Effect of root-extracts of Ficus benghalensis (Banyan) in memory, anxiety, muscle co-ordination and seizure in animal models

Background Ficus benghalensis L. (Banyan) is a commonly found tree in Eastern Nepal. Its different plant parts are used for various neurological ailments. This study was performed in mice to see its effects in various neuropharmacological parameters. Methods Passive-avoidance (memory), Open-field (anxiety), Pentobarbital-induced Sleep potentiation (sleep), Rota-rod (muscle-co-ordination), Pentylenetetrazol-Induced and Maximal Electroshock Seizure Tests were performed. Sample size was calculated using G*Power 3.1.9.2. Aqueous root extracts (Soxhlet method) of Ficus benghalensis 100 mg/kg and 200 mg/kg with negative and positive controls were used. The experimental results were represented as Mean ± SD. P-value was set at <0.05. Oneway analysis of variance (ANOVA) or Mann-Whitney U test was appropriately used. Results Passive-avoidance test showed 200 mg/kg group spent significantly less. Time (0.00s + 0.00s) in shock-zone than Normal Saline-group (9.67 s + 14.36 s, P = 0.000) or Diazepam-group (41.07 s + 88.24 s, P = 0.000). Open-field test showed 200 mg/kg group spent significantly longer Time (24.77 s + 12.23 s) in central-square than either Normal Saline group (15.08 s + 6.81 s, P = 0.000) or Diazepam-group (15.32 s + 5.12 s, P = 0.000). In Rota-rod test, 200 mg/kg group fell off the rod significantly (P = 0.000) earlier (33.01 s + 43.61 s) than both Normal Saline (>120 s) and Diazepam (62.07 s + 43.83 s) PTZ model showed that 100 mg/kg significantly (P = 0.004) delayed seizure-onset (184.40s + 36.36 s) compared to Normal Saline (101.79 s + 22.81 s), however, in MES model 200 mg/kg significantly (P = 0.000) prolonged tonic hind-limb extension (17.57 s + 2.15 s) compared to Normal Saline (13.55 s + 2.75 s) or Phenytoin (00.00s + 00.00s). Conclusion Aerial roots of Ficus benghalensis have memory-enhancing, anxiolytic, musclerelaxant, and seizure-modifying effect. Electronic supplementary material The online version of this article (doi:10.1186/s12906-016-1413-5) contains supplementary material, which is available to authorized users.


Background
According to the WHO (World Health Organization), 70-80 % of the population in developing countries still relies on non-conventional medicine mainly of herbal sources in their primary healthcare. Even in allopathy, pharmacological screening of plants often provide basis for developing new lead molecules [1,2]. The recent advancements in phyto-chemistry and molecular biology have renewed and rejuvenated our interest in herbal medicines. Again, anecdotal claims of 'no side effects' , easy access and cheap price have lured people from all walks of life to turn back to nature [3].
Nepal, a repository of wild flora and fauna, harbors 2.2 % of world's flowering plants in spite of sharing only 0.1 % of the total land area of the world. Out of the 7000 species of higher plants available in Nepal, recent literature claims that, more than 1400 are of medicinal value [4][5][6].
In traditional system of medicine, various plant parts of Ficus benghalensis L.such as stem bark, aerial roots, underground roots, vegetative buds, leaves, fruits and latex have been used in various nervous disorders i.e. seizure, insomnia, anxiety etc. [8,9]. Basu and Lal described many neuropharmacological effects of the plant [10].
Several of its pharmacological activities have also been scrutinized under scientific light. In Passive-avoidance test and plus-maze tests on scopolamine-induced amnesia in young mice, Chandra et al. [11] found that aqueous extracts of Ficus benghalensis L.bark did have cognitive enhancing activity.
Deraniyagaia [12] however could not verify significant sedative effects of aqueous leaf extract of Ficus benghalensis L.
It is shocking to find very scarce scientific studies especially from our country trying to verify our local traditional claims and uses. Few scientific studies done elsewhere may not be the answer to our different climatic and social perspectives. We, therefore, undertook this study to scrutinize whether the aqueous extracts of aerial roots of Ficus benghalensis L.do have any effect in memory, anxiety, sleep, muscle co-ordination, and seizure in animal-models.

