Antidepressant Activity of Methanol Stem Bark Extract of Ficus platyphylla Del ( Moraceae ) in Mice

Background & Aim: Depression is a heterogeneous mood disorder affecting both people in developing and developed countries. The drugs used in its management are associated with adverse effects and delayed response which compromises their therapeutic benefits. This makes it worthwhile to look for antidepressants plants with proven advantage and favourable benefit-to-risk ratio. Ficus platyphylla is used traditionally in West Africa for the management of mental illnesses. The aim of this study was to evaluate the antidepressant potential of the methanol stem bark extract of F. platyphylla. Experimental: Thin layer chromatographic finger prints of the extract were established. The oral median lethal dose of the extract was estimated using OECD 420 guidelines. Antidepressant activity of the extract at doses of 125-500 mg/kg was evaluated using tail suspension test (TST) and forced swim test (FST). The Effects of the extract on motor co-ordination deficit and locomotion were evaluated using beam walking assay (BWA) and open field test (OFT) respectively. Results: The chromatographic profile suggested the presence of steroids, tannins, flavonoids, alkaloids and saponins. The LD50 was found to be ≥2000 mg/kg orally. The extract significantly (p<0.01) and dose dependently (125, 250 and 500 mg/kg) decrease the duration immobility in the TST and FST. There is no significant increase in the number of lines crossed and on the number of foot slips in the OFT and BWA respectively. Recommended applications/industries: The methanol stem bark extract of F. platyphylla possesses antidepressant activity. The phytoconstituents found present in the plant assumed to be responsible for the activity can be characterized and isolated to serve as lead compounds in the treatment of depression. Type: Original Research Topic: Medicinal Plants Received July 5 2017 Accepted February 2


Introduction
Depression is a severe disorder affecting millions of people which is the fourth leading cause of disability worldwide (WHO, 2001) with approximately over half of depressed patients experiencing suicidal thoughts and suicidal attempts (Vikran and Swati, 2011).Antidepressant drugs used in the management of depression have been proven to be effective.However, they are associated with some disadvantages including various unwanted side effects, severe interactions and delayed response (Ronald, 2014).Additionally, reports have shown that some patients do not show response to any given treatment better than placebo.
However, naturally originated drugs were found to be effective with lower suicidal risk and minimal side effects profile (Dhingra and Sharma, 2006).This made search for new treatment modalities from medicinal plants in management of psychiatric disorders to progress tremendously.Therefore, medicinal plants such as F. platyphylla with folkloric claims used in psychiatric abnormalities are useful alternatives in the search for novel antidepressant from plant origin.The plant F. platyphylla (FP) Del.-Holl belongs to the family Moraceae.It is a tall tree of about 18 metres height and 6 meters in diameter popularly known as flake rubber tree, red Kano rubber tree and gutta-percha tree in English, "Ogbagba" in Nupe and "Gamji" in Hausa.Ficus platyphylla is an epiphyte, widely available in crown of open and wooded savannah, believed to have evolved from Senegal to Northern and Southern Nigeria (Nnabuk et al., 2012).It was reported to be used in northern Nigeria for the management of epilepsy and other psychiatric disorders (Chindo et al., 2009).
A scientific evidence proposed the plant F. platyphylla to contain phytoconstituents with anticonvulsant activities (Chindo et al., 2014).The analgesic, anti-inflammatory (Amos et al., 2008) and fertility enhancing (Ugwah-Oguejiofor et al., 2011) effects of the plant have also been scientifically validated.Additionally, an ethnobotanical survey among Hausa tribes of Kaduna State reported the use of F. platyphylla in the management of depression (Shehu et al., 2017).Thus, this suggested the screening of the methanol stem bark extract of F. platyphylla for antidepressant potentials.

Plant Collection and Extraction
The stem barks, leaves and fruits of plant F.platyphylla (SBFP) were collected.The plants were taken to Herbarium Site of Department of Biological Sciences, Ahmadu Bello University Zaria where it was identified and authenticated.A specimen voucher number 900106 was assigned there in the Department.
The SBFP were transported to Department of Pharmacognosy and Drug Development where they were dried under shade with occasional weighing until fixed weight obtained.The plant materials were grounded using mortar and pestle.About 1000 grams of powdered SBFP was extracted with 2.5 liters of methanol via soxhlet extraction.The solution was concentrated under temperature 45ºC which resulted in a brownish mass identified as methanol stem bark extract of F.platyphylla (MEFP).The extract was stored in desiccator until needed for the work.Aqueous solution was freshly prepared for each study using distilled water.

