Antioxidant activity of Citrus limon essential oil in mouse hippocampus

Abstract

Context: Citrus limon (L.) Burms (Rutaceae) has been shown in previous studies to have various biological functions (anti-inflammatory, antiallergic, antiviral, antimutagenic, and anticarcinogenic). However, traditional uses in folk medicine suggest that C. limon may have an effect on the central nervous system (CNS).

Objective: This study investigated the effects of C. limon essential oil (EO) on lipid peroxidation level, nitrite content, glutathione reduced (GSH) concentration, and antioxidant enzymes [superoxide dismutase (SOD), catalase, and glutathione peroxidase (GPx)] activities in mice hippocampus.

Materials and methods: Swiss mice were treated with the suspension of 0.5% Tween 80, in distilled water used as vehicle (i.p., control group) and with EO in three different doses (0.05, 0.1, or 0.15 g/kg, i.p., EO 50, EO 100, and EO 150 groups, respectively). After the treatments, all groups were observed for 24 h. The enzyme activities as well as the lipid peroxidation, nitrite, and GSH concentrations in mice hippocampus were measured using spectrophotometric methods and the results were compared with values obtained from control group.

Results: EO of C. limon treatment significantly reduced the lipid peroxidation level and nitrite content but increased the GSH levels and the SOD, catalase, and GPx activities in mice hippocampus.

Discussion and conclusion: Our findings strongly support the hypothesis that oxidative stress in hippocampus can occur during neurodegenerative diseases, proving that hippocampal damage induced by the oxidative process plays a crucial role in brain disorders, and also imply that a strong protective effect could be achieved using EO of C. limon as an antioxidant.

Keywords::

• Citrus limon
• oxidative stress
• essential oil
• hippocampus
• mice

Introduction

Lemon [Citrus limon (L.) Burms (Rutaceae)] has many important natural chemical components, including citric acid, ascorbic acid, minerals, and flavonoids. Although its health-related properties have always been associated with its vitamin C content, it has recently been shown that flavonoids also play a role in this respect. Some authors suggest that flavonoids have different biological functions, including antioxidative, anti-inflammatory, antiallergic, antiviral, antiproliferative, antimutagenic, and anticarcinogenic activities (Elangovan et al., 1994; Benavente-Garcia et al., 1997). Therefore, although the new Citrus cultivars have mainly been selected and developed for fresh consumption, the particular characteristics of their flavonoid contents have led to their use in pharmacological and food technology areas (Ortuño et al., 1997; Del Río et al., 1998).

Lemon fruit contains very important natural chemical components, including phenolic compounds (flavonoids) and other nutrients and non-nutrients (vitamins, minerals, dietary fiber, essential oils (EO), and carotenoids). The health-promoting effects and properties have been associated with the contents of vitamin C and flavonoids, due to their natural antioxidant characteristics (Tanaka et al., 1996; Rice-Evans et al., 1997). Overall, lemon fruits, rich in flavonoids, are a very important part of a balanced diet, particularly for their role in prevention of diseases (Miyake et al., 1998), such as obesity, diabetes, blood lipid lowering, cardiovascular diseases, and certain types of cancer (Monforte et al., 1995; Miyake et al., 1997). Thus, C. limon EO may possess a modulatory role in the treatment of neurodegenerative diseases, since their phenolic compound can interrupt cellular oxidative processes in the central nervous system (CNS). The effects of EO of C. limon in the CNS have not yet been determined. Therefore, it would be important to conduct these studies in order to clarify its brain action mechanism.

Pharmacological studies with EO of C. limon can reveal its effects on lipid peroxidation level, nitrite content, glutathione reduced (GSH) concentration, and antioxidant enzymes [superoxide dismutase (SOD), catalase, and glutathione peroxidase (Gpx)] activities. Once those effects are established, they may justify the use of this EO in preclinical and clinical studies in the treatment of neurodegenerative diseases. Thus, the aim of this study was to investigate the effects of C. limon EO on lipid peroxidation level, nitrite content, GSH concentration, and antioxidant enzymes activities in mice hippocampus.

Materials and methods

Plant materials

C. limon was identified and collected by Chistiane Mendes Feitosa in February 2010, at the city of Picos, state of Piaui, Brazil; their excicatae was deposited at the Graziella Barroso Herbarium of the Federal University of Piaui under the voucher number 26.453. The sample of C. limon EO leaves were prepared by Laboratory of Chemistry of Natural Products of that University (Matos et al., 1996).

