IN VITRO TOXICOLOGICAL EVALUATION STRATEGIES FOR PEANUT SKIN EXTRACT FOR UPCYCLING

Peanut (Arachis hypogaea L.) is the second most cultivated legume worldwide. It is widely used to produce oil, butter, and paste, generating approximately 1 million tons of peanut skin waste annually. Although often discarded as an agro-industrial byproduct, peanut skin is a recognized source of phenolic compounds. However, the process of upcycling faces challenges related to safety, stability, and regulatory compliance. Peanut skin has shown high cytotoxicity in cell viability assays in our previous work, raising doubts regarding its safety as food ingredient. Although the quantification of exogenous agents was found to be within legal limits, the impact of their endogenous compounds is unclear. Therefore, this project aims to evaluate the cytotoxicity in Caco-2 cells and bioactive profile of peanut skin extract from processed peanuts before and after in vitro gastrointestinal digestion as a mitigation strategy for potential toxic fractions. A hydroethanolic extract (HEE) from the high-oleic Runner variety was produced and then subjected to in vitro digestion using the INFOGEST protocol, yielding digested hydroethanolic extract (DHE). Afterward, these extracts (HEE and DHE) were characterized by analyses of total phenolic content, showing 422.8 µg of GAE/mg extract and 92.9 µg of GAE/mg digested extract, respectively, resulting in 21.2% phenolic bioaccessibility and ORAC assay to assess the potencial antioxidant, showing 3306.1 µmoL of Trolox/mg extract and 959.5 µmoL of Trolox/mg digested extract, respectively. Studies suggest that intestinal conditions such as duodenal alkaline pH and interactions with enzymes and bile salts compromise the stability and solubility of phenolic compounds, reducing their bioaccessibility. Consequently, this degradation lowers polyphenol content and antioxidant capacity during the intestinal phase, as shown above. Subsequently, the samples were undergone in vitro toxicological evaluation through cell viability by MTT assay. Regarding in vitro toxicological evaluation through assays of cell viability, the digested extracts exhibited lower cytotoxicity than the undigested extracts, as the concentration of compound required to inhibit 50% of a biological process in vitro (IC50 - Half-Maximal Inhibitory Concentration) presented a concentration of 31.05 µg/mL for HEE, whereas the DHE showed IC50 at a concentration of 132.7 µg/mL. In conclusion, simulated digestion decreased the phenolic compound content and consequently the antioxidant capacity of the peanut skin extract compared to the undigested extract due to lower bioaccessibility; however, it also reduced cytotoxicity, as indicated by the IC50 values. This reduction in cytotoxicity allows the addition of a higher concentration of digested extract (DHE) than undigested extract (HEE) without inducing cell damage. Furthermore, compared to the digestion control, DHE demonstrated higher cell viability, suggesting a protective effect of the peanut skin extract on intestinal cells.

Acknowledgments: CAPES - Finance Code 001, PROEX/CAPES and FAPESP - project Grant number: 2020/07015-7.