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Cerebrosides with antiproliferative activity from Euphorbia peplis L.
Fitoterapia 81 (2010) 97–103
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Fitoterapia j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / f i t o t e
Cerebrosides with antiproliferative activity from Euphorbia peplis L. Francesca Cateni a,⁎, Jelena Zilic a, Marina Zacchigna a, Giuseppe Procida b a b
Department of Pharmaceutical Sciences, University of Trieste, P.zle Europa, 1, 34127 Trieste, Italy Department of Materials and Natural Resources, University of Trieste, Via A. Valerio 6, 34127 Trieste, Italy
a r t i c l e
i n f o
Article history: Received 25 March 2009 Accepted in revised form 23 July 2009 Available online 22 August 2009 Keywords: Euphorbia peplis L. Euphorbiaceae Cerebrosides Glycosphingolipid
a b s t r a c t Two new cerebrosides have been isolated from the whole plants of Euphorbia peplis L. The structures were established by FT-IR spectroscopy, FAB MS, EI-MS, ESI-MS, 1D and 2D NMR spectroscopy. The structures of the cerebrosides were characterized as 1-O-β-D-glycosides of phytosphingosines, which comprised a common long-chain base, (2S, 3S, 4R, 8Z)-2-amino-8 (Z)-octadecene-1,3,4triol with 2-hydroxy fatty acids of varying chain lengths (C25, C22) linked to the amino group. The isolated compounds were shown to possess significant antiproliferative properties against cultured human tumor cell lines KB and IMR-32. © 2009 Elsevier B.V. All rights reserved.
1. Introduction Euphorbia peplis L. (Family Euphorbiaceae) is a perennial herbaceous plant with a milky juice, distributed mainly in North Italy. Previous chemical studies on E. peplis have revealed the presence of macrocyclic diterpenes for the treatment and prophylaxis of prostate cancer, inflammation and protein kinase C (PKC) related conditions [1–3]. From the lipophilic fraction of Euphorbia peplis have been isolated and identified triacylglycerols and phospholipids [4]. In our previous studies, we reported the isolation and structure elucidation of biologically active glyceroglycolipids obtained from the less polar fraction of the MeOH extract of the plant E. peplis [5]. In a continuation of those studies, conducted for the considerable interest and importance connected with the determination of the composition of the mixtures of glyceroglycolipids and glycosphingolipids, we performed the isolation and characterization of cerebrosides with an interesting antifungal and antitubercular activity [6]. As part of our current interest on bioactive substances from Euphorbiaceae [5], we have chemically examined a fraction previously isolated from the plant Euphorbia peplis. The present paper describes the isolation and structure determination of two new cerebrosides (1, 2) from E. peplis (Fig. 1). The isolated compounds have been tested against ⁎ Corresponding author. Tel.: +39 040 5583722; fax: +39 040 52572. E-mail address: [email protected] (F. Cateni). 0367-326X/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.fitote.2009.08.022
human epithelial tumor cell lines KB and human neuroblastoma cell lines, IMR-32, showing a pharmacological interesting cell toxicity profile in both cell lines.
2. Results and discussion The MeOH extract of the whole plant of E. peplis was subjected to silica gel column chromatography (CC), using CHCl3 and MeOH as eluents. The fraction eluted with MeOH was purified over a CC on silica gel, eluted with CHCl3/MeOH (5:1) and CHCl3/MeOH (5:2), to give four subfractions (A–D). Fraction C, eluted with CHCl3/MeOH (5:1.5), was subjected to CC on RP-18 (MeOH), separately, yielding the pure compounds 1 and 2. Compound 1 was obtained as an amorphous solid, showing the presence of hydroxyl and amide NH (3500–3200 cm− 1) and amide carbonyl (1640 cm− 1) in the FT-IR spectrum. The 1 H- and 13C-NMR spectra of 1 (Table 1) indicated the presence of a sugar moiety (δH 4.2, 1H, d, J = 7.5 Hz, anomeric proton; δC 105.0), an amide function (δH 8.5, 1H, d, J = 9.0 Hz, NH; δC 175.8), and long-chain aliphatic and olefinic groups (δH 0.8, t, J = 7.0 Hz, CH3; δH 1.2, br s, CH2, δH 5.4, 2H; δC 129.6, 130.4). The data were suggestive of a glycosphingolipid structure. The 13C-NMR spectrum of 1 (Table 1) was assigned by a combination of distortionless enhancement by polarization transfer, heteronuclear multiple quantum coherence and heteronuclear multiple bond correlation experiments.
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Fig. 1. Chemical structures of cerebrosides 1, 2.
Important long-range correlations were observed between C-1″ and Hab-1; C-1 and H-1″, H-2, H-3; C-2 and NH and C-1′ and NH and H-2′ (Fig. 2). The 1H-NMR spectrum of the heptaacetate derivative (1a) of 1 was much clearer, with well resolved signals. 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 signal of the anomeric proton of a β-D-galactopyranose appeared at δ 4.2 as a doublet (J1,2 = 7.5 Hz, diaxial); 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 (Table 1). Signals from two olefinic protons at δ 5.4 (each dt, J = 9.6, 6.4 Hz), two methyl groups at δ 0.8 (t, J = 7.2 Hz) and the long-chain methylene protons at δ 1.2 (br s) and 1.2 (br s) suggested the presence of two long aliphatic chains, one of which possessed a cis double bond. A doublet at δ 8.6 (J = 9.0 Hz) was assigned to the NH of the amide moiety. The spectrum of 1a also showed signals of two oxygenated methylene protons as two doublets of doublets at δ 3.7 (J = 11.0, 3.4 Hz, Ha-1) and 3.88 (J = 11.0, 3.3 Hz, Hb-1) and four methine protons as a triplet of triplets at δ 4.3 (J = 9.0, 3.4 Hz, H-2), a doublet of doublets at δ 5.1 (J = 9.0, 3.3 Hz, H-3), a doublet of triplets at δ 4.89 (J = 9.0, 3.3 Hz, H-4) and a triplet at δ 5.15 (J = 5.0, H-2′). These data, together with the other 1H–1H COSY correlations of 1a (Fig. 2), supported the structure as 1-β-D-galactopyranoside of a 3,4-dihydroxysphingosine-type ceramide possessing a
2-hydroxy fatty acid acyl group. The 13C-NMR of 1a (Table 1) was assigned by a combination of DEPT, HMQC and HMBC experiments. In particular, the long-range correlations which were observed in the HMBC spectrum (Fig. 2) also supported the substitution pattern in 1a. 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 1H-NMR data of the cerebrosides isolated from E. peplis with that of synthetic analogs as reported in literature in terms of the signals due to 1-H to 4-H [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, 2 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 1α- and 1βconfigurations of the OH on the pyrano ring. 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,
F. Cateni et al. / Fitoterapia 81 (2010) 97–103
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Table 1 NMR spectral data of 1, 1a and 2 in C5D5N (δ in ppm, multiplicities, J in Hz). Position
1
1a
1
13
13
8.5 (d, 9.0) 4.6 (dd, 12, 5.2) 4.5 (m) 5.2 (m) 4.3 (dd, 3.6, 5) 4.3 (obs) 2.4 (m) 1.8 (m) 1.7 (m) 1.6 (m) 2.2 (m) 5.4 (m) 5.4 (m) 2.2 (m) 1.3 (m) 1.2 (m) 1.2 (m) 0.8 (t, 7) – 4.5 (dd, 7.6, 5.0) 2.2 (m) 1.9 (m) 1.9 (m) 1.7 (m) 1.3 (m)
– 70.5
– 67.2
51.8 76.2 72.5 34.0
8.6 (d, 9.0) 3.7 (dd, 11.0, 3.4) 3.8 (dd, 11.0, 3.3) 4.3 (tt, 9.0, 3.4) 5.1 (dd, 9.0, 3.3) 4.9 (dt, 9.0, 3.3) 1.6 (m)
26.2
1.6 (m)
27.8
27.8 130.4 129.6 27.7 29.7–30.4 32.3 23.2 14.7 175.8 72.5 35.8
1.9 (m) 5.4 (dt, 9.6, 6.3) 5.4 (dt, 9.6, 6.4) 1.9 (m) 1.3 (m) 1.2 (m) 1.2 (m) 0.8 (t, 7.2) – 5.1 (t, 5.0) 1.8 (m)
26.4 128.6 130.5 27.2 29.7–30.4 32.3 23.2 14.2 170.0 71.8 31.7, 31.9
26.5
1.9 (m)
26.6
29.8–30.4
1.3 (m)
29.8–30.4
1.2 (m) 1.2 (m) 0.8 (t, 7) 7.6 (br s) 6.8 (br s) 6.0 (br s)
32.3 23.2 14.1 – – –
1.2 (m) 1.2 (m) 0.8 (t, 7.2)
32.3 23.2 14.2
4.2 (d, 7.5) 3.5 (dd, 9.7, 7.5) 3.5 (dd, 9.7, 3.3) 3.8 (dd, 3.3, 0.8) 3.5 (m) 3.7 (dd, 11.4, 5.1) 3.7 (dd, 11.4, 6.9) 6.4 (br s)
105.0 73.0 73.0 70.0 70.0 62.0
4.6 (dd, 8.0) 5.2 (dd, 3.9, 10.2) 5.4 (dd, 10.2, 3.9) 5.3 (dd, 3.4, 1.2) 4.4 (m) 4.1 (dd, 12.5, 2.5) 4.2 (dd, 12.5, 4.0)
100.3 74.1 72.6 68.9 68.4 66.6
1.2 (br s) 1.9, 2.0, 2.0 (2×), 2.0,2.1, 2.2, all s 170.1, 170.2, 170.5, 170.9
22.7–29.7 20.5 (3×), 20.6, 20.7, 20.8, 21.0
H
Ceramide NH 1a 1b 2 3 4 5a 5b 6a 6b 7 8 9 10 11–15 16 17 18 1′ 2′ 3′a 3′b 4′a 4′b 5′–22′(21′) 22′ 23′ 24′ 25′ OH-2′ OH-3 OH-4 Galactose 1″ 2″ 3″ 4″ 5″ 6″a 6″b OH-6″ (CH2)n COCH3 COCH3
2
1
C
H
comparison with members of the NBS library and with retention times of α and β-D-galactopyranoside and methyl α- and β-D-glucopyranoside standard TMS derivatives.
