From the Spectroscopic Reassessment of Authentic Alkaloid Samples to the Molecular Networking-Guided Discovery of Criophylline-Related Analogues from Callichilia inaequalis.

The molecular network-guided exploration of the alkaloid extract of Callichilia inaequalis stems revealed a cluster attributed tentatively to dimeric monoterpene indole alkaloids of the rare criophylline subtype, initiating the dual study reported herein. A patrimonial-themed portion of this work was aimed at performing a spectroscopic reassessment of criophylline (1), a monoterpene bisindole alkaloid for which the nature of the inter-monomeric connectivity and configurational assignments have remained dubious. A targeted isolation of the entity annotated as criophylline (1) was undertaken to strengthen the available analytical evidence. An extensive set of spectroscopic data was acquired from the authentic sample of criophylline (1a) isolated earlier by Cavé and Bruneton. These spectroscopic studies proved the samples to be identical, and the complete structure of criophylline could be assigned, half a century after it was first isolated. The absolute configuration of andrangine (2) was also ascertained based on a TDDFT-ECD approach from the authentic sample. The forward-looking aspect of this investigation resulted in the characterization of two new criophylline derivatives from C. inaequalis stems, namely, 14'-hydroxycriophylline (3) and 14'-O-sulfocriophylline (4). Their structures, including absolute configurations, were elucidated by analysis of NMR and MS spectroscopic data and by ECD analysis. Notably, 14'-O-sulfocriophylline (4) is the first sulfated monoterpene indole alkaloid to have been reported. The antiplasmodial activity against the chloroquine-resistant strain of Plasmodium falciparum FcB1 was determined for criophylline and its two new analogues.

Five new cassane diterpenes from the seeds and bark of Erythrophleum suaveolens.

Five new cassane-type diterpenoid heterosides, i. e. two cassane-type amides (1-2), two erythrophlamine-type amine esters (3-4) and a non­nitrogenous erythrophlamine analogue (5) were isolated from the root barks (1-2) and the seeds (3-5) of Erythrophleum suaveolens. Their structures were unambiguously established by interpretation of their HRESIMS, 1D and 2D NMR data, and chemical degradation for sugar determination. Compounds 3-5 were evaluated for their cytotoxicity against a panel of three cell lines, revealing modest to strong activities.

Streamlined targeting of Amaryllidaceae alkaloids from the bulbs of Crinum scillifolium using spectrometric and taxonomically-informed scoring metabolite annotations.

Four undescribed alkaloids have been isolated from the bulbs of the previously unstudied Crinum scillifolium. These compounds were targeted following a state-of-the-art molecular networking strategy comprising a dereplication against in silico databases and re-ranking of the candidate structures based on taxonomically informed scoring. The unreported structures span across a variety of Amaryllidaceae alkaloids appendages. Their structures were unambiguously elucidated by thorough interpretation of their HRESIMS and 1D and 2D NMR data, and comparison to literature data. DFT-NMR calculations were performed to support the determined relative configurations of scillitazettine and scilli-N-desmethylpretazettine and their absolute configurations were mitigated by comparison between experimental and theoretically calculated ECD spectra. The lack of a methyl group on the nitrogen atom in the structure of scilli-N-desmethylpretazettine series is highly unusual in the pretazettine/tazettine series but the most original structural feature in it lies in its 11α disposed hydrogen, which is new to pretazettines. The antiplasmodial as well as the cytotoxic activities against the human colon cancer cell line HCT116 were evaluated, revealing mild to null activities.

Structural Characterisation of Acetogenins from Annona muricata by Supercritical Fluid Chromatography Coupled to High-Resolution Tandem Mass Spectrometry.

INTRODUCTION: Acetogenins are plant polyketides known to be cytotoxic and proposed as antitumor candidates. They are also suspected to be alimentary neurotoxins. Their occurrence as complex mixtures renders their dereplication and structural identification difficult using liquid chromatography coupled to tandem mass spectrometry and efforts are required to improve the methodology. OBJECTIVE: To develop a supercritical fluid chromatography (SFC) high-resolution tandem mass spectrometry method, involving lithium post-column cationisation, for the structural characterisation of Annonaceous acetogenins in crude extracts. METHODOLOGY: The seeds of Annona muricata L. were extracted with methanol. Supercritical fluid chromatography of the extract, using a 2-ethylpyridine stationary phase column, was monitored using a high-resolution quadrupole time-of-flight mass spectrometer. Lithium iodide was added post-column in the make-up solvent. For comparison, the same extract was analysed using high-pressure liquid chromatography coupled to the same mass spectrometer, with a column based on solid core particles. RESULTS: Sensitivity was similar for both HPLC and SFC approaches. Retention behaviour and fragmentation pathways of three different isomer groups are described. A previously unknown group of acetogenins was also evidenced for the first time. CONCLUSION: The use of SFC-MS/MS allows the reduction of the time of analysis, of environmental impact and an increase in the chromatographic resolution, compared to liquid chromatography. This new methodology enlightened a new group of acetogenins, isomers of montanacin-D. Copyright © 2017 John Wiley & Sons, Ltd.

