bs_bs_banner
Geriatr Gerontol Int 2014
ORIGINAL ARTICLE: BIOLOGY
Olive leaf extract decreases age-induced oxidative stress in major organs of aged rats Jale Çoban,1 Serdar Öztezcan,1 Semra Dog˘ru-Abbasog˘lu,2 Ilknur Bingül,2 Kübra Yes¸il-Mizrak1 and Müjdat Uysal2 1
Department of Biochemistry, Yeditepe University Medical Faculty and 2Department of Biochemistry, Istanbul Medical Faculty, Istanbul University, Istanbul, Turkey
Aim: Olive leaf (Olea europaea L.) extract (OLE) is a powerful anti-oxidant rich in polyphenols. As oxidative stress plays an important role in aging, we investigated the effect of OLE on oxidative stress in the liver, heart and brain of aged rats. Methods: Young (age 3 months) and aged (age 20 months) Wistar rats were used. Aged rats received OLE (500 and 1000 mg/kg/day) in drinking water for 2 months. Malondialdehyde (MDA), diene conjugate (DC), protein carbonyl (PC), glutathione (GSH), vitamin E and vitamin C levels, and superoxide dismutase (SOD), glutathione peroxidase (GSH-Px) and glutathione transferase (GST) activities were determined. Results: MDA, DC and PC levels increased in tissues of aged rats. GSH levels decreased in the liver, but not in the heart and brain. There was no change of other anti-oxidant parameters in tissues. Hepatic SOD and GSH-Px protein expressions also remained unchanged. OLE treatment caused decreased tissue MDA, DC and PC levels, and increased hepatic GSH levels in aged rats. Other anti-oxidant parameters, hepatic SOD and GSH-Px protein expressions did not alter in aged rats by OLE treatment. Conclusion: The present results suggest that OLE seems to be useful for decreasing oxidative stress in examined tissues by acting as an anti-oxidant itself without affecting the anti-oxidant system. Geriatr Gerontol Int 2014; ••: ••–••. Keywords: aging, olive leaf extract, oxidative stress, rats, tissues.
Introduction Free radicals-induced oxidative stress plays an important role in the aging process.1 Oxidative stress parameters have been found to increase in tissues, such as the liver,2–8 heart3,4,6–8 and brain,2,3,7,8 with increasing age. Several reports are also available in the literature regarding non-enzymatic and enzymatic anti-oxidants in the liver,2,5–9 heart6–9 and brain5,7–10 during aging. It has been found that pro-oxidant–anti-oxidant balance is altered in an organism during aging. Therefore, several authors have investigated the effect of anti-oxidants in protecting the tissues against age-induced oxidative stress.5–9
Accepted for publication 10 October 2013. Correspondence: Dr Müjdat Uysal MD, Department of Biochemistry, Istanbul Medical Faculty, Istanbul University, Çapa, 34093, Istanbul, Turkey. Email: [email protected]
© 2014 Japan Geriatrics Society
Olive tree (Olea europaea L.) leaves have been used widely in traditional remedies in Mediterranean countries.11 Animal studies have shown that olive leaves have antihypertensive, anti-atherogenic, anti-inflammatory, hypoglycemic and hypocholesterolemic effects.11 These effects are attributed to the anti-oxidant components of olive leaves.11,12 The main constituent of olive leaf is oleuropein, which is thought to be responsible for its pharmacological effects. In addition, olive leaf contains triterpenes, flavonoids, chalcones and tannins. Therefore, olive leaf is considered to be one of the most potent natural anti-oxidants by its chemical content.11–13 Several experimental studies have shown that olive leaf extract (OLE) or its constituents, such as oleuropein, had protective effects in atherosclerosis,14 diabetes,15 hypertension,16 cardiotoxicity,17,18 neurotoxicity,19,20 gastric lesions21,22 and cancers.23,24 OLE attenuated cardiac, hepatic and metabolic changes in rats with metabolic syndrome.25 It prevented hepatic fat accumulation in a rat model of non-alcoholic steatosis,26 and modulated cold restraint stress-induced oxidative doi: 10.1111/ggi.12192
|
1
J Çoban et al.
changes in rat liver.27 Although some studies are available on the beneficial effects of polyphenolic compounds obtained from various sources,28–30 there is no study investigating the effect of OLE in tissues against age-induced oxidative stress. For this reason, we wanted to investigate the effect of OLE treatment on pro-oxidant and anti-oxidant status in the liver, heart and brain tissues of aged rats. Malondialdehyde (MDA), diene conjugate (DC) and protein carbonyl (PC) levels, as well as glutathione (GSH), vitamin E and vitamin C levels, and Cu,Znsuperoxide dismutase (SOD), glutathione peroxidase (GSH-Px) and glutathione transferase (GST) activities together with protein expressions of SOD and GSH-Px enzymes were determined. These parameters were also measured in tissues of aged rats treated with OLE.
Methods Materials Chemicals were supplied from Sigma-Aldrich (St. Louis, MO, USA). OLE (liquid) was provided by the Bio-Olive Ltd Company (Ayvalık, Turkey). For the preparation of OLE, olive leaves were collected from the Ayvalık region in Turkey. Samples were dried and crushed. After crushing, 10 g of olive leaf powder was extracted with 70% (v/v) aqueous ethanol for 24 h at room temperature by a shaking incubator, which was fixed to 180 r.p.m. The extracts were filtered with Whatman No. 1 filter paper. To remove ethanol, the filtrates were evaporated by using a rotary evaporator under reduced pressure at 38°C. Thus, obtained liquid OLE is dark green in color, clear and has a slightly sweet taste. This liquid extract was kept at 4°C during the period of administration. The total phenol and oleuropein content of OLE liquid were analyzed by using the Folin–Ciocalteu method31 and Hewlett-Packard high-performance liquid chromatography with a diode array detector, respectively. Total phenol content was calculated as 196.8 mg gallic acid equivalent /g OLE. Oleuropein content was also detected as 97.0 mg/g OLE.
Animals and treatment Young (age 3 months; 200.6 ± 18.2 g) and aged (age 20 months; 498.2 ± 48.6 g) male Wistar rats were obtained from Yeditepe University Animal Research Center, Istambul, Turkey. The animals were allowed free access to food and water, and were kept in wire-bottomed stainless steel cages. The Animal Ethics Review Committee of Yeditepe University approved the experimental protocol. 2
|
At the start of the experiment, the young animals were 3 months-of-age and the aged animals were 20 monthsof-age. Rats were given OLE (6 or 12 mL OLE liquid/L in drinking water) for 2 months. In the present study, the consumption of OLE was roughly equivalent to 500 and 1000 mg/kg/day in aged rats.
Preparation of tissues At the end of the experimental period, rats were fasted overnight and anesthesized with sodium pentobarbital (50 mg/kg; i.p.). Blood was collected in dry tubes by cardiac puncture, and serum was obtained by centrifugation for biochemical analysis. Liver, heart and brain tissues of rats were quickly removed, and washed in 0.9% NaCl and kept at −70°C until they were analyzed. Tissues were homogenized in ice-cold 0.15 mol/L KCl (10%; w/v). Homogenates were centrifuged at 600 g for 10 min at 4°C, and then supernatants were recentrifuged at 10 000 g for 20 min to obtain postmitochondrial fractions.
Determination of lipid peroxides and PC levels Lipid peroxidation was assessed by two methods in the tissue homogenates. MDA levels were measured by thiobarbituric acid test.32 The breakdown product of 1,1,3,3-tetraethoxypropane was used as the standard. DC levels were determined in tissue lipid extracts at 233 nm spectrophotometrically, and calculated using a molar extinction coefficient of 2.52 × 104 mol/L1 cm1.32 The oxidative protein damage was spectrophotometrically detected by the quantification with 2,4dinitrophenylhydrazine of protein carbonyl groups. PC levels were calculated from the maximum absorbance (360 nm) using a molar absorption coefficient of 22 000 mol/L1 cm1. The results were expressed as nmol carbonyl per mg protein.33
Determination of non-enzymatic and enzymatic anti-oxidants in tissues GSH levels were measured with 5,5-dithiobis-(2nitrobenzoate) at 412 nm in tissue homogenates.34 Vitamin E and vitamin C levels were also measured in tissue homogenates by the method of Desai35 and Omaye et al.,36 respectively. Anti-oxidant enzyme activities were determined in postmitochondrial fractions. Cu,Zn-SOD activity was assayed by its ability to increase the effect of riboflavinsensitized photo-oxidation of o-dianisidine.37 GSH-Px38 and GST39 activities were measured using cumene hydroperoxide and 1-chloro-2,4-dinitrobenzene as substrates, respectively. Protein levels were determined using bicinchoninic acid.40 © 2014 Japan Geriatrics Society
Effect of olive leaf extract on aged rats
Determination of the expressions of SOD and GSH-Px in the liver
Statistical analyses
Western blotting was carried out to study the protein expressions of SOD and GSH-Px proteins as previously described.41 Then, 50 μg/well proteins were loaded onto 12% sodium dodecylsulfate-polyacrylamide gel electrophoresis and transferred to polyvinylidene fluoride membranes. After blocking, membranes were incubated for 16 h at 4°C with rabbit polyclonal antibody against SOD (sc-11407; Santa Cruz Biotechnology, Santa Cruz, CA, USA) or GSH-Px protein (sc-30147; Santa Cruz Biotechnology). They were further incubated for 1.5 h at room temperature with goat anti-rabbit immunoglobulin G conjugated to horseradish peroxidase (sc-2004; Santa Cruz Biotechnology). The chemiluminescence signals were visualized by exposing the membranes to Kodak Biomax X-ray film (Sigma-Aldrich Chemie GmbH, Taufkrichen, Germany) for 1–5 min, and then the bands were quantitated using the image analysis software. Values of western blot (SOD and GSH-Px) were normalized with an internal standard (actin, sc-1616R; Santa Cruz Biotechnology).