C
50.1 74.2 73.4 27.8
1
13
8.5 (d, 7.9) 4.7 (dd) 4.5 (m) 5.2 (m) 4.3 (dd) 4.2 (obs) 1.8 (m) 2.4 (m) 1.6 (m) 1.7 (m) 2.2 (m) 5.4 (m) 5.4 (m) 2.2 (m) 1.3 (m) 1.2 (m) 1.2 (m) 0.8 (t, 7) – 4.5 (dd, 8.0, 4.0) 2.0 (m) 2.2 (m) 1.7 (m) 1.9 (m) 1.3 (m) 0.8 (t, 7.0)
– 70.6
7.6 (br s) 6.8 (br s) 6.0 (br s)
– – –
4.2 (d, 7.5) 3.5 (dd, 9.7, 7.5) 3.5 (dd, 9.7, 3.3) 3.8 (dd, 3.3, 0.8) 3.5 (m) 3.7 (dd, 11.4, 5.2) 3.7 (dd, 11.4, 6.9) 6.3 (br s)
105.0 73.0 73.0 70.0 70.0 62.0
H
C
51.8 76.0 72.5 34.0 26.2 27.9 130.6 129.6 27.7 29.9–30.3 32.4 23.2 14.6 175.9 72.5 35.8 26.4 23.1–32.0 14.6
Recently, a new cerebroside possessing a galactose unity as polar head was isolated and identified from E. platyphyllos L. [11].
Fig. 2. Selected 1H–1H COSY (bold lines) and Selected HMBC (full-line arrows) Correlations of 1.
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Fig. 3. LCB-DMDS Derivatives of 1, 2.
The fatty acid methyl ester was identified by GC/MS as methyl 2-hydroxypentacosanoate. The MS spectrum of 1 showed a molecular ion [M + H]+ peak at m/z 858 and fragment ion at m/z 696 [858 - Glc]. 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]25 D -11.0°) [12–14]. 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 LCB-1 was treated with dimethyl disulfide (DMDS) and I2 and the product subjected to electron impact (EI)-MS analysis [15]. 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 at C-8-C-9 in the long-chain base (Fig. 3). The double bond in 1 was determined to be cis (Z) by the upfield shifted carbon chemical shifts of C-7 (δ 27.8) and C-10 (δ 27.7) [14] and the relative small coupling constant of H-8 at δ 5.4 (dt, J = 9.6, 6.3 Hz) and H-9 at δ 5.4 (dt, J = 9.6, 6.4 Hz) in 1a. Therefore 1 was characterized as (2S, 3S, 4R, 8Z)-1-O(β- D -galactopyranosyl)-2N-[(2′R)-2′-hydroxypentacosanoilamino]-8 (Z)-octadecene-1,3,4-triol (1). Cerebroside 2 showed strong hydroxy (3414 cm− 1) and amide (1645, 1543 cm− 1) absorptions in the FT-IR spectrum. The positive FAB mass spectrum of cerebroside 2 exhibited (M + H)+ ion peak at m/z 816 (2). The 1H and 13C-NMR spectra of cerebroside 2 exhibited the characteristic signals of a sphingosine-type cerebroside possessing 2-hydroxy fatty acid and β-galactopyranose moieties (Fig. 1, Table 1). When cerebroside 2 was methanolyzed with methanolic hydrochloric acid, fatty acid methyl ester (FAM) was obtained together with long-chain base (LCB) and methyl galactopyranoside. Comparison of the 1H-NMR and 13C-NMR data of 2 (Table 1) with those of 1 indicated that the major difference was in the length of the FAM. On the basis of mass spectrometry analysis, the FAM was characterized as methyl 2-hydroxy docosanoate (FAM-2). The stereochemistry of the ceramide moiety was determined by comparison of the 1H-NMR data of the cerebrosides isolated from E. peplis with that of synthetic analogs as reported in literature in terms of the signals due to 1-H to 4-H [16]. The absolute configuration of the galactopyranose moiety in 2 was determined to be the D-form using the Hara method [7]. Close similarity of the spectral data with those of 1 described above suggested that 2 is a higher homologue of 1.
