Neuroprotective Effect of Terpenoids Recovered from Olive Oil By-Products

Abstract

The neuroprotective potential of 32 natural extracts obtained from olive oil by-products was investigated. The online coupling of supercritical fluid extraction (SFE) and dynamic adsorption/desorption allowed the selective enrichment of olive leaves extracts in different terpenoids’ families. Seven commercial adsorbents based on silica gel, zeolite, aluminum oxide, and sea sand were used with SFE at three different extraction times to evaluate their selectivity towards different terpene families. Collected fractions were analyzed by gas chromatography coupled to quadrupole-time-of-flight mass spectrometry (GC-QTOF-MS) to quantify the recoveries of monoterpenes (C10), sesquiterpenes (C15), diterpenes (C20), and triterpenes (C30). A systematic analysis of the neuroprotective activity of the natural extracts was then carried out. Thus, a set of in vitro bioactivity assays including enzymatic (acetylcholinesterase (AChE), butyrylcholinesterase (BChE)), and anti-inflammatory (lipoxidase (LOX)), as well as antioxidant (ABTS), and reactive oxygen and nitrogen species (ROS and RNS, respectively) activity tests were applied to screen for the neuroprotective potential of these extracts. Statistical analysis showed that olive leaves adsorbates from SS exhibited the highest biological activity potential in terms of neuroprotective effect. Blood–brain barrier permeation and cytotoxicity in HK-2 cells and human THP-1 monocytes were studied for the selected olive leaves fraction corroborating its potential.

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

“…Differentiated THP-1 macrophages were induced to inflammation through LPS, and two non-toxic concentrations of OL-SS [20 and 40 μg/mL, as in ( 14 )] were evaluated. As it can be observed in Figure 1 , cells treated only with LPS (Control +) showed a relevant increase in all pro-inflammatory cytokines measured (TNF-α, IL-6, and IL-1β), in comparison to non LPS-induced cells (Control –) after 24 h of incubation.…”

“…Among them, the triterpenoids (β-amyrin, α-amyrin, and uvaol) were associated with the neuroprotective potential of OL-SS, and they have been reported as the main compounds in olive leaves (48) with several associated biological properties (49,50). In addition, these compounds showed promising results due to their BBB permeability, similar to pharmacological drugs as galantamine (14). It has to be noted that the OL-SS fraction is complex and more compounds present in the extract could act synergistically with the identified triterpenoids, and previous studies have demonstrated that complex olive leave extracts have higher bioactive properties due to the presence of additive and/or synergistic effects of their phytochemicals (51,52).…”

“…Olives leaves-sea sand adsorbate (OL-SS) tested along this work was obtained as previously described by Suárez Montenegro et al ( 14 ). Briefly, shade-dried olive leaves were ground with a knife mill and sieved to 500–1,000 μm particle size.…”

“…Briefly, cells were grown in RPMI 1640 culture medium supplemented with 10% FBS, 100 U/mL penicillin, 100 µg/mL streptomycin, 2 mM L-glutamine and 0.05 mM β-mercaptoethanol at 37 • C in 95% humidified air containing 5% CO 2 . Then, cells were seeded in 24-well-plates at a density of 5 × 10 5 cells/mL and monocytes were differentiated to macrophages by maintaining the cells with 100 ng/mL of PMA for 48 h. After differentiation, cells were washed with PBS and incubated with 0.05 µg/mL of LPS for inducing inflammation for 24 h, with the presence of different non-toxic concentrations of the OL-SS (20 and 40 µg/mL), previously described by the group (14). Then, the supernatants were collected and frozen at −20 • C. Positive controls were performed inducing cells with LPS but without sample, whereas negative controls indicated cells without LPS or extract stimulation.…”

“…Moreover, other food and food byproducts, such as phenolic derivatives and terpenoids from olive leaves, have been deeply investigated, showing a wide range of healthy properties such as in vitro and in vivo anti-inflammatory, antioxidant and anticarcinogenic activities (11)(12)(13). In this regard, a recently published study from our group has demonstrated that olive leaves extracts and fractions obtained using supercritical fluid extraction (SFE) and dynamic adsorption/desorption with different adsorbents have neuroprotective potential (14). This work enabled the association of in vitro neuroprotective bioactivities (inhibition of acetylcholinesterase (AChE), butyrylcholinesterase (BChE), and lipoxygenase (LOX) enzymes; antioxidant (ABTS) activity; and ROS and RNS scavenging capacity) to the C30 terpene content (ursolic acid derivatives, erythrodiol, and uvaol, among others).…”