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ORIGINAL ARTICLE
Year : 2015  |  Volume : 6  |  Issue : 1  |  Page : 37-39  

Assessment of aqueous extract of Lemon verbena on anxiety-like behavior in rats


Neurophysiology Research Center, Hamadan University of Medical Sciences, Hamadan, Iran

Date of Web Publication20-May-2015

Correspondence Address:
Mojgan Veisi
Neurophysiology Research Center, Hamadan University of Medical Sciences, Hamadan
Iran
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Source of Support: The study was supported by a grant (No. 9207232346) by Hamadan University of Medical Sciences, Iran., Conflict of Interest: None


DOI: 10.4103/0976-9234.157390

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   Abstract 

Aim: The purpose of the present study was to evaluate the anxiolytic effects of the aqueous extract of Lemon verbena on rats. Materials and Methods: In this study, aqueous extract of Lemon verbena leaves was prepared, and then male Wistar rats (200-230 g) were divided randomly into six experimental groups. Each group received a single intraperitoneal (IP) injection of saline, aqueous extract of Lemon verbena (10 mg/kg, 100 mg/kg, 500 mg/kg, 1,000 mg/kg)or diazepam, respectively. The level of anxiety was asserted by an elevated plus maze (EPM) 20 min after treatment. Statistical Analysis Used: Results were analyzed by one-way analysis of variance (ANOVA) and Tukey's post-hoc test. The results were expressed as mean ± standard error of mean (SEM). The differences were considered significant at P < 0.05. Results: The results of this study showed that there was a significant difference in the number of entrances into the open arms, spent time in the open arms and number of total entrances into the open and closed arms of the EPM between control, diazepam and extract-receiving groups (10 mg/kg, 100 mg/kg, 500 mg/kg, 1,000 mg/kg) and control. The numbers of entrances into the open arms, spent time in the open arms in extract receiving groups were significantly less than control group. Conclusion: These results indicated that acute administration of aqueous extract of Lemon verbena (≥10 mg/kg) increased anxiety-like behavior in rats in the EPM.

Keywords: Anxiety, elevated plus maze, rat, vervain


How to cite this article:
Veisi M, Shahidi S, Komaki A, Sarihi A. Assessment of aqueous extract of Lemon verbena on anxiety-like behavior in rats. J Pharm Negative Results 2015;6:37-9

How to cite this URL:
Veisi M, Shahidi S, Komaki A, Sarihi A. Assessment of aqueous extract of Lemon verbena on anxiety-like behavior in rats. J Pharm Negative Results [serial online] 2015 [cited 2019 Sep 22];6:37-9. Available from: http://www.pnrjournal.com/text.asp?2015/6/1/37/157390


   Introduction Top


Anxiety is a common psychiatric disorder in society and includes an unpleasant feeling in a person. The effects of various plants have been studied to reduce anxiety, including Melissa officinalis[1],[2] and Passiflora incarnata Linneaus. [3] Anxiolytic activity of these plants is dependent on the activity of flavonoids. [2],[3] Isolated flavonoids from plants that are used traditionally for tranquilizers have a selective affinity for central benzodiazepine receptors (BDZ-Rs). [4],[5] Lemon verbena (Lippia citriodora) is a perennial shrub belonging to the family of Verbenaceae. This plant is rich in flavonoids [6] and its antioxidant effect has been proved by several studies. [7],[8],[9] Lemon verbena leaves are used in herbal tea or in standard tea in place of actual lemon (as is common with Moroccan tea). [10] Lemon verbena has also been used traditionally to reduce stress and anxiety; [11] however, no scientific study has been done to prove this claim. The aim of the present study was to evaluate the effect of aqueous extract of Lemon verbena on anxiety-like behavior in male rats by using the elevated plus maze (EPM).


   Materials and methods Top


Preparation of the extract

Dried leaves of Lemon verbena were purchased from Mehr Giahe Kosar Company (Mashhad, Iran). The chopped leaves were soaked in 2 L of distilled water for 12 h at room temperature. Then the aqueous material was filtered, dried by hot air ovens (50°C), and turned into a powder. The powder of aqueous extract of Lemon verbena was stored in a refrigerator. At the time of injection, the required amount of extract powder was weighed, and dissolved in a certain amount of normal saline.

Animals and experimental groups

Male Wistar rats (weighing 220-250 g) were bought from the Pasteur Institute of Iran. All animals were kept in a room under controlled conditions of 24 ± 1°C and 12/12 reversed light-dark cycle. They were housed in triple groups in polypropylene cages with free access to water and food ad libitum. All procedures were done according to the guide for the caring and using of laboratory animals published by United States Nation Institutes of Health (NIH Publication No. 85-23, revised 1985). The rats were randomly divided into four experimental groups, one control group, and one diazepam-receiving group of eight (n = 8). The control group received 0.5 mL of intraperitoneal (IP) normal saline and the four experimental groups received IP Lemon verbena aqueous extract of 10 mg/kg, 100 mg/kg, 500 mg/kg, and 1000 mg/kg, and the diazepam-receiving group received 0.3 mg/kg of IP diazepam.

