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Indigo is a bis-indolic alkaloid that has antioxidant and anti-inflammatory effects reported in literature and is a promissory compound for treating chronic inflammatory diseases. This fact prompted to investigate the effects of this alkaloid in the experimental model of Duchenne muscular dystrophy. The main aim of this study was to evaluate the potential role of the indigo on oxidative stress and related signaling pathways in primary skeletal muscle cell cultures and in the diaphragm muscle from mdx mice. The MTT and Neutral Red assays showed no indigo dose-dependent toxicities in mdx muscle cells at concentrations analyzed (3.12, 6.25, 12.50, and 25.00 μg/mL). Antioxidant effect of indigo, in mdx muscle cells and diaphragm muscle, was demonstrated by reduction in 4-HNE content, H(2)O(2) levels, DHE reaction, and lipofuscin granules. A significant decrease in the inflammatory process was identified by a reduction on TNF and NF-κB levels, on inflammatory area, and on macrophage infiltration in the dystrophic sample, after indigo treatment. Upregulation of PGC-1α and SIRT1 in dystrophic muscle cells treated with indigo was also observed. These results suggest the potential of indigo as a therapeutic agent for muscular dystrophy, through their action anti-inflammatory, antioxidant, and modulator of SIRT1/PGC-1α pathway. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s12192-022-01282-0.

This study is aimed at investigating the effects of LEDT, at multiple wavelengths, on intracellular calcium concentration; on transient receptor potential canonical channels; on calcium-binding protein; on myogenic factors; on myosin heavy chains; on Akt signaling pathway; on inflammatory markers; and on the angiogenic-inducing factor in dystrophic muscle cell culture experimental model. Dystrophic primary muscle cells were submitted to LEDT, at multiple wavelengths (420 nm, 470 nm, 660 nm, and 850 nm), and evaluated after 48 h for cytotoxic effects and intracellular calcium content. TRPC-1, TRPC-6, Calsequestrin, MyoD, Myogenin, MHC-slow, MHC-fast, p-AKT, p-mTOR, p-FoxO1, Myostatin, NF-κB, TNF-α, and VEGF levels were evaluated in dystrophic primary muscle cells by western blotting. The LEDT, at multiple wavelengths, treated-mdx muscle cells showed no cytotoxic effect and significant lower levels in [Ca2 +]i. The mdx muscle cells treated with LEDT showed a significant reduction of TRPC-1, NF-κB, TNF-α and MyoD levels and a significant increase of Myogenin, MHC-slow, p-AKT, p-mTOR, p-FoxO1 levels, and VEGF levels. Our findings suggest that different LEDT wavelengths modulate the Akt-signaling pathways and attenuate pathological events in dystrophic muscle cells, and a combined multiwavelength irradiation protocol may even provide a potentially therapeutic strategy for muscular dystrophies.

Background Ca2+ dysregulation and oxidative damage appear to have a central role in Duchenne muscular dystrophy (DMD) progression. The current study provides muscle cell-specific insights into the effect of Tempol on the TRPC 1 channel; on the positive and negative regulators of muscle cell differentiation; on the antioxidant enzymatic system; on the activators of mitochondrial biogenesis; and on the inflammatory process in the dystrophic primary muscle cells in culture.The Trypan Blue, MTT and Live/Dead Cell assays showed that Tempol (5 mM) presented no cytotoxic effect on the dystrophic muscle cells. The Tempol treated-Our findings demonstrated that Tempol decreased intracellular calcium and oxidative stress in primary dystrophic muscle cells, promoting a cross-talk between TRPC-1, mTOR, PGC-1α and PPARδ.

The study aimed to contribute to establish parameters for analyzing the physiological quality of seed-clove basil (Ocimum gratissimum L.), since the species is not among those described in the Rules for Testing Seeds. Seeds of basil, clove, gathered on the campus of UENP-CLM, Bandeirantes, PR, analyzed for water content, germination, accelerated aging water and saline tetrazolium and seedling emergence. The temperature of 30 ° C for the germination test and the sand substrate for the emergence test are conditions and procedures for evaluating the physiological quality of seeds of O. gratissimum. The aging test, regardless of the type of solution used, the test is correlated with seedling emergence in soil. The low germination and seed viability of O. gratissimum occurring due to deterioration and lack of internal tissue.O estudo teve como propósito contribuir para o estabelecimento de parâmetros para análise da qualidade fisiológica de sementes de alfavaca-cravo (Ocimum gratissimum L.), uma vez que a referida espécie não se encontra entre as descritas nas Regras para Análise de Sementes. Foram utilizadas sementes de alfavaca-cravo, colhidas no Campus da UENP-CLM, Bandeirantes-PR, submetidas a análises de teor de água, germinação, envelhecimento acelerado com água e solução salina, tetrazólio e emergência de plântulas. A temperatura de 30 ºC para o teste de germinação e o substrato areia para o teste de emergência de plântulas são condições e procedimentos apropriados para avaliação da qualidade fisiológica de sementes de O. gratissimum. O teste de envelhecimento, independente do tipo de solução utilizada, apresenta correlação com o teste de emergência de plântulas em solo. O baixo potencial germinativo e viabilidade das sementes de O. gratissimum ocorrem em função da deterioração e ausência dos tecidos internos.

