Evaluation of ethanol leaf extract of Ocimum sanctum in experimental models of anxiety and depression

Abstract

Context: Recent studies reveal the co-occurrence of both anxiety and depressive disorders in many clinical conditions, which has introduced the concept of mixed anxiety and depressive disorders (MADD).

Objective: The study evaluated the ethanol leaf extract of Ocimum sanctum (OS) Linn. (Labiatae), a prominent medicinal plant, against both anxiety and depressive disorder, to evaluate its potency in combating MADD.

Materials and methods: Swiss albino mice weighing 20–25 g were used. Gross behavior was observed through Digiscan animal activity monitor. Depression was studied through tail suspension test (TST) and forced swim test (FST). Anxiety experiments included light dark test, elevated plus maze test, and holeboard test. Further, rotarod test was also used to study any defects in motor coordination.

Discussion and conclusion: OS at 200 mg/kg showed motor-depressant activity as evaluated with locomotor activity and stereotypy measures. OS at 50 mg/kg shortened the immobility time in the TST and FST, respectively, indicating a possible antidepressant activity. Further, a diminution in the anxiety response at a dose of 50 mg/kg, p.o. body weight was also observed against light dark, elevated plus maze, and holeboard tests, which signifies its antianxiety activity. No defects were observed in the motor coordination of the mice in the rotarod test. Thus, the OS extract shows antianxiety and antidepressant properties at the same dose and can be a potential therapeutic agent against mixed anxiety and depressive syndrome.

Keywords::

• Mixed anxiety and depressive disorders
• elevated plus maze
• forced swim test
• holeboard test
• light dark test
• tail suspension test

Introduction

Depression and anxiety are the most prevalent chronic psychiatric disorders. The World Health Organization (WHO) has envisaged that depression will become the second leading cause of premature death or disability worldwide by the year 2020 (WHO, 2001). A number of recent studies have shown that depression predicts the onset of number of clinical conditions including hypertension, coronary heart disease, cancer, neurological disorders, hypothyroidism, as well as diabetes mellitus (Reus, 2008) and constitutes the most potent risk factors for suicide, a leading cause of death worldwide.

Similarly in any given year, 40 million adults are affected by anxiety disorder and can also precipitate or aggravate cardiovascular and psychiatric disorders (Weissman et al., 1990). Approximately two-thirds of the anxious or depressed patients respond to the currently available treatments but the magnitude of improvement is still disappointing; besides, they also produce various systemic side effects and exhibit dependence and tolerance on chronic treatment (Baldessarini, 2006).

In 1992, the International Classification of Diseases (ICD-10) introduced the concept of mixed anxiety and depressive disorder (MADD) (Barkow et al., 2004). Recent studies have reported that depression and anxiety may occur together with the association of subthreshold depressive symptoms and subthreshold depressive anxiety representing comorbid “pure” conditions. Anxiety may also predispose depression (or vice versa), or symptoms of anxiety and depression may be external manifestations of one underlying cause. So, drugs having properties to combat both anxiety and depression, with few side effects, might be useful for such clinical conditions. In order to overcome these adverse effects, extensive investigations have searched for novel and better-tolerated molecules from plant sources.

Herbal medicines play an important role in healthcare programs worldwide. The search for novel pharmacotherapy from medicinal plants for psychiatric illnesses has progressed significantly in the past decade and their therapeutic potential has been assessed in a variety of animal models (Zhang, 2004).

Indian medicinal plants and their derivatives have been an invaluable source of therapeutic agents to treat various disorders including psychiatric illness (Walter & Rey, 1999). Ocimum sanctum (OS) Linn. (Labiatae), popularly known as Tulsi in Hindi and Holy Basil in English, is an indigenous to India. The plant is distributed and cultivated throughout the India. It is an erect, much branched softly pubescent undershrub, 30–60 cm high with red or purple subquadrangular branches. Leaves are simple, opposite, oblong, with entire or dentate margins, minutely gland dotted, with slender, hairy petioles.

