Isolation and Structure Elucidation of Cerebrosides from Euphorbia Platyphyllos L

A new cerebroside (1), together with three known ones (2–4) have been isolated from the whole plants of Euphorbia platyphyllos L. The structures were established by FT-IR spectroscopy, FAB-MS, advanced two-dimensional NMR, including H-NMR, H,H-COSY, HMQC and HMBC experiments and chemical reactions. The structures of the cerebrosides were characterized as 1-O-β-Dglycosides of phytosphingosines, which comprised a common long-chain base, (2S,3S,4R,8Z)-2-aminooctadec-8-ene-1,3,4-triol (1–3) and (2S,3S,4E,8E)-2aminooctadeca-4,8-diene-1,3-diol (4) with 2-hydroxy fatty acids of varying chain lengths (C16, C24, C26:1, C28:1) linked to the amino group. The isolated compounds have been evaluated for their antifungal and antitubercular activities.

1D and 2D 1 H-NMR spectroscopy, DQF-COSY and HMQC indicated that the head group consists of a single galactose residue in the β configuration.The galactose configuration was determined by the characteristic chemical shifts, the spin-spin splitting and the multiplicity of the characteristic resonance of the H-4″ proton, as well as by the splittings of the other ring protons.The absolute configuration of the galactopyranose moiety was determined to be the D-form using the Hara method [7].
The stereochemistry of the ceramide moiety was determined by comparison of the 1 H-NMR data of the cerebrosides isolated from Euphorbia platyphyllos L. with that of synthetic analogs as reported in literature in terms of the signals due to H-1 to H-4 [8].Methanolysis [9] of 1 yielded methyl galactoside, fatty acid methyl ester (FAM) and a trihydroxy long-chain base (LCB).
Methyl glycosides obtained from methanolysis of 1 were converted to trimethylsilyl derivatives using N,O-bis(trimethylsilyl)trifluoroacetamide (BSTFA) containing 1% trimethylchlorosilane (TMCS) for 3 h at 70°C and the silylated derivatives obtained were analyzed by gas chromatograph mass spectrometer (GC-MS) system.The monosaccharides presented two GC peaks due to the 1αand 1β-configurations of the OH on the pyrano ring (Table 2).The mass spectra of saccharides with the pyrano ring are mainly characterized by the m/z 204 fragment ion [e.g.galactopyranose as trimethylsilyl (TMS)]; in good agreement with the data reported in literature [10].In fact, this fragment is normally used as key ion to identify sugar compounds (especially monosaccharides as TMS) in complex extracts.Identification of galactose was carried out by study of the MS spectra, comparison with members of the NBS library and with retention times of methyl α and β-D-galactopyranoside and methyl αand β-D-glucopyranoside standard TMS derivatives.This is the first example of the presence of a galactose unity as part of a cerebroside isolated from Euphorbiaceae.
The fatty acid methyl ester was identified by GC/MS as methyl 2hydroxyoctacosenoate.The MS spectrum of 1 showed a molecular ion [M + H] + peak at m/z 898 and a fragment ion at m/z 736 [898-Gal] + .The absolute configuration at C(2) of the 2-hydroxy fatty acid was presumed to be R from the specific rotation of the fatty acid methyl ester ([α] D −11.0 ºC) [11][12][13].
On the other hand, on the basis of mass spectrometry analysis the LCB component of 1 was suggested to be 2-amino-1,3,4-trihydroxy-8-octadecene (LCB-1).In order to establish the position of the double bond, the FAM-1 and LCB-1 were treated with dimethyl disulfide (DMDS) and I 2 and the products subjected to electron impact EI-MS analysis [14].The EI-MS spectrum of the DMDS derivative showed a molecular ion at m/z 409 and significant fragments ions at m/z 221 and 187 arising from selective fragmentation at C(8)=C(9) position of C18 chain, thus confirming the position of the double bond at C(8)=C (9) in the long-chain base (Figure 3).The Δ 8 double bond in 1 was determined to be cis (Z) by the upfield shifted carbon chemical shifts of C-7 (δ 27.74) and C-10 (δ 27.62 ) [13] and the relative small coupling constant of H-8 at δ 5.43 (dt, J = 10.8, 7.2 Hz) and H-9 at δ 5.46 (dt, J = 10.8, 6.7 Hz).The FAM obtained from the methanolysis of the cerebroside 1 exhibited 13 C-NMR signals at about 130.1 and 130.2 expected for monounsaturated fatty acid methyl ester.The resonance at about 27.8, 27.9 confirms the Z geometry of the double bond in the long-chain fatty acid (Table 1).The position of the double bond in the monounsaturated fatty acid methyl ester was determined by EI-MS analysis of the corresponding dimethyl disulfide (DMDS) derivative [14].The F. Cateni, J. Zilic, and M. Zacchigna: characteristic fragments at m/z 373 and 173, obtained from the cleavage between the sulphide carbons, indicated the position of the double bond in 1.
Compounds 2-4 were found to be identical in all of their characteristics, with the cerebrosides previously isolated from Euphorbia nicaeensis All.[15] and Euphorbia peplis L. [16].The extract containing the complex mixture of cerebrosides 2-4 exhibited cytotoxic activity against KB cells and moderate antifungal and antitubercular activities.

