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Barthelemy K. Attioua*, Ramiarantsoa Harisolo, Jean Brice Boti, Vincent A. Adiko, Félix Z. Tonzibo, Léon A. Djakoure
Laboratoire de chimie Organique Biologique, UFR Sciences des Structures de la Matière et Technologie, Université de Cocody-Abidjan, Côte d’Ivoire.
stationary phase. The elution was performed with a at m/z 242.29294, formula C15H15NO2. See Table 2 for
mixture of CH2Cl2-EtOAc then EtOAc – MeOH, following a additional NMR spectral data.
gradient of polarity. For the first fractionation, we used a
Aurentiamide acetate (5): white amorphous solid, mp
chromatography column with the sizes: 3.5 cm of
=180-182°C, UV max (MeOH) nm: 242, 293 and 302 nm;
diameter (d) and 17.5 cm of height (h). Elution with a
mixture of CH2Cl2 - EtOAc (90: 10 v/v) gave fraction I, that IR max (KBr) in cm-1: 1670 (HNC=O) and 1634 (HNC=O),
with a mixture of CH2Cl2-EtOAc (50:50 v/v) to EtOAc - 1741 (COOCH3). 1H and 13C NMR (CDCl3) (ppm): 7.15 to
MeOH (99:01 v/v) gave the fraction II and the fraction III 8.12 (aromatic protons), 170.1 (C-14), 2.01 (br s, H-15,
+
was obtained with a mixture of EtOAc - MeOH (95:5 to OCOCH3). HRESIMS: [M+H] at m/z 445.53782 formula
85:15 v/v). The fraction I (200 mg) was purified by C27H28N2O4. See table 2 for additional NMR spectral data.
recristallization in MeOH to give 63.2 mg of compound (1) RESULTS AND DISCUSSION
(Rf 0.5 in CH2Cl2- EtOAc 85:15 v/v). The fraction II (1325
mg) was purified by column chromatography (d: 1.51 cm, The fractionation by aluminum oxide neutral gel (100-300
h: 10 cm) on aluminum oxide neutral gel. The elution with mesh) column chromatography of the EtOAc – NH4OH
a mixture of CH2Cl2-EtOAc (55:45 v/v) gave sub-fraction crude extract from the leaves and stems of C. lobatus
II1, then a mixture of CH2Cl2-EtOAc (40:60 v/v) the sub- gave three fractions (I, II and III). Future purification of
fraction II2 and with CH2Cl2-EtOAc (25:75 v/v) the sub- these fractions by a combination of column
fraction II3. These sub-fractions were purified by chromatography and recristallisation methods has led to
recristallization in cyclohexane. The sub-fraction II1 gave the isolation of five alkaloids (figure 1).
compound (2) (25 mg, Rf 0.7 in CH2Cl2- CH3OH 95:5 v/v),
the sub-fraction II2 gave compound (3) (18 mg, Rf 0.55 in
CH2Cl2- CH3OH 95:5 v/v) and the sub-fraction II3, the
compound (4) (21 mg, Rf 0.5 in CH2Cl2- CH3OH 95:5 v/v).
The latest fraction (III) 500 mg, was purified by column
chromatography (d: 1.51 cm, h: 10 cm) on aluminum
oxide gel. The elution with CH2Cl2 - EtOAc (10:90 v/v) gave
the sub-fraction III1. This sub-fraction was then purified by
recristallization in cyclohexane to afford compound (5)
(15 mg, Rf 0.45 in CH2Cl2- CH3OH 95:5 v/v). All these
compounds were soluble in chloroform.
Palmitamide (1): White powder; mp: 105-106°C; UV max
(MeOH) 215 nm; IR max (KBr) in cm-1: 1647 (strong,
H2NC=O), 3375-3310 (N-H), 2918 and 2849 (CH2, CH3). 1H
and 13C NMR (CDCl3) (ppm): 2.24 (t, J=7.5 Hz, H-2), 1.60
(m, H-3), 1.25 (br s, H-4 to H-15, CH2), 0.87 (t, J=6.9Hz, H-
16, CH3), 5.40 (br s, H-17, NH2); 175.7 (C-1, C=O), 36.0 (C- Figure 1: structures of Palmitanoide (1), Onosmin B (2), N-(2-
2), 29.2 -29.6 (C-4 to C-13, CH2), 14.1 (C-16, CH3). hydroxy-1-phenylpropyl)benzamide (3), Onosmin A (4) and
3 2
+
HRESIMS: [M+H] at m/z 256.45918, formula C16H33NO. Aurentiamide acetate (5). Black arrow: COSY ( JH-H) and HMB ( JC-
3 4
See Table 1 for additional NMR spectral data. H, JC-H and JC-H) correlations.
Onosmin B (2): white amorphous solid; mp: 139-141ᵒC; This is in confirming with the results of alkaloid tests
realized with Dragendorff and Ehrlich reagents. The
UV max (MeOH) nm: 242, 293 and 302 nm; IR max (KBr) in
-1 compound (1) was Palmitamide or Hexadecanamide, an
cm : 3345 (sharp, N-H), 3012 (wide, N-H), 728 and 987
aliphatic alkaloid derivative. It was isolated as a white
(benzyl), 1698 (strong, C=O). 1H and 13C NMR (CDCl3)
powder with a melting point (mp: 105-106°C), UV
(ppm): 3.77 (br s, H-8, O-CH3), 51.6 (C-8, O-CH3), 6.31 to
absorption band of 215 nm. The IR spectrum on KBr disc
7.77 (aromatic protons). HRESIMS: [M+H]+ at m/z
showed stretching band of the carbonyl H2NC=O at 1647
256.13371, formula C16H17NO2. See table 1 for additional
cm-1. For the N-H, absorption was observed between
NMR spectral data.
