71.9 75.6 72.5 71.4 17.3
Quinovose (Qui)
dH (J in Hz)
4.84, d (7.4) 4.02 4.13 4.19 4.03 4.91 4.27
4.91, d (7.9) 4.05 4.00 4.06 3.82 1.55, d (6,5)
95.6 76.3 78.2 71.4 79.5 62.2
6.17, d (7.9) 4.33 4.09 4.17 4.01 4.39 4.30
95.2 76.4 77.9 71.4 79.3 61.9
6.17, d (7.9) 4.32 4.04 4.17 4.00 4.39 4.30
101.5 72.2 83.8 83.1 68.7 18.7
6.43, brs 4.89 4.28 4.50 4.60 1.76
101.2 71.9 83.5 82.7 68.4 18.9
6.43, brs 4.89 4.27 4.50 4.60 1.78
GlcIII 1 2 3 4 5 6a 6b
106.5 75.8 77.5 71.8 78.5 62.9
5.39, d (8.1) 4.15 4.14 4.15 3.94 4.49 4.27
106.2 75.6 77.9 71.9 78.6 62.6
5.34, d (7.8) 4.15 4.14 4.15 3.94 4.49 4.28
Xyl 1 2 3 4 5a 5b
106.6 76.7 78.2 71.3 67.7
5.31, d (7.2) 4.10 4.06 4.12 4.26 3.50
106.2 76.8 78.2 71.4 67.3
5.31, d (7.0) 4.10 4.07 4.12 4.26 3.50
Rha 1 2 3 4 5 6
Table 3 1 H NMR (600 MHz) and 13C NMR (150 MHz) data of the quinovosyl moieties attached at C-21 of compounds 1–2 in pyridine-d5 (d in ppm, J in Hz).a
Assignments were based on the HMBC, HSQC, COSY, TOCSY, NOESY, and DEPT experiments. nd, not determinated. a Overlapped proton NMR signals are reported without designated multiplicity.
at C-3 and C-28 of the aglycon of 2 were identical to that of 1, and that the two saponins differ only by the length of the monoterpene-quinovosyl moiety at C-21, as already suggested by the mass spectrum of 2, which displayed 312 mass units less than that of 1, accounting for the mass of one quinovose and one monoterpene unit. Therefore, the sugar chains at C-3 and C-28 were established as b-D-fucopyranosyl-(1 ! 6)-b-D-glucopyranoside, and b-D-xylopyranosyl-(1 ! 4)-[b-D-glucopyranosyl-(1 ! 3)]-a-L-rhamnopyranosyl-(1 ! 2)-b-D-glucopyranoside, respectively. Extensive analysis of 1D and 2D NMR spectra of 2 allowed to identify the unit attached at C-21 as {(2E,6S)-6-O-{4-O-[(2E,6S)-2,6-dimethyl6-O-(b-D-quinovopyranosyl)octa-2,7-dienoyl]-b-D-
1
2
dC
dH (J in Hz)
dC
dH (J in Hz)
QuiI 1 2 3 4 5 6
99.6 75.9 76.3 77.5 70.4 18.7
4.90, d (7.8) 4.02 4.21 5.37 3.71 1.37, d (6.1)
99.3 75.6 76.1 77.2 70.1 18.7
4.91, d (7.7) 4.02 4.22 5.37 3.71 1.37, d (6.1)
QuiII 1 2 3 4 5 6
99.6 75.6 76.3 75.9 72.8 19.0
4.88, d (7.8) 4.03 4.23 5.67 3.77 1.62, d (5.9)
99.3 75.5 75.9 76.8 72.5 19.2
4.89, d (7.6) 4.03 4.23 3.75 3.73 1.62, d (5.4)
QuiIII 1 2 3 4 5 6
97.2 75.9 76.4 77.1 70.4 19.0
4.97, d (8.0) 4.02 4.23 3.75 3.73 1.60, d (5.4)
Assignments were based on the HMBC, HSQC, COSY, TOCSY, NOESY, and DEPT experiments. nd, not determinated. a Overlapped proton NMR signals are reported without designated multiplicity.
quinovopyranosyl}-2,6-dimethylocta-2,7-dienoyl}, a unit identical to that of gummiferaosides A and B (Cao et al., 2007), coriarioside E (Noté et al., 2010), and lebbeckoside B (Noté et al., 2015). Consequently, the structure of zygiaoside B (2) was established as 3-O-[b-D-fucopyranosyl-(1 ! 6)-b-D-glucopyranosyl]-21-O{(2E,6S)-6-O-{4-O-[(2E,6S)-2,6-dimethyl-6-O-(b-D-quinovopyranosyl)octa-2,7-dienoyl)-b-D-quinovopyranosyl]octa-2,7-dienoyl} acacic acid 28-O-b-D-xylopyranosyl-(1 ! 4)-[b-D-glucopyranosyl(1 ! 3)]-a-L-rhamnopyranosyl-(1 ! 2)-b-D-glucopyranosyl ester, a new acacic acid derivative. Compound 3, isolated as an amorphous powder, was identified as 3-O-{b-D- fucopyranosyl-(1 ! 6)-[b-D-glucopyranosyl-(1 ! 2)]b-D-glucopyranosyl}-21-O-{(2E,6S)-6-O-{4-O-[(2E,6S)-2,6-dimethyl-6-O-(b-D-quinovopyranosyl)octa-2,7-dienoyl]-4-O[(2E,6S)-2,6-dimethyl-6-O-(b-D-quinovopyranosyl)octa-2,7dienoyl]-b-D-quinovopyranosyl}-2,6-dimethylocta-2,7-dienoyl} acacic acid 28-O-a-L-arabinofuranosyl-(1 ! 4)-[b-D-glucopyranosyl-(1 ! 3)]-a-L-rhamnopyranosyl-(1 ! 2)-b-D-glucopyranosyl ester, named coriarioside A (Noté et al., 2009) and compound 4, isolated as an amorphous powder, was identified as 3-O-[b-Dxylopyranosyl-(1 ! 2)-b-D-fucopyranosyl-(1 ! 6)-b-D-glucopyranosyl]-21-O-{(2E,6S)-6-O-{4-O-[(2E,6S)-2,6-dimethyl-6-O-(b-Dquinovopyranosyl)octa-2,7-dienoyl]-4-O-[(2E,6S)-2,6-dimethyl6-O-(b-D-quinovopyranosyl)octa-2,7-dienoyl]-b-D-quinovopyranosyl}-2,6-dimethylocta-2,7-dienoyl}acacic acid 28-O-b-D-xylopyranosyl-(1 ! 4)-[b-D-glucopyranosyl-(1 ! 3)]-a-L-rhamnopyranosyl-(1 ! 2)-b-D-glucopyranosyl ester, named lebbeckoside A by comparison of their respective spectral data with those reported in the literature (Noté et al., 2015). The pro-apoptotic activity of compounds 1–2 was evaluated using Annexin V-FITC binding assay on the A431 human epidermoid cancer cell. In result, percentage of apoptotic cells following 24 h of treatment was increased in a concentrationdependent manner for A431 cell (Fig. 3). Computed concentrations of each compound to induced half-maximal effects (EC50) on cell apoptosis were determined as being 9 mM and 249 mM for
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Fig. 3. Effects of compounds 1–2 on the apoptosis rate. Cells were exposed to compounds (1–2) at the indicated concentrations and incubated for 24 h. Cell apoptosis rate was assessed by flow cytometry using the Annexin V-FITC/ PI staining assay (n = 3). *P < 0.05 and ***P < 0.001 vs. vehicle-treated cells.
