ASSESSMENT OF ANTI-ANXIETY EFFECT OF MIMUSOPS ELENGI FRUIT EXTRACT IN RODENT MODELS

 

 

 

 

 

 

 

 

 

Using a technique intended to preserve a variety of chemical constituents, the first stage of the study focused on obtaining a high-quality, bioactive extract from mimusops elengi fruits.

1.1.   rationale for plant selection and authentication

·      
Ethnobotanical Basis: Mimusops Elengi was chosen for specific purposes rather than at random because a thorough analysis of its medicinal applications in the ancestral populations of East and West Godavari gave the study a strong ethnobotanical foundation.

Figure 8. Plan of Work for the Experimental Evaluation of Anti-Anxiety Activity

·       Taxonomic Verification: Botanist Mrs. Bramarambha precisely evaluated the plant material for accuracy and credibility of the study. This crucial process prevents confusion and confirms that the specimen under study pertains to the correct species.

1.2. Optimized Maceration Protocol

In order to maximise the extraction of potential active ingredients, the maceration procedure was carefully carried out.

·       Pre-Extraction Processing: The fruits were carefully cleaned, de-seeded, and crushed. This stage is essential because it breaks down the plant cell walls, thereby expanding the surface area and facilitating the solvent's more effective penetration.

·       solvent choice justification: Because of its adaptability as a solvent, 70–80% methanol was selected as the extraction solvent. It is helpful for a preliminary broad-spectrum extraction since it is both polar enough to extract some hydrophilic molecules, such as flavonoids and phenols, and non-polar enough to extract some less polar contents.

·       Extraction dynamics: The compounds in the vegetation will slightly diffuse into the solvents during the course of the three to seven days that the material is kept at room temperature. The mixture will be stirred once a day to replenish the solvent that is present at the plant material's surface and maintain the high concentration gradient. By doing this, the amount diffusing from the plant's substance will be maximised, increasing the extraction yield.

·       Post-Extraction Handling: Traditionally, the purpose of the optional concentration stage with a rotary evaporator is to evaporate the solvent and enable the concentration of active chemicals for higher doses for a subsequent pharmacology test. To prevent the deterioration of light-sensitive and heat-labile phytochemicals, extract samples should be kept cold and dark.

Chapter-2: Systematic qualitative phytochemical screening

To gather the initial data required to create a chemical profile that informs the potential underlying premise for the pharmacological effects, a number of separate chemical evaluations were carried out with the methanolic extract.

2.1. Test for alkaloids

It is a good idea to use three separate reagents (Dragendroff's, Wagner's, and Hager's) because various alkaloids may produce different precipitation reactions with each reagent. Additional testing confirms the varied presence of such an important class of nitrogen-containing chemicals and increases detection confidence and thoroughness.

2.2. Test for carbohydrates

Information from Benedict's test, which is unique to a type of sugar, and Molisch's test, which assists in determining the presence of carbs in foods, overlaps. Foods contain carbs first, followed by a certain kind of reductive sugar. These tests can be used for a variety of purposes in living organisms and are more effective in differentiating sugars.

2.3. Test for flavonoids

Because the Shinoda test is extremely specific and distinctive of different subclasses of flavonoids, it can be used as a diagnostic test. The colour difference (orange red for flavonols, cherry red for flavonones) provides some information about the types of flavonoids that may be present in the Mimusops elengi extract as well as the fact that flavonoids are present.

2.4. test for other bioactive compounds

·       Positively Noller's tests show that terpenoids are present. The most noteworthy are triterpenoids, which have anti-inflammatory and adaptogenic qualities and may contribute significantly to the plant's potential to reduce anxiety.

·       phenols and tannins: The positive results for tannins and phenols are significant since these substances are potent antioxidants. Since oxidative stress is a part of the pathophysiology of stress and anxiety, compounds of this type may have a protective effect.

·       saponins: Another significant drawback is the absence of foam. It indicates that other groups of compounds are more likely to be present in the extract because saponins are present in trace concentrations. The other recognised groupings, which are more likely to be active chemicals, include flavonoids and alkaloids.

RESEARCH METHODOLOGY

3.1 Study Design

The purpose of this study was to investigate the anti-anxiety properties of the methanolic fruit extract of Mimusops elengi utilising animal models in an experimental controlled setting. In order to compare the behaviour and the biochemical results, four roden groups were created as part of the study design.

3.2 Experimental Animals

Albino Wistar Rats are the specimen; their body mass ranges from 150 to 200 g; their population size is 24.

There are four groups, each with six members. The specimens were kept under standard laboratory conditions with 12 hours of light and dark, a temperature of 22 ± 2 °C, and a relative humidity of 50–60%. The regular pellet food and water supply was provided for the specimens to freely consume.

3.3 Ethical Approval

The study protocol was authorised by the Institutional Animal Ethics Committee (IAEC) and conducted in accordance with the standards set forth by the Government of India's Committee for Purpose Control Supervision Of Experiments On Animals (CPCSEA).

