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Riverine communities in the Brazilian Amazon region use a variety of plants to treat snakebites. These plants can be effective against secondary infections, one of the main complications of snakebites. The aim of this study was to determine whether plants traditionally used to treat snakebites in the Brazilian Amazon may also have antimicrobial and antioxidant activities, and if so, which classes of chemicals may be responsible for these activities. Aqueous extracts of nine plants were tested in microdilution assays and the more active were prepared using solvents (hexane, methanol and water) and decoction, and nine assays were performed. Assays to determine the antioxidant activity of the most active species were carried out, as well as phytochemistry studies to determine the active components of this species. Bellucia dichotoma exhibited the greatest antimicrobial potential, particularly the hexane, methanol and decoction extracts. In comparative TLC, extracts of this species showed characteristics of terpenoids, compounds with double bonds and flavonoids. In 1H NMR, characteristic signals of sterols such as β-sitosterol, stigmasterols or triterpenes were observed, as well as signals indicating the presence of aromatic hydrogens, characteristic of aromatic substances, and sugars. The methanol extracts and decoction were considered active in the antioxidant assay.

Aims: Pectis species, belonging to Asteraceae, are aromatic and medicinal herbaceous plants, distributed in the Americas, the West Indies, and the Pacific islands, with lemon, cumin, or oregano aroma, used in infusions to treat several diseases or as spices. Methodology: In this study, the composition of Pectis elongata from the Amazon was correlated with other Neotropical Pectis oils, including their traditional uses and biological properties. Pectis elongata oils were obtained by hydrodistillation and analyzed by GC and GC-MS. Results: Citral (39.1% of neral and 47.7% of geranial), perilla aldehyde (51.7% to 81.8%), and limonene (33.7% to 43.7%) were identified as their primary constituents of the Pectis elongata oils from the Amazon. It is assumed the existence of at least two chemotypes for the variability of the oils of P. elongata: Citral (neral plus geranial) and perilla aldehyde plus limonene. The main C10-skeletal monoterpenes found in Pectis oils can be depicted according to their biosynthetic pathways: neral and geranial arranged in an acyclic-type skeleton, and limonene, perilla aldehyde, cumin aldehyde, carvone, p-cymene, and thymol in a p-menthane-type skeleton. Conclusion: There is a particular interest in the world's citral-rich essential oils, such as Pectis elongata oil and other similar Pectis oils, given their application in human health and food preservation.

A new consumer profile for pharmaceutical and cosmetic products has motivated research into natural raw materials in the development of “green” products such as herbal medicines and biocosmetics. However, various limitations have been encountered in the marketing of these products, for example the quality control of the natural raw materials used by the industrial market. This study aims to evaluate the sensory and physicochemical parameters of murumuru (Astrocaryum murumuru Mart.), bacuri (Platonia insignis Mart.), tucuma (Astrocaryum vulgare Mart.), and ucuuba (Virola sebifera Aubl.) butters for applications in pharmaceutical and cosmetic bioproducts. The acidity and saponification as well as the iodine and peroxide indexes were evaluated and fatty acid profiles for the samples obtained by GC-MS. The sensory properties of the butters showed the appearance of solid to soft cream, color (yellow, brown, buttercup, and ochre), and characteristic odor. The melting temperatures of all butters ranged between 31 oC and 49 °C. The acidity, saponification, iodine and peroxide indexes for the butters were of 5.82 – 17.73 mg (NaOH or KOH) g−1, 181.10 – 573.55 mg KOH g−1, 2.78 – 44.96 gl2 100 g−1, and 1.39 – 9.30 meq kg−1, respectively. From analyses of the fatty acid profiles, the major components identified were lauric acid in murumuru (40%) and ucuuba butters (73%), myristic acid in tucuma butter (53%), and palmitic acid in bacuri butter (42%). In general, the results of the analyses differed from the specifications of the supplier reports and official compendia. These findings highlight the importance of quality control in natural raw materials to ensure their functionality in pharmaceutical and cosmetic bioproducts.

Myrciaria dudia, também conhecida como camu-camu, é uma planta que tem ocorrência espontânea na Amazônia e vem ganhando destaque devido aos avanços ocorridos na área científica e na busca da população por terapias e alimentos mais saudáveis. O objetivo deste artigo foi realizar um levantamento bibliográfico sobre produtos oriundos de Myrciaria dubia. O levantamento foi realizado em plataformas on line lens.org, Instituto Nacional da Propriedade Industrial (INPI) e na base de dados do Portal Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES), bem como de produtos comercializados na plataforma de busca Google. Os parâmetros de busca utilizados foram os termos "Myrciaria dubia" ou "camu-camu", com intervalo temporal de busca entre 01/01/1990 e 10/05/2019. Os registros de patentes mais depositados para uso de Myrciaria dubia estão relacionados a cosméticos principalmente com ação antioxidante para a pele. Dos 21 pedidos de patentes, apenas quatro são de patentes concedidas. Sobre os artigos científicos, dos 212 relacionados com a espécie, nove referiam-se a desenvolvimento de produtos em publicações de 2003 a 2019, onde a revista que mais publicou foi a Food Science and Technology. Dentre os produtos encontrados nas publicações observa-se um avanço para a área de alimentos e cosméticos. No que se refere aos produtos comercializados pela Internet, exeste uma variação de produtos como na forma de pó (19), cápsulas (14), licor (3), chocolate (1), geleia (1) e muda (1). O valor dos produtos variando entre R$ 12,50 e R$ 199,00. Observamos que os produtos oriundos de Myrciaria dubia estão sendo difundidos e vem crescendo devido principalmente ao seu alto teor de bioativos com pespectivas de uso pelas indústrias de alimentos, cosméticos e farmacêuticas.