Plant extraction of C. limon EO

Leaves (0.3 kg) from “limoeiro” were collected on the same day and hour, from flowering plants. The same leaf samples collected were steam distilled for 1 h. Oils obtained from these leaves were analyzed (Matos et al., 1996) by GC/MS using a GC-17 A/MS-QP505A (Shimadzu, Japan) instrument under the following conditions: column: dimethylpolysiloxane DB-1 fused-silica capillary column (30 m × 0.25 mm); carrier gas, helium (1 mL/min); injector temperature, 35–180°C at 4°C/min, then 180–250°C at 10°C/min; mass spectra electron impact, 70 eV. Individual components were identified by spectrometric analyses using two computer library MS searches, and Kovacs indices as a preselection aid. Visual mass spectra comparison data from the literature were used for confirmation.

Animals procedures

Male Swiss mice (25–30 g) were used. All animals were maintained at a controlled temperature (23 ± 1°C) and a 12-h dark/light cycle. The EO was emulsified with 0.5% Tween 80. Control animals were treated with a suspension of 0.5% Tween 80, in distilled water used as vehicle. All experiments were performed according to the Guide for the Care and Use of Laboratory Animals, from the US Department of Health and Human Services, Washington DC, 1985. The project was previously approved by the Animal’s Ethics Committee, of the Federal University of Piaui. All doses are expressed in milligrams per kilogram and were administered in a volume of 10 mL/kg injected intraperitoneally (i.p.). In a set of experiments, the treatment with EO [0.05 g/kg (EO 50), 0.1 g/kg (EO 100), or 0.15 g/kg (EO 150)] was delivered orally by gavage after suspension in polyoxyethylenesorbitan monooleate (Tween 80—0.5% v/v in saline; Synth, Brazil) in a constant volume of 10 mL/kg. Mice were treated for 30 consecutive days and 24 h after the drug administration, the animals were sacrificed by decapitation, and the hippocampus was dissected. After dissection, the area was used for determination of lipid peroxidation level, nitrite content, GSH concentration, and antioxidant enzymes. Tests were run between 8:00 a.m. and 6:00 p.m. in an illuminated room with controlled temperature and attenuated sound.

Determination of lipid peroxidation levels and nitrite content

For all of the experimental procedures, 10% (w/v) homogenates of the area of the brain investigated were prepared for all groups. Lipid peroxidation levels in the EO 50 (n = 7), EO 100 (n = 7), EO 150 (n = 7), and control (n = 10) groups were analyzed by method previously described by Draper and Hadley (1990). The results above were expressed as nmol of malondialdehyde (MDA)/g wet tissue.

To determine nitrite contents of EO 50 (n = 7), EO 100 (n = 7), EO 150 (n = 7), and control (n = 10) groups, the 10% (w/v) was determined based on the Griess reaction (Green et al., 1981). The results above were expressed as nM.

Determination of SOD activity

The hippocampus was ultrasonically homogenized in 1 mL 0.05 M sodium phosphate buffer, pH 7.0. Protein concentration was measured by the method of Lowry et al. (1951). The 10% homogenates were centrifuged (800 g, 20 min), and the supernatants were used to assay SOD, catalase, and GPx activities. SOD activity in the EO 50 (n = 7), EO 100 (n = 7), EO 150 (n = 7), and control (n = 10) groups was assayed by using xanthine and xanthine oxidase to generate superoxide radicals (Flohé & Otting, 1984). SOD activity in the samples was determined from this curve, and the results were expressed as U/mg of protein.

Determination of catalase activity

Catalase activity was measured in the EO 50 (n = 7), EO 100 (n = 7), EO 150 (n = 7), and control (n = 10) groups by the method that uses H₂O₂ to generate H₂O and O₂ (Chance & Maehly, 1955). Protein concentration was measured by the method of Lowry et al. (1951). Results are expressed as mmol/min/mg of protein (Maehly & Chance, 1954).

Determination of GPx activity GSH concentration

GPx was measured in the EO 50 (n = 7), EO 100 (n = 7), EO 150 (n = 7), and control (n = 10) groups using the method described by Sinet et al. (1975) and the protein concentration was measured according to the method described by Lowry et al. (1951). Results are expressed as mU/mg of protein.

