Olive leaf extract prevents obesity, cognitive decline, and ...

Ethical approval

Animal use and procedures were in accordance with the National Institute of Health guideline and approved by the Animal Care and Use Committee of Nippon Medical School (approval no. 30-029). We conducted all efforts to minimize animal pain and discomfort. This study was carried out in compliance with the ARRIVE guidelines.

You can find more information on our web, so please take a look.

Animals

We used 120 male C57BL/6J mice (Sankyo Lab Service, Tokyo, Japan) aged 10 weeks, weighing 20-22 g, and kept under controlled conditions of 24 °C and 50% humidity. The mice were fed tap water and food ad libitum, with a standard day-night cycle of 12 h induced by switching room lights on between 8:00 a.m. and 8:00 p.m.

Olive leaf extract

We used OLEAVITA for the olive leaf extract (Phytodia S.A.S., Illkirch-Graffenstaden, France). OLEAVITA is an ethanol/water extract of olive leaves and has been shown to contain 5% or more of both oleanolic acid and oleuropein12.

Rearing conditions

In this study, we examined whether olive leaf extract administration was effective in preventing cognitive decline and depressive behaviors in physically inactive (PI) mice fed a high-fat diet (HFD). We divided mice into the following three groups:

1. 1.

   Mice reared in a standard cage and fed a standard diet (control).

2. 2.

   Mice reared in divided cages and fed an HFD (PI + HDF).

3. 3.

   Mice reared in divided cages and fed an HFD containing olive leaf extract (PI + HFD + Ole).

The divided cages used in the present study were created to suppress the mice's daily physical activity by subdividing a standard mouse cage into six compartments using plastic boards 42,43. We used a Western diet (F2WTD) as an HFD and a standard diet as per a Western diet (F-93G-MC). Both diets were purchased from Oriental Yeast Co. LTD. (Tokyo, Japan). The control mice were fed with a standard diet prepared as a control for the Western diet. The nutrient and calorie content per 100 g diet of the Western and standard diet was as follows. Standard diet (protein, 20.0 g; fat, 7.0 g; carbohydrate 63.0 g; calorie, 415.0 kcal); Western diet (protein, 17.8 g; fat, 20.0 g; carbohydrate 49.0 g; calorie, 450.8 kcal). Other nutrients (vitamins, minerals, etc.) according to weight, were the same among the two diets.

To prepare the standard diet for feeding to the mice, water was added, and then it was mixed, kneaded, and rounded into pasty sections, after which it was cut and dried. For the HFD, we added 200 g of butter to 800 g of a Western diet, mixed it uniformly, and added 80 ml of water to the kneaded diet, after which we mixed it again, then cut it into small sections and dried it. The HFD diet containing olive leaf extract was made by adding 1 g of olive leaf extract per 1000 g of HFD. Mice were fed these diets for 10 weeks and then subjected to the behavioral tests mentioned below (Fig. 1A). For another experiment, mice were reared under two different experimental conditions for 10 weeks, including (1) mice reared in divided cages and fed an HFD (PI + HFD) and (2) mice reared in divided cages and fed an HFD containing oleanolic acid and oleuropein (PI + HFD + OA + OP). The HFD containing oleanolic acid and oleuropein was made by adding 100 mg of both oleanolic acid and oleuropein per 1000 g of HFD. Ten weeks later, these mice were also subjected to behavioral tests followed by dissection (Fig. 3A).

Behavioral tests

Ten weeks after study initiation, we conducted four behavioral tests, including the FST, SPT, CFCT. For evaluation of depression-like behaviors, the SFT and FST were conducted, according to the methods of Covington et al.44 and Porsolt et al.45, respectively. For the SFT, mice were accustomed to drinking from two bottles in their home cages for 3 days before the test. The mice were then subjected to 3 days of a free-choice test between water and a 1% sucrose solution. The bottle positioning was changed daily from the left to the right to eliminate any potential effects of positioning on water consumption. Intake of water and sucrose during a 12-h dark cycle was measured by weighing the bottles before and after the test. The sucrose preference ratio was calculated as a percentage as follows: [sucrose consumption/water and sucrose consumption × 100].

For the FST, mice were placed in a water-filled (25ºC) cylinder with a height of 2 cm and a diameter of 15 cm and video-recorded for 6 min. Immobility times were determined using analytical software based on how long mice floated during the last 4 min of the test (Smart Junior ver.1.0.06, Panlab Inc., Spain).

For examining long-term memory, the CFCT was performed according to published protocols with slight modifications46. The mice were placed in a test shock box (model MK-450MSQ, Muromachi Kikai CO. LTD, Japan) and were subjected to three electric foot shocks (0.8 mA, 2-s duration, and 2-min interval) 2 min later. Mice were left in the apparatus for 1 min more and then returned to their home cages. Twenty-four hours later, the mice were again placed in the test box and video recorded for 3 min to measure immobility times using the same software.

A Y-maze test was conducted to examine working memory based on the methods of Rubaj et al.47 with slight modifications. The Y maze consisted of three equally spaced arms (length: 40 cm, height: 12 cm, width: 3 cm). Mice were placed at the end of one arm and were allowed to traverse within the apparatus freely, while being video-recorded for 8 min. Complete entry was determined when a mouse’s hind paws had entirely entered any arm of the maze. The ‘right’ choice was defined as consecutive entry into three different arms. Spontaneous alternation was calculated as the percentage of correct entries to the total number of entries.

