http://informahealthcare.com/phb ISSN 1388-0209 print/ISSN 1744-5116 online Editor-in-Chief: John M. Pezzuto Pharm Biol, Early Online: 1–6 ! 2014 Informa Healthcare USA, Inc. DOI: 10.3109/13880209.2014.923003
ORIGINAL ARTICLE
Evaluation of p-cymene, a natural antioxidant Talita Mendes de Oliveira1, Rusbene Bruno Fonseca de Carvalho2, Iwyson Henrique Fernandes da Costa1, Guilherme Antoˆnio Lopes de Oliveira1, Alexandre Araujo de Souza2, Sidney Gonc¸alo de Lima2, and Rivelilson Mendes de Freitas1
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Department of Biochemistry and Pharmacology, Laboratory of Research in Experimental Neurochemistry of Post-Graduation Program in Pharmaceutics Science, Federal University of Piauı´, Teresina – Piauı´, Brazil and 2Department of Chemistry, Federal University of Piauı´, Teresina – Piauı´, Brazil Abstract
Keywords
Context: Several studies have demonstrated that essential oils and their major components have antioxidant activity. p-Cymene is a monoterpene and a major constituent of essential oils of various species of plants. Objective: This paper evaluated the antioxidant potential of p-cymene in the hippocampus of mice by determining the levels of thiobarbituric acid reactive substances (TBARS), nitrite content, and activity of catalase (CAT) and superoxide dismutase (SOD). Materials and methods: Swiss mice were intraperitoneally treated with 0.05% Tween 80 dissolved in 0.9% saline solution, ascorbic acid 250 mg/kg, and p-cymene at doses of 50, 100, and 150 mg/kg, respectively. After treatment, all groups were observed for 24 h, afterwards, the groups were euthanized for removal of the brain and dissection of the hippocampus. Results: The results of treatment with p-cymene were a significant decrease in lipid peroxidation and nitrite content at a dose of CYM 50: 65.54%, CYM 100: 73.29%, CYM 150: 89.83%, and CYM 50: 71.21%; CYM 100: 68.61% and CYM 150:67%, respectively, when compared with the control group. The results showed that at all tested doses, p-cymene produces an increase in SOD and catalase activity significantly at a dose of CYM 50: 22.7%, CYM 100: 33.9%, CYM 150: 63.1%, and CYM 50: 119.25%, CYM 100: 151.83% and CYM 150: 182.70%, respectively, when compared with the vehicle-treated group. Discussion and conclusion: The result of this study shows that p-cymene has an antioxidant potential in vivo and may act as a neuroprotective agent in the brain. This compound may present a new strategy in the development of treatment for many diseases in which oxidative stress plays an important pathophysiological role.
Catalase, lipid peroxidation, monoterpene, nitrite, superoxide dismutase
Introduction Since the beginning of human civilization, plants have been used for medicinal purposes, for treatment, prevention, and curing of several diseases (Campeˆlo et al., 2011; Maciel et al., 2002; Mengues et al., 2011; Veiga Ju´nior et al., 2005). In Brazil, due to its extensive plant genetic diversity, with more than 55 000 species cataloged from a total estimated between 350 000 and 550 000 species (Dias, 1996; Rodrigues, 2008), and a privileged geographical location, there are several studies with natural substances derived from plants to treat numerous pathologies (Almeida et al., 2012; Campeˆlo et al., 2011; Freitas, 2001). Among these diseases, a special investigation is necessary on the antioxidant potential in vitro (Costa Ju´nior et al, 2011; Nogueira Neto et al., 2013;
Correspondence: Rivelilson Mendes de Freitas, Programa de Po´s-graduac¸a˜o em Cieˆncias Farmaceˆuticas/Nu´cleo de Tecnologia Farmaceˆutica do Centro de Cieˆncias da Sau´de da Universidade Federal do Piauı´, Ininga, Teresina, CEP 64 049-550 Piauı´, Brazil. Tel./fax: +55 86 3215 5870. E-mail: [email protected]
History Received 17 October 2013 Revised 7 April 2014 Accepted 07 May 2014 Published online 3 December 2014
Silva et al., 2012) and in vivo (Freitas et al., 2005), since oxidative stress may account for the pathophysiology of several diseases (Santos et al., 2011). In this context, while biological compounds are generated and called reactive oxygen species (ROS), which in excess may cause cellular damage, culminating in a process called oxidative stress. This process results in an imbalance in the state of oxidation–reduction in favor of oxidation. In this sense, we can cite the enzymes superoxide dismutase (SOD), catalase, and glutathione peroxidase which promotes detoxification of superoxide radicals, hydrogen peroxide, and lipid hydroperoxides, respectively (Costa, 2012; Wickens, 2001). Endogenous enzymes, some vitamins, and minerals participate in antioxidant defense mechanisms. Antioxidants may slow or prevent oxidative damage in human tissues produced by ROS and reactive nitrogen species (RNS). Thus, it may be defined as a diverse group of natural molecules, which may be present at low concentrations compared with biomolecules with antioxidant effect (Gutteridge & Halliwell, 2010; Halliwell, 1991; SiqueiraCatania et al., 2009).
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Figure 1. Molecular structure of p-cymene (1-methyl-4-isopropyl benzene).
Several studies show antioxidant activity of monoterpenes, a-terpineol (Bicas et al., 2011), a-thujone (Mothana et al., 2001), 1,8-cineole, a-pinene (Wannes et al., 2010), c-terpinene (Ruberto & Baratta, 2000), R-(+)-a-pinene, S-()-b-pinene, a-pinene (racemic mixture), a-terpinene, limonene, and linalool (Apel et al., 2001, 2008). Among monoterpenes, we can highlight p-cymene (1-methyl-4-isopropyl-benzene) as the biological precursor of carvacrol and a major constituent of the essential oil of Protium heptaphyllum species, with more than 80% of this species found in the Amazon region (Santana et al., 2011; Siani et al., 1999; Figure 1). Recent studies have shown that p-cymene rich species, such as P. heptaphyllum (Aubl.) Marchand, Protium kleinii Cuatrec, (Burseraceae), Hyptis pectinata (L.) Poit, (Lamiaceae), and Zataria multiflora Boiss, (Lamiaceae), show antinociceptive activity in rodents (Bispo et al., 2011; Oliveira et al., 2005; Otuki et al., 2011; Ramezani et al., 2004; Santana et al., 2011). Given the above information, the objective of this study was to evaluate the antioxidant activity of p-cymene in the hippocampus of adult mice treated acutely by determining the levels of thiobarbituric acid reactive substances (TBARS), nitrite content, activity of catalase (CAT), and SOD.
Materials and methods Animals Adult male Swiss mice were used, 2 months of age, weighing 25–30 g, from the Central Animal Facility of the Center for Agricultural Sciences, at Federal University of Piauı´. The experimental units received water and Purina standard type food ad libitum and they were maintained under controlled lighting (12 h cycle light/dark) and temperature (25 ± 2 C). All experiments were performed in accordance with the Guide for the Care and Use of Laboratory Animals, from the US Department of Health and Human Services, Washington DC, 1985. This project was approved by the Ethics Committee on Animal Experimentation of the Federal University of Piauı´ (013/2011 CEEA/UFPI).
