Preliminary analysis of Brazilian Amazonian plant extracts using bench-top assays in a through-put basis aiming the identification of potential antioxidant natural product

Plants are a source of compounds that are used for the treatment of human and veterinary diseases. Brazil is one of the richest countries in the world in terms of biodiversity. The present study evaluated extracts using thin-layer chromatography to identify antioxidant activity and determine the presence of groups of compounds, in a large-scale basis. A total of 1,260 aqueous and organic extracts were obtained from plants that were collected in the Amazon and Atlantic rain forests. Thin-layer chromatography was performed to evaluate the presence of alkaloids, anthraquinones, cardioactive glicosides, terpenes, and phenolics and determine antioxidant and radical scavenging activity using the following reagents: Dragendorff's reagent, KOH, Kedde's reagent, sulphuric acid, NP reagent, ß-carotene, and 2,2-diphenyl-1-picrylhydrazyl (DPPH). Of the 1,260 extracts, 837 (66.43%) presented a ß-carotene/bleaching response, and 1,205 (95.63%) presented a radical scavenging response. Alkaloids were found in 203 extracts (16.11%). Anthraquinones were found in 14 extracts (1.11%), cardenolides were found in eight extracts (0.63%). The present findings shows the importance of the Brazilian Amazon plants as sources of antioxidant and radical scavenging active compounds.(AU)

Casinga-cheirosa organic extract impairment over Balb-c male mice behavioral phenotype

ABSTRACT Laetia suaveolens (Poepp.) Benth., Salicaceae, popularly known as "casinga-cheirosa", "caferana", or "laranjinha", is native to Brazil but not endemic to this country. A crude organic extract was obtained from the leaves and stem and intraperitoneally administered in male Balb-c mice. Its behavioral effects were evaluated in the open field and elevated plus maze in a two-stage experiment that assessed ten different parameters related to behavior as locomotion, emotionality, and anxiety. In the first stage of the experiment, intraperitoneal the crude organic extract administration dose-dependently impaired locomotion and emotionality 30–120 min after administration. A significant decrease in defecation was observed, which was related to emotionality. No alterations in the elevated plus maze were found; thus, this apparatus was not used in the next stage of the experiment. In the second stage, the previously determined non-lethal dose of 0.1563 g/kg was intraperitoneally administered, which impaired locomotion and rearing frequency and increased immobility time. Necropsy revealed smooth intestine hemorrhage. Rutin, leucoside, nicotiflorin, guaijaverin, and astragalin were isolated from the crude organic extract. This is the first time that these compounds have been identified in L. suaveolens. In conclusion, the crude organic extract impaired locomotion and emotionality and caused hemorrhage in male Balb-c mice, indicating that its consumption can be harmful to humans and animals. The present results provide a basis for further studies on the pharmacology, toxicology, and natural product chemistry of the crude organic extract.

Identification key for lycophytes and ferns from the Picinguaba and Santa Virgínia Nuclei, Parque Estadual da Serra do Mar, Ubatuba, SP, Brazil / Chave de identificação para licófitas e samambaias dos Núcleos Picinguaba e Santa Virgínia, Parque Estadual da Serra do Mar, Ubatuba, SP, Brasil

Abstract This work contributes to the knowledge of the fern and lycophytes diversity in the Picinguaba and Santa Virginia Nuclei, Parque Estadual da Serra do Mar (PESM), Ubatuba, São Paulo, Brazil, as well as to provide subsidies to identification of the found taxa in these areas. In total, 30 collecting expeditions were conducted and the vouchers were deposited at UEC Herbarium. We have identified 135 species distributed in 53 genera and 19 families for both areas. The most representative families were Polypodiaceae (28 species) and Dryopteridaceae (19 species), whereas Asplenium (12 species), Blechnum and Elaphoglossum (8 species each) were the most important genera. Epiphytic species predominated (62 overall), probably due to the presence of favorable habitats. Among the taxa, 41 are considered endemic from Brazil, 35 of them endemic to the Brazilian Atlantic Forest. Five species are classified as vulnerable according to the "Official list of endangered species in the State of São Paulo": Elaphoglossum macahense (Fée) Rosenst., Anetium citrifolium (L.) Splitg., Asplenium muellerianum Rosenst., Cyathea glaziovii (Fée) Domin, and Thelypteris angustifolia (Willd.) Proctor, which emphasizes the importance of preservation areas such as the Parque Estadual da Serra do Mar. An identification key has been elaborated to help with the recognition of these groups in Picinguaba and Santa Virgínia Nuclei.