Dissection and sample collection

After completing all behavioral tests, mice were weighed and sacrificed by decapitation, and blood samples were collected. Brain, epididymal adipose fat, soleus muscle, and EDL muscle were then harvested, and the hippocampus was quickly isolated from the brain. Subsequently, the weights of the soleus, EDL, and epididymal adipose tissue were measured. Blood samples were centrifuged at 1500×g for 20 min to separate plasma. Collected tissues and plasma were then frozen by liquid nitrogen and stored at − 80°C until analysis.

Plasma corticosterone and adiponectin concentration

Plasma corticosterone and adiponectin concentrations were measured using the Corticosterone ELISA Kit (Cayman Chem., USA) and Circulex Mouse Adiponectin ELISA Kit (MBL Life Science, Japan), respectively, according to the user manuals.

Superoxide dismutase activity, total radical trapping antioxidant parameter, and lipid peroxide levels

Hippocampi were homogenized in 100-mM Tri-HCl buffer (pH 7.4) using a Bioruptor UCD-250 (Cosmo Bio Inc., Japan). The homogenate was centrifuged at 750×g for 10 min. A portion of the supernatant was used to analyze superoxide dismutase (SOD) activity using a SOD activity analysis kit according to the supplier’s instructions (Dojin Science Inc., Japan). The remainder of the supernatant was used to collect the cytosolic fraction using a ProteoExtract Subcellular Proteome Extraction Kit (Merck Millipore Co., USA) according to the kit’s manual. Collected cytosolic fractions were filtered using an ultrafree MC filter (Merck Millipore Co., USA, pore size = 3000 A) to remove protein from the cytosolic fraction and were then used to measure the total radical trapping antioxidant parameter (TRAP) according to the methods of Mikami et al.48. TRAP level was defined as the level of water-soluble tocopherol Trolox (Aldrich, USA), which is known to trap two radicals per molecule. The rest of the homogenate was then used to measure LPO levels by a lipid hydroperoxide analysis kit (Cayman Chemical, Inc., USA).

RNA extraction and real-time PCR

The hippocampal and muscular samples of mice were homogenized in TRIzol Reagent (Invitrogen, CA, USA) on ice, and total RNA was extracted according to the manufacturer’s instructions. Total RNA was quantified using absorption at 260 nm and the 260:280 nm ratio in order to assess concentration and purity, respectively. Complementary DNA (cDNA) was synthesized using 1 μg of total RNA in a 20-μL reaction with oligo (dT)12–18 primers (Invitrogen) and the ReverTra Ace cDNA reverse transcription kit (Toyobo, Osaka, Japan) according to the manufacturer’s instructions. Quantitative real-time PCR was performed with the SsoAdvanced Universal SYBR Green Supermix (Bio-Rad) and a CFX Connect real-time PCR system (Bio-Rad) to quantify the mRNA levels of TGR5, BDNF, PGC-1α, Sirt1 and mTOR. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as the endogenous control. The oligonucleotide sequences used for analysis are shown in Table S1. The 2-ΔΔCt method was used to analyze relative mRNA expression values 49. Sample analysis for each gene was performed in duplicate.

Mitochondrial DNA copy number

For quantifying adipose tissue and soleus mtDNA, total DNA was extracted from the adipose tissue and soleus muscle using the phenol–chloroform method. Approximately 20 mg of the adipose tissue or soleus muscle was dissolved in 200 μL of lysate buffer containing 10 mM Tris–HCl (pH 8.0), 150 mM NaCl, 10 mM EDTA (pH 8.0), 0.1% SDS, and 2.5% proteinase K. Next, it was vortexed, incubated at 56 °C for 90 min, and centrifuged at 2,000g for 10 min. The supernatant was mixed with an equal volume of phenol/chloroform/isoamyl alcohol, vortexed, and centrifuged at 2,000g for 10 min. Then, the supernatant that was obtained was mixed with two volumes of 100% ethanol by slow inversion and centrifuged at 10,000g for 10 min. After the supernatant was removed, the precipitate was dissolved in 70% ethanol by slow inversion and centrifuged at 10,000g for 10 min. Subsequently, after the supernatant was removed, the precipitate was left for 5 min at room temperature, dissolved in Tris–EDTA (TE) buffer (pH 8.0), and stored at − 20 °C until analysis. DNA concentration and purity were analyzed using absorption at 260 nm and the 260:280 nm absorption ratio, respectively. Quantitative real-time PCR (10 ng DNA) was performed with the SsoAdvanced Universal SYBR Green Supermix. 18S ribosomal RNA (18S rRNA) was used as the nuclear DNA (nDNA) control. The sequences of the oligonucleotide used for mtDNA and nDNA analysis are shown in Table S1. The 2-ΔΔCt method was used to analyze the relative mtDNA to nDNA copy number ratio49. Sample analysis for each gene was performed in duplicate.

Lipolysis in adipocytes in response to oleanolic acid and oleuropein

We examined the lipolytic effects of oleanolic acid and oleuropein on primary cultured adipocytes according to the modified methods of Rodbel50. Briefly, the collected adipocytes from the mice fed with the standard diet mentioned above were incubated in an MEM medium containing 10% fetal bovine serum for 3 h and then incubated with 10 μM oleanolic acid or 1 μM oleuropein for 4 h. In another experiment, since oleanolic works via TRG5 and oleuropein dose via PPARγ, one hour before adding oleanolic acid or oleuropein to the medium, adipocytes were added 200 μM SBI-115 (TGR5 antagonist) or 10 μM GW9662 (PPARγ antagonist) to the medium and incubated ​for 4 h. The adipocytes and medium were then collected, and the concentration of free glycerol released from adipocytes into the medium was measured using a glycerol assay kit (Sigma-Aldrich, USA). The amount of released glycerol was calculated as pg per mg of protein, with the amount of adipocyte protein measured using a Pierce 660-nm protein assay reagent (Thermo Scientific, USA).