Setting
Laboratory of Department of Clinical Pharmacology and Therapeutics, BPKIHS, Dharan, Eastern Developmental Region, Nepal.
Experiments were conducted 2014 March-May when the average temperature was between 11 to 21 C.

Sample, sampling technique and animals
Sample size was calculated using sample-size calculating software G*Power version 3.1.9.2 (Program written, concept and design by Franz, Universitat Kiel, Germany. Freely available windows application software). With power of 80 %, 0.05 statistical level of significance and effect size of 0.8, sample size for each test was calculated to be 24. Therefore, for six different tests, sample size was 6 × 24 = 144.
One hundred and fourty four Swiss albino mice were randomly assigned into one of the four experimental groups.
The mice were produced in the breeding house of Department of Clinical Pharmacology and Therapeutics, BPKIHS, Nepal.
The mice had free access to food and water and were fasted for 12 h before the experiments.

Design of the study
It was a Quantitative experimental study in mice.

Inclusion criteria
Swiss albino mice of either sex. Weighing 20-30 g.

Exclusion criteria
Apparently, free of any disease or handicap.
Study flow chart ( Fig. 1) Test drug preparation (Retention sample number: DDRP-1) (Fig. 2) A botanist authenticated a Ficus benghalensis L. tree in the premise of BPKIHS, eastern part of Nepal, with Latitude and Longitude 26°49′ 0″ N and 87°17′ 0″ E respectively. Sample specimen with Retention sample number DDRP-1, was deposited to National Herbarium & Plant Laboratories, Nepal under Department of Plant Resources, Thapathali, Kathmandu. In February 2014, about 2.5 kg aerial roots of the tree was carefully collected, thoroughly washed with tap-water, shadedried for several days and pulverized to fine powder in a mixer (Panasonic MX AC 400 Mixer Grinder, 550 W Power Consumption, Jaipur, India). About 2 kg of resulting crude root-powder was extracted in several batches using soxhlet apparatus (JAIN SCIENTIFIC GLASS WORKS AMBALA CANTT; Extraction Pot: 250 ml; Soxhlet chamber size: 100 ml; Heater: DICA India). About 250 ml of Distilled water was used in each batch of extraction. Each batch was extracted for 24 h. Thus produced aqueous root extract was heated in 50 C for a brief time interval, stopped just before the apparently saturated solution precipitated and left in room temperature till the moisture dried. Two kg crude root powder yielded 102.68 g extract (5.13 %) by soxhlet method [26]. Thus resulted dried powder extract was safely stored in a dry air-tight plastic container till the day of experiment [27]. On the experiment-day, 20 mg/ml and 10 mg/ml solutions in distilled water were prepared by serial dilution such that 1 ml/100 g mouse body weight could be injected to the mice in test-drug group for the desired test dose of 200 mg/kg and 100 mg/kg respectively. On the day of experiment, the previous-day solutions were discarded and fresh solutions prepared.

Timing of the drug administration
All paroral (PO) drugs were carefully administered with the help of oro-gastric tube 60 min prior to the intended test. All parenteral drugs were given Intrperitioneal (IP) [28][29][30].
Drugs and chemicals used in experimental models Passive-avoidance test (Fig. 3) In a dark, sound-attenuated experimental room was the study chamber for Passive-avoidance Test, which consisted of grid metal floor of 34 × 34 × 20 cm dimension.
In the metal floor, 20 mA was being run. A shock free  Prior to the actual documentation of the parameters every mouse underwent a training session in which each was left on the live metal grid for sometimes till it became apparent that it had identified shock-zone (Grid-metal floor) and shock-free zone (Petri-dish) where it would be safe from electric shock. This was the principle of avoidance. Those mice, who did not learn this avoidance technique in 5 consecutive training sessions were discarded. Learning was assumed when the mouse stayed on the petri-dish for at least 60s. Then, a learnt mouse who took oral drug 1 h back was placed on the Petri-dish and following parameters were documented for 5 consecutive minutes [30,32].

1.
Step-down latency (Initial duration for which the animal remained in SFZ till it moved to the shock zone). 2.
Step-down error (Number of unsuccessful attempts made to go to the shock zone). 3. Total time spent in the shock zone.