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p-cymene (50, 100, and 150 mg/kg, i.p.; CYM 50, 100, and 150 groups, respectively, n ¼ 7). The treated and control animals were sorted in cages containing a maximum of six animals and placed into a reserved environment, under direct observation for 24 h to monitor and record any signs of toxicity of the compound. After evaluation of the parameters related acute toxicity during the observation period, the animals were euthanized by administration of sodium pentobarbital (500 mg/kg, i.p.) for removal and dissection of the hippocampus on both sides of the brain for neurochemical studies: determine the levels of TBARS, nitrite content, and antioxidant enzymes SOD and CAT. Method for determining the levels of TBARS The extent of lipid peroxidation was measured by determining the levels of TBARS. The method was previously described by Draper and Hadley (1990). The homogenate was prepared at 10% (w/v) in sodium phosphate buffer 50 mmol/l, pH 7.4 with hippocampal areas of all groups, vehicle (n ¼ 7), AA 250 (n ¼ 7), and CYM 50, 100, and 150 (n ¼ 7). The results were expressed as nmol of MDA/g of tissue. Method for determining the content of nitrite and catalase (CAT) activity The content of nitrite in the groups: vehicle (n ¼ 7), AA 250 (n ¼ 7), and CYM 50, 100, and 150 (n ¼ 7) were determined based on the Griess reaction (Green et al., 1981). The results were expressed in mmol/l. CAT activity was measured in the groups: vehicle (n ¼ 7), AA 250 (n ¼ 7), and CYM 50, 100, and 150 (n ¼ 7); using the basic principle of measuring the production rate of O2 and H2O. Protein concentration was determined by the method of Lowry and colleagues. The results were expressed in mmol/min/mg protein (Lowry et al., 1951). Method for determining the SOD activity The hippocampal homogenates 10% were centrifuged (800 g, 20 min) and the supernatants used to determine the activity of SOD. SOD activity in the vehicle group (n ¼ 7), AA 250 (n ¼ 7), and CYM 50, 100, and 150 (n ¼ 7) was investigated through the rate of reduction of cytochrome C by superoxide radicals, using xanthine–xanthine oxidase system as a source of superoxide anion (O2) (Arthur & Boyne, 1985). The results were expressed as U/mg protein. One unit (U) of SOD activity corresponds to 50% inhibition of the reaction of O2-with cytochrome C. Protein concentration in the homogenates was obtained by the method of Lowry et al. (1951).
Treatment of experimental groups
Statistical analyses
The p-cymene was emulsified with 0.05% Tween 80 (Sigma, St. Louis, MO) and dissolved in 0.9% saline (vehicle). The 35 animals were sorted into five groups and were intraperitoneally treated (i.p.). The first group was treated with vehicle, 0.01 ml/g (0.05% Tween 80 in 0.9% saline i.p., negative control group, n ¼ 7). The second group received ascorbic acid at a dose of 250 mg/kg (positive control, i.p., 250 AA group, n ¼ 7). And the other three groups were treated with
All results were presented as mean ± standard error of mean (SEM). Data were evaluated by analysis of variance (ANOVA) followed by Student–Neuman–Keuls as the post hoc test. Data were analyzed by applying the GraphPad Prism 5.0 (San Diego, CA); experimental groups were compared with the vehicle group and positive control (ascorbic acid). Differences were considered statistically significant when p50.05.
Antioxidant effects of p-cymene
DOI: 10.3109/13880209.2014.923003
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Table 1. Antioxidant potential evaluation of p-cymene in the hippocampus of adult mice by determining the levels of TBARS, nitrite content, CAT and SOD.
Groups
TBARS levels (nmol of MDA/g wet tissue)
Nitrite content (mM)
SOD activity (U/mg protein)
CAT activity (mmol/min/mg of protein)
1.24 ± 0.03 0.39 ± 0.02a 0.42 ± 0.03a 0.33 ± 0.01a,b,c 0.12 ± 0.03a,b,c,d
79.17 ± 1.88 53.25 ± 1.88a 22.79 ± 1.29a.b 25.01 ± 1.04a.b 26.12 ± 1.22a.b
2.28 ± 0.14 2.29 ± 0.14 2.80 ± 0.07a.b 3.08 ± 0.04a.b 3.73 ± 0.10a.b
13.87 ± 0.55 33.86 ± 2.44a 30.41 ± 1.725a 34.93 ± 1.19a 39.21 ± 0.35a
Vehicle AA 250 CYM 50 CYM 100 CYM 150
Values represent the mean ± EPM. The number of animals used in the experiments (n ¼ 7 per group). a p50.05 compared with the vehicle group. b p50.05 when compared with AA 250. c p50.05 when compared with CYM 50. d p50.05 when compared with CYM 100 (ANOVA and t Student–Newman–Keuls as the post hoc test)
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Results and discussion Levels of lipid peroxidation in mice hippocampus treated with p-cymene acutely Table 1 presents the results of effects of treatment with p-cymene lipid peroxidation, in the hippocampus of adult mice. In the AA group (0.39 ± 0.02), a significant decrease was observed of 50.77% lipid peroxidation compared with the vehicle (1.24 ± 0.03) in the hippocampus of adult mice. In turn, treatment with p-cymene at a dose of 50 mg/kg (0.42 ± 0.03) produced a significant reduction in lipid peroxidation 65.54% when compared with the control group. However, there was no significant change observed when compared with the group treated with AA 250. A significant reduction was also detected in the group treated with p-cymene at a dose of 100 mg/kg (0.33 ± 0.01), compared with the values of the vehicle group (73.29%), AA 250 (16.57%), and CYM 50 (22.49%). In the group that received 150 mg/kg (0.12 ± 0.01) was observed a significant decrease compared with the vehicle group (89.83%), and AC 250 (68.26%), CYM 50 (70, 5%), and CYM 100 (61.95%). Nitrite content in mice hippocampus treated with p-cymene acutely In Table 1, the results of the effects of p-cymene in the nitrite content in the hippocampus of adult mice are shown. In group AA, 250 (53.2 ± 1.88) was observed, a significant decrease in the content of 32.73% nitrite group compared with the vehicle (79.1 ± 1.88) in the hippocampus of adult mice. In turn, treatment with p-cymene at a dose of 50 mg/kg (22.79 ± 1.29) produced a significant reduction of 71.21% when compared with the vehicle group, 57.20% AA 250. A significant reduction was also detected in the group treated with p-cymene at a dose of 100 mg/kg (25.01 ± 1.04) compared with the values of the vehicle group (68.61%) and AA 250 (53.03%). In the group receiving 150 mg/kg (26.12 ± 1.22) was observed, a significant decrease compared with the vehicle group (67%) and AA 250 (50.94%). SOD activity in mice hippocampus treated with p-cymene acutely The results presented in Table 1 show that p-cymene at tested concentrations were able to increase the activity of SOD significantly compared with the vehicle group (2.28 ± 0.14)
[CYM 50: 22.7% (2.81 ± 0.07); CYM 100: 33.9% (3.09 ± 0.04); CYM 150: 63.1% (3.73 ± 0.10)]. In turn, there was a significant increase in SOD at all concentrations [CYM 50: 22.4% (2.81 ± 0.07), CYM 100: 34.60% (3.08 ± 0.04); CYM 150: 62.70% (3.73 ± 0.10)] compared with the 250 AA group (2.29 ± 0.14). Catalase activity in mouse hippocampus treated with p-cymene acutely In Table 1, the effect of treatment with p-cymene in catalase activity in the hippocampus of adult mice is shown. The results showed that at all tested doses (50, 100, and 150 mg/kg), p-cymene produces an increase in catalase activity relative to the vehicle-treated group (13.87 ± 0.55) [CYM 50: 119.25% (30.41 ± 1.72); CYM 100: 151.83% (34.93 ± 1.72), 150: 182.70% (39.21 ± 0.35)]. Oxidation is a metabolic process that leads to the production of energy required for essential cell activities. However, oxygen metabolism in living cells also leads to the production of free radicals (Roesler et al., 2007; Stieven et al., 2009). Antioxidants that may sequester these free radicals have a high therapeutic potential in diseases. Antioxidants are substances capable of preventing the deleterious effects of oxidation, inhibiting the beginning of lipid peroxidation, sequestering free radicals and/or chelating metal ions (Silva et al., 2006). The brain continually produces ROS (Campeˆlo, 2011; Castagne et al., 1999) in the absence of an efficient defense mechanism; these ROS cause the peroxidation of polyunsaturated fatty acids of cell membranes (Freitas, 2001; Ruberto & Baratta, 2000). The brain is particularly susceptible to peroxidation due to the simultaneous presence of high levels of polyunsaturated fatty acids and iron (Caˆmpelo, 2011; Halliwell & Chirico, 1993), which is the target of free radical damage. Several methods are described in the literature for the evaluation of lipid peroxidation, such as determination of reactive species with thiobarbituric acid (TBARS), which is used to quantify lipid peroxidation which is a cell membrane damage caused by oxidative stress (Vicentino & Menezes, 2007) and reported in chronic degenerative diseases such as Alzheimer’s disease, Parkinson’s disease, atherosclerosis, complications of diabetes mellitus, and aging, are all related to oxidative stress (Sorg, 2004).
