The present study was aimed to evaluate the protective effect of hesperidin against immobilization-stress-induced alterations in biochemical, behavioral, and mitochondrial functions in mice. In many instances neuroscientists have reported that acute immobilization stress for 6 h resulted in anxiety and impaired locomotor activity due to excess oxidative-nitrergic stress, depletion of antioxidant defense mechanisms, and mitochondrial dysfunction in animals. In the present study, 6 h of acute immobilization stress had significantly altered the behavioral (anxiety and memory) and biochemical parameters coupled with mitochondrial dysfunction in Swiss albino mice. Fourteen days of pretreatment with Hesperidin (50 and 100 mg/kg, p.o.) significantly and dose-dependently inhibited the behavioral and biochemical alterations and mitochondrial dysfunction caused by acute immobilization stress. Furthermore, pre-treatment of
Stress is a very crucial factor in the maintenance of health and disease [
Natural products such as bioflavonoids possess very good antioxidant property [
With this background, the present study was designed to investigate the possible neuroprotective effect of Hesperidin against acute immobilization-stress-induced anxiety-like behavior and associated oxidative damage in mice. The functional interaction of Hesperidin with nitrergic signaling was investigated using nitric oxide precursor,
Hesperidin,
Male Swiss albino mice weighing 20–25 g were purchased from Bioneeds Preclinical Services, Nelamangala, Bangalore. They were housed under standard laboratory conditions and maintained on 12 h light/dark cycle and had free access to food and water. Animals were acclimatized to laboratory conditions before the experiment. Each group consists of 10 animals each. All the experiments were carried out between 9:00 am and 17:00 pm. The experimental protocols were approved by Institutional Animal Ethics Committee of PES College of Pharmacy, Bangalore, India (no. PESCP/IAEC/06/2006-07) and conducted according to the committee for the purpose of control and supervision of experimentation on animals (CPCSEA) guidelines for the use and care of experimental animals.
The suspension of Hesperidin in 0.5% CMC (carboxy methyl cellulose) in distilled water,
Illustration of the protocol used for the study.
On the 15th day all the animals (except naïve) were immobilized for 6 h by taping all the four limbs on a board, after putting them on their backs using zinc oxide hospital tape. Release was affected by unraveling the tape after moistening with acetone in order to minimize pain or discomfort. In unstressed group, the mice were kept in animal cage with soft bedding in the experimental room [
Animals were kept in actophotometer for 3 minutes for acclimatization, followed by 5 minutes of actual recording. The apparatus was placed in a dark, light-sound-attenuated and ventilated testing room. Each animal was observed over a period of 5 min in a square (30 cm) closed arena equipped with infrared-light-sensitive photocells using digital photoactometer and values were expressed as counts per 5 min [
The mirror chamber consisted of a wooden chamber having a mirror cube enclosed within it. The container box was
During the 5 min test session, the following parameters were noted: (a) latency to enter the mirror chamber and (b) average time spent per entry in mirror chamber. An anxiogenic response was defined as decreased number of entries and time spent in the mirror chamber [
The elevated plus maze test is described elsewhere. The apparatus comprises of two open arms (35 cm × 5 cm) and two closed arms (30 cm × 5 cm × 15 cm) that extend from a common central platform (5 cm × 5 cm). The floor and walls of the closed arms are wooden and painted black. The entire maze is elevated to height of 50 cm above the floor level. Mice of 18–22 g weight were housed in pair for 10 days prior to testing in the apparatus. During this time animals were handled by the investigator on alternate days to reduce stress; each group consists of 10 animals each.
On the 15th day after immobilization stress, each mouse was placed in the center of the maze facing one of the open arms. During a 5-minute test period the following measures were taken: the number of entries into and time spent in the open and the closed arms and time spent in central zone and rearing. The procedure was conducted preferably in a sound-attenuated room [
All the animals were sacrificed by decapitation on the same day immediately after behavioral assessents. The brains were removed, rinsed in isotonic saline, and weighed. 10% (w/v) tissue homogenates were prepared with 0.1 M phosphate buffer (pH 7.4). The postnuclear fractions were obtained by centrifugation of the homogenate at 12000 ×g for 20 min at 4°C.
The quantitative measurement of lipid peroxidation in the whole brain was assessed as per the method of Wills [
The amount of malondialdehyde formed was measured by the reaction with thiobarbituric acid at 532 nm using Perkin Elmer lambda 20 spectrophotometer. The results were expressed as nanomol of malondialdehyde per mg protein using the molar extinction coefficient of chromophore (1.56 × 105 M−1 cm−1).
