Ramdas Bhat1*, Suchethana P. S.2, Vinishia Saldanha2, Yuktha S. K.2,
Neema Bhallaram Patel2, A. R. Shabaraya3
1Associate Professor, Department of Pharmacology, Srinivas College of Pharmacy, Valachil,
Mangalore 574143, Karnataka, India.
2Student, Department of Pharmacology, Srinivas College of Pharmacy, Valachil,
Mangalore 574143, Karnataka, India.
3Principal and Director, Srinivas College of Pharmacy, Valachil, Mangalore 574143, Karnataka, India.
*Corresponding Author E-mail: ramdas21@gmail.com
ABSTRACT:
Background and Objective: Anxiety, a complex psychological and physiological state, negatively impacts various aspects of life. This study aimed to evaluate the anti-anxiety effects of the hydroalcoholic extract of Ficus religiosa leaves in experimental animals. Methodology: The anxiolytic effects were investigated in mice using the elevated plus-maze and light-dark models. Two oral doses of 100mg/kg and 200mg/kg were administered, with diazepam (1mg/kg i.p.) as the standard. Parameters measured included total time and entries in open/closed arms and light/dark chambers. Results: Animals treated with the hydroalcoholic extract at 200 mg/kg exhibited a significant increase in time and entries in the open arms of the elevated plus-maze. Additionally, there was an increase in time spent on the illuminated side of the light–dark test compared to control animals. Conclusion: The study demonstrated significant anxiolytic activity in Swiss albino mice treated with the hydroalcoholic extract of Ficus religiosa leaves. The exact mechanism remains unclear, suggesting a potential association with specific compounds in the leaves. Further research is required to identify and assess the active chemical constituents responsible for this anxiolytic activity.
KEYWORDS: Hydroalcoholic extract of Ficus religiosa leaves (HEFRL), Anti-anxiety, Elevated plus maze (EPM), Light and dark chamber (LDC), Swiss albino mice.
INTRODUCTION:
Anxiety, a complex psychological and physiological state, significantly impacts individuals' quality of life1. Its prevalence is substantial, with anxiety disorders affecting around 18.1% during teenage years and a lifetime prevalence of 28.8%2. Traditional drug treatments target neurotransmitter pathways, including adrenergic and serotonergic systems.
Selective Serotonin Reuptake Inhibitors (SSRIs) and benzodiazepines are commonly used, with SSRIs preferred due to their broader efficacy3.
In India, there's a growing interest in alternative medicine, particularly plant-derived remedies4. Ficus religiosa (Bo tree), also known as Peepal, is a revered plant with therapeutic applications in Ayurveda and Unani. Its various parts contain phytoconstituents like phenols, tannins, flavonoids, and β-sitosteryl-D-glucoside5. Despite its extensive use, there's a lack of reported data on the antianxiety activity of Ficus religiosa leaves.
Figure 1: Preparation of Hydroalcoholic Extract of Ficus religiosa leaves.
The present study aims to fill this gap by evaluating the antianxiety properties of the hydroalcoholic extract of Ficus religiosa leaves (HEFRL). This exploration aligns with the broader trend of investigating natural remedies, reflecting the ongoing interest in merging traditional knowledge with contemporary research.
METHODOLOGY:
Collection and authentication of Ficus religiosa leaves:
The Ficus religiosa were collected from the Uppinangadi, Dakshina Kannada District and Authenticated by Dr. H S Shenoy Principal Scientist and Head of Botany Division of Pilikula Development Authority Moodushedde Post, Mangalore -575028.
Preparation of Hydroalcoholic Extract of Ficus religiosa leaves (HEFRL):
The powdered leaves that had been shade-dried were extracted using the Maceration process, which used 70% (v/v) ethanol and water. 500grams of leaf remains were macerated with water and 70% (v/v) ethanol for 72 hours to create the crude hydroalcoholic extract. In the rotary flash evaporator, the extract was filtered and concentrated at 45°C under low pressure. After obtaining a greenish-dark powder, it was stored at 4-5°C in an airtight container until needed later6.
Phytochemical screening:
A phytochemical examination was carried out for both extracts as per standard methods7.
Animals:
The experiment involved healthy Swiss albino mice (18 to 25g), obtained from the animal house of Srinivas College of Pharmacy. They were kept in standard conditions and acclimatized for a week. Housed in sanitized cages with sterile bedding, they had free access to a standard diet and water. The Institutional Animal Ethics Committee approved the protocol (Approval no SCP/IAEC/F150/P226/2023), ensuring compliance with animal care guidelines. All procedures followed CPCSEA directives under the Ministry of Animal Welfare, Government of India.
Preparation of Stock Solution of the Extract for Dosing:
The HEFRL was weighed and dissolved in distilled water. Each time fresh preparation of the extract was prepared before administration. The extract was administered post orally at a constant volume of 100mg/kg and 200mg/kg for each animal.
