Toxicological and pharmacological effects of Eugenia brasiliensis Lam. (Myrtaceae) leaves in mice.

ETHNOPHARMACOLOGICAL RELEVANCE: Eugenia brasiliensis Lam., popularly known as "grumixama" or "Brazilian cherry", is widely used in folk medicine with astringent, diuretic, energizing, anti-rheumatic, and anti-inflammatory properties. AIM OF THE STUDY: Despite its traditional use, detailed toxicological studies of Eugenia brasiliensis are few. Thus, in the current study, we evaluate the toxicological effects of hydroalcoholic extract of Eugenia brasiliensis (HEEb) and its antinociceptive and anti-inflammatory activity. MATERIALS AND METHODS: We used male, and female Swiss mice. Acute toxicity study was performed following the Organization for Economic Cooperation and Development (OECD) guideline 425, and subacute toxicity was assessed following OECD guideline 407. We observed behavioral responses, in addition to hematological, biochemical, and histological evaluations. The antinociceptive and anti-inflammatory activity of HEEb were assessed using the Carrageenan-induced mechanical allodynia and paw edema model. Mechanical allodynia, levels of inflammatory cytokines, and oxidative damage were evaluated. RESULTS: The treatment with HEEb was not able to generate important toxicological alterations. Moreover, doses of 100 and 300 mg/kg of HEEb were able to reduce mechanical allodynia, paw edema, and inflammatory cytokines (TNF-α, IL-1ß, and IL-6), decrease malondialdehyde and increase superoxide dismutase enzyme activity in the paw. CONCLUSIONS: This study demonstrated that HEEb does not present important toxic effects. Additionally, an important antinociceptive, anti-inflammatory, and antioxidant potential were observed.

Buscando novos antimicrobianos: avaliação da atividade antibacteriana de extratos de Eugenia brasiliensis / Searching for new antimicrobials: evaluation of the antibacterial activity of Eugenia brasiliensis extracts

Objetivo: Devido à atual resistência bacteriana aos antimicrobianos e à necessidade de buscar novas substâncias com essa atividade, principalmente em produtos naturais, esse trabalho objetivou avaliar e comparar o potencial antibacteriano de extratos de folhas da planta Eugenia brasiliensis, ainda pouco estudada, obtidas por coletas sazonais. Métodos: As coletas foram realizadas trimestralmente no Jardim Botânico do RJ (JBRJ) e os extratos etanólicos preparados com o pó das folhas secas. Foram utilizadas cepas de referência ATCC (American Type Culture Collection) Gram positivas de Staphylococcus aureus: 25.923 (Beta Lactamase -) e 29.213 (Beta Lactamase +), e duas Gram negativas, Escherichia coli (28.922) e Pseudomonas aeruginosa (27.853). A técnica de escolha foi a microdiluição em placa de 96 poços, pois permite avaliar a concentração inibitória mínima (CIM) e posteriormente a concentração bactericida mínima (CBM). Resultados: A melhor atividade antibacteriana (1 mg/mL) foi identificada nos extratos obtidos nos meses mais quentes (menores CIM para bactérias Gram positivas). A CBM foi compatível com a CIM na maioria das estações/cepas bacterianas, indicando possível atividade bactericida dos extratos. Conclusão: A pesquisa demonstrou uma atividade antibacteriana promissora para Gram positivos e existência de variação sazonal, sugerindo um bom potencial para o uso de E. brasiliensis como antimicrobiano. Os resultados também indicam que, no futuro, outros estudos devem ser realizados, como a prospecção química destes extratos.

Objective: Due to the current bacterial resistance to antimicrobials and the need to search for new substances with this activity, mainly in natural products, this work aimed to evaluate and compare the antibacterial potential of Eugenia brasiliensis leaf extracts, still little studied, obtained by seasonal collections. Methods: Leaf collections were made quarterly at the Jardim Botânico do RJ (JBRJ) and ethanol extracts prepared with dry leaf powder. Reference strains ATCC (American Type Culture Collection) Gram positive from Staphylococcus aureus were used: 25923 (Beta Lactamase -) and 29213 (Beta Lactamase +), and two Gram negative strains, Escherichia coli (28.922) and Pseudomonas aeruginosa (27.853). The technique of choice was microdilution in a 96-well plate, as it allows the assessment of the minimum inhibitory concentration (MIC) and subsequently the minimum bactericidal concentration (CBM). Results: The best antibacterial activity (1 mg/mL) was identified in the extracts obtained in the warmer months (lower MICs for Gram positive bacteria). CBM was compatible with MIC in most seasons/ bacterial strains, indicating possible bactericidal activity of the extracts. Conclusion: The research demonstrated a promising antibacterial activity for Gram positive and seasonal variation, suggesting a good potential for the use of E.brasiliensis as an antimicrobial. The results also indicate that in the future, other studies should be carried out, such as the chemical prospecting of these extracts.