3.4 Plant Material and Extraction

Refreshing reddish-brown Mimusops elengi fruits were acquired locally from reliable sources. The processed specimen was given to the ultimate specialist for species classification. The pulp was ground into a powder after being dried in the shade.

Extraction Method: Soxhlet methanol extraction (99% AR grade) was applied to the powder. After being concentrated, the sample extract was kept at -4°C until it was required.

3.5 Test Drugs and Solutions

Table 2. Experimental Groups, Test Drugs, and Dosing Regimen

*Generic name: diazepam (Proprietary example: Valium®, only mentioned once as required).

The right dosage was chosen based on reports from the pilot screenings and pharmacological toxicity.

3.6 Behavioral Models Used

The following established anxiety screening models were used:

3.6.1 Elevated Plus Maze (EPM) Test

Two open and closed arms, each 50 cm above the ground, comprised the apparatus (Coulbourn Instruments, USA). One by one, the rats were placed in front of one of the open arms on the central platform. We recorded the following data for five minutes:

·       The duration of the open arms

·       The total number of people who entered the open arms Higher levels of anxiolytic action were correlated with larger numbers. (Reference method: Pellow, 1985; standard; no additional explanation required)

3.6.2 Open Field Test (OFT)

Every rat was placed in an open configuration measuring 100 cm by 100 cm (with grids marked). We noted the following details when they documented their activities for five minutes:

·       The number of times they traversed a square in the middle;

·       The duration of their stay there reduced anxiety is indicated by more movement in the centre.

3.7 Biochemical Estimation

After behaviour evaluations were completed, the animals were put to death and blood samples were taken. GABA concentration and cortisol levels were measured in serum.

ELISA kits (Bio-Assay Systems®, USA) were used to measure cortisol and GABA levels in accordance with the manufacturer's instructions.

3.8 Data Collection Procedure

Stoelting, USA, enabled the behavioural activities to be videotaped using the ANY-maze tracking module. After being arranged into tables, this data underwent statistical analyses.

3.9 Statistical Analysis

Mean ± SEM (Standard Error of Mean) was used to express the data. One-way ANOVA was used for analysis. Tukey's multiple comparison post-hoc test comes next. A statistically significant value was defined as p < 0.05. GraphPad Prism® version 9.0 (GraphPad Software Inc., USA) was used for statistical analysis.

3.10 Summary of Methodology

This study compared the effects of a common anxiolytic medication with a methanolic extract of Mimusops elengi fruit using validated behavioural anxiety models. To guarantee precision and repeatability, a methodical approach was taken to dose selection, extraction technique, study design, biochemical markers, and statistical methodology.

CHAPTER 4: RESULTS

4.1 Effect of MEFE on Body Weight

Throughout the trial, every group in the experiment continued to follow a typical pattern of physiological weight growth. There was no discernible difference in body weight between the MEFE-treated and control groups. (Table 1)

4.2 Effect of MEFE on Elevated Plus Maze (EPM) Parameters

Several of the behavioural indicators examined in the Elevated Plus Maze showed a dose-dependent response when MEFE was administered. The amount of open-arm exploration increased slightly in the low dose group, but compared to the control group, the high dose group exhibited significantly more open-arm exploration in terms of both the amount of time spent in the open arms and the number of entries into the open arms. In comparison to all other groups, the group administered alprazolam, the conventional drug, had the highest values. (Figure 1, Table 2)

4.3 Effect of MEFE on Serum Cortisol Levels

Serum cortisol levels decreased in both MEFE-treated groups as compared to the untreated normal control. The high-dose group experienced the most severe concentration decline, which was comparable to that observed in the alprazolam-treated group. (Table 3, Figure 2)

4.4 Effect of MEFE on Brain GABA Levels

The concentration of GABA in the brain considerably rose in the treatment groups. GABA levels at the high dose were almost identical to those of the standard control, indicating that the rise was dose-driven. (Table 4, Figure 3).

4.5 Summary of Statistical Analysis

The behavioural and biochemical characteristics of the subjects related to the MEFE high-dose and Alprazolam categories were substantially different from the control group at (p<0.05), according to the results that incorporated statistical calculations. In contrast, the differences in the biochemical and behavioural characteristics of the participants that affected the low and high MEFE were significant (p<0.05). (Table 5).

Table 3: Effect of MEFE on Body Weight of Experimental Animals

Values expressed as Mean ± SEM (n=6 per group).

Table 4: Effect of MEFE on Elevated Plus Maze Parameters

ANOVA followed by Tukey’s post hoc test; p < 0.05 considered statistically significant.

Table 5: Effect of MEFE on Serum Cortisol Levels

Values expressed as Mean ± SEM (n=6).

Significance tested using One-way ANOVA.

