Stability of Leaf Yerba Mate (Ilex paraguariensis) Metabolite Concentrations over the Time from the Prism of Secondary Sexual Dimorphism.

The yerba mate leaf metabolic composition depends mainly on genetics, sex, plant and leaf age, light intensity, harvest time, climate, and fertilization. In yerba mate, the secondary sexual dimorphism (SSD), the leaf metabolic SSD association with the frequency of leaf harvests, and the stability of the metabolites in the two genders over the years is not known. It was hypothesized that (1) the SSD in the metabolite segregation would differ among the winter and summer growth pauses, (2) females would show lower metabolite concentrations, and (3) the metabolic concentrations would show stability over the years on the same plants, not obligatorily associated with the SSD stability expression. Variations in theobromine, caffeine, chlorogenic and caffeic acids were correlated to the increasing time since the previous harvest, especially in females. However, the frequency of the metabolic SSD were associated with the studied growth pauses, rejecting the first hypothesis. No regular gender superiority was expressed in the yerba mate leaf secondary metabolites, rejecting our second hypothesis, even though more cases of superior female metabolite accumulation were identified. The stability of the leaf protein was preserved over the four years, with no SSD cases observed. The leaf methylxanthines were time stable, while the decrease in the phenolic content occurred with tree aging, which was not associated with the SSD expression, partially proving our third hypothesis. The novelty was related to the time stability of the leaf metabolic SSD observed over the winter and summer growth pauses, and over the four consecutive years without a regular expression of the male- or female-biased concentrations in the studied metabolites. To demystify the random metabolic gender responses in yerba mate, gender-orientated experiments with a high number of tree repetitions must be conducted, including clonal plants grown in various environments, such as monoculture and agroforestry, or on plantations in different climates and altitudes.

Multigenomic modifications in human circulating immune cells in response to consumption of polyphenol rich extract of yerba mate (Ilex paraguariensis A. St.-Hil.) are suggestive of cardiometabolic protective effects.

Mate is a traditional drink obtained from the leaves of yerba mate and rich in a diversity of plant bioactive compounds including polyphenols, particularly chlorogenic acids. Studies, even though limited, suggest that consumption of mate is associated with health effects, including prevention of cardiometabolic disorders. Molecular mechanisms underlying the potential health properties are still largely unknown, especially in humans. The aim of this study was to investigate nutrigenomic effects of mate consumption and identify regulatory networks potentially mediating cardiometabolic health benefits. Healthy middle-aged men at risk for cardiovascular disease consumed a standardized mate extract or placebo for 4 weeks. Global gene expression, including protein coding and non-coding RNAs profiles were determined using microarrays. Biological function analyses were performed using integrated bioinformatic tools. Comparison of global gene expression profiles showed significant change following mate consumption with 2635 significantly differentially expressed genes, among which 6 are miRNAs and 244 are lncRNAs. Functional analyses showed that these genes are involved in regulation of cell interactions and motility, inflammation or cell signaling. Transcription factors, such as MEF2A, MYB or HNF1A, could have their activity modulated by mate consumption either by direct interaction with polyphenol metabolites or by interactions of metabolites with cell signaling proteins, like p38 or ERK1/2, that could modulate transcription factor activity and regulate expression of genes observed. Correlation analysis suggests that expression profile is inversely associated with gene expression profiles of patients with cardiometabolic disorders. Therefore, mate consumption may exert cardiometabolic protective effects by modulating gene expression towards a protective profile.

Evaluation of Reproductive Toxicology of Aqueous Extract of Yerba Mate (Ilex paraguariensis A. St.-Hil.), a Traditional South American Beverage.

