Article pubs.acs.org/JAFC
Identification of the Environmental Neurotoxins Annonaceous Acetogenins in an Annona cherimolia Mill. Alcoholic Beverage Using HPLC-ESI-LTQ-Orbitrap Jessica Le Ven,† Isabelle Schmitz-Afonso,*,‡ Guy Lewin,† Alain Brunelle,‡ David Touboul,‡ and Pierre Champy† †
Laboratoire de Pharmacognosie, CNRS UMR 8076 BioCIS, Faculté de Pharmacie, Université Paris-Sud, 5 rue J.-B. Clément, 92296 Châtenay-Malabry, France ‡ Centre de Recherche de Gif, Institut de Chimie des Substances Naturelles, CNRS, avenue de la terrasse, 91198 Gif-sur-Yvette Cedex, France S Supporting Information *
ABSTRACT: Epidemiological and toxicological studies have suggested Annonaceaeous acetogenins to be environmental neurotoxins responsible for sporadic atypical parkinsonism/dementia in tropical areas. These compounds are present in the tropical genus Annona (Annonaceae), known for its fruit-yielding cultivated species such as Annona cherimolia. This species is widely cultivated in South America, Spain, and Portugal and yields acetogenins in its seeds, stems, and roots. The presence of these compounds in the pulp of its fruit and in derived food products is unclear. An innovative and sensitive methodology by HPLC-ESI-LTQ-Orbitrap with postcolumn infusion of lithium iodide was used to identify the presence of low levels of acetogenins in an A. cherimolia Mill. fruit-based commercial alcoholic beverage. More than 80 representatives were detected, and the 31 most intense acetogenins were identified. All together these findings indicate that this species should be considered as a risk factor within the framework of a worldwide problem of food toxicity. KEYWORDS: acetogenins, Annona cherimolia Mill., Annonaceae, annonacin, dereplication, environmental neurotoxins, mass spectrometry, HPLC-MS/MS
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above-mentioned structural features and global lipophilicity.12,14 In agreement with clinical and anatomopathological observations, toxicological studies showed that these compounds induce hyperphosphorylation and redistribution of Tau and cell death in striatal and cortical neuronal primary cultures.14−17 Annonacin, the major AAG of A. muricata, induces neurodegeneration after subchronic systemic intoxication in rats and Tau hyperphosphorylation in neurons of mice transfected with the human mutated Tau gene.18−20 Chemical homogeneity among members of the Annonaceae family suggests that all edible species might be sources of AAGs. Indeed, phytochemical studies of A. muricata, A. squamosa, and Asimina triloba Dunnal. (paw paw) revealed significant quantities of AAGs in their fruits.17,21−24 In addition, soursop processed nectars from different origins were also shown to contain AAGs.21−23,25 The fruit of A. cherimolia is cultivated and eaten in South America and Asia and also in southern Europe, with Spain as the main producer and an annual volume of 300 tons for the Portuguese island of Madeira only.26 However, it is unclear whether the species is a dietary source of AAGs. Indeed, the chemical composition of this fruit is poorly known apart from essential oil and kaurane diterpenes.27−29 According to the literature, 27 AAGs have been isolated from the seed, stem, and
INTRODUCTION The tropical family Annonaceae has economic importance in the food-processing industry and fresh fruit local markets, mainly with members of the genus Annona. Species such as Annona cherimolia Mill. (annona, chirimoya), Annona muricata L. (soursop), and Annona squamosa L. (custard apple) are appreciated for their edible fruits and derived food products.1 These plants are also widely used in traditional medicine.2 Case-control studies3−5 have suggested consumption of the fruits of Annonaceae and herbal infusions derived from these plants as the cause of the high prevalence of sporadic atypical Parkinsonism/dementia clusters reported in Guadeloupe (French West Indies)6,7 and New Caledonia.8 They might also be involved in atypical Parkinsonism observed in some communities in London9 and on the island of Guam.10 Phytochemical and toxicological studies have identified Annonaceous acetogenins (AAGs), a class of polyketides specific to the Annonaceae,11,12 as candidate neurotoxins. The French food safety agency expressed its concern about this issue.13 AAGs constitute a chemically homogeneous class of polyketides with a common skeleton consisting of an α,β-saturated or unsaturated γ-methyl-γ-lactone, optionally hydroxylated, substituted in position 2 by a long alkyl chain of 32 or 34 carbons. The aliphatic chain most often bears one or two tetrahydrofuranic cycles (THF) flanked by adjacent hydroxyls, free hydroxyls (possibly acetylated), ketones, epoxides, or double bonds. These compounds are potent inhibitors of mitochondrial complex I, with structure−activity relationships based on the © XXXX American Chemical Society
