An evaluation of the quality of Annona muricata leaf products.

OBJECTIVES: Annona muricata, also known as graviola, is traditionally used for the treatment of a range of disorders including cancer. Interest in A. muricata use has increased in recent years. This study investigated the quality and safety of a selection of commercially available A. muricata leaf products. METHODS: Seven commercially available products were purchased via online shopping sites. Each product was assessed for quality indicators including weight variation, quantification of the bioactive constituent annonacin, presence of annonaceous acetogenins and contaminants. The samples were evaluated by thin-layer chromatography, high-performance liquid chromatography, liquid chromatography-mass spectroscopy, low-resolution mass spectrometry and nuclear magnetic resonance spectroscopy. Microbial analysis was carried out in accordance with the British Pharmacopoeia. Heavy metals were analysed by inductive coupled plasma mass spectrometry. KEY FINDINGS: Of the seven products analysed, one product contained less than half of the content stated on the label. The labelled dosage recommendation varied between products. There was a high variation in annonacin concentration (1.05-3.09 mg/g) and the presence of annonaceous acetogenins. One of the products was found to have a total aerobic microbial count above the United States Pharmacopoeia limit. CONCLUSIONS: The variation in the indicators of quality and safety of commercially available A. muricata leaf products tested have implications for clinicians and people living with cancer who use these herbal products.

Dereplication of Acetogenins from Annona muricata by Combining Tandem Mass Spectrometry after Lithium and Copper Postcolumn Cationization and Molecular Networks.

Annonaceous acetogenins are natural products held responsible for atypical Parkinsonism due to chronic consumption in traditional medicine or as food, leading to the development of analytical strategies for their complete chemical characterization in complex mixtures. Characterization by tandem mass spectrometry (MS/MS) of acetogenins using collision-induced dissociation from lithium adducts provides additional structural information compared to protonated or sodiated species such as ketone location on the acetogenin backbone. However, very low intensity diagnostic ions together with the lack of extensive structural information regarding position of OH and THF substituents limit this approach. Copper adducts led to diagnostic fragment ions that allow us to identify the position of oxygen rings and hydroxyl substituents. Fragmentation rules were established on the basis of acetogenin standards allowing the identification of 45 over the 77 analogues observed in an extract of Annona muricata by LC-MS/MS using postcolumn infusion of copper sulfate (CuSO4) solution. Molecular networks that were generated thanks to specific fragmentations obtained with copper led to the distinction of THF ring position or to the identification of hydroxylated lactone, for instance.

Alkaloid and acetogenin-rich fraction from Annona crassiflora fruit peel inhibits proliferation and migration of human liver cancer HepG2 cells.

Plant species from Annonaceae are commonly used in traditional medicine to treat various cancer types. This study aimed to investigate the antiproliferative potential of an alkaloid and acetogenin-rich fraction from the fruit peel of Annona crassiflora in HepG2 cells. A liquid-liquid fractionation was carried out on the ethanol extract of A. crassiflora fruit peel in order to obtain an alkaloid and acetogenin-rich fraction (AF-Ac). Cytotoxicity, proliferation and migration were evaluated in the HepG2 cells, as well as the proliferating cell nuclear antigen (PCNA), vinculin and epidermal growth factor receptor (EGFR) expression. In addition, intracellular Ca2+ was determined using Fluo4-AM and fluorescence microscopy. First, 9 aporphine alkaloids and 4 acetogenins that had not yet been identified in the fruit peel of A. crassiflora were found in AF-Ac. The treatment with 50 µg/mL AF-Ac reduced HepG2 cell viability, proliferation and migration (p < 0.001), which is in accordance with the reduced expression of PCNA and EGFR levels (p < 0.05). Furthermore, AF-Ac increased intracellular Ca2+ in the HepG2 cells, mobilizing intracellular calcium stores, which might be involved in the anti-migration and anti-proliferation capacities of AF-Ac. Our results support the growth-inhibitory potential of AF-Ac on HepG2 cells and suggest that this effect is triggered, at least in part, by PCNA and EGFR modulation and mobilization of intracellular Ca2+. This study showed biological activities not yet described for A. crassiflora fruit peel, which provide new possibilities for further in vivo studies to assess the antitumoral potential of A. crassiflora, especially its fruit peel.

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

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.

Correlation Between Acetogenin Content and Antiproliferative Activity of Pawpaw (Asimina triloba [L.] Dunal) Fruit Pulp Grown in Korea.

