Estudo químico e atividade mutagênica e antiradicalar e Paepalanthus chiquitensis Herzog (ERIOCAULACEAE)

Este trabalho descreve o estudo fitoquímico, a avaliação das atividades mutagênica e antiradicalar e a determinação dos teores de flavonoides e fenóis totais dos extratos metanólicos e da fração acetato de etila de Paepalanthus chiquitensis Herzog, pertencente à família Eriocaulaceae. As análises por HPLC-UV-PDA permitiram inferir, qualitativamente, que o extrato metanólico de capítulos possui maior concentração de naftopiranonas, enquanto que os escapos possuem maior concentração de flavonoides. Os fingerprints por HPLC-ESI-IT-MSn permitiram identificar dezenove flavonoides e seis naftopiranonas pela análise dos padrões de fragmentações por ESI-MSn, modo negativo, além da comparação com dados existentes na literatura. No estudo fitoquímico, foram utilizadas técnicas cromatográficas usuais, principalmente para substâncias polares, como (Cromatografia de Permeação em gel, Sephadex LH-20, Cromatografia Líquida de Alta Eficiência e Extração em Fase Sólida - SPE), que forneceram misturas de flavonoides identificados por métodos espectroscópicos (Espectrometria de Massas, Ultravioleta e Ressonância Magnética Nuclear). Foram, portanto, identificados dois flavonóides considerados inéditos na literatura (PGC2B e PGC3). A atividade antiradicalar dos extratos metanólicos e das frações acetato de etila foram avaliadas utilizando-se o ensaio DPPH (1,1-difenil-1-picril-hidrazila) e, como padrões, o ácido gálico e a quercetina. Este estudo revelou uma melhor atividade antiradicalar da fração acetato de etila (IC50 = 1,82 ± 0,0374 μg mL-1 para escapos, e 2,05 ± 0,0133 μg mL-1, para capítulos) quando comparadas com os padrões de ácido gálico (IC50 = 4,37 ± 0,0377 μg mL-1) e quercetina (IC50 = 5,12 ± 0,0079μg mL-1). Foi observada uma maior concentração de fenóis totais na fração acetato de etila de escapos...This research describes the phytochemical study, the evaluation of the mutagenic and antiradicalar activities, and the determination of the total levels from flavonoids and phenols of the methanolic extract and ethyl acetate fraction of Paepalanthus chiquitensis Herzog, belonging to the Eriocaulaceae family. The analysis by HPLC-UV-PDA allowed inferring qualitatively that the methanolic extract of the capitulae has a higher concentration of naphythopyranones while scapes have a higher concentration of flavonoids. The fingerprints by HPLC-ESI-IT-MSn allowed to identify nineteen flavonoids and six naphythopyranones by the analisys of the fragmentation patterns by ESI-MSnin the negative mode for flavonoids and naphythopyranones, besides the comparison with to the literature data. In the phytochemical study, usual chromatographic techniques were used, especially for polar compounds -such as Gel Permeation Chromatography, Sephadex LH-20, High Performance Liquid chromatography and Solid Phase Extraction- to provide mixtures of flavonoids which were identified by spectroscopic methods (Mass Spectrometry, Ultraviolet and Nuclear Magnetic Resonance). That allowed to identify two flavonoids unpublished in the literature (PGC2 and PGC3). The antiradical activity of the methanolic extracts and ethyl acetate fractions were evaluated using DPPH assay, with gallic acid and quercetin as antiradical patterns. This study revealed a better antiradical activity of ethyl acetate fractions (IC50 = 1.82 ± 0.0374 μg mL-1, for scapes and 2.05 ± 0.0133 μg mL-1 for capitulae), when compared to the standards galic acid (IC50 = 4.37 ± 0.0377 μg mL-1) and quercetin patterns (IC50 = 5.12 ± 0.0079μg m L-1). Was observed a higher content of total phenols in the ethyl acetate fractions of the scapes (106.54 mg/g of total phenols presents in the... (Complete abstract click electronic access below)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP

Development of "patches" containing the association of Artemether - Lumefantrine for the treatment of malaria caused by "Plasmodium falciparum"