The results were expressed as mean ± SD. Experimental groups were compared using Kruskal–Wallis variance analysis test followed by post-hoc Mann–Whitney U-test. In all cases, P < 0.05 was considered to be statistically significant.
Results Effect of OLE on bodyweight Bodyweight gain in young rats was significantly increased during 2 months (10.2 ± 2.1 g/per week). However, the weight gain and bodyweight did not alter in untreated and OLE-treated aged rats during the experimental period. Final bodyweights were detected as 282.2 ± 30.7 g in the young group; 505 ± 42.8 g in the untreated-aged group; 512 ± 49.6 g in the OLE 500-treated aged group; and 503 ± 47.2 g in the OLE 1000-treated aged rats at the end of the experimental period.
Determinations in serum
Effect of aging on pro-oxidant–anti-oxidant balance
Serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), lactate dehydrogenase (LDH) and creatine kinase (CK) activities were determined on a Cobas Integra 800 autoanalyzer (Roche Diagnostics, Mannheim, Germany).
MDA, DC and PC levels increased significantly in the liver, heart and brain tissues of aged rats as compared with young rats (Table 1). Significant decreases in GSH levels were detected in the liver, but not in the heart and brain of aged rats. However, vitamin E and vitamin C
Table 1 The effect of olive leaf extract (treatment (500 and 1000 mg/kg/day for 2 months) malondialdehyde, diene conjugate and protein carbonyl levels in the liver, heart and brain tissues of aged rats
Liver Young rats Aged rats Aged rats Aged rats Heart Young rats Aged rats Aged rats Aged rats Brain Young rats Aged rats Aged rats Aged rats
Treatment
MDA (nmol/g)
DC (μmol/g)
PC (nmol/mg protein)
– – OLE (500 mg/kg/day) OLE (1000 mg/kg/day)
33.1 ± 3.82 50.0 ± 10.6† 36.6 ± 4.50‡ 32.3 ± 4.70‡
2.55 ± 0.24 3.96 ± 0.80† 2.48 ± 0.30‡ 2.42 ± 0.42‡
1.37 ± 0.22 2.39 ± 0.41† 1.40 ± 0.28‡ 1.28 ± 0.32‡
– – OLE (500 mg/kg/day) OLE (1000 mg/kg/day)
16.5 ± 1.69 23.3 ± 5.03† 20.8 ± 4.60† 17.8 ± 4.88‡
1.62 ± 0.17 2.09 ± 0.49† 1.61 ± 0.12‡ 1.56 ± 0.13‡
1.40 ± 0.19 2.20 ± 0.36† 1.50 ± 0.28‡ 1.48 ± 0.32‡
– – OLE (500 mg/kg/day) OLE (1000 mg/kg/day)
57.7 ± 4.88 69.9 ± 8.68† 66.7 ± 8.12† 58.3 ± 7.90‡
2.80 ± 0.36 3.57 ± 0.54† 3.06 ± 0.48 2.87 ± 0.32‡
1.26 ± 0.14 2.08 ± 0.32† 1.60 ± 0.22†‡ 1.38 ± 0.26‡
Data presented as mean ± SD; n = 8, each. †P < 0.05 as compared with young rats; ‡P < 0.05 as compared with aged rats (Kruskal–Wallis test; post-hoc Mann–Whitney U-test). DC, diene conjugate; MDA, malondialdehyde; OLE, olive leaf extract; PC, protein carbonyl. © 2014 Japan Geriatrics Society
|
3
J Çoban et al.
Table 2 Effect of olive leaf extract treatment (500 and 1000 mg/kg/day for 2 months) on glutathione, vitamin E and vitamin C levels in liver, heart and brain tissues of aged rats
Liver Young rats Aged rats Aged rats Aged rats Heart Young rats Aged rats Aged rats Aged rats Brain Young rats Aged rats Aged rats Aged rats
Treatment
GSH (μmol/g)
Vitamin E (nmol/g)
Vitamin C (μmol/g)
– – OLE (500 mg/kg/day) OLE (1000 mg/kg/day)
5.16 ± 0.71 3.78 ± 0.61† 4.97 ± 0.53‡ 5.18 ± 0.52‡
29.3 ± 3.24 28.4 ± 4.54 31.7 ± 6.63 30.8 ± 5.70
1.19 ± 0.20 1.02 ± 0.20 1.10 ± 0.21 1.08 ± 0.26
– – OLE (500 mg/kg/day) OLE (1000 mg/kg/day)
1.58 ± 0.25 1.34 ± 0.50 1.62 ± 0.25 1.66 ± 0.40
31.1 ± 2.80 31.1 ± 2.80 31.7 ± 5.17 30.9 ± 4.22
1.04 ± 0.05 1.03 ± 0.08 1.04 ± 0.07 1.06 ± 0.07
– – OLE (500 mg/kg/day) OLE (1000 mg/kg/day)
2.43 ± 0.26 2.40 ± 0.27 2.43 ± 0.27 2.42 ± 0.32
11.3 ± 1.05 12.1 ± 1.37 11.7 ± 1.24 12.0 ± 1.38
1.11 ± 0.11 1.09 ± 0.10 1.02 ± 0.18 1.08 ± 0.16
Data presented as mean ± SD; n = 8, each. †P < 0.05 as compared with young rats; ‡P < 0.05 as compared with aged rats (Kruskal–Wallis test; post-hoc Mann–Whitney U-test). GSH, glutathione; OLE, olive leaf extract.
Table 3 Effect of olive leaf extract treatment (500 and 1000 mg/kg/day for 2 months) on superoxide dismutase, glutathione peroxidase and glutathione transferase activities in liver, heart and brain tissues of aged rats
Liver Young rats Aged rats Aged rats Aged rats Heart Young rats Aged rats Aged rats Aged rats Brain Young rats Aged rats Aged rats Aged rats
Treatment
SOD (U/mg protein)
GSH-Px (nmol/min/mg protein)
GST (nmol/min/mg protein)
– – OLE (500 mg/kg/day) OLE (1000 mg/kg/day)
20.5 ± 2.06 19.9 ± 3.58 20.6 ± 2.00 21.0 ± 3.02
942.6 ± 114.4 888.9 ± 84.0 1008.4 ± 168.9 970.0 ± 126.0
607.6 ± 83.2 598.8 ± 60.6 608.7 ± 55.5 620.2 ± 58.7
– – OLE (500 mg/kg/day) OLE (1000 mg/kg/day)
17.8 ± 1.10 17.2 ± 2.02 17.9 ± 3.83 18.0 ± 3.90
280.3 ± 40.9 251.7 ± 33.3 273.0 ± 34.7 276.2 ± 38.2
67.7 ± 10.0 69.4 ± 13.9 72.9 ± 14.4 70.3 ± 12.5
– – OLE (500 mg/kg/day) OLE (1000 mg/kg/day)
10.3 ± 2.10 11.2 ± 1.40 10.1 ± 1.84 10.8 ± 2.00
48.0 ± 6.10 43.0 ± 11.6 50.7 ± 4.78 49.3 ± 7.20
114.1 ± 11.7 114.1 ± 21.6 107.2 ± 18.1 112.0 ± 16.2
Data presented as mean ± SD; n = 8, each. GSH-Px, glutathione peroxidase; GST, glutathione transferase; OLE, olive leaf extract; SOD, superoxide dismutase.
levels as well as SOD, GSH-Px and GST activities remained unchanged in these tissues (Tables 2,3). Protein expressions of SOD and GSH-Px did not alter in the liver of aged rats as compared with young rats (Fig. 1). 4
|
Effect of OLE treatment on pro-oxidant–anti-oxidant balance in aged rats High MDA, DC and PC levels in the liver, heart and brain tissues of aged rats returned to those of young rats © 2014 Japan Geriatrics Society
Effect of olive leaf extract on aged rats
by OLE 1000 treatment. OLE 500 treatment was also observed to decrease PC levels in examined tissues of aged rats. However, OLE 500 also decreased cardiac DC, but not MDA levels in aged rats. In addition, no significant changes in MDA and DC levels were detected in the brain tissues of aged rats by OLE 500 treatment (Table 1). (a) Young
Aged+ OLE 500
Aged
Aged+ OLE 1000
OLE 500 and OLE 1000 treatments caused significant increases in GSH levels in the liver of aged rats, but heart and brain GSH levels remained unchanged. Vitamin E and vitamin C levels, and SOD, GSH-Px and GST activities were found unchanged in these tissues after OLE 500 and OLE 1000 treatments (Tables 2,3). SOD and GSH-Px protein expressions did not alter in aged rats as a result of OLE treatments (Fig. 1). In contrast, there was no change in serum ALT, AST, LDH and CK activities in aged and OLE-treated aged rats (Table 4).