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H
(A)
H +L
+
H
CH2 +L
L-H
+
L-H
+
HNO2
H
H
(B)
H + NO2
+
CH2
H + NO2
HNO2
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H
Figure 2. Possible mechanisms of antioxidant reactions of p-cymene. (A) Formation of lipid moiety and (B) formation of radical nitrite (NO2).
The study showed that p-cymene prominently inhibited the amount of TBARS formed. Thus, this monoterpene was able to reduce the levels of lipid peroxidation at all tested doses, indicating a protective antioxidant effect. It is noteworthy that the doses of CYM 100 and 150 indicated an important antioxidant protection over that produced by ascorbic acid compound used as a standard, as shown in Table 1. Based on the results obtained in the study, it was proposed a possible mechanism of antioxidant reaction of p-cymene against the formation of lipid moiety as shown in Figure 2. It is important to notice that there are two possible antioxidant reactions of p-cymene in the formation of lipid radical and formation of nitrite radical (Figure 2) because the compound has two positions with benzylic hydrogens. Nitric oxide (NO) and nitrogen dioxide (NO2) are RNS (Sorg, 2004; Souza et al., 2007). Studies have shown that neurodegenerative diseases generate changes in nitric oxide metabolism, and interact with glutamate receptors to produce part of its stimulating action on the central nervous system (CNS) (Dymond & Crandall, 1976; Freitas, 2009; Silva et al., 2006; Souza et al., 2007; Xavier et al., 2007). In the nervous system, NO participates in various signaling processes, in tissue defense against pathogens, but is also a harmful agent when in excess, evoking oxidative reactions/reducing, producing RNS (Freitas, 2009; Migliorea & Coppede`b, 2009; Sueishi et al., 2011). In regard to the content of nitrite, results show a significant decrease of this ion at all tested doses, being more significant than that produced by ascorbic acid, indicating that the p-cymene can be highly effective against the formation of reactive species derived from nitrogen culminating in a neuroprotective role. The decrease in nitrite content may be a consequence of inhibiting the formation of radical scavenges reactive species derived from oxygen and products of lipid peroxidation (Caˆmpelo, 2011; Souza et al., 2007). The ROS levels are normally controlled by an antioxidant defense system as enzymes: Mn–SOD (superoxide dismutase containing manganese), Cu–Zn–SOD (superoxide dismutase containing copper-zinc), CAT, and GPX (glutathione peroxidase) these have been shown to be a significant decrease in
the products of lipid peroxidation (Gomes, 2011; Siow et al., 2007; Vander et al., 1997). The enzyme SOD catalyzes the dismutation of superoxide to hydrogen peroxide, which is degraded by the action of CAT, GPX (Bravard et al., 2002; Gomes, 2011). In this sense, the monoterpene was able to significantly increase both the activity of SOD and catalase, demonstrating a positive change toward broadening the neuroprotective defenses of the brain. The increase of this activity suggests that the p-cymene has a potential antioxidant related to modulation of enzymatic activity against the formation of hydrogen peroxide. In this study, it was not possible to verify a dose-dependent effect between the different doses of CYM evaluated in vivo.
Conclusion Treatment with p-cymene significantly reduced the level of lipid peroxidation, nitrite content, suggesting an antioxidant role in vivo since it was able to reduce the formation of reactive species derived from oxygen and nitrogen. Furthermore, the p-cymene increased the activity of CAT and SOD activities in mice hippocampus, suggesting that its antioxidant role may be due to positive modulation of the activity of these enzymes. Therefore, the results of this study show that the p-cymene has an antioxidant potential by in vivo methods used. This compound may constitute a new strategy in the development of treatments for various diseases in which oxidative stress plays an inherent in their physiology. However, more studies should be performed to investigate the mechanism responsible for the antioxidant activity of p-cymene, and correlate the pharmacological activity with its chemical structure.
Declaration of interest The authors report no conflicts of interest. The authors alone are responsible for the content and writing of this article.
References Almeida AAC, Costa JP, Carvalho RBF, et al. (2012). Evaluation of acute toxicity of a natural compound (+)-limonene epoxide and its anxiolytic-like action. Brain Res 1448:56–62.
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DOI: 10.3109/13880209.2014.923003
Apel MA, Limberger RP, Sobral M, et al. (2001). Chemical composition of the essential oil of Siphoneugena reitzii D. Legr. J Essent Oil Res 13:429–30. Apel MA, Neves BM, Bauermann L, et al. (2008). Evaluation of antioxidant activity of volatile oil Siphoneugena reitzii and monoterpenes isolated. In: Proceedings of the 31st Annual Meeting of the Brazilian Chemical Society. Arthur JR, Boyne R. (1985). Superoxide dismutase and glutathione peroxidase activities in neutrophils from selenium deficient and copper deficient cattle. Life Sci 36:1569–75. Bicas JL, Neri-Numa IA, Ruiz ALTG, et al. (2011). Evaluation of the antioxidant and antiproliferative potential of bioflavors. Food Chem Toxicol 49:1610–15. Bispo MD, Moura˜o RH, Franzotti EM, et al. (2011). Antinociceptive and antiedematogenic effects of the aqueous extract of Hyptis pectinata leaves in experimental animals. J Ethnopharmacol 76:81–6. Bravard A, Ageron-Blanc A, Alvarez S, et al. (2002). Correlation between antioxidant status, tumorigenicity, and radiosensitivity in sister rat cell lines. Carcinogenesis 23:705–11. Campeˆlo LML, Almeida AAC, Freitas RL, et al. (2011). Antioxidant and antinociceptive effects of Citrus limon essential oil in mice. J Biomed Biotechnol 2011:1–8. Campeˆlo LML, Sa´ CG, Almeida AAC, et al. (2011). Sedative, anxiolytic and antidepressant activities of Citrus limon (Burn) essential oil in mice. Pharmazie 66:623–7. Campeˆlo LML. (2011). Pharmacological evaluation of the essential oil of Citrus limon (Burm) central nervous system: A study behavioral, neurochemical and histological [Master dissertation] Federal University of Piauı´. Castagne V, Gautschi M, Lefevre K, et al. (1999). Relationships between neuronal death and the cellular redox status. Focus on the developing nervous system. Prog Neurobiol 59:397–423. Costa DA. (2012). Preclinical study of biochemical and pharmacological effects of the cyano-carvone in mice: Allowance for the development of phyto [Master dissertation]. Federal University of Piauı´. Costa Ju´nior JS, Ferraz ABF, Filho BAB, et al. (2011). Evaluation of antioxidant effects in vitro of garcinielliptone FC (GFC) isolated from Platonia insignis Mart. J Med Plants Res 5:293–9. Dias BFS. (1996). The Implementation of the Convention on Biological Diversity in Brazil: Challenges and Opportunities. Sa˜o Paulo, Brazil: Tropical Foundation for Research and Technology Andre´ Tosello. Draper HH, Hadley M. (1990). Malondialdehyde determination as an index of lipid peroxidation. Methods Enzymol 186: 421–31. Dymond AM, Crandall PH. (1976). Oxygen availability and blood flow in the temporal lobes during spontaneous epileptic seizures in man. Brain Res 102:191–6. Freitas RM, Sousa FCF, Vasconcelos SMM, et al. (2005). Oxidative stress in the hippocampus after pilocarpine-induced status epilepticus in Wistar rats. FEBS J 272:1307–12. Freitas RM. (2001). Antioxidant properties and therapeutic potential of essential oils. In: Sousa DP. Medicinal Essential Oils: Chemical, Pharmacological and Therapeutic Aspects. New York: New Science Publishers, 1–9. Freitas RM. (2009). The evaluation of effects of lipoic acid on the lipid peroxidation, nitrite formation and antioxidant enzymes in the hippocampus of rats after pilocarpine-induced seizures. Neurosci Lett 455:140–4. Gomes DBV. (2011). Study of antioxidant activity of liquid cashew (Anacardium occidentale L.) and technical in nature for pharmaceutical formulations [Master dissertation]. Federal University of Piauı´. Green LC, Tannenbaum SR, Goldman P. (1981). Nitrate synthesis in the germfree and conventional rat. Science 212:56–8. Gutteridge JMC, Halliwell B. (2010). Antioxidants: Molecules, medicines, and myths. Biochem Biophys Res Commun 393:561–4. Halliwell B, Chirico S. (1993). Lipid peroxidation: Its mechanism, measurement, and significance. Am J Clin Nutr 57:724S–5S. Halliwell B. (1991). Reactive oxygen species in living systems: Source, biochemistry and role in human disease. Am J Med 91:14–22. Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. (1951). Protein measurement with follin phenol reagent. J Biol Chem 19: 265–75.
