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Assessment of nutraceutical potential of a chestnut by-product towards circular economy – In-vitro versus in-vivo studies
* 1 , 1 , 2, 3 , 4 , 4 , 5, 6, 7 , 2, 3 , 2, 3 , 8 , * 8
1  REQUIMTE/LAQV - Instituto Superior de Engenharia do Porto
2  Nutrition, Food Science and Gastronomy Department, School of Pharmacy and Food Science, University of Barcelona, 08028 Barcelona, Spain
3  Consorcio CIBER, M.P. Fisiopatología de la Obesidad y la Nutrición (CIBERObn), Instituto de Salud Carlos III (ISCIII), Madrid, Spain
4  Department of Pharmaceutical and Pharmacological Sciences, University of Padova, Via Marzolo 5, 35121 Padova, Italy
5  i3S, Institute for Research and Innovation in Health, University of Porto, 4200-135 Porto, Portugal
6  INEB, Institute of Biomedical Engineering, University of Porto, 4200-135 Porto, Portugal
7  CESPU, Institute for Research and Advanced Training in Health Sciences and Technologies, 4585-116 Gandra, Portugal
8  REQUIMTE/LAQV, Polytechnic of Porto, School of Engineering, 4249-015 Porto, Portugal
Academic Editor: Adriana Borriello


The increasing demand for nutraceuticals has pressured the industry to seek new pro-healthy compounds. Agro-industrial residues generated across the food supply chain have been explored as sources of added-value molecules, reducing their environmental impacts, and creating additional economic revenue towards the implementation of circular economy [1]. Chestnut (Castanea sativa) shells (CS) are an exceptional source of antioxidants [2,3]. Although the European legislation on nutraceuticals’ validation remains vague, an in-depth assessment of in-vitro and in-vivo bioactivity should be accomplished, with metabolomics arising as a valuable tool. This study explores the nutraceutical potential of CS extract prepared by Subcritical Water Extraction (SWE) after in-vitro simulated digestion and in-vivo bioavailability assays on rats (50 and 100 mg/kg body weight, per os administered once daily for 7 days) using metabolomic techniques by LC-ESI-LTQ-Orbitrap-MS and LC/DAD-ESI-MS. Antioxidant activity was evaluated by spectrophotometric assays, while hypoglycemic and neuroprotective properties were assessed using commercial kits. The extract optimization and safety on intestinal cells were attested in our previous work [2]. The relationship between metabolomic fingerprinting and potential oxidative stress biomarkers was ascertained by multivariate analysis.

The results unveiled higher phenolic concentrations retained after intestinal digestion, reaching 40% of bioaccessibility. The metabolomic profiling sustained the antioxidant, hypoglycemic, and neuroprotective effects observed before and after in-vitro digestion probably ascribed to phenolic acids and hydrolyzable tannins metabolites. Considering animal studies, metabolites from phase I and II reactions were identified in rat tissues, mainly derived from phenolic acids, flavonoids, and lignans. The multivariate analysis predicted the outstanding contribution of phenolic metabolites to the in-vivo antioxidant responses. This study supports the use of CS extract as potential anti-aging ingredient for prevention of oxidative stress-triggered diseases in nutraceuticals.

Keywords: chestnut shells; nutraceutical use; metabolomics; in-vitro digestion model; in-vivo assays.