Elevated plus maze model

The EPM has been accepted as an animal model for testing anxiolytic substances. [12],[13],[14],[15] The EPM apparatus consists of two closed arms (50 × 10 × 40 cm) and two open arms (50 × 10 cm). The maze is elevated 50 cm from the floor. [16] Twenty minutes after the treatment with normal saline (for the control group) or the Lemon verbena extract (10 mg/kg, 100 mg/kg, 500 mg/kg, 1000 mg/kg), diazepam (0.3 mg/kg) was injected IP, rats were placed at the center of the maze so that their heads were toward the open arms. The number of entrances into the open arms, time spent in the open arms, and number of total entrances into the open and closed arms were recorded for 10 min by using closed circuit camera. The tests were performed between 9 AM and noon. An increase in both the number of entrances into the open arms and time spent in the open arms are indicators of anxiety reduction in rats. [17]

Statistical analysis

The obtained results for different groups were analyzed by one-way (ANOVA) and if applicable, by Tukey's post-hoc test. The results were expressed as mean ± standard error of mean (SEM). The differences were considered significant at P < 0.05.


   Results Top


The results of this study showed that there was a significant difference between the number of entrances into the open arms [F (5, 42) =20.399; P < 0.001] [Figure 1], spent time in the open arms [F (5, 42) =14.916; P < 0.001] [Figure 2], and total number of entrances into the open and closed arms [F (5, 42) =22.166; P < 0.001] [Figure 3] of extract-receiving groups (10 mg/kg, 100 mg/kg, 500 mg/kg, 1000 mg/kg), the control group, and the diazepam-receiving group (P < 0.001). 
Figure 1: The number of entrances into the open arms in the effect of Lemon verbena extract (10, 100, 500, 1000 mg/kg) on anxiety. ***P < 0.001, (ANOVA), in comparison to saline. ###P < 0.001 in comparison to Diazepam. Values represent the means ± SEM. (n = 8)

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Figure 2: Time spent in the open arms in the effect of Lemon verbena extract (10 mg/kg, 100 mg/kg, 500 mg/kg, 1000 mg/kg) on anxiety. ***P < 0.001, (ANOVA), in comparison to saline. ###P < 0.001 in comparison to Diazepam. Values represent the means ± SEM. (n = 8)

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Figure 3: Total number of entrance into the open and close arms in the effect of Lemon verbena extract (10 mg/kg, 100 mg/kg, 500 mg/ kg, 1000 mg/kg) on anxiety. ***P < 0.001, (ANOVA), in comparison to saline. ###P < 0.001 in comparison to Diazepam. Values represent the means ± SEM. (n = 8)

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   Discussion Top


The fear of height induces anxiety in the rats when they are placed on the EPM. Anxiolytic agents increase the number of entrances and spent time in the open arms by the animals. [18] In this study, results indicated that treatment with aqueous extract of Lemon verbena with doses ≥100 mg/kg caused a decrease in the number of entrances and spent time in the open arms by the animal. However, there was a reduction in the motor activity of rats. On the other hand, observations showed that administration of doses of aqueous extract of Lemon verbena dependently caused oversleeping in rats. Previous studies showed that flavonoids caused a significant reduction of walking time and increased the number of sleeping animals. [19] Flavonoids have a selective affinity for central benzodiazepine receptors (BDZ-Rs). [4],[5] Benzodiazepines in low doses cause anxiolytic effect, including diazepam (0.3 mg/kg), but with high doses show a more hypnotic effect, so it is possible that the doses less than 10 mg/kg of extract induce anxiolytic behavior in rats.


   Conclusion Top


These results indicate that acute administration of aqueous extract of Lemon verbena with doses 10 mg/kg, 100 mg/kg, 500 mg/kg and 1,000 mg/kg would increase anxiety-like behaviors in rat in EPM.


   Acknowledgment Top


This work was a part of a thesis to achieve a Master of Sciences degree and it was done in the Neurophysiology Research Center of Hamadan University of Medical Sciences, Iran.

