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Some Secondary Metabolites in Leaves of Echinodorus Grandiflorus (Cham

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Some Secondary Metabolites in Leaves of Echinodorus Grandiflorus (Cham The Open Agriculture Journal, 2008, 2, 75-79 75 Open Access Some Secondary Metabolites in Leaves of Echinodorus grandiflorus (Cham. & Schldl.) Micheli in Brazil W.M. Joaquim1, E.O. Ono*,2 and M.L. Salatino3 1Universidade do Vale do Paraíba - UNIVAP, São José dos Campos, SP, Brazil 2Universidade Estadual Paulista - UNESP, Departamento de Botânica, Instituto de Biociências de Botucatu, C.P. 510, CEP 18618-000, Botucatu, SP, Brazil 3Universidade de São Paulo - USP, Departamento de Botânica, Instituto de Biociências, São Paulo, SP, Brazil Abstract: The paper aimed to study the influence of naphthalene acetic acid (NAA) and ethephon on total phenols, fla- vonoids and tannins as a function of seasonality in Echinodorus grandiflorus plants. The experiment used adventitious plantlets from the flower stem with 4 to 5 leaves, planted in 10-liter pots. The leaf spray treatments applied were: control, NAA 100 mg L-1, and ethephon 100 mg L-1. Treatments were applied fifteen days after collecting the leaf blades, once at each season of the year. The results showed that plant growth regulators did not influence E. grandiflorus’ phenology and morphology. The ethephon did not affect synthesis of total phenols and flavonoids. The NAA caused a decrease in the synthesis of total phenols and flavonoids in some periods. Tannin synthesis is not expressive in E. grandiflorus. The study presented information about ecological aspects and chemical and mechanical defenses of Echinodorus grandiflorus. Keywords: Ethephon, flavonoids, NAA, phenols, tannins, secondary metabolites, plant growth regulators. INTRODUCTION plants under natural conditions. However, literature regard- ing agronomic aspects is scarce for most plants, or merely Echinodorus is a genus that belongs to family Alismata- only a few aspects about cultivation are mentioned, as is the ceae, which is found exclusively in the Americas, with a case with Echinodorus grandiflorus (Cham. & Schldl.) center of dispersion in South America, occurring in several Micheli (“chapéu-de-couro”, Amazon swordplant, burhead). regions of Brazil. Besides Echinodorus, the family is also represented among us by the genus Sagittaria [1]. Popularly The action of plant growth regulators on adventitious known as “chapéu-de-couro”, it is frequently used by the plantlets originated on the flower stem of the plant, a repro- population as diuretic, antirheumatic, antiinflammatory, and ductive strategy for the perpetuation of the species, would be as an agent to fight uric acid and skin problems [2]. For this an interesting approach, since there are no papers in the reason, it has been the target of extractivism. literature relating the action of plant growth regulators on contents of total phenols, flavonoids, and tannins in leaves of With regard to ecological aspects, Echinodorus grandi- E. grandiflorus plants. florus (Cham. & Schldl.) is important in aquatic ecosystems because it provides refuges for aquatic fauna reproduction, Both total phenols and flavonoids and tannins are secon- feeding, and because it prevents erosion on the banks of dary metabolites with great potential against herbivory, also those systems [1]. acting as fungicides and having a number of therapeutic uses. Because synthetic medicines cost so much the population has resumed the use of medicinal plants due to their low cost In view of the lack of information concerning the action and easy access, and also because this practice is a tradition, of plant growth regulators on adventitious plantlets of E. passed from generation to generation. This fact has called the grandiflorus, we gave priority in this work to an approach attention of researchers seeking to obtain information of a about the influence of the application of naphthalene acetic scientific nature [3], both for a knowledge of their therapeu- acid (NAA) and ethephon (ET) over the contents of total tic action and their implications, and for the establishment of phenols, flavonoids, and tannins as a function of seasonality, cropping conditions, which would make plants accessible to in the plant's leaf blades. We also aimed to evaluate the in- populations in urban centers that generally have no contact fluence of plant growth regulators on aspects of the plant's with natural vegetation. In addition, the large-scale growing phenology. The importance of these studies in Echinodorus of medicinal plants is one of the basic steps, highly impor- grandiflorus plants can be justified by a lack of studies with tant for the quality of the herbal medicine, since it is so hard the objectives here proposed and because the secondary to find satisfactory and homogeneous quantities of these metabolites studied play a role against herbivory. MATERIALS AND METHODS *Address correspondence to this author at the Botany Department, Bio- The experiment was conducted at the Medicinal Plants science