Received: March 9, 2014 Revised: August 3, 2014 Accepted: August 3, 2014
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dx.doi.org/10.1021/jf501174j | J. Agric. Food Chem. XXXX, XXX, XXX−XXX
Journal of Agricultural and Food Chemistry
Article
performed at a flow rate of 5 μL/min. Mass measurements were obtained with a mass precision below 3 ppm using an external calibration. The LOD (UV, MS) of purified annonacin (6) was evaluated in the same conditions, based on triplicate injection (10 μL) of the targeted concentration, with a signal-to-noise ratio above 3. AAGs Classification. After structural identification, the AAGs contained in the extract were ranked using the classification established by Cavé et al.11 The first rank classification type is based on the layout of the aliphatic chain: A-type (mono-THF), B-type (adjacent bis-THF), or C-type (nonadjacent bis-THF). These THF cycles are always flanked by two adjacent hydroxyl groups, except for a few representatives with one hydroxyl only, placed on the methyl-terminal end. E-type AAGs contain no THF, but epoxides and/or double bonds. The second rank classification is the subtype depending on the nature of the lactone ring. The most frequently referenced are subtype 1a (α,βunsaturated γ-methyl-γ-lactone) and subtype 1b (α,β-unsaturated γ-methyl-γ-lactone with a hydroxyl on the C-4 position). Data Analyses. The search for AAGs formulas corresponding to lithiated adducts was carefully achieved following the method described below. [M + H]+, [M + Na]+, and [M + K]+ ions were excluded on the basis of the raw formulas assigned for each of these ions (precision better than 3 ppm). The characteristic structural fragments of lithiated adducts were sought according to the method established in our previous publication.31 To validate that these compounds are AAGs, characteristic fragments of AAGs were first sought in the MS2 spectra of C35 and C37 compounds corresponding to the AAGs: loss of CO (−27.9949 amu), CO2 (−43.9898 amu), and cleavage on β of the lactone. Second, other characteristic structural fragments were searched, according to the classification established by Laprévote et al.: terminal CH3-containing X-fragments, produced by an α-cleavage of hydroxyl group; lactone-containing B-fragments for cleavage of a THF group; terminal CH3-containing Y-fragments by breaking of bis-THF (B-type AAGs) and nonadjacent-THF groups (C-type AAGs); and finally lactone-containing c-fragments for the cleavage of an epoxide cycle.25,31 Numeral subscripts used with these letters distinguish the different ions belonging to the same series (e.g., X1 is the fragment produced by α-cleavage to the first hydroxyl group, starting from the methyl end) (Figure 1). Fragments corresponding to successive water
root of this species, including 3 artifactual compounds.12,30 Our previous study showed the absence of AAGs in fruits originating from Spain.29 We recently developed an innovative approach for the detection of AAGs, using reversed-phase highperformance liquid chromatography coupled to a hybrid linear trap/Orbitrap mass spectrometer (LTQ-Orbitrap) with postcolumn infusion of lithium.25 Under these conditions, intense lithium adducts of AAGs are preferably formed, with enhanced detection compared to other metabolites, providing useful fragment ions for structural studies. This technique was here used to investigate an industrial alcoholic beverage prepared with A. cherimolia originating from Madeira (Portugal). In the total ethyl acetate (EtOAc) extract, numerous AAGs could be detected, and those yielding the most intense [M + Li]+ ions could be identified or structurally characterized.
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MATERIALS AND METHODS
Chemical Reagents. Ethyl acetate (EtOAc), methanol (MeOH, LC-MS grade), acetonitrile (HPLC grade), and H2SO4 were purchased from VWR-Prolabo (Fontenay-sous-Bois, France). Lithium iodide (LiI) and sodium sulfate (Na2SO4) were purchased from Sigma-Aldrich (Saint Quentin-Fallavier, France). Milli-Q water was prepared in house. Standards. Diepomuricanin-B (1) (C35H62O4; monoisotopic mass = 546.4648 Da), corossolin (3) (C35H64O6; monoisotopic mass = 580.4703 Da), annonacin (6) and cis-annonacin (7) (C35H64O7; monoisotopic mass = 596.4652 Da), trans-corossolone (13) and cis-corossolone (14) (C35H62O6; monoisotopic mass = 585.4701 Da) were obtained as previously described.12,31 Stock solutions were prepared in methanol at a concentration of 1 μM. Extracted Material. A. cherimolia Mill. alcoholic beverage (brand Licor de Annona, J. Faria and Filhos, LDA; no batch number), packaged in a glass bottle (total volume = 0.7 L), was purchased in Madeira (Portugal). Its composition was “water, 20° alcohol, sugar and annona”. Extraction. The beverage (0.5 L) was acidified with H2SO4 (0.5 N) to pH 3 and then extracted (L/L) three