Protective effect of olive leaf extract on hippocampal injury induced by transient global cerebral ischemia and reperfusion in Mongolian gerbils.
Keywords: Olive leaf Neuroprotective effects Oxidative stress Hippocampus Gerbils
The beneficial effects of antioxidant nutrients, as well as complex plant extracts, in cerebral ischemia/reperfusion brain injury are well known. Mediterranean diet, rich in olive products, is associated with lower incidence of cardiovascular disease, cancer, inflammation and stroke. In this study, the possible neuroprotective effect of standardized dry olive leaf extract (OLE) is investigated for the first time. Transient global cerebral ischemia in Mongolian gerbils was used to investigate the OLE effects on different parameters of oxidative stress and neuronal damage in hippocampus. The biochemical measurements took place at different time points (80min, 2,4 and 24 h) after reperfusion. The effects of applied OLE were compared with effects of quercetin, a known neuroprotective plant flavonoid. Pretreatment with OLE (100 mg/kg, per os) significantly inhibited production of superoxide and nitric oxide, decreased lipid peroxidation, and increased superoxide dismutase activity in all time points examined. Furthermore, OLE offered histological improvement as seen by decreasing neuronal damage in CAl region of hippocampus. The effects of applied OLE were significantly higher than effects of quercetin (100 mg/kg, per os). Our results indicate that OLE exerts a potent neuroprotective activity against neuronal damage in hippocampus after transient global cerebral ischemia, which could be attributed to its antioxidative properties.
[C] 2011 Elsevier GmbH. All rights reserved.
Mediterranean diet rich in olive drupes and olive oil, is associated with the lower incidence of cardiovascular disease, cancer, inflammation and stroke (Trichopoulou and Critselis 2004; Fung et al. 2009). Olive (Olea europaea L.) phenolics are powerful antioxidants, both in vitro and in vivo and it is known that olive oil represents a key healthy component of Mediterranean diet (Fito et al. 2007). Moreover, it has been reported that dietary intake of virgin olive oil, thanks to its minor constituents, shows neuroprotective effects (Gonzalez-Correa et al. 2007).
Not only olive oil, but olive leaf also has different beneficial effects on human health (El and Karakaya 2009). The main constituent of the olive leaf is oleuropeine, one of iridoide monoterpenes, which is thought to be responsible for its pharmacological effects. Furthermore, the olive leaf contains triterpenes (oleanolic and maslinic acid), flavonoides (luteolin, apigenine, rutin), and chalcones (olivin, olivin-diglucoside). It has been traditionally used in hypertonia, arteriosclerosis, rheumatism, gout, diabetes mellitus, and fever (Fleming 2000). A number of papers have been published reporting different pharmacological effects of olive leaf and its constituents, but none of them has focused on neuroprotective activity of total olive leaf extract (OLE) in an global ischemia animal model.
Cerebral ischemia and reperfusion (I/R) is known to induce the generation of reactive oxygen species (ROS), which, in turn, leads to oxidative damage of membrane lipids, proteins and nucleic acids (Chan 2001). In global cerebral ischemia, increased production of ROS has been regarded as an underlying factor for mediating delayed neuronal death, especially to pyramidal neurons in the hippocampal CA1 area (Hara et al. 2000). This has raised attention to testing possible beneficial effects of different antioxidants, especially those from plant sources, on cerebral injury after stroke. The beneficial effects of antioxidant nutrients in cerebral ischemia and recirculation brain injury are previously reported (Ikeda et al. 2003). Neuroprotective effects of single phenolics, such as resveratrol from grape and red wine, curcumin from turmeric, apocynin from Picrorhiza kurroa, and epi-gallocatechin from green tea were evaluated (Sun et al. 2008). However, the reports of neuroprotection by natural compounds from plants frequently refer to complex extracts like those of Ginkgo biloba (Lee et al. 2002) and green tea (Hong et al. 2001) and not to a single compounds.
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In light of the above considerations and since OLE is known for its anti-oxidative activity (El and Karakaya 2009), we investigated, for the first time, the possible neuroprotective effect of total OLE in the hippocampus of Mongolian gerbils subjected to transient global cerebral I/R.