SOD (23 kDa) GSH-Px (23 kDa) Actin (43 kDa)
SOD (relative densitometric unit)
(b) 1,2 1 0,8 0,6 0,4 0,2 0
Young
Aged
Aged + OLE 500
Aged + OLE 1000
Young
Aged
Aged + OLE 500
Aged + OLE 1000
GSH-Px (relative densitometric unit)
(c) 1,4 1,2 1 0,8 0,6 0,4 0,2 0
Figure 1 (a) Western blot analysis of superoxide dismutase (SOD), glutathione peroxidase (GSH-Px) and actin expression, and densitometric quantification of (b) SOD and (c) GSH-Px in the livers of untreated and olive leaf extract (OLE)-treated (500 and 1000 mg/kg/day for 2 months) aged rats. Results are the average values of four rats (mean ± SD).
Discussion The role of oxidative damage in normal aging is supported by studies in experimental animals. Rats are one of the most suitable animals for aging studies. Oxidative stress parameters, such as MDA levels, protein carbonyls and DNA damage together with non-enzymatic and enzymatic anti-oxidant systems, have been investigated in young and aged rats.2–10 Although there are some controversial results, a shift towards an oxidant milieu in the cellular pro-oxidant– anti-oxidant balance is generally observed. Some discrepancies might be due to the difference in the susceptibility of organs and tissues to oxidative damage, as well as the techniques used for assessing oxidative stress in aged rats. In the current study, MDA, DC and PC levels increased in the liver, heart and brain, whereas GSH levels decreased only in the liver of aged rats. I contrast, no change was found in vitamin E and C levels as well as SOD, GSH-Px and GST activities in these tissues of aged rats. These results are in accordance with our previous studies.5,7,8 OLE and individual phenolic compounds in OLE show strong in vitro anti-oxidant activities.13,19,42,43 As it is known, several methods, such as 2,2-diphenyl-1picrylhydrazyl and 2,2-azino-bis(3-ethylbenzthiazoline6-sulfonic acid) radical scavenging activities, ferric reducing antioxidant capacity, oxygen radical absorbance capacity and nitrite scavenging activity, can be
Table 4 Effect of olive leaf extract treatment (500 and 1000 mg/kg/day for 2 months) on serum alanine aminotransferase, aspartate aminotransferase, lactate dehydrogenase and creatine kinase activities in aged rats
Serum Young rats Aged rats Aged rats Aged rats
Treatment
ALT (U/L)
AST (U/L)
LDH (U/L)
CK (U/L)
– – OLE (500 mg/kg/day) OLE (1000 mg/kg/day)
42.2 ± 5.86 41.8 ± 7.25 39.2 ± 6.82 40.8 ± 6.36
142.6 ± 18.8 138.6 ± 20.6 132.2 ± 28.6 140.2 ± 25.4
640.6 ± 96.8 680.2 ± 106.5 620.4 ± 96.6 710.4 ± 00.8
220.7 ± 33.6 260.4 ± 42.6 242.8 ± 36.5 253.7 ± 40.5
Data presented as mean ± SD; n = 8, each. ALT, alanine aminotransferase; AST, aspartate aminotransferase; CK, creatine kinase; LDH, lactate dehydrogenase; OLE, olive leaf extract. © 2014 Japan Geriatrics Society
|
5
J Çoban et al.
used to measure in vitro anti-oxidant activities of compounds or extracts.13,43 Some investigators have measured anti-oxidant activities of OLE or its individual phenolics by using these assays.13,19,42,43 OLE contains many phenolic compounds that possess anti-oxidant properties. It appears that the overall anti-oxidant action provided by OLE is much stronger than each of the phenolic compounds present.13 OLE or its constituents, especially oleuropein, have tissue protective effects as an anti-oxidant when administered therapeutically. OLE ameliorated non-alcoholic steatosis in spontaneously hypertensive rats,26 and hepatic and cardiac injury in a model of diet-induced obesity and diabetes in rats.25 OLE decreased hepatic MDA levels, and increased catalase and SOD activities in rats exposed to cold restraint stress.27 OLE treatment also attenuated atherosclerosis by lowering serum lipids, inhibiting low-density lipoprotein oxidation and suppressing inflammatory factors in atherosclerotic rabbits.17 Oleuropein, an OLE constituent, was found to restore changes in oxidative and anti-oxidative parameters in serum and erythrocytes of alloxan-diabetic rabbits.15 Significant decreases in serum glucose and lipid levels, as well as increases in SOD and GSH-Px activities, were detected in a genetic rat model of hypertension as a result of triterpenoids isolated from olive leaves.16 Likewise the liver, OLE has cardioprotective17,18 and neuroprotective19,20 effects. Oleuropein reduced cardiac infarct size, and the circulating lipid levels in normal and hypercholesterolemic rabbits subjected to ischemia.17 Serum cardiac marker activities, cardiac MDA, DC and nitrotyrosine levels, and inducible nitric oxide synthase expression together with the histopathological amelioration were also found in doxorubicin-treated rats as a result of oleuropein treatment.18 OLE attenuated the increases in nitric oxide, superoxide anions and lipid peroxide levels, and hippocampal neuronal damage after transient global cerebral ischemia in Mongolian gerbils,19 and caused significant protection against leadinduced brain damage through inhibition of apoptosis, oxidative stress and inflammation.20 In light of these observations, we investigated whether OLE treatment was protective against the oxidative stress in tissues of aged rats. In the present study, total OLE extract was used, and aged rats received OLE in drinking water for 2 months. The effective doses of OLE (approximately 500 and 1000 mg/kg bodyweight/day) were chosen according to previous studies.24–26 In the current study, OLE, especially OLE 1000, was detected to diminish the high levels of hepatic MDA, DC and PC levels in aged rats. OLE treatments elevated GSH levels in the liver, but not the heart and brain. There was no change in vitamin E and vitamin C levels, and SOD, GSH-Px and GST activities as a result of OLE treatments. In addition, we 6
|
also detected that protein expressions of hepatic SOD and GSH-Px did not alter in OLE-treated rats as compared with aged rats. According to this, the present results suggest that OLE treatment diminished ageinduced oxidative stress in the liver, heart and brain tissues by acting as an anti-oxidant itself without affecting the anti-oxidant system. In conclusion, the present results suggest that OLE treatment seems to be useful for decreasing oxidative stress in the liver, heart and brain tissues in aged rats. This anti-aging effect could be related to its strong radical scavenging action.
Acknowledgment This work was supported by the Research Fund of University of Istanbul (Project No: BYP-14443).
Disclosure statement The authors declare no conflict of interest.
