An oleuropein rich-olive (Olea europaea L.) leaf extract reduces β-amyloid and tau proteotoxicity through regulation of oxidative- and heat shock-stress responses in Caenorhabditis elegans

Food and Chemical Toxicology 162 (2022) 112914 Available online 8 March 2022 0278-6915/© 2022 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/). An oleuropein rich-olive (Olea europaea L.) leaf extract reduces β-amyloid and tau proteotoxicity through regulation of oxidativeand heat shock-stress responses in Caenorhabditis elegans Jose M. Romero-M´ arquez a , 1 , María D. Navarro-Hortal a , 1 , Victoria Jim´ enez-Trigo a , Laura Vera-Ramírez a , b , Tamara J. Forbes-Hern´ andez a , Adelaida Esteban-Mu˜ noz c , Francesca Giampieri d , e , f , Pedro Bull´ on g , Maurizio Battino d , h , Cristina S´ anchez-Gonz´ alez a , i , ** , Jos´ e L. Quiles a , f , * a Department of Physiology, Institute of Nutrition and Food Technology ‘‘Jos´ e Mataix Verdú”, Biomedical Research Centre, University of Granada, Avda. del Conocimiento s.n, 18100, Armilla, Spain b Department of Genomic Medicine, GENYO: Centre for Genomics and Oncology (Pfizer-University of Granada and Andalusian Regional Government), PTS Granada, 18016, Spain c Department of Nutrition and Bromatology, University of Granada, 18071, Granada, Spain d Department of Clinical Sciences, Polytechnic University of Marche, Ancona, 60131, Italy e Department of Biochemistry, Faculty of Sciences, King Abdulaziz University, Jeddah, Saudi Arabia f Research Group on Foods, Nutritional Biochemistry and Health, Universidad Europea del Atl´ antico, Isabel Torres, 21, 39011, Santander, Spain g Department of Periodontology, Dental School, University of Seville, C/Avicena, s/n, 41009, Seville, Spain h International Joint Research Laboratory of Intelligent Agriculture and Agri-products Processing, Jiangsu University, Zhenjiang, China i Sport and Health Research Centre, University of Granada, C/. Men´ endez Pelayo 32. 18016 Armilla, Granada, Spain ARTICLE INFO Handling Editor: Dr. Jose Luis Domingo Keywords: Alzheimer disease DAF-16/FOXO HSP-16.2 IIS pathway RNAi SKN-1/NRF2 ABSTRACT Olive tree-derived products have been associated with numerous benefits for health. The aim of the present study was to characterize an olive leaf extract enriched in oleuropein (OLE) concerning phenolic content and profile as well as antioxidant capacity. Short-term and long-term toxicity, including oxidative stress, was in vivo evaluated in the experimental model Caenorhabditis elegans. Moreover, the potential therapeutic effect of the extract against Aβ inducedand tau protein induced-toxicity was also evaluated in C. elegans. OLE treatment did not exert toxicity. On the contrary, the extract was able to ameliorate oxidative stress and proteotoxicity related to Aβ and tau aggregation. The potential molecular mechanisms present behind the observed results explored by RNAi technology revealed that DAF-16/FOXO and SKN-1/NRF2, elements of the insulin insulin-like signalling pathway, as well as HSP-16.2 overexpression were involved. 1. Introduction Olive tree (Olea europaea) leaves are considered as a waste from the olive grove, which are discarded in two stages: during the pruning process and as a result of the mechanical harvesting of the olive fruit (Espeso et al., 2021). It has been estimated that the total amount of olive leaves that could be gathered in Spain alone is around 750,000 tons per year (Manzanares et al., 2017). Because of this production magnitude, this by-product should be re-used, promoting the circular economy. Olive leaf has been used in traditional medicine since it is a valuable source of bioactive compounds (Romani et al., 2019). The most predominant compound in the olive leaf extract is the oleuropein. The presence of this secoiridoid is the reason for the characteristic bitter taste of olive cultivars (Acar-Tek and A˘ gagündüz, 2020). Oleuropein first is transformed into its aglycone form, and then into hydroxytyrosol, together with glucose and elenolic acid (Romani et al., 2019). The * Corresponding author. Department of Physiology, Institute of Nutrition and Food Technology ‘‘Jos´ e Mataix Verdú”, Biomedical Research Centre, University of Granada, Avda. del Conocimiento s.n, 18100, Armilla, Spain. ** Corresponding author. Sport and Health Research Centre, University of Granada, C/. Men´ endez Pelayo 32. 18016 Armilla, Granada, Spain. E-mail addresses: [email protected] (C. S´ anchez-Gonz´ alez), [email protected] (J.L. Quiles). 