Abstract
This study was designed to investigate the antioxidant effects of Naringin, in ischemia/reperfusion (I/R)-induced skeletal muscle injury in rats. The rats were randomly allocated into three groups including control, I/R and I/R + Naringin groups. Muscle tissues of I/R groups revealed significantly higher antioxidant enzyme activities, and increased levels of malondialdehyde, as specific a marker of the lipid peroxidation and tissue damage, compared to the control group (p < 0.05). Levels of these parameters in muscle revealed significant reductions in the I/R + Naringin group compared to the I/R group (p < 0.05). Histopathological examination of ischemia muscles in the I/R group showed significant degeneration and inflammation compared to the control group, whereas ischemic muscles of Naringin-administered group showed significant reduction in degeneration and inflammation compared to the I/R group (p < 0.05). We suggest that the protective effect of Naringin may reduce the I/R injury in cases of extremity injuries with acute vascular complications, extremity surgery with prolonged tourniquet application.
Introduction
A number of surgical procedures, such as free flap transfer, transplantation, extremity revascularisation and abdominal aortic surgery, inevitably cause skeletal muscle ischemia/reperfusion (I/R) injury, which lead to severe postoperative complicationsCitation1,Citation2. Although skeletal muscle appears to be relatively more resistant to warm ischemia than many other tissues, prolonged arterial occlusion may cause ischemic injuryCitation3. Skeletal muscle I/R injury often results in the loss of contractile function and in severe cases can lead to disability, limb amputation and even death. They are continuously produced by the body’s normal use of oxygen such as respiration and some cell-mediated immune functions. Reactive oxygen species (ROS) include free radicals such as superoxide anion radicals , hydroxyl radicals
and non-free radical species such as hydrogen peroxide (H2O2) and singlet oxygen (1O2)Citation4–7. Antioxidant compounds had a large biological activities spectrumCitation8–11 and can scavenge free radicals and increase shelf life by retarding the process of lipid peroxidation, which is one of the major reasons for deterioration of food and pharmaceutical products during processing and storageCitation12–15. Antioxidants can also protect the human body from free radicals and ROS effectsCitation16–19 and retard the progress of many chronic diseases as well as lipid peroxidationCitation20–22. They protect against detrimental effects of ROS and free radicals in the human bodyCitation23–25. The first-line defense mechanism includes antioxidants such as superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GSH-Px)Citation26–28. These enzymes catalyse the conversion of ROS into less-reactive speciesCitation29,Citation30.
Natural antioxidants are found in almost all plants, microorganisms, fungi and even in animal tissuesCitation31–34. Phenolic compounds are secondary plant metabolites and naturally present in almost all plant materials, including food products of plant origin. These compounds are thought to be an integral part of both human and animal dietsCitation35–38. The majority of natural antioxidants are phenolic compounds, and the most important groups of natural antioxidants are flavonoids, which including flavanones, flavanols and flavanonolsCitation39–43.
Naringin (4′,5,7-trihydroxyflavanone 7-rhamnoglucoside) is a major antioxidant compound and active flavanone glycoside of grapefruit and many citrus herbs as well. Naringin is reported to possess anti-ulcer, superoxide scavenging and antioxidant activitiesCitation29,Citation30. When Naringin is administered orally, it is hydrolysed by intestinal microflora to yield a major metabolite Naringenin (4,5,7-trihydrosyflavanone), which is the absorbable formCitation44,Citation45. Naringin had anti-ulcerCitation46 and aortic dilatationCitation47. Moreover, it demonstrated inhibition of breast cancer cell proliferation and delay of mammary tumerogenesisCitation48.
In this study, we evaluated the antioxidant effects of Naringin against skeletal muscle I/R injury in a rat model. We analysed several antioxidant enzymes including SOD, CAT, GSH-Px activities, and malondialdehyde (MDA) level in muscle. We also assessed plasma levels of creatine kinase (CK) and lactate dehydrogenase (LDH) as indicators of muscle damage in blood. Also we evaluated histological parameters and leukocyte infiltration in samples gastrocnemius muscle.
