Abstract
The antioxidant effects of ellagic acid (EA) and hesperidin (HES) against skeletal muscle ischemia/reperfusion injury (I/R) were performed. Hindlimb ischemia has been induced by tourniquet occlusion for 2 h on left hindlimb. At the end of ischemia, the tourniquate has been removed and initiated reperfusion for 2 h. EA (100 mg/kg) has been applied orally before ischemia/reperfusion in the EA + I/R group. HES (100 mg/kg) has been given orally in the HES + I/R group. The left gastrocnemius muscle has been harvested and stored immediately at −80 °C until assessed for the levels of MDA and antioxidant enzymes activities. MDA level has statistically increased in I/R group (p < 0.05) compared to other groups. The muscle tissue antioxidant enzymes activities were lower than the other groups in the I/R group (p < 0.05). EA and HES treatments significantly reversed the damage level in I/R, also activity of tissue SOD increased in the EA + I/R and HES + I/R groups.
Introduction
Reperfusion injury occurs when the tissues reintake molecular oxygen after ischemia. It is quite an important clinical condition, which includes a large spectrum of vascular events such as organ transplantation, thrombolytic therapy, limb trauma and cardiovascular surgery. Ischemia/Reperfusion (I/R) affects many different organs such as the brain, heart, lung and skeletal muscle in varying degreesCitation1. Skeletal muscle I/R can start numerous detrimental events in tissue such as release and production of oxygen-derived radicals by the cells when exposed to ischemia, degradation of phospholipids by peroxidation, damage in endothelial and parenchymal cells, cytosolic calcium overload and edema due to increased permeability in the microcirculationCitation2. Reactive oxygen species (ROS) are known as reactive oxygen derivatives occurred from free radicals such as superoxide anion radicals ( and hydroxyl radicals (OH•)Citation3–6. ROS assault to membrane lipids and this event results in lipid peroxidationCitation7–10. Also, they can affect the cellular proteins, lipids, nucleic acids and other potential susceptible substances. This process results eventually with excess production of free radicals and organ dysfunction, also ROS can lead to many diseasesCitation11–14. They are mainly responsible for I/R injury. Different treatment strategies that are applied to reduce I/R injury include various antioxidant vitamins, bioflavonoids and drugsCitation15.
Ellagic acid (EA) is a polyphenolic compounds that exist in plants and fruits such as strawberries, walnuts, nuts and blueberriesCitation16. It was reported that EA shows different pharmacological effects, including anti-inflammatory, antioxidant and chemoprevention inhibition of tumorigenesisCitation17–20. Hesperidin (HES), a natural flavonoid, exists in fruits and vegetablesCitation21. HES also have wide pharmacological effects such as anti-inflammatory, antioxidant, anticancer and antiallergic effectsCitation22–24.
The main purpose of this study is to investigate the potential antioxidant effects of EA and HES against skeletal muscle injury induced I/R in rats. MDA level, as a specific biomarker of lipid peroxidation, and antioxidant enzyme activities such as SOD, CAT and GSH-Px, were measured to show the potential protective capacities of EA and HES.
Materials and methods
Chemicals
Ellagic acid, hesperidin and sodium carboxymethylcellulose (CMC) were obtained from Sigma Chemicals, St. Louis, MO and stored in dark at 2–4 °C until use.
Animals
The experimental permission for this study was confirmed by the Experimental Animal Local Ethic Committee of the Veterinary Faculty of Atatürk University (2016–2013). In this experimental study, 30 adult Sprague-Dawley rats between 200 and 230 g weight were used. We kept the rats in standard polypropylene cages (four rats/cage) in a temperature-controlled room (22 ± 2 °C), humidity (55 ± 5%), alternating 12 h light–dark cycles and gave water ad libitum. Also, they were acclimatized before the experiment for 1 week. Animals were fasted 8 h before the study.