Animals
Albino mice (Swiss type weighing 18-22 grams) of both sexs were bought from the Animal House Facility of Pharmacology and Therapeutics Department, Ahmadu Bello University Nigeria.They were kept in improvised propylene cages under natural day and light cycle.The animals were given laboratory animal feeds and water freely available.All tests protocols carried out were as approved by the University Animal ethical committee and conducted within 7.00 to 5.00 hours.

Drugs and Chemicals
Some of the chemicals used for the experiment were Imipramine (Tofranil GSK brand), Diazepam (Roche, France), Methanol (Fluka-Aldrich)

Phytochemistry
Phytochemical tests were done on the methanol extract of the Ficus platyphylla (MEFP) using thin layer chromatographic finger printing technique (Marston and Hostettmann, 1991).

Acute Toxicity Studies
Acute toxic dose (LD 50 ) was determined using Organization for Economic Co-operation and Development (OECD 420) guidelines in mice.In this method, two groups each of three mice were fasted 3 hours before extract administration, then fasted body weight was used to calculate doses administered.Food was further withheld for 1-2 hours after the MEFP has been administered.The extract was administered in one oral dose using canula.A start dose of 5000 mg/kg (limit test) was used for one mouse in the first phase and observed for 48 hours.The mouse died and the main test was conducted where another mouse was dose at 2000 mg/kg then observed as above.The mouse survived, and an additional four mice were dosed and all five mice were each observed within 30minutes post treatment, then occasionally within 24hours with observation most at first 4 hours and then every day for 14days.Changes in skin and fur, eyes and mucosa, behaviour pattern and central nervous systems activities were observed.Animals were further observed for shaking, seizures, salivation, diarrhoea, wilting, sedation and coma.Time of onset of toxic symptoms and disappearance were also noted.Individual weights of animals was determined weekly for two weeks.

Tail suspension test in mice
Mice were kept in propylene cages for10 days under natural day and night cycle prior to test, with food and water ad libitum.They were taken to test area and adapted for 1 hour.Forty animals divided into five groups each of eight mice were used.Groups 1, 2 and 3 received graded doses of MEFP (125, 250 and 500 mg/kg) orally 1 hour prior to test.Group 4 and 5 were administered distilled water (10 ml/kg) and imipramine (15 mg/kg) respectively.For the test, each mouse was hung on the edge of a shelf 58 cm high above a table surface with a masking tape placed about 1cm from the tip of the tail.Immobility time was recorded over 6 minutes period (Steru et al., 1985).

Forced swim test
Forty mice were taken to laboratory one day before the test and kept individually in cages with food and water freely available.On the day of test, mice were grouped into five each of eight animals.Groups 1, 2 and 3 received 125, 250 and 500 mg/kg of MEFP orally.Groups 4 and 5 were treated with distilled water (10 ml/kg) and imipramine (15 mg/kg) respectively.One hour after treatment, each mouse was forced to swim inside a vertical Plexiglass cylinder of 24 cm height, 12 cm in diameter containing about 12 cm level of water at temperature of 25 degrees.The immobility time was measured over 5 minutes period.An animal was considered immobile when it floated passively in the water in a hunched but upright position (Alpermann et al., 1992).

Test for locomotor behaviour (Open field test)
Forty animals were shared into five groups each of eight animals.The first, second and third groups received 125, 250 and 500 mg/kg of MEFP orally 1 hour prior to test.Group 4 and 5 received distilled water (10 ml/kg) and imipramine (15 mg/kg) respectively.Each mouse was placed in white wooden open field apparatus (70×70×35 cm, length × breadth × height) of which one wall is made up of transparent plexiglass and also a plexiglass floor divided into 16 visible squares (15×15 cm) with a central square.Peripheral and central square crossing behavior of each mouse was recorded for 5 minutes with the aid of a camera suspended at the top of the apparatus.The open field inner surface area was cleaned with 10% alcohol between tests (Rex et al., 1996(Rex et al., , 1998)).

Test for motor co-ordination deficit (Beam walking assay)
Mice were forced to walk from one point on a ruler of length 80 cm, width 3 cm placed 30 cm above a table by a wooden support to a targeted goal box (Magaji et al., 2008).Each mouse was tested thrice and made to be aware that there was a goal box that could be reached.A ruler was used because the mouse easily crosses it with minimum anxiety.About forty mice that successfully attempted the walk were randomly grouped into five groups.The first, second and third groups received 125, 250 and 500 mg/kg of MEFP orally one hour prior to test.The fourth and fifth groups received distilled water (10 ml/kg) and diazepam (0.25 mg/kg) respectively via the oral route.An hour posttreatment, each mouse was placed at one end of the beam which was 60 cm long, 8 mm in diameter and elevated 30 cm above a table and allowed to walk to the targeted goal box.Fallen mice were returned to the point of fall, a maximum of one minute was allowed on beam.The number of foot slips (one or both hind limbs slipped from the beam) was counted (Stanley et al., 2005).