Lipolysis in vivo via olive leaf extract, oleanolic acid and oleuropein

We examined the lipolysis effect of olive leaf extract, oleanolic acid, oleuropein in vivo using the mice fed a standard diet and fasted for 16 h. Mice were intraperitoneally administered oleanolic acid (100 mg/kg of body weight) or oleuropein (100 mg/kg of body weight), and then their blood samples were collected from the tail vein at 1, 2, and 4 h after the injection. In another experiment, the mice were orally administered olive leaf extract (2,000 mg/kg of body weight) dissolved in water using a gavage probe, and their blood samples were collected from the tail vein at 1, 2, and 4 h after the injection. We determined the administered dose of oleanolic acid based on the assumption that olive leaf extract contained 5% of in-weight % oleanolic acid. Immediately afterwards, blood was centrifuged, and the plasma was then collected and stored at − 80 °C until analysis. The lipolytic effects of olive leaf extract, oleanolic acid and oleuropein were examined by measuring plasma glycerol levels using a glycerol assay kit.

Endurance running under atmospheric and low-oxygen conditions

We investigated whether administration of olive leaf extract improved endurance exercise capacity. Male ICR mice aged 8 weeks were divided into two groups: (1) mice fed a standard diet (control) and (2) mice fed a standard diet containing olive leaf extract. Both groups of the mice were reared in the standard cage at four mice per cage. Before starting the feeding experiment, all the mice were subjected to running on the treadmill at the speed of 10 m/min, 5 min per day during a week to habituate them to treadmill running. During the feeding experiment, the mice ran on a treadmill at a treadmill speed of 10 m/min for 10 min two times per week. After 5 weeks of feeding the diet containing olive leaf extract, an endurance exercise test was then performed on the treadmill under atmospheric conditions. One week later, the same endurance exercise test was conducted on a closed treadmill filled with 16% oxygen gas. During both tests, the treadmill speed and inclination angle were 20 m/min and 10°, respectively (Fig. 5A). We measured the time until mice became exhausted. We defined as exhaustion time when mice did not return to the treadmill lane from the resting platform three consecutive times. Immediately after the endurance test, blood samples were collected under anesthesia from the tail vein to measure blood lactate levels. Afterward, the mice were dissected by decapitation, and the soleus, EDL and gastrocnemius muscles were collected and stored at − 80 °C.

Statistical analysis

All data are expressed as mean ± SE and were analyzed using GraphPad Prism 8 (MDF Co., Ltd, Tokyo, Japan). Group comparisons were performed using a one-way ANOVA followed by Dunnett’s and Tukey’s post hoc tests. Time-course changes were compared between pre-exercise and post-exercise levels using a one-way ANOVA followed by t test comparisons. Comparisons of two groups were performed using Student’s t test for unpaired data. The differences between groups were considered statistically significant at p < 0.05.

Abstract

Olive tree by-products have been deeply studied as an invaluable source of bioactive compounds. Several in vitro and in vivo studies showed that olive leaf extract (OLE) has anti-inflammatory and antioxidant properties. Here, we wanted to assess the valuable benefits of two less-studied OLE components—3,4-DHPEA-EDA (Oleacin, OC) and 3,4-DHPEA-EA (Oleuropein-Aglycone, OA)—directly purified from OLE using a cost-effective and environmentally sustainable method, in line with the principles of circular economy. OLE, OC and OA were then tested in human cellular models involved in acute and chronic inflammation and in the pathogenesis of viral infections, i.e., lipopolysaccharide (LPS)-treated monocyte/macrophages (THP-1) and endothelial cells (HUVECs), senescent HUVECs and Poly(I:C)-treated small airway epithelial cells (hSAECs). Results showed that OC and OA are efficient in ameliorating almost all of the pro-inflammatory readouts (IL-1β, TNF-α, IL-8, ICAM, VCAM) and reducing the release of IL-6 in all the cellular models. In hSAECs, they also modulate the expression of SOD2, NF-kB and also ACE2 and TMPRSS2, whose expression is required for SARS-CoV-2 virus entry. Overall, these data suggest the usefulness of OLE, OC and OA in controlling or preventing inflammatory responses, in particular those associated with viral respiratory infections and aging.

Keywords:

olive leaf extract, oleacin, oleuropein-aglycone, antioxidant, anti-inflammatory, senescence, respiratory infections

1. Introduction

The human and animal health effects of chemical compounds that can be found in the fruit and leaf of Olea europaea L. tree have been known for a long time, especially those of oleuropein and hydroxytyrosol [1,2,3]. Olive leaf extract (OLE) showed similar properties to extra-virgin olive oil (EVOO), one of the main components of the Mediterranean Diet, whose qualities have long been studied [4,5]. Many in vitro and in vivo reports throughout the years demonstrated that OLE and EVOO reduce oxidative stress and inflammation [6,7,8], but their valuable effects were also found in cardiovascular diseases [9,10,11], metabolic disorders [12,13,14] and bacterial infections [15,16].