1 These authors contributed equally to this manuscript. Contents lists available at ScienceDirect Food and Chemical Toxicology journal homepage: www.elsevier.com/locate/foodchemtox https://doi.org/10.1016/j.fct.2022.112914 Received 30 December 2021; Received in revised form 2 March 2022; Accepted 6 March 2022 Food and Chemical Toxicology 162 (2022) 112914 2 positive health effects of olive leaves and their compounds have been extensively demonstrated. Benefits concerning blood pressure, inflammatory status, serum lipid profile (Lockyer et al., 2017) and glycaemia (Acar-Tek and A˘ gagündüz, 2020; Al-Azzawie and Alhamdani, 2006) have been attributed to them, as well as antioxidant (Acar-Tek and A˘ gagündüz, 2020; Al-Azzawie and Alhamdani, 2006,), cardioprotective (Wu et al., 2018) and anticancer (Mijatovic et al., 2011) effects. Furthermore, olive biophenols (Omar et al., 2018) have demonstrated interference with the amyloid aggregation path in vitro and, for oleuropein, also in vivo (Leri et al., 2019; Omar et al., 2018). The increase of the incidence of neurodegenerative disorders such as dementias poses a severe challenge for the society and the healthcare system. Alzheimer’s disease (AD) is the most common form of dementia, which contributes to 60–70% of the cases (World Health Organization, 2021a). Among all the diseases, AD and other dementias are the seventh leading cause of death globally (World Health Organization, 2020) and the second one in high-income countries (World Health Organization, 2020). Moreover, it is considered as one of the major causes of disability and dependency in older people (World Health Organization, 2021a), which leads to high physical, psychological, social and economic impacts (World Health Organization, 2021a). Currently, more than 55 million people live with dementia worldwide, and there are nearly 10 million new cases every year. As the proportion of older people in the population is increasing in nearly every country, this number is expected to rise to 78 million in 2030 and 139 million in 2050 (World Health Organization, 2021b). AD is a proteinopathy characterized by accumulation of hyperphosphorylated Tau and β-amyloid together with an oxidative stress increase among other features (Cordero et al., 2018; Quiles et al., 2020). Regarding the disease etiology, genetics and environmental factors are involved in this multifactorial pathology. Among them, the main modifiable risk factor is the diet (Caruso et al., 2021; Godos et al., 2020). Numerous studies have associated a neuroprotective effect with the intake of foods included in the Mediterranean Diet (Berti et al., 2018) such as the virgin olive oil and its bioactive compounds (Berr et al., 2009; Robles-Almazan et al., 2018). The increase of AD incidence along with the population aging and the lack of effective pharmacotherapy to counteract the pathology have raised the scientific community interest in the search of compounds able to prevent, delay or treat the AD. In this sense, there is a strong need for the development of new tools and the olive leaf, and its compounds could be a promising and excellent approach. According to that, the aim of the present research was to investigate an olive leaf extract enriched in oleuropein that has been processed according to the European Pharmacopoeia (European Pharmacopoeia, 2007). This extract has been authorized to be used as an ingredient for nutritional supplements in human nutrition. Firstly, the characterization in terms of antioxidant capacity and polyphenols profile was made. Moreover, Caenorhabditis elegans was used to evaluate in vivo toxicity of the extract. This model was also used to evaluate the potential therapeutic effect of the novel formulation against AD-related proteinopathies such Aβ inducedand tau protein induced-toxicity as well as to describe the molecular basis of the protective effects observed. 2. Materials and methods 2.1. Chemicals and reagents Reagents were purchased from Thermo Fisher (Waltham, Massachusetts, USA), Sigma-Aldrich (St. Louis, Missouri, USA), Merck (Darmstadt, Germany) or Roche (Basel, Switzerland). All reagents were of analytical grade and double distilled deionized water was obtained from a Milli-Q purification system from Millipore (Milford, MA, USA). 2.2. Extract preparation Dry extract from Olea Europaea leaves enriched 40% in oleuropein (OLE) was a kind gift by Natac (Madrid, Spain). The dry extract was directly diluted in a solution composed of ethanol/Milli-Q water (25:75, v/v) for its use. This extraction medium was experimentally calculated to use the lowest concentration of ethanol to solubilize OLE with no toxicity for the experimental model. One-hundred μ g/mL of extract was used in most of the experiments with worms. (European Pharmacopoeia, 2007). Fig. S1 present a certificate of analysis in Spanish provided by the company. This analysis certifies that the content in oleuropein of the extract is a 43.68%. 