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ORIGINAL ARTICLE
Year : 2013  |  Volume : 3  |  Issue : 2  |  Page : 96-101

Evaluation of anxiolytic activity of vanillin in wistar albino rats


Department of Pharmacology, Kasturba Medical College, Manipal University, Mangalore, India

Date of Submission12-May-2012
Date of Acceptance22-Jun-2012
Date of Web Publication3-Jun-2013

Correspondence Address:
Mukta N Chowta
Department of Pharmacology, Manipal University, KMC l, Attavar, Mangalore - 575 001
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/2231-0738.112828

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   Abstract 

Background and Objectives: Vanillin is one of the primary chemical components of the extract of the vanilla bean. Vanillin has been claimed to possess various beneficial effects like anti-mutagenic, anti-nociceptive, anti-invasive and metastasis inhibiting potential by suppressing enzymatic activity of matrix metalloproteinase-9. However, literature research revealed no scientific data on its anxiolytic activity. Hence, this study was designed to evaluate the anxiolytic activity of vanillin in wistar albino rats. Materials and Methods: The rats were divided into five groups (n = 6). Vanillin administered at the dose of 10,100,200 mg/kg/day, orally was compared with the standard drug Diazepam (1.0 mg/kg/day, oral) fed for the latter 10 days. The two pharmacologically validated models, elevated plus maze and bright and dark arena were used. The data presented was analyzed using Kruskal Wallis followed by Mann-Whitney Test. P <0.05 was considered as statistically significant. Result: Vanillin significantly reduced the time spent in closed arm, increased the entries into open arm both in chronic and acute model of elevated plus maze (P < 0.05) in all three doses (10,100,200 mg/kg) used. Time spent in open arm, percentage ratio of open arm entries and number of rears in open arm also increased. Maximum effect was seen with 100 mg/kg. In bright and dark arena test there was an increase in number of entries, time spent and rears in bright chamber both in acute and chronic study at all doses (P < 0.05). Conclusion: The present study demonstrates the anxiolytic activity of vanillin in wistar albino rats.

Keywords: Anxiolytic, diazepam, vaniilin


How to cite this article:
Bhagwat V, Chowta MN, Shoeb A, Maskeri R, Venkatesh V, Rai A. Evaluation of anxiolytic activity of vanillin in wistar albino rats. Int J Nutr Pharmacol Neurol Dis 2013;3:96-101

How to cite this URL:
Bhagwat V, Chowta MN, Shoeb A, Maskeri R, Venkatesh V, Rai A. Evaluation of anxiolytic activity of vanillin in wistar albino rats. Int J Nutr Pharmacol Neurol Dis [serial online] 2013 [cited 2019 Nov 19];3:96-101. Available from: http://www.ijnpnd.com/text.asp?2013/3/2/96/112828


   Introduction Top


Anxiety is a cardinal symptom of many psychiatric disorders and an almost inevitable component of many medical and surgical conditions. Indeed it is a universal human emotion, closely allied with appropriate fear presumably serving psycho biologically adaptive purposes. [1] Anxiety is a normal emotional behavior. When it is severe or chronic, however, it becomes pathological and can precipitate or aggravate cardiovascular and psychiatric disorders. Although many drugs are available in modern medicine to treat anxiety disorders, they produce various systemic side effects or exhibit tolerance upon chronic use. [2]

Despite the advent of new molecules in the pharmacotherapy of anxiety, it is unfortunate that this disorder goes undiagnosed and untreated. Although the currently prescribed molecules provide some improvement in the clinical condition of the patient, it is at the cost of having to bear the burden of their adverse effects. The use of alternate medicines is increasing worldwide day by day in various diseases. Hence, finding newer and safer therapeutic agents would benefit the existing treatment modalities.

