Old Meets New: Mass Spectrometry-Based Untargeted Metabolomics Reveals Unusual Larvicidal Nitropropanoyl Glycosides from the Leaves of Heteropterys umbellata.

The Aedes aegypti (Diptera: Culicidae) mosquito is the vector of several arboviruses in tropical and subtropical areas of the globe, and synthetic pesticides remain the most widely used combat strategy. This study describes the investigation of secondary metabolites with larvicidal activity from the Malpighiaceae taxon using a metabolomic and bioactivity-based approach. The workflow initially consisted of a larvicidal screening of 394 extracts from the leaves of 197 Malpighiaceae samples, which were extracted using solvents of different polarity, leading to the selection of Heteropterys umbellata for the identification of active compounds. By employing untargeted mass spectrometry-based metabolomics and multivariate analyses (PCA and PLS-DA), it was possible to determine that the metabolic profiles of different plant organs and collection sites differed significantly. A bioguided approach led to the isolation of isochlorogenic acid A (1) and the nitropropanoyl glucosides karakin (2) and 1,2,3,6-tetrakis-O-[3-nitropropanoyl]-beta-glucopyranose (3). These nitro compounds exhibited larvicidal activity, possibly potentialized by synergistic effects of their isomers in chromatographic fractions. Additionally, targeted quantification of the isolated compounds in different extracts corroborated the untargeted results from the statistical analyses. These results support a metabolomic-guided approach in combination with classical phytochemical techniques to search for natural larvicidal compounds for arboviral vector control.

Nanoemulsions and Solid Microparticles Containing Pentyl Cinnamate to Control Aedes aegypti.

The Aedes aegypti mosquito is a vector of severe diseases with high morbidity and mortality rates. The most commonly used industrial larvicides have considerable toxicity for non-target organisms. This study aimed to develop and evaluate liquid and solid carrier systems to use pentyl cinnamate (PC), derived from natural sources, to control Ae. aegypti larvae. The liquid systems consisting of nanoemulsions with different lecithins systems were obtained and evaluated for stability over 30 days. Microparticles (MPs) were obtained by the spray drying of the nanoemulsions using maltodextrin as an adjuvant. Thermal, NMR and FTIR analysis indicated the presence of PC in microparticles. Indeed, the best nanoemulsion system was also the most stable and generated the highest MP yield. The PC larvicidal activity was increased in the PC nanoemulsion system. Therefore, it was possible to develop, characterize and obtain PC carrier systems active against Ae. aegypti larvae.

Combination of GC-MS Molecular Networking and Larvicidal Effect against Aedes aegypti for the Discovery of Bioactive Substances in Commercial Essential Oils.

Dengue is a neglected disease, present mainly in tropical countries, with more than 5.2 million cases reported in 2019. Vector control remains the most effective protective measure against dengue and other arboviruses. Synthetic insecticides based on organophosphates, pyrethroids, carbamates, neonicotinoids and oxadiazines are unattractive due to their high degree of toxicity to humans, animals and the environment. Conversely, natural-product-based larvicides/insecticides, such as essential oils, present high efficiency, low environmental toxicity and can be easily scaled up for industrial processes. However, essential oils are highly complex and require modern analytical and computational approaches to streamline the identification of bioactive substances. This study combined the GC-MS spectral similarity network approach with larvicidal assays as a new strategy for the discovery of potential bioactive substances in complex biological samples, enabling the systematic and simultaneous annotation of substances in 20 essential oils through LC50 larvicidal assays. This strategy allowed rapid intuitive discovery of distribution patterns between families and metabolic classes in clusters, and the prediction of larvicidal properties of acyclic monoterpene derivatives, including citral, neral, citronellal and citronellol, and their acetate forms (LC50 < 50 µg/mL).

Residual Larvicidal Activity of Quinones against Aedes aegypti.

