DETECTION AND QUANTIFICATION OF PHYTOCHEMICAL MARKERS OF Ilex paraguariensis BY LIQUID CHROMATOGRAPHY

Ilex paraguariensis (yerba-mate) is used as a beverage, and its extract requires adequate quality control methods in order to guarantee quality and safe use. Strategies to develop and optimize a chromatographic method to quantify theobromine, caffeine, and chlorogenic acid in I. paraguariensis extracts were evaluated by applying a quality by design (QbD) model and ultra high-performance liquid chromatography (UHPLC). The presence of these three phytochemical markers in the extracts was evaluated using UHPLC-MS and was confirmed by the chromatographic bands in the total ion current traces (m/z of 181.1 [M+H]+, 195.0 [M+H]+, and 353.0 [M−H]−, respectively). The developed method was then transferred to a high-performance liquid chromatography (HPLC) platform, and the three phytochemical markers were used as external standards in the validation of a method for analyses of these compounds in extracts using a diode array detector (DAD). The validated method was applied to quantify the chlorogenic acid, caffeine, and theobromine in the samples. HPLC-DAD chromatographic fingerprinting was also used in a multivariate approach to process the entire data and to separate the I. paraguariensis extracts into two groups. The developed method is very useful for qualifying and quantifying I. paraguariensis extracts.

Flora arbórea da bacia do rio Tibagi (Paraná, Brasil): Celastrales sensu Cronquist

A study of the tree species of the order Celastrales sensu Cronquist from the Tibagi river basin, Paraná state, Brazil, is presented, based on herbarium material. This basin is subdivided into three zones, from north to south: lower Tibagi (BT), mid Tibagi (MT) and upper Tibagi (AT), each with different environmental conditions and vegetation types. The order Celastrales is represented in the basin by 15 tree species belonging to three families: Aquifoliaceae, Celastraceae and Icacinaceae. Icacinaceae has only two species, Citronella gongonha and C. paniculata. The former is distinguished by a glabrous ovary and leaves that usually bear thorns. Aquifoliaceae has six species: Ilex brasiliensis, I. brevicuspis, I. chamaedryfolia, I. dumosa, I. paraguariensis and I. theezans. These species are found mainly in AT and MT and are distinguished by leaf size, indument, apices and margins, and by sepal features. Celastrales is represented by seven species and two genera; Plenckia populnea, a Brazilian savannah species found only in MT, and six species of Maytenus (M. evonymoides, M. robusta, M. dasyclada, M. salicifolia, M. ilicifolia and M. aquifolia) distinguished by leaf size and margins, branch shape and number of flowers per inflorescence.

Influência da floresta alterada na distribuição espacial de três espécies da Floresta Ombrófila Mista avaliada pela geoestatística

Os objetivos deste trabalho foram analisar e representar a distribuição espacial dos indivíduos de três espécies da Floresta Ombrófila Mista (FOM), sob distúrbio ocasionado por ações antrópicas, utilizando geoestatística e interpolações por krigagem, bem como verificar a influência das alterações da floresta no resultado obtido com essa metodologia. Os dados foram obtidos de levantamento fitossociológico realizado em 2007, contendo 45 unidades amostrais (ua) de 500 m², em que foram mensurados indivíduos com DAP > 10 cm. O fragmento, localizado em General Carneiro, PR, passou por ciclos de exploração de madeira, erva-mate, xaxim, pastoreio e coleta de sementes, que causaram abertura de grandes clareiras, contendo alta densidade de taquaras. Posteriormente, a área foi transformada em Reserva Particular do Patrimônio Natural. A condição de floresta alterada interferiu nos resultados para distribuição espacial dos indivíduos de D. sellowiana, M. scabrella e I. paraguariensis (xaxim, bracatinga e erva-mate) por ua. Apesar de ser detectada alta dependência espacial (>75%), foi também observada quebra da continuidade espacial para distribuição dessas espécies. Conclui-se que, nessa condição, geoestatística e krigagem não são indicadas devido aos usos inadequados dado à floresta, podendo ser utilizadas outras técnicas de interpolação para evitar as extrapolações de médias obtidas na amostragem da área total do fragmento. No entanto, por apresentar sensibilidade às alterações das florestas, análises geoestatísticas devem ser experimentadas quanto à sua utilização na avaliação da capacidade de resposta da floresta a eventos impactantes de grande magnitude.