Materials and methods
Olive leaf extract EFLA[R] 943, standardized to 18-26% of oleuropein, was purchased from Frutarom Switzerland Ltd. (Wadenswil, Switzerland). The extract was manufactured from the dried leaves of Olea europaea L., applying an ethanol (80% m/m) extraction procedure. After a patented filtration process (EFLA[R] Hyperpure), the crude extract was dried. Stability and microbiological purity were confirmed by the manufacturer. The extract was further analyzed in our previous study, and it was detected that its total phenols content, determined by Folin-Ciocalteau assay, was 197.8 jxg GAE per g of dry extract; total flavonoids and tannins content was 0.29% and 0.52%, respectively. HPLC analysis (Fig. 1 and Table 1) revealed a complex mixture of phenolic compounds: oleuropein, luteolin-7-O-glucoside, apigenine-7-O-glucoside, quercetin and caffeic acid (Dekanski et al. 2009). In this study, the same batch of EFLA[R] 943 is used. Quercetin, butylated hydroxytoluene (BHT) and 2,2-diphenyl-1-picrylhydrazyl radical (DPPH) were purchased from Sigma (St. Louis, MO, USA), PEG 400 from BASF, Germany. All other chemicals used for biochemical analyses were from Sigma.
Table 1 Quantitative determination of flavonoids, phenolcarbonic acids and oleuropein in olive leaf extract. Compound name (a) Amount mg % Caffeic acid (1) 0.013 0.02 Vanilin (2) Not found Rutin (3) Not found Luteolin-7-O-glucoside (4) 0.027 0.04 Apigenin-7-O-glucoside (5) 0.046 0.07 Oleuropein (6) 13.147 19.8 Quercetin (7) 0.027 0.04 Luteolin (8) Trace (a) Apigenin (9) Not found Chryseriol (1O) Trace (b) Reproduced from Dekanski et al. (2009) with permission. (a) The numbers refer to the compounds marked on the HPLC chromatogram (Fig. 1). (b) Determination was not possible - in the extract under the limit of quantitative analysis
Determination of antioxidant activity in vitro
Antioxidant capacity of the both olive leaf extract and quercetin was measured using the DPPH assay based on the scavenging ability to 2,2-diphenyl-1-picrylhydrazyl (DPPH) stable radical (Goupy et al 1999). Butylated hydroxytoluene (BHT) was used as a positive control. Briefly, the samples in different concentrations were mixed with DPPH solution and ethanol. After vortexing, the tubes were left in the dark at room temperature after which the absorbance was measured at 517 nm using a UV-vis spectrophotometer HP 8453 (Agilent Technologies, Santa Clara, CA). Each measurement was performed in triplicate under identical conditions. Antioxidant activities were expressed as the [IC.sub.50] values, i.e., the concentration of antioxidant required to cause 50% reduction in the original concentration of DPPH.
Adult male Mongolian gerbils (Meriones unguiculatus, 55-65 g) were used in this study. Groups of four gerbils per cage (Erath, FRG), were housed in an air-conditioned room, at the temperature of 23 [+ or -] 2[degrees]C, with 55 [degrees] 10% humidity, and with lights on 12h/day (7:00-19:00). The gerbils were given commercial food and tap water ad libitum. All experimental manipulations were performed during the light phase, between 9:00 and 15:00, under identical conditions. Animals used for procedures were treated in strict accordance with the NIH Guide for Care and Use of Laboratory Animals (1985) and the European Communities Council Directive (86/609/EEC), as well as with approval of the local Ethical Committee. All efforts were made to minimize animal suffering and to reduce the number of animals used.
Occlusion of common carotid arteries
Global ischemia occurs when cerebral blood flow is reduced throughout the most part or the entire brain. Since mature gerbils lack posterior communicating arteries, those normally connect the posterior circulation of the brain from the vertebral arteries with the anterior circulation from the carotid arteries within the circle of Willis, occlusion of both common carotid arteries results, reproducibly, in global forebrain ischemia. The Mongolian gerbils were anaesthetized by diethyl ether and placed in the dorsal position.The neck area was shaved, and then both common carotid arteries were exposed carefully by blunt dissection and clamped for 10 min with microaneurysm clips. After the clips were removed, reperfusion was confirmed visually, and the skin was sutured by 3-4 loose silk stitches. For sham-operated animals, both common carotid arteries were exposed but not occluded. Post-ischemic temperature was carefully monitored. Since the changes in body temperature are known to have impact on the consequences of global ischemia, it was maintained at 37 [+ or -] 0.3[degrees]C throughout the surgical procedure by a feedback-controlled heating pad (TR-100, PS-100, Fine Science Tool, North Vancouver, Canada). Gerbils were allowed to recover in their home cages for 2 h under a Xenon heating lamp and then returned to animal quarters.