Vanilla (Vanilla planifolia), a monocotyledonous orchid native of Central America, is grown for the attractive aroma produced by its fruit. [3] Synthetic vanilla contains only one organic component - vanillin - the flavour and fragrance that we most associate with vanilla. Vanillin is one of the primary chemical components of the extract of the vanilla bean. It is a fine, white to slightly yellow crystal, usually needle-like, having an odour and taste suggestive of vanilla. Synthetic vanillin is used as a flavouring agent in foods, beverages, and pharmaceuticals. [4]

Other studies on vanillin has demonstrated that it has anti-mutagenic, [5] anti-invasive [6],[7] and metastasis inhibiting potential [6],[7] by suppressing enzymatic activity of matrix metalloproteinase-9. [7] It has also shown to have anti-nociceptive property in acetic acid induced visceral inflammation pain model, [8] this property has been attributed to α-adrenergic and opioid receptor mediate action. Based on the above activities on different receptors we undertook the above study to evaluate anxiolytic activity of vanillin in wistar albino rats.


   Materials and Methods Top


Animals

Male, Wistar albino rats weighing 150 to 180 g (90 to 110 days old) bred in the central animal house of Kasturba Medical College, Mangalore, were used for the study. They were housed in clean, clear, polypropylene cages in groups of four and maintained at 24.0 ± 2°C with 12 hrs light and dark cycle and had free access to food and water ad libitum. Animals were kept in experimental lab for seven days prior to experiment to acclimatize laboratory conditions. Each rat was used only once. Experiments were conducted between 9:00 to 14:00 hrs. The experimental protocol was approved by the Institutional Animal Ethical Committee (IAEC) and the study was conducted according to the Indian National Science Academy Guidelines for the use and care of experimental animals.

Drugs and dosage

Vanillin [IUPAC name 4-hydroxy-3-methoxybenzaldehyde, chemical formula (CH 3 O) (OH) C 6 H 3 CHO, molecular weight of 152.15] obtained from HiMedia laboratories, India. Standard anxiolytic drug, Diazepam (Ranbaxy Ltd. India). All the drugs were suspended in 14% DMSO and administered in a dose of 10, 100 and 200 mg/kg. Each drug solution was prepared freshly just before the administration. Drugs and vehicles were administered orally (10 ml/kg). The doses of each drug was selected on the basis of earlier findings of vanillin. [8] Drugs, dosage and number of animals used per treatment are shown in [Table 1]. In acute study, drugs/vehicle was administered 60 min prior to experiment. In chronic study they were administered once daily for 10 days and the last dose was given on the 10 th day, one hour in prior to experiment.
Table 1: The animals are divided into fi ve groups with six animals in each group

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Apparatus

Elevated plus maze

The wooden maze consists of two open arms (length 50 cm × breadth 10 cm) and two closed arms of the same size (height 40 cm). The arms of the same type are opposite to each other, with a central square of 10 cm. The maze is elevated to height of 50 cm above the floor. [9]

Bright and dark arena

The apparatus consists of an open top wooden box. Two distinct chambers, a black chamber (20 × 30 × 35 cm) painted black and illuminated with dimmed red light and a bright chamber (30 × 30 × 35 cm) painted white and brightly illuminated with 100 W white light source located 17 cm above the box. The two chambers are connected through a small open doorway (7.5 × 5 cm) situated on the floor level at the centre of the partition. [9]

Behavioral assessment

Each animal was tested initially in elevated plus maze and then, in light and dark box in a single setting. In acute study observations were done after one hour of drug or vehicle administration. In chronic study observations were done 60 min after the last dose on the 10 th day of drug or vehicle administration, each animal was placed in the centre square of the plus maze, facing one of the open arms. The number of entries into and the time spent in open and closed arms and the number of rears in each arm in a five-minute period was noted. [9]

Following the elevated plus maze test, the animal was placed at the centre of the brightly lit arena in the light and dark box. The number of entries into and the time spent in the bright arena, the number of rears in the bright and dark arenas and the duration of immobility was noted during the 5 minutes of observation.