The number of documented dengue cases has increased dramatically in recent years due to transmission through the Aedes aegypti mosquito bite. Vector control remains the most effective measure to protect against this and other arboviral diseases including Zika, chikungunya and (urban) yellow fever, with an established vaccine only available for yellow fever. Although the quinone class shows potential as leading compounds for larvicide development, limited information restricts the development of optimized structures and/or formulations. Thus, in this contribution we investigated the larvicidal and pupicidal activity of three quinone compounds isolated from a Connarus suberosus root wood ethyl acetate extract together with 28 quinones from other sources. Eight quinones demonstrated larvicidal activity, of which tectoquinone (4) proved to be the most active (LC50 1.1 µg/mL). The essential residual effect parameter of four of these quinones was evaluated in laboratory trials, with tectoquinone (4) and 2-ethylanthraquinone (7) presenting the most prolonged activity. In small-scale field residual tests, tectoquinone (4) caused 100% larvae mortality over 5 days, supporting its selection for formulation trials to develop a prototype larvicide to control Ae. aegypti.

Natural resources used in the traditional medicine of the Marinaú community, Caxiuanã forest, Brazil.

Background: The diversity of Conservation Units in Brazil, ranging from Strict Protection areas like National Parks to Sustainable Use areas such as National Forests, supports the coexistence of human populations with extensive traditional knowledge of local natural resources. This traditional and local knowledge plays a crucial role in their subsistence and has significant potential to contribute to bioprospecting initiatives, as well as to enhance research and strategies for biological conservation. The São Sebastião de Marinaú community, situated within the Caxiuanã National Forest, offers valuable opportunities for ethnobiological studies. Methods: The field study was carried out with 48 informants from the São Sebastião de Marinaú Community, Caxiuanã National Forest (CNF), Brazil, a Sustainable Use Conservation Unit. It involved participant observation, semi-structured interviews, and guided tours. Indices of Use Value (UV), Fidelity Level (FL), and Consensus Factor (ICF) indicated plant species with therapeutic potential. Results: A total of 944 uses associated with 154 plants and 21 animals were reported. Statistical tests pointed out that factors such as gender and age are not determinants in the richness of known plants among the residents. The plants are distributed among 59 botanical families and 126 genera. They predominantly use species native to Brazil (69%), among which 47 are endemic to the Amazon. The medicines are prepared mainly by decoction of leaves and bark. According to UV, veronica (Dalbergia monetaria L.f.) was the most important. The animals used are all vertebrates, and paca (Cuniculus paca Linnaeus, 1766,) was the most cited. Bile and lard are the parts most used in the recipes. They mentioned 116 diseases, especially those of the digestive system. The ceruzeiro (Allantoma lineata (Mart. ex O.Berg) Miers) had a high consensus of local use, and no additional studies on this species exist. Conclusion: This study underscores the vital role of traditional communities in sustainable conservation units, as their involvement is crucial for preserving plant and animal species essential to local traditional medicine. Such research also promotes the recognition of non-timber products as valuable raw materials with potential applications in the chemical and pharmaceutical industries. Additionally, mapping the occurrence and use of species in vulnerable conditions aids in developing effective conservation strategies for these resources.

Bioprospection for new larvicides against Aedes aegypti based on ethnoknowledge from the Amazonian São Sebastião de Marinaú riverside community.