Oil in water nano-emulsions are drug delivery systems constituted by liquid lipophilic nano-droplets dispersed through the external aqueous phase, often reaching the kinetic stability with surfactant as stabilizers. Essential oils can be the oily phase or the source of bioactive compounds. In this study, the essential oil of Aeollanthus suaveolens-a plant used in folk medicine due to its psychopharmacological effects-was used for preparation of fine nano-emulsions by a low-energy titrating method. Monodisperse small nano-droplets (ca. 70 nm; PdI 0.200) were assembled by using blends of non-ionic surfactants, indicating modulation on surfactant system lead to altering the physical property. In a separate set of experiments, we investigated the role of this modulation on biological properties of the optimal nano-emulsion. The zebrafish embryos were more susceptible to the nano-emulsion than the bulk essential oil, showing the improved bioactivity due to nano-sizing. Therefore, adult zebrafish was treated, and paralysis was observed in the groups treated with the nano-emulsion, being this finding in accordance with hypnosis. At the same essential oil dose, another behavior was observed, suggesting that expected dose-dependent effects associated to sedative-hypnotics can be achieved by nano-sizing of psychoactive essential oils. This paper contributes to the state-of-art drug delivery systems by opening perspectives for novel sedative-hypnotics nano-emulsified essentials oils that can reach hypnotic effects at considerably lower dose, when compared with bulk materials, being useful for further completed dose-response studies.Graphical abstract.

The objective of this work was to evaluate the use of Myrcia sylvatica Hydrolate (MSH) as a sedative under conditions of simulated transport of Colossoma macropomum juveniles. Simulated a 17 h tran...

Passiflora nitida Kunth. (maracujá-do-mato) é uma espécie nativa do Brasil com ocorrência nas regiões norte, nordeste e centro-oeste. O seu fruto é consumido principalmente in natura, sendo pouco explorado quanto as suas propriedades funcionais. O objetivo deste estudo, foi extrair substâncias pécticas do mesocarpo de Passiflora nitida Kunth. O mesocarpo foi desidratado, triturado e obtido um pó de fina granulação (farinha), o qual, foi caracterizado quanto a granulometria, índice de intumescência, acidez titulável, cinzas e teor de umidade. Para a extração de substâncias pécticas foi utilizado 4g da farinha, em meio ácido. Foi usado o ácido cítrico comercial e suco de limão a 5% a 60ºC e 80ºC por 60 minutos e 120 minutos. Os experimentos foram realizados em triplicata e submetidos à análise de variância. O rendimento de substâncias pécticas com suco de limão por 60 minutos a 80ºC e 120 minutos a 60ºC foi de 17,9% e 14,3%, respectivamente. Para o ácido cítrico comercial, por 60 min a 80ºC e 120 minutos a 60ºC foi de 12,9% e 12,2%, respectivamente. O mesocarpo de Passiflora nitida poderá ser uma fonte para obtenção de substâncias pécticas.

Pectis elongata is found in the northern and northeastern regions of Brazil. It is considered a lemongrass due to its citric scent. The remarkable citral content and the wide antimicrobial properties and bioactive features of this terpene make this essential oil (EO) eligible for several industrial purposes, especially in cosmetics and phytotherapics. However, to address the problems regarding citral solubility, nano-emulsification is considered a promising strategy thanks to its improved dispersability. Thus, in this paper we propose a low-energy approach for the development of citral-based nano-emulsions prepared with P. elongata EO. The plant was hydrodistillated to produce the EO, which was characterized with a gas chromatograph coupled to mass spectrometry. The nano-emulsion prepared by a non-heated water titrating (low-energy) method was composed of 5% (w/w) EO, 5% (w/w) non-ionic surfactants and 90% (w/w) deionized water and was analyzed by dynamic light scattering. Levels of citral of around 90% (neral:geranial—4:5) were detected in the EO and no major alteration in the ratio of citral was observed after the nano-emulsification. The nano-emulsion was stable until the 14th day (size around 115 nm and polydispersity index around 0.2) and no major alteration in droplet size was observed within 30 days of storage. Understanding the droplet size distribution as a function of time and correlating it to concepts of compositional ripening, as opposing forces to the conventional Ostwald ripening destabilization mechanism, may open interesting approaches for further industrial application of novel, low-energy, ecofriendly approaches to high citral essential oil-based nano-emulsions based on lemongrass plants.

Lippia alba (Mill.) N.E.Br. ex Britton & P. Wilson, Verbenaceae, is considered a great source of a bioactive volatile oil. Due to the wide range of known chemotypes, its chemical analysis is very important. Among the several activities of this volatile oil, a potential larvicidal action against Culicidae species is highlighted. However, the low water miscibility of volatile oils limits their application in aqueous media. Oil in water nano-emulsions are in the spotlight of novelty to solve this main problem. Thus, the aim of the present study was to obtain this nanostructured system with L. alba volatile oil (citral chemotype) and evaluate its larvicidal activity against Aedes aegypti and Culex quinquefasciatus larvae. The major compounds were geranial (30.02%) and neral (25.26%). Low mean droplet size (117.0 ± 1.0 nm) and low polydispersity index (0.231 ± 0.004) were observed and no major changes were observed after seven days of storage. LC50 values against C. quinquefasciatus and A. aegypti third-instar larvae were respectively 38.22 and 31.02 ppm, while LC90 values were, respectively, 59.42 and 47.19 ppm. The present study makes use of a low energy, solvent-free and ecofriendly method with reduced costs. Thus, this paper contributes significantly to phyto-nanobiotechnology of larvicidal agents, opening perspectives for the utilization of L. alba volatile oil in integrated practices of vector control. Keywords: Colloidal system, Droplet size distribution, Miniemulsion, Larvicide

In Brazil, the infusion from leaves of Lippia alba (Verbenaceae), popularly known as Erva-cidreira, is used for the treatment of infections, digestive disorders, cough, asthma, colds and anxiolytic, among others. It has been used in phytotherapy practices of communities of Santarem, Para State, Brazil, cultivated in a public program of medicinal plants and phytotherapy. In this work, the local influence of seasonality and planting time on essential oil yield and composition were evaluated, as well as in the development of a topical formulation to determine the oil efficacy in inhibition the Staphylococcus epidermidis strain. The plant was sampled during the dry and rainy seasons, three times a day, the oils extracted and its constituents identified by GC and GC-MS. The formulation of topical use was prepared with the incorporation of the oil (10%) to an anionic cream, followed by analysis of its organoleptic characteristics. The antibacterial activity of the topical formulation was evaluated by the Well Diffusion Method, using the S. epidermidis strain. The oil yield in the plant was from 2.2 to 4.3%, and its main constituents were geranial, neral, elemol, p-cymene, limonene, carvone, and geraniol, with the quantitative variation depending on the seasonality and the collection time. The results for the seasonal and circadian study were compatible with the data variance obtained by Principal Component Analysis (PCA) and Hierarchical Cluster Analysis (HCA), with the formation of two distinct groups of primary constituents for the rainy and dry seasons. The formulated product maintained about 45% of the primary constituents of the oil (neral and geranial) for various days. It was homogeneous after centrifugation test, having shown excellent stability at 5 °C and 25 °C. Storage of the formulated product at higher temperatures appears to be a limiting factor for the antimicrobial action of the oil.

