Natural Aspirin-Like Compounds from White Willow (Salix alba) Bark Extract Prevent Structural Changes of Human Hemoglobin during In Vitro Non-Enzymatic Glycation and Fructation, Preserving Its Peroxidase and Esterase Activity

Abstract

Proteins undergo continuous changes under the action of various intrinsic and extrinsic factors, leading to alteration of several intracellular metabolic pathways and the development of various clinical disorders. Non-enzymatic glycosylation is one of the main factors responsible for the progression of diabetic complications and the aging process. Although there are currently many effective therapies in the prevention and treatment of these diseases, in the last decade, there has been an increasing trend of replacing synthetic drugs by natural compounds, in order to reduce the side effects that may occur and the production costs. It is well known that aspirin (acetylsalicylic acid) inhibits the glycation process of serum proteins by acetylating N-terminal amino groups and lysine residues in their structure. Therefore, the main purpose of our research was to analyze the non-enzymatic glycation and fructation process of hemoglobin through spectrometric and electrophoretic techniques, in order to reveal how this process could influence the three-dimensional structure and biological function of the protein, and the effect of some natural aspirin-like compounds on the peroxidase and esterase activity of hemoglobin during fructose and glucose binding. In this way, a preliminary phytochemical characterization of a bark extract of white willow (Salix alba) was performed in order to evaluate the content of total phenolics, flavonoids, and salicylic derivatives, as well as the antioxidant activity. Then, human erythrocytes isolated from whole peripheral blood were incubated with different concentrations of fructose/glucose (10, 50, 100 mM) and S. alba extract for 5, 7, 10 and 14 days. The results obtained from the THz spectra confirmed that fructose was more reactive than glucose, so the glycation process took place more slowly than fructation. Also, the presence of S. alba extract showed an antiglycosylating effect, but not a total inhibition of the glycation process. In addition, enzymatic determinations proved that willow bark extract restored the peroxidase and esterase activities to the control levels. Our data indicated that salicylic compounds can be successfully used as substitutes for aspirin, one of the main synthetic compounds with anti-inflammatory and anti-glycosylating roles. Salicin, salicylic acid and other salicylic compounds possess strong antioxidant properties, which give them the ability to participate in the glycosylation process to block the formation of advanced glycation end products (AGEs).