Bioassay-guided Fractionation of Anti-Leishmania amazonensis Extracts from Ampelozizyphus amazonicus by High-speed Countercurrent Chromatography.

Ampelozizyphus amazonicus Ducke, known as "saracura-mirá" in the Amazon rainforest, is valued for its traditional use in malaria prevention and treatment. The plant's roots and bark are employed for these purposes, while the wood is often overlooked. Given the global importance of leishmaniasis, research focused on A. amazonicus anti-Leishmania amazonensis potential. The ethyl acetate extract from the bark (EAEB) exhibited the most effective inhibition of intracellular amastigote growth with IC50 7.0 µg.mL-1 but showed high toxicity (CC50 9.0 µg.mL-1). The wood ethanol (EW) and ethyl acetate (EAEW) extracts demonstrated promising results against L. amazonensis with IC50 15.8 µg.mL-1 and 10.0 µg.mL-1, CC50 50.5 µg.mL-1 and 22.7 µg.mL-1, respectively. High-speed countercurrent chromatography combined with mass spectrometry  off-line detection was employed for the bioassay-guided fractionation of EAEB and EAEW using HEMWat as solvent system. These extracts showed to be rich in saponins and triterpenes, besides minor glycosylated flavonoids. HSCCC was effective in obtaining high purity fractions, leading to the identification of a di-glycosylated triterpene saponin from EAEB, and melaleucic acid from EAEW. These findings highlight A. amazonicus as a potential source for developing new therapeutic alternatives for leishmaniasis and HSCCC as a technique enabling better separation efficiency of complex saponin matrices.

Alkaloids from Siparuna (Siparunaceae) are predicted as the inhibitors of proteolysis and plasma coagulation caused by snake venom and potentially counteract phospholipase A2 activity of Bothrops jararaca.

ETHNOPHARMACOLOGICAL RELEVANCE: Snakebite envenomation (SBE) is the world's most lethal neglected tropical disease. Bothrops jararaca is the species that causes the greatest number of SBEs in the South and Southeastern of Brazil. The main symptoms are local (inflammation, edema, hemorrhage, and myonecrosis) and systemic (hemorrhage, hemostatic alterations with consumptive coagulopathy, and death) effects. Species of the genus Siparuna, Siparunaceae, are used in folk and traditional medicine to treat SBE. However, limited information is available concerning Brazilian Siparuna species against SBE. AIM OF THE STUDY: To investigate the correlation between the compounds present in the extracts of five Siparuna species as potential agents against proteolytic activity, plasma coagulation, and phospholipase A2 (PLA2) activity caused by B. jararaca venom, using data obtained by UHPLC-MS/MS, biological activity, and multivariate statistics. MATERIALS AND METHODS: The ethanol extracts from leaves of S. ficoides, S. decipiens, S. glycycarpa, S. reginae, and S. cymosa were fractionated by liquid-liquid extraction using different solvents of increasing polarity (hexane, dichloromethane, ethyl acetate, and n-butanol), affording their respective extracts, totaling 25 samples that were assayed through in vitro plasma coagulation and proteolytic activity assays. Moreover, the extracts were analyzed by UHPLC-MS/MS, using electrospray ionization (ESI) and atmospheric-pressure chemical ionization (APCI) in negative and positive ionization modes. The data was processed in MZmine v. 2.53 and evaluated by multivariate statistical tests (PLS) using the software UnscramblerX v. 10.4. These data were also used to build molecular networks (GNPS), and some ions of interest could be annotated using the library of molecules on the GNPS platform. RESULTS: A total of 19 extracts inhibited B. jararaca-induced plasma coagulation, with emphasis on S. cymosa and S. reginae (800 s). The inhibition of the proteolytic activity was also promising, ranging from 16% (S. glycycarpa) to 99% (S. cymosa, S. decipiens, and S. reginae). In addition, most extracts from S. cymosa and S. reginae inhibited 70-90% of PLA2 activity. Based on data from positive mode APCI analyses, it was possible to obtain a statistic model with reliable predictive capacity which exhibited an average R2 of 0.95 and a Q2 of 0.88, indicating a robust fit. This process revealed five ions, identified as the alkaloids: coclaurine (1), stepholidine (2) O-methylisopiline (3), nornantenine (4) and laurolitsine (5). This is the first study to evidence the potential antivenom of alkaloids from Siparuna species. CONCLUSIONS: Altogether, our results give support to the popular use of Siparuna extracts in SBE accidents, suggesting their potential as an alternative or complementary strategy against envenoming by B. jararaca venom. The predicted ions in the chemometric analysis for the assayed activities can also be correlated with the blocking activity and encourage the continuation of this study for possible isolation and testing of individual compounds on the used models.

Myrtucommulones and Related Acylphloroglucinols from Myrtaceae as a Promising Source of Multitarget SARS-CoV-2 Cycle Inhibitors.

