Proteomic analyses of venom from a Spider Hawk, Pepsis decorata.

Background: The composition of the venom from solitary wasps is poorly known, although these animals are considered sources of bioactive substances. Until the present moment, there is only one proteomic characterization of the venom of wasps of the family Pompilidae and this is the first proteomic characterization for the genus Pepsis. Methods: To elucidate the components of Pepsis decorata venom, the present work sought to identify proteins using four different experimental conditions, namely: (A) crude venom; (B) reduced and alkylated venom; (C) trypsin-digested reduced and alkylated venom, and; (D) chymotrypsin-digested reduced and alkylated venom. Furthermore, three different mass spectrometers were used (Ion Trap-Time of Flight, Quadrupole-Time of Flight, and Linear Triple Quadruple). Results: Proteomics analysis revealed the existence of different enzymes related to the insect's physiology in the venom composition. Besides toxins, angiotensin-converting enzyme (ACE), hyaluronidase, and Kunitz-type inhibitors were also identified. Conclusion: The data showed that the venom of Pepsis decorata is mostly composed of proteins involved in the metabolism of arthropods, as occurs in parasitic wasps, although some classical toxins were recorded, and among them, for the first time, ACE was found in the venom of solitary wasps. This integrative approach expanded the range of compounds identified in protein analyses, proving to be efficient in the proteomic characterization of little-known species. It is our understanding that the current work will provide a solid base for future studies dealing with other Hymenoptera venoms.

A new cyclodipeptide from Tetragonisca angustula honey active against Neospora caninum and in silico study.

A new cyclic natural compound formed by succinic acid and two alanine amino acid units was isolated from the Tetragonisca angustula honey extract. The chemical structure of 1 was established based on spectroscopic data analysis, including one- (1H and 13C NMR) and two-dimensional NMR techniques (1H-1H-COSY, HSQC and HMBC). A primary culture model previously infected with Neospora caninum was used to evaluate 1 for two time intervals (24 and 72 h), showing a reduction (40-56%) of the number of tachyzoites in the first 24 h and until 72 h, a dose-dependent reduction in parasite proliferation (25-50%). Glial cells treated with 1 did not demonstrate toxicity at concentrations up to 25 ug/mL. Treated and infected cultures showed an increase in NO when compared to control cells in 24 h and 72 h. In silico studies suggest that the new compound may affect DNA synthesis and impair -protein production.

Chemistry and Bioactivity of the Genus Persea - A Review.

This review provides the first comprehensive appraisal of bioactive compounds and their biological activities in Persea species from 1950 to 2023. Relevant articles from reputable databases, including PubMed, Web of Science, Science Direct and Google Scholar were collected, leading to the isolation of about 141 metabolite compounds, mainly flavonoids, terpenoids, fatty alcohols, lignoids, and γ-lactone derivatives. These compounds exhibit diverse biological activities, including insecticidal, antifeedant, nematicidal, antibacterial, antifungal, antiviral, cytotoxic, anti-inflammatory, and antioxidant properties. The review emphasizes the significant chemical and pharmacological potential of different Persea species, encouraging further research in various fields and medicine. Valuable insights into potential applications of Persea plants are provided.

Two new dilactonized glycerol glycosides of the dual anticholinesterase active extract from Ocotea daphnifolia using bioguided fractionation and molecular docking studies.

The dual inhibition of acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) is considered as an important strategy for the treatment of Alzheimer's disease. In this study, we applied the bioguided fractionations of Ocotea daphinifolia ethyl acetate active extract to furnish a fraction with high inhibitory activity for AChE and BuChE (82% and 92%, respectively). High-performance liquid chromatography semipreparative purification of this fraction provided two new natural products: 1-ß-D-galactopyranosyl-glycerol-2,3-heptanedionate, (1) whose complete chemical structural elucidation was made with spectrometric analysis (MS, 1D, and 2D NMR) and its minor derivative 1-ß-D-gulopyranosyl-glycerol-2,3-heptanedionate; (2) which could be characterized by 2D 1 H-13 C heteronuclear single-quantum correlation spectra analysis. Investigation of the intermolecular interactions with cholinesterases was carried out by molecular docking studies, and results suggested that both compounds are capable to interact with the catalytic site of both enzymes. Compounds 1 and 2 interact with residues of catalytic domains and the peripheral anionic binding site of AChE and BuChE. The results are comparable to those achieved with rivastigmine and galantamine. Thus, this study provides evidence for consideration of the glycosylglycerol from O. daphnifolia as new valuable dual cholinesterases inhibitor.

