Pharmacological Research 49 (2004) 5966

In vitro antioxidant properties of morphine

Ilhami Gln a, , Skr Beydemr a , H. Ahmet Alici b , Mahfuz Elmasta c , s M. Emin Bykokuro lu d g
b

Department of Chemistry, Science and Arts Faculty, Atatrk University, 25240 Erzurum, Turkey Department of Anaesthesiology and Reanimation, Medical Faculty, Atatrk University, 25240 Erzurum, Turkey c Department of Chemistry, Science and Arts Faculty, Gaziosmanpaa University, 64240 Erzurum, Turkey s d Department of Pharmacology, Medical Faculty, Atatrk University, 25240 Erzurum, Turkey Accepted 31 July 2003

Abstract Morphine is implicated in diverse functions, from development to immune modulation in the central and peripheral nervous systems. It has also been used extensively in the clinical management of pain due to its potent analgesic effect. This study was designed to evaluate the in vitro antioxidant capacity of morphine using different antioxidant tests, including total antioxidant activity, reducing power, free radical scavenging, superoxide anion radical scavenging, hydrogen peroxide scavenging and metal chelating activities. Morphine exhibited strong total antioxidant activity. The concentrations of 25, 50 and 75 g ml1 of morphine showed 79.1, 84.3 and 92.3% inhibition on peroxidation of linoleic acid emulsion, respectively. On the other hand, at 75 g ml1 concentration of standard antioxidant, such as butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT) and -tocopherol, exhibited 88.7, 94.5 and 70.4% inhibition on peroxidation of linoleic acid emulsion, respectively. In addition, morphine had effective reducing power, free radical scavenging, superoxide anion radical scavenging, hydrogen peroxide scavenging and metal chelating activities at the same concentrations (25, 50 and 75 g ml1 ). These various antioxidant activities were compared to standard antioxidants such as BHA, BHT and -tocopherol. 2003 Elsevier Ltd. All rights reserved.
Keywords: Antioxidant activity; Morphine; In vitro

1. Introduction Exogenous chemical and endogenous metabolic processes in the human body or in food system might produce highly reactive oxygen species (ROS), which are capable of oxidizing biomolecules, resulting in tissue damage and cell death. ROS include a number of chemically reactive molecules derived from oxygen such as hydrogen peroxide (H2 O2 ), superoxide (O2 ), and hydroxyl radical (OH ). ROS are formed and degraded by all aerobic organisms and can readily react with most biomolecules including proteins lipids and lipoproteins and DNA [1]. Oxidative damage plays a signicantly pathological role in human diseases such as arthritis, atherosclerosis, cirrhosis, emphysema and cancer. Also, excessive generation of ROS induced by various stimuli and which exceed the antioxidant capacity of the organism leads to a variety of pathophysiological processes
author. Tel.: +90-442-2314444; fax: +90-442-2360948. E-mail address: igulcin@atauni.edu.tr (I. Gln). 1043-6618/$ see front matter 2003 Elsevier Ltd. All rights reserved. doi:10.1016/j.phrs.2003.07.012
Corresponding

such as inammation, diabetes, genotoxicity and cancer [2]. Antioxidants are reported to prevent oxidative damage by free radical and ROS, and may prevent the occurrence of disease, cancer and aging. When the mechanism of antioxidant protection becomes unbalanced by exogenous factors such as tobacco smoke, ionising radiation, certain pollutants, organic solvents and pesticides, and endogenous factors such as normal aerobic respiration, stimulated polymorphonuclear leukocytes and macrophages, and peroxisomes may occur, resulting in above-mentioned diseases and accelerating aging. Antioxidants can interfere with the oxidation process by reacting with free radicals, chelating, catalytic metals, and also by acting as oxygen scavengers [3,4]. However, antioxidant supplements or foods containing antioxidants may be used to help the human body reduce oxidative damage [5]. The most commonly used antioxidants at the present time are BHA, BHT, propyl gallate and tert-butylhydroquinone [6,7]. However, they have been suspected of being responsible for liver damage and carcinogenesis in laboratory animals. Therefore, the development and utilization of more effective antioxidants are desired [2,8].