2.3. Total phenolic and flavonoids content analysis Total phenolic content of the OLE was measured by the FolinCiocalteu method (Singleton et al., 1999). Briefly, samples were reacted for 5 min with the Folin-Ciocalteu reagent. Next, sodium carbonate (Na 2 CO 3 ) was added to the mixture and incubated for 2 h at room temperature. Gallic acid was used as standard, and the absorbance was measured at 760 nm. Phenolic profile was also determined by LC-MS to validate that the extract was mainly rich in oleuropein (Fig. S2 and Fig. S3). The determination of flavonoids content was performed as previously described (Navarro-Hortal et al., 2021). In this context, samples were reacted with NaNO 2 for 6 min and then, were incubated with AlCl 3 for 5 min. Next, NaOH was added and immediately the absorbance was read at 510 nm. Catechin was used as standard. For both analyses, a Synergy Neo2 microplate reader (Biotek, Winooski, Vermont, U.S.A.) was used to measure the absorbances. Every determination was carried out at least three times. The results are expressed as mg of gallic acid equivalent/g of dry extract and mg of catechin equivalent/g of dry extract for total phenolic and flavonoid content, respectively. 2.4. Total antioxidant capacity Total antioxidant capacity of the OLE was assessed by three different methods: ABTS, DPPH and FRAP. First, ABTS assay was made following the protocol described by Re et al. (1999), which is based on the reduction of the free radical 2,2′-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) by the antioxidants present in samples. The absorbance values were measured at 734 nm. Next, a DPPH assay was made following the protocol described by (Kumaran and Karunakaran, 2007). This test is based on the colorimetric measurement of the free radical 2,2-diphenyl-1-picryl-hydrazyl-hydrate (DPPH) reduction which is progressively lost when it is reduced by the antioxidant compounds of the samples. The absorbance was measured spectrophotometrically at 517 nm of wavelength. For the FRAP assay, the protocol described by Deighton et al., was followed. This assay evaluates the ability of the sample to reduce the ferric to ferrous ion. The absorbance when the iron, complexed with 2,4,6-tripyridyl-s-triazine (TPTZ), changes its color, was measured at 593 nm (Deighton et al., 2000). A Synergy Neo2 microplate reader (Biotek, Winooski, Vermont, U.S.A.) was used to measure the absorbances. Every determination was performed at least three times. Results are expressed as mM of trolox equivalents/g dry extract. 2.5. Caenorhabditis elegans strains and maintenance Caenorhabditis elegans strains used in this work were N2 Bristol (wild type), CL4176 (dvIs27 [myo-3p:A-Beta (1–42):let-851 3′UTR) +rol-6 (su1006)] X), CL802 (smg-1(cc546) I; rol-6(su1006) II), BR5706 (bkIs10 [aex-3p:hTau V337M +myo-2p:GFP]), LD1 (ldIs7 [skn-1b/c:GFP +rol6(su1006)]), TJ356 (zIs356[daf-16p:daf-16a/b:GFP +rol-6(su1006)]), TJ375 (gpIs1[hsp-16.2:GFP]), CF1553 (mu1s84[pAD76(sod-3:GFP) + rol-6(su1006)]) and CL2166 (dvIs19 [(pAF15)gst-4p:GFP:NLS] III). All strains were routinely maintained in an incubator (VELP Scientifica FOC 120 E, Usmate, Italy) at 20 ◦C, except CL4176 and CL802, which were maintained at 16 ◦C. Worms were grown on nematode growth medium J.M. Romero-M´ arquez et al. Food and Chemical Toxicology 162 (2022) 112914 3 (NGM) agar plates with Escherichia coli OP50 as food source, according to standard protocols. The worm strains and the bacteria were acquired from the Caenorhabditis Genetics Center (Minneapolis, MI, USA). Agesynchronized animals were obtained by isolating embryos from gravid hermaphrodites using a bleaching method. 2.6. Lethality test OLE acute toxicity assay was performed in wild-type N2 synchronized L4 larvae. In this context, age-synchronized worms were placed in NGM plates until L4 larvae state. At this moment, L4 worms were placed in plates which contained increasing concentrations of the extract dissolved in NGM (0, 0.1, 1, 10, 100, 1000 μ g/mL) without food. After 24 h, nematodes were scored as live or dead using a Motic dissecting microscope (Motic Inc., LTD., Hong Kong, China). Worms were considered dead when there was no response to repeated touches with platinum wire. At least, three independent assays for each concentration were made and the experiment was performed in triplicate with a minimum of 5 worms per plate. Results were expressed as percentage of survival in 24 h. The non-lethal submaximal concentration (100 μ g/mL) was selected for further experiments. 2.7. Pharyngeal pumping assay Worm metabolism was assessed by pharyngeal pumping assay in wild-type N2 synchronized young adults. Briefly, OLE was dissolved until a final concentration of 100 μ g/mL in NGM plates. Then, worms were incubated at 20 ◦C on E. coli sown plates with or without the treatment for 96 h. Next, worms were moved to different NGM plates to evaluate the number of contractions per minute of the terminal bulb of the pharynx using a Motic microscope (Motic Inc. LTD. Hong Kong, China). The experiment was performed three times and ten worms per group were evaluated (n =10). Results are expressed as the mean number of contractions per minute. 2.8. Growth test The effect of the OLE on worm developments was carried out similarly as described for the pharyngeal pumping assay. Briefly, four-dayold worms from treatment or control groups were photographed on a microscope (Motic Inc., LTD. Hong Kong, China) and the pictures were used to measure the body lengths using Motic Images Plus 3.0 (Motic Inc., LTD. Hong Kong, China). A minimum of three replicates were carried out with 40 worms per each one. The results are expressed as the mean of body length of worms. 