Pawpaw (Asimina triloba [L.] Dunal) is widely cultivated in Korea for its fruit, which contains bioactive compounds, such as acetogenins. In this study, we investigated the acetogenin content and antiproliferative activity of pawpaw fruit pulp against various cancer cell lines and evaluated the relationship between these two variables at different maturation stages. Unripe fruit had higher antiproliferative activity than ripe fruit, and the activity level depended on acetogenin content. In addition, the presence of specific acetogenins was related to inhibition of certain cancer cell types. The unripe fruit methanol and ethanol extracts (URFM and URFE, respectively) that were rich in acetogenins strongly inhibited the growth of HT-1080, HeLa, and AGS cells by >50% at concentrations of less than 115 µg/mL. These findings indicate that URFM and URFE have therapeutic potential for the treatment of cancer, and our study establishes a basis for further mechanistic studies of the antiproliferative activity of pawpaw fruit. However, it is necessary to further study the anticancer activity of acetogenins from pawpaw fruit using in vivo activity approaches. PRACTICAL APPLICATION: Pawpaw (Asimina triloba) contains acetogenins that can inhibit the growth of cancer cells. In our study, we demonstrate that the antiproliferative activity is higher in unripe than in ripe fruit and depends on acetogenin content. Our results indicate that the extract of unripe pawpaw fruit has value not only as a functional food, but has therapeutic potential for the treatment of cancer as a naturally derived substance that may be less toxic than conventional chemotherapy drugs.

The fruit of Annona squamosa L. as a source of environmental neurotoxins: From quantification of squamocin to annotation of Annonaceous acetogenins by LC-MS/MS analysis.

Annonaceous acetogenins (AAGs) are neurotoxins possibly responsible for atypical Parkinsonism/dementia clusters, via the consumption of edible Annonaceae fruits. Their presence was investigated in fruit pulps of Annona squamosa from different locations. Qualitative analysis of other AAGs was performed. We here report the identification of squamocin in batches from Asia, the Caribbean Basin and the Indian Ocean. This molecule was quantified by HPLC-UV, evidencing a content of 13.5-36.4mg/fruit. HPLC-ESI-Q-TOF allowed the detection of 25 different raw formulas matching with AAGs. LC-MS/MS methodological development was performed using 4 representative standards. The main AAGs could be annotated, including bullatacin (rolliniastatin-2) and annonacin. This study evidences a remarkable homogeneity for the main AAGs within the species, and discrepancies for minor compounds. These findings indicate that A. squamosa should be considered a risk factor for neurodegenerative disorders.

1 H qNMR Quantification of Annonaceous Acetogenins in Crude Extracts of Annona muricata L. Fruit Pulp.

INTRODUCTION: Annonaceous acetogenins (AAGs) constitute a group of environmental neurotoxins, possibly implicated in sporadic atypical Parkinsonism/dementia complexes. The recent evidencing of complex mixtures of AAGs in edible fruits and derived food products requires efficient and practical analytical tools for an estimation of human exposure. OBJECTIVE: To develop a simple method for the direct quantitation of the majority of AAGs (sub-types 1a and 1b) within crude extracts, using commonly available 1 H-NMR spectrometers, for food control. METHODOLOGY: Method development was carried out on 400 MHz and 300 MHz spectrometers, for routine application on fruits crude extracts of Annona muricata L. The method was validated with annonacin and squamocin as reference compounds. Two internal standards (ISs), fumaric acid and dimethyl fumarate, were successfully used, in deuterated methanol (CD3 OD) and deuterated chloroform (CDCl3 ), respectively. RESULTS: Quantitation was carried out using signals corresponding to the deshielded ethylenic protons characterising most AAGs, at δ 7.18 or δ 6.98 ppm in CDCl3 . The limit of quantification (LOQ) was 2.5 mM, with acceptable accuracy, and the limit of detection (LOD) was 0.5 mM. The AAGs contents measured in seven distinct fruit samples of Annona muricata ranged from 14 µmol to 226 µmol of AAGs per 100 g fresh pulp (i.e. 0.14 mmol to 1.3 mmol of AAGs per fruit). CONCLUSION: A simple, accurate and specific method for quantification of AAGs content was developed and validated for routine application to fruit pulp crude extracts. Copyright © 2017 John Wiley & Sons, Ltd.

Structure and Function of Macroalgal Natural Products.

Since the initial discovery of marine phyco-derived secondary metabolites in the 1950s there has been a rapid increase in the description of new algal natural products. These metabolites have multiple ecological roles as well as commercial value as potential drugs or lead compounds. With the emergence of resistance to our current arsenal of drugs as well as the development of new chemotherapies for currently untreatable diseases, new compounds must be sourced. As outlined in this chapter algae produce a diverse range of chemicals many of which have potential for the treatment of human afflictions.In this chapter we outline the classes of metabolites produced by this chemically rich group of organisms as well as their respective ecological roles in the environment. Algae are found in nearly every environment on earth, with many of these organisms possessing the ability to shape the ecosystem they inhabit. With current challenges to climate stability, understanding how these important organisms interact with their environment as well as one another might afford better insight into how they respond to a changing climate.