Bothrops leucurus snake venom protein profile, isolation and biological characterization of its major toxin PLA2s-like.

Bothrops leucurus is considered as a snake of medical interest in the State of Bahia, Brazil. However, so far, there are no studies that provide a refined mapping of the composition of this venom. The aim of this work was to better understand the protein composition of B. leucurus snake venom and to isolate and biologically characterize the most abundant toxin, a basic PLA2-like. Shotgun proteomics approach identified 137 protein hits in B. leucurus venom subdivided into 19 protein families. The new basic PLA2-like toxin identified was denominated Bleu-PLA2-like, it and other proteoforms represents about 25% of the total proteins in the venom of B. leucurus and induces myotoxicity, inflammation and muscle damage. Immunoreactivity assays demonstrated that B. leucurus venom is moderately recognized by bothropic and crotalic antivenoms, and on the other hand, Bleu-PLA2-like and its proteoforms are poorly recognized. Our findings open doors for future studies in order to assess the systemic effects caused by this snake venom in order to better understand the toxinological implications of this envenomation and, consequently, to assist in the clinical treatment of victims.

Activation of the Kynurenine Pathway and Production of Inflammatory Cytokines by Astrocytes and Microglia Infected With Neospora caninum.

In the central nervous system, astrocytes and microglia contribute to homeostasis, regulating the immune response to infectious agents. Neospora caninum is an obligate intracellular protozoan that infects different animal species and it is encysted in their nervous tissue while triggering an immune response modulated by glia. This study aimed to evaluate the infection of primary cultures of rat glial cells by N. caninum through the catabolites of tryptophan, the expression of inflammatory mediators and the integrity of neural tissue. Infection with this coccidium resulted in morphological and functional changes, particularly astrogliosis and microgliosis, and increased the expression of the inflammatory mediators TNF, IL1ß, IL-10, and arginase, as well as mRNA for CCL5 and CCL2, molecules involved in the CNS chemotaxis. The infection with N. caninum in glial cells also triggered the activation of the tryptophan pathway, characterized by increased kynurenine 2,3 monooxygenase (KMO) mRNA expression, and by the production of the excitotoxin quinolinic acid (QUIN). Moreover, glia-neuron co-cultures, when exposed to the secretome derived from N. caninum infected glial cells, presented greater neurons distribution and formation of neurite extensions, associated to morphological changes in astrocytes compatible with neuro-preservation. Considering that the tryptophan catabolism is associated to immune response, these findings suggest that glial activation in N. caninum infection should be responsible for modulating the inflammatory status in an attempt to restore the nervous system homeostasis, since excessive inflammatory response can cause irreversible damage to tissue preservation.

Acetylcholinesterase inhibitory activity of Ocotea pomaderroides extracts: HPLC-MS/MS characterization and molecular modeling studies.

The present work describes the acetylcholinesterase inhibitory activity of Ocotea pomaderroides extracts besides the chemical composition of chromatographic fractions. The hexane, dichloromethane and ethyl acetate extract soluble fractions showed high Electrophorus electricus acetylcholinesterase (EelAChE) inhibition (92.18, 71.86 and 74.25%, respectively) while the butanolic and aqueous extracts showed moderate to low activities (44.48 and 20.74%, respectively). The high-performance liquid chromatography coupled with electrospray ionization multiple-stage mass spectrometry (HPLC-ESI-MSn) analysis led to the identification of the alkaloids and flavonol glycoside derivatives present in these extracts. The binding profile of the alkaloids and their atomic effect on 3D structure of Electrophorus electricus AchE (EelAChE) were assessed with molecular modeling.