Thus, the structure of 2 was proposed to be 1-O-(β-Dgalactopyranosyl)-(2S, 3S, 4R, 8Z)-2-[(2′R)-2′-hydroxydocosanoilamino]-8 (Z)-octadecene-1,3,4-triol (2). Euphorbia peplis accumulates complex sphingoglycolipids in which dihydroxy [17] and trihydroxy (1, 2) long-chain bases (LCBs) occur as part of the cerebroside moiety. Compounds 1 and 2 are new, since they have been isolated for the first time in Euphorbiaceae; analogous cerebrosides with a glucose moiety as head group, instead of galactose, have been isolated from Euphorbia sororia [18] and Euphorbia nicaeensis All. [17]. The antiproliferative activity of compounds 1 and 2 was tested in vitro against two tumor cell lines (human KB and IMR-32) and expressed as IC50 values. IC50 is the concentration (µM) required to inhibit tumor cell proliferation by 50% after 72 h exposure of the cells to a tested compound. The measured IC50 values for compounds 1 and 2 are summarized in Table 2. Cisplatinum was used as a reference compound [19]. KB and IMR-32 cells were exposed at 1.25, 2.50, 5.00 and 10 µg mL− 1 solutions of each compounds. After 72 h of treatment, the cerebrosides 1 and 2 exhibited growth inhibition in both cell lines with IC50 values ranging from 4.63 to 10.09 µM. 3. Experimental 3.1. Chemistry 3.1.1. General experimental procedures Optical rotations were determined on a Jasco P-1020 polarimeter (Jasco, Italy). Elementary analyses were carried out on a Carlo Erba model 1016 analyzer. 1H and 13C NMR spectra were recorded on a Varian Unity spectrometer operating at 400 MHz (1H) and 100 MHz (13C), respectively. The chemical shift values were reported in parts per million units and the coupling constants were in Hz. FAB MS was recorded on Kratos MS 80 RFA mass spectrometer using a beam of Argon/Xenon (2–8 Kv), methanol as solvent and glycerol as the matrix. IR spectra were recorded on a Table 2 IC50 values for compounds 1 and 2 against KB and IMR-32 cell lines. Compound
KB IC50 (µM)
IMR-32 IC50 (µM)
1 2 Cisplatinum
4.63 7.20 0.37
10.09 6.61 0.5
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Jasco IR-700 infrared spectrophotometer. Silica gel column chromatography was carried out using silica gel Kieselgel 60 (230–400 Mesh, 60 Å Merck), reversed-phase chromatography was carried out using Lichroprep RP-18 (40–63 µm, Merck). GC–MS experiments were carried out on Fisons GC 8000 (Carlo Erba, 20090 Milan, Italy) instrument, equipped with a split/splitless injector (1:20); the capillary gas chromatograph system was coupled directly to a MD 800 mass spectrometer (Carlo Erba, 20090 Milan, Italy). A SPB 5 fused silica capillary column, 30 m × 0.32 mm I.D., 0.25 µm film thickness (Supelco, Inc., Bellefonte, PA) was employed. Sugar standards were purchased from Fluka/Aldrich/Sigma (Milano, Italy). All solvents were distilled before use. TLC were carried out using Kieselgel 60 F254 (20 × 20 cm; 0.2 mm, Merck) and RP-18 F254 (10 × 10 cm, Merck); spots were detected by spraying with 10% aq. H2SO4 followed by heating. 3.1.2. Plant material E. peplis was collected from wild stock growing in Carso Triestino (Trieste), Italy in May 2007. A voucher specimen was deposited at the Herbarium of the Department of Biology (TSB) of the University of Trieste (Italy) (TSB-51803). 3.1.3. Extraction and isolation Dried, ground whole plants of E. peplis (500 g) were extracted with MeOH and evaporated to a crude residue (25.4 g), which was fractionated on a silica gel column with CHCl3 and MeOH, respectively. The fraction eluted with MeOH was subjected to silica gel column chromatography using mixtures of CHCl3/MeOH (5:1) and (5:2), respectively, yielding four subfractions (A–D). Subfraction C, eluted with CHCl3/MeOH (5:1.5), was further chromatographed on RP-18 (MeOH) affording 1 (34.1 mg) and 2 (54.4 mg). ð2S; 3S; 4R; 8ZÞ‐1‐O‐ðβ‐D‐galactopyranosylÞ‐2N ‐½ð2′RÞ‐2′‐hydroxy‐19′ðZÞ‐pentacosanoilamino‐8ðZÞ ‐octadecene‐1; 3; 4‐triol ð1Þ Nujol A colorless solid. [α]24 D + 11.3° (c 0.2, pyridine). FT-IR νmax cm : 3317 (broad), 2920, 2850, 1590, 1074, 1036. Positiveion FAB MS: m/z = 880 [M + Na, 78%]+, 858 [M + H, 20%]+, 840 [M + H − H2O, 12%]+, 719 [M + Na − C6H11O5 + H, 24%]+, 696 [M + H − C6H11O5, 100%]+, 678 [M + H − Glc, 80%]+, 500 [M + Na − C25H49O2 + H, 100%]+, 476 [M + H − C25H50O2, 10%]+. Negative-ion FAB MS: m/z = 856 [M − H, 100%]−, 694 [M − H − C6H11O5, 20%]−, 677 [M − H − Glc, 18%]−. 1H- and 13 C-NMR data are reported in Table 1. Anal. calcd. for C49H95O10N: C, 68.57; H, 11.08; N, 1.63. Found: C, 68.93; H, 10.99; N, 1.71. −1
ð2S; 3S; 4R; 8ZÞ‐1‐O‐ðβ‐D‐galactopyranosylÞ‐2N ‐½ð2′RÞ‐2′‐hydroxydocosanoilamino‐8ðZÞ‐octadecene ‐1; 3; 4‐triol ð2Þ Amorphous powder. [α]24 D + 4.8° (c 0.2, pyridine). FT-IR cm− 1: 3413, 1646, 1540. Positive-ion FAB MS: m/z = 816 [M + H, 83%]+, 798 [M + H − H2O, 35%]+, 654 [M + H − C6H11O5, 95%]+, 636 [M + H − Glc, 40%]+, 476 [M + H − C22H43O2, 10%]+, 500 [M + Na − C22H43O2, 100%]+, Negativeion FAB MS: m/z = 814 [M − H, 100%], 652 [M − H − C6H11O5, 23%]−. ESI-MS: m/z = 838 [M + Na, 50%]+, 658 [M + Na − Glc, 40%]+. 1H- and 13C-NMR data are reported in Table 1. νKBr max
101
Anal. calcd. for C46H89O10N: C, 67.69; H, 10.91; N, 1.72. Found: C, 67.96; H, 10.03; N, 1.84. 3.1.4. Methanolysis of 1, 2 Compound 1 (34.1 mg) was refluxed with 0.9 M HCl in 82% aq. MeOH (10 mL) for 18 h. The mixture was extracted with n-hexane and the combined organic phases were washed with water and dried over Na2SO4. Removal of the solvent gave a colorless wax (18.0 mg) which was chromatographed on silica gel [hexane/EtOAc (20:1, 5:1)] to yield fatty acid methyl ester as a colorless wax (11.5 mg). The compound 2 was methanolyzed using the same method described above. The esters were analyzed by GC–MS. The results were as follows: FAM-1 (2-hydroxypentacosanoic acid methyl ester), EI-MS: m/z= 413 [M]+, 381 [M - CH3OH]+, 354 [M - CH3COO]+; FAM-2 (2-hydroxydocosanoic acid methyl ester), EI-MS: m/z= 370 [M]+, 338 [M - CH3OH]+, 311 [M CH3COO]+. The aq. MeOH layer was neutralized with NH4OH and extracted with EtOAc. The combined EtOAc extract was washed with H2O, dried over Na2SO4 and evaporated to give the longchain base (LCB) as a slightly yellow wax (15.6 mg). The aq. MeOH layer was then evaporated to dryness and chromatographed on silica gel [CH2Cl2/MeOH/H2O (lower layer) (20:3:1, 10:3:1, 7:3:1)] to give methyl galactopyranoside (α- and β-anomer) as a colorless solid (4.2 mg). TLC [silica gel, CH2Cl2/MeOH/H2O (lower layer) (10:3:1)] of the resulting methyl galactopyranoside (α- and β-anomer) was identical to that of the standard methyl α-D-galactopyranoside and methyl β-D-galactopyranoside. 3.1.5. GC–MS analysis of TMS ethers of methyl glycosides from 1, 2 The mixture of methyl glycosides obtained by column chromatography of the aq. MeOH layer derived from methanolysis of 1 and 2 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). Methyl glycosides (GC–MS): methyl α- and β-galactopyranosides were detected. Methyl α-Dgalactopyranoside: C7H14O6, m/z = 194 [M]+, TMS derivative, m/z = 482 [M]+, 204, 191, 217, tR (min): 13.188. Methyl β-Dgalactopyranoside: C7H14O6, m/z = 194 [M]+, TMS derivative, m/z = 482 [M]+, 204, 191, 217, tR (min): 14.307. Methyl α-Dglucopyranoside: C7H14O6, m/z = 194 [M]+, TMS derivative, m/z = 482 [M]+, 204, 191, 217, tR (min): 12.770. Methyl β-Dglucopyranoside: C7H14O6, m/z = 194 [M]+, TMS derivative, m/z = 482 [M]+, 204, 191, 217, tR (min): 14.008. 3.1.6. Dimethyl disulfide derivatives of LCBs from cerebrosides 1 and 2 LCB-1 (10 mg) was dissolved in carbon disulfide (1 mL) and dimethyl disulfide (1 mL) and iodine (20 mg) were
