Cytotoxicity of an unprecedented brominated oleanolide and a new furoceramide from the Cameroonian spice, Echinops giganteus

Abstract A preliminary study on Echinops giganteus (Asteraceae) showed that the methanolic extract has interesting cytotoxicities against a panel of cancer cell lines. From this extract, a lignan, a flavonoid and a polyacetylenic thiophene identified were three times less cytotoxic than the extract. In the search of the metabolites responsible for the bioactivity, a new harvested E. giganteus was subjected to a phytochemical study using chromatographic methods. In the course of the work, two new compounds: a brominated oleanolide (1) and a tetrahydrofurano-ceramide (2) were obtained along with β-amyrin acetate (3), 2-(penta-1,3-diynyl)-5-(4-hydroxybut-1-ynyl)-thiophene (4), 2-(penta-1,3-diynyl)-5-(3,4-dihydroxybut-1-ynyl)-thiophene (5) and 4-hydroxy-2,6-di-(3′,4′-dimethoxyphenyl)-3,7-dioxabicyclo-(3.3.0)octane (6). Their structures were determined on the basis of NMR spectroscopy and mass spectrometry data in conjunction with those reported in the literature. The cytotoxicity of 1, 2 and 5 was evaluated by employing resazurin assay against a panel of cancer cell lines with IC50 values in range 6.12 ± 0.46–46.96 ± 3.61 μM.


Introduction
our preliminary studies on Echinops giganteus revealed interesting cytotoxicities for the metabolic extract, explaining why this spice and culinary ingredient is commonly used in the western part of Cameroon by traditional healers as a remedy for the cancer treatment (Kuete et al. 2013). Thus, the bioactivity observed prompted us to undergo a cytotoxicity-guided isolation in order to identify metabolites responsible for the activity. Six secondary metabolites were isolated; among them a polyacetylene thiophene nevertheless, the antiproliferative effect observed against the tested cancer cells turned to be weaker than that observed for the crude extract (Kuete et al. 2013). As continuation of the work, we wanted to identify more plant-derived thiophenes according to the cytotoxicity reported previously for related compounds (Zhongjun et al. 2009). Polyacetylenic mono- (Hymete et al. 2005) and polythiophenes (Nakano et al. 2011) have formerly been identified from Echinops species together with sesquiterpenes (El Sayed 2001), flavonoids (Singh et al. 2006), alkaloids (Su et al. 2004) and a lignan (Tene et al. 2004). Besides, bioactivities such as larvicidal (Nakano et al. 2004, Nakano et al. 2011, antimycobacterial (Tekwu et al. 2012), phototoxicity (Wang et al. 2007), antifeeding (Fokialakis et al. 2006) and NQo1 [NAd(P)H:quinone oxidoreductase 1] inducer (Shi et al. 2010) were reported for Echinops species or their isolated secondary metabolites. Thus, rhizomes and aerial part of E. giganteus newly collected were subjected to phytochemical investigation from which a brominated triterpene and a tetrahydrofuranyl ceramide were identified as new compounds. Alongside, four known metabolites were further obtained including another polyacetylenic thiophene, β-amyrin acetate and two metabolites formerly identified during the first study (Kuete et al. 2013). Their structures were established based on their recorded physical data in conjunction with those reported in the literature. We herein present the structure elucidation of the new secondary metabolites and their cytotoxicity towards a panel of cancer cell lines.
Compound 2 was obtained as a colourless gum. The molecular formula C 43 H 83 No 4 was deduced from its HR-ESI-MS which gave a pseudo-molecular peak of [M + Na] + at m/z 700.6252 (Calcd 700.6220). The NMR spectra (Section 3) showed resonances of a fatty acid chain including those of two methyl groups at δ 0.88 and the sequence of the CH 2 groups between δ 1.20 and δ 1.40. Furthermore, downfield signals of one CH 2 and four CH groups group were observed at (δ 3.96, 4.56)/72.0 and (δ 4.82/52.3, 4.68/72.9, 4.36/75.1 and 4.17/86.6), respectively. In addition, resonances assigned to three exchangeable protons were revealed at δ 7.74, 7.76 and 8.57 suggesting the presence of three free oH groups. The proton at δ 8.57 showed HMBC correlations ( Figure S15, supporting information) with carbon atoms at δ 52.3, 75.1 and 176.2 suggesting the presence of two free oH groups and one amide function. Thus, a ceramide skeleton was subsequently suggested for compound 2 from the aforementioned data (Nana et al. 2012). Inspection of HMBC spectrum showed correlations of H-1b (δ 4.56) with C-3 (δ 75.1) and C-4 (δ 86.6) consistent with a 4H-furane ring formed by C-1, C-2, C-3 and C-4 with C-1 and C-4, involved in the ether function. This assumption was further supported by carbon shifts of C-1, C-3 and C-4 which are more downfield than those usually observed for the same carbon atoms (δ 63.2, 76.4 and 74.1, respectively) in linear ceramides (Gao et al. 2001;Tchoukoua et al. 2013). Resonances of a double bond were also revealed at δ 5.48/130.8 and δ 5.48/131.5 in the NMR spectra of 2. The assignment of the double bond geometry was tentatively performed based on the chemical carbons shifts of the allylic CH 2 group. Previous studies demonstrated that the carbon shift of the allylic CH 2 groups resonate between 32 and 33 ppm when the geometry of olefinic bond is trans (Tazoo et al. 2007) and 27.5 ppm for the cis geometry (Cateni et al. 2008). Methanolysis of 2 furnished a long chain base which was identified as an ammonium salt of (E)-4-amino-2-(henicos-15-en-1-yl)tetrahydrofuran-3-ol at m/z 454.3 on the basis of the HPLC-MS spectrum. This information also indicated the presence of double bond in the long chain base (LCB) skeleton. The position of this latter was located between C-20 and C-21 from the mass fragment found at m/z 301.2 due to the allylic cleavage ( Figure S16, supporting information). The relative configurations of chiral centres were tentatively established with the NoE correlations ( Figure S15, supporting information). Thus, spatial correlations were displayed between signals H-2 (δ 4.82), H-1b (δ 4.56) and H-3 (δ 4.36) whereas, the proton resonating at δ 3.96 (H-1a) showed similar correlations with H-4 (δ 4.17) and H-N (δ 8.57). From the same data, the relative configuration at C-2' could not be determined.
The ESI tandem mass fragmentation of 2 (Figures S18 and S19, supporting information) revealed two interesting peaks at m/z 300 from the Mc Lafferty mechanism type and m/z 385 from the radical cleavage of the side chain attached to the furan ring. These two fragments supported the presence of two oH groups, a furan ring and an amide function. Moreover, the peak at m/z 576 resulting from the water loss and the allylic cleavage confirmed the position of the double bond. In Figure S20 (supporting information), the fragment observed at m/z 283 corresponded to the α-hydroxyl fatty acid portion.
Compounds 1, 2 and 5 displayed selective cytotoxicity towards the tested cancer cell lines. However, the three compounds were active on the two leukaemia cell lines, and the IC 50 values were better than that of doxorubicin towards CEM/AdR5000 (Human T-cell lymphoblast-like cell line) ( Table 1). The lowest IC 50 values were obtained with compound 2 (6.12 μM) towards CEM/AdR5000 cell line while doxorubicin showed weak cytotoxicity. This kind of hypersensitivity of multidrug-resistant cells has been termed collateral sensitivity (Saeed et al. 2013) and indicates that compounds 2 and 5 may be promising candidates for treating refractory and unresponsive tumours in the clinic. Whilst compound 1 displayed IC 50 of 52.82 μM on u87MG.ΔEGFR, none of the other tested compounds was active on this line. Apart from their activity against CEM/AdR5000 cell line, none of the compounds was as active as doxorubicin. It is worth to note that the three compounds were also less cytotoxic on the normal AML12 hepatocyte cells than on leukaemia cells. Considering the low activity of the three compounds on carcinoma cells, more hemi-synthetic derivatives would be of interest to explore further anti-leukaemic agents. Based on a previous study demonstrating that halogenation (F, Br and I) of a natural product could improve its cytotoxicity (Saeed et al. 2013), it is noteworthy to mention that brominated pentacyclic triterpene opens possibilities for exploration of new halogenated phytochemicals in cancer chemotherapy.