Reduced glutathione (GSH) in the brain was estimated according to the method of Ellman [
Nitrite is the stable end product of nitric oxide (NO) in the living system. Accumulation of nitrite was measured in cell free supernatants from brain homogenates by spectrophotometer assay based on Greiss reagent 15 (1% sulphanilamide/0.1% naphthylethylenediamine dihydrochloride/2.5% phosphoric acid) and incubated at room temperature for 10 min to yield a chromophore. Absorbance was measured at 543 nm spectrophotometrically.
The nitrite concentration was calculated from a standard curve using sodium nitrite as standard and expressed as micromolar nitrite per mL homogenate [
The protein content was measured according to the method of Lowry et al. using bovine serum albumin as standard [
Catalase activity was assayed by the method of Luck [
Rat brain mitochondria were isolated by the method of Berman and Hastings [
Homogenates were centrifuged at 13,000 g for 5 min at 4°C. Pellet was resuspended in isolation buffer with EGTA and spun again at 13,000 g for 5 min. The resulting supernatants were transferred to new tubes and topped off with isolation buffer with EGTA and again spun at 13,000 g for 10 min. Pellets containing pure mitochondria were resuspended in isolation buffer without EGTA.
NADH dehydrogenase activity was measured spectrophotometrically (UV-Pharmaspec 1700 Shimadzu, Japan) by the method of King and Howard [
Succinate Dehydrogenase (SDH) activity was measured spectrophotometrically (UV-Pharmaspec 1700 Shimadzu, Japan) according to King [
The method employed in the present study is based on the
Cytochrome oxidase activity was assayed according to the method of Sottocasa et al. in striatal mitochondria [
All the values are expressed as mean ± SEM. The data were analyzed by using one-way analysis of variance (ANOVA) followed by Tukey’s test.
The naïve animals showed consistent and stable locomotor activity and anxiety-like behavior. The 6 h acute immobilization stress has significantly reduced the locomotor activity (as indicated by decreased ambulatory movements) and induced anxiety-like behaviors (delayed latency to enter the mirror chamber, decreased number of entries and time spent in the mirror chamber, and decreased number of entries and time spent in open arm in elevated plus maze test) as compared to the unstressed (naïve) group (
Effect of Hesperidin on immobilization-stress-induced altered locomotor activity. IS: immobilization stress, Hes: Hesperidin, and pr: protein, all the values are expressed as mean ± SEM. a
Effect of Hesperidin on immobilization-stress-induced anxiety-like behavior in elevated plus maze test (time spent in open arm). IS: immobilization stress, Hes: Hesperidin, and pr: protein, all the values are expressed as mean ± SEM. a
Effect of Hesperidin on immobilization-stress-induced anxiety-like behavior in Elevated plus maze test (No. of entries in to open arm). IS: immobilization stress, Hes: Hesperidin, and pr: protein, all the values are expressed as mean ± SEM. a
Effect of Hesperidin on immobilisation stress induced anxiety-like behavior in mirror chamber test. IS: immobilization stress, Hes: Hesperidin, and pr: protein, all the values are expressed as mean ± SEM. a
Effect of naringin on immobilisation stress induced anxiety-like behaviour in mirror chamber test (No. of entries). IS: immobilization stress, Hes: Hesperidin, and pr: protein, all the values are expressed as mean ± SEM. a
Pretreatment with
In conclusion, pretreatment with Hesperidin (50 and 100 mg/kg) offered significant protection against acute immobilization-stress-induced behavioral and oxidative damage and possible mechanism of action involves modulation of nitric oxide pathway.
6 h acute immobilization stress has significantly increased the malondialdehyde (MDA) and nitrite concentration and depleted reduced glutathione (GSH) and catalase activity as compared to unstressed (naïve) animals. Fourteen days of pretreatment with Hesperidin (50 and 100 mg/kg) (
Effects of Hesperidin on immobilization-stress-induced altered oxidative and biochemical parameters in mice (percentage of naïve in parentheses).