Yerba mate (Ilex paraguariensis A. St.-Hil.; Aquifoliaceae) is a popular tonic and stimulant beverage that is widely consumed in different South American countries. Estimates indicate the consumption of >1 L per day in southern Brazil and Uruguay. Despite its relatively high consumption, data on reproductive toxicity during critical periods of gestation remain unclear. Thus, we evaluated the effects of an aqueous extract of I. paraguariensis leaves ("chimarrão" [IPC]) at two critical periods of gestation in Wistar rats: preimplantation embryonic stage and fetal organogenesis. Pregnant Wistar rats were orally treated with IPC (3, 30, and 300 mg/kg) from days 1 to 7 or 8 to 21 of pregnancy. The respective control groups received vehicle. During treatment, clinical signs of maternal toxicity, maternal body weight, and food and water intake were monitored. The rats were killed on days 8 and 20 of pregnancy, and the following parameters were evaluated: weight of the maternal uterus, weight of the liver, weight of the kidneys, weight of the spleen, total embryo implantation, preimplantation loss, the mean of live fetuses, the percentage of dead fetuses, fetus weight, and fetal malformation. The aqueous extract of the leaves of I. paraguariensis L. did not present any deleterious effects on preimplantation embryos or the organogenesis of offspring from female Wistar rats. These safety data provide evidence that IPC may be safe for consumption during gestation.

90-Day Oral Toxicity Assessment of Tropaeolum majus L. in Rodents and Lagomorphs.

Tropaeolum majus L., popularly known as nasturtium, is a species widely used in the form of infusions and salads. In the last years, the antihypertensive, diuretic, and calcium and potassium sparing activities of T. majus preparations were shown. Moreover, no preclinical 90-day oral toxicity studies were conducted. Thus, this study evaluated the toxicity of the hydroethanolic extract obtained from T. majus (HETM) leaves in female and male mice, rats, and rabbits. Swiss mice and Wistar rats were treated with HETM (75, 375, and 750 mg/kg). The doses of rabbits (30, 150, and 300 mg/kg) were calculated by allometric extrapolation. The control groups received vehicle. The animals were orally treated, daily, for 90 days. At the end, the animals were anesthetized, and body weight gain, relative weight of liver, kidney, and spleen, and histopathological changes were evaluated. Serum hematological and biochemical parameters were also analyzed. No alterations were found in body and organ weights or in histopathological and biochemical evaluation. Hematological analyses revealed small changes in lymphocytes and neutrophil counts in rats after administration of 750 mg/kg of HETM. These results showed that 90-day use of T. majus is safe in rodents and lagomorphs.

Lipid-lowering and antiatherogenic effects of Vitex megapotamica (Spreng.) Moldenke in a mice experimental model.

ETHNOPHARMACOLOGICAL RELEVANCE: Vitex megapotamica (Spreng.) Moldenke is a deciduous tree, native of South America. Its leaves are traditionally used to treat cardiovascular diseases. This activity is related to the presence of flavonoids, the major compounds of the crude extract. AIM OF THE STUDY: This study investigated the effects of the oral administration of crude extract and standardized fractions from V. megapotamica leaves on lipid profile and on the formation of atherosclerotic plaque in C57BL/6 LDLr-KO mice treated with high-fat diet (HFD). MATERIALS AND METHODS: Male C57BL/6 LDLr-KO mice were fed with HFD (cholesterol, 1.25%) for 30 days. They were treated with hydroethanolic extract (500 or 1000mg/kg/day) or fractions (125 or 250mg/kg/day). After 30 days of treatment, it was evaluated the serum lipid profile, atherogenic index, and atherosclerotic plaque. RESULTS: All doses of the hydroethanolic extract reduced significantly the levels of total cholesterol, triglycerides, LDL-c and the atherogenic index. The n-butanolic fraction also reduced significantly the levels of total cholesterol, triglycerides, LDL-c and the atherogenic index, at all doses, with exception for the triglycerides, which only the lower dose was effective. The residual fraction reduced significantly the levels of total cholesterol, LDL-c and the atherogenic index, at all doses, with exception for the atherogenic index, which only the higher dose was effective. The atherosclerotic plaque formation was impaired only by the lower dose of the hydroethanolic extract. CONCLUSIONS: Overall, our data suggest that V. megapotamica has potential for the treatment of dyslipidemias.

Daily Intake of Chlorogenic Acids from Consumption of Maté (Ilex paraguariensis A.St.-Hil.) Traditional Beverages.

The main objective of this study was to investigate the daily intake of chlorogenic acids (CGAs) and methylxanthines by consumers of maté traditional beverages (MTBs), terere and chimarrão (Ilex paraguariensis A.St.-Hill). In the studied population (450 citizens from Toledo, PR, Brazil), 63% consume the chimarrão and 37% terere, with weighted mean daily intakes estimated at 648-2160 and 244-746 mL, respectively. For every 100 mL of beverage consumed, the average amount of total phenol compounds extracted was 673.6 mg for chimarrão and 1184.9 mg for terere. Regarding CGAs composition, caffeoylquinic acids (CQAs) constitute about 38.4% for chimarrão and 55.3% for terere, and dicaffeoylquinic acids (diCQAs) represent 61.6 and 44.7% of the extracted compounds, respectively. The daily intake of phenolic compounds by MTB consumers was estimated for chimarrão (512.5-1708.5 mg/day) and terere (583.0-1779.7 mg/day). These results allow us to conclude that MTBs are important dietary sources of CGAs, mainly CQAs and di-CQAs.

Genetic and Phytochemical Analysis to Evaluate the Diversity and Relationships of Mate (Ilex paraguariensis A.St.-Hil.) Elite Genetic Resources in a Germplasm Collection.

The aim of this study was to evaluate the phytochemical and genetic diversity, relationships and identification of mate (Ilex paraguariensis A.St.-Hil.) elite genetic resources belonging to the Brazilian germplasm collection and mate breeding program. Mate has been studied due to the presence of phytochemical compounds, especially methylxanthines and phenolic compounds. The samples were collected from the leaves of 76 mate elite genetic resources (16 progenies × 5 localities). Total DNA was extracted from mate leaves and 20 random primers were used for DNA amplification. Methylxanthines (caffeine and theobromine) and phenolic compounds (chlorogenic, neochlorogenic, and criptochlorogenic acids) were quantified by HPLC. The genetic divergence estimated was higher within (92%) than among (8%) the different populations. Analysis of genetic distance between origins provided the formation of two groups by UPGMA cluster analysis, with higher polymorphism (94.9%). The average content of caffeine ranged from 0.01 to 1.38% and theobromine of 0.10 - 0.85% (w/w). The caffeoylquinic acids concentrations (1.43 - 5.38%) showed a gradient 3-CQA > 5-CQA > 4-CQA. The coefficient of genetic variation (CVg) was of low magnitude for all mono-caffeoylquinics acids. Significant correlations (positive and negative) were observed between the phytochemical compounds. Genetic diversity analysis performed by RAPD markers showed a greater intra-populational diversity; genetic resources with low caffeine and higher theobromine content were identified and can be used in breeding programs; the correlation between methylxanthines and phenolic compounds can be used as a good predictor in future studies.

Isolamento e atividade antibacteriana de fungos endofíticos de Piper glabratum Kunth / Isolation and antibacterial activity of Piper glabratum Kunth endophytic fungi

Devido a capacidade dos endófitos em colonizar diferentes espécies de plantas, inclusive as medicinais, produzindo substâncias de interesse biotecnológico como antibióticos, o objetivo deste trabalho foi isolar fungos endofíticos de folhas de Piper glabratum Kunth e avaliar a atividade antibacteriana contra bactérias patogênicas humanas. O isolamento dos fungos endofíticos foi realizado utilizando-se a desinfestação superficial com hipoclorito de sódio 3%. Para avaliação da atividade antibacteriana, foi obtido extrato de 20 fungos endofíticos isolados que foram testados o extrato diretamente na fase aquosa, a fração obtida com acetato de etila e com clorofórmio P.A. Foi utilizado o teste de difusão em discos contra as bactérias patogênicas humanas Escherichia coli, Staphylococcus aureus, Salmonella enterica e Shigella flexneri. Em placas de Petri com meio Muller Hinton (MH) contendo as bactérias foram inseridos quatro discos de papel estéreis inoculados com 10 µL de cada extrato metabólito. Avaliou-se a atividade antimicrobiana pela formação de halos de inibição. Foi possível isolar 98 de P. glabratum, com frequência de isolamento de 98%. Dentre os 20 fungos avaliados quanto a atividade antimicrobiana, catorze isolados apresentaram atividade antibacteriana para pelo menos uma das quatro bactérias patogênicas testadas, destacando-se potencial de inibição para a bactéria S. aureus a partir dos testes realizados diretamente com o extrato bruto e com acetato de etila.

Endophytes are able to colonize different species of plants, including medicinal plants, producing substances of biotechnological interest such as antibiotics. Therefore, the aim of this study was to isolate endophytic fungi from Piper glabratum Kunth leaves and evaluate the antibacterial activity against human pathogenic bacteria. The isolation of endophytic fungi was performed using the surface disinfection method with 3% sodium hypochlorite. In order to evaluate the antibacterial activity, extracts from 20 endophytic fungi were tested, with the extract directly into the aqueous phase and the fraction obtained with ethyl acetate and chloroform P.A. The method of disk diffusion was used against human pathogenic bacteria Escherichia coli, Staphylococcus aureus, Shigella flexneri and Salmonella enterica. In Petri dishes with medium Muller Hinton (MH) containing bacteria, four sterile paper disks inoculated with 10 µL of each metabolite extract were inserted. The antimicrobial activity was evaluated due to the formation of the inhibition halo. It was possible to isolated 98 endophytic fungi from P. glabratum with isolation frequency of 98%. The results of the test showed that extracts from 20 endophytic fungi have inhibited the activity against fourteen isolates and antibacterial activity for at least one of the four tested pathogenic bacteria, emphasizing the potential for inhibition of S. aureus from the tests performed directly with the crude extract and ethyl acetate.

Renoprotective Effects of Vitex megapotamica (Spreng.) Moldenke in C57BL/6 LDLr-Null Mice Undergoing High Fat Diet.

Although Vitex megapotamica (Spreng.) Moldenke is used in Brazilian folk medicine as hypolipidemic drug no study has been conducted to evaluate the effects of this species in an experimental model of atherosclerosis. So, the aim of this study was to evaluate the possible renoprotective activity of methanolic extract obtained from Vitex megapotamica (MEVM) using C57BL/6 LDLr-null mice submitted to high fat diet (HFD). MEVM was orally administered at doses of 30, 100, and 300 mg/kg, for three weeks, starting from the 2nd week of HFD. Systolic blood pressure (SBP) and diuretic activity were measured weekly. At the end of experiments the serum lipids, atherogenic index serum (AIS), oxidative stress, and markers of renal function were determined. HFD induced a significant increase in the systolic blood pressure, dyslipidemia, increase in AIS, and lipid peroxidation accompanied by an important reduction in renal function. Treatment with MEVM was able to prevent increase in SBP, total cholesterol, triglycerides, AIS, urea, and creatinine levels in LDLr-null mice. These effects were accompanied by a significant reduction in oxidative stress and renal injury. The data reported here support the potential of Vitex megapotamica as candidate to be an herbal medicine used in cardiovascular or renal diseases.

Ethno-medicinal study of plants used for treatment of human ailments, with residents of the surrounding region of forest fragments of Paraná, Brazil.

ETHNOPHARMACOLOGICAL RELEVANCE: This study aims to document the traditional knowledge on the use of medicinal plants in the neighborhood of the protected area "Parque Estadual da Cabeça do Cachorro", a fragment of seasonal semideciduous forests. This vegetation is intensely fragmented and disturbed; despite its importance there are few records of the traditional knowledge of medicinal species. MATERIAL AND METHODS: Twenty-four residents in the neighborhood of the protected area "Parque Estadual da Cabeça do Cachorro" were interviewed. The residents were questioned about preparation techniques, recommended doses, ways of administration and healing properties of various parts of the plants and were invited to walk through the park to collect in situ some species of plants. The recognized medicinal species were identified and traditional knowledge was systematized. Quantitative indices (Informant Consensus Factor - FIC and Use Value - UV) were calculated. RESULTS: 115 species of medicinal plants belonging to 54 botanical families were cited. Asteraceae (n=14), Fabaceae (n=11), Myrtaceae (n=6), Bignoniaceae, Solanaceae and Verbenaceae (n=5) were the most species-rich. The highest use values were calculated for Achyrocline satureioides, Aristolochia triangularis and Bauhinia forficata (0.63). Moreover, the informants consensus about usages of medicinal plants ranges from 0.024 to 0.663, which shows high level of agreements among the informants for gastro-intestinal and respiratory system diseases. Furthermore, for the first time, new traditional medicinal uses of Asteraceae (Chromolaena pedunculosa Hook. & Arn.), Commelinaceae (Tradescantia fluminensis Vell.) and Polypodiaceae (Microgramma vacciniifolia Langsd. & Fisch.) species were reported. CONCLUSIONS: Present study revealed that the residents of the surrounding region of forest fragments of Paraná are rich in ethno-medicinal knowledge and rely on plant-based remedies for common health problems. As in many parts of Brazil knowledge of the past is combined with new knowledge that has recently been incorporated emphasizing the cultural changes that this area is experiencing. Despite the use of different species of plants are crucial to their way of life, there is concern that these rich popular knowledge may disappear in the future as a result of a possible modernization of this area.

Amides from Piper as a Diuretic: Behind the Ethnopharmacological Uses of Piper glabratum Kunth.

Several species of the genus Piper are known in Brazilian folk medicine as having diuretic activity. So, we propose to investigate the acute diuretic activity and the possible toxic effects of Piper glabratum Kunth, popularly known as false Jaborandi. Additionally, we propose to check whether there is any correlation between the biological activities of the crude extract (MEPG) and its 2-methoxy-4,5-methylenedioxy-trans-cinnamoyl-pyrrolidine (MMCP) in Wistar rats. The MEPG was fractioned by chromatography column and the MMCP was identified by analyses of (1)H and (13)C RMN spectral data and correlations. Both MEPG and MMCP were assayed for diuretic activity. The preparations obtained were orally administered in a single dose to rats. The urine excretion, pH, density, conductivity, and content of Na(+), K(+), Cl(-), and HCO3 (-) were measured in the urine of saline-loaded animals. Additionally, acute toxicity of the extract was also evaluated. MMCP at doses of 30 mg/kg was able to increase the urine volume, pH, and HCO3 (-) excretion. Moreover, high dosage of MEPG showed important liver toxicity and elevated mortality when injected intraperitoneally. The results indicate that the MMCP shows important diuretic properties when administered in Wistar rats. Additionally, MEPG can induce important acute toxicity if given in high doses.

Cardiovascular protective effects of Casearia sylvestris Swartz in Swiss and C57BL/6 LDLr-null mice undergoing high fat diet.

ETHNOPHARMACOLOGICAL RELEVANCE: Although Casearia sylvestris Swartz is used in Brazilian folk medicine to treat obesity, no study has been conducted to evaluate the effects of this species in an experimental model of dyslipidemia and atherosclerosis. So, the aim of this study was to evaluate possible hypolipemiant and antiatherogenic activity of the methanolic extract obtained from Casearia sylvestris (MECS) using Swiss and C57BL/6 LDLr-null mice undergoing high fat diet (HFD). MATERIAL AND METHODS: Dyslipidemia and atherogenesis were induced by the administration of commercial HFD for 4 weeks. The MECS was administered orally at doses of 250 and 500mg/kg, once a day, for two weeks, starting from the 2nd week of HFD. The gain in body weight and systolic blood pressure (SBP) were measured weekly over the four week study. At the end of the experiments the levels of triglyceride (TG), total cholesterol (TC), high-density lipoprotein cholesterol (HDL-C), low-density lipoprotein cholesterol (LDL-C) and very low-density lipoprotein cholesterol (VLDL-C) were measured by colorimetric method. Aldosterone, vasopressin and angiotensin converting enzyme (ACE) activity were also evaluated in collected serum. The renal function, atherogenic index serum (AIS) and in vitro antiplatelet activity were investigated. Additionally, histopathological analyzes were performed to determine the intima-media thickness (IMT) and intima media ratio (IMR) in aorta samples. RESULTS: The HFD induced dyslipidemia and major structural changes in the aortic wall, including raising of the systolic blood pressure in LDLr-null mice. In addition, we observed an increase in lipid peroxidation accompanied by a reduction of serum nitrite. The treatment with MECS was able to prevent the increase of SBP, TC, LDL-C, VLDL-C and triglycerides levels and increase HDL-C in Swiss and LDLr-null mice. These effects were accompanied by a significant reduction in oxidative stress. Moreover, AIS, IMT and IMR were significantly reduced in MECS-treated mice, and the extract was able to reduce platelet aggregation in vitro. CONCLUSION: This study demonstrated that MECS reduces the serum lipids and oxidative stress when orally administered to Swiss and LDLr-null mice. In addition, it was able to prevent arterial thickening induced by HFD and to inhibit platelet aggregation in vitro.