Experimental design
The rats were weighed and anesthetized with Sodium Pentothal 40–50 mg/kg dose intraperitoneally and the sevoflurane (1,1,1,3,3,3-hexafluoro-2-(fluoromethoxy)propane). They were randomly allocated into five groups of six rats. Group 1 (Sham group, n = 6), which was subjected to all operative processes, except ischemia reperfusion. Five hundred microliters of CMC (0.5%) were given orally per 100 g weight to this group. Group 2 (ischemia group, n = 6); 0.5% CMC was administered prior to ischemia period for 1 week and was exposed to ischemia for 2 h. Group 3 (I/R group, n = 6); rats were exposed to ischemia for 2 h and reperfusion for 2 h. Also, 0.5% CMC was performed. Group 4 (EA + I/R group, n = 6); I/R was applied. A solution of EA 100 mg/kg as used in the previous studyCitation25 was suspended into 0.5% CMC solution and administered before I/R period for 1 week. Group 5 (I/R + HES group, n = 6); a solution of HES 100 mg/kg as used in the previous studyCitation26 was suspended into 0.5% CMC solution and administered before I/R period for 1 week. Later the rats were subjected to I/R.
Surgery and specimens collection
Surgery was performed at the Research Laboratory of Animals Experimental, Atatürk University. Operative procedure was similar in all groups but the tourniquate was not applied to the sham group. The animals were placed on the carton in dorsal recumbency position and their limbs immobilized with sticky band. Body temperature was maintained with a heating lamb. They were anesthetized with thiopental sodium prior to ischemia (40–50 mg/kg, intraperitoneally) and additional doses were given when it is necessary throughout the ischemic period. The decline of cyanosis and temperature in the extremity was accepted as ischemia. Later then, tourniquate was removed and reperfusion started. The extremities such as normalization of the temperature, edema and change in color to pink were evaluated as reperfusion. Muscle tissue was harvested for biochemical evaluations after completing the experiment. The gastrocnemius muscles were cleaned in cold isotonic saline. Euthanasia was performed at the end of experiment using an overdose of thiopental sodium (300 mg/kg, ip) to all rats.
Biochemical assays
MDA level, SOD, CAT and GSH-Px activities were detected in the samples obtained from the skeletal muscle. Each tissue was frozen at −80 °C until the date of analysis. Then, the tissues were homogenized. GSH-Px activity measurement was performed according to the method of Matkovics et al.Citation27 as described previouslyCitation28. The results were expressed as Units per milligrams of protein tissue. CAT activity in muscle tissue was measured using spectrophotometric methodsCitation29,Citation30. Results were expressed as units per grams of protein. The total SOD activity determination was made kinetically by using the method described by Sun et al.Citation31. The method is based on the inhibition of nitroblue tetrazolium (NBT) reductionCitation32–34 via the xanthine–xanthine oxidase enzyme system, which is an generator. One unit of SOD was defined as the SOD concentration causing 50% inhibition in the NBT reduction rate. SOD activity was expressed as endotoxin units per milligrams of protein. MDA levels were determined spectrophotometrically using thiobarbituric acid (TBA) reaction as described by Placer et al.Citation35. The origin of this method is related to the measurement of the red-pink color produced by the interaction of barbituric acid with MDA. A portion of the MDA is formed during the peroxidation, but majority of the MDA occurs as a result of combustion of lipid peroxides in heating step after the acidified environment. Results were measured using a standard calibration curve and were calculated as nanomoles per grams of protein.
Statistical analysis
Data were analyzed with IBM SPSS Statistics for Windows, Version 22.0 (IBM Corp., Armonk, NY). Groups were compared with analysis of variance (ANOVA), Bonferroni’s multiple comparison tests were performed. Results of the study were given as Mean ± SEM. p < 0.05 was accepted as statistically significant.
Results and discussion
The effects of skeletal muscles I/R were quite devastating. Ischemia induces the damage but this damage increases paradoxically in reperfusion, which causes most significant damage in the muscle and other tissues. The consequence of the I/R injury depends on many factors and is important to understand the pathophysiology of I/R injuryCitation36,Citation37. The effectiveness of many antioxidant agents has been investigated for palliative I/R injuryCitation38,Citation39. Scientific data have shown that skeletal muscle I/R injury is an unavoidable event on several surgical procedures. The reblood supply to ischemic tissues results with the intensive formation of ROS, which causes damage to the biological molecules such as nucleic acids, membrane lipids and enzymesCitation40. ROS can cause tissue damage and it leads to various diseasesCitation5,41–44. ROS, which involves free radicals such as OH• and O2•− radicals, and nonfree radical species such as H2O2 and singlet oxygen (1O2) were known as different forms of activated oxygenCitation45–48. On the other hand, cellular injury can initiate with various defensive mechanisms by ROS. Firstly, defensive mechanisms contain antioxidant enzymes including CAT, SOD and GSH-Px. Consequently, these enzymes produce the less reactive species. Normally, the pro-oxidant and antioxidant activities are balanced in all physiological processesCitation49–51. If this balance is disrupted toward pro-oxidant activity, it causes hazardous damage in functional tissues and organs. Free radicals assault on polyunsaturated fatty acids in cell membraneCitation52,Citation53. Antioxidants prevent and stop the free radical activity against lipid peroxidation. Free radicals can alter the structure and feature of biological molecules such as proteins, lipids, carbohydrates and DNACitation54–56. Antioxidants can scavenge oxygen-derived radicals by delaying the process of polyunsaturated fatty acids peroxidationCitation57,Citation58. Consequently, the human body can be protected by antioxidants against the damage, depending on the free radicals and ROSCitation59–61.