Were accomplished the effect of plant extracts of clove basil (Ocimum gratissimum L.), horsetail (Equisetum hyemale L.), coriander (Coriandrum sativum L.) and tobacco (Nicotiana tabacum L.) on Brevicoryne brassicae (L., 1758) and Myzus persicae (Sulzer, 1776) aphids in cabbage Brassica oleracea (L.). The treatments consisted of plant extracts prepared fresh and dry (concentrations of 2.5; 5.0; and 10%) and the controls insecticide acephate and water. These solutions were sprayed on cabbage discs placed on agar in Petri dishes, containing twenty adult aphids. In sequence, the Petri dishes were sealed with plastic film and this procedure was repeated for the two aphid species studied. The assessment of the number of live nymphs and adults occurred at 1, 12, 24, and 72 hours after installation. The extracts of coriander and tobacco prepared in a concentration of 10% showed toxic effects similar to the organophosphate insecticide acephate, on adults and nymphs of the aphids Brevicoryne brassicae and Myzus persicae. Coriander revealed a promising alternative that deserves detailed studies regarding the performance of its active ingredients and dosage determination in order to provide a safe herbal product to control insects.Avaliou-se o efeito de extratos vegetais de alfavaca-cravo (Ocimum gratissimum L.), cavalinha (Equisetum hyemale L.), coentro (Coriandrum sativum L.) e fumo (Nicotiana tabacum L.) sobre os pulgões Brevicoryne brassicae (L., 1758) e Myzus persicae (Sulzer, 1776) em couve Brassica oleracea (L.). Os tratamentos consistiram de extratos vegetais preparados a fresco e seco (nas concentrações de 2,5; 5,0 e 10%), do padrão inseticida acefato e de água. As soluções assim obtidas foram pulverizadas em discos de couve colocados sobre agar em placas de Petri, contendo vinte pulgões adultos. Na sequência, as placas de Petri foram vedadas com filme plástico transparente, sendo este procedimento repetido para as duas espécies de afídeos. A avaliação do número de ninfas e adultos vivos ocorreu 1, 12, 24 e 72 horas após a pulverização dos tratamentos. Os extratos de coentro e fumo preparados na concentração de 10% demonstraram ação tóxica semelhante à do inseticida organofosforado acefato, sobre adultos e ninfas dos pulgões Brevicoryne brassicae e Myzus persicae. O coentro evidenciou-se como alternativa promissora que merece estudos detalhados, no tocante a atuação de seus princípios ativos e determinação de dosagens, a fim de disponibilizar um produto botânico seguro para o controle de insetos.

