Behavioral and Biochemical Effects of Mukia madrespatana Following Single Immobilization Stress on Rats

Background and Objectives: Elevated oxidative stress has been shown to play an important role in the diagnosis and prognosis of stress and memory-related complications. Mukia madrespatana (M. madrespatana) has been reported to have various biological and antioxidant properties. We intended to evaluate the effect of M. madrespatana peel on single immobilization stress-induced behavioral deficits and memory changes in rats. Materials and Methods: M. madrespatana peel (2000 mg/kg/day, orally) was administered to control and immobilize stressed animals for 4 weeks. Anxiolytic, antidepressant, and memory-enhancing effects of M. madrespatana were observed in both unstressed and stressed animals. Results: Lipid peroxidation was decreased while antioxidant enzymes were increased in both unstressed and stressed animals. Acetylcholine level was increased while acetylcholinesterase activity was decreased in both M. madrespatana treated unstressed and stressed rats. There was also an improvement in memory function. Serotonin neurotransmission was also regulated in M. madrespatana treated rats following immobilization stress with anxiolytic and anti-depressive effects. Conclusion: Based on the current study, it is suggested that M. madrespatana has strong antioxidant properties and may be beneficial as dietary supplementation in stress and memory-related conditions.


Introduction
Stress is a major threat to the body that alters homeostasis at the cellular level. The immobilization stress model is an easy and continent method for inducing both psychological and physical stresses in mammals [1]. Stress may lead to a variety of central nervous system irregularities that include anxiety, depression, learning, and memory functions [2]. According to earlier reports, there is restricted mobility, an anxiety-like effect, and deterioration of antioxidant enzyme activities following immobilization stress [3].
The activation of hypothalamic pituitary adrenal (HPA)-axis by a stress situation induced greater release of glucocorticoid, which damaged neurons and their functions [4]. The increased levels of glucocorticoid stimulated the release of glutamate in the cortex and limbic part of the brain [5]. The

Plant Material and Chemicals
Fresh fruits of Mukia maderaspatana (M. madrespatana) were collected from the area of the field near Multan City, Pakistan. The plant material was recognized and authenticated by the taxonomist (Dr. Zafar Ullah Zafar, Department of Botany, Bahauddin Zakariya University, Multan) and a voucher (R.R. Stewart F.W. Pak.706 (3)) was retained in an herbarium. Fresh fruits of M. madrespatana were washed and peeled off. Peel of M. madrespatana was air-dried at room temperature and then ground into a fine powder using an electric grinder and stored in an airtight container. The selection of low and high doses of M. madrespatana fruit peel powder in the present study based on the previous report in which the whole plant of M. madrespatana with various extracts (aqueous and ethanolic) was used with various dosages, i.e., 100-200 mg/kg body weight [24] and 2000 mg/kg body weight [25,26]. The required amount of powder was weighed and mixed well in deionized water (3 mL) and fed to animals by the gavage technique. Every time a fresh drug dose was prepared for oral administration. All chemicals used in the current study were purchased from Chemical Co. Sigma-Aldrich, St. Louis, MO, USA.

Acute Toxicity Test
The toxicity procedure was performed as reported previously [27]. Twelve animals (n = 4) were used for the acute toxicity test as reported previously [28]. Two sets of rats (n = 4) were accommodated overnight in a fasting condition with tap water and food was withdrawn for 4-5 h after oral intake of M. madrespatana fruit peel powder. Starting a dose of 125 mg/kg body weight was given to the first set and a maximum dose of 2000 mg/kg body weight was given to the second set. The third set of rats (n = 4) was drinking tap water and all the rodents were observed individually. Clinical signs were examined. Body posture, tremors, locomotion, pain response, vocalization, body weight, water intake, etc. were also observed. No death was seen with given doses. According to Economic Co-operation and Development (OECD) guidelines, the LD50 value considered above 125 mg/kg and 2000 mg/kg body weight. Number of animals in sub-groups used in the present study was already reported [28]. Animals of the stressed groups were immobilized on metal wire grids for 2 h (at 7:00-9:00 am) in a separate room. Meanwhile, unstressed groups were left in their home cages. For 24 h post-immobilization stress (on day 30), behavioral activities include: (i) an elevated plus maze (EPM) test, (ii) light/dark activity (LDA) test, (iii) forced swim test (FST), and (iv) Morris water maze (MWM) test (acquisition and short term memory) were conducted between 9:00 a.m. to 6:00 p.m. with the help of blind observers. On day 31, long-term memory of all animals was assessed using MWM. After that, animals were decapitated and the hippocampi (brain region) were removed [29], kept frozen at −20 • C, and utilized for further biochemical analysis 24 h after the decapitation.