Resumo Este trabalho visa contribuir para o conhecimento da diversidade de licófitas e samambaias ocorrentes nos núcleos Picinguaba e Santa Virgínia do Parque Estadual da Serra do Mar (PESM), Ubatuba, SP, Brasil, bem como fornecer subsídios para a identificação dos táxons encontrados nestas áreas. Ao todo, foram realizadas 30 expedições de coleta e o material botânico encontra-se depositado no Herbário UEC. Foram identificadas 135 espécies, 19 famílias e 53 gêneros para as duas áreas. As famílias mais representativas foram Polypodiaceae (28 espécies) e Dryopteridaceae (19 espécies), enquanto Asplenium (12 espécies), Blechnum e Elaphoglossum (8 espécies cada) foram os gêneros melhor representados. As espécies epífitas predominaram (62 ao todo), provavelmente devido à presença de hábitats favoráveis. Dos táxons encontrados, 41 são considerados endêmicos do Brasil, sendo 35 deles endêmicos da Mata Atlântica brasileira; cinco espécies são classificadas como vulneráveis, conforme a "Lista oficial de espécies ameaçadas de extinção no Estado de São Paulo": Elaphoglossum macahense (Fée) Rosenst., Anetium citrifolium (L.) Splitg., Asplenium muellerianum Rosenst., Cyathea glaziovii (Fée) Domin e Thelypteris angustifolia (Willd.) Proctor, o que ressalta a importância de áreas de preservação como o Parque Estadual da Serra do Mar, no contexto conservacionista. Uma chave de identificação foi elaborada para contribuir no reconhecimento destes grupos nos núcleos Picinguaba e Santa Virgínia.

In vitro activity of Amazon plant extracts against Enterococcus faecalis

Previous studies analyzing 2,200 plant extracts indicated anti-enterococcal activity in 25 extracts obtained from Brazilian forests' plants. In the present study, these extracts were subjected to microdilution broth assay (MDBA) and disk diffusion assay (DDA) using planktonic Enterococcus faecalis ATCC® 29212TM and were submitted to phytochemical analysis in TLC and HPLC. Three extracts obtained from Ipomoea alba (MIC < 40 µg/mL), Diclinanona calycina (MIC < 40 µg/mL) and Moronobea coccinea (40 < MIC < 80 µg/mL; MBC = 80 µg/mL) showed significant bactericidal activity in the MDBA and four extracts obtained from I. alba (14.04 ± 0.55 mm diameter) S. globulifera (14.43 ± 0.33 mm and 12.18 ± 0.28 mm diameter) and Connarus ruber var. ruber (13.13 ± 0.18 mm diameter) were active in DDA. Residues H2O obtained from Psidium densicomum (mean of 16.78 mm diameter) and from Stryphnodendron pulcherrimum (mean of 15.97 mm diameter) have shown an improved antibacterial activity after fractionation if compared to that obtained from the respective crude extracts. Antioxidant activity was observed in some residues of the active extracts. TLC analysis showed that phenolic compounds are likely to be found in active extracts. Three molecules were isolated from S. globulifera and were identified by 13C NMR lupeol, α-amyrin and 3β-hydroxyglutin-5-ene. The present chemical and biological findings suggest that these extracts are a potential source of new anti-Enterococcus compounds to be introduced in endodontic therapy.

