Journal of Pharmaceutical and Biomedical Analysis 70 (2012) 13–25 Contents lists available at SciVerse ScienceDirect Journal of Pharmaceutical and Biomedical Analysis journal homepage: www.elsevier.com/locate/jpba Bioactive alkaloid extracts from Narcissus broussonetii: Mass spectral studies Jean Paulo de Andrade a , Natalia Belén Pigni a , Laura Torras-Claveria a , Strahil Berkov a,b , Carles Codina a , Francesc Viladomat a , Jaume Bastida a,∗ a b Department of Natural Products, Plant Biology and Soil Science, Faculty of Pharmacy, Av. Diagonal 643, 08028 Barcelona, Spain AgroBioInstitute, 8 Dragan Tzankov Blvd., Sofia 1164, Bulgaria a r t i c l e i n f o Article history: Received 15 February 2012 Received in revised form 21 April 2012 Accepted 4 May 2012 Available online 14 May 2012 Keywords: Amaryllidaceae Narcissus Dinitrogenous alkaloids Pretazettine Antiprotozoal studies a b s t r a c t Plants of the Amaryllidaceae family are a well-known source of tetrahydroisoquinoline alkaloids with a wide range of biological activities, including antiviral, antitumoral, antiparasitic, psychopharmacological, and acetylcholinesterase inhibitory, among others. Recent advances in the use of GC or LC coupled to MS have allowed a chemically guided isolation of uncommon and bioactive alkaloids. In the present work, analytical methods were applied to study the alkaloid profile of Narcissus broussonetii, a plant endemic to North Africa. Using the GC–MS technique and an in-home mass fragmentation database, twentythree alkaloids were identified, including the very rare dinitrogenous alkaloids obliquine, plicamine, and secoplicamine. Applying LC–ESI-LTQ-Orbitrap-MS, fragmentation profiles were found to be similar for obliquine and plicamine but different for secoplicamine. Pretazettine, a potent cytotoxic alkaloid, was also isolated from N. broussonetii, although its identification by GC–MS was only possible after a BSTFAderivatization. The silylated crude methanolic extract only showed the presence of pretazettine–TMS, confirming that tazettine was formed after the alkaloid extraction. The same observation was made in Narcissus cultivars in which tazettine had been detected as the major alkaloid. As part of an ongoing project on MS of Amaryllidaceae alkaloids, the silylated tazettine and pretazettine were studied by GC–MS/MS, and found to differ in their fragmentation routes. Finally, the EtOAc extract of N. broussonetii showed notable in vitro activity against Trypanosoma cruzi, with an IC50 value of 1.77 ␮g/ml. © 2012 Elsevier B.V. All rights reserved. 1. Introduction Narcissus is the most common genus of the Amaryllidaceae family in the Iberian Peninsula and North Africa, comprising 80–100 wild species. The alkaloids found in Amaryllidaceae species possess putative pharmacological properties such as antiprotozoal, antiviral, antitumoral, and acetylcholinesterase inhibitory activity [1–3]. The well-known Amaryllidaceae alkaloid, galanthamine, is a marketed drug for Alzheimer’s disease therapy (Razadyne® , formerly Reminyl® ). Alkaloids like narciclasine, pretazettine and others bearing haemanthamine- and lycorine-type skeletons have demonstrated interesting antitumoral and/or apoptotic effects [4–6]. Some compounds of the lycorine series have shown interactions with the human cytochrome P450 3A4 [7]. From a biosynthetic point of view, the Amaryllidaceae alkaloids in the genus Narcissus are grouped in eight skeleton types formed from the common precursor O-methylnorbelladine [2]. The development of metabolite profiling methods using capillary electrophoresis, gas chromatography (GC), gas chromatography coupled to mass spectrometry (GC–MS) or ∗ Corresponding author. Tel.: +34 934020268; fax: +34 934029043. E-mail address: jaumebastida@ub.edu (J. Bastida). 0731-7085/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.jpba.2012.05.009 high-performance liquid chromatography (HPLC) has allowed the identification and quantification of many Amaryllidaceae alkaloids [8–11]. In biological samples, liquid chromatography–electrospray ionization–tandem mass spectrometry (LC–ESI-MS/MS) has been successfully used to quantify alkaloids bearing the galanthaminetype skeleton [12]. It has been found that Amaryllidaceae alkaloids, with a few exceptions, can be analyzed by GC–MS without any previous derivatization, and they show a mass spectral fragmentation pattern very similar to those recorded by direct insertion probe [13]. Furthermore, the high resolution ability of the capillary column allows the separation of more than 60–70 compounds from complex mixtures, while the identification of known alkaloids is achieved through the specific mass fragmentation by electron impact mass spectrometry (EI-MS) and retention indices using deconvolution software [14]. These features facilitate the development of an in-home spectral database of known compounds, allowing their rapid identification and the isolation of compounds showing unusual EI-MS fragmentations. Nevertheless, a drawback of GC–MS is the lack of information about some unstable compounds (or those in N-oxide form) together with the low stability of several molecular ions, as found in homolycorine-type alkaloids. Compounds such as haemanthamine may undergo thermal degradation and hence show different mass fragmentation under GC conditions [13]. 