et al.
assay during 15 months, totalizing 15 experiments. For each
experiment we investigated 4 samples in six distinct temperatures (45 oC, 55 oC, 65 oC, 75 oC, 85 oC, 95 oC). Considering
this range of data, we used the Excel software for determine
the Average and Standard Deviation of the data, which were
showed in the figures 4 and 6.
We also investigated the angiogenic response of the nonrubber constituents of natural rubber, ie. the serum fraction
in the absence of rubber particles. The latex stabilized with
ammonium was submitted to a coagulation process by addition of acetic acid (2.0% v/v). The coagulation takes place
when pH falls below 5.0, and the latex is separated into two
parts: the rubber fraction and the serum fraction (nonrubber
constituents). The serum fraction was purified in a column
of DEAE-cellulose (5 × 40 cm) eluted with stepwise gradient of NH4HCO3/NaCl. From this purification, three fractions were obtained by exclusion based on the charge affinity with the DEAE column. Each fraction was dialyzed and
lyophilized.
The first fraction (FrHB1) eluted from the separation column had the most evident angiogenic effects in the CAM
assays [29]. Therefore, we submitted this most active fraction (FrHB1) to the thermal treatment and evaluated the angiogenic response. The lyophilized (FrHB1) fraction was
weighted and dissolved in distilled water (2.5mg/mL). Then,
a 3 µL droplet of this solution was pipetted on the filter disc
papers (0.5 cm diameter). These discs were left to dry at
room temperature and after been dried the testing samples
were heated in an oven in temperatures ranging from 45-
105oC to simulate the thermal treatment performed with the
NR membranes. After the heating process, the sample containing serum fraction was placed on chorioallantoic membrane for evaluation of the angiogenic activity