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Liquid chromatography coupled with high-resolution mass spectrometry data-independent acquisition (LC-HRMS/DIA), including MSE, enable comprehensive metabolomics analyses though they pose challenges for data processing with automatic annotation and molecular networking (MN) implementation. This motivated the present proposal, in which we introduce DIA-IntOpenStream, a new integrated workflow combining open-source software to streamline MSE data handling. It provides 'in-house' custom database construction, allows the conversion of raw MSE data to a universal format (.mzML) and leverages open software (MZmine 3 and MS-DIAL) all advantages for confident annotation and effective MN data interpretation. This pipeline significantly enhances the accessibility, reliability and reproducibility of complex MSE/DIA studies, overcoming previous limitations of proprietary software and non-universal MS data formats that restricted integrative analysis. We demonstrate the utility of DIA-IntOpenStream with two independent datasets: dataset 1 consists of new data from 60 plant extracts from the Ocotea genus; dataset 2 is a publicly available actinobacterial extract spiked with authentic standard for detailed comparative analysis with existing methods. This user-friendly pipeline enables broader adoption of cutting-edge MS tools and provides value to the scientific community. Overall, it holds promise for speeding up metabolite discoveries toward a more collaborative and open environment for research.

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Ocotea duckei , known as Louro - de - cheiro, belongs to the Lauraceae family and presents lignoid yangambine (YAN) as the main plant marker. This work aimed to develop and validate an analytical method by high performance liquid chromatography for the quantification of YAN. The sample used was the crude eth anolic extract (CEE) obtained from aerial parts. In the developed method, a C18 column was used. The mobile phase was composed of acetonitrile and water (45:55), whereas the method parameters included mobile phase flow rate at 0.8 mL/min, oven temperature at 40°C, and monitoring at 205 nm. In the validation, the parameters of selectivity, linearity, precision, accuracy, robustness, limits of detection and quantification were evaluated. As a result, the developed method is in accordance with the guidelines f or validation of analytical methods and presented satisfactory chromatographic parameters for YAN determination. Thus, the present analytical methodology can be applied in the quality control of O. duckei raw materials.

Ocotea duckei , conocida como Louro - de - cheiro, pertenece a la familia Lauraceae y presenta la yangambina lignoide (YAN) como principal marcador vegetal. Este trabajo tuvo como objetivo desarrollar y val idar un método analítico por cromatografía líquida de alta resolución para la cuantificación de YAN. La muestra utilizada fue el extracto etanólico crudo (EEC) obtenido de partes aéreas. En el método desarrollado se utilizó una columna C18. La fase móvil c onsistió en acetonitrilo y agua (45:55), mientras los parámetros del método incluyeron el caudal de la fase móvil a 0,8 m L /min, la temperatura del horno a 40°C y la monitorización a 205 nm. En la validación se evaluaron los parámetros de selectividad, line alidad, precisión, exactitud, robustez, límites de detección y cuantificación. Como resultado, el método desarrollado está de acuerdo con las pautas para la validación de métodos analíticos y presentó parámetros cromatográficos satisfactorios para la deter minación de YAN. Por lo tanto, la presente metodología analítica se puede aplicar en el control de calidad de las materias primas de O. duckei.