Immobilization Stress Procedure
The animals were immobilized on wire grids of 10" × 9" fitted with a Perspex plate of 9" × 6.5". The previously described procedure was used for the immobilization of animals [30]. Immobilization was produced by pressing the forelegs of the rat through the gaps in the metal grids and taping them together with Zinc Oxide plaster tape. Hind limbs were also taped and the head of the animal rested on the Perspex plate. Unstressed groups were kept in their home cages during the immobilization period.

Elevated Plus Maze (EPM) Test
Elevated plus-maze is used for the measurement of anxiolytic activity of test compounds and, for the said purpose we used, the apparatus of plus shape with the total dimensions of 110 × 10 × 15 cm.
The closed and open arms are distinguished with the central 10 cm arena. For the testing purpose, we used the protocol established previously [31]. The animal was permitted to visit on the elevated stage for 5 min and behavioral assessment was done by its time and frequency spent in open arm vs. closed arm. The top-mounted camera system (Logitech C-310, Basel, Switzerland) recorded the videos. Between each trial, the apparatus was cleaned with 70% isopropyl alcohol to wash out the smell of the previous trial.

Light-Dark Activity (LDA) Test
A customized-made apparatus of acrylic glass was used for measuring an animal's anxiety behavior in a two compartmental system of light and dark environment. The dimension of the apparatus is 40 × 30 × 35 cm where light and dark compartments are equally divided, i.e., 20 cm each and central door of 10 × 10 cm allows free passage of animals between the compartments. The total duration of the test is 5 min in which the duration of the animal's time spent in each compartment was noted with the lateral camera recording system. Subsequently, the recorded videos were analyzed by a blinded observer to the experiments with the help of a stopwatch.

Forced Swim Test (FST)
The forced swim test (FST) has a widely employed method for evaluating the depressant/antidepressant behavior of rodents [31]. In the experiment, we used a glass tank with a dimension of 45 cm in height and 30 cm in the radius for assessing the antidepressant activity of the test substances. The apparatus was full of water (25 • C) up to~35 cm where the feet of the animal should not get the support from the basement and become, simultaneously, inescapable from the tank [28]. In a trial session of 15 min each, all animals were allowed to force swim. In an experimental session, 24 h after the trial session, the treated rats were challenged to the environment of inescapability and difference between the states of mobility (climbing and swimming) vs. immobility was observed by lateral recording, which was, subsequently, analyzed by a blinded observer. The rat will be considered in a phase of depression where it spent most of the time in a phase of immobility and makes virtually no efforts to escape and merely attempt to retain its head above the water.

Morris Water Maze (MWM) Test
In this test, we assessed the impact of M.M. fruit peel powder on the retention of spatial memory in rats. For the purpose, we used a customized circular tank of grey color with a dimension of 150 cm in radius and 50 cm in height [32]. The tank was filled with water and the temperature was maintained at 25°C throughout the experiment. The tank was equally divided into quadrants, i.e., north-east (NE), south-east (SE), (north-west (NW), and south-west (SW), and an opaque color platform made of acrylic glass was placed in the SW quadrant of the tank. The distal cues with distinct shapes were placed in all four quadrants of tank walls, which were helpful for the animal to navigate in the tank and direction. Before starting the experiment, the color of the water was changed to murky with the addition of nonhazardous white ink and the platform was submerged 1-2 cm in water. The test was performed in two sessions: (1) the training session and (2) test phases, which is comprised of retention of short (1 h after training) and long (24 h after training) term retention of memory [33]. The animal was placed all the time from the same quadrant, i.e., NE and headed for the wall of the tank. In the training phase, each rat was allowed 2 min time to navigate and find the hidden platform and, if located within the cut-off time, the animal was permissible to stay at the platform for 10-20 s to navigate the surroundings. Otherwise, after 2 min, the animal was directed to the platform by manually guiding it to the platform. One hour after the training phase, short-term retention of memory was performed on the trained rats, and latency to reach the platform was noted. The videos were recorded on the top-mounted camera and, subsequently, analyzed by Any-Maze (V 6.01) video tracking software (Wood Dale, IL, USA).