2.9. Reproduction and fertility test The effect of OLE on worm fertility and egg viability was carried out in L4 aged-synchronized N2 worms. In this context, age-synchronized worms were placed in NGM plates until L4 larvae state. At this moment, L4 worms were individually placed in 24 well plates containing or not the OLE dissolved in NGM (100 μ g/mL) with E. coli OP50 as a source of food. After 24 h, worms from both groups were moved to a different non-treated NGM 24 well plate seeded with bacteria. Worms were continuously moved every day until they stopped laying eggs. For every single worm, eggs were counted the same day of adult removal and the larvae number were counted the day after by using a dissection microscope (Motic Inc., LTD. Hong Kong, China). At least 15 worms were used per group and the experiment was repeated at least three times. Results are expressed as the mean of the total number of eggs or larvae per group. 2.10. Lifespan analysis Lifespan analysis was assessed to evaluate long-term toxicity of the extract. OLE was dissolved until a final concentration of 100 μ g/mL in fresh NGM plates. Then, 120 aged-synchronized worms were lifelong grown from embryos at 20 ◦C on supplemented or not supplemented plates and with a bacterial lawn until the last worm died. The day one of adulthood was considered as the first day of the experiment. Worms were daily transferred to fresh plates containing or not the extract to separate larvae from adults and to avoid worm starving. Animal survival was scored every day. Death was assumed when there was no response to mechanical stimulus. The worms dragged out of the dish or with extruded internal organs were considered as censored. Results are presented as a Kaplan-Meier survivorship curve. 2.11. Intracellular reactive oxygen species (ROS) content under induced oxidative stress conditions In vivo ROS content was measured in worms by using the 2ʹ,7ʹDichlorofluorescin Diacetate (DCFDA) method, as previously described by Navarro-Hortal and coworkers (Navarro-Hortal et al., 2021). Briefly, synchronized embryos were lifelong cultured in bacteria seeded NGM plates with or without 100 μ g/mL of the OLE for 48 h at 20 ◦C. Next, worms were washed three times with M9 buffer to remove bacteria. To induce oxidative stress, collected worms were incubated or not with 2.5 mM of 2,2′-azobis-2-amidinopropane dihydrochloride (AAPH) for 15 min at 20 ◦C. Then, AAPH was removed from the worms with 3 times M9 washed. Finally, worms were incubated with 25 μ M of DCFDA for 2 h at 20 ◦C. This dye can penetrate through the cell membrane and emits fluorescence when it reacts with free radicals. The intensity of fluorescence was measured using a COPAS BioSorter® flow cytometer (Union Biom´ etrica, Belgium, Europe). Results are expressed as the mean of the fluorescence intensity, which is related to the ROS content (AU). A minimum of three replicates were carried out with at least 180 worms per group and experiment. 2.12. Amyloid-β toxicity induced-paralysis test Paralysis assay was performed by using CL4176 strain, a temperature sensitive transgenic strain that expresses human amyloid β 1–42 peptide in muscle cells. In this context, animals were incubated for 48 h at 16 ◦C from embryos on NGM containing or not 100 μ g/mL of the OLE. To initiate the β-amyloid (Aβ) induced paralysis, worms were temperatureup-shifted from 16 to 25 ◦C. After 20 h, paralysis was scored at 2 h intervals until 32h. The strain CL802 was used as negative control in the assay. Nematodes were scored as paralyzed when there was no response to repeated touches with platinum wire. The experiment was performed in triplicate with, at least, 20 worms per group. Results are expressed as percentage (%) of non-paralyzed worms. 2.13. Amyloid-β aggregate staining To visualize Aβ aggregates, worms of the CL4176 strain were stained with Thioflavin T. Worms used in Thioflavin T staining were grown as described for the paralysis assay. In this context, after 26 h of temperature raised from 16 ◦C to 25 ◦C on paralysis assay, worms were collected and washed 3 times with M9 buffer. First, a fixer solution (4% paraformaldehyde/M9 buffer, pH 7.4) was used to fix worms at 4 ◦C. After 24 h, worms were permeabilized at 37 ◦C with a solution made with 5% fresh β-mercaptoethanol, 1% Triton X-100 and 125 mM Tris (pH 7.4) for 24 h. Next, permeabilized solution was removed with 2 washed with M9 and worms were stained with 0.125% Thioflavin T in 50% ethanol for 30 min. Finally, Thioflavin T excess was removed with sequential ethanol washes (50%, 75%, 90%, 75%, and 50% v/v) for 2 min each one. Finally, stained worms with Thioflavin T were observed under a Nikon epi-fluorescence microscope (Eclipse Ni, Nikon, Tokyo, Japan) and images were acquired at 40×magnification using the GFP filter with a Nikon DS-Ri2 camera (Tokyo, Japan). CL802 were considered as negative control and untreated CL4176 were the positive J.M. Romero-M´ arquez et al. Food and Chemical Toxicology 162 (2022) 112914 4 control. 