The main constituents of OLE are polyphenols; they are produced in leaves as protective molecules (phytoalexins) against leaf-eating insects, microbes and fungi [17,18]. Polyphenol total concentration in Olea europea L. tree decreases during summer, progressively increases in autumn and reaches its higher level at the beginning of winter. Therefore, olive leaves can be valorised more efficiently as olive by-products during EVOO production (defoliation of olives delivered to the mill) or during pruning [19].

Oleuropein, an ester of elenolic acid and 3,4-dihydroxyphenyl ethanol belonging to the secoiridoids family, is the major phenolic component found in olive leaves followed by other secoiridoids derived from 2-(4-hydroxyphenyl) ethanol (tyrosol). Oleuropein content depends upon the cultivar, ripening stage and extraction methods [20,21]. Notably, oleuropein concentration in olives is higher as compared to leaves but it is hydrolysed during olive oil production [22]. Therefore, the beneficial properties of EVOO are due to the oleuropein degradation products: its aglycone 3,4-DHPEA-EA (oleuropein aglycone) and dialdehydic forms 3,4-DHPEA-EDA (oleacein) and 3,4-DHPEA (hydroxytyrosol) [23]. However, there are many difficulties in isolating oleacein and oleuropein aglycone as pure compounds from olives, not to mention the high-priced extraction process.

Polyphenols have long been studied as antioxidant and anti-inflammatory agents; in the last decade, their efficacy has been demonstrated in the reduction of inflammaging. [24,25,26]. Inflammaging is a subclinical systemic inflammation observed during the aging process. Aging also entails an acquired immune system impairment (immune senescence). The main culprit of both inflammaging and immune senescence is the acquisition of a senescence-associated secretory phenotype (SASP) by senescent cells [27]. The main feature of SASP is the release of high levels of pro-inflammatory cytokines [24]. Altogether, these conditions contribute to the development of age-related diseases.

The experience and research following the COVID-19 pandemic showed that in elderly men with age-related comorbidities, viral infections could cause high mortality. In most of these patients, SARS-CoV-2 induces an uncontrolled local and systemic hyperinflammation (cytokine storm) with serious harmful acute respiratory distress syndrome and local bacterial/fungal superinfections, due to impaired microbicide capacity [28] and multi-organ failure [29,30,31]. In viral infections, this hyperinflammation is also promoted by the imbalance of redox homeostasis. This phenomenon has also been demonstrated for SARS-CoV-2 which spoils the redox homeostasis in the airways, leading to an increased replication and the entrance of SARS-CoV-2 into host cells [32].

Timely targeted strategies based on antioxidant drugs and viral-induced cytokine inhibitors would certainly improve the clinical outcome of infectious diseases, also in elderly people.

In light of the above-mentioned issues to obtain sufficient amounts of pure compounds from olive leaves, their beneficial effect in chronic inflammation associated with aging and acute inflammation associated with viral infection has still been poorly explored. Of note, our research group has developed a new environmentally friendly extraction method to obtain pure bioactive compounds from olive leaves, in line with circular economy principles and the 2030 Agenda for Sustainable Development [24,31,32,33].

chenlv Product Page

Therefore, here, we wanted to explore the anti-inflammatory properties of (i) a total olive leaf aqueous extract (OLE) and (ii) its less-studied derivatives oleuropein aglycone and oleacein, both produced in our laboratory, in in vitro human models of acute and chronic inflammation. For this purpose, we used (i) human LPS-treated primary vascular endothelial cells (HUVECs) and the monocytic cell line THP-1, (ii) replicative senescent HUVECs (RS-HUVECs)—the most studied and representative of SASP—and (iii) Poly(I:C)-treated primary small airway epithelial cells (hSAECs) as a model to study the molecular mechanisms underlying respiratory viral infections.

2. Materials and Methods

2.1. Sample Collection and Preparation

Olive leaves were collected in November from cultivar Leccino in the Marche region (Italy). Trees were grown without any kind of chemical treatment. Samples were washed with distilled water, dried and lyophilised using a benchtop freeze drier (Virtis Wizard 2.0 instrument, SP Industries, New York, NY, USA). Olive leaves were vacuum-sealed and stored at room temperature in the dark. Upon use, olive leaves were ground with a homogeniser to obtain an uneven powder which was immediately used for extraction ( ).

2.2. Olive Leave Extraction Procedure

An amount of 5. gr of olive leaf powder was macerated in 50 mL PBS at 4 °C for 24 h in the dark to maximise the extraction and avoid degradation. The resulting extract was centrifuged at 1500 RPM for 10 min to separate coarse particles and then filtered through a 0.22 μm membrane filter to ensure sterility. The extract was aliquoted and stored at −20 °C until use for cell treatments.

2.3. OLE Composition

The Folin–Ciocalteu assay, with minor modifications, was used to determine OLE’s total phenolic content (TPC). Different concentrations of Gallic acid were used as standard. An amount of 1 mL of Folin–Ciocalteau reagent diluted 1:10 in distilled water was mixed with 0.1 mL of OLE or standard. An amount of 0.9 mL of Na2CO3 solution (7.5% w/v) was added precisely after 3 min and incubated at room temperature for 90 min, protected from light. The TPC amount was determined by measuring the optical density (OD) at 760 nm using a microplate reader (MPT Reader, Invitrogen, Milano, Italy).