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Experimental procedure - induction of global cerebral ischemia
The gerbils were divided into experimental groups (n=6 per group). Control, non-treated groups were intact and sham-operated while the treated groups were submitted to 10 min ischemia. According to the drug treatment plan, 30 min before the occlusion, negative control group of gerbils (PEG) was given 10% PEG 400 intragastrically (ig) by metal tube for gavage, OLE group was given l00mg/kg of OLE previously dissolved in 10% PEG 400, and QUE group (positive control) was given 100 mg/kg of QUE also dissolved in 10% PEG 400. All solutions were prepared on the day of experiment. Intact gerbils were not submitted to any type of surgical procedure and served as a control for operation stress, while sham-operated gerbils were exposed to the same surgical intervention as ischemic gerbils, but without occlusion of common carotid arteries. The animals were allowed to survive from 80 min up to 24 h for biochemical evaluation, and 24 h for histological evaluation (Fig. 2).
For biochemical evaluation, animals were decapitated and the brains immediately removed at 80 min, 120 min, 4 h and 24 h after 10 min ischemia. Both hippocampi from individual animals were quickly isolated and homogenized in the ice-cold buffer containing 0.25 M sucrose, 0.1 mM EDTA, 50 mM K-Na phosphate buffer, pH 7.2. Homogenates were centrifuged twice at 1580 xg for 15min at 4[degrees]C. The supernatant (crude mitochondrial fraction) obtained by this procedure was then frozen and stored at -70[degrees]C. Nitric oxide (NO) production was quantified by measuring nitrite, a stable oxidation end product of NO by Griess' method (Guevara et al. 1998). [O.sub.2] - was measured by the reduction of nitro blue tetrazolium (NBT), as previously described (Spitz and Oberley 1989). The assay of superoxide dismutase (SOD) activity by the adrenaline method (Fridovich 1995) was used. Total SOD activity includes the activity of two SOD isoforms - SOD1 (Cu/ZnSOD) citoplasmatic and SOD2 (MnSOD) mitochondrial isoforms. In this way we measured the activity of total endogenous antioxidative status/capacity. Mal-ondialdehyde (MDA), the product of polyunsaturated free fatty acids, reacts with thiobarbituric acid and it represents index of lipid peroxidation (ILP). 1LP was measured spectrophotometrically as thiobarbituric acid reactive species. The content of protein in the hippocampus homogenates was measured by the method of Lowry et al. (1951) using bovine serum albumin as a standard. Each biochemical assay was performed in triplicate under identical conditions.
Histological staining for nerve cells was performed to evaluate hippocampal neural damage. The gerbils (treated with PEG, OLE or QUE as explained) were euthanized by decapitation 24 h after I/R. The brains were removed, fixed in buffered 10% formalin, dehydrated in graded alcohol solutions and embedded in paraffin. Brain coronal sections (7 [micro] m of thickness) at the level approximately 6 mm posterior to ventral pole of the brain were taken with a microtome (Reichart, Austria). Sections were stained with 0.5% cresyl violet acetate and analysed using a light microscope (Zeiss Axio-scop 2). The morphology of neurons in the dorsal hippocampus of both hemispheres was examined. Quantitative analysis of morphological changes was carried out by counting the number of both viable and damaged neurons in the CA1 region of the hippocampus. Cells with round nuclei and visible nucleoli were considered undamaged, while dark shrunken cells were considered damaged. The quantification of ischemic brain damage was done by counting the basophilic cells seen in medial, intermediate and lateral sectors of the hippocampal CA1 regions. Extent of cell damage in CA1 hippocampal regions was quantified as the mean number of the persisting neurons in the coronal sections. Three defined 300 [[micro]m.sup.2] fields CA1 hippocampal regions were recorded with camera MC 100095 (Carl Zeiss Jena) and counted for persisting neurons in a computer-assisted image analysis system (KS 300, Carl Zeiss Jena). The percentage of remaining neurons in the CA1 region was calculated in all experimental groups.