The study aimed to establish the lethal concentration at 96 h (LC50-96h) of hydrolate of Lippia alba (citral chemotype) (LAH) in juvenile tambaqui ( Colossoma macropomum ), as well as to identify the anaesthetic induction and recovery times. The acute toxicity test LC50-96h was performed in five animals per replicate (three replicates per treatment). The concentrations tested were 5, 6, 7 and 8% hydrolate diluted in water, and a control treatment containing only water. During the LC50-96h test, the physical and chemical parameters of the water were evaluated (temperature, dissolved oxygen, pH, and electrical conductivity). Water was also collected at the beginning and end of the experiment for the analysis of alkalinity, hardness, total ammonia, Na+, K+ and Cl− levels. Gills were collected from the survival fish to determine the density of gill mucous cells (DGMC). The concentrations of 10, 15, 20 and 25% of the hydrolate diluted in water were used for anaesthesia tests, which lasted up to 30 min (15 animals/treatment). After anaesthesia, the recovery time was evaluated. The toxicity test indicated LC50-96h 7.43% (confidence interval: 6.9–8.2%). During LC50-96h, ion loss and increased mucous gill cell density was observed in treatments with LAH. However, a long exposure time (up to 96 h) at lower concentrations (6, 7 and 8%) of the by-product caused osmoregulatory (ion loss) and cellular (increased DGMC) disturbances. The data also indicated that increasing the concentration of hydrolate proportionally decreases the time for anaesthesia induction. Anaesthesia recovery time was not significantly affected by LAH concentration. The concentrations of 20 and 25% of LAH showed the best times for anaesthesia induction in juvenile tambaqui (1.76 min and 2.01 min, respectively).

Aniba rosiodora has been exploited since the end of the nineteenth century for its essential oil, a valuable ingredient in the perfumery industry. This species occurs mainly in Northern South America, and the morphological similarity among different Aniba species often leads to misidentification, which impacts the consistency of products obtained from these plants. Hence, we compared the profiles of volatile organic compounds (essential oils) and non-volatile organic compounds (methanolic extracts) of two populations of A. rosiodora from the RESEX and FLONA conservation units, which are separated by the Tapaj&oacute, s River in Western Par&aacute, State. The phytochemical profile indicated a substantial difference between the two populations: samples from RESEX present &alpha, phellandrene (22.8%) and linalool (39.6%) in their essential oil composition, while samples from FLONA contain mainly linalool (83.7%). The comparison between phytochemical profiles and phylogenetic data indicates a clear difference, implying genetic distinction between these populations.

This study investigated the acute toxicity (LC50-24 hr ) effects of the essential oil of Cymbopogon citratus for adult Argulus sp. and Dolops discoidalis, before and during oviposition. In vitro acute toxicity (LC50-24 hr ) was tested using 20, 40, 60, 80, 100, 120, 140 and 160 μg/L of C. citratus essential oil, and two control groups (one with cultivation tank water and one with cultivation tank water + alcohol) were used. Specimens of Argulus sp. and D. discoidalis submitted to acute toxicity were evaluated using histological procedures. The major chemical compounds of C. citratus essential oil were geranial (47.5%), neral (35.6%) and myrcene (6.7%). The LC50-24 hr for Argulus sp. adults was 67.97 μg/L, while for D. discoidalis it was 59.55 µg/L. In the oviposition of both species of argulids, maximum mortality began with treatments of 140 μg/L, while the LC50-24 hr for Argulus sp. and D. discoidalis was 83.98 μg/L and 82.48 μg/L, respectively. In both argulid species exposed to C. citratus essential oil, morphological alterations were observed only in the eyes, and they occurred in the ommatidium and rhabdomeres and were dependent on the concentration of C. citratus essential oil and the parasite species.

portuguesO objetivo deste estudo foi avaliar a eficacia do oleo essencial de Lippia alba quimiotipo citral (EOLA) como aditivo em transporte simulado com diferentes densidades de carga de juvenis de tambaqui (Colossoma macropomum). O transporte simulado de juvenis de tambaqui em tres densidades de carga (30, 60 e 90 peixes / L) foi realizado apenas com agua (WC) ou 20 μL.L-1 EOLA. Niveis de oxigenio dissolvido, pH, condutividade e temperatura da agua foram medidos no inicio e no final do transporte simulado. O tratamento com EOLA nao alterou a sobrevivencia ou os niveis de oxigenio dissolvido no final da simulacao. O pH da agua no final da simulacao com o EOLA foi menor que o do WC, mas nao mudou com a densidade de carga. A condutividade da agua aumentou com a densidade de carga, no entanto foi menor nos grupos com EOLA. A adicao de EOLA pode ser util no transporte de longo prazo em altas densidades de carga, mas sao necessarios mais estudos relacionados a fisiologia do tambaqui EnglishThe objective of this study was to evaluate the efficacy of the citral chemotype of Lippia alba essential oil (EOLA) as an additive in a simulated transport with different loading densities of tambaqui (Colossoma macropomum) juveniles. Simulated transport of tambaqui juveniles at three loading densities (30, 60 and 90 fish/L) was carried out either with only water (WC) or 20 μL.L-1 EOLA. Dissolved oxygen levels, pH, conductivity and temperature of the water were measured at the beginning and end of the simulated transport. Treatment with EOLA did not change survival or dissolved oxygen levels at the end of simulation. The pH of the water at the end of simulation with EOLA was lower than in that of WC but did not change with loading density. Water conductivity increased with loading density; however, it was lower in the groups with EOLA. The addition of EOLA can be useful in long-term transportation at high loading densities, but further studies related to tambaqui physiology are required