Materials and methods
Animals
The study was conducted with 24-male Spraque–Dawley albino rats (purchased from Central Animal House of the Atatürk University, Erzurum, Turkey) weighing between 250 and 300 g. The rats were kept at room temperature and provided with free access to standard chow and tap water. This study received approval from the Animal Research Ethical Committee of the Atatürk University of Erzurum, 2012. Under Ketamine and Xylazine (50 mg/kg of body weight and 10 mg/kg of body weight) mixture anaesthesia intraperitoneally (i.p.), the rats were placed in supine position. Latex tourniquets were applied to the root of one of the hindlimbs. Total ischemia was characterized by the absence of an arterial pulse distal to the tourniquet, and cyanosis and coldness of the corresponding limb. Ischemia was maintained for 2 h, with the muscle temperature kept at 36 ± 1 °C using heat lamps. At the end of the ischemia period, the tourniquet was removed; initiating hindlimb reperfusion for 2 h. Supplementary Ketamine and Xylazine mixture anaesthesia was given i.p. if required.
Experimental design
The rats were divided into three groups of eight animals as follows: control group: animals were anaesthetized and placed under the heat lamps for the same time of ischemia without application of the tourniquet. I/R group: animals were subjected to 2 h of hindlimb ischemia and 2-h reperfusion and then sacrificed. IR + Naringin (I/R + NAR) group: animals were subjected to 2 h of hindlimb ischemia and 2-h reperfusion and then sacrificed; however, this group received Naringin (400 mg/kg, p.o.), which was administered three times with an 8-h interval before ischemia. At the time of sacrifice under anaesthesia, blood samples were obtained from abdominal aorta for the determination of serum CK and LDH levels. Muscle tissue samples were obtained from the gastrocnemius muscle and frozen at −80 °C for GSH-Px, CAT SOD activities and MDA level analyses. Muscle biopsies were also fixed in buffered formalin for histological analysis.
Biochemical studies
Measurement of malondialdehyde activity
The tissues weighed were homogenized on ice with Tris-HCl buffer [0.2 mM pH 7.4, (1:10 w/v)] (IKA Ultra-Turrax T25 basic homogenizer, Germany). The measurement tissue MDA was made in these examples. Homogenate was centrifuged at 5000 × g for 55 min. Then, the supernatant, the upper clean portion, was stored in aliquots at −80 °C for the measurements of the SOD, CAT and GSH-Px activities later on. UV-VIS Shimadzu 1600 was used in the spectrophotometric measurements.
Measurement of antioxidant enzymes activities
CAT (EC 1.11.1.6) enzyme activity was measured spectrophotometrically using the method of AebiCitation49 at 240 nm. The buffer was adjusted to 0.500 OD adding hydrogen peroxide (H2O2) to phosphate buffer (50 mM). The fall in the absorbance with the addition of the sample was recorded in every 15 sec. The rate of consumed H2O2 in 1 min was expressed as k/g protein [k = (2.3 x log (OD1/OD2))/30 s].
To measure the SOD (E.C. 1.15.1.1) enzyme activity the reduction of the with nitroblue tetrazolium (NBT) was assessed spectrophotometrically at 560 nmCitation50. Inhibition of the enzyme activity 50% was taken and expressed as U/mg protein. SOD inhibition was calculated from the following formula:
GSH-Px (EC 1.11.1.9) enzyme activity was measured according to the method of PagliaCitation51, in which the oxidation of reduced glutathione (GSH) to oxidized glutathione (GSSG) was measured in the presence of H2O2. Absorbance decrease during of the oxidation to the NADP+ of the NADPH was followed at 340 nm, and the enzyme activity was calculated. The amount of micromoles NADPH oxidized per unit time was expressed as U/mg protein.
Determination of malondialdehyde quantity
MDA level was measured according to the method of Esterbauer and Cheeseman spectrophotometrically at 532 nm at –95 °CCitation52. Results were calculated according to the results of the standard graph and expressed as nmol/g in wet tissue.
