**2. 3,4-DHPEA-EA and acute inflammation**

*Premise* - The inflammatory reaction is characterized by an initial increase in blood flow to the site of injury, enhanced vascular permeability, production of mediators such us prostaglandins, leukotrienes, histamine, bradykinin, platelet-activating factor (PAF) and the ordered and directional influx and selective accumulation of different effector cells from the peripheral blood at the site of injury. Influx of antigen non-specific but highly destructive cells (neutrophils) is one of the earliest stages of the inflammatory response. Carrageenaninduced local inflammation is commonly used to evaluate anti-inflammatory effects of nonsteroidal drugs (NSAIDs). Therefore, carrageenan-induced local inflammation (pleurisy) is a useful model to assess the contribution of mediators involved in cellular alterations during the inflammatory process. In particular, the initial phase of acute inflammation (0-1h) which is not inhibited by NSAIDs such as indomethacin or aspirin, has been attributed to the release of histamine, 5-hydroxytryptamine and bradykinin, followed by a late phase (1-6 h) mainly sustained by prostaglandin release and attributed to the induction of inducible cyclo-oxygenase (COX-2) in the tissue (Nantel *et al.*, 1999). It appears that the onset of the carrageenan-induced acute inflammation has been linked to neutrophil infiltration and the production of neutrophil-derived free radicals, such as hydrogen peroxide, superoxide and hydroxyl radical, as well as the release of other neutrophil-derived mediators. Free radicals are produced in small amounts by normal cellular processes as part of the mitochondrial electron transport chain and the microsomal cytochrome P-450 system. They are formed during traumatic or hypoxic injuries as a consequence of insufficient oxygenation. Reactive oxygen species (ROS) and reactive nitrogen species (RNS) can react with and subsequently damage proteins, nucleic acids, lipids, and extracellular matrix proteins. During the inflammatory response, ROS and RNS modulate phagocytosis, secretion, gene expression, and apoptosis. Indeed, under pathological circumstances such as acute lung injury and sepsis, excess production of neutrophil-derived ROS and RNS may influence neighbouring endothelial or epithelial cells, contributing to the amplification of inflammatory tissue injury (Fialkow *et al.*, 2007). Furthermore, oxidative stress elicits the activation of the redox-sensitive transcription factors such as nuclear factor-κB (NF-κB) and AP-1, that play a central and crucial role in inducing the expression of inflammatory cytokines and intercellular adhesion molecule (ICAM-1) (Chen *et al.*, 2004) and the activation of the redox-sensitive protein kinases such as the mitogen-activated protein kinase (MAPK) superfamily (Li *et al.*, 2002). Thus, the study model was designed to evaluate the effects of 3,4-DHPEA-EA in a mice model of acute inflammation (0.1 ml of saline containing 2% λcarrageenan was injected into the pleural cavity). In particular, we investigated the effects of 3,4-DHPEA-EA on the lung injury associated with carrageenan induced pleurisy. In order to gain a better insight into the mechanism of action of 3,4-DHPEA-EA, we have also investigated the effects on: 1) lung damage (histology), 2) polymorphonuclear (PMN) infiltration (myeloperoxidase [MPO] activity), 3) ICAM-1 and platelet-adhesion-molecule (P-selectin) expression, 4) nitrotyrosine and poly-ADP-ribose (PAR) formation, 5) proinflammatory cytokines production, tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β), 6) lipid peroxidation (malondialdehyde [MDA] levels), and 7) nitric oxide (NO) synthesis (nitrite-nitrate concentration).