Methoxylated flavonols from Vellozia dasypus Seub ethyl acetate active myeloperoxidase extract: in vitro and in silico assays.

This study aimed to evaluate the effect of a methoxylated fraction from Vellozia dasypus Seub on myeloperoxidase (MPO)-chlorinating activity and subsequent in silico assays for binding profile prediction. Therefore, the ethyl acetate extract of aerial parts from Vellozia dasypus Seub was fractionated on open-column chromatography containing SiO2 and eluted with solvent in crescent polarity to yield a fraction with a mixture of flavonols quercetin 3-O-methyl ether (1) and 6-C-methyl quercetin 3-O-methyl ether (2). Their chemical structures were proposed by HPLC coupled to photodiode array (HPLC-DAD) and mass spectrometer using electrospray ionization multistage analysis (HPLC-MS/MS). The fraction enriched with compounds 1 and 2 inhibited more efficiently the in vitro MPO-chlorinating activity (IC50 = 40 µg/mL) than the ethyl acetate extract (IC50 = 64.0 µg/mL). Molecular docking studies revealed that these compounds interact with MPO active pocket similarly to trifluoromethyl-substituted aromatic hydroxamate, a well-known MPO inhibitor, co-crystallized at the MPO binding site (PDB ID: 4C1M). Molecular dynamics trajectories confirmed that these two molecules interact with the MPO binding site with a similar energetic pattern when compared to the crystallographic ligand. Taken together, these data expand the sources of phenolic natural compounds that may be further investigated against inflammation-related diseases. Communicated by Ramaswamy H. Sarma.

Homology modeling, docking, molecular dynamics and in vitro studies to identify Rhipicephalus microplus acetylcholinesterase inhibitors.

Rhipicephalus microplus is an important ectoparasite of cattle, causing considerable economical losses. Resistance to chemical acaricides has stimulated the search for new antiparasitic drugs, including natural products as an eco-friendly alternative of control. Flavonoids represent a class of natural compounds with many biological activities, such as enzyme inhibitors. Acetylcholinesterase is an essential enzyme for tick survival that stands out as an important target for the development of acaricides. This work aimed to predict this 3D structure by homology modeling and use the model to identify compound with inhibitory activity. The model of R. microplus AChE1 (RmAChE1) was constructed using MODELLER program. The optimization and molecular dynamic investigation were performed in GROMACS program. The model developed was used, by molecular docking, to evaluate the anticholinesterase activity of flavonoids (quercetin, rutin, diosmin, naringin and hesperidin) and an acaricide synthetic (eserine). Additionally, in vitro inhibition of AChE and larval immersion tests were performed. The model of RmAChE1 showed to be sterically and energetically acceptable. In molecular dynamics simulations, the 3D structure remains stable with Root Mean Square Deviation = 3.58 Å and Root Mean Square Fluctuation = 1.43 Å. In molecular docking analyses, only eserine and quercetin show affinity energy to the RmAChE (Gridscore: -52.17 and -39.44 kcal/mol, respectively). Among the flavonoids, quercetin exhibited the best in vitro inhibition of AChE activity (15.8%) and mortality of larvae tick (30.2%). The use of in silico and in vitro techniques has shown that quercetin showed promising anti-tick activity and structural requirements to interact with RmAChE1. Communicated by Ramaswamy H. Sarma.

Hydroxycinnamic acid-spermidine amides from Tetragonisca angustula honey as anti-Neospora caninum: In vitro and in silico studies.