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added. The reaction mixture was then kept at 60 °C for 48 h in a small sealed vial. The reaction was subsequently quenched with 5% aq. Na2S2O3 and the mixture was extracted with n-hexane. The n-hexane layer was dried over Na2SO4, filtered and concentrated to give the dimethyl disulfide derivative of LCB-1 as a light yellow oil (9.3 mg). The LCBs DMDS derivatives of cerebrosides 1 and 2 were subjected to MS analysis. The results were as follows: LCB-1 DMDS derivative, EI-MS: m/z = 221, 187. LCB-2 DMDS derivative, EI-MS: m/z = 221, 187. 4. Pharmacology 4.1. Cells and cytotoxic assays 4.1.1. Cell cultures KB, human oral epidermoid carcinoma cell line (ECACC no. 86103004), and IMR-32, human adenocarcinoma cell line (ECACC no. 86041809) were cultured according to standard procedure [19]. Vials of the original line were maintained in liquid N2; cells were obtained, routinely subcultured once a week, and used for the reported experiment. The cell lines were maintained in Eagle's minimum essential medium (MEM) [20] supplemented with 10% newborn calf serum (Hyclone) for KB and fetal calf serum (Euroclone) for IMR-32, with 10 mL− 1 penicillin and streptomycin solution (Sigma Chemical Co., St. Louis, MO) (100 U mL− 1 penicillin G and 100 µg mL− 1 streptomycin) and buffered with 3 mM tris [hydroxymethyl]methyl-2-aminoethane sulfonic acid, 3 nM N,N-bis [2-hydroxyethyl]-2-aminoethanesulfonic acid, 3 mM N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid and 3 mM Tricine (Sigma Chemical Co.). The cell population doubling time was ca. 24 and 20 h for KB and IMR-32, respectively. Cells from confluent monolayers were removed with 2–3 mL of 0.05% (KB cells) or 0.25% (IMR-32 cells) trypsin solution (Sigma Chemical Co.). 4.2. Growth inhibition of KB and IMR-32 cells derivatives For the valuation of cytostatic activity, KB and IMR-32 cells were sown at a density of 2.5×104 cells per mL, in 0.2 mL per well in a 96-well plate (Corning Costar, Milano, Italy). After 24 h, derivatives were dissolved in sterile DMSO and solutions diluted in culture medium up to obtained opportune concentration (1.25, 2.50, 5.00 and 10.00 µg mL− 1); nutritive medium of every well was substituted with 0.2 mL of solution. After 72 h incubation at 37 °C, cellular vitality was evaluated with a colorimetric assay based on the quantification with sulforhodamine B (SRB – Sigma Chemical Co.) of cellular protein component [21]. Briefly, adherent cell cultures were fixed in situ by addition of 50 µl of cold 50% (v/v) trichloroacetic acid (TCA) and were kept for 60 min at 4 °C. The supernatant was then discarded and the plates were washed two times with bi-distilled water and air-dried. SRB solution (0.4% w/v in 1% acetic acid) was added and the cells were allowed to stain for 30 min at room temperature. Unbound SRB was removed by washing three times with 1% acetic acid. Then the plates were air-dried. Bound stain was dissolved with unbuffered 10 mM Tris base (tris-hydroxymethylaminomethane) (Sigma Chemical Co.) and the optical density was read at 570 nm with an automated microplate reader
EL311s spectrophotometer (BIO-TEK Instruments, INC. Winooski, Vermount, USA). Each experiment was performed in quintuplicate and repeated twice. Cytostatic activity was evaluated as percentage of cellular growth inhibition in culture treated with compounds to respect to the growth observed in control culture. IC50 and parallelism test were performed with the aim of PCS program [22]. 4.3. Statistical analysis Data were analyzed using Student's t-test. Significance was accepted with P < 0.05. Values of IC50 were obtained with PCS program [22]. Acknowledgements The authors are grateful to Dr. Vito Scarcia, Department of Biomedical Sciences, University of Trieste, Italy for the in vitro cytostatic activity assays. References [1] Aylward JH, Gordon PP. Diterpenes obtained from Euphorbiaceae for the treatment of prostate cancer. Au. Pat. WO 2002011743; 2002. [2] Aylward JH, Gordon PP, Suhrbier A, Turner KA. Euphorbiaceae macrocyclic diterpenes for the treatment of inflammation. Au. Pat. WO 2001093885; 2001. [3] Aylward JH, Gordon PP, Suhrbier A, Turner KA. Macrocyclic diterpenes for treatment and prophylaxix of PKC-related conditions. Au. Pat. WO 2001093884; 2001. [4] Ivanova A, Khozin-Goldberg I, Kamenarska Z, Nechev J, Cohen Z, Popov S, et al. Lipophilic compounds from Euphorbia peplis L. — a halophytic plant from the Bulgarian Black Sea coast. Z Naturforsch C: J Biosci 2003;58: 783–8. [5] Cateni F, Falsone G, Zilic J. Terpenoids and glycolipids from Euphorbiaceae. Mini Rev Med Chem 2003;3:425–37. [6] Cateni F, Zilic J, Falsone G, Scialino G, Banfi E. New cerebrosides from Euphorbia peplis L.: antimicrobial activity evaluation. Bioorg Med Chem Lett 2003;13:4345–50. [7] Hara S, Okabe H, Mihashi K. Gas–liquid chromatographic separation of aldose enantiomers as trimethylsilyl ethers of methyl 2-(polyhydroxyalkyl)-thiazolidine-4(R)-carboxylates. Chem Pharm Bull 1987;35: 501–6. [8] Yoda H, Oguchi T, Takabe K. An expeditious and pratical synthetic process for phytosphingosine and tetrahydroxy-LCB from D-glutamic acid. Tetrahedron Asymmetry 1996;7:2113–6. [9] Gaver RC, Sweely CC. Methods for methanolysis of sphingolipids and direct determination of long-chain bases by gas chromatography. J Am Oil Chem Soc 1965;42:294–8. [10] Medeiras PM, Simoneit BRT. Analysis of sugars in environmental samples by gas chromatography–mass spectrometry. J Cromatogr A 2007;1141:271–8. [11] Cateni F, Zilic J, Zacchigna M. Isolation and structure elucidation of cerebrosides from Euphorbia Platyphyllos L. Sci Pharm 2008;76:451–69. [12] Higuchi R, Natori T, Komori T. Biologically active glycosides from Asteroidea, XX. Glycosphingolipids from the starfish Asterina pectinifera, 1. Isolation and characterization of acanthacerebrosides B and structure elucidation of related, nearly homogeneous cerebrosides. Liebigs Ann Chem 1990:51–5. [13] Shibuya H, Kawashima K, Sakagami M, Kawanishi H, Shimamura M, Ohashi K, et al. Sphingolipids and glycerolipids. I. Chemical structure and ionophoretic activities of soyacerebrosides I and II from soybean. Chem Pharm Bull 1990;38:2933–8. [14] Kang SS, Kim JS, Son KH, Kim HP, Chang HW. Cyclooxygenase-2 inhibitory cerebrosides from Phytolaccae radix. Chem Pharm Bull 2001;49:321–3. [15] Scribe P, Guezennect J, Dagaut J, Pepe C, Saliot A. Identification of the position and the stereochemistry of the double bond in monounsaturated fatty acid methyl esters by gas chromatography/mass spectrometry of dimethyl disulfide derivatives. Anal Chem 1988;60:928–31. [16] Cateni F, Zacchigna M, Zilic J, Di Luca G. Total synthesis of a natural cerebroside from Euphorbiaceae. Helv Chim Acta 2007;90:282–9.
F. Cateni et al. / Fitoterapia 81 (2010) 97–103 [17] Cateni F, Zilic J, Falsone G, Hollan F, Frausin F, Scarcia V. Preliminary biological assay on cerebroside mixture from Euphorbia nicaeensis All. Isolation and structure determination of five glucocerebrosides. Il Farmaco 2003;58:809–17. [18] Zhang W, Xu J, Zhang X, Yao X, Ye W. Sphingolipids with neuritogenic activity from Euphorbia sororia. Chem Phys Lipids 2007;148:77–83. [19] Frausin F, Cocchietto M, Bergamo A, Scarcia V, Furlani A, Sava G. Tumour cell uptake of the metastasis inhibitor ruthenium complex NAMI-A and its in vitro effects on KB cells. Cancer Chemother Pharmacol 2002;50: 405–11.
