EVALUATION OF ANTIDEPRESSANT ACTIVITY OF ETHANOLIC EXTRACT OF LAGERSTROEMIA SPECIOSA LEAVES IN ALBINO WISTAR RATS

Objective: The objective of the study was to evaluate the antidepressant activity of ethanolic extract of dried leaves of Lagerstroemia speciosa L. (EELS) on acute restraint stress (ARS)-induced depression-like behavior and biochemical alterations in albino Wistar rats. Methods: Thirty rats were randomly divided into five experimental groups. Group-I (normal control) rats received normal saline (2.0 ml/kg, p.o.) daily for 14 days; Group-II (stress control) rats received normal saline (2.0 ml/kg, p.o.) daily for 14 days and subjected to restraint stress on the 13th day. Group-III (standard drug-treated) rats received imipramine (15 mg/kg, p.o.) daily for 14 days and subjected to restraint stress on the 13th day. Groups-IV and V rats were treated with EELS (100 mg/kg and 300 mg/kg, p.o.) daily for 14 days subjected to ARS on the 13th day. Stress-like behavior was assessed by subjecting the rats to behavioral paradigms such as tail-suspension test (TST) and open field test (OFT), 40 min post-restraint stress procedure. Pretest of 10 min for forced swim test (FST) was also given to each rat simultaneously. Then, 23.5 h later, the relevant samples were administered and the main test performed 30 min later. Oxidative stress parameters such as superoxide dismutase (SOD), catalase (CAT), malondialdehyde (MDA), and extent of lipid peroxidation (LPO) were analyzed in restraint stress-induced animals and control group, following FST on the 15th day. Statistical Analysis: Expression of data was done as a mean standard error of the mean. The normally distributed data were subjected to one-way analysis of variance followed by Dunnett’s test. *p<0.05 was considered statistically significant. Results: It was observed that L. speciosa L. showed a significant dose-dependent decrease in duration of immobility time in TST and FST when compared with the control group in a dose-dependent manner. The results of OFT also showed a dose-dependent increase in locomotor activity. In addition to behavioral tests, EELS also normalized oxidative stress markers such as CAT, SOD, MDA, and LPO in a dose-dependent manner. Conclusion: The results suggest that the ethanolic extract of L. speciosa L. leaves possesses significant antidepressant property, may be recommended as a supplement for the antidepressant activity.


INTRODUCTION
Depression is a heterogeneous disorder that affects a person's mood, physical health, and behavior. It is caused not only by changing lifestyle as perceived by the general public but also by some of the allopathic drugs, for example, an anti-hypertensive drug, reserpine that depletes neuronal storage granules of norepinephrine (NE), serotonin, and dopamine (DA), cause clinically significant depression in more than 15% of patients. Patients with major depression have symptoms that reflect changes in brain monoamine neurotransmitters, specifically NE, serotonin, and DA. According to a World Health Report, about 450 million people suffer from a mental or behavioral disorder, yet only a small proportion of them receive even the basic treatment [1]. Depression accounts for about 12% of the global burden of disease which is expected to rise to 15% by 2020. The major problems of existing allopathic antidepressant drugs include delayed clinical benefit, serious side effects, and a response rate of <50%. Commonly used drugs for depression are monoamine oxidase (MAO) inhibitors and tricyclic antidepressants (TCAs). They increase the synaptic concentration of at least two of three neurotransmitters, namely, 5-hydroxytryptamine or serotonin (5-HT), NE, and DA. The combined effect of serotonin selective reuptake inhibitor (SSRI) and serotonin reuptake transporter inhibitor increases the synaptic concentration of 5-HT and its duration of action. Ethanolic extracts of most of the herbal drugs are rich in chemical constituents such as phenolic compounds, flavonoids, triterpenoids, and steroids. Ethanolic extracts have less harmful effects, availability, and lower cost of medicinal plants versus synthetic substances make them as outstanding and simple selection in the treatment of nervous diseases. Therefore, identification and validation of plant-derived substances for the treatment of various depressive disorders attract the attention of researchers [2].
Lagerstroemia speciosa L. is also known as the crepe myrtle or Pride of India or Banaba belongs to the family (Lythraceae). Lagerstroemia is named for the Swedish merchant, Magnus von Lagerstrom, was honored by Linnaeus, speciosa means showy, referring to the flowers varying in color from pink to purple. The tree is native to Southeast Asia and is called Banaba in the Philippines [3]. The flowers and leaves are used to make an herbal tea, as they contain corosolic acid, a chemical that has an insulin-like effect of lowering glucose levels in the body. It is also utilized in weight loss management [4].

Collection and authentication of plant
The twigs (leaves, flowers, and fruits) of L. speciosa L. were collected from Mahapalika Garden, Bhayandar East, Thane. Sample specimen voucher was submitted to Dr. Rajendra D. Shinde, Director, Blatter Herbarium of St. Xavier's College, Mumbai. The leaves were compared and authenticated by comparison with the Blatter Herbarium Specimen No. JF-1532 of J. Fernandez.