OS is known to possess various therapeutic properties, and mentioned as one of the most noteworthy plant in various medicinal systems. OS has been reported to possess anticarcinogenic (Karthikeyan et al., 1999), antidiabetic (Gupta et al., 2006), antihelminthic (Asha et al., 2001), anti-inflammatory (Godhwani et al., 1987), antioxidative (Geetha & Vasudevan, 2004), antibacterial (Singh et al., 2005), antistress (Gupta et al., 2007), and antiulcer (Dharmani et al., 2004) properties. OS leaves contain 0.7% volatile oil comprising about 71% eugenol and 20% methyl eugenol. Additional components are carvacrol, sesquiterpine hydrocarbon caryophyllene, apigenin, luteolin, ocimumosides A and B, ocimarin, apigenin-7-O-glucuronide, orientin, olludistin, and ursolic acid (Gupta et al., 2007).

In our previous studies, we found some traditional medicinal plant extracts such as Bacopa monerra Linn. (Scrophulariaceae) and Panax quinquefolium Linn. (Araliaceae) to possess promising activity in MADD (Chatterjee et al., 2010). Therefore, our aim was to further evaluate whether OS extract can be effective against MADD as well. According to the pharmacological profile of OS, it is reasonable to assume that these extracts might have some other neuroactive properties (Bhattacharyya et al., 2008). Therefore, the present study was designed to investigate the anxiolytic and antidepressant effects of these extracts by using various experimental anxiety and depression paradigms in rodents.

Materials and methods

Animals

All experimental protocols were approved by our Institutional Animal Ethical Committee (Approval No. 93/09/Pharmacol/IAEC) following the guidelines of Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA), which complies with International norms of Indian National Science Academy (INSA). Albino male Swiss mice weighing 20–25 g were employed in the study. Mice were housed in six per cage at constant temperature (22°C ± 2°C) and 12 h light/dark (8:00 a.m. to 8:00 p.m.). Mice were fed standard laboratory food (provided by N.L.A.C., C.D.R.I., Lucknow) and water was given ad libitum. Each animal was used once in the behavior tests.

Preparation of extract

The leaves of OS were collected from Lucknow in the month of November 2008 and identified by Dr. S.C. Agarwal, Head, Botany division of Central Drug Research Institute, Lucknow. Voucher specimen (No. 0038) is kept in the herbarium of the institute. Dried powdered leaves of OS (200 g) were placed in glass percolator with ethanol (1.2 L) and were allowed to stand at room temperature for about 16 h. The percolate was collected. This process of extraction was repeated four times. The combined extract was filtered, concentrated under vacuum using rotavapor at 40°C, and the weight of extract obtained was 16.46 g.

Drugs and treatment schedule

Imipramine (IMP), the standard antidepressant drug, and diazepam (DZP), standard antianxiety drug, was obtained from Sigma (St. Louis, MO) and Ranbaxy Laboratories Ltd. (India), respectively. All compounds were dissolved in 0.9% physiological saline and freshly prepared. Compounds were administered orally at a rate of 0.1 mL/10 g.

Treatment schedule and behavioral observations

Mice were divided into different groups and each group consisted of 12 mice.

Spontaneous motor activity

In order to study the spontaneous motor activity, mice were treated with graded doses of OS (25, 50, 100, and 200 mg/kg, p.o.) 1 h prior to subjecting them to the Digiscan Animal Activity Monitor.

Depression experiments

Graded doses (25, 50, and 100 mg/kg, p.o.) of OS were administered to mice 1 h prior to the tail suspension test (TST). The dose of OS identified to be effective in TST was used in forced swim test (FST) model to confirm its antidepressant activity. IMP (60 mg/kg, p.o.) was used as the standard antidepressant drug. Further, we also evaluated the effect of standard anxiolytic drug DZP (1.5 mg/kg, p.o.) in TST model of depression.

Anxiety experiments

Graded doses (50, 100, and 200 mg/kg, p.o.) of OS were administered 1 h prior to subjecting mice for light dark test. The dose identified as effective in the light dark model was considered as effective anxiolytic dose and used in other models of anxiety. DZP (1.5 mg/kg, p.o.) was used as the standard anxiolytic drug. Further, we have also evaluated the effect of standard antidepressant drug, IMP (60 mg/ kg, p.o.) in the light dark model of anxiety.

Rotarod test

The maximum dose used in the behavioral studies, that is, 200 mg/kg, p.o. was used in the rotarod model for muscle coordination.