Institute, São Paulo State University - UNESP, C.P. 510, 18618- Garden, at Universidade do Vale do Paraíba - UNIVAP 000, Botucatu, SP, Brazil; E-mail: [email protected] 1874-3315/08 2008 Bentham Open 76 The Open Agriculture Journal, 2008, Volume 2 Joaquim et al. Campus, São José dos Campos (SP, Brazil), in the period Henshaw [5], and the extracts were submitted to the total from March 2002 to February 2003 (23°14' S, 45°51' W). phenols quantification method of Folin-Ciocalteu [6]. Ab- The Echinodorus grandiflorus experiment was installed sorbance readings were done in a spectrophotometer at 760 in a nursery 20 meters long by 4 meters wide, at a 2-meter nm, according to a methodology proposed by Waterman and 2 Mole [6]. A standard curve, prepared with gallic acid solu- height, totaling an area of 100 m , completely covered with a -1 black polypropylene shading (50% shade cloth), up to a tions at 50, 100, 150, 200, 250, 300, and 350 g mL was height of 30 cm above the ground [4], thus providing the plotted to determine the concentration of total phenols in the most suitable light intensity for the development of E. gran- sample. This method is based upon the reducing effect of diflorus adventitious plantlets. Three benches 4 meters long phenolic hydroxyls present in the extract. by 1.30 meter wide were constructed inside the nursery, The methodology proposed by Deutsches Arzneibuch spaced at 3 meters from one another. (1978), cited by Motta [7] was used to dose flavonoids. A standard curve prepared with quercetin solutions at 20, 40, The E. grandiflorus adventitious plantlets used in the -1 experiment came from plants grown in a lowland area near 60, 80, 100, 120, 140, and 160 μg mL was used to deter- the experiment site. After adventitious plantlets were emit- mine the concentration of flavonoids present in the samples. ted, they were removed from the mother plant and placed in Tannins were dosed according to the modified colorimet- ® Styrofoam trays, which remained in a greenhouse until ric method of Watermann and Mole [6], which is based on transplanted to the final pots used during the experiment. the precipitation of tannin with BSA (bovine serum albumin The water-draining holes in 10-L white plastic pots were solution). A standard curve prepared with tannic acid solu- sealed with Durepoxi® resin. The pots were filled with sub- tions at 0.075, 0.150, 0.250, 0.300, 0.375, 0.450, and 0.60 -1 strate consisting of 20% vermicompost, 40% rice hulls, and mL was used to determine the concentration of tannins 40% dirt, and fertilized according to a soil chemical analysis present in the samples. performed by the Soils Sector of the Natural Resources De- Phenological observations such as flowering period and partment of Faculdade de Ciências Agronômicas de Botu- emission of adventitious plantlets were made every fifteen catu (Botucatu Agricultural Sciences College), of Universi- days, before collecting the leaf blades of E. grandiflorus. dade Estadual Paulista - UNESP, Brazil. A completely randomized experimental design was used, E. grandiflorus plantlets 10 cm in height with 4 to 5 containing 3 treatments with 5 replicates of 5 pots each, leaves were transplanted to the pots containing substrate, totaling 75 pots. The results were subjected to analysis of which was kept saturated with water up to the edge of the variance and the means of the treatments were compared by pot; the water was replaced as the level decreased [4]. Tukey’s test at 0.05 significance level. Treatments consisted of: control; NAA (naphthalene acetic acid) at 100 mg L-1; and ethephon (2- chloroethyl- RESULTS AND DISCUSSION phosphonic acid) at 100 mg L-1. The treatment solutions Flowering and Emission of Adventitious Plantlets of were added of 0.5 mL nonionic surfactant adjuvant (Extra- Echinodorus grandiflorus von®), and the leaf-spray treatments were applied with a hand sprayer 40 days after the plantlets were planted in the During the experiment, it was observed in all treatments pots. The other plant growth regulator applications were that the Echinodorus grandiflorus plants began flowering in performed at every 15 days from the leaf blade collections, spring (end of October), ending practically in the beginning which were made at the 4 seasons of the year. of March (summer), with a significantly higher intensity from November to January. The first collection of leaf blades from E. grandiflorus plants was made 15 days after the treatments, for chemical In Rio Grande do Sul (southern region of Brazil), Rego analysis of total phenols, flavonoids, and tannins. All leaf [1] reported that an interruption of E. grandiflorus flowering blades collected for the phenols and flavonoids analyses was observed in winter, followed by significant senescence were packaged in paper bags and placed to dry in a model of leaves; the same was verified by Vieira and Lima [8] in 315SE - FANEM drying oven at 60ºC, at the Chemistry Viçosa (Minas Gerais State), in the southeastern region of Laboratory of Instituto de Engenharia, of Universidade do Brazil.
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