Protein determination
The analysis of protein samples extracted and the supernatant were studied according to method of Lowry et al.Citation53
Measurement of creatine kinase and lactate dehydrogenase levels
Serum CK and LDH enzyme levels were determined using autoanalyser in routine biochemistry laboratory.
Histological assessments
The tissues fixed in a 10% neutral-buffered formalin solution were embedded in paraffin and used for histopathological examination. Five micrometres (5 μm) of thick sections were cut, deparaffinized, hydrated and stained with haematoxylin and eosin. Rat gastrocnemius muscle was scored for the histopathological evaluation. Using light microscopy, to demonstrate the deterioration in epimysium and perimysium, increased cellularity in perimysium; degeneration in the arterial wall, coagulation in vessels and vasodilatation on tissue samples were scored semi-quantitatively according to grade 0 (−), grade 1 (+), grade 2 (++) and grade 3 (+++) damage.
Statistical analysis
The obtained results were presented as means ± SEM. First, non-parametric Kruskal–Wallis analysis of variance test was applied to evaluate the data. Then, pairwise comparisons were performed with Mann–Whitney U test. p < 0.05 was considered significant. Calculations were performed using SPSS 15.0 package program compatible with Windows.
Results
Results of oxidative stress markers and histological evaluation scores of the in each group were shown in and and . The tissue MDA levels in the I/R group were significantly higher than the control and Naringin-treated groups. This situation shows that the I/R model causes damage of the cellular lipids in tissue. The difference between the I/R and the control and Naringin-treated groups was also significant (p < 0.01; ). The muscle tissue GSH-Px levels in the I/R group were significantly lower than the control and Naringin-treated group (p < 0.05). The difference between the I/R group and control group and Naringin-treated group was significant (p < 0.05; ). I/R injury to the muscle significantly decreased SOD activity muscle tissues compared with the Naringin-treated group. The difference between the control group and I/R group and Naringin-treated group was significant (p < 0.01; ). SOD levels were significantly improved by Naringin treatment. Also, there were significant differences in the Naringin-treated and I/R groups (p < 0.01). CAT activity in muscle tissue decreased markedly after reperfusion compared with the control group. A significant decrease in the activity of CAT was observed in I/R group compared with control and Naringin-treated group. The difference between the control-, I/R- and Naringin-treated groups was also significant (p < 0.05, ). The difference between the control group and I/R group and Naringin-treated group was significant at the same time (p < 0.05). The plasma levels of CK in the I/R (ischemia/reperfusion injury in the skeletal muscle) group were significantly higher (p < 0.01) than that of the control group, however the difference between the control group, I/R and Naringin-treated group significantly (p < 0.01). The difference between the I/R and the Naringin-treated groups was also significant (p < 0.05, ). The plasma levels of LDH in the control group were lower than the I/R and Naringin-treated groups. There were any the differences between in control, I/R and Naringin-treated groups ().
Figure 1. (A) The effects of ischemia/reperfusion (I/R) and treatment with Naringin on the MDA levels. *Between the I/R and control, I/R + NAR, p < 0.01. (B) The effects of ischemia/reperfusion (I/R) and treatment with Naringin on the GSH-Px levels. *Between the I/R and control, I/R + NAR, p < 0.05. (C) The effects of ischemia/reperfusion (I/R) and treatment with Naringin on the SOD levels. *Between the control and I/R and I/R + NAR, p < 0.01, **Between the I/R and I/R + NAR, p < 0.01. (D) The effects of ischemia/reperfusion (I/R) and treatment with Naringin eon the CAT levels. *Between the control and I/R, I/R + NAR, p < 0.05, **Between the I/R and I/R + NAR, p < 0.05.
Figure 2. (A) Creatine kinase levels. *Between the control and I/R, I/R + NAR, p < 0.01, **Between the I/R and I/R + NAR, p < 0.05. (B) Lactate dehydrogenase (LDH) as indicators of muscle damage in blood levels of all groups. (C) Leukocyte infiltration of the all groups (the average leukocyte counts/0.25 mm2). *Between the control and I/R, p < 0.05, **Between the I/R and I/R + NAR, p < 0.05.