Acute ingestion of yerba mate infusion (Ilex paraguariensis) inhibits plasma and lipoprotein oxidation

Yerba mate extract (Ilex paraguariensis) is a Source of phenolic compounds that possesses in vitro antioxidant activities and may contribute to a reduction in the risk of cardiovascular disease. In this Study we examined the acute effects of the consumption of mate infusion on ex vivo plasma and low-density lipoprotein (LDL) oxidation, plasma antioxidant capacity, and platelet aggregation. Twelve healthy fasted subjects ingested 500 mL. of mate infusion and blood samples were collected before and I h after mate intake. Lipid peroxidation of plasma and LDL was monitored by the measurement of cholesteryl-ester hydroperoxides (CE-OOH) and cholesterol oxides. The plasma antioxidant capacity was measured as ferric-reducing antioxidant potential (FRAP). Platelet aggregation was evaluated in platelet-rich plasma Stimulated with adenosine diphosphate and coagulation was tested in platelet-poor plasma. Ingestion of mate infusion diminished the ex vivo oxidizability of both plasma and LDL particles. After mate intake, the CE-OOH levels were around 50% lower in plasma oxidized with copper or 2,2`-azobis[-2-amidine-propane-hydrochloride] (AAPH) and the lag time to plasma oxidation increased 2-fold (P < 0.05). Copper- and AAPH-induced LDL peroxidation were also inhibited by around 50% and 20%, respectively, after mate Consumption (P < 0.05). The levels of various oxysterols were significantly reduced in oxidized-plasma and LDL (P < 0.05) and FRAP increased by 7.7% after mate intake (P < 0.01). However. mate consumption did not inhibit platelet aggregation or blood coagulation. In summary, intake of yerba mate infusion improved the antioxidant capacity and the resistance of plasma and LDL particles to ex vivo lipid peroxidation. (C) 2008 Elsevier Ltd. All rights reserved.

Effect of potassium fertilization on yield and nutrition of yerba mate (Ilex paraguariensis)

Yerba mate (Ilex paraguariensis) is a tree species native to the subtropical regions of South America, and is found in Brazil predominantly in the southern region. Despite the historical importance in this region, so far, studies on crop nutrition to improve yields are scarce. Thus, this study evaluated the effect of potassium rates on K soil availability, and the yield and nutritional status of yerba mate. The experiment was conducted in São Mateus do Sul, State of Paraná, on a Humox soil, where K2O rates of 0, 20, 40, 80, 160, and 320 kg ha-1 were tested on 7-year-old plantations. The experiment was harvested 24 months after installation by removing approximately 95 % of the canopy that had sprouted from the previous harvest. The soil was evaluated for K availability in the layers 0-10, 0-20, 10-20, and 20-40 cm. The plant parts leaf fresh matter (LM), twigs (TW), thick branches (BR) and commercial yerba mate (COYM), i.e., LM+TW, were analyzed. In addition, the relationship between fresh matter/dry matter (FM/DM) and K concentration in LM, AG and BR were evaluated. The fertilization increased K availability in all evaluated soil layers, indicating good mobility of the nutrient even at low rates. Yerba mate responded positively to increasing K2O rates with higher yields of all harvested components. The crop proved K-demanding, with a maximum COYM yield of 28.5 t ha-1, when 72 mg dm-3 K was available in the 0-20 cm layer. Yerba mate in the plant production stage requires soil K availability at medium to high level; in clayey soil with low K availability, a rate of 300 kg ha-1 K2O should be applied at 24 month intervals to obtain high yields. A leaf K concentration of 16.0 g ha-1 is suitable for yerba mate in the growth stage.

Plantae Paraguariensis

Se enumeran 50 especies fanerógamas colectadas en Paraguay. Junto a numerosas citas de desigual interés corológico, se describe una especie nueva: Erythroxylum perpusillum Fdez. Casas.

Estudo comparativo de dellineamentos experimentais para estimativas de parâmetros genéticos em erva-mate (Ilex paraguariensis A. St. - Hil.)