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BACKGROUND: Species within the Ocotea genus (Lauraceae), have demonstrated an interesting profile of bioactivities. Renowned for their diverse morphology and intricate specialized metabolite composition, Ocotea species have re-emerged as compelling candidates for bioprospecting in drug discovery research. However, it is a genus insufficiently studied, particularly regarding anti-inflammatory activity. PURPOSE: To investigate the anti-inflammatory activity of Ocotea spp. extracts and determine the major markers in this genus. METHODS: Extracts of 60 different Ocotea spp. were analysed by an ex vivo anti-inflammatory assay in human whole blood. The experiment estimates the prostaglandin E2 levels, which is one of the main mediators of the inflammatory cascade, responsible for the classical symptoms of fever, pain, and other common effects of the inflammatory process. Untargeted metabolomics analysis through liquid chromatography coupled with high-resolution mass spectrometry was performed, along with statistical analysis, to investigate which Ocotea metabolites are correlated with their anti-inflammatory activity. RESULTS: The anti-inflammatory screening indicated that 49 out of 60 Ocotea spp. extracts exhibited significant inhibition of PGE2 release compared to the vehicle (p < 0.05). Furthermore, 10 of these extracts showed statistical similarity to the reference drugs. The bioactive markers were accurately identified using multivariate statistics combined with a fold change (> 1.5) and adjusted false discovery rate analysis as unknown compounds and alkaloids, with a majority of aporphine and benzylisoquinolines. These alkaloids were annotated with an increased level of confidence since MSE spectra were compared with comprehensive databases. CONCLUSION: This study represents the first bioprospecting report revealing the anti-inflammatory potential of several Ocotea spp. The determination of their anti-inflammatory markers could contribute to drug discovery and the chemical knowledge of the Ocotea genus.

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Sporothrix brasiliensis with low susceptibility isolates were described from the Brazilian zoonotic sporotrichosis hyperendemics. The aim of this work was to evaluate distinct fractions of Ocotea pulchella, Ocotea notata, Myrciaria floribunda, and Hypericum brasiliense plant extracts against itraconazole-sensitive and low susceptibility S. brasiliensis isolates. Crude extracts were tested against clinical isolates and the ATCC MYA4823 to determine the minimum inhibitory concentrations (MICs) and fungicidal or fungistatic activities (MFC). A high MICs and MFCs amplitude (1 - > 128 µg/mL) were obtained for seven extracts. The highest antimicrobial activities against sensitive S. brasiliensis were displayed by the ethyl acetate extracts of O. notata (MIC = 2-128 µg/mL) and M. floribunda (MIC = 1-8 µg/mL). A fungicidal effect was observed for all fraction extracts. Ocotea spp. and M. floribunda ethyl acetate extracts provide promising profiles against itraconazole-sensitive or low susceptibility S. brasiliensis. Future studies will determine if these extracts can contribute as alternative therapies to this neglected zoonosis.

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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.

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This article describes the morpho-anatomies of the leaves and stems of Ocotea paranaensis Brotto, Baitello, Cervi & Santos (Lauraceae) using light and scanning electron microscopy. The main anatomical features characterizing the species are the presence of simple non-glandular trichomes in the leaves and stems, large secretory cells in the leaves, flat-convex petioles with two small lateral ribs, brachysclereids in the stem cortex and pith, and the presence of starch grains and various types of calcium oxalate crystals in the pith. Histochemical tests indicated the presence of lignin in stone cells, fibers, and xylem. Lipophilic contents were found in the secretory cells. Phenolic compounds were detected in the epidermis, hypodermis, phloem, and xylem. The present study's findings can contribute to the taxonomy and authentication of O. paranaensis. RESEARCH HIGHLIGHTS: This article is the first morpho-anatomical study of Ocotea paranaensis. Anatomy and histochemistry of the leaves and stems were studied by light microscopy, FESEM and EDS. The findings of this study would aid in the species identification and taxonomy.

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Introduction: Yangambin and epi-yangambin are the main lignans found in Louro-de-Cheiro [Ocotea fasciculata (Nees) Mez], a tree native to the Atlantic forests of northeastern Brazil whose leaves and bark are widely used in folk medicine. The present study investigated the leishmanicidal and immunomodulatory effects of both lignans in in vitro models of infection by Leishmania amazonensis or Leishmania braziliensis, both etiological agents of Cutaneous Leishmaniasis in Brazil. Methods: Bone marrow-derived mouse macrophages were infected with L. amazonensis or L. braziliensis and then treated for 48 h at varying concentrations of yangambin or epi-yangambin. Results: Yangambin and epi-yangambin were found to reduce the intracellular viability of either Leishmania species in a concentration-dependent manner, with respective IC50 values of: 43.9 ± 5 and 22.6 ± 4.9 µM for L. amazonensis, compared to IC50 values of 76 ± 17 and 74.4 ± 9.8 µM for L. braziliensis. In this context, epi-yangambin proved more selective and effective against in vitro infection by L. amazonensis. However, both lignans were found to distinctly modulate the production of inflammatory mediators and other cytokines by macrophages infected by either of the Leishmania species evaluated. While yangambin increased the production of IL-10 by L. braziliensis-infected macrophages, both compounds were observed to lower the production of NO, PGE2, IL-6 and TNF-α in both Leishmania species. Discussion: The present results serve to encourage the development of novel studies aimed at screening natural bioactive compounds with the hope of discovering new therapeutic options for the treatment of Cutaneous Leishmaniasis.