Table 1. Comparison of the effects of ischemia/reperfusion (I/R) on the histological evaluation scores of the each group.
Discussion
I/R injury in skeletal muscle is a clinical condition, which affects the morbidity of the patients negatively. Therefore, developing new drugs to decrease the adverse effects of this injury in skeletal muscle is important. Application of the tourniquet is a method used to facilitate the surgeon by providing a bleeding-free surgical area as much as possible during operations. However, this method has a pitfall since it may cause I/R injury. Thus, it would be useful to administer the drugs to prevent a possible I/R injury during the tourniquet application. Therefore, the possible beneficial effects of the Naringin with antioxidant activity were investigated in hindlimb I/R model in rat in this study.
As can seen in other flavonoids, Naringin has characteristics of metal chelating, antioxidant and free radical scavenging abilitiesCitation47,Citation54 and also has anti-mutagenic effectsCitation55 and lipid peroxidation. It is shown that Naringin has antioxidant effects as GSH and inhibits H2O2, which starts lipid peroxidationCitation56,Citation57. Recently, it was demonstrated that Naringin plays an important role in regulation of the antioxidant capacity by increasing the CAT and SOD activities via regulating the gene expressions of GSH-Px, SOD and CATCitation58. All of those effects might have contributed to the in vivo effect of Naringin to decrease I/R-induced oxidative damage in rat skeletal muscle in this study. It was reported higher reduction in MDA level in the Naringin group of 400 mg/kg compared with the Naringin groups of 100 and 200 mg/kg in renal I/R modelCitation59. As seen in other I/R studiesCitation60,Citation61, tissue MDA level increased in skeletal muscle due to I/R injury in our study. In our study, MDA level in I/R + NAR group was found to be significantly lower compared with I/R-only group. Evaluation of our results and previous studies has showed that Naringin has the effect attenuating lipid peroxidation by the oxidative damage. Naringin with free radical scavenging effect reduced the MDA levels by the decrease the lipid peroxidation initiated by free radical.
SOD, CAT, GSH-Px and GSH are the basic defense mechanisms against oxidative stress. Rajadural and coworkers have reported that Naringin increased SOD and CAT activitiesCitation62. Similarly, Ozyurt and coworkers have proved that caffeic acid phenethyl ester (CAPE) protect rat skeletal muscle and enhanced SOD and CAT activities against I/R-induced oxidative stressCitation63. As the lower SOD activity compared with the control group was reported in 6-h ischemia and 4-h reperfusion of hindlimb tourniquet modelCitation64. Sing et al. demonstrated that Naringin (400 mg/kg) attenuated the decrease in SOD activity and decreased the MDA level compared with I/R group in their kidney model of 45 min of ischemia and 24 h of reperfusionCitation59. In our study, CAT activity was found to be decreased in I/R group compared with the control group. On the other hand, CAT activities of I/R + NAR group were significantly (p < 0.05) higher compared with the I/R group. In our study, SOD activity was found to be decreased in I/R group compared with the control group; SOD activities of I/R + NAR group was significantly (p < 0.01) higher compared with the I/R group.
Skeletal muscle tissue GSH-Px activity increased in I/R group compared with control group in a study of 2-h ischemia by applying tourniquet and 3-h reperfusion in hindlimp of ratsCitation59. In our study, GSH-Px activity significantly decreased in I/R group compared with the control group. GSH-Px takes part in the detoxification of the H2O2. The decrease in GSH-Px activity may be due to increased free radical formation in I/R group. This decrease in GSH-Px activity was attenuated in I/R + NAR group. In our study, skeletal muscle SOD activity was found to be decreased in I/R group compared with the control group.