2.4. UHPLC-UV-ESI-HRMS Analysis

The chemical characterisation of OLE main compounds was carried out at the Department of Health Sciences of the University Magna Graecia of Catanzaro. UHPLC/UV-ESI-HRMS analyses for quantification of the extract were performed by reverse-phase ultra-high-performance liquid chromatography followed by high-resolution mass spectrometry, with ionisation in negative mode. Chromatography separation was performed using a Dionex Ultimate 3000 RS (Thermo Scientific—Rodano, MI, Italy), equipped with a Hypersil Gold C18 column (100 × 2.1 mm, 1.9 µm particle size, Thermo Scientific) and a mobile phase consisting of the following solvents: 98% A (H2O + 0.1% formic acid) and 2% B (methanol). The UV/VIS detector was set at 235, 254, 280 and 330 nm. Mass detection was performed by a high-resolution Q-Exactive orbitrap mass spectrometer (Thermo Scientific, Rodano, MI, Italy). Heated electrospray ionisation (HESI) was selected in negative polarity, with the following operating conditions: 70,000 resolving power (defined as FWHM at m/z 200), IT 100 ms, ACG target = 1 × 106 and scan range (100–900 m/z). In each scan, the negative exact mass [M-H]− of biophenols precursors was selected (Hydroxytyrosol 153.0557 m/z; Oleacein 319.1187 m/z; Oleuropein Aglycone 377.1248 m/z; Oleuropein 539.1770 m/z) in Parallel Reaction Monitoring (PRM). MS/MS analyses were performed according to the following operating conditions: resolution: 35.000; AGC target = 1 × 105; maximum IT 200 ms; collision energy (stepped NCE): 20,30,40. The quadrupole isolation window was set to 2.0 m/z. High-purity nitrogen was used as the sheath gas (30 arb units) and auxiliary gas (10 arb units). Xcalibur software (version 4.1, Thermo Fisher Scientific) was used for instrument control, data acquisition and data analysis.

2.5. Preparation of 3,4-DHPEA-EA (Oleuropein Aglycone) and 3,4-DHPEA-EDA (Oleacein)

Oleuropein aglycone (3,4-DHPEA-EA) was obtained from the controlled hydrolysis of oleuropein extracted from olive leaves as previously described [33,34]. Briefly, oleuropein (1.0 mM) dissolved in aqueous CH3CN (10 mL) was refluxed for 8 h at 80 °C in the presence of 10 mol% of Er(OTf)3. The hydrolysate was cooled, 5 mL of water was added and the mixture was extracted with CH2Cl2. After drying on Na2SO4, the organic solvent was removed in vacuo and the crude product was purified by flash chromatography (total yield = 70%).

Oleacein (3,4-DHPEA-EDA) was obtained via one step semi-synthesis under microwave-assisted aqueous Krapcho decarbomethoxylation of OLE extracted from the olive leaves of Olea europaea L., cultivar Coratina, as previously described [23]. Briefly, a water solution of oleuropein (0.1 mM in 0.5 mL of water) was put into a 3.0 mL glass vial, NaCl (2.0 equivalents) was added, it was sealed and then reacted in a Synthos 3000 Microwave oven by Anton Paar. At the end of the reaction (20 min), the oleuropein conversion was complete. The reaction crude was recovered by adding 0.5 mL of EtOH and purified by flash column chromatography to give pure oleacein (48% total yield).

2.6. Cell Culture

Primary Human Umbilical Vein Endothelial Cells (HUVEC) were obtained from a pool of donors and purchased from Clonetics (Lonza, Basel, Switzerland). Cells were maintained in a humidified atmosphere at 37 °C and 5% CO2, seeded at a density of 5000/cm2 in T75 flasks (Corning Costar, Sigma Aldrich, St. Louis, MO, USA), with endothelial growth medium (EGM-2), composed of endothelial basal medium (EBM) and the SingleQuots Bullet kit (Lonza, Switzerland). The medium was changed every 48 h and cells were trypsinised when approximately 80% confluent.

Replicative senescence was achieved after several replicative passages (measured as cumulative population doubling, cPD). cPD was calculated as the sum of PD changes, using the formula (log10 (F) − log10 (I))/log10, where F is the number of cells at the end of a passage, and I is the number of seeded cells. HUVECs were classified as young or senescent based on cPD, senescence-associated (SA)-β-Galactosidase activity and p16ink4a expression as previously described by our research group [24].

Human monocytic THP-1 cells were purchased from ATCC (Rockville, MD, USA) and grown in RPMI-1640 culture medium supplemented with 2-mercaptoethanol to a final concentration of 0.05 mM and with 10% heat-inactivated foetal bovine serum, 1% penicillin/streptomycin and 1% L-glutamine (all from Euroclone, Milano, Italy) at 37 °C in 5% CO2 in a humidified incubator. The cells were seeded at a density of 2 × 105 cells/ml in T75 flasks.

Primary human small airway epithelial cells (hSAEC) were purchased from ATCC (PCS-301-010) and grown to confluence in a humidified atmosphere at 37 °C and 5% CO2, seeded at a density of 5000/cm2 with Airway Epithelial Cell Basal Medium (ATCC. PCS-300-030) and the bronchial epithelial cells growth kit (ATCC, PCS-030-040). The medium was changed every 48 h and cells were trypsinised when approximately 80% confluent.

All cell lines have been tested for Mycoplasma.