#### **2.1. Materials and methods**

288 Olive Germplasm – The Olive Cultivation, Table Olive and Olive Oil Industry in Italy

R'OOC

O O

R= OH: R'=Me; oleuropein R=OH: R'=H; demethyloleuropeine R= H: R'=Me; ligstroside

R

HO

In particular, the anti-inflammatory properties of olive oil phenolic compounds seem to overlap with those attributed to non-steroidal anti-inflammatory drugs (Procopio A, *et al.*, 2009). The majority of phenolic compounds found in olive oil or table olives are derived from the hydrolysis of oleuropein, the major phenolic constituent of the leaves and unprocessed olive drupes of *Olea europaea* and responsible for the bitter taste of immature and unprocessed olives. Concentrations of up to 9.0 mg/l of oleouropein and 5.6 mg/l of its hydrolysis product hydroxytyrosol, have been detected in some preparations of olive oil (Montedoro *et al.*, 1992). Oleuropein, a glucoside with hydroxyaromatic functionality, has recognized several pharmacological properties including antioxidant, anti-inflammatory, anti-atherogenic, anti-cancer, antidiabetic, antimicrobial, and antiviral, and for these reasons, it is commercially available as food supplement in Mediterranean countries (Miles *et al.*, 2005; Covas, 2008; Omar, 2010). A more efficient anti-inflammatory role of the aglyconic 3,4-DHPEA-EA compared with the glycosidic form of oleuropein possibly derives from the greater lipophilicity of the former, a property that should allow better cell membrane incorporation and/or interaction with other lipids (Saija *et al.*, 1998). We focused on the antinflammatory properties of 3,4-DHPEA-EA, a hydrolysis product obtained from Oleuropein by the action of β-glucosidase on the parent glucoside, has been evaluated in a mice model of acute inflammation (carrageenan-induced pleurisy) and in a mice model of

O O

OGluc

O

MeOOC

R= OH: 3,4-DHPEA-EA R= H: *p*-HPEA-EA

CHO

R= OH: 3,4-DHPEA R= H: *p*-HPEA

R

OH

HO

O

chronic inflammation (collagen-induced arthritis) (Impellizzeri *et al.*, 2011a-b).

*Premise* - The inflammatory reaction is characterized by an initial increase in blood flow to the site of injury, enhanced vascular permeability, production of mediators such us prostaglandins, leukotrienes, histamine, bradykinin, platelet-activating factor (PAF) and the ordered and directional influx and selective accumulation of different effector cells from the peripheral blood at the site of injury. Influx of antigen non-specific but highly destructive cells (neutrophils) is one of the earliest stages of the inflammatory response. Carrageenaninduced local inflammation is commonly used to evaluate anti-inflammatory effects of non-

**2. 3,4-DHPEA-EA and acute inflammation** 

**Figure 1.** Chemical structures.

R

HO

*Animals* - Male CD mice, weight 20-25 g; Harlan Nossan, Milan, Italy, were used in these studies. The animals were housed in a controlled environment and provided with standard rodent chow and water. Animal care was in compliance with Italian regulations on the protection of animals used for experimental and other scientific purposes (D.lgs 116/92) as well as with EEC regulations (O.J. of E.C. L358/1 12/18/1986).

*Carrageenan-induced pleurisy -* Carrageenan-induced pleurisy was induced as previously described (Cuzzocrea *et al.*, 1999). Mice were anaesthetized with isoflurane and subjected to a skin incision at the level of the left sixth intercostals space. The underlying muscle was dissected and saline (0.1 ml) or saline containing 2% λ-carrageenan (0.1 ml) was injected into the pleural cavity. The skin incision was closed with a suture and the animals were allowed to recover. At 4 h after the injection of carrageenan, the animals were killed by inhalation of CO2. The chest was carefully opened and the pleural cavity rinsed with 1 ml of saline solution containing heparin (5 U ml-1) and indomethacin (10 µg ml-1). The exudate and washing solution were removed by aspiration and the total volume measured. Any exudate, which was contaminated with blood, was discarded.

Oleuropein an Olive Oil Compound in Acute and Chronic Inflammation Models: Facts and Perspectives 291

