Free radical scavenging activity of the marine mangrove Rhizophora apiculata bark extract with reference to naphthalene induced mitochondrial dysfunction

Chemico-Biological Interactions 163 (2006) 170–175

Free radical scavenging activity of the marine mangrove Rhizophora apiculata bark extract with reference to naphthalene induced mitochondrial dysfunction K. Vijayavel ∗ , C. Anbuselvam, M.P. Balasubramanian Department of Pharmacology and Environmental Toxicology, Dr. ALM Post Graduate Institute of Basic Medical Sciences, University of Madras, Taramani, Chennai 600 113, Tamil Nadu, India Available online 23 June 2006

Abstract Rhizophora apiculata bark extract was tested for its free radical scavenging activity and protective role against mitochondrial dysfunction in naphthalene stressed rats. Lipid peroxidation activity was increased and activity of mitochondrial enzymes (cytochrome-c-oxidase, NADH-dehydrogenase, ␣-ketoglutarate dehydrogenase and succinate dehydrogenase) and glutathione was decreased in the liver and kidney of rats intoxicated with naphthalene when compared to control rats. Intraperitoneal administration of plant extract significantly reduced the lipid peroxidation, increased the activity of mitochondrial enzymes and increased glutathione to near control levels. These results suggest that the sulfated polysaccharides in R. apiculata play a protective role through their free radical scavenging properties. © 2006 Elsevier Ireland Ltd. All rights reserved. Keywords: Rhizophora apiculata; Naphthalene; Oxidative stress; Mitochondrial enzymes

1. Introduction Naphthalene is a major byproduct of petroleum distillation and fractionation. It is principally used as an intermediate in the production of phthalic anhydride, tanning agents, carbaryl, moth repellent, deodorizer, etc. Naphthalene undergoes extensive microsomal metabolism to form 1,2-dihydro-1, 2-dihydroxynaphthalene [1]. These metabolites have the potential to generate free radicals responsible for depleting tissue antioxidants and inducing lipid peroxidation (LPO) [2]. Toxicity is frequently mediated by the toxic metabolites that directly affect the biochemical pathways of the cell, which can result in ∗

Corresponding author at: Pacific Biosciences Research Center, Kewalo Marine Laboratory, 41 Ahui Street, Honolulu, HI 96813, United States. E-mail address: [email protected] (K. Vijayavel).

oxidative stress. Oxidative stress can consequently result in altered enzyme activity in the mitochondria, leading to mitochondrial dysfunction. Mitochondria are the powerhouses of the cell that provide energy for metabolism and maintenance of calcium homeostasis within the cell. Alterations in mitochondrial function may adversely affect the survival of an organism [3]. Reactive oxygen radicals play a crucial role in the initiation of LPO, the complex process in which polyunsaturated fatty acids in the mitochondrial membranes undergo changes by a chain of reactions to form lipid hydroperoxides, which decompose double bonds of unsaturated fatty acids and thereby destructing membrane lipids [4]. During the lipid peroxidation process, the activity of different mitochondrial membrane-bound enzymes are changed which ultimately leads to the changes in membrane permeability [5]. Reactive oxygen species (ROS) may lead to irreversible damage to

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mitochondrial enzymes, membrane lipids and proteins resulting in mitochondrial dysfunction. Mitochondrial susceptibility to oxidative stress is suggested by a decline in the activity of their enzyme systems. The resultant mitochondrial decay may eventually cause inadequate energy production and the loss of cell homeostasis. Such changes could result in unwarranted decline in energy production [6]. Sulfated polysaccharides (SPS) comprise a complex group of macromolecules with a wide range of biological properties. These anionic polymers are widespread in nature occurring in a various organisms. The Rhizophora sps of mangroves possess various pharmacological and biomedicinal properties such as antiviral R. apiculata [7]; antioxidants, R. mangle [8]; anti-ulcerogenic, R. mangle [9] and antibacterial, R. mangle [10]. SPS are used as a therapy for many diseases associated with impaired energy metabolism and their dietary supplementation could confer potent antioxidant defense and ameliorate oxidative stress [11]. The critical role of antioxidants in preventing oxidative stress induced by naphthalene toxicity is well documented [12,2,13] nevertheless; there is paucity of information for the use of sulfated polysaccharides as effective antioxidants. Hence, we are interested in investigating the protective role of Rhizophora apiculata bark extract in naphthalene-induced oxidative stress and mitochondrial dysfunction in rats.