Tetragonisca angustula honey was fractioned in a SiO2 column to furnish three fractions (A-C) in which four hydroxycinnamic acid-Spermidine amides (HCAAs), known as N', N″, N‴-tris-p-coumaroyl spermidine, N', N″-dicaffeoyl, N‴-coumaroyl spermidine, N', N″, N‴-tris-caffeoyl spermidine and N', N″-dicaffeoyl and N‴-feruloyl spermidine were identified in the fractions B and C by electrospray ionization tandem mass spectrometry. A primary culture model previously infected with Neospora caninum (72 h) was used to evaluate the honey fractions (A-C) for two-time intervals: 24 and 72 h. Parasitic reduction ranged from 38% on fraction C (12.5 µg/ml), after 24 h, to 54% and 41% with fractions B and C (25 µg/ml) after 72 h of treatment, respectively. Additionally, HCAAs did not show any cell toxicity for 24 and 72 h. For infected cultures (72 h), the active fractions B (12.5 µg/ml) and C (25 µg/ml) decreased their NO content. In silico studies suggest that HCAAs may affect the parasite's redox pathway and improve the oxidative effect of NO released from infected cells. Here, we presented for the first time, that HCAAs from T. angustula honey have the potential to inhibit the growth of N. caninum protozoa.

Gastroprotective Activity of Neoglaziovia variegata (Arruda) Mez. (Bromeliaceae) in Rats and Mice.

Neoglaziovia variegata (Arruda) Mez (Bromeliaceae) is a medicinal plant popularly known as "caroá." The leaves are made up of highly resistant fibers, which is of great commercial value to the handicraft and textile industry. Some studies have demonstrated that ethanolic extract of N. variegata have gastroprotective properties. This study aimed to investigate the gastroprotective activity and cytoprotective mechanisms of ethyl acetate (Nv-AcOEt), hexane (Nv-Hex), and chloroform (Nv-CHCl3) fractions of N. variegata leaves. The gastroprotective activity of Nv-AcOEt, Nv-Hex, and Nv-CHCl3 was evaluated using the ethanol and ethanol/HCl-induced gastric injury model. To elucidate the gastroprotective mechanisms, the functions of prostaglandins (PGs), nitric oxide (NO), and KATP channels were evaluated. In addition, the nonprotein sulfhydryl groups and the mucus content in the gastric tissues were analyzed. All fractions of N. variegata leaves at oral doses of 100, 200, and 400 mg/kg significantly decreased ethanol and ethanol/HCl-induced gastric lesions, leading to gastroprotection, accompanied by an increase in reduced glutathione (GSH) and gastric mucus. Gastroprotective activity of Nv-AcOEt was inhibited after pretreatment with ibuprofen and N(G)-nitro-L-arginine (L-NOARG). Gastroprotective effect of Nv-Hex and Nv-CHCl3 was also inhibited after pretreatment with L-NOARG and with glibenclamide. The results indicate that N. variegata (Arruda) Mez exhibits promising gastroprotective activity with the possible participation of NO, PGs, mucus, sulfhydryl groups, and KATP.

In vitro anthelmintic evaluation of three alkaloids against gastrointestinal nematodes of goats.

This study assessed the in vitro anthelmintic activity of the alkaloids berberine, harmaline and piperine on gastrointestinal nematodes (GIN) of goat and their possible cytotoxic effects in Vero cells. The anthelmintic evaluation was performed using the egg hatch (EHA) and larval motility (LMA) assays. Cytotoxicity was determined using the 3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyl-tetrazolium bromide (MTT) assay. The alkaloids berberine and piperine inhibited the hatching of GIN eggs in more than 90 %. Piperine was the most active compound against goat GIN eggs with an EC50 (effective concentration 50 %) of 0.0074 mM (0.0021 mg/mL), while the EC50 of berberine was 1.32 mM (0.49 mg/mL). Harmaline (EC50 = 1.6 mM - 0.34 mg/mL) showed moderate ovicidal action (80.30 %). In LMA, piperine and harmaline reduced larval motility in 2.75 and 25.29 %, respectively. Larvicidal efficacy was evidenced only with the alkaloid berberine, which showed a percentage of inhibition of larval motility of 98.17 % (2.69 mM =1.0 mg/mL). In the MTT assay, all alkaloids showed low toxicity to Vero cells, with a percentage of cell viability greater than 50 % in all concentrations tested. These results suggest that berberine and piperine have anthelmintic potential on goat gastrointestinal nematodes with low toxicity to mammalian cells.

γ-Lactones from Persea americana and Persea fulva - in Vitro and in Silico Evaluation of Trypanosoma cruzi Activity.

