The highest levels were observed at the end of the first month of storage, except for the fruits treated with CaCl(2), where the maximal values were achieved at the end of the second month.
The storage time as well as the fruit treatment prior to storage influenced total anthocyanin content. The anthocyanin levels were quantified by either comparison with an external standard of cyanidin 3-rutinoside (based on the peak area) or individual calculation from the peak areas based on standard curves of each anthocyanin type. The fruits were exposed to cold storage ( $5^$ C) after the following treatments: spraying with wax spraying with $1.5$ % CaCl(2) spraying with wax and $1.5$ % CaCl(2) covering boxes with 25 $\mu$ c thickness low-density polyethylene film. The concentration of anthocyanins in fruits of "Assaria" pomegranate, a sweet Portuguese cultivar typically grown in Algarve (south Portugal), was monitored during storage under different conditions. Consequently, grape seed extracts at 4% and 20% may be useful as antibacterial agents to prevent the deterioration of food products. The grape seed extracts at 1% and 2% concentrations were also ineffective.
The grape bagasse extracts and methanol (control) had no inhibitory effects on the fifteen bacteria tested. subtilis, while the acetone:water:acetic acid (90:9.5:0.5) extract at 4% was effective against most of the test bacteria. The acetone:water:acetic acid (90:9.5:0.5) extract was found to be more effective than the other one, whose extracts at 4% concentration were inactive against A. The grape seed extracts at 20% concentration inhibited all the bacteria except B. These extracts at 1%, 2%, 4% and 20% concentrations were tested for their antibacterial effects by using the paper disc diffusion method against some food spoilage and pathogenic bacteria including Aeromonas hydrophila ATCC 7965, Bacillus amyloliquefaciens ATCC 3842, Bacillus brevis FMC 3, Bacillus cereus FMC 19, Bacillus megaterium DSM 32, Bacillus subtilis IMG 22, Enterobacter aerogenes CCM 2531, Enterococcus feacalis ATCC 15753, Escherichia coli DM, Klebsiella pneumoniae FMC 5, Listeria monocytogenes Scott A, Mycobacterium smegmatis RUT, Proteus vulgaris FMC 1, Pseudomonas aeruginosa ATCC 27853 and Staphylococcus aureus COWAN 1. The bagasse extracts contained 45.44 mg GAE/g and 29.55 mg GAE/g with ethyl acetate:methanol:water (60:30:10) and ethanol:water (95:5), respectively. In the grape seeds extracts the content of total phenolic compounds was found to be 627.98 mg gallic acid equivalent (GAE)/g with acetone:water:acetic acid (90:9.5:0.5) and 667.87 mg GAE/g with ethyl acetate:methanol:water (60:30:10). Powdered grape seeds and bagasse were extracted with petroleum ether to remove fatty material and then re-extracted with different solvent mixtures to determine their total phenolics and antibacterial activities. Taken together, our results demonstrated that WGP exerts potent anti-inflammatory activity through the inhibition of iNOS and COX-2 by regulating NF κ B and p38 MAPK pathway. Furthermore, we found that WGP inhibited LPS-induced phosphorylation of p38 mitogen-activated protein kinase (MAPK). Moreover, WGP prevented nuclear translocation of nuclear factor- κ B (NF κ B) p65 subunit by reducing inhibitory κ B- α (I κ B α ) and NF κ B phosphorylation. Consistently, WGP significantly reduced LPS-stimulated expression of proinflammatory cytokines such as tumor necrosis factor α (TNF- α ) and interleukin- (IL-) 1 β. Our data indicate that WGP significantly reduced NO, PGE2, and ROS production and also inhibited the expression of proinflammatory mediators such as inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) protein expressions. We used nitric oxide (NO) and prostaglandin E2 (PGE2) and reactive oxygen species (ROS) assays to examine inhibitory effect of WGP and further investigated the mechanisms of WGP suppressed LPS-mediated genes and upstream expression by Western blot and confocal microscopy analysis. In the present study, the anti-inflammatory effect and underlying mechanisms of wild grape seeds procyanidins (WGP) were examined using lipopolysaccharide- (LPS-) stimulated RAW 264.7 cells.
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