International Journal of Nutrition, Pharmacology, Neurological Diseases

: 2013  |  Volume : 3  |  Issue : 3  |  Page : 269--275

Protective effect of gallic acid on immobilization induced stress in encephalon and myocardium of male albino Wistar rats

Shabir Ahmad Rather1, Nadanam Saravanan2,  
1 Department of Biochemistry and Biotechnology, Faculty of Science, Annamalai University, Annamalai Nagar, Tamil Nadu, India
2 Division of Biochemistry, Rani Mayyamai College of Nursing, Faculty of Medicine, Annamalai University, Annamalai Nagar, Tamil Nadu, India

Correspondence Address:
Nadanam Saravanan
Division of Biochemistry, Rani Mayyamai College of Nursing, Faculty of Medicine, Annamalai University, Annamalai Nagar - 608 002, Tamil Nadu


Objectives: The aim was to elucidate the effect of gallic acid (GA) on encephalon/brain and myocardium/heart of rats subjected to immobilization stress (IS). Materials and Methods: IS was induced by placing the rats in 20 cm × 7 cm plastic tubes for 2 h/day for 21 days. Rats were post-orally treated with GA at 10 mg/kg body weight daily for three weeks. Followed by sacrifice, brain and heart tissues were removed carefully for biochemical estimations, and H and E staining for histopathological studies. Results: In IS, significant ( P < 0.05) increase in lipid peroxidation (LPO) and a significant ( P < 0.05) decrease in antioxidant activities showed shrunken neurons in brain and myocardial edema as an evidence of major tissue damage in stressed rats. The data revealed that IS produced a severe oxidative damage in the brain and myocardium, and treatment with GA distinctly reduced these stress-induced changes compared to stressed rats. GA (10 mg/kg) to control rats did not show any significant effect. Conclusions: We concluded that GA inhibits LPO and preserved the antioxidant levels as an evidence of resuming the structural integrity of brain and heart tissues. So, GA may be valuable for the prevention and treatment of stress related disorders.

How to cite this article:
Rather SA, Saravanan N. Protective effect of gallic acid on immobilization induced stress in encephalon and myocardium of male albino Wistar rats.Int J Nutr Pharmacol Neurol Dis 2013;3:269-275

How to cite this URL:
Rather SA, Saravanan N. Protective effect of gallic acid on immobilization induced stress in encephalon and myocardium of male albino Wistar rats. Int J Nutr Pharmacol Neurol Dis [serial online] 2013 [cited 2020 Aug 13 ];3:269-275
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Stress is a condition of disturbed homeostasis, harmony, and equilibrium caused by physical or psychological stressors. Stress causes increased release of catecholamines and glucocorticoids as well as excitatory amino acids in brain by the activation of sympathoadrenal and hypothalamic pituitary adrenal (HPA) axis. [1] Exposure to stress alters central neurotransmitters and neurohormonal factors, mostly connected with pituitary-adrenal axis and lead to free radical generation, [2],[3] which in turn leads to oxidative stress and tissue damage. [4] Imbalance between the excitatory amino acids and inhibitory neurotransmission in the brain is the main cause of seizure development in experimental and clinical conditions. [5] The brain has 100 billion neurons and groups of neurons operate like small factories to perform their special jobs like thinking, learning, remembering, hear, and smell. [6] Brain is the target for stress because of its high-sensitivity to stress-induced degenerative conditions. The higher amounts of polyunsaturated fatty acids and inadequate endogenous antioxidant, defense mechanisms in brain makes it vulnerable to free radical attacks. [7],[8] Immobilization stress (IS) produced lipid peroxidation (LPO) intermediates in the myocardium has recently been recognized as a major cardiovascular risk-factor. [9],[10] Stress is a common phenomenon among the heart patient complaints as there is a clear link between brain and heart. [11] Production of reactive oxygen species (ROS) during IS cause imbalance between oxidant and antioxidant status. Free radicals and peroxides like hydrogen peroxide (H 2 O 2 ), hydroxyl radical (HO• ), and superoxide anion radical (O2• ) cause membrane LPO, damage proteins and DNA, [12] and play an important role in tissue injury. [13] Oxidative damage caused by the increased production of free radicals ultimately cause diseases like cancer, diabetes, cardiovascular diseases, atherosclerosis, Parkinson's, and Alzheimer's diseases in humans, and also cause destruction of neuronal tissues. [14],[15]