Following each trial, the apparatus was cleaned with hydrogen peroxide to mask the odour left by the animal in the previous experiment. Hand operated counters and stop watches were used to score the behavior of animals.

Statistical analysis

The data was analysed by the Kruskal-Wallis Test followed by Mann Whitney Test. P < 0.05 was considered to be statistically significant.


   Results Top


Elevated plus maze

Acute study: The results given in [Table 2] indicate that the diazepam (1.0 mg/kg) treated rats showed a significant increase in the percentile ratio of open arm to total arm entries (0.63 ± 0.02), time spent in the open arms (215.5 ± 12.8). They showed a reduction in the time spent in the closed arms (59.5 ± 13.8). Vanillin treated rats exhibited a significant (P < 0.05) increase in the percentile ratio of open arm to total arm entries (0.65 ± 0.085), time spent in the open arms (173.50 ± 19.59) but decrease the time spent in the closed arms (126.50 ± 19.60) at a dose 100 mg/kg. There was a significant (P < 0.05) increase in number of rears in open arm at a dose of 100 mg/kg (8.16 ± 2.20).
Table 2: Effect of diazepam and vanillin on behavior of rats in elevated plus maze (acute study)

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Chronic study: The results given in [Table 3] indicate that diazepam (1.0 mg/kg) treated rats significantly (P < 0.05) increased the number of open arm entries (5.50 ± 0.34), percentile ratio of open arm entries (0.60 ± 0.011), time spent in open arm (229.6 ± 14.38) and number of rears in the open arms (6.16 ± 0.87) and reduction in time spent in closed arm (59.0 ± 11.33) as compared to control. Vanillin treated rats exhibited a significant (P < 0.05) increase in number of open arm entries (6.83 ± 1.04), percentile ratio of open arm to total arm entries (0.77 ± 0.05), time spent in open arm (173.50 ± 19.59) and number of rears in open arm (10.33 ± 1.89) at a dose of 100 mg/kg. There were a significant (P < 0.05) increase in percentile ratio of open arm to total arm entries (0.77 ± 0.05) and reduction in time spent in closed arm (35.16 ± 32.58) as compared to diazepam at a dose of 100 mg/kg.
Table 3: Effect of diazepam and vanillin on behaviour of rats in elevated plus maze (chronic study)

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Bright and dark arena

Acute study: The results given in [Table 4] indicate that the diazepam (1.0 mg/kg) and vanillin at all the doses, treated rats significantly (P < 0.05) increased the number of bright chamber entries, time spent and the rears in bright arena as compared to control. Number of rears in bright chamber was significant (P < 0.05) at 100 mg/kg vanillin as compared to diazepam.
Table 4: Effect of vanillin on behavior of rats in light and dark box (acute study)

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Chronic study: The results given in [Table 5] indicate a significant (P < 0.05) increase the number of bright chamber entries, time spent and number of rears in bright arena in diazepam treated group of animals when compared to vehicle treated group. Vanillin at all the doses (10, 100, 200 mg/kg) of repeated administration significantly increased the number of entries, time spent, and number of rears in bright arena when compared with control. Vanillin at dose of 100 and 200 mg/kg significantly (P < 0.01) increase the time spent in bright chamber when compared with diazepam [Figure 1], [Figure 2], [Figure 3] and [Figure 4].
Figure 1: Effect of diazepam and vanillin on behavior of rats in elevated plus maze (acute study)

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Figure 2: Effect of diazepam and vanillin on behaviour of rats in elevated plus maze (chronic study)

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Figure 3: Effect of vanillin on behavior of rats in light and dark box (acute study)

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Figure 4: Effect of vanillin acid on behavior of rats in light and dark box (chronic study)

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Table 5: Effect of vanillin acid on behaviour of rats in light and dark box (chronic study)

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   Discussion Top


The two experimental models of anxiety, elevated plus maze and bright and dark arena, are based on the assumption that unfamiliar, non-protective and brightly lit environmental stress provokes inhibition of normal behavior. This normal behavioral inhibition is further augmented in the presence of fear or anxiety like state.