Orientador: Mary Ann FoglioTese (doutorado) - Universidade Estadual de Campinas, Instituto de BiologiaResumo: Este trabalho teve como objetivo desenvolver sistemas terapêuticos transdérmicos contendo a associação dos fármacos arteméter (Art) e lumfantrina (Lum), com o propósito de ampliar o acesso ao tratamento da malária em áreas endêmicas afastadas, sem acesso a hospitais. Art-Lum são considerados os fármacos de primeira linha para o tratamento da malária (WHO, 2018), nos quais o Art possui rápida e potente ação, diminuindo a parasitemia nas primeiras horas de administração e a Lum atua mais lentamente, eliminando os parasitas residuais. A via de liberação transdérmica é uma via alternativa favorável, com vantagens em relação à via oral, que incluem evitar o metabolismo da primeira passagem, prevenir a degradação gastrointestinal e, adicionalmente, melhorar a adesão de crianças - principais vítimas da malária no mundo e muitas vezes são resistentes à via oral ou parenteral - ao tratamento. Dentre os sistemas desenvolvidos, encontram-se filmes, sistemas líquidos cristalinos (LCs), membranas obtidas pela técnica de eletrofiação (ME) e também microagulhas (MNs) contendo nanosuspensões (NNs) de fármacos. As formulações de Art e Lum foram desenvolvidas separadamente e em associação. Os perfis de liberação dos fármacos foram realizados em célula de Franz e quantificados por UPLC/MS. As MNs de Lum apresentaram o melhor resultado de permeação para Lum com 217 µg/cm2/24 hrs e os filmes de Art, com o maior perfil de liberação para Art com 6000 µg/cm2/24 hrs. Para selecionar os sistemas de liberação de escolha, foram realizados estudos in vitro com Plasmodium falciparum 3D7. Nestes estudos, verificou-se que a cultura celular não evoluiu na presença de LCs, ME e bioadesivos. Foi possível testar in vitro apenas as formulações de MNs, prosseguindo-se, assim, para os estudos de biodisponibilidade e avaliação da atividade antimalárica in vivo em modelo murino de infecção por Plasmodium yollei com as MNs desenvolvidas. Para os experimentos com in vivo, foram utilizados camundongos C57BL/6J. Os estudos de biodisponibilidade foram realizados em animais sadios (sem infecção), onde avaliou-se dois esquemas de tratamento: a) dose única, sendo aplicada 1 MN Art-Lum no 1° dia de tratamento e removida após 72 hrs e b) 1x/dia/3dias, que incluiu a troca diária de MNs nos 3 dias de tratamento. Nestes estudos, observou-se o Cmax de Lum a 4?g/mL de plasma, no tempo de 72 hrs, quando utilizou-se esquema de aplicação das MNs 1x/dia/3dias. O Cmax de Art foi de 375?g/mL e diidroartemisinina, metabólito ativo de Art, de 3125?g/mL, ambos no tempo de 6 hrs, indicando que as MNs são capazes de prolongar o tempo de permanência do fármaco no organismo. Nos estudos de atividade antimalárica, os camundongos foram infectados com 106 eritrócitos Plasmodium yoelii 17XNLe foram tratados com MNs Art-Lum a partir do dia 0 da infecção no esquema de aplicação das MNs por 1x/dia/3dias, tendo demonstrado uma redução da parasitemia de 99,5% até o dia 12 pós infecção. Os presentes resultados sugerem que o tratamento utilizando MNs Art-Lum mantém os animais tratados abaixo de 0,5% de parasitemia, ou seja, são capazes de abolir a infecçãoAbstract: Herein we report the development of transdermal drug delivery systems containing an association of artemether (Art) and lumefantrine (Lum)-loaded, with the focus on expanding the options to malaria treatment in communities isolated from the cities, without access to hospitals. Art-Lum are considered standard golden for malaria treatment (WHO, 2018). Art has fast onset action, reducing the parasitemia in a few hours after administration. Lum acts more slowly, eliminating the residual parasites. The transdermal delivery route is a favorable alternative pathway with advantages over oral or parenteral routes that include avoiding first passage metabolism, preventing gastrointestinal degradation and, additionally, increase the children¿s adhesion, which are the main victims from malaria around the world. Among the developed systems, there are liquid crystalline systems (LCs), electrospinning membranes (EM) and microneedles (MNs) nanosuspensions (NNs) loaded arteméter (Art) ¿ lumefantrine (Lum). The formulations of Art and Lum were developed separately and in the association. The permeation profiles of the drugs were studied and quantified by UPLC/MS. The best results for Lum were achieved with MNs (217 µg/cm2/24 hrs). For Art films, the best permeation profile was (~6000 µg/cm2/24 hrs). To select the suit drug delivery system, in vitro studies with Plasmodium falciparum 3D7 were performed. In these studies, it was verified that the cell culture did not evolve in the presence of LCs, ME and bioadhesives. Only formulations of MNs were possible to be tested in vitro, and thus, the bioavailability and evaluation studies of the antimalarial activity in vivo in a murine model of Plasmodium yoleii infection with the MNs developed were carried out. For the in vivo experiments, C57BL / 6J mice were used. The bioavailability studies were performed in healthy animals (without infection), in which two treatment regimens were evaluated: a) single dose, in which 1 MN Art-Lum was applied on the 1st day of treatment and removed after 72 hrs and b) 1x /day /3 days, which included the daily exchange of MNs during the 3 days of treatment. In these studies, the Cmax of Lum was observed at 4?g / mL of plasma, during the period of 72 hrs, when the application scheme of the MNs 1x / day / 3 days was used. Art Cmax was 375 ?g / mL and the Cmax of dihydroartemisinin, Art's active metabolite, was 3125 ?g / mL, both at 6 hrs, indicating that the MNs are able to prolong the time the drug stays in the blood. In the antimalarial activity studies, the mice were infected with 106 Plasmodium yoelii 17XNL erythrocytes and treated with Art-Lum MNs from day 0 of the infection in the MNs application scheme 1x/day/3 days, which demonstrated a 99.5% reduction in parasitemia by day 12 post-infection. The present results suggest that the treatment using Art-Lum MNs keeps the treated animals below 0.5% of parasitemia, that is, they are able to abolish the infectionDoutoradoFármacos, Medicamentos e Insumos para SaúdeDoutora em Ciências2014/16008-3; 2016/18384-8FAPES