Antioxidant effects of p-cymene
5
Maciel MAM, Pinto AC, Veiga Ju´nior VF, et al. (2002). Medicinal plants: The need for multidisciplinary studies. New Chem 25:429–38. Mengues SS, Mentz LA, Schenkel EP. (2011). Use of medicinal plants in pregnancy. Rev Bras Farmacogn 11:21–35. Migliorea L, Coppede`b F. (2009). Environmental-induced oxidative stress in neurodegenerative disorders and aging. Mutat Res 674: 73–84. Mothana RAA, Hasson SS, Schultze W, et al. (2001). Phytochemical composition and in vitro antimicrobial and antioxidant activities of essential oils of three endemic Soqotraen Boswellia species. Food Chem 126:1149–54. Nogueira Neto JD, Sousa DP, Freitas RM. (2013). Evaluation of antioxidant potential in vitro of nerolidol. Rev Cieˆnc Farm Ba´sica Apl 34:125–30. Oliveira FA, Costa CLS, Chaves MH, et al. (2005). Attenuation of capsaicin-induced acute and visceral nociceptive pain by alpha- and beta-amyrin, a triterpene mixture isolated from Protium heptaphyllum resin in mice. Life Sci 77:2942–52. Otuki MF, Lima FV, Malheiros A, et al. (2011). Evaluation of the antinociceptive action caused by ether fraction and a triterpene isolated from resin of Protium kleinii. Life Sci 69:2225–36. Ramezani M, Hosseinzadeh H, Samizadeh S. (2004). Antinociceptive effects of Zataria multiflora Boiss fractions in mice. J Ethonopharmacol 9:167–70. Rodrigues PA. (2008). Gastroprotective and antioxidant activity of extracts and chemical constituents of Byrsonima sericea DC and Plectranthus grandis Cramer (Willensem) [Doctoral thesis]. State University of Ceara´. Roesler R, Malta LG, Carrasco LC, et al. (2007). Antioxidant activity of native fruits. Cieˆnc Tecnol Aliment 27:53–60. Ruberto G, Baratta MT. (2000). Antioxidant activity of selected essential oil components in two lipid model systems. Food Chem 69:167–74. Santana MF, Quintans-Ju´nior LJ, Cavalcanti SCH, et al. (2011). p-Cymene reduces orofacial nociceptive response in mice. Rev Bras Farmacogn 21:1138–43. Santos PS, Costa JP, Tome´ AR, et al. (2011). Oxidative stress in rat striatum after pilocarpine-induced seizures is diminished by alphatocopherol. Eur J Pharmacol 668:65–71. Siani AC, Garrido IS, Carvalho ES, Ramos MFS. (1999). Evaluation of anti-inflammatory-related activity of essential oils from the leaves and resin of species of Protium. J Ethnopharmacol 66: 57–69. Silva AP, Cerqueira GS, Nunes LC, Freitas RM. (2012). Effects of an aqueous extract of Orbignya phalerata Mart on locomotor activity and motor coordination in mice and as antioxidant in vitro. Pharmazie 67: 260–3. Silva DA, Silva TMS, Lins ACS, et al. (2006). Chemical constituents and antioxidant activity of Sida galheirensis Ulbr. (Malvaceae). Quim Nova 29:1250–4. Siow RC, Ishii T, Mann G.E. (2007). Modulation of antioxidant gene expression by 4-hydroxynonenal: Atheroprotective role of the Nrf2/ARE transcription pathway. Redox Rep 12:11–15. Siqueira-Catania A, Barros CR, Ferreira SRG. (2009). Vitamins and minerals with antioxidant properties and cardiometabolic risk: Controversies and perspectives. Arq Bras Endocrinol Metabol 53: 550–9. Sorg O. (2004). Oxidative stress: A theoretical model or biological reality? C R Biol 327:649–62 Souza WM, Brehmer F, Nakao LS, et al. (2007). Uleine action on nitric oxide production in RAEC and B16F10. Rev Bras Farmacogn 17: 191–6. Stieven AC, Moreira JJS, Ferraz C. (2009). Essential oils of uvaia (Eugenia pyriformis Cambess): Evaluation of microbial and antioxidant activities. Eclectic Chem 34:7–16. Sueishi Y, Hori M, Kita M, Kotake Y. (2011). Nitric oxide (NO) scavenging capacity of natural antioxidants. Food Chem 129: 866–70. Vander Jagt DL, Hunsaker LA, Vander Jagt TJ, et al. (1997). Inactivation of glutathione reductase by 4-hydroxynonenal and other endogenous aldehydes. Biochem Pharmacol 53:1133–40. Veiga Ju´nior VF, Pinto AC, Maciel MAM. (2005). Medicinal plants: Safe cure? New Chem 28:519–28
6
T. M. de Oliveira et al.
Pharmaceutical Biology Downloaded from informahealthcare.com by Selcuk Universitesi on 12/21/14 For personal use only.
Vicentino ARR, Menezes FS. (2007). Antioxidant activity of vegetable dyes, sold in pharmacies and suitable for various types of diseases by the DPPH method. Rev Bras Farmacogn 17:384–7. Wannes WA, Mhamdi B, Sriti J, et al. (2010). Antioxidant activities of the essential oils and methanol extracts from myrtle (Myrtus communis var. italica L.) leaf, stem and flower. Food Chem Toxicol 48:1362–70.
Pharm Biol, Early Online: 1–6
Wickens AP. (2001). Ageing and the free radical theory. Respir Physiol 128:379–91. Xavier SM, Barbosa CO, Barros DO, et al. (2007). Vitamin C antioxidant effects in hippocampus of adult Wistar rats after seizures and status epilepticus induced by pilocarpine. Neurosci Lett 420:76–9.