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Morphine is implicated in diverse functions, from development to immune modulation in the central and peripheral nervous system. Also, it was investigated that the effect of morphine on the release of tumour necrosis factor-alpha from murine neonatal microglia. Microglial cell cultures did not release tumour necrosis factor-alpha when incubated with morphine alone [9]. Morphine has been used extensively in the clinical management of pain due to their potent analgesic effect. Moreover, morphine has protective effects in peroxynitriteinduced apoptosis of primary rat neonatal astrocytes [10]. Also, the effects of morphine produce analgesia, euphoria, sedation and a decreased ability to concentrate. The cause of pain persists, but even small doses of morphine increase the threshold to pain and modify the perception of anxious stimulation such that it is no longer experienced as pain [11]. In addition, the effect of morphine (106 M) on the oxidant and antioxidant activity in cells of the immunophagocytic system was studied as in vitro. Morphine caused different changes in production of active forms of oxygen and antioxidant activity of the enzymes such as superoxide dismutase and gluthatione reductase in neutrophils, monocytes and lymphocytes in human peripheral blood [12]. Furthermore, morphine had acute decreases in cerebrospinal uid gluthatione levels after intracerebroventricular for cancer pain [13]. Therefore, the objectives of this study were to investigate the total antioxidant activity, reducing power, free radical scavenging, superoxide anion radical scavenging, hydrogen peroxide scavenging, and metal chelating activities of morphine. An important goal of this research was in vitro antioxidative effects of morphine were compared with same dose of commercial and standard antioxidants such as -tocopherol, BHA and BHT, commonly used by the pharmaceutical industry.

of morphine was dissolved in 10 ml water. 25, 50 and 75 g ml1 of morphine or standards samples in 2.5 ml of potassium phosphate buffer (0.04 M, pH 7.0) was added to linoleic acid emulsion (2.5 ml) in potassium phosphate buffer (0.04 M, pH 7.0). Five millilitres linoleic acid emulsion consists of 17.5 g Tween-20, 15.5 l linoleic acid, and 0.04 M potassium phosphate buffer (pH 7.0). On the other hand, 5.0 ml control consists of 2.5 ml linoleic acid emulsion and 2.5 ml potassium phosphate buffer (0.04 M, pH 7.0). The mixed solution was incubated at 37 C in a glass ask and in the dark. After the mixture was stirred for 3 min, the peroxide value was determined by reading the absorbance at 500 nm in a spectrophotometer (8500 II, Bio-Crom GmbH, Zurich, Switzerland), after reaction with FeCl2 and thiocyanate at intervals during incubation. During the linoleic acid oxidation, peroxides formed. These compounds oxidize Fe2+ to Fe3+ . The latter Fe3+ ions form complex with SCN , which had maximum absorbance at 500 nm. Therefore, high absorbance indicates high linoleic acid oxidation. The solutions without morphine or standards were used as blank samples. All data about total antioxidant activity are the average of duplicate analyses. The inhibition of lipid peroxidation in percentage was calculated by following equation: Inhibition (%) = A0 A1 A0 100

where A0 was the absorbance of the control reaction and A1 was the absorbance in the presence of the sample of morphine. 2.3. Reducing power The reducing power of morphine was determined according to the method of Oyaizu [15]. The different concentrations of morphine (25, 50 and 75 g ml1 ) in 1 ml of distilled water was mixed with phosphate buffer (2.5 ml, 0.2 M, pH 6.6) and potassium ferricyanide [K3 Fe(CN)6 ] (2.5 ml, 1%). The mixture was incubated at 50 C for 20 min. A portion (2.5 ml) of TCA (10%) was added to the mixture, which was then centrifuged for 10 min at 1000 g (MSE Mistral 2000, UK). The upper layer of solution (2.5 ml) was mixed with distilled water (2.5 ml) and FeCl3 (0.5 ml, 0.1%), and the absorbance was measured at 700 nm in a spectrophotometer (8500 II, Bio-Crom GmbH). Higher absorbance of the reaction mixture indicated greater reducing power. 2.4. Superoxide anion scavenging activity Measurement of superoxide anion scavenging activity of morphine was based on the method described by Liu et al. [16] with slight modication [8]. Superoxide radical is generated in PMSNADH systems by oxidation of NADH and assayed by the reduction of nitroblue tetrazolium (NBT). In this experiment, the superoxide radical was generated in 3 ml of TrisHCl buffer (16 mM, pH 8.0) containing 1 ml of