Novel object recognition task (NORT)
It involved the use of open field arenas (44 × 44 × 17 cm 2 ) kept in a sound attenuated cubicle with reduced light.The NORT was divided into familiarization, training and retention sessions carried out over three days.On day one, each mouse was exposed (habituated) to open field apparatus for 30 minutes.On day two (training session), mice were exposed to a 10 minutes' trial in the presence of two identical objects made up of cylindrical glassware of diameter 3 cm and height 8 cm packed with white cotton placed oppositely 12 cm from the walls.Exploration time for the objects by each mouse was hand-scored using stopwatches.Mice with exploration time less than 3 seconds were not used for the test.On day three (test session), mice were subjected to a trial choice where familiar object F and a novel object N were placed oppositely by the corner for 10 minutes.The exploration time of the two objects F and N was scored for 5 minutes.After each test, apparatus and objects were cleaned with 70% ethanol solution.Each mouse was placed in the centre of the apparatus facing the wall and allowed to freely explore it and the objects (Sahay et al., 2011).
Exploration of object is taken as the animal moving its nose close to the object at a distance of about 2 cm.In addition, if animal placed the fore paws on the objects but not climbing it is considered exploration.Data from this test were expressed as (1) exploration time (in seconds) of each object which was calculated as time spent exploring familiar or novel object by total exploration time of both objects and (2) a discrimination index (DI) between objects, calculated as the exploration time difference between the novel object (N) and the familiar object (F) divided by the total exploration time of both objects (DI = [N-F)/(N+F)].A high significant discrimination index reflects good recognition memory.

Statistical analysis
Values were expressed as mean ± SEM.The differences in the mean duration of immobility, climbing and swimming from FST, mean duration of immobility from TST, mean exploration time and mean discrimination index from NORT, mean number of foot slips from BWA and mean count of line crossed from OFT among different treated groups were analysed using one-way ANOVA followed by Bonferroni post hoc test using SPSS version 23.0.A value of p <0.05 was considered significant.

Results and discussion
The present study attempted to provide some scientific rationale on ethnomedicinal use of methanol stem bark extract of F.platyphylla in the treatment of depressive illnesses using the TST and FST.The methanol stem bark extract of F.platyphylla decreased the duration immobility in both TST (Figure 1) and FST (Figure 2) in the treated mice.Significant response was obtained at 125 mg/kg (p< 0.05), and at doses of 500 and 1000 mg/kg (p< 0.01) in the TST.Significant response ( < 0.05) was also obtained at doses of 250, 500 and 1000 mg/kg tested in the FST followed by increased climbing activity with no swimming modifications (Figure 3).Both behavioural models are widely accepted for assessing pharmacological antidepressant activity (Bourin, 1990;Porsolt et al., 1977).The FST is the most utilized pharmacological model for screening novel antidepressants in rodents with acute or immediate treatment (Cryan et al., 2002).
The test is considered sensitive and relatively specific to all types of antidepressants like tricyclics, serotonin selective reuptake inhibitors and MAO inhibitors (Detke et al., 1995).Although all antidepressant drugs were observed to reduce immobility in the FST, two distinctive behavioural patterns were reported.It has been practically ascertained that swimming is more with serotonergic compounds like fluoxetine, while climbing is linked to compounds like tricyclic antidepressants with selective effects on noradrenergic transmission (Vazquez-Palacios et al., 2004).In this study, treatment with MEFP dose dependently decreased immobility which was accompanied by an increase in climbing activity.Several studies indicated that immobility, swimming and climbing behaviours are displayed by different classes of antidepressant agents.The activity pattern showed by the MEFP and imipramine of increased climbing without swimming modifications is the pattern showed by agents that act via the adrenergic pathways.However, FST has not been viewed as a consistent model for observing selective serotonin reuptake inhibitory action, but are generally reported as active in the TST with greater pharmacological activity as compared to FST (Cryan et al., 2005).The ability of the MEFP to reduce duration immobility in the TST has further ascertain the antidepressant activity and the utilization of adrenergic pathways.
Control mice when placed in the open field arena exhibited exploratory behaviour marked by the number of lines crossed while the mice treated with methanol stem bark extract of Ficusplatyphylla unlike diazepam did not alter the number of lines crossed (Figure 4).Similarly, the methanol stem bark extract of F.platyphylla did not impair motor coordination at the tested doses (Figure 5).Thus, antidepressant effect of MEFP was further observed not to be associatedwith any motor and stimulant effects, due to its insignificant change inlocomotion of mice in comparison with control in the beam walking assay and open field test.This indicates thatthe extract neither stimulate nor inhibit central nervous system (CNS) within the tested dose range.Several studies have also shown its lack of excitatory or inhibitory effects on the CNS (Chindo et al., 2014).The result from the OFT further eliminated the probability of false positiveresults in FST and TST.This ensured that the increased active behaviours in the FST and TST were not as a result of CNS stimulatory effect and confirmed the specificity of antidepressant like effect of the extract.It has also further explained the dose dependent effect of the extract on immobility and climbing behaviours resulting in increased response on increasing the dose.The methanol stem bark extract of F.platyphylla had insignificant effect on the exploration time (Figure 6) as well as the discrimination index (Figure 7).The connections between novelty and behaviour has been considered worthy of attention by many neuropharmacologists. Novelty encompasses distortion from expected likelihood of an event on the basis of both prior information and internal estimated probable circumstances (Antunes and Biala, 2012).Additional interesting aspect about novelty is to know that rodents can be affected by a novel stimulus.The novel tests are utilized to determine effects of various pharmacological therapies as well as brain alterations (Leger et al., 2013).The three tested doses of MEFP did not show any effect on exploratory behaviour as well as on the discrimination index.
Phytochemical tests of methanol stem bark extract of F.platyphylla revealed the presence of flavonoids, tannins, saponins, alkaloids and other phenolic compounds (Table 1, Plate I).These secondary metabolites are reported to have wide biological modifications including central nervous system activities (Emamghoreishi and Talebianpour, 2009;Priyanka et al., 2012;Ashok et al., 2013;Mamta et al., 2013).Thus, these phytoconstituents may be responsible for the reported antidepressant effect of F. The LD 50 was found to be ≥2000 mg/kg orally in mice with insignificant change in body weights of mice.Symptoms of coma and death were reported when the extract was given at 5000 mg/kg (Table 2).The signs of symptoms observed at the dose of 5000 mg/kg are an indication of its potential toxicity when used at higher doses.
Table 2. Median Lethal (LD50) values for methanol stem bark extract of Ficus platyphylla.Data was analyzed using One-way ANOVA followed by Dunett's test, then presented as mean± SEM, n=5.