*Immunohistochemical localization of ICAM-1, P-selectin, nitrotyrosine and PAR -* At the end of the experiment, the tissues were fixed in 10% (w/v) PBS-buffered formaldehyde and 8 µm sections were prepared from paraffin embedded tissues. After deparaffinization, endogenous peroxidase was quenched with 0.3% (v/v) hydrogen peroxide in 60% (v/v) methanol for 30 min. The sections were permeablized with 0.1% (w/v) Triton X-100 in PBS for 20 min. Non-specific adsorption was minimized by incubating the section in 2% (v/v) normal goat serum in PBS for 20 min. Endogenous biotin or avidin binding sites were blocked by sequential incubation for 15 min with biotin and avidin, respectively. Sections were incubated overnight with anti-nitrotyrosine rabbit polyclonal antibody (Upstate, 1:500 in PBS, v/v), anti-PAR antibody (BioMol, 1:200 in PBS, v/v), anti-ICAM-1 antibody (Santa Cruz Biotechnology, 1:500 in PBS, v/v) or with anti-P-selectin polyclonal antibody (Santa Cruz Biotechnology, 1:500 in PBS, v/v). Sections were washed with PBS, and incubated with secondary antibody. Specific labeling was detected with a biotin-conjugated goat anti-rabbit IgG and avidin-biotin peroxidase complex (Vector Laboratories, DBA). In order to confirm that the immunoreaction for the nitrotyrosine was specific, some sections were also incubated with the primary antibody (anti-nitrotyrosine) in the presence of excess nitrotyrosine (10 mM) to verify the binding specificity. To verify the binding specificity for PAR, ICAM-1, P-selectin, some sections were also incubated with only the primary antibody (no secondary) or with only the secondary antibody (no primary). In these situations no positive staining was found in the sections indicating that the immunoreaction was positive

*MPO activity -* MPO activity, an indicator of PMN accumulation, was determined as previously described (Mullane *et al.*, 1985). At the specified time following injection of carrageenan, lung tissues were obtained and weighed, each piece homogenized in a solution containing 0.5% (w/v) hexadecyltrimethyl-ammonium bromide dissolved in 10 mM potassium phosphate buffer (pH 7) and centrifuged for 30 min at 20,000 x *g* at 4°C. An aliquot of the supernatant was then allowed to react with a solution of tetramethylbenzidine (1.6 mM) and 0.1 mM H2O2. The rate of change in absorbance was measured spectrophotometrically at 650 nm. MPO activity was defined as the quantity of enzyme degrading 1 µmol of peroxide min-1 at 37°C and was expressed in milliunits per gram

*MDA measurement -* MDA levels in the lung tissue were determined as an indicator of lipid peroxidation as previously described (Ohkawa *et al.*, 1979). Lung tissue collected at the specified time, was homogenized in 1.15% (w/v) KCl solution. A 100 µl aliquot of the homogenate was added to a reaction mixture containing 200 µl of 8.1% (w/v) SDS, 1.5 ml of 20% (v/v) acetic acid (pH 3.5), 1.5 ml of 0.8% (w/v) thiobarbituric acid and 700 µl distilled water. Samples were then boiled for 1 h at 95°C and centrifuged at 3,000 x *g* for 10 min. The

*Materials -* Unless otherwise stated, all compounds were obtained from Sigma-Aldrich Company Ltd. (Poole, Dorset, U.K.). 3,4-DHPEA-EA was obtained from the controlled

absorbance of the supernatant was measured using spectrophotometry at 650 nm.

in all the experiments carried out.

weight of wet tissue.

*Experimental Design* - Mice were randomly allocated into the following groups: (i) CAR + saline group. Mice were subjected to carrageenan-induced pleurisy (N = 10); (ii) CAR + 3,4- DHPEA-EA group (100 µM/kg). Same as the CAR + saline group but 3,4-DHPEA-EA (100 µM/kg, i.p.) were administered 30min after to carrageenan (N = 10); (iii) CAR + 3,4-DHPEA-EA group (40 µM/kg). Same as the CAR + saline group but 3,4-DHPEA-EA (40 µM/kg, i.p.) were administered 30min after to carrageenan (N = 10); (iv) Sham + saline group. Shamoperated group in which identical surgical procedures to the CAR group was performed, except that the saline was administered instead of carrageenan (N = 10); (v) Sham + 3,4- DHPEA-EA group. Same as the Sham+saline group but 3,4-DHPEA-EA (100 µM /kg, i.p.) were administered 30min after to carrageenan (N = 10). The doses of 3,4-DHPEA-EA (40 and 100 µ M /kg, i.p.) used here were based on previous in vivo studies (Procopio A, *et al.*, 2009).