zygiaosides A–B, respectively. Furthermore, it is worthy to note that the proapoptotic function of triterpenoid saponins isolated from Albizia genus has been previously reported for glaberrimosides isolated from Albizia glaberrima (Noté et al., 2016), and for adianthifoliosides A and D isolated from Albizia adianthifolia (Haddad et al., 2004). These later compounds and the present saponins share the following structural features: a common aglycone unit, acacic acid, with various oligosaccharide moieties at C-3 and C-28 and an acyl group at C-21. As for adianthifoliosides, the next steps will be to further explain the apoptotic route induced by zygiaoside A (1). In addition, this study contributes to complete the knowledge of the composition of Albizia spp, which are natural sources of complex bioactive triterpenoid saponins. Furthermore, our findings, especially for the monoterpenequinovosyl units, established in compounds 1 and 2, and identical to those found in the related compounds isolated from Albizia coriaria, A. gummifera, and A. lebbeck, strongly suggest a close relationship between these four species of Albizia genus. 3. Experimental section 3.1. General experimental procedures Optical rotations were measured on a Jasco P-2000 polarimeter. H NMR (600 MHz) and 13C NMR (150 MHz) spectra were recorded at room temperature in pyridine-d5 using a Bruker 600 MHz spectrometer. Chemical shifts are given in d (ppm) value relative to TMS as internal standard. HRESIMS spectra were recorded on a microTOF ESI-TOF mass spectrometer (Agilent) operating in positive mode. Analytical HPLC was performed on Varian 920-LC apparatus equipped with an autosampler, a pump, a diode array detector (DAD), and Galaxie software. Semipreparative HPLC was performed on a Gilson apparatus equipped with Trilution LC software using a Nucleodur 100-5 C18ec (21 250 mm, 5 mm) column purchased from Machery-Nagel (Germany). Thin layer chromatography (TLC) was performed on precoated silica gel plates (60 F254, Merck) using the system solvent n-BuOH-AcOHH2O, 65:15:25 as eluent. The spots were observed after spray with Komarowsky reagent, which is a mixture (5:1) of phydroxybenzaldehyde (2% in MeOH) and Ethanolic H2SO4 (50%). Vacuumliquid chromatography (VLC) was carried out using RP-18 silica gel 60 (25–40 mm) and silica gel 60 (15–40 mm and 40–63 mm). 1
3.2. Plant material The roots of Albizia zygia were harvested at Nkolbisson, Yaoundé peripheral quarter, in Cameroon in November 2013
under the guidance of Mr. Victor Nana, botanist of the National Herbarium of Cameroon (NHC), where a voucher specimen (2338/ SRFK) was deposited. 3.3. Extraction and isolation Air-dried and finely powdered roots of A. zygia (300 g) were extracted with 70% EtOH in a Soxhlet apparatus. The hydroalcoholic solution was then evaporated to dryness under reduced pressure to yield a brown residue (7.03 g). The residue was suspended in 200 ml of water and partitioned between H2O and satured n-BuOH (3 300 ml). The n-BuOH phase was evaporated to dryness affording 5.20 g of a brown gum which was taken in a minimum amount of water (10 ml) and then submitted to vacuumliquid chromatography (VLC) using RP-18 (25–40 mm) eluting with H2O, 50% MeOH, and 100% MeOH. The 100% MeOH extract was evaporated to dryness affording 3.40 g crude saponin mixture that was then submitted to VLC using silica gel 60 (15–40 mm), eluted with CHCl3-MeOH-H2O (80:20:2, 70:30:5, 60:33:7, and 60:40:10) to give three main fractions (Z1- Z3). Fraction Z3 (600.2 mg) was subjected to VLC on silica gel 60 (15–40 mm), eluted with CHCl3MeOH-H2O (80:20:2, 70:30:5, 60:33:7, and 60:40:10) affording four main subfractions (AZ31- AZ34). Subfraction AZ34 (150.7 mg) was purified by semipreparative HPLC using gradient system of CH3CN-H2O (20 ml/min) to yield compounds 1 (tR, 24.80 min, 7.6 mg), 2 (tR, 14.97 min, 6.7 mg), 3 (tR, 21.87 min, 3.2 mg), and 4 (tR, 23.14 min, 3.6 mg). 3-O-[b-D-fucopyranosyl-(1 ! 6)-b-D-glucopyranosyl]-21-O{(2E,6S)-6-O-{4-O-[(2E,6S)-2,6-dimethyl-6-O-(b-D-quinovopyranosyl)octa-2,7-dienoyl]-4-O-[(2E,6S)-2,6-dimethyl-6-O-(b-D-quinovopyranosyl)octa-2,7-dienoyl]-b-D-quinovopyranosyl}-2,6dimethylocta-2,7-dienoyl}acacic acid 28-O-b-D-xylopyranosyl(1 ! 4)-[b-D-glucopyranosyl-(1 ! 3)]-a-L-rhamnopyranosyl(1 ! 2)-b-D-glucopyranosyl ester (1), white amorphous powder; [a]25D + 29.25 (c 0.06, MeOH); 1H NMR (600 MHz, C5D5N) and 13C NMR (150 MHz, C5D5N), see Tables 1–4. Positive HR-ESI–MS: m/z 2353.1597, 2354.15812, 2355.1637 and doubly charged ion peaks at m/z 1185.5927, 1186.1004, 1186.5977 [M + 2(NH4)]2+. 3-O-[b-D-fucopyranosyl-(1 ! 6)-b-D-glucopyranosyl]-21-O{(2E,6S)-6-O-{4-O-[(2E,6S)-2,6-dimethyl-6-O-(b-D-quinovopyranosyl)octa-2,7-dienoyl)-b-D-quinovopyranosyl]octa-2,7-dienoyl} acacic acid 28-O-b-D-xylopyranosyl-(1 ! 4)-[b-D-glucopyranosyl(1 ! 3)]-a-L-rhamnopyranosyl-(1 ! 2)-b-D-glucopyranosyl ester (2), white amorphous powder; [a]25D 18.75 (c 0.16, MeOH); 1H NMR (600 MHz, C5D5N) and 13C NMR (150 MHz, C5D5N), see Tables 1–4; positive HR-ESI–MS: m/z 2042.00732 [M +NH4]+ and doubly charged ion peak at m/z 1029.51814 [M + 2(NH4)]2+.
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Table 4 1 H NMR (600 MHz) and 13C NMR (150 MHz) data of the monoterpene moieties attached at C-21 of compounds 1–2 in pyridine-d5 (d in ppm, J in Hz).a Monoterpene (MT)
MT1 1 2 3 4a 4b 5a 5b 6 7 8a 8b 9 10 MT2 1 2 3 4a 4b 5a 5b 6 7 8a 8b 9 10 MT3 1 2 3 4a 4b 5a 5b 6 7 8a 8b 9 10
1
2
dC
dH (J in Hz)
dC
dH (J in Hz)
168.1 128.5 142.4 24.0
– – 6.90, t (7.3) 2.47 nd 1.78 nd – 6.24, dd (10.9, 17.6) 5.47, d (17.6) 5.27, d (10.9) 1.93, s 1.51, s
168.1 128.5 142.1 23.5
– – 6.90, t (7.5) 2.44 nd 1.78 nd – 6.24, dd (10.8, 17.5) 5.43, d (17.5) 5.25, d (10.8) 1.85, s 1.55, s
– – 7.00, t (7.1) 2.42 nd 1.76 nd – 6.27, dd (10.9, 17.6) 5.45, d (17.6) 5.25, d (10.9) 1.85, s 1.56, s
168.1 128.5 143.3 23.8
parameters were as follows: ionization potential, 70 eV; ion source temperature, 230 C; solvent delay 4.0 min, mass range 100–700. The following sugars were detected for 1 and 2: D-glucose, Dquinovose, D-fucose, D-xylose, and L-rhamnose. 3.5. Pro-apoptotic evaluation
40.8 80.1 144.3 115.2 13.0 24.2
168.1 128.5 143.1 23.8 40.8 80.1 144.3 115.4 13.0 24.1
167.8 128.5 143.3 24.0 40.8 80.1 143.5 115.8 13.0 23.8
40.3 80.1 143.9 114.8 12.7 23.7
40.3 80.1 144.1 115.0 12.7 23.6
– – 7.06, t (7.3) 2.43 nd 1.79 nd – 6.27, dd (10.8, 17.6) 5.49, d (17.6) 5.30, d (10.8) 1.90, s 1.56, s
– – 7.08, t (7.4) 2.46 nd 1.79 nd – 6.23, dd (11.0, 17.2) 5.40, d (17.2) 5.29, d (11.0) 1.96, s 1.57, s
Assignments were based on the HMBC, HSQC, COSY, TOCSY, NOESY, and DEPT experiments. nd, not determinated. a Overlapped proton NMR signals are reported without designated multiplicity.