Dose Fixation:
A dose of 100mg/kg and 200mg/kg body weight was chosen as per previous works8.
Anxiolytic Activity:
Preparation of animals: The animals were selected in such a way that they were free from illness, injury, and disease and kept in their cages for at least 5 days before dosing to allow for acclimatization to the laboratory conditions. Only those animals which are healthy having weights 18-30g were selected and maintained at standard laboratory conditions.
Preparation and administration of doses: The test doses were prepared in distilled water and administered intraperitoneally. The standard dose was administered orally. In all cases, the concentrations were prepared in 1 ml/30g of body weight. The test substances were administered in a single dose after fasting for 3 to 4 hours.
Elevated Plus Maze: The anxiety levels of the animals were observed using a plus maze apparatus, comprising two open arms (16 x 5cm) and two closed arms (16 x 5 x 12cm) with an open roof, elevated 25cm above the floor. Before the experiment, the animals were fasted for 18 hours. The dose administration was timed so that each mouse experienced the plus maze 45 minutes after receiving the dose. Placing each mouse in the center of the elevated plus maze, with its head directed towards the open arms, the 5-minute experiment recorded several behavioural aspects: a) the preference of the animal for its initial entry into open or closed arms, b) the number of entries into the open arm, and c) the average time spent by the animal in the open arm. The anti-anxiety activity was assessed by noting the average time spent by the animals in the open arms. Throughout the experiment, the animals were allowed to interact socially, and precautions were taken to prevent external stimuli from disturbing them9,10.
Light dark chamber test:
The Light Dark Chamber Test (40, 41) was conducted to assess anxiolytic effects in mice based on the model described by Crawley and Goodwin (1980) and Crawley (1981). Mice tend to explore novel environments but avoid brightly lit open fields and dark corners. Anxiolytic treatment is expected to result in increased crossings between the two chambers and heightened locomotor activity. The test apparatus included a light and dark chamber divided by a Photocell-equipped zone. A polypropylene animal cage (44×21×21cm) was dark-ended with black spray covering one-third of the cage and bright two-thirds. The cage, placed on animex® activity monitors, tracked total locomotor activity. A small partition with a 13cm long×5cm high opening separated the dark chamber from the light chamber. Male mice were introduced into the cage, and 30 minutes before the experiment, they were treated intraperitoneally with test drugs or vehicle. Observation continued for 10 minutes, with groups of 3 animals for each dose. Behavioural measurements included the number of entries into the dark and light chambers and the time spent in each chamber. The procedure occurred preferably in a sound-attenuated room, and observations were made from an adjacent room using a web camera (CyberPix S-300) connected to the computer system9,10.
Experimental Design:
The Swiss albino mice (22- 27gms) of either sex was selected. The mice were divided into following groups (n=6) for each model as follows:
Group I: Vehicle control (Saline 0.9%ml/kg)
Group II: Standard group (Diazepam-1mg/kg) (i.p)
Group III: Hydroalcoholic extract of Ficus religiosa leaves (100mg/Kg) (p.o)
Group IV: Hydroalcoholic extract of Ficus religiosa leaves (200mg/Kg) (p.o).
All animals were pretreated for 20 days, except diazepam-treated animals. On the 21st day, animals were treated 30 minutes before the evaluation.
Statistical Analysis:
The results, presented as Mean±SEM, underwent statistical analysis using one-way ANOVA. After the ANOVA, Dunnett's multiple comparison tests were employed. A significance level of "P" value 0.05 or less was considered for statistical significance. All the analysis were carried out in GraphPad Prism version 9.5.0.
RESULT:
Preliminary phytochemical screening:
Results of the preliminary phytochemical investigation of HEFRL are shown in table 1.
Table 1: Results of Preliminary phytochemical screening of HEFRL.
|
S. No. |
Test |
Result |
|
1 |
Test for alkaloids |
|
|
a. Dragendorff’s test |
+ |
|
|
b. Mayer’s test |
+ |
|
|
c. Wagner’s test |
+ |
|
|
d. Hager’s Test |
+ |
|
|
2 |
Carbohydrates |
|
|
a. Molisch’s Test |
+ |
|
|
b. Benedict’s Test |
+ |
|
|
c. Fehling’s Test |
+ |
|
|
3 |
Glycosides |
|
|
a. General test |
+ |
|
|
b. Borntrager’s Test - |
+ |
|
|
c. Killer Killiani’s test |
- |
|
|
d. Legal’s Test |
+ |
|
|
4 |
Flavonoids |
|
|
a. Lead Acetate Test |
+ |
|
|
b. Shinoda Test |
+ |
|
|
5 |
Steroids |
|
|
Salkowaski Test |
+ |
|
|
6 |
Tannis & Phenolic compounds |
|
|
FeCl3 Test |
+ |
|
|
7 |
Amino Acids |
|
|
Ninhydrin Test |
- |
|
|
8 |
Proteins |
|
|
Ethanol Test |
- |
|
|
9 |
Saponins |
|
|
Foam Test |
- |
|
|
10 |
Fixed Oils & Fats |
- |
|
11 |
Volatile Oil |
+ |
Screening of Hydroalcoholic extract of Ficus religiosa leaves for its anxiolytic activity:
In this study, anxiolytic models namely elevated plus maze model and light and dark chamber were employed.