Ethnopharmacological relevance In the region of Western Para, Amazonia, Brazil, Philodendron megalophyllum is widely used for the treatment of envenomations caused by bites from venomous snakes. The traditional use of plants is usually done through oral administration of an infusion (decoction) soon after the bite occurs. The efficiency of aqueous extracts of P. megalophyllum was demonstrated for blocking the activity of the venom of Bothrops sp., but only for a pre-incubation protocol (venom:extract), which fails to simulate the real form of use of this species. In this context, the objective of this research was to evaluate the anti-snakebite potential of the aqueous extract of P. megalophyllum to inhibit for the biological activity induced by Bothrops atrox venom (BaV) using traditional treatment methods. Material and methods Initially, an aqueous extract using the stem of P. megalophyllum (AEPm) was prepared following the standard procedure used by the residents of the rural area along the Tapajos River (Eixo Forte region) in Santarem, PA, Brazil. The phytochemical profile of AEPm was conducted using thin layer chromatography (TLC) and phenolic compounds were quantified through colorimetric trials. The cytotoxicity of AEPm was evaluated using the MRC-5 human fibroblast line, and the antioxidant potential was measured using DPPH methods and cell culture. AEPm antimicrobial action was evaluated by the 96-well plate microdilution and the minimum inhibitory concentration (MIC) methods using 18 types of microorganisms including bacteria that are present in the oral cavity of snakes. AEPm blocking potential was tested against BaV activity in vitro (fibrinolytic) and in vivo (defibrinating and hemorrhagic). In order to test for an interaction between BaV and AEPm SDS-PAGE electrophoresis was conducted. Results The presence of coumarins, fatty acids, and hydrolysable tannins were detected in the AEPm. The colorimetric trials showed that AEPm had a high concentration of condensed tannins (20.1 ± 1.2%). The potential of AEPm for blocking of hemorrhagic and fibrinolytic activity of BaV showed a maximum reduction of 86.1% and 96.5%, respectively, for the pre-incubation protocol (1:10, venom:extract). However, when the extract was administered orally there was no significant blocking of these activities. The interaction of BaV and AEPm showed a modification of the profile of proteic bands when compared to the pattern of bands obtained from the BaV alone. The AEPm was not considered toxic, demonstrated antioxidant activity, and was capable of reducing the growth of 10 of the 18 studied microorganisms. Conclusion Although the stem of P. megalophyllum is indicated by traditional medicine techniques as effective against snakebites, the extract, when tested orally was not able to significantly inhibit (p ˃ 0.05) hemorrhage and defibrinating activity induced by the B. atrox venom. On the other hand, the extract yielded a promising result with respect to antioxidant and antimicrobial potential, and after further studies it could be used as a complementary treatment for localized action and secondary infections that frequently occur with snakebites from the genus of Bothrops sp.

The essential oil of Eugenia uniflora has been attributed anti-depressive, antinociceptive, antileishmanial, larvicidal, antioxidant, antibacterial, and antifungal activities. It is known that the cultivation of this plant can be affected by seasonality, promoting alteration in the oil composition and its biological activities. This study aims to perform the annual evaluation of the curzerene-type oil of E. uniflora and determine its antioxidant activity. The oil yield from the dry season (1.4 &plusmn, 0.6%) did not differ statistically from that of the rainy season (1.8 &plusmn, 0.8%). Curzerene, an oxygenated sesquiterpene, was the principal constituent, and its percentage showed no significant difference between the two periods: dry (42.7% &plusmn, 6.1) and rainy (40.8 &plusmn, 5.9%). Principal component and hierarchical cluster analyses presented a high level of similarity between the monthly samples of the oils. Also, in the annual study, the yield and composition of the oils did not present a significant correlation with the climatic variables. The antioxidant activity of the oils showed inhibition of DPPH radicals with an average value of 55.0 &plusmn, 6.6%. The high curzerene content in the monthly oils of E. uniflora suggests their potential for use as a future phytotherapeutic alternative.

O fruto de Myrciaria dubia é rico em bioativos, este apresenta alto teor de ácido ascórbico e compostos fenólicos, por este motivo, o fruto vem ganhando destaque pela sua capacidade antioxidante. O objetivo deste estudo foi avaliar se diferentes extratos obtidos de cascas do fruto de M. dubia influencia na atividade antioxidante. Extratos hidroalcoólico 70% (EHA) e glicólicos 10 e 20% (EG10 e EG20) foram obtidos de cascas a partir de um pó moderadamente grosso com perda por dessecação de 13,53%, cinzas totais de 1,95%, cinzas insolúveis em ácidos de 0,057%, pH 2,53±0,05 e índice de intumescência de 1,1ml. Os extratos foram caracterizados quanto ao teor de ácido ascórbico e polifenóis totais e a atividade antioxidante determinada por DPPH e ABTS+. O EHA apresentou 7011±39 mg/100ml de ácido ascórbico e 7,75% de polifenóis totais, enquanto que, nos EG10 e EG20 o teor de ácido ascórbico foi 3034±0 e 6137±0 mg/100mL e de polifenóis totais de 2,65 e 3,55% respectivamente. Os extratos apresentaram capacidade antioxidante DPPH com IC50 de 11,80, 48,95 e 25,26 µg/mL e ABTS+ de 92,91±0,205, 74,59±0,259 e 77,21±1,34% nos extratos EHA, EG10 e EG20, respectivamente. Os extratos apresentam conteúdos importantes de compostos bioativos, porém, o solvente influenciou no conteúdo e na atividade biológica, sendo o EHA com maior teor de bioativos o que refletiu também na sua maior capacidade antioxidante.

Myrcia sylvatica (G. Mey.) DC., Myrtaceae, is an aromatic species that occurs in savanna areas of the Lower Amazon River, Brazil. Its essential oil showed an excellent yield, presenting a moss green coloration and a woody and spicy scent. The seasonal and circadian study of the leaf oils and the analysis of oil composition of the fruits were performed by GC and GC-MS. The primary compounds identified in fruit oil were δ-cadinene, β-selinene, 1-epi-cubenol, cubenol, α-calacorene, β-pinene, and trans-muurola-3,5-diene. In the leaf oils, the main compounds found were β-selinene, 1-epi-cubenol, cadalene, mustakone, δ-cadinene, α-calacorene, trans-calamenene, cubenol and caryophyllene oxide. Analyses of PCA and HCA applied to the samples of leaf oils presented a quantitative variation in their compositions, attributed to the rainy and dry periods. Also, it was observed a significant influence on the volatile constituents of the oils in the rainy season, depending on the time of collection. Thus, it was confirmed that the seasonal variation in the oil composition from leaves of M. sylvatica should be due to the influence of the climatic parameters, during the plant collection.