Procedures

Spontaneous motor activity

Gross open-field activity was studied using Digiscan Infrared Photocell system (Omnitech Electronics, Columbus, OH) (Sanberg et al., 1987) in 42 × 42 × 30 cm Plexiglass arenas, fitted into infrared beam containing metallic grid. Activity of animals was observed by the interruption of infrared beams.

1. Horizontal activity: The total number of beam interruptions that occurred in the horizontal sensor in the duration of 2 min.

2. Stereotypy counts: If the animal breaks the same beam (or set of beams) repeatedly, then the monitor considers that the animal exhibit stereotypy, which typically involves grooming, licking, head bobbing, and so on.

Prior to the experiment, animals were habituated in the Test Box for 15 min. After the initial habituation process, the activity of the control and treated animals were monitored for 2 min at 30 min intervals for duration of 1 h.

Depression models

Tail suspension test

The TST was performed according to the method described (Steru et al., 1985). In brief, the mice were individually suspended 60 cm above the surface of table with an adhesive tape placed 1 cm away from the tip of the tail. After 1 min acclimatization, immobility duration was recorded for 5 min. Mice were considered immobile only when they hung passively and were completely motionless.

Forced swim test

FST in mice is a behavioral despair model (Porsolt et al., 1977). The mice were placed individually in glass cylinders (20 cm height, 10 cm diameter) containing 10 cm depth of water at 25°C. After 5 min, the animals were removed from water, dried, and returned back to their home cages. They were again placed in the cylinder 24 h later and after the initial 1 min acclimatization period, the total duration of immobility was measured for 5 min. Mice were considered to be immobile when they were floating motionless. The duration of swimming was measured by digital counter.

Anxiety

Light dark test

The apparatus consisted of a Plexiglass box with two compartments (20 cm × 20 cm each), one of which was illuminated with a white light while the other remained dark. Each animal was placed at the junction of the light dark compartment, facing the illuminated compartment. The time spent in illuminated places was recorded for 5 min (Young & Johnson, 1991). After each test, the box was carefully cleaned up with a wet tissue paper (10% ethanol solution).

Elevated plus maze

This test has been widely validated to measure anxiety in rodents (Lister, 1987). This apparatus was made of stainless steel and consisted of two open arms (30 cm × 5 cm) and two closed arms (30 cm × 5 cm) with 25 cm walls. The arms extended from a central platform (5 cm × 5 cm). The maze was elevated 38.5 cm from the room floor. All the four arms consist of infrared beams fitted at regular distance. Mice were treated with OS (50 mg/kg, p.o.) and DZP (1.5 mg/kg, p.o.) 1 h prior to the experiment. Each animal was placed at the center of the maze, facing one of the open arms. The time spent in enclosed and open arms was recorded for 5-min test. The movement of animals across the arms is calculated by interruption of beams, which was analyzed by Maze tracking software (Columbus Instruments, Columbus, OH). After each test, the maze was carefully cleaned up with a wet tissue paper (10% ethanol solution).

Holeboard test

The apparatus was composed of a transparent Plexiglass arena (42 × 42 × 30 cm) with 16 equidistant holes 2.5 cm in diameter in the floor (Moreira et al., 2000). The center of each hole was 10 cm from the nearest wall of the box. The floor of the box was positioned 15 cm above the ground. An animal was placed in the center of the holeboard and allowed to freely explore the apparatus for 3 min. The number of head-dipping was recorded. A head dip was scored if both eyes disappeared into the hole.

Rotarod test in mice

Rotarod test is commonly used for evaluation of neuromuscular coordination in mice and the protocol was used as described by Dunham and Miya (1957) and studied in the Rotamex 4/8 apparatus (Columbus Instruments). Basically, the rotarod consists of a rod that is coated with polypropylene foam to provide friction and to prevent animals from slipping off the rod. The distance between the rod and floor is kept 15 cm to avoid intentional jumping of mice. The rod is driven by a motor and the rotational speed can be regulated, which is maintained at 8 rpm in our study. Animals were trained on the rotarod for duration of 2 min per trial, with three trials per day for 2 days. On the third day, mice were given trials before and after treatment of extract. The OS extract was used at the highest dose of 200 mg/kg, p.o. in present study to evaluate any defects in motor coordination.

