Estudo das atividades biológicas in vitro da 5-(2,3-Dihidroxi-3-Metilbutilox)-6,7- Metilenedioxicumarina isolada de Pterocaulon lorentzii Malme E Pterocaulon alopecuroides DC. com ênfase na atividade anti-helmíntica em Nematoides gastrintestinais de ovinos

Orientador: Prof. Dr. Obdulio Gomes MiguelOrientadora: Profª Drª Marilis Dallarmi MiguelTese (doutorado) - Universidade Federal do Paraná, Setor de Ciências da Saúde, Programa de Pós-Graduação em Ciências Farmacêuticas. Defesa: Curitiba, 06/09/213Bibliografia: f. 95-108Área de concentração: Insumos, medicamentos e correlatosResumo: Infecções gastrintestinais causadas por nematoides constituem um dos principais fatores limitantes para a ovinocultura no Brasil e em todo o mundo. A saúde dos rebanhos depende de um controle antiparasitário eficiente e a utilização de plantas medicinais como alternativa fitoterápica na medicina veterinária vêm ganhando espaço e se mostrando próspera. Tendo em vista os aspectos sociais, econômicos e ambientais este trabalho tem por objetivo estudar numa abordagem multidisciplinar a efetividade dos extratos, frações e da 5-(2,3-dihidroxi-3-metilbutiloxi)-6,7-metilenedioxicumarina isolada das espécies Pterocaulon alopecuroides DC. e Pterocaulon lorentzii Malme. Como foco buscou-se a efetividade destas espécies em ensaios antiparasitários in vitro contra nematoides gastrintestinais de ovinos além dos seus potenciais antioxidantes e da toxicidade. As partes aéreas secas e estabilizadas de ambas espécies foram submetidas a extração com etanol e particionadas com solventes com gradiente de polaridade crescente hexano-clorofórmio-acetato de etila em aparelho de soxhlet modificado. A partir da cromatografia líquida em coluna da fração clorofórmio e elucidação estrutural por RMN 1H e 13C foi identificada a cumarina 5-(2,3-dihidroxi-3- metilbutiloxi)-6,7-metilenedioxicumarina. Os extratos brutos, as frações e a cumarina foram submetidos a bioensaios in vitro para avaliação de toxicidade, atividade antioxidante, e atividade anti-helmíntica. Na avaliação da ecotoxicidade frente ao microcrustáceo Artemia salina as amostras testadas apresentaram atividade apenas em concentrações superiores a 1000?g/mL, consideradas não tóxicas neste ensaio. Da mesma forma, na avaliação da atividade hemolítica, tanto no teste em sangue de carneiro como em ágar sangue os extratos, as frações e a cumarina não apresentaram atividade. O potencial antioxidante foi avaliado por três metodologias. No teste de inibição do complexo fosfomolibdênio destacou-se a fração acetato de etila de P. alopecuroides que apresentou 36,4% de atividade em relação à vitamina C e 137,7% em relação à rutina. A fração acetato de etila de P. lorentzii também apresentou maior atividade em relação às demais amostras testadas ARR(%) de 101,7% em relação à vitamina C. No método de reação com o radical DPPH as frações acetato de etila de ambas espécies mostraram as melhores atividades, EC50 10,74 ?g/mL para P. alopecuroides e EC50 7,63 ?g/mL para P. lorentzii, em relação ao controle vitamina C (EC50 2,48 ?g/mL) e rutina (EC50 9,43 ?g/mL). As demais amostras, extrato bruto, fração hexano, fração clorofórmio e cumarina não apresentaram atividade significativa. No teste de reação ao ácido tiobarbitúrico (TBARS) as amostras mais expressivas foram os extratos brutos das duas espécies em relação ao controle positivo BHT cujo IA% 0,169 ± 0,0087. O extrato bruto de P. alopecuroides apresentou IA% 0,419 ± 0,0517 e o extrato bruto de P. lorentzii IA% 0,213 ± 0,0094. Já a 5-(2,3-dihidroxi-3-metilbutiloxi)-6,7- metilenedioxicumarina apresentou IA% 0,462 ± 0,0184. Na avaliação da atividade antihelmíntica pelo método da eclodibilidade de ovos de nematoides a cumarina apresentou atividade muito expressiva na inibição do desenvolvimento e eclosão dos ovos com EC50 3,2 ?g/mL, seguido das frações clorofórmio 17,2 ?g/mL (P. alopecuroides) e 9,4 ?g/mL (P. lorentzii) enquanto que as demais frações e extratos apresentaram atividades menores. Da mesma forma a cumarina apresentou maior