ORIGINAL ARTICLE
Evaluation of p-cymene, a natural antioxidant Talita Mendes de Oliveira1, Rusbene Bruno Fonseca de Carvalho2, Iwyson Henrique Fernandes da Costa1, Guilherme Antoˆnio Lopes de Oliveira1, Alexandre Araujo de Souza2, Sidney Gonc¸alo de Lima2, and Rivelilson Mendes de Freitas1
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Department of Biochemistry and Pharmacology, Laboratory of Research in Experimental Neurochemistry of Post-Graduation Program in Pharmaceutics Science, Federal University of Piauı´, Teresina – Piauı´, Brazil and 2Department of Chemistry, Federal University of Piauı´, Teresina – Piauı´, Brazil Abstract
Keywords
Context: Several studies have demonstrated that essential oils and their major components have antioxidant activity. p-Cymene is a monoterpene and a major constituent of essential oils of various species of plants. Objective: This paper evaluated the antioxidant potential of p-cymene in the hippocampus of mice by determining the levels of thiobarbituric acid reactive substances (TBARS), nitrite content, and activity of catalase (CAT) and superoxide dismutase (SOD). Materials and methods: Swiss mice were intraperitoneally treated with 0.05% Tween 80 dissolved in 0.9% saline solution, ascorbic acid 250 mg/kg, and p-cymene at doses of 50, 100, and 150 mg/kg, respectively. After treatment, all groups were observed for 24 h, afterwards, the groups were euthanized for removal of the brain and dissection of the hippocampus. Results: The results of treatment with p-cymene were a significant decrease in lipid peroxidation and nitrite content at a dose of CYM 50: 65.54%, CYM 100: 73.29%, CYM 150: 89.83%, and CYM 50: 71.21%; CYM 100: 68.61% and CYM 150:67%, respectively, when compared with the control group. The results showed that at all tested doses, p-cymene produces an increase in SOD and catalase activity significantly at a dose of CYM 50: 22.7%, CYM 100: 33.9%, CYM 150: 63.1%, and CYM 50: 119.25%, CYM 100: 151.83% and CYM 150: 182.70%, respectively, when compared with the vehicle-treated group. Discussion and conclusion: The result of this study shows that p-cymene has an antioxidant potential in vivo and may act as a neuroprotective agent in the brain. This compound may present a new strategy in the development of treatment for many diseases in which oxidative stress plays an important pathophysiological role.
Catalase, lipid peroxidation, monoterpene, nitrite, superoxide dismutase
Introduction Since the beginning of human civilization, plants have been used for medicinal purposes, for treatment, prevention, and curing of several diseases (Campeˆlo et al., 2011; Maciel et al., 2002; Mengues et al., 2011; Veiga Ju´nior et al., 2005). In Brazil, due to its extensive plant genetic diversity, with more than 55 000 species cataloged from a total estimated between 350 000 and 550 000 species (Dias, 1996; Rodrigues, 2008), and a privileged geographical location, there are several studies with natural substances derived from plants to treat numerous pathologies (Almeida et al., 2012; Campeˆlo et al., 2011; Freitas, 2001). Among these diseases, a special investigation is necessary on the antioxidant potential in vitro (Costa Ju´nior et al, 2011; Nogueira Neto et al., 2013;
Correspondence: Rivelilson Mendes de Freitas, Programa de Po´s-graduac¸a˜o em Cieˆncias Farmaceˆuticas/Nu´cleo de Tecnologia Farmaceˆutica do Centro de Cieˆncias da Sau´de da Universidade Federal do Piauı´, Ininga, Teresina, CEP 64 049-550 Piauı´, Brazil. Tel./fax: +55 86 3215 5870. E-mail: [email protected]
History Received 17 October 2013 Revised 7 April 2014 Accepted 07 May 2014 Published online 3 December 2014
Silva et al., 2012) and in vivo (Freitas et al., 2005), since oxidative stress may account for the pathophysiology of several diseases (Santos et al., 2011). In this context, while biological compounds are generated and called reactive oxygen species (ROS), which in excess may cause cellular damage, culminating in a process called oxidative stress. This process results in an imbalance in the state of oxidation–reduction in favor of oxidation. In this sense, we can cite the enzymes superoxide dismutase (SOD), catalase, and glutathione peroxidase which promotes detoxification of superoxide radicals, hydrogen peroxide, and lipid hydroperoxides, respectively (Costa, 2012; Wickens, 2001). Endogenous enzymes, some vitamins, and minerals participate in antioxidant defense mechanisms. Antioxidants may slow or prevent oxidative damage in human tissues produced by ROS and reactive nitrogen species (RNS). Thus, it may be defined as a diverse group of natural molecules, which may be present at low concentrations compared with biomolecules with antioxidant effect (Gutteridge & Halliwell, 2010; Halliwell, 1991; SiqueiraCatania et al., 2009).
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Figure 1. Molecular structure of p-cymene (1-methyl-4-isopropyl benzene).
Several studies show antioxidant activity of monoterpenes, a-terpineol (Bicas et al., 2011), a-thujone (Mothana et al., 2001), 1,8-cineole, a-pinene (Wannes et al., 2010), c-terpinene (Ruberto & Baratta, 2000), R-(+)-a-pinene, S-()-b-pinene, a-pinene (racemic mixture), a-terpinene, limonene, and linalool (Apel et al., 2001, 2008). Among monoterpenes, we can highlight p-cymene (1-methyl-4-isopropyl-benzene) as the biological precursor of carvacrol and a major constituent of the essential oil of Protium heptaphyllum species, with more than 80% of this species found in the Amazon region (Santana et al., 2011; Siani et al., 1999; Figure 1). Recent studies have shown that p-cymene rich species, such as P. heptaphyllum (Aubl.) Marchand, Protium kleinii Cuatrec, (Burseraceae), Hyptis pectinata (L.) Poit, (Lamiaceae), and Zataria multiflora Boiss, (Lamiaceae), show antinociceptive activity in rodents (Bispo et al., 2011; Oliveira et al., 2005; Otuki et al., 2011; Ramezani et al., 2004; Santana et al., 2011). Given the above information, the objective of this study was to evaluate the antioxidant activity of p-cymene in the hippocampus of adult mice treated acutely by determining the levels of thiobarbituric acid reactive substances (TBARS), nitrite content, activity of catalase (CAT), and SOD.
Materials and methods Animals Adult male Swiss mice were used, 2 months of age, weighing 25–30 g, from the Central Animal Facility of the Center for Agricultural Sciences, at Federal University of Piauı´. The experimental units received water and Purina standard type food ad libitum and they were maintained under controlled lighting (12 h cycle light/dark) and temperature (25 ± 2 C). All experiments were performed in accordance with the Guide for the Care and Use of Laboratory Animals, from the US Department of Health and Human Services, Washington DC, 1985. This project was approved by the Ethics Committee on Animal Experimentation of the Federal University of Piauı´ (013/2011 CEEA/UFPI).