References 1 Harman D. Aging: overview. Ann N Y Acad Sci 2001; 928: 1–21. 2 Tian L, Cai Q, Wei H. Alterations of antioxidant enzymes and oxidative damage to macromolecules in different organs of rats during aging. Free Radic Biol Med 1998; 24: 1477–1484. 3 Wolf FI, Fasanella S, Tedesco B et al. Peripheral lymphocyte 8-OHdG levels correlate with age-associated increase of tissue oxidative DNA damage in Sprague-Dawley rats. Protective effects of caloric restriction. Exp Gerontol 2005; 40: 181–188. 4 Wong YT, Ruan R, Tay FEH. Relationship between levels of oxidative DNA damage, lipid peroxidation and mitochondrial membrane potential in young and old F344 rats. Free Radic Res 2006; 40: 393–402. 5 Parıldar-Karpuzog˘lu H, Mehmetçik G, Özdemirler-Erata G, Dog˘ru-Abbasog˘lu S, Koçak-Toker N, Uysal M. The effect of taurine treatment on prooxidant-antioxidant balance in livers and brains of old rats. Pharmacol Rep 2008; 60: 673–678. 6 Xu J, Rong S, Xie B et al. Procyanidins extracted from the lotus seedpod ameliorate age-related antioxidant deficit in aged rats. J Gerontol A Biol Sci Med Sci 2010; 65: 236– 241. 7 Aydın AF, Küçükgergin C, Özdemirler-Erata G, Koçak-Toker N, Uysal M. The effect of carnosine treatment on prooxidant-antioxidant balance in liver, heart and brain tissues of male aged rats. Biogerontology 2010; 11: 103–109. 8 Çoban J, Bingül I, Yes¸il-Mızrak K, Dog˘ru-Abbasog˘lu S, Öztezcan S, Uysal M. Effects of carnosine plus vitamin E and betaine treatments on oxidative stress in some tissues of aged rats. Curr Aging Sci 2013; 6: 199–205. © 2014 Japan Geriatrics Society
Effect of olive leaf extract on aged rats 9 Jayakumar T, Thomas PA, Geraldine P. Protective effect of an extract of the oyster mushroom, Pleurotus ostreatus, on antioxidants of major organs of aged rats. Exp Gerontol 2007; 42: 183–191. 10 Siqueira IR, Fochesatto C, de Andrade A et al. Total antioxidant capacity is impaired in different structures from aged brain. Int J Devl Neurosci 2005; 3: 663–671. 11 El SN, Karakaya S. Olive tree (Olea europaea) leaves: potential beneficial effects on human health. Nutr Rev 2009; 67: 632–638. 12 Kontogianni VG, Gerothanassis IP. Phenolic compounds and antioxidant activity of olive extracts. Nat Prod Res 2012; 26: 186–189. 13 Lee OH, Lee BY. Antioxidant and antimicrobial activities of individual and combined phenolics in Olea europaea leaf extract. Bioresour Technol 2010; 101: 3751–3754. 14 Wang L, Geng C, Jiang L et al. The anti-atherosclerotic effect of olive leaf extract is related to suppressed inflammatory response in rabbits with experimental atherosclerosis. Eur J Nutr 2008; 47: 235–243. 15 Al-Azzawie HF, Alhamdani MS. Hypoglycemic and antioxidant effect of oleuropein in alloxan-diabetic rabbits. Life Sci 2006; 78: 1371–1377. 16 Somova LI, Shode FO, Ramnanan P, Nadar A. Antihypertensive, antiatherosclerotic and antioxidant activity of triterpenoids isolated from Olea europaea, subspecies africana leaves. J Ethnopharmacol 2003; 84: 299–305. 17 Andreadou I, Iliodromitis EK, Mikros E et al. The olive constituent oleuropein exhibits anti-ischemic, antioxidative, and hypolipidemic effects in anesthetized rabbits. J Nutr 2006; 136: 2213–2219. 18 Andreadou I, Sigala F, Iliodromitis EK et al. Acute doxorubicin cardiotoxicity is successfully treated with the phytochemical oleuropein through suppression of oxidative and nitrosative stress. J Mol Cell Cardiol 2007; 42: 549–558. 19 Dekanski D, Selakovic V, Piperski V, Radulovic Z, Korenic A, Radenovic L. Protective effect of olive leaf extract on hippocampal injury induced by transient global cerebral ischemia and reperfusion in Mongolian gerbils. Phytomedicine 2011; 18: 1137–1143. 20 Seddik L, Bah TM, Aoues A, Slimani M, Benderdour M. Elucidation of mechanisms underlying the protective effects of olive leaf extract against lead-induced neurotoxicity in Wistar rats. J Toxicol Sci 2011; 36: 797–809. 21 Dekanski D, Janic´ijevic´-Hudomal S, Ristic´ S et al. Attenuation of cold restraint stress-induced gastric lesions by an olive leaf extract. Gen Physiol Biophys 2009; 28: 135–142. 22 Alirezaei M, Dezfoulian O, Neamati S, Rashidipour M, Tanideh N, Kheradmand A. Oleuropein prevents ethanolinduced gastric ulcers via elevation of antioxidant enzyme activities in rats. J Physiol Biochem 2012; 68: 583–592. 23 Hamdi HK, Castellon R. Oleuropein, a non-toxic olive iridoid, is an anti-tumor agent and cytoskeleton disruptor. Biochem Biophys Res Commun 2005; 334: 769–778. 24 Abdel-Hamid NM, El-Moselhy MA, El-Baz A. Hepatocyte lysosomal membrane stabilization by olive leaves against chemically induced hepatocellular neoplasia in rats. Int J Hepatol 2011; 2011: 736581. doi: 10.4061/2011/736581 25 Poudyal H, Campbell F, Brown L. Olive leaf extract attenuates cardiac, hepatic, and metabolic changes in high carbohydrate-, high fat-fed rats. J Nutr 2010; 140: 946– 953.
© 2014 Japan Geriatrics Society
26 Omagari K, Kato S, Tsuneyama K et al. 2010) Olive leaf extract prevents spontaneous occurrence of non-alcoholic steatohepatitis in SHR/NDmcr-cp rats. Pathology 2010; 42: 66–72. 27 Dekanski D, Ristic´ S, Radonjic´ NV et al. Olive leaf extract modulates cold restraint stress-induced oxidative changes in rat liver. J Serb Chem Soc 2011; 76: 1207–1218. 28 Queen BL, Tollefsboll TO. Polyphenols and aging. Curr Aging Sci 2010; 3: 34–42. 29 Cañuelo A, Gilbert-López B, Pacheco-Liñán P, Martínez-Lara E, Siles E, Miranda-Vizuete A. Tyrosol, a main phenol present in extra virgin olive oil, increases lifespan and stress resistance in Caenorhabditis elegans. Mech Ageing Dev 2012; 133: 563–574. 30 Charles AL, Meyer A, Dal-Ros S et al. Polyphenols prevents aging-related impairment in skeletal muscle mitochondrial function through decreased reactive oxygen species production. Exp Physiol 2013; 98: 536–545. 31 Liu LC, Wang CJ, Lee CC et al. Aqueous extract of Hibiscus sabdariffa L. decelerates acetaminophen-induced acute liver damage by reducing cell death and oxidative stress in mouse experimental models. J Sci Food Agric 2010; 90: 329– 337. 32 Buege JA, Aust JD. Microsomal lipid peroxidation. Methods Enzymol 1978; 52: 302–310. 33 Reznick AZ, Packer L. Oxidative damage to proteins: spectrophotometric method for carbonyl assay. Methods Enzymol 1994; 233: 357–363. 34 Beutler E, Duron O, Kelly BM. Improved method for the determination of blood glutathione. J Lab Clin Med 1979; 61: 882–888. 35 Desai DI. Vitamin E analysis methods for animal tissues. Methods Enzymol 1984; 105: 138–147. 36 Omaye ST, Turnbull JD, Sauberlich HE. Selected methods for the determination of ascorbic acid in animal cells, tissues and fluids. Methods Enzymol 1979; 62: 3–8. 37 Mylorie AA, Collins H, Umbles C, Kyle J. Erythrocyte superoxide dismutase activity and other parameters of copper status in rats ingesting lead acetate. Toxicol Appl Pharmacol 1986; 82: 512–520. 38 Lawrence RA, Burk RF. Glutathione peroxidase activity in selenium–deficient rat liver. Biochem Biophys Res Commun 1976; 71: 952–958. 39 Habig WH, Jacoby WB. Assays for differentation of glutathione s-transferases. Methods Enzymol 1981; 77: 398–405. 40 Smith PK, Krohn RI, Hermanson GT et al. Measurement of protein using bicinchoninic acid. Anal Biochem 1985; 150: 76–85. 41 Aydın AF, Küskü-Kiraz Z, Dogru-Abbasoglu S, Güllüoglu M, Uysal M, Koçak- Toker N. Effect of carnosine against thioacetamide-induced liver cirrhosis in rat. Peptides 2010; 31: 67–71. 42 Komes D, Bescak-Cvitanovic A, Horzic D, Rusak G, Likic S, Berendika M. Phenolic composition and antioxidant properties of some traditionally used medicinal plants affected by the extraction time and hydrolysis. Phytochem Anal 2011; 22: 172–180. 43 Hayes JE, Allen P, Brunton N, O’Grady MN, Kerry JP. Phenolic composition and in vitro antioxidant capacity of four commercial phytochemical products: olive leaf extract (Olea europaea L.), lutein, sesamol and ellagic acid. Food Chem 2011; 126: 948–955.
