1. INTRODUCTION:

As reported by the WHO study, mood disorders are the second greatest cause globally of disability adjusted life years and the primary determinant of years lived with illness in every age group. Approximately 60% of patients who take medication for this condition report improvement. The symptoms of major depressive disorder (MDD), often known as "depression," include a persistently low mood, a lack of enthusiasm in previously enjoyed activities, an altered appetite, physical complaints (such as aches and pains), modifications to psychomotor functioning (such as agitation), diminished energy and fatigue, feelings of inadequacy or regret, trouble concentrating, thoughts of suicide, and overall cognitive impairments.^1,2

Following the criteria put forth by the DSM-IV, MDD is currently defined as a clinical presentation defined by multiple episodes of major depression lacking a past of manic, mixed, or hypomanic phases. For an acceptable assessment, five of the following DSM-IV signs are required to be present consistently for at least a 2-week period:

(i) A poor mood,

(ii) A lack of motivation or fulfillment,

(iii) Changes in hunger or weight,

(iv) Trouble sleeping (sleeplessness or hyposomnia),

(v) Trouble with motor skills,

(vi) Lack of vitality or stamina,

(vii) Senses of inadequacy

(viii) Trouble concentrating or thinking clearly, indecision, and

(ix) Suicidal thoughts.

It is thought that mood control is influenced by three primary compounds, which are chemically referred to as monoamines.. Primary functions of these chemicals include acting as neurotransmitters, which transfer impulses from nerves to brain receptors. The following neurotransmitters are involved in antidepressant action: Serotonin that controls many aspects of behavior, including hunger, sleep, memory, social interactions, and libido. Norepinephrine, which plays a role in stress regulation of B.P. and HR and affects alertness and motor performance. Dopamine, a neurotransmitter essential for reward and pleasure signaling, motivation, arousal, and decision-making.³

The two most popular kinds of antidepressants, especially for initial therapy, are SSRIs and SNRIs, respectively.⁴Some of the mental illnesses that fall under the umbrella term "anxiety disorder" include diseases characterized by excessive dread or concern; they include generalized anxiety disorder (GAD), panic disorders, complex phobia, social nervousness disorder, sporadic mutism, anxiety when alone, and particular obsessions. Some people may suffer from depression and another anxiety condition at simultaneously at a time; this is not uncommon, and it is strongly connected to PTSD and OCD.

Anxieties are mediated by the GABAergic and 5HT3 neurotransmission systems.^5,6 Furthermore, SSRIs are well-known to have powerful antidepressant properties and are beneficial in treating GAD. While benzodiazepines (BDZs) are known to have certain positive effects, they also come with a long list of negative side effects, such as drowsiness, muscular relaxation, physical dependency, memory problems, and medication interactions.⁷ The demand for innovative, safer, and more effective therapies is significant since existing medications have extremely limited effectiveness in many disorders.

Drugs used to treat anxiety Benzodiazepines, Beta blockers and Azapirones. The BDZs are able to enhance the inhibitory effect of GABA by binding to the GABA-A receptor subunit and then activating the chloride channel. They ended up being second-line choices once it became apparent that SSRIs were more tolerant and effective than BDZs and TCAs, despite being first-line possibilities due to their tolerance and equivalent effectiveness. People whose reactions to antidepressants have been less than satisfactory are now the ones most often prescribed BDZs.⁸

Beta blockers and azapirones have even fewer uses. Since beta blockers may alleviate physical manifestations of anxiety in around 30 to 60 minutes, they are often given as a single dosage to patients suffering from performance-related nervousness. Nevertheless, it is important to note that these medications do not address the mental or emotional aspects of anxiety. It is believed that azapirones change regulation of serotonin neuronal firing rates after binding to the 5-HT1A receptors.⁹ They are usually tolerated well, lack a dependency like BDZs, and take action in two to four weeks on average. In case of GAD, azapirones have never failed to alleviate symptoms. It is more reasonable to treat GAD with antidepressants since the disorder often involves a depressive element.

Although western modern medicine and medicinal herbs are distinct, the two eventually come together. The current trend in healthcare is utilizing synthetic medications, which initially were based on chemicals derived mostly from plants. Herbal treatments have the potential to function as supplementary or a substitute therapies.¹⁰

It is also reported to have anti-oxidant activity, seizures, memory, enhance focus, and alleviate anxiety brought on by stress. The medication is classified as a nootropic, which means it may improve cognitive function.

Our research aims to assess the anti-depressant and anxiolytic effects of plant extracts of B. monniera in rats. In the study, the EEBM is used along with anti-depressant drugs like fluoxetine and anxiolytic drugs like diazepam.