Biochemical Analysis
Decapitation of all animals was done on the same day after behavioral tests. Brain samples were detached and the hippocampus was removed, weighed (averagely 76 mg), and washed with 0.9% saline solution and used for various biochemical tests 24 h after the decapitation. A tissue homogenate 10% (wt./vol.) was prepared with phosphate buffer (0.1 M, pH 7.4) and got by centrifugation (12,000× g) for 20 min at 4 • C for all biochemical estimations.

Determination of Malondialdehyde (MDA)
The method of Chow & Tappel [34] was used for the analysis of malondialdehyde with some changes in the procedure. 0.3 µL hippocampus homogenate was added in a 2 mL trichloro-acetic acid-thio-barbituric acid (TCA-TBA) solution and then boiled in a water bath for 20 min. The mixture was then cooled down and centrifuged (35,000 RPM) for 10 min. Light pink-colored supernatant was collected and 532 nm absorbance was used to note the absorbance.

Determination of Superoxide Dismutase (SOD) Activity
The previously reported method was used for the analysis of SOD [35]. A total of 300 µL of hippocampus homogenate was centrifuged with 0.75 mL ethanol and 0.15 mL of chloroform. After centrifugation, 500 µL supernatant was taken and then 0.5 mL EDTA and 1.0 mL of 0.1 M carbonate-bicarbonate buffer (pH 10.2) were added. By adding 0.5 mL of an epinephrine reaction, absorbance was taken at 480 nm, and the percentage inhibition of SOD was calculated.

Determination of Catalase (CAT) Activity
A total of 100 µL hippocampus supernatants mixed with 1.4 mL solution comprised of 400 µL of hydrogen peroxide and 1 mL of phosphate buffer (0.01 M, pH 7.0). The reaction was stopped 1 min after by adding 2 mL of the reagent (dichromate acetic acid). To calculate the activity of CAT, absorbance was taken at 620 nm, as described previously [36].

Determination of Glutathione Peroxidase (GPx) Activity
The method of Flohe & Gunzler [37] was used to measure the activity of GPx as µmol/min/g of the hippocampus. A total of 1.0 mL of reaction solution that contained 0.3 mL phosphate buffer (0.1 M, pH 7.4), 0.2 mL of glutathione reduced, 0.1 mL of sodium azide, 0.1 mL of hydrogen peroxide, and 0.3 mL of homogenate was prepared. The reaction solution was incubated for 15 min at 37 • C by adding 0.5 mL of TCA. The reaction was terminated and the solution was centrifuged (1500× g) for 15 min and filtrate was removed. A total of 0.2 mL phosphate buffer and 0.7 mL 5,5'-dithiobis-(2-nitrobenzoic acid (DTNB) were mixed with 0.1 mL of filtrate. The absorbance of the solution was recorded at 420 nm.

Determination of Acetylcholinesterase (AChE) Activity
The method of Ellman et al. [38] was used to estimate the activity of AChE. A total of 0.4 mL hippocampus homogenate, 2.6 mL phosphate buffer, and 0.1 mL DTNB were mixed by bubbling air and put down into a spectrophotometer. When the reaction solution was steady, the basal reading followed by the addition of 5.2 µL of acetylthiocholine iodide in a cuvette contained a reaction solution. Absorbance was recorded at 412 nm. The activity of AChE was mentioned as µmol/min/g of tissue.
2.6.6. Determination of 5-HT and 5-HIAA 5-hydroxy indole acetic acid (5-HIAA) and 5-hydroxytryptamine (5-HT) levels in the hippocampus were estimated using the method described by Samad et al. [28]. Reversed-phase High-Performance Liquid Chromatography (HPLC) with an electrochemical detector (Shimadzu LEC 6A detector) was performed to detect levels of biogenic amines in brain samples. The electro-chemical (EC) detector was operated at a potential of +0.8 V. The stationary phase used for separation is a 5-µ Shim-pack octa-decyl silane (ODS) column having an internal diameter of 4.0 mm and a length of 150 mm. The mobile phase that passes through a column with a pump pressure of 2000-3000 psi contains octyl sodium sulfate (0.023%) in 0.1 M phosphate buffer at pH 2.9.