Experimental animals
Female albino Wistar rats (200-300 g) were used for the study. The animals were obtained from SA-FORD, Plot No.: V-10, MIDC, Taloja, Navi Mumbai, Dist. Raigad Maharashtra -410 208. The use of these animals and the study protocols were approved by CPCSEA recognized the Institutional Animal Ethical Committee (IAEC) of Oriental College of Pharmacy, Sanpada, Navi Mumbai -400 705 under protocol no. OCP/IAEC/2018-19/08. Rats were kept at the animal house in polypropylene cages (three in each cage), at 22±2°C, with 12:12 h dark: light cycle. They were provided with commercial rat feed and water given ad libitum. The animals were allowed to acclimatize for 7 days before the study.

Selection of doses
In the literature survey, it was found that the ethanolic extract of L. speciosa L. was safe. LD 50 of the ethanolic extract is reported to be 3000 mg/kg [7]. The plant is often eaten by animals, which is also an indicator to prove it is less toxic. Thus, for the purpose of the research study, the doses of ethanolic extract of Lagerstroemia speciosa (EELS) were finalized 100 mg/kg and 300 mg/kg.

Drugs and chemicals
Imipramine hydrochloride (Abbott, Mumbai, India) was used as reference standards for the antidepressant activity.

Experimental design
Thirty rats were randomly divided into five experimental groups. Group-I (normal control) rats received normal saline (2.0 ml/kg, p.o.) daily for 14 days; Group-II (stress control) rats received normal saline (2.0 ml/kg, p.o.) daily for 14 days and subjected to restraint stress on the 13 th day. Group-III (standard drug-treated) rats received imipramine (15 mg/kg, p.o.) daily for 14 days and subjected to restraint stress on the 13 th day. Groups-IV and V rats were treated with EELS (100 mg/kg and 300 mg/kg, p.o.) daily for 14 days subjected to acute restraint stress (ARS) on the 13 th day.
Depression-like behavior was assessed by subjecting the rats to behavioral paradigms such as tail-suspension test (TST) and open field test (OFT), 40 min post-restraint stress procedure. Pretest of 10 min for forced swim test (FST) was also given to each rat simultaneously. Then, 23.5 h later, the relevant samples were administered and the main test performed 30 min later.
Oxidative stress parameters such as superoxide dismutase (SOD), catalase (CAT), malondialdehyde (MDA), and extent of lipid peroxidation (LPO) were analyzed in restraint stress-induced animals and control group, following FST on the 15 th day.

Procedure for ARS
ARS was accomplished by placing rats in an individual plastic rodent restraint device for 12 h. This restrained all physical movements without subjecting the animal to pain. Animals were deprived of food and water during the entire period of exposure to stress. After 12 h, the animals were released from their enclosure and 40 min post-release, the animals were subjected to behavioral tests and then to biochemical estimations. In the normal control group, the rats were kept in the animal cage in the experimental room [8,9].

TST
The animals were suspended individually by the end of the tail with micropore adhesive tape (approximately 1 cm) with the head 50 cm from the bottom in a suspension box 40 min post-restraint stress procedure. Rats were suspended for a total of 6 min. During the final 4 min interval of the test, the duration of immobility was recorded. Rats were considered immobile only when they will be hung passively and absolutely motionless. Antidepressant decreases the immobility of the rat in this test [10].

OFT
Open-field apparatus was made as reported, each rat was placed in the center of the open field, and its behavior observed for 5 min. The parameters evaluated were the total number of squares crossed, the number of outer squares (those adjacent to the walls) crossed, and the number of inner squares crossed; the three measures referred to as total (TL), peripheral (PL), and central locomotion, respectively. The numbers of leanings (one or two paws in contact with the wall), rearing (the mouse standing on its two hind paws without touching the walls), grooming (face cleaning, paw licking, fur licking, head scraping, and rubbing), and defecations were also recorded. At the end of each test, the whole area was cleaned with a wet sponge and a dry paper towel [11].

FST
On day 14, all the rats were allowed to swim individually for 10 min for adaptation. Then, 23

Kanase and Vishwakarma
30 cm of freshwater maintained at 25°C±1°C. Water in the cylinder was changed after each animal to prevent behavioral alteration among animals due to used water. Each animal showed vigorous movement during the initial 2 min period of the test. Duration of immobility will be manually recorded during the next 4 min of a total 6 min testing period by the observer. Rats were considered to be immobile when they floated in an upright position, making only small movements to keep their heads above the water level. Following the swimming session, rats were dried using a cotton towel and returned to home cages after the experiment. A decrease in the duration of immobility is indicative of antidepressant-like effect, whereas an increase of immobility time, when compared with the control group, is associated with depressivelike effects [12,13].