Herbal medicines are used by around 80% of population throughout the world. [16] Plants are important resource of antioxidants and have an outstanding role in the traditional medicine in different countries. The prevention of diseases has been associated with intake of natural antioxidants mostly available in fruits and vegetables. The protective effects of plants can be due to the existence of anthocyanins, flavonoids, and other phenolic compounds. [17],[18] Previous reports from our laboratory have shown plant extracts and its active principles protect the rats against stress to some extent. [19],[20] GA [3,4,5-trihydroxybenzoic acid, Scheme 1] [SUPPORTING:1] is a polyphenol mainly abundant in processed beverages such as red wines and green teas, [21] leaves of beriberry and flowers of water lily. [22],[23] GA possesses strong free radical scavenging and antioxidant activities. [24] It has also been reported for antibacterial, antiviral, anti-inflammatory, [25],[26],[27] anticancer, and anti-apoptotic actions. [28],[29] So, in view of the above facts, the present analysis was undertaken to study the protective effect of gallic acid (GA) in reducing the extent of tissue damage by virtue of its free radical scavenging and antioxidant properties in immobilization induced-stress rats.

 Materials and Methods


The study protocol was approved by the Institutional Animal Ethics Committee of Rajah Muthiah Medical College and Hospital (Reg. No. 160/1999/CPCSEA, Proposal number: 886), Annamalai University, Annamalai Nagar. Adult male albino Wistar rats of 150-180 g body weight were procured from Central Animal House, Rajah Muthiah Institute of Health Sciences, Annamalai University, Tamil Nadu, India. Rats were housed individually in hygienic metabolic cages and were kept under controlled environmental conditions. They were fed on standard pellet diet (Amrut Laboratory Animal Feed, Pranav Agro Industries Ltd., Bangalore, India) and water adlibitum.

Drugs and chemicals

GA was purchased from Sigma-Aldrich Co. St. Louis, Missouri, USA or Himedia Laboratories Pvt, Ltd., Mumbai, India. All other chemicals and solvents used were of analytical grade.

Experimental design

The IS was induced in rats by putting them in 20 cm × 7 cm plastic tubes for 2 h/day for three weeks. [30] There are several 3 mm holes at the both ends of the tubes which allow sufficient air for breathing and the rats were unable to move.GA was dissolved in saline and rats were post-orally treated with GA at 10 mg/kg body weight [31] using an intragastric tube, daily after 2 h IS for 3 weeks.All the rats were randomly divided in to four groups. Six animals were used for each group. Group I: Control rats; Group II: Control rats treated with GA (10 mg/kg); Group III: Stressed rats; Group IV: Stressed rats treated with GA (10 mg/kg). Following each stress session, rats were returned to home cages and were able to access food and water freely for the remainder of the day. After the experimental period, all the rats were anesthetized and then sacrificed by cervical decapitation between 10:00 am and 11:00 am.

Estimation of LPO

The concentration of thiobarbituric acid reactive substances (TBARS) in the tissues was estimated by the method of Niehaus and Samuelson. [32] In this method, malondialdehyde and other TBARS react with thiobarbituric acid in an acidic condition to generate a pink colorchromophore which was read at 535 nm. Values were expressed as mmol/100 g of tissue.

Lipid hydroperoxide (LOOH) in the tissues was estimated by the method of Jiang et al. [33] Oxidation of ferrous ion (Fe ++ ) under acidic conditions in the presence of xylenol orange leads to the formation of a chromophore with an absorbance maximum at 560 nm. The values were expressed as mmol/100 g of tissue.