Ethnopharmacological relevance Extracts of leaves and bark of Aniba fragrans are used as tea (decoction) to treat snakebites in communities in the Brazilian Amazon. The aqueous extract of the leaves of A. fragrans has been proven to be effective against Bothrops venom, but only when pre-incubated with the venom. This study sought to assess the potential of different types of extract of this species to inhibit the biological activities of Bothrops atrox venom (BaV) when used the same way as in folk medicine. The main classes of secondary metabolites and the concentrations of phenolics in the extracts were also determined. Materials and methods Four types of extract of A. fragrans were prepared: aqueous extract of the leaf (AEL), aqueous extract of the bark (AEB), hydroalcoholic leaf extract (HLE) and extract of the residue from hydrodistillation of the leaf (ERHL). The phytochemical profiles of the aqueous extracts were determined using thin layer chromatography (TLC), and the concentrations of phenolics were measured by colorimetric assays. To investigate the potential of the extracts to inhibit the biological activities of BaV, in vitro tests for antiphospholipase and antifibrinolytic activities were performed. In vivo tests for antihemorrhagic and antidefibrinating activities were also carried out, as well as antimicrobial tests for activity against the main bacteria found in the oral cavity of snakes. Interaction between the extracts and the proteins in BaV was assessed by electrophoresis (SDS-PAGE) and Western blot (WB). The cytotoxicity of the extracts was assessed in a strain of MRC-5 human fibroblasts. Results Terpenoids, flavonoids and condensed and hydrolysable tannins were detected in all the extracts. Metabolites such as coumarins, fatty acids and alkaloids were present in some extracts but not in others, indicating different phytochemical profiles. Phenolics content varied between extracts, and there were more tannins in AEB and HLE. In the in vitro tests, the extracts inhibited the phospholipase and fibrinolytic activities of BaV in the two ratios of venom to extract used. HLE exhibited effective antimicrobial action as it inhibited growth of 11 of the 15 bacteria investigated, including Morganella morganii, the main bacteria described in the oral cavity of snakes. The extracts failed to inhibit the defibrinating activity of BaV, and only the Bothrops antivenom had a significant effect (96.1%) on this activity. BaV-induced hemorrhage was completely inhibited by AEL and AEB when the pre-incubation (venom:extract) protocol was used. When administered orally, as in folk medicine, both AEB and AEL produced significant inhibition of hemorrhagic activity (maximum inhibition 46.5% and 39.2%, respectively). SDS-PAGE and WB of the extracts pre-incubated with BaV showed that the main proteins in the venom had been precipitated by the extracts. None of the four extracts showed cytotoxic effects in the tests carried out with a human fibroblast cell line. Conclusion In addition to being effective in reducing hemorrhage when administered orally, the extracts displayed a high antimicrobial potential against microorganisms involved in secondary infections at the site of the snakebite. Once the extracts have been tested in accordance with the appropriate regulations, this species could potentially be used to produce a phytomedicine for complementary treatment of the secondary infections due to bacteria that aggravate the local signs and symptoms after snakebite envenomation.

The extracts of Plathymenia reticulata and Connarus favosus are widely used in the folk medicine. The potential protective effects of these extracts have been evaluated against cadmium in the yeast Saccharomyces cerevisiae, and against mercurial contamination in zebrafish Danio rerio. In yeast, both extracts efficiently protected the Δycf1 mutant strain exposed to cadmium chloride restoring the growth, the expression of stress-response genes and decreasing the level of oxidative stress. In zebrafish, the supplementation of methylmercury-contaminated diet with both plant extracts similarly protected fish through the suppression of the methylmercury-induced lipid peroxidation, decrease of acetylcholinesterase activity, and restoring the expression levels of stress-response genes. This study particularly demonstrates the protective potential of both aqueous extracts against methylmercury, and could represent an interesting alternative for the Amazonian fish-eating communities to cope with the impact of chronic exposure to contaminated diets.

Differences in snake venom composition occur across all taxonomic levels and it has been argued that this variation represents an adaptation that has evolved to facilitate the capture and digestion of prey and evasion of predators. Bothrops atrox is a terrestrial pitviper that is distributed across the Amazon region, where it occupies different habitats. Using statistical analyses and functional assays that incorporate individual variation, we analyzed the individual venom variability in B. atrox snakes from four different habitats (forest, pasture, degraded area, and floodplain) in and around the Amazon River in Brazil. We observed venom differentiation between spatially distinct B. atrox individuals from the different habitats, with venom variation due to both common (high abundance) and rare (low abundance) proteins. Moreover, differences in the composition of the venoms resulted in individual variability in functionality and heterogeneity in the lethality to mammals and birds, particularly among the floodplain snakes. Taken together, the data obtained from individual venoms of B. atrox snakes, captured in different habitats from the Brazilian Amazon, support the hypothesis that the differential distribution of protein isoforms results in functional distinctiveness and the ability of snakes with different venoms to have variable toxic effects on different prey.

Lippia alba hydrolate (LAH) is produced as a secondary product in essential oil extraction. The objective of this study was to evaluate the use of LAH in a simulation of the transport of tambaqui (Colossoma macropomum) juveniles. A series of 17 hr simulations were performed using load densities of 30, 60 and 90 juveniles per L and four LAH concentrations of 0.4%, 1%, 2% and 5%, with a control of just water. Fish survival, temperature, dissolved oxygen, conductivity, pH, ammonia excretion, alkalinity water hardness and net Na+, K+, Cl- fluxes, were all evaluated before and after the transport simulations. Mucous cell densities in the gills were also determined microscopically, post simulation. The results showed that addition of 5% LAH achieved better survival during the simulation than the water control. Dissolved oxygen was significantly lower with an increased load density. Also, conductivity, NH4 excretion and net ionic fluxes (K+ and Cl−) were lower with 5% LAH, although mainly where higher load densities were tested. Mucous cell density was lower in LAH concentrations of 2% and 5%, again with higher load densities. In view of these findings, it is suggested that 5% LAH is an effective sedative for use in the transport of tambaqui, mainly with high load densities. This work is the first study of LAH and highlights its potential applications in aquaculture management.