O presente trabalho teve por objetivo comparar a eficiência do látice em relação aos delineamentos em blocos casualizados (DBC) e inteiramente casualizados (DIC) quanto aos caracteres altura e produção de massa verde em erva-mate (Ilex paraguariensis A. St. - Hil.). O experimento, conduzido na Fazenda Experimental do Canguiri, da Universidade Federal do Paraná, em Pinhais, PR, foi composto de cinco procedências, quatro com 13 e uma com 12 progênies de meias-irmãs. Cada progênie continha 54 plantas (nove parcelas de seis plantas). O experimento seguiu o modelo látice 8 x 8 com nove repetições balanceadas. As análises foram conduzidas usando-se tanto este modelo quanto os modelos DBC e DIC. Com relação ao caráter altura, a estimativa da eficiência do DBC em relação ao DIC (Êb/i) foi de 48,6%; do látice em relação ao DBC (Êl/b), de 61%; e do látice em relação ao DIC (Êl/i), de 139%. A estimativa da correlação intraclasse entre plantas dentro das parcelas (c²) foi de 9,4% para o DBC e zero para o látice. Para o caráter produção de massa verde, Êb/i, Êl/b e Êl/i foram de 14,3; 81,7; e 108%, respectivamente; e c², 16,9% para o DBC e 3,1% para o látice. Essas estimativas, associadas a uma estimativa de F de Snedecor altamente significativa dos dois caracteres, permitiram concluir que a capacidade de teste do DBC foi satisfatória para o caráter altura de plantas, mas insatisfatória para o caráter produção de massa verde.

Caracterização química da erva mate (Ilex paraguariensis) : aplicação de diferentes processos de extração e influência das condições de plantio sobre a composição química

O objetivo deste trabalho foi estudar a composição química das folhas de erva-mate, sob diferentes condições agronômicas e técnicas de extração. Os métodos de extração usados foram maceração, ultra-som, extração com líquido pressurizado e extração com fluído supercrítico. Foram investigadas as variáveis que podem influenciar no processo de extração, tais como temperatura, pressão, polaridade do solvente, tempo de extração, massa de amostra, entre outras. A identificação dos compostos foi realizada por cromatografia gasosa acoplada à espectrometria de massas. Todos os métodos de extração utilizados mostraram-se eficientes para a obtenção dos extratos, com as diferenças sendo mais quantitativas do que qualitativas . Entre os métodos de extração que utilizam solventes orgânicos, a extração com líquido pressurizado mostrou-se mais eficiente, produzindo maior rendimento em massa de extrato e maior concentração de alguns dos compostos de interesse, com as vantagens de redução de solvente e tempo de extração. A composição química da erva-mate é influenciada pelas condições agronômicas de plantio, bem como pelas condições de extração de suas folhas. A melhor condição agronômica avaliada, ou seja, aquela que produziu maior quantidade de extrato, foi o cultivo das plantas a pleno sol, adubadas com nitrogênio e com idade de poda de 18 meses. A variável mais importante das técnicas de extração utilizadas foi a polaridade do solvente. Solventes de maior polaridade produziram maior rendimento em extrato. A análise cromatográfica dos extratos obtidos permitiu identificar cerca de 50 compostos qualitativamente e 6 quantitativamente, destacando-se a cafeína, fitol, ácido palmítico, ácido esteárico, esqualeno e vitamina E.

Efeitos da temperatura e do mate (Ilex paraguariensis) sobre o processo de carcinogênese de esôfago em ratos wistar machos

Pós-graduação em Biologia Geral e Aplicada - IBB

Indirect and expedite assessment of Ilex paraguariensis commercial yield.

A produção de biomassa comercial é uns dos principais indicadores para seleção de progênies e clones de erva-mate. Técnicas tradicionais para se obter tais informações dependem da colheita das árvores e apresentam, dentre outras limitações, elevado custo e reduzida praticidade. Assim, objetivou-se avaliar a eficiência de métodos indiretos, por meio de estimativa de biomassa comercial e escore de produtividade em função de diferentes procedências, sexo e morfotipos. Em um teste de progênies e procedências instalado em 1997, foram avaliadas em agosto de 2015 (dois anos após a última colheita) duas metodologias de análise visual. Para tanto, participaram cinco avaliadores treinados que determinaram, para cada planta, uma estimativa de biomassa comercial (kg), e uma nota, com base em um escore de produtividade (0-10). Para avaliação da produtividade pelas técnicas tradicionais, todas as plantas foram podadas e tiveram sua biomassa comercial (folhas e ramos finos menores de 7 mm de diâmetro) colhida e avaliada por meio de pesagem (Kg.planta-1). As avaliações foram realizadas em experimento instalado em blocos ao acaso, com cinco repetições, sete procedências e 126 progênies, totalizando 5292 plantas avaliadas. Os métodos avaliados foram eficientes na estimativa da biomassa comercial. Os avaliadores apresentaram boa acuidade nas estimativas, expressando de forma eficiente a maior produtividade determinada por comparação de médias entre as procedências, sexo das matrizes e morfotipos. As maiores correlações foram verificadas na análise geral das médias e a estimativa de biomassa comercial é a melhor metodologia para estimar a biomassa comercial aferida em plantas adultas de erva-mate.