In the elevated plus maze, the open arms are more fear provoking than the closed arms. The ratio of entries, time spent and rearing behavior in open arms to closed arms reflects the safety of closed arms with relative fearfulness of open arms. [10] The reduction in entry, time spent, rearing in open arms, ratio of open arm to total arm entries and increased defecation are the indications of high level of fear or anxiety. Anxiolytic drugs increase the proportion of entries, time spent and rearing in open arms. They also increase the ratio of open arm to total arm entries.

In the light and dark box paradigm, the brightly lit environment is a noxious environment stressor that inhibits the exploratory behavior of rodents. Reduction in the number of entries, time spent and rearing behavior in the light chamber were regarded as markers of anxiety. [11] Rearing reflects an exploratory tendency of the animal that can be reduced due to a high level of fear. [12]

In the present study, rats that received both in acute and chronic study diazepam showed a significant increase in the time spent and the rears in open arms and the percentage ratio of open/total arm entries. They showed a decrease in time spent in closed arms of elevated plus maze. They also showed an increase in the time spent and the rears in bright arena in the bright and dark arena paradigm. All these suggest decreased fear, an increased exploratory behavior and the behavioral dis-inhibitory effect of the standard anxiolytic. Diazepam and Vanillin are known to act through the GABA receptors; the role of GABA in anxiety is well established. [13]

The test compound, vanillin increased the time spent and rearing in open arms and also increased the percentile ratio of open arm to total arm entries in the elevated plus maze paradigm. The anti-anxiety effects of vanillin in the elevated plus maze were comparable with those following the administration of diazepam in this task-ie, activity and latency ratios increased compared with vehicle. In bright and dark paradigm, the test drug significantly increased the time spent in light arena; rears in both light and dark arena and transition between chambers were comparable with those following the administration of diazepam. All these behavioral changes in both paradigms are suggestive of decreased fear, decreased aversion to bright light and increased exploratory behavior of the animal.

Vanillin, a compound widely used in foods, beverages, cosmetics and drugs, has been reported to exhibit multifunctional effects such as anti-mutagenic, anti-angiogenetic, anti-colitis, anti-sickling, and anti-analgesic effects. Tai A et al. by using multiple antioxidant assays shown that vanillin also possess strong antioxidant activity. This antioxidant activity may explain the anxiolytic like action of vanillin in rats.

Oxidative stress (OS) represents a loss of balance in oxidation-reduction reactions.

It is characterized by the reduced ability of the antioxidant defense system to efficiently eliminate the excess of the oxygen-derived species production, eliciting the toxicity of oxygen and its detrimental effects. [14] GABAergic and serotoninergic systems are considered among the principal regulatory systems of anxiety. However, recent studies have shown that patients with anxiety disorders have higher activity levels of the enzymes like superoxide dismutase and glutathione peroxidase as well as higher lipid peroxidation activity. [14] Hence, oxidative metabolism also regarded as a plausible pathway that can affect the regulation of anxiety. When the production of ROS prevails over the brain defense systems, the lipid-rich constitution of brain may favor lipid peroxidation, constituting a free radical chain reaction that may result in decrease in membrane fluidity and damage in membrane proteins inactivating receptors, enzymes and ion channels, even disrupting membrane integrity resulting eventually in cell death. In addition to oxidative damage of neuronal membrane lipids and proteins, oxidation of other sensitive components such as nucleic acids and neurotransmitters can occur.

As the test drug possesses anxiolytic-like effect similar to that of diazepam, it can be further studied by using other models of anxiety like open field and rota rod to determine its possible use in human beings.


   Conclusion Top


The present study demonstrates anxiolytic activity of vanillin at a dose of 100 mg/kg was comparable to diazepam. Vanillin's antianxiety activity could be due to its antioxidant property. Further studies are required to elucidate and pinpoint the mechanism of action of vanillin in anxiety.

