In vitro Neuroprotective Potential and Lipidomics Study of Olive Leaves Extracts Enriched in Triterpenoids

Abstract

Alzheimer's Disease (AD) is the most common form of dementia that is associated with extracellular amyloid beta (Aβ) plaque formation. Genetic, environmental, and nutrition factors have been suggested as contributors to oxidative stress and neuroinflammation events that are connected to AD etiology, and secondary metabolites, such as triterpenes, have shown promising results in AD prevention. In this work, the neuroprotective and anti-inflammatory potential of an olive leaves fraction enriched in triterpenoid compounds obtained using supercritical fluid extraction (SFE) and dynamic adsorption/desorption using sea sand as adsorbent has been performed. In addition, a comprehensive lipidomics study of the response of SH-SY5Y neuroblastoma cell line to this fraction was carried out using advanced analytical methodologies, namely, charged-surface hybrid chromatography-quadrupole-time of flight mass spectrometry (CSH-Q-TOF MS/MS). The use of freely available lipidomic annotation tools and databases, and stringent cut-off filters allowed the annotation of more than 250 intracellular lipids. Advanced bioinformatics and statistical tools showed a number of phosphatidylcholines and phosphatidylethanolamines significantly increased, which could explain the protection against the cell death caused by Aβ1–42. Moreover, several triacylglycerols were found decreased. These results suggest triterpenoids from olive leaves as good neuroprotective candidates, and open a new gate for future experiments using in vivo models to corroborate this hypothesis.

Study of the potential neuroprotective effect of Dunaliella salina extract in SH-SY5Y cell model

“…SH-SY5Y cells were grown, maintained, and differentiated as described by Medeiros et al [9], with some modifications performed by our group [12]. In order to evaluate the in vitro toxicity of the DS extract in this cell line, the two highest non-toxic concentrations obtained for HK-2 cell line (10 and 20 µg/mL) were evaluated in SH-SY5Y cells.…”

“…The next day, the growth medium was removed by aspiration; cells were washed with PBS, trypsinized, and washed again with PBS. Then, cell pellets were centrifuged at 1,500 rpm for 7 min and proteins were extracted using 500 μL of the same lysis buffer as in [12,15]. Samples were incubated for 60 min at 4 °C, sonicated for 30 min at 0 °C in ice-cold water bath, and then centrifuged at 10,000 g at 4 °C for 15 min.…”

“…Volumes of 3 μL (for ESI positive) and 5 μL (for ESI negative) were injected into a HPLC model 1290 (Agilent Technologies, Germany) coupled to a quadrupole Q-TOF series 6540 (Agilent Technologies, Germany) equipped with an Agilent Jet Stream thermal orthogonal ESI source. Agilent Mass Hunter Qualitative Analysis software (B.10.0) was used for MS control and data acquisition, and samples were analyzed using the same HPLC-MS conditions as previously described [12]. Method blanks and pooled mixtures of all control and DS-treated samples were included as quality control samples and were subjected to iterative MS/MS with mass error tolerance of 20 ppm and retention time (RT) exclusion tolerance of ± 0.2 min to increase the coverage of the MS/MS spectra acquired.…”

“…In-house m/z retention time libraries, the public LipidBlast MS/MS spectra library, and the NIST19 MS/MS database were used for metabolite annotation. The parameters used for processing were the same as in [12]. CUDA, CE (22:1), LPC (17:0), PG (17:0/17:0), Cer (d18:1/17:0), MG (17:0/0:0/0:0), DG (18:1/2:0/0:0), TG (17:0/17:1/17:0)-d5, and palmitic acid-d3 internal standard compounds were used for retention time correction in CSH-Q-TOF MS/MS data; and CUDA, dlalanine-d 3 , dl-glutamic acid-d 3 , choline-d 9 , 15N 2 -l-arginine, l-methionine-d 8 , and Val-Tyr-Val internal standard compounds were used for retention time correction in HILIC-Q-TOF MS/MS data.…”

“…Previous works have analyzed the lipidomic composition of this cell line [10] and, more recently, the potential changes in mitochondrial phospholipid classes of SH-SY5Y transfected cells to simulate an AD model [11]. Moreover, the effect of an olive extract on these cells has also been evaluated in terms of lipid alterations [12]. However, a comprehensive metabolomics study including the analysis of polar and non-polar compounds of these neuron-like cells treated with carotenoids-enriched extract has never been accomplished.…”