3.4. Acid hydrolysis of compounds and determination of the absolute configuration of the sugar residues Each saponin (2 mg) was hydrolyzed with 2 ml of 2 M HCl at 85 C during 2 h. After cooling, the solvent was removed under reduced pressure. The sugar mixture was extracted from the aqueous phase (10 ml) and washed with CH2Cl2 (3 5 ml). The combined CH2Cl2 extracts were washed with water to give after evaporation the aglycone moiety. The sugars were first analyzed by TLC over silica gel (CHCl3–MeOH H2O, 8:5:1) by comparison with standard samples. The absolute configuration of each monosaccharide was determined from GC–MS analysis of their trimethylsilylated derivatives by comparison with authentic samples using the method previously described (Chaabi et al., 2010). GC analysis was performed with a capillary TR-5MS SQC (15 m x 0.25 mm x 0.25 mm) column. Operating conditions were as follows: carrier gas, helium with a flow rate of 1 ml/min; column temperature, 1 min in 150 C, 150–220 C at 4 C/min; injector temperature, 250 C; volume injected 1 ml of the trimethylsilylated sugar in methylene chloride (0.1%); split ratio, 1:50. The MS operating
3.5.1. Cell lines and culture conditions The A431 human epidermoid cancer cell was purchased from ATCC (LGC Standards, Molsheim, France) and cultivated in DMEMbased media (Sigma-Aldrich, Saint-Quentin-Fallavier, France), supplemented with 10% (v/v) fetal bovine serum (BioWhittaker, Verviers, Belgium), 2 mMultraglutamine, 50 mM non-essential amino acids, 50 U/ml penicillin and 50 mg/ml streptomycin (Sigma-Aldrich). The culture was kept at 37 C in a humidified incubator equilibrated with 5% CO2. Before confluency adherent cells were trypsinized and subcultured twice a week. 3.5.2. Pro-apoptotic evaluation Cells were treated with the two purified saponins (1–2) in ranging concentrations (1 to 15 mM) then collected for apoptosis induction estimation. A minimum of 5000 cells was acquired per sample and analyzed on the InCyte software (Guava/Millipore/ Merck, CA, USA). Apoptosis rates were assessed by capillary cytometry (Guava EasyCyte Plus, Millipore Merck) using Annexin V-FITC (ImmunoTools, Germany) and PI (MiltenyiBiotec Inc., USA) according to the manufacturer’s recommendations. Gates were drawn around the appropriate cell population using a forward scatter (FSC) versus side scatter (SSC) acquisition dot plot to exclude debris. Final concentration of DMSO applied to cells during incubation with tested samples was always 0.5%. In the tested setup that concentration had no adverse effects on cell viability, nor cell morphology. To discriminate between negative and positive events in the analysis, a non-stained control sample from each culture condition always accompanied acquisition of the stained cells to define their cut off. Negative control, i.e. sample with cells without compounds but with the same amount of DMSO as for diluted compounds, as well as positive control with 50 mM Celastrol, a natural pentacyclic triterpenoid (Enzo Life Sciences, Farmingdale, US), were included for each experimental set. Cytometers performances are checked weekly using the Guava easy Check Kit 4500-0025 (Merck/Millipore/Guava Hayward, CA, USA). 3.5.3. Statistical analysis Data, presented as bar graphs, were expressed as means S.E. M. of at least three independent experiments. Statistical evaluation was performed with the one-way ANOVA test followed by the post-hoc Bonferroni test using GraphPad Prism software (Prism version 5.04 for Windows, GraphPad Software, CA, USA); a p-value less than 0.05 was considered as significant (*), less than 0.01 very significant (**) and less than 0.001 highly significant (***). Acknowledgments This work was financially supported by the World Academy of sciences (TWAS) (grant no: 3240277733). The authors are also grateful to Mr. Victor Nana of the National Herbarium of Cameroon (NHC) for the identification and collection of plant. References Abdalla, M.A., Laatsch, H., 2012. Flavonoids from sudanese Albizia zygia (Leguminosae, subfamily mimosoideae), a plant with antimalarial potency. Afr. J. Tradit. Complement. Altern. Med. 9, 56–58. Arbonnier, M., 2009. Arbres, arbustes et lianes des zones d’Afrique de l’Ouest; MNHN (Eds) Quæ, Paris. p. 383.
O.P. Noté et al. / Phytochemistry Letters 18 (2016) 128–135 Cao, S., Norris, A., Miller, J.S., Ratovoson, F., Razafitsalama, J., Andriantsiferana, R., Rasmison, V.E., Tendyke, K., Suh, T., Kingston, D.G.I., 2007. Cytotoxic triterpenoid saponins of Albizia gummifera from the rain forest. J. Nat. Prod. 70, 361–366. Chaabi, M., Chabert, P., Vonthron-Sénécheau, C., Weniger, B., Ouattara, M., Corstjens, H., Sente, I., Declercq, L., Lobstein, A., 2010. Acylated flavonol pentaglycosides from Baphia nitida leaves. Phytochem. Lett. 3, 70–74. Erb, P., Ji, J., Wernli, M., Kump, E., Glaser, A., Büchner, S.A., 2005. Role of apoptosis in basal cell and squamous cell carcinoma formation. Immunol. Lett. 100, 68–72. Haddad, M., Laurens, V., Lacaille-Dubois, M.A., 2004. Induction of apoptosis in a leukemia cell line by triterpene saponins from Albizia adianthifolia. Bioorg. Med. Chem. 12, 4725–4734. Konstantopoulou, M., Lord, M.G., Macfarlane, A.W., 2006. Treatment of invasive squamous cell carcinoma with 5-percent imiquimod cream. Dermatol. Online J. 12, 10. Lacaille-Dubois, M.A., Pegnyemb, D.E., Noté, O.P., Mitaine-Offer, A.C., 2011. A review of acacic acid-type saponins from leguminosae-mimosoideae as potent cytotoxic and apoptosis inducing agents. Phytochem. Rev. 10, 565–584. Laird, S.A., Betafor, M., Enanga, M., Fominyam, C., Itoe, M., Litonga, E., Mafani, J., Menyoli, J., Meseke, J., Mukete, W., Motia, M., Ndumbe, P., Nkefor, J., Nning, J., Ndam, N., Sunderland, T., Tchouto, P., Wana, M., 1997. Medicinal Plants of the Limbe Botanic Garden, Limbe Botanic Garden, Cameroon. Lenta, B.N., Vonthron-Sénécheau, C., Soh, R.F., Tantangmo, F., Ngouela, S., Kaiser, M., Tsamo, E., Anton, R., Weniger, B., 2007. In vitro antiprotozoal activities and cytotoxicity of some selected Cameroonian medicinal plants. J. Ethnopharmacol. 111, 8–12. Lewis, K.G., Weinstock, M.A., 2004. Nonmelanoma skin cancer mortality (1988– 2000): the Rhode Island follow-back study. Arch. Dermatol. 140, 837–842. Liu, R., Ma, S., Yu, S., Pei, Y., Zhang, S., Chen, X., Zhang, J., 2010. Albizosides D and E, two new cytotoxic triterpene saponins from Albizia chinensis. Carbohydr. Res. 345, 1877–1881.
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Luo, J.G., Ma, L., Kong, L.Y., 2008. New triperpenoid saponins with strong alphaglucosidase inhibitory activity from the roots of Gypsophila. Bioorg. Med. Chem. 16, 2912–2920. Noté, O.P., Mitaine-offer, A.C., Miyamoto, T., Paululat, T., Mirjolet, J.F., Duchamp, O., Pegnyemb, D.E., Lacaille-Dubois, M.A., 2009. Cytotoxic acacic acid glycosides from the roots of Albizia coriaria. J. Nat. Prod. 72, 1725–1730. Noté, O.P., Chabert, P., Pegnyemb, D.E., Weniger, B., Lacaille-Dubois, M.A., Lobstein, A., 2010. Structure elucidation of new acacic acid-type saponins from Albizia coriaria. Magn. Reson. Chem. 48, 829–836. Noté, O.P., Jihu, D., Antheaume, C., Zeniou, M., Pegnyemb, D.E., Guillaume, D., Chneiwess, H., Kilhoffer, M.C., Lobstein, A., 2015. Triterpenoid saponins from Albizia lebbeck (L.) Benth and their inhibitory effect on the survival of high grade human brain tumor cells. Carbohydr. Res. 404, 26–33. Noté, O.P., Azouaou, S.A., Simo, L., Antheaume, C., Guillaume, D., Pegnyemb, D.E., Muller, C.D., Lobstein, A., 2016. Phenotype-specific apoptosis induced by three new triterpenoid saponins from Albizia glaberrima (Schumach. & Thonn.) Benth. Fitoperapia 109, 80–86. Shu, L., Cheung, K.L., Khor, T.O., Chen, C., Kong, N., 2010. Phytochemicals: cancer chemoprevention and suppression of tumor onset and metastasis. Cancer Metastasis Rev. 29, 483–502. Ushida, K., Fujita, S., Ohashi, G., 2006. Nutritional significance of the selective ingestion of Albizia zygia gum exudate by wild chimpanzees in Bossou, Guinea. Am. J. Primatol. 68, 143–151. Yang, S.Y., Wang, H.M., Wu, T.W., Chen, Y.J., Shieh, J.J., Lin, J.H., Ho, T.F., Luo, R.J., Chen, C.Y., Chang, C.C., 2013. Subamolide B isolated from medicinal plant Cinnanomum subavenium induces cytotoxicity in human Cutaneous squamous cell carcinoma cells through mitochondrial and CHOP-dependent cell death pathways. Evidence-Based Compl. Altern. Med. 2013, 1–13.