atividade na inibição da migração de larvas de nematóides em ágar com EC50 3,1 ?g/mL e as frações clorofórmio 104,1?g/mL (P. alopecuroides) e 64,7 ?g/mL (P. lorentzii). Os resultados obtidos sugerem a aplicação dos extratos e frações de P. alopecuroides e P. lorentzii e a 5-(2,3-dihidroxi-3-metilbutiloxi)-6,7-metilenedioxicumarina no desenvolvimento de preparações farmacêuticas de uso veterinário.Abstract: Gastrointestinal infection caused by nematodes are the main limiting factor for sheep breeding in Brazil and around the world. Livestock healthiness depends on an efficient anthelmintic control and the use of medicinal plants as an herbal alternative in veterinary medicine is gaining space and showing prosperous. Considering the social, economic and environmental scope this work aims to study in a multidisciplinary approach, the effectiveness of extracts, fractions and 5-(2,3-dihydroxy-3-methylbutyloxy)-6,7- methylenedioxicoumarin isolated from Pterocaulon alopecuroides DC. and Pterocaulon lorenztii Malme species. This study was focused on the effectiveness of these two species in vitro anthelmintic assays against sheep gastrointestinal nematodes as well as their antioxidant and toxicity potential. The dried and stabilized aerial parts of both species were subjected to extraction with ethanol and partitioned with solvents of increasing polarity gradient hexane-chloroform-ethyl acetate in modified soxhlet apparatus. From the liquid column chromatography of chloroform fraction an the structure elucidation by NMR 1H and NMR 13H the 5-(2,3-dihydroxy-3-methylbutyloxy)- 6,7-methylenedioxicoumarin was identified. The crude extracts, fractions and the coumarin were submitted to in vitro bioassays to evaluate toxicity, antioxidant activity, and anthelmintic activity. In the ecotoxicity assessment against Artemia salina tested samples showed activity only at concentrations over 1000?g/mL considered non-toxic in this assay. Likewise, in the hemolytic activity assay test in sheep blood and blood agar, extracts, fractions and the coumarin showed no activity. The scavenging activity was evaluated by three methods. In the phosphomolybdenium complex inhibition test the ethyl acetate fraction of P. alopecuroides showed 36.4% activity compared to vitamin C and 137.7% compared to rutin. The ethyl acetate fraction of P. lorentzii also showed higher activity in comparison to other samples tested with ARR (%) 101.7% compared to vitamin C. In DPPH reaction methods ethyl acetate fractions of both species showed the best activities, EC50 10.74 mg/mL for P. alopecuroides and EC50 7.63 mg/mL for P. lorentzii compared to the control Vitamin C (EC50 2.48g/mL) and rutin (EC50 9.43 g/mL). The remaining samples, crude extract, hexane fraction, chloroform fraction and the coumarin showed no significant activity. The crude extracts of the two species showed the most significant results in thiobarbituric acid reaction test (TBARS) in relation to the positive control BHT (AI 0.169 ± 0.0087%). The crude extract of P. alopecuroides presented IA 0.419% ± 0.0517 and the crude extract of P. lorentzii IA 0.213% ± 0.0094. The 5-(2,3-dihydroxy-3-methylbutyloxy)-6,7-methylenedioxicoumarin showed0020cIA 0.462% ± 0.0184. In the evaluation of anthelmintic activity by the egg hatch test (EHT) the coumarin showed very significant activity in inhibiting sheep nematoid egg development hatching with EC50 3.2 mg/mL, followed by chloroform fractions 17.2?g/mL (P. alopecuroides) and 9.4 mg/mL (P. lorentzii) while the other fractions and extracts showed lower activity. As well as in the EHT the same coumarin showed higher effectiveness in inhibiting nematode larvae migration in agar with a EC50 3.1 g/mL followed by the chloroform fractions 104.1g/mL (P. alopecuroides) and 64.7 mg/mL (P. lorentzii). The obtained results suggest the application of extracts and fractions of P. alopecuroides and P. lorentzii and 5-(2,3-dihydroxy-3-methylbutyloxy)-6,7- methylenedioxicoumarin in the development of pharmaceutical preparations for veterinary use