The LABEXTRACT plant extract bank, featuring diverse members of the Myrtaceae family from Brazilian hot spot regions, provides a promising avenue for bioprospection. Given the pivotal roles of the Spike protein and 3CLpro and PLpro proteases in SARS-CoV-2 infection, this study delves into the correlations between the Myrtaceae species from the Atlantic Forest and these targets, as well as an antiviral activity through both in vitro and in silico analyses. The results uncovered notable inhibitory effects, with Eugenia prasina and E. mosenii standing out, while E. mosenii proved to be multitarget, presenting inhibition values above 72% in the three targets analyzed. All extracts inhibited viral replication in Calu-3 cells (EC50 was lower than 8.3 µg·mL-1). Chemometric analyses, through LC-MS/MS, encompassing prediction models and molecular networking, identified potential active compounds, such as myrtucommulones, described in the literature for their antiviral activity. Docking analyses showed that one undescribed myrtucommulone (m/z 841 [M - H]-) had a higher fitness score when interacting with the targets of this study, including ACE2, Spike, PLpro and 3CLpro of SARS-CoV-2. Also, the study concludes that Myrtaceae extracts, particularly from E. mosenii and E. prasina, exhibit promising inhibitory effects against crucial stages in SARS-CoV-2 infection. Compounds like myrtucommulones emerge as potential anti-SARS-CoV-2 agents, warranting further exploration.

Antioxidant activity of natural compounds of Stachytarpheta cayennensis (Rich.) Vahl, Verbenaceae, by scavenger of mitochondrial reactive oxygen species

The etiology of a range of diseases is associated with an excessive generation of reactive oxygen species (ROS). Exacerbated ROS production leads to mitochondrial dysfunction, cellular damage and apoptosis. Nowadays, many strategies are being developed for the targeted delivery of antioxidants compounds to mitochondria, trying to minimize the damages. Production of ROS was investigated by the molecular probes CM-H2DCFDA and Amplex Red. The purpose of this work is to evaluate the antioxidant capacity of verbascoside, martinoside, betulinic acid from the Stachytarpheta cayennensis and quercetin by an in vitro assay with isolated mitochondria from mice's brain The results showed that all compounds tested exhibited a scavenger effect on the ROS generated by the isolated mitochondria, which displayed a dependent dose increase.

Estudo etnofarmacognóstico da saracuramirá (Ampelozizyphus amazonicus Ducke), uma planta medicinal usada por comunidades quilombolas do Município de Oriximiná-PA, Brasil / Ethnopharmacology Study of Saracuramirá (Ampelozizyphus amazonicus Ducke) in the "Quilombola" communities of Oriximiná, Pará State, Brazil

Um levantamento etnobotânico realizado em comunidades quilombolas de Oriximiná, Pará, destacou a saracuramirá (SAR), Ampelozizyphus amazonicus Ducke, com vasto uso popular no tratamento da malária, como tônica e depurativa. Por este motivo, o presente trabalho objetivou realizar um estudo etnofarmacognóstico da SAR nas respectivas comunidades. Por meio de uma análise etnobotânica quantitativa, foi verificado que SAR apresentou-se dentre as 10 espécies mais versáteis pela elevada importância relativa (1,3), dentre as cinco espécies com maior importância cultural pelo elevado índice de saliência (0,311) e a espécie com maior concordância de uso principal para malária (85,7 por cento). Uma análise do índice de espuma e do índice de hemólise para SAR demonstra a presença de saponinas com elevado índice de espuma (833) e uma baixa atividade hemolítica (CH50 2,6 mg mL-1). Para realizar uma análise das agliconas das saponinas de SAR, a bebida preparada pelo método tradicional quilombola (BMT) foi hidrolisada e, após reação com diazometano, foi analisada por cromatografia gasosa. Dois sinais majoritários foram caracterizados por espectrometria de massas, um referente a um triterpeno de esqueleto damarânico, característico das saponinas da SAR, e outro referente ao éster metílico do ácido betulínico. Partindo das informações de uso popular da SAR, foi avaliada in vitro a atividade inibidora da acetilcolinesterase. Apesar de BMT não ter mostrado atividade neste ensaio, é possível supor que as indicações de uso desta planta pelos quilombolas como fortificante e contra malária podem estar relacionadas a uma possível atividade adaptógena e imunoestimulante, dada à presença das saponinas e do ácido betulínico em BMT.