An increase in
Step-down latency and decrease in either step-down error or time spent in the shock zone indicated learning and memory [30,32].
Diazepam 1 mg/kg IP [30], the positive control of the test, was administered 15 min prior to the test.
Open field test (Fig. 4) In a dark, sound-attenuated experimental room was the open-field apparatus vaguely illuminated with a zero Watt red-bulb. Open Field consisted of a wooden box (40 cm × 40 cm with 30 cm high walls) with black floor, subdivided into nine (3 vertical X 3 horizontal) equal squares, by 4 (2 horizontal and 2 vertical) white lines. One hour after the oral drug administration, the mouse was placed in the open field. There, it was left undisturbed for about 1 min so that it could acclimatize to this new surroundings. Then, following parameters were documented in the next 5 min [33]. are recorded.
In an anxious situation, a rodent tends to reduce exploratory activities like ambulation, rearing (an animal standing on the rear paws) and shows spontaneous preference to the periphery of the apparatus. Anxiety decreases all three parameters [33].
Diazepam 1 mg/kg IP [30], the positive control of the test, was administered 15 min prior to the test.

Pentobarbital-induced sleep potentiation test
One hour after the oral drug administration, every mouse received Pentobarbital 50 mg/kg IP [34] The time that elapsed between loss and recovery of righting reflex was taken as the sleeping time. The mouse was considered as being awake if it could right itself, i.e. return to upright position. Diazepam 3 mg/kg IP [30], the positive control of the test, was administered, 15 min prior to the Pentobarbital administration.

Rota-rod test (Fig. 5)
A Rota-rod Treadmill device (Techno, India) was used. One hour after the oral administration of the drug, the mouse was placed on a horizontal rotating rod revolving at 15 revolution per minute [35]. Before that, mice were trained to adjust to the revolving rod. Only those mice which demonstrated their ability to remain on the revolving rod for at least 1 min were used for the test [30]. 'Fall-off time' when the mouse fell from the rotating rod was noted. For mice, who did not fall, observation was made till 2 complete minutes [31]. Diazepam 4 mg/kg IP [28], the positive control of the test, was administered 15 min prior to the test.

Anticonvulsant effect Pentylenetetrazol (PTZ) induced seizure test
One hour after the oral drug administration, 60 mg/ kg [30] PTZ IP (Intraperitoneal) was administered to all mice. The documented parameters were latency to seizure-onset and the number of mice not exhibiting seizure. Seizure was defined as jerky movements of whole body or convulsion [28]. Each mouse was observed for one whole hour for the occurrence of seizure. Diazepam 4 mg/kg [31] IP, the positive control of the test, was administered 15 min prior to the test.
Maximal Electroshock (MES) Seizure Test (Fig. 6) One hour after the oral drug administration, the mouse was subjected to alternating current of 150 mA, 50 Hz for 0.2 s duration. Current was produced by Convulsiometer (Techno, India) and was applied through a pair of electrodes attached to either ear [36]. Each animal was observed for 2 complete minutes [30]. Parameters observed and documented were a. Duration of tonic hind limb extension. b. Number of animals not showing seizure (animals protected against seizure).
Phenytoin 20 mg/kg PO [30] was the positive control of the test.

Statistical analysis
The experimental results were represented as Mean ± SD (Standard Deviation). SPSS (Statistical Package for the Social Sciences) version 21 {Property of International Business Machines (IBM) Corporation, SPSS is the registered trademarks of the corporation (IBM)} was used. For normally distributed dependent variable, parametric-test-one way analysis of variance (ANOVA) followed by Tukey's multiple comparison was applied. For those, not normally distributed, non-parametric test-Mann-Whitney U test was employed for comparison. Statistical level of significance was set at P-value < 0.05.

Ethical considerations
Prior to the conduction of the experiment, ethical clearance was obtained from the Ethical Committee on Animal Research of BPKIHS. Maximum precaution was taken to reduce the pain and injury to the animals in the course of experiment yet not compromising the standard of the experiment. Appropriate help was procured from expert experimental animal handlers during the course of study.