An Isoflavone From Dipteryx Alata Vogel Is Active Against The In Vitro Neuromuscular Paralysis Of Bothrops Jararacussu Snake Venom And Bothropstoxin I, And Prevents Venom-induced Myonecrosis.

Snakebite is a neglected disease and serious health problem in Brazil, with most bites being caused by snakes of the genus Bothrops. Although serum therapy is the primary treatment for systemic envenomation, it is generally ineffective in neutralizing the local effects of these venoms. In this work, we examined the ability of 7,8,3'-trihydroxy-4'-methoxyisoflavone (TM), an isoflavone from Dipteryx alata, to neutralize the neurotoxicity (in mouse phrenic nerve-diaphragm preparations) and myotoxicity (assessed by light microscopy) of Bothrops jararacussu snake venom in vitro. The toxicity of TM was assessed using the Salmonella microsome assay (Ames test). Incubation with TM alone (200 μg/mL) did not alter the muscle twitch tension whereas incubation with venom (40 μg/mL) caused irreversible paralysis. Preincubation of TM (200 μg/mL) with venom attenuated the venom-induced neuromuscular blockade by 84% ± 5% (mean ± SEM; n = 4). The neuromuscular blockade caused by bothropstoxin-I (BthTX-I), the major myotoxic PLA2 of this venom, was also attenuated by TM. Histological analysis of diaphragm muscle incubated with TM showed that most fibers were preserved (only 9.2% ± 1.7% were damaged; n = 4) compared to venom alone (50.3% ± 5.4% of fibers damaged; n = 3), and preincubation of TM with venom significantly attenuated the venom-induced damage (only 17% ± 3.4% of fibers damaged; n = 3; p < 0.05 compared to venom alone). TM showed no mutagenicity in the Ames test using Salmonella strains TA98 and TA97a with (+S9) and without (-S9) metabolic activation. These findings indicate that TM is a potentially useful compound for antagonizing the neuromuscular effects (neurotoxicity and myotoxicity) of B. jararacussu venom.

Nebivolol Reduces Central Blood Pressure In Stage I Hypertensive Patients: Experimental Single Cohort Study.

Assessment of central blood pressure (BP) has grown substantially over recent years because evidence has shown that central BP is more relevant to cardiovascular outcomes than peripheral BP. Thus, different classes of antihypertensive drugs have different effects on central BP despite similar reductions in brachial BP. The aim of this study was to investigate the effect of nebivolol, a β-blocker with vasodilator properties, on the biochemical and hemodynamic parameters of hypertensive patients. Experimental single cohort study conducted in the outpatient clinic of a university hospital. Twenty-six patients were recruited. All of them underwent biochemical and hemodynamic evaluation (BP, heart rate (HR), central BP and augmentation index) before and after 3 months of using nebivolol. 88.5% of the patients were male; their mean age was 49.7 ± 9.3 years and most of them were overweight (29.6 ± 3.1 kg/m2) with large abdominal waist (102.1 ± 7.2 cm). There were significant decreases in peripheral systolic BP (P = 0.0020), diastolic BP (P = 0.0049), HR (P < 0.0001) and central BP (129.9 ± 12.3 versus 122.3 ± 10.3 mmHg; P = 0.0083) after treatment, in comparison with the baseline values. There was no statistical difference in the augmentation index or in the biochemical parameters, from before to after the treatment. Nebivolol use seems to be associated with significant reduction of central BP in stage I hypertensive patients, in addition to reductions in brachial systolic and diastolic BP.

Predictive Value Of Phase I Trials For Safety In Later Trials And Final Approved Dose: Analysis Of 61 Approved Cancer Drugs.