A. Elevated Plus Maze Model (EPM):
EPM was proposed as a model to test for anxiolytic activity. The test has been proposed for selective identification of anxiolytic drugs. Anxiolytic compounds decrease anxiety and increase open-arm exploration time. In EPM, out of animals treated with two doses of HEFRL (100mg/kg and 200mg/kg), animals treated with a high dose (200mg/kg) showed an increase in the number of entries in the open arm, which was significant when compared with the control. Animals treated with diazepam (1mg/kg), as expected, showed a significant increase in the number of entries to the open arm. Animals treated with a high dose also showed a significant decrease in the number of entries to the closed arm when compared with the control. Similarly, out of animals treated with two doses of HEFRL (100 mg/kg and 200mg/kg), animals treated with a high dose (200mg/kg) showed an increase in the time spent at the open arm, which was significant when compared with the control. Similarly, animals treated with diazepam (1 mg/kg), as expected, showed a significant increase in the time spent at the open arm as shown in table 2 and figure 2, 3, 4 and 5. Animals treated with a high dose also showed a significant decrease in the time spent in the closed arm when compared with the control.
Table 2: Effect of HEFRL on the EPM model.
|
Group No. |
Drug Treatment |
Dose (mg/kg) |
Number of Entries (Mean ± SEM) |
Time spent in seconds. (Mean ± SEM) |
||
|
Open arm |
Closed arm |
Open arm |
Closed arm |
|||
|
I |
Control |
1ml |
4.167 ± 0.307 |
6.16 ± 0.543 |
135.167 ± 7.731 |
186.50 ± 7.549 |
|
II |
Diazepam |
1 |
11.16 ± 0.167*** |
3.167 ±0.477*** |
176.833 ±7.812*** |
125.0 ± 7.638*** |
|
III |
Test 1 |
100 |
4.50 ± 0.428ns |
5.667 ± 0.615ns |
136.0 ± 4.163ns |
170.50 ± 14.40ns |
|
IV |
Test 2 |
200 |
6.33 ± 0.422*** |
3.33 ± 0.211*** |
174.66 ± 1.202*** |
120.66 ± 9.06*** |
Note: Data in each column is represented as Mean ± SEM; *** indicates P < 0.001 when compared to the control group."ns" indicated non-significant differences.
Figure 2: Comparative profile of EPM entries in the open arm following oral HEFRL administrations of 100mg/kg and 200 mg/kg.
Figure 3: Comparative profile of EPM entries in the closed arm following oral HEFRL administrations of 100 mg/kg and 200 mg/kg.
Figure 4: Comparative profile of time in open arm in EPM following oral HEFRL administrations of 100mg/kg and 200 mg/kg.
Figure 5: Comparative profile of time in closed arm in EPM following oral HEFRL administrations of 100 mg/kg and 200 mg/kg.
Light and Dark Chamber has been described as simple behavior model in mice to detect compounds with anxiolytic effects. In LDC, out of all animals treated with two doses of HEFRL, animals treated with a high dose(200mg/kg) showed an increased number of entries in the light chamber and a decrease in the number of entries in the dark chamber when compared with the control, respectively. Similarly, animals treated with diazepam (1 mg/kg) as expected showed an increased number of entries to the light chamber, respectively.Out of all animals treated with two doses of HEFRL (100 mg/kg and 200 mg/kg), animals treated with a high dose(200mg/kg) showed reduced time spent in the dark chamber and a concomitant increase in time spent in the light chamber when compared with the control.Similarly, animals treated with diazepam (1 mg/kg) as expected showed reduced time spent in a dark chamber with a concomitant increase in time in a light chamber respectively as shown in table 3 and figure 6,7,8 and 9.