Orientador: Marcos José Salvador Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia Resumo: O objetivo do estudo químico, biológico e biotecnológico de duas espécies de Annonaceae (Annona cacans, Annona mucosa, Annona squamosa, ou Guatteria hispida) foi buscar substâncias bioativas e fotoativadas nestas matrizes vegetais (plantas in natura e obtidas por cultura de células). O estudo dos extratos brutos metanólicos foi realizado monitorando o espectro de absorção na região de interesse em terapia fotodinâmica (600 a 800 nm), na análise do espectro de fluorescência (modo emissão e excitação), na avaliação da eficiência fotoquímica e nos resultados dos bioensaios, incluindo a avaliação do efeito biológico fotoinduzido por radiação laser a 660 nm em modelo experimental in vitro de terapia fotodinâmica antimicrobiana (PDT-antimicrobiana). Para tanto, procedeu-se a adequação de metodologia para os ensaios biologicos em PDT-antimicrobiana e o desenvolvimento de procedimentos analíticos, incluindo o estudo fitoquímico biomonitorado, obtendo-se inicialmente o perfil químico (CCDC, CG, CLAE, RMN ou ESI-MS). Os resultados possibilitaram a identificação de 11 alcalóides já descritos em literatura para espécies de Annonaceae diretamente nos extratos brutos (desreplicação). Após a etapa de triagem e por comparação entre os resultados obtidos foi possível definir a G. hispida e A. squamosa como as espécies mais promissoras para as quais continuou-se o estudo. As demais espécies apresentaram baixa absorção e fluorescência na região de interesse em PDT e baixa eficiência fotoquímica. Os extratos das duas espécies escolhedas com substâncias que não puderam ser identificadas na etapa de desreplicação foram submetidos ao fracionamento biomonitorado e para as substâncias isoladas e purificadas realizou-se a sua determinação estrutural utilizando-se métodos espectroscópicos de análise o que possibilitou a identificação de 5 alcalóides: os alcalóide Isomoschatolina e 9-metoxi-isomoschatolina foram isolados apenas das amostras de G. hispida e o alcalóide Palmatina isolado apenas do extrato de A. squamosa, enquanto os alcalóides Anonaina e Liriodenina foram isolados dos extratos das duas espécies. No estudo biotecnológico procedeu-se o estabelecimento e desenvolvimento de culturas de células das duas espécies vegetais mais promissoras nos bioensaios, visando, além do estudo fitoquímico, a manipulação de fatores físicos e químicos para a produção de metabólitos secundários e o estudo comparativo dos metabólitos bioativos nas culturas de células e aqueles obtidos de partes da planta in natura. Foram obtidos calos friáveis e livres de oxidação para as duas espécies. O estudo comparativo dos espectros de ESI-MS/MS dos extratos bioativos mostrou similaridades entre a composição dos extratos da planta in natura com o obtido por cultura de células e possibilitou a quantificação por UHPLC-MS de alguns alcalóides já descritos para espécies de Annonaceae nas amostras estudadas, incluindo os alcalóides asimilobina, Liriodenina, lisicamina e Palmatina que apresentam potencial para aplicação em PDT antimicrobiana observando-se similaridades nos resultados em termos químicos e biológicos. Em colaboração com o "Laboratoire Jean Perrin, Campus Jussieu, Université Pierre et Marie Curie, em Paris, França", foram realizados alguns estudos dos alcalóides isoquinolínicos, tais como, Isomoschatolina (Gh1) e Palmatina (As3) sozinhos e associados à ciclodextrina. Os estudos sugerem que esses alcalóides quando complexados são melhor direcionados no interior das células tumorais e se concentram predominantemente nos lisossomos Abstract: he aim of the study chemical, biological and biotechnological of two Annonaceae¿s species (Annona Cacans, Annona mucosa, Annona squamosa or Guatteria hispida) went for bioactive substances and photoactivated these plant matrices (plants in nature and obtained by cell culture). The study of methanolic crude extract was carried out by monitoring the absorption spectrum in the region of interest in photodynamic therapy (600 to 800 nm), the fluorescence spectrum analysis (mode emission and excitation), the evaluation of the photochemical efficiency and the results of bioassays including assessment of the biological effect photoinduced by laser radiation at 660 nm in an experimental model in vitro antimicrobial photodynamic therapy (PDT-antimicrobial). Therefore, we proceeded to fit methodology for biological testing PDT-antimicrobial and development of analytical procedures including the study bioactivity Phytochemistry, obtaining initially a chemical profile (CCDC, GC, HPLC, NMR and ESI-MS ). The results allowed the identification of 11 alkaloids already described in literature for Annonaceae species directly in crude extracts (dereplication). After the step of screening and by comparing the results obtained it was possible to define G hispida and. A. squamosa as the most promising species for which continued the study. The other species showed low absorption and fluorescence in the region of interest in PDT and low photochemical efficiency. The extracts of the two select species with substances that could not be identified in dereplication stage underwent biomonitored fractionation and isolated substances and purified held its structural determination using spectroscopic methods of analysis which enabled the identification of 5 alkaloids: alkaloid Isomoschatoline and 9-methoxy-isomoschatoline were isolated only from samples G. hispida and Palmatine alkaloid isolated only from A. squamosa extract while Anonaina and liriodenine alkaloids have been isolated from two species of the extracts. In biotechnology study proceeded to the establishment and development of cell cultures of the two most promising species in bioassays, aimed beyond the phytochemical study, the handling of physical and chemical factors for the production of secondary metabolites and the comparative study of bioactive metabolites in cell cultures and those obtained from the fresh plant parts. Friable callus and oxidation free were obtained for the two species. The comparative analysis of ESI-MS / MS spectra of the bioactive extracts showed similarities between the composition of the plant extracts in nature to that obtained by cultivating cells and allowed the quantitation UHPLC-MS some alkaloids already described for species of Annonaceae in samples studied, including Asimilobine alkaloids, Liriodenine, Lysicamine and Palmatine that have potential for use in antimicrobial PDT observing similarities in results in chemical and biological terms. In collaboration with the "Laboratoire Jean Perrin, Campus Jussieu, Université Pierre et Marie Curie in Paris, France," were carried out some studies of isoquinoline alkaloids, such as Isomoschatoline (GH1) and Palmatine (AS3) alone and associated with the cyclodextrin. Studies suggest that these alkaloids are best directed when complexed within tumor cells and predominantly concentrated in the lysosomes Doutorado Biologia Vegetal Doutora em Biologia Vegetal FAPESP 2011/18315-2 CNPQ CAPES FAEPEX