2.7. Cell Viability Assay

Cell viability was assessed through the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. Cells were grown for 24 h in 12-well plates at a density of 5000/cm2 (HUVEC and hSAEC) or 2 × 105 cells/mL (THP-1) and then treated with different amounts of OLE (different doses in terms of TPC) or with different concentrations of Oleacein and Oleorupein-Aglycone for 24 h. As concerns Oleacin, the concentration range tested was 1–100 μM (0.32–32 μg/mL), whereas Oleuropein-Aglycone was 1–100 μM (0.36–36 μg/mL). Untreated cells were used as the control group. MTT solution (5 mg/mL) was added in each well (10 µL/100 µL Medium) and incubated for 2.5 h. Insoluble formazan salts produced were solubilised by adding DMSO (400 μL). The absorbance was measured at OD 540 nm using a microplate reader (MPT Reader, Invitrogen, Milano, Italy). Data are expressed as a percentage, according to the equation (T/C) × 100%, where T and C represent the mean OD of treated cells and untreated cells (control group), respectively.

2.8. Cell Treatments

Based on the results of the viability assay, young HUVECs were seeded at a density of 5000/cm2; after 24 h, cells were pre-treated with OLE (4 μg/mL) or with Oleacein (5 μM) or Oleorupein-Aglycone (5 μM) for 2 h and then stimulated with lipopolysaccharide (LPS) (500 ng/mL) for three hours to induce an inflammatory response. Untreated cells were used as a control to ensure the inflammatory effect of LPS. THP-1 cells were seeded at a concentration of 2 × 105/mL in suspension and treated with the same conditions after 24 h. RS-HUVECs were seeded at a density of 5000/cm2 and treated with OLE or with the single compounds for 24 h to evaluate their effect on the basal inflammatory status. hSAEC cells were seeded at a density of 5000/cm2 and pre-treated with OLE (4 μg/mL) or with the single compounds (1 μM OC, 5 μM OA) for 2 h, followed by a 24 h treatment with Poly(I:C) (5 μg/mL), according to the literature [35,36].

2.9. RNA Isolation and mRNA Expression by RT-qPCR

Total RNA was isolated using the Total RNA Purification kit (Norgen Biotek Corp., #37500, Thorold, ON, Canada), following the manufacturer’s instructions and stored at −80 °C until use. Total RNA quantification was determined by spectrophotometric quantification with Nanodrop ONE (NanoDrop Technologies, Wilmington, DE, USA) and reverse transcribed using PrimeScriptTM RT Reagent Kit with gDNA eraser (Cat. #RR037B, Takara), following manufacturer’s instructions. mRNA expression was evaluated by RT-qPCR using TB GreenTM Premix Ex TaqTM (Cat#RR420A, Takara) in a Rotor-Gene Q (Qiagen). The following primers were all acquired from Merck Millipore (Darmstadt, Germany): β-actin (FW: 5′-AACTGGAACGGTGGTCAAGGTGAC-3′, RV: 5′CAAGGGACTTCCTGTAACAATGC-3′), IL-1β(FW: 5′-AGATGATAAGCCCACTCTACAG-3′, RV: 5′-ACATTCAGCACAGGACTCTC-3′), IL-6 (FW: 5′-TGCAATAACCACCCCTGACC-3′, RV: 5′-GTGCCCATGCTACATTTGCC-3′), IL-8 (FW: 5′-GGACAAGAGCCAGGAAGAAA-3′, RV: 5′-CCTACAACAGACCCACACAATA-3′), NF-kB (FW: 5′-ACAGCTGGATGTGTGACTGG-3′, RV: 5′-TCCTCCGAAGCTGGACAAAC-3′), TNF-α (FW: 5′-AAGCCTGTAGCCCACGTCGTA-3′, RV: 5′-GGCACCACTAGTTGGTGGTCTTTG-3′), ICAM1 (FW: 5′-AGCCAGGAGACACTGCAGACA-3′, RV: 5′-TGGCTTCGTCAGAATCACGTT-3′), VCAM (FW: 5′-GGGAAGATGGTCGTGATCCTT-3′, RV: 5′-TCTGGGGTGGTCTCGATTTTA-3′), ACE2 (FW: 5′-GGGATCAGAGATCGGAAGAAGAAA-3′, RV: 5′-AGGAGGTCTGAACATCATCAGTG-3′), TMPRSS2 (FW: 5′-ACTCTGGAAGTTCATGGGCAG-3′, RV: 5′-TGAAGTTTGGTCCGTAGAGGC-3′), SOD2 (FW: 5′-GTTGGGGTTGGCTTGGTTTC-3′, RV: 5′-ATAAGGCCTGTTGTTCCTTGC-3′). mRNA quantification was assessed using the 2−ΔΔCt method. β-actin was used as endogenous control.

2.10. Western Blot

Cell lysates were obtained using RIPA buffer (10 mM Tris, pH 7.2, 150 mM NaCl, 1.0% Triton X-100, 0.1% SDS, 5 mM EDTA, pH 8.0) with a protease inhibitor cocktail (Roche Applied Science, Indianapolis, IN, USA). Bradford assay was used to evaluate protein concentration in each sample. Proteins (25 μg) were analysed with SDS-PAGE and transferred to a nitrocellulose membrane (Bio-Rad, Hercules, CA, USA). Then, 5% skim milk was used to block the membrane, which was then incubated overnight with the primary antibody. Rabbit anti-phospho-NF-κB (Cell Signaling Technology, Danvers, MA, USA), rabbit anti-ACE2 (Cell Signaling), mouse anti-TMPRSS2 (Santa Cruz Biotechnology, Dallas, TX, USA), mouse anti-β-actin (Santa Cruz Biotechnology, Dallas, TX, USA) and rabbit anti-GAPDH (Cell Signaling Technology, Danvers, MA, USA) were used as primary antibodies. Horseradish peroxidase-conjugated antibodies anti-mouse or anti-rabbit (The Jackson Laboratory, Bar Harbor, ME, USA) were used as secondary antibodies. Protein bands were visualised by using the Clarity ECL chemiluminescence substrate (Bio-Rad) with Uvitec Imager (UVItec, Cambridge, UK) and then quantified using ImageJ software 4.1.