Open-field test (Table 2) Number of squares crossed
One-way ANOVA (Additional file 1: Table S14)  Mean time (s) spent in central-square (Fig. 8) Mann-Whitney U test (Additional file 1:

Mean number of rearing
Mann-Whitney U test (Additional file 1: Table S21) showed non-significant (p > 0.05) influence of aqueous extracts of aerial roots of Ficus benghalensis L.on mean number of rearing among Swiss-albino mice.
Pentobarbital-induced sleep potentiation test (Table 3) One way ANOVA (Additional file 1: Table S24) revealed a significant (P = 0.00) effect on mean total sleep time. Multiple Comparisons (Additional file 1: Rota-rod test ( Fig. 9 and Table 4) Mann-Whitney U test (Additional file 1: MES test (Fig. 11 and Tables 7 and 8) No animal died in MES seizure model.

Discussion
To our knowledge, this is the first report on the detailed neuropharmacological aspects of root extracts of Ficus benghalensis L. from Nepal.
Preliminary experiments demonstrated that the high doses (2000 mg/kg) were tolerated without any acute signs of toxicity or mortality. Therefore, one tenth of this dose [37] (i.e. 200 mg/kg) was considered as the highest evaluation dose for pharmacological studies.

Effect of aqueous extracts of aerial roots of Ficus benghalensis L.in learning and memory
In the Passive-avoidance test, we tried to explore the effect of the drug in short-term memory of the mice. Among 3 parameters, step-down latency and step-down error were statistically not significant. Third parameter-Time spent in shock zone pointed towards salutary effects of high dose of the extract in short-term memory of the mice. Central cholinergic receptors are often involved in memory [38]. Therefore, it must have affected the central muscarinic receptor which are involved in Alzheimer brain [39].
Chandra P et al. found that aqueous extract from the bark of Ficus benghalensis L. mitigated scopolamineinduced memory deficits in the Passive-avoidance test [11]. Other taxonomically close plants also have proven antiamnestic effects [40,41].

Effect of aqueous extracts of aerial roots of Ficus benghalensis L.in anxiety
The open field test is a valid animal model of anxietylike behavior and is based on the conflict between the exploration of a new environment and the aversion to open spaces from which escape is prevented by a surrounding wall [42].
Animals removed from their acclimatized cage and placed in the open field express fear/anxiety and therefore tend to avoid the aversive centre and spend more time in the protective corners and periphery (thigmotaxis). An increase in central locomotion or in  time spent in the central part of the field can be interpreted as an anxiolytic-like effect while the contrary, that is a decrease of these variables, is associated with anxiogenic effects [33]. In our study, mice treated with higher dose of the extract spent more time in the central-square even when compared to Diazepam suggesting anxiolytic effect even better than the conventional axiolytic. To our knowledge, there is no published report regarding effect of Ficus benghalensis L.plant parts in anxiety. However, many other plants of same genus do have proven anxiolytic effects like. Ficus exasperata Vahl [43] Ficus religiosa [44] Ficus pumila [45].
Healing anxiety with herbs has been a topic of hot debate in recent years. Natural products are being explored as an alternative for anxiety disorder because of public acceptance of plants and unwanted sideeffects of popular anti-anxiety drugs like Benzodiazepines [46].
Several traditional herbal medicines are already known to possess anxiolytic activity Ginseng. Therefore, it most also influence level and activities of several central neurotransmitters noradrenaline, serotonin, gamma-amino butyric acid (GABA), Benzodiazepine (BDZ) affecting anxiety [47].

Effect of aqueous extracts of aerial roots of Ficus benghalensis L.in Pentobartal-induced sleep
We evaluated effect of our test drug in sleep induced by Pentobarbital in mice and compared the duration of resulted sleep with standard sedative drug Benzodiazepine. Taking mean total sleep time as parameter, we could not arrive at a statistically significant effect in sleep.