Statistical analysis

The results were expressed as mean ± SEM. The statistical significance was determined by Student’s t-test and one-way analysis of variance (ANOVA) followed by Dunnett’s test wherever applicable, using Prism software version 3.0. P < 0.05 was considered to be statistically significant.

Results

Open-field locomotor activity test

Graded doses of OS extract (25, 50, 100, and 200 mg/kg, p.o.) were studied in Digiscan animal activity monitor in order to determine the effect on spontaneous motor activity and stereotypy counts in mice (Figure 1A). At the 60 min time point, the activity of mice treated with OS at doses 25, 50, 100, and 200 mg/kg did not show any statistical significant difference from the vehicle-treated control groups. However, after 1 h of observations, the highest dose of 200 mg/kg showed significant reduction [F(4, 55) = 2.792, (P < 0.05)] in the horizontal activity counts when compared with the vehicle-treated group as shown in Figure 1A. The doses of 25, 50, and 100 mg/ kg did not show any significant reduction in the horizontal activity counts when compared with control.

Figure 1.  (A) Bar diagram representing the horizontal activity counts of mice in Digiscan animal activity monitor. Results are represented as mean ± SEM with n = 8 in each group. *P < 0.05 when compared with control group. (B) Bar diagram representing the stereotypy counts of mice in Digiscan animal activity monitor. Results are represented as mean ± SEM with n = 8 in each group. *P <0.05, ***P < 0.001 when compared with control group.

The effect of OS at stereotypy counts was also observed as shown in Figure 1B. At the 60 min time point, the lower doses of extract did not show any significant difference when compared with the vehicle-treated control mice. However, the highest dose of 200 mg/kg shows significant decrease in stereotypy counts (P < 0.001) when compared with vehicle-treated mice. After 1 h of observation, the stereotypy counts at the dose of 25 and 50 mg/ kg remained insignificant. However, higher doses of 100 and 200 mg/ kg showed a significant reduction in the stereotypy counts (P < 0.05 for 100 mg/kg and P < 0.001 for 200 mg/kg).

Depression models

Effect of OS at graded doses in TST

Graded doses of OS extract (25, 50, and 100 mg/kg, p.o.) were studied in the tail suspension model of depression in mice (Figure 2). OS at 25 mg/kg had no significant effect on the immobility time when compared with the vehicle-treated groups. Further, higher doses of 50 and 100 mg/ kg produced a significant diminution in the immobility duration by 55% and 54%, respectively [F(4,  55) = 28.73, P < 0.001]. IMP (60 mg/kg, p.o.), the standard antidepressant, also produced a significant decrease (90%) in the immobility time (P < 0.001). The dose of 50 mg/kg, p.o. was selected as the efficacious dose and the activity was confirmed in other models of depression. Further, in order to test the efficacy of DZP in TST, mice were treated with DZP at a dose of 1.5 mg/ kg, p.o., which is the anxiolytic dose in anxiety models. The immobility duration was significantly (P < 0.01) increased by 29% when compared with control.

Figure 2.  Bar diagram representing the immobility duration (in sec) of mice in tail suspension test and forced swim test. Results are represented as mean ± SEM with n = 8 in each group. **P <0.01, ***P < 0.001 when compared with control group.

Effect of OS in FST

The dose of 50 mg/kg of OS was used to assess the degree of immobility in mice exposed to FST (Figure 2). The immobility duration in OS- and IMP-treated mice was reduced by 42 and 91%, respectively [F(2, 21) = 51.53, P < 0.001].

Anxiety

Effect of OS at graded doses in light dark model

The administration of OS extract at graded doses (50, 100, and 200 mg/kg, p.o.) in mice induced a significant increment of the time spent by mice on the illuminated side of the light dark apparatus (Figure 3). The dose of 50 mg/kg increased in the time spent in the illuminated chamber by 63%, which was significant (P < 0.001) in comparison with the vehicle-treated groups. The dose of 100 mg/kg also significantly (P < 0.05) increased the time spent in light chamber by 56%. At further higher doses of 200 mg/kg, the time spent in light chamber gets reduced by 87.30%. Similar effects were also observed in animals treated with standard anxiolytic drug, DZP, which increases the time spent in light chamber by 66% [F(4, 55) = 13.67; P < 0.001]. The dose of 50 mg/kg produced maximal effect in the light dark model of anxiety and was used in other anxiety models for confirmation of its anxiolytic potential. IMP was administered orally at a dose of 60 mg/kg, p.o., the dose used in standard depression model, in order to evaluate its anxiolytic potential. The mice showed no increase in the time spent in light chamber when compared with control group as shown in Figure 3.

