Antioxidant and Anti-inflammatory Properties of Peanut Skin Extracts

Peanut skins have been regarded as a low economic waste by-product of the peanut industry but they contain high levels of bioactive compounds including catechins and procyanidins, which could benefit human health. The antioxidant activities of peanut skins have been reported but, no attempt has been undertaken to study the relationship between antioxidant and anti-inflammatory properties of peanut skins. The purpose of this research was to (i) evaluate the antioxidant activity, (ii) investigate anti-inflammatory properties and (iii) identify the mechanism of action that may be responsible for peanut skin's anti-inflammatory activity in vitro. To achieve an optimized amount of phenolic compounds, polar solvents such as aqueous ethanol or acetone were used for extraction purposes. Antioxidant activity was evaluated using Hydrophilic Oxygen Radical Absorbance Capacity (H-ORAC) and Total Phenol analyses. Peanut skin extracts (PSE) extracted with acetone/water had similar ORAC and total phenolic values (3060 micromol Trolox/100 g and 290 mg GAE/g) than PSE extracted with ethanol/water (2620 micromol Trolox/100 g and 250 mg GAE/g, respectively). A RAW 264.7 mouse macrophage cell line was used to examine the anti-inflammatory properties of PSE. RAW 264.7 cells were treated with three concentrations of PSE (1, 2.5 and 5% (v/v)) and induced with an inflammatory marker, lipopolysaccharide (LPS). Western blotting analysis of COX-2 expression was significantly affected by PSE. PGE2 was measured by enzyme linked immunosorbant assay (ELISA). Increasing concentrations of PSE induced with LPS showed a decrease in PGE2 concentration. This suppression of pro-inflammatory markers was found to be dose-dependent and demonstrates that PSE has anti-inflammatory properties. To investigate the mechanism of action, the anti-inflammatory effects of PSE on inducible nitric oxide synthase (iNOS) and nuclear factor-kappa B (NF-kappaB) were identified as pathways of interest, because of its role as starting the onset of inflammation. We also wanted to investigate if iNOS and NF-kappaB could be further inhibited at a longer incubation, 12 and 18 hours. The Greiss assay was used to determine nitrite levels in culture media. There was an increase in NO production between 12 and 18 hours when RAW 264.7 macrophages were stimulated with the positive control LPS, however, PSE reduced NO production in a dose-dependent manner suggesting that inhibition had occurred. Isolation of nuclear fractions was performed for the NF-kappaB transcription factor assay. PSE significantly decreased NF-kappaB activity between 12 and 18 hours by inhibiting the RelA (p65) heterodimer. PCR analysis of PSE showed nucleotide changes compared to the published iNOS sequence, suggesting that a single nucleotide polymorphism (SNP) was present Therefore, a suggested molecular mechanism of action for PSE would be the inhibition of NF-kappaB activity. This study highlights the need to investigate the relationship between antioxidant and anti-inflammatory properties of a waste by-product that has a potential use as an anti-inflammatory agent.