14 J.P. de Andrade et al. / Journal of Pharmaceutical and Biomedical Analysis 70 (2012) 13–25 The growing interest in Amaryllidaceae alkaloids after the therapeutic success of galanthamine has also prompted the study of these compounds using metabolomic approaches. GC–MS analysis was performed to investigate the alkaloid profile of N. broussonetii, a plant species with remarkable in vitro anti-Trypanosoma cruzi activity. Since the dinitrogenous alkaloids obliquine, plicamine, and secoplicamine were not very well detected by GC–MS, they were also analyzed by LC–ESI-MS/MS. The sole presence of pretazettine rather than tazettine in the methanolic macerates was confirmed by previous N,O-bis(trimethylsilyl)trifluoroacetamide (BSTFA) derivatization and also corroborated in Narcissus cultivars in which tazettine had been detected as the major alkaloid. The GC–MS/MS spectra of silylated tazettine and pretazettine are also discussed. 2. Experimental 2.1. Used chemicals and standards Methanol (MeOH), diethyl ether (Et2 O), n-hexane (n-Hex), ethyl acetate (EtOAc), chloroform (CHCl3 ), acetone (Me)2 CO, sulfuric acid (H2 SO4 ) and ammonia (NH3 ) of analytical grade were used for the extraction and isolation procedure, being purchased from SDS (France). MeOH, CHCl3 , and acetic acid (MeCOOH) of HPLC grade were used in GC–MS (/MS) and HPLC procedures, being also purchased from SDS (France). Deuterated methanol (CD3 OD) and deuterated chloroform (CDCl3 ) with trimethylsilane (TMS) as the internal standard were used for recording nuclear magnetic resonance (NMR) spectra. Tazettine and pretazettine isolated from N. broussonetii were used as positive standards for derivatization and/or GC–MS (/MS) study. The hydrocarbon mixture (C9–C36, Restek, Cat no. 31614) was supplied by Teknokroma (Spain). The solutions of BSTFA and pyridine were purchased from Sigma–Aldrich (St. Louis, MO, USA). 2.2. General experimental procedures Nuclear magnetic resonance (NMR) spectra were recorded in a Varian Gemini 300 MHz or Varian VNMRS 400 MHz spectrometer. Chemical shifts were reported in ı units (ppm) and coupling constants (J) were expressed in Hz. A Jasco-J-810 Spectrophotometer was used to run CD spectra, all recorded in MeOH. Silica gel SDS chromagel 60 A CC (6–35 ␮m) was used for vacuum liquid chromatography (VLC), and silica gel 60 F254 SDS for analytical and semi-preparative thin layer chromatography (TLC). Spots on chromatograms were detected under UV light (254 nm) and with Dragendorff’s reagent. 2.3. Plant material N. broussonetii Lag. was collected on Wad Mouzeg dar Bouaza beach, near Casablanca (Morocco), in October 2008. A voucher specimen was deposited at the University of Barcelona Herbarium (BCN 58745). Narcissus cultivars ‘Toto’ and ‘Pencrebar’ were obtained from Ludwig Ltd. (The Netherlands). (25%, v/v) and extracted with n-Hex (5× 500 ml) to give the n-Hex extract (274 mg), followed by extraction with EtOAc (7× 500 ml) to provide the EtOAc extract (1.2 g). Finally, the basic solution was extracted with an EtOAc–MeOH mixture (3:1) but no alkaloids were detected. A rapid alkaloid extraction was performed using 50 mg of dried bulbs from Narcissus cultivars ‘Toto’ and ‘Pencrebar’ in screw-top 1.5 ml Eppendorf tubes (6 tubes for each cultivar). The maceration procedure was carried out with 1 ml of MeOH adjusted to pH 8 with NH3 (25%, v/v). After 2 h of extraction at room temperature assisted by 15 min ultrasonic baths every 30 min, the samples were centrifuged at 10,000 rpm for 2 min. Three tubes of each cultivar were used for alkaloid extraction as follows: 500 ␮l aliquots of methanolic macerate were acidified with 750 ␮l of H2 SO4 (2%, v/v) and the neutral material was removed with CHCl3 (3× 700 ␮l). The aqueous fraction was then basified with 250 ␮l of NH3 (25%, v/v) and the alkaloids were extracted with CHCl3 (3× 700 ␮l). Finally, the purified alkaloid extract was dried under N2 and re-dissolved in 300 ␮l of CHCl3 for GC–MS analysis. The alkaloid extraction of Narcissus cultivars was carried out to confirm the presence of tazettine by GC–MS. The other three glass tubes for each cultivar were reserved for the derivatization process, transferring aliquots of 500 ␮l to vials to be dried under N2 . The derivatization method is explained below in Section 2.6. 2.5. GC–MS The EI-MS spectra were obtained on an Agilent 6890N GC 5975 inert MSD operating in EI mode at 70 eV (Agilent Technologies, Santa Clara, CA, USA). A DB-5 MS column (30 m × 0.25 mm × 0.25 ␮m, Agilent Technologies) was used. The temperature program was: 100–180 ◦ C at 15 ◦ C min−1 , 1 min hold at 180 ◦ C and 180–300 ◦ C at 5 ◦ C min−1 and 40 min hold at 300 ◦ C. The injector temperature was 280 ◦ C. The flow rate of carrier gas (helium) was 0.8 ml min−1 . The split ratio was 1:20 for the analysis of the N. broussonetii extracts, 1:10 for extracts from the Narcissus cultivars ‘Toto’ and ‘Pencrebar’, and 1:5 for isolated compounds. 2.6. Derivatization method Five mg of the EtOAc extract from N. broussonetii was dissolved in 150 ␮l of pyridine and derivatized with 150 ␮l of BSTFA for 2 h at 70 ◦ C. After cooling, 300 ␮l of CHCl3 was added and the samples were analyzed by GC–MS. Derivatization of the crude extracts from Narcissus ‘Toto’ and ‘Pencrebar’ was also carried out using the same method and quantities. For the GC–MS/MS study, one mg of pretazettine and tazettine were each dissolved in 200 ␮l of pyridine and derivatized at 70 ◦ C with 200 ␮l of BSTFA for 2 h. The derivatized solution was diluted up to 1 ml with CHCl3 and 100 ␮l of diluted solution was evaporated to dryness with N2 and dissolved in 300 ␮l of CHCl3 before GC–MS/MS injection. 