In this study, significant increases in the activities of SOD, GSH-Px and CAT were determined in I/R + NAR group compared with the I/R only group. The increases in these antioxidant enzymes in Naringin-supplemented I/R group may be due to the facilitating effect of Naringin in the stimulation of the expression of the antioxidant enzymes due to increased formation of ROS during reperfusion period after ischemia. Thus, similar to the Ozyurt et al.’s findings, these compensatory increases in the antioxidant enzyme activities in Naringin-supplemented group would have decreased the oxidative stress due to I/R injuryCitation63.
LDH (E.C.1.1.1.27) catalyses the interconversion of pyruvate and lactate with concomitant interconversion of NADH and NAD+.
Ozaki et al. have shown that serum LDH activity increases after hepatic reperfusionCitation65. Kuzon et al. have demonstrated that the tissue lactate level was not stable during 7-h ischemia followed by 4-h reperfusion in skeletal muscleCitation66. In our study, serum lactate level seemed to be elevated after 2-h ischemia followed by 2-h reperfusion in I/R and I/R + NAR groups compared with the control group; however, these increases were not statistically significant. On the other hand, Prem et al. have reported increased blood CK activity in their study in which they applied 2-h ischemia followed by 30 min of reperfusion in rat model of I/R study in skeletal musclesCitation67. CK (E.C. 2.7.3.2) catalyses the conversion of creatine and utilizes adenosine triphosphate (ATP) to create phosphocreatine and adenosine diphosphate (ADP). Also, it is a marker in blood that reflects muscle damage anywhere in the body. Clinically, CK is assayed in blood tests as a marker of damage of CK-rich tissue such as in myocardial infarction, muscular dystrophy, autoimmune myositis and in acute renal failure.
In our study, serum CK activity of the I/R group increased dramatically compared with the control group. The dramatic increase in serum CK activity in I/R group was attenuated in I/R + NAR group.
The histological examination of the skeletal muscles of the control group rats was normal. The order of the collagen fibbers was deteriorated in epimysium, and there was an apparent increase in connective tissue cells among collagen fibbers in I/R group. In addition, there was an intensive leukocyte migration from the capillaries to the epimysium in I/R group. Histological appearance was similar to I/R group in I/R + NAR group. However, leukocyte infiltration in skeletal muscle increased in I/R group compared with the control group (), and this increase was attenuated in I/R + NAR group. It has been shown with our results that Naringin has beneficial effects.
Conclusion
To conclude, the results of this study showed that I/R causes skeletal muscle damage by increasing oxidative stress and Naringin attenuates this I/R injury probably because of its antioxidant capacity scavenging free radicals. In large scale it seems that further studies should be carried out in order to use Naringin in the treatment of I/R injury in clinical conditions.
Declaration of interest
The authors report no conflicts of interest.
This study was produced from the doctoral thesis. Also, this study was supported by Research Fund of Atatürk University (2011/281).
Acknowledgements
The authors are grateful to the Research Fund of Atatürk University for financial support.
References
- Avci G, Kadioglu H, Sehirli AO, et al. Curcumin protects against ischemia/reperfusion injury in rat skeletal muscle. J Surg Res 2012;172:39–46.
- Beyersdorf F, Unger A, Wildhirt A, et al. Studies of reperfusion injury in skeletal muscle: preserved cellular viability after extended periods of warm ischemia. J Cardiovasc Surg 1991;32:664–76.
- Bushell AJ, Klenerman L, Taylor S, et al. Ischaemic preconditioning of skeletal muscle. 1. Protection against the structural changes induced by ischaemia/reperfusion injury. J Bone Joint Surg Br 2002;84:1184–8.
- Gulcin I. Antioxidant and antiradical activities of l-carnitine. Life Sci 2006;78:803–11.
- 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.
- 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.
- Öztaşkın N, Çetinkaya Y, Taslimi P, et al. Antioxidant and acetylcholinesterase inhibition properties of novel bromophenol derivatives. Bioorg Chem 2015;60:49–57.
- 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.