GSH concentration was measured in the EO 50 (n = 7), EO 100 (n = 7), EO 150 (n = 7), and control (n = 10) groups using the method described by Sedlak and Lindsay (1968). The hippocampus was homogenized in 0.02 M ethylenediaminetetraacetic acid (EDTA). Results are expressed as μg/g tissue wet weight.

Statistical analyses

Results of neurochemical determinations were compared using ANOVA and the Student-Newman-Keuls t-test as post hoc test, because these results show a parametric distribution. In all situations, statistical significance was at P ≤ 0.05. The statistical analyses were performed with the software GraphPad Prism, Version 3.00 for Windows, GraphPad Software (San Diego, CA).

Results

GCMS analysis showed a mixture of monoterpenes among which limonene (52.77%), geranyl acetate (9.92%), and trans-limonene oxide (7.13%) were the main compounds in C. limon EO (Table 1).

Table 1.  Chemical composition and retention indices of the constituents of the Citrus limon essential oil.

RT (min)^a	Compounds^b	(%)	IK^c
4.785	Limonene	52.77	1025.5
6.365	Linalool	1.73	1100
7. 137	cis-Limonene oxide	2.68	1129.3
7.253	trans-Limonene oxide	7.13	1133.7
7.686	Citronellal	2.77	1150
10.141	Neral	6.85	1238.5
11.030	Geranial	5.49	1268.9
13.062	NI	6.62	1337.8
13.857	Nerol	4.04	1363.3
14.441	Geranyl acetate	9.92	1384.2
	Total identified	93.38

NI = Not identified.

^aRetention time.

^bCompounds listed in order of elution from an DB-5MS column.

^cKovats indices were calculated against n-alkanes (C9–C18) on a DB-5MS column.

Effects of C. limon EO on lipid peroxidation levels, nitrite concentrations, and GSH concentration in mice hippocampus are presented in Figure 1. Lipid peroxidation was markedly decreased 12, 13, and 27% in EO 150 [P < 0.001], EO 100 [P < 0.001], and EO 50 [P < 0.001] groups, when compared with corresponding values for the control group, respectively (Figure 1). No group that received injections of EO (0.1 g/kg) [P > 0.05] or (0.15 g/kg) [P > 0.05] showed alterations in lipid peroxidation level, when compared with the EO 50 group. However, the group treated with EO (0.15 mg/kg) showed decreases of 17 and 15% in lipid peroxidation level, when compared with the EO 50 [P < 0.001] and EO 100 [P < 0.001] groups, respectively.

Figure 1.  Citrus limon essential oil effects on status of lipid peroxidation, nitrite content, and glutathione reduced (GSH) levels in mice hippocampus. Results are mean ± SEM for the number of animals used in experimental procedures. The differences in experimental groups were determined by analysis of variance (ANOVA). ^aP < 0.05 as compared with control animals (ANOVA and Student-Newman-Keuls t-test); ^bP < 0.05 as compared with EO 50 group (ANOVA and Student-Newman-Keuls t-test); and ^cP < 0.05 as compared with EO 100 group (ANOVA and Student-Newman-Keuls t-test).

Post hoc comparison of means indicated significant decreases of 34, 30, and 23% in nitrite content of mice hippocampus pretreated with EO 150 [P < 0.001], when compared with the control, EO 50, and EO 100 groups, respectively (Figure 1). In groups treated with EO 100 [P < 0.001] and EO 50 [P < 0.05], important decreases of 15 and 6% in nitrite content of mice hippocampus were observed, when compared with the control group, respectively. In addition, in EO 100 group [P < 0.01] it was observed a significant decrease of 10% in nitrite content of mice hippocampus, when compared with the EO 50 group.

GSH concentration was not markedly changed in EO 100 and EO 50 groups, when compared with corresponding values for the control group [P > 0.05] (Figure 1). However, the group treated with EO (0.15 g/kg) showed an increase of 4% in GSH concentration in mice hippocampus, when compared with the control group [P < 0.01].

Figure 2 shows the effects of C. limon EO on SOD, catalase, and GPx activities in mice hippocampus. SOD activities in mice hippocampus was not markedly altered in EO 50 group [P > 0.05], when compared with the control group (Figure 2). Post hoc comparison of means indicated significant increases of 40, 46, and 28% in SOD activities [P < 0.0001] of hippocampus of mice pretreated with EO 150 as compared with the control, EO 50, and EO 100 groups, respectively (Figure 2). In addition, post hoc comparison of means indicated significant (9 and 14%) increases in hippocampal SOD activity of mice pretreated with EO 100 [P < 0.0001] when compared with control and EO 50 groups, respectively.

