Targeted and non-targeted analysis of annonaceous alkaloids and acetogenins from Asimina and Annona species using UHPLC-QToF-MS

Highlights

• Pharmacological investigations of Asimina/Annona species revealed the presence of several bioactive compounds.

• UHPLC-QToF-MS technique was applied for rapid screening of target and non-target compounds in complex matrices.

• Thirty-five standard compounds were characterized and detected from methanolic extracts of Asimina and Annona species.

• About ninety-six non-targeted compounds were identified based on accurate mass from mass spectrum.

• The developed analytical method would be a valuable tool for herbal identification and quality control.

Abstract

In current work, targeted and non-targeted analysis of alkaloids and acetogenins from methanolic extracts of Asimina, Annona species and dietary supplements have been performed using UHPLC-QToF in positive ion mode. Thirty-five standard compounds (twelve alkaloids and twenty-three acetogenins) were used for the analysis. The fragment ions produced by collision induced dissociation (CID) revealed the characteristic cleavage and provided structural information. Aporphine alkaloids and acetogenins are the major groups found in Asimina and Annona species. An untargeted analysis based on high-resolution mass spectrometry was carried out to profile the alkaloids and acetogenins from Asimina species (As. triloba, As. parviflora). Magnoflorine, being a major alkaloid from twigs of As. triloba samples, was used as an example to discuss the fragmentation patterns. In (+)-ESI-MS, magnoflorine gave [M]⁺ ions at m/z 342.1705. The fragment ions at m/z 297.1127 [M-(CH₃)₂NH]⁺, 282.0886 [M-(CH₃)₃NH]⁺, 265.0865 [M-(CH₃)₂NH-CH₃OH]⁺, 237.0916 [M-(CH₃)₂NH-CH₃OH-CO]⁺, and 222.0681 [M-(CH₃)₂NH-CH₃OH-CO-CH₃]⁺ resulted from the [M]⁺ molecular ion. One dietary supplement claiming to contain paw paw (As. triloba) was also analyzed and showed a similar profile to twigs of As. triloba. A total of 131 compounds including standard compounds were identified from the different parts of As. triloba and As. parviflora samples. These compounds can be used to distinguish Asimina species. However, for definite identification of these unknown components, further investigation is required. This may provide a model for the rapid screening and structural characterization of bioactive constituents from plant extracts in a single analysis.

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.