Título da Tese: Efeitos inibitórios de bebidas preparadas com variedades de Camellia sinensis sobre a absorção de amido e triglicerídeos

Orientadora: Profa Dra. Rosane Marina Peralta

Data da Defesa:


 INTRODUCTION AND OBJECTIVES – Camellia sinensis is the plant that gives rise to green, black, white and oolong teas. The names are associated with the parts of the plant used and the post-harvest drying and fermentation processes. These beverages that are prepared with them have several beneficial health effects, which are attributed to their polyphenol content, mainly catechins and catechin derivatives, including (−)-epigallocatechin-3-gallate (EGCG), epigallocatechin (EGC) and epicatechin-3-gallate (ECG). C. sinensis extracts have been proposed to be useful in the prevention and/or treatment of various disorders, including cancer, cardiovascular disease, diabetes mellitus, and hepatitis. A new variety of C. sinensis produced in Kenya has attracted the attention of many researchers. Thanks to the high content of anthocyanins of this variety, the leaves have a purple color and the drink obtained by infusion is called purple tea. It is known that anthocyanins are phenolic compounds belonging to the flavonoid family, commonly found in red, purple and blue flowers, fruits and tubers. It has been suggested that regular consumption of anthocyanins may reduce the risk of various diseases such as dyslipidemia and diabetes, resulting in potential health benefits. Delayed digestion of starch by inhibiting intestinal amylases and alpha-glucosidase can be considered an appropriate treatment to control postprandial hyperglycemia. Likewise, delayed absorption of fatty acids from dietary triglycerides, due to inhibition of pancreatic lipase, can be considered a therapeutic approach to the control of dyslipidemia. The objectives of this work were: Chapter 1: to provide an up-to-date, comprehensive, and critical review of the inhibitory action of anthocyanins on the absorption of starch and triglycerides, mainly as a result of their ability to inhibit the digestive enzymes amylases and lipases. Chapter 2. Compare the inhibitory effects of different C. sinensis beverages on starch digestion using the mouse starch tolerance test. Chapter 3. Compare the phenolic profiles and various bioactivities (antioxidant, anti-inflammatory, cytotoxic, antibacterial activities) of beverages prepared from green tea and purple tea with emphasis on their effects on fat absorption.
METHODS- For the review work, we searched the specialized literature for studies that used anthocyanins in the treatment of diabetes, obesity and/or metabolic syndrome. The selected works were evaluated according to the type of research and classified as: classical methods for evaluating the inhibitory activity of anthocyanins on amylases and lipases and methods for evaluating the molecular interactions of inhibitors with enzymes. For the experimental work, that is, chapters 2 and 3, the following materials were used: 2,2-diphenyl-1-picrylhydrazyl (DPPH), 6-hydroxy-2,5,7,8-tetramethyl-chroman-2-carboxylic acid (trolox), thiobarbituric acid, sulforhodamine B, salivary and pancreatic amylases, pancreatic lipase, which were purchased from Sigma-Aldrich. Acarbose and orlistat were purchased from local pharmacies. Green, oolong, white and black teas were purchased from in the local market (Maringá, Brazil), and Kenyan purple tea (purple tea) was purchased from Justea.com, Vancouver, Canada. To carry out the experiments, the extracts were prepared mimicking consumption where the leaves were placed in contact with water for 15 minutes promoting infusion. The extracts were filtered, frozen and lyophilized and stored at -20 ºC until use. The extracts were characterized and quantified for their phenolic compounds profiles by HPLC-MS and the total anthocyanins content was quantified by the pH differential method. The inhibitory potential of the extracts on the pancreatic α-amylase was evaluated by in vitro and in vivo methods. In the in vitro method, the inhibitory effect on pancreatic alpha-amylase was evaluated by quantifying the release of reducing sugars from starch by the 3,5 dinitrosalicylic acid method in the absence and presence of different concentrations of the extracts. In in vivo assays, extracts derived from the various teas were administered to mice by gavage at doses of up to 500 mg/kg before starch administration. Acarbose, a classic inhibitor of starch hydrolysis and digestion, was administered in doses up to 50 mg/kg before starch. Plasma glucose was determined at 0, 30, 60, 90 and 120 min after starch administration. Blood samples were collected from the tail vein and analyzed using a glucometer (AccuChek®). For the study described in Chapter 3, experiments were carried out using only green and purple tea extracts. The extracts were analyzed for antioxidant activity, anti-inflammatory activity, cytotoxicity against four human tumor cell lines and antimicrobial activity. Pancreatic lipase inhibitory activity by purple and green tea extracts was evaluated by in vitro and in vivo methods. For the in vitro pancreatic lipase assay, p-nitrophenyl-palmitate was used as substrate. For lipase inhibition in vivo the extracts were administered by gavage to mice prior to the addition of an olive oil load. Plasma levels of triglycerides were determined at 0, 90, 180, 270 and 360 minutes after administration of olive oil. The determination of the concentration of triglycerides in blood was performed using an AccutrendPlus® Roche triglyceride meter. The inhibitory effects of the extracts were compared to those caused by orlistat. Statistical analyses of experimental data were performed using the GraphPad Prism (version 8.0) and Scientist software (MicroMath Scientific Software, Salt Lake City, UT).
MAIN RESULTS, DISCUSSION AND CONCLUSION – In chapter 1, it was concluded that in vivo studies and especially clinical studies are highly necessary to confirm and give relevance to the numerous data already obtained under in vitro conditions. Efforts are also needed to allow a more secure attribution of the observed effects to well-defined molecular species, since, in general, experiments have been conducted with extracts that contain a great diversity of compounds. In Chapter 2, it was observed that purple tea was the most potent inhibitor of pancreatic α-amylase, a property that caused a very significant reduction in starch digestion and consequent reduction in the postprandial glycemic peak. A stimulatory effect of α-amylase was observed in vitro at lower concentrations of green tea, which may explain its lower inhibitory effect on starch digestion in vivo. Our results allow us to conclude that purple tea exerts a beneficial postprandial antihyperglycemic action in healthy or diabetic individuals. In the third chapter, it was observed that in terms of the analyzed bioactivities (antioxidant, anti-inflammatory, antibacterial and antifungal) the extracts of green and purple teas were very similar. However, purple tea had a significantly superior inhibitory activity on the pancreatic lipase, causing a marked inhibition of triglyceride digestion. We can generally conclude that purple tea was an efficient inhibitor of starch digestion (Chapter 2) and triglycerides digestion (Chapter 3) when compared to other C. sinensis beverages. These results, when combined, suggest that regular consumption of purple tea may be helpful in managing obesity and diabetes.
Keywords: starch, amylases, anthocyanins, Camellia sinensis, digestive enzymes, lipases, triglycerides.

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