The data are presented as means [+ or -]SD. The statistical significance of differences between groups regarding biochemical assays' results was assessed by one-way ANOVA (with p < 0.05 as significant, p < 0.01 as higher significant, and p < 0.001 as highly significant). For histopathological evaluation, the difference between proportions was assessed by t-test and p value less than 0.05 was considered statistically significant.
In vitro antioxidant activity
Before in vivo experiments, we investigated in vitro antioxidant potential of olive leaf extract and quercetin, as well as synthetic antioxidant BHT, as positive control. Radical scavenging activity was assessed using the 2,2-diphenyl-l-picrylhydrazyl (DPPH). The concentrations of the extract, quercetin or BHT that causes 50% neutralization of DPPH radicals (IC50 values) were 33.1 [micro]g/ml, 16.3 [micro]g/ml and 18.2 [micro]g/ml, respectively.
Oxidative stress parameters in the gerbil hippocampus
Results obtained from our preliminary experiments, comparing mean values of oxidative stress parameters (nitrite levels, [O sub.2] production, SOD activity, and ILP) in intact and sham-operated animals represent the effect of surgical procedure on the gerbil brain. Since there was no significant difference between mean values in these two groups (data not shown), it means that only surgical procedure is not sufficient to trigger oxidative stress in the gerbil brain. Applied PEG (solvent), OLE (treatment) and QUE (positive control) per se did not affect the measured oxidative stress parameters (data not shown). We considered sham-operated group of animals as control (presented as dash line in Figs. 3-6). Our treatment groups were: 10-min ischemia + PEG, 10-min ischemia + QUE, and 10-min ischemia + OLE. Measurements of oxidative stress parameters were done 80 min, 120 min, 4 h and 24 h after reperfusion, thus we detected the pattern and the dynamics of oxidative stress after 10-min ischemia in gerbil hippocampus (Figs. 3-6).
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Post-ischemic nitrite levels ([mu]M/mg proteins) in hippocampal homogenates are presented in Fig. 3. Cerebral ischemia resulted in generally higher nitrite production with the highest nitrite level detected 4h after reperfusion (Fig. 3; black bars, p < 0.001). Pretreatment with QUE and OLE significantly decreased nitrite levels in all tested time points (Fig. 3; gray and white bars, respectively). Pretreatment with OLE was more effective than QUE, but decreased nitrite levels were still above control. The most significant effect of pretreatment with OLE was observed 4 and 24 h after reperfusion (p < 0.001; Fig. 3).
Production of [O sub.2]-([mu] M NBT/mg prot.) in hippocampal homogenates is presented in Fig.4. Cerebral ischemiaperse induced generally higher [O sub.2]- levels (Fig. 4; black bars, p < 0.001). The highest [O sub.2]- levels were obtained, again, 4 h after 10-min ischemia, but with the tendency of decreasing detected 24 h after reperfusion. Pretreatment with QUE and OLE significantly decreased [O sub.2]- levels in all time points (Fig. 4; gray and white bars, respectively). Pretreatment with OLE was more effective, but decreased [O sub.2]- levels were still above control. The most significant effect of pretreatment with OLE was observed 4 h after reperfusion (p < 0.001; Fig. 4), when the post-ischemic nitrite levels reached the highest scores.
The results presented in Fig. 5, show SOD activity (U/mg prot.) in hippocampal homogenates. Ischemia induced significant decrease in SOD activity that was the greatest 4 h after reperfusion (Fig. 5; black bars, p < 0.001). Pretreatment with QUE significantly increased SOD activity, while pretreatment with OLE was more effective and returned SOD activity to the control levels in all tested time-points after reperfusion (Fig. 5; gray and white bars, respectively).
