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2011 - Sandra Cristina Girotto Pinto

MSc. Thesis

Author: Sandra Cristina Girotto Pinto

Postgraduate Program in Pharmaceutical Sciences - State University of Maringá

Date of Defense: August 25, 2011

Advisor: Prof. Dr.  João Carlos Palazzo de Mello

Examination Board: Profa. Dra. Elaine Rosely Lepri

                            Profa. Dra. Maria da Conceição Torrado Truiti


Title: Stryphnodendron adstringens: phytochemistry and biological analysis in vitro and in vivo

Abstract: The extracts from the barks of Stryphnodendron adstringens have excellent wound healing activity demonstrated in healthy laboratory animals. This is a good indicator that the extract of this plant would have effective action in the treatment of chronic wounds in diabetics rats. The aim of this study was the isolation and structural identification of tannins of a semipurified fraction obtained from Stryphnodendron adstringens using different chromatographic methods, evaluation in vitro the antioxidant capacity of crude (CE) and semipurified extracts (Ethyl-acetate –EAF and Aqueous -AF Fractions) by free radical DPPH method, and evaluation the wound healing activity of CE in diabetic rats. The isolation of the compounds was performed by column chromatography, high speed countercurrent chromatography (HSCCC) and TLC. The identification of the compounds carried out by 1 13 1 spectroscopic methods of nuclear magnetic resonance (NMR) 1D ( H and C), 2D NMR ( H- 1 H COSY, HMQC and HMBC), mass spectrometry and optical. The antioxidant capacity was determined by DPPH assay, to evaluate the IC50 (µg/mL). The wound healing activity was assessed at 4, 7, 12 and 17 days of treatment with a gel containing 1% of CE across the length and thickness of the re-epithelialized area, analysis of skin permeation by photoacoustic spectroscopy, number of cells and fibers in metaphase collagen (types I and III). From column chromatography 21 subfractions were obtained. Subfractions F4, F5, F6 and F7 were chromatographed on HSCCC for separation of compounds. The compounds 4′- O-methyl-gallocatechin, gallocatechin, epigallocatechin and 4′-O-methyl-gallocatechin- (4α→8)-4′-O-methyl-gallocatechin were identified. The antioxidant capacity of the samples were: CE=3.99, EAF=3.47, AF=5.19 and vitamin C = 5.19, and the CE and the EAF have an antioxidant capacity about 1.3 and 1.5 times greater than vitamin C, respectively. The evaluation of wound healing activity showed the formation of a re-epithelialized area greater in animals treated with CE at 4 and 7 days and thicker at 17 days, as the proliferation of metaphases after 4 and 17 days. The stimulation of maturation of collagen fibers (type I) at 17 days of treatment was highest in treatment with the gel containing 1% CE. In assessing the wound healing activity, proved the permeation of EB in all periods analyzed and the gel containing EB showed activity on the tissue repair, providing more rapid and strong healing at 17 days of treatment with regard to control. The analysis by Western Blotting confirms the presence of larger blood vessels at 7 days in large quantities by the expression of VEGEFR.

Keywords: Stryphnodendron adstringens, Wound healing, Diabetes mellitus, Condensed tannins, Antioxidant capacity in vitro, VEGFR.



Dissertation PDFSandra Cristina Girotto Pinto

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