Growth and Evaluation of Phenolic Compounds in Physalis angulata L. at Two Different Periods in the Bahia Reconcavo, Brazil

Physalis angulata L. has aroused the interest of the researchers, since its properties come from the great therapeutic potential attributed to the commonly called substances of physalins. However, its cultivation in the study region lacks important information on the species adaptation to local soil and climate characteristics. The aim of this study was to evaluate the growth and concentration of phenolic compounds in P. angulata L. at two different periods in the Bahia Reconcavo. The experimental design was completely randomized blocks with 10 replicates. We evaluated the stem height and diameter, number of leaves and fruits, dry mass of leaves, stems and roots, leaf area, specific leaf area, leaf area ratio, leaf mass ratio and total phenolics, total flavonoids and antioxidant activity in the different organs of the plant. The growth was favorable from April to June. However, the concentration of phenolic compounds was higher in the period from December to February. The cultivation period influenced the growth and concentration of phenolic compounds in P. angulata L. in the Bahia Reconcavo.


Introduction
The Physalis production in Brazil is concentrated in the South, however, other species of the genus, such as Physalis angulata L., have been studied in the Northeast region, especially in the semi-arid region, mainly due to its medicinal potential, seeds conservation and differential expression of genes in seeds and seedlings submitted to abiotic stress (M.Souza, C. Souza, Barroso, & Pelacani, 2014;Souza, 2015;Souza et al., 2016).The species occurs in almost all country (Stehmann et al., 2015), but in Bahia there are records in the municipalities of Seabra, Água Quente, Mucugê and Rio de Contas (Matos, 2000).
The interest in the species comes from the great therapeutic potential attributed to the substances commonly known as physalins (Tomassini, 2000) and its proven anti-inflammatory, immunotherapy, leishmanicidal action with potential in vitro and in vivo by inhibiting cutaneous leishmaniasis and induction of cell death by apoptosis of human oral cancer cells (Bastos, Silveira, Salgado, Picanço-Diniz, & Nascimento, 2008;Guimarães et al., 2009;Lee et al., 2009).
The Physalis cultivation may be an alternative for the small and medium rural producers, since the obtaining of these substances that have therapeutic properties (Yu et al., 2010;Sun et al., 2011) is linked to extractive practices that generates the reduction or even the risk of extinction of the species.According to Betemps, Fachinello, Lima, Galarça, and Rufato (2014), the study of Physalis sowing time is necessary, since they provide important information about the adaptation to the local soil and climate characteristics, allowing the appropriate management of the species.Until now, data reporting the best planting period for the growth and production of phenolic compounds in P. angulata L. have not been found.
Thus, the aim of this study was to evaluate the growth and production of phenolic compounds in P. angulata L. in two different periods in the Bahia Reconcavo, Brazil.

Localization and Conditions of Plant Growth
The seeds of P. angulata L. were obtained from fruits, from plants grown in a greenhouse at the Forest Garden Experimental Unit of the State University of Feira de Santana, Bahia.However, the experiment was conducted at the experimental station of the Bahia Reconcavo Federal University, located in the municipality of Cruz das Almas, BA, (12º40′19″ S and 39º06′22″ W, 220 m of altitude).
The local climate is hot and humid tropical with a dry season in the summer, As type, according to the classification of Köppen.The region has an average annual rainfall of 1,224 mm, average annual temperature of 22.4 ºC and 80% of relative humidity (Almeida, 1991).
The sowing in the greenhouse and the transplanting to the field were carried out in two different periods, in March and November of 2016.The transplants to the field were carried out in April and December 2016.The first and second evaluations were carried out from April to June 2016 and from December 2016 to February 2017, respectively.
The seeds were sown in plastic tubes with a capacity of 327 g containing soil, sand and organic compound in the proportion (2:1:1), irrigated daily and kept in a greenhouse until the day of transplanting.Upon reaching an average size of 20 cm, the seedlings were transferred to the experimental field in pits with width, length and depth of 40 × 40 × 40 cm, respectively.In spacing of 2 m × 1 m.The experimental design was completely randomized blocks with 10 replicates.