2. Materials and methods 2.1. Chemicals Polyoxyethylenesorbitan monolaurate (Tween-20), -tocopherol, 1,1-diphenyl-2-picryl-hydrazyl (DPPH), 3(2-pyridyl)-5,6-bis(4-phenyl-sulfonic acid)-1,2,4-triazine (ferrozine), nicotinamide adenine dinucleotide (NADH), BHA, BHT and trichloracetic acid (TCA) were purchased from Sigma (Sigma-Aldrich GmbH, Sternheim, Germany). Ammonium thiocyanate was purchased from Merck. Morphine was obtained from Department of Pharmacology, Atatrk University. All other chemicals were analytical grade and obtained from either Sigma or Merck. 2.2. Total antioxidant activity determination The antioxidant activity of morphine was determined according to the thiocyanate method [14]. Ten milligrams

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NBT (50 M), 1 ml NADH (78 M) and sample solution of morphine (from 25 to 75 g ml1 ) in water were mixed. The reaction was started adding 1 ml of phenazine methosulphate (10 M) (PMS) to the mixture. The reaction mixture was incubated at 25 C for 5 min, and the absorbance at 560 nm in a spectrophotometer (8500 II, Bio-Crom GmbH) was measured against blank samples. l-Ascorbic acid was used as a control. Decrease in absorbance of the reaction mixture indicated increased superoxide anion scavenging activity. The percentage inhibition of superoxide anion generation was calculated using the following formula: Inhibition (%) = A0 A 1 A0 100

where A0 was the absorbance of the control, and A1 was the absorbance in the presence of the sample of morphine and standards. The control did not contain FeCl2 and ferrozine, complex formation molecules [19]. 2.7. Scavenging of hydrogen peroxide The ability of the morphine to scavenge H2 O2 was determined according to the method of Ruch et al. [20]. A solution of H2 O2 (4 mM) was prepared in phosphate buffer (pH 7.4). H2 O2 concentration was determined spectrophotometrically from absorption at 230 nm in a spectrophotometer (8500 II, Bio-Crom GmbH). Different concentration of morphine (2575 g ml1 ) in distilled water was added to a H2 O2 solution (0.6 ml, 40 mM). Absorbance of H2 O2 at 230 nm was determined after 10 min against a blank solution containing in phosphate buffer without H2 O2 . The percentage of scavenging of H2 O2 of morphine and standard compounds: %Scavenged [H2 O2 ] = A0 A1 A0 100

where A0 was the absorbance of the control (l-ascorbic acid), and A1 was the absorbance of morphine and standards. 2.5. Free radical scavenging activity The free radical scavenging activity of morphine was measured by DPPH using the method of Shimada et al. [17]. Briey, 0.1 mM solution of DPPH in ethanol was prepared and 1 ml of this solution was added to 3 ml of morphine solution in water at different concentrations (2575 g ml1 ). The mixture was shaken vigorously and allowed to stand at room temperature for 30 min. Then the absorbance was measured at 517 nm in a spectrophotometer (8500 II, BioCrom GmbH). Lower absorbance of the reaction mixture indicated higher free radical scavenging activity. The percent DPPH scavenging effect was calculated using the following equation: DPPH scavenging effect (%) A 0 A1 100 = 100 A0 where A0 was the absorbance of the control reaction and A1 was the absorbance in the presence of the standard sample or morphine. 2.6. Metal chelating activity The chelation of ferrous ions by the morphine and standards was estimated by the method of Dinis et al. [18]. Briey, the samples (2575 g ml1 ) were added to a solution of 2 mM FeCl2 (0.05 ml). The reaction was initiated by the addition of 5 mM ferrozine (0.2 ml) and the mixture was vigorously shaken and left standing at room temperature for 10 min. After the mixture had reached equilibrium, the absorbance of the solution was then measured spectrophotometrically at 562 nm in a spectrophotometer (8500 II, Bio-Crom GmbH). All tests and analyses were run in triplicate and averaged. The percentage of inhibition of Fe2+ ferrozine complex formation was given below: %Inhibition = A0 A1 A0 100