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Phytochemistry Letters journal homepage: www.elsevier.com/locate/phytol
Mini review
Two new triterpenoid saponins from the roots of Albizia zygia (DC.) J.F. Macbr. Olivier Placide Notéa,b,* , Line Simoa,b , Joséphine Ngo Mbingb , Dominique Guillaumec , Sarah Ali Aouazoua , Christian Dominique Mullera , Dieudonné Emmanuel Pegnyembb , Annelise Lobsteina a Pharmacognosie et Molécules Naturelles Bioactives, Laboratoire d’Innovation Thérapeutique, UMR 7200, CNRS-Université de Strasbourg, Faculté de Pharmacie, 74 Route du Rhin, F-67401 Illkirch Cedex, France b Laboratoire de Pharmacochimie des Substances Naturelles, Département de Chimie Organique, Faculté de Sciences, Université de Yaoundé, BP 812, Yaoundé, Cameroon c Laboratoire de Chimie Thérapeutique, UMR7312, 51 Rue Cognacq-Jay, 51100 Reims, France
A R T I C L E I N F O
Article history: Received 6 July 2016 Received in revised form 12 September 2016 Accepted 20 September 2016 Available online xxx Keywords: Albizia zygia Mimosaceae Triterpenoid saponins NMR Pro-apoptotic activity Human epidermoid cancer cell A431
A B S T R A C T
As part of our search of new apoptosis-inducing triterpenoid saponins from Cameroonian Albizia genus, phytochemical investigation of the roots of Albizia zygia led to the isolation of two new oleanane-type saponins, named zygiaosides A–B (1–2), together with two known saponins, coriarioside A (3) and lebbeckoside A (4). Their structures were established on the basis of extensive 1D and 2D NMR (1H, 13C NMR, DEPT, COSY, HSQC-TOCSY, NOESY, HSQC and HMBC) and HRESIMS studies, and by chemical evidence. The apoptotic effect of saponins 1–2 was evaluated on the A431 human epidermoid cancer cell. Cytometric analyses showed that saponins 1–2 induced apoptosis of human epidermoid cancer cell (A341) in a dose-dependent manner. ã 2016 Published by Elsevier Ltd on behalf of Phytochemical Society of Europe.
Contents 1. 2. 3.
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Results and discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Experimental section . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . General experimental procedures . . . . . . . . . . . . . . . . . . . . . . . . . 3.1. 3.2. Plant material . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Extraction and isolation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.3. 3.4. Acid hydrolysis of compounds and determination of the absolute Pro-apoptotic evaluation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.5. 3.5.1. Cell lines and culture conditions . . . . . . . . . . . . . . . . . . . Pro-apoptotic evaluation . . . . . . . . . . . . . . . . . . . . . . . . . 3.5.2. 3.5.3. Statistical analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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1. Introduction
* Corresponding author at: Laboratoire de Pharmacochimie des Substances Naturelles, Département de Chimie Organique, Faculté des Sciences, Université de Yaoundé, BP 812, Yaoundé, Cameroun. E-mail addresses: [email protected], [email protected] (O.P. Noté).
Cutaneous squamous cell carcinoma represents the second most common skin cancer (Konstantopoulou et al., 2006), carrying a significant risk of metastasis and accounting for the majority of several thousand deaths attributable to non-melanoma skin cancer
http://dx.doi.org/10.1016/j.phytol.2016.09.010 1874-3900/ã 2016 Published by Elsevier Ltd on behalf of Phytochemical Society of Europe.
O.P. Noté et al. / Phytochemistry Letters 18 (2016) 128–135
Its leaves are used against diarrhoea (Arbonnier, 2009). In the South Western and Western regions of Cameroon, it is used for the treatment of several diseases such as fever and eczema. Remedies from root barks are also used for the treatment of venereal diseases (Laird et al., 1997). The seeds are eaten by chimpanzees and are known to be a source of gum exudate rich in mineral salts (Ushida et al., 2006). The methanolic extract of its stem bark has been reported to exhibit strong activity against Plasmodium falciparum K1 strain and Trypanosoma brucei rhodesienses (Lenta et al., 2007). Previous phytochemical studies of A. zygia stem bark reported the presence of flavonoids (Abdalla and Laatsch, 2012). In the present investigation on A. zygia roots, we report the isolation, structure characterization of two new triterpenoid saponins, named zygiaosides A–B (1–2), together with two known saponins, coriarioside A (3) and lebbeckoside A (4). The new isolated saponins (1–2) were evaluated for their pro-apoptotic effect on the A431 human epidermoid cancer cell, results are reported herein.
each year (Lewis and Weinstock, 2004). Surgical treatment for non-melanoma skin cancers is successful for the majority of patients. However, patients with advanced disease, frequently suffer chemotherapy treatment failure (Yang et al., 2013). Therefore, novel anti-skin cancer agents are sorely needed. Some natural products have been proved to be powerful anti-cancer agents. Several studies have described their mechanism of actions, through different pathways such as cell cycle arrest and/or induction of apoptosis in addition to inhibition of cell survival (Shu et al., 2010). Most cancer cells develop ways to evade apoptosis, or exhibit defective apoptosis mechanisms, thus allowing uncontrollable cell development (Erb et al., 2005). In these cases, apoptosis process becomes therefore the major target of anti-cancer chemotherapeutics. Triterpenoid saponins, commonly described in Albizia genus, are complex glycosides of acacic acid-type saponins. They have been reported to inhibit the growth of tumor cells, and thus appear as a new potential class of anticancer natural triterpenoid saponins (Lacaille-Dubois et al., 2011). In order to discover new bioactive acacic acid glycosides, we have investigated the saponins content of the roots of Albizia zygia. Albizia zygia (DC.) J.F. Macbr. (Mimosaceae) is a medium-sized (1 m of diameter) tree up to 25 m high distributed in Central and Southern Africa, occurring from Senegal to Cameroon and in Soudan (Arbonnier, 2009). In traditional medicine, its roots barks are used against cough, while its stem bark is used as a purgative, disinfectant, aphrodisiac, and to treat gastritis, toothache, conjunctivitis, as well as to fight worms and overcome female sterility.
2. Results and discussion The air-dried powdered roots of A. zygia (300 g) were extracted with aq-EtOH 70% using a Soxhlet apparatus. After evaporation of the solvent, the resulting brown residue was partitioned between water and water-saturated n-BuOH. The n-BuOH fraction was then submitted to vacuum-liquid chromatography (VLC) on reversedphase silica gel yielding three main fractions that were subjected to VLC on silica gel. Purification of the eluated subfractions by
29
11
13 14
10 9 5
4
6
8 27
7
O
21
17
MT1 O
22
O 28
16
O
OH
15
OH
O
O
OH
O HO
Fuc
18
26
1
2 3
12
30 20
19
25
129
OH
23
O
Qui I
24 OH
O
HO HO
GlcI
O
HO HO
OH
O
GlcII
Xyl
HO HO
O
O
OH
O
O
O
O
OH
HO O
O
MT2 O
Rha OH
O
GlcIII
O OH
O
HO HO
MT3
OH
O
Qui III
Qui II
OH
HO OR
HO OH
MT-Qui
R (1) (2)
MT-Qui H
Fig. 1. Structures of compounds 1–4.