In the present study, five known γ-lactones (majoranolide B - 1, majorenolide - 2, majorynolide - 3, lincomolide D - 4, and isolinderanolide E - 5), as well as a new one (perseanolide - 6), were isolated from Persea fulva and P. americana. All isolated compounds exhibited potential activity against trypomastigote forms of Trypanosoma cruzi, whereas compounds 2 (EC50 of 4.8 µM) and 6 (EC50 of 3.6 µM) displayed superior activity than the positive control benznidazole (EC50 of 16.4 µM), with selectivity index (SI) values of 17.8 and >55.6, respectively (benznidazole, SI>12.2). Molecular docking studies were performed for 1-6 against six T. cruzi molecular targets. Using this approach, we observed that, even though perseanolide (6) showed favorable docking to several studied targets, the results were especially promising for hypoxanthine phosphoribosyl transferase (PDB 1TC1). As PDB 1TC1 is associated to the transference of a monophosphorylated ribose from phosphoribosylpyrophosphate (PRPP) in the ribonucleotide synthesis pathway, this interaction may affect the survival of T. cruzi in mammalian cells. The data herein also indicate that possible intermolecular interactions between 6 and PDB 1TC1 derive from (i) hydrogen bonds in the α,ß-unsaturated-γ-lactone unity and (ii) hydrophobic interactions in the long-chain alkyl group. Based on our results, perseanolide (6), reported for the first time in this work, can auspiciously contribute to future works regarding new trypanocidal agents.

In vitro and in silico studies of the larvicidal and anticholinesterase activities of berberine and piperine alkaloids on Rhipicephalus microplus.

Rhipicephalus microplus is responsible for high economic losses in livestock and its control has become difficult due to the establishment of tick populations resistant to commercial acaricides. This study aimed to evaluate the in vitro larvicidal effect of the alkaloids berberine and piperine, and also to investigate their inhibitory mechanisms against the acetylcholinesterase enzyme. The effects of the alkaloids on larvae were observed through the immersion test at the following concentrations: 1.5; 3; 6; 12; 16 and 24 mM. Berberine and piperine presented larvicidal activity greater than 95 %, not differing from 100 % for the positive fipronil control (p > 0.05). Of the two alkaloids, piperine had a lower effective concentration (EC), with an EC50 of 6.04 mM. The acetylcholinesterase enzyme used in the study was obtained from R. microplus larvae (RmAChE) and the anticholinesterase activity was determined spectrophotometrically. The highest anticholinesterase activity, measured as inhibition concentration (IC), was observed for berberine (IC50 = 88.13 µM), while piperine showed lower activity (IC50 > 200 µM). Docking studies in RmAChE, followed by 10 ns molecular dynamics simulation, suggest that berberine stabilizes the RmAChE at lower Root-Mean-Square Deviation (RMSD) than Apo protein. Few hydrogen-bond interactions between berberine and RmAChE residues were balanced by hydrophobic and π-type interactions. Berberine fills preferentially the peripheral anionic site (PAS), which correlates with its non-competitive mechanism. These results suggest that berberine and piperine alkaloids have an in vitro acaricidal action on R. microplus larvae, and the likely mechanism of action of berberine is related to RmAChE inhibition when accessing the PAS residues. These findings could help the study of new natural products that could inhibit RmAChE and aid in the development of new acaricides.

Identification of flavonoids in Hymenaea martiana Hayne (Fabaceae) by HPLC-DAD-MSn analysis.

Hymenaea martiana is a native tree known in Brazil as 'jatobá' and used in folk medicine to treat pain and inflammation. The aim of this work was to identify the flavonoids present in the crude ethanolic extract and ethyl acetate fraction using HPLC-DAD and LC-MSn analysis. The ethanolic extract was partitioned to obtain the ethyl acetate fraction. The analysis of astilbin content also was carried out by HPLC analysis. HPLC-DAD-ESI/MSn analysis of the ethanolic extract and ethyl acetate fraction revealed the presence of eleven peaks in the chromatograms, and all these peaks were identified: taxifolin, eucryphin, astilbin and 3 diastereoisomers, engeletin and 2 diastereoisomers, quercitrin and 2,6,3',4'-tetrahydroxy-2-benzylcoumaran-3-one. The ethyl acetate fraction had a higher astilbin concentration (151.87 µg/mL) than the ethanolic extract (40.13 µg/mL). In conclusion, the species could be considered a good source of flavonoids, which can be related to the main chemotaxonomic markers for the genus Hymenaea.

