2014 - Fernanda Giacomini Bueno
Ph.D. Thesis
Author: Fernanda Giacomini Bueno
Postgraduate Program in Pharmaceutical Sciences - State University of Maringá
Area of Knowledge: Pharmacognosy
Date of Defense: December 05, 2014
Advisor: Prof. Dr. João Carlos Palazzo de Mello
Examination Board: Prof. Dra. Francine Guimarães Baesso
Prof. Dra. Luzmarina Hernandes
Prof. Dr. Andréia Cristina Conegero Sanches
Prof. Dr. João Ernesto de Carvalho
Title: Poincianella pluviosa: Study of cell viability and proliferation of fibroblasts and keratinocytes in vitro, and evaluation of cicatrizant activity in diabetic and normoglycemic rats
Abstract: Wound healing is a dynamic and complex process that due to diseases such as diabetes mellitus may have an impaired tissue repair. Due to the high costs and decreased life quality, the search for new drugs to improve the healing process has been outstanding. This research aimed to evaluate the wound healing activity of the crude extract (CE) of Poincianella pluviosa and subfractions through bioguided study with in vitro model, as well as the action on wounds skin in normoglycemic and diabetic rats. In the bioguided study, in vitro cell viability and proliferation of keratinocytes (HaCaT) and fibroblasts (pNDHF) were evaluated, and the fraction with activity was characterized by solid 13C NMR and s LC-MS/MS. The healing activity was measured in normoglycemic mice at 4, 7, 10 and 14 days of treatment with the gel containing 1% EB by evaluating the length and thickness of the re-epithelialized surface of skin, cutaneous permeation analysis by photoacoustic spectroscopy technique (PAS), number of cells in metaphase and collagen fibers (type I and III) and quantification of proteins (vascular endothelial growth factor (VEGF), cyclooxygenase 2 (COX-2) and superoxide dismutase 2 (SOD-2)). The healing activity in diabetic rats was evaluated at 4, 7, 10, 14 and 21 days with the same gel by evaluating the length and thickness of the reepithelialized surface and quantification of proteins. In vitro evaluations have shown that CE and subfraction FC1 stimulated mitochondrial activity in both types of cells and the proliferation of keratinocytes. The characterization of subfraction FC1 showed the presence of polyphenols and hydrolysable tannins. The evaluation of the effect of the gel containing 1% CE in normoglycemic mice reveladed migration of keratinocytes on day 4 and their proliferation in the following days, with a significantly (p<0.005) increased amount of cells in metaphase after 7 days of treatment. The collagen type I fibers were formed more rapidly on treatment with CE. PAS showed that there was CE permeation through the skin. With relation to the proteins, VEGF was stimulated at day 4 and SOD-2 and COX-2 at day 7. The results suggest that CE promoted the regulation of proteins, early angiogenesis and contributed to accelerate the processes involved in wound healing. This led to an increase in re-epithelialized surface with significant mitotic proliferation. The maturation of collagen fibers was increased, which could influence the strength of the extracellular matrix. Accordingly, the results obtained from P. pluviosa crude extract might become an efficient and promising herbal for enhancing the wound healing process.
Keywords: Poincianella pluviosa, in vitro keratinocytes and fibroblasts, in vivo wound healing, normoglycemic and diabetic rats.
Thesis PDF: Fernanda Giacomini Bueno
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