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semiprep-HPLC afforded two new triterpenoid saponins, named zygiaosides A–B (1–2), together with two known saponins, coriarioside A (3) and lebbeckoside A (4) (Fig. 1). Zygiaoside A (1) was obtained as a white, amorphous powder. Its positive HR-ESI–MS gave a pseudo-molecular ion peak at m/z 2354.1581 [M + NH4]+ with its isotopic peak at m/z 2353.1597, 2355.1637 and the corresponding doubly charged ion peaks at m/z 1185.5927, 1186.1004, and 1186.5977 [M + 2(NH4)]2+. Consequently, compound 1 was assigned a C113H178O50 molecular formula. Upon acid hydrolysis with 2.0 M HCl, 1 gave an acacic acid lactone unit, which was identified with an authentic sample, together with glucose (Glc), xylose (Xyl), fucose (Fuc), rhamnose (Rha), and quinovose (Qui), which were identified by co-TLC with authentic samples (Section 3). The absolute configuration of these sugar residues was determined to be D for Glc, Xyl, Qui, and Fuc, and L for Rha based on GC analysis of their trimethylsilyl thiazolidine derivatives (Section 3) (Chaabi et al., 2010). 1H NMR spectrum of 1 showed seven angular methyl groups as singlets at dH 0.89, 1.02, 1.08, 1.10, 1.11, 1.23, and 1.79 (each 3H, s), one olefinic proton at dH 5.67 (1H, brs), and sugar proton signals at dH 4.84-6.42. Its 13C NMR spectrum showed two olefinic carbon signals at dC 123.3 and 143.4, evidencing that 1 was an oleanane type triterpenoid saponin. 1D (1H, 13C NMR, DEPT) and 2D (COSY, HSQC and HMBC) NMR techniques permitted the unambiguous assignment of all 1H and 13 C NMR signals of the aglycone of 1 identified as acacic acid (3b, 16a, 21b-trihydroxyolean-12-ene-28-oic acid) by comparison of its 1H and 13C NMR signals with those reported in the literature (Table 1) (Cao et al., 2007; Noté et al., 2009; Liu et al., 2010). Substitutions at C-3 and C-28 of acacic acid were evidenced by the observed glycosylation-induced shifts: C-3 at dC 89.4 and C-28 at dC 175.5. Substitution at C-21 was ascertained from the acylationinduced shift observed for C-21 (dC 77.3) establishing that 1 was a 21-acyl 3, 28-bidesmosidic acacic acid derivative with sugar chains linked to C-3 and C-28 of the aglycon through ether and ester bond, respectively, and with an acyl group attached at C-21 (Fig. 2). The 1H NMR spectrum of 1 showed nine anomeric protons at dH 4.84 [d, J = 7.0 Hz, glucose (GlcI)], 4.92 [d, J = 8.1 Hz, fucose (Fuc)], 6.17 [d, J = 7.9 Hz, glucose (GlcII)], 6.43 [brs, rhamnose (Rha)], 5.39 [d, J = 8.1 Hz, glucose (GlcIII)], 5.31 [d, J = 7.2 Hz, xylose (Xyl)], 4.90 [d, J = 7.8 Hz, quinovose (QuiI)], 4.88 [d, J = 7.8 Hz, quinovose (QuiII)], and 4.97 [d, J = 8.0 Hz, quinovose (QuiIII)], which correlated with nine anomeric carbon atom resonances at dC 105.4, 105.9, 95.6, 101.5, 106.5, 106.6, 99.6, 99.6, and 97.2, respectively, in the HSQC spectrum (Tables 2 and 3). The 1H and 13C NMR data (Tables 2 and 3) of the monosaccharide residues were assigned starting, either from the readily identifiable anomeric proton of each hexosyl or pentosyl unit, or from the CH3-proton doublet of each 6deoxyhexosyl unit, by means of COSY, HSQC-TOCSY, HSQC, NOESY, and HMBC spectra obtained for this compound. Data from the above experiments indicated that these nine sugar residues were in their pyranose form. The anomeric centers of the D-glucose, Dfucose, D-quinovose, and D-xylose units in their pyranose form were each determined to have a b-configuration based on large 3JH1,H-2 values. The a-anomeric configuration of the L-rhamnose was judged by the broad singlet of its anomeric proton and the chemical shift value of C-5 (d 68.7) (Luo et al., 2008). In addition, the 1H NMR spectrum of compound 1 exhibited three olefinic proton signals at dH 6.90 (d, J = 7.3 Hz), 7.00 (d, J = 7.1 Hz), and 7.08 (d, J = 7.4 Hz), and three groups of one-substituted olefin proton signals, one group at dH 6.24 (dd, J = 10.9; 17.6 Hz), 5.27 (d, J = 10.9 Hz) and 5.47 (d, J = 17.6 Hz), another group at dH 6.27 (dd, J = 10.9; 17.6 Hz), 5.25 (d, J = 10.9 Hz) and 5.45 (d, J = 17.6 Hz), and the other group at dH 6.23 (dd, J = 11.0; 17.2 Hz), 5.29 (d, J = 11.0 Hz) and 5.40 (d, J = 17.2 Hz), indicating that compound 1 had three units of monoterpenoid moieties (Liu et al., 2010).
Table 1 1 H NMR (600 MHz) and 13C NMR (150 MHz) data of the aglycone part of compounds 1–2 in pyridine-d5 (d in ppm, J in Hz).a N C
1a 1b 2a 2b 3 4 5 6a 6b 7a 7b 8 9 10 11a 11b 12 13 14 15a 15b 16 17 18 19a 19b 20 21 22a 22b 23 24 25 26 27 28 29 30
1
2
dC
dH (J in Hz)
dC
dH (J in Hz)
39.0
1.61 nd 1.92 nd 3.18, dd (10.7, 4.1) – 0.63, d (12.6) 1.51 1.30 1.63 1.55 – 1.75 – 2.47 2.03 5.67, brs – – 2.24, d (4.4) 2.08, nd 5.67 – 3.50 3.00 1.51 – 5.37 2.24 2.09 1.23, s 1.11, s 0.89, s 1.10, s 1.79, s – 1.02, s 1.08, s
38.7
1.62 1.03 1.91 2.41 3.17 dd (10.5, 4.0) – 0.62, d (12.6) 1.51 1.31 1.62 1.56 – 1.75 – 2.44 2.03 5.66, brs – – 2.24, nd 2.09, nd 5.66 – 3.50 3.02 1.50 – 5.39 2.23 2.08 1.22, s 1.10, s 0.88, s 1.11, s 1.79, s – 1.08, s 1.09, s
27.0 89.4 39.4 56.1 18.7 33.7 40.0 47.2 37.2 24.0 123.3 143.4 42.1 36.2 75.8 52.6 41.3 48.3 35.4 77.3 36.7 28.5 17.1 16.0 17.6 27.5 175.5 29.5 19.5
26.7 89.1 39.4 55.8 18.3 33.6 40.1 46.9 37.2 23.8 123.0 143.3 42.2 35.9
52.6 40.9 48.2 35.4 77.2 36.4 28.2 16.8 15.7 17.3 27.1 174.4 29.2 19.2
Assignments were based on the HMBC, HSQC, COSY, HSQC-TOCSY, NOESY, and DEPT experiments. nd, not determinated. a Overlapped proton NMR signals are reported without designated multiplicity.
Extensive analysis of 1D and 2D NMR spectra of 1, revealed that the sugar moiety at C-28 was identical to that of lebbeckosides A–B (Noté et al., 2015), and the monoterpene-quinovosyl moiety at C21 was identical to that of gummiferaoside C (Cao et al., 2007), coriarioside A (Noté et al., 2009), coriariosides C-D (Noté et al., 2010), and lebbeckoside A (Noté et al., 2015). Hence, the units attached at C-28 and C-21 of the aglycon of 1 were established as b-D-xylopyranosyl-(1 ! 4)-[b-D-glucopyranosyl-(1 ! 3)]-a-Lrhamnopyranosyl-(1 ! 2)-b-D-glucopyranoside, and {(2E,6S)-6-O{4-O-[(2E,6S)-2,6-dimethyl-6-O-(b-D-quinovopyranosyl) octa-2,7dienoyl]-4-O-[(2E,6S)-2,6-dimethyl-6-O-(b-D-quinovopyranosyl) octa-2,7-dienoyl]-b-D-quinovopyranosyl}-2,6-dimethylocta-2,7dienoyl}, respectively. In the same way, extensive analysis of 1D and 2D NMR spectra of 1, revealed that the sugar moiety at C-3 was identical to that of coriarioside E (Noté et al., 2010). Hence, the unit attached at C-3 of the aglycone was established as b-D-fucopyranosyl-(1 ! 6)-b-Dglucopyranoside. Consequently, the structure of zygiaoside A (1) was elucidated as 3-O-[b-D- fucopyranosyl-(1 ! 6)-b-D-glucopyranosyl]-21-O-{(2E,6S)-6-O-{4-O-[(2E,6S)-2,6-dimethyl-6-O-(b-Dquinovopyranosyl)octa-2,7-dienoyl]-4-O-[(2E,6S)-2,6-dimethyl6-O-(b-D-quinovopyranosyl)octa-2,7-dienoyl]-b-D-quinovopyranosyl}-2,6-dimethylocta-2,7-dienoyl}acacic acid 28-O-b-D-xylopyranosyl-(1 ! 4)-[b-D-glucopyranosyl-(1 ! 3)]-a-L-
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131
O O O
O
OH
OH
O
O
HO OH
HO HO
OH
O
O
O OH
O
OH
HO O
O
HO HO
O O
HO HO
O
O
O
OH O
OH HO HO
O OH
O
OH
O HO O
OH O
O OH
O
HMBC
HO OH
NOESY Fig. 2. Key HMBC and NOESY correlations of compound 1.