Evaluation of Plant Growth
The following growth characteristics were evaluated weekly in 8 plants of each block: height of the main branch, diameter of the stem, number of leaves and fruits.The height was measured with millimeter ruler from the neck to the apex of the plant (terminal bud); the diameter of the stem was measured using a digital caliper (cm) from the neck of the plant and the number of leaves and fruits were done by manual counting.
After 60 days of cultivation, the plants were collected and separated in leaves, stems and roots, placed in properly identified paper bags and taken to drying in a greenhouse with forced air circulation at 40 ± 5 °C, until constant mass, to determine the dry masses of leaves (DML), stem (DMS) and roots (DMR).A precision analytical balance of 10 -4 g was used.For the collection of the roots in the field, a 2 mm mesh screen was used to separate the soil from the roots.
The leaf area per plant was determined by the ratio of the dry mass of ten leaf discs and the total dry mass of the leaves.The leaf discs were obtained with the aid of a metal perforator of known area, avoiding the central vein as described in Benincasa (2004).The foliar area ratio (FAR), foliar mass ratio (FMR) and specific leaf area (SLA) were determined from total leaf area (TLA), expressed in cm 2 , of the plant dry mass (PDM) and leaves dry mass (LDM), expressed according to Benincasa (2004).

Total Flavonoid, Phenolic and Antioxidant Activity
The plant material was crushed for the analysis of total phenols, total flavonoids and antioxidant activity.The extractive solution of the leaves, stems and roots were obtained by extraction under reflux with hydroalcoholic solution (ethyl alcohol and water, 40% v/v) for 30 minutes, developed and validated by Petry, Souza, Bassani, Petrovick and González-Ortega (1998).

Total Flavonoids
The determination of flavonoids followed the methodology proposed by Marques et al. (2012), of the extractive solution were transferred to 10 ml volumetric flasks, in which 2.0 ml of AlCl 3 ethanolic solution (5%, m/v) were added.The volume was adjusted with the hydroalcoholic solution (40%, v/v).After addition of the AlCl 3 solution (5%, m/v) we waiting 30 m and the readings were carried out in a spectrophotometer (WPA Lightwave II, Biochrom, China) at 408 nm.The tests were carried out in triplicate.The total flavonoid content was calculated from the calibration curve of quercetin used as standard and expressed in milligrams of quercetin equivalents per gram of dry extract (mgQE/gDE).

Total Phenolics
The total phenol contents of the extracts were determined using the Folin-Ciocalteau reagent and gallic acid as standard (Slinkard & Singleton, 1977).The extract sample (0.5 mL) and 2 mL of sodium carbonate (75 g L -1 ) were added to 2.5 mL of 10% (v/v) Folin-Ciocalteau reagent.After 30 min of reaction at room temperature and protected from light, the readings were carried out in a spectrophotometer (WPA Lightwave II, Biochrom, China) at 765 nm.The tests were carried out in triplicate.The total phenolic compounds content was calculated from the calibration curve of gallic acid and expressed in milligrams of gallic acid equivalents per gram of dry extract (mgGAE/gDE).

Antioxidant Capacity Determined by DPPH Radical Assay
The determination of antioxidant activity by the capture of free radical DPPH (2,2 diphenyl-1-picrylhydrazyl) followed the methodology proposed by Rufino et al. (2007).From the extract obtained above, five different concentrations (1.00, 3.00, 5.00, 7.00, 10.00 g L -1 ) were prepared in triplicate in test tubes.In the dark environment, a 300 μL aliquot of each test tube extract dilution was transferred with 3.9 mL of the DPPH radical and homogenized on a tube shaker.We was used 300 μL of the control solution (methyl alcohol, acetone and water) with 3.9 mL of the DPPH radical and then homogenized the solution.The DPPH solution was prepared on the same day before the analysis and stored in volumetric flask covered within an aluminum paper protected from light until analysis.The methyl alcohol was used as the blank to calibrate the spectrophotometer.The whole procedure was done in triplicate, and the spectrophotometer (WPA Lightwave II, Biochrom, China) at readings at 517 nm.

Statistical Analysis
The results were statistically analyzed, and the analysis of variance was carried out.The averages of the treatments were compared by the F test at 5% probability.The Sisvar 4.6 software was used (Ferreira, 2011).

Climatic Data during the Study Period
The climatological data of the study period are shown in Figure 1, in which the monthly values of the rainfall and the monthly average of the temperature collected during the experiments are found in the municipality of Cruz das Almas in the Bahia Reconcavo, Brazil.The period considered the driest was January (15 mm) and the rainiest was May (145 mm).   ) can be inf parts.

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