INTERAÇÕES PLANTA-MEDICAMENTO: IMPORTÂNCIA E MECANISMO DE AÇÃO

As plantas medicinais são amplamente utilizadas na cura de doenças. Ultimamente, com o avanço tecnológico, o interesse pelas plantas medicinais foi revigorado, uma vez que foram desenvolvidos métodos e equipamentos para a verificação da segurança e eficácia do seu uso. Entretanto, pesquisas sobre as possíveis interações planta-medicamento ainda não são expressivas a ponto de estarem presente na prática clínica. Muitos pacientes não relatam aos profissionais da saúde o uso de plantas medicinais. Persiste a crença que produtos oriundos de plantas não causam efeitos adversos. O objetivo deste trabalho é apresentar interações planta-medicamento relatadas em artigos científicos através de levantamento bibliográfico em bases de dados, contribuindo assim para a prática clínica

C-glycosyl flavones and a comparative study of the antioxidant, hemolytic and toxic potential of Jatropha multifida leaves and bark

The ethyl acetate extract from Jatropha multifida (Euphorbiaceae) leaves yielded two C-glycosyl flavones. Their structures were elucidated through spectroscopic methods, including UV, IR, 1D and 2D NMR, and compared with the related known compounds. The structures of the two flavonoids were determined as Vitexin (1) and Isovitexin (2). The ethanol extracts of leaves and bark and their fractions did not interfere in the integrity of erythrocytes, not even 1 and 2. In the Brine shrimp lethality method, bark extracts showed greater toxic potential than the leaf extracts. Both flavonoids are not toxic. The Phosphomolybdenum and DPPH assays were used in order to investigate the antioxidant activity of both compounds and fractions of leaf and bark extracts. The ethyl acetate fraction of bark showed excellent activity, with IC50 17.23 μg/mL-1, equivalent to the standard values, Vitamin C and Rutin. Compounds 1 - 2 demonstrated good activity with IC50 values of 54.37 and 87.27μg/mL-1.  In the Phosphomolybdenum test, the ethyl acetate fraction of bark showed 86.18% of antioxidant activity compared with Rutin, and the chloroform fraction of leaves, 103.29%. In all tests the bark extracts were more bioactive than the leaf extracts

C-glycosyl flavones and a comparative study of the antioxidant, hemolytic and toxic potential of Jatropha multifida leaves and bark

The ethyl acetate extract from Jatropha multifida (Euphorbiaceae) leaves yielded two C-glycosyl flavones. Their structures were elucidated through spectroscopic methods, including UV, IR, 1D and 2D NMR, and compared with the related known compounds. The structures of the two flavonoids were determined as Vitexin (1) and Isovitexin (2). The ethanol extracts of leaves and bark and their fractions did not interfere in the integrity of erythrocytes, not even 1 and 2. In the Brine shrimp lethality method, bark extracts showed greater toxic potential than the leaf extracts. Both flavonoids are not toxic. The Phosphomolybdenum and DPPH assays were used in order to investigate the antioxidant activity of both compounds and fractions of leaf and bark extracts. The ethyl acetate fraction of bark showed excellent activity, with IC50 17.23 μg/mL-1, equivalent to the standard values, Vitamin C and Rutin. Compounds 1 - 2 demonstrated good activity with IC50 values of 54.37 and 87.27μg/mL-1.  In the Phosphomolybdenum test, the ethyl acetate fraction of bark showed 86.18% of antioxidant activity compared with Rutin, and the chloroform fraction of leaves, 103.29%. In all tests the bark extracts were more bioactive than the leaf extracts

Phytochemical constituents and preliminary toxicity evaluation of leaves from Rourea induta Planch. (Connaraceae)