*Histological examination -* Lung tissues samples were taken 4 h after injection of carrageenan. Lung tissues samples were fixed for 1 week in 10 % (w/v) PBS-buffered formaldehyde solution at room temperature, dehydrated using graded ethanol and embedded in Paraplast (Sherwood Medical, Mahwah, NJ, USA). Sections were then deparaffinized with xylene, stained with hematoxylin and eosin. All sections were studied using Axiovision Zeiss (Milan, Italy) microscope.

*Measurement of cytokines* **-** TNF-α and IL-1β levels were evaluated in the exudate 4 h after the induction of pleurisy by carrageenan injection as previously described (Cuzzocrea *et al.*, 1999). The assay was carried out using a colorimetric commercial ELISA kit (Calbiochem-Novabiochem Corporation, Milan, Italy).

*Measurement of nitrite-nitrate concentration* **-** Total nitrite in exudates, an indicator of NO synthesis, was measured as previously described (Cuzzocrea *et al.*, 2001). Briefly, the nitrate in the sample was first reduced to nitrite by incubation with nitrate reductase (670 mU/ml) and β-nicotinamide adenine dinucleotide 3'-phosphate (NADPH) (160 µM) at room temperature for 3 h. The total nitrite concentration in the samples was then measured using the Griess reaction, by adding 100 µl of Griess reagent (0.1% w/v) naphthylethylendiamide dihydrochloride in H2O and 1% (w/v) sulphanilamide in 5% (v/v) concentrated H3PO4; vol. 1:1) to the 100 µl sample. The optical density at 550 nm (OD550) was measured using ELISA microplate reader (SLT-Lab Instr., Salzburg, Austria). Nitrite concentrations were calculated by comparison with OD550 of standard solutions of sodium nitrite prepared in H2O.

which was contaminated with blood, was discarded.

(Milan, Italy) microscope.

Novabiochem Corporation, Milan, Italy).

*Carrageenan-induced pleurisy -* Carrageenan-induced pleurisy was induced as previously described (Cuzzocrea *et al.*, 1999). Mice were anaesthetized with isoflurane and subjected to a skin incision at the level of the left sixth intercostals space. The underlying muscle was dissected and saline (0.1 ml) or saline containing 2% λ-carrageenan (0.1 ml) was injected into the pleural cavity. The skin incision was closed with a suture and the animals were allowed to recover. At 4 h after the injection of carrageenan, the animals were killed by inhalation of CO2. The chest was carefully opened and the pleural cavity rinsed with 1 ml of saline solution containing heparin (5 U ml-1) and indomethacin (10 µg ml-1). The exudate and washing solution were removed by aspiration and the total volume measured. Any exudate,

*Experimental Design* - Mice were randomly allocated into the following groups: (i) CAR + saline group. Mice were subjected to carrageenan-induced pleurisy (N = 10); (ii) CAR + 3,4- DHPEA-EA group (100 µM/kg). Same as the CAR + saline group but 3,4-DHPEA-EA (100 µM/kg, i.p.) were administered 30min after to carrageenan (N = 10); (iii) CAR + 3,4-DHPEA-EA group (40 µM/kg). Same as the CAR + saline group but 3,4-DHPEA-EA (40 µM/kg, i.p.) were administered 30min after to carrageenan (N = 10); (iv) Sham + saline group. Shamoperated group in which identical surgical procedures to the CAR group was performed, except that the saline was administered instead of carrageenan (N = 10); (v) Sham + 3,4- DHPEA-EA group. Same as the Sham+saline group but 3,4-DHPEA-EA (100 µM /kg, i.p.) were administered 30min after to carrageenan (N = 10). The doses of 3,4-DHPEA-EA (40 and 100 µ M /kg, i.p.) used here were based on previous in vivo studies (Procopio A, *et al.*, 2009). *Histological examination -* Lung tissues samples were taken 4 h after injection of carrageenan. Lung tissues samples were fixed for 1 week in 10 % (w/v) PBS-buffered formaldehyde solution at room temperature, dehydrated using graded ethanol and embedded in Paraplast (Sherwood Medical, Mahwah, NJ, USA). Sections were then deparaffinized with xylene, stained with hematoxylin and eosin. All sections were studied using Axiovision Zeiss