2.4. Extraction procedure 2.7. GC–MS/MS Fresh bulbs (2 kg) of N. broussonetii were macerated thoroughly with MeOH at room temperature for 48 h (4× 2.0 l), then the combined macerate was filtered and the solvent evaporated to dryness under reduced pressure. The bulb crude extract (70.7 g) was acidified to pH 3 with dilute H2 SO4 (2%, v/v) and the neutral material was removed using Et2 O (4× 500 ml). EtOAc (4× 500 ml) was used to carry out a first alkaloid extraction in acid media but with negative results. The aqueous solution was basified up to pH 9–10 with NH3 GC–MS/MS spectra were obtained on a Thermo Scientific Trace GC Ultra operating in EI mode at 70 eV coupled with an ITQ 900 Ion Trap detector (Thermo Scientific, Hemel Hempstead, United Kingdom). An HP-5 MS column (30 m × 0.25 mm × 0.25 ␮m, Agilent Technologies) was used. The temperature program was: 180–300 ◦ C at 5 ◦ C min−1 , 1 min hold at 180 ◦ C and 5 min hold at 300 ◦ C. The flow rate of carrier gas (helium) was 0.8 ml min−1 . The analyses were carried out in splitless mode. Nineteen alkaloids were obtained during the phytochemical isolation procedure. Homolycorine (4, 25 mg) and lycorine (1, 124 mg) 2.75 0.19 Traces Traces 0.92 8.32 – 0.10 Traces 20.30 Traces 55.61 1.15 9.01 1.18 Traces – – – ap.100 % in EtOAc % in n-Hex MS data 238(100), 211(6), 196(8), 168(6), 154(3), 106(4), 77(3) 222(38), 167(8), 165(9), 164(14), 138(20), 137(9), 111(13) 300(3), 191(8), 110(9), 109(100), 108(15), 94(3), 82(2), 42(2) 250(100), 192(13), 191(11), 165(4), 164(3), 139(2), 124(7) 302(20), 301(100), 286(33), 270(34), 246(23), 231(73), 217(19), 123(22) 280(7), 264(13), 263(17), 262(20), 252(15), 238(0.5), 204(7), 191(14), 132(8), 107(6) 268(15), 244(32), 215(100), 203(56), 189(22), 128(23), 115(26), 71(11), 56(20) 300(15), 260(5), 231(100), 227(10), 211(15), 197(10), 152(8), 115(9), 141(8) 299(3), 191(1), 179(1), 110(9), 109(100), 108(17), 94 (2), 82(2), 44(4) 316(15), 298(23), 247(100), 230(12), 201(15), 181(11), 152(7) 284(52), 233(48), 211(45), 201(80), 199(70), 181(69), 173(71), 115(100), 56(71) 206(<1), 178(2), 109(100), 150(1), 108(22), 94(3), 82(3) 286(19), 268(24), 250(15), 227(79), 226(100), 211(7), 147(15) 192(<1), 164(2), 110(8), 109(100), 108(23), 94(3), 82(3) 314(23), 245(100), 225(14), 201(83), 139(16), 70(18) 328(20), 270(34), 269(57), 268(83), 252(35), 251(33), 250(100), 226(55), 43(36) 342(22), 341(100), 327(4), 270(6), 258(7), 242(4), 212(2), 121(3), 107(4), 77(4) 432(100), 379(38), 348(23), 272(29), 253(22), 228(48), 216(39), 121(13), 107(13), 77(12) 355(34), 344(19), 343(100), 258(5), 254(4), 226(5), 139(2), 120(5), 107(5), 77(3) 257 (35) 223 (100) 331 (–) 251 (43) 317 (–) 281 (100) 287 (97) 315 (21) 317 (–) 331 (31) 317 (59) 315 (–) 287 (31) 301 (–) 329 (27) 329 (16) 448 (–) 464 (–) 462 (–) [M]+ R.T. a, 15 Identification: a compounds identified using in-home MS database; b NIST 05 database; recursive procedure, HR-MS and literature data. The compounds marked with * together with pretazettine (15) and homolycorine-N-oxide (8) were identified after isolation and NMR experiment. The compounds marked with ** were also isolated in the course of phytochemical procedure. Values less than 0.10 are described as “traces”. R.T.: retention time. 2.10. Isolation of alkaloids Compound The alkaloids were identified by comparing their GC–MS spectra and Kovats retention indices (RI) with our own library database. This library has been continually updated and reviewed with alkaloids repeatedly isolated by our group and identified using other spectroscopic techniques such as NMR, UV, CD and MS [2,15–21]. The alkaloids pretazettine and homolycorine-N-oxide were identified after NMR experiments since they could not be identified by GC–MS analysis. Their NMR spectral data were in agreement with those previously reported [16,22]. Additionally, the dinitrogenous alkaloids obliquine, plicamine, and secoplicamine were isolated for the first time in a Narcissus species, being therefore added to our library database after their identification by 2D NMR experiments. Their spectral data were in agreement with those previously published [17,23]. Galanthindole was identified by comparison of its 1 H NMR data and EI mass fragmentation with those previously reported [24]. Mass spectra were deconvoluted using AMDIS 2.64 software (NIST). Kovats retention indices (RI) of the compounds were recorded with a standard calibration n-hydrocarbon mixture (C9–C36) using AMDIS 2.64 software. The proportion of each individual compound in the alkaloid fractions analyzed by GC–MS (Table 1) is expressed as a percentage of the total alkaloids (TIC – total ion current). The area of the GC–MS peaks depends not only on the concentration of the corresponding compound but also on the intensity of their mass spectral fragmentation. Although data given in Table 1 do not express a real quantification, they can be used for a relative comparison of the alkaloids, which is the aim of this work. Table 1 GC–MS data for Narcissus broussonetii alkaloids. Values are expressed as a relative percentage of TIC. 2.9. Identification of alkaloids by GC–MS 19.28 20.16 22.20 22.49 22.77 22.83 23.03 23.96 24.17 25.22 25.79 26.52 26.53 27.38 27.42 28.31 47.25 49.43 54.92 The LC–MS/MS analysis of the dinitrogenous alkaloids obliquine, plicamine, and secoplicamine were performed on an Accela