Anti-Streptococcal activity of Brazilian Amazon Rain Forest plant extracts presents potential for preventive strategies against dental caries

Caries is a global public health problem, whose control requires the introduction of low-cost treatments, such as strong prevention strategies, minimally invasive techniques and chemical prevention agents. Nature plays an important role as a source of new antibacterial substances that can be used in the prevention of caries, and Brazil is the richest country in terms of biodiversity. Objective: In this study, the disk diffusion method (DDM) was used to screen over 2,000 Brazilian Amazon plant extracts against Streptococcus mutans. Material and Methods: Seventeen active plant extracts were identified and fractionated. Extracts and their fractions, obtained by liquid-liquid partition, were tested in the DDM assay and in the microdilution broth assay (MBA) to determine their minimal inhibitory concentrations (MICs) and minimal bactericidal concentrations (MBCs). The extracts were also subjected to antioxidant analysis by thin layer chromatography. Results: EB271, obtained from Casearia spruceana, showed significant activity against the bacterium in the DDM assay (20.67±0.52 mm), as did EB1129, obtained from Psychotria sp. (Rubiaceae) (15.04±2.29 mm). EB1493, obtained from Ipomoea alba, was the only extract to show strong activity against Streptococcus mutans (0.08 mg/mL<MIC<0.16 mg/mL; MBC=0.16 mg/mL) in the MBA. Conclusions: The active extracts, discovered in the Amazon rain forest, show potential as sources of new antibacterial agents for use as chemical coadjuvants in prevention strategies to treat caries. .

Influence of the intraperitoneal administration of antitumor Abarema auriculata extract on mice behavior

The organic extract EB689, obtained from the stem of Abarema auriculata (Benth.) Barneby & J.W.Grimes, Fabaceae, commonly known as "saboeiro-ferro", was chemically studied, as well as its influence over behavioral effects such as locomotion, emotionality and anxiety, after intra-peritonial administration were assessed. The open-field and elevated-plus maze were used in experiments divided into two stages. The first stage aimed for the identification of the main effects over behavior using a reduced number of animals against half-fold diluted doses of EB689. The same variables were also tested in a second stage of the experiment using the non-lethal intra-peritoneal dose of 4.8 mg/kg in a larger number of animals. It was observed that EB689 clearly decreased locomotion, which was probably caused by internal hemorrhage causing hypovolemic shock. Although it is the first time lupeol and eucryphin are described in A. auriculata, it is still not clear if they are involved in the toxicology of A. auriculata. The undesirable effects of EB689 are better understood, the basis for further pharmacological assays aiming antitumor activity are supported.

New trends in dentistry: plant extracts against Enterococcus faecalis. The efficacy compared to chlorhexidine

Enterococcus faecalis is an important pathogen associated with endodontic diseases, and its elimination and control are of paramount importance, as it represents one of the major causes of failure in the treatment of endodontic disease. Twenty-five plant extracts obtained from Brazilian forests were found to be effective against planktonic E. faecalis and were subjected to two traditional antibacterial assays, the microdilution broth assay (MDBA) and the disk diffusion assay (DDA), using chlorhexidine (CHX) as a control. Seven out of 25 extracts showed significant antibacterial activity and were tested in a biofilm assay, and three of these extracts were subjected to chemical fractionation. Residues were tested for their antibacterial activity, and the first chemical findings were described based on thin layer chromatography (TLC). Extracts obtained from Ipomoea alba, Symphonia globulifera and Moronobea coccinea showed significant bactericidal activity in the MDBA. The same I. alba and S. globulifera extracts, as well as the extract obtained from Connarus ruber var. ruber, showed significant activity in the DDA. RH2O obtained from Psidium densicomum and Stryphnodendron pulcherrimum showed better antibacterial activity compared to the respective crude extracts and CHX. TLC analysis showed that phenolic compounds and triterpenes represent the first findings of chemical groups that may occur in all species. The results of the present study include the discovery of six active extracts against planktonic E. faecalis and support further testing via assays involving biofilm formation, as well as the determination of the compounds' chemical profiles, as their activity was significantly better than that observed for CHX.