*Measurement of cytokines* **-** TNF-α and IL-1β levels were evaluated in the exudate 4 h after the induction of pleurisy by carrageenan injection as previously described (Cuzzocrea *et al.*, 1999). The assay was carried out using a colorimetric commercial ELISA kit (Calbiochem-

*Measurement of nitrite-nitrate concentration* **-** Total nitrite in exudates, an indicator of NO synthesis, was measured as previously described (Cuzzocrea *et al.*, 2001). Briefly, the nitrate in the sample was first reduced to nitrite by incubation with nitrate reductase (670 mU/ml) and β-nicotinamide adenine dinucleotide 3'-phosphate (NADPH) (160 µM) at room temperature for 3 h. The total nitrite concentration in the samples was then measured using the Griess reaction, by adding 100 µl of Griess reagent (0.1% w/v) naphthylethylendiamide dihydrochloride in H2O and 1% (w/v) sulphanilamide in 5% (v/v) concentrated H3PO4; vol. 1:1) to the 100 µl sample. The optical density at 550 nm (OD550) was measured using ELISA microplate reader (SLT-Lab Instr., Salzburg, Austria). Nitrite concentrations were calculated

by comparison with OD550 of standard solutions of sodium nitrite prepared in H2O.

*Immunohistochemical localization of ICAM-1, P-selectin, nitrotyrosine and PAR -* At the end of the experiment, the tissues were fixed in 10% (w/v) PBS-buffered formaldehyde and 8 µm sections were prepared from paraffin embedded tissues. After deparaffinization, endogenous peroxidase was quenched with 0.3% (v/v) hydrogen peroxide in 60% (v/v) methanol for 30 min. The sections were permeablized with 0.1% (w/v) Triton X-100 in PBS for 20 min. Non-specific adsorption was minimized by incubating the section in 2% (v/v) normal goat serum in PBS for 20 min. Endogenous biotin or avidin binding sites were blocked by sequential incubation for 15 min with biotin and avidin, respectively. Sections were incubated overnight with anti-nitrotyrosine rabbit polyclonal antibody (Upstate, 1:500 in PBS, v/v), anti-PAR antibody (BioMol, 1:200 in PBS, v/v), anti-ICAM-1 antibody (Santa Cruz Biotechnology, 1:500 in PBS, v/v) or with anti-P-selectin polyclonal antibody (Santa Cruz Biotechnology, 1:500 in PBS, v/v). Sections were washed with PBS, and incubated with secondary antibody. Specific labeling was detected with a biotin-conjugated goat anti-rabbit IgG and avidin-biotin peroxidase complex (Vector Laboratories, DBA). In order to confirm that the immunoreaction for the nitrotyrosine was specific, some sections were also incubated with the primary antibody (anti-nitrotyrosine) in the presence of excess nitrotyrosine (10 mM) to verify the binding specificity. To verify the binding specificity for PAR, ICAM-1, P-selectin, some sections were also incubated with only the primary antibody (no secondary) or with only the secondary antibody (no primary). In these situations no positive staining was found in the sections indicating that the immunoreaction was positive in all the experiments carried out.

*MPO activity -* MPO activity, an indicator of PMN accumulation, was determined as previously described (Mullane *et al.*, 1985). At the specified time following injection of carrageenan, lung tissues were obtained and weighed, each piece homogenized in a solution containing 0.5% (w/v) hexadecyltrimethyl-ammonium bromide dissolved in 10 mM potassium phosphate buffer (pH 7) and centrifuged for 30 min at 20,000 x *g* at 4°C. An aliquot of the supernatant was then allowed to react with a solution of tetramethylbenzidine (1.6 mM) and 0.1 mM H2O2. The rate of change in absorbance was measured spectrophotometrically at 650 nm. MPO activity was defined as the quantity of enzyme degrading 1 µmol of peroxide min-1 at 37°C and was expressed in milliunits per gram weight of wet tissue.