ETHNOPHARMACOLOGICAL RELEVANCE: Vector-borne diseases represent a huge global burden impacting health systems. Aedes aegypti is the main vector of arboviral diseases including dengue, Zika, chikungunya and urban yellow fever in both tropical and subtropical areas. Ethnopharmacological investigations provide potential avenues for developing new vector control strategies. AIM OF THE STUDY: The objective of this study is to document the São Sebastião de Marinaú riverside community's ethnoknowledge of local plants used to control mosquitoes and perform bioguided fractionation to isolate the compounds active against the arboviral disease vector Ae. aegypti. MATERIALS AND METHODS: Semi-structured interviews were conducted with residents of the Marinaú community located in the Caxiuanã National Forest, in the Amazon biome, Pará, Brazil. The plants used to control mosquitoes were subjected to phytochemical studies guided by Ae. aegypti assays. Extracts were obtained from seven species using distinct organic solvents. Active extracts and fractions were separated by chromatographic techniques. Isolated compounds were characterized by NMR, LC/MS and GC/MS. Sample activity against Ae. aegypti larvae and pupae was evaluated after 24, 48 and 72 h exposure. The extracts were also investigated against adult female mosquitoes. The LC50 values were determined by diluting each sample to obtain different concentrations in the respective activity range. RESULTS: The Marinaú community uses more than ten plants as a repellent, most of which are trees native to the region. The primary applications of these plants to protect against insect bites were: burning plants (fumigation), application of body oils and bathing in macerated plants. Carapa guianensis is the predominant species used as a repellent. Extracts from Diospyros guianensis fruits, Carapa guianensis seed shells and Aspidosperma nitidum wood demonstrated Ae. aegypti larvicidal activity. The C. guianensis seed shell extract demonstrated a residual larvicidal effect. Plumbagin, stigmasterol, ß-sitosterol, betulinic, ursolic and oleanolic acids, and betulin were identified in the D. guianensis extract. The plumbagin, ursolic and oleanolic acids displayed larvicidal activity. Oleanolic, ursolic and betulinic acids, and betulin were considered pupicidal. Aricine, the major alkaloid isolated from A. nitidum wood, also presented larvicidal activity. CONCLUSIONS: Ten plant species traditionally used by the Marinaú community to afford protection against mosquitoes were reported. C. guianensis, D. guianensis and A. nitidum extracts were considered larvicidal against Ae. aegypti. Four triterpenes stood out as very active compounds against pupae. Aricine, an indole alkaloid, displayed larvicidal activity. Therefore, traditional knowledge of Amazonian plants combined with bioguided fractionation constitutes a strategy for the development of eco-friendly insecticides to control Ae. aegypti, an arbovirus vector.

Combining chemometric and phytochemical tools to isolate and characterize activity of Vismia gracilis compounds against Aedes aegypti.

Vismia gracilis extracts were tested against Aedes aegypti to assess mortality and behavioural effects. The leaf hexanic extract (L-Hex) presented increased larvicidal activity with exposure period: LC50 46.48 µg/mL (24 h) and LC50 20.57 µg/mL (48 h). Eight compounds were annotated/isolated from the L-Hex active extract, 4 benzophenones and 4 anthraquinones. Considering chemometric findings, the benzophenone moiety, tested as the commercial benzophenone, promoted larval mortality (LC50 16.35 µg/mL). Both the L-Hex extract and benzophenone induced intestinal damage in larvae. Benzophenone also promoted toxicity and behavioural effects in female adults. These findings highlighted the potential use of this class of compounds for developing vector-control products.

A Molecular Networking Strategy: High-Throughput Screening and Chemical Analysis of Brazilian Cerrado Plant Extracts against Cancer Cells.

Plants have historically been a rich source of successful anticancer drugs and chemotherapeutic agents, with research indicating that this trend will continue. In this contribution, we performed high-throughput cytotoxicity screening of 702 extracts from 95 plant species, representing 40 families of the Brazilian Cerrado biome. Activity was investigated against the following cancer cell lines: colon (Colo205 and Km12), renal (A498 and U031), liver (HEP3B and SKHEP), and osteosarcoma (MG63 and MG63.3). Dose-response tests were conducted with 44 of the most active extracts, with 22 demonstrating IC50 values ranging from <1.3 to 20 µg/mL. A molecular networking strategy was formulated using the Global Natural Product Social Molecular Networking (GNPS) platform to visualize, analyze, and annotate the compounds present in 17 extracts active against NCI-60 cell lines. Significant cytotoxic activity was found for Salacia crassifolia, Salacia elliptica, Simarouba versicolor, Diospyros hispida, Schinus terebinthifolia, Casearia sylvestris var. lingua, Magonia pubescens, and Rapanea guianensis. Molecular networking resulted in the annotation of 27 compounds. This strategy provided an initial overview of a complex and diverse natural product data set, yielded a large amount of chemical information, identified patterns and known compounds, and assisted in defining priorities for further studies.

Machaerium acutifolium compounds with larvicidal activity against Aedes aegypti.