Figure 3.  Bar diagram representing the time (in sec) spent in light chamber in light dark test. Results are represented as mean ± SEM with n = 8 in each group.*P < 0.05, **P < 0.01, ***P < 0.001 vs. control group.

Effect of OS in elevated plus maze

The oral administration of OS extract at 50 mg/kg, p.o., significantly (P < 0.05) increased the time spent in open arms by 55% (Figure 4), whereas DZP showed a 78% increase [F(2, 33) = 26.58; P < 0.001].

Figure 4.  Bar diagram representing the time (in sec) spent in open arms (elevated plus maze)/no. of holepokings (holeboard test). Results are represented as mean ± SEM with n = 8 in each group.*P < 0.05, ***P < 0.001 vs. control group.

Effect of OS in holeboard test

The administration of different doses of OS extract in mice induced a significant increment of the number of pokings by mice on the holeboard apparatus (Figure 4). The mice treated with OS 50 mg/kg, p.o., and DZP produced a significant increase in the number of holepokings by 27 and 30%, respectively [F(2, 33) = 7.42; P < 0.001].

Rotarod test

OS at the highest dose, 200 mg/kg, p.o., studied in the experiments was tested for rotarod. Both the control group and OS-treated group spend 120 sec in the rotating rod, without falling down even once. This confirms the finding that the extract had no motor impairments.

Discussion

Anxiety and depressive disorders accompany most of the clinical conditions including cardiovascular disorder, thyroid disorders, and postpartum condition. Also, depression is more prevalent in anxious individuals. In view of this, there is an urgent need of a drug that can overcome both these symptoms. Therefore, the plant-based products that can be identified to possess both antianxiety and antidepressant effects might prove useful as a therapeutic agent in these disorders.

This study was aimed to analyze the behavioral effects of the ethanol leaf extract of OS, a plant known for its various therapeutic properties in Indian traditional medicine. In our study, it was demonstrated that OS was able to induce antidepressant as well as anxiolytic-like effects without impairing the neuromuscular tone in mice.

The spontaneous locomotor activity observations indicate no significant effect of OS at lower doses. However, OS at higher doses caused a significant diminution of horizontal activity and stereotypy counts, in comparison with the control group, which might be due to the hypnotic nature of the drugs at high doses.

We have also studied the antidepressant effects of the OS extract in tail suspension and forced swim models of depression, which provides a rapid and reliable behavior screening test for antidepressants. The immobility has been expected to reflect a state of “behavioral despair and variants” or “failure to adapt to stress” (Willner & Muscat, 1991). It was observed that OS produces its maximal effect at the dose of 50 mg/kg, p.o. and produces a diminution of immobility time in mice exposed to both the models, comparable with that of standard antidepressant drug, IMP.

Further, we have evaluated the anxiolytic potential of the OS extract in various models of anxiety. The light dark test has been widely used for modeling anxiety, and it has been developed for predicting the efficacy of clinically used compounds for treating this disease (Young & Johnson, 1991). It has been assumed that the time mice spend in the illuminated side of the box is the most useful and consistent parameter of anxiety (Young & Johnson, 1991). Administration of OS produces a significant response in the light dark test of anxiety, as the time spent by animals in the light chamber increases significantly as compared with the control group, indicating an anxiolytic activity of the extract. However, the dose of 100 mg/kg shows a relatively weaker anxiolytic effect, which further reduces at 200 mg/kg, which might be due to motor-depressant effects of OS at these doses.