*MDA measurement -* MDA levels in the lung tissue were determined as an indicator of lipid peroxidation as previously described (Ohkawa *et al.*, 1979). Lung tissue collected at the specified time, was homogenized in 1.15% (w/v) KCl solution. A 100 µl aliquot of the homogenate was added to a reaction mixture containing 200 µl of 8.1% (w/v) SDS, 1.5 ml of 20% (v/v) acetic acid (pH 3.5), 1.5 ml of 0.8% (w/v) thiobarbituric acid and 700 µl distilled water. Samples were then boiled for 1 h at 95°C and centrifuged at 3,000 x *g* for 10 min. The absorbance of the supernatant was measured using spectrophotometry at 650 nm.

*Materials -* Unless otherwise stated, all compounds were obtained from Sigma-Aldrich Company Ltd. (Poole, Dorset, U.K.). 3,4-DHPEA-EA was obtained from the controlled

hydrolysis of oleuropein extracted from olive leaves by means the patented method reported by Procopio *et al.* (2009). All other chemicals were of the highest commercial grade available. All stock solutions were prepared in non-pyrogenic saline (0.9% NaCl; Baxter, Italy, UK).

Oleuropein an Olive Oil Compound in Acute and Chronic Inflammation Models: Facts and Perspectives 293

**Figure 2.** Effect of 3,4-DHPEA-EA (Ole aglycone) on histological alterations of lung tissue 4 h after carrageenan-induced injury and on PMN infiltration in the lung. Lung sections taken from carrageenantreated mice treated with vehicle demonstrated edema, tissue injury (B) as well as infiltration of the

demonstrating the normal architecture of the lung tissue (A). The histological score (D) was made by an independent observer. MPO activity, index of PMN infiltration, was significantly elevated at 4 h after carrageenan (CAR) administration in vehicle-treated mice (E), if compared with sham mice (E). 3,4- DHPEA-EA significantly reduced MPO activity in the lung (E). The figure is representative of at least 3 experiments performed on different experimental days. Data are expressed as mean ± S.E.M. from

No significant increase of TNF-α and IL-1β exudates levels was found in the sham animal (Fig. 4A,B). NO levels were also significantly increased in the exudate obtained from mice administered carrageenan (Fig. 4C). Treatment of mice with 3,4-DHPEA-EA significantly reduced NO exudates levels (Fig. 4C). No significant increase of NO exudates levels was

Effects of 3,4-DHPEA-EA on carrageenan-induced nitrotyrosine formation, lipid peroxidation and poly-ADP-ribosyl polymerase (PARP) activation - Immunohistochemical analysis of lung sections obtained from mice treated with carrageenan revealed positive staining for nitrotyrosine (Fig. 5B). In contrast, no positive staining for nitrotyrosine was found in the lungs of carrageenan-treated mice, which had been treated with 3,4-DHPEA-EA (100 µM/kg) (Fig. 5C). In addition, at 4 hours after carrageenan-induced pleurisy, MDA levels were also measured in the lungs as an indicator of lipid peroxidation. As shown in Figure 5D, MDA levels were significantly increased in the lungs of carrageenan-treated mice. Lipid peroxidation was significantly attenuated by the intraperitoneal injection of 3,4-DHPEA-EA (Fig. 5D). At the same time point (4 h after carrageenan administration),

tissue with neutrophils (B). Carrageenan-treated animals treated with 3,4-DHPEA-EA (C) demonstrated reduced lung injury and neutrophil infiltration. Section from sham animals

n = 10 mice for each group. \*\*, P < 0.01 versus sham group. ##, P < 0.01 versus carrageenan.

found in the sham animal (Fig. 4C).

*Statistical evaluation* **-** All values in the figures and text are expressed as mean � standard error (s.e.m.) of the mean of *n* observations. For the in vivo studies n represents the number of animals studied. In the experiments involving histology or immunohistochemistry, the figures shown are representative of at least three experiments (histological or immunohistochemistry coloration) performed on different experimental days on the tissue sections collected from all the animals in each group. The results were analyzed by one-way ANOVA followed by a Bonferroni post-hoc test for multiple comparisons. A *p*-value less than 0.05 were considered significant and individual group means were then compared with Student's unpaired t test. A *p*-value of less than 0.05 was considered significant.