BACKGROUND: Plant extracts and isolated compounds are known for their insecticidal activity. The Aedes aegypti mosquito has a significant medical impact as it transmits a number of arboviruses and is able to develop resistance to the commercially available insecticides. This study investigates larvicidal compounds isolated from Machaerium acutifolium, designated by the Brazilian Forest Service as a sustainable species. RESULTS: A M. acutifolium trunk ethyl acetate extract was fractionated using chromatographic methods with full structural elucidation by mass spectrometry (MS), nuclear magnetic resonance and specific rotation analyses revealing: one new 3-arylcoumarin derivative 1; two flavonoids 2 and 3; a trans-stilbene 4, and an unprecedented natural indene 5. The larvicidal activity against Ae. aegypti after 24 h exposure was: crude extract (median lethal dose, LC50 205 mg L-1 ), fraction C (LC50 27 mg L-1 ) and 5 (LC50 24 mg L-1 ). CONCLUSION: A M. acutifolium extract showed larvicidal activity, which increased with prolonged exposure, demonstrating LC50 75 mg L-1 after 72 h. Although the flavonoids 2 and 3 and trans-stilbene 4 were deemed inactive according to the adopted mortality limit, additional tests revealed their ability to cause 65% Ae. aegypti larvae mortality, suggesting they could contribute to the larvicidal activity. Compound 5, identified by liquid chromatography-MS, was over eight-fold more toxic to larvae than the crude extract after 24 h. Therefore, 5 constitutes a structural model for new prototypes to control Ae. aegypti. These data reinforce the potential of natural products as a source of commercial alternatives for vector control strategies, respecting both sustainability and eco-friendly principles. © 2020 Society of Chemical Industry.

Antileishmanial compounds from Connarus suberosus: Metabolomics, isolation and mechanism of action.

Leishmaniasis is a disease impacting public health worldwide due to its high incidence, morbidity and mortality. Available treatments are costly, lengthy and toxic, not to mention the problem of parasite resistance. The development of alternative treatments is warranted and natural products demonstrate promising activity. This study investigated the activity of Connarus suberosus extracts and compounds against Leishmania species. Several C. suberosus extracts were tested against L. amazonensis promastigotes. Active and inactive extracts were analyzed by UHPLC-MS and data evaluated using a metabolomics platform, revealing an unknown neoflavonoid (connarin, 3), isolated together with the pterocarpans: hemileiocarpin (1) and leiocarpin (2). The aforementioned compounds (1-3), together with the benzoquinones: rapanone (4), embelin (5) and suberonone (6) previously isolated by our group from the same species, were tested against: (i) L. amazonensis and L. infantum promastigotes, and (ii) L. amazonensis intracellular amastigotes, with the most active compound (3) also tested against L. infantum amastigotes. Cytotoxicity against murine peritoneal macrophages was also investigated. Compounds 2 and 3 presented an IC50 33.8 µM and 11.4 µM for L. amazonensis promastigotes; and 44.3 µM and 13.3 µM for L. infantum promastigotes, respectively. For L. amazonensis amastigotes, the IC50 of 2 was 20.4 µM with a selectivity index (SI) of 5.7, while the IC50 of 3 was 2.9 µM with an SI of 6.3. For L. infantum amastigotes, the IC50 of 3 was 7.7 µM. Compounds 2 and 3 presented activity comparable with the miltefosine positive control, with compound 3 found to be 2-4 times more active than the positive control, depending on the Leishmania species and form. The extracts and isolated compounds showed moderate toxicity against macrophages. Compounds 2 and 3 altered the mitochondrial membrane potential (ΔΨm) and neutral lipid body accumulation, while 2 also impacted plasma membrane permeabilization, culminating in cellular disorder and parasite death. Transmission electron microscopy of L. amazonensis promastigotes treated with compound 3 confirmed the presence of lipid bodies. Leiocarpin (2) and connarin (3) demonstrated antileishmanial activity. This study provides knowledge of natural products with antileishmanial activity, paving the way for prototype development to fight this neglected tropical disease.