Instrumentation
Column chromatography (CC) and thin layer chromatography (TLC) were performed over silica gel 63-43 μm, 60 Å and 60 F 254 , respectively. 1d and 2d NMR data were recorded with a Bruker dRX-400 MHz. IR spectra were carried out on a Perkin-Elmer (FT-IR system spectrum BX spectrometer) using KBr discs. HR-ESI-MS and ESI-MS/MS were recorded, respectively, on a variant jEoL and a Xevo G2-S QTof mass spectrometer.

Plant material
Cultivated plant materials (rhizomes and aerial parts) were harvested from in a farm in dschang, Western Region of Cameroon in March 2013. The plant sample has been compared with the voucher specimen 23647/SRF-Cam and identified as E. giganteus at the Cameroonian National Herbarium, Yaoundé.
of 200 μL/well. After 72 h, resazurin (Sigma-Aldrich, Schnelldorf, Germany) (20 μL, 0.01% w/v) in distilled H 2 o was added to each well and the plates were incubated at 37 °C for 4 h. Fluorescence was measured on an Infinite M2000 ProTM plate reader (Tecan, Crailsheim, Germany) using an excitation wavelength of 544 nm and an emission wavelength of 590 nm. Each assay was done at least twice with six replicates each. The viability was evaluated based on a comparison with untreated cells. IC 50 values represent the compound concentrations required to inhibit 50% of cell proliferation and were calculated from a calibration curve by linear regression using Microsoft Excel (Kuete, Krusche, et al. 2011).

Conclusion
The present investigation described a second phytochemical study on E. giganteus which led to the identification of six secondary metabolites, among them two new and four known.
The new compounds included a furoceramide and a brominated pentacyclic triterpene. Another interesting finding in this article is that otherwise highly drug-resistant CEM/ AdR5000 revealed sensitivity and collateral sensitivity, respectively, towards these three compounds, which could represent valuable lead compounds for the generation of semi-synthetic derivatives to specifically target multidrug-resistant tumour cells.

Supporting information
The NMR data of compounds 1 and 2, and the ESI fragmentation mechanism of 2 have been published online as supporting information.