The animals well tolerated the experimental procedure and have not died during the experiment. Results are presented as mean ± SEM in all groups. The MDA levels of the muscle tissue were as follows: sham group, 16.26 ± 0.54; ischemia group, 48.70 ± 2.17; I/R group, 58.43 ± 3.70; EA group 32.29 ± 3.30 and hesperidin group 43.61 ± 2.08. The tissue MDA levels in the I/R group were markedly higher than the sham, ischemia, ellagic acid and hesperidin groups. The difference between all the groups was also statistically significant (p < 0.01) ().
Figure 1. The effects of ischemia, I/R and the treatment with EA and hesperidin (HES) on the MDA levels ((a) p < 0.01; (b) p < 0.05 and (c) p < 0.05). The comparison between the sham group with other groups are denoted by “a”. The comparison between the ischemia group with IR, EA and HES groups is denoted by “b”. The comparison between the IR group with EA, and HES groups is denoted by “c”.
The SOD levels of the muscle tissue were decreased as follows: sham group (15.11 ± 0.36); ischemia group (16.57 ± 0.71); I/R group (12.54 ± 0.40); EA group 21.53 ± 1.18; hesperidin group (22.71 ± 0.60). Muscle tissues SOD activity decreased in I/R group significantly compared to sham group. SOD levels were significantly increased in EA and hesperidin groups and there were significant differences in EA and hesperidin groups (p < 0.01) ().
Figure 2. The effects of ischemia, I/R and the treatment with EA and hesperidin (HES) on the SOD activity ((a) p < 0.01; (b) p < 0.05 and (c) p < 0.05). The comparison between the sham group with EA, and HES groups is denoted by “a”. The comparison between the ischemia group with IR, EA, and HES groups is denoted by “b”. The comparison between the IR group with EA, and HES groups is denoted by “c”.
The CAT levels of the muscle tissue were as follows: sham group (247.94 ± 12.05); ischemia group (244.03 ± 9.88); I/R group (237.97 ± 3.97); EA group (241.84 ± 2.06) and HES group (244.36 ± 1.72). CAT levels were enhanced in EA and HES groups but there were no significant differences in sham, ischemia and I/R compared to EA and HES groups ().
Figure 3. The effects of ischemia, I/R and the treatment with EA and hesperidin (HES) on the CAT activity.
Finally, the GSH-Px levels of the muscle tissue were as follows: sham group (41.52 ± 3.12); ischemia group (36.32 ± 3.00); I/R group (39.11 ± 3.25); EA group (49.25 ± 5.99) and HES group (55.16 ± 3.75). By comparing all the groups, it can be seen that there were significant differences between I/R and HES groups (p < 0.05) ().
Figure 4. The effects of ischemia, I/R and the treatment with EA, the treatment with hesperidin (HES) on the GSH-Px activity ((a) p < 0.05). The comparison between the ischemia group with other groups is denoted by “a”.