rhamnopyranosyl-(1 ! 2)-b-D-glucopyranosyl ester, a new acacic acid derivative. Zygiaoside B (2), obtained as a white amorphous powder, exhibited in its HRESIMS spectrum (positive-ion mode) a pseudomolecular ion peak at m/z 2042.0073 [M +NH4]+ with its corresponding doubly charged ion peak at m/z 1029.5181 [M + 2 (NH4)]2+. Consequently, compound 2 was assigned a C97H154O44 molecular formula. As already observed for 1, acid hydrolysis of 2 also afforded an acacic acid lactone, identified by co-TLC with authentic sample, together with D-glucose, D-xylose, D-fucose, Lrhamnose, and D-quinovose units which were identified by GC analysis of their trimethylsilyl thiazolidine derivatives (Section 3). Comparison of 1D and 2D NMR data of 2 with those of 1 indicated that 2 had acacic acid as aglycon while the major difference regarding the aglycone moiety was the absence of one monoterpene-quinovosyl (MT-Qui) unit in 2, as already suggested by its
mass spectrum, which displayed 312 mass unit less than that of 1. The observation of glycosylation- and acylation-induced shifts at dC 89.1 (C-3 of Agly), dC 77.2 (C-21 of Agly), and dC 174.4 (C-28 of Agly) in the 13C NMR spectrum of 2 suggested that it should be also a 21-acyl 3,28-bidesmosidic acacic acid derivative with sugar chains linked to C-3 and C-28 through an ether and ester bond, respectively, and with an acyl group attached at C-21. The 1H NMR spectrum of 2 showed eight anomeric protons signals at dH 4.84 [d, J = 7.4 Hz, glucose (GlcI)], 4.91 [d, J = 7.7 Hz, fucose (Fuc)], 6.17 [d, J = 7.9 Hz, glucose (GlcII)], 6.43 [brs, rhamnose (Rha)], 5.34 [d, J = 7.8 Hz, glucose (GlcIII)], 5.31 [d, J = 7.0 Hz, xylose (Xyl)], 4.89 [d, J = 7.6 Hz, quinovose (QuiI)], and 4.91 [d, J = 7.7 Hz, quinovose (QuiII)], which gave correlations with eight anomeric carbon resonances at dC 105.1, 105.6, 95.2, 101.2, 106.2, 106.2, 99.3, and 99.3, respectively in the HSQC spectrum (Tables 2 and 3). Comparison of their NMR spectra revealed that the units attached
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Table 2 1 H NMR (600 MHz) and 13C NMR (150 MHz) data of the sugar moieties attached at C-3 and C-28 of compounds 1–2 in pyridine-d5 (d in ppm, J in Hz).a Position
1
dC 3-O-sugars GlcI 1 2 3 4 5 6a 6b Fuc 1 2 3 4 5 6 28-O-sugars GlcII 1 2 3 4 5 6a 6b
105.4 75.8 78.7 71.7 76.3 70.4
105.9 71.9 75.8 72.4 71.4 17.6
2
dH (J in Hz)
4.84, d (7.0) 4.03 4.13 4.18 4.05 4.90 4.27
4.92, d (8.1) 4.06 4.01 4.08 3.82 1.54, d (6.4)
dC
105.1 75.6 78.6 71.5 76.4 70.1
105.6 71.9 75.6 72.5 71.4 17.3
Quinovose (Qui)
dH (J in Hz)
4.84, d (7.4) 4.02 4.13 4.19 4.03 4.91 4.27
4.91, d (7.9) 4.05 4.00 4.06 3.82 1.55, d (6,5)
95.6 76.3 78.2 71.4 79.5 62.2
6.17, d (7.9) 4.33 4.09 4.17 4.01 4.39 4.30
95.2 76.4 77.9 71.4 79.3 61.9
6.17, d (7.9) 4.32 4.04 4.17 4.00 4.39 4.30
101.5 72.2 83.8 83.1 68.7 18.7
6.43, brs 4.89 4.28 4.50 4.60 1.76
101.2 71.9 83.5 82.7 68.4 18.9
6.43, brs 4.89 4.27 4.50 4.60 1.78
GlcIII 1 2 3 4 5 6a 6b
106.5 75.8 77.5 71.8 78.5 62.9
5.39, d (8.1) 4.15 4.14 4.15 3.94 4.49 4.27
106.2 75.6 77.9 71.9 78.6 62.6
5.34, d (7.8) 4.15 4.14 4.15 3.94 4.49 4.28
Xyl 1 2 3 4 5a 5b
106.6 76.7 78.2 71.3 67.7
5.31, d (7.2) 4.10 4.06 4.12 4.26 3.50
106.2 76.8 78.2 71.4 67.3
5.31, d (7.0) 4.10 4.07 4.12 4.26 3.50
Rha 1 2 3 4 5 6
Table 3 1 H NMR (600 MHz) and 13C NMR (150 MHz) data of the quinovosyl moieties attached at C-21 of compounds 1–2 in pyridine-d5 (d in ppm, J in Hz).a
Assignments were based on the HMBC, HSQC, COSY, TOCSY, NOESY, and DEPT experiments. nd, not determinated. a Overlapped proton NMR signals are reported without designated multiplicity.
at C-3 and C-28 of the aglycon of 2 were identical to that of 1, and that the two saponins differ only by the length of the monoterpene-quinovosyl moiety at C-21, as already suggested by the mass spectrum of 2, which displayed 312 mass units less than that of 1, accounting for the mass of one quinovose and one monoterpene unit. Therefore, the sugar chains at C-3 and C-28 were established as b-D-fucopyranosyl-(1 ! 6)-b-D-glucopyranoside, and b-D-xylopyranosyl-(1 ! 4)-[b-D-glucopyranosyl-(1 ! 3)]-a-L-rhamnopyranosyl-(1 ! 2)-b-D-glucopyranoside, respectively. Extensive analysis of 1D and 2D NMR spectra of 2 allowed to identify the unit attached at C-21 as {(2E,6S)-6-O-{4-O-[(2E,6S)-2,6-dimethyl6-O-(b-D-quinovopyranosyl)octa-2,7-dienoyl]-b-D-
1
2
dC
dH (J in Hz)
dC
dH (J in Hz)
QuiI 1 2 3 4 5 6
99.6 75.9 76.3 77.5 70.4 18.7
4.90, d (7.8) 4.02 4.21 5.37 3.71 1.37, d (6.1)
99.3 75.6 76.1 77.2 70.1 18.7
4.91, d (7.7) 4.02 4.22 5.37 3.71 1.37, d (6.1)
QuiII 1 2 3 4 5 6
99.6 75.6 76.3 75.9 72.8 19.0
4.88, d (7.8) 4.03 4.23 5.67 3.77 1.62, d (5.9)
99.3 75.5 75.9 76.8 72.5 19.2
4.89, d (7.6) 4.03 4.23 3.75 3.73 1.62, d (5.4)
QuiIII 1 2 3 4 5 6
97.2 75.9 76.4 77.1 70.4 19.0
4.97, d (8.0) 4.02 4.23 3.75 3.73 1.60, d (5.4)
Assignments were based on the HMBC, HSQC, COSY, TOCSY, NOESY, and DEPT experiments. nd, not determinated. a Overlapped proton NMR signals are reported without designated multiplicity.