 
   References Top

1.Baldessarini RJ. Drug therapy of depression and anxiety disorders. In: Brunton LL, Lazo JS, Parker KL, editors. Goodman and Gilman's the pharmacological basis of therapeutics, New York: McGraw Hill; 2006. p. 452-4.  Back to cited text no. 1
    
2.Pari L, Maheshwari JU. Hypoglycaemic effects of Musa sapientum L in alloxan induced diabetic rats. J Ethnopharmacol 1999;38:1-5.  Back to cited text no. 2
    
3.Odoux E, Escoute J, Verdeil JL, Brillouet JM. Localization of β-D_Glucosidase activity and Glucovanillin in vanilla Bean (Vanilla planifolia Andrews). Ann Bot 2003;92:437-44.  Back to cited text no. 3
    
4.Sweetman SC. Martindale: The complete drug reference. 36 th ed. London: Pharmaceutical Press; 2009.  Back to cited text no. 4
    
5.Kinga AA, Shaughnessy DT, Murea K, Leszczynska J, Ward WO, Umbach DM, et al. Antimutagenicity of cinnamaldehyde and vanillin in human cells: Global gene expression and possible role of DNA damage and repair. Mutat Res 2007;616:60-9.  Back to cited text no. 5
    
6.Lirdprapamongkol K, Sakurai H, Kawasaki N, Choo M, Saitoh Y, Aozuka Y, et al. Vanillin suppresses in vitro invasion and in vivo metastasis of mouse breast cancer cells. Eur J Pharm Sci 2005;25:57-65.  Back to cited text no. 6
    
7.Liang J, Wu S, Lo H, Hsiang C, Ho T. Vanillin inhibits matrix metalloproteinase-9 expression through down-regulation of nuclear factor-kappaB signaling pathway in human hepatocellular carcinoma cells. Mol Pharmacol 2009;75:151-7.  Back to cited text no. 7
    
8.Park S, Sim Y, Choi S, Seo Y, Kwon M, Lee J, et al. Anti-nociceptive profiles and mechanisms of orally administered vanillin in the mice. Arch Pharm Res 2009;32:1643-9.  Back to cited text no. 8
    
9.Krishna HN, Sangha RB, Misra N, Pai M. Antianxiety activity of NR-ANX-C, a polyherbalpreparation in rats. Indian J Pharmacol 2006;38:330-5.  Back to cited text no. 9
    
10.Pellow G, Chopin P, File SE, Briley M. Validation of open-closed arm entries in elevated plus maze as a measure of anxiety in the rat. J Neurosci methods 1985;14:149-67.  Back to cited text no. 10
    
11.Costall B, Domeney AM, Gerrard PA, Kelly ME, Naylor RJ. Zacopride: Anxiolytic profile in rodent and primate models of anxiety. J Pharm Pharmacol 1988;40:302-5.  Back to cited text no. 11
    
12.Van der poel AM. A note on "stretched attention", A behavioral element indicative of an approach-avoidance conflict in rats. Animal Behav 1979;27:446-50.  Back to cited text no. 12
    
13.Pemminati S, Swati B, Shreyasi C, Chandrasekhar R, GopalaKrishna HN, Pai MR. Anxiolytic activity of ethanolic extract of leaves of Ocimum sanctum in rats. Drug Invent Today 2010;2:115-8.  Back to cited text no. 13
    
14.Akihiro T, Takeshi S, Futoshi Y, Hideyuki I. Evaluation of antioxidant activity of vanillin by using multiple antioxidant assays. Biochim Biophys Act 2010;1810:170-7.  Back to cited text no. 14
    


    Figures

  [Figure 1], [Figure 2], [Figure 3], [Figure 4]
 
 
    Tables

  [Table 1], [Table 2], [Table 3], [Table 4], [Table 5]


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