EA shows effective scavenging action on free radicals in vitro, as well as the protective effects against lipid peroxidationCitation62. HES is a potential antioxidant agent, with strong superoxide radical scavenging activity against free radicalsCitation63. A lot of scientists have evaluated the antioxidant activity and O2•−, OH• and H2O2 scavenging activities of HESCitation64,Citation65. Previous studies have shown that oxidative damage was attenuated by EA and HES after I/R in animalsCitation64,Citation66,Citation67. Several studies have reported various antioxidants that can reduce the tissue MDA levelCitation1. The intestinal ischemia reperfusion study has reported a significant decrease of MDA level in EA groupCitation67. Also, the decrease of MDA levels in HES group was in accordance with the results of the lung ischemia reperfusion study by Bayomy et al.Citation66 The results obtained from the present study suggest that I/R injury increased MDA levels in muscle tissue. In contrast, EA and HES led to decrease in the MDA levels in muscle tissue. Depending on these results, in the I/R group, we can clearly say that greater muscle damage occurred only due to ischemia. Also, EA and HES have antioxidative effects that attenuated the muscle damage reducing lipid peroxidation levels. Normally, living organisms are protected from the dangerous effects of O2•− by SOD, which converts O2•− into H2O2Citation68–70. Whereas, during reperfusion of ischemic tissues, these natural defences may not be overcome and H2O2 is converted into OH•, this transformation has the capacity to damage a wide range of biomolecules species containing amino acids, membrane proteins and nucleic acidsCitation71–73. CAT is an oxidoreductase that catalyzes the conversion of H2O2 to H2O and O2. Also, it can protect the cells from damage induced by I/RCitation74. It has reported that HES treatment increased SOD, CAT and GSH-Px activities and reduced the tissue damage caused by oxidative stress-induced ischemia and I/RCitation75. It was suggested that HES has positive effects on SOD, CAT and GSH-Px activities and has protective effects against oxidative stress in rat brainCitation76. In another study, it has been demonstrated that EA enhanced increased tissue SOD, CAT and GSH-Px activitiesCitation77. According to our results, it has been detected that SOD, CAT and GSH-Px activities increased in EA and HES groups compared to ischemia and I/R groups. The results show the most important point that the EA and HES with antioxidant properties also have cell protective effects against lipid peroxidation.
Conclusion
We have detected the antioxidant effects of EA and HES against the skeletal muscle injury-induced I/R. Further studies should be performed to use these drugs, which have clinically protective effects against the I/R injury in skeletal muscle.
Declaration of interest
The authors report there is no conflict of interest.
Acknowledgements
The authors are grateful to Central Research Laboratory of Ağrı İbrahim Çeçen University for completely support of the work successfully approved by the Unit of Scientific Research Projects (project number: SYO.14.002). IG and SHA would like to extend their sincere appreciation to the Research Chairs Program at King Saud University for providing funding to this research.
References
- Ozyurt H, Ozyurt B, Koca K, Ozgocmen S. Caffeic acid phenethyl ester (CAPE) protects rat skeletal muscle against ischemia-reperfusion-induced oxidative stress. Vasc Pharmacol 2007;47:108–12
- Land WG. The role of postischemic reperfusion injury and other nonantigen-dependent inflammatory pathways in transplantation. Transplantation 2005;79:505–14
- Gulcin I. Antioxidant activity of food constituents: an overview. Arch Toxicol 2012;86:345–91
- Bae JH, Park YJ, Namiesnik J, et al. Effects of artificial lighting on bioactivity of sweet red pepper (Capsicum annuum L.). Int J Food Sci Technol 2016;51:1378–85