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of Dodgson and Price [16]. Uronic acid estimation was carried out according to the procedures of Knutson and Jeans [17] using galacturonic acid as a reference. Crude fucoidan from the mangrove extract was measured for fucose content by the method of Doner and Whistler [18]. 2.3. Free radical scavenging activity This assay was based on the scavenging of stable 1,1-diphenyl-2-picrylhydrazyl (DPPH) radicals by the radical scavenging components in RA extract. The radical scavenging activity of bark extract was assayed by the DPPH (1,1-diphenyl-2-picrylhydrazyl) method as described by Abe et al. [19]. A 0.5 ml aliquot of 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140 and 150 mg l−1 ethanol dissolved in RA extract was mixed with 0.25 ml of ethanolic 0.5 mM DPPH solution and 0.5 ml of 100 mM acetate buffer (pH 5.5). The decrease in absorbance is due to the reduction of the DPPH radical, which was measured using a UV–vis spectrophotometer at 517 nm (UV-1200, Shimazu Co., Japan). The percentage inhibition of DPPH* was calculated based on the formula (Acontrol − Atest )/Acontrol × 100, where Acontrol is the absorbance of the control (DPPH solution without the test sample) and Atest is the absorbance of the test sample. The antioxidant activity of the test samples was expressed as the effective concentration of the extract (mg L−1 ) required for scavenging the free radicals. All tests were performed in triplicate.

2. Materials and methods 2.4. Experimental animals 2.1. Plant material and extract preparation Fresh bark of Rhizophora apiculata (RA) was collected from the Pitchavaram mangrove forest (latitude 11◦ 27 N; longitude 79◦ 47 E), Tamil Nadu, India. Barks were washed in distilled water, shadow dried and coarsely powdered. The dried bark powder was packed in a permeable cellulose thimble and subjected to continuous Soxhlet extraction with ethanol. The resulting extract was concentrated to a dry residue using a rotary evaporator and refrigerated until use. 2.2. Sulfated polysaccharide estimation The crude extract was ignited to ash at 550 ◦ C in a muffle furnace for a period of 6 h and the percentage of ash content was calculated based on the weight of dried sample. Total sugar content was calculated according to the method of Dubois et al. [14]. Protein content (nitrogen) was estimated by Kjeldahl technique [15]. Sulphate content was measured by BaCl2 -gelatin method

Male Wistar strain albino rats weighing about 120 ± 150 g was obtained from the Fredrick Institute for Plant Protection and Toxicology, Padappai, Chennai, India. The animals were maintained in a 12 h light and dark at 22 ± 3 ◦ C cycle and fed with a commercial pelleted diet (M/s. Hindustan Foods Ltd., Bangalore, India) and had free access to water. 2.5. Experimental design The experiments were conducted according to the ethical norms approved by the Ministry of Social Justices and Empowerment, Government of India and Institutional Animal Ethics Committee Guidelines. The rats were randomized into three groups with six animals each. Group I rats served as control, which received corn oil in addition to food and water. Group II rats were administered naphthalene dissolved in corn oil (435 mg/kg body weight intraperitoneally) (naphthalene was procured from Sigma Chemical Co. Ltd., St. Louis, MO,

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USA), group III rats were treated with RA bark extract in 0.9% saline (120 mg/kg body weight orally) and naphthalene in corn oil intraperitoneally (435 mg/kg body weight intraperitoneally) daily for a period of 15 days. At the end of the experiment, the rats were anesthetized with sodium pentobarbitone (35 mg/kg body weight intraperitoneally) and sacrificed by cervical decapitation and blood was collected to obtain serum. Liver was quickly excised and placed in ice-cold 0.9% NaCl solution. 2.6. Biochemical analysis Portions (100 mg) of liver and kidney tissues were homogenized in 5 ml ice-cold 0.25 M sucrose solution to isolate the mitochondria according to the method of Johnson and Lardy [20]. A 10% (w/v) homogenate was prepared in 0.25 M sucrose solution and centrifuged at 600 × g for 10 min. The supernatant fraction was decanted and centrifuged at 15,000 × g for 5 min. The resultant mitochondrial pellet was then washed and resuspended again in 0.25 M sucrose. Lipid peroxidation (LPO) was determined in the liver by the method of Ohkawa et al. [21] by following a reaction of thiobarbituric acid (TBA) with malonyldialdehyde (MDA) formed by the peroxidation of lipids. Cytochrome-coxidase activity was assayed by measuring the oxidation of reduced cytochrome-c as a decrease in absorbance at 550 nm spectrophotometrically by the method of Wharton and Tzagoloff [22]. Nicotinamide adenine dinucleotide (NADH)-dehydrogenase activity was assessed according to the method of Minakami et al. [23]. The activity of ␣-ketoglutarate dehydrogenase was assayed according to the method of Reed and Mukherjee [24] where the ferrocyanide produced by the decarboxylation of ␣-ketoglutarate with ferricyanide as electron acceptor was determined spectrophotometrically. The activity of succinate dehydrogenase was assayed by the method of Slater and Bonner [25] in which the rate of reduction of potassium ferricyanide was measured in the presence of potassium cyanide. Glutathione (GSH) was estimated in the cytosolic fraction by the method of Moron et al. [26] by reading the optical density in a spectrophotometer (412 nm) of the yellow substance formed when 5 5 -dithio-2-nitro benzoic acid (DTNB) is reduced by GSH. Mitochondrial protein content was estimated by the method of Lowry et al. [27] using bovine serum albumin as standard.