 
   References Top

1.
Ibarra A, Feuillere N, Roller M, Lesburgere E, Beracochea D. Effects of chronic administration of Melissa officinalis L. extract on anxiety-like reactivity and on circadian and exploratory activities in mice. Phytomedicine 2010;17:397-403.  Back to cited text no. 1
    
2.
Kennedy DO, Wake G, Savelev S, Tildesley NT, Perry EK, Wesnes KA, et al. Modulation of mood and cognitive performance following acute administration of single doses of Melissa officinalis (Lemon balm) with human CNS nicotinic and muscarinic receptor-binding properties. Neuropsychopharmacology 2003;28:1871-81.  Back to cited text no. 2
    
3.
Dhawan K, Kumar S, Sharma A. Anti-anxiety studies on extracts of Passiflora incarnata Linneaus. J Ethnopharmacol 2001;78:165-70.  Back to cited text no. 3
    
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Medina JH, Viola H, Wolfman C, Marder M, Wasowski C, Calvo D, et al. Overview-flavonoids: A new family of benzodiazepine receptor ligands. Neurochem Res 1997;22:419-25.  Back to cited text no. 4
    
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Medina JH, Viola H, Wolfman C, Marder M, Wasowski C, Calvo D, et al. Neuroactive flavonoids: New ligands for the benzodiazepine receptors. Phytomedicine 1998;5:235-43.  Back to cited text no. 5
    
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Gomes PC, Oliveira HR, Vicente AM, Ferreira MF. Production, transformation and essential oils composition of leaves and stems of Lemon verbena [Aloysia triphylla (L′Herit.) Britton] grown in Portugal. Rev Bras Pl Med 2006;8:130-5.  Back to cited text no. 6
    
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Carrera-Quintanar L, Funes L, Viudes E, Tur J, Micol V, Roche E, et al. Antioxidant effect of lemon verbena extracts in lymphocytes of university students performing aerobic training program. Scand J Med Sci Sports 2012;22:454-61.  Back to cited text no. 7
    
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Funes L, Carrera-Quintanar L, Cerdán-Calero M, Ferrer MD, Drobnic F, Pons A, et al. Effect of lemon verbena supplementation on muscular damage markers, proinflammatory cytokines release and neutrophils′ oxidative stress in chronic exercise. Eur J Appl Physiol 2011;111:695-705.  Back to cited text no. 8
    
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Valentão P, Fernandes E, Carvalho F, Andrade PB, Seabra RM, de Lourdes Basto M. Studies on the antioxidant activity of Lippia citriodora infusion: Scavenging effect on superoxide radical, hydroxyl radical and hypochlorous acid. Biol Pharm Bull 2002;25:1324-7.  Back to cited text no. 9
    
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Aloysia citrodora, from Wikipedia, the free encyclopedia. Available from: http://en.wikipedia.org/wiki/Aloysia_citrodora. [Last accessed on 2014 Apr 20].  Back to cited text no. 10
    
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Pascual ME, Slowing K, Carretero E, Sánchez Mata D, Villar A. Lippia: Traditional uses, chemistry and pharmacology: A review. J Ethnopharmacol 2001;76:201-14.  Back to cited text no. 11
    
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Hogg S. A review of the validity and variability of the elevated plus-maze as an animal model of anxiety. Pharmacol Biochem Behav 1996;54:21-30.  Back to cited text no. 12
    
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Carobrez AP, Bertoglio LJ. Ethological and temporal analyses of anxiety-like behavior: The elevated plus-maze model 20 years on. Neurosci Biobehav Rev 2005;29:1193-205.  Back to cited text no. 13
    
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Dawson GR, Tricklebank MD. Use of the elevated plus maze in the search for novel anxiolytic agents. Trends Pharmacol Sci 1995;16:33-6.  Back to cited text no. 14
    
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Pellow S, Chopin P, File SE, Briley M. Validation of open: Closed arm entries in an elevated plus-maze as a measure of anxiety in the rat. J Neurosci Methods 1985;14:149-67.  Back to cited text no. 15
    
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Kulkarni SK, Reddy DS. Animal behavioral models for testing antianxiety agents. Methods Find Exp Clin Pharmacol 1996;18:219-30.  Back to cited text no. 16
    
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Parasuraman S, Sujithra J, Syamittra B, Yeng WY, Ping WY, Muralidharan S, et al. Evaluation of sub-chronic toxic effects of petroleum ether, a laboratory solvent in Sprague-Dawley rats. J Basic Clin Pharm 2014;5:89-97.  Back to cited text no. 17
    
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Kumar S, Sharma A. Anti-anxiety activity studies of various extracts of Turnera aphrodisiaca ward. J Herb Pharmacother 2005;5:13-21.  Back to cited text no. 18
    
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Jiang JG, Huang XJ, Chen J, Lin QS. Comparison of the sedative and hypnotic effects of flavonoids, saponins, and polysaccharides extracted from Semen Ziziphus jujube. Nat Prod Res 2007;21:310-20.  Back to cited text no. 19
    


    Figures

  [Figure 1], [Figure 2], [Figure 3]


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