- Arabaci B, Gülçin İ, Alwasel S. Capsaicin: a potent inhibitor of carbonic anhydrase isoenzymes. Molecules 2014;19:10103–14.
- 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, Beydemir S. Phenolic compounds as antioxidants: carbonic anhydrase isoenzymes inhibitors. Mini Rev Med Chem 2013;13:408–30.
- 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.
- 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 (Weinheim) 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.
- 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.
- 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.
- Göçer H, Gülçin İ. Caffeic acid phenethyl ester (CAPE): correlation of structure and antioxidant properties. Int J Food Sci Nutr 2011;62:821–5.
- 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 I, Buyukokuroglu ME, Oktay M, Kufrevioglu OI. On the in vitro antioxidative properties of melatonin. J Pineal Res 2002;33:167–71.
- Lai LS, Chou ST, Chao WW. Studies on the antioxidative activities of hsian-tsao (Mesona procumbens Hemsl) leaf gum. J Agric Food Chem 2001;49:963–8.
- Gülçin İ, Mshvildadze V, Gepdiremen A, Elias R. Screening of antiradical and antioxidant activity of monodesmosides and crude extract from Leontice smirnowii tuber. Phytomedicine 2006;13:343–51.
- Gulcin İ. Antioxidant activity of caffeic acid (3,4-dihydroxycinnamic acid). Toxicology 2006;217:213–20.
- Kalın P, Gülçin İ, Gören AC. Antioxidant activity and polyphenol content of Vaccinium macrocarpon. Rec Nat Prod 2015;9:496–502.
- Gulcin I, Sat IG, Beydemir S, Kufrevioglu OI. Evaluation of the in vitro antioxidant properties of broccoli extracts (Brassica oleracea L.). Ital J Food Sci 2004;16:17–30.
- Cetin C, Kose AA, Aral E, et al. Protective effect of Fucoidin (a neutrophil rolling inhibitor) on ischemia reperfusion injury: experimental study in rat epigastric island flaps. Annal Plast Surg 2001;47:540–6.
- 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.
- Işık M, Korkmaz M, Bursal E, et al. Determination of antioxidant properties of Gypsophila bitlisensis. Int J Pharmacol 2015;11:366–71.
- Dhalla NS, Elmoselhi AB, Hata T, Makino N. Status of myocardial antioxidants in ischemia-reperfusion injury. Cardiovasc Res 2000;47:446–56.
- 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.
- 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.
- Şerbetçi Tohma H, Gülçin İ. Antioxidant and radical scavenging activity of aerial parts and roots of Turkish liquorice (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.
- Gülçin İ, Elias R, Gepdiremen A, et al. Antioxidant activity of bisbenzylisoquinoline alkaloids from Stephania rotunda: cepharanthine and fangchinoline. J Enzyme Inhib Med Chem 2010;25:44–53.
- Büyükokuroğlu ME, Gülçin İ. In vitro antioxidant and antiradical properties of Hippophae rhamnoides. L. Pharmacog Mag 2009;4:189–95.
- Talaz O, Gülçin İ, Göksu S, Saracoglu N. Antioxidant activity of 5,10-dihydroindeno[1,2-b]indoles containing substituents on dihydroindeno part. Bioorg Med Chem 2009;17:6583–9.
- Gülçin İ. Antioxidant activity of l-adrenaline: a structure-activity insight. Chem Biol Interact 2009;179:71–80.
- Gülçin İ, Elias R, Gepdiremen A, et al. Antioxidant secoiridoids from fringe tree (Chionanthus virginicus L.). Wood Sci Technol 2009;43:195–212.
- Ak T, Gülçin İ. Antioxidant and radical scavenging properties of curcumin. Chem Biol Interact 2008;174:27–37.
- Chen YT, Zheng RL, Jia ZJ, Ju Y. Flavonoids as superoxide scavengers and antioxidants. Free Radic Biol Med 1990;9:19–21.
- Ameer B, Weintraub RA, Johnson JV, et al. Flavanone absorption after Naringin, hesperidin, and citrus administration. Clin Pharmacol Ther 1996;60:34–40.
