A New Cytotoxic Ceramide from Dalbergia boehmii Taub. Stem Bark

Background: Dalbergia boehmii (Leguminosae) , is a medicinal plant used in the Northen part of Cameroon, against digestive tract diseases and fever. Objectives: This work which is part of the search for bioactive molecules through natural products, aimed to isolate, characterize and evaluate the cytotoxicity of compounds from D. boehmii . Materials and Methods: Chemical investigations were carrying out on stem barks of Dalbergia boehmii using chromatographic techniques. Structures elucidation of isolated compounds were done using 1D and 2D NMR as well as EI-MS and literature data. Some isolated compounds were evaluated in vitro against MCF-7 cell lines for their cytotoxicity activity, using the MTT colorimetric method. Results: The chemical investigation leaded to the isolation of a new ceramide derivative named Dalbergiamide (1), a triterpene identified as olean-12-ene-3,11-dione (2) isolated here for the first time from Dalbergia genus, along with two known compounds, glucoside 3-O - β - D-sitosterol (3) and glucoside 3-O - β -D-stigmasterol (4). Compounds 1 and 2 were found to be cytotoxic at 50 μM with IC 50 value of 17.13 μM and 35.52 ± 2.95 μM respectively. Conclusion: These promising results suggested more in-depth studies should be carried out on this plant in order to assess its potential in the fight against cancer.


INTRODUCTION
Dalbergia species belonging to Leguminosae family, is very appreciated in wood crafts because of the variety of pigmented colors of their wood, are also valued for their use in traditional medicine. [1][4] Phytochemical studies showed that plants from Dalbergia genus contain various classes of compounds as phenolic compounds, stibenes, terpenoids, glycosides, miscellaneous, ...etc. [5,6]s part of our continued screening program of Cameroon medicinal plant constituents especially Dalbergia boehmii, we carried out investigation on the stem barks.Dalbergia boehmii which is known as Ngalayhi in Fulfulde in the Northen Cameroon, is a small to medium-sized deciduous tree or a shrub, used traditionally to treat digestive tract diseases and fever. [6,7]Previous chemical investigation carried on leaves and hearthwood of D. boehmii leaded to the isolation of thirteen phenolic compounds, mostly isoflavonoids and stilbenes. [6]Further to this, the phytochemical study of the stem barks gave four compounds including a new ceramide named dalbergiamide, a triterpene: olean-12-ene-3,11-dione and two phytosterols:

Experimental Section
General experimental procedures 1 H NMR and 13 C NMR spectra were recorded on a Bruker AM 400 (600) and AMX 500 NMR (Avance) spectrometer respectively at 500 MHz and 125 MHz using pyridine-d 5 and CDCl 3 as solvents.JEOL JMS-600H spectrometer was used for EI-MS spectra recording; TMS was used as internal standard and chemical shifts are in ppm.Silica gel was used for column chromatography (CC) monitored by TLC on alumina precoated silica gel sheets (60F 254 ).Spots were visualized using UV lamp (254 and 386 nm) followed by spraying ceric sulphate solution and heating at 105°C.

Plant material
Identificated by Dr Tsabang Nole in Touboro in the North region of Cameroon, stem barks of Dalbergia boehmii were collected in September 2012, and authentificated at the Cameroon National Herbarium where a Voucher specimen (66887/HNC) has been deposited.

Extraction and purification
The pulverized barks (3 kg) was macerated in 9 L of a mixture of CH 2 Cl 2 /MeOH (1:1) at room temperature, to give after filtration and solvent evaporation under reduced pressure 159.7 g of a brown crude extract.Silica gel column chromatography (CC) of 150.0 g of this crude extract using a gradient of Hexane-EtOAc (1→0 to 0→1) then EtOAc-MeOH (1→0 to 0→1) was performed.210 fractions were collected and pooled according to their TLC profiles leading nine fractions (A-I).The purification of A (6.0 g) on silica gel CC furnished 15.0 mg of compound 2 using a mixture of Hexane-EtOAc (9:1) as eluent.Fraction I (8.0 g) was also subjected to CC of silica gel eluted with EtOAc (100%) yielding 20.0 mg of a mixture of compound 3 and 4 while fraction B furnished compound 1 (2.0 mg) when eluted with Hexane-EtOAc (8:2

Cytotoxicity assay
Cytotoxicity activity of isolated compounds 1 and 2 was evaluated in vitro against MCF-7 cell lines using the MTT colorimetric standard method according to Scudiere et al. [8]
In vitro cytotoxicity activity of dalbergiamide (1) and olean-12-ene-3,11dione (2) was evaluated against MCF-7 cell lines using MTT colorimetric method, and were found to be active with IC 50 value 17.13 μM and 35.52 ± 2.95 μM respectively when tested at the concentration of 50 μM.