Conjugated dienes (CD's) in the lipid extract of tissues were analysed by the method of Recknagel and Glende. [34] LPO is associated with the rearrangement of double bonds in polyunsaturated fatty acids leading to the formation of CD, which absorb light at 233 nm. The oxidation index of the lipid sample at 233 nm and 215 nm was computed which reflect the diene content and the extent of peroxidation.CD content is expressed as mmoles/mg tissue.

Estimation of antioxidants

The activity of superoxide dismutase (SOD) was assayed by the method of Kakkar et al. [35] The assay is based on the inhibition of the formation of nicotinamide adenine dinucleotide -phenazinemetho sulphate-nitrobluetetrazoliumformazon. The reaction was initiated by the addition of NADH. After incubation for 90 s adding glacial acetic acid stopped the reaction. The colour developed at the end of the reaction was extracted into n-butanol layer and measured. The specific activity of the enzyme was expressed as units/min/mg protein for tissue.

Catalase (CAT) was assayed colorimetrically by the method of Sinha. [36] Dichromate in acetic acid was converted to perchromic acid and then to chromic acetate when heated in the presence of H 2 O 2 . The chromic acetate formed was measured at 620 nm. The CAT preparation was allowed to split H 2 O 2 for different periods of time.The reaction was stopped at different time intervals by the addition of a dichromate-acetic acid mixture and the remaining H 2 O 2 was determined colorimetrically as chromic acetate. One unit of activity was expressed as a μmol of H 2 O 2 consumed/min/mg protein.

Glutothione peroxidase (GPx) was estimated by the method of Rotruck et al. [37] A known amount of enzyme preparation was allowed to react with H 2 O 2 in the presence of reduced glutathione for a specified time period, then the remaining GSH content was allowed to react with 5,5'-dithio-bis-2-nitrobenzoic acid (DTNB) and the yellow color developed was measured at 412 nm. One unit of activity was expressed as μg of glutathione consumed/min/mg protein.

Vitamin-C was estimated by the method of Roe and Kuether. [38] The ascorbic acid was converted to dehydroascorbic acid by mixing with acid washed norit and was then coupled with 2,4-Dinitrophenylhydrazine in the presence of thiourea, a mild reducing agent. The coupled DNPH was converted into an orange red-colored complex when treated with sulphuric acid, which was read colorimetrically at 520 nm. Values were expressed as mg/100 g of tissue.

Vitamin-E was estimated by the method of Baker et al. [39] To 0.5 mL of tissue sample, 1.5 mL of ethanol and 2.0 mL of petroleum ether were added, mixed and centrifuged. It was evaporated to dryness at 8°C. To this was added 0.2 mL of 2,2-dipyridyl solution and 0.2 mL of ferric chloride solution. Mixed well and kept in dark for 5 min and added 4 mL of butanol. The intense red color developed was read at 520 nm. Values were expressed as mg/100 g of tissue.

Histopathological studies

Histopathological studies were done by removing the heart and brain in two rats from each group. After perfusion, heart and brain were isolated and processed for paraffin sectioning. The sections (3-5 μm) were stained with H and E, and were analyzed for histopathological studies.

Statistical analysis

Data were analyzed by one way analysis of variance (ANOVA) followed by Duncan's multiple range test (DMRT) using a commercially available statistics software package (Statistical Package for the Social Sciences for Windows, V. 16.0, Chicago, USA). Results were presented as means ± SD P values < 0.05 were regarded as statistically significant.



LPO markers (TBARS, LOOH and CD's) level increased significantly ( P < 0.05) in the brain and heart of stressed rats compared to control rats. In stressed rats, LPO was found higher in brain than in the heart tissue. Oral treatment with GA (10 mg/kg) significantly ( P < 0.05) reduced LPO compared to stressed rats [Table 1].{Table 1}

Enzymatic and non-enzymatic antioxidants

[Table 2] represents the SOD, CAT and GPx activities in the brain and heart of control and experimental rats. These activities decreased significantly ( P < 0.05) in stressed rats compared to control rats, and lesser enzymatic activities were found in the brain than in the heart of stressed rats. GA at 10 mg/kg significantly ( P < 0.05) increased these activities compared to stressed rats.{Table 2}