In vitro cytotoxic effects of Brazilian plant extracts on squamous cell carcinoma of the oral cavity

Squamous cell carcinoma (SCC) is the most prevalent cancer of the oral cavity and the fifth most prevalent of all malignancies in males. Many researchers have attempted to develop new treatments that will improve the prognosis of SCC patients. Over 20 percent of the world's biodiversity is located within the Brazilian forests, but little is known about the chemical and/or pharmacological potential of these plants. Certain extracts obtained from Amazon and Atlantic Forest plants have previously been shown to have cytotoxic activity against various cancers. The aim of this study was to screen these extracts for cytotoxic activity against oral SCC cells. The extracts were analyzed for activity against the KB-ADL#12 cell line at various concentrations up to a maximum dose of 100 µg/mL. Comparisons with a control group were performed using one-way ANOVA. Significant cytotoxicity was induced by the extracts obtained from the aerial parts of Picrolemma sprucei (Simaroubaceae), from the leaves and stems of Laetia suaveolens (Salicaceae), from the aerial parts of Abarema auriculata (Fabaceae-Mimosoideae) and from the stem of A. auriculata.

A composição florística e a diversidade de pteridófitas diferem entre a Floresta de Restinga e a Floresta Ombrófila Densa das Terras Baixas do Núcleo Picinguaba/PESM, Ubatuba/SP? / Ferns composition and diversity differ between Restinga and Lowland Rainforest areas in the Serra do Mar?

As pteridófitas constituem um componente importante da flora tropical, representando aproximadamente 10 por cento do total de plantas vasculares de florestas tropicais, sendo o grupo dominante entre a vegetação herbácea. O objetivo do trabalho foi verificar se as comunidades de pteridófitas de duas áreas de Mata Atlântica, Floresta de Restinga (FRS) e Floresta Ombrófila Densa de Terras Baixas (FTB), situadas sobre solos notavelmente distintos e separadas por uma grande rodovia, difeririam em sua composição florística e diversidade. Estas duas fisionomias estão localizadas no Núcleo Picinguaba do Parque Estadual da Serra do Mar, Ubatuba, São Paulo (23° 31' a 23° 34' S e 45° 02' a 45° 05' W). Em cada área delimitamos sete parcelas de 20 × 20 m, nas quais os indivíduos foram coletados, identificados e recenseados. Efetuamos três métodos de análise multivariada (TWINSPAN, UPGMA e PCO) para verificar a existência de grupos florísticos; calculamos o índice de Payandeh para definirmos o nível de agregação de cada espécie em cada uma das duas áreas (FRS e FTB); obtivemos espécies indicadoras para cada área; e realizamos análises de diversidade (riqueza, equabilidade e heterogeneidade). Nas duas áreas amostramos 3.482 indivíduos, dos quais 1.366 ocorrem na FRS e 2.116 na FTB. Encontramos 14 espécies na FRS e 29 na FTB; cinco espécies foram compartilhadas entre as duas áreas. As análises de grupos florísticos mostraram a formação de dois blocos bem definidos (FRS × FTB). Na FRS, relativamente mais espécies apresentaram distribuição agregada e menos espécies apresentaram distribuição aleatória em relação à FTB. Encontramos quatro espécies indicadoras para a FRS e oito para a FTB. As curvas resultantes da rarefação por indivíduos indicaram forte diferença de riqueza específica entre as duas comunidades, favorável à FTB. As diferenças encontradas podem estar relacionadas às características físico-químicas do solo (mais fértil e menos tóxico na FTB), à dinâmica da água (as espécies vegetais da FRS estão, de modo geral, sujeitas ao estresse hídrico) e ao impacto causado pela estrada que corta o Parque. Esses aspectos poderiam estar relacionados a diferentes formas de ocupação de nichos e ao fato de que algumas espécies em ambientes com recursos limitados e distribuídos heterogeneamente no espaço, como é o caso da Restinga estudada, eliminariam as demais.