In an ethnobotanical survey conducted within "Quilombola" communities of Oriximiná, Pará State, "saracuramirá" (SAR), Ampelozizyphus amazonicus Ducke, stood out as one of the most cited species with wide popular use, especially for malaria, as tonic and depurative. The aim of this paper was to carry out an ethnopharmacognostic study of SAR in these communities. Quantitative ethnobotanical analysis of the data showed that SAR stands out as one of the 10 most versatile species with a high relative importance (1,3); it remains among the five species with the greatest cultural importance, demonstrated by the high salience index (0,311), being the species with the highest major use agreement to malaria (85,7 percent). Analysis of the foam and hemolysis indexes of SAR shows the presence of saponins with high foam index (833) and low hemolytic activity (HD50 2,6 mg mL-1). In order to analyze SAR saponin aglycones, the drink was prepared by the traditional quilombola method (BMT), was hydrolyzed, and analyzed by gas chromatography after reaction with diazomethane. Two major peaks were characterized by mass spectrometry, one referring to a dammarane triterpene skeleton, characteristic from the SAR saponins, and the other identified as the methyl ester of betulinic acid. Owing to the popular usage of SAR, its in vitro acetylcholinesterase inhibitory activity was evaluated, but with negative results. However, it is possible to suggest that the indications of this plant as a tonic and for treating malaria may be related to an adaptogen and immunostimulant effect due to the presence of saponins and betulinic acid in BMT.

Evaluation of antimicrobial, antioxidant and phototoxic activities of extracts and isolated compounds from Stachytarpheta cayennensis (Rich.) Vahl, Verbenaceae / Avalição da atividade antimicrobiana, antioxidante e fototóxica dos extratos e compostos isolados de Stachytarpheta cayennensis (Rich.) Vahl, Verbenaceae

Stachytarpheta cayennensis (Rich.) Vahl, Verbenaceae, plant extract, is a Brazilian medicinal plant externally used in folk medicine for purulent ulcers, skin lesions and internally for inflammations, fever, renal disorders and atherosclerosis. S. cayennesis was studied to identify potential bioactive compounds that may justify their therapeutic use against skin lesions and atherosclerosis. The antioxidant, antimicrobial and phototoxicity capacities of the crude ethanolic extract, fractions and isolated compounds from roots of S. cayennesis were evaluated through in vivo and in vitro tests. Strains of Saccharomyces cerevisiae, an eukaryotic cell model, were used to assess both the phototoxicity and the capacity to protect against the lethal oxidative stress caused by menadione and hydrogen peroxide. The extract, fractions and the two major isolated compounds, verbascoside and betulinic acid, of S. cayennensis were able to increase the tolerance and decrease the lipid peroxidation of S. cerevisiae to reactive oxygen species (ROS). The antioxidant activity was also evaluated by scavenging of 2,2-diphenyl-1-picrylhydrazyl (DPPH•). Verbascoside exhibited a moderate antimicrobial activity against Streptococcus pyogenes, S.epidermidis and Staphylococcus aureus. Neither the ethanolic extract nor fractions showed phototoxicity, indicating that the S. cayennensis extract is safe for use in the treatment of skin lesions and as an active cosmetic ingredient.

Stachytarpheta cayennensis (Rich.) Vahl, Verbenaceae, é uma planta utilizada na medicina popular brasileira contra úlceras e lesões de pele. Internamente é usada contra inflamações, febre, doenças renais e aterosclerose. Essa planta foi estudada com o objetivo de identificar os compostos bioativos majoritários que possam justificar seu uso terapêutico contra lesões de pele e arteriosclerose. A atividade antioxidante do extrato bruto etanólico, partições e os compostos majoritários isolados das raízes de S. cayennesis foi avaliada através de testes in vivo e in vitro. In vitro a atividade antioxidante foi avaliada pelo teste fotocolorimétrico do radical 2,2-difenil-1-picrilidrazil (DPPH•). In vivo, Saccharomyces cerevisiae, um modelo de célula eucariótica, foi utilizado tanto para avaliar a fototoxicidade quanto a capacidade antioxidante contra as espécies reativas de oxigênio (EROS) menadiona e peróxido de hidrogênio. O extrato, partições e os dois compostos majoritários isolados, verbascosídeo e ácido betulínico foram capazes de aumentar a sobrevivência e diminuir a peroxidação lipídica de S. cerevisiae contra EROS. Verbascosídeo apresentou atividade antimicrobiana moderada contra Streptococcus pyogenes, S.epidermidis e Staphylococcus aureus. O extrato etanólico e as partições testadas não apresentaram fototoxicidade, indicando que S. cayennensis é uma planta segura para o tratamento de lesões de pele e como possível ingrediente em cosméticos.

Investigation of the antimycobacterial activity of 36 plant extracts from the Brazilian Atlantic Forest / Análise da atividade antimicobacteriana de 36 extratos vegetais da Mata Atlântica brasileira

Thirty-six plant extracts from the brazilian Atlantic Forest were tested for their antimycobacterial activity against Mycobacterium tuberculosis H37Rv and M. kansasii, using the method REMA in seriate concentrations of 100 to 0.20 µg/mL. Among the thirty six extracts tested, five were active against M. tuberculosis, and three of these extracts also showed activity against M. kansasii. Cytotoxicity test with VERO cells was performed with the five extracts active against M. tuberculosis. Only the extract of Peschiera affinis was identified as non-toxic in the concentration of 100µg/mL.