- Topal F, Topal M, Gocer H, et al. Antioxidant activity of taxifolin: an activity-structure relationship. J Enzyme Inhib Med Chem 2016;31:674–83
- Topal M, Gocer H, Topal F, et al. Antioxidant, antiradical and anticholinergic properties of cynarin purified from the illyrian thistle (Onopordum illyricum L.). J Enzyme Inhib Med Chem 2016;31:266–75
- Gulcin I, Beydemir S. Phenolic compounds as antioxidants: carbonic anhydrase isoenzymes inhibitors. Mini Rev Med Chem 2013;13:408–30
- Sehitoglu MH, Han H, Kalin P, et al. Pistachio (Pistacia vera L.) gum: a potent inhibitor of reactive oxygen species. J Enzyme Inhib Med Chem 2015;30:264–9
- Kalın P, Gülçin İ, Gören AC. Antioxidant activity and polyphenol content of Vaccinium macrocarpon. Rec Nat Prod 2015;9:496–502
- Aksu K, Topal F, Gülçin I, et al. Acetylcholinesterase inhibitory and antioxidant activities of novel symmetric sulfamides derived from phenethylamines. Arch Pharm 2015;348:446–55
- Lai LS, Chou ST, Chao WW. Studies on the antioxidative activities of Hsian-tsao (Mesona procumbens Hems L.) leaf gum. J Agric Food Chem 2001;49:963–8
- Gulcin I, Buyukokuroglu ME, Oktay M, Kufrevioglu OI. On the in vitro antioxidative properties of melatonin. J Pineal Res 2002;33:167–71
- Gulcin I, Elias R, Gepdiremen A, Boyer L. Antioxidant activity of lignans from fringe tree (Chionanthus virginicus L.). Eur Food Res Technol 2006;223:759–67
- Çakmakçı S, Topdaş EF, Kalın P, et al. Antioxidant capacity and functionality of oleaster (Elaeagnus angustifolia L.) flour and crust in a new kind of fruity ice cream. Int J Food Sci Technol 2015;50:472–81
- Punz A, Nanobashvili J, Neumayer C, et al. Multivitamin administration before ischemia reduces ischemia-reperfusion injury in rabbit skeletal muscle. Clin Nutr 1999;18:219–26
- Sellappan S, Akoh CC, Krewer G. Phenolic compounds and antioxidant capacity of Georgia-grown blueberries and blackberries. J Agric Food Chem 2002;50:2432–8
- Buniatian GH. Stages of activation of hepatic stellate cells: effects of ellagic acid, an inhibiter of liver fibrosis, on their differentiation in culture. Cell Proliferat 2003;36:307–19
- Festa F, Aglitti T, Duranti G, et al. Strong antioxidant activity of ellagic acid in mammalian cells in vitro revealed by the comet assay. Anticancer Res 2001;21:3903–8
- Lei F, Xing DM, Xiang L, et al. Pharmacokinetic study of ellagic acid in rat after oral administration of pomegranate leaf extract. J Chromatogr B Analyt Technol Biomed Life Sci 2003;796:189–94
- Townsend DM, Tew KD. The role of glutathione-S-transferase in anti-cancer drug resistance. Oncogene 2003;22:7369–75
- Justesen U, Knuthsen P, Leth T. Quantitative analysis of flavonols, flavones, and flavanones in fruits, vegetables and beverages by high-performance liquid chromatography with photo-diode array and mass spectrometric detection. J Chromatogr A 1998;799:101–10
- Aboobaker VS, Balgi AD, Bhattacharya RK. In vivo effect of dietary factors on the molecular action of aflatoxin B1: role of non-nutrient phenolic compounds on the catalytic activity of liver fractions. In Vivo 1994;8:1095–108
- Garg A, Garg S, Zaneveld LJD, Singla AK. Chemistry and pharmacology of the citrus bioflavonoid hesperidin. Phytother Res 2001;15:655–69
- Ebrahimi A, Schluesener H. Natural polyphenols against neurodegenerative disorders: potentials and pitfalls. Ageing Res Rev 2012;11:329–45
- Ueda H, Kawanishi K, Moriyasu M. Effects of ellagic acid and 2-(2,3,6-trihydroxy-4-carboxyphenyl)ellagic acid on sorbitol accumulation in vitro and in vivo. Biol Pharm Bull 2004;27:1584–7
- Suresh M, Kumar SNK, Kumar SA, et al. Hesperidin safeguards hepatocytes from valproate-induced liver dysfunction in Sprague-Dawley rats. Biomed Prevent Nutr 2014;4:209–17
- Matkovics B, Sz Szabó L, Varga I. Determination of enzyme activities in lipid peroxidation and glutathione pathways (in Hungarian). Laboratóriumi Diagnosztika 1988;15:248–50
