Potent anti-inflammatory activity of betulinic acid treatment in a model of lethal endotoxemia.

Betulinic acid (BA) is a lupane-type triterpene with a number of biological activities already reported. While potent anti-HIV and antitumoral activities were attributed to BA, it is considered to have a moderate anti-inflammatory activity. Here we evaluated the effects of BA in a mouse model of endotoxic shock. Endotoxemia was induced through intraperitoneally LPS administration, nitric oxide (NO) and cytokines were assessed by Griess method and ELISA, respectively. Treatment of BALB/c mice with BA at 67 mg/kg caused a 100% survival against a lethal dose of lipopolysaccharide (LPS). BA treatment caused a reduction in TNF-α production induced by LPS but did not alter IL-6 production. Moreover, BA treatment increased significantly the serum levels of IL-10 compared to vehicle-treated, LPS-challenged mice. To investigate the role of IL-10 in BA-induced protection, wild-type and IL-10(-/-) mice were studied. In contrast to the observations in IL-10(+/+) mice, BA did not protect IL-10(-/-) mice against a lethal LPS challenge. Addition of BA inhibited the production of pro-inflammatory mediators by macrophages stimulated with LPS, while promoting a significant increase in IL-10 production. BA-treated peritoneal exudate macrophages produced lower concentrations of TNF-α and NO and higher concentrations of IL-10 upon LPS stimulation. Similarly, macrophages obtained from BA-treated mice produced less pro-inflammatory mediators and increased IL-10 when compared to non-stimulated macrophages obtained from vehicle-treated mice. In conclusion, we have shown that BA has a potent anti-inflammatory activity in vivo, protecting mice against LPS by modulating TNF-α production by macrophages in vivo through a mechanism dependent on IL-10.

Cytotoxicity of the diterpene 14-O-methyl-ryanodanol from Erythroxylum passerinum in an astrocytic cells model.

Plant secondary metabolites, such as, specifically, alkaloids and terpenes, may present psychoactive properties that modify the function of the central nervous system (CNS) and induce neurotoxicity. Neurotoxicity involves the response of glial cells, mainly astrocytes, which play a fundamental role in the control of homeostasis of the CNS. Some Erythroxylum species are indigenous to the state of Bahia in Brazil. This study investigated the cytotoxic activity of the diterpene AEP-1, extracted from the fruit of E. passerinum in a GL-15 cell line, astrocytic, glial cells model. The effects on cell viability, analyzed by the MTT assay, demonstrated a dose-dependent cytotoxic effect, with maximum effect at 500 µg/mL of AEP-1, and with a reduction of about 40 and 47% on cellular viability after 24 h and 72 h treatment, respectively. Evidence for induction of apoptosis by AEP-1 was first obtained when GL-15 glial cells were incubated with 250 µg/mL AEP-1 causing reniform and/or pyknotic nuclei and apoptotic bodies revealed by chromatin staining with Hoechst 33258. Increase in DNA fragmentation was also observed by comet assays in cells incubated with 500 µg/mL of AEP-1. Moreover, cells exposed to a sub toxic dose of AEP-1 (250 µg/mL) showed significant changes in morphology--contraction of the cytoplasm and expansion of cellular projections--signifying the presence of astrocytic cytoskeletal protein and glial fibrillary acidic protein (GFAP). These findings indicated astrocytic cells as the target for terpene AEP-1 and suggest the involvement of glial cells with psychoactive symptoms observed in humans and animals after consumption of fruits of plants of the genus Erythroxylum.

Immunomodulatory and antibacterial activities of extracts from Rutaceae species / Atividade imunomoduladora e antibacteriana de extratos de espécies da família Rutaceae

Rutaceae is a taxon with species very well distributed in Brazilian semi-arid area, commonly used in folk medicine. Species from this genus have diverse biological activity described in literature. In this work, immunomodulatory and bactericidal activity are described for chloroform and ethyl acetate extracts of three of them (Esenbeckia grandiflora Mart., Pilocarpus spicatus A.St.-Hil. and Galipea simplicifolia Schult.). Initially all the samples had their cytotoxicity evaluated, aiming to determine the LC50. The immunomodulatory potential was evaluated in cultures of murine splenocytes stimulated or not with concanavalin A and in a mixed lymphocyte reaction (MLR) using splenocytes from BALB/c (H-2d) mice immunized with splenocytes from C57Bl/6 (H-2b) mice. Four samples had higher values of lymphoproliferation inhibition in concanavalin A-stimulated cultures and were evaluated in MLR. The antibacterial activity of extracts was also evaluated and the minimal inhibitory concentrations (MIC) for two active samples were 1.0 and 5.0 mg/ml for extracts from Esenbeckia grandiflora Mart. and Galipea simplicifolia Schult., respectively. Thus, our results reinforce data of literature relating biological activity for many species of the Rutaceae family and encourage studies with these species aiming to discover active compounds, candidates to new medicines.