A maioria das plantas ativas é tóxica em doses elevadas, portanto, é importante a investigação da toxicidade preliminar dos extratos das plantas. A espécie Rourea induta é uma droga potencial que não apresenta estudo fitoquímico ou biológico descrito na literatura. Assim, um estudo fitoquímico e análises toxicológicas foram realizados com o extrato etanólico obtido das folhas de Rourea induta Planch., Connaraceae. Foram obtidos um hidrocarboneto de cadeia longa, n-tetracosano, e quatro flavonóides, quercetina e três derivados glicosilados, quercetina-3-O-α-arabinofuranosideo, quercetina-3-O-β-xilosideo e quercetina-3-O-β-galactosideo. Esta é a primeira vez que estes compostos são isolados nesta espécie. As estruturas foram elucidadas por espectroscopia de RMN-13C, RMN-¹H, ultravioleta e infravermelho. A avaliação da toxicidade dos extratos foi analisada pelo método da Artemia salina e atividade hemolítica. Nenhuma das amostras testadas apresentou um potencial tóxico pelos métodos analisados.Most active plants are toxic at high doses and it is therefore important to investigate the preliminary toxicity of plant extracts. The Rourea induta species is a potential drug with no phytochemical or biological studies registered in the literature. Thus, a phytochemical study and a toxicity analysis of the ethanolic extract obtained from the leaves of Rourea induta Planch., Connaraceae, was run. A long chain hydrocarbon, n-tetracosane, and four flavonoids were identified: quercetin, and three glycosylated derivates, quercetin-3-O-α-arabinofuranoside, quercetin-3-O-β-xyloside and quercetin-3-O-β-galactoside. This is the first time these have been isolated in this species. The structures were elucidated by 13C NMR, ¹H NMR, UV and IR spectroscopy. The toxicity evaluation of extracts was performed by the brine shrimp method and determination of hemolytic activity. The samples demonstrated no toxic potential by the analyzed methods

PHYTOTOXICITY AND ANTIOXIDANT ACTIVITY OF Jacaranda micrantha (Bignoniaceae) FLOWERS

Foi estudado o efeito fitotóxico, tóxico e antioxidante de frações semipurificadas das flores de Jacaranda micrantha (Binoniaceae). Na avaliação da atividade fitotóxica, foram realizados bioensaios de germinação e crescimento com a espécie-alvo Lactuca sativa. Para o ensaio de toxicidade preliminar utilizou- se o microcrustáceo Artemia salina, e para o teste de atividade antioxidante o método do complexo fosfomolibdênico. A fração clorofórmica apresentou atividade antioxidante de 30% em relação à vitamina C e 61% em relação à rutina. Os extratos utilizados não apresentaram toxicidade frente a Artemia salina, a inibição no crescimento do hipocótilo e da radícula de Lactuca sativa pela fração hexânica e fração remanescente evidencia que estas apresentam potencial para serem utilizadas como modelo de herbicidas, uma vez que não causam toxicidade em organismos vivos

Analgesic, Anti-Inflammatory, and Antioxidant Activities of Byrsonima duckeana

Background. Byrsonima is a promising neotropical genus, rich in flavonoids and triterpenes, with several proven pharmacological properties. Nevertheless, Byrsonima duckeana W. R. Anderson is an Amazonian species almost not studied. Objective. To assess the antioxidant, anti-inflammatory, and analgesic activities of Byrsonima duckeana leaves. Materials and Methods. We analyzed an ethanol extract and its fractions for polyphenol content and UHPLC-MS/MS, phosphomolybdenum, DPPH, TBARS antioxidant tests, formalin-induced pain, carrageenan-induced peritonitis, acetic acid-induced abdominal writhings, and hot plate assays. Results. All the samples showed high polyphenol content and antioxidant capacity in the phosphomolybdenum, DPPH, and TBARS tests. We identified ethyl gallate, quinic acid, gallic acid, catechin, epicatechin, quercetrin, and quercetin in the samples. B. duckeana was able to reduce leukocyte migration in the carrageenan-induced peritonitis by 43% and the licking time in the formalin test by 57%. In the acetic acid-induced writhing test, the chloroform (FCL) and ethyl acetate (FEA) fractions were the most active samples. FEA was selected for the hot plate test, where all the dosages tested (5, 50, and 200 mg·kg−1) showed significant analgesic activity. Conclusion. B. duckeana has interesting analgesic and antioxidant activities, due to its high phenolic content, especially phenolic acids

Coumarin isolation and comparative study of biological activities of Pterocaulon alopecuroides DC and Pterocaulon lorentzii Malme