The elevated plus maze is currently one of the most frequently used models of animal anxiety (Hogg, 1996). The indices of anxiety in this test, percent of open-arm entries, and time spent in the open arm are sensitive to agents thought to act via the GABA-A receptor complex, justifying the use of DZP as a positive control in this study. In agreement with previously published reports, DZP increased the percentage of open-arm entries and the time spent in the open arms (Crawley & Goodwin, 1980), confirming its anxiolytic effects. The leaf extract of OS had similar effects on these parameters. Further, in the holeboard test, OS extract could significantly increase the number of nose poking, signifying an anxiolytic effect of the extract. The holeboard test provides a simple method for measuring the response of an animal to an unfamiliar environment and is widely used to assess emotionality, anxiety, and/or responses to stress in animals (Moreira et al., 2000). Head-dipping behavior (Takeda et al., 2002) is sensitive to changes in the emotional state of the animal, and has been suggested to express an anxiolytic state in animals, which may be reflected by an increase in head-dipping behavior. These observations clearly indicate that leaf extract of OS exerts an anxiolytic activity. Further, we have also tested the efficacy of standard antidepressant drugs in anxiety model and standard anxiolytic drug in depression model and have found them ineffective in ameliorating the symptom of other models at the same dose. However, OS shows both anxiolytic and antidepressant activity at the same dose.

OS did not cause alteration in motor coordination on the rotarod test in the protocol studied, suggesting that the decreased locomotor action observed may not be exerted through peripheral neuromuscular blockage or centrally mediated impairment of motor function (Adzu et al., 2002).

The bioassay-graded fractionation of the ethanol leaf extract of OS had previously revealed the presence of 10 compounds (Gupta et al., 2007). Ocimumosides A and B, ocimarin, together with eight other known substances, apigenin, apigenin-7-O-β-d-glucopyranoside, apigenin-7-O-β-d-glucuronic acid, apigenin-7-O-β-d-glucuronic acid 6"-methyl ester, luteolin-7-O-β-d-glucuronic acid 6"-methyl ester, luteolin-7-O-β-d-glucopyranoside, luteolin-5-O-β-d-glucopyranoside, and 4-allyl-1-O-β-d-glucopyronosyl-2-hydroxybenzene and two known cerebrosides. Out of which, ocimumoside A and B and 4-allyl-1-O-β-d-glucopyronosyl-2-hydroxybenzene had been identified as the potential antistress agents in our previous studies (Gupta et al., 2007). These compounds could normalize various biochemical and structural stress-induced abnormalities in rats, by normalizing hyperglycemia, corticosterone levels, creatine kinase, and adrenal hypertrophy. Moreover, ocimumoside A was the most potent antistress constituent found in this extract. It is quite possible that this constituent mediates the possible antidepressant and anxiolytic-like effects observed in our studies.

In this regard, it is also important to note that OS has been used traditionally as an adaptogen. Adaptogens are helpful in attaining the general hemostatic response under various physiological conditions. The stress response, which has psychological and biological components, may be a common pathway leading to affective and/or anxiety reactions. Thus OS extract showing both antianxiety and antidepressant-like activity in our study pointing toward such overall normalization of the system that causes these clinical conditions.

The wide variety of neuropharmacological actions of this plant extract opens up interesting avenues for further research (Shader & Greenblatt, 1995; Samson et al., 2006). The activity of these extract both as an anxiolytic and antidepressant needs further evaluation. This offers new perspectives in the treatment of these diseases, as there is compelling evidence that symptoms of anxiety and depression overlap with one another (Shader & Greenblatt, 1995). Many antidepressants have been reported to be of use in anxiety disorders and anxiolytics in depression (Haefely, 1992). There are several reports pertaining to both anxiolytic and antidepressant effects of serotonergic drugs (Yocca, 1990; Labrid et al., 1992; Murphy et al., 1995). These reports show that anxiety and depressant may share some common etiological factors and drugs showing both anxiolytic and antidepressant activities are to be extensively studied for their therapeutic beneficial uses.

Based on our studies, we conclude that the OS demonstrates significant anxiolytic and antidepressant potential and also rules out any side effect of the extract on motor coordination. The anxiolytic and antidepressant effect of OS at the same dose may be beneficial as a therapy for cases of MADDs. Thus, our results fortify the ethnopharmacological importance of OS in psychiatric disorders like anxiety, depression, and MADD; however, more experimentation on pharmacological mechanisms and neurotransmitter analysis is required for a definitive conclusion.

Acknowledgements

The authors are grateful to the CSIR, New Delhi, India for providing financial support. We sincerely acknowledge Mrs. Shibani Sen Gupta for her technical support.

Declaration of interest

The authors report no declaration of interests.