A família Rutaceae apresenta espécies vegetais muito bem distribuídas no Semi-Árido Brasileiro e comumente usadas em medicina popular. Espécies dessa família tem diversas atividades biológicas descritas na literatura. Neste trabalho, atividades imunomoduladora e bactericida são descritas para o extrato acetato de etila e clorofórmico de três espécies da família (Esenbeckia grandiflora Mart., Pilocarpus spicatus A.St.-Hil. e Galipea simplicifolia Schult.). Todas as amostras foram inicialmente avaliadas quanto à sua citotoxicidade, com objetivo de determinar a LC50. O potencial imunomodulador foi avaliado em culturas de esplenócitos murinos estimulados ou não com concanavalina A e também em reação mista linfocitária (RML), usando também esplenócitos de camundongos BALB/c (H-2d) imunizados com esplenócitos de camundongos C57Bl/6 (H-2b). Quatro amostras tiveram os mais elevados valores percentuais de inibição da proliferação de linfócitos ativados pela concanavalina A e foram avaliados em RML. A atividade antibacteriana dos extratos foi também avaliada e a concentração mínima inibitória (CMI) para duas amostras ativas foi de 1.0 e 5.0 mg/mL, respectivamente para as espécies Esenbeckia grandiflora Mart. and Galipea simplicifolia Schult. Assim, os dados aqui apresentados reforçam informações da literatura científica relacionados à atividade biológica para muitas espécies da família Rutaceae e incentivam outros estudos com estas visando descobrir substâncias ativas, potenciais candidatas a novos fármacos.

Antimalarial activity of betulinic acid and derivatives in vitro against Plasmodium falciparum and in vivo in P. berghei-infected mice.

Malaria is one of the most important tropical diseases and mainly affects populations living in developing countries. Reduced sensitivity of Plasmodium sp. to formerly recommended antimalarial drugs places an increasing burden on malaria control programs as well as on national health systems in endemic countries. The present study aims to evaluate the antimalarial activity of betulinic acid and its derivative compounds, betulonic acid, betulinic acid acetate, betulinic acid methyl ester, and betulinic acid methyl ester acetate. These substances showed antiplasmodial activity against chloroquine-resistant Plasmodium falciparum parasites in vitro, with IC(50) values of 9.89, 10.01, 5.99, 51.58, and 45.79 microM, respectively. Mice infected with Plasmodium berghei and treated with betulinic acid acetate had a dose-dependent reduction of parasitemia. Our results indicate that betulinic acid and its derivative compounds are candidates for the development of new antimalarial drugs.

Immunomodulatory activity of extracts from Cordia superba Cham. and Cordia rufescens A. DC. (Boraginaceae), plant species native from Brazilian Semi-arid / Atividade imunomoduladora de extratos de Cordia superba Cham. and Cordia rufescens A. DC. (Boraginaceae), espécies de plantas nativas do semi-árido brasileiro

The family Boraginaceae is widely distributed in Brazil and in the Northeastern region some species are popularly used to treat symptoms of rheumatism, painful menstruation and dyspepsia. In this work we studied Cordia superba Cham. and C. rufescens A. DC., native from Brazilian Semi-arid region, in order to investigate their immunomodulatory activity. Six extracts were prepared from aerial parts of C. superba and C. rufescens. The cytotoxicity was evaluated using splenocytes from BALB/c mice. The immunomodulatory activity was determined by in vitro assays using activated mouse macrophages and lymphocytes. Peritoneal macrophages obtained from BALB/c mice were stimulated with IFN-gamma and LPS in the presence/absence of the samples. The NO production was measured indirectly through Griess method. Three samples inhibited the production of nitric oxide in values near 50 percent at a concentration of 100 µg/mL. To evaluate the effects of the extracts on lymphocytes, splenocytes from BALB/c mice were incubated with the samples and concanavalin A. Proliferation inhibition was determined by analysis of ³H-thymidine uptake. Samples from the two species had a strong inhibitory activity on lymphocyte proliferation and IL-2 production. Two chloroform extracts prepared from aerial parts of C. rufescens had the lowest IC50 values (7.6 and 11.0 µg/mL).

A família Boraginaceae é amplamente distribuída no Brasil e na região nordeste algumas espécies são usadas popularmente no tratamento de reumatismo, dores menstruais e dispepsias. Neste trabalho foram estudadas as espécies Cordia superba Cham. and C. rufescens A. DC., nativas da região semi-árida brasileira, objetivando investigar a atividade imunomoduladora. Seis extratos foram preparados a partir de partes aéreas das espécies. A citotoxicidade foi avaliada usando culturas de esplenócitos de camundongos BALB/c. A atividade imunomoduladora foi determinada por ensaios in vitro usando macrófagos e linfócitos murinos ativados. Macrófagos peritoneais obtidos de camundongos BALB/c foram estimulados com IFN-gama and LPS na presença/ausência das amostras. A produção de NO foi medida indiretamente através do método de Griess. Três amostras inibiram a produção de NO em valores próximos a 50 por cento (100 µg/mL). Os efeitos das amostras sobre os linfócitos foram avaliados cultivando esplenócitos de camundongos BALB/c em presença destas amostras e de concanavalina A. A proliferação foi determinada pela análise da incorporação de ³H-tritiada. Amostras de duas espécies apresentaram uma forte atividade inibidora sobre a proliferação de linfócitos e sobre a produção de IL-2. Dois extratos clorofórmicos (partes aéreas de C. rufescens) tiveram os menores valores de IC50 (7,6 and 11,0 µg/mL).

Anti-leishmanial and immunomodulatory activities of extracts from Portulaca hirsutissima and Portulaca werdermannii.

Ethyl acetate and chloroform extracts from aerial parts of Portulaca werdermannii and P. hirsutissima were tested in lymphoproliferation assays and axenic cultures of Leishmania amazonensis and Trypanosoma cruzi. Both extracts of P. werdermannii and P. hirsutissima had a potent inhibitory activity on lymphocyte proliferation. On the contrary, only the chloroformic extract of both plants inhibited L. amazonensis growth, without effect on T. cruzi cultures. These results indicate these Portulaca species as potential sources of new active molecules for the treatment of leishmaniasis and immune-mediated pathologies.