quinovopyranosyl}-2,6-dimethylocta-2,7-dienoyl}, a unit identical to that of gummiferaosides A and B (Cao et al., 2007), coriarioside E (Noté et al., 2010), and lebbeckoside B (Noté et al., 2015). Consequently, the structure of zygiaoside B (2) was established as 3-O-[b-D-fucopyranosyl-(1 ! 6)-b-D-glucopyranosyl]-21-O{(2E,6S)-6-O-{4-O-[(2E,6S)-2,6-dimethyl-6-O-(b-D-quinovopyranosyl)octa-2,7-dienoyl)-b-D-quinovopyranosyl]octa-2,7-dienoyl} acacic acid 28-O-b-D-xylopyranosyl-(1 ! 4)-[b-D-glucopyranosyl(1 ! 3)]-a-L-rhamnopyranosyl-(1 ! 2)-b-D-glucopyranosyl ester, a new acacic acid derivative. Compound 3, isolated as an amorphous powder, was identified as 3-O-{b-D- fucopyranosyl-(1 ! 6)-[b-D-glucopyranosyl-(1 ! 2)]b-D-glucopyranosyl}-21-O-{(2E,6S)-6-O-{4-O-[(2E,6S)-2,6-dimethyl-6-O-(b-D-quinovopyranosyl)octa-2,7-dienoyl]-4-O[(2E,6S)-2,6-dimethyl-6-O-(b-D-quinovopyranosyl)octa-2,7dienoyl]-b-D-quinovopyranosyl}-2,6-dimethylocta-2,7-dienoyl} acacic acid 28-O-a-L-arabinofuranosyl-(1 ! 4)-[b-D-glucopyranosyl-(1 ! 3)]-a-L-rhamnopyranosyl-(1 ! 2)-b-D-glucopyranosyl ester, named coriarioside A (Noté et al., 2009) and compound 4, isolated as an amorphous powder, was identified as 3-O-[b-Dxylopyranosyl-(1 ! 2)-b-D-fucopyranosyl-(1 ! 6)-b-D-glucopyranosyl]-21-O-{(2E,6S)-6-O-{4-O-[(2E,6S)-2,6-dimethyl-6-O-(b-Dquinovopyranosyl)octa-2,7-dienoyl]-4-O-[(2E,6S)-2,6-dimethyl6-O-(b-D-quinovopyranosyl)octa-2,7-dienoyl]-b-D-quinovopyranosyl}-2,6-dimethylocta-2,7-dienoyl}acacic acid 28-O-b-D-xylopyranosyl-(1 ! 4)-[b-D-glucopyranosyl-(1 ! 3)]-a-L-rhamnopyranosyl-(1 ! 2)-b-D-glucopyranosyl ester, named lebbeckoside A by comparison of their respective spectral data with those reported in the literature (Noté et al., 2015). The pro-apoptotic activity of compounds 1–2 was evaluated using Annexin V-FITC binding assay on the A431 human epidermoid cancer cell. In result, percentage of apoptotic cells following 24 h of treatment was increased in a concentrationdependent manner for A431 cell (Fig. 3). Computed concentrations of each compound to induced half-maximal effects (EC50) on cell apoptosis were determined as being 9 mM and 249 mM for
O.P. Noté et al. / Phytochemistry Letters 18 (2016) 128–135
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Fig. 3. Effects of compounds 1–2 on the apoptosis rate. Cells were exposed to compounds (1–2) at the indicated concentrations and incubated for 24 h. Cell apoptosis rate was assessed by flow cytometry using the Annexin V-FITC/ PI staining assay (n = 3). *P < 0.05 and ***P < 0.001 vs. vehicle-treated cells.
zygiaosides A–B, respectively. Furthermore, it is worthy to note that the proapoptotic function of triterpenoid saponins isolated from Albizia genus has been previously reported for glaberrimosides isolated from Albizia glaberrima (Noté et al., 2016), and for adianthifoliosides A and D isolated from Albizia adianthifolia (Haddad et al., 2004). These later compounds and the present saponins share the following structural features: a common aglycone unit, acacic acid, with various oligosaccharide moieties at C-3 and C-28 and an acyl group at C-21. As for adianthifoliosides, the next steps will be to further explain the apoptotic route induced by zygiaoside A (1). In addition, this study contributes to complete the knowledge of the composition of Albizia spp, which are natural sources of complex bioactive triterpenoid saponins. Furthermore, our findings, especially for the monoterpenequinovosyl units, established in compounds 1 and 2, and identical to those found in the related compounds isolated from Albizia coriaria, A. gummifera, and A. lebbeck, strongly suggest a close relationship between these four species of Albizia genus. 3. Experimental section 3.1. General experimental procedures Optical rotations were measured on a Jasco P-2000 polarimeter. H NMR (600 MHz) and 13C NMR (150 MHz) spectra were recorded at room temperature in pyridine-d5 using a Bruker 600 MHz spectrometer. Chemical shifts are given in d (ppm) value relative to TMS as internal standard. HRESIMS spectra were recorded on a microTOF ESI-TOF mass spectrometer (Agilent) operating in positive mode. Analytical HPLC was performed on Varian 920-LC apparatus equipped with an autosampler, a pump, a diode array detector (DAD), and Galaxie software. Semipreparative HPLC was performed on a Gilson apparatus equipped with Trilution LC software using a Nucleodur 100-5 C18ec (21 250 mm, 5 mm) column purchased from Machery-Nagel (Germany). Thin layer chromatography (TLC) was performed on precoated silica gel plates (60 F254, Merck) using the system solvent n-BuOH-AcOHH2O, 65:15:25 as eluent. The spots were observed after spray with Komarowsky reagent, which is a mixture (5:1) of phydroxybenzaldehyde (2% in MeOH) and Ethanolic H2SO4 (50%). Vacuumliquid chromatography (VLC) was carried out using RP-18 silica gel 60 (25–40 mm) and silica gel 60 (15–40 mm and 40–63 mm). 1
3.2. Plant material The roots of Albizia zygia were harvested at Nkolbisson, Yaoundé peripheral quarter, in Cameroon in November 2013
under the guidance of Mr. Victor Nana, botanist of the National Herbarium of Cameroon (NHC), where a voucher specimen (2338/ SRFK) was deposited. 3.3. Extraction and isolation Air-dried and finely powdered roots of A. zygia (300 g) were extracted with 70% EtOH in a Soxhlet apparatus. The hydroalcoholic solution was then evaporated to dryness under reduced pressure to yield a brown residue (7.03 g). The residue was suspended in 200 ml of water and partitioned between H2O and satured n-BuOH (3 300 ml). The n-BuOH phase was evaporated to dryness affording 5.20 g of a brown gum which was taken in a minimum amount of water (10 ml) and then submitted to vacuumliquid chromatography (VLC) using RP-18 (25–40 mm) eluting with H2O, 50% MeOH, and 100% MeOH. The 100% MeOH extract was evaporated to dryness affording 3.40 g crude saponin mixture that was then submitted to VLC using silica gel 60 (15–40 mm), eluted with CHCl3-MeOH-H2O (80:20:2, 70:30:5, 60:33:7, and 60:40:10) to give three main fractions (Z1- Z3). Fraction Z3 (600.2 mg) was subjected to VLC on silica gel 60 (15–40 mm), eluted with CHCl3MeOH-H2O (80:20:2, 70:30:5, 60:33:7, and 60:40:10) affording four main subfractions (AZ31- AZ34). Subfraction AZ34 (150.7 mg) was purified by semipreparative HPLC using gradient system of CH3CN-H2O (20 ml/min) to yield compounds 1 (tR, 24.80 min, 7.6 mg), 2 (tR, 14.97 min, 6.7 mg), 3 (tR, 21.87 min, 3.2 mg), and 4 (tR, 23.14 min, 3.6 mg). 3-O-[b-D-fucopyranosyl-(1 ! 6)-b-D-glucopyranosyl]-21-O{(2E,6S)-6-O-{4-O-[(2E,6S)-2,6-dimethyl-6-O-(b-D-quinovopyranosyl)octa-2,7-dienoyl]-4-O-[(2E,6S)-2,6-dimethyl-6-O-(b-D-quinovopyranosyl)octa-2,7-dienoyl]-b-D-quinovopyranosyl}-2,6dimethylocta-2,7-dienoyl}acacic acid 28-O-b-D-xylopyranosyl(1 ! 4)-[b-D-glucopyranosyl-(1 ! 3)]-a-L-rhamnopyranosyl(1 ! 2)-b-D-glucopyranosyl ester (1), white amorphous powder; [a]25D + 29.25 (c 0.06, MeOH); 1H NMR (600 MHz, C5D5N) and 13C NMR (150 MHz, C5D5N), see Tables 1–4. Positive HR-ESI–MS: m/z 2353.1597, 2354.15812, 2355.1637 and doubly charged ion peaks at m/z 1185.5927, 1186.1004, 1186.5977 [M + 2(NH4)]2+. 3-O-[b-D-fucopyranosyl-(1 ! 6)-b-D-glucopyranosyl]-21-O{(2E,6S)-6-O-{4-O-[(2E,6S)-2,6-dimethyl-6-O-(b-D-quinovopyranosyl)octa-2,7-dienoyl)-b-D-quinovopyranosyl]octa-2,7-dienoyl} acacic acid 28-O-b-D-xylopyranosyl-(1 ! 4)-[b-D-glucopyranosyl(1 ! 3)]-a-L-rhamnopyranosyl-(1 ! 2)-b-D-glucopyranosyl ester (2), white amorphous powder; [a]25D 18.75 (c 0.16, MeOH); 1H NMR (600 MHz, C5D5N) and 13C NMR (150 MHz, C5D5N), see Tables 1–4; positive HR-ESI–MS: m/z 2042.00732 [M +NH4]+ and doubly charged ion peak at m/z 1029.51814 [M + 2(NH4)]2+.