2.14. Locomotive behavior analysis related to tau proteotoxicity To evaluate an extensive field of the pathophysiology of the AD, we analyze the effect of the OLE in behavior parameters in a C. elegans model of tauopathy. BR5706 strain shows a constitutive pan-neuronal expression of pro-aggregant human Tau protein which results in the deposition of aggregates and locomotion defects mainly noted from day one of adulthood. In this context, BR5706 worms were incubated at 20 ◦C from eggs on NGM containing or not 100 μ g/mL of the extract for 72h. Then, animals were forced to swim to stimulate worm movement. For this purpose, at least 50 worms were transferred to a slide with a drop of M9 and worm movement was recorded, tracked, and analyzed with WormLab Imaging System (MBF Bioscience, Williston, Vermont, EE. UU). The swimming speed, wavelength and wavelength dynamic amplitude were evaluated as representative parameters of locomotive behavior. The experiment was carried out in triplicate. 2.15. Expression analysis of DAF-16/FOXO, SKN-1/NRF2, HSP-16.2, GST-4 and SOD-3 in green fluorescence protein (GFP)-reporter transgenic strains To deepen the molecular mechanisms operating under the observed effects of the OLE, transgenic strains with GFP-reporter for dauer formation (DAF)-16/FOXO, transcription factor skinhead (SKN)-1/NRF2, heat shock protein 16.2 (HSP 16.2), glutathione S-transferase (GST)-4 and superoxide dismutase (SOD)-3 were used. LD1 worms present GFPreporter of SKN-1/NRF2 which is present in ASI chemosensory neurons in a constitutive way and migrates to cell nuclei in response to oxidative stress. TJ356 worms present a GFP-reporter of DAF-16/FOXO and the translocation of this gen to the cell nucleus can be observed by fluorescence microscopy. HSP-16.2 gene expression was evaluated in the anterior pharynx bulb of the transgenic strain TJ375 which becomes activated under oxidative stress. GST-4 is also fused with GFP on the whole body of CL2166 similarly with SOD-3 protein is expressed in the transgenic CF1553. For all gene expression experiments with the GFP-reporter strains, worms were grown on plates with or without the OLE at 100 μ g/mL for 48 h. Next, animals were moved to slides and immobilized with sodium azide (1M). Nikon epi-fluorescence microscopy (Eclipse Ni, Nikon, Tokyo, Japan) was used to catch worm images using the GFP filter fitted with a Nikon DS-Ri2 camera (Tokyo, Japan). Pictures were taken at 10× magnification except for the TJ375 strain which was 40X. NIS-Elements BR software (Nikon, Tokyo, Japan) was used to analyze images and the background signal was subtracted from all readings. A semi-quantitative scale was used for the TJ356 strain, assigning the value ‘1′to worms with cytosolic expression of DAF-16:GFP, ‘2′to the intermediate status, and ‘3′to the nuclear location. SKN-1:GFP fluorescence intensity was measured in the gut area below the pharynx on LD1 worms. Anterior area of the pharyngeal bulb was measured to analyze HSP-16.2:GFP expression in TJ375 worms. Finally, the whole worm body was measured to analyze fluorescence intensity related to SOD-3:GFP and GST-4:GFP expression in CF1553 and CL2166 worms, respectively. Experiments were performed in triplicate, with at least 20 worms per experiment and group. 2.16. RNAi experiments by feeding For the RNAi experiments, E. coli HT115 expressing SKN-1/NRF2, HSP 16.2 (Sources BioScience, Nottingham, UK), DAF-16/FOXO, SOD2 and SOD-3 (Cultek SL, Madrid, Spain) dsRNA were seed on NGM plates containing 1 mM Isopropyl β-D-1-thiogalactopyranoside (IPTG) and 25 μ g/mL carbenicillin. Next, age-synchronized L3-L4 worms (F0) grown in standard conditions were moved to RNAi plates for the specific gene until the second day of adulthood (fertile age). Then, embryos (F1) were isolated from gravid hermaphrodites using a bleaching method and placed into RNAi plates for the specific gene containing or not the extract and used for RNAi experiments. Once F1 embryos of CL4176 and BR5706 were obtained, the paralysis assay and locomotive behavior test with RNAi technology followed the protocols shown above for the respective test. F1 worms of the CL4176 and BR5706 were used for RNAi experiments to deepen the molecular mechanisms operating under the observed effects of the extract on amyloid-β toxicity induced-paralysis test and tau toxicity-related locomotive behavior tests, respectively. 2.17. Verification of the RNAi effect in GFP-reporter transgenic strains To verify whether an RNAi specific gene was effective for the particular gene inhibition in RNAi experiments, F1 worms of LD1, TJ356, CF1553 and TJ375 F1 exposed or not to their specific RNAi inhibitor were used. Once F1 embryos of the mentioned strains above were obtained, verification RNAi experiments were made according to GFPreporter transgenic strains experiment protocols. 