Título da Dissertação: Atividade antioxidante e antimicrobiana do polissacarídeo da erva mate (Ilex paraguaniensis).

Orientadora: Profa. Dra. Rosane Marina Peralta

Data da Defesa: 19/02/2018



INTRODUCTION: The yerba mate (YM) (Ilex paraguariensis A. St. Hil.) is associated with numerous health benefits among which it is important to mention its antioxidant properties. Different chemical components responsible for the effects of the beverage have been identified. A polysaccharide from yerba mate leaves was purified and its chemical structure identified as a rhamnogalacturonan I (RG-I). Natural polysaccharides including rhamnogalacturonan-I (RG-I) and rhamnogalacturonan-II (RG-II) type pectins from different origins have gained the attention in the last years due to a variety of pharmacological and biological activities. The objective of this work was to evaluate the antioxidant, antimicrobial and cytotoxic potentials against tumoral cells and porcine liver cells of this polysaccharide.
METHODOLOGY: One hundred (100) gram of dried powdered mate were extracted twice with of ethanol. The residue was subjected to a hot extraction at 80º C, for 15 min, and autoclaved for 20 min. The soluble fraction was precipitated with 3 volumes of ethanol and centrifuged. To remove the protein fraction, the material was treated using the Sevag method, dialyzed for 24 h against distilled water and removed DNA contamination. The DNA-free polysaccharide was precipitated with ethanol and centrifuged. The presence of phenolics and proteins in the yerba mate polysaccharide was evaluated by using the Folin-Ciocalteu method and Bradford´s method. The UV-Vis spectra of the solution between 190 and 800 nm, and the FTIR analysis were obtained using an average of 128 scans in the range between 500 cm-1 and 4000 cm-1 with a spectral resolution of 2 cm-1. Five different methods were used to evaluate the antioxidant activity of the YM polysaccharide: reduction power of the ferric ion (FRAP), oxygen radical absorbance radical assay (ORAC), reduction of the 2,2-diphenyl-1-picrylhydrazyl radical (DPPH), reduction of the 2,2-azino-bis (3-ethylbenzothiazoline-6-sulphonate) cation (ABTS), and hydroxyl radical scavenging activity. Standard curves were constructed with trolox (r2 = 0.99) and the results were expressed as mmol trolox equivalents (TE)/mg lyophilisate material. The results were expressed as IC50 values. The antimicrobial activity evaluation were realized with four Gram-negative bacteria, four Gram-positive bacteria and Seven fungal isolates. MIC determinations were performed by the microdilution method and the rapid p-iodonitrotetrazolium chloride (INT) colorimetric assay. The cytotoxicity activities was performed in human tumor cell lines and in non-tumor liver cells primary culture. The results were expressed as GI50 values. Three repetitions of the sample and triplicates for each concentration were carried out in all the assays. The results were reported as mean ± standard error.
RESULTS AND DISCUSSION: An amount of 1.2 g of the dried polysaccharide was obtained starting from 100 g of yerba mate. Phenolics and proteins were not detected by chemical methods. Significant contamination by proteins, peptides, DNA and phenolics is thus unlikely. FTIR spectroscopy identifying vibrations and glycosidic linkages typical of polysaccharide structures. The IC50 values of the scavenging activities were 1.25±0.10 mg/mL, 0.41±0.05 mg/mL and 3.36±0.31 mg/mL, to DPPH, ABTS and hydroxyl assays, respectively. FRAP and ORAC, expressed as equivalents of Trolox, were 20.84±1.61 μM TE/mg and 556.30±12.83 μM TE/mg, respectively. The mechanism by which polysaccharides act as antioxidants is still not a consensus among researchers. While some researchers strongly relate the structures of the polysaccharides to their antioxidant activity, others suggest that the antioxidant activities of complex carbohydrates should be attributed to their phenolic and protein components or contaminants rather than to the carbohydrate moieties, especially when the analyses were carried out using crude or semi-purified polysaccharides. In general, functional groups such as –OH, –COOH and C=O, largely found in polysaccharides in addition to anionic and cationic functional groups, such as uronic acids, have been related to the antioxidant activities of polysaccharides. The polysaccharide had prominent antibacterial effects against Gram-negative bacteria and against Gram-positive bacteria. The polysaccharide had also prominent antifungal effects. It was reported elsewhere that the YM polysaccharide prevents lethality caused by polymicrobial sepsis in mice, an action that was attributed to a reduction in neutrophil infiltration. The mechanisms involved in the antimicrobial activity of polysaccharides are worthy of further investigations. The YM polysaccharide showed antibacterial activity against all tested strains, except to E. coli. This result suggests that the inhibition of iron absorption by the bacteria is a possible mechanism of the antibacterial activity of polysaccharides. No toxicity of the YM polysaccharide against normal (non-tumor) porcine liver primary cells was observed. The same is valid for the four tumoral human cell lines, MCF-7, NCI-H460, HeLa and HepG2 (GI50 > 400 mg/mL). A polysaccharide can act directly on the tumor cells or by enhancing the immune function of the organism, exerting thus an indirect antitumor activity. Different rhamnogalacturan I and II type polysaccharides (RG-I and RG-II) have been described as possessing potentially antitumoral activites. Our results suggest that the YM polysaccharide does not act directly on the tumour cell. However, it could be able to act indirectly if one takes into account what was found for the RG-II-type polysaccharide from green tea leaves, presented antitumor and anti-metastatic activities via activation of macrophages and natural killer cells. Additional approaches are necessary to clarify this question.
CONCLUSION: The results indicate that the YM polysaccharide possesses antioxidant activities corroborated by different in vitro methods. The YM polysaccharide also presents antibacterial, antifungal and cytotoxic activities, what helps to explain mitigation of sepsis caused by this macromolecule. For these and other reasons the YM polysaccharide can be considered to be potentially useful for the pharmaceutical and food industries.
KEY WORDS: antioxidant activity, antimicrobial activity, antitumoral activity, polysaccharide, yerba mate.

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