where A0 was the absorbance of the control, and A1 was the absorbance in the presence of the sample of morphine and standards. 2.8. Statistical analysis All analyses were performed in triplicate. The data were recorded as mean S.D. and analysed by SPSS (version 9.0 for Windows 98, SPSS Inc.). One-way analysis of variance was performed by ANOVA procedures. Signicant differences between means were determined by Duncans Multiple Range tests. The data presented in percentage were arranged by the correct mathematical transformation (Arcsin) before ANOVA procedures. P values <0.05 were regarded as signicant and P values <0.01 very signicant.

3. Results and discussion 3.1. Total antioxidant activity determination in linoleic acid emulsion Numerous antioxidant methods and modications have been proposed to evaluate antioxidant activity and to explain how antioxidants function. Of these, total antioxidant activity, reducing power, DPPH assay, metal chelating, active oxygen species such as H2 O2 , O2 and OH quenching assay are most commonly used for the evaluation of an antioxidant molecule [10]. Total antioxidant activity of morphine was determined by the thiocyanate method. Morphine exhibited effective and powerful antioxidant activity at all concentrations. The effects of various concentrations of morphine (from 25 to 75 g ml1 ) on peroxidation of linoleic acid emulsion

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2.5
Control TOC-75 g/ mL MOR-25 g/mL MOR-50 g/mL MOR-75 g/mL

2 Absorbance (517 nm)

1.5

0.5

0 0 10 20 30 40 50 60 Incubation Time (Hours)

Fig. 1. Determination of antioxidant activity of different concentrations of morphine and -tocopherol in the linoleic acid emulsion was determined by the thiocyanate method. TOC: -tocopherol; MOR: morphine; BHA: butylated hydroxyanisole; BHT: butylated hydroxytoluene.

are shown in Fig. 1. The antioxidant activity of morphine was increased with an increasing concentration. The different concentrations of morphine (25, 50 and 75 g ml1 ) showed higher antioxidant activities than that of 75 g ml1 -tocopherol (70.4%). The percentage inhibition of peroxidation of 25, 50 and 75 g ml1 concentrations of morphine in linoleic acid system was 79.1, 84.3 and 92.3%, respectively. On the other hand, percentage inhibition of 75 g ml1 concentrations of BHA, BHT, and -tocopherol was found as 88.7, 94.5 and 70.4%, respectively. 3.2. Reducing power Fig. 2 shows the reductive capability of morphine compared to BHA, BHT and -tocopherol. For the measurements of the reductive ability, we investigated the

Fe3+ Fe2+ transformation in the presence of morphine samples using the method of Oyaizu [15]. The reducing capacity of a compound may serve as a signicant indicator of its potential antioxidant activity. The antioxidant activity of an antioxidant compound have been attributed to various mechanisms, among which are prevention of chain initiation, binding of transition metal ion catalysts, decomposition of peroxides, prevention of continued hydrogen abstraction, reductive capacity and radical scavenging. Like total antioxidant activity, the reducing power of morphine was increased with increasing concentration. At the all concentrations, morphine showed higher activities than BHT and -tocopherol and these differences were statistically signicant (P < 0.05). Although, reductive capability of morphine was lower than BHA, difference was statistically insignicant (P > 0.05). Reducing power

3 Absorbance (700 nm)

TOC

BHA BHT MOR

0 0 10 20 30 40 50 Concentration (g/mL) 60 70

Fig. 2. Reducing power of different concentrations of morphine, BHA, BHT and -tocopherol by spectrophotometric detection of the Fe3+ Fe2+ transformation. TOC: -tocopherol; MOR: morphine; BHA: butylated hydroxyanisole; BHT: butylated hydroxytoluene.