2. MATERIALS AND METHODS:

2.1. Extraction of Bacopa monnieri:

To remove moisture, the entire Bacopa monnieri (L) plant material was maintained in an oven at 40ºC and shade dried at room temperature. A mechanical grinder was then used to finely grind the dried plant. After being sieved, the powder was stored in airtight containers pending the completion of the extraction procedure. The conventional maceration procedure was used to obtain the extracts of Bacopa monnieri (L). Ethanol was used to dissolve the dried plant powder of Bacopa monnieri (L).¹¹ For seventy-two hours, the mixture was kept apart at room temperature and sometimes shaken. Following the maceration process, the mixture was filtered and the supernatant was decanted. By letting the filtrate evaporate the solvent completely, the extract was concentrated until it was completely dry. The extract was weighed and let to evaporate in the air.

2.2 Animals:

The laboratory supplied the Albino Wistar rats, which range in weight from 150 to 250grams for both sexes. The rats were held in a controlled environment with a temperature of 25±2°C and a moisture content of 75± 5%. They went through a 12-hour light-dark cycle. The mice were housed in sets of six in individual 408 × 280 × 150 polyvinyl enclosures. The animals were housed in a typical farm setting with free and unfettered availability of food and water, and they were granted seven days to adjust to their new surroundings before experimentation began. A total of twelve rats were used for the trials; the night before, they were fasted.

Table 1: Phytochemical screening of ethanolic extracts of Bacopa monnieri

I	Negative control group-Saline administered 2ml/kg
II	Reserpine at 1 mg/kg (This group excluded in Anxiolytic activity)
III	Standard drug-Fluoxetine at 5 mg/kg (Diazepam at 5 mg/kg for anxiolytic activity)
IV	Low-dose treatment - Bacopa monnieri at 200 mg/kg p.o.
V	High-dose treatment - Bacopa monnieri at 400 mg/kg orally

To determine the phytochemical ingredient profile of plant extract, various phytochemical assays were conducted. It was done to screen Bacopa monnieri (L) for various phytochemicals, including proteins, carbohydrates, amino acids, glycosides, anthraquinone glycosides, flavonoids, and tannins. The ethanol extract was utilized for phytochemical screening (Table 1).

2.3 Acute Toxicity Study (LD₅₀):

An acute toxicity investigation was performed on an ethanolic Bacopa monnieri extract in accordance with OECD standards 425.¹² It was observed that the test extract was not lethal to the rats even at 2000mg/kg b.w. doses. Each of the two of the five groups served as a control. The animals were allowed to drink water whenever they wanted during a 24-hour fast. In one study, participants were given a single oral dosage of 2000 mg/kg b.w. of an EEBM (10mL/kg b.w. in NS), while another group served as a control and was given normal saline (0.9%) as a vehicle. The extract was given to the subject orally. Extreme caution was used in order to detect any toxicity in the animals, such as enhanced physical activity, saliva production, abrupt convulsions, coma, or death, throughout the initial three hours following extract treatment. After that, every day for two weeks straight. The dosage was considered unsafe if it caused death in two out of three animals. After one animal died, the unsafe dosage would be confirmed by giving the same dose again.

2.4 Anti-depressant activity:

2.4.1 Forced Swimming Test (FST):

In a glass beaker with dimensions of 11cm in diameter and 15cm in height, loaded with potable water to a depth of 6 cm, and kept at 27±2°C, rats were made to swim independently for 15 minutes. This was considered the "pre-test" period. The next day, during a "test-session," each mouse was subjected to the same swimming conditions for another six minutes. Two sessions of testing were performed: one before (0 day) and one after (8 day) the pharmacological therapy. If a mouse floats still or moves just its head to maintain itself above water, we say that it is immobile. For the latter four minutes of the whole six-minute test, the total amount of time spent immobile was recorded.^13,14

2.4.2 Tail Suspension Test (TST):

The rats were hung 40cm in the air by their tails. Even if they tried to stand up straight again, the rats remained in an immobile position. The duration of baseline immobility was 6minutes. The next week, the rats were given medication. The longest stretch of immobility, lasting 6 minutes, was documented on the eighth day.^15,16

2.5 Anxiolytic activity:

2.5.1 Elevated Plus Maze (EPM):

Two open arms (30cm x 10cm x 2.5cm) and two closed arms (30cm x 10cm x 15cm) protruded from an integral center platform (10cm x 10cm) comprised the EPM device. On opposite sides of the room stood two sets of matching arms. The whole setup was placed 50cm above the ground. The therapy was administered to the animals thirty minutes prior to the scheduled session start time. We used diazepam and extract as treatments in our mice.¹⁷ Within 30 minutes of ingestion, the mice were moved to the middle of the four arms and given complete freedom to go around the labyrinth as they wished. As part of the experiment, we recorded how many times subjects entered and exited the open arms. For 10 minutes, every mouse was monitored.