LC (Thermo Scientific, Hemel Hempstead, United Kingdom) system coupled with ESI-LTQ-Orbitrap–MS. The chromatographic method was optimized using a Luna C18 (2)-HST column (100 mm × 2.00 mm, 2.5 ␮m particle size; Phenomenex® , Torrance, CA, USA) at a constant solvent flow rate of 150 ␮l/min with aqueous MeCOOH (0.05%, v/v) as solvent A and MeOH (100%) as solvent B. An increasing linear gradient (v/v) of solvent B was applied (min, %B): (0, 55), (30, 65), (31, 100), (36, 55) and (46, 55). The compound obliquine and a mixture of plicamine and secoplicamine were dissolved in 100 ␮l CHCl3 and a volume of 5 ␮l was injected for each. An LTQ Orbitrap Velos mass spectrometer (Thermo Scientific, Hemel Hempstead, United Kingdom) equipped with an ESI source in positive mode was used to acquire mass spectra of obliquine, plicamine, and secoplicamine in profile mode with a setting of 15,000 resolution at m/z 400. Operating parameters were as follows: source voltage, 3.5 kV; sheath gas, 40; auxiliary gas, 10 (arbitrary units); sweep gas, 10 (arbitrary units); and capillary temperature, 275 ◦ C. The collision energy for MSn experiments (expressed as a % of 5 V) varied between 20 and 80. The isolation width (m/z) was 2 and the Activation Q was 0.25 for all MSn experiment. All the MSn experiments were done using CID activation with the exception of the MS2 spectra, which required a higher energy CID (HCD). The MS2 spectra of secoplicamine were performed using both CID activation and HCD. The mass range was from m/z 100 to 600 in FTMS experiments. Calibration was done using LTQ Velos ESI positive ion calibration solution. Data analyses were performed using XCalibur software. Ismine (19) ** Trisphaeridine (18)a, ** O-Methyllycorenine (7)a, ** Anhydrolycorine (3)b Papyramine (9)/6-epi-papyramine (10)a, ** Galanthindole (20)a, ** Maritidine (13)a 6-Deoxytazettine (17)b Lycorenine (6)a, ** Tazettine (14)a, ** Haemanthidine (11)/6-epi-haemanthidine (12)a, ** Homolycorine (4)a, ** Lycorine (1)a, ** 8-O-Demethylhomolycorine (5)a, ** 3-epi-Macronine (16)a, ** 2-O-Acetyllycorine (2)a Obliquine (21)* Secoplicamine (23)* Plicamine (22)* 2.8. LC–ESI-LTQ-Orbitrap-MS 2.55 0.27 Traces Traces Traces 6.13 0.15 Traces Traces 66.40 Traces 2.53 2.35 14.49 3.50 Traces 0.77 Traces Traces ap. 100 J.P. de Andrade et al. / Journal of Pharmaceutical and Biomedical Analysis 70 (2012) 13–25 16 J.P. de Andrade et al. / Journal of Pharmaceutical and Biomedical Analysis 70 (2012) 13–25 Fig. 1. Biogenetic pathway of the identified alkaloids in Narcissus broussonetii. * isolated alkaloids. The alkaloids are listed based in the skeleton-type [2]. classified as “Miscellaneous” due to still uncertain biogenesis. precipitated spontaneously from the n-Hex and EtOAc extracts, respectively, after re-suspension in MeOH. The n-Hex extract was subjected to VLC (3 cm × 4.5 cm) over silica gel, eluting with n-Hex, EtOAc and EtOAc–MeOH (1:1, v/v) in increasing order of polarity. Fractions of 100 ml were collected (100 in total) and combined according to their TLC profiles monitored by UV light 254 nm and Dragendorff’s reagent. From the first fractions (17–34) the alkaloids ismine (19, 4 mg), trisphaeridine (18, 2.5 mg) and again homolycorine (4, 9 mg) were isolated after purification by semi-preparative TLC using n-Hex–EtOAc (7:2, v/v). All remaining fractions from the n-Hex extract showed a similar alkaloid profile in GC–MS to those observed in the EtOAc extract and so were not purified and quantified. The EtOAc extract was subjected to VLC (5 cm × 5 cm) over silica gel, eluting with n-Hex, EtOAc and EtOAc–MeOH (1:1, v/v) in increasing order of polarity. Fractions of 200 ml were collected (150 in total) and combined according to their TLC profiles monitored by UV light 254 nm and Dragendorff’s reagent. Combined fractions were submitted to GC–MS and separately processed in three groups. From the first group (fractions 27–31), tazettine (14, 230 mg) precipitated spontaneously and obliquine (21, 7.2 mg) was isolated along with 3-epi-macronine (16, 5.4 mg) after optimization of a semi-preparative TLC using n-Hex–acetone–EtOAc (7:3:0.5, v/v/v). From the second group (fractions 32–43), plicamine (22) and secoplicamine (23), which were isolated as a mixture (7 mg), lycorenine (6, 2 mg), O-methyllycorenine (7, 1.5 mg) and tazettine (14, 40 mg) were isolated after purification using a semi-preparative TLC run with n-Hex–acetone–EtOAc (6:2:0.5, v/v/v). Lycorenine (6, 2.5 mg) and O-methyllycorenine (7, 2 mg) were purified once more by semi-preparative TLC using n-Hex–acetone–EtOAc (4:2:0.5, v/v/v). The last group (fractions 60–110, 350 mg) was submitted to a new VLC (3 cm × 4.5 cm) resulting in 100 fractions of 100 ml each. After purification by alkaloids semi-preparative TLC using EtOAc–acetone–MeOH (3:2:1, v/v/v), pretazettine (15, 7 mg), papyramine and 6-epi-papyramine (9 and 10, 5 mg), haemanthidine and 6-epi-haemanthidine (11 and 12, 3 mg) together with homolycorine-N-oxide (8, 1.5 mg) were isolated. Finally, using semi-preparative TLC optimized with an EtOAc–acetone–MeOH (3:0.5:1, v/v/v) mixture, 8-Odemethylhomolycorine (5, 5 mg) was isolated along with a trace of the alkaloid galanthindole (20, around 1 mg). 