Table 3: Effect of HEFRL on Light and dark chamber model
|
Group No. |
Drug Treatment |
Dose (mg/kg) |
Number of entries (Mean±SEM) |
Time spent in seconds (Mean ± SEM) |
||
|
Light |
Dark |
Light |
Dark |
|||
|
I |
Control |
1ml |
2.500 ± 0.342 |
16.833 ± 1.515 |
0.417 ± 0.031 |
9.833 ± 0.749 |
|
II |
Diazepam |
1 |
15.167 ± 1.352*** |
9.500 ± 0.224*** |
5.833 ± 0.477*** |
3.167 ± 0.477*** |
|
III |
Test 1 |
100 |
3.500 ± 0.428ns |
15.667 ± 0.843ns |
0.567 ± 0.105ns |
8.0 ± 0.632ns |
|
IV |
Test 2 |
200 |
8.167 ± 0.601*** |
10.167±0.543*** |
2.333 ± 0.333*** |
5.167 ± 0.703*** |
Note: Data in each column is represented as Mean ± SEM; *** indicates P < 0.001 and when compared to the control group."ns" indicated non-significant differences.
Figure 6: Comparative profile of the number of entries in the light chamber in the LDC following oral HEFRL treatment at doses of 100/kg mg and 200 mg/kg.
Figure 7: Comparative profile of the number of entries in the dark chamber in the LDC following oral HEFRL treatment at doses of 100/kg mg and 200 mg/kg.
Figure 8: Comparative analysis of the amount of time spent in the light chamber in the LDC following oral HEFRL administration at doses of 100mg/kg. and 200 mg/kg.
Figure 9: Comparative analysis of the amount of time spent in the dark chamber in the LDC following oral HEFRL administration at doses of 100mg/kg. and 200 mg/kg.
DISCUSSION:
The last two decades have presented significant challenges in minimizing the side effects of psychiatric drugs for anxiety and depression11. In response to this, herbal medicines are emerging as a promising therapeutic strategy4. Despite the frequent use of the synthetic drug diazepam as a standard anxiolytic, its associated side effects prompt the exploration of alternative solutions. Conventional drug treatments for anxiety typically target the adrenergic and serotonergic pathways in the brain. However, the chronic use of synthetic drugs, particularly benzodiazepines like diazepam, is associated with limitations and potential side effects, prompting the exploration of alternative solutions such as herbal medicine4,12.
The study evaluates the anxiolytic properties of the hydroalcoholic extract of Ficus religiosa leaves (HEFRL) using behavioral models like the Elevated Plus Maze (EPM) and Light and Dark Chamber (LDC) tests in Swiss albino mice. The phytochemical screening of HEFRL revealed the presence of various compounds, including alkaloids, carbohydrates, glycosides, flavonoids, steroids, tannins, phenolic compounds, and volatile oil. In the EPM model, animals treated with a high dose of HEFRL showed a significant increase in the number of entries and time spent in the open arm and a significant decrease in the number of entries and time spent in the closed arm when compared with the control. Similarly, in the LDC model, animals treated with a high dose of HEFRL showed an increased number of entries in the light chamber and a decrease in the number of entries in the dark chamber when compared with the control.
The anxiolytic effect of HEFRL may be attributed to the presence of various phytochemicals that could interact with the central nervous system and help reduce anxiety levels. The specific mechanisms through which HEFRL exerts its anxiolytic effects were not detailed in the provided text. However, the presence of compounds such as flavonoids, tannins, and phenolic compounds in the extract may contribute to its anxiolytic properties. Flavonoids have been reported to possess anxiolytic effects through their interactions with neurotransmitter systems in the brain, such as the gamma-aminobutyric acid (GABA) and serotonin systems. Tannins and phenolic compounds are also known for their potential to modulate neurotransmitter activity and exhibit anxiolytic effects. Therefore, it is plausible that the combination of these phytochemicals in Ficus religiosa leaves could contribute to its anxiolytic activity.
CONCLUSION:
The study confirms the significant anxiolytic effects of the hydroalcoholic extract of Ficus religiosa leaves in Swiss albino mice, as demonstrated by the Elevated Plus Maze and Light and Dark models. The obtained data is sufficient and conclusive, supporting the traditional knowledge regarding the anxiolytic properties of Ficus religiosa leaves. Preliminary phytochemical studies identified various constituents, including alkaloids and flavonoids. Although the exact mechanism remains unclear, further research is warranted to pinpoint the active compounds responsible for the observed anxiolytic activity.
ETHICAL STATEMENT:
The study protocol was approved by IAEC, Srinivas College of Pharmacy, Valachil, Mangalore (Ref no. SCP/IAEC/F150/P226/2023).
CONFLICT OF INTEREST:
None.
ACKNOWLEDGMENT:
We would like to thank the Staff and Management of Srinivas College of Pharmacy.
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Received on 16.01.2024 Revised on 13.05.2024 Accepted on 03.07.2024 Published on 07.12.2024 Available online on December 30, 2024 Res.J. Pharmacology and Pharmacodynamics.2024;16(4):263-268. DOI: 10.52711/2321-5836.2024.00045 ©A and V Publications All right reserved
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