Determination of Acetylcholine (ACh)
The level of ACh concentration was determined as described by Reference [39] and was presented as µmol/g of the hippocampus. The tissue sample was boiled to release the bound ACh and inactivate the enzyme. The reaction mixture was then mixed with ferric chloride 1% (1000 µL) to form a brown color complex and read at 540 nm against the reagent blank.

Statistical Analysis
Data of exploratory activity in the novel and familiar environment were analyzed by One-Way ANOVA. All the biochemical and behavioral (Post stress) data were analyzed by Tukey's test and this was followed by two-way ANOVA. All the data analyzed by SPSS ver. 20.0 (IBM, Chicago, IL, USA). p < 0.05 was taken as significant.  Values are means ± standard deviation (n = 5) 4 weeks after the administration of the drug. Significant differences by Tukey's test * p < 0.05 from control animals following one-way ANOVA.  Values are means ± standard deviation (n = 5) 4 weeks after the administration of the drug. Significant differences by Tukey's test * p < 0.05 from control animals following one-way ANOVA.  Values are mean ± standard deviation (n = 5). Data were analyzed by Tukey's test following two-way ANOVA. Statistical difference is expressed as * p < 0.05 versus respective control and + p < 0.05 versus unstressed rats.  Values are mean ± standard deviation (n = 5). Data were analyzed by Tukey's test following two-way ANOVA. Statistical difference is expressed as * p < 0.05 versus respective control and + p < 0.05 versus unstressed rats.  Values are mean ± standard deviation (n = 5). Data were analyzed by Tukey's test following two-way ANOVA. Statistical difference is expressed as * p < 0.05 versus respective control and + p < 0.05 versus unstressed rats. Values are mean ± standard deviation (n = 5). Data were analyzed by Tukey's test following two-way ANOVA. Statistical difference is expressed as * p < 0.05 versus respective control and + p < 0.05 versus unstressed rats.       Values are mean ± standard deviation (n = 5). Data were analyzed by Tukey's test following two-way ANOVA. Statistical difference is expressed as * p < 0.05 versus respective control and + p < 0.05 versus unstressed animals.  Values are mean ± standard deviation (n = 5). Data were analyzed by Tukey's test following two-way ANOVA. Statistical difference is expressed as * p < 0.05 versus respective control and + p < 0.05 versus unstressed animals. Values are mean ± standard deviation (n = 5). Data was analyzed by Tukey's test following two-way ANOVA. Statistical difference is expressed as * p < 0.05 versus respective control and + p < 0.05 versus unstressed rats.   Values are mean ± standard deviation (n = 5). Data were analyzed by Tukey's test following two-way ANOVA. Statistical difference is expressed as * p < 0.05 versus respective control and + p < 0.05 versus unstressed animals.  Values are mean ± standard deviation (n = 5). Data were analyzed by Tukey's test following two-way ANOVA. Statistical difference is expressed as * p < 0.05 versus respective control and + p < 0.05 versus unstressed animals.

Discussion
The present study aimed to investigate the effect of repeated administration of M. madrespatana fruit peel powder on single immobilization-induced behavioral and biochemical changes in the rat brain (brain region). The first of the study showed that normal (unstressed) animals increased exploratory activity dose-dependently. In the present study, psychological deficits and cognitive alteration disrupted serotonin neurotransmission and imbalanced oxidant-antioxidant levels were found in immobilized rats. Acute immobilization stress significantly decreased the time spent in open arm and lightbox in EPM and LDA, respectively, which indicates anxiety-like symptoms. The