An integrated approach using UHPLC-PDA-HRMS and 2D HSQC NMR for the metabolic profiling of the red alga Laurencia: dereplication and tracing of natural products.

The global metabolic profile of Laurencia crude red algal extracts was addressed by applying high-throughput analytical techniques, namely UHPLC­PDA­HRMS and 2D HSQC NMR. An integrated platform including software tools and databases, such as Xcalibur, ToxID, ACD/Labs and MarinLit, has been developed to mine the complex analytical data towards the accelerated identification of known metabolites and the detection of new natural products at the early stages of phytochemical analysis. In parallel, a searchable 'in-house' Laurencia-focused NMR database incorporating chemical structures, NMR spectroscopic data and reported biological activities has been generated. The screening strategy has been developed as a tool to prioritize the crude extracts to be further subjected to phytochemical analysis by tracing the presence of new natural products among the pool of known compounds. The successful application of this integrated methodology in the crude extract of Laurencia chondrioides led to the rapid detection of two new C15 bromoallene acetogenins (1 and 2), which were subsequently isolated and characterized.

Activity-guided identification of acetogenins as novel lipophilic antioxidants present in avocado pulp (Persea americana).

Avocado fruit is a rich source of health-related lipophilic phytochemicals such as monounsaturated fatty acids, tocopherols, carotenes, acetogenins and sterols. However, limited information is available on the contribution of specific phytochemicals to the overall antioxidant capacity (AOC) of the fruit. Centrifugal partition chromatography was used as fractionation tool, guided by an in vitro chemical assay of oxygen radical absorbance capacity (ORAC). Subsequent experiments focused on isolation and characterization of the chemical nature of the main contributors to lipophilic AOC of avocado pulp. ORAC values obtained for acetogenins were contrasted with results from an isolated kidney mitochondria membrane lipid peroxidation bioassay. The present study established that lipophilic AOC of the pulp was significantly higher than its hydrophilic AOC. Our results confirmed the presence of acetogenins in the fractions with highest lipophilic AOC, and for the first time linked them as contributors to lipophilic-ORAC values. Further HPLC-PDA/MS-TOF analysis led to structural elucidation of two novel acetogenins, not previously reported as present in avocado pulp, along with five already known related-compounds. Antioxidant properties observed for avocado pulp acetogenins by the ORAC assay suggested that, in the presence of an emulsifying agent, acetogenins could serve as novel lipophilic antioxidants in a food matrix. Results from isolated mitochondria lipid peroxidation bioassay, indicated that L-ORAC values which may have relevance for food matrix applications, should not be interpreted to have a direct relevance in health-related claims, compounds need to be evaluated considering the complexity of biological systems.

Comprehensive characterization of Annonaceous acetogenins within a complex extract by HPLC-ESI-LTQ-Orbitrap® using post-column lithium infusion.

Annonaceous acetogenins (AAGs) are a homogenous class of polyketides proposed as environmental neurotoxins. Previous dereplication studies of AAGs were limited by the use of low-resolution mass spectrometers. Only poor information in terms of structures was provided due to the limited fragmentation of protonated or sodium cationized species. An innovative approach, using reversed-phase high-performance liquid chromatography coupled to a hybrid linear ion trap/orbitrap mass spectrometer (LTQ-Orbitrap®), was therefore performed. Sensitivity was enhanced by post-column infusion of lithium, since AAGs have a high affinity for this cation. High level of structural information was obtained from low-energy-collision-induced dissociation fragmentation experiments of lithium-cationized AAGs ([M + Li](+) ions) as demonstrated with purified standards. The method was then applied to a total ethyl-acetate extract prepared from commercial soursop nectar (Annona muricata L.). The sensitivity, mass accuracy and specific fragmentation patterns proved to be particularly useful for characterization of the AAGs. Typical structural identification procedure and unexpected observations for specific structural types are illustrated, with major and minor compounds.

Anti-tumor activity of Annona squamosa seeds extract containing annonaceous acetogenin compounds.

