Antiedema and antinociceptive potential of the essential oil of Pectis elongata Kunt (Asteraceae) from the Brazilian Amazon.

ETHNOPHARMACOLOGICAL RELEVANCE: Pectis elongata, a herbaceous species that is known in northern Brazil as "cominho" or "limãozinho", is traditionally used in the region for the treatment of fevers, colds, hypotension, genitourinary and gastric disorders, and pain. AIM OF THE STUDY: Determine the chemical composition and acute oral toxicity and evaluate whether Pectis elongata essential oil (PeEO) has antiedema and antinociceptive activity. MATERIALS AND METHODS: The chemical composition was determined using gas chromatography coupled to mass spectrometry (GC/MS) and the degree of toxicity determined by the LD50. The antiedema and antinociceptive potential was evaluated via the λ-carrageenan-induced paw edema test and formalin test, respectively. RESULTS: Citral (geranial and neral) was characterized as a major component of the PeEO, representing 89% of the total identified compounds. According to OECD criteria (2002), the EO was considered non-toxic since it presented LD50 values over 2000 mg kg-1. Its antiedema potential was observed at doses of 200 and 400 mg kg-1 (p ≤ 0.05). At a dose of 400 mg kg-1, PeEO also showed antinociceptive potential (p ≤ 0.05), both in the neurogenic phase and in the inflammatory phase. CONCLUSIONS: PeEO, which is rich in citral, did not induce any characteristic signs of acute oral toxicity and was also efficient in reducing carrageenan-induced paw edema, in addition to presenting antinociceptive potential and acting on both central and peripheral pain. It is thus a promising candidate for the development of a new herbal medicine with anti-inflammatory and analgesic action.

Evaluation of the quality of Amazonian butters as sustainable raw materials for applications in bioproducts

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.

Nano-emulsification of Aeollanthus suaveolens Mart. Ex Spreng essential oil modifies its neuroeffects?

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.

Lethal concentration of Cymbopogon citratus (Poaceae) essential oil for Dolops discoidalis and Argulus sp. (Crustacea: Argulidae).

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.

Citral chemotype of the Lippia alba essential oil as an additive in simulated transport with different loading densities of tambaqui juveniles / Óleo essencial de Lippia alba quimiotipo citral como aditivo em transporte simulado de juvenis de Tambaqui com diferentes densidades

ABSTRACT: The 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.

RESUMO: O objetivo deste estudo foi avaliar a eficácia do óleo 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 três densidades de carga (30, 60 e 90 peixes / L) foi realizado apenas com água (WC) ou 20 μL.L-1 EOLA. Níveis de oxigênio dissolvido, pH, condutividade e temperatura da água foram medidos no início e no final do transporte simulado. O tratamento com EOLA não alterou a sobrevivência ou os níveis de oxigênio dissolvido no final da simulação. O pH da água no final da simulação com o EOLA foi menor que o do WC, mas não mudou com a densidade de carga. A condutividade da água aumentou com a densidade de carga, no entanto foi menor nos grupos com EOLA. A adição de EOLA pode ser útil no transporte de longo prazo em altas densidades de carga, mas são necessários mais estudos relacionados à fisiologia do tambaqui.

Evaluation of the anti-snakebite, antimicrobial and antioxidant potential of Philodendron megalophyllum Schott (Araceae), traditionally used in accidents caused by snakes in the western region of Pará, Brazil

Ethnopharmacological relevance In the region of Western Pará, 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 Tapajós River (Eixo Forte region) in Santarém, 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

Evaluation of the anti-snakebite, antimicrobial and antioxidant potential of Philodendron megalophyllum Schott (Araceae), traditionally used in accidents caused by snakes in the western region of Pará, Brazil

Ethnopharmacological relevance In the region of Western Pará, 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 Tapajós River (Eixo Forte region) in Santarém, 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

Protective Effects of Plathymenia reticulata and Connarus favosus Aqueous Extracts against Cadmium- and Mercury-Induced Toxicities.

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.

Anesthetic potential of the essential oils of Lippia alba and Lippia origanoides in Tambaqui juveniles / Potencial anestésico do óleo essencial de Lippia Alba e Lippia origanoides em juvenis de Tambaqui

ABSTRACT: 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.

