Targeted and non-targeted analysis of annonaceous alkaloids and acetogenins from Asimina and Annona species using UHPLC-QToF-MS
Graphical abstract
Introduction
Phytochemical and pharmacological investigations on Asimina/Annona species revealed the presence of several bioactive compounds including alkaloids, acetogenins, phenolic compounds, terpenes and essential oils [1]. Chemical studies of the plant (family Annonaceae) have increased in the last two decades due to the presence of annonaceous molecules (e.g., isoquinoline alkaloids and acetogenins) with pharmacological uses [2]. Approximately 500 alkaloids have been identified in 138 Annonaceae species in 43 genera. Most of these alkaloids present in Annonaceae possess isoquinoline derived structures including benzyltetrahydroisoquinolines (coclaurine, higenamine), protoberberines (coreximine, jatrorrhizine, berberine, corydaline, tetrahydropalmatine), proaporphines (pronuciferine, stepharine), aporphines (anonaine, asimilobine, glaucine, magnoflorine, isocorydine, norisocorydine, norcorydine, lysicamine, nornuciferine), and oxoaporphines (liriodenine, anolobine, norushinsunine, lanuginosine) [1], aporphinoids are the largest group of compounds occurring in the Annonaceae plants [1].
In addition, 593 annonaceous acetogenins (ACGs) had been identified, from 51 species in 13 genera [1] and most of them occur in tropic and subtropic regions of the world. Acetogenins found in the plant family Annonaceae are an important group of long-chain fatty acid derivatives. Chemically they contain one to three tetrahydrofuran (THF) and/or tetrahydropyran (THP) rings and have a long aliphatic chain on one side (belonging to a series of C35-C37 compounds) and aliphatic chain ending in an α, β-unsaturated γ-lactone (or ketolactone) on the other side. Various double bonds, hydroxyls, carbonyls and acetyls are located throughout the long unbranched aliphatic regions of the molecule [3,4]. Paw paw is the only temperate member of the Annonaceae family [5,6], which includes several delicious tropical fruits, such as custard apple (Annona reticulata L.), cherimoya (An. cherimoya Mill.), sweetsop or sugar apple (An. squamosa L.), atemoya (An. squamosa x An. cherimoya), soursop (An. muricata L.), and biriba (Rollinia mucosa Baill.) [7].
Paw paw (As. triloba) might be sometimes confused with the completely different papaya fruit (Carica papaya) [8]. Members of the Annonaceae family, such as paw paw, cherimoya, soursop, custard apple, and sweetsop, contain substantial amounts of acetogenins. Annonaceous acetogenins (ACGs) exhibited a wide range of biological activities and some have strong cytotoxic effect against various cancer cell lines [[9], [10], [11]]. Leaf and fruit extracts of As. triloba contain acetogenins, including the neurotoxin, annonacin [12]. The seeds and bark contain asimitrin [13] and other acetogenins including asimin, asiminacin and asiminecin [12,14]. Acetogenins as well as alkaloids have been found in the bark, twig, fruit, seed, root and leaf of the paw paw tree (As. triloba) [5,6].
Based on previous literature, an HPLC-MS method was developed for three acetogenins: asimicin, bullatacin, and bullatalicin from ripe pawpaw pulp extract [15]. The study by Yang et al. [16] used HPLC-DAD method for the determination of eight acetogenins from Annona squamosa seeds [16]. In another study, MALDI-TOF MS or HPLC-ESI-LTQ-Orbitrap® using post-column lithium infusion was developed to assess the presence of acetogenins for A. muricata [17,18]. A spectrophotometric method for total phenolic content and HPLC-DAD for chemical fingerprinting analysis was applied for the As. triloba fruits [19]. A pharmacokinetic study of annonacin (Annona muricata, Asimina triloba) was performed on rat plasma and brain [20,21]. The concentrations of the two neurotoxins, annonacin and squamocin were determined from the fruit pulp of the As. triloba using LC–MS [22]. The phytochemical content and quality characteristics of paw paw pulp from ten varieties was studied for the characterization of three phenolic acids and flavonols, particularly (-)-epicatechin, B-type procyanidin dimers and trimers [23]. A quantitative liquid chromatography/tandem mass spectrometry (LC/MS/MS) method was established for the quality control of the acetogenins in the extracts of the paw paw tree (Asimina triloba). The concentrations of the three major compounds (bullatacin, asimicin and trilobacin) were evaluated [24]. Ripe fruit from four paw paw cultivars (Asimina triloba) were analyzed for headspace volatiles and sensory properties. The volatile components of paw paws were found to be mainly ethyl and methyl esters of fatty acids. Ethyl hexanoate was found to be in the highest concentration in the fruit from all cultivars [25]. A droplet-liquid microjunction-surface sampling probe coupled to UPLC-PDA-HRMS/MS system was employed to detect acetogenins in situ from A. triloba. The seeds, fruit pulp, twigs, leaves, and flowers of A. triloba were all examined for major acetogenins [26]. No previous phytochemical work has been reported with species of A. parviflora except for one paper where five bioactive compounds (asimicilone, 6-cis-docosenamide, asimicin, (+)-syringaresinol, and β-sitosterol-β-D-glucopyranoside) were isolated and identified [27]. The presence of acetogenins was investigated in fruit pulps of Annona squamosa from different locations. LC-MS/MS method development was performed using four standards (bullatacin (rolliniastatin-2), squamocin, annonacinone and annonacin). Qualitative analysis of other ACGs was also performed [28]. Liriodenine content was analyzed by TLC image analysis and HPLC-DAD method from twenty-eight plant materials of Magnoliaceae, Annonaceae and Nelumbonaceae in Thailand [29]. The levels of reticuline, norreticuline and N-methylcoclaurine were determined in three Annona species (A. reticulata, A. squamosa, A. muricata) [30]. Previous studies [[15], [16], [17], [18], [19], [20], [21], [22], [23], [24], [25], [26], [27], [28], [29], [30]] have evaluated the analysis to specific type of plant or acetogenins or alkaloids from Annona or Asimina species. Most of the previous work was focused on target acetogenins or alkaloids with limited information on alkaloids from As. triloba samples. In this regard, metabolomics provides an efficient tool for investigating the alkaloids and acetogenins in a single analysis including health promoting compounds.