Trinta e seis extratos vegetais originários da Mata Atlântica foram testados quanto à sua atividade antimicobacteriana frente ao M. tuberculosis H37Rv e M. kansasii, utilizando o método REMA em concentrações seriadas de 100 a 0,20 µg/mL. Dentre os trinta e seis extratos testados, cinco mostraram atividade frente ao M. tuberculosis, e destes apenas, três mostraram atividade ao M. kansasii, que apresentou susceptibilidade a outros dez. O teste de citotoxicidade com células VERO foi realizado com os cinco extratos ativos frente ao M. tuberculosis em que identificou-se a não toxicidade em apenas um extrato (Peschiera affinis) na concentração de 100 µg/mL.

Effect of different extracts from the Brazilian Atlantic Forest on the Pdr5p ATPase activity / Efeito de extratos de plantas da Floresta Atlântica Brasileira sobre a atividade da Pdr5p ATPase

In the current study, we tested the effect of 27 plant extracts and fractions from different botanical families on the activity of Pdr5p from yeast plasma membrane, responsible for the multidrug resistance phenotype in yeast cells. Some of the extracts were able to produce a good inhibition in the fixed concentration (200 µg/mL) and were selected for a deeper investigation. Dose-response curves were obtained for the crude ethanol extracts of Bathysa australis (A. St.-Hill.) Benth. & Hook f., Mabea fistulifera Mart. and Virola oleifera (Schott) A. C. Sm. with concentrations ranging up to 400 µg/mL. The lower IC50 value was obtained for Virola oleifera, 22.8 µg/mL, followed by Bathysa australis, 35.3 µg/mL, and Mabea fistulifera, 42.5 µg/mL. After fractionation of the crude extracts by liquid-liquid partition with different organic solvents and each fraction was tested again, only some of the fractions retained the ability to inhibit the enzymatic activity. When analyzed by HPLC/DAD, the active fractions showed the presence of flavonoid derivatives, already reported for their ability to inhibit Pdr5p ATPase activity, as well as other classes of secondary metabolites such as lignans and alkaloids.

No presente estudo, testamos o efeito de 27 extratos e frações de plantas de diferentes famílias botânicas sobre a atividade da proteína Pdr5p de membranas plasmáticas de leveduras, responsável pelo fenótipo de resistência a múltiplas drogas em leveduras. Alguns dos extratos foram capazes de produzir uma boa inibição na concentração fixa de 200 µg/mL e foram selecionados para uma investigação mais aprofundada. Curvas de dose-resposta foram obtidas para os extratos brutos etanólicos de Bathysa australis (A. St.-Hill.) Benth. & Hook f., Mabea fistulifera Mart. e Virola oleifera (Schott) A. C. Sm., com concentrações até 400 µg/mL. O menor valor de IC50 foi obtido para Virola oleifera, 22,8 µg/mL, seguido por Bathysa australis, 35,3 µg/mL e Mabea fistulifera, 42,5 µg/mL. Após o fracionamento dos extratos brutos por partição líquido-líquido com diferentes solventes orgânicos, cada fração foi novamente testada, sendo que apenas algumas das frações mantiveram a habilidade de inibir a atividade enzimática. Quando analisadas por HPLC/DAD, as frações ativas demonstraram a presença de derivados de flavonóides, que já demonstraram ter a habilidade de inibir a atividade ATPasica da Pdr5p, assim como outras classes de metabólitos secundários, tais como lignanas e alcalóides.

Substâncias fenólicas com atividade antioxidante de Pseudopiptadenia contorta (Leguminosae-Mimosoideae)

O extrato bruto em acetona/ água das folhas de Pseudopiptadenia contorta foi purificado rendendo frações e substâncias puras. A atividade antioxidante foi avaliada para o extrato e frações, empregando-se o teste químico do DPPH. As frações contendo taninos condensados e substâncias fenólicas de baixo peso molecular mostraram ser as responsáveis pela atividade antioxidante do extrato bruto das folhas de P. contorta.

Lactaçäo em ratas tratadas com extrato etanólico de Vitex Polygama: observaçöes preliminares / Lactation in female rats treated with an ethanolic extract of Vitex Polygama: preliminary observations

Vitex Polygama (Verbenacea) pertence ao mesmo gênero do Vitex Agnus Castus (Verbenacea), cujo extrato etanólico tem sido indicado para o tratamento de mastodinia, havendo estudos que lhe imputam efeito sobre a lactaçäo. Este trabalho verifica o efeito do extrato etanólico de V. Polygama na lactaçäo de ratas...