times with EtOAc (3 × 300 mL) followed by centrifugation for 15 min at 4000 rpm to remove the emulsion produced by sugars. The organic phases were combined, dried over anhydric Na2SO4, and evaporated to dryness under reduced pressure to obtain a sample of 141 mg (yield = 4.7 × 10−2% w/v). The extract was kept at 4 °C. Aliquots were extemporaneously dissolved in MeOH (1 mg/mL) and filtered using an Acrodisc CR 4 mm syringe filter (0.45 μm PTFE membrane) before injection in the chromatographic system. High-Performance Liquid Chromatography with Tandem Mass Spectrometry (HPLC-MS/MS). HPLC was performed on an HPLC Ultimate 3000 system (Dionex, Voisins-le-Bretonneux, France) consisting of a degasser, a quaternary pump, an autosampler, a column oven, and a photodiode array detector. Separation was achieved on an octadecyl column (Sunfire, 100 × 2.1 mm, 3.5 μm particle size; Waters, Guyancourt, France), equipped with a guard column. Column oven temperature was set at 30 °C. Elution was conducted with a mobile phase consisting of water (A) and acetonitrile (B), following the gradient 40 to 100% B in 25 min, then maintaining 100% B for 7 min at a flow rate of 0.3 mL/min. Injection volume was 10 μL (full loop) for standards and extract solutions. After LC separation, UV detection was operated at 210 nm. The HPLC system was interfaced to a hybrid linear trap/Orbitrap mass spectrometer (LTQ-XL Orbitrap, Thermo Scientific, Les Ulis, France) equipped with an electrospray (ESI) ionization source. Accessible ranges were m/z 250−1200 for fullscan MS experiment and m/z 160−700 for MS2 experiments. Mass resolutions (fwhm) were fixed at 60000 and 30000 for MS and MS2 experiments, respectively. The source parameters in the positive ion mode were as follows: spray voltage set at 5 kV, capillary temperature at 300 °C, capillary voltage at 45 V, and tube lens voltage at 165 V. The optimal collision energy was 50 au, and activation time was 33 ms. Postcolumn infusion of lithium iodide (2 mM in MeOH) was
Figure 1. Fragmentation patterns for the [M + Li]+ adduct ions of AAGs identified in the Annona cherimolia alcoholic beverage. losses are not specific to the number of hydroxyls and therefore were not used. The relative intensity of the fragment corresponding to the β-cleavage of the lactone can differentiate subtype 1a (35 ng. This is less than the concentrations measured in samples of A. muricata leaf herbal teas.21 Moreover, this amount is negligible in comparison to the fruit pulps of A. muricata and Asimina triloba.17,23,24 This is the first evidence of the presence of AAGs in a dietary product derived from A. cherimolia. These results are not in agreement with our previous study made by MALDI-TOF MS (LOD ∼ 1.5 × 10−7 M for annonacin), which showed an absence of AAGs in the pulp of the fruit.21,22,29 This might be due to the lower sensitivity of the method used here. However, this discrepancy could also be attributed to genetic or external factors. Indeed, 5-fold seasonal variations in AAGs content have been measured for the twigs of Asimina triloba.33 Varieties and cultural conditions also are likely to influence secondary metabolites contents. Our previous study on A. cherimolia was performed with fruits harvested in southern Spain (Mediterranean climate, 38° N latitude), whereas the fruits used for preparation of the beverage studied here originated from Madeira (tropical climate, 32° N latitude). Nevertheless, a contamination of the beverage with seed fragments cannot be excluded. A. cherimolia thus appears as a minor source of exposure to environmental neurotoxins. Its consumption and that of derived products should, however, be considered as a risk factor in surveys about sporadic atypical Parkinsonism and dementia.
(C37H66O7, Rt = 16−24 min; C37H66O8, Rt = 11−20 min; C37H68O8, Rt = 17−19 min). However, AAGs with the formula C37H68O8 were not detected, although several representatives were identified in the species (asimicin, itrabin).12 The other formulas identified in our analysis were C37H64O7 (Rt = 13− 25 min), C37H64O8 (Rt = 13−22 min), C37H66O9 (Rt = 7− 14 min), and C37H68O9 (Rt = 8−11 min). Due to numerous coeluting compounds and low ionic intensities, only AAGs with the formulas C37H66O7 and C37H66O8 could be structurally characterized. However, C37 AAGs belonging to E-, A-, and C-types were detected in the other most prominent groups. B-Type: Adjacent Bis-THF. As for C35 AAGs, the C37 AAGs bearing seven oxygen atoms (here with three degrees of unsaturation; C37H66O7) are those with the peaks of the highest intensity (3.8 × 106). The extracted ion chromatogram shows at least seven peaks, with the major one at Rt = 19.7 min (29) (Figure 2l). The MS2 spectrum of this compound is identical to that of the AAG at Rt = 19.1 min (28). Both compounds correspond to AAGs of subtype 1a (X4 fragment, relative intensity