Utilizing the high chromatographic resolution and separation capabilities of UHPLC with QToF-MS provides the structural characterization from accurate mass measurement for both MS and MS-MS experiments. This technique offers a significant advantage for rapid screening of target and non-target compounds in complex matrices. In the present study, the structural characteristics of thirty-five known compounds (alkaloids and acetogenins) from methanolic extracts of dried seed, bark, twigs and mixed parts of As. triloba, leaf, stem, root of As. parviflora, seed and leaf of Annona species (An. squamosa, An. muricata, An. montana and An. X atemoya which is a hybrid of An. squamosa X An. cherimoya), and one dietary supplement claiming to contain paw paw (As. triloba) have been studied using UHPLC-QToF-MS in positive ion mode. Herein, is described an investigation of the fragmentation pathways leading to the identification of key diagnostic fragment ions for thirty-five compounds (alkaloids and acetogenins). The identification of diagnostic fragment ions is important to the data mining of new compounds (that is, ones that have not yet been identified). MS-MS experiments lessen the ambiguities in the identification of compounds. Because of the many isomers, even accurate mass MS/MS is insufficient for complete identification, requiring chromatographic separation and comparison of standards. Some compounds with similar masses were further characterized and confirmed from their retention times obtained from standard compounds. This technique also provides fast screening of alkaloids and acetogenins from various Asimina/Annona species and dietary supplement in a single analysis. To the best of our knowledge, this study is the first on the simultaneous analysis of alkaloids and acetogenins composition of different parts of Asimina species based on a high-resolution mass spectrometric method.
Section snippets
Chemicals
Thirty-five compounds were used as reference standards (Fig. 1). The alkaloids, squamolone (1) and liriodenine (11) were purchased from LabNetwork (South Portland, Maine, USA), higenamine (2), coclaurine (3), stepharine (4), magnoflorine (5), anolobine-9-O-β-D-glucopyranoside (6), nornuciferine (8), norushinsunine (9), anolobine (10), lysicamine (12) were isolated at the National Center for Natural Products Research (NCNPR, University of Mississippi, University, Mississippi, USA); the identity
Results and discussion
Acetogenins lack a specific chromophore for UV/Vis detection, therefore mass specific detection was employed. Optimized chromatographic conditions were achieved after several trials with methanol, acetonitrile and water in different proportions as the mobile phase. In order to determine the optimal ionization method for analytes, positive and negative ion modes were investigated with the same UHPLC mobile phase at a flow rate of 0.23 mL/min. All compounds showed good detection in positive ion
Conclusion
In conclusions, UHPLC-QToF-MS has been applied to the identification and characterization of acetogenins and alkaloids from Asimina and Annona species. Thirty-five standard compounds were characterized and detected from methanolic extracts of Asimina and Annona species. The compounds were identified by comparing the retention times and the MS and MS/MS data with reference standards. The non-targeted compounds (about ninety-six) were identified based on accurate mass. For all compounds, MS in
Conflict of interest
The authors declare no conflict of interest.
Acknowledgements
This research is supported in part by “Science Based Authentication of Dietary Supplements” funded by the Food and Drug Administration grant number 2U01FD004246-06, the United States Department of Agriculture, Discovery & Development of Natural Products for Pharmaceutical & Agricultural Applications No. 58-6060-6-015. The authors would also like to thank Dr. Jon F. Parcher for his help in correcting grammar and support.
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2021, Journal of Chromatography B: Analytical Technologies in the Biomedical and Life SciencesCitation Excerpt :Indeed, the Mannich-like reaction involving two tyrosine derivatives, dopamine, and 4-hydroxyphenylacetaldehyde, yields the stereospecific product (S)-norcoclaurine that is the precursor for benzylisoquinoline alkaloid biosynthesis [35,36]. Besides, norcoclaurine has been reported in A. squamosa [31]. For alkaloids 2, 3, and 4, at m/z 286.147 established the molecular formula as C17H19NO3 (6DBE, calcd 286.1443, Δm/z theoretical ≤ −8.67 ppm.
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Authors share equal contribution.