Grumixama (Eugenia brasiliensis Lam.) cultivated in the Cerrado has high content of bioactive compounds and great antioxidant potential / Grumixama (Eugenia brasiliensis Lam.) cultivada no Cerrado possui altos teores de compostos bioativos e elevado potencial antioxidante

ABSTRACT: Grumixama (Eugenia brasiliensis Lam.) is a Brazilian berry native to the Atlantic Rainforest. Information on nutritional and bioactive profiles of the fruit cultivated in the Brazilian Savannah (Cerrado) is not available in literature. The aim of this study was to investigate the physical and proximate composition; mineral, total phenolics, total tannins and total carotenoids contents; and antioxidant capacity (DPPH and FRAP assays) of the dark purple grumixama cultivated in the Cerrado area (Goiás State). Results showed a similar physical and proximate composition to those of grumixama native to the Atlantic Rainforest. However, grumixama from Cerrado presented the highest dietary fiber and magnesium contents. In addition, the Cerrado grumixama showed higher content of total phenolics (with a large amount of tannins), carotenoids and antioxidant capacity than those of grumixama native to the Atlantic Rainforest, cherry, blueberry, and fruits native to the Cerrado. Thus, the consumption of whole fruit (peel, pulp and seed) in healthy diets and its use as an ingredient for functional food products should be encouraged.

RESUMO: Grumixama (Eugenia brasiliensis Lam.) é uma 'berry' nativa da Mata Atlântica. Informações sobre o perfil nutricional e bioativo do fruto cultivado no Cerrado não estão disponíveis na literatura. O objetivo deste estudo foi investigar a composição física e centesimal; o conteúdo em minerais, fenólicos totais, taninos totais e carotenoides totais; e a capacidade antioxidante (ensaios DPPH and FRAP) da grumixama roxa cultivada na região do Cerrado (Estado de Goiás). Os resultados mostraram uma composição física e centesimal similar à da grumixama nativa da Mata Atlântica. Entretanto, a grumixama cultivada no Cerrado apresentou teores de fibra alimentar e magnésio superiores. Além disso, a grumixama do Cerrado apresentou maiores teores de compostos fenólicos totais (com grande quantidade de taninos) e carotenoides e maior capacidade antioxidante do que os teores da grumixama nativa da Mata Atlântica, cereja, mirtilo e frutos nativos do Cerrado. Assim, o consumo do fruto inteiro (casca, polpa e semente) em dietas saudáveis e seu emprego como ingrediente em produtos alimentícios funcionais devem ser estimulados.

Photoinduced Antibacterial Activity of the Essential Oils from Eugenia brasiliensis Lam and Piper mosenii C. DC. by Blue Led Light.

The objective of this work was to evaluate the phytochemical composition and the antibacterial and antibiotic-modulating activities of the essential oils of Eugenia brasiliensis Lam (OEEb) and Piper mosenii C. DC (OEPm) singly or in association with blue LED (Light-emitting diode) light. The antibacterial and antibiotic-modulatory activities of the essential oils on the activity of aminoglycosides were evaluated to determine the minimum inhibitory concentration (MIC, µg/mL) in the presence or absence of exposure to blue LED light. The chemical analysis showed α-pinene and bicyclogermacrene as major constituents of OEPm, whereas α-muurolol was the main compound of OEEb. Both OEEb and OEPm showed MIC ≥ 512 µg/mL against the strains under study. However, the association of these oils with the blue LED light enhanced the action of the aminoglycosides amikacin and gentamicin. In conclusion, the association of aminoglycosides with the blue LED light and essential oils was effective against resistant bacteria.

Short communication: Biogenic amine formation during fermentation in functional sheep milk yogurts.