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[20] Eagle H. Amino acid metabolism in mammalian cell cultures. Sciences 1959;130:432–7. [21] Skehan P, Storeng R, Scudiero D, Monks A, McMahon J, Vistica D, et al. New colorimetric cytotoxicity assay for anticancer-drug screening. J Natl Cancer Inst 1990;82:1107–12. [22] Tallarida RJ, Murray RB. Manual of Pharmacological Calculation with Computer Programs. 2nd ed. New York: Springer Verlag; 1986.
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Fitoterapia j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / f i t o t e
Cerebrosides with antiproliferative activity from Euphorbia peplis L. Francesca Cateni a,⁎, Jelena Zilic a, Marina Zacchigna a, Giuseppe Procida b a b
Department of Pharmaceutical Sciences, University of Trieste, P.zle Europa, 1, 34127 Trieste, Italy Department of Materials and Natural Resources, University of Trieste, Via A. Valerio 6, 34127 Trieste, Italy
a r t i c l e
i n f o
Article history: Received 25 March 2009 Accepted in revised form 23 July 2009 Available online 22 August 2009 Keywords: Euphorbia peplis L. Euphorbiaceae Cerebrosides Glycosphingolipid
a b s t r a c t Two new cerebrosides have been isolated from the whole plants of Euphorbia peplis L. The structures were established by FT-IR spectroscopy, FAB MS, EI-MS, ESI-MS, 1D and 2D NMR spectroscopy. The structures of the cerebrosides were characterized as 1-O-β-D-glycosides of phytosphingosines, which comprised a common long-chain base, (2S, 3S, 4R, 8Z)-2-amino-8 (Z)-octadecene-1,3,4triol with 2-hydroxy fatty acids of varying chain lengths (C25, C22) linked to the amino group. The isolated compounds were shown to possess significant antiproliferative properties against cultured human tumor cell lines KB and IMR-32. © 2009 Elsevier B.V. All rights reserved.
1. Introduction Euphorbia peplis L. (Family Euphorbiaceae) is a perennial herbaceous plant with a milky juice, distributed mainly in North Italy. Previous chemical studies on E. peplis have revealed the presence of macrocyclic diterpenes for the treatment and prophylaxis of prostate cancer, inflammation and protein kinase C (PKC) related conditions [1–3]. From the lipophilic fraction of Euphorbia peplis have been isolated and identified triacylglycerols and phospholipids [4]. In our previous studies, we reported the isolation and structure elucidation of biologically active glyceroglycolipids obtained from the less polar fraction of the MeOH extract of the plant E. peplis [5]. In a continuation of those studies, conducted for the considerable interest and importance connected with the determination of the composition of the mixtures of glyceroglycolipids and glycosphingolipids, we performed the isolation and characterization of cerebrosides with an interesting antifungal and antitubercular activity [6]. As part of our current interest on bioactive substances from Euphorbiaceae [5], we have chemically examined a fraction previously isolated from the plant Euphorbia peplis. The present paper describes the isolation and structure determination of two new cerebrosides (1, 2) from E. peplis (Fig. 1). The isolated compounds have been tested against ⁎ Corresponding author. Tel.: +39 040 5583722; fax: +39 040 52572. E-mail address: [email protected] (F. Cateni). 0367-326X/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.fitote.2009.08.022
human epithelial tumor cell lines KB and human neuroblastoma cell lines, IMR-32, showing a pharmacological interesting cell toxicity profile in both cell lines.
2. Results and discussion The MeOH extract of the whole plant of E. peplis was subjected to silica gel column chromatography (CC), using CHCl3 and MeOH as eluents. The fraction eluted with MeOH was purified over a CC on silica gel, eluted with CHCl3/MeOH (5:1) and CHCl3/MeOH (5:2), to give four subfractions (A–D). Fraction C, eluted with CHCl3/MeOH (5:1.5), was subjected to CC on RP-18 (MeOH), separately, yielding the pure compounds 1 and 2. Compound 1 was obtained as an amorphous solid, showing the presence of hydroxyl and amide NH (3500–3200 cm− 1) and amide carbonyl (1640 cm− 1) in the FT-IR spectrum. The 1 H- and 13C-NMR spectra of 1 (Table 1) indicated the presence of a sugar moiety (δH 4.2, 1H, d, J = 7.5 Hz, anomeric proton; δC 105.0), an amide function (δH 8.5, 1H, d, J = 9.0 Hz, NH; δC 175.8), and long-chain aliphatic and olefinic groups (δH 0.8, t, J = 7.0 Hz, CH3; δH 1.2, br s, CH2, δH 5.4, 2H; δC 129.6, 130.4). The data were suggestive of a glycosphingolipid structure. The 13C-NMR spectrum of 1 (Table 1) was assigned by a combination of distortionless enhancement by polarization transfer, heteronuclear multiple quantum coherence and heteronuclear multiple bond correlation experiments.
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Fig. 1. Chemical structures of cerebrosides 1, 2.
Important long-range correlations were observed between C-1″ and Hab-1; C-1 and H-1″, H-2, H-3; C-2 and NH and C-1′ and NH and H-2′ (Fig. 2). The 1H-NMR spectrum of the heptaacetate derivative (1a) of 1 was much clearer, with well resolved signals. 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 signal of the anomeric proton of a β-D-galactopyranose appeared at δ 4.2 as a doublet (J1,2 = 7.5 Hz, diaxial); 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 (Table 1). Signals from two olefinic protons at δ 5.4 (each dt, J = 9.6, 6.4 Hz), two methyl groups at δ 0.8 (t, J = 7.2 Hz) and the long-chain methylene protons at δ 1.2 (br s) and 1.2 (br s) suggested the presence of two long aliphatic chains, one of which possessed a cis double bond. A doublet at δ 8.6 (J = 9.0 Hz) was assigned to the NH of the amide moiety. The spectrum of 1a also showed signals of two oxygenated methylene protons as two doublets of doublets at δ 3.7 (J = 11.0, 3.4 Hz, Ha-1) and 3.88 (J = 11.0, 3.3 Hz, Hb-1) and four methine protons as a triplet of triplets at δ 4.3 (J = 9.0, 3.4 Hz, H-2), a doublet of doublets at δ 5.1 (J = 9.0, 3.3 Hz, H-3), a doublet of triplets at δ 4.89 (J = 9.0, 3.3 Hz, H-4) and a triplet at δ 5.15 (J = 5.0, H-2′). These data, together with the other 1H–1H COSY correlations of 1a (Fig. 2), supported the structure as 1-β-D-galactopyranoside of a 3,4-dihydroxysphingosine-type ceramide possessing a