The hemiparasitic species Psittacanthus plagiophyllus Eichl. (Loranthaceae), also known as erva de passarinho, is used in folk medicine in the Santarém region in the state of Pará, Brazil, to treat gastritis and a variety of inflammatory disorders. In view of the lack of pharmacological studies on this species in the literature and the fact that it is used constantly by the local population, this study sought to standardize the extract of the leaves of P. plagiophyllus (AEPp) and to assess its anti-inflammatory potential in in vivo tests.Quality control and standardization of AEPp were performed following the 5th edition of the Brazilian Pharmacopeia. To assess the in vivo anti-inflammatory activity of AEPp, the carrageenan-induced and dextran-induced rat paw edema models were initially used. To investigate the effect of AEPp on carrageenan-induced leukocyte recruitment and exudate production, the air pouch inflammation model was used. In addition, the antioxidant activity of AEPp was assessed in vitro by the DPPH radical scavenging method.The chromatographic profile of AEPp indicated the presence of flavonoids, coumarins and hydrolyzable and condensed tannins. Measurement of phenolics revealed the following percentages in the extract: 12.62±0.18% total phenolics, 5.39±0.01% total tannins, 12.54±0.24% hydrolyzable tannins, 8.37±0.32% condensed tannins and 1.23±0.02% total flavonoids. In 500 and 1000mg/kg doses (p.o.) AEPp had significant edema-inhibiting activity (p0.01) in both the models used, suggesting that the extract may act in vascular and cell events in the inflammatory response and exert an inhibitory effect on mediators responsible for edema. In all the doses tested [250, 500 and 1000mg/kg (p.o.)], AEPp inhibited total leukocyte and neutrophil migration and reduced the amount of exudate in the air pouch in a dose-dependent manner. Both effects were statistically significant (p0.01). The extract also reduced the DPPH radical (maximum reduction 93.13±1.71% at a concentration of 75µg/mL), indicating that it has antioxidant activity. AEPp, therefore, exhibited considerable in vivo anti-inflammatory activity and in vitro antioxidant activity. This may be due to its high phenolics content.These findings provide evidence to support the use of P. plagiophyllus in folk medicine to treat inflammatory disorders.

Venom variability is commonly reported for venomous snakes including Bothrops atrox. Here, we compared the composition of venoms from B. atrox snakes collected at Amazonian conserved habitats (terra-firme upland forest and varzea) and human modified areas (pasture and degraded areas). Venom samples were submitted to shotgun proteomic analysis as a whole or compared after fractionation by reversed-phase chromatography. Whole venom proteomes revealed a similar composition among the venoms with predominance of SVMPs, CTLs, and SVSPs and intermediate amounts of PLA2s and LAAOs. However, when distribution of particular isoforms was analyzed by either method, the venom from varzea snakes showed a decrease in hemorrhagic SVMPs and an increase in SVSPs, and procoagulant SVMPs and PLA2s. These differences were validated by experimental approaches including both enzymatic and in vivo assays, and indicated restrictions in respect to antivenom efficacy to variable components. Thus, proteomic analysis at the isoform level combined to in silico prediction of functional properties may indicate venom biological activity. These results also suggest that the prevalence of functionally distinct isoforms contributes to the variability of the venoms and could reflect the adaptation of B. atrox to distinct prey communities in different Amazon habitats. Biological significance In this report, we compared isoforms present in venoms from snakes collected at different Amazonian habitats. By means of a species venom gland transcriptome and the in silico functional prediction of each isoform, we were able to predict the principal venom activities in vitro and in animal models. We also showed remarkable differences in the venom pools from snakes collected at the floodplain (varzea habitat) compared to other habitats. Not only was this venom less hemorrhagic and more procoagulant, when compared to the venom pools from the other three habitats studied, but also this enhanced procoagulant activity was not efficiently neutralized by Bothrops antivenom. Thus, using a functional proteomic approach, we highlighted intraspecific differences in B. atrox venom that could impact both in the ecology of snakes but also in the treatment of snake bite patients in the region.

Ethnopharmacological importance The effectiveness of aqueous extract of Bellucia dichotoma Cogn. (Melastomataceae) specimems collected in Santarem, PA, against some biological activities of Bothrops atrox venom (BaV) has been scientifically proven. Here, we analyzed the components and assessed the anti-snakebite potential of aqueous extracts of bark of B. dichotoma collected in Manaus, AM, (AEBd-MAO) and Santarem, PA, (AEBd-STM), both in Brazil. Materials and methods The phytochemical profiles of the aqueous extracts were identified using thin layer chromatography (TLC), and the concentrations of phenolics were determined by colorimetric assay. The inhibitory potential of the extracts was tested against the phospholipase A 2 , coagulant and gelatinolytic activities of BaV in vitro and its defibrinating and edema-inducing activities in vivo. Interaction between BaV and the extracts was investigated using SDS-Page electrophoresis and Western blotting. Extract cytotoxicity and antioxidant potential were assessed using the human fibroblast cell line MRC-5 and the DPPH assay in cell culture, respectively. Results While there was no difference between the phytochemical profiles of the extracts, AEBd-MAO had higher concentrations of total phenolics, total tannins and hydrolysable tannins. The extracts inhibited 100% of the phospholipase and coagulant activity of BaV when pre-incubated. Without pre-incubation, however, there was no reduction in phospholipase activity, although significant inhibition of coagulant activity was observed. In the doses used in folk medicine, without pre-incubation, both extracts inhibited 100% of the coagulant activity of BaV. In vivo, the extracts were unable to inhibit the defibrinating activity of the venom but were effective in inhibiting its edema-inducing activity. In the profiles of the extracts pre-incubated with BaV, not all the protein bands revealed by SDS-PAGE and Western blot were observed. Both extracts had a high antioxidant potential and neither had a cytotoxic effect. Conclusion Although the concentrations of phenolics in each extract were different, the anti-snakebite potential was similar for the concentrations of extract tested. Our findings are of importance for the quality control of this raw material, which, once tested in accordance with Brazilian National Health Surveillance Agency recommendations, may be suitable for use as a phytomedicine to complement treatment of the local effects induced by Bothrops venoms.