Revisiting Previously Investigated Plants: A Molecular Networking-Based Study of Geissospermum laeve.

Three new monoterpene indole alkaloids (1-3) have been isolated from the bark of Geissospermum laeve, together with the known alkaloids (-)-leuconolam (4), geissolosimine (5), and geissospermine (6). The structures of 1-3 were elucidated by analysis of their HRMS and NMR spectroscopic data. The absolute configuration of geissolaevine (1) was deduced from the comparison of experimental and theoretically calculated ECD spectra. The isolation workflow was guided by a molecular networking-based dereplication strategy using an in-house database of monoterpene indole alkaloids. In addition, five known compounds previously undescribed in the Geissospermum genus were dereplicated from the G. laeve alkaloid extract network and were assigned with various levels of identification confidence. The antiparasitic activities against Plasmodium falciparum and Leishmania donovani as well as the cytotoxic activity against the MRC-5 cell line were determined for compounds 1-5.

"Squalenoylcurcumin" nanoassemblies as water-dispersible drug candidates with antileishmanial activity.

Curcumin, a natural polyphenolic compound, showed antiparasitic potential, including trypanocidal and leishmanicidal activity, in several in vitro and in vivo models. The molecule is well tolerated in humans. However, it is insoluble in water and displays poor oral bioavailability as a result of low absorption. New derivatives of curcumin were prepared by esterification of one or two of its phenolic groups with 1,1',2-tris-norsqualenic acid. These "squalenoylcurcumins" were formulated as water-dispersible nanoassemblies of homogeneous size, and they proved to be stable. Squalenoylcurcumins were inactive against Trypanosoma brucei brucei trypomastigotes, even as nanoassemblies, in contrast with curcumin. However, against Leishmania donovani promastigotes, the activities of the squalenoylcurcumins and their nanoassemblies were enhanced relative to that of curcumin. In L. donovani axenic and intramacrophagic amastigotes, they showed activity in the range of miltefosine, with good selectivity indexes. In regard to their dispersibility in water and to the safety of curcumin, these nanoassemblies are promising candidates for preclinical study toward the treatment of visceral leishmaniasis.

Establishing high temperature gas chromatographic profiles of non-polar metabolites for quality assessment of African traditional herbal medicinal products.

The quality assessment of African traditional herbal medicinal products is a difficult challenge since they are complex mixtures of several herbal drug or herbal drug preparations. The plant source is also often unknown and/or highly variable. Plant metabolites chromatographic profiling is therefore an important tool for quality control of such herbal products. The objective of this work is to propose a protocol for sample preparation and gas chromatographic profiling of non-polar metabolites for quality control of African traditional herbal medicinal products. The methodology is based on the chemometric assessment of chromatographic profiles of non-polar metabolites issued from several batches of leaves of Combretum micranthum and Mitracarpus scaber by high temperature gas chromatography coupled to mass spectrometry, performed on extracts obtained in refluxed dichloromethane, after removal of chlorophyll pigments. The method using high temperature gas chromatography after dichloromethane extraction allows detection of most non-polar bioactive and non-bioactive metabolites already identified in leaves of both species. Chemometric data analysis using Principal Component Analysis and Partial Least Squares after Orthogonal Signal Correction applied to chromatographic profiles of leaves of Combretum micranthum and Mitracarpus scaber showed slight batch to batch differences, and allowed clear differentiation of the two herbal extracts.

Pyrone and unusually furanone-substituted flavones from the leaves of Hoslundia opposita.

Two new unusual 6-furanoflavones, hoslunfuranine (5) and 5-O-methylhoslunfuranine (6), were isolated from the leaves of Hoslundia opposita Vahl.. Four known methylpyranoflavonic analogues [hosloppin (1), hoslundin (2), 5-O-methylhoslundin (3), oppositin (4)], all specific of the species, were also obtained. Their structures were established on the basis of their spectroscopic data. In vitro cytotoxic, trypanocidal, and leishmanicidal activities of compounds 1 and 3 to 6 were evaluated. Compounds 4 and 6 exhibited leishmanicidal potential in the micromolar range.

Composition, and antimicrobial and remarkable antiprotozoal activities of the essential oil of rhizomes of Aframomum sceptrum K. Schum. (Zingiberaceae).