2-hydroxy fatty acid acyl group. The 13C-NMR of 1a (Table 1) was assigned by a combination of DEPT, HMQC and HMBC experiments. In particular, the long-range correlations which were observed in the HMBC spectrum (Fig. 2) also supported the substitution pattern in 1a. 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 1H-NMR data of the cerebrosides isolated from E. peplis with that of synthetic analogs as reported in literature in terms of the signals due to 1-H to 4-H [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, 2 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 1α- and 1βconfigurations of the OH on the pyrano ring. 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,
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99
Table 1 NMR spectral data of 1, 1a and 2 in C5D5N (δ in ppm, multiplicities, J in Hz). Position
1
1a
1
13
13
8.5 (d, 9.0) 4.6 (dd, 12, 5.2) 4.5 (m) 5.2 (m) 4.3 (dd, 3.6, 5) 4.3 (obs) 2.4 (m) 1.8 (m) 1.7 (m) 1.6 (m) 2.2 (m) 5.4 (m) 5.4 (m) 2.2 (m) 1.3 (m) 1.2 (m) 1.2 (m) 0.8 (t, 7) – 4.5 (dd, 7.6, 5.0) 2.2 (m) 1.9 (m) 1.9 (m) 1.7 (m) 1.3 (m)
– 70.5
– 67.2
51.8 76.2 72.5 34.0
8.6 (d, 9.0) 3.7 (dd, 11.0, 3.4) 3.8 (dd, 11.0, 3.3) 4.3 (tt, 9.0, 3.4) 5.1 (dd, 9.0, 3.3) 4.9 (dt, 9.0, 3.3) 1.6 (m)
26.2
1.6 (m)
27.8
27.8 130.4 129.6 27.7 29.7–30.4 32.3 23.2 14.7 175.8 72.5 35.8
1.9 (m) 5.4 (dt, 9.6, 6.3) 5.4 (dt, 9.6, 6.4) 1.9 (m) 1.3 (m) 1.2 (m) 1.2 (m) 0.8 (t, 7.2) – 5.1 (t, 5.0) 1.8 (m)
26.4 128.6 130.5 27.2 29.7–30.4 32.3 23.2 14.2 170.0 71.8 31.7, 31.9
26.5
1.9 (m)
26.6
29.8–30.4
1.3 (m)
29.8–30.4
1.2 (m) 1.2 (m) 0.8 (t, 7) 7.6 (br s) 6.8 (br s) 6.0 (br s)
32.3 23.2 14.1 – – –
1.2 (m) 1.2 (m) 0.8 (t, 7.2)
32.3 23.2 14.2
4.2 (d, 7.5) 3.5 (dd, 9.7, 7.5) 3.5 (dd, 9.7, 3.3) 3.8 (dd, 3.3, 0.8) 3.5 (m) 3.7 (dd, 11.4, 5.1) 3.7 (dd, 11.4, 6.9) 6.4 (br s)
105.0 73.0 73.0 70.0 70.0 62.0
4.6 (dd, 8.0) 5.2 (dd, 3.9, 10.2) 5.4 (dd, 10.2, 3.9) 5.3 (dd, 3.4, 1.2) 4.4 (m) 4.1 (dd, 12.5, 2.5) 4.2 (dd, 12.5, 4.0)
100.3 74.1 72.6 68.9 68.4 66.6
1.2 (br s) 1.9, 2.0, 2.0 (2×), 2.0,2.1, 2.2, all s 170.1, 170.2, 170.5, 170.9
22.7–29.7 20.5 (3×), 20.6, 20.7, 20.8, 21.0
H
Ceramide NH 1a 1b 2 3 4 5a 5b 6a 6b 7 8 9 10 11–15 16 17 18 1′ 2′ 3′a 3′b 4′a 4′b 5′–22′(21′) 22′ 23′ 24′ 25′ OH-2′ OH-3 OH-4 Galactose 1″ 2″ 3″ 4″ 5″ 6″a 6″b OH-6″ (CH2)n COCH3 COCH3
2
1
C
H
comparison with members of the NBS library and with retention times of α and β-D-galactopyranoside and methyl α- and β-D-glucopyranoside standard TMS derivatives.
C
50.1 74.2 73.4 27.8
1
13
8.5 (d, 7.9) 4.7 (dd) 4.5 (m) 5.2 (m) 4.3 (dd) 4.2 (obs) 1.8 (m) 2.4 (m) 1.6 (m) 1.7 (m) 2.2 (m) 5.4 (m) 5.4 (m) 2.2 (m) 1.3 (m) 1.2 (m) 1.2 (m) 0.8 (t, 7) – 4.5 (dd, 8.0, 4.0) 2.0 (m) 2.2 (m) 1.7 (m) 1.9 (m) 1.3 (m) 0.8 (t, 7.0)
– 70.6
7.6 (br s) 6.8 (br s) 6.0 (br s)
– – –
4.2 (d, 7.5) 3.5 (dd, 9.7, 7.5) 3.5 (dd, 9.7, 3.3) 3.8 (dd, 3.3, 0.8) 3.5 (m) 3.7 (dd, 11.4, 5.2) 3.7 (dd, 11.4, 6.9) 6.3 (br s)
105.0 73.0 73.0 70.0 70.0 62.0
H
C
51.8 76.0 72.5 34.0 26.2 27.9 130.6 129.6 27.7 29.9–30.3 32.4 23.2 14.6 175.9 72.5 35.8 26.4 23.1–32.0 14.6
Recently, a new cerebroside possessing a galactose unity as polar head was isolated and identified from E. platyphyllos L. [11].
Fig. 2. Selected 1H–1H COSY (bold lines) and Selected HMBC (full-line arrows) Correlations of 1.
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Fig. 3. LCB-DMDS Derivatives of 1, 2.
The fatty acid methyl ester was identified by GC/MS as methyl 2-hydroxypentacosanoate. The MS spectrum of 1 showed a molecular ion [M + H]+ peak at m/z 858 and fragment ion at m/z 696 [858 - Glc]. 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]25 D -11.0°) [12–14]. 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 LCB-1 was treated with dimethyl disulfide (DMDS) and I2 and the product subjected to electron impact (EI)-MS analysis [15]. 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 at C-8-C-9 in the long-chain base (Fig. 3). The double bond in 1 was determined to be cis (Z) by the upfield shifted carbon chemical shifts of C-7 (δ 27.8) and C-10 (δ 27.7) [14] and the relative small coupling constant of H-8 at δ 5.4 (dt, J = 9.6, 6.3 Hz) and H-9 at δ 5.4 (dt, J = 9.6, 6.4 Hz) in 1a. Therefore 1 was characterized as (2S, 3S, 4R, 8Z)-1-O(β- D -galactopyranosyl)-2N-[(2′R)-2′-hydroxypentacosanoilamino]-8 (Z)-octadecene-1,3,4-triol (1). Cerebroside 2 showed strong hydroxy (3414 cm− 1) and amide (1645, 1543 cm− 1) absorptions in the FT-IR spectrum. The positive FAB mass spectrum of cerebroside 2 exhibited (M + H)+ ion peak at m/z 816 (2). The 1H and 13C-NMR spectra of cerebroside 2 exhibited the characteristic signals of a sphingosine-type cerebroside possessing 2-hydroxy fatty acid and β-galactopyranose moieties (Fig. 1, Table 1). When cerebroside 2 was methanolyzed with methanolic hydrochloric acid, fatty acid methyl ester (FAM) was obtained together with long-chain base (LCB) and methyl galactopyranoside. Comparison of the 1H-NMR and 13C-NMR data of 2 (Table 1) with those of 1 indicated that the major difference was in the length of the FAM. On the basis of mass spectrometry analysis, the FAM was characterized as methyl 2-hydroxy docosanoate (FAM-2). The stereochemistry of the ceramide moiety was determined by comparison of the 1H-NMR data of the cerebrosides isolated from E. peplis with that of synthetic analogs as reported in literature in terms of the signals due to 1-H to 4-H [16]. The absolute configuration of the galactopyranose moiety in 2 was determined to be the D-form using the Hara method [7]. Close similarity of the spectral data with those of 1 described above suggested that 2 is a higher homologue of 1.