Vitamin-C and vitamin-E levels in the brain and heart of control and experimental rats are shown in [Figure 1] and [Figure 2], respectively. Vitamin-C and vitamin-E levels decreased significantly ( P < 0.05) in both the tissues of stressed rats compared to control rats. Treatment with GA (10 mg/kg) significantly ( P < 0.05) increased these levels compared to stressed rats. However, higher activities of these antioxidants were observed in the brain than in the heart of stressed rats.{Figure 1}{Figure 2}


[Figure 3]a and b represent the light micrograph of control and treated rat brain, respectively, showing normal architecture. [Figure 3]c shows the neuron structures in stressed rat brain, revealed some degree of alteration and less distinct tissue delamination. Furthermore, many neurons were shrunken, pyknotic, and darkly stained with small nuclei. Stressed rats treated with GA (10 mg/kg) did not show any evidence of tissue damage or neuronal alterations [Figure 3]d.{Figure 3}

[Figure 4]a and b shows a light micrograph of control and control treated rat hearts, respectively, showing normal architecture. In stressed rats, there was an evidence of mild myocardial edema on light microscopy [Figure 4]c. Stressed rats after treatment with GA (10 mg/kg) showed normal morphology without any evidence of myocardial edema [Figure 4]d.{Figure 4}


LPO is caused via ROS in the brain, which lead to membrane damage, plays an important role in tissue injury and in the pathogenesis of neurodegenerative diseases. [40],[41] Enzymatic antioxidants like CAT, SOD, and GPx are present in all the cells including neurons and help to detoxify ROS. [8] Our results showed decreased activities of SOD, CAT, GPx, and increased levels of LPO in the tissues (brain and heart) of IS rats was in an agreement with earlier studies. [42] Minimal activities of enzymatic antioxidants in the brain and heart of stressed rats might be due to the interaction of these antioxidants with oxygen radicals. [43] Post-oral treatment decreased the LPO levels and increased the enzymatic antioxidant activities in the brain and heart of IS rats, showed the anti-lipid peroxidative effect of GA.

In the present study, stress has depleted the glutathione based antioxidant defense and decrease the activities of vitamin-C and vitamin-E. [44],[45] Vitamin-C and vitamin-E interacts directly with free radicals, thus preventing oxidative damage. [46] Lipid soluble Vitamin-E prevents LPO chain reactions in cellular membranes by interfering with the propagation of lipid radicals. Increased free radical scavenging activity under IS condition might have decreased the enzymatic and non-enzymatic antioxidant levels in the brain and heart. GA acid treatment might have scavenged the free radicals and hence increased levels of enzymatic and non-enzymatic antioxidants in heart and brain, shows the antioxidant and hence anti-stress activity of GA.

Stress induced production of free radicals in brain with catecholamine metabolism cause elevated catecholamine levels which undergo auto-oxidation to generate ROS. [47],[48] It is reported that IS increased LPO and decreased important antioxidant levels in the tissues like liver and kidney. [17] Oxidative stress initiated by inequity in oxidants and antioxidants in the hypothalamus might intervene cell damage and may be accountable for the neuronal disorders during stress. [49] It is also reported that chronic restraint stress cause myocardial edema and acute stress induces cell damage in heart of rats. [50],[51] In the current study, IS caused LPO with decreased important enzymatic and non-enzymatic antioxidants in brain and heart tissues. These alterations were accompanied with shrunken neurons with pyknotic and darkly stained small nuclei in brain, and with myocardial edema. Oral treatment of GA to IS rats reversed these changes, which in turn might be responsible for the maintenance of cellular integrity.


The present study showed that GA preserved the structural integrity by maintaining the oxidant and antioxidant balance in the heart and brain of stressed rats. This proves the two important actions of GA in oxidative stress i.e. quenching of ROS and enhancing the cellular antioxidant defense system. So, this study may have a significant impact on reducing the stress based consequences in a current stress full life.


The financial assistance to Mr. Shabir Ahmad Rather, in the form of Meritorious Fellowship by the University Grants Commission, New Delhi, is gratefully acknowledged.


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