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Fig. 3. Comparison of superoxide anion radical scavenging activity of morphine, BHA, BHT and -tocopherol by the PMSNADHNBT method at the same concentration (25 g ml1 ). TOC: -tocopherol; MOR: morphine; BHA: butylated hydroxyanisole; BHT: butylated hydroxytoluene.

of morphine and standard compounds followed the order: BHA > morphine > BHT > -tocopherol. 3.3. Superoxide anion scavenging activity Superoxide is produced endogenously by avoenzymes, e.g. xanthine oxidase activated in ischemia-reperfusion. During experimental renal ischemia-reperfusion in rabbits, morphine signicantly inhibited the generation of superoxide anion radicals (O2 ) by resting and opsonized zymosanstimulated phagocytes in renal venous blood [21]. Opioid peptides rapidly stimulate superoxide production by human polymorphonuclear leukocytes and macrophages [22]. Morphine had a particularly reducing effect on polymorphonuclear superoxide anion production [23]. In PMSNADHNBT system, superoxide anion derived from dissolved oxygen by PMSNADH coupling reaction reduces NBT. The decrease of absorbance at 560 nm with antioxidants indicates the consumption of superoxide anion in the reaction mixture. Fig. 3 shows the percentage inhibition of superoxide radical generation by 25 g ml1 morphine and comparison with the same concentration of BHA, BHT and -tocopherol. Morphine had strong superoxide radical scavenging activity and exhibited higher superoxide radical scavenging activity than BHA, but lower than BHT and -tocopherol (P < 0.05). The percentage inhibition of superoxide generation by 25 g ml1 morphine was found as 74.0%. This ratio at this concentration for BHA, BHT and -tocopherol was found as 69.6, 82.2 and 75.4% inhibition of superoxide radical generation, respectively. Superoxide radical scavenging activity of those samples followed the order: BHT > -tocopherol > morphine > BHA. 3.4. Free radical scavenging activity The model of scavenging the stable DPPH radical is a widely used method to evaluate antioxidant activities in a relatively short time compared with other methods. The effect

of antioxidants on DPPH radical scavenging was thought to be due to their hydrogen donating ability. DPPH is a stable free radical and accepts an electron or hydrogen radical to become a stable diamagnetic molecule [24]. Morphine is chemically similar to phenol-based free radical scavengers such as BHA, BHT and -tocopherol. The reduction capability of DPPH radicals was determined by the decrease in its absorbance at 517 nm induced by antioxidants. It is visually noticeable as a discolouration from purple to yellow. Hence, DPPH is usually used as a substrate to evaluate antioxidative activity of antioxidants. Fig. 4 illustrates a signicant decrease (P < 0.01) in the concentration of DPPH radical due to the scavenging ability of the morphine and standards. We used BHA, BHT and -tocopherol as standard radical scavengers. The scavenging effect of morphine and standards on the DPPH radical decreased in the order of BHA > -tocopherol > BHT > morphine and were 79, 78, 76 and 66% at the concentration of 75 g ml1 , respectively. These results indicated that morphine have a noticeable effect on scavenging free radicals. Free radical scavenging activity was also increased with an increasing concentration. It was reported that oxidative stress, which occurs when free radical formation exceeds the bodys ability to protect itself, forms the biological basis of chronic condition [25]. Based on the data obtained from this study, morphine is a powerful free radical inhibitor or scavenger, as well as a primary antioxidant that reacts with free radicals, which may limit free radical damage occurring in the human body.