Conclusion
This study showed that the methanol stem bark extract of F. platyphylla possesses significant antidepressant effects; and provided some scientific basis for the folkloric use of the plant in depressive illnesses.Certainly, further studies are required for isolation and identification of the bioactive compound(s) responsible for the observed activity.

Figure 2 .
Figure 2. Effects of methanol stem bark extract of Ficus platyphylla (FP) and imipramine (Imip) on the FST in mice.Each column represents the mean ± S.E.M. of 8 animals.Data was analysed using one-way ANOVA followed by Bonferroni post hoc test, *** <0.001 significantly different from distilled water treated group.

Figure 4 .
Figure 4. Effects of methanol stem bark etract of Ficus platyphylla (FP) and Diazepam (Dia) on the Number of Lines Crossed by mice in the OFT.Animals were acutely treated with the extract (125, 250, or 500 mg/kg, p.o.), distilled water (10 ml/kg), or diazepam (10 mg/kg, p.o).Each column represents the mean ± S.E.M. of 8 animals.Data analysis was performed using One-way ANOVA followed by Bonferroni post hoc test, **= P≤ 0.01, significantly different from distilled water-treated animals.

Figure 5 .
Figure 5. Effects of methanol stem bark extract of Ficus platyphylla (FP) and Diazepam on Motor Coordination Deficit in mice.Each column represents the mean ± S.E.M. of 6 animals.Data analysis was performed using One-way ANOVA followed by Bonferroni post hoc test, ***=P≤ 0.001, significantly different from distilled water treated animals.

Figure 6 .
Figure 6.Mean Exploration Time of Identical Objects and Novel Object in the 5-min Acquisition Phase of the Novel Object Recognition Task (NORT) following acute Methanol Stem Bark Extract of Ficus platyphylla (125, 250 and 500 mg/kg p.o.), Imipramine 15 mg/kg and Distilled water 10 ml/kg Administration in Mice.Data are expressed as mean ± S.E.M and were analysed by one-way ANOVA followed by Bonferoni posthoc test, (n=8).

Figure 7 .
Figure 7. Effects of methanol stem bark extract of Ficus platyphylla (FP) (125, 250 and 500 mg/kg p.o.), Imipramine 15 mg/kg and Distilled water 10 ml/kg on the discrimination index (DI).Data were expressed as the mean±S.E.M. and were analysed by one-way ANOVA followed by Bonferoni post-hoc test, (n=8).

Table 1 .
Phytochemical constituents present in the methanol stem bark extract of Ficusplatyphylla.