Fig. 1. The levels of total sugar, sulphate, uronic acid, protein and crude fucoidan in Rhizophora apiculata bark extract. Values are expressed in terms of mean ± S.D. for three observations.

mean values were determined by one-way analysis of variance (ANOVA). The significance is given as p < 0.05, p < 0.01 and p < 0.001. 3. Result and discussion Fig. 1 presents the percentage of total sugar, sulphate, uronic acid, protein and crude fucoidan in RA extract. The DPPH scavenging activity of RA extract is presented in Fig. 2. The minimum concentration (120 mg) of the extract that exhibited maximum free radical scavenging activity (100%) was chosen for the experimental study. The levels of LPO, mitochondrial enzymes and GSH of liver and kidney of control and experimental groups are presented in Table 1. The LPO activity increased significantly (p < 0.001) when compared to control. Mitochondrial enzymes and GSH decreased remarkably in the liver and kidney of naphthalene-exposed rats when compared to control. Administration of RA extract to the naphthalene-stressed rats reduced the LPO activity significantly (p < 0.001) and increased the mitochondrial enzymes and GSH levels to near control level. Aerobic cells are constantly exposed to reactive oxygen radical species, which induce LPO and inactivate membrane-bound enzymes [4]. During the course of microsomal metabolism, naphthalene produces hydroquinone intermediates capable of generating free

2.7. Analysis of data Values are expressed as mean ± S.D. for six rats in each group and significance of the differences between

Fig. 2. Free radical scavenging capacity of Rhizophora apiculata bark extract on DPPH (1,1-diphenyl-2-picrylhydrazyl).

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Table 1 Effect of Rhizophora apiculata bark extract on lipid peroxidation, mitochondrial enzymes and glutathione in liver and kidney of naphthalene intoxicated rats Parameters

Lipid peroxidation (nmol of MDA released per mg protein) Cytochrome-c-oxidase (O.D. × 10−2 per min per mg protein) NADH-dehydrogenase (␮mol of NADH oxidized per min per mg protein) ␣-Ketoglutarate dehydrogenase (␮mol of potassium ferrocyanide liberated per min per mg protein) Succinate dehydrogenase (␮mol of succinate oxidized per min per mg protein) Glutathione (␮g/mg protein)

Liver

Kidney

Group I

Group II

Group III

Group I

Group II

Group III

125.81 ± 1.32

193.42 ± 1.14†

188.24 ± 1.49†

125.84 ± 1.26

183.28 ± 1.05†

174.82 ± 1.28†

8.62 ± 0.52

3.82 ± 0.13†

7.92 ± 0.35#

8.35 ± 0.16

4.62 ± 0.26#

6.45 ± 0.24†

31.11 ± 1.67

14.26 ± 0.33†

28.39 ± 1.56†

41.42 ± 1.71

28.6 ± 1.25†

7.53 ± 0.18

3.16 ± 0.05†

6.14 ± 0.06†

6.25 ± 0.16

4.91 ± 0.37#

5.13 ± 0.20†

35.46 ± 1.2*

34.57 ± 1.85

23.16 ± 0.9#

30.86 ± 1.15†

25.11 ± 0.93

17.64 ± 0.91#

21.27 ± 0.02#

12.68 ± 0.88

6.29 ± 0.6#

11.36 ± 0.41†

11.42 ± 0.06

8.27 ± 0.05*

10.42 ± 0.01†

Values are expressed as mean ± S.D. for six rats in each group. Group II compared with Group I and Group III compared with Group II. * Values are statistically significant at p < 0.05. # Values are statistically significant at p < 0.01. † Values are statistically significant at p < 0.001.