Phase I trials use a small number of patients to define a maximum tolerated dose (MTD) and the safety of new agents. We compared data from phase I and registration trials to determine whether early trials predicted later safety and final dose. We searched the U.S. Food and Drug Administration (FDA) website for drugs approved in nonpediatric cancers (January 1990-October 2012). The recommended phase II dose (R2PD) and toxicities from phase I were compared with doses and safety in later trials. In 62 of 85 (73%) matched trials, the dose from the later trial was within 20% of the RP2D. In a multivariable analysis, phase I trials of targeted agents were less predictive of the final approved dose (OR, 0.2 for adopting ± 20% of the RP2D for targeted vs. other classes; P = 0.025). Of the 530 clinically relevant toxicities in later trials, 70% (n = 374) were described in phase I. A significant relationship (P = 0.0032) between increasing the number of patients in phase I (up to 60) and the ability to describe future clinically relevant toxicities was observed. Among 28,505 patients in later trials, the death rate that was related to drug was 1.41%. In conclusion, dosing based on phase I trials was associated with a low toxicity-related death rate in later trials. The ability to predict relevant toxicities correlates with the number of patients on the initial phase I trial. The final dose approved was within 20% of the RP2D in 73% of assessed trials.

Pyrimidine-5'-nucleotidase Campinas, A New Mutation (p.r56g) In The Nt5c3 Gene Associated With Pyrimidine-5'-nucleotidase Type I Deficiency And Influence Of Gilbert's Syndrome On Clinical Expression.

Pyrimidine-5'-nucleotidase type I (P5'NI) deficiency is an autosomal recessive condition that causes nonspherocytic hemolytic anemia, characterized by marked basophilic stippling and pyrimidine nucleotide accumulation in erythrocytes. We herein present two African descendant patients, father and daughter, with P5'N deficiency, both born from first cousins. Investigation of the promoter polymorphism of the uridine diphospho glucuronosyl transferase 1A (UGT1A) gene revealed that the father was homozygous for the allele (TA7) and the daughter heterozygous (TA6/TA7). P5'NI gene (NT5C3) gene sequencing revealed a further change in homozygosity at amino acid position 56 (p.R56G), located in a highly conserved region. Both patients developed gallstones; however the father, who had undergone surgery for the removal of stones, had extremely severe intrahepatic cholestasis and, liver biopsy revealed fibrosis and siderosis grade III, leading us to believe that the homozygosity of the UGT1A polymorphism was responsible for the more severe clinical features in the father. Moreover, our results show how the clinical expression of hemolytic anemia is influenced by epistatic factors and we describe a new mutation in the P5'N gene associated with enzyme deficiency, iron overload, and severe gallstone formation. To our knowledge, this is the first description of P5'N deficiency in South Americans.

Effects Of Partial Inhibition Of Respiratory Complex I On H2o 2 Production By Isolated Brain Mitochondria In Different Respiratory States.

The aim of this work was to characterize the effects of partial inhibition of respiratory complex I by rotenone on H2O2 production by isolated rat brain mitochondria in different respiratory states. Flow cytometric analysis of membrane potential in isolated mitochondria indicated that rotenone leads to uniform respiratory inhibition when added to a suspension of mitochondria. When mitochondria were incubated in the presence of a low concentration of rotenone (10 nm) and NADH-linked substrates, oxygen consumption was reduced from 45.9 ± 1.0 to 26.4 ± 2.6 nmol O2 mg(-1) min(-1) and from 7.8 ± 0.3 to 6.3 ± 0.3 nmol O2 mg(-1) min(-1) in respiratory states 3 (ADP-stimulated respiration) and 4 (resting respiration), respectively. Under these conditions, mitochondrial H2O2 production was stimulated from 12.2 ± 1.1 to 21.0 ± 1.2 pmol H2O2 mg(-1) min(-1) and 56.5 ± 4.7 to 95.0 ± 11.1 pmol H2O2 mg(-1) min(-1) in respiratory states 3 and 4, respectively. Similar results were observed when comparing mitochondrial preparations enriched with synaptic or nonsynaptic mitochondria or when 1-methyl-4-phenylpyridinium ion (MPP(+)) was used as a respiratory complex I inhibitor. Rotenone-stimulated H2O2 production in respiratory states 3 and 4 was associated with a high reduction state of endogenous nicotinamide nucleotides. In succinate-supported mitochondrial respiration, where most of the mitochondrial H2O2 production relies on electron backflow from complex II to complex I, low rotenone concentrations inhibited H2O2 production. Rotenone had no effect on mitochondrial elimination of micromolar concentrations of H2O2. The present results support the conclusion that partial complex I inhibition may result in mitochondrial energy crisis and oxidative stress, the former being predominant under oxidative phosphorylation and the latter under resting respiration conditions.