2013 - Ana Luiza Sereia
MSc. Thesis
Author: Ana Luiza Sereia
Postgraduate Program in Pharmaceutical Sciences - State University of Maringá
Area of Knowledge: Pharmacognosy
Date of Defense: February 26, 2013
Advisor: Prof. Dr. João Carlos Palazzo de Mello
Examination Board: Prof. Dr. Claudio Celestino de Oliveira
Prof. Dr. Gisely Cristiny Lopes
Title: Development of analytical method by capillary electrophoresis for identification and quantification of polyphenols in a semipurified fraction of Trichilia catigua A. Juss. (Meliaceae)
Abstract: The species Trichilia catigua A. Juss., popularly known as “catuaba”, shows several biological activities including as an antioxidant, antinociceptive, vasodilator (aphrodisiac), anti-inflammatory and antidepressant. Considering the economic and scientific interest of this species, the physicochemical characterization of the bark, a crude extract (EB) and an ethyl-acetate fraction (FAE) of T. catigua was performed, and a methodology using capillary electrophoresis (CE) for the separation, identification and quantification of polyphenols in FAE was developed and validated. The pharmacopeia tests performed for physicochemical quality control of the bark were: determination of foreign matter (DME), water content (UMI), total ash (CT), acid-insoluble ash (CIA), granulometric analysis (AG) and determination of extractives (TE). The results were: DME: 25.9%, UMI: 11.61% ± 0.25 (CV%=2.17), CT: 5.15% ± 0.066 (CV%=1.29), CIA: unquantifiable, AG: d50 0.237 mm and TE: 25.99% ± 0.87(m v-1) (CV%=3.34). The bark, EB and FAE were submitted to quantitative analyses to determine the contents of total polyphenolics (PT) and total tannins (TT), and antioxidant activity using the DPPH scavenging method (IC50). The PT contents found in the bark, EB and FAE were 9.54% ± 0.24 (CV%=2.50), 42.34% ± 0.27 (CV%=0.63) and 68.86% ± 1.79 (CV%=2.60), respectively. The TT contents were 7.07% ± 0.07 (CV%=0.93), 31.42% ± 0.37 (CV%=1.16) and 49.72% ± 2.10 (CV%=4.22), in the same order. The IC50 (µg ml-1) found for EB and FAE were 6.72 ± 0.28 (CV%=4.17) and 4.47 ± 0.07 (CV%=1.58), respectively. For the development of the EC analytical methodology, the influence of the variables wavelength, voltage, concentration and pH of borate buffer and the use of cyclodextrins (CDs) as additives were evaluated. The sample solution was subjected to solid phase extraction. The electrophoretic conditions that enabled the separation of the peaks of catechin, epicatechin, chrologenic acid, PB1, PB2, and cinchonains Ia, Ib, IIa and IIb were: fused silica capillary of 375 µm D.E. x 50 µm D.I. with length of 50/60.2 cm, borate buffer 80 mmol l-1; pH 8.80; 2-hydroxypropyl-β-CD 10 mmol l-1; 30 kV for 5.7 min, 5 kV for 2 min and 30 kV for 7.3 min at 25 ºC; injection: 5 s (0.5 psi); detection: 214 nm; FAE: 1 mg ml-1. The method thus developed showed selectivity and linearity over the range 20-120 µg ml-1 of gallic acid (internal standard), repeatability 2.53%, intermediate precision 2.92%, accuracy 100.46%, limits of detection 5.19 µg ml-1 and limits of quantification 17.29 µg ml-1. The method was not robust under the conditions evaluated. The sample solution and the gallic-acid solution showed stability below 72 h, at room temperature or under refrigeration. This method is the first to use CE technology for quantification of polyphenols in a semipurified fraction of T. catigua.
Keywords: Trichilia catigua; analytical development; capillary electrophoresis; polyphenols
Dissertation PDF: Ana Luiza Sereia
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