The essential oil from the rhizomes of Aframomum sceptrum (Zingiberaceae) was analyzed by GC/MS, and its major constituents were found to be ß-pinene (12.7%), caryophyllene oxide (10.0%), and cyperene (6.0%). The oil was also evaluated for antimicrobial activities, in comparison with ß-pinene, caryophyllene oxide, and the leaf essential oil of Melaleuca alternifolia (Myrtaceae). The A. sceptrum essential oil exhibited bacteriostatic activity against the Gram-positive bacteria Bacillus subtilis, Staphylococcus epidermidis, and S. aureus, but not against Gram-negative bacteria. Moreover, it showed mild fungicidal activity against Candida albicans and Aspergillus fumigates, and remarkable antiprotozoal activity against Trypanosoma brucei brucei (MLC of 1.51 µl/ml) and Trichomonas vaginalis (IC(50) of 0.12±0.02 and MLC of 1.72 µl/ml).

Natural lipophilic inhibitors of mitochondrial complex I are candidate toxins for sporadic neurodegenerative tau pathologies.

Annonacin, a natural lipophilic inhibitor of mitochondrial complex I has been implicated in the etiology of a sporadic neurodegenerative tauopathy in Guadeloupe. We therefore studied further compounds representing the broad biochemical spectrum of complex I inhibitors to which humans are potentially exposed. We determined their lipophilicity, their effect on complex I activity in submitochondrial particles, and their effect on cellular ATP levels, neuronal cell death and somatodendritic redistribution of phosphorylated tau protein (AD2 antibody against pS396/pS404-tau) in primary cultures of fetal rat striatum. The 24 compounds tested were lipophilic (logP range 0.9-8.5; exception: MPP(+) logP=-1.35) and potent complex I inhibitors (IC(50) range 0.9 nM-2.6 mM). They all decreased ATP levels (EC(50) range 1.9 nM-54.2 microM), induced neuronal cell death (EC(50) range 1.1 nM-54.5 microM) and caused the redistribution of AD2(+) tau from axons to the cell body (EC(5) range 0.6 nM-33.3 microM). The potency of the compounds to inhibit complex I correlated with their potency to induce tau redistribution (r=0.80, p<0.001). In conclusion, we propose that the widely distributed lipophilic complex I inhibitors studied here might be implicated in the induction of tauopathies with global prevalence.

Annonacin, a natural mitochondrial complex I inhibitor, causes tau pathology in cultured neurons.

A neurodegenerative tauopathy endemic to the Caribbean island of Guadeloupe has been associated with the consumption of anonaceous plants that contain acetogenins, potent lipophilic inhibitors of complex I of the mitochondrial respiratory chain. To test the hypothesis that annonacin, a prototypical acetogenin, contributes to the etiology of the disease, we investigated whether annonacin affects the cellular distribution of the protein tau. In primary cultures of rat striatal neurons treated for 48 h with annonacin, there was a concentration-dependent decrease in ATP levels, a redistribution of tau from the axons to the cell body, and cell death. Annonacin induced the retrograde transport of mitochondria, some of which had tau attached to their outer membrane. Taxol, a drug that displaces tau from microtubules, prevented the somatic redistribution of both mitochondria and tau but not cell death. Antioxidants, which scavenged the reactive oxygen species produced by complex I inhibition, did not affect either the redistribution of tau or cell death. Both were prevented, however, by forced expression of the NDI1 nicotinamide adenine dinucleotide (NADH)-quinone-oxidoreductase of Saccharomyces cerevisiae, which can restore NADH oxidation in complex I-deficient mammalian cells and stimulation of energy production via anaerobic glycolysis. Consistently, other ATP-depleting neurotoxins (1-methyl-4-phenylpyridinium, 3-nitropropionic, and carbonyl cyanide m-chlorophenylhydrazone) reproduced the somatic redistribution of tau, whereas toxins that did not decrease ATP levels did not cause the redistribution of tau. Therefore, the annonacin-induced ATP depletion causes the retrograde transport of mitochondria to the cell soma and induces changes in the intracellular distribution of tau in a way that shares characteristics with some neurodegenerative diseases.

A new adjacent bis-tetrahydrofuran annonaceous acetogenin from the seeds of Uvaria chamae.

A novel acetogenin, joolanin ( 1), along with eight known acetogenins, squamocin ( 2), desacetyluvaricin ( 3), chamuvarinin ( 4), tripoxyrollin ( 5), diepoxyrollin ( 6), dieporeticanin-1 ( 7), dieporeticanin-2 ( 8) and dieporeticenin ( 9) were isolated from the seeds of Uvaria chamae (Annonaceae). Compound 1, the first adjacent bis-tetrahydrofuranic ring acetogenin bearing a ketone group at C-5, shows significant cytotoxicity towards the KB 3 - 1 cell line (IC (50) = 0.4 nM).