The present study evaluated biogenic amine (BA) content during the fermentation period in functional sheep milk yogurts. Four treatments were prepared and assessed: natural (NSY), prebiotic (PreSY), probiotic (ProSY), and synbiotic (SynSY). Biogenic amines (putrescine, cadaverine, spermidine, spermine, and tyramine), proteolysis activity, and pH were measured during each hour of fermentation. Grumixama pulp was added to all formulations as a technological strategy and potential substrate for bacteria during fermentation. The yogurt and probiotic bacteria were viable (≥7 log cfu·mL-1) on d 0. The pH levels of the functional sheep milk yogurts had a more pronounced decrease than did the control of NSY. However, all yogurt samples underwent gradual decreases in pH until final fermentation. Proteolytic activity remained constant in all treatments during fermentation. The NSY, PreSY, ProSY, and SynSY presented the same behavior for all BA, with differences in concentration. Putrescine, cadaverine, and spermidine contents decreased, whereas spermine remained constant and tyramine increased. We conclude that fermentation of functional sheep milk yogurts can produce tyramine.

Wild Brazilian species of Eugenia genera (Myrtaceae) as an innovation hotspot for food and pharmacological purposes.

The Myrtaceae is one of the most common plant families in Brazil, including >1000 species of native fruit spread from North to South, where around 50% of all species are endemic to the Atlantic Rain Forest Biome. Most Brazilian species belong to Eugenia, Campomanesia, Psidium and Myrciaria genera. In general, they are characterized by the presence of leaves opposite, simple, entire, with pellucid glands containing ethereal oils and produces berry-like, fleshy fruits with a wide diversity of beneficial characteristics. Several parts of these plants are widely used in regional folk medicine as an astringent, anti-inflammatory, antihypertensive, to treat gastrointestinal disorders, and so on. Some species have already been studied and most of their metabolic effects are attributed to the presence of polyphenols, carotenoids as well as sesquiterpenes and monoterpenes. In addition, the Myrtaceae family has great economic potential, because of the sensory characteristics which encourages their commercial exploitation plus the presence of phytochemicals which play an important role in managing several degenerative chronic diseases besides representing a hotspot of technological innovation for food, cosmetic and pharmaceutical purposes. In this context, this review discusses about the importance of some Eugenia genera representatives such as Eugenia uniflora, Eugenia stipitata, Eugenia brasiliensis, Eugenia pyriformis and Eugenia dysenterica, emphasizing its profile of bioactive compounds as well as the knowledge of the nutritional and functional fruit potential, providing information for further studies aiming to stimulate the cultivation of Brazilian wild berries, for consumption and new food ingredients.

Eugenia brasiliensis leaves extract attenuates visceral and somatic inflammatory pain in mice.

ETHNOPHARMACOLOGICAL RELEVANCE: Eugenia brasiliensis Lam. (Myrtaceae) is a Brazilian tree distributed throughout Atlantic rain forest, since Bahia until Santa Catarina state, and is popularly known as "grumixaba, grumixameira, cumbixaba, ibaporoiti, and cereja-brasileira". The bark and leaves of Eugenia brasiliensis are used in folk medicine as adstringent, diuretic, energizing, anti-rheumatic and anti-inflammatory. This study aimed at investigating the chemical composition, antinociceptive and anti-inflammatory effect of the hydroalcoholic extract of Eugenia brasiliensis (HEEb). MATERIAL AND METHODS: Chemical composition of the HEEb was determined by High Performance Liquid Chromatography/ESI-Mass Spectrometry (HPLC-ESI-MS/MS). The antinociceptive and anti-inflammatory effects of HEEb (30-300 mg/kg) was verified in mice after oral administration by intra-gastric gavage (i.g.) 60 min prior to experimentation. It was investigated whether HEEb decreases visceral pain and leukocyte migration induced by an intraperitoneal (i.p.) injection of acetic acid (0.6%). We also evaluated whether HEEb decreases nociceptive behavior induced by formalin (including paw edema and temperature), prostaglandin E2 (PGE2), histamine, and compound 48/80. Finally, we evaluated the effect of HEEb in the chronic inflammatory (mechanical and thermal hypersensitivity) pain induced by complete Freund's adjuvant (CFA), as well as quantifying the concentration of the pro-inflammatory cytokines TNF-α and IL-6 in the paw by ELISA method. RESULTS: Seven polyphenols were identified in HEEb by HPLC-ESI-MS/MS analysis. HEEb treatment alleviated nocifensive behavior and leukocyte migration caused by acetic acid. Moreover, HEEb also reduced the inflammatory pain and paw temperature induced by formalin, as well as it decreased nociceptive behavior induced by histamine and compound 48/80. Finally, acute and repeated treatment of animals with HEEb (100 mg/kg, i.g.) markedly reduced the mechanical and thermal (heat) hypersensitivity, besides decrease paw edema and temperature induced by CFA, and this effect was evident until the day 7. Moreover, repeated treatment with HEEb (100 mg/kg, i.g.) significantly reduced the levels of IL-6 and TNF-α in the paw when compared to the CFA group. CONCLUSIONS: This is the first report showing that HEEb presents antinociceptive and anti-inflammatory effects in the visceral and somatic inflammatory pain in mice, possibly involving the inhibition of histamine receptors and pro-inflammatory cytokines activated pathways. Our results are of interest because they support the use of Eugenia brasiliensis as a potential source of phytomedicine for inflammatory diseases and pain.