Figure 2.  Citrus limon essential oil effects on superoxide dismutase, catalase, and glutathione peroxidase activities in mice hippocampus. Results are mean ± SEM for the number of animals used in experimental procedures. The differences in experimental groups were determined by analysis of variance (ANOVA). ^aP < 0.05 as compared with control animals (ANOVA and Student-Newman-Keuls t-test); ^bP < 0.05 as compared with EO 50 group (ANOVA and Student-Newman-Keuls t-test); and ^cP < 0.05 as compared with EO 100 group (ANOVA and Student-Newman-Keuls t-test).

Catalase activities in mice hippocampus were markedly increased (18 and 29%) in EO 50 and EO 100 groups [P < 0.01], when compared with the control group, respectively (Figure 2). Post hoc comparison of means indicated significant increases of 59%, 36%, and 24% in hippocampus of mice pretreated with EO 150 in catalase activities [P < 0.001], when compared with control, EO 50, and EO 100 groups, respectively (Figure 2).

GPx activities in mice hippocampus were not markedly changed in EO 50 and EO 100 groups [P > 0.05], when compared with the control group (Figure 2). Post hoc comparison of means indicated significant increases of 10, 9, and 7% in hippocampus of mice pretreated with EO 150 in GPx activities [P < 0.001], when compared with control, EO 50, and EO 100 groups, respectively (Figure 2).

Discussion

Most of the studies with EO were carried out in organs and systems other than the CNS (Viana et al., 2000; Gottlieb et al., 2006). In the present work, lipid peroxidation level, nitrite content, GSH concentration, and antioxidant enzymes were determined in mice hippocampus, after the EO of C. limon administration. We have examined whether the treatment with EO (0.15 g/kg) decreases the lipid peroxidation level in mice hippocampus. Reactive oxygen species (ROS) generation is currently observed in brain (Castagne et al., 1999). In the absence of an efficient defense mechanism, these ROS cause peroxidation of membrane polyunsaturated fatty acids (Halliwell & Gutteridge, 1999). Brain is particularly susceptible to peroxidation due to the simultaneous presence of high levels of polyunsaturated fatty acids and iron (Halliwell & Gutteridge, 1989), which is the target of free radical damage. We have recorded the reduction in lipid peroxidation level in mice hippocampus, after 24 h of EO treatment. This is reflected by decrease in TBARS level that may be related to scavenge of free radicals by EO of C. limon.

It has been demonstrated that neurodegenerative diseases produced changes in nitric oxide metabolism and interacts with glutamatergic receptors to produce part of its stimulatory action on the CNS (Dymond & Crandall, 1976; Xavier et al., 2007; Freitas, 2009). The fall in nitrite content, after pretreatment with EO of C. limon, is most readily explained as a consequence of inhibiting formation of radicals, scavenges ROS, and lipid peroxidation products (Xavier et al., 2007). Increased free radicals and elevated nitrite levels observed in neurodegenerative disease may cause lipid peroxidation (Freitas et al., 2005; Ferreira et al., 2009).

SOD, catalase, and GPx activities can protect against neuronal damage involved in physiopathology of neurodegenerative diseases. It has been demonstrated that pretreatment with EO of C. limon (0.15 g/kg) produces an increase in antioxidant enzymatic activities in mouse hippocampus. The increase in antioxidant enzymes activities, after treatment with EO of C. limon, is most readily explained as a necessary consequence of inhibiting ROS formation and can induce the GSH (Michiels et al., 1994; Kudin et al., 2004).

Results of animal studies demonstrate that EO of medicinal plants can reduce damage to neurons caused by free radicals that are produced by inflammatory process in neurodegenerative diseases. A growing body of evidence has suggested that ROS generation may underlie the neurotoxic effects of neurodegenerative diseases (Barros et al., 2007; Liang et al., 2007; Oliveira et al., 2007; Militão et al., 2010). Several plant-derived products that exhibit direct antioxidant activity are also being shown to have anticonvulsant activity (McCord, 1995; Hsieh et al., 1999). Recently, our group detected antioxidant properties from lipoic acid, α-tocopherol, and ascorbic acid in pilocarpine-induced seizures in rats (Barros et al., 2007; Xavier et al., 2007; Santos et al., 2008; Militão et al., 2010). Using these data, the present study also investigated whether the EO of C. limon antioxidant actions would be involved in oxidative stress in mice hippocampus. For that, the activities of antioxidant enzymes, as well as the levels of nitrite, GSH, and lipid peroxidation were measured in brain hippocampus of mice.