2.11. Antiprotozoal in vitro assay T. cruzi. Rat skeletal myoblasts (L-6 cells) were seeded in 96well microtiter plates at 2000 cells/well/100 ml in RPMI 1640 medium with 10% FBS and 2 mM l-glutamine. After 24 h, 5000 trypomastigotes of T. cruzi (Tulahuen strain C2C4 containing the ␤galactosidase (Lac Z) gene) were added in 100 ml per well with 2× of a serial drug dilution. The plates were incubated at 37 ◦ C in 5% CO2 for 4 days. For measurement of the IC50 , the substrate CPRG/Nonidet was added to the wells. The color reaction that developed during the following 2–4 h was read photometrically at 540 nm. IC50 values were calculated from the sigmoidal inhibition curve. Trypanosoma brucei rhodesiense. Serial drug dilutions in supplemented Minimum Essential Medium were added to microtiter plates. Trypomastigotes of T. brucei rhodesiense STIB 900 were added to each well and the plates were incubated for 72 h. Viability was assessed by Alamar Blue and read in a fluorescence scanner (Millipore Cytofluor 2300). Fluorescence development was expressed as a percentage of the control, and IC50 values determined. Leishmania donovani. Mouse peritoneal macrophages were seeded in RPMI 1640 medium with 10% heat-inactivated FBS into Lab-tek 16-chamber slides. After 24 h, L. donovani amastigote were added and the medium containing free amastigotes was replaced J.P. de Andrade et al. / Journal of Pharmaceutical and Biomedical Analysis 70 (2012) 13–25 17 Fig. 2. Tentative fragmentation of obliquine (21). by fresh medium 4 h later. The following day, the medium was replaced by fresh medium containing different drug concentrations. The slides were incubated at 37 ◦ C under a 5% CO2 atmosphere for 96 h. The ratio of infected to non-infected macrophages was determined microscopically, expressed as a percentage of the control and the IC50 value calculated by linear regression. Plasmodium falciparum. Antiplasmodial activity was determined using NF54 (sensitive to all known drugs) and K1 (resistant to chloroquine and pyrimethamine) strains of P. falciparum. Briefly, infected human red blood cells were exposed to serial drug dilutions in microtiter plates for 48 h. Viability was assessed by measuring the incorporation of [3H]-hypoxanthine by liquid scintillation counting. IC50 values were calculated from the sigmoidal inhibition curves. The EtOAc extract and the isolated alkaloids lycorine (1), tazettine (14), homolycorine (4), 8-O-demethylhomolycorine (5), O-methyllycorenine (7), papyramine/6-epi-papyramine (9 and 10) and obliquine (21) were evaluated against these protozoal parasites following the methodology mentioned above and published in previous work [25–27]. 3. Results and discussion 3.1. GC–MS results The n-Hex and EtOAc fractions showed compounds with EI-MS fragmentation patterns characteristic of Amaryllidaceae alkaloids. The GC–MS data from the n-Hex and EtOAc extracts are presented in Table 1 and the alkaloids are shown in Fig. 1. Lycorine (1), tazettine (14), homolycorine (4), and to a lesser extent, 8-O-demethylhomolycorine (5) and galanthindole (20) were the main alkaloids observed in N. broussonetii. Tazettine (14) was the main alkaloid observed by GC–MS, isolated in a higher amount than any other alkaloid, although tazettine is in fact an artifact from pretazettine. Pretazettine showed no EI mass fragmentation under GC–MS conditions due to its rapid conversion to tazettine in the chromatographic column. A previous BSTFA-derivatization carried out in the present work was able to avoid this conversion (Section 3.3). Another notable divergence was observed with lycorine (1), which appeared at only 2.35% in the EtOAc extract analyzed by GC–MS due to its precipitation before injection into the chromatographic column. It should be emphasized that the quantities of each isolated alkaloid only refer to the amounts obtained, purified and quantified (Section 2.10). Not all the fractions were studied: those from the n-Hex extract, which was found to contain known alkaloids by GC–MS, were not quantified. A list of the alkaloids from N. broussonetii observed by GC–MS analysis is included in Table 1. However, it should be borne in mind that in these GC–MS studies: (i) alkaloids that precipitate could not be quantified accurately (lycorine and homolycorine); (ii) some alkaloids underwent thermal degradation (haemanthamine derivatives), and (iii) some alkaloids could not be detected without previous silylation (pretazettine). The GC–MS results also showed the presence of compounds with unknown EI fragmentation patterns. The complete phytochemical procedure, therefore, allowed the isolation of the alkaloid obliquine (21, 7.2 mg), followed by the isolation of a mixture (7.0 mg) of plicamine (22) and secoplicamine (23). The structures of these dinitrogenous alkaloids were elucidated by 2D NMR and high resolution mass spectrometry (HR-ESI-MS). Compounds 22 and 23 18 J.P. de Andrade et al. / Journal of Pharmaceutical and Biomedical Analysis 70 (2012) 13–25 Fig. 3. ESI-MS/MS of the [M+H]+ ions of obliquine (21). have previously been found in Galanthus plicatus subsp. byzantinus [23] and, together with 21, in Cyrthanthus obliquus [17]. These compounds have an unusual modification in the basic tazettine-type skeleton, in which the oxygen atom at position 5 is replaced by a nitrogen atom, which has a pendant 4-hydroxyphenethyl moiety as a substituent. Biogenetically, this replacement probably occurs via aminoaldehyde followed by Schiff base formation with a tyramine molecule [23]. Galanthindole (20) is another example of an unusual compound that has been classified as a new skeleton-type [24], although the possibility that it is an artifact from the homolycorine series should be considered. 