Plant essential oils against bacteria isolated from fish: an in vitro screening and in vivo efficacy of Lippia origanoides / Óleos essenciais de plantas contra bactérias isoladas de peixes: uma triagem in vitro e eficácia in vivo de Lippia origanoides

ABSTRACT: The use of natural products, such as essential oils (EOs), is a potential novel approach to treat fish bacterial infections with a lower risk of developing resistance. There has been a number of studies reporting the activity of EOs as those obtained from the species Achyrocline satureioides, Aniba parviflora, Aniba rosaeodora, Anthemis nobilis, Conobea scoparioides, Cupressus sempervirens, Illicium verum, Lippia origanoides, and Melaleuca alternifolia against bacteria. However, there are few studies investigating the effect of these EOs against fish bacteria. Therefore, the aim of this study was to evaluate the in vitro antibacterial activity of EOs against the following fish bacteria, Aeromonas hydrophila, Citrobacter freundii, and Raoultella ornithinolytica. Additionally, the in vivo antibacterial activity of the EO L. origanoides was evaluated against experimentally induced A. hydrophila infection of silver catfish (Rhamdia quelen). The EO of L. origanoides was chosen as it showed the highest in vitro antibacterial activity, with minimum inhibitory concentrations ranging from 0.2 to 0.8 mg mL-1. This EO also presented a therapeutic success of 58.33%, on a 30 day A. hydrophila infection. Therefore, we suggested that the EO of L. origanoides may be a viable alternative as a treatment for A. hydrophila infection.

RESUMO: O uso de produtos naturais, como óleos essenciais (OEs), é uma nova abordagem potencial para o tratamento de infecções bacterianas em peixes com um baixo risco de desenvolvimento de resistência. Existem estudos reportando a atividade de OEs como aqueles obtidos das espécies Achyrocline satureioides, Aniba parviflora, Aniba rosaeodora, Anthemis nobilis, Conobea scoparioides, Cupressus sempervirens, Illicium verum, Lippia origanoides e Melaleuca alternifolia contra bactérias. Porém, existem poucos estudos investigando o efeito desses OEs supracitados contra bactérias isoladas de peixes. Portanto, o objetivo desse estudo foi avaliar a atividade antibacteriana in vitro desses OEs contra bactérias isoladas de peixes (Aeromonas hydrophila, Citrobacter freundii e Raoultella ornithinolytica). Assim como avaliar a atividade antibacteriana in vivo do OE de L. origanoides contra uma infecção por A. hydrophila experimentalmente induzida em jundiá (Rhamdia quelen). O OE de L. origanoides foi escolhido porque apresentou a melhor atividade antibacteriana in vitro, com concentrações inibitórias mínimas variando de 0,2 a 0,8 mg mL-1. Esse OE apresentou sucesso terapêutico de 58,33% no dia 30 pós-infecção. Portanto, sugerimos que o OE de L. origanoides possa ser uma alternativa viável no tratamento da infecção por A. hydrophila em peixes.

Assessment of the anti-snakebite properties of extracts of Aniba fragrans Ducke (Lauraceae) used in folk medicine as complementary treatment in cases of envenomation by Bothrops atrox.

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.

Assessment of the anti-snakebite properties of extracts of Aniba fragrans Ducke (Lauraceae) used in folk medicine as complementary treatment in cases of envenomation by Bothrops atrox

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 hydro distillation 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. Bay-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.

Assessment of the anti-snakebite properties of extracts of Aniba fragrans Ducke (Lauraceae) used in folk medicine as complementary treatment in cases of envenomation by Bothrops atrox

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 hydro distillation 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. Bay-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.

Anesthesia and sedation of map treefrog (Hypsiboas geographicus) tadpoles with essential oils / Anestesia e sedação de girinos da perereca geográfica Hypsiboas geographicus com óleos essenciais

ABSTRACT: The goal of this study was to investigate the sedative and anesthetic properties of essential oils (EOs) in map treefrog tadpoles (Hypsiboas geographicus) and to determine the sedation and deep anesthesia induction times as well as the recovery time. The tadpoles were exposed to one of the EOs from three plant species: Aniba rosaeodora (EOAR - 25, 50, 100 or 200µL L-1), Lippia origanoides (EOLO - 13, 25, 50, 100 or 200µL L-1), and Lippia alba (either chemotype citral [EOL-C - 25, 50, 100 or 200µL L-1] or linalool [EOL-L - 50, 75, 100 or 200µL L-1]) (n = 8 per replicate). The tadpoles exposed to 25 and 50µL L-1 EOL-C and EOL-L, respectively, were not anesthetized within 30min (the maximum time of observation), and those exposed to 200µL L-1 EOLO did not recover within 30min. Sedation, deep anesthesia and recovery times showed a concentration-dependent relationship for all EOs tested, with the exception of the recovery with EOLO. The results allowed concluding that all investigated EOs can be used to anesthetize tadpoles of H. geographicus, but the use of EOLO must not exceed 100µL L-1.