2.5.2 Light/dark box test (LDB):

The test was conducted using a rectangular box with two sections, one measuring 46 × 27 × 30cm and the other 18 × 27cm. There is a tiny opening measuring 7.5 x 7.5 cm on the compartmental divider that allows for passage. The smaller chamber had a dark red lighting (60 W; 4 lx), while the larger chamber had a white lighting (60-W, 400 lx). The rats were given diazepam, extract, or a vehicle. We then watched the rats for 5 minutes as they sat in the center of the open box. Duration in the light zone, initial crossing delay, and transition durations between the two compartments were measured right away by evaluation.^18,19

2.6 Statistics:

The mean±SEM was used to state all the data. We used one-way ANOVA and Dunnett's test for our statistical study. Comparing the data to the control group revealed a significant difference at *P < 0.05.

3. RESULTS AND DISCUSSION:

3.1 Anti-depressant activity:

The EEBM was prepared using soxhlation method, The extract was obtained with a yield rate of 7.5%. In the qualitative phytochemical assessment of Bacopa monnieri flowers extract it was positive for Carbohydrates, Proteins, Glycosides, Tannins, Alkaloids and Flavonoids.

Ethanolic root extract of Bacopa monnieri was tested on Wistar rats up at 2000mg/kg bd.wt. There were no toxicity or fatalities seen in the animals up to a dosage of 2000mg/kg. In our research, several physical and behavioral traits were noted. Additionally, we tracked variables including caloric intake and food and drink intake. We continued to monitor the animals for another 14 days, and they were all remained safe. We conclude that the extract is tolerable up to a dosage of 2000mg/kg bd.wt. Testing showed that 2000mg/kg, bd.wt. was within safer limits and the tolerated by the animals based on the toxicity studies mentioned above. Therefore, dosage was set as 1/10^thof the lethal dose i.e., 200 mg/kg, bd.wt. second dose selected was double the dose of 1/10^thi.e. 400mg/kg bd.wt.

We assessed the total body mass and individual organ weights of the animals in every group at the conclusion of the study session. The rats in the trial group and the untreated group did not vary significantly in the acute toxicity test. The body weights of rats in acute toxicity study are mentioned in Table 2.

Table 2: Body weights of Rats

Groups	Day 0	Day 7	Day 14
Control	218.00 ± 3.74	215.00 ± 2.24	233.00 ± 2.00
EEBM (2000 mg/kg)	234.00 ± 4.30	255.00 ± 6.12	260.00 ± 7.58

3.1.1 FST:

In this method, reserpine at oral dose of 2mg/kg was used to induce depression.

From the results below, it was found that, after the treatment with ethanolic extracts of Bacopa monnieri at doses of 200 and 400mg/kg orally and fluoxetine at 5 mg/kg reduced the immobility duration of rats (Table 3).

Table 3: Anti-depressant activity of Bacopa monnieri in FST

Groups	Treatment	Dose (per kg)	Immobility period (s)
I	Negative control-Saline administered	1ml/kg	140.33±6.62
II	Reserpine	2mg	73.75±1.38
III	Fluoxetine	5mg	81.5±1.54*
IV	EEBM	200mg	93.5±2.58*
V	EEBM	400mg	99.45±1.31*

3.1.2 Tail Suspension Test:

Rats were treated with reserpine at 2mg/kg dose to induce depression like effects and the time was recorded. From the results below, it was found that, the treatment with ethanolic extracts of Bacopa monnieri at doses of 200 and 400mg/kg orally and imipramine at 5 mg/kg reduced the immobility duration of rats (Table 4).

Table 4: Anti-depressant activity of Bacopa monnieri in Tail suspension test

Groups	Treatment	Dose	Immobility period (s)
I	Negative control-Saline administered	1ml/kg	148.5±5.62
II	Reserpine	2mg/kg	82.75±1.38
III	Imipramine	5mg/kg	95.35±4.56*
IV	EEBM	200mg/kg	106.4±3.24*
V	EEBM	400mg/kg	114.3±3.58*

The current study set out to assess the presence of phytochemicals and also to ascertain the anti-depressant and anxiolytic properties of the ethanolic extracts of Bacopa monnieri in rats. The standard FST and TST were used for anti-depressant activity and standard EPM and LDB test were used as anxiolytic test models.The preliminary phytochemical analysis of the extract indicatedthe presence of sugar, proteins, flavonoids, alkaloids and tannins when tested with the standard test methods.