- Şentürk M, Gülçin İ, Çiftci M, Küfrevioğlu Öİ. Dantrolene inhibits human erythrocyte glutathione reductase. Biol Pharm Bull 2008;31:2036–9
- Gülçin İ, Beydemir Ş, Hisar O. The effect of α-tocopherol on the antioxidant enzymes activities and lipid peroxidation of rainbow trout (Oncorhynchus mykiss). Acta Vet Hung 2005;53:425–33
- Taysi S, Akçay F, Uslu C, et al. Trace elements and some extracellular antioxidant proteins levels in serum of patients with laryngeal cancer. Biol Trace Elem Res 2003;91:11–18
- Sun Y, Oberley LW, Li Y. A simple method for clinical assay of superoxide dismutase. Clin Chem 1988;34:497–500
- Gülçin İ, Alici HA, Cesur M. Determination of in vitro antioxidant and radical scavenging activities of propofol. Chem Pharm Bull 2005;53:281–5
- Gülçin İ, Beydemir Ş, Şat İG, Küfrevioğlu Öİ. Evaluation of antioxidant activity of cornelian cherry (Cornus mas L.). Acta Aliment Hung 2005;34:193–202
- Gülçin İ. The antioxidant and radical scavenging activities of black pepper (Piper nigrum) seeds. Int J Food Sci Nutr 2005;56:491–9
- Placer ZA, Cushman LL, Johnson BC. Estimation of product of lipid peroxidation (malonyl dialdehyde) in biochemical systems. Anal Biochem 1966;16:359–64
- Giannoudis PV. Current concepts of the inflammatory response after major trauma: an update. Injury 2003;34:397–404
- Gillani S, Cao J, Suzuki T, Hak DJ. The effect of ischemia reperfusion injury on skeletal muscle. Injury 2012;43:670–5
- Dong X, Xing Q, Li Y, et al. Dexmedetomidine protects against ischemia-reperfusion injury in rat skeletal muscle. J Surg Res 2014;186:240–5
- Takhtfooladi HA, Takhtfooladi MA, Karimi P, et al. Influence of tramadol on ischemia-reperfusion injury of rats' skeletal muscle. Int J Surg 2014;12:963–8
- Grace PA. Ischaemia-reperfusion injury. Br J Surg 1994;81:637–47
- Gulcin I, Buyukokuroglu ME, Oktay M, Kufrevioglu OI. Antioxidant and analgesic activities of turpentine of Pinus nigra Arn. subsp. pallsiana (Lamb.) Holmboe. J Ethnopharmacol 2003;86:51–8
- Göçer H, Akıncıoğlu A, Öztaşkın N, et al. Synthesis, antioxidant, and antiacetylcholinesterase activities of sulfonamide derivatives of dopamine-related compounds. Arch Pharm (Weinheim) 2013;346:783–92
- Bursal E, Köksal E, Gülçin İ, et al. Antioxidant activity and polyphenol content of cherry stem (Cerasus avium L.) determined by LC-MS/MS. Food Res Int 2013;51:66–74
- Gülçin İ, Elmastaş M, Aboul-Enein HY. Antioxidant activity of clove oil – a powerful antioxidant source. Arab J Chem 2012;5:489–99
- Çetinkaya Y, Göçer H, Menzek A, Gülçin İ. Synthesis and antioxidant properties of (3,4-dihydroxyphenyl)(2,3,4-trihydroxyphenyl)methanone and its derivatives. Arch Pharm 2012;345:323–34
- Gülçin İ, Beydemir S, Topal F, et al. Apoptotic, antioxidant and antiradical effects of majdine and isomajdine from Vinca herbacea Waldst. and kit. J Enzyme Inhib Med Chem 2012;27:587–94
- Göçer H, Gülçin İ. Caffeic acid phenethyl ester (CAPE): correlation of structure and antioxidant properties. Int J Food Sci Nutr 2011;62:821–5
- Gulcin İ. Antioxidant and antiradical activities of L-carnitine. Life Sci 2006;78:803–11
- Gülçin İ. Antioxidant activity of caffeic acid (3,4-dihydroxycinnamic acid). Toxicology 2006;217:213–20
- Polat Köse L, Gülçin İ, Gören AC, et al. LC-MS/MS analysis, antioxidant and anticholinergic properties of galanga (Alpinia officinarum Hance) rhizomes. Ind Crops Prod 2015;74:712–21
- Bursal E, Gülçin İ. Polyphenol contents and in vitro antioxidant activities of lyophilized aqueous extract of kiwifruit (Actinidia deliciosa). Food Res Int 2011;44:1482–9
- Gülçin İ, Topal F, Çakmakçı R, et al. Pomological features, nutritional quality, polyphenol content analysis and antioxidant properties of domesticated and three wild ecotype forms of raspberries (Rubus idaeus L.). J Food Sci 2011;76:C585–93
- Gülçin İ, Topal F, Oztürk Sarikaya SB, et al. Polyphenol contents and antioxidant properties of medlar (Mespilus germanica L.). Rec Nat Prod 2011;5:158–75