radicals that induce mitochondrial oxidative stress. The LPO was found to be increased with decreasing oxidized glutathione and mitochondrial enzymes in liver and kidney of naphthalene stressed rats when compared to control. Toxicity mediated increase in the generation of free radicals in mitochondria predictably elevates the oxidative damage in the mitochondria leading to the fall in their enzyme levels. Mitochondrial membrane contains relatively large amounts of polyunsaturated fatty acids in its phospholipids, which could probably be a reason for increase in LPO activity in naphthalene stressed rats. The mitochondrial enzymes viz. cytochrome-coxidases, NADH-dehydrogenase, ␣-KDH and SDH catalyze the oxidation of a number of substrates via the citric acid cycle yielding reducing equivalents. These reducing equivalents are channeled through the mitochondrial electron transport chain, which provides energy needed for many cellular functions. The inner and outer mitochondrial membranes contain unsaturated lipids that are more susceptible to attack by oxidants [28]. The mitochondrial membrane damage leads to the inhibition of mitochondrial enzymes and this might further affect the mitochondrial substrate oxidation resulting in

reduced oxidation of substrates. This can further reduce the rate of transfer of reducing equivalents to molecular oxygen and thereby depleting energy production [6]. Cytochrome-c-oxidase, the terminal part of the mitochondrial respiratory chain requires phospholipid for its optimal activity and any change in the lipid composition of mitochondrial membrane has been reported to decrease the activity of the enzyme. The increased LPO activity in group 2 rats could explain the fall in cytochrome-c-oxidase during naphthalene intoxication. The other reason can be due to oxidative stress induced by naphthalene since it is reported that cellular stress produces mitochondrial depletion involving the coding regions of cytochrome-c-oxidase in rat liver [29]. NADH-dehydrogenase, a flavin-linked dehydrogenase plays an important role in the distribution of electrons from NADH to other coenzymes via the electron transport reaction. If the electrons stop flowing through the chain, the proton-motive force will dissipate and ATP production cannot continue. Thus, the major role of oxygen for all aerobic organisms is simply to act as a sink for electrons [30]. The decrease in the activity of NADHdehydrogenase can be ascribed due to the shortage in the formation of reducing equivalents which in turn causes

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the depletion of mitochondrial GSH accompanied by a functional loss of the activity of cytochrome-c-oxidase. Unproductive electron transport and increased superoxide production results in oxidative damage to the mitochondria, thus compromising their ability to meet cellular energy demands. Retardation in oxidative phosphorylation and ATP production in mitochondria can be well related to the consequence of xenobiotic stress which would diminish the flux of reducing equivalents and gluconeogenic intermediates across the mitochondrial barrier and thereby the consequent decrease in the activity of ␣-ketoglutarate dehydrogenase. This inhibition is mainly due to the reaction of active oxygen species with the vicinal thiol group of a protein [31]. Succinate dehydrogenase (SDH) is associated with the electron transport chain due to its ability to transfer electrons to respiratory chain [32]. SDH also contains a number of cysteine-rich sulfur clusters and can be inhibited by a number of agents that modify sulfhydryl groups. Naphthalene might have directly interacted with sulfhydryl groups on SDH resulting in the decline in the activity and thereby limiting the ability of the mitochondria to meet the energy demands of the cell and disrupting cellular energy homeostasis as suggested by Martensson et al. [33]. GSH, which is not synthesized in mitochondria is required for mitochondrial function and hence is imported from the cytosol [34]. GSH therefore plays a critical role in cell viability through the regulation of mitochondrial inner membrane permeability by maintaining sulfhydryl groups in the reduced state [5]. This suggests that the oxidative stress and GSH depletion affect the redox status in the mitochondria resulting in protein inactivation by oxidation of protein thiols. Hence, the downregulation of the above discussed mitochondrial enzyme expression in the liver and kidney of naphthalene stressed rats might be due to the reduction in the number glutathione substrate receptors involved in the mitochondrial electron transport, which is best correlated. Administration of RA extract significantly attenuated the LPO activity and resulted in the increased activity of mitochondrial enzymes in the liver and kidney of naphthalene intoxicated rats. RA extract was able to prevent the formation of mitochondrial LPO peroxidation products, which could be attributed to the antioxidative action of SPS and thereby neutralizing naphthalene toxicity. Mitochondrial GSH plays a critical role in maintaining cell viability through the regulation of mitochondrial inner membrane permeability by maintaining sulfhydryl groups in the reduced state [35]. Sulfated polysaccharides in the RA extract might have been

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