|
7
Geriatr Gerontol Int 2014
ORIGINAL ARTICLE: BIOLOGY
Olive leaf extract decreases age-induced oxidative stress in major organs of aged rats Jale Çoban,1 Serdar Öztezcan,1 Semra Dog˘ru-Abbasog˘lu,2 Ilknur Bingül,2 Kübra Yes¸il-Mizrak1 and Müjdat Uysal2 1
Department of Biochemistry, Yeditepe University Medical Faculty and 2Department of Biochemistry, Istanbul Medical Faculty, Istanbul University, Istanbul, Turkey
Aim: Olive leaf (Olea europaea L.) extract (OLE) is a powerful anti-oxidant rich in polyphenols. As oxidative stress plays an important role in aging, we investigated the effect of OLE on oxidative stress in the liver, heart and brain of aged rats. Methods: Young (age 3 months) and aged (age 20 months) Wistar rats were used. Aged rats received OLE (500 and 1000 mg/kg/day) in drinking water for 2 months. Malondialdehyde (MDA), diene conjugate (DC), protein carbonyl (PC), glutathione (GSH), vitamin E and vitamin C levels, and superoxide dismutase (SOD), glutathione peroxidase (GSH-Px) and glutathione transferase (GST) activities were determined. Results: MDA, DC and PC levels increased in tissues of aged rats. GSH levels decreased in the liver, but not in the heart and brain. There was no change of other anti-oxidant parameters in tissues. Hepatic SOD and GSH-Px protein expressions also remained unchanged. OLE treatment caused decreased tissue MDA, DC and PC levels, and increased hepatic GSH levels in aged rats. Other anti-oxidant parameters, hepatic SOD and GSH-Px protein expressions did not alter in aged rats by OLE treatment. Conclusion: The present results suggest that OLE seems to be useful for decreasing oxidative stress in examined tissues by acting as an anti-oxidant itself without affecting the anti-oxidant system. Geriatr Gerontol Int 2014; ••: ••–••. Keywords: aging, olive leaf extract, oxidative stress, rats, tissues.
Introduction Free radicals-induced oxidative stress plays an important role in the aging process.1 Oxidative stress parameters have been found to increase in tissues, such as the liver,2–8 heart3,4,6–8 and brain,2,3,7,8 with increasing age. Several reports are also available in the literature regarding non-enzymatic and enzymatic anti-oxidants in the liver,2,5–9 heart6–9 and brain5,7–10 during aging. It has been found that pro-oxidant–anti-oxidant balance is altered in an organism during aging. Therefore, several authors have investigated the effect of anti-oxidants in protecting the tissues against age-induced oxidative stress.5–9
Accepted for publication 10 October 2013. Correspondence: Dr Müjdat Uysal MD, Department of Biochemistry, Istanbul Medical Faculty, Istanbul University, Çapa, 34093, Istanbul, Turkey. Email: [email protected]
© 2014 Japan Geriatrics Society
Olive tree (Olea europaea L.) leaves have been used widely in traditional remedies in Mediterranean countries.11 Animal studies have shown that olive leaves have antihypertensive, anti-atherogenic, anti-inflammatory, hypoglycemic and hypocholesterolemic effects.11 These effects are attributed to the anti-oxidant components of olive leaves.11,12 The main constituent of olive leaf is oleuropein, which is thought to be responsible for its pharmacological effects. In addition, olive leaf contains triterpenes, flavonoids, chalcones and tannins. Therefore, olive leaf is considered to be one of the most potent natural anti-oxidants by its chemical content.11–13 Several experimental studies have shown that olive leaf extract (OLE) or its constituents, such as oleuropein, had protective effects in atherosclerosis,14 diabetes,15 hypertension,16 cardiotoxicity,17,18 neurotoxicity,19,20 gastric lesions21,22 and cancers.23,24 OLE attenuated cardiac, hepatic and metabolic changes in rats with metabolic syndrome.25 It prevented hepatic fat accumulation in a rat model of non-alcoholic steatosis,26 and modulated cold restraint stress-induced oxidative doi: 10.1111/ggi.12192
|
1
J Çoban et al.
changes in rat liver.27 Although some studies are available on the beneficial effects of polyphenolic compounds obtained from various sources,28–30 there is no study investigating the effect of OLE in tissues against age-induced oxidative stress. For this reason, we wanted to investigate the effect of OLE treatment on pro-oxidant and anti-oxidant status in the liver, heart and brain tissues of aged rats. Malondialdehyde (MDA), diene conjugate (DC) and protein carbonyl (PC) levels, as well as glutathione (GSH), vitamin E and vitamin C levels, and Cu,Znsuperoxide dismutase (SOD), glutathione peroxidase (GSH-Px) and glutathione transferase (GST) activities together with protein expressions of SOD and GSH-Px enzymes were determined. These parameters were also measured in tissues of aged rats treated with OLE.
Methods Materials Chemicals were supplied from Sigma-Aldrich (St. Louis, MO, USA). OLE (liquid) was provided by the Bio-Olive Ltd Company (Ayvalık, Turkey). For the preparation of OLE, olive leaves were collected from the Ayvalık region in Turkey. Samples were dried and crushed. After crushing, 10 g of olive leaf powder was extracted with 70% (v/v) aqueous ethanol for 24 h at room temperature by a shaking incubator, which was fixed to 180 r.p.m. The extracts were filtered with Whatman No. 1 filter paper. To remove ethanol, the filtrates were evaporated by using a rotary evaporator under reduced pressure at 38°C. Thus, obtained liquid OLE is dark green in color, clear and has a slightly sweet taste. This liquid extract was kept at 4°C during the period of administration. The total phenol and oleuropein content of OLE liquid were analyzed by using the Folin–Ciocalteu method31 and Hewlett-Packard high-performance liquid chromatography with a diode array detector, respectively. Total phenol content was calculated as 196.8 mg gallic acid equivalent /g OLE. Oleuropein content was also detected as 97.0 mg/g OLE.
Animals and treatment Young (age 3 months; 200.6 ± 18.2 g) and aged (age 20 months; 498.2 ± 48.6 g) male Wistar rats were obtained from Yeditepe University Animal Research Center, Istambul, Turkey. The animals were allowed free access to food and water, and were kept in wire-bottomed stainless steel cages. The Animal Ethics Review Committee of Yeditepe University approved the experimental protocol. 2
|
At the start of the experiment, the young animals were 3 months-of-age and the aged animals were 20 monthsof-age. Rats were given OLE (6 or 12 mL OLE liquid/L in drinking water) for 2 months. In the present study, the consumption of OLE was roughly equivalent to 500 and 1000 mg/kg/day in aged rats.
Preparation of tissues At the end of the experimental period, rats were fasted overnight and anesthesized with sodium pentobarbital (50 mg/kg; i.p.). Blood was collected in dry tubes by cardiac puncture, and serum was obtained by centrifugation for biochemical analysis. Liver, heart and brain tissues of rats were quickly removed, and washed in 0.9% NaCl and kept at −70°C until they were analyzed. Tissues were homogenized in ice-cold 0.15 mol/L KCl (10%; w/v). Homogenates were centrifuged at 600 g for 10 min at 4°C, and then supernatants were recentrifuged at 10 000 g for 20 min to obtain postmitochondrial fractions.
Determination of lipid peroxides and PC levels Lipid peroxidation was assessed by two methods in the tissue homogenates. MDA levels were measured by thiobarbituric acid test.32 The breakdown product of 1,1,3,3-tetraethoxypropane was used as the standard. DC levels were determined in tissue lipid extracts at 233 nm spectrophotometrically, and calculated using a molar extinction coefficient of 2.52 × 104 mol/L1 cm1.32 The oxidative protein damage was spectrophotometrically detected by the quantification with 2,4dinitrophenylhydrazine of protein carbonyl groups. PC levels were calculated from the maximum absorbance (360 nm) using a molar absorption coefficient of 22 000 mol/L1 cm1. The results were expressed as nmol carbonyl per mg protein.33
Determination of non-enzymatic and enzymatic anti-oxidants in tissues GSH levels were measured with 5,5-dithiobis-(2nitrobenzoate) at 412 nm in tissue homogenates.34 Vitamin E and vitamin C levels were also measured in tissue homogenates by the method of Desai35 and Omaye et al.,36 respectively. Anti-oxidant enzyme activities were determined in postmitochondrial fractions. Cu,Zn-SOD activity was assayed by its ability to increase the effect of riboflavinsensitized photo-oxidation of o-dianisidine.37 GSH-Px38 and GST39 activities were measured using cumene hydroperoxide and 1-chloro-2,4-dinitrobenzene as substrates, respectively. Protein levels were determined using bicinchoninic acid.40 © 2014 Japan Geriatrics Society
Effect of olive leaf extract on aged rats
Determination of the expressions of SOD and GSH-Px in the liver
Statistical analyses
Western blotting was carried out to study the protein expressions of SOD and GSH-Px proteins as previously described.41 Then, 50 μg/well proteins were loaded onto 12% sodium dodecylsulfate-polyacrylamide gel electrophoresis and transferred to polyvinylidene fluoride membranes. After blocking, membranes were incubated for 16 h at 4°C with rabbit polyclonal antibody against SOD (sc-11407; Santa Cruz Biotechnology, Santa Cruz, CA, USA) or GSH-Px protein (sc-30147; Santa Cruz Biotechnology). They were further incubated for 1.5 h at room temperature with goat anti-rabbit immunoglobulin G conjugated to horseradish peroxidase (sc-2004; Santa Cruz Biotechnology). The chemiluminescence signals were visualized by exposing the membranes to Kodak Biomax X-ray film (Sigma-Aldrich Chemie GmbH, Taufkrichen, Germany) for 1–5 min, and then the bands were quantitated using the image analysis software. Values of western blot (SOD and GSH-Px) were normalized with an internal standard (actin, sc-1616R; Santa Cruz Biotechnology).
