2016 - Andressa Blainski
Ph.D. Thesis
Author: Andressa Blainski Pinha
Postgraduate Program in Pharmaceutical Sciences - State University of Maringá
Area of Knowledge: Pharmacognosy
Date of Defense: October 20, 2016
Advisor: Prof. Dr. João Carlos Palazzo de Mello
Examination Board: Prof. Dr. Andreas Hensel
Proaf. Dra. Denise Brentan da Silva
Prof. Dr. Galdino Andrade Filho
Profa. Dra. Mara Lane Carvalho Cardoso
Title: Pharmacognostic Quality Control with Quantitative Chromatographic Analyses, Statistical Mixture Design with Antibacterial Evaluation and Phytochemical Study of Limonium brasiliense (Baicuru).
Abstract: Limonium brasiliense (Boiss.) Kuntze (Plumbaginaceae), known as baicuru, is a native plant of the south coast of Brazil and the region of Rio da Prata. Its rhizomes are employed mainly in the treatment of premenstrual syndrome, menstrual disorders and genitourinary infections. Previous studies have reported a large concentration of the phenolic compounds, tannins and flavonoids in the genus Limonium. The latest works show antiestrogen effect in immature female rats and low toxical effect in non-clinical models. In present work, pharmacognostic quality control data from three different materials were compared in order to collaborate with the determination of minimum quality parameters for this species. It was analyzed pharmacopoeial and non- pharmacopoeial parameters such as: particle size (d50 = 0.21 to 0.48 mm), loss on drying (11.1% to 12.4%), total ash (4.9% to 5.7%), dry residue from acetone:water (7:3, v/v) extract (30.6% to 39.5%), total polyphenol content (8.5% to 15.8%) and chromatographic fingerprint by high performance liquid chromatography (HPLC). The crude extract and its ethyl acetate and aqueous fractions shown a good antioxidant potential (IC50 = 6.87, 5.91 and 6.92 μg/mL, respectively). The chromatographic fingerprint was obtained by thin-layer chromatography and by HPLC from a developed and validated method for the separation of polyphenols and quantification of gallocatechin (GC) and epigallocatechin (EGC) in ethyl acetate fraction. The analytical method was specific, precise and accurate. The statistical mixture design (simplex centroid) evaluated the influence of the solvents (water, methanol, acetone and ethanol). A confirmatory experiment was tested with acetone:water (7:3, v/v). Statistical models can be used to predict the total polyphenols and GC content. For dry residue, it was not possible to determine a predictive model even after testing the full cubic model. For EGC, there is a higher (about 20 fold) selectivity of water for phenols of lower molecular weight. The simplex centroid results showed variety response: the highest dry residue was provided for water:acetone:ethanol mixture (36.6%±0.96 [2.6%]), polyphenols content for pure acetone (37.5%±0.46 [1.2%]) and gallo- and epigallocatechin for pure water (7.9%±0.39 [5.0%] and 19.6%±0.69 [3.5%], respectively). Principal component and hierarchical clustering analysis of the chromatographic fingerprints were disturbed by displacement retention time of some peaks, but the ultraviolet spectra from each peaks indicate the presence homogeneously of flavan-3-ols characteristic to tannins. The mass spectrometry analysis (LC-DAD-MS) confirmed the presence of gallic acid, gallocatechin, epigallocatechin and prodelphinidins gallate. Besides, the extracts produced by simplex centroid were tested for activity against to Vancomycin Resistant Enterococcus faecium (VREfm), Methicillin Resistant Staphylococcus aureus (MRSA) and Klebsiella pneumoniae Carbapenemase (KPC)-producing K. pneumoniae. The ethyl acetate fractions showed better potential than crude extracts, in special from the ternary extracts of water:acetone:ethanol against VREfm (19 μg/mL) and of methanol:acetone:ethanol against MRSA (39 μg/mL). A moderate activity was noted against KPC from different ethyl acetate fractions (625 μg/mL). The phytochemistry study enabled the isolation and identification of phenolic substances, such as myricetin-3-O-α-L-rhamnoside, myricetin-3-O-β-D-galactoside-6̎-gallate, gallocatechin, epigallocatechin, epigallocatechin-3-O-gallate, epigallocatechin-3-O-gallate-(4β→8)-epigallocatechin-3-O-gallate and monomers and B-Type dimers with doubly bonded structures as epigallocatechin-3-O-(2'→O→4")-gallate, epigallocatechin-3-O-(2'→O→3")-gallate (4β→8) epigallocatechin-3-O-gallate and samarangenin B.
Keywords: Limonium brasiliense, HPLC/UV-Vis, simplex centroid, multidrug-resistant bacteria, NMR of tannins, pharmacognostic control.
Thesis PDF: Andressa Blainski
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