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p-cymene (50, 100, and 150 mg/kg, i.p.; CYM 50, 100, and 150 groups, respectively, n ¼ 7). The treated and control animals were sorted in cages containing a maximum of six animals and placed into a reserved environment, under direct observation for 24 h to monitor and record any signs of toxicity of the compound. After evaluation of the parameters related acute toxicity during the observation period, the animals were euthanized by administration of sodium pentobarbital (500 mg/kg, i.p.) for removal and dissection of the hippocampus on both sides of the brain for neurochemical studies: determine the levels of TBARS, nitrite content, and antioxidant enzymes SOD and CAT. Method for determining the levels of TBARS The extent of lipid peroxidation was measured by determining the levels of TBARS. The method was previously described by Draper and Hadley (1990). The homogenate was prepared at 10% (w/v) in sodium phosphate buffer 50 mmol/l, pH 7.4 with hippocampal areas of all groups, vehicle (n ¼ 7), AA 250 (n ¼ 7), and CYM 50, 100, and 150 (n ¼ 7). The results were expressed as nmol of MDA/g of tissue. Method for determining the content of nitrite and catalase (CAT) activity The content of nitrite in the groups: vehicle (n ¼ 7), AA 250 (n ¼ 7), and CYM 50, 100, and 150 (n ¼ 7) were determined based on the Griess reaction (Green et al., 1981). The results were expressed in mmol/l. CAT activity was measured in the groups: vehicle (n ¼ 7), AA 250 (n ¼ 7), and CYM 50, 100, and 150 (n ¼ 7); using the basic principle of measuring the production rate of O2 and H2O. Protein concentration was determined by the method of Lowry and colleagues. The results were expressed in mmol/min/mg protein (Lowry et al., 1951). Method for determining the SOD activity The hippocampal homogenates 10% were centrifuged (800 g, 20 min) and the supernatants used to determine the activity of SOD. SOD activity in the vehicle group (n ¼ 7), AA 250 (n ¼ 7), and CYM 50, 100, and 150 (n ¼ 7) was investigated through the rate of reduction of cytochrome C by superoxide radicals, using xanthine–xanthine oxidase system as a source of superoxide anion (O2) (Arthur & Boyne, 1985). The results were expressed as U/mg protein. One unit (U) of SOD activity corresponds to 50% inhibition of the reaction of O2-with cytochrome C. Protein concentration in the homogenates was obtained by the method of Lowry et al. (1951).
Treatment of experimental groups
Statistical analyses
The p-cymene was emulsified with 0.05% Tween 80 (Sigma, St. Louis, MO) and dissolved in 0.9% saline (vehicle). The 35 animals were sorted into five groups and were intraperitoneally treated (i.p.). The first group was treated with vehicle, 0.01 ml/g (0.05% Tween 80 in 0.9% saline i.p., negative control group, n ¼ 7). The second group received ascorbic acid at a dose of 250 mg/kg (positive control, i.p., 250 AA group, n ¼ 7). And the other three groups were treated with
All results were presented as mean ± standard error of mean (SEM). Data were evaluated by analysis of variance (ANOVA) followed by Student–Neuman–Keuls as the post hoc test. Data were analyzed by applying the GraphPad Prism 5.0 (San Diego, CA); experimental groups were compared with the vehicle group and positive control (ascorbic acid). Differences were considered statistically significant when p50.05.
Antioxidant effects of p-cymene
DOI: 10.3109/13880209.2014.923003
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Table 1. Antioxidant potential evaluation of p-cymene in the hippocampus of adult mice by determining the levels of TBARS, nitrite content, CAT and SOD.
Groups
TBARS levels (nmol of MDA/g wet tissue)
Nitrite content (mM)
SOD activity (U/mg protein)
CAT activity (mmol/min/mg of protein)
1.24 ± 0.03 0.39 ± 0.02a 0.42 ± 0.03a 0.33 ± 0.01a,b,c 0.12 ± 0.03a,b,c,d
79.17 ± 1.88 53.25 ± 1.88a 22.79 ± 1.29a.b 25.01 ± 1.04a.b 26.12 ± 1.22a.b
2.28 ± 0.14 2.29 ± 0.14 2.80 ± 0.07a.b 3.08 ± 0.04a.b 3.73 ± 0.10a.b
13.87 ± 0.55 33.86 ± 2.44a 30.41 ± 1.725a 34.93 ± 1.19a 39.21 ± 0.35a
Vehicle AA 250 CYM 50 CYM 100 CYM 150
Values represent the mean ± EPM. The number of animals used in the experiments (n ¼ 7 per group). a p50.05 compared with the vehicle group. b p50.05 when compared with AA 250. c p50.05 when compared with CYM 50. d p50.05 when compared with CYM 100 (ANOVA and t Student–Newman–Keuls as the post hoc test)
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Results and discussion Levels of lipid peroxidation in mice hippocampus treated with p-cymene acutely Table 1 presents the results of effects of treatment with p-cymene lipid peroxidation, in the hippocampus of adult mice. In the AA group (0.39 ± 0.02), a significant decrease was observed of 50.77% lipid peroxidation compared with the vehicle (1.24 ± 0.03) in the hippocampus of adult mice. In turn, treatment with p-cymene at a dose of 50 mg/kg (0.42 ± 0.03) produced a significant reduction in lipid peroxidation 65.54% when compared with the control group. However, there was no significant change observed when compared with the group treated with AA 250. A significant reduction was also detected in the group treated with p-cymene at a dose of 100 mg/kg (0.33 ± 0.01), compared with the values of the vehicle group (73.29%), AA 250 (16.57%), and CYM 50 (22.49%). In the group that received 150 mg/kg (0.12 ± 0.01) was observed a significant decrease compared with the vehicle group (89.83%), and AC 250 (68.26%), CYM 50 (70, 5%), and CYM 100 (61.95%). Nitrite content in mice hippocampus treated with p-cymene acutely In Table 1, the results of the effects of p-cymene in the nitrite content in the hippocampus of adult mice are shown. In group AA, 250 (53.2 ± 1.88) was observed, a significant decrease in the content of 32.73% nitrite group compared with the vehicle (79.1 ± 1.88) in the hippocampus of adult mice. In turn, treatment with p-cymene at a dose of 50 mg/kg (22.79 ± 1.29) produced a significant reduction of 71.21% when compared with the vehicle group, 57.20% AA 250. A significant reduction was also detected in the group treated with p-cymene at a dose of 100 mg/kg (25.01 ± 1.04) compared with the values of the vehicle group (68.61%) and AA 250 (53.03%). In the group receiving 150 mg/kg (26.12 ± 1.22) was observed, a significant decrease compared with the vehicle group (67%) and AA 250 (50.94%). SOD activity in mice hippocampus treated with p-cymene acutely The results presented in Table 1 show that p-cymene at tested concentrations were able to increase the activity of SOD significantly compared with the vehicle group (2.28 ± 0.14)
[CYM 50: 22.7% (2.81 ± 0.07); CYM 100: 33.9% (3.09 ± 0.04); CYM 150: 63.1% (3.73 ± 0.10)]. In turn, there was a significant increase in SOD at all concentrations [CYM 50: 22.4% (2.81 ± 0.07), CYM 100: 34.60% (3.08 ± 0.04); CYM 150: 62.70% (3.73 ± 0.10)] compared with the 250 AA group (2.29 ± 0.14). Catalase activity in mouse hippocampus treated with p-cymene acutely In Table 1, the effect of treatment with p-cymene in catalase activity in the hippocampus of adult mice is shown. The results showed that at all tested doses (50, 100, and 150 mg/kg), p-cymene produces an increase in catalase activity relative to the vehicle-treated group (13.87 ± 0.55) [CYM 50: 119.25% (30.41 ± 1.72); CYM 100: 151.83% (34.93 ± 1.72), 150: 182.70% (39.21 ± 0.35)]. Oxidation is a metabolic process that leads to the production of energy required for essential cell activities. However, oxygen metabolism in living cells also leads to the production of free radicals (Roesler et al., 2007; Stieven et al., 2009). Antioxidants that may sequester these free radicals have a high therapeutic potential in diseases. Antioxidants are substances capable of preventing the deleterious effects of oxidation, inhibiting the beginning of lipid peroxidation, sequestering free radicals and/or chelating metal ions (Silva et al., 2006). The brain continually produces ROS (Campeˆlo, 2011; Castagne et al., 1999) in the absence of an efficient defense mechanism; these ROS cause the peroxidation of polyunsaturated fatty acids of cell membranes (Freitas, 2001; Ruberto & Baratta, 2000). The brain is particularly susceptible to peroxidation due to the simultaneous presence of high levels of polyunsaturated fatty acids and iron (Caˆmpelo, 2011; Halliwell & Chirico, 1993), which is the target of free radical damage. Several methods are described in the literature for the evaluation of lipid peroxidation, such as determination of reactive species with thiobarbituric acid (TBARS), which is used to quantify lipid peroxidation which is a cell membrane damage caused by oxidative stress (Vicentino & Menezes, 2007) and reported in chronic degenerative diseases such as Alzheimer’s disease, Parkinson’s disease, atherosclerosis, complications of diabetes mellitus, and aging, are all related to oxidative stress (Sorg, 2004).