The results were expressed as mean ± SD. Experimental groups were compared using Kruskal–Wallis variance analysis test followed by post-hoc Mann–Whitney U-test. In all cases, P < 0.05 was considered to be statistically significant.
Results Effect of OLE on bodyweight Bodyweight gain in young rats was significantly increased during 2 months (10.2 ± 2.1 g/per week). However, the weight gain and bodyweight did not alter in untreated and OLE-treated aged rats during the experimental period. Final bodyweights were detected as 282.2 ± 30.7 g in the young group; 505 ± 42.8 g in the untreated-aged group; 512 ± 49.6 g in the OLE 500-treated aged group; and 503 ± 47.2 g in the OLE 1000-treated aged rats at the end of the experimental period.
Determinations in serum
Effect of aging on pro-oxidant–anti-oxidant balance
Serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), lactate dehydrogenase (LDH) and creatine kinase (CK) activities were determined on a Cobas Integra 800 autoanalyzer (Roche Diagnostics, Mannheim, Germany).
MDA, DC and PC levels increased significantly in the liver, heart and brain tissues of aged rats as compared with young rats (Table 1). Significant decreases in GSH levels were detected in the liver, but not in the heart and brain of aged rats. However, vitamin E and vitamin C
Table 1 The effect of olive leaf extract (treatment (500 and 1000 mg/kg/day for 2 months) malondialdehyde, diene conjugate and protein carbonyl levels in the liver, heart and brain tissues of aged rats
Liver Young rats Aged rats Aged rats Aged rats Heart Young rats Aged rats Aged rats Aged rats Brain Young rats Aged rats Aged rats Aged rats
Treatment
MDA (nmol/g)
DC (μmol/g)
PC (nmol/mg protein)
– – OLE (500 mg/kg/day) OLE (1000 mg/kg/day)
33.1 ± 3.82 50.0 ± 10.6† 36.6 ± 4.50‡ 32.3 ± 4.70‡
2.55 ± 0.24 3.96 ± 0.80† 2.48 ± 0.30‡ 2.42 ± 0.42‡
1.37 ± 0.22 2.39 ± 0.41† 1.40 ± 0.28‡ 1.28 ± 0.32‡
– – OLE (500 mg/kg/day) OLE (1000 mg/kg/day)
16.5 ± 1.69 23.3 ± 5.03† 20.8 ± 4.60† 17.8 ± 4.88‡
1.62 ± 0.17 2.09 ± 0.49† 1.61 ± 0.12‡ 1.56 ± 0.13‡
1.40 ± 0.19 2.20 ± 0.36† 1.50 ± 0.28‡ 1.48 ± 0.32‡
– – OLE (500 mg/kg/day) OLE (1000 mg/kg/day)
57.7 ± 4.88 69.9 ± 8.68† 66.7 ± 8.12† 58.3 ± 7.90‡
2.80 ± 0.36 3.57 ± 0.54† 3.06 ± 0.48 2.87 ± 0.32‡
1.26 ± 0.14 2.08 ± 0.32† 1.60 ± 0.22†‡ 1.38 ± 0.26‡
Data presented as mean ± SD; n = 8, each. †P < 0.05 as compared with young rats; ‡P < 0.05 as compared with aged rats (Kruskal–Wallis test; post-hoc Mann–Whitney U-test). DC, diene conjugate; MDA, malondialdehyde; OLE, olive leaf extract; PC, protein carbonyl. © 2014 Japan Geriatrics Society
|
3
J Çoban et al.
Table 2 Effect of olive leaf extract treatment (500 and 1000 mg/kg/day for 2 months) on glutathione, vitamin E and vitamin C levels in liver, heart and brain tissues of aged rats
Liver Young rats Aged rats Aged rats Aged rats Heart Young rats Aged rats Aged rats Aged rats Brain Young rats Aged rats Aged rats Aged rats
Treatment
GSH (μmol/g)
Vitamin E (nmol/g)
Vitamin C (μmol/g)
– – OLE (500 mg/kg/day) OLE (1000 mg/kg/day)
5.16 ± 0.71 3.78 ± 0.61† 4.97 ± 0.53‡ 5.18 ± 0.52‡
29.3 ± 3.24 28.4 ± 4.54 31.7 ± 6.63 30.8 ± 5.70
1.19 ± 0.20 1.02 ± 0.20 1.10 ± 0.21 1.08 ± 0.26
– – OLE (500 mg/kg/day) OLE (1000 mg/kg/day)
1.58 ± 0.25 1.34 ± 0.50 1.62 ± 0.25 1.66 ± 0.40
31.1 ± 2.80 31.1 ± 2.80 31.7 ± 5.17 30.9 ± 4.22
1.04 ± 0.05 1.03 ± 0.08 1.04 ± 0.07 1.06 ± 0.07
– – OLE (500 mg/kg/day) OLE (1000 mg/kg/day)
2.43 ± 0.26 2.40 ± 0.27 2.43 ± 0.27 2.42 ± 0.32
11.3 ± 1.05 12.1 ± 1.37 11.7 ± 1.24 12.0 ± 1.38
1.11 ± 0.11 1.09 ± 0.10 1.02 ± 0.18 1.08 ± 0.16
Data presented as mean ± SD; n = 8, each. †P < 0.05 as compared with young rats; ‡P < 0.05 as compared with aged rats (Kruskal–Wallis test; post-hoc Mann–Whitney U-test). GSH, glutathione; OLE, olive leaf extract.
Table 3 Effect of olive leaf extract treatment (500 and 1000 mg/kg/day for 2 months) on superoxide dismutase, glutathione peroxidase and glutathione transferase activities in liver, heart and brain tissues of aged rats
Liver Young rats Aged rats Aged rats Aged rats Heart Young rats Aged rats Aged rats Aged rats Brain Young rats Aged rats Aged rats Aged rats
Treatment
SOD (U/mg protein)
GSH-Px (nmol/min/mg protein)
GST (nmol/min/mg protein)
– – OLE (500 mg/kg/day) OLE (1000 mg/kg/day)
20.5 ± 2.06 19.9 ± 3.58 20.6 ± 2.00 21.0 ± 3.02
942.6 ± 114.4 888.9 ± 84.0 1008.4 ± 168.9 970.0 ± 126.0
607.6 ± 83.2 598.8 ± 60.6 608.7 ± 55.5 620.2 ± 58.7
– – OLE (500 mg/kg/day) OLE (1000 mg/kg/day)
17.8 ± 1.10 17.2 ± 2.02 17.9 ± 3.83 18.0 ± 3.90
280.3 ± 40.9 251.7 ± 33.3 273.0 ± 34.7 276.2 ± 38.2
67.7 ± 10.0 69.4 ± 13.9 72.9 ± 14.4 70.3 ± 12.5
– – OLE (500 mg/kg/day) OLE (1000 mg/kg/day)
10.3 ± 2.10 11.2 ± 1.40 10.1 ± 1.84 10.8 ± 2.00
48.0 ± 6.10 43.0 ± 11.6 50.7 ± 4.78 49.3 ± 7.20
114.1 ± 11.7 114.1 ± 21.6 107.2 ± 18.1 112.0 ± 16.2
Data presented as mean ± SD; n = 8, each. GSH-Px, glutathione peroxidase; GST, glutathione transferase; OLE, olive leaf extract; SOD, superoxide dismutase.
levels as well as SOD, GSH-Px and GST activities remained unchanged in these tissues (Tables 2,3). Protein expressions of SOD and GSH-Px did not alter in the liver of aged rats as compared with young rats (Fig. 1). 4
|
Effect of OLE treatment on pro-oxidant–anti-oxidant balance in aged rats High MDA, DC and PC levels in the liver, heart and brain tissues of aged rats returned to those of young rats © 2014 Japan Geriatrics Society
Effect of olive leaf extract on aged rats
by OLE 1000 treatment. OLE 500 treatment was also observed to decrease PC levels in examined tissues of aged rats. However, OLE 500 also decreased cardiac DC, but not MDA levels in aged rats. In addition, no significant changes in MDA and DC levels were detected in the brain tissues of aged rats by OLE 500 treatment (Table 1). (a) Young
Aged+ OLE 500
Aged
Aged+ OLE 1000
OLE 500 and OLE 1000 treatments caused significant increases in GSH levels in the liver of aged rats, but heart and brain GSH levels remained unchanged. Vitamin E and vitamin C levels, and SOD, GSH-Px and GST activities were found unchanged in these tissues after OLE 500 and OLE 1000 treatments (Tables 2,3). SOD and GSH-Px protein expressions did not alter in aged rats as a result of OLE treatments (Fig. 1). In contrast, there was no change in serum ALT, AST, LDH and CK activities in aged and OLE-treated aged rats (Table 4).