Characterization of a flavonol-rich antioxidant fraction from Spondias purpurea L. pulp and the effect of its incorporation on cellulose acetate-based film.

BACKGROUND: Active packaging containing natural flavonoid has recently emerged as a result of its potential to inhibit the oxidation of foods by interacting with it and/or its surrounding environment, with the aim of counteracting oxidation reactions and extending the shelf-life of foods. The plant Spondias purpurea L. is widely found in northeastern Brazil and is known to contain bioactive flavonoids. The present study aimed to obtain a flavonoid-rich fraction from the pulp of S. purpurea L. and incorporate it into the cellulose acetate film to obtain biodegradable films with antioxidant properties. RESULTS: The fractionation in SiO2 open-column chromatography of the S. purpurea pulp crude extract furnished an antioxidant active fraction containing the flavonols quercetin 3-O-rutinoside and kaempferol 3-O-rutinoside as the major compounds. This active fraction was incorporated (10, 20 and 30 g kg-1 ) into the substance produced with the casting method for cellulose acetate films. The films produced were characterized concerning mechanical properties, water vapor permeability (WVP) and antioxidant activity. CONCLUSION: The incorporation of the active flavonoid fraction from S. purpurea in the cellulose acetate films decreases WVP and elongation at break, at the same time as increasing antioxidant activity, tensile strength and elastic modulus. Thus, the S. purpurea pulps may be an alternative as a source of antioxidants for use in cellulose acetate films. © 2020 Society of Chemical Industry.

Identification of Sassafras albidum alkaloids by high-performance thin-layer chromatography tandem mass spectrometry and mapping by desorption electrospray ionization mass spectrometry imaging.

Sassafras albidum is an important tree species that occurs across North America. The presence of benzylisoquinoline and aporphine alkaloids has been previously described; however, the spatial distribution of these compounds within S. albidum and other plants of Lauraceae family is still unclear. Mass spectrometry imaging has become an important tool in analysis of plants metabolites, uncovering important contributions about the functional role, biosynthetic pathway, and accumulation of these compounds in the plant. This work aimed to identify further alkaloids present in S. albidum roots, twigs, and leaves by high-performance thin-layer chromatography coupled to desorption electrospray ionization multistage mass spectrometry (HPTLC DESI-MSn ) and to map the spatial distribution of these compounds by DESI-MS imaging. A total of 12 alkaloids were indentified in the roots and twigs, and six of them were detected for the first time in S. albidum. A high number of alkaloids was found in S. albidum roots; however, alkaloids were not detected in the leaves. Cross sections of roots and twigs were blotted onto TLC plates assisted by heating and solvent extraction, and these imprints were analyzed by DESI-MS imaging. The profile of alkaloid spatial distribution in DESI-MS images showed different accumulation patterns across and within different plant parts. Most alkaloids displayed higher intensities in the outer-most layer of the roots and twigs. The detailed spatial localization pattern of these alkaloids analyzed by DESI-MS imaging in different plant parts could contribute to a better understanding of the profile of distribution, accumulation, and biosynthesis of benzylisoquinoline and aporphine alkaloids.