Variability in snake venom composition has been frequently reported and correlated to the adaptability of snakes to environmental conditions. Previous studies report plasticity for the venom phenotype. However, these observations are not conclusive, as the results were based on pooled venoms, which present high individual variability. Here we tested the hypothesis of plasticity by influence of confinement and single diet type in the venom composition of 13 adult specimens of Bothrops atrox snakes, maintained under captivity for more than three years. Individual variability in venom composition was observed in samples extracted just after the capture of the snakes. However, composition was conserved in venoms periodically extracted from nine specimens, which presented low variability restricted to the less abundant components. In a second group, composed of four snakes, drastic changes were observed in the venom samples extracted at different periods, mostly related to snake venom metalloproteinases (SVMPs), the core function toxins of B. atrox venom, which occurred approximately between 400 and 500 days in captivity. These data show plasticity in the venom phenotype during the lifetime of adult snakes maintained under captive conditions. Causes or functional consequences involved in the phenotype modification require further investigations.

The aim of this study was to investigate the anesthetic effect of the essential oils (EOs) of Lippia alba (citral and linalool chemotypes, EOLA-C and EOLA-L respectively) and Lippia origanoides (chemotype carvacrol, EOLO-Ca) on tambaqui (Colossoma macropomum) juveniles. The tested concentrations of the L. alba and L. origanoides EOs were: 25, 50, 100, and 200 μL/L. Time required to reach the stages of sedation, deep anesthesia, and recovery, as well as ventilatory frequency, were monitored for each EO concentration. The results indicated that all EO concentrations tested induced tambaqui anesthesia and that the ventilatory frequency was altered by the EOs. L. origanoides showed the shortest time to induce sedation and anesthesia, but had long recovery times. L. alba EO (citral 100 μL/L and linalool 200 μL/L) can be used to efficiently anaesthetise tambaqui juveniles with an efficient recovery. RESUMO: O objetivo deste estudo foi investigar o efeito anestésico dos óleos essenciais (EOs) de Lippia alba (quimiotipos citral e linalool - EOLA-C e EOLA-L respectivamente) e Lippia origanoides (quimiotipo carvacrol- EOLO-Ca) em juvenis de tambaqui (Colossoma macropomum). As concentrações testadas dos EOs de L. alba e L. origanoides foram: 25, 50, 100 ou 200 μL/L. O tempo para atingir os estágios de sedação, anestesia profunda, recuperação e frequência ventilatória foram monitorados em cada concentração de EO. Os resultados indicam que todas as concentrações testadas dos OE induziram a anestesia do tambaqui e que a frequência ventilatória foi alterada pelos EOs. L. origanoides mostrou o tempo mais rápido para induzir sedação e anestesia, mas os tempos de recuperação foram longos. EO L. alba (citral 100 μL/L e linalol 200 μL/L) pode ser utilizado como anestésico em juvenis de tambaqui, pela eficiência em anestesiar e recuperar.

EnglishAnesthetics are effective to reduce or minimize stress in handling operations related to fish cultures. Aiming to avoid residual effects of synthetic drugs, several natural agents, such as essential oils (EOs), have been tested. The aim of this study was to determine the optimal concentration of the EO of Ocimum micranthum for induction of anesthesia in juveniles of silver catfish (Rhamdia quelen) and grass carp (Ctenopharyngodon idella). Results demonstrated that the most suitable concentration to sedate silver catfish and carp is 25μL/L, while to anesthetize 200μL/L is more suitable for silver catfish and 100μL/L for grass carp, because these concentrations induce deep anesthesia in less than three min and recovery in less than five min. Analysis of the EO constituents showed methyl chavicol, a compound with carcinogenic potential, as the major component (58.2%). Therefore, the EO of O. micranthum is not recommended for fish anesthesia if the fish are to be used as food. Key words: anesthesia; silver catfish; grass carp; sedation portuguesOs anestesicos sao eficazes para reduzir ou minimizar o estresse nas operacoes de manuseio relacionadas a piscicultura. Com o objetivo de evitar efeitos residuais de farmacos sinteticos, varios agentes naturais, como oleos essenciais (OEs), tem sido testados. O objetivo deste estudo foi determinar a concentracao otima do OE de Ocimum micranthum para a inducao de anestesia em juvenis de jundia (Rhamdia quelen) e carpa capim (Ctenopharyngodon idella). Os resultados demonstram que a concentracao mais adequada para sedar jundias e carpas capim e 25μL/L. Enquanto que para anestesiar 200μL/L e mais adequado para jundias e 100μL/L para carpas capim, pois essas concentracoes induzem anestesia profunda em menos de tres minutos e recuperacao em menos de cinco minutos. A analise dos constituintes do OE identificou o metil chavicol, um composto com potencial carcinogenico, como componente majoritario (58,2%). Portanto, o OE de O. micranthum nao e recomendado para anestesia de peixes usado como alimento. Palavras-chave: anestesia; jundia; carpa-capim; sedacao

This work analyzed the chemical compositions and evaluated the antioxidant and cytotoxic activities of essential oils (EO) of Eugenia patrisii (Epat), Eugenia stipitata (Esti), Myrcia splendens (Mspl), Myrcia sylvatica (Msyl), Psidium guajava (Pgua), and Psidium guineense (Pgui-1 and Pgui-2) from the Brazilian Amazon. Sesquiterpenoids were found in high concentrations in the oils of E. patrisii and M. splendens, which were rich in E-caryophyllene (32.0% and 45.8%); E. stipitata and M. sylvatica, which displayed germacrene D (11.8%) and germacrene B (24.5%); and P. guajava that showed epi-β-bisabolol (16.1%) as the main compound. However, P. guineense samples (Pgui-1 and Pgui-2) were rich in monoterpenoids such as limonene (Pgui-1: 30.2%; Pgui-2 30.4%) and α-pinene (Pgui-1: 22.5%; Pgui-2: 17.7%). The samples showed a weak and moderate antioxidant activities in the DPPH assay, displaying inhibition rates from 11.5% to 38.6% (at 10 mg/mL). All samples were cytotoxic against human cancer cells by the MTT method. Epat oil showed higher activity against melanoma (SKMEL-19, IC505.8 µg/mL), gastric (AGP01, IC503.2 µg/mL), and colon (HCT116, IC506.7 µg/mL). Meanwhile, the samples Pgua and Pgui were more active against breast cancer cells (MCF7, IC5012.4 µg/mL and 11.6 µg/mL, respectively).