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This review describes the chemical composition and biological properties of essential oils from plants of the Ocotea genus, from different origin. This taxon belongs to the Laureaceae family, which in turn, is best known for medicinal use, often related to the essential oils. The text includes studies about Ocotea species distributed mainly on the South American continent, but also reporting species located in North America and Africa. Brazil, Colombia, Costa Rica and Ecuador are countries with highest number of species mentioned in the review. Also, the major components of each essential oil, its chemical structures, as well as the description and extent of biological activities related to essential oils are detailed in this review. Finally, the text discusses the chemical and biological aspects of these studies in a comparatively way, also informing additional data such as yield, biome of origin and pharmacobotanical location.

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The Ocotea genus exhibits a variety of pharmacological, antibacterial and antioxidant effects. The aim of the present study was to evaluate the antioxidant potential and antibacterial properties of the ethyl acetate fraction of Ocotea nutans leaves. Isolation and identification of the phenolic compounds from the fraction was also carried out. The isolated compounds were characterised by one- and two-dimensional nuclear magnetic resonance analysis and identified as vitexin (1) and isovitexin (2). The ethyl acetate fraction of Ocotea nutans leaves demonstrated considerable antioxidant potential. The observed minimum inhibitory concentration of 250 µg.mL-1 was classified as a moderate antibacterial activity against Enterococcus faecalis. Findings from this study demonstrate the utility of this plant as a potential source of antioxidant and antibacterial agents.

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The growing interest in the discovery of new compounds from medicinal plants justifies the study of phytochemical components of these plants and their biological activities. The genus Ocotea exhibits a variety of pharmacological, antimicrobial and antioxidant effects. The aim of the present study was to evaluate the antioxidant potential and antimicrobial properties of the ethyl acetate fraction of Ocotea paranaensis leaves. Isolation and identification of the phenolic compounds from the fraction was also carried out. The isolated compounds were characterized by one and two-dimensional nuclear magnetic resonance analysis and identified as (-) epicatechin (1), quercetin (2), kaempferol (3) and hyperin (4). The ethyl acetate fraction of Ocotea paranaensis leaves demonstrated considerable antioxidant potential. The observed minimum inhibitory concentration of 500 µg/mL was classified as a moderate antibacterial activity against Staphylococcus aureus. Findings from this study demonstrate the utility of this plant as a potential source of antioxidant and antimicrobial agents.

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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.

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Abstract Essential oils from four Ocotea species collected in southern Brazil were evaluated for chemical composition using gas chromatography coupled with mass spectrometry. The primary compound identified in O. acutifolia essential oil was an unsaturated tetracyclic diterpene, phyllocladene (67.7%), followed by a sesquiterpene hydrocarbon, ß-selinene (18.0%). The sesquiterpene fraction was predominant in oils from two collections of O. puberula; ß-caryophyllene (25.2%) and globulol (22.6%) were the major compounds identified in collections 1 and 2, respectively. O. silvestris essential oil contained predominantly germacrene D and bicyclogermacrene. These compounds were also predominant in essential oil from O. indecora leaves collected from shady habitats. By contrast, essential oil extracted from O. indecora grown under direct sunlight contained mainly oxygenated sesquiterpenes, such as guaiol (30.2%), α-eudesmol (27.6%), and ß-eudesmol (12.7%). Chemotaxis assays showed that Ocotea essential oils had no significant inhibitory activity on leukocyte migration compared with a chemotactic stimulant (lipopolysaccharide from Escherichia coli). However, the oils exhibited antifungal activity against Candida parapsilosis, with a minimum inhibitory concentration of 500 µg/mL. To our knowledge, this is the first study to investigate the in vitro antifungal and antichemotactic activities of essential oils from Ocotea species native to southern Brazil