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H
(A)
H +L
+
H
CH2 +L
L-H
+
L-H
+
HNO2
H
H
(B)
H + NO2
+
CH2
H + NO2
HNO2
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H
Figure 2. Possible mechanisms of antioxidant reactions of p-cymene. (A) Formation of lipid moiety and (B) formation of radical nitrite (NO2).
The study showed that p-cymene prominently inhibited the amount of TBARS formed. Thus, this monoterpene was able to reduce the levels of lipid peroxidation at all tested doses, indicating a protective antioxidant effect. It is noteworthy that the doses of CYM 100 and 150 indicated an important antioxidant protection over that produced by ascorbic acid compound used as a standard, as shown in Table 1. Based on the results obtained in the study, it was proposed a possible mechanism of antioxidant reaction of p-cymene against the formation of lipid moiety as shown in Figure 2. It is important to notice that there are two possible antioxidant reactions of p-cymene in the formation of lipid radical and formation of nitrite radical (Figure 2) because the compound has two positions with benzylic hydrogens. Nitric oxide (NO) and nitrogen dioxide (NO2) are RNS (Sorg, 2004; Souza et al., 2007). Studies have shown that neurodegenerative diseases generate changes in nitric oxide metabolism, and interact with glutamate receptors to produce part of its stimulating action on the central nervous system (CNS) (Dymond & Crandall, 1976; Freitas, 2009; Silva et al., 2006; Souza et al., 2007; Xavier et al., 2007). In the nervous system, NO participates in various signaling processes, in tissue defense against pathogens, but is also a harmful agent when in excess, evoking oxidative reactions/reducing, producing RNS (Freitas, 2009; Migliorea & Coppede`b, 2009; Sueishi et al., 2011). In regard to the content of nitrite, results show a significant decrease of this ion at all tested doses, being more significant than that produced by ascorbic acid, indicating that the p-cymene can be highly effective against the formation of reactive species derived from nitrogen culminating in a neuroprotective role. The decrease in nitrite content may be a consequence of inhibiting the formation of radical scavenges reactive species derived from oxygen and products of lipid peroxidation (Caˆmpelo, 2011; Souza et al., 2007). The ROS levels are normally controlled by an antioxidant defense system as enzymes: Mn–SOD (superoxide dismutase containing manganese), Cu–Zn–SOD (superoxide dismutase containing copper-zinc), CAT, and GPX (glutathione peroxidase) these have been shown to be a significant decrease in
the products of lipid peroxidation (Gomes, 2011; Siow et al., 2007; Vander et al., 1997). The enzyme SOD catalyzes the dismutation of superoxide to hydrogen peroxide, which is degraded by the action of CAT, GPX (Bravard et al., 2002; Gomes, 2011). In this sense, the monoterpene was able to significantly increase both the activity of SOD and catalase, demonstrating a positive change toward broadening the neuroprotective defenses of the brain. The increase of this activity suggests that the p-cymene has a potential antioxidant related to modulation of enzymatic activity against the formation of hydrogen peroxide. In this study, it was not possible to verify a dose-dependent effect between the different doses of CYM evaluated in vivo.
Conclusion Treatment with p-cymene significantly reduced the level of lipid peroxidation, nitrite content, suggesting an antioxidant role in vivo since it was able to reduce the formation of reactive species derived from oxygen and nitrogen. Furthermore, the p-cymene increased the activity of CAT and SOD activities in mice hippocampus, suggesting that its antioxidant role may be due to positive modulation of the activity of these enzymes. Therefore, the results of this study show that the p-cymene has an antioxidant potential by in vivo methods used. This compound may constitute a new strategy in the development of treatments for various diseases in which oxidative stress plays an inherent in their physiology. However, more studies should be performed to investigate the mechanism responsible for the antioxidant activity of p-cymene, and correlate the pharmacological activity with its chemical structure.
Declaration of interest The authors report no conflicts of interest. The authors alone are responsible for the content and writing of this article.
References Almeida AAC, Costa JP, Carvalho RBF, et al. (2012). Evaluation of acute toxicity of a natural compound (+)-limonene epoxide and its anxiolytic-like action. Brain Res 1448:56–62.
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DOI: 10.3109/13880209.2014.923003
Apel MA, Limberger RP, Sobral M, et al. (2001). Chemical composition of the essential oil of Siphoneugena reitzii D. Legr. J Essent Oil Res 13:429–30. Apel MA, Neves BM, Bauermann L, et al. (2008). Evaluation of antioxidant activity of volatile oil Siphoneugena reitzii and monoterpenes isolated. In: Proceedings of the 31st Annual Meeting of the Brazilian Chemical Society. Arthur JR, Boyne R. (1985). Superoxide dismutase and glutathione peroxidase activities in neutrophils from selenium deficient and copper deficient cattle. Life Sci 36:1569–75. Bicas JL, Neri-Numa IA, Ruiz ALTG, et al. (2011). Evaluation of the antioxidant and antiproliferative potential of bioflavors. Food Chem Toxicol 49:1610–15. Bispo MD, Moura˜o RH, Franzotti EM, et al. (2011). Antinociceptive and antiedematogenic effects of the aqueous extract of Hyptis pectinata leaves in experimental animals. J Ethnopharmacol 76:81–6. Bravard A, Ageron-Blanc A, Alvarez S, et al. (2002). Correlation between antioxidant status, tumorigenicity, and radiosensitivity in sister rat cell lines. Carcinogenesis 23:705–11. Campeˆlo LML, Almeida AAC, Freitas RL, et al. (2011). Antioxidant and antinociceptive effects of Citrus limon essential oil in mice. J Biomed Biotechnol 2011:1–8. Campeˆlo LML, Sa´ CG, Almeida AAC, et al. (2011). Sedative, anxiolytic and antidepressant activities of Citrus limon (Burn) essential oil in mice. Pharmazie 66:623–7. Campeˆlo LML. (2011). Pharmacological evaluation of the essential oil of Citrus limon (Burm) central nervous system: A study behavioral, neurochemical and histological [Master dissertation] Federal University of Piauı´. Castagne V, Gautschi M, Lefevre K, et al. (1999). Relationships between neuronal death and the cellular redox status. Focus on the developing nervous system. Prog Neurobiol 59:397–423. Costa DA. (2012). Preclinical study of biochemical and pharmacological effects of the cyano-carvone in mice: Allowance for the development of phyto [Master dissertation]. Federal University of Piauı´. Costa Ju´nior JS, Ferraz ABF, Filho BAB, et al. (2011). Evaluation of antioxidant effects in vitro of garcinielliptone FC (GFC) isolated from Platonia insignis Mart. J Med Plants Res 5:293–9. Dias BFS. (1996). The Implementation of the Convention on Biological Diversity in Brazil: Challenges and Opportunities. Sa˜o Paulo, Brazil: Tropical Foundation for Research and Technology Andre´ Tosello. Draper HH, Hadley M. (1990). Malondialdehyde determination as an index of lipid peroxidation. Methods Enzymol 186: 421–31. Dymond AM, Crandall PH. (1976). Oxygen availability and blood flow in the temporal lobes during spontaneous epileptic seizures in man. Brain Res 102:191–6. Freitas RM, Sousa FCF, Vasconcelos SMM, et al. (2005). Oxidative stress in the hippocampus after pilocarpine-induced status epilepticus in Wistar rats. FEBS J 272:1307–12. Freitas RM. (2001). Antioxidant properties and therapeutic potential of essential oils. In: Sousa DP. Medicinal Essential Oils: Chemical, Pharmacological and Therapeutic Aspects. New York: New Science Publishers, 1–9. Freitas RM. (2009). The evaluation of effects of lipoic acid on the lipid peroxidation, nitrite formation and antioxidant enzymes in the hippocampus of rats after pilocarpine-induced seizures. Neurosci Lett 455:140–4. Gomes DBV. (2011). Study of antioxidant activity of liquid cashew (Anacardium occidentale L.) and technical in nature for pharmaceutical formulations [Master dissertation]. Federal University of Piauı´. Green LC, Tannenbaum SR, Goldman P. (1981). Nitrate synthesis in the germfree and conventional rat. Science 212:56–8. Gutteridge JMC, Halliwell B. (2010). Antioxidants: Molecules, medicines, and myths. Biochem Biophys Res Commun 393:561–4. Halliwell B, Chirico S. (1993). Lipid peroxidation: Its mechanism, measurement, and significance. Am J Clin Nutr 57:724S–5S. Halliwell B. (1991). Reactive oxygen species in living systems: Source, biochemistry and role in human disease. Am J Med 91:14–22. Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. (1951). Protein measurement with follin phenol reagent. J Biol Chem 19: 265–75.