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We found that 10-min ischemia significantly increased ILP up to 24 h (Fig. 6; black bars, p < 0.001). The highest ILP was obtained, again, 4h after reperfusion, and after 24 h ILP decreased. Pretreatment with QUE and OLE significantly decreased ILP (Fig. 6; gray and white bars, respectively). Pretreatment with OLE was more effective, but decreased ILP was still above control. The most significant effect of pretreatment with OLE was observed 4 and 24 h after reperfusion (p < 0.001; Fig. 6).
Hippocampal neuronal injury
For histological evaluation of neuronal hippocampal damage 24 h after reperfusion, coronal brain sections were stained with cresyl violet (Fig. 7). Viable neurons were microscopically identified by the presence of typical nuclei with the clear nucleoplasm and a distinct nucleolus, surrounded by purple-stained cytoplasm. Dark blue stained shrunken neurons with pycnotic cell shape and nuclear condensation were considered damaged (Eke et al. 1990).
Light microscopy examination of the sections from the control group revealed the regular structure of the hippocampus. The nuclei of pyramidal neurons in CA1 region were clear, round or oval in shape, with distinct nucleoli. They were arranged in 4-5 layers (Fig. 7A).
Significant morphological changes of pyramidal neurons in the hippocampal CA1 region were observed in the ischemia + PEG experimental group. Numerous neurons (31.4 [+ or -] 3.1%; Fig. 7E) showed robust morphological changes, including the shrinkage of perikarions and increased intensity of cytoplasm staining. Nuclei of damaged nerve cells were dark, irregular in shape and shrunken in comparison with clear round nuclei of the viable neurons. The signs of extracellular edema were also evident, as seen by clear space around both capillaries and neurons (Fig. 7B).
In the OLE and QUE pretreated animal groups, significantly smaller degree of morphological damage of CA1 pyramidal neurons was seen compared to the PEG group (p<0.01; Fig. 7E). QUE pretreatment decreased the number of damaged neurons to 11.3 [+ or -]2.7% (Fig. 7C and E), while OLE pretreatment showed the highest protection of hippocampal neurons, since only 5.2 [+ or -] 1.5% of damaged neurons was seen (Fig. 7D and E). The difference between OLE and QUE group was also statistically significant (p<0.01; Fig. 7E).
Cerebral ischemia and reperfusion is known to induce oxidative stress in brain, and consequently neuronal death, especially in CA1 region of the hippocampus. This study presents, for the first time, the biochemical evidence of reduction of ischemia-induced oxidative stress by olive leaf extract - OLE (l00mg/kg) in gerbil hippocampus and its neuroprotective efficacy at the histological level. The effects of OLE were significantly higher than the effects of quercetin (100 mg/kg), a known neuroprotective plant flavonoid.
In vitro antioxidant activity of both OLE and quercetin was in the range of antioxidant activity of BHT. The high DPPH radical scavenging capacity of OLE is not surprising, since results obtained using a variety of in vitro assays showed its great antioxidative potential (Hayes et al. 2011). It is known that the structure of phenolic compounds is a key determinant of their radical scavenging activity. Hence, the strong antioxidant activity of OLE could be due to the presence of several phenolic compounds and hydroxy1 groups in their structure.
The in vivo dose of OLE used in the present study was calculated according to a clinical study in which OLE EFLA[R] 943 at l000mg daily effectively reduced blood pressure in patients with stage-1 hypertension (Susalit et al. 2011). Also, clinical study tested effectiveness of quercetin in a similar daily dose (Edwards et al. 2007). For the extrapolation of the dosage from humans to gerbils, we used food intake rather than body weight as a criterion (Rucker and Storms 2002).
Oleuropein, the main component of OLE, is rapidly absorbed after oral administration, with maximum plasma concentration occurring after 2 h (Del Boccio et al. 2003). In a short-term kinetic study in humans, it has been shown that quercetin is also rapidly absorbed and that a peak plasma values are achieved approximately 2 h after single ingestion of black currant juice (Erlund et al. 2006). According to this data, we had chosen tested time points after reperfusion to obtain more detailed information on dynamic changes in the oxidative stress rate in the hippocampus.
Our experiments showed time dependent changes of oxidative stress parameters in the hippocampus after 10-min ischemia. The same pattern of changes was observed for all parameters examined. The highest increase of NO, [O sub.2] - and MDA production, as well as a decrease of SOD activity was seen 4 h after reperfusion.