This study aimed to evaluate the sedative and anesthetic potential of the essential oil (PHEO) and hydrolate (PHH) from the fruit of Protium heptaphyllum and the isolated compounds (IC) of PHEO in tambaqui (Colossoma macropomum) juveniles. Fish were placed in aquaria with different concentrations of PHEO, PHH or IC (sabinene, terpinen-04-ol, γ-terpinene and α-pinene) to evaluate sedative and anesthetic efficacy. In another experiment, fish were transported for 22 h with 10% anesthetic concentrations added to the water of transport. No similarities regarding the major compounds of PHEO and PHH were observed. No mortality was observed during the experiments, and PHH did not show any sedative or anesthetic effect. PHEO induced sedation at 250 mg L−1 and deep anesthesia at 500, 750 and 1000 mg L−1. The recovery time increased with increasing concentration of PHEO. There was a significant difference in the time to induce deep anesthesia between PHEO and IC. Water conductivity and mucous cell density were lower in the transport with 50 mg L−1 PHEO. Tambaqui transported with 50 mg L−1 PHEO and 11.6, 17.4, and 23.2 mg L−1 terpinen-04-ol presented lower net whole body K+ and Cl− effluxes. PHEO is an alternative as sedative and anesthetic for tambaqui juveniles, and the concentration of 500 mg L−1 was the best for deep anesthesia. The use of 50 mg L−1 PHEO was effective for the transport of this species because it reduced net K+ and Cl− effluxes and did not stimulate proliferation mucous cells.

A true sustainable and ecofriendly concept of utilization of natural products must involve not only the obtainment of plant material in consonance with biodiversity preservation, but also the potential lower risk to the environment and lower consume of energy in the transformation of the plant into novel products. We therefore describe an elegant study in which the fruits of Pterodon emarginatus, a high promising Brazilian species that can be obtained through a sustainable use of biodiversity, are the natural raw material for the preparation of nano-emulsions. The essential oil was prepared by hydrodistillation, using a classical extraction with a non-toxic solvent instead of organic solvents. Moreover, the utilization of low energy emulsification method at room temperature and rational design with blends of non-ionic surfactants allowed the preparation of nano-emulsions with desirable properties. An optimal nano-emulsion with droplet size around 110−130 nm, low polydispersity index (around 0.14 – 0.20) and high kinetic stability was obtained. The process of preparation of this nano-emulsion was reproducible and it presented stability towards temperature (from 25 Co to 80 Co) and also long-term physical stability for 540 days in storage. Moreover, toxicity in vitro assays were also performed in order to evaluate the potential risks of the optimal nano-emulsion. The results showed that it could be considered non-irritant. Therefore, the present study shows the feasibility of classical concepts of nano-emulsion stabilization to generate a novel nano-emulsion with desirable properties.

Parasitic diseases have an enormous health and economic impact and are a particular problem in tropical regions of the world. Disease caused by protozoa, such as trypanosomiasis, are the cause of most parasite related morbidity and mortality. Thus, the aim of this study was to verify the trypanocidal effectiveness of Lippia alba and Lippia origanoides against Trypanosoma evansi in vitro and in vivo. L. alba and L. origanoides were used in vitro on trypomastigotes at different concentrations (0.5, 1.0 and 2.0 %) and exposure times (0, 1, 3, 6 and 9 h). The three concentrations tested showed trypanocidal activity in vitro, completely eliminating the parasites in small concentration after 6 h of assay. In vivo tests were performed using mice as the experimental model. T. evansi infected mice were treated with L. alba and L. origanoides with dose of 1.5 mL kg−1 during 5 days. These protocols did not provide curative efficacy, however the mice treated with L. origanoides showed a significant increase in the longevity when compared to control group. Active compounds present in essential oils, such as L. origanoides, may potentiate the treatment of trypanosomosis when associated with other trypanocidal drugs.

Anaesthetic substances are necessary to reduce fish stress during aquaculture activities. The objectives of this study were: (i) to determine the efficacy of essential oils (EOs) of Myrcia sylvatica (EOMS) and Curcuma longa (EOCL) as anaesthetics for Colossoma macropomum and (ii) to evaluate the effects of rapid anaesthesia and long-term sedation (6 h) with these oils. Therefore, the main primary stress indicator (cortisol) and secondary factors (biochemical indices, hepatic metabolism, oxidative biomarkers) were measured. Sedation with the EOCL resulted in lower cortisol levels compared to control group. Total cholesterol levels were lower in fish sedated with EOMS than in control. Lactate levels were higher in fish anaesthetized with both EOs and sedated with EOCL compared to control. Both EOs increased hepatic glycogen levels after anaesthesia and EOMS increased this parameter after sedation compared to control. Anaesthesia and sedation with EOs resulted in lower levels of lipid peroxidation (LPO) compared to control. In turn, the activity of some antioxidant enzymes evaluated (superoxide dismutase, catalase, glutathione peroxidase, glutathione reductase and glutathione-S-transferase), the content of non-protein thiols and total reactive antioxidant potential were higher in tissues of fish anaesthetized and sedated with EOs compared to control. This induction of antioxidant capacity in the tissues could be due to the antioxidant property exerted by these EOs. Thus, EOMS and EOCL are recommended for anaesthesia and sedation of fish because in spite of inducing anaerobic metabolism, these EOs did not alter most biochemical parameters, reduced the LPO and increased the antioxidant capacity in vital tissues.

This study investigated the efficacy of essential oils of Lippia alba (EOLA) citral chemotype and Lippia origanoides (EOLO) and their effects on ventilatory frequency (VF) of silver catfish, Rhamdia quelen. Fish were exposed to 50-300 μL L−1 of EOLA and 25-300 μL L−1 of EOLO to determine induction times to sedation, anesthesia, and recovery. Moreover, VF was determined in fish exposed to 5 or 10 μL L−1 of EOLA and of EOLO for 8 h. The increasing concentration of essential oils proportionally decreased sedation and anesthesia induction times. The highest EOLA concentration increased VF of fish from the control group at 1 h of exposure, but VF decreased at both EOLO concentrations after 2 h. The EOLA citral chemotype and EOLO were effective sedatives and anesthetics for silver catfish. However, EOLO was the most suitable sedative for additional studies regarding fish transport as it reduced VF and did not induce VF increase in silver catfish. The EOLA citral chemotype and EOLO are effective sedatives and anesthetics for silver catfish. Moreover, the EOLO is recommended for transport of silver catfish, because it maintains the ventilatory frequency constant, avoiding a possible metabolic stress.