3.1. Plant selection
The 14 species employed in this study were selected from a survey of flora and herbaceous trees and shrubs found in a rural area in the municipality of Altinho (08°29′23″S and 36°03′34″W), in the state of Pernambuco (NE Brazil). From this sample, we performed a sampling of inventoried species, evaluated the cytotoxic activity in vitro and selected those with the best anti-proliferative and proliferative results.
The survey of the herbaceous flora was performed in three different anthropogenic zones: areas where Zea mays
L. and Opuntia ficus-indica
(L.) Mill. were under cultivation and a native pasture area. For each area, 100 1 × 1 m plots were sampled for a total of 300 plots. The survey was conducted between November 2007 and July 2008 and a total of 119 species were registered in these three areas. More details of the survey can be found in Santos et al
The floristic and phytosociological survey of the woody-shrubby layer was performed on a fragment of native vegetation located in a hilly area using the point-Quadrant method. For this method, three parallel lines of 500 meters, were laid out 10 meters apart. For each point, four vertices were created at an angle of 90 degrees, and individuals with a diameter ≥3 cm above ground level and closer to each vertex were sampled. A total of 150 points were demarcated and 600 individuals sampled for a total of 48 identified taxa. Annona muricata, an exotic species, was also included in this study due to its popularity in cancer treatment by local inhabitants in this region.
All material collected was identified by experts and incorporated into the Professor Vasconcelos Sobrinho Herbarium of the Universidade Federal Rural de Pernambuco and duplicates sent to the Herbarium Dardano de Andrade Lima (Agronomic Institute of Pernambuco) and to Sergio Tavares (Universidade Federal Rural de Pernambuco).
3.3. Determination of tannins
The determination of tannins was performed by the radial diffusion method [21
]. This method consists of the reaction between tannins and proteins in an agarose gel forming a measurable and visible ring. For this reaction, a solution of 50 mM acetic acid and 60 µM ascorbic acid were prepared and adjusted to pH 5.0 [21
]. This solution was then used to prepare the gel by adding 1% agarose (type I, Sigma-Aldrich). This solution was then heated until complete agarose homogenization and the solution cooled to 45 °C. Next, 0.1% bovine serum albumin (BSA) fraction V free fatty acid (Sigma-Aldrich) was added. Quickly, the gel was distributed in aliquots of 10 mL in 9.0 cm in diameter Petri dishes. Four-millimeter diameter wells were made on the gel 2.0 cm apart and from the plate edges, each having a volumetric capacity of 8 µL. With the help of a micropipette, three successive aliquots of 8 µL of each extract were added to the wells. All samples were processed in authentic triplicates.
To obtain the standard curve, an aqueous solution of tannic acid at a concentration of 25 mg/mL was prepared and aliquots of 2, 4, 8, 12, 16, 20, and 24 µL were placed in wells, in triplicate, and divided between more than one well whenever the aliquots were larger than the capacity of the well.
3.4. Antiproliferative activity
evaluation of antiproliferative activity was carried out on two cancer cell lines (HEp-2 and NCI-H292). NCI-H292 is a mucoepidermoid cell line derived from human lung carcinoma, and HEp-2 cells are derived from primary tumors of the human larynx. The HEp-2 and NCI-H292 cell lines were maintained in a suitable medium (Dulbecco’s modified Eagle’s Minimum Essential Medium [Sigma]) with the addition of 10% fetal bovine serum (Sigma) and 1% L-glutamine (200 mM). Cell viability was determined by 0.4% Trypan blue (Merck). Cell counting was performed on a Leitz inverted microscope using a hemocytometer. The cell suspensions were distributed in 96-well culture plates (198 μL in each well). These were incubated at 37 °C and 5% humidity in an appropriate incubator. After 24 h of incubation, the extracts were added (at a concentration of 50 µg/mL) and the plates again incubated at 37 °C [22
After 72 hours, 3-[4,5-dimethylthiazol-2-yl]-2,5-difeniltetrazole (MTT) bromide (25 μL) was added to each well at a concentration of 5 mg/mL in PBS. The plates were then left for two hours in an incubator (37 °C). Subsequently, the culture medium and MTT were removed by aspiration, and dimethylsulfoxide (100 μL) was added to each well to dissolve the crystals that formed [23
]. To verify the percentage of inhibition, optical readings were performed on a Multiscan-type automatic plate reader at 595 nm. All measurements were performed in triplicate.
3.5. Quantification of antioxidant activity using the DPPH method (2,2-diphenyl-2-picrylhydrazyl)
Six different concentrations were prepared from the extracts (250, 200, 150, 100, 50, and 25 or 100, 50, 25, 20, 15, and 10 µg/mL) with the objective of obtaining an exponential curve. This variation depended on the antioxidant power of the extract, given that higher concentrations of certain species saturated the DPPH solution, leading to similar absorbance values and poor curve shape.
The protocol was adapted from Cotelle et al
] and McCune and Johns [25
] and quantified the antioxidant activity using the (2,2-diphenyl-2-picrylhydrazyl) (DPPH) assay A 40-µM DPPH solution in methanol was prepared for this assay. Next, for each concentration, plant extract (0.5 mL) was removed and mixed with the DPPH standard solution (3.0 mL) in a test tube. After 30 minutes, the absorbance of this solution was read at 517 nm. A duplicate reading was performed for each concentration.
The positive control in this assay was ascorbic acid, used at concentrations of 5, 10, 15, 20, 25, 30, 40, and 50 µg/mL, and subjected to the same aforementioned procedures for the quantification of antioxidant activity.
With these different concentrations, the inhibitory concentration (IC50) was calculated, which corresponds to the concentration required to increase or decrease the initial DPPH concentration by 50%. To calculate the IC50, the concentrations of the samples and the positive controls (µg/mL) were plotted on the abscissa, and the percentage of the remaining DPPH (% DPPHREM) on the ordinate, to obtain a first-order exponential curve and an equation from which the effective concentration could be calculated.