The mitochondrial complex I inhibitor rotenone triggers a cerebral tauopathy.

Reduced activity of the mitochondrial respiratory chain--particularly complex I--may be implicated in the etiology of both Parkinson's disease and progressive supranuclear palsy, although these neurodegenerative diseases differ substantially as to their distinctive pattern of neuronal cell loss and the predominance of cerebral alpha-synuclein or tau protein pathology. To determine experimentally whether chronic generalized complex I inhibition has an effect on the distribution of alpha-synuclein or tau, we infused rats systemically with the plant-derived isoflavonoid rotenone. Rotenone-treated rats with a pronounced metabolic impairment had reduced locomotor activity, dystonic limb posture and postural instability. They lost neurons in the substantia nigra and in the striatum. Spherical deposits of alpha-synuclein were observed in a few cells, but cells with abnormal cytoplasmic accumulations of tau immunoreactivity were significantly more numerous in the striatum of severely lesioned rats. Abnormally high levels of tau immunoreactivity were found in the cytoplasm of neurons, oligodendrocytes and astrocytes. Ultrastructurally, tau-immunoreactive material consisted of straight 15-nm filaments decorated by antibodies against phosphorylated tau. Many tau+ cell bodies also stained positive for thioflavin S, nitrotyrosine and ubiquitin. Some cells with abnormal tau immunoreactivity contained activated caspase 3. Our data suggest that chronic respiratory chain dysfunction might trigger a form of neurodegeneration in which accumulation of hyperphosphorylated tau protein predominates over deposits of alpha-synuclein.

Annonacin, a lipophilic inhibitor of mitochondrial complex I, induces nigral and striatal neurodegeneration in rats: possible relevance for atypical parkinsonism in Guadeloupe.

In Guadeloupe, epidemiological data have linked atypical parkinsonism with fruit and herbal teas from plants of the Annonaceae family, particularly Annona muricata. These plants contain a class of powerful, lipophilic complex I inhibitors, the annonaceous acetogenins. To determine the neurotoxic potential of these substances, we administered annonacin, the major acetogenin of A. muricata, to rats intravenously with Azlet osmotic minipumps (3.8 and 7.6 mg per kg per day for 28 days). Annonacin inhibited complex I in brain homogenates in a concentration-dependent manner, and, when administered systemically, entered the brain parenchyma, where it was detected by matrix-associated laser desorption ionization-time of flight mass spectrometry, and decreased brain ATP levels by 44%. In the absence of evident systemic toxicity, we observed neuropathological abnormalities in the basal ganglia and brainstem nuclei. Stereological cell counts showed significant loss of dopaminergic neurones in the substantia nigra (-31.7%), and cholinergic (-37.9%) and dopamine and cyclic AMP-regulated phosphoprotein (DARPP-32)-immunoreactive GABAergic neurones (-39.3%) in the striatum, accompanied by a significant increase in the number of astrocytes (35.4%) and microglial cells (73.4%). The distribution of the lesions was similar to that in patients with atypical parkinsonism. These data are compatible with the theory that annonaceous acetogenins, such as annonacin, might be implicated in the aetiology of Guadeloupean parkinsonism and support the hypothesis that some forms of parkinsonism might be induced by environmental toxins.

Chronic systemic complex I inhibition induces a hypokinetic multisystem degeneration in rats.

In Parkinson's disease, nigral dopaminergic neurones degenerate, whereas post-synaptic striatal target neurones are spared. In some atypical parkinsonian syndromes, both nigral and striatal neurones degenerate. Reduced activity of complex I of the mitochondrial respiratory chain has been implicated in both conditions, but it remains unclear if this affects the whole organism or only the degenerating brain structures. We therefore investigated the differential vulnerability of various brain structures to generalized complex I inhibition. Male Lewis rats infused with rotenone, a lipophilic complex I inhibitor [2.5 mg/kg/day intraveneously (i.v.) for 28 days], were compared with vehicle-infused controls. They showed reduced locomotor activity and loss of striatal dopaminergic fibres (54%), nigral dopaminergic neurones (28.5%), striatal serotoninergic fibres (34%), striatal DARPP-32-positive projection neurones (26.5%), striatal cholinergic interneurones (22.1%), cholinergic neurones in the pedunculopontine tegmental nucleus (23.7%) and noradrenergic neurones in the locus ceruleus (26.4%). Silver impregnation revealed pronounced degeneration in basal ganglia and brain stem nuclei, whereas the hippocampus, cerebellum and cerebral cortex were less affected. These data suggest that a generalized mitochondrial failure may be implicated in atypical parkinsonian syndromes but do not support the hypothesis that a generalized complex I inhibition results in the rather selective nigral lesion observed in Parkinson's disease.