- Motilva V, Alarcon de la Lastra C, Martin MJ. Ulcer-protecting effects of naringenin on gastric lesions induced by ethanol in rat: role of endogenous prostaglandins. J Pharm Pharmacol 1994;46:91–4.
- Rojas D, Sanchez VR, Somoza B, et al. Vasodilatory effect of naringenin in rat aorta. Phytother Res 1996;10:123–5.
- So FV, Guthrie N, Chambers AF, et al. Inhibition of human breast cancer cell proliferation and delay of mammary tumorigenesis by flavonoids and citrus juices. Nutr Cancer 1996;26:167–81.
- Aebi H. Catalase. Methods Enzym Anal 1974;10:673–7.
- Sun Y, Oberley LW, Li Y. A simple method for clinical assay of superoxide dismutase. Clin Chem 1988;34:497–500.
- Paglia DE, Valentine WN. Studies on the quantitative and qualitative characterization of erythrocyte glutathione peroxidase. J Lab Clin Med 1967;70:158–69.
- Esterbauer H, Cheeseman KH. Determination of aldehydic lipid peroxidation products: malonaldehyde and 4-hydroxynonenal. Meth Enzymol 1990;186:407–21.
- Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. Protein measurement with the Folin phenol reagent. J Biol Chem 1951;193:265–75.
- Bolcal C, Yildirim V, Doganci S, et al. Protective effects of antioxidant medications on limb ischemia reperfusion injury. J Surg Res 2007;139:274–9.
- Gulcin I. Antioxidant properties of resveratrol: a structure-activity insight. Innov Food Sci Emerg 2010;11:210–18.
- Elmastaş M, Gulcin I, Beydemir S, et al. A study on the in vitro antioxidant activity of juniper (Juniperus communis L.) seeds extracts. Anal Lett 2006;39:47–65.
- Gülçin İ, Alici HA, Cesur M. Determination of in vitro antioxidant and radical scavenging activities of propofol. Chem Pharm Bull 2005;53:281–5.
- Gulcin I, Berashvili D, Gepdiremen A. Antiradical and antioxidant activity of total anthocyanins from Perilla pankinensis decne. J Ethnopharmacol 2005;101:287–93.
- Singh D, Chander V, Chopra K. Protective effect of Naringin, a bioflavonoid on glycerol-induced acute renal failure in rat kidney. Toxicology 2004;201:143–51.
- Koksal E, Gulcin I, Beyza S, et al. In vitro antioxidant activity of silymarin. J Enzyme Inhib Med Chem 2009;24:395–405.
- Granger DN. Role of xanthine oxidase and granulocytes in ischemia-reperfusion injury. Am J Physiol 1998;255:1269–75.
- Rajadurai M, Stanely Mainzen Prince P. Preventive effect of Naringin on lipid peroxides and antioxidants in isoproterenol-induced cardiotoxicity in Wistar rats: biochemical and histopathological evidences. Toxicology 2006;228:259–68.
- Ozyurt H, Ozyurt B, Koca K, Ozgocmen S. Caffeic acid phenethyl ester (CAPE) protects rat skeletal muscle against ischemia-reperfusion-induced oxidative stress. Vascul Pharmacol 2007;47:108–12.
- Petrasek PF, Homer-Vanniasinkam S, Walker PM. Determinants of ischemic injury to skeletal muscle. J Vasc Surg 1994;19:623–31.
- Ozaki M, Nakamura M, Teraok S, Ota K. Ebselen a novel antioxidant compound, protects the rat liver from ischemia-reperfusion injury. Transplant Int 1997;10:96–102.
- Kuzon WM Jr., Walker PM, Mickle DA, et al. An isolated skeletal muscle model suitable for acute ischemia studies. J Surg Res 1986;41:24–32.
- Prem JT, Eppinger M, Lemmon G, et al. The role of glutamine in skeletal muscle ischemia/reperfusion injury in the rat hind limb model. Am J Surg 1999;178:147–50.