Hypolipemiant and antioxidant effects of Eugenia brasiliensis in an animal model of coconut oil-induced hypertriglyceridemia.

We investigated the effects of chronic administration of crude hydroalcoholic extract (CHE) and crude acetone extract (CAE) obtained from leaves of Eugenia brasiliensis species on hypertriglyceridemia and oxidative stress caused by the chronic administration of coconut oil. Rats received CHE or CAE (50, 100 or 150mg/kg, orally) for 30days, plus coconut oil (2mL, orally) or saline for 15th. Triglyceride levels, liver cell lipid accumulation, thiobarbituric acid reactive substances (TBA-RS), total sulfhydryl content and the activities of antioxidant enzymes catalase (CAT), superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) were evaluated in the blood and liver of rats. Results showed that chronic administration of CHE or CAE was able to prevent hypertriglyceridemia and decrease the lipid droplets in liver cells, as well as the increase in TBA-RS, the reduction in total sulfhydryl content and CAT activity in the blood and prevent total or partial the increase in CAT and reduction in SOD and GSH-Px activities in the liver. These findings indicate that both extracts may have hypolipidemic and antioxidant effects.

Evaluation of seasonal chemical composition, antibacterial, antioxidant and anticholinesterase activity of essential oil from Eugenia brasiliensis Lam.

This study describes the seasonal composition and the antibacterial, antioxidant and anticholinesterase activity of the essential oil from Eugenia brasiliensis leaves. Analysis by using GC allowed the identification of 40 compounds. It was observed that the monoterpenes varied more (42%) than the sesquiterpenes (14%), and that the monoterpene hydrocarbons suffered the greatest variation throughout the year (64%). Major compounds were spathulenol in the spring (16.02 ± 0.44%) and summer (18.17 ± 0.41%), τ-cadinol in the autumn (12.83 ± 0.03%) and α-pinene (15.94 ± 0.58%) in the winter. Essential oils were tested for their antibacterial activity, and the best result was obtained from the autumn oil, with MIC = 500 µg mL(- 1) against Staphylococcus saprophyticus and Pseudomonas aeruginosa. Antioxidant activity was evaluated using DPPH, lipid peroxidation and iron-reducing power assays, as well as the anticholinesterase activity. Both tests showed a weak performance of the essential oils.

Anatomia foliar de Eugenia brasiliensis Lam. (Myrtaceae) proveniente de áreas de restinga e de floresta / Foliar anatomy of Eugenia brasiliensis Lam. (Myrtaceae) from restinga and forest areas

O presente trabalho apresenta a anatomia foliar de Eugenia brasiliensis Lam., cujo óleo essencial possui propriedades anti-reumática, diurética, anti-inflamatória, além de ser ativo contra o Tripanosoma cruzi. O estudo foi feito, comparando-se folhas coletadas em áreas de restinga e de floresta, registrando-se as diferenças estruturais resultantes da influência dos fatores ambientais. Anatomicamente, a folha de E. brasiliensis é hipostomática, glabra, dorsiventral, com tendência à isobilateralidade. A epiderme apresenta notável espessamento da cutícula e seus estratos. Em posição subepidérmica, ocorrem numerosas cavidades secretoras de óleos essenciais, nas duas faces da lâmina foliar. No parênquima lacunoso situam-se drusas de oxalato de cálcio. As folhas provenientes da restinga apresentam, em relação àquelas coletadas na floresta, maior quantidade de estruturas secretoras de óleos essenciais, maior concentração de substâncias ergásticas, maior espessamento da cutícula e da lâmina foliar, maior quantidade de estômatos, mesofilo mais compacto, fibras esclerenquimáticas e elementos xilemáticos com maior grau de lignificação e maior densidade da rede vascular. Os dados obtidos são indicativos que a escolha do local de coleta de E. brasiliensis é relevante, tendo em vista que, na área da restinga, observa-se maior produção de óleos essenciais e demais substâncias associadas aos seus efeitos terapêuticos.