5-(2,3-Dihidroxy-3-methylbuthyloxy)-6,7-methylenedioxycoumarin was isolated from the chloroform extract of the two Asteraceae species Pterocaulon alopecuroides DC. and Pterocaulon lorentzii Malme. The structure was elucidated through IR and 1H and 13C  NMR analyses. The extracts and the isolated compound did not exhibit toxic activity, as determined through the brine shrimp lethality method, and did not interfere with the integrity of erythrocytes, as demonstrated through a hemolytic assay. The antioxidant activities were investigated through three methods. In the phosphomolybdenum test, the ethyl acetate fraction of P. alopecuroides exhibited an antioxidant activity of 137.7% compared with rutin (positive control), and the ethyl acetate fraction of P. lorentzii exhibited an antioxidant activity of 101.7% compared with vitamin C (positive control). The two ethyl acetate fraction also exhibited excellent activity through the DPPH assay: P. alopecuroides and P. lorentzii exhibited IC50 values of 10.74 μg/ml and 7.63 μg/ml, respectively. In the TBARS bioassay, the crude extracts showed the more significant results: IA% 0.419 ± 0.0517 for P. alopecuroides and IA% 0.213 ± 0.0094 for P. lorentzii

Phytochemical constituents and preliminary toxicity evaluation of leaves from Rourea induta Planch. (Connaraceae)

Most active plants are toxic at high doses and it is therefore important to investigate the preliminary toxicity of plant extracts. The Rourea induta species is a potential drug with no phytochemical or biological studies registered in the literature. Thus, a phytochemical study and a toxicity analysis of the ethanolic extract obtained from the leaves of Rourea induta Planch., Connaraceae, was run. A long chain hydrocarbon, n-tetracosane, and four flavonoids were identified: quercetin, and three glycosylated derivates, quercetin-3-O-α-arabinofuranoside, quercetin-3-O-β-xyloside and quercetin-3-O-β-galactoside. This is the first time these have been isolated in this species. The structures were elucidated by 13C NMR, ¹H NMR, UV and IR spectroscopy. The toxicity evaluation of extracts was performed by the brine shrimp method and determination of hemolytic activity. The samples demonstrated no toxic potential by the analyzed methods

Empagliflozin in Patients with Chronic Kidney Disease

Background The effects of empagliflozin in patients with chronic kidney disease who are at risk for disease progression are not well understood. The EMPA-KIDNEY trial was designed to assess the effects of treatment with empagliflozin in a broad range of such patients. Methods We enrolled patients with chronic kidney disease who had an estimated glomerular filtration rate (eGFR) of at least 20 but less than 45 ml per minute per 1.73 m(2) of body-surface area, or who had an eGFR of at least 45 but less than 90 ml per minute per 1.73 m(2) with a urinary albumin-to-creatinine ratio (with albumin measured in milligrams and creatinine measured in grams) of at least 200. Patients were randomly assigned to receive empagliflozin (10 mg once daily) or matching placebo. The primary outcome was a composite of progression of kidney disease (defined as end-stage kidney disease, a sustained decrease in eGFR to < 10 ml per minute per 1.73 m(2), a sustained decrease in eGFR of & GE;40% from baseline, or death from renal causes) or death from cardiovascular causes. Results A total of 6609 patients underwent randomization. During a median of 2.0 years of follow-up, progression of kidney disease or death from cardiovascular causes occurred in 432 of 3304 patients (13.1%) in the empagliflozin group and in 558 of 3305 patients (16.9%) in the placebo group (hazard ratio, 0.72; 95% confidence interval [CI], 0.64 to 0.82; P < 0.001). Results were consistent among patients with or without diabetes and across subgroups defined according to eGFR ranges. The rate of hospitalization from any cause was lower in the empagliflozin group than in the placebo group (hazard ratio, 0.86; 95% CI, 0.78 to 0.95; P=0.003), but there were no significant between-group differences with respect to the composite outcome of hospitalization for heart failure or death from cardiovascular causes (which occurred in 4.0% in the empagliflozin group and 4.6% in the placebo group) or death from any cause (in 4.5% and 5.1%, respectively). The rates of serious adverse events were similar in the two groups. Conclusions Among a wide range of patients with chronic kidney disease who were at risk for disease progression, empagliflozin therapy led to a lower risk of progression of kidney disease or death from cardiovascular causes than placebo