Antianxiety Activity of Ethanolic Extract of Triticum aestivum in Acute Stress Induced Wistar Albino Rats by Light and Dark Apparatus

Swapna Mahapatra¹, Prasanta Kumar Nayak², Subrat Kumar Sahany³ and Saroj Shekhar Rath^4*

^*Correspondence: Saroj Shekhar Rath, Department of Pediatrics, MKCG Medical College, Berhampur, Odisha, India, Email:

Author info »

Introduction

Stress has become an integral part of day to day life. Stress targets nervous system along with the immune system, metabolic and cardiovascular system etc which are affected by both adaptive and mal-adaptive responses to stress [1]. Stress induced generation of reactive oxygen species (ROS) in brain is a contributing factor for alteration in motor, visceral, endocrine and behavioural performances. So, many researchers have explained that anti-oxidants ameliorate neurobehavioral and endocrine function by counteracting oxidative damage [2]. Triticum aestivum, commonly known as wheat grass has been used since ancient times in folk medicine for its medicinal properties. A number of scientific reports show that juice of wheat grass has potent anti-ulcer [3], antioxidant [4], and anti-arthritic [5], antidiabetic [6] effects. Recently its neuroprotective effect on β-amyloid induced cell death and memory impairment has been studied in rats [7]. Basing on this, present study was undertaken to evaluate the anxiolytic effect of ethanolic extract of the test drug, Triticum aestivum against acute stress in wistar albino rats.

Methodology

In the present study, Thirty wistar albino rats of either sex weighing between 100-150 gm were selected. The animals were randomly divided into five (5) different groups of six rats in each (n=6) group. All animals were hygienically housed at room temperature and under standard laboratory condition of 12hr light and dark cycle in the animal house of department of pharmacology, M.K.C.G. Medical College, Berhampur, Odisha. The study was conducted after taking permission from Institutional Animal Ethical Committee (IAEC). The study period was three months.

Before the experiment, all animals were acclimatized to standard laboratory conditions for 7 days and had free access to food and water throughout the period of experiment. They were used only once in the experiment. All experiments were carried out in the day time from 10:00hr and 16:00hr. In our study 300 grams of Wheat grass powder in pure form was procured from Girme’s Wheatgrass pvt. Ltd. The powder was subjected to soxhlet extraction with 99.99% ethanol for 24hrs.The alcoholic extract was then subjected to evaporation in a beaker on a water bath maintained at 500c till a thick paste of extract remained in the beaker. It was stored in refrigerator at 40C and used throughout the experiment. The yield [1] obtained was 8.7%. During the time of experiment fresh solution was prepared with 5% DMSO for daily administration.

For evaluating the antianxiety activity, the test drug or reference standard drug or vehicle was administered to rats before the induction of stress as per the treatment protocol for 14 days. Stress was induced to rats for short time (Acute stress) and these rats were treated as stress control group for comparison.

Acute stress induction [8]

Immobilization of rats was done in a wire mesh for 2 hours called restraint stress.

For testing anxiety, laboratory model used is Black & White Test Box /Light-Dark Box test [9]. It consists of a wooden box measuring 44X21X21 cm. One third of it, was darkened with black spray over its all surfaces. A partition containing a 13cm long X 5cm high opening separates the dark one third, from bright two third of the box. The light compartment was illuminated with a 60 watt bulb, located 40cm above the centre of it (Table 1). Rats were individually placed in light compartment of the box and loco motor activity was observed for 10min as follows.

Table 1: Experimental design for light and dark box test.

✓Number of exploratory rearing in the light and dark compartments. ✓ Number of transmissions between the two compartments. ✓ Time spent in the light and dark compartments. ✓ Latency of the initial movement from the light and dark compartments.

Following behavioural tests, rats from each laboratory test model groups, were sacrificed by cervical dislocation. Whole brain was dissected out and weighed individually .Then it was homogenized with 10ml of Normal saline. The brain homogenate was subjected estimation of MDA and SOD. Stomach was dissected out by dividing at gastro-esophageal junction and gastroduodenal junction. Then stomach was opened along the greater curvature and washed gently in running water.

The gastric mucosa was displayed on a wax platform and coded to eliminate bias. Using a magnifying glass the ulcer scores were recorded. Scoring:0 - Normal coloured stomach, 0.5 - Red coloration,1- Spot ulceration,1.5 - Hemorrhagic streak, 2 – ulcers, 3-Perforations. Ulcer index was calculated for each rat by adding the scores and recorded.

Statistical analysis

The statistical software Graphpad prism 5 was used for statistical calculation. The parametric data were analysed by one way ANOVA followed by Tukey’s multiple comparison‘t’–test. The data of Non-parametric type were analysed using Kruskal Wallis one way ANOVA followed by Dunn’s multiple comparison for comparison of 3 or more group. The p<0.05 was considered as significant.

Results

The latency of initial movement with acute stress control rats was significantly shorter [p<0.01] than normal control groups. [Acute:3.2 ± 0.48 vs 6.8 ± 1.0 esc]. Diazepam, the reference standard drug, prolonged the latency of initial movement in acute stress model rats [8.8 ± 0.70] significantly [p<0.001]. So, also with TAE- 200 mg/kg treated stressed rats which is comparable to that of normal control rats [p>0.05] (Table 2).

