Phytochemistry and bioprospection of antioxidant and anti-inflammatory activities from unexplored Brasilian native fruits.

Abstract

In the search for alternative bioactive molecules, Brazilian native fruit species are part of a rich collection of unexplored substances, primarily for their high content of compounds with biological properties. Some examples include carotenoids and flavonoids, which have promising antioxidant and anti-inflammatory activities and have been the subject of studies by our research group (FOP/UNICAMP and ESALQ/USP). There is a need for bioprospecting new molecules and understanding their anti-inflammatory mechanisms, as drugs currently available on the market have adverse effects that may prevent certain medical and dental treatments. Thus, the search for novel molecular patterns that modulate the inflammatory process through mechanisms differing from those of the classical pathways, is of paramount necessity and of great interest to the pharmaceutical industry. This study aimed to investigate the phytochemical profile of unexplored Brazilian native fruits and their antioxidant and anti-inflammatory activity. These properties are of medical and dental interest and add value to these rare exotic species, stimulate their consumption to promote health, and lead to discovery of new compounds, which may generate a functional / therapeutic product with additional social and economic developments. Five Brazilian native fruit species were selected, namely: Psidium myrtoides (Araçá-icica); Citharexylum myrianthum (Jacatuaba); Smilax quinquinervia (Pitanga amarela de cipó); Eugenia stipitata (Araçá-boi) and Byrsonima lancifolia (Muricí cuaçú). The fruits will be harvested at pre-determined times, their pulps processed to obtain hydroethanolic extracts (80:20, v/v), lyophilized and stored at -20°C. The five extracts will be submitted to phytochemical analysis by GC/MS, HPLC/DAD, LC-MS/MS and tested for their antioxidant activity (peroxide and hypochlorous acid sequestering capacity, superoxide radical, ORAC and antioxidant on line activity by HPLC-DAD-UV). The anti-inflammatory activity of all extracts will be evaluated through cytotoxicity testing in RAW 264.7 macrophages and analysis of gene expression related to inflammation. The extract(s) showing the best activity will be selected for further analysis (1st selection). The selected extract(s) will be submitted to in vitro assays of neutrophil chemotaxis, expression of adhesion and rolling molecules, and determination of inflammatory mediators in RAW 264.7 cells. The extracts with the best activity will be selected (2nd selection) and submitted to fractionation using silica gel, resulting in 6 fractions per extract with different polarity degrees. The extracts and their corresponding fractions will be submitted to biomonitoring, besides secretome and microRNA analysis, aiming a new selection (3rd selection), which will determine the most active extract/fraction in in vivo preclinical testing in mice: carrageenan-induced neutrophil migration, expression of adhesion and rolling molecules, measurement of inflammatory mediators and intravital microscopy. Finally, the compounds present in the selected fractions will be identified by chromatographic techniques (GC/MS, HPLC/DAD and LC-MS/MS). Thus, this project is expected to provide information about the phytochemical profile and antioxidant and anti-inflammatory properties of native fruit species, add value to the Brazilian flora diversity, stimulate interest to the agro-industrial production and exploitation of the functional properties of native fruits.