Discussion
The present study aimed to investigate the effect of repeated administration of M. madrespatana fruit peel powder on single immobilization-induced behavioral and biochemical changes in the rat brain (brain region). The first of the study showed that normal (unstressed) animals increased exploratory activity dose-dependently. In the present study, psychological deficits and cognitive alteration disrupted serotonin neurotransmission and imbalanced oxidant-antioxidant levels were found in immobilized rats. Acute immobilization stress significantly Stress-related psychiatric illnesses are evident as the malfunction of the antioxidant system. Oxidative stress plays a major role in the development of stress-induced behavioral deficits (anxiety and depression) in both human models [40] and experimental animal models [41,42]. An uncontrollable stress situation produces behavioral, neurochemical, and biochemical impairment [30,31]. The present study shows that immobilization stress-induced behavioral deficits such as depression-like symptoms measured by FST, anxiety-like behavior measured by LDA/EPM, and memory impairment (STM and LTM) measured by MWM tests. Behavioral decline (anxiety and depression) and cognitive impairment are the significant features of stress-induced neuronal oxidative stress [28,31]. In the present work, rats exposed to immobilization stress exhibited anxiety-like symptoms by decreasing time spent in an open arm ( Figure 2) and light compartment (Figure 3). Similarly, depression-like symptoms were exhibited by increased immobility time while decreasing swimming and climbing time in FST ( Figure 4). It is believed that free radical production at the central and periphery level deteriorates the anatomy of the cell and produces peroxidation of the cell membrane's lipid contents [43]. Damage of neuronal cells leads to physiological malfunction of the brain, which may be linked with various behavioral deficits (anxiety and depression). Extensive studies also relate stress-induced oxidative stress to neuroinflammation. It is reported earlier that a stress situation also provokes the synthesis of inflammatory cytokines [44], which may worsen the normal situation and alter the behavioral responses. The activation of inflammatory events following physical and psychological stress burden along with the environmental stress produces cytokines and other inflammatory mediators, which are involved in the development of behavioral impairment and neurodegenerative disorders [45]. Therefore, anxiety-/depression-like behavior observed in the present study is due to increased oxidative stress markers ( Figure 6) and inflammatory responses in rats experienced to immobilization stress. It is apparent that repeated administration of M. madrespatana peel powder dose-dependently increased exploratory activity in the novel and familiar environment in normal/unstressed animals ( Figure 1). Moreover, the selected dose of M. madrespatana exhibited increased time spent in lightbox and open arm of LDA ( Figure 2) and EPM (Figure 3), respectively, and decreased immobility and increased swimming and climbing time ( Figure 4) in both unstressed and stressed animals. It is indicated that the selected dose of M. madrespatana increased motor behavior in unstressed animals (Figures 1-3) as well as in stressed (Figures 2 and 3) animals, and produced anxiolytic and antidepressant effects. Increased oxidative stress stimulates the hypothalamic-pituitary-adrenal axis (HPA-axis), which causes the further release of glucocorticoids [46]. The enhanced release of glucocorticoids may also involve stress-induced behavioral alteration [46,47].
It is well documented that an imbalance between pro-oxidant and antioxidant causes various diseases such as anxiety and depression [48]. The natural cellular antioxidant enzyme SOD, CAT, and GPx fight against ROS production [49] and produce protective effects against various threats/diseases [50]. The present study also showed that single immobilization stress imbalanced the normal natural environment at the cellular level and increased lipid peroxidation/malondialdehyde ( Figure 6) and decreased antioxidant enzymes (SOD, CAT, and GPx, Figure 7). Numerous studies also showed free radical production become enhanced and the antioxidant mechanism becomes suppressed in anxiety and depression in humans [40] as well as in animal studies [51]. Various treatments are available, which produce anxiolytic and anti-depressive effects by increasing the antioxidant mechanism and diminishing oxidative stress markers. The available treatment, which is reported until the date are 5-HT-1A agonist such as buspirone [47], 8-OH-DPAT [52], etc. Various parts of plants such as Allium cepa peel [31], Banana fruit pulp, and peel [51], etc., and an active compound, which are present in various plants such as quercetin [40], gallic acid [42], etc. Previously, it was reported that leaf extract of M. madrespatana have anxiolytic [53] anti-inflammatory [54] and antioxidant [53] effects. Having potent antioxidant M. madrespatana enhanced free radical scavenging activity and improved the antioxidant enzyme system [53,54]. The present study showed that M. madrespatana peel powder improves immobilization stress-induced antioxidant enzymes by increasing SOD, CAT, and GPx ( Figure 7) and decreasing MDA levels ( Figure 6). These results indicating that single immobilization stress-induced oxidative stress is protected by repeated administration of M. madrespatana peel powder possibly via its antioxidant effects.