3375 and 3310 cm-1. The two strong bands at 2918 cm-1
N-(2-Hydroxy-1-phenyl-propyl)-benzamide (3): White and 2849 cm-1 could be attributed to the methyl (CH3) and
crystals; mp: 157-158°C; UV λmax (MeOH) nm: 293, 310; IR methylene (CH2) groups. NMR spectra were recorded in
νmax (KBr) cm−1: 3369, 2957 (middle, CH3), 1638 (strong, CDCl3 and spectral data are reported in table 1. The 1H
C=O). HRESIMS: [M+H]+ at 256.32024, formula C16H17NO2. NMR spectrum exhibited one methyl group at 0.87 ppm
Onosmin A (4) was white amorphous solid, mp 187-189°C; (t; H-16, J=6,9 Hz). The singlet at 1.25 ppm (br s)
indicated an aliphatic chain (CH2)n. Amide protons
UV max (MeOH) nm: 242, 293 and 302 nm; IR max (KBr) in
-1
cm : 3345 (sharp, N-H), 3012 (wide, N-H), 728 and 987 (H2NC=O) gave wide signal between 5.52 and 5.37 ppm.
13
1
(benzyl), 1699 (strong, C=O), 3015 (O-H of acid). H and C NMR spectra confirmed the presence of amide group
13
C NMR (CDCl3) (ppm): 169.8 (COOH), HRESIMS: [M+H]+ with the peak at 175.7 ppm (H2NC=O).
stretching of the carbonyl C=O (HNC=O) at 1670 and 1634 Secotrachylobanoic Acids, J. Nat. Prod., 54, 1991, 1625-
cm-1, that from the carbonyl of the ester group (COOCH3) 1633.
appeared at 1741 cm-1. The 1H NMR spectrum in CDCl3 6. Sutthivaiyakit S, Nareeboon P, Ruangrangsi N, Ruchirawat
exhibited the chemical shifts of aromatic protons S, Pisutjaroenpong S, Mahidol C, Labdane and pimarane
between 8.12 and 7.02 ppm. Protons of amide group diterpenes from Croton joufra, Phytochemistry, 56, 2001,
gave the wide signal at 8.12 ppm (H-8, H-11) and these 811-814.
of the acetate (CH3-COO) gave a singlet at 2.01 ppm (H- 7. Barbosa PR, Fascio M, Martins D, Silva Guedes ML, Roque
15). The 13C NMR spectrum showed carbonyl signals at F, Triterpenes of Croton betulaster (Euphorbiaceae),
168.5 ppm (C-7, HNC=O), 171.8 ppm (C-10, HNC=O) and Biochem. System. and Ecology, 31, 2003, 307–308.
170.1 ppm (CH3O-C=O). The aromatic carbons are 8. Cai Y, Evans FJ, Roberts MF, Phillipson JD, Zenk MH, Gleba
observed between 125.7 and 139.1 ppm. The structure YY, Polyphenolic compounds from Croton lechleri,
of this compound was clearly established thanks to COSY, Phytochemistry, 30, 1991, 2033–2040.
HSQC and HMBC spectral data. The HRESIMS spectrum 9. Aboagye FA, Sam GH, Massiot G,Lavaud, Julocrotine C, A
exhibited the molecular ion peak [M+H]+ at m/z glutarimide alkaloid from Croton membranaceus,
445.53782 with the molecular formula C27H28N2O4 Fitoterapia, 71, 2000, 461-462.
(M=444.54 g/mol). Aurentiamide acetate was previously
10. Attioua B, Contribution à l’étude phytocimique des
isolated from C. hieronymi Griseb17 and Patrinia villosa
feuilles et tiges de Croton lobatus (Euphorbiaceae), Ph.D.,
Juss18. Dissertation, University of Strasbourg, 2005, 135.
CONCLUSION 11. Chabert P, Attioua B, Weniger B, Brouillard R, Croton
Investigation of the EtOAc-NH4OH crude extract from C. lobatus, an African Medicinal plant: Spectroscopic and
chemical elucidation of its many constituents, BioFactors,
lobatus aerial parts has led to the isolation of five
27, 2006, 69-78.
alkaloids: Palmitanoide (1), Onosmin B (2), N-(2-hydroxy-
1-phenylpropyl) benzamide (3), Onosmin A (4) and 12. Attioua B, Chabert P, Weniger B, Anti-plasmodial Activity
Aurentiamide acetate (5). Their structures were of Constituents Isolated from Croton lobatus
determined thanks by NMR, UV, IR and HRESIMS (Euphorbiaceae), Pharmaceut. Biol., 45, 2007, 1-4.
spectroscopic data. 13. Shihai X, Yang K, Guo S, Yingping L, Studies on chemical
constituents from Acropora pulchra, Tianran Chanwu
Acknowledgements: The authors wish to thank the AUF Yanjiu Yu Kaifa, 15, 2003, 109-112.
(Agence Universitaire de la Francophonie) for its financial
support, Cyril Antheaume and Patrick Wehrung for NMR 14. Peng N, Yaoming H, Zhao J, Feng Y, Zhong Y, Chemical
and MS analyses respectively. Special thanks to retired composition of the essential oils of two Alpinia species
from Hainan Island, China, Zeitschrift fuer Naturforschung,
Professor L. Aké Assi who kindly performed the botanical
J. Biosci.,59, 2004, 157-160.
determinations.
15. Dembitsky VM, Shkrob I, Rozentsvet OA, Fatty acid amides
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