DISCUSSION
Compound 1 was isolated for the first time from natural sources, and is structure has been elucidated as follow: obtained as a white solid (m.p. 129-131°C), its molecular formula C 41 H 81 O 5 N was determined from rigorous analysis of its NMR spectra (1D & 2D) and its EI-MS spectrum which exhibited molecular ion peak [M] + at m/z 667.2 and characteristic fragments ions at m/z 647.
[13][14][15] In the 1 H NMR spectrum of compound 1, a signal of a proton exchangeable with D 2 O is observed at δ H 8.57 (d, J = 9.0 Hz) advising the presence of the group -NH-CO-.Moreover, 13 C-NMR spectrum showed at δ C 53.0 a signal of C-N and a C=O signal at δ C 175.2 confirming the presence of a secondary amide function in this compound. [15,16] 1H NMR spectrum also exhibited resonances for terminal methyls of two aliphatic chains at δ H 0.87 (6H, t, J = 6.9 Hz), an azomethine proton at δ H 5.12 (1H, m), signals at δ H 4.51 (m) and 4.42 (m) for an hydroxymethylene group, three carbinylic protons at δ H 4.29 (H-4), 4.36 (m, H-3) and 4.61 (m, H-2ʹ), as well as signals of four -OH groups at δ H 6.21 (1H, brs), 6.68 (2H, brs) and 7.68 (brs).Two alkyl long chains were also observed as a broad signal at δ H 1.20-1.34.Further extensive analysis of the 1 H NMR spectrum, reveals signals of two olefinic protons at δ H 5.54 (m) and 5.49 (m), suggesting the presence of a disubstituted double bond.As expected, instead the characteristic signals of secondary amide group, the 13 C NMR spectrum of compound 1 exhibited two carbon signals at δ C 130.8 and 130.7 corresponding to a double bond, with a trans configuration, that was determined by the chemical shifts of its allylic carbon atoms appearing at δ C 32.9 and 33.3. [17,18]This double bond was located in the fatty acid moiety at C-15ʹ by EI-MS analysis.Signals of an oxymethylene at δ C 62.1 and three oxymethines at δ C 76.8, 73.1 and 72.5 were also visible.Additionally, several signals of carbon atoms in the range of δ C 34.2-22.9related to -CH 2 -groups and a signal at δ C 14.3 corresponding to two terminals -CH 3 were also deduced from 13 C NMR spectrum, confirming that compound 1 belongs to the class of ceramide.
In the 1 H-1 H COSY spectrum several correlations are observed.It is the case of the correlation of the two -CH 2 -protons at δ H 4.42 and 4.51 with the methine proton at δ H 5.12, which also showed correlation with the methine at δ H 4.36.This last methine proton at δ H 4.36 also correlated with the methine proton at δ H 4.29, suggesting three hydroxyl groups respectively on C-1, C-3 and C-4.This spectrum also exhibited correlations between the two olefinic protons at δ H 5.54 and 5.49, as well as, respective correlations between these protons and their adjacent methylenes at δ H 1.98 and 2.16.The correlation of H-2ʹ at δ H 4.61 and the methylene protons H-3' at δ H 2.03 and 2.22 was also visible.Interpretation of the HMBC spectrum revealed correlations of the proton of amide group at δ H 8.57 and -C=O group at δ C 175.2 and the -CH-NH at δ C 53.0; correlations of H-2 (δ H 5.12) and C-1 (δ C 62.1) and C-3 (δ C 76.8); correlations of H-3 (δ H 4.36) and C-1 (δ C 62.1), C-2 (δ C 53.0), and C-4 (δ C 73.1), confirming positions of -OH groups on C-1, C-3 and C-4 (Figure 2). [15]Correlations between H-2ʹ (δ H 4.61) -C=O (δ C 175.2) and C-3ʹ (δ C 35.7) were also visible confirming the α-hydroxy fatty acid side chain.The length of the long chain base and the fatty acid was determined by EI-MS, which showed significant fragment ion peaks at m/z 384, 357 and 339 that were congruent to the long chain base moiety (Figure 3).The length of the fatty acid chain was deduced from the fragment at m/z 280 resulting from the cleavage in α of the carbonyl group of the amide function.This fragment, combined with the ones observed at m/z 57 and 83, resulting from the α-cleavages of the double bond, confirm the emplacement of this olefine bond on the fatty acid chain.All these assignments were congruent with the proposed structure.Finally, considering the steric hindrance and biogenesis of sphingolipids, absolute stereochemistry of the phytosphingosine moiety can be generally determine using chemical shift of the carbon C-2 to C-4. [17] Thus, according to the 13 C NMR data of compound 1, the relative stereochemistries of C-2 (δ C 53.0), C-3 (δ C 76.8), and C-4 (δ C 73.1), were found to be 2S, 3S, and 4R. [17,18]From the above analysis the structure of compound 1 was set as (2ʹR, 15ʹE)-2ʹ-hydroxy-N-[(2S,3S,4R)-1,3,4trihydroxyhenicosan-2-yl]eicos-15ʹ-enamide named dalbergiamide.The new compound dalbergiamide (1) and the triterpene olean-12ene-3,11-dione (2) were tested for their cytotoxic activities against MCF-7 cells.These two compounds were slightly active at 50 μM, the most remarkable cytotoxic activity against breast cancer cells MCF-7 being recorded for compound 1 with IC 50 value of 17.13 ±0.21 μM while compound 2 was less active with IC 50 value 35.52 ± 2.95 μM.Previous studies have proved the activity of ceramides against cancer cell lines [19] and established their mechanism of action.Ceramides have been shown to act by blocking the cell cycle, following the up-regulation of p27 kip1 thus causing apoptosis. [20]This could explain why the new ceramide, dalbergiamide (1) showed promising cytotoxic activity against MCF-7 cancer cell lines.[23][24][25] Others studies have shown that triterpenes with an oleanane type skeleton displayed cytotoxic activity against cancer cell lines. [16]