Antioxidant effects of p-cymene
5
Maciel MAM, Pinto AC, Veiga Ju´nior VF, et al. (2002). Medicinal plants: The need for multidisciplinary studies. New Chem 25:429–38. Mengues SS, Mentz LA, Schenkel EP. (2011). Use of medicinal plants in pregnancy. Rev Bras Farmacogn 11:21–35. Migliorea L, Coppede`b F. (2009). Environmental-induced oxidative stress in neurodegenerative disorders and aging. Mutat Res 674: 73–84. Mothana RAA, Hasson SS, Schultze W, et al. (2001). Phytochemical composition and in vitro antimicrobial and antioxidant activities of essential oils of three endemic Soqotraen Boswellia species. Food Chem 126:1149–54. Nogueira Neto JD, Sousa DP, Freitas RM. (2013). Evaluation of antioxidant potential in vitro of nerolidol. Rev Cieˆnc Farm Ba´sica Apl 34:125–30. Oliveira FA, Costa CLS, Chaves MH, et al. (2005). Attenuation of capsaicin-induced acute and visceral nociceptive pain by alpha- and beta-amyrin, a triterpene mixture isolated from Protium heptaphyllum resin in mice. Life Sci 77:2942–52. Otuki MF, Lima FV, Malheiros A, et al. (2011). Evaluation of the antinociceptive action caused by ether fraction and a triterpene isolated from resin of Protium kleinii. Life Sci 69:2225–36. Ramezani M, Hosseinzadeh H, Samizadeh S. (2004). Antinociceptive effects of Zataria multiflora Boiss fractions in mice. J Ethonopharmacol 9:167–70. Rodrigues PA. (2008). Gastroprotective and antioxidant activity of extracts and chemical constituents of Byrsonima sericea DC and Plectranthus grandis Cramer (Willensem) [Doctoral thesis]. State University of Ceara´. Roesler R, Malta LG, Carrasco LC, et al. (2007). Antioxidant activity of native fruits. Cieˆnc Tecnol Aliment 27:53–60. Ruberto G, Baratta MT. (2000). Antioxidant activity of selected essential oil components in two lipid model systems. Food Chem 69:167–74. Santana MF, Quintans-Ju´nior LJ, Cavalcanti SCH, et al. (2011). p-Cymene reduces orofacial nociceptive response in mice. Rev Bras Farmacogn 21:1138–43. Santos PS, Costa JP, Tome´ AR, et al. (2011). Oxidative stress in rat striatum after pilocarpine-induced seizures is diminished by alphatocopherol. Eur J Pharmacol 668:65–71. Siani AC, Garrido IS, Carvalho ES, Ramos MFS. (1999). Evaluation of anti-inflammatory-related activity of essential oils from the leaves and resin of species of Protium. J Ethnopharmacol 66: 57–69. Silva AP, Cerqueira GS, Nunes LC, Freitas RM. (2012). Effects of an aqueous extract of Orbignya phalerata Mart on locomotor activity and motor coordination in mice and as antioxidant in vitro. Pharmazie 67: 260–3. Silva DA, Silva TMS, Lins ACS, et al. (2006). Chemical constituents and antioxidant activity of Sida galheirensis Ulbr. (Malvaceae). Quim Nova 29:1250–4. Siow RC, Ishii T, Mann G.E. (2007). Modulation of antioxidant gene expression by 4-hydroxynonenal: Atheroprotective role of the Nrf2/ARE transcription pathway. Redox Rep 12:11–15. Siqueira-Catania A, Barros CR, Ferreira SRG. (2009). Vitamins and minerals with antioxidant properties and cardiometabolic risk: Controversies and perspectives. Arq Bras Endocrinol Metabol 53: 550–9. Sorg O. (2004). Oxidative stress: A theoretical model or biological reality? C R Biol 327:649–62 Souza WM, Brehmer F, Nakao LS, et al. (2007). Uleine action on nitric oxide production in RAEC and B16F10. Rev Bras Farmacogn 17: 191–6. Stieven AC, Moreira JJS, Ferraz C. (2009). Essential oils of uvaia (Eugenia pyriformis Cambess): Evaluation of microbial and antioxidant activities. Eclectic Chem 34:7–16. Sueishi Y, Hori M, Kita M, Kotake Y. (2011). Nitric oxide (NO) scavenging capacity of natural antioxidants. Food Chem 129: 866–70. Vander Jagt DL, Hunsaker LA, Vander Jagt TJ, et al. (1997). Inactivation of glutathione reductase by 4-hydroxynonenal and other endogenous aldehydes. Biochem Pharmacol 53:1133–40. Veiga Ju´nior VF, Pinto AC, Maciel MAM. (2005). Medicinal plants: Safe cure? New Chem 28:519–28
6
T. M. de Oliveira et al.
Pharmaceutical Biology Downloaded from informahealthcare.com by Selcuk Universitesi on 12/21/14 For personal use only.
Vicentino ARR, Menezes FS. (2007). Antioxidant activity of vegetable dyes, sold in pharmacies and suitable for various types of diseases by the DPPH method. Rev Bras Farmacogn 17:384–7. Wannes WA, Mhamdi B, Sriti J, et al. (2010). Antioxidant activities of the essential oils and methanol extracts from myrtle (Myrtus communis var. italica L.) leaf, stem and flower. Food Chem Toxicol 48:1362–70.
Pharm Biol, Early Online: 1–6
Wickens AP. (2001). Ageing and the free radical theory. Respir Physiol 128:379–91. Xavier SM, Barbosa CO, Barros DO, et al. (2007). Vitamin C antioxidant effects in hippocampus of adult Wistar rats after seizures and status epilepticus induced by pilocarpine. Neurosci Lett 420:76–9.