RESUMO: O objetivo deste estudo foi investigar as propriedades sedativas e anestésicas de óleos essenciais (OEs) em girinos da perereca Hypsiboas geographicus e determinar os tempos de indução à sedação e anestesia profunda, bem como o de recuperação. Os girinos foram expostos a um dos OEs de três espécies de plantas: Aniba rosaeodora (OEAR - 25, 50, 100 ou 200µL L-1), Lippia origanoides (OELO - 13, 25, 50, 100 ou 200µL L-1 ) ou Lippia alba quimiotipos citral (OEL-C - 25, 50, 100 ou 200µL L-1) ou linalol (OEL-L - 50, 75, 100 ou 200µL L-1) (n = 8 cada repetição). Girinos expostos a 25 e 50µL L-1 OEL-C e OEL-L, respectivamente, não foram anestesiados dentro de 30min (tempo máximo de observação) e aqueles expostos a 200µL L-1 OELO não recuperaram dentro de 30min. Os tempos de sedação, anestesia profunda e recuperação apesentaram uma relação concentração-resposta para todos os OEs testadas, exceto a recuperação com OELO. Os resultados permitem concluir que todos os OEs investigados podem ser usados para anestesiar os girinos de H. geographicus, mas o uso de OELO não deve ser superior a 100µL L-1.

Trypanocidal action of Lippia alba and Lippia origanoides essential oils against Trypanosoma evansi in vitro and in vivo used mice as experimental model.

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.

Experimental Bothrops atrox envenomation: Efficacy of antivenom therapy and the combination of Bothrops antivenom with dexamethasone.

Bothrops atrox snakes are the leading cause of snake bites in Northern Brazil. The venom of this snake is not included in the antigen pool used to obtain the Bothrops antivenom. There are discrepancies in reports on the effectiveness of this antivenom to treat victims bitten by B. atrox snakes. However, these studies were performed using a pre-incubation of the venom with the antivenom and, thus, did not simulate a true case of envenomation treatment. In addition, the local lesions induced by Bothrops venoms are not well resolved by antivenom therapy. Here, we investigated the efficacy of the Bothrops antivenom in treating the signs and symptoms caused by B. atrox venom in mice and evaluated whether the combination of dexamethasone and antivenom therapy enhanced the healing of local lesions induced by this envenomation. In animals that were administered the antivenom 10 minutes after the envenomation, we observed an important reduction of edema, dermonecrosis, and myonecrosis. When the antivenom was given 45 minutes after the envenomation, the edema and myonecrosis were reduced, and the fibrinogen levels and platelet counts were restored. The groups treated with the combination of antivenom and dexamethasone had an enhanced decrease in edema and a faster recovery of the damaged skeletal muscle. Our results show that Bothrops antivenom effectively treats the envenomation caused by Bothrops atrox and that the use of dexamethasone as an adjunct to the antivenom therapy could be useful to improve the treatment of local symptoms observed in envenomation caused by Bothrops snakes.

Standardization and anti-inflammatory activity of aqueous extract of Psittacanthus plagiophyllus Eichl. (Loranthaceae).

ETHNOPHARMACOLOGICAL IMPORTANCE: 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. MATERIAL AND METHODS: 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. RESULTS: 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 (p<0.01) 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 (p<0.01). 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. CONCLUSION: These findings provide evidence to support the use of P. plagiophyllus in folk medicine to treat inflammatory disorders.

The potential of aqueous extracts of Bellucia dichotoma Cogn. (Melastomataceae) to inhibit the biological activities of Bothrops atrox venom: A comparison of specimens collected in the states of Pará and Amazonas, Brazil.