3.2 Anxiolytic activity:

3.2.1 EPM:

The amount of time used to explore the open arms was considerably enhanced in the experimental group relative to the control group when administered 200 and 400mg/kg of EEBM, correspondingly (p<0.05). The frequency of responses in the EPM's open arms likewise went up as a result (Table 5).

Table 5: Anti-depressant activity of Bacopa monnieri in EPM

Groups	Treatment	Dose (per kg)	Time spent In open arm (s)	Open arm entries
I	Negative control group - Normal distilled water administered	2ml	385.00 ± 1.70	9.60 ± 1.03
II	Standard drug - Diazepam	Diazepam at 5mg	326.40 ± 1.96*	18.20 ± 1.56*
III	Test group 1 - EEBM	200mg orally	290.00 ± 1.41*	13.20 ± 1.56
IV	Test group 2 -EEBM	400mg orally	300.80 ± 1.28*	17.20 ± 1.28*

3.2.2 LDB Test:

There was a notable improvement in both latency time and light chamber time when administered 200 and 400 mg/kg of extract, respectively. However, at 200mg/kg, there is a substantial increase in the frequency of light/dark switches (p<0.05) (Table 6). The frequency of transitions and duration stayed in the light chamber were both enhanced by diazepam (5mg/kg), but the latency time was dramatically lowered compared to 200mg/kg (Table 7).

Table 6: Effect of Bacopa monnieri on Latency time

Groups	Treatment	Dose (mg/kg)	Latency time (s)
I	Negative control group - Normal distilled water administered	2ml/kg	16.60±1.43
II	Standard drug - Diazepam	5mg/kg	30.80±1.46*
III	Test group 1 - EEBM	200mg/kg	33.20±1.28*
IV	Test group 2 -EEBM	400mg/kg	42.90±1.59*

Table 7: Effect of Bacopa monnieri on transition number and time spent in light parts

Groups	Treatment	Dose (per kg)	Transition number	Time in light part (s)
I	Negative control group - Normal distilled water administered	2ml	9.60± 1.03	73.60± 2.13
II	Standard drug - Diazepam	5mg	18.20± 1.56*	96.2± 1.77*
III	Low-dose group	200 mg	16.20± 1.36*	110.8± 1.93*
IV	High-dose group	400 mg	21.20± 1.26*	122.4± 1.52*

In this study, based on our findings and as described in the results above, we can assume that the ethanolic extract of Bacopa monnieri showed comparable outcome in treating the anti-depressant activity and anxiolytic activity. This study showed significant reduction of depressant activity induced by reserpine in the TST and FST which indicates that EEBM possesses significant antidepressant activity. The extracts of Bacopa monnieri showed promising anxiolytic effects in the EPM and the LDB test as the rats spent comparatively more time in the open arms after administration of the extract.

4. CONCLUSION:

In conclusion, the extract showed promising anti-depressant and anxiolytic effects on par with the standard drugs used to treat these disorders. Further research is warranted to understand the effect of the extract on the mono amine transmission involved in the clinical depression and to understand the underlying mechanism.

5. LIST OF ABBREVIATIONS:

EEBM: Ethanolic Extract of Bacopa monnieri; OECD: Organization for Economic Co-operation and Development; WHO: World Health Organization; DSM-IV: Diagnostic and Statistical Manual of Mental Health, Fourth Edition; MDD: Major Depressive Disorder; SSRIs: Selective Serotonin Reuptake Inhibitors; SNRIs: Serotonin and Norepinephrine Reuptake Inhibitors; GAD: Generalized Anxiety Disorder; PTSD: Post-Traumatic Stress Disorder; OCD: Obsessive-Compulsive Disorder; BDZs: Benzodiazepines; FST: Forced Swimming Test; TST: Tail Suspension Test; EPM: Elevated Plus Maze; LDB: Light/dark Box test; ANOVA: Analysis of Variance; B.P.: Blood Pressure; HR: Heart Rate

6. ACKNOWLEDGEMENT:

Authors are very Thankful to School of Pharmacy, GITAM Deemed to be University, Hyderabad and Department of Pharmacy, Chaitanya Deemed to be University, Gandipet, Himayathnagar, Hyderabad for their constant support for our entire Research work and manuscript publishing

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Received on 06.05.2023 Modified on 21.12.2023

Research J. Pharm. and Tech 2024; 17(6):2587-2591.

DOI: 10.52711/0974-360X.2024.00404