2.18. Statistical analysis Kolmogorov-Smirnov test was used to study the normality of the variables as well as the Levene test was used to study the homogeneity of variance. For normally distributed variables, the T-student test was employed. Non-normally distributed variables were analyzed by nonparametric tests (Kruskal-Wallis and Mann-Whitney-U). Data are expressed as mean ±SEM from at least 3 independent experiments unless otherwise stated. Significance was considered for P <0.05. For lifespan curves, the Log-Rank test was used to evaluate the differences among survival distributions of cohorts. Statistical analysis was performed by SPSS 24.0 (IBM, Armonk, NY, USA). 3. Results 3.1. Total phenolics content, phenolics profile, total flavonoids content and total antioxidant capacity of OLE Table 1 presents data on total phenolics content, total flavonoids and total antioxidant capacity measured by three different methods of OLE. Figs. S2 and S3 show phenolics profile of the OLE analyzed by LC-MS. Quantitative analysis provided by supplier certified a concentration in oleuropein in our extract sample of 43 mg/100 mg. 3.2. Toxicological characterization of oleuropein rich extract As a first approach to the in vivo effects of the olive leaf oleuropeinrich extract OLE, different tests were used to evaluate the toxicity in C. elegans. First, oleuropein rich extract short-term toxicity was evaluated by the 24h-lethality test. As shown in Fig. 1A, all tested concentrations were non-lethal for the exposed worms, with a survival percentage near to 100% in every dosage. According to these results, non-lethal submaximal concentration (100 μ g/mL) of extract was selected for the rest of the experiments. Subsequently, pharyngeal pumping (Fig. 1B) and growth (Fig. 1C) were evaluated as an overview Table 1 Total phenolics content, total flavonoids content and total antioxidant capacity of the Olea Europaea leaves extract 40% rich in oleuropein (OLE). Parameter Mean ±SEM Total phenolics content (mg galic/g extr) 212.1 ±13.9 Total flavonoids content (mg catechin/g extr) 388.1 ±34.7 FRAP (mM trolox/g extr) 3.5 ±0.2 DPPH (mM trolox/g extr) 2.8 ±0.1 ABTS (mM trolox/g extr) 2.3 ±0.2 ABTS:2,2′-azinobis (3-ethylbenzothiazoline-6-sulfonic acid); DPPH: 2,2diphenyl-1-picryl-hydrazyl-hydrate; FRAP: Ferric Reducing Antioxidant Power. J.M. Romero-M´ arquez et al. Food and Chemical Toxicology 162 (2022) 112914 5 Fig. 1. Short-term in vivo toxicity characterization of the oleuropein rich-olive leaf extract (OLE) in the Caenorhabditis elegans wild type N2 strain. A) Lethality; B) Pharyngeal pumping; C) Growth; D) Reproduction and fertility; E) Reactive oxygen species (ROS) content. For each parameter, columns with different lower-case letters mean statistically significant differences (P <0.05). Results are expressed as mean ±SEM. AAPH: 2,2’-azobis-2-amidinopropane dihydrochloride. Fig. 2. Kaplan-Meier representation for the long-term in vivo toxicity characterization of the oleuropein rich-olive leaf extract (OLE) in the Caenorhabditis elegans wild type N2 strain. Mean and maximal lifespan as well as Log-Rank test are presented inside the curve. J.M. Romero-M´ arquez et al. Food and Chemical Toxicology 162 (2022) 112914 6 of worm’s metabolism and adequate development of nematodes. Results showed that there were no differences neither in pharyngeal pump rate nor in body length between treated and untreated worms. In the same way, as represented in Fig. 1D, treatment with the extract did not affect the ability of worms’ egg-laying (reproduction) or eggs viability (progeny). Additionally, long-term toxicity of the extract was evaluated on worms using survival curves. In this context, lifespan in the worms was not affected by OLE, as presented in Fig. 2. 3.3. Effect of oleuropein rich extract on AAPH-induced oxidative stress To test the effect of OLE regarding oxidative stress (also considered as a marker of toxicity), DCFDA was used to measure ROS after induction with the stressor AAPH. As it can be shown in Fig. 1E, AAPH induction markedly elevated ROS content in worms compared to the unexposed controls. In contrast, OLE treatment avoided AAPH related ROS enhancement, leading to similar values than those found in nonAAPH induced control group. 3.4. OLE effect on Aβ toxicity and accumulation Paralysis test was carried out to assess the effect of OLE on Aβ toxicity using CL4176 which expresses human amyloid β 1–42 peptide in muscle cells. Results, represented in Fig. 3, showed that OLE led to a delayed paralysis in treated worms. The bigger differences on non-paralyzed worm score were obtained in the range between 24 and 28 h after the temperature upshift. Furthermore, this result was associated with the findings obtained by thioflavin T staining. Thioflavin T is a well-known reagent used to specifically dye Aβ aggregation. As shown in Fig. 3, a large number of Aβ deposits can be observed in the positive control. In contrast, extract-treated worms images reflect a clear effect of OLE, with less accumulation of Aβ deposits. These results demonstrated that OLE treatment diminished