Biochemical estimation
Preparation of brain tissue homogenate All the animals were sacrificed by euthanasia using the CO 2 chamber as per rodent euthanasia guidelines, after behavioral observations. The brains were quickly removed, washed in ice-cold sterile isotonic saline, and weighed. A 10% (w/v) tissue homogenates were prepared with 0.1 Mphosphate buffer (pH 7.4). The supernatant was obtained by centrifugation of the homogenate at 1000 rpm for 20 min at 5°C and used for further biochemical estimation [14].

CAT activity
The supernatant (50 µl) was added to a cuvette containing 2.95 ml of 19 mM/L solution of H 2 O 2 prepared in potassium phosphate buffer. The change in absorbance was monitored at 240 nm wavelength at the 1 min interval for 3 min. The presence of CAT decomposes H 2 O 2 leading to a decrease in absorbance [15].

Sodium oxide dismutase activity
The SOD activity in the supernatant was measured by the method of Misra and Fridovich. The supernatant (500 µl) was added to 0.800 ml of carbonate buffer (100 mM, pH 10.2) and 100 µl of epinephrine (3 mM). The change in absorbance of each sample was then recorded at 480 nm in spectrophotometer for 2 min at an interval of 15 s. Parallel blank and standard were run for determination of SOD activity. The reaction mixtures are diluted 1/10 just before taking the readings in a spectrophotometer [16].

Determination of MDA formation
To 1 ml of suspension medium was taken from the 10% tissue homogenate. To 0.5 ml of 30% TCA will be added to it, followed by 0.5 ml of 0.8% thiobarbituric acid (TBA) reagent. The tubes were then be covered with aluminum foil and kept in shaking water bath for 30 min at 80°C. After 30 min, tubes were taken out and kept in icecold water for 30 min. These were then be centrifuged at 3000 rpm for 15 min.
The absorbance of the supernatant was read at 540 nm at room temperature against an appropriate blank. Blank consists of 1 ml distilled water, 0.5 ml of 30% TCA and 0.5 ml of 0.8% TBA [17].

Determination of LPO assay
To 0.2 ml of the test sample, 0.2 ml of SDS, 1.5 ml of acetic acid, and 1.5 ml of TBA were added. The mixture was made up to 4 ml with water and then heated in a water bath at 95°C for 60 min. After cooling, 1 ml of water and 5 ml of n-butanol/pyridine mixture were added and agitated smartly. After centrifugation at 4000 rpm for 10 min, the organic layer was taken and its absorbance was scan at 532 nm. The level of lipid peroxides was expressed as nmoles of MDA released/g wet tissue [17].

Statistical analysis
The data obtained from animal experiments were analyzed with InStat Software by GraphPad (version 3.10). It was expressed as mean±standard error of the mean. For statistical analysis, the data were subjected to analysis of variance followed by Dunnett's t-test. Results were considered to be statistically significant at p≤0.05. Significance levels were as follows: *Indicates p≤0.05 as significant; **indicates p≤0.01 as highly significant; ***indicated p≤0.001 as very significant.

Antidepression evaluation
TST Both the doses of the ethanolic extract of leaves of L. speciosa L. showed a dose-dependent decrease in immobility time when compared against    Table 4 and Fig. 1.

OFT
Both the doses of the ethanolic extract of leaves of L. speciosa L. showed a dose-dependent increase in locomotor activity when compared against stress control as well as against imipramine which was used as a standard. The results of TST are shown in Table 5 and Fig. 2.

FST
Both the doses of the ethanolic extract of leaves of L. speciosa L. showed a dose-dependent decrease in immobility time when compared against stress control as well as against imipramine which was used as a standard. The results of TST are shown in Table 6 and Fig. 3.

CAT activity
Evaluation of CAT activity revealed that stressed rats presented a significant decrease in CAT activity, which was significantly prevented by EELS (100 mg/kg and 300 mg/kg) pre-treatment when compared to unstressed group, as shown in Table 7 and Fig. 4.

SOD activity
In the rats pretreated with EELS 100 mg/kg and 300 mg/kg p.o., the level of SOD was significantly increased (p<0.001) as compared to ARS rats. Table 7 and Fig. 5 show significant and dose-dependent recovery on ARS induced reduced level of SOD in the animal due to EELS.

MDA formation
The results depicted in Table 7 and Fig. 6 illustrate that ARS significantly increased MDA level in rat brain as compared to unstressed rats. The results indicated that EELS (100 mg/kg and 300 mg/kg) pre-treatment and imipramine significantly abolished the increase in MDA level caused by ARS.

LPO assay
Quantitative measurement of LPO in the whole brain was assessed based on the amount of MDA formed, the statistical analysis revealed that ARS produced a significant increase in MDA level whereas EELS (100 mg/kg and 300 mg/kg) pre-treatment significantly abolished the increase in MDA level compared to stressed animals. The results are shown in Table 7 and Fig. 7.