ETHNOPHARMACOLOGICAL IMPORTANCE: The effectiveness of aqueous extract of Bellucia dichotoma Cogn. (Melastomataceae) specimems collected in Santarém, 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 Santarém, 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 A2, 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.

Experimental Bothrops atrox envenomation: efficacy of antivenom therapy and the combination of Bothrops antivenom with dexamethasone

Bothrops atrox snakes are the leading cause of snake bites in Northern Brazil. The venom of this snake is not included in the antigen pool used to obtain the Bothrops antivenom. There are discrepancies in reports on the effectiveness of this antivenom to treat victims bitten by B. atrox snakes. However, these studies were performed using a pre-incubation of the venom with the antivenom and, thus, did not simulate a true case of envenomation treatment. In addition, the local lesions induced by Bothrops venoms are not well resolved by antivenom therapy. Here, we investigated the efficacy of the Bothrops antivenom in treating the signs and symptoms caused by B. atrox venom in mice and evaluated whether the combination of dexamethasone and antivenom therapy enhanced the healing of local lesions induced by this envenomation. In animals that were administered the antivenom 10 minutes after the envenomation, we observed an important reduction of edema, dermonecrosis, and myonecrosis. When the antivenom was given 45 minutes after the envenomation, the edema and myonecrosis were reduced, and the fibrinogen levels and platelet counts were restored. The groups treated with the combination of antivenom and dexamethasone had an enhanced decrease in edema and a faster recovery of the damaged skeletal muscle. Our results show that Bothrops antivenom effectively treats the envenomation caused by Bothrops atrox and that the use of dexamethasone as an adjunct to the antivenom therapy could be useful to improve the treatment of local symptoms observed in envenomation caused by Bothrops snakes.

Physiological responses of Rhamdia quelen (Siluriformes: Heptapteridae) to anesthesia with essential oils from two different chemotypes of Lippia alba

The aim of this study was to evaluate if Lippia alba has different chemotypes according to the chemical composition of the essential oil (EO) considering collection site, and if the EO may have different effects on blood and plasma parameters in silver catfish, Rhamdia quelen, during and immediately after anesthesia. The citral (EO-C) and linalool (EO-L) chemotypes were identified, and both presented similar anesthetic effects for silver catfish. Fish were exposed to two concentrations of each EO, which induced slow and fast anesthesia (100 and 300 µL L-1, respectively). Blood ions did not change at any time of anesthesia induction and recovery and, therefore, the electrolyte balance was not altered. Blood gases oscillated through all exposure and recovery times, but there was an increase in pO2 after 10 min recovery in fish anesthetized with EO-C. Glucose increased in fish exposed to both EOs when compared with the control group. Overall, exposure to both EOs (except 100 µL L-1 EO-L at most times) reduced plasma cortisol levels compared to the control and/or ethanol groups. However, as plasma creatinine levels in fish anesthetized with EO-C were higher than control fish, the use of EO-L is preferable.(AU)

O objetivo deste estudo foi avaliar se Lippia alba apresenta diferentes quimiotipos de acordo com a composição química do óleo essencial (OE), considerando local de coleta e se o OE causa diferentes efeitos nos parâmetros sanguíneos e plasmáticos em jundiá, Rhamdia quelen, durante e imediatamente após a anestesia. Os quimiotipos citral (OE-C) e linalol (OE-L) foram identificados e ambos apresentaram efeito anestésico semelhante para jundiá. Os peixes foram expostos a duas concentrações de cada OE, que induziram anestesia lenta e rápida (100 e 300 mL L-1, respectivamente). Íons sanguíneos não se alteraram em nenhum tempo e consequentemente, o equilíbrio eletrolítico não foi alterado. Os gases sanguíneos oscilaram durante todo tempo de exposição e recuperação, mas houve aumento na pO2 após 10 min de recuperação em peixes anestesiados com OE-C. Níveis sanguíneos de glicose aumentaram nos peixes expostos a ambos OEs quando comparados com o grupo controle. De um modo geral, a exposição a ambos OEs (exceto 100 µL L-1 OE-L na maioria dos tempos) reduziu o cortisol plasmático comparado aos grupos controle e etanol. No entanto, como os níveis de creatinina plasmática em peixes anestesiados com OE-C foram maiores que nos peixes controle, é preferível o uso do OE-L.(AU)