The present paper presents the foliar anatomy of Eugenia brasiliensis Lam. The essential oils produced by this species present anti-reumathic, diuretic and anti-inflammatory properties and show activity against Tripanosoma cruzi. This study was carried out by comparison between leaves developed in restinga and forest areas, in order to register the main differences in the structure of this organ related to environmental conditions. Anatomically, the leaf of E. brasiliensis is hipostomatic, glabrous, with dorsiventral mesophyll, whit tendency towards the isobilateral type. Thickness of the cuticle and cutinized wall is noteworthy. In sub-epidermical position, there are many secretory cavities that produce essential oils, in both sides of the foliar blade. In the spongy parenchyma there are calcium oxalate druses crystals. E. brasiliensis leaves developed in restinga area present, in relation to that proceeding from forest, a larger amount of secretory structures, a higher ergastic substances concentration, thicker cuticle and foliar blade, more abundant stomata, a more compact mesophyll, sclerenchyma fibers and xilem elements with a deeper lignification and a greater development of the vascular system. The data obtained in this research suggest that the choice of the area where E. brasiliensis should be collected is important, because in restinga area it presents a greater production of essential oils and other substances associated with therapeutic effects.

Aplicação de cromatografia centrífuga de contra-corrente na purificação de ácido ursólico das folhas de Eugenia brasiliensis Lam. / Aplication of counter-current chromatography in the purification of ursolic acid from leavesof Eugenia brasiliensis Lam.

Os ácidos triterpênicos são metabólitos comuns na família Myrtaceae, especialmente no gênero Eugenia. O ácido ursólico foi descrito como um dos principais constituintes, nas folhas de Eugenia brasiliensis, coletada no Sudoeste do Brasil. Uma partição prévia, por solventes, do extrato etanólico ou do extrato clorofórmico de E. brasiliensis, seguida por uma purificação por cromatografia de contra-corrente de alta velocidade (CCCAV), conduziu ao isolamento do ácido ursólico com alto grau de pureza (> 97 por cento). Esta substância, também foi isolada por cromatografia convencional de coluna aberta (rendimento de 0.22 por cento a partir do extrato etanólico), e caracterizada por 13C-RMN, GC-EM e co-injeção com padrão comercial em CG-DIC, na forma do éster metílico. A técnica de CCCAV, usualmente usada para triterpenos glicosilados, foi aqui aplicada para a aglicona. As fases móvel e estacionária, no experimento de CCCAV, foram geradas pela mistura de n-hexano : acetato de etila : metanol : água, na proporção 10:5:2,5:1. A seleção do sistema de solventes (fases estacionária e móvel) foi determinada pela máxima distribuição eqüitativa do ácido ursólico em ambas as fases, medida por densitometria e monitorada por cromatografia em camada delgada, CCD, usando-se ácido ursólico comercial como referência.

Triterpene acids are common metabolites in the Myrtaceae family, especially in the genus Eugenia. Ursolic acid was found in Eugenia brasiliensis collected in Southeastern Brazil. A previous solvent partition of the ethanol or chloroform extracts of the leavesof E. brasiliensis, followed by rapid high-speed counter-current chromatography (HSCCC) afforded ursolic acid in high purity (> 97 percent). This compound was also purified apart by conventional column chromatography (yield of 0.22 percent from the ethanolic extract) and characterized by 13C-NMR, GC-MS and co-injection of its methyl ester with standards in GC-FID. The HSCCC technique, usually applied to triterpene glycosides, was here applied successfully to an aglycone, to which examples are rarely described. The mobile and stationary phase for the HSCCC experiment were derived from the two-phase solvent system composed by n-hexane : ethyl acetate : methanol : water in the proportion of 10:5:2.5:1. The choice of the developing solvent system for optimum HSCCC separation was determined by TLC coupled to densitometric measurements of ursolic acid in both stationary and mobile phase, generated by the upper and lower layer of the system above. Commercial ursolic acid was used as standard.