Kanase and Vishwakarma
The introduction of drugs such as amitriptyline, fluvoxamine, imipramine, citalopram, venlafaxine, and others has modernized the treatment of depression. The marvellous efficacy of imipramine in these depressive disorders has paved the way for the introduction and use of newer antidepressant agents. However, the safety factor in respect of the imipramine antidepressant drugs has been rather intriguing and hence a definite need is visualized for the introduction of safer antidepressant drugs having no troublesome adverse effects [20].
The ARS was used as a model of depression induced by stress, which has both emotional and physical components in combination to distress the brain's intracellular redox status. The main purpose of using restraint is that it induces an inescapable physical and mental stress which is not accompanied by a conditioned response. It is important to clarify that this study was performed in female rats because women are more susceptible to the development of depressive disorder followed to lifetime stress events than men [21].
The behavior despair test (TST and FST) is quite sensitive and relatively specific to all major classes of antidepressant drugs, including tricyclics, SSRI, MAO inhibitors, and atypicals. Imipramine is a presynaptic uptake inhibitor of both nor-adrenaline and serotonin. Since catecholamines and 5-HT have been implicated in the etiology of

DISCUSSION
The incidence of depression in the community is very high and is associated with a lot of diseases. Hence, it is very important to report these problems and find effective remedies. Several drugs are available, all are associated with some limitations and there is a crucial need for alternative medications for these disorders. Medical therapies with herbs may be effective alternatives in the treatment of depression, and the research of their effects has progressed significantly since the past decade [18,19].

Kanase and Vishwakarma
depression, the positive effect of these drugs in TST and FST seems to be due to increased availability of these neurotransmitters at the post-synaptic receptor sites following their reuptake inhibition. In the current study, ARS significantly increased the immobility time in the FST and TST indicating depressive-like behavior which showed that restraint stress-induced depressive-like behavior as evidenced by increased immobility time in FST and TST. This immobility reflects a state of despair in animals and is claimed to reproduce a condition similar to depression in humans. Animals after antidepressant treatment feel amazing even in desperate situations, and they spend less time with immobility. In the present study, EELS in the dose of 100 and 300 mg/kg showed a significant dose-dependent antidepressantlike effect in behavior despair test (TST and FST), as they reduced the immobility time when compared against stress control as well as against standard imipramine (15 mg/kg) showing that in both the doses, it has significant antidepressant activity.
Similarly, restraint stress caused a significant decrease in central and PL crossings in the OFT, indicating depression-like behavior. The dose of 100 mg/kg and 300 mg/kg of EELS increased the number of central and PL crossings. The observed results showed a significant dose-dependent antidepressant-like effect in OFT, as they increase in locomotor activity when compared against stress control as well as against standard imipramine (15 mg/kg) showing that in both the doses, it has significant antidepressant activity.
The exact mechanism by which extract of EELS leaves exert antidepressant action is not precisely known. The leaves of EELS contain phenolic compounds and many flavonoids such as quercetin, isoquercetin, and kaempferol. Phenolic compounds and flavonoids have been reported to inhibit catechol-O-methyltransferase and MAO enzymes in the brain. Hence, it increases the concentration of catecholamines in synaptosomes and thus attenuates depression. Oxidative stress is one of the mechanisms involved in the pathogenesis of depression. Many natural products have an antioxidant property which is involved in reversing the deleterious effects of neuronal communication and behavior. CAT and SOD are one of the free radicals suggested, role in stress and depression either by modulating the release of other neurotransmitters, decrease in SOD activity, and decrease in CAT activity induced as a result of ARS, is an index of pro-oxidative conditions. In the present study, 12 h restraint stress-induced significant oxidative damage, as indicated by decreased SOD and decreased CAT, which was significantly normalized by EELS pre-treatment in a dose-dependent manner [22].
MDA and LPO are considered as a critical mechanism in causing cell injury during oxidative stress. Several studies have shown that restraint stress significantly elevated the concentration of MDA levels in the hippocampus of rats. The present study results show a significant concentration of MDA level, evidenced by an increased amount of MDA, which was significantly normalized by EELS pre-treatment in a dose-dependent manner. Thus, the antidepressant-like effect of EELS could be associated with its capacity to prevent the MDA and lipid peroxidative damage, induced by ARS [23].

CONCLUSION
It can be concluded from the study that the ethanolic extract of L. speciosa L. leaves possesses significant antidepressant property, which is probably due to flavonoids which play an active role in providing antidepressant-like effect. L. speciosa L. plant can be used for the treatment of neurological disorders and may be recommended as a supplement for the antidepressant activity.

AUTHORS' FUNDING
The authors received no specific funding for this research project work.