2.11. ELISA

HUVEC and hSAEC supernatants were collected after each treatment, centrifuged and stored at −80 °C until use. Interleukin-6 (human) ELISA Kit (Invitrogen, Waltham, MA, USA) was used to measure the concentration of human IL-6 released, according to manufacturer’s protocol. In the case of THP-1 cells, IL-6 was measured in cell lysate obtained at the end of each treatment using RIPA buffer and protein concentration was determined with Bradford assay.

2.12. Statistical Analysis

Data are shown as mean ± SD or frequency (%) of at least three independent biological replicates. A paired sample t test was used for RT-qPCR, ELISA and densitometry data analysis. Data analysis was performed using IBM SPSS Statistics for Windows, version 25 (IBM Corp, Armonk, NY, USA). Statistical significance was defined as a two-tailed p-value < 0.05.

4. Discussion

In recent years, olive tree by-products, such as olive leaves, have been deeply studied for their invaluable source of bioactive compounds useful as anti-inflammatory and antioxidant agents. The olive leaf total extract (OLE) and its derived compounds, mainly oleuropein and hydroxytyrosol, have been tested in several in vitro and in vivo systems showing similar properties to EVOO. They can reduce inflammation and oxidative species production in experimental models of gastric and intestinal diseases [7,8,16]; they also exert beneficial effects in metabolic syndrome, atherosclerosis and cardiovascular diseases by suppressing the inflammatory response, reducing lipid peroxidation and attenuating hypertension [11,13,14,42].

Accordingly, here we have demonstrated the valuable benefits of OLE and two of its less-studied components—OC and OA—that we directly purified from OLE using a cost-effective and environmentally sustainable method. Specifically, these molecules efficiently reduced the inflammatory response of (i) two of the cell types always involved in host responses to pathogens, i.e., monocyte/macrophages and endothelial cells (LPS-treated HUVECs and THP-1) [43], (ii) small airway epithelial cells, central to the pathogenesis of lung injury following environmental agent inhalation (hSAECs treated with Poly(I:C) to mimic a viral infection) and (iii) endothelial senescent cells (RS-HUVECs), characterised by a SASP phenotype which negatively affects the dynamics of the host response to pathogens [44]. Indeed, even if the beneficial effects of OLE on the endothelium have been greatly investigated [11,13,42], the modulatory effect of OC and OA on endothelial cell activation due to severe infection has been little explored. Of note, our results showed that in both LPS-stimulated HUVECs and THP-1, pre-treatment with OLE, OC and to a lesser extent with OA was effective in reducing the mRNA expression of most of the cytokines and in the case of endothelial cells all the adhesion molecules tested. Notably, all treatments still successfully reduced IL-8 mRNA and IL-6 release in the conditioned medium. Due to the pivotal role played by these cytokines in LPS-induced inflammatory response, these data highlight the efficacy of both OC and OA as anti-inflammatory compounds even if OLE, which indeed contains a mix of more active compounds is the most effective.

Based on these data, we were encouraged to analyse the effects of the above-mentioned extract and compounds in endothelial senescent cells, an in vitro model of chronic inflammation. It has been now widely recognised that aging is associated with progressive cellular senescence and dysfunction. One of the main features of senescent cells is the SASP phenotype, which exerts a detrimental paracrine effect within the tissues (even in the lung [45]) on one hand and contributes to inflammaging on the other one [27], thus playing a role in the development of Age-Related Diseases (ARDs). In this framework, we demonstrated for the first time that OLE and its compounds can inhibit the synthesis and the release of pro-inflammatory cytokines characterising the SASP phenotype and furthermore the expression of adhesion molecules. Therefore, it can be assumed that they could be useful to reduce leucocyte recruitment, progressive tissue dysfunction and also systemic inflammation in vivo during aging ( ). Moreover, our results showed that OLE can restore RS-HUVECs’ antioxidant activity, by upregulating SOD2 mRNA expression ( A), an enzyme that prevents the age-dependent endothelial cell dysfunction and apoptosis by serving as a first line of defence against superoxide anion radical toxicity [46]. The possibility of using OLE and its components to control both endothelial dysfunction and oxidative stress could represent valid help to promote what has been called the Health Aging process [47].