Aβ production and/or accumulation in the Aβ human-transgenic model. To explore the molecular mechanisms underlying the effects found in the paralysis assay, RNAi technology was used. In this context, paralysis assay was conducted as before but with worms subjected in parallel to RNAi for different genes. As can be observed in Fig. 4, the protective effect of oleuropein rich extract against Aβ-induced paralysis was partially lost in silenced DAF-16 worms. Similar results were obtained for silenced SKN-1 worms. In the same way, extract treated worms exposed to HSP-16.2 RNAi significantly reduced the non-paralyzed worm score. In contrast, the protective effect of the extract was not modified after the knockout of SOD-2 and SOD-3 enzymes by RNAi Fig. 3. Effects of the oleuropein rich-olive leaf extract (OLE) on Aβ-induced paralysis phenotype in CL4176 transgenic strain. A) Paralysis curve shown non-paralyzed CL4176 nematodes (%) since 20h after the increase of temperature. B) Thioflavin T staining shows representative images of the CL4176 nematodes collected at 26h after the increase of temperature (40×magnification). Aβ aggregates are remarked with white arrow. Different lower-case letters mean statistically differences (p < 0.05) between groups for each time. Results are expressed as mean ±SEM. Fig. 4. Effect of the different RNAi (SKN-1, DAF-16, HSP-16.2, SOD-3 and SOD2) on the oleuropein rich-olive leaf extract (OLE) treated CL4176 nematodes in a paralysis test. * means statistically differences (p <0.05) with respect to OLEtreated group unexposed to RNAi. Results are expressed as mean ±SEM. J.M. Romero-M´ arquez et al. Food and Chemical Toxicology 162 (2022) 112914 7 feeding. 3.5. Effect of OLE on transgenic GFP reporter strains of DAF-16, SKN-1, HSP-16.2, GST-4 and SOD-3 To test the effect of OLE on genes blocked by RNAi, transgenic strains in which the targeted gene was marked with GFP were treated with the extract and expression assessed by fluorescence microscopy. Thus, GFP fluorescence for DAF-16, SKN-1, HSP-16.2, GST-4 and SOD-3 transgenic strains after OLE treatment is presented in Fig. 5. Worms from the TJ356 strain were individually classified in cytosolic, intermediate or nuclear according to the presence of nucleation of DAF-16:GFP (Fig. 5B). As shown in Fig. 5A, the assayed dosage of OLE did not induce the nuclear translocation of the transcription factor nor increased the expression of SKN-1:GFP in the LD1 strain (activation of this transcription factor is also mediated by its nucleation (Figure C). On the other hand, CF1553 worms treated with the oleuropein rich extract increased the fluorescence intensity of SOD-3:GFP (Fig. 5A and D). In the same way, OLE increased the expression of HSP-16.2:GFP compared to untreated TJ375 worms (Fig. 5A and E). In contrast, expression of GST-4:GFP was reduced by oleuropein rich extract in comparison with untreated CL2166 worms (Fig. 5A and F). To verify that the RNAi feeding technology was effective on different tests, specific transgenic GFP reporter strains were exposed to their respective RNAi. As shown in Fig. 6, DAF-16:GFP, SKN-1:GFP, SOD-3: GFP and HSP-16.2:GFP expression in the specific GFP reporter strain was reduced by RNAi exposure. 3.6. Effect of oleuropein rich extract on locomotive behavior related to tau proteotoxicity To test more features associated with AD besides Aβ aggregation, tau aggregation was analyzed by investigating swimming locomotive behavior. By using the WormLab station and software, swimming speed, wavelength and wavelength dynamic amplitude parameters were chosen (Fig. 7). Swimming speed is the traveling speed of an animal Fig. 5. Effects of the oleuropein rich olive leaf extract (OLE) on GFP-reporter transgenic strains. A) Quantification of the GFP expression on the different transgenic strains gene reporters treated with OLE. B) Illustrative images of the different status of DAF-16:GFP (cytosolic, intermediate or nuclear) in the strain TJ356 (10× magnification). C) Illustrative images of SKN-1:GFP expression in the strain LD1 for each group (10×magnification). D) Illustrative images of SOD-3:GFP expression in the strain CF1553 for each group (10×magnification). E) Illustrative images of HSP-16:2:GFP expression in the strain TJ375 for each group (40×magnification). F) Illustrative images of GST-4:GFP expression in the strain CL2166 for each group (10×magnification). Results are expressed as mean ±SEM. * means statistically significant differences with the control group (P <0.05). Fig. 6. Verification of the effect of RNAi in the transgenic strains LD1 (SKN-1: GFP), TJ356 (DAF-16:GFP), CF1553 (SOD-3:GFP) and TJ375 (HSP-16.2:GFP). Results are expressed as percentage with respect to the control group of the same strain without the RNAi for the specific gene. * Means statistically significant differences with the control group (P <0.05). J.M. Romero-M´ arquez et al. Food and Chemical Toxicology 162 (2022) 112914 8 measured over a two-stroke interval, whereas wavelength determines the