OLE pretreatment significantly inhibited post-ischemic production of superoxide anion and nitric oxide, inhibited lipid peroxidation, and increased SOD activity in the gerbil hippocampus in all tested time points. Furthermore, it offered histological improvement as seen by decreasing neuronal damage in hippocampal CA1 region, 24 h after reperfusion.
The occurrence of oxidative stress correlates well with delayed neuronal loss of hippocampal CA1 pyramidal neurons, suggesting that ROS formation may cooperate in a series of molecular events that link ischemic injury to the neuronal cell death. In this study, we did not investigate neuronal death in CA1 region of hippocampus, but rather post-ischemic neuronal changes (stage IV, according to Eke et al. 1990), since 24 h is time not sufficient to develop delayed neuronal death (Kirino 1982). More than 30% of CA1 hippocampal neurons 24 h after reperfusion showed robust ischemic changes, which were significantly attenuated by pretreatment with both OLE and QUE, the effect of OLE being significantly better.
Having in mind known effects of OLE constituents, these results are not surprising. It is already reported that oleuropein protects against oxidative damage in ischemic-reperfused myocardium in rabbits. Pretreatment with oleuropein (10 and 20mg/kg, per os) reduced the infarct size and decreased MDA plasma concentration. Also, oleuropein kept the SOD activity stable at different time points of ischemia and reperfusion and therefore left the myocardium in an "antioxidant" state (Andreadou et al. 2006).
The role of QUE in the stroke prevention was already proven in a prospective clinical study (Keli et al. 1996). In addition, it was suggested that the group of quercetin-related flavonoids could become lead molecules for the development of neuroprotective compounds with multitarget anti-ischemic effects (Dajas et al. 2003). Cho et al. (2006) demonstrated that QUE (50mg/kg i.p.) reduced neuronal damage caused by global ischemia. All of above mentioned was the reason why we selected QUE as positive control in our study.
Another OLE constituent - caffeic acid also has the protective effect on both early and delayed injuries after focal cerebral ischemia in rats; and this effect may partly relate to 5-lipoxygenase inhibition (Zhou et al. 2006). Furthermore, a protective effect against hydrogen peroxide-induced oxidative damage on rat brain tissue was observed through the treatment with low doses of caffeic acid (Pereira et al. 2006).
All so far known effects of OLE constituents are of great importance in their possible synergistic effects in neuroprotection.
Since purification of herbal extracts to single compounds usually results in loss of potent biological activity, we may also suppose that the association of various substances in OLE, with their complex interactions and different cellular and molecular targets might be responsible for the impressive neuroprotective activity of the extract documented here. Additionally, the obvious advantage of total OLE is that it is an easily attainable product of olive leaves, without purification of any of the fractions of the extract needed in order to apply it in possible prevention of neuronal damage after stroke.
In conclusion, our results indicate that OLE exerts a potent neuroprotective activity. It ameliorates oxidative stress and hippocampal neuronal damage after transient global cerebral ischemia in Mongolian gerbils. Concerning the different mechanisms of action of OLE phytochemicals (olive phenolics), we can presume that its neuroprotective effect is more complex and thus it is worthy of further investigation.
Conflict of interest
None of the co-authors has any conflict of interest to declare.
This study was supported by the Serbian Ministry of Science and Technological Development (Grant No. 41005) and MMA Grant (VMA/06-10/B.4).
0944-7113/$ - see front matter [C] 2011 Elsevier GmbH. All rights reserved. doi: 10.1016/j.phymed.2011.05.010
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Dragana Dekanski (a), *, Vesna Selakovic (b), Vesna Piperski (a), Zeljka Radu!ovic (a), Andrej KorenicS LidijaRadenovic (c)
(a) Biomedical Research, R&D Institute, Galenika a.d., Pasterova 2, 11000 Belgrade, Serbia
(b) Institute for Medical Research, Military Medical Academy, Belgrade, Serbia
(c) Faculty of Biology, University of Belgrade, Serbia
* Corresponding author. Tel.: +381 11 3610 314; fax: +381 113610 053.
E-mail address: firstname.lastname@example.org (D. Dekanski).