Table 2: Effect of drugs on latency of initial movement in rats exposed to acute stress.

The vehicle treated rats when exposed to acute stress, the time spent in light box was significantly reduced and stay in dark box was significantly longer than that of normal control group of rats in respective compartments. [Light box: 6.16 ± 0.47 vs 26.17 ± 3.83 sec; Dark box: 593.8 ± 0.47 vs 573.8 ± 3.83 sec respectively]. Diazepam treated group of rats showed a longer stay (77.17 ± 0.51) sec in the light box in comparison to stress control rats [p<0.001]. The stressed rats pre-treated with TAE-150 mg/kg also showed a significant change in time spent in light and dark boxes both in acute stress model [p<0.05]. On Pre-treatment with TAE-200 mg/kg, the time spent in light box in stressed rats was significantly longer [p<0.001] (43.17 ± 3.0). This reveals TAE-200 mg/kg has significant anxiolytic effect just like Diazepam (Table 3).

Table 3: ffect of different treatments on time spent in light and dark compartments.

Stress control rats in dark box showed a highly significant increase in exploratory rearing [p<0.001] compared to normal control rats (18 ± 0.48 vs. 7.5 ± 0.56) (Table 4). Diazepam and TAE-200 mg/kg treated stressed rats had a significantly higher number of exploratory rearing (8.2 ± 0.95 and 9.3 ± 0.42 respectively) in comparison to stress control rats [p<0.05] (Table 5). There was no significant change in the brain MDA and SOD levels of rats on exposure to acute stress [p>0.05] (Table 6).

Table 4: Effect of acute stress on exploratory behaviour in rats in light and dark test.

Table 5: Effect of drugs on behaviour of rats exposed to acute stress.

Table 6: Effect of different drugs on ulcer index of rats exposed to acute stress.

Discussion

The word stress has been associated with sensation of discomfort. It is a Psycho-physiological process induced by stressor agents and a physiological reaction that induces loss of homeostasis and rupture of psychological balance resulting in various physical and mental disorders [10,11]. Stress induces a variety of CNS disorders such as anxiety, depression, anorexia and elevated corticosterone level as well as plasma glucose concentrations in animals and humans [12,13] .Even It is reported that prolonged anxiety, emotional stress and trauma are known to cause severe gastric irritation [14]. The report of Imrana Tabassum et al, 2010 states that, restraint stress for a brief period (2-6hr) can induce a series of dysfunction such as cognitive impairment, anxiety, depression, amnesia and insomnia [12]. Hence in this study, the acute restraint stress was given in wire mesh for 2hrs. Wistar albino rats were selected in this research work because of its easy availability, easy to handle and good experimental performance [15].

Plants having antianxiety activity [16] are Abies pindrow, Achillea millefolium, Aloysia polystachya etc. The test drug, Triticum aestivum, has protective role of on Aβ-induced apoptosis in SH-SY5Y cells and cognitive dysfunctions in Sprague-Dawley (SD) rats [17]. Hence this study was undertaken to explore the neurological effects of ethanolic extract of Triticum aestivum against stress. Light and Dark box test is widely used tool to measure anxiety like behaviour in rodents and based on a conflict between the natural aversion of brightly illuminated area and on their spontaneous exploratory behaviour in novel experiment. This is a sensitive test to explore the anxiolytic effect of test drugs [18]. Diazepam[19] (1mg/kg) selected as reference standard drugs for anxiety test model.

The rats exposed to acute restraint stress when subjected to Light and Dark box test, they entered into dark compartment from light compartment quickly as compared to normal control rats (Table 2). Again the time spent in the light box was brief in comparison to the non-stressed control rats (Table no-3). The number of exploratory rearing in both stress model rats were significantly more than non-stressed control rats (Table-5). These are the indications of high level of fear and anxiety. Also in comparison to the stress control rats, the time spent in Light box was longer and decrease in number of exploratory rearing in both compartments was observed with rats pre-treated with Diazepam and TAE-200mg/kg. These activities of the test drug are indices of its anxiolytic activity against stress. Similar observations were reported by a study in the plant Nymphaea alba in Light and Dark box test [20].

Stressful life events adversely affect the Gastric ulcer formation, principally via acid secretions [21]. The rats exposed to acute stress showed a significant increase in scores of ulcer index and severe hemorrha gic gastric lesions (Table-6). Pre-treatment with reference standard drug and TAE (200mg/kg) decreased the scores of ulcer index which is comparable to study [3]. Several studies have reported the anti-stress, gastro protective effects [12] of medicinal plants are due to their phenolic content, flavonoid etc. In this study [3] the test drug Triticum aestivum is rich in chlorophyll, minerals like magnesium, selenium, zinc, chromium, antioxidants like beta-carotene, Vit-E, Vit-C etc. These constituents might be responsible for its beneficial effects.