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Table 4 1 H NMR (600 MHz) and 13C NMR (150 MHz) data of the monoterpene moieties attached at C-21 of compounds 1–2 in pyridine-d5 (d in ppm, J in Hz).a Monoterpene (MT)
MT1 1 2 3 4a 4b 5a 5b 6 7 8a 8b 9 10 MT2 1 2 3 4a 4b 5a 5b 6 7 8a 8b 9 10 MT3 1 2 3 4a 4b 5a 5b 6 7 8a 8b 9 10
1
2
dC
dH (J in Hz)
dC
dH (J in Hz)
168.1 128.5 142.4 24.0
– – 6.90, t (7.3) 2.47 nd 1.78 nd – 6.24, dd (10.9, 17.6) 5.47, d (17.6) 5.27, d (10.9) 1.93, s 1.51, s
168.1 128.5 142.1 23.5
– – 6.90, t (7.5) 2.44 nd 1.78 nd – 6.24, dd (10.8, 17.5) 5.43, d (17.5) 5.25, d (10.8) 1.85, s 1.55, s
– – 7.00, t (7.1) 2.42 nd 1.76 nd – 6.27, dd (10.9, 17.6) 5.45, d (17.6) 5.25, d (10.9) 1.85, s 1.56, s
168.1 128.5 143.3 23.8
parameters were as follows: ionization potential, 70 eV; ion source temperature, 230 C; solvent delay 4.0 min, mass range 100–700. The following sugars were detected for 1 and 2: D-glucose, Dquinovose, D-fucose, D-xylose, and L-rhamnose. 3.5. Pro-apoptotic evaluation
40.8 80.1 144.3 115.2 13.0 24.2
168.1 128.5 143.1 23.8 40.8 80.1 144.3 115.4 13.0 24.1
167.8 128.5 143.3 24.0 40.8 80.1 143.5 115.8 13.0 23.8
40.3 80.1 143.9 114.8 12.7 23.7
40.3 80.1 144.1 115.0 12.7 23.6
– – 7.06, t (7.3) 2.43 nd 1.79 nd – 6.27, dd (10.8, 17.6) 5.49, d (17.6) 5.30, d (10.8) 1.90, s 1.56, s
– – 7.08, t (7.4) 2.46 nd 1.79 nd – 6.23, dd (11.0, 17.2) 5.40, d (17.2) 5.29, d (11.0) 1.96, s 1.57, s
Assignments were based on the HMBC, HSQC, COSY, TOCSY, NOESY, and DEPT experiments. nd, not determinated. a Overlapped proton NMR signals are reported without designated multiplicity.
3.4. Acid hydrolysis of compounds and determination of the absolute configuration of the sugar residues Each saponin (2 mg) was hydrolyzed with 2 ml of 2 M HCl at 85 C during 2 h. After cooling, the solvent was removed under reduced pressure. The sugar mixture was extracted from the aqueous phase (10 ml) and washed with CH2Cl2 (3 5 ml). The combined CH2Cl2 extracts were washed with water to give after evaporation the aglycone moiety. The sugars were first analyzed by TLC over silica gel (CHCl3–MeOH H2O, 8:5:1) by comparison with standard samples. The absolute configuration of each monosaccharide was determined from GC–MS analysis of their trimethylsilylated derivatives by comparison with authentic samples using the method previously described (Chaabi et al., 2010). GC analysis was performed with a capillary TR-5MS SQC (15 m x 0.25 mm x 0.25 mm) column. Operating conditions were as follows: carrier gas, helium with a flow rate of 1 ml/min; column temperature, 1 min in 150 C, 150–220 C at 4 C/min; injector temperature, 250 C; volume injected 1 ml of the trimethylsilylated sugar in methylene chloride (0.1%); split ratio, 1:50. The MS operating
3.5.1. Cell lines and culture conditions The A431 human epidermoid cancer cell was purchased from ATCC (LGC Standards, Molsheim, France) and cultivated in DMEMbased media (Sigma-Aldrich, Saint-Quentin-Fallavier, France), supplemented with 10% (v/v) fetal bovine serum (BioWhittaker, Verviers, Belgium), 2 mMultraglutamine, 50 mM non-essential amino acids, 50 U/ml penicillin and 50 mg/ml streptomycin (Sigma-Aldrich). The culture was kept at 37 C in a humidified incubator equilibrated with 5% CO2. Before confluency adherent cells were trypsinized and subcultured twice a week. 3.5.2. Pro-apoptotic evaluation Cells were treated with the two purified saponins (1–2) in ranging concentrations (1 to 15 mM) then collected for apoptosis induction estimation. A minimum of 5000 cells was acquired per sample and analyzed on the InCyte software (Guava/Millipore/ Merck, CA, USA). Apoptosis rates were assessed by capillary cytometry (Guava EasyCyte Plus, Millipore Merck) using Annexin V-FITC (ImmunoTools, Germany) and PI (MiltenyiBiotec Inc., USA) according to the manufacturer’s recommendations. Gates were drawn around the appropriate cell population using a forward scatter (FSC) versus side scatter (SSC) acquisition dot plot to exclude debris. Final concentration of DMSO applied to cells during incubation with tested samples was always 0.5%. In the tested setup that concentration had no adverse effects on cell viability, nor cell morphology. To discriminate between negative and positive events in the analysis, a non-stained control sample from each culture condition always accompanied acquisition of the stained cells to define their cut off. Negative control, i.e. sample with cells without compounds but with the same amount of DMSO as for diluted compounds, as well as positive control with 50 mM Celastrol, a natural pentacyclic triterpenoid (Enzo Life Sciences, Farmingdale, US), were included for each experimental set. Cytometers performances are checked weekly using the Guava easy Check Kit 4500-0025 (Merck/Millipore/Guava Hayward, CA, USA). 3.5.3. Statistical analysis Data, presented as bar graphs, were expressed as means S.E. M. of at least three independent experiments. Statistical evaluation was performed with the one-way ANOVA test followed by the post-hoc Bonferroni test using GraphPad Prism software (Prism version 5.04 for Windows, GraphPad Software, CA, USA); a p-value less than 0.05 was considered as significant (*), less than 0.01 very significant (**) and less than 0.001 highly significant (***). Acknowledgments This work was financially supported by the World Academy of sciences (TWAS) (grant no: 3240277733). The authors are also grateful to Mr. Victor Nana of the National Herbarium of Cameroon (NHC) for the identification and collection of plant. References Abdalla, M.A., Laatsch, H., 2012. Flavonoids from sudanese Albizia zygia (Leguminosae, subfamily mimosoideae), a plant with antimalarial potency. Afr. J. Tradit. Complement. Altern. Med. 9, 56–58. Arbonnier, M., 2009. Arbres, arbustes et lianes des zones d’Afrique de l’Ouest; MNHN (Eds) Quæ, Paris. p. 383.
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