Biological Activity Evaluation
The fraction B containing the complex mixture of cerebrosides and the individual isolated compound 1 were tested for the antimicrobial, antitubercular and antifungal activities (Tables 3, 4).The biological activity evaluation of compounds 2-4 has been reported in a previous paper [16].Reference drugs are reported.
No activity was exhibited against Gram positive nor Gram negative reference strains.We registered an interesting antitubercular activity of the fraction B containing the complex mixture of cerebrosides 1-4 with a MIC of 40 μg/mL against a human clinical M. tuberculosis strain and MICs of 80 μg/mL against the reference strain and two additional clinical human isolates.Compound 1 has shown an interesting antitubercular activity with MICs of 40 μg/mL on reference strain and on three clinical isolates and MICs of 80 μg/mL against clinical strains H331, H242, H172; all strains being sensitive to ciprofloxacin with MICs ranging from 0.125 to 0.25 μg/mL (Table 3).
As concerning antifungal activity, the complex mixture of cerebrosides had a MIC of 8 μg/mL on C. neoformans clinical strain after 24 h incubation and a MIC of inhibiting activity against the three different species of Candida studied, having a MIC of 64 μg/mL after 24 h incubation and a MIC of 128 μg/mL after 48 h incubation.No pure cerebroside showed any activity against Candida spp.; C. neoformans was slightly sensitive after 24 h incubation time with MIC of 64 μg/mL for compound 1 (Table 4).Sugar standards were purchased from Fluka/Aldrich/Sigma (Milano, Italy).

Plant Material
E. platyphyllos was collected from wild stock growing in Carso Triestino

Extraction and Isolation
Dried, ground whole plants of E. platyphyllos (300 g) were extracted with MeOH and evaporated to a crude residue (15.6 g), which was fractionated on a silica gel column with CHCl 3 and MeOH, respectively.The fraction eluted with MeOH was subjected to silica-gel column chromatography using mixtures of

GC-MS Analysis of TMS Ethers of Methyl Glycoside from 1
The mixture of methyl glycosides obtained by column chromatography of the aq.MeOH layer derived from methanolysis of 1 was converted to their trimethylsilyl derivatives using BSTFA containing 1% TMCS for 3 h at 70°C.0.1 µl of silylated mixture was analyzed by gas chromatograph mass spectrometer (GC-MS) system.
The chromatographic conditions were: column temperature was programmed from 100°C to 260°C at 8°C/min, with 15 min of final isotherm, injector temperature 280°C, carrier gas (helium) flow rate 1.5 ml/min.Transfer line temperature was kept at 270°C.The mass spectrometer scanned from m/z 100 to m/z 600 at 1.0 second cycle time.The ion source was set at 180°C and spectra were obtained by electron impact (70 eV).

GC-MS Analysis of TMS Ethers of Methyl Glycosides from 2-4
The mixture of methyl glycosides obtained by column chromatography of the aq.MeOH layer derived from methanolysis of 2-4 was trimethylsilylated and the reaction mixture was analyzed by GC-MS in the same manner as described for 1.

Dimethyl Disulfide Derivative of FAMs from cerebrosides 1,3
FAM-1 (6 mg) was dissolved in carbon disulfide (0.5 ml) and dimethyl disulfide (0.5 ml) and iodine (10 mg) were added.The reaction mixture was then kept at 60°C for 48h in a small sealed vial.The reaction was subsequently quenched with 5% aq.

(
Trieste), Italy in May 2006.A voucher specimen was deposited at the Herbarium of the Department of Biology (TSB) of the University of Trieste (Italy).
Characteristics of methyl glycosides analyzed by GC-MS as TMS derivatives.
Gram-positive and Gramnegative and tubercular bacteria.
Tab. 4. Antifungal activity of cerebrosides against Candida species and Cryptococcus neoformans.MIC (μg/mL)identical to the cerebrosides isolated from Euphorbia nicaeensis All. and Euphorbia peplis L. Besides, the pure isolated cerebrosides show a synergistic antifungal activity when present in mixture.As concerning antitubercolar activity, only compounds 1 and 3 are responsible for this effect.