Fluorescence-coupled techniques for determining rose bengal in dermatological formulations and their application to ex vivo skin deposition studies

Rose Bengal (RB) is a fluorescent dye with several potential biomedical applications, particularly in dermatology. Due to RB’s poor physicochemical properties, several advanced delivery systems have been developed as a potential tool to promote its permeation across the skin. Nevertheless, no validated quantitative method to analyse RB within the skin is described in the literature. Considering RB exhibits a conjugated ring system, the current investigation proposes fluorescence-based techniques beneficial for qualitatively and quantitatively determining RB delivered to the skin. Notably, the development and validation of a fluorescence-coupled HPLC method to quantify RB within the skin matrix are herein described for the first time. The method was validated based on the ICH, FDA and EMA guidelines, and the validated parameters included specificity, linearity, LOD, LLOQ, accuracy and precision, and carry-over and dilution integrity. Finally, the method was applied to evaluate RB’s ex vivo permeation and deposition profiles when loaded into dermatological formulations. Concerning qualitative determination, multiphoton microscopy was used to track the RB distribution within the skin strata, and fluorescence emission spectra were investigated to evaluate RB’s behaviour when interacting with different environments. The analytical method proved specific, precise, accurate and sensitive to analyse RB in the skin. In addition, qualitative side-analytical techniques were revealed to play an essential role in evaluating the performance of RB’s dermatological formulation

Quantification of Flavonoids, Naphthopyranones and Xanthones in Eriocaulaceae Species by LC-PDA

The linearity, stability, accuracy and inter-day precisions of the assay method were evaluated in methanolic aerial-part extracts of Paepalanthus giganteus and Syngontnhus nitens from the Eriocaulaceae family. Their small capitulae hinder morphological analysis, and thus complicate taxonomic studies of these species, which present anti-ulcer, antimutagenic and antioxidant activities. Taxonomic studies of these plants revealed that the Paepalanthus genus presents flavonols and naphthopyranones while the Syngontnhus genus has flavone and xanthone as majority compounds. The prepared samples were analyzed quantitatively by High Performance Liquid Chromatography with PDA detection for the presence of quercetin, luteolin, 3,6-dimethoxy-1,5,7-tri-hydroxyxanthone and paepalantine. The substances were recovered from these samples at rates from 98.01 to 99.99%. The coefficient of variation in the quantitative analysis of the sample com- pounds was under 5%. The linearity of the method was determined by linear regression. The analysis of the samples spiked with known amounts of analyte demonstrated that the response was proportional to the concentrations of the sam- ples with re-spective determination coefficients of r2 = 0.9999 (luteolin and 3,6-dimethoxy-1,5,7-tri-hydroxyxanthone) and r2 = 0.9998 (quercetin and paepalantine) for the linear range of the analytical calibration curves of the samples. The detection limits were 0.07 μg?mL–1 for quercetin and luteolin, 0.06 μg?mL–1 for 3,6-dimethoxy-1,5,7-tri-hydroxyxanthone and 0.10 μg?mL–1 for paepalantine. The quantification limits were 0.23 μg?mL–1 for quercetin and luteolin, 0.20 μg?mL–1 for 3,6-dimethoxy-1,5,7-tri-hydroxyxanthone and 0.33 μg?mL–1 for paepalantine by LC. The method was considered sen- sitive for quantification of the quercetin, luteolin, 3,6-dimethoxy-1,5,7-tri-hydroxyxanthone and paepalantine in plant samples.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq