Optimizing the Vanillin-Acid Sulfuric Method to Total Saponin Content in Leaves of Yerba Mate Clones.

Yerba mate (Ilex paraguariensis) is a forest species consumed in the form of non-alcoholic beverages in South America, with applications in foods, cosmetics, and pharmaceutical industries. The species leaves are globally recognized for their important bioactive compounds, including, saponins. We adjusted the vanillin-acid sulfuric method for determining spectrophotometrically the total saponin in yerba mate leaves. Seeking to maximize the extraction of saponins from leaves, a Doehlert design combined with Response Surface Methodology (RSM) was used, considering ethanol:water ratios and ultrasound times. In addition, the same methodology was used for the analysis of times and temperatures in the vanillin-sulfuric acid reaction heating. The contents of total saponin in mature leaves were compared in four yerba mate clones. The extraction was maximized using 40 % ethanol:60 % water and 60 minutes of ultrasound assisted extraction (UAE) without heating. For the reaction conditions, 70 °C for 10 minutes heating is recommended, and UV/Vis reading from 460 to 680 nm. Using the optimized methodology, total saponin contents ranged from 28.43 to 53.09 mg g-1 in the four yerba mate clones. The significant difference in saponin contents between clones indicate great genetic diversity and potential for clones' selection and extraction of these compounds from yerba mate leaves.

1H NMR Mixture Design-Fingerprints and ASCA Analysis in Ilex paraguariensis: Model Stability in Search of a Global Metabolome.

The effects of experimental repetitions and solvent extractors on the 1H NMR fingerprinting of yerba mate extracts, obtained from two genders and two light environments, were analyzed in-depth by ANOVA-simultaneous component analysis (ASCA). Different solvents were used according to a mixture design based on ethanol, dichloromethane, and hexane and their combinations. The number of experimental repetitions significantly affected the ASCA results. Increasing repetitions led to decreases in the percentage effect variance values and an increase in the percentage residual variance. However, secondary sexual dimorphism, light availability, and their interaction effects became more significant with decreasing p-values at or above the 95% confidence level. The choice of a solvent extractor significantly affects the chemical profile and can lead to distinct conclusions regarding the significance of effect values. Pure solvents yielded different conclusions about the significance of factorial design effects, with each solvent extracting unique metabolites and maximizing information for specific effects. However, the use of binary solvent mixtures, such as ethanol-dichloromethane, proved more efficient in extracting sets of compounds that simultaneously differentiate between different experimental conditions. The mixture design-fingerprint strategy provided satisfactory results expanding the range of extracted metabolites with high percentage of residual variances and low explained percentage effect variances in the ASCA models. Ternary and even higher-ordered mixtures could be good alternative extracting media for work-intensive procedures. Our study underscores the significance of experimental design and solvent selection in metabolomic analysis, improving the accuracy, robustness, and interpretability of metabolomic models, leading to a better understanding of the chemical composition and biological implications of plant extracts.

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.

Accumulation Capacity of Nickel and Zinc in Yerba Mate Cultivated in Soils with Contrasting Parent Materials.

Yerba mate (Ilex paraguariensis St. Hill.) has shown a relatively high capacity for micronutrient absorption and could be a candidate for biofortification and combating a lack of micronutrients. To further evaluate the accumulation capacity of Ni and Zn, yerba mate clonal seedlings were grown in containers under five rates of Ni or Zn (0, 0.5, 2, 10, and 40 mg kg-1) with three soils originating from different parent material (basalt, rhyodacite, and sandstone). After 10 months, plants were harvested, divided into component parts (leaves, branches, and roots), and evaluated for 12 elements. The use of Zn and Ni enhanced seedling growth under rhyodacite- and sandstone-derived soils at the first application rate. Application of Zn and Ni resulted in linear increases based on Mehlich I extractions; recovery of Ni was smaller than Zn. Root Ni concentration increased from approximately 20 to 1000 mg kg-1 in rhyodacite-derived soil and from 20 to 400 mg kg-1 in basalt- and sandstone-derived soils; respective increases in leaf tissue were ~ 3 to 15 mg kg-1 and 3 to 10 mg kg-1. For Zn, the maximum obtained values were close to 2000, 1000, and 800 mg kg-1 for roots, leaves, and branches for rhyodacite-derived soils, respectively. Corresponding values for basalt- and sandstone-derived soils were 500, 400, and 300 mg kg-1, respectively. Although yerba mate is not a hyperaccumulator, this species has a relatively high capacity to accumulate Ni and Zn in young tissue with the highest accumulation occurring in roots. Yerba mate showed high potential to be used in biofortification programs for Zn.

Manganese hyperaccumulation capacity of Ilex paraguariensis A. St. Hil. and occurrence of interveinal chlorosis induced by transient toxicity.

Manganese (Mn) toxicity is common in plants grown on very acid soils. However, some plants species that grow in this condition can take up high amounts of Mn and are referred to as hyperaccumulating species. In this study, we evaluated the capacity of Ilex paraguariensis to accumulate Mn and the effect of excessive concentrations on plant growth and nutrition. For this, a container experiment was conducted using soils from different parent materials (basalt and sandstone), with and without liming, and at six doses of applied Mn (0, 30, 90, 270, 540 and 1,080 mg kg-1). Clonal plants grown for 203 days were harvested to evaluate yield, and leaf tissue samples were evaluated for Mn and other elements. Without liming and with high Mn doses, leaf Mn concentrations reached 13,452 and 12,127 mg kg-1 in sandstone and basalt soils, respectively; concentrations in excess of 10,000 mg kg-1 are characteristic of hyperaccumulating plants. Liming reduced these values to 7203 and 8030 mg kg-1. More plant growth accompanied increased Mn leaf concentrations, with a growth reduction noted at the highest dose in unlimed soils. Elemental distribution showed Mn presence in the mesophyll, primarily in vascular bundles, without high Mn precipitates. Interveinal chlorosis of young leaves associated with high Mn concentration and lower Fe concentrations was observed, especially in sandstone soil without liming. However, the occurrence of this symptom was not associated with decreased plant growth.

Plant growth, nutrients and potentially toxic elements in leaves of yerba mate clones in response to phosphorus in acid soils

ABSTRACT Native to subtropical region of South America, yerba mate is responsive to P under some conditions, but the degree of influence of genetic and soil on the growth and composition of the leaf is unknown. The aim of study was to evaluate plant growth, nutrients and potentially toxic elements in leaves of yerba mate clones in response to P application in acid soils. In greenhouse condition, two yerba mate clone seedlings were grown (210 days) in pots, each clone in a completely randomized design in factorial scheme (with and without P; four acid soils). The elemental composition of leaves and the growth of plants were determined. Phosphorus promoted plant growth, but this was not accompanied by increased P in leaf tissue in all conditions tested. The P effect on the elemental composition varied: decrease/null (N, K, Mg, Mn, Cu, Ni, B, Mo, Al, Cd); increase/null (C/N, C, Ca, Fe, V); increase/decrease/null (Zn, Ba, Pb) and; null (Cr). The soils affect the elemental composition of the leaves, especially Mn, with accumulation greater than 1000 mg kg-1. The Ba, Pb, Al and Zn in the leaves varied among clones. Yerba mate response to P was affected by edaphic and plant factors.

Plant growth, nutrients and potentially toxic elements in leaves of yerba mate clones in response to phosphorus in acid soils.

Native to subtropical region of South America, yerba mate is responsive to P under some conditions, but the degree of influence of genetic and soil on the growth and composition of the leaf is unknown. The aim of study was to evaluate plant growth, nutrients and potentially toxic elements in leaves of yerba mate clones in response to P application in acid soils. In greenhouse condition, two yerba mate clone seedlings were grown (210 days) in pots, each clone in a completely randomized design in factorial scheme (with and without P; four acid soils). The elemental composition of leaves and the growth of plants were determined. Phosphorus promoted plant growth, but this was not accompanied by increased P in leaf tissue in all conditions tested. The P effect on the elemental composition varied: decrease/null (N, K, Mg, Mn, Cu, Ni, B, Mo, Al, Cd); increase/null (C/N, C, Ca, Fe, V); increase/decrease/null (Zn, Ba, Pb) and; null (Cr). The soils affect the elemental composition of the leaves, especially Mn, with accumulation greater than 1000 mg kg-1. The Ba, Pb, Al and Zn in the leaves varied among clones. Yerba mate response to P was affected by edaphic and plant factors.

Vegetative rescue and ex vitro system production of Tibouchina sellowiana clonal plants by cutting and mini-cutting / Resgate vegetativo e sistema ex vitro de produção de mudas de Tibouchina sellowiana por estaquia e miniestaquia

ABSTRACT: We aimed to evaluate the rooting potential of Tibouchina sellowiana through the experiments: I - Cuttings from current-year shoots and epicormic shoots were submitted to IBA concentrations: 0, 500, 1000, 1500 and 2000mg L-1, in a factorial arrangement 2 x 5 (two types of cuttings x five IBA concentrations), with four replicates and 20 cuttings each; II - mini-stumps of Tibouchina sellowiana were submitted to successive shoots collecting during the four seasons, in a split-plot design, with five replications of ten mini-stumps per experimental unit. From the shoots of mini-stumps, mini-cuttings were produced, which were initially kept in greenhouse and later transferred to full sun, in a 4 x 5 factorial arrangement (four seasons x five collections per season), with four replicates of 12 mini-cuttings. Superiority of epicormic shoots cuttings was reported when compared to the current-year shoots, which showed the highest rooting and leaves maintenance (42.50% and 55.00%, respectively), eliminating the use of IBA. High survival of mini-stumps (over 80%) and the mini-cuttings production (170mini-cuttings m-2 month-1) in clonal mini-garden and the mini-cuttings survival (above 80%) in the greenhouse demonstrated the technical feasibility, with summer as the most appropriate time to collect mini-cuttings.

RESUMO: Objetivou-se avaliar o potencial de enraizamento de Tibouchina sellowiana por meio dos experimentos: I - Estacas provenientes de brotações do ano e epicórmicas foram submetidas às concentrações de IBA: 0, 500, 1000, 1500 e 2000mg L-1, em arranjo fatorial 2 x 5 (dois tipos de estacas x cinco concentrações de IBA), com quatro repetições de 20 estacas cada; II - Minicepas de Tibouchina sellowiana foram submetidas à coletas sucessivas de suas brotações durante as quatro estações do ano, em modelo de parcelas subdivididas no tempo, com cinco repetições de dez minicepas por unidade experimental. A partir das brotações das minicepas foram produzidas miniestacas, as quais foram inicialmente mantidas em casa de vegetação e posteriormente transferidas para pleno sol, em arranjo fatorial 4 x 5 (quatro estações do ano x cinco coletas por estação), com quatro repetições de 12 miniestacas. Verificou-se superioridade das estacas de brotações epicórmicas em comparação às brotações do ano, as quais apresentaram o maior enraizamento e retenção foliar (42,50% e 55,00%, respectivamente), dispensando o uso de IBA. A elevada sobrevivência das minicepas (superior a 80%) e produção de miniestacas (170 miniestacas m-2 mês-1) em minijardim clonal e sobrevivência (acima de 80%) de miniestacas em casa de vegetação demonstram a viabilidade da técnica, sendo o verão a época mais adequada para coleta de miniestacas.