ETHNOPHARMACOLOGICAL RELEVANCE: Seeds of Annona squamosa L. have been used in the south of China as a folk remedy to treat "malignant sores" (cancer). AIM OF THE STUDY: To investigate the chemical constituents and the anti-tumor activity of the standardized A. squamosa seeds extract in vitro and in vivo. MATERIALS AND METHODS: Annonaceous acetogenin profiles of the standardized extract were determined by using Fourier transform infrared (FT-IR) and high performance liquid chromatography (HPLC) techniques. The anti-tumor activity of the extract was tested by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) cytotoxicity in vitro and H(22) hepatoma cells transplantation tumor model in vivo. RESULTS: The FT-IR spectroscopy showed the presence of annonaceous acetogenin compounds in the extract. Two major annonaceous acetogenins: 12, 15-cis-squamostatin-A and bullatacin were identified and quantified by HPLC. The seed extract showed significant anti-tumor activity against four human tumor cell lines, especially for MCF-7 (IC(50). 0.25 µg/ml) and Hep G2 (IC(50). 0.36 µg/ml) cells in vitro. The extract inhibited the growth of H(22) tumor cells in mice with a maximum inhibitory rate of 69.55% by oral administration. CONCLUSION: A. squamosa seed extract showed significant anti-tumor activities against human hepatoma cells in vitro and in vivo, indicating a potential for developing the extract as a novel anti-liver cancer drug.

MALDI-TOF MS profiling of annonaceous acetogenins in Annona muricata products for human consumption.

Annonaceous acetogenins are proposed as environmental neurotoxicants consumed through medicinal and alimentary habits and responsible for atypical parkinsonian syndromes observed in tropical areas. Potential sources of exposure still have to be determined, as, to date, only a few batches of products for human consumption were searched for these compounds. To assess the presence of acetogenins, we propose a fast, sensitive and accurate method of screening, using MALDI-TOF MS, with minimal sample preparation. Development of the technique is discussed. Its application to leaves of herbal tea, pulp and bottled nectar of Annona muricata is presented.

Identification of annonaceous acetogenins in the ripe fruit of the North American pawpaw ( Asimina triloba ).

The North American pawpaw [ Asimina triloba (L.) Dunal] is a tree fruit in the early stages of commercial production in the United States. This plant contains annonaceous acetogenins in the twigs, unripe fruit, seeds, roots, and bark tissues, which display antitumor, pesticidal, antimalarial, anthelmintic, piscicidal, antiviral, and antimicrobial effects, suggesting many potentially useful applications. However, commercial development of these compounds, based on twig extracts, has been problematic due to limited availability of biomass for extraction. Additionally, acetogenin compounds contained in fruit of pawpaw relatives (soursop or Annona muricata ) and tea made from the leaves of these plants may lead to an increased risk of atypical Parkinsonism later in life with overconsumption of these compounds. Therefore, the objectives of this study were (1) to determine if extracts of ripe pawpaw fruit pulp displayed acetogenin activity, (2) to identify potential acetogenin compounds in the fruit tissue, and (3) to determine if the acetogenin activity varied in diverse pawpaw genotypes and closely related Annona species. Extracts of ripe fruit had total extract weights and bioactivity using the brine shrimp bioassay similar to those from 'NC-1' pawpaw twig tissue. Pulp from soursop, cherimoya, and several additional pawpaw cultivars ('Mitchell', 'Overleese', 'NC-1','Zimmerman', 'Wells', and 'Sunflower') also displayed bioactivity, but peach or banana pulp did not. Ripe pawpaw pulp extract subjected to HPLC-MS analysis identified three prominent acetogenins: asimicin, bullatacin, and bullatalicin. This study points to pawpaw fruit pulp serving as a new biomass source for the extraction of acetogenin compounds for product development. An assessment of the potential human health risk of overconsumption of fruit and acetogenin bioavailability and degradation studies should be pursued.

Supercritical fluid CO2 extraction and simultaneous determination of eight annonaceous acetogenins in Annona genus plant seeds by HPLC-DAD method.

Annonaceous acetogenins (ACGs) isolated from Annonaceae plants exhibited a broad range of biological bioactivities such as cytotoxic, antitumoral, antiparasitic, pesticidal and immunosuppresive activities. However, their structures were liable to change at more than 60 degrees C and their extraction yields were low using traditional organic solvent extraction. In the present study, all samples from Annona genus plant seeds were extracted by supercritical carbon dioxide under optimized conditions and a high-performance liquid chromatography (HPLC) method was established for simultaneously determining eight ACGs. All of the eight compounds were simultaneously separated on reversed-phase C(18) column (250 mm x 4.6 mm, 5 microm) with the column temperature at 30 degrees C. The mobile phase was composed of (A) methanol and (B) distilled water, the flow rate was 1.0 ml/min and the detection wavelength was set at 220 nm. All calibration curves showed good linear regression (gamma>0.9995) within the test range. The established method showed good precision and accuracy with overall intra-day and inter-day variations of 0.87-2.53% and 1.91-3.42%, respectively, and overall recoveries of 95.81-105.39% for the eight compounds analyzed. The established method can be applied to evaluate the intrinsic quality of Annonaceae plant seeds. The determination results recover the content-variation regularities of various ACGs in different species, which are helpful to choose the good-quality Annonaceae plant seeds for anticancer lead compound discovery.