Effects of plant-derived polyphenols on glycation and oxidative stress related to vascular diseases

There is a large amount of evidence indicating that oxidative stress and glycation play a crucial role in aging as well as in neurodegenerative and cardiovascular diseases. Oxidative stress is linked with reactive oxygen species (ROS), whose harmful effects can lead to several pathological conditions like vascular alterations, diabetes complications and inflammation. Moreover, ROS and oxidative stress can directly contribute to glycation reactions with the formation of reactive α-oxoaldehydes and advanced glycation end-products (AGEs). The glycation process is characterized by non-enzymatic reactions between aldehyde or keto groups of sugars and free amino-residues of proteins, which lead to the formation of AGEs. Glycation and AGEs cause the irreversible modification of the proteins structure and the consequent loss of their functionality, causing detrimental effect in vasculature. Thus, it can be reasonable that compounds which counteract the effect of oxidative stress and glycation may assume an important role in the prevention of vascular damage. Natural phenolic compounds and flavonoids have received attention for their biological effects, such as antioxidant and antiglycative activities. Despite the health claims of polyphenols, it is known that their bioavailability can vary dramatically depending on many factors. The effects of the polyphenols, and of their in vivo circulating metabolites, will ultimately depend on the extent to which they associate with cells, either by interactions at the membrane or more importantly by their cellular uptake. Therefore, in order to assess the potential activity of natural polyphenols, it is primarily important to evaluate the intestinal permeability and bioavailability. In this research, human colorectal carcinoma cells (HT-29) were used as experimental model of intestinal cells, to evaluate the polyphenols ability to cross the cellular membrane and permeate into the cell. The polyphenols studied in this research are characteristic phytoconstituents of several medicinal plants widely used in traditional medicine such as: baicalein (from Scutellaria baicalensis G.), eupatorin (Eupatorium semiserratum DC.), galangin (Alpinia officinarum L.), magnolol (Magnolia officinalis L.), myricetin (Myrica rubra S. Z.), oleuropein (Olea europaea L.), and silybin (Silybum marianum L.). The aims of the PhD project were firstly to evaluate the HT-29 intestinal cells permeability of natural compounds, and secondly, to investigate the in vitro antiglycation activity of polyphenols and their activity against endothelial cellular damage related to oxidation and glycation, using HUVEC cells as experimental model for endothelium function. The results showed that the ability of polyphenols to cross the HT-29 cell membrane depends on many factors, including molecular size and hydrophilic properties of each compound. The flavonoids baicalein, eupatorin and galangin were able to cross the HT-29 cell membranes and undergo biotransformation process; conversely, oleuropein seemed to not been taken up in the HT-29 cells. The results of AGEs determination showed that most of the polyphenols, at the higher concentration, displayed antiglycative activity in all experimental conditions, and that baicalein, eupatorin and galangin showed the highest activity in counteract the AGEs formation. In the experimental model involving HUVEC, the aim was to evaluate the potential role of several compounds related to hyperglycemia and glycation, such as glycated albumin, glyoxal and glucose at high concentration, in inducing cytotoxicity and oxidative stress. Moreover, the antioxidant activity of the selected natural compounds against the oxidative stress induced by those factors was assessed using the DCFH-DA assay. The results from ROS detection assay showed that the glycation products known as glycated albumin and glyoxal were not able to directly induce oxidative stress in HUVEC in the assay conditions, whereas glucose at high concentration displayed an effect of oxidative stress induction. Therefore, high glucose was used as oxidative stressor in the polyphenols evaluation assay. Baicalein, eupatorin and galangin were selected as natural compounds with a potential antioxidant activity, and their activity on ROS formation in HUVEC induced by high glucose was evaluated. The highest antioxidant activity was seen after the pre-treatment with the polyphenols at the concentration of 5 μM. All together, the results obtained motivate further researches on the activities of polyphenols against oxidative and glycative damages in order to understand their potential role in the prevention of vascular diseases