Serotonin neurotransmission is an important element of the stress response [55]. It was reported earlier that acute stress enhances 5-HT function [40] and increases 5-HT levels in the whole brain [31,40] and various regions of the rat's brain (such as hippocampus, [56]). Dysfunction of serotonergic neurotransmission produces various psychiatric ailments including anxiety [57], depression [58], and cognitive impairment [59]. Increased levels of 5-HT enhancing cognitive function [31,40] and exhibiting anti-depressive behavior [60], however, reduced concentration of 5-HT in the brain [30], and in the hippocampus [33] by altering memory function. Results of the present study showed that the administration of M. madrespatana decreased 5-HT and 5-HIAA levels in immobilized rats than water-treated rats ( Figure 9) and proved its anxiolytic property. Apart from that, the reduction of 5-HT and its metabolite was smaller in M. madrespatana-treated stressed animals than unstressed animals ( Figure 9). In the present study, 5-HT turnover was found comparable in M. madrespatana-treated unstressed animals and streamed animals ( Figure 9). Although memory improvement ( Figure 5) was found in both M. madrespatana-treated stressed rats and unstressed rats, it was more improved in M. madrespatana-stressed rats than unstressed rats, which may be due to a smaller decrease of 5-HT and 5-HIAA levels in M. madrespatana-stressed animals (Figure 9). Similarly, the anti-depressive effect of M. madrespatana was observed in both stressed and unstressed rats. It is reported earlier that the whole plant of M. madrespatana contains an alkaloid, terpenoid, flavonoid, and phenolic compounds [61] and the potential role of M. madrespatana as an antioxidant is due to the presence of these phytochemicals, which are involved in its anxiolytic, antidepressant, and memory-enhancing effects.
AChE is a degradative enzyme and a biomarker to determine the cognitive function. ACh is a key neurochemical, which is concerned with memory and learning [62]. Previously, it has reported that acute stress increases the release of ACh in the hippocampus [63] and modulates the genes that regulate ACh availability after stress and blockade of AChE [64]. Single immobilization increases the ACh availability in the synapse and enhances memory function [64] as observed increased levels of ACh in our study ( Figure 10). The previous report showed that single immobilization stress decreased AChE activity [31] and decreased latency escape, which signifies improved memory in these rats. The present result on AChE activity ( Figure 8) and ACh levels ( Figure 10) are consistent with the previous report [31,40]. Although acute immobilization stress increased oxidative stress (Figure 5), memory improvement as observed in the present study may be due to increased cholinergic function and antioxidant effect of M.M. Previously, various phytochemicals such as gallic acid [42], quercetin [40], and curcumin [65] decreased AChE activity and increased the availability of ACh with improved memory function. It may be postulated that M.M. peels involved in neuronal plasticity by its free radical scavenging potential increase the ACh levels ( Figure 10), and produce an inhibitory effect on AChE activity (Figure 8), as observed in the present results. Single immobilization stress and M. madrespatana peel powder have the potential to improve cognitive function via its antioxidant effects, which is the novelty of the present study.

Conclusions
It is concluded that the present research supports the antioxidant potential of M. madrespatana as reported previously, which protects against single immobilization stress-induced anxiety and depression-like behaviors in rats. Besides, M. madrespatana also involved in the regulation of serotonin function and produces anxiolytic, antidepressant, and memory-enhancing effects. These consequences offer the pharmacological indication of folkloric practices of this plant for some neurological disorders. It is suggested that daily intake of M. madrespatana peel could be an enhanced antioxidant system of the body and a potential medication for the daily stress-based situation. However, in the future, more studies could be performed to evaluate the effect of M. madrespatana on female rats and using chronic stress conditions.

Conflicts of Interest:
The authors report no conflict of interest.