Ocotea daphnifolia: phytochemical investigation, in vitro dual cholinesterase inhibition, and molecular docking studies

This study aimed to evaluate the anticholinesterase activities of extracts and fractions of Ocotea daphnifolia in vitro and characterize its constituents. The effects of hexane, ethyl acetate, and ethanolic extracts on acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) activity were determined with a spectrophotometry assay. All extracts inhibited cholinesterase activity, and the ethanolic extract (2 mg/mL) exhibited the highest inhibition of both enzymes (99.7% for BuChE and 82.4% for AChE). The ethanolic extract was fractionated by column chromatography resulting in 14 fractions that were also screened for their anticholinesterase effects. Fraction 9 (2 mg/mL) showed the highest activity, inhibiting AChE and BuChE by 71.8% and 90.2%, respectively. This fraction was analyzed by high-performance liquid chromatography high-resolution mass spectrometry which allowed the characterization of seven glycosylated flavonoids (containing kaempferol and quercetin nucleus) and one alkaloid (reticuline). In order to better understand the enzyme-inhibitor interaction of the reticuline toward cholinesterase, molecular modeling studies were performed. Reticuline targeted the catalytic activity site of the enzymes. Ocotea daphnifolia exhibits a dual cholinesterase inhibitory activity and displays the same pattern of intermolecular interactions as described in the literature. The alkaloid reticuline can be considered as an important bioactive constituent of this plant.

Anti-tick effect and cholinesterase inhibition caused by Prosopis juliflora alkaloids: in vitro and in silico studies.

We investigated the in vitro acaricide activity of the methanolic extract (ME) and alkaloid-rich fraction (AF) of Prosopis juliflora on Rhipicephalus microplus and correlated this effect with acetylcholinesterase (AChE) inhibition. The acaricide activity was evaluated using adult and larval immersion tests. Also, we studied the possible interaction mechanism of the major alkaloids present in this fraction via molecular docking at the active site of R. microplus AChE1 (RmAChE1). Higher reproductive inhibitory activity of the AF was recorded, with effective concentration (EC50) four times lower than that of the ME (31.6 versus 121 mg/mL). The AF caused mortality of tick larvae, with lethal concentration 50% (LC50) of 13.8 mg/mL. Both ME and AF were seen to have anticholinesterase activity on AChE of R. microplus larvae, while AF was more active with half-maximal inhibitory concentration (IC50) of 0.041 mg/mL. The LC-MS/MS analyses on the AF led to identification of three alkaloids: prosopine (1), juliprosinine (2) and juliprosopine (3). The molecular docking studies revealed that these alkaloids had interactions at the active site of the RmAChE1, mainly relating to hydrogen bonds and cation-pi interactions. We concluded that the alkaloids of P. juliflora showed acaricide activity on R. microplus and acted through an anticholinesterase mechanism.

Rapid structural characterisation of benzylisoquinoline and aporphine alkaloids from Ocotea spixiana acaricide extract by HPTLC-DESI-MSn.

INTRODUCTION: Lauraceae alkaloids are a structurally diverse class of plant specialised secondary metabolites that play an important role in modern pharmacotherapy, being useful as well as model compounds for the development of synthetic analogues. However, alkaloids characterisation is challenging due to low concentrations, the complexity of plant extracts, and long processes for accurate structural determinations. OBJECTIVE: The use of high-performance thin layer chromatography coupled with desorption electrospray ionisation multistage mass spectrometry (HPTLC DESI-MSn ) as a fast tool to identify alkaloids present in Ocotea spixiana extract and evaluate the extract's acaricide activity. METHODS: Ocotea spixiana twigs were extracted by conventional liquid-liquid partitioning. HPTLC analysis of the ethyl acetate extract was performed to separate isobaric alkaloids prior to DESI-MSn analysis, performed from MS3 up to MS7 . The extract's acaricide activity against Rhipicephalus microplus was evaluated by in vitro (larval immersion test) and in silico tests. RESULTS: HPTLC-DESI-MSn analysis was performed to identify a total of 13 aporphine and four benzylisoquinoline-type alkaloids reported for the first time in O. spixiana. In vitro evaluation of the extract and the alkaloid boldine showed significant activity against R. microplus larvae. It was established in silico that boldine had important intermolecular interactions with R. microplus acetylcholinesterase enzyme. CONCLUSION: The present study demonstrated that HPTLC-DESI-MSn is a useful analytical tool to identify isoquinoline alkaloids in plant extracts. The acaricide activity of the O. spixiana ethyl acetate extract can be correlated to the presence of alkaloids.