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Biomarkers of anti-inflammatory and neuroprotective activity investigated by untargeted UPLC-ESI-QTOF-MS metabolomics analyses

Abstract

Screening for bioactive metabolites is frequently carried out in pharmaceutical drug discovery, and metabolomics arises as a modernized tool applied in the Natural Products (NP) research field for accessing plant metabolites through faster real-time analyses. Thus, this research aimed to perform a comprehensive LC/MS-based metabolomics study to investigate the anti-inflammatory potential of Ocotea (Lauraceae) species as well as the neuroprotective activity of the popularly known Ayahuasca beverage and its matrix plants. It is well known the chief role of PGE2 in acute inflammatory diseases, and recent publications have also indicated that higher COX 2 expression and increased circulating PGE2 levels play a significant role in cancer progression through lymphangiogenic growth. On the other hand, neural damage is involved in Parkinson disease (PD) progression and in other neurodegenerative diseases, and neuroprotective candidates for medical treatment are utterly needed to develop therapies that can prevent the natural progression of the disease. Thus, new drugs able to elicit neuroprotective activity or to inhibit PGE2 release are potential targets for drug discovery and development. Formerly, 60 different Ocotea species and the matrix plants of Ayahuasca beverage were extracted following current metabolomics extraction protocol. For biological activity, an ex-vivo anti-inflammatory screening was realised using human blood LPS-induced inflammation for quantitative PGE2 determination in plasma by UPLCMS/MS. The dexamethasone and indomethacin were used as positive controls. For the neuroprotective evaluation, the Ayahuasca beverage, the extracts from its matrix plants (Banisteriopsis caapi and Psychotria viridis), its fractions and its main alkaloids were evaluated on the viability of SH-SY5Y neuroblastoma cells in an in vitro PD model. For the metabolomics analyses, quality control samples were prepared and all samples were subjected to UPLC-ESI-TOF-MS. The MS E data acquisition and treatment were performed and the dereplication was done using the in-house together with the UNIFI software databases (Waters® ) and the Dictionary of Natural Products (DNP) for MS 1 and molecular formula match. The level 2 identification was performed for the Ocotea study by the obtained MSE product ions, which were compared with a spectral library in the literature for tentative compound identification. The Ayahuasca study was performed via level 3 according to current metabolomics standards. The treated data were analysed by multivariate statistical analysis for biomarkers investigation. The metabolic fingerprints generated allowed the dereplication of the majority of the extracted compounds found on the evaluated extracts. Under the Ocotea metabolomics study, it was observed quantitatively more aporphine alkaloids and glycosylated flavonoids in most of the Ocotea extracts. Moreover, 12 out of 60 Ocotea spp. evaluated have exhibited high satisfactory PGE2 inhibition. Regarding the neuroprotective evaluation, the lower doses of the active samples from Ayahuasca and its base plants showed to be able to stimulate neuronal cell proliferation and/or display the most efficacious neuroprotection profile. Intriguingly, the hydroalcoholic fractions exhibited enhanced neuroprotective effects when compared to other samples and isolated alkaloids. The general metabolomic analysis allowed determining the biomarkers of anti-inflammatory and neuroprotective activity, besides possibly new compounds with no hits in chemical databases. The biomarkers findings of this research might be potential neuroprotective or antiinflammatory lead-structures. In addition, this is the first report regarding the chemical composition and bioactivity of several endemic Ocotea spp. of the Brazilian territory. And also, this work found that the Ayahuasca beverage and its base plants have potential applicability for PD treatment and other neurodegenerative diseases. Keywords: Ocotea. Ayahuasca. Parkinson’s disease. LPS-induced PGE2 inhibition.