3.2. LC–ESI-LTQ-Orbitrap-MS Under GC–MS conditions, dinitrogenous compounds eluted much later than the other alkaloids and their [M]+ ions appeared in very low abundance (<1%). Due to the interesting skeletal arrangement presented by these alkaloids and the difficulties for their study by GC–MS, we performed an LC–ESI-LTQ-Orbitrap-MS analysis. Compounds 21 and 22 have a closed ring D like tazettine and pretazettine, even though they have a carbonyl group at C-12 forming a cyclic amide function. In contrast, compound 23 shows an open ring D with an N-methyl-N-formylamino moiety attached to C-4a and a carbonyl group at C-11. Considering the MSn of the main fragments from compound 21, the MS2 [449.20→] showed the two most abundant ion peaks at m/z 329.1504 [C18 H21 N2 O4 ]+ and m/z 121.0652 [C8 H9 O]+ , which suggests that compound 21 initially tends toward losing the substituent 4-hydroxyphenethyl (see Figs. 2 and 3). All other fragments displayed less than 50% of relative abundance. In some fragments, the ion peaks at m/z 389.1871 [C24 H25 N2 O3 ]+ and m/z 364.1553 [C21 H18 NO3 ]+ are in agreement with the loss of 60 amu (C2 H4 O2 ) and 85 amu (C4 H7 NO), respectively. The loss of 85 amu after a Retro-Diels Alder (RDA) process in ring C is in agreement with related compounds Latifaliumin A and B [29]. The MS3 [449.20 → 364.15] showed a very abundant fragment at m/z 332.1279 [C21 H18 NO3 ]+ along with less abundant peaks at m/z 244.0966 [C14 H14 NO3 ]+ , m/z 212.0703 [C13 H10 NO2 ]+ and m/z 121.0648 [C8 H9 O]+ . Interestingly, the only abundant ion peak shown by MS4 [449.20 → 364.15 → 332.13] was at m/z 212.0707. An ion peak at m/z 121.0650 was also observed, although in low abundance and these results suggest a straightforward fragmentation of J.P. de Andrade et al. / Journal of Pharmaceutical and Biomedical Analysis 70 (2012) 13–25 Fig. 4. Tentative fragmentation of plicamine (22). Fig. 5. ESI-MS/MS of the [M+H]+ ions of plicamine (22). 19 20 J.P. de Andrade et al. / Journal of Pharmaceutical and Biomedical Analysis 70 (2012) 13–25 Fig. 6. Tentative fragmentation of secoplicamine (23). m/z 332.1283 to m/z 212.0707 through the loss of the substituent 4-hydroxyphenethyl. The MS3 [449.20 → 329.15] showed an ion peak at m/z 269.1287 [C16 H17 N2 O2 ]+ , followed by ion peaks at m/z 244.0970 and m/z 212.0707. The fragment at m/z 269.1287 agrees with the loss of 60 amu (C2 H4 O2 ) from the ion peak at m/z 329.15. The most interesting result was observed with the MS4 spectra. The MS4 [449.20 → 329.15 → 244.10] along with MS4 [449.20 → 364.15 → 244.10] spectra showed the ion peak at m/z 212.0703, which means that the fragment [C13 H10 NO2 ]+ (calculated mass of 212.0706) arose from three different routes, since it was also observed in MS4 [449.20 → 364.15 → 332.13] spectra. The very minor fragment at m/z 135.0443 [C8 H7 O2 ]+ coming from the ion peak at m/z 329.15 was also observed, as in other related Fig. 7. ESI-MS/MS of the [M+H]+ ions of secoplicamine (23). J.P. de Andrade et al. / Journal of Pharmaceutical and Biomedical Analysis 70 (2012) 13–25 compounds [29]. All the proposed fragments showed an error of around ±1 mDa (Fig. 2). A carbonyl group at C-6 and the methoxy substituent at the ␣-position at C-3 are the only differences presented by compounds 21 and 22. Again, the loss of the 4-hydroxyphenethyl substituent gave ion peaks at m/z 121.0653 [C8 H9 O]+ and m/z 343.1299 [C18 H19 N2 O5 ]+ as was observed in the MS2 of compound 22 (see Figs. 4 and 5). This first fragmentation seemed to be more predominant for compound 22 than compound 21 since the remaining fragments showed no more than 20% of relative abundance using the same collision energy in both cases. Other similarities were observed in their fragmentation mode. The MS2 [463.20→] spectra showed minor fragments at m/z 378.1345 [C22 H20 NO5 ]+ , m/z 258.0767 [C14 H12 NO4 ]+ and m/z 226.0504 [C13 H8 NO3 ]+ . The MS3 [463.20 → 378.13] showed ion peaks at m/z 346.1078 [C21 H16 NO4 ]+ , m/z 258.0762 and m/z 226.0502. The MS4 [463.20 → 378.13 → 346.11] along with MS4 [463.20 → 378.13 → 258.08] and MS4 [463.20 → 343.13 → 258.08] spectra only showed an ion peak at m/z 226.05 (m/z 226.0497, m/z 226.0498 and m/z 226.0500, respectively), confirming that they yielded the same fragment [C13 H8 NO3 ]+ through different routes, which was in agreement with a calculated mass of 226.0499 amu. In the case of the fragmentation of the ion peaks at m/z 378.13 and m/z 346.11, the substituent 4-hydroxyphenethyl should be eliminated as neutral loss. It was also difficult to observe this ion peak in the MS3 and MS4 of compound 21. Other very small fragments were observed in the MS2 , such as the ion peak at m/z 369.1454 [C20 H21 N2 O5 ]+ and m/z 311.1032 [C17 H15 N2 O4 ]+ . The ion peak at m/z 369.1454 is in agreement with the loss of C6 H6 O residue and the ion peak at m/z 311.1026 was observed in the MS3 [463.20 → 343.13], which is in agreement with the loss of a methanol residue. All the proposed fragments showed an error of around ± 1 mDa (Fig. 4). The MS2 [465.20→] for compound 23 showed an abundant ion peak at m/z 433.1761 [C25 H25 N2 O5 ]+ and minor fragments at m/z 348.1232 [C21 H18 NO4 ]+ and m/z 211.0755 [C14 H11 O2 ]+ (see Figs. 6 and 7). The first loss of the 4-hydroxyphenethyl observed in compounds 21 and 22 was not observed in compound 23, even when applying stronger collision