magnitude of the body’s waviness. Furthermore, the wavelength dynamic amplitude provides an estimate of the type of body bend (deep or flat) and of the intensity of the “stretching” effort in a stroke. Overall, proaggregatory tau worms treated with OLE showed better locomotive parameters, with a higher speed and wavelength and a lower stretching effort (dynamic amplitude) compared with control groups. To explore the molecular mechanisms underlying the effects found in the locomotive behavior assay, RNAi technology was used. In this context, the same experiment as before was conducted with the extract and subjected to RNAi for different genes (SKN-1, DAF-16, HSP-16.2, SOD-3 and SOD-2). As can be observed in Fig. 8, the protective effect of OLE against locomotive alterations related to tau-neurotoxicity was modified in different knockout worms. Thus, swimming speed was reduced in all studied knockout extract-treated worms except in those knocked out SOD-3 worms (Fig. 8A). Concerning the worm’s wavelength, the waviness was reduced in all knockout extract-treated worms studied (Fig. 8B). Finally, the stretching effort was increased in all studied knockout extract-treated worms except in knockout SOD-3 worms (Fig. 8C). 4. Discussion Olive leaves consumption has been associated with various health benefits including protection against neurodegenerative diseases in preclinical models and in humans (Giacometti and Grubi´ c-Kezele, 2020; Lockyer et al., 2017; Sarbishegi et al., 2018). Those effects could be attributed to their great phytochemicals content, especially oleuropein (Romani et al., 2019; Vogel et al., 2014). UPLC-QTOF-MS/MS methodology was used to confirm that the concentrated extract was mainly rich in oleuropein and, with a small amount of its derivative hydroxytyrosol. These data would serve to assign the results observed in the present investigation to the high content of oleuropein of the extract used in this study. Concerning total phenolic content, results were similar to those found by Orak et al., in different methanolic olive leaves extracts from Turkey and Italy (Orak et al., 2019). However, OLE showed higher values for the three different methods of antioxidant capacity (ABTS, FRAP and DPPH) (Nicolì et al., 2019; Orak et al., 2019; Zaïri et al., 2020) as well as higher total flavonoids content (Nicolì et al., 2019). In contrast, Zaïri et al. showed that an aqueous olive leaf extract from Turkey presented higher phenolic and flavonoid content compared to our results (Zaïri et al., 2020). Those differences could be attributed to the variations in the geographic location, stage of harvesting, storage conditions as well as different extraction procedures and solvents used. C. elegans is a very useful model to investigate the effect of food byproducts in some biomedical research areas such as toxicology. To our knowledge, evidence about the olive leaves extract toxicity on C. elegans is very limited and this is the first investigation providing evidence on a 40% concentrated oleuropein extract concerning toxicity and AD features. According to literature, Luo et al. showed that food clearance (a similar form to measure worm metabolism/pharyngeal pumping) was not reduced (400 μ g/mL) or even increased (300, 500 and 600 μ g/mL) in nematodes treated with a methanolic olive leaves extract. In the same way, egg laying was increased in worms treated with 400 μ g/mL of the extract (Luo et al., 2019). In the present research, pharyngeal pumping as well as reproduction and fertility tests were not affected by a lower Fig. 7. Effects of OLE on Tau-induced altered locomotive behavioral phenotype in BR5706 transgenic strain. A) Swimming speed B) Wavelength. C) Dynamic amplitude/stretching effort. *Means statistically differences (p <0.05) with respect to the OLE-treated group unexposed to RNAi. Results are expressed as mean ± SEM. The picture also shows a representation of the process followed with WormLab system and software to process locomotive worm’s video. J.M. Romero-M´ arquez et al. Food and Chemical Toxicology 162 (2022) 112914 9 concentration of olive leaf treatment (100 μ g/mL). In fact, OLE treatment did not exert lethality at all assayed dosages (0, 0.1, 1, 10, 100, 1000 μ g/mL). Likewise, C. elegans has been used to study oleuropein toxicity by Feng et al. Similarly, to results from the present research, worm length and body width were not affected by the exposure to differing concentrations (0, 21.6, 97.2, 237.6 μ g/mL) of a pure oleuropein extract. In the same way, lifespan was not affected by the lowest concentration of oleuropein but was increased by the rest of dosages Fig. 8. Effect of the different RNAi (SKN-1, DAF-16, HSP-16.2, SOD-3 and SOD-2) on OLE treated BR5706 nematodes in the locomotive behavioral test. A) Swimming speed B) Wavelength. C) Dynamic amplitude/stretching effort. *Means statistically differences (p <0.05) with respect to the OLE-treated group unexposed to RNAi. Results are expressed as mean ±SEM. J.M. Romero-M´ arquez et al.