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Ocotea quixos (Lam.) Kosterm. is an aromatic tree native to Ecuador, whose leaves are used to prepare aromatic beverages to which different health benefits are attributed. In this study, Ocotea quixos leaves were collected in the Amazon region in different environmental conditions and subjected to hydrodistillation to isolate the essential oil. The collection variables used were type of soil, amount of shade, and height; in addition, the presence of twig and leaf age and moisture were used as variables. Chemical composition was analyzed by means of gas chromatography equipped with a flame ionization detector and gas chromatography coupled to mass spectrometry. A wide variety of chemical compositions were detected in the samples. In total, forty-seven compounds were identified, which represented between 97.17% and 99.89% of the total composition. The constituents were mainly grouped into aliphatic sesquiterpene hydrocarbons (33.03-55.89%), other compounds (8.94-47.83%), and oxygenated monoterpenes (1.97-39.66%). The main constituents were found to be (E)-cinnamyl acetate (5.96-41.65%), (E)-methyl cinnamate (0.38-37.91%), and trans-caryophyllene (8.77-37.02%). The statistical analysis suggested the existence of two essential oil chemotypes and a direct correlation between environmental conditions and chemical composition of the essential oils.

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One new aporphine, dicentrine-ß-N-oxide (1), together with five related known alkaloids dehydrodicentrine (2), predicentrine (3), N-methyllaurotetanine (4), cassythicine (5), and dicentrine (6) were isolated from the leaves of Ocotea puberula (Lauraceae). Antiprotozoal activity of the isolated compounds was evaluated in vitro against trypomastigote forms of Trypanosoma cruzi. Among the tested compounds, alkaloid 1 exhibited higher potential with EC50 value of 18.2 µM and reduced toxicity against NCTC cells (CC50 >200 µM - SI>11.0), similar to positive control benznidazole (EC50 of 17.7 µM and SI=10.7). Considering the promising results of dicentrine-ß-N-oxide (1) against trypomastigotes, the mechanism of parasite death caused by this alkaloid was investigated. As observed, this compound reached the plasma membrane electric potential directly after 2 h of incubation and triggered mitochondrial depolarization, which probably leads to trypomastigote death. Therefore, dicentrine-ß-N-oxide (1), reported for the first time in this work, can contribute to future works for the development of new trypanocidal agents.

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ETHNOPHARMACOLOGICAL RELEVANCE: Ocotea odorifera (Vell.) Rohwer has been used in traditional medicine in the south of Brazil for the treatment of inflammatory-related conditions, such as rheumatism. However, there is not any scientific evidence for popular use. AIMS OF THE STUDY: To investigate the O. odorifera anti-inflammatory potential and identification of the main active compounds through metabolomic approaches. MATERIALS AND METHODS: In order to in vivo evaluate the inhibition of the main inflammatory pathways, the leaf decoction, leaf extract, its fractions and the essential oils from leaves and branches were submitted to the ear oedema and the neutrophils recruitment assays. The samples were chemically investigated by UHPLC-HRMS or GC-MS. The multivariate statistical analysis (PLS-DA) was used to determine the substances correlated with the anti-inflammatory properties. RESULTS: The in vivo studies indicated a promissory anti-inflammatory effect on both oedema and neutrophil recruitment for some samples including the decoction; hydroethanolic, ethyl acetate, and chloroform fractions; and the essential oils. According to the PLS-DA, the S-(+)-reticuline was evidenced as one of the three compounds of the plant most correlated with both anti-inflammatory mechanisms. Thus, S-(+)-reticuline was isolated and the anti-inflammatory activity was confirmed. Moreover, for the first time, the dual inhibition of oedema and neutrophil recruitment was uncovered and reported. Another compound positively correlated with the anti-inflammatory activity is likely to be a new compound since zero hit on the comprehensive mass database were encountered. The compounds found in the essential oils also showed significant anti-inflammatory activity, and thus indeed the plant has different classes of active substances. CONCLUSIONS: The decoction of O. odorifera and different fractions from its ethanolic extract demonstrated anti-inflammatory activity through dual inhibition of oedema and neutrophil recruitment. Thus, corroborating the popular medicinal use of the decoction of leaves from O. odorifera as an anti-inflammatory medicine. Besides, reticuline, one of the main active compounds, was isolated and proved to display the dual mechanism of action, indicating the O. odorifera as a promising source of active compounds for the treatment of inflammatory conditions.