SOD (23 kDa) GSH-Px (23 kDa) Actin (43 kDa)
SOD (relative densitometric unit)
(b) 1,2 1 0,8 0,6 0,4 0,2 0
Young
Aged
Aged + OLE 500
Aged + OLE 1000
Young
Aged
Aged + OLE 500
Aged + OLE 1000
GSH-Px (relative densitometric unit)
(c) 1,4 1,2 1 0,8 0,6 0,4 0,2 0
Figure 1 (a) Western blot analysis of superoxide dismutase (SOD), glutathione peroxidase (GSH-Px) and actin expression, and densitometric quantification of (b) SOD and (c) GSH-Px in the livers of untreated and olive leaf extract (OLE)-treated (500 and 1000 mg/kg/day for 2 months) aged rats. Results are the average values of four rats (mean ± SD).
Discussion The role of oxidative damage in normal aging is supported by studies in experimental animals. Rats are one of the most suitable animals for aging studies. Oxidative stress parameters, such as MDA levels, protein carbonyls and DNA damage together with non-enzymatic and enzymatic anti-oxidant systems, have been investigated in young and aged rats.2–10 Although there are some controversial results, a shift towards an oxidant milieu in the cellular pro-oxidant– anti-oxidant balance is generally observed. Some discrepancies might be due to the difference in the susceptibility of organs and tissues to oxidative damage, as well as the techniques used for assessing oxidative stress in aged rats. In the current study, MDA, DC and PC levels increased in the liver, heart and brain, whereas GSH levels decreased only in the liver of aged rats. I contrast, no change was found in vitamin E and C levels as well as SOD, GSH-Px and GST activities in these tissues of aged rats. These results are in accordance with our previous studies.5,7,8 OLE and individual phenolic compounds in OLE show strong in vitro anti-oxidant activities.13,19,42,43 As it is known, several methods, such as 2,2-diphenyl-1picrylhydrazyl and 2,2-azino-bis(3-ethylbenzthiazoline6-sulfonic acid) radical scavenging activities, ferric reducing antioxidant capacity, oxygen radical absorbance capacity and nitrite scavenging activity, can be
Table 4 Effect of olive leaf extract treatment (500 and 1000 mg/kg/day for 2 months) on serum alanine aminotransferase, aspartate aminotransferase, lactate dehydrogenase and creatine kinase activities in aged rats
Serum Young rats Aged rats Aged rats Aged rats
Treatment
ALT (U/L)
AST (U/L)
LDH (U/L)
CK (U/L)
– – OLE (500 mg/kg/day) OLE (1000 mg/kg/day)
42.2 ± 5.86 41.8 ± 7.25 39.2 ± 6.82 40.8 ± 6.36
142.6 ± 18.8 138.6 ± 20.6 132.2 ± 28.6 140.2 ± 25.4
640.6 ± 96.8 680.2 ± 106.5 620.4 ± 96.6 710.4 ± 00.8
220.7 ± 33.6 260.4 ± 42.6 242.8 ± 36.5 253.7 ± 40.5
Data presented as mean ± SD; n = 8, each. ALT, alanine aminotransferase; AST, aspartate aminotransferase; CK, creatine kinase; LDH, lactate dehydrogenase; OLE, olive leaf extract. © 2014 Japan Geriatrics Society
|
5
J Çoban et al.
used to measure in vitro anti-oxidant activities of compounds or extracts.13,43 Some investigators have measured anti-oxidant activities of OLE or its individual phenolics by using these assays.13,19,42,43 OLE contains many phenolic compounds that possess anti-oxidant properties. It appears that the overall anti-oxidant action provided by OLE is much stronger than each of the phenolic compounds present.13 OLE or its constituents, especially oleuropein, have tissue protective effects as an anti-oxidant when administered therapeutically. OLE ameliorated non-alcoholic steatosis in spontaneously hypertensive rats,26 and hepatic and cardiac injury in a model of diet-induced obesity and diabetes in rats.25 OLE decreased hepatic MDA levels, and increased catalase and SOD activities in rats exposed to cold restraint stress.27 OLE treatment also attenuated atherosclerosis by lowering serum lipids, inhibiting low-density lipoprotein oxidation and suppressing inflammatory factors in atherosclerotic rabbits.17 Oleuropein, an OLE constituent, was found to restore changes in oxidative and anti-oxidative parameters in serum and erythrocytes of alloxan-diabetic rabbits.15 Significant decreases in serum glucose and lipid levels, as well as increases in SOD and GSH-Px activities, were detected in a genetic rat model of hypertension as a result of triterpenoids isolated from olive leaves.16 Likewise the liver, OLE has cardioprotective17,18 and neuroprotective19,20 effects. Oleuropein reduced cardiac infarct size, and the circulating lipid levels in normal and hypercholesterolemic rabbits subjected to ischemia.17 Serum cardiac marker activities, cardiac MDA, DC and nitrotyrosine levels, and inducible nitric oxide synthase expression together with the histopathological amelioration were also found in doxorubicin-treated rats as a result of oleuropein treatment.18 OLE attenuated the increases in nitric oxide, superoxide anions and lipid peroxide levels, and hippocampal neuronal damage after transient global cerebral ischemia in Mongolian gerbils,19 and caused significant protection against leadinduced brain damage through inhibition of apoptosis, oxidative stress and inflammation.20 In light of these observations, we investigated whether OLE treatment was protective against the oxidative stress in tissues of aged rats. In the present study, total OLE extract was used, and aged rats received OLE in drinking water for 2 months. The effective doses of OLE (approximately 500 and 1000 mg/kg bodyweight/day) were chosen according to previous studies.24–26 In the current study, OLE, especially OLE 1000, was detected to diminish the high levels of hepatic MDA, DC and PC levels in aged rats. OLE treatments elevated GSH levels in the liver, but not the heart and brain. There was no change in vitamin E and vitamin C levels, and SOD, GSH-Px and GST activities as a result of OLE treatments. In addition, we 6
|
also detected that protein expressions of hepatic SOD and GSH-Px did not alter in OLE-treated rats as compared with aged rats. According to this, the present results suggest that OLE treatment diminished ageinduced oxidative stress in the liver, heart and brain tissues by acting as an anti-oxidant itself without affecting the anti-oxidant system. In conclusion, the present results suggest that OLE treatment seems to be useful for decreasing oxidative stress in the liver, heart and brain tissues in aged rats. This anti-aging effect could be related to its strong radical scavenging action.
Acknowledgment This work was supported by the Research Fund of University of Istanbul (Project No: BYP-14443).
Disclosure statement The authors declare no conflict of interest.