Conclusion

Triticum aestivum exhibited anti-anxiety effects in stress induced anxiety model and this effect may be mediated by central monoaminergic neurotransmitter system like 5-HT and Dopamine. In our study, Triticum also showed significant anti-ulcerogenic activity in stressed rats. Thus, Triticum aestivum may provide an alternative to conventional therapy for attenuating the behavioural impairments like anxiety in stress as well as it may come up with safe and effective treatment of ulcer as well.

References

1. Mc Ewen. The neurobiology of stress: From serendipity to clinical relevance. Brain Res 2000; 886:172-89.

Indexed at, Google Scholar, Cross Ref

2. Krishnamoorthy M, Sasikumar JM, Shamna R, et al. Antioxidant activities of bark extract from mangroves, Bruguiera cylindrica (L.) Blume and Ceriops decandra Perr. Indian J Pharmacol 2011; 43:557.

Indexed at, Google Scholar, Cross Ref

3. Ketan Shah, Devang Sheth, Pravin Tirgar. Anti-ulcer activity of Triticum aestivum on ethanol induced mucosal damage (cyto-protective activity) in wistar rats. Pharmacologyonline 2011; 2:929-35.

 Google Scholar

4. Mates MJ, Jimenez S, Fransisca M. Role of reactive oxygen species in apoptosis: Implication for cancer therapy. Int J Biochem Cell Biol 2000; 32:157-170.

 Google Scholar, Cross Ref

5. Nenonen M, Helve TA, Rauma AL, et al. Uncooked, lactobacilli-rich, vegan food and rheumatoid arthritis. Br J Rheumatol 1998; 37:274-281.

Indexed at, Google Scholar, Cross Ref

6. Mohan Y, Jesuthankaraj GN, Ramasamy Thangavelu N. Antidiabetic and antioxidant properties of Triticum aestivum in streptozotocin-induced diabetic rats. Adv Pharmacol Sci 2013; 2013.

Indexed at, Google Scholar, Cross Ref

7. Jung-Hee Jang, Chang-Yul Kim, Sun Ha Lim. Neuroprotective effects of Triticum aestivum L. against β-Amyloid-induced cell death and memory impairments. Phytotherapy Res 2010; 74-86.

Indexed at, Google Scholar, Cross Ref

8. Mitchell PJ, Redfern PH. Animal models of depressive illness: the importance of chronic drug treatment. Current Pharm Design 2005; 11:171-203.

Indexed at, Google Scholar, Cross Ref

9. Gerhard Vogel, Wolfgang HV. Drug discovery and evaluation. 1996; 622
10. Patil MB, Jalalpure SS, Ashraf A. Preliminary phytochemical investigation and wound healing activity of the leaves of Argemone mexicana Linn. (Papaveraceae). Indian Drugs Bombay 2001; 38:288-293.
11. Dhingra D, Parle M, Kulkarni SK. Memory enhancing activity of Glycyrrhiza glabra in mice. J Ethnopharmacol 2004; 91:361-365.

Indexed at, Google Scholar, Cross Ref

12. Tabassum I, Siddiqui ZN, Rizvi SJ. Effects of Ocimum sanctum and Camellia sinensis on stress-induced anxiety and depression in male albino Rattus norvegicus. Indian J Pharmacol 2010; 42:283.

Indexed at, Google Scholar, Cross Ref

13. Vander AJ, Sherman JH, Luciano DS. Defence mechanisms of the body: Immunology, Foreign chemicals and stress. In: Human Physiology: The mechanisms of body function. 5th Edn 1990; 274.

 Google Scholar

14. https://catalogue.library.ulster.ac.uk/items/211896?query=Adaptations&resultsUri=items%3Fquery%3DAdaptations%26sort%3Dauthor&sort=author
15. Mahendra P, Bisht S. Anti-anxiety activity of Coriandrum sativum assessed using different experimental anxiety models. Indian J Pharmacol 2011; 43:574.

Indexed at, Google Scholar, Cross Ref

16. Gilhorta N, Dhingra D. A review on anti-anxiety plants. Natural Product Radi 2008; 476-483.
17. Jung-Hee Jang, Chang-Yul Kim, Sun Ha Lim. Neuroprotective effects of Triticum aestivum L. against β-Amyloid-induced cell death and memory impairments. Phytotherap Res 2010; 74-86.

Indexed at, Google Scholar, Cross Ref

18. Crawley JN. Neuropharmacologic specificity of a simple animal model for the behavioral actions of benzodiazepines. Pharmacol Biochem Behav 1981; 15:695-699.

Indexed at, Google Scholar, Cross Ref

19. T.S Nagaraja, R.Mahmood. Evaluation of Anxiolytic effect of Erythrina mysorensis Gamb. in mice. Indian Journal of pharmacology 2012; 44 (4) : 489-92.

Indexed at, Google Scholar, Cross Ref

20. Thippeswamy BS, Mishra B, Veerapur VP, et al. Anxiolytic activity of Nymphaea alba Linn. in mice as experimental models of anxiety. Indian J Pharmacol 2011; 43:50.

Indexed at, Google Scholar, Cross Ref

21. Moynihan JA, Alder R. Psychoneuroimmunology: Animal models of disease. Psycho Med Am Psycho Society 1996; 58:546-58.

Indexed at, Google Scholar, Cross Ref