3.5. Metal chelating activity The chelating of ferrous ions by morphine was estimated by the method of Dinis et al. [18]. Ferrozine can quantitatively form complexes with Fe2+ . In the presence of chelating agents, the complex formation is disrupted with the result that the red colour of the complex is decreased. Measurement of colour reduction, therefore, allows estimation

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100
DPPH Scavenging Effects (%)

75
TOC BHA

50

BHT MOR

25

0 0 25 50 75 Concentration (g/mL)
Fig. 4. Free radical scavenging activity of different concentrations of morphine, BHA, BHT and -tocopherol by 1,1-diphenyl-2-picrylhydrazyl radicals. TOC: -tocopherol; MOR: morphine; BHA: butylated hydroxyanisole; BHT: butylated hydroxytoluene.
100

Metal Chelating Effects (%)

75 TOC BHA 50 BHT MOR 25

0 0 25 50 Concentration (g/mL) 75

Fig. 5. Metal chelation effect of different concentrations of morphine, BHA, BHT and -tocopherol on ferrous ions. TOC: -tocopherol; MOR: morphine; BHA: butylated hydroxyanisole; BHT: butylated hydroxytoluene.

Fig. 6. Hydrogen peroxide scavenging activity of 25 g ml1 of morphine, BHA, BHT and butylated hydroxyanisole; BHT: butylated hydroxytoluene.

-tocopherol. TOC:

-tocopherol; MOR: morphine; BHA:

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of the chelating activity of the coexisting chelator. In this assay morphine and standard antioxidant compounds interfered with the formation of ferrous and ferrozine complex, suggesting that they have chelating activity and capture ferrous ion before ferrozine. Iron can stimulate lipid peroxidation by Fenton reaction, and also accelerates peroxidation by decomposing lipid hydroperoxides into peroxyl and alkoxyl radicals that can themselves abstract hydrogen and perpetuate the chain reaction of lipid peroxidation [19,26]. As shown in Fig. 5, the formation of the Fe2+ ferrozine complex is not complete in the presence of morphine, indicating that morphine chelates iron. The absorbance of Fe2+ ferrozine complex was linearly decreased in a dosedependent manner (from 25 to 75 g ml1 ). The difference between morphine and the control was statistically signicant (P < 0.01). The percentage of metal chelating capacity of 75 g ml1 morphine, -tocopherol, BHA, and BHT were found as 73, 75, 69 and 66%, respectively. The metal scavenging effect of morphine and standards decreased in the order of -tocopherol > morphine > BHA > BHT. Metal chelating capacity is signicant since it reduced the concentration of the catalysing transition metal in lipid peroxidation. It was reported that chelating agents, which form -bonds with a metal, are effective as secondary antioxidants because they reduce the redox potential thereby stabilizing the oxidized form of the metal ion. The data obtained from Fig. 6 reveal that morphine demonstrates a marked capacity for iron binding, suggesting that their action as peroxidation protector may be related to its iron binding capacity. 3.6. Scavenging of hydrogen peroxides The ability of morphine to scavenge H2 O2 was determined according to the method of Ruch et al. [20]. Fig. 6 shows the percentage H2 O2 scavenging effect by 25 g ml1 of morphine and comparison with the same doses of BHA, BHT, and -tocopherol. Morphine had strong H2 O2 scavenging activity when compared to control (P < 0.01). The percentage H2 O2 scavenging effect by the same concentration (25 g ml1 ) of morphine, BHA, BHT and -tocopherol was found as 25, 88, 87 and 93%, respectively. H2 O2 scavenging activity of those samples followed the order: -tocopherol > BHA > BHT > morphine. H2 O2 is highly important because of its ability of penetrate biological membranes. H2 O2 itself is not very reactive, but it can sometimes be toxic to cell because it may give rise to hydroxyl radical in the cells. Thus, removing H2 O2 is very important for the protection of living systems.

to its antioxidant activity [7]. On the basis of the results of this study, it clearly indicates that morphine had powerful in vitro antioxidant capacity against various antioxidant systems in vitro. From our results, the antioxidant activity of morphine was concentration dependent, with stronger inhibition of lipid peroxidation. From the above assays, the possible mechanism of antioxidant activity of morphine includes reductive ability, metal chelator, hydrogen donating ability and scavengers of hydrogen peroxide, superoxide and free radicals.

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