References 1 Harman D. Aging: overview. Ann N Y Acad Sci 2001; 928: 1–21. 2 Tian L, Cai Q, Wei H. Alterations of antioxidant enzymes and oxidative damage to macromolecules in different organs of rats during aging. Free Radic Biol Med 1998; 24: 1477–1484. 3 Wolf FI, Fasanella S, Tedesco B et al. Peripheral lymphocyte 8-OHdG levels correlate with age-associated increase of tissue oxidative DNA damage in Sprague-Dawley rats. Protective effects of caloric restriction. Exp Gerontol 2005; 40: 181–188. 4 Wong YT, Ruan R, Tay FEH. Relationship between levels of oxidative DNA damage, lipid peroxidation and mitochondrial membrane potential in young and old F344 rats. Free Radic Res 2006; 40: 393–402. 5 Parıldar-Karpuzog˘lu H, Mehmetçik G, Özdemirler-Erata G, Dog˘ru-Abbasog˘lu S, Koçak-Toker N, Uysal M. The effect of taurine treatment on prooxidant-antioxidant balance in livers and brains of old rats. Pharmacol Rep 2008; 60: 673–678. 6 Xu J, Rong S, Xie B et al. Procyanidins extracted from the lotus seedpod ameliorate age-related antioxidant deficit in aged rats. J Gerontol A Biol Sci Med Sci 2010; 65: 236– 241. 7 Aydın AF, Küçükgergin C, Özdemirler-Erata G, Koçak-Toker N, Uysal M. The effect of carnosine treatment on prooxidant-antioxidant balance in liver, heart and brain tissues of male aged rats. Biogerontology 2010; 11: 103–109. 8 Çoban J, Bingül I, Yes¸il-Mızrak K, Dog˘ru-Abbasog˘lu S, Öztezcan S, Uysal M. Effects of carnosine plus vitamin E and betaine treatments on oxidative stress in some tissues of aged rats. Curr Aging Sci 2013; 6: 199–205. © 2014 Japan Geriatrics Society
Effect of olive leaf extract on aged rats 9 Jayakumar T, Thomas PA, Geraldine P. Protective effect of an extract of the oyster mushroom, Pleurotus ostreatus, on antioxidants of major organs of aged rats. Exp Gerontol 2007; 42: 183–191. 10 Siqueira IR, Fochesatto C, de Andrade A et al. Total antioxidant capacity is impaired in different structures from aged brain. Int J Devl Neurosci 2005; 3: 663–671. 11 El SN, Karakaya S. Olive tree (Olea europaea) leaves: potential beneficial effects on human health. Nutr Rev 2009; 67: 632–638. 12 Kontogianni VG, Gerothanassis IP. Phenolic compounds and antioxidant activity of olive extracts. Nat Prod Res 2012; 26: 186–189. 13 Lee OH, Lee BY. Antioxidant and antimicrobial activities of individual and combined phenolics in Olea europaea leaf extract. Bioresour Technol 2010; 101: 3751–3754. 14 Wang L, Geng C, Jiang L et al. The anti-atherosclerotic effect of olive leaf extract is related to suppressed inflammatory response in rabbits with experimental atherosclerosis. Eur J Nutr 2008; 47: 235–243. 15 Al-Azzawie HF, Alhamdani MS. Hypoglycemic and antioxidant effect of oleuropein in alloxan-diabetic rabbits. Life Sci 2006; 78: 1371–1377. 16 Somova LI, Shode FO, Ramnanan P, Nadar A. Antihypertensive, antiatherosclerotic and antioxidant activity of triterpenoids isolated from Olea europaea, subspecies africana leaves. J Ethnopharmacol 2003; 84: 299–305. 17 Andreadou I, Iliodromitis EK, Mikros E et al. The olive constituent oleuropein exhibits anti-ischemic, antioxidative, and hypolipidemic effects in anesthetized rabbits. J Nutr 2006; 136: 2213–2219. 18 Andreadou I, Sigala F, Iliodromitis EK et al. Acute doxorubicin cardiotoxicity is successfully treated with the phytochemical oleuropein through suppression of oxidative and nitrosative stress. J Mol Cell Cardiol 2007; 42: 549–558. 19 Dekanski D, Selakovic V, Piperski V, Radulovic Z, Korenic A, Radenovic L. Protective effect of olive leaf extract on hippocampal injury induced by transient global cerebral ischemia and reperfusion in Mongolian gerbils. Phytomedicine 2011; 18: 1137–1143. 20 Seddik L, Bah TM, Aoues A, Slimani M, Benderdour M. Elucidation of mechanisms underlying the protective effects of olive leaf extract against lead-induced neurotoxicity in Wistar rats. J Toxicol Sci 2011; 36: 797–809. 21 Dekanski D, Janic´ijevic´-Hudomal S, Ristic´ S et al. Attenuation of cold restraint stress-induced gastric lesions by an olive leaf extract. Gen Physiol Biophys 2009; 28: 135–142. 22 Alirezaei M, Dezfoulian O, Neamati S, Rashidipour M, Tanideh N, Kheradmand A. Oleuropein prevents ethanolinduced gastric ulcers via elevation of antioxidant enzyme activities in rats. J Physiol Biochem 2012; 68: 583–592. 23 Hamdi HK, Castellon R. Oleuropein, a non-toxic olive iridoid, is an anti-tumor agent and cytoskeleton disruptor. Biochem Biophys Res Commun 2005; 334: 769–778. 24 Abdel-Hamid NM, El-Moselhy MA, El-Baz A. Hepatocyte lysosomal membrane stabilization by olive leaves against chemically induced hepatocellular neoplasia in rats. Int J Hepatol 2011; 2011: 736581. doi: 10.4061/2011/736581 25 Poudyal H, Campbell F, Brown L. Olive leaf extract attenuates cardiac, hepatic, and metabolic changes in high carbohydrate-, high fat-fed rats. J Nutr 2010; 140: 946– 953.
© 2014 Japan Geriatrics Society
26 Omagari K, Kato S, Tsuneyama K et al. 2010) Olive leaf extract prevents spontaneous occurrence of non-alcoholic steatohepatitis in SHR/NDmcr-cp rats. Pathology 2010; 42: 66–72. 27 Dekanski D, Ristic´ S, Radonjic´ NV et al. Olive leaf extract modulates cold restraint stress-induced oxidative changes in rat liver. J Serb Chem Soc 2011; 76: 1207–1218. 28 Queen BL, Tollefsboll TO. Polyphenols and aging. Curr Aging Sci 2010; 3: 34–42. 29 Cañuelo A, Gilbert-López B, Pacheco-Liñán P, Martínez-Lara E, Siles E, Miranda-Vizuete A. Tyrosol, a main phenol present in extra virgin olive oil, increases lifespan and stress resistance in Caenorhabditis elegans. Mech Ageing Dev 2012; 133: 563–574. 30 Charles AL, Meyer A, Dal-Ros S et al. Polyphenols prevents aging-related impairment in skeletal muscle mitochondrial function through decreased reactive oxygen species production. Exp Physiol 2013; 98: 536–545. 31 Liu LC, Wang CJ, Lee CC et al. Aqueous extract of Hibiscus sabdariffa L. decelerates acetaminophen-induced acute liver damage by reducing cell death and oxidative stress in mouse experimental models. J Sci Food Agric 2010; 90: 329– 337. 32 Buege JA, Aust JD. Microsomal lipid peroxidation. Methods Enzymol 1978; 52: 302–310. 33 Reznick AZ, Packer L. Oxidative damage to proteins: spectrophotometric method for carbonyl assay. Methods Enzymol 1994; 233: 357–363. 34 Beutler E, Duron O, Kelly BM. Improved method for the determination of blood glutathione. J Lab Clin Med 1979; 61: 882–888. 35 Desai DI. Vitamin E analysis methods for animal tissues. Methods Enzymol 1984; 105: 138–147. 36 Omaye ST, Turnbull JD, Sauberlich HE. Selected methods for the determination of ascorbic acid in animal cells, tissues and fluids. Methods Enzymol 1979; 62: 3–8. 37 Mylorie AA, Collins H, Umbles C, Kyle J. Erythrocyte superoxide dismutase activity and other parameters of copper status in rats ingesting lead acetate. Toxicol Appl Pharmacol 1986; 82: 512–520. 38 Lawrence RA, Burk RF. Glutathione peroxidase activity in selenium–deficient rat liver. Biochem Biophys Res Commun 1976; 71: 952–958. 39 Habig WH, Jacoby WB. Assays for differentation of glutathione s-transferases. Methods Enzymol 1981; 77: 398–405. 40 Smith PK, Krohn RI, Hermanson GT et al. Measurement of protein using bicinchoninic acid. Anal Biochem 1985; 150: 76–85. 41 Aydın AF, Küskü-Kiraz Z, Dogru-Abbasoglu S, Güllüoglu M, Uysal M, Koçak- Toker N. Effect of carnosine against thioacetamide-induced liver cirrhosis in rat. Peptides 2010; 31: 67–71. 42 Komes D, Bescak-Cvitanovic A, Horzic D, Rusak G, Likic S, Berendika M. Phenolic composition and antioxidant properties of some traditionally used medicinal plants affected by the extraction time and hydrolysis. Phytochem Anal 2011; 22: 172–180. 43 Hayes JE, Allen P, Brunton N, O’Grady MN, Kerry JP. Phenolic composition and in vitro antioxidant capacity of four commercial phytochemical products: olive leaf extract (Olea europaea L.), lutein, sesamol and ellagic acid. Food Chem 2011; 126: 948–955.
|
7