energy. The ion peak at m/z 433.1761 was formed after the loss of a methanol residue from the methoxy group at C-3. The ion peak at m/z 348.1232 arose from m/z 433.18 after an RDA process in ring C, which led to the loss of [C4 H7 NO] residue. This sequence is suggested by the MS3 [465.20 → 433.18]. A careful look at this fragmentation revealed very small ion peaks at m/z 211.0754 and m/z 181.0649 [C13 H9 O]+ . The MS3 [465.20 → 211.07] spectra confirmed that the ion peak at m/z 181.0649 arose from m/z 211.07. Both these ion peaks are 21 Fig. 8. (A) GC–MS chromatogram of EtOAc extract with main alkaloids identified. (B) Section of chromatogram of TMS-derivatized EtOAc extract with identification of silylated tazettine and pretazettine along with 6-epimers of papyramine and haemanthidine. fragments from tazettine, crinine and well-known haemanthamine-type alkaloids [28,29]. Regarding the ion peak at m/z 348.1232, the MS3 [465.20 → 348.12] showed the presence of both fragments at m/z 228.0656 [C13 H10 NO3 ]+ and m/z 121.0650 [C8 H9 O]+ , which was indicative of the loss of 4-hydroxyphenethyl residue. The fragments proposed for 23 are shown in Fig. 6 with an error of around ±1 mDa. 3.3. BSTFA-derivatization Tazettine (14), one of the most widely distributed Amaryllidaceae alkaloids, is considered to be an artifact from pretazettine due to a rearrangement during the routine acid–base alkaloid extraction [30,31]. In the course of the phytochemical procedure tazettine was isolated as a major alkaloid although a small quantity Fig. 9. Chromatogram of Narcissus cultivar ‘Toto’ with selected ions at m/z 256 (continuous line) and m/z 319 (discontinuous line). In detail, identification of epimers A and B of pretazettine–TMS. No signal from tazettine–TMS was observed. 22 J.P. de Andrade et al. / Journal of Pharmaceutical and Biomedical Analysis 70 (2012) 13–25 Fig. 10. Tentative fragmentation of tazettine–TMS assigned after GC–MS/MS experiments. of pretazettine (15, 7 mg) was also isolated and identified by NMR. Pretazettine shows an EI mass fragmentation and retention index identical to those observed in tazettine, which indicates that it also converts to tazettine in GC–MS analysis. The well-known instability of pretazettine, especially in strong basic conditions, is due to its trans B–D ring fusion resulting in a relatively strained molecule, while the cis B–D fusion of tazettine allows more flexibility. The driving force for the B-ring opening would appear to be the relief of this internal strain. The completion of the rearrangement may be considered an intramolecular crossed-Cannizzaro reaction with subsequent hemiaketal formation [31,32]. The BSTFA-derivatization introduces a bulky group as a substituent at position 6, thus blocking the reaction. It is accepted that pretazettine is converted into tazettine in extraction processes, although the presence of small quantities of tazettine in the initial plant extract cannot be ruled out. Aiming to clarify their detection, we submitted the isolated tazettine and pretazettine together with both alkaloid and crude methanolic extracts to a derivatization process. Fig. 8 compares the EtOAc extract before and after the derivatization process and pretazettine–TMS was only identified after derivatization. To check the presence of tazettine in the crude extract, a rapid maceration in MeOH was carried out with bulbs of N. broussonetii and two Narcissus cultivars “Toto” and “Pencrebar” previously found to contain important quantities of tazettine by GC–MS. This extraction procedure, with a slightly basic pH (pH 8) and ultrasonic bath-assisted, is frequently used in metabolomic studies [11]. In the silylated crude extracts of both Narcissus cultivars and N. broussonetii, no peak for silylated tazettine was observed, which strongly suggests that 100% of the tazettine arose from pretazettine (Fig. 9). Previously published work [30,31] has raised this hypothesis, but the unique presence of pretazettine as a real metabolite in the crude plant extract (without any traces of tazettine) has been confirmed here for the first time using analytical methods. 3.4. GC–MS/MS Silylated tazettine and pretazettine demonstrated different fragmentation patterns by GC–MS/MS analysis. Tazettine–TMS displayed a molecular ion peak at m/z 403, a base peak at m/z 319 and an abundant peak at m/z 298. Its MS fragmentation, rationalized following Duffield et al. [28], is proposed in Fig. 10. GC–MS/MS experiments showed that the ion fragment at m/z 388 is formed from the molecular ion as a result of the loss of a methyl group, most probably from the O-TMS substituent at position 11, while the base peak at m/z 319 appeared after an RDA process in ring C. Further loss of Si(CH3 )2 OH from the ion fragment at m/z 388 produced an ion at m/z 313, from which the ion at m/z 298 arose after the loss of a methyl radical. The loss of 28 amu from the m/z 298 corresponded to a CO group, which led to a fragment at m/z 270. An MS/MS experiment with the ion at m/z 319 displayed peaks at m/z 304 and m/z 230 with low abundance, which was in agreement with the loss of a methyl radical from O-TMS together with the subsequent expulsion of dimethylsilane residue. Although the 1 H NMR of pretazettine confirmed the purity of the sample, two peaks with similar fragmentation patterns were observed by GC–MS, differing only in the relative abundance of fragments. This suggests the co-existence of C-6 epimers