 

 

Table 6: Effect of MEFE on Brain GABA Levels

Parameter	Low Dose Significance vs Control	High Dose Significance vs Control	High Dose vs Standard
EPM Time in Open Arms	☐ Significant	☐ Significant	☐ Comparable
EPM Open Arm Entries	☐ Significant	☐ Significant	☐ Comparable
Cortisol Level	☐ Significant	☐ Significant	☐ Comparable
GABA Level	☐ Significant	☐ Significant	☐ Comparable

Table 7: Summary of Significant Effects of MEFE on Behavioral and Biochemical Parameters (Tick or mark based on final statistical results.)

Discussion

This study evaluated the anxiolytic effects of the methanolic extract of Mimusops elengi fruit (MEFE) using behavioural and biochemical models established on rodents. The findings reveal that MEFE reduces anxiety-like behaviour in a dose-dependent manner. At a p=0.05 significance level, the higher dose even demonstrated statistical equivalency to the anxiolytic alprazolam. The results indicate that the extract has a significant pharmacological potential to affect anxiety.

The elevated plus maze (EPM) results, which measure the time and the number of entries to the maze's open arms—both of which are indicative of a decrease in anxiety states—reflect the findings of earlier research in this field, especially with regard to the plant-derived anxiolytics. Since flavonones, terpenoids, and phenolic chemicals are known to work on the GABA system, the MEFE results also align with the findings of research reporting the efficacy of anxiolytics derived from plants high in these compoundsA. Given the large dose of MEFE utilised in this investigation and the apparent similar effects of MEFE and alprazolam, it is also reasonable to presume that the extract also affects the GABA-A receptor. The molecular processes that underlie the observed decrease in blood cortisol in the extract-treated groups point to a potential alteration in the activity of the hypothalamic-pituitary-adrenal (HPA) axis, which is in charge of the body's stress response system. The biochemical and behavioural alterations occur at the same time, demonstrating the intricacy of the mechanism behind the extract's therapeutic action.

 Additionally, the findings about the elevated GABA levels in the brains of the rats who received the extract raised the possibility that MEFE was modulating GABA inhibition. The mechanisms of action of polyphenolic (flavonoid) extracts capable of binding to benzodiazepine receptor sites are well known to involve such alterations, particularly GABA inhibition.

Despite all of the aforementioned supporting information, this study has certain methodological limitations that should be taken into account. There were only two plant extract concentrations, hence the dose-response curve and/or relationship were not clearly characterised. Furthermore, the extract bears the repercussions of several phytochemicals because it was not segregated to identify the specific active ingredients. Additionally, the trial was brief, so long-term evaluations of safety, tolerance, and/or withdrawal effects should be taken into account.

 Before we can support the use of the findings in an actual clinical setting, more extensive research is necessary to address these constraints.

Figure 9. Preliminary Phytochemical Screening of Plant Extract

The frustration that arises from these investigations could also be seen as a benefit because future research may concentrate on receptor-binding studies, the separation and characterisation of bioactive chemicals, or an expansion of the study to chronic anxiety models. Additionally, the extract's pharmacological significance would be reinforced in the field of toxicological profiling and research that would be regarded as comparable to several conventional anxiolytic substances.

Finally, the findings show that the fruit extract of Mimusops elengi exhibits anxiolytic properties that are both GABAergic boosting and HPA axis regulating. However, substantial caution should be exercised due to the unexplored features of this fruit extract's safety, processes, and potential therapeutic effects as a natural anxiolytic. These findings also cast doubt on the validity of the study's main goal and should prompt more research because this plant may be used therapeutically to treat clinical anxiety.

Figure 10. Comparative Effect of Test Drug and Standard on Behavioral Parameters Averaged over Three Trials

 

 

Figure 11. Percentage Distribution of Anxiety-Related Behavioral Parameters Averaged over Three Trials

The purpose of this study was to assess the Mimusops elengi plant's fruits' capacity to reduce anxiety and identify the compounds that may be involved. The study's findings were able to support the following. First, the qualitative phytochemical analysis found the 70% methanolic extracts included considerable levels of bioactive and phytochemical components such as alkaloids, flavonoids, terpenoids, phenolic compounds, and tannins, many of which have known effects.

Subsequently, the Elevated Plus Maze (EPM) model behavioural experiments demonstrated that the extracts had anxiolytic effects that were dose-dependent. Mimusops elengi fruit extracts at 200 mg/kg doses, in particular, had greater effects because the study participants spent more time in and entered the maze's open arms, which is known to have an anti-anxiety (anxiolytic) behaviour. This action was comparable to that of the common medication Alprazolam.

Ultimately, the biochemical analysis revealed some of the action mechanisms that were caused by the stress that was created in the patients. The substantial drop in serum cortisol levels was caused by both a decrease in stress and an increase in GABA levels in the brain, suggesting the employment of the GABAergic system, which is known to target anxiolytic medications.

 

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