Abstract
Acetylcholinesterase and tyrosinase are two enzymes that are involved in Alzheimer’s disease and other neurodegenerative diseases. Acetylcholinesterase hydrolyzes acetylcholine, a neurotransmitter in the human brain into choline and acetate, resulting in the decline of acetylcholine levels at the synaptic cleft. Tyrosinase catalyzes the oxidation of tyrosine and dopamine, producing reactive oxygen and nitrogen species during the reaction that leads to oxidative stress and eventually resulting in cell death. Blueberries are micro-nutrient rich fruit that have many health benefits. Some of their health benefits include antioxidant, anti inflammatory, cardiovascular health, weight loss and more. Both polar and nonpolar components of blueberries contribute to their health benefits. Effective extraction methods will help identify the bioactive compounds as well as development of safe and effective healthy supplements derived from blueberries. For these reasons we applied three different solvent systems to extract the polar components of blueberries: mixed solvent of acetone, methanol, water, and formic acid(40:40:19:1 v/v/v/v); methanol only, and ethanol only. We demonstrated that blueberry extract can inhibit tyrosinase at the levels 0.17 mg/mL, 1.43 mg/mL, and 1.61 mg/mL for the solvent mixture, methanolic and ethanolic respectively as well as acetylcholinesterase at the levels of1.20 mg/mL, 3.04 mg/mL and 5.98 mg/mL for the solvent mixture, methanolic and ethanolic respectively. While the methanolic based extraction method showed the stronger inhibition overall on both enzymes, ethanolic extraction also showed significant inhibition, suggesting that the non-toxic ethanol can be used to effectively extract the biological active compounds from blueberries. Two blueberry sources were used to compare the differences of sources on polar components of blueberries: fresh berries from local market, and lyophilized whole berry powder from US Highbush Blueberry Council (USHBC). The extracts were analyzed using NMR to identify compounds in the six extracts: fresh fruit solvent mix, fresh fruit EDS, fresh fruit MDS,USHBC solvent mix, USHBC EDS and USHBC MDS. It was found that all extracts were similar at a 95% confidence interval using PCA to analyze the spectra from NMR. To further study the mechanism of inhibition on tyrosinase, we carried out the inhibition kinetics with the three blueberry extracts. This study demonstrated that tyrosinase inhibited all three blueberry extracts in a mixed manner. A similar study was performed on the pure compounds found in blueberries such as chlorogenic acid, gallic acid, quercetin, epigallocatechin gallate and catechin. These compounds can inhibit tyrosinase at levels of 8.34 mg/L, 4.55 mg/mL, 0.59 mg/mL, 0.40mg/mL, and 0.08 mg/mL respectively. Quercetin, epigallocatechin gallate and catechin were tested for their inhibition relationship with tyrosinase. All three compounds inhibited tyrosinase in a mixed manner, similar to our extracts. Molecular docking was performed using AutoDock Vina to study the enzyme and compound interaction.