of pretazettine, as reported in alkaloids bearing crinine- and haemanthamine-type skeletons [16,31]. In the GC–MS spectrum of epimer A (Fig. 11) of silylated pretazettine, a molecular ion peak was observed at m/z 403 along with a base and abundant peak at m/z 319 and m/z 388, respectively. The GC–MS/MS data for pretazettine showed that, similarly to tazettine, the ions at m/z 388 and m/z 319 arose from the molecular ion (at m/z 403). The ion peak at m/z 388 may also be formed after the loss of a methyl radical from the methoxyl group at C-3. Further elimination of the (CH3 )3 SiO residue resulted in the formation of an ion at m/z 298 from which an ion fragment at m/z 256 was formed after the loss of C3 H7 N. The MS spectrum of pretazettine showed a characteristic J.P. de Andrade et al. / Journal of Pharmaceutical and Biomedical Analysis 70 (2012) 13–25 23 Fig. 11. Tentative fragmentation of the epimer (A) and epimer (B) from pretazettine–TMS assigned after GC–MS/MS experiments. ion at m/z 372 [M−31]+ arising from the ion at m/z 388 [M−15]+ , indicating a cyclization process [33]. This rearrangement process has been frequently observed in silylated hydroxypyrene derivatives showing an aromatic ring at the alpha position of the carbon that bears the hydroxyl group undergoing the silylation reaction [34,35]. A tentative fragmentation of pretazettine assigned after GC–MS/MS analysis is presented in Fig. 11. 3.5. In vitro assay The EtOAc extract and some isolated compounds were tested against the parasitic protozoa T. cruzi, T. brucei rhodesiense, L. donovani and P. falciparum. This assay was performed as described by Labraña et al. [27]. Only the EtOAc extract showed significant in vitro activity against T. cruzi, with an IC50 of 1.77 ␮g/ml 24 J.P. de Andrade et al. / Journal of Pharmaceutical and Biomedical Analysis 70 (2012) 13–25 (ref. value of 0.349 ␮g/ml for Benznidazole). Although the alkaloidrich extract demonstrated notable activity, the main alkaloids tazettine (14), lycorine (1) and homolycorine (4), and those found to a lesser extent, such as 8-O-demethylhomolycorine (5), Omethyllycorenine (7), papyramine/6-epi-papyramine (9 and 10) and obliquine (21), showed no significant activity against T. cruzi. Benaissa and Dr. Abdelaziz Elamrani for their helpful collaboration in the collection of N. broussonetii and Dr. Nehir Ünver for providing us with the 1 H NMR and EI-MS spectra of galanthindole. J.P.A. is thankful to the Agencia Española de Cooperación Internacional para el Desarollo (BECAS-MAEC-AECID) for a doctoral fellowship. References 4. Conclusion The alkaloids identified in N. broussonetii are commonly found in Narcissus species, with the exception of galanthindole and the dinitrogenous obliquine, plicamine, and secoplicamine, which are reported here for the first time in the Narcissus genus. The main alkaloids were lycorine, homolycorine and tazettine (arising from pretazettine). Also noteworthy is the absence of crinine-type alkaloids in this plant species, as occurs in all the Narcissus species studied to date. The alkaloids obliquine, plicamine, and secoplicamine, along with some related compounds, have been the subject of several total syntheses, prompted by the particular interest of their distinctive dinitrogenous skeletal arrangement [36,37]. GC–MS/MS analysis of dinitrogenous alkaloids using BSTFA-derivatization was carried out to improve their elution and detection, but without any success. Using an LC–ESI-LTQ-Orbitrap-MS a very similar fragmentation mode was observed for obliquine and plicamine, which yielded the fragments [C13 H10 NO2 ]+ and [C13 H8 NO3 ]+ , respectively, through different routes. The compounds obliquine and plicamine showed a very similar structure, particularly with respect to the closed ring D, which seemed to influence their fragmentation pattern, since the fragments corresponding to the loss of the substituent 4-hydroxyphenethyl appeared as base peaks. Otherwise, the compound secoplicamine showed a pendant 4hydroxyphenethyl moiety on the core structure and the remaining fragments presented very low abundance. It is accepted that the conversion of pretazettine to tazettine in a routine alkaloid extraction is near total, without ruling out the presence of small quantities of tazettine in the initial plant extract. A BSTFA-derivatization step confirmed that all the tazettine present in the plant extract arose from pretazettine, and was therefore absent as a natural alkaloid. The crude methanolic extract obtained by a fast extraction in a slightly basic environment from N. broussonetii and two Narcissus cultivars previously found to contain tazettine as a major alkaloid revealed the sole presence of pretazettine. As part of an ongoing project on the chemical aspects of Amaryllidaceae alkaloids, silylated pretazettine and tazettine were submitted to an MS/MS study. In contrast with the dinitrogenous alkaloids, the GC–MS/MS analysis proved to be an important tool for the study of their fragmentation. Silylated tazettine and pretazettine showed distinct fragmentation routes in GC–MS/MS, although a Retro-Diels Alder process formed the base peak at m/z 319 in both. Finally, N. broussonetii exhibited important in vitro activity against T. cruzi, which could be due to a synergic action of some of the identified alkaloids, since they showed no significant activity when tested individually. 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