- Gulcin İ, Berashvili D, Gepdiremen A. Antiradical and antioxidant activity of total anthocyanins from Perilla pankinensis decne. J Ethnopharmacol 2005;101:287–93
- Gulcin İ, Mshvildadze V, Gepdiremen A, Elias R. The antioxidant activity of a triterpenoid glycoside isolated from the berries of Hedera colchica: 3-O-(beta-D-glucopyranosyl)-hederagenin. Phytother Res 2006;20:130–4
- Gulcin İ. Comparison of in vitro antioxidant and antiradical activities of L-tyrosine and L-Dopa. Amino Acids 2007;32:431–8
- Köksal E, Bursal E, Dikici E, et al. Antioxidant activity of Melissa officinalis leaves. J Med Plants Res 2011;5:217–22
- Gülçin İ. Antioxidant activity of eugenol: a structure-activity relationship study. J Med Food 2011;14:975–85
- Gülçin İ, Huyut Z, Elmastaş M, Aboul-Enein HY. Radical scavenging and antioxidant activity of tannic acid. Arab J Chem 2010;3:43–53
- Innocenti A, Gülçin İ, Scozzafava A, Supuran CT. Carbonic anhydrase inhibitors. Antioxidant polyphenol natural products effectively inhibit mammalian isoforms I-XV. Bioorg Med Chem Lett 2010;20:5050–3
- Gülçin İ, Bursal E, Şehitoğlu HM, et al. Polyphenol contents and antioxidant activity of lyophilized aqueous extract of propolis from Erzurum, Turkey. Food Chem Toxicol 2010;48:2227–38
- Ozkaya A, Celik S, Yuce A, et al. The effects of ellagic acid on some biochemical parameters in the liver of rats against oxidative stress induced by aluminum. Kafkas Univ J Veterinary Faculty 2010;16:263–8
- Cho J. Antioxidant and neuroprotective effects of hesperidin and its aglycone hesperetin. Arch Pharm Res 2006;29:699–706
- Kamaraj S, Anandakumar P, Jagan S, et al. Hesperidin attenuates mitochondrial dysfunction during benzo(a)pyrene-induced lung carcinogenesis in mice. Fundament Clin Pharmacol 2011;25:91–8
- Kamaraj S, Ramakrishnan G, Anandakumar P, et al. Antioxidant and anticancer efficacy of hesperidin in benzo(a)pyrene induced lung carcinogenesis in mice. Invest New Drugs 2009;27:214–22
- Bayomy NA, Elshafhey SH, ElBakary RH, Abdelaziz EZ. Protective effect of hesperidin against lung injury induced by intestinal ischemia/reperfusion in adult albino rats: histological, immunohistochemical and biochemical study. Tissue Cell 2014;46:304–10
- Boyuk A, Onder A, Kapan M, et al. Ellagic acid ameliorates lung injury after intestinal ischemia-reperfusion. Pharmacogn Mag 2011;7:224–8
- Gülçin İ, Kirecci E, Akkemik E, et al. Antioxidant and antimicrobial activities of an aquatic plant: Duckweed (Lemna minor L.). Turk J Biol 2010;34:175–88
- Şerbetçi Tohma H, Gülçin İ. Antioxidant and radical scavenging activity of aerial parts and roots of Turkish licorice (Glycyrrhiza glabra L.). Int J Food Propert 2010;13:657–71
- Balaydın HT, Gülçin İ, Menzek A, et al. Synthesis and antioxidant properties of diphenylmethane derivative bromophenols including a natural product. J Enzyme Inhib Med Chem 2010;25:685–95
- Anaya-Prado R, Toledo-Pereyra LH, Lentsch AB, Ward PA. Ischemia/reperfusion injury. J Surg Res 2002;105:248–58
- Gülçin İ. Antioxidant properties of resveratrol: a structure-activity insight. Innov Food Sci Emerg 2010;11:210–18
- Oztaskın N, Çetinkaya Y, Taslimi P, et al. Antioxidant and acetylcholinesterase inhibition properties of novel bromophenol derivatives. Bioorg Chem 2015;60:49–57
- Gurlek A, Celik M, Parlakpinar H, et al. The protective effect of melatonin on ischemia-reperfusion injury in the groin (Inferior epigastric) flap model in rats. J Pineal Res 2006;40:312–17
- Wang JC, Zhu HL, Yang ZJ, Liu ZP. Antioxidative effects of hesperetin against lead acetate-induced oxidative stress in rats. Indian J Pharmacol 2013;45:395–8
- El-Sayed EM, Abo-Salem OM, Abd-Ellah MF, Abd-Alla GM. Hesperidin, an antioxidant flavonoid, prevents acrylonitrile-induced oxidative stress in rat brain. J Biochem Mol Toxicol 2008;22:268–73
- Yuce A, Atessahin A, Ceribasi AO, Aksakal M. Ellagic acid prevents cisplatin-induced oxidative stress in liver and heart tissue of rats. Basic Clin Pharmacol Toxicol 2007;101:345–9