Ferns are an important component of the tropical flora, representing about 10 percent of all vascular plants of tropical forests, constituting the dominant group among the herbaceous vegetation. The aim of this study was to determine whether the ferns communities in two areas of Atlantic Forest, Restinga (FRS) and Lowland Ombrophilous Dense (FTB) forests, located on soils remarkably distinct and separated by a highway, differ in their floristic composition and diversity. These two faces are located in Picinguaba locality, Parque Estadual da Serra do Mar, Ubatuba, São Paulo (23° 31' to 23° 34' S and 45° 02' to 45° 05' W). We delimited seven plots of 20 × 20 m in each area, collected, identified and listed the fern individuals. We performed three methods of multivariate analysis (TWINSPAN, UPGMA and PCO) to verify the existence of floristic groups; calculated the Payandeh index to define the level of aggregation of each species in each of the two areas (FRS and FTB); obtained indicator species for each area; and performed diversity analysis (richness, evenness and heterogeneity). The two areas sampled 3482 individuals, 1366 of which occur in FRS and 2116 in FTB. We found 14 species in FRS and 29 in FTB; these areas shared five species. We found two well-defined floristic blocks (FRS × FTB) in cluster analysis. In the FRS, relatively most species showed aggregated distribution and fewer species showed random distribution in relation to FTB. We found four indicator species for FRS and eight for FTB. The curves resulting from the individual-based rarefaction indicated a strong difference in species richness between the two communities, in favor to FTB. These differences may be related to physical and chemical characteristics of soil (more fertile and less toxic in the FTB), water dynamics (plant species in the FRS are generally subject to water stress) and the sharp break caused by highway that cuts through the park. These aspects could be related to different forms of occupation of niches and to the fact that some species in environments with limited resources and heterogeneously distributed in space, such as the studied Restinga, would eliminate the others.

Antibacterial activity of Brazilian Amazon plant extracts

Infections caused by multiresistant bacteria are a widespread problem, especially in intensive care units. New antibiotics are necessary, and we need to search for alternatives, including natural products. Brazil is one of the hottest spots in the world in terms of biodiversity, but little is known about the chemical and pharmacological properties of most of the plants found in the Amazon rain forest and the Atlantic Forest. We screened 1,220 organic and aqueous extracts, obtained from Amazon and Atlantic rain forest plants, against Staphylococcus aureus, Enterococcus faecalis, Pseudomonas aeruginosa and E. coli. Seventeen organic and aqueous extracts obtained from 16 plants showed activity against both Gram-positive bacteria. None of the extracts showed relevant activity against the Gram-negative E. coli and Pseudomonas aeruginosa.

Antibacterial and cytotoxic activity of Brazilian plant extracts - Clusiaceae

Twelve extracts obtained from nine plants belonging to six different genera of Clusiaceae were analyzed against Gram-negative (Escherichia coli and Pseudomonas aeruginosa) and Gram-positive (Staphylococcus aureus and Enterococcus faecalis) bacteria using the microdilution broth assay. Tovomita aff. longifolia, T. brasiliensis, Clusia columnaris, Garcinia madruno, Haploclathra paniculata, and Caraipa grandifolia extracts showed significant results against the bacteria. The organic extract obtained from the leaves of T. aff. longifolia showed minimal inhibitory concentration (MIC) = 70 µg/ml and minimal bactericidal concentration (MBC) = 90 µg/ml against E. faecalis and the organic extract made with the stem of C. columnaris showed MIC = 180 µg/ml and MBC = 270 µg/ml against P. aeruginosa. None of the antibacterial extracts showed lethal activity against brine shrimp nauplii. On the other hand, both aqueous and organic extracts obtained from the aerial organs of Vismia guianensis that were cytotoxic to brine shrimp nauplii did not show a significant antibacterial activity in the assay.