Thus, the structure of 2 was proposed to be 1-O-(β-Dgalactopyranosyl)-(2S, 3S, 4R, 8Z)-2-[(2′R)-2′-hydroxydocosanoilamino]-8 (Z)-octadecene-1,3,4-triol (2). Euphorbia peplis accumulates complex sphingoglycolipids in which dihydroxy [17] and trihydroxy (1, 2) long-chain bases (LCBs) occur as part of the cerebroside moiety. Compounds 1 and 2 are new, since they have been isolated for the first time in Euphorbiaceae; analogous cerebrosides with a glucose moiety as head group, instead of galactose, have been isolated from Euphorbia sororia [18] and Euphorbia nicaeensis All. [17]. The antiproliferative activity of compounds 1 and 2 was tested in vitro against two tumor cell lines (human KB and IMR-32) and expressed as IC50 values. IC50 is the concentration (µM) required to inhibit tumor cell proliferation by 50% after 72 h exposure of the cells to a tested compound. The measured IC50 values for compounds 1 and 2 are summarized in Table 2. Cisplatinum was used as a reference compound [19]. KB and IMR-32 cells were exposed at 1.25, 2.50, 5.00 and 10 µg mL− 1 solutions of each compounds. After 72 h of treatment, the cerebrosides 1 and 2 exhibited growth inhibition in both cell lines with IC50 values ranging from 4.63 to 10.09 µM. 3. Experimental 3.1. Chemistry 3.1.1. General experimental procedures Optical rotations were determined on a Jasco P-1020 polarimeter (Jasco, Italy). Elementary analyses were carried out on a Carlo Erba model 1016 analyzer. 1H and 13C NMR spectra were recorded on a Varian Unity spectrometer operating at 400 MHz (1H) and 100 MHz (13C), respectively. The chemical shift values were reported in parts per million units and the coupling constants were in Hz. FAB MS was recorded on Kratos MS 80 RFA mass spectrometer using a beam of Argon/Xenon (2–8 Kv), methanol as solvent and glycerol as the matrix. IR spectra were recorded on a Table 2 IC50 values for compounds 1 and 2 against KB and IMR-32 cell lines. Compound
KB IC50 (µM)
IMR-32 IC50 (µM)
1 2 Cisplatinum
4.63 7.20 0.37
10.09 6.61 0.5
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Jasco IR-700 infrared spectrophotometer. Silica gel column chromatography was carried out using silica gel Kieselgel 60 (230–400 Mesh, 60 Å Merck), reversed-phase chromatography was carried out using Lichroprep RP-18 (40–63 µm, Merck). GC–MS experiments were carried out on Fisons GC 8000 (Carlo Erba, 20090 Milan, Italy) instrument, equipped with a split/splitless injector (1:20); the capillary gas chromatograph system was coupled directly to a MD 800 mass spectrometer (Carlo Erba, 20090 Milan, Italy). A SPB 5 fused silica capillary column, 30 m × 0.32 mm I.D., 0.25 µm film thickness (Supelco, Inc., Bellefonte, PA) was employed. Sugar standards were purchased from Fluka/Aldrich/Sigma (Milano, Italy). All solvents were distilled before use. TLC were carried out using Kieselgel 60 F254 (20 × 20 cm; 0.2 mm, Merck) and RP-18 F254 (10 × 10 cm, Merck); spots were detected by spraying with 10% aq. H2SO4 followed by heating. 3.1.2. Plant material E. peplis was collected from wild stock growing in Carso Triestino (Trieste), Italy in May 2007. A voucher specimen was deposited at the Herbarium of the Department of Biology (TSB) of the University of Trieste (Italy) (TSB-51803). 3.1.3. Extraction and isolation Dried, ground whole plants of E. peplis (500 g) were extracted with MeOH and evaporated to a crude residue (25.4 g), which was fractionated on a silica gel column with CHCl3 and MeOH, respectively. The fraction eluted with MeOH was subjected to silica gel column chromatography using mixtures of CHCl3/MeOH (5:1) and (5:2), respectively, yielding four subfractions (A–D). Subfraction C, eluted with CHCl3/MeOH (5:1.5), was further chromatographed on RP-18 (MeOH) affording 1 (34.1 mg) and 2 (54.4 mg). ð2S; 3S; 4R; 8ZÞ‐1‐O‐ðβ‐D‐galactopyranosylÞ‐2N ‐½ð2′RÞ‐2′‐hydroxy‐19′ðZÞ‐pentacosanoilamino‐8ðZÞ ‐octadecene‐1; 3; 4‐triol ð1Þ Nujol A colorless solid. [α]24 D + 11.3° (c 0.2, pyridine). FT-IR νmax cm : 3317 (broad), 2920, 2850, 1590, 1074, 1036. Positiveion FAB MS: m/z = 880 [M + Na, 78%]+, 858 [M + H, 20%]+, 840 [M + H − H2O, 12%]+, 719 [M + Na − C6H11O5 + H, 24%]+, 696 [M + H − C6H11O5, 100%]+, 678 [M + H − Glc, 80%]+, 500 [M + Na − C25H49O2 + H, 100%]+, 476 [M + H − C25H50O2, 10%]+. Negative-ion FAB MS: m/z = 856 [M − H, 100%]−, 694 [M − H − C6H11O5, 20%]−, 677 [M − H − Glc, 18%]−. 1H- and 13 C-NMR data are reported in Table 1. Anal. calcd. for C49H95O10N: C, 68.57; H, 11.08; N, 1.63. Found: C, 68.93; H, 10.99; N, 1.71. −1
ð2S; 3S; 4R; 8ZÞ‐1‐O‐ðβ‐D‐galactopyranosylÞ‐2N ‐½ð2′RÞ‐2′‐hydroxydocosanoilamino‐8ðZÞ‐octadecene ‐1; 3; 4‐triol ð2Þ Amorphous powder. [α]24 D + 4.8° (c 0.2, pyridine). FT-IR cm− 1: 3413, 1646, 1540. Positive-ion FAB MS: m/z = 816 [M + H, 83%]+, 798 [M + H − H2O, 35%]+, 654 [M + H − C6H11O5, 95%]+, 636 [M + H − Glc, 40%]+, 476 [M + H − C22H43O2, 10%]+, 500 [M + Na − C22H43O2, 100%]+, Negativeion FAB MS: m/z = 814 [M − H, 100%], 652 [M − H − C6H11O5, 23%]−. ESI-MS: m/z = 838 [M + Na, 50%]+, 658 [M + Na − Glc, 40%]+. 1H- and 13C-NMR data are reported in Table 1. νKBr max
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Anal. calcd. for C46H89O10N: C, 67.69; H, 10.91; N, 1.72. Found: C, 67.96; H, 10.03; N, 1.84. 3.1.4. Methanolysis of 1, 2 Compound 1 (34.1 mg) was refluxed with 0.9 M HCl in 82% aq. MeOH (10 mL) for 18 h. The mixture was extracted with n-hexane and the combined organic phases were washed with water and dried over Na2SO4. Removal of the solvent gave a colorless wax (18.0 mg) which was chromatographed on silica gel [hexane/EtOAc (20:1, 5:1)] to yield fatty acid methyl ester as a colorless wax (11.5 mg). The compound 2 was methanolyzed using the same method described above. The esters were analyzed by GC–MS. The results were as follows: FAM-1 (2-hydroxypentacosanoic acid methyl ester), EI-MS: m/z= 413 [M]+, 381 [M - CH3OH]+, 354 [M - CH3COO]+; FAM-2 (2-hydroxydocosanoic acid methyl ester), EI-MS: m/z= 370 [M]+, 338 [M - CH3OH]+, 311 [M CH3COO]+. The aq. MeOH layer was neutralized with NH4OH and extracted with EtOAc. The combined EtOAc extract was washed with H2O, dried over Na2SO4 and evaporated to give the longchain base (LCB) as a slightly yellow wax (15.6 mg). The aq. MeOH layer was then evaporated to dryness and chromatographed on silica gel [CH2Cl2/MeOH/H2O (lower layer) (20:3:1, 10:3:1, 7:3:1)] to give methyl galactopyranoside (α- and β-anomer) as a colorless solid (4.2 mg). TLC [silica gel, CH2Cl2/MeOH/H2O (lower layer) (10:3:1)] of the resulting methyl galactopyranoside (α- and β-anomer) was identical to that of the standard methyl α-D-galactopyranoside and methyl β-D-galactopyranoside. 3.1.5. GC–MS analysis of TMS ethers of methyl glycosides from 1, 2 The mixture of methyl glycosides obtained by column chromatography of the aq. MeOH layer derived from methanolysis of 1 and 2 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). Methyl glycosides (GC–MS): methyl α- and β-galactopyranosides were detected. Methyl α-Dgalactopyranoside: C7H14O6, m/z = 194 [M]+, TMS derivative, m/z = 482 [M]+, 204, 191, 217, tR (min): 13.188. Methyl β-Dgalactopyranoside: C7H14O6, m/z = 194 [M]+, TMS derivative, m/z = 482 [M]+, 204, 191, 217, tR (min): 14.307. Methyl α-Dglucopyranoside: C7H14O6, m/z = 194 [M]+, TMS derivative, m/z = 482 [M]+, 204, 191, 217, tR (min): 12.770. Methyl β-Dglucopyranoside: C7H14O6, m/z = 194 [M]+, TMS derivative, m/z = 482 [M]+, 204, 191, 217, tR (min): 14.008. 3.1.6. Dimethyl disulfide derivatives of LCBs from cerebrosides 1 and 2 LCB-1 (10 mg) was dissolved in carbon disulfide (1 mL) and dimethyl disulfide (1 mL) and iodine (20 mg) were