Treatment | Reduced GSH | LPO | Catalase | Nitrite ( |
---|---|---|---|---|
Naïve | 0.094 ± 0.004 (100) | 0.120 ± 0.003 (100) | 0.806 ± 0.005 (100) | 261.3 ± 9.4 (100) |
Control (IS) | 0.014 ± 0.004 (14.89)a | 0.634 ± 0.004 (674.46)a | 0.141 ± 0.01 (17.49)a | 686.4 ± 21.5 (262.68)a |
Hes-50 | 0.038 ± 0.003 (40.42)b | 0.462 ± 0.004 (491.48)b | 0.421 ± 0.002 (52.23)b | 425.8 ± 15.2 (162.95)b |
Hes-100 | 0.061 ± 0.004 (64.89)b, c, e | 0.243 ± 0.006 (258.51)b, c, e | 0.502 ± 0.001 (62.28)b, c, e | 341.6 ± 19.4 (130.73)b, c, e |
0.013 ± 0.005 (13.82)a | 0.619 ± 0.007 (658.51)a | 0.125 ± 0.003 (15.51)a | 694.9 ± 16.2 (265.93)a | |
0.095 ± 0.002 (101.06)b, c, d, e | 0.129 ± 0.001 (137.23)b, c, d, e | 0.785 ± 0.001 (97.39)b, c, d, e | 260.51 ± 17.5 (119.59)b, c, d, e | |
0.016 ± 0.003 (17.02) a | 0.597 ± 0.003 (635.10)a | 0.135 ± 0.005 (16.75)a | 668.2 ± 23.2 (255.72)a | |
0.019 ± 0.004 (20.21)a | 0.588 ± 0.006 (625.53)a | 0.142 ± 0.003 (17.62)a | 657.6 ± 15.5 (251.66)a | |
0.096 ± 0.003 (102.12)b, c, d, e | 0.120 ± 0.004 (127.6)b, c, d, e | 0.795 ± 0.004 (98.64)b, c, d, e | 294.8 ± 14.4 (112.82)b, c, d, e | |
0.108 ± 0.005 (114.89)b, c, d, e | 0.109 ± 0.004 (115.9)b, c, d, e | 0.816 ± 0.003 (101.24)b, c, d, e, f | 272.2 ± 19.5 (104.17)b, c, d, e |
IS: immobilization stress, Hes: Hesperidin, and pr: protein, all the values are expressed as mean ± SEM. a
Treatment with Hesperidin (50 and 100 mg/kg) significantly attenuated elevated lipid peroxidation nitrite activity, restored reduced glutathione and catalase activity as compared to control (IS) (
A combination of
There were no significant alterations in mitochondrial enzyme complexes-I, II, IV and mitochondrial redox activity of naïve group. However, 6 h of immobilization stress has significantly impaired the mitochondrial complexes I (NADH dehydrogenase), II (succinate dehydrogenase), and IV (cytochrome oxidase) enzyme activity as well as mitochondrial redox activity in the immobilization stress (IS) control group as compared to the naïve group (Figure
Effect of Hesperidin on immobilization stress induced mitochondrial dysfunction in mice (% naïve). IS: immobilization stress, Hes: Hesperidin, and pr: protein, all the values are expressed as mean ± SEM (% of naïve group), a
Pretreatment with
Stress is a physical and/or environmental and/or psychological stimulus that is capable of altering the physiological homeostasis of the body, and hence coping up with such stress condition makes it as a crucial determinant in health and disease [
Stress activates hypothalamus-pituitary-adrenal axis (HPA) and also causes changes in catecholamine levels and thereby influences several neurological functions at both central and peripheral levels. Any kind of stress influences brain functions by causing long-term changes in the multiple neural systems, which leads to various neurodegenerative disorders [
In the present study, Hesperidin (50 and 100 mg/kg, p.o.) offered significant protection against immobilization stress induced neurobehavioral alterations (locomotor activity and anxiety-like behavior) and oxidative damage to the brain, suggesting its neuroprotective effect against stressful conditions.
Immobilization stress causes drastic increase in the production of reactive oxygen species and consequent oxidative damage with a proportionate decrease in
In accordance with the above findings, in the present study, Hesperidin elevated the antioxidant enzyme defense system against immobilization stress-induced oxidative damage. The possible mechanism behind the protective effect of Hesperidin is inhibition of NO synthesis in the brain.
Mitochondria are considered as a major source of ROS, which include superoxide anion (
Hesperidin (50 and 100 mg/kg) pretreatment has significantly restored the mitochondrial enzyme activity suggesting its role in mitochondrial enzyme functions. Further,
The findings of the present study suggest that Hesperidin (50 and 100 mg/kg) is a bioflavonoid found to ameliorate the immobilization-stress-induced oxidative-nitrergic stress, biochemical alterations, mitochondrial dysfunction, and associated neurobehavioral alterations in mice and also suggest the involvement of nitrergic pathway in the protective effect of Hesperidin against immobilization-stress-induced neurobehavioral alterations.
The authors report no conflicts of interests.