Neurodegenerative diseases have been found to trigger the activation of membrane phospholipases, proteases, and nucleases, which cause degradation of membrane phospholipids, proteolysis of cytoskeletone proteins, and protein phosphorylation (Ono et al., 2000; Freitas et al., 2003; Akbas et al., 2005; Obay et al., 2008). In fact, in accordance to those studies, a recent work has shown that neurodegenerative diseases lead to an increase in lipid peroxidation and nitrite levels (Freitas, 2009; Santos et al., 2010). Treatment of animals with (0.05, 0.1, and 0.15 g/kg) of EO of C. limon significantly prevented elevations in lipid peroxidation and nitrite. It is also known that neurodegenerative diseases followed by an increase in lipid peroxidation and nitrite in brain tissue might be diminished by substances with antioxidant properties (Walz et al., 2000; Barros et al., 2007; Oliveira et al., 2007). Thus, our results suggest that, at least in part, the EO of C. limon bioprotective activities are also probably related to its antioxidant effects against lipid peroxidation in mice hippocampus.

ROS, such as superoxide, hydroxyl radical, and hydrogen peroxide (H₂O₂), are normally produced in the brain. The conversion of H₂O₂ to H₂O can be made by catalase and GPx, which involves the GSH, a cofactor of this enzyme. When produced in excess, ROS can also cause enzyme inactivation (Sharma et al., 2007). In fact, the current study showed that catalase activity in the EO 150 group was significantly higher than in the control group, demonstrating an alteration in neuroprotective brain defenses. EO treatment with lower doses (0.05 and 0.1 g/kg) preserved catalase activity at normal levels. These findings suggest that EO of C. limon could help the brain cells to counteract the neurodegenerative-induced ROS overproduction and the oxidative damage.

GSH is an essential tripeptide and endogenous antioxidant found in all animal cells. It reacts with the free radicals and can protect the brain from singlet oxygen, hydroxide radicals, and superoxide radical damage (Oliveira et al., 2007). Our results showed that the treatment of mice with EO of C. limon resulted in decreased levels of GSH in hippocampus. Interestingly, EO treatment with lower doses (0.05 and 0.1 g/kg) did not produce changes in GSH content, but the highest dose (EO 150) promoted increased GSH levels, suggesting that EO of C. limon could be possibly inducing an increase in GSH levels in mice hippocampus. In a similar way, in previous studies conducted by our group (Freitas et al., 2005; Xavier et al., 2007; Santos et al., 2010), we have found that antioxidant compounds themselves were able to induce an increased GSH level in mice hippocampus.

Nevertheless, whether EO of C. limon increases GSH levels, preventing depletion or inducing its synthesis, requires further investigation. Thus, our results are in agreement with previous studies that indicate increased oxidative stress during brain disorders (Oliveira et al., 2007; Ferreira et al., 2009), but they contrast with assays that show increased oxidative stress and decreased antioxidant defense during anticonvulsant therapy (Ono et al., 2000; Vinson et al., 2001; De Sousa et al., 2007).

Conclusions

In a previous study conducted by our group (data not shown), we have reported pharmacological activity from EO of C. limon at doses of 0.05 (32.41 mM/kg), 0.1 (64.82 mM/kg), and 0.15 g/kg (97.23 mM/kg). In the present study, the highest dose of EO of C. limon was needed for effective bioprotective actions against oxidative stress. The acute oral toxicity of EO of C. limon was already evaluated in mice. The results obtained and the LD₅₀ (~1.732 ± 0.1 g/kg) value represent a relative low toxicity from EO of C. limon, which was particularly related to their possible CNS depressor properties. In spite of the knowledge that EO constitution of monoterpenes are often considered to have low toxicity (Tanaka et al., 1996; De Sousa et al., 2007), we assume that additional studies are recommended to further ascertain the safety of EO of C. limon use.

Acknowledgement

We would like to thank the National Council of Technological and Scientific Development (CNPq/Brazil) [grant number 503354/2009-0] and the Research Supporting Foundation of State of Piaui (FAPEPI/Brazil) for the financial support.

Declaration of interest

The authors report no declarations of interest.