These data are also important in light of the recent pandemic of COVID-19 that showed that inflammaging could be relevant for severe lung viral infections pathogenesis and progression because the case fatality rate grows exponentially with age and comorbidities [48]. Indeed, SARS-CoV-2 infection can cause systemic hyperinflammation [49] accompanied by the so-called “cytokine storm” and possibly the subsequent multi-organ failure. In this context, we wanted also to investigate the potential role of OLE and its bioactive compounds in modulating small airway epithelial cells’ activation upon viral infection. We exploited Poly(I:C)-treated hSAEC cells, which outline a model system that reliably mirrors the physiology and architecture of the lung epithelium, thus representing an effective tool to study the molecular mechanisms underlying respiratory viral infections, such as SARS-CoV-2 [50,51]. Our findings mostly agreed with the other models of bacterial-induced acute inflammation, because OLE was able to reduce the synthesis and release of all the pro-inflammatory cytokines and markers tested; concerning OC and OA, we observed for the first time that they inhibited NF-kB, TNF-α and IL-1β mRNA expression, respectively, and both IL-6 release and NF-kB phosphorylation ( ). The radical scavenging activity of OLE has long been known [52]. Here, we showed that OLE and OA reduced the transcription of SOD2, which was upregulated upon Poly(I:C)-stimulation as an NF-kB responsive gene [53] ( ), highlighting the contribution of these compounds in restoring the impaired redox homeostasis in the context of acute viral infections [41]. On the contrary, we could not appreciate SOD1 modulation, in line with the literature [54] (Supplementary Figure S3).

Finally, because several in silico computational studies reported an efficient molecular interaction between olive leaf metabolites and SARS-CoV-2 main viral targets [55], we wanted to test the antiviral activity of OC and OA, by analysing their ability to counteract the increasing expression of SARS-CoV-2 receptor ACE2 for entry and the serine protease TMPRSS2 for S protein priming upon Poly(I:C) stimulation [56,57]. Our results showed opposite trends between ACE2 and TMPRSS2 at mRNA or protein expression levels: OLE, OC and OA were effective in reducing ACE2 mRNA expression, whereas they could not significantly reduce TMPRSS2; on the contrary, OLE and OC were efficient in decreasing TMPRSS2 protein expression, whereas only OA could lower ACE2 ( ). The intricate role of ACE2 in the pathophysiology of respiratory viral infections is yet to be unravelled. It has been demonstrated that ACE2 and TMPRSS2 are differentially expressed depending on genetics, age and comorbidities, with significant upregulation in the elderly, smokers and COPD patients [41,57]. Therefore, the reduction in ACE2 mRNA expression levels after OLE, OC and OA treatment could be explained as a positive effect to counteract an inflammatory status; however, because ACE2 also exerts a protective effect in the lungs, by targeting angiotensin II [58], we can hypothesise the existence of a post-transcriptional regulatory mechanism that ensures a basal level of ACE2 protein expression in our cellular system. On the other hand, it has been shown that TMPRSS2-mediated ACE2 cleavage is even more harmful to the host and that an increased expression of TMPRSS2—due to genetics, age or comorbidities—could exacerbate the course of COVID-19 [59]. Our findings showed that although OLE, OC and OA were not effective in downregulating TMPRSS2 mRNA expression ( A), OLE and OC could successfully modulate the protein expression ( C). Therefore, we can assume that using OLE or its bioactive compounds could ameliorate infection prognosis.

5. Conclusions

Overall, this work strengthens the already existing data on the beneficial effects of olive leaf extract intake as an anti-oxidant, anti-bacterial and anti-inflammatory agent [14,60,61,62], also in upper respiratory illnesses [63]. In addition, it suggests its use to modulate the cytokine storm occurring during several forms of viral infections, especially in aged individuals. Our data also invite researchers (i) to deeply investigate the role and (ii) consider the eventual use of the two less-studied active OLE derivatives, oleacin and oleuropein-aglycone, in controlling inflammation. Their production from OLE is simple, safe, cost-effective and with a low environmental impact. OC and OA could, therefore, be formulated for oral administration in case of systemic inflammation, i.e., inflammaging, and infectious diseases. Moreover a specific formulation for aerosol intake to reduce or prevent local airway infection can be also hypothesised.

In fact, these bioactive compounds as a supplement may reduce the doses or the time of administration of conventional drugs, while maintaining their efficacy. However, while oral use of OLE has already been tested in several human clinical trials and commercial supplements are available, in vivo studies for OC and OA are still very few in humans and also in mice [64,65]. Hence, further studies are needed to test their toxicity and the best formulation for their administration. OC and OA bioactive and mucoadhesive formulations for systemic and local treatments are currently under investigation in our laboratory.

Supplementary Materials

The following supporting information can be downloaded at https://www.mdpi.com/article/10.3390/antiox12081509/s1, Figure S1: UHPLC-UV-ESI-HRMS analysis. Figure S2: hSAEC-induced inflammation. Figure S3: SOD1 mRNA expression.

Funding Statement

Ministero dell’Università e della Ricerca (MUR), Fondo Speciale per la Ricerca (FISR): IP 02077—OLEA-ACT (Olive leaf extract active ingredients against COVID-19 activity)—2020.

Author Contributions

Conceptualisation, A.P. and M.R.R.; investigation, A.S., C.G., S.B., A.G., D.R., G.M. and S.D.V.; methodology, A.S., C.G. and S.B.; formal analysis, A.S., C.G. and S.B.; data curation, A.S., C.G., S.B., A.G. and J.S.; writing—original draft preparation, A.S., C.G. and M.R.R.; writing—review and editing, A.G., J.S., D.P., A.P. and A.D.P.; visualisation, C.G. and S.B.; supervision, M.R.R.; project administration, M.R.R.; funding acquisition, M.R.R. and A.P. All authors have read and agreed to the published version of the manuscript.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

Data are contained within the article and Supplementary material.

Conflicts of Interest

The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results.

Footnotes

Disclaimer/Publisher’s Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.

Contact us to discuss your requirements of Olive Leaf Extract Supplier. Our experienced sales team can help you identify the options that best suit your needs.