Investigation of the bio-active phytochemical constituents of highbush blueberry fruit (Vaccinium corymbosum L. cv. Bluecrop)

Blueberries are well known for their antioxidant property, due to the polyphenolics present in these fruits. The antioxidant activity of blueberries is usually measured by colorimetric assays involving metals such as iron and copper. We found that the interaction of the protein bound iron in Total Antioxidant Status (TAS) assay, and the amount of iron in highbush blueberries did not affect the antioxidant activity measured by the TAS assay. TAS assay and bathocuproine copper antioxidant (BCA) assay were both compared using ascorbic acid. Both the assays were efficient in assessing the antioxidant activity of ascorbic acid with a linear regression of R2> 90 whereas, BCA assay has lower limit for sample detection and is easy to use in 96-well plates compared to TAS assay based on our observations. Blueberries were also found to have various bioactivities such as bacterial anti-adhesion, anti-inflammatory, anti-angiogenic, anti-Alzheimers and anti-cancer properties. A schematic bio-assay guided fractionation was designed to find the inhibition of vascular endothelial growth factor (VEGF) production by blueberry polyphenolics and the activity was initially reported in whole blueberries extract (WBE) in both U937 and Eahy.926 cells, followed by highest activity in ethyl acetate fraction (flavonoidal), 70% acetone fraction (low molecular weight proanthocyanidin fraction), and 50% ethanolic fraction (proanthocyanidins and hydroxycinnamates) using enzyme linked immun-sorbent assay (ELISA). The major peak from the 50% ethanolic fraction with a distinctive wavelength lambdamax of 238.6 nm and 323.2 nm was separated and analyzed using UV, IR, LC-MS, GC-MS and NMR and was identified as ethyl ferulate. Ethyl ferulate is reported for the first time in blueberries. Traces of catechin, epicatechin and unknown peaks were found by GC-MS after silylation of 50% ethanolic fraction. Cytotoxicity studies were also performed on U937 for the effective inhibitory concentrations of the WBE fraction and were found to be non toxic. We also extracted the whole PAC (proanthocyanidin concentrate) separately from the highbush blueberries and assayed it for bacterial anti-adhesion activity and it was found to be active at a concentration of 94 microg/mL.