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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.

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Abstract INTRODUCTION: Aedes aegypti is the main vector of dengue and yellow fever. Recently, the use of plant-sourced larvicides has gained momentum. METHODS: The hydroethanolic extracts and fractions ofOcotea nutansleaves and stems were bioassayed to determine the larvicidal efficacy of these samples. RESULTS: S-HEX (hexane fraction from the crude stem extract) demonstrated high potential for controlling third-stage larvae, with an LC50 of 14.14 µg.mL-1 (concentration required to inhibit 50% of the treated larvae). CONCLUSIONS Extracts from O. nutans were effective against third-stage larvae ofA. aegyptiafter 24 h of exposure.

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BACKGROUND: Opportunistic fungal infections are increasingly common, with Candida albicans being the most common etiological agent; however, in recent years, episodes of candidiasis caused by non-albicans Candida species have emerged. Plants belonging to the Lauraceae family have shown remarkable antifungal effects. This study assessed the anti-Candida activity of Ocotea glomerata extracts and fractions, time of death and the synergistic effects with conventional antifungals. The possible mechanism of action was also addressed. METHODS: Minimal inhibitory concentrations (MIC) were determined by broth microdilution technique, and the mechanism of action was assessed by ergosterol, sorbitol, cell viability, reactive oxygen species (ROS) generation and phosphatidylserine externalization tests. RESULTS: All the tested extracts evidenced antifungal activity, but the methanol extract was revealed to be the most effective (MIC = 3.12 µg/mL) on C. krusei. The combination of methanol extract with ketoconazole and fluconazole revealed a synergistic effect for C. krusei and C. albicans, respectively. Fractions 1 and 5 obtained from the methanol extract had fungicidal activity, mainly against C. krusei. Methanol extract did not reveal effects by ergosterol and sorbitol assays; however, it led to an increase in intracellular ROS levels, decreased cell viability, and consequently, cell death. CONCLUSION: O. glomerata methanol extract may be viewed as a rich source of biomolecules with antifungal activity against Candida spp.

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The Ocotea puberula bark powder (OPBP) was evaluated as an effective adsorbent for the removal of crystal violet (CV) from colored effluents. OPBP was characterized and presented a surface with large cavities, organized as a honeycomb. The main functional groups of OPBP were O-H, N-H, C=O, and C-O-C. The adsorption of CV on OPBP was favorable at pH 9 with a dosage of 0.75 g L-1. The Avrami model was the most suitable to represent the adsorption kinetic profile, being the estimated equilibrium concentration value of 3.37 mg L-1 for an initial concentration of 50 mg L-1 (CV removal of 93.3%). The equilibrium was reached within 90 min. The data were better described by the Langmuir isotherm, reaching a maximum adsorption capacity of 444.34 mg g-1 at 328 K. The Gibbs free energy ranged from - 26.3554 to - 27.8055 kJ mol-1, and the enthalpy variation was - 11.1519 kJ mol-1. The external mass transfer was the rate-limiting step, with Biot numbers ranging from 0.0011 to 0.25. Lastly, OPBP application for the treatment of two different simulated effluents was effective, achieving a removal percentage of 90%.

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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.

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