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added. The reaction mixture was then kept at 60 °C for 48 h in a small sealed vial. The reaction was subsequently quenched with 5% aq. Na2S2O3 and the mixture was extracted with n-hexane. The n-hexane layer was dried over Na2SO4, filtered and concentrated to give the dimethyl disulfide derivative of LCB-1 as a light yellow oil (9.3 mg). The LCBs DMDS derivatives of cerebrosides 1 and 2 were subjected to MS analysis. The results were as follows: LCB-1 DMDS derivative, EI-MS: m/z = 221, 187. LCB-2 DMDS derivative, EI-MS: m/z = 221, 187. 4. Pharmacology 4.1. Cells and cytotoxic assays 4.1.1. Cell cultures KB, human oral epidermoid carcinoma cell line (ECACC no. 86103004), and IMR-32, human adenocarcinoma cell line (ECACC no. 86041809) were cultured according to standard procedure [19]. Vials of the original line were maintained in liquid N2; cells were obtained, routinely subcultured once a week, and used for the reported experiment. The cell lines were maintained in Eagle's minimum essential medium (MEM) [20] supplemented with 10% newborn calf serum (Hyclone) for KB and fetal calf serum (Euroclone) for IMR-32, with 10 mL− 1 penicillin and streptomycin solution (Sigma Chemical Co., St. Louis, MO) (100 U mL− 1 penicillin G and 100 µg mL− 1 streptomycin) and buffered with 3 mM tris [hydroxymethyl]methyl-2-aminoethane sulfonic acid, 3 nM N,N-bis [2-hydroxyethyl]-2-aminoethanesulfonic acid, 3 mM N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid and 3 mM Tricine (Sigma Chemical Co.). The cell population doubling time was ca. 24 and 20 h for KB and IMR-32, respectively. Cells from confluent monolayers were removed with 2–3 mL of 0.05% (KB cells) or 0.25% (IMR-32 cells) trypsin solution (Sigma Chemical Co.). 4.2. Growth inhibition of KB and IMR-32 cells derivatives For the valuation of cytostatic activity, KB and IMR-32 cells were sown at a density of 2.5×104 cells per mL, in 0.2 mL per well in a 96-well plate (Corning Costar, Milano, Italy). After 24 h, derivatives were dissolved in sterile DMSO and solutions diluted in culture medium up to obtained opportune concentration (1.25, 2.50, 5.00 and 10.00 µg mL− 1); nutritive medium of every well was substituted with 0.2 mL of solution. After 72 h incubation at 37 °C, cellular vitality was evaluated with a colorimetric assay based on the quantification with sulforhodamine B (SRB – Sigma Chemical Co.) of cellular protein component [21]. Briefly, adherent cell cultures were fixed in situ by addition of 50 µl of cold 50% (v/v) trichloroacetic acid (TCA) and were kept for 60 min at 4 °C. The supernatant was then discarded and the plates were washed two times with bi-distilled water and air-dried. SRB solution (0.4% w/v in 1% acetic acid) was added and the cells were allowed to stain for 30 min at room temperature. Unbound SRB was removed by washing three times with 1% acetic acid. Then the plates were air-dried. Bound stain was dissolved with unbuffered 10 mM Tris base (tris-hydroxymethylaminomethane) (Sigma Chemical Co.) and the optical density was read at 570 nm with an automated microplate reader
EL311s spectrophotometer (BIO-TEK Instruments, INC. Winooski, Vermount, USA). Each experiment was performed in quintuplicate and repeated twice. Cytostatic activity was evaluated as percentage of cellular growth inhibition in culture treated with compounds to respect to the growth observed in control culture. IC50 and parallelism test were performed with the aim of PCS program [22]. 4.3. Statistical analysis Data were analyzed using Student's t-test. Significance was accepted with P < 0.05. Values of IC50 were obtained with PCS program [22]. Acknowledgements The authors are grateful to Dr. Vito Scarcia, Department of Biomedical Sciences, University of Trieste, Italy for the in vitro cytostatic activity assays. References [1] Aylward JH, Gordon PP. Diterpenes obtained from Euphorbiaceae for the treatment of prostate cancer. Au. Pat. WO 2002011743; 2002. [2] Aylward JH, Gordon PP, Suhrbier A, Turner KA. Euphorbiaceae macrocyclic diterpenes for the treatment of inflammation. Au. Pat. WO 2001093885; 2001. [3] Aylward JH, Gordon PP, Suhrbier A, Turner KA. Macrocyclic diterpenes for treatment and prophylaxix of PKC-related conditions. Au. Pat. WO 2001093884; 2001. [4] Ivanova A, Khozin-Goldberg I, Kamenarska Z, Nechev J, Cohen Z, Popov S, et al. Lipophilic compounds from Euphorbia peplis L. — a halophytic plant from the Bulgarian Black Sea coast. Z Naturforsch C: J Biosci 2003;58: 783–8. [5] Cateni F, Falsone G, Zilic J. Terpenoids and glycolipids from Euphorbiaceae. Mini Rev Med Chem 2003;3:425–37. [6] Cateni F, Zilic J, Falsone G, Scialino G, Banfi E. New cerebrosides from Euphorbia peplis L.: antimicrobial activity evaluation. Bioorg Med Chem Lett 2003;13:4345–50. [7] Hara S, Okabe H, Mihashi K. Gas–liquid chromatographic separation of aldose enantiomers as trimethylsilyl ethers of methyl 2-(polyhydroxyalkyl)-thiazolidine-4(R)-carboxylates. Chem Pharm Bull 1987;35: 501–6. [8] Yoda H, Oguchi T, Takabe K. An expeditious and pratical synthetic process for phytosphingosine and tetrahydroxy-LCB from D-glutamic acid. Tetrahedron Asymmetry 1996;7:2113–6. [9] Gaver RC, Sweely CC. Methods for methanolysis of sphingolipids and direct determination of long-chain bases by gas chromatography. J Am Oil Chem Soc 1965;42:294–8. [10] Medeiras PM, Simoneit BRT. Analysis of sugars in environmental samples by gas chromatography–mass spectrometry. J Cromatogr A 2007;1141:271–8. [11] Cateni F, Zilic J, Zacchigna M. Isolation and structure elucidation of cerebrosides from Euphorbia Platyphyllos L. Sci Pharm 2008;76:451–69. [12] Higuchi R, Natori T, Komori T. Biologically active glycosides from Asteroidea, XX. Glycosphingolipids from the starfish Asterina pectinifera, 1. Isolation and characterization of acanthacerebrosides B and structure elucidation of related, nearly homogeneous cerebrosides. Liebigs Ann Chem 1990:51–5. [13] Shibuya H, Kawashima K, Sakagami M, Kawanishi H, Shimamura M, Ohashi K, et al. Sphingolipids and glycerolipids. I. Chemical structure and ionophoretic activities of soyacerebrosides I and II from soybean. Chem Pharm Bull 1990;38:2933–8. [14] Kang SS, Kim JS, Son KH, Kim HP, Chang HW. Cyclooxygenase-2 inhibitory cerebrosides from Phytolaccae radix. Chem Pharm Bull 2001;49:321–3. [15] Scribe P, Guezennect J, Dagaut J, Pepe C, Saliot A. Identification of the position and the stereochemistry of the double bond in monounsaturated fatty acid methyl esters by gas chromatography/mass spectrometry of dimethyl disulfide derivatives. Anal Chem 1988;60:928–31. [16] Cateni F, Zacchigna M, Zilic J, Di Luca G. Total synthesis of a natural cerebroside from Euphorbiaceae. Helv Chim Acta 2007;90:282–9.
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