Effect of aqueous extracts of aerial roots of Ficus benghalensis L.in muscle-co-ordination
Skeletal muscle relaxants are drugs that act peripherally at neuromuscular junction/muscle fibre itself or centrally in the cerebrospinal axis to reduce muscle tone and/or cause paralysis [48]. We tried to evaluate the muscle relaxing property of the mice by documenting how long they could remain in the rotating rod after administering the test drug and compared the fall-off time with that of the standard drug Diazepam. Mice ingesting higher dose of test drug fell from the revolving rod significantly earlier than the standard muscle relaxant Diazepam. To our knowledge there is no published article concerning effect of Ficus benghalensis L.on muscle contraction. However, aerial parts of Ficus carica [49], leaves, stem-bark and root-bark of Ficus sycomorus [50]   and different parts of Ficus religiosa [40] do have proven muscle relaxant property.

Effect of aqueous extracts of aerial roots of Ficus benghalensis L.in seizure
Seizure is a paroxysmal event having various manifestations, ranging from dramatic convulsion to an experiential phenomena. It is due to abnormal excessive or synchronous neuronal activity in the brain [51]. Different ion channels (voltage-gated Na + channels, voltage-gated Ca 2+ channels, voltage-gated K + channels); receptors (excitatory receptors like AMPA (α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid) receptors, NMDA (N-methyl-D-aspartate) receptors and inhibitory receptors like GABA receptors and their subtypes, are involved in seizure [47]. Predictive of effectiveness in nonconvulsive (absence or myoclonic) seizures [48], PTZ is known to produce clonic seizure [52] by antagonizing [53] GABA A (Gamma-Aminobutyric Acid) receptors. Convulsion so produced is effectively blocked by Diazepam which increases frequency of opening of GABA A receptor [30].
Electroshock results in a group of neurons firing at an abnormal, excessive, and synchronized manner mimicking tonic phase of Grand mal epilepsy [54,55]. The tonic phase (especially extensor) is selectively abolished only by drugs effective in generalized tonic-clonic seizures and partial seizure [48,56].
Our study at low dose significantly delayed PTZ seizure onset. On the other hand, at high dose it increased MES seizure duration. This apparent anomaly in different seizure types is explained by difference in genesis and resulting effective drug in the two seizure type [57]. However, even MES and PTZ models together may not detect all anti-seizure activities [52]. Therefore, few anti-seizure properties of the test drug might have been missed.
Though not in Ficus benghalensis, few studies analyzing the effect of other species of same Ficus genus have been published which showed mixed results [58,59].

Limitations and scope for further study
Though our test-result has brought many things under light, we assume certain shortcomings in our endeavor.
Single acute dose-effect was studied. Sub-acute and chronic dose-effect may not be predicted from these result.
We studied only two most relevant doses of test drug. More doses are needed to establish statistically significant dose-effect relationship.
Distinct climate, soil and environment of the Eastern Nepal might have produced different properties that may reduce the global applicability of our findings.
Individual differences in anatomy, organ function, drug absorption and metabolism among animals are among the myriad of other differences that could have given us different information.
In lack of phytochemical analysis accompanied by causeeffect establishment of individual phyto-constituents, we remain skeptical about extrapolating our report to other plant-parts. Further in-depth research disclosing the phyto-constituents responsible for each neuropharmacological activity along with their correct mode of action is desperately needed.
In spite of all these, we consider our study has high internal and external validity; and our findings can be replicated elsewhere.

Suggestions
Though technically more demanding, phyto-molecular identification followed by in-vitro receptor-ligand binding studies may be the answer to this dispute.
In any particular effect, the use of antagonists of putative neurotransmitters involved might have shed more lights on the mechanism of the observed effect e.g. In Pentobarbital-induced sleep potentiation test, adding of serotonergic antagonist or anti-glutamate as other positive controls might have cleared the dilemma regarding neurotransmitters concerned.
Step down error. Table S6.

Comparison of mean
Step-down error. Table S7. One-Way ANOVA Test-mean Step down error. Table S8. Tukey HSD post hoc test comparing mean Step down error. Table S9. Time spent in shock zone in s. Table S10. Comparison of mean Time spent in shock-zone. Table  S11. Mann-Whitney U test comparing mean time spent in shock-zone (s).  Table S25. Tukey HSD post hoc tests-comparing mean total sleep time. Table S26. Fall-off time (s). Table S27. Comparison of mean Fall-off time. Table S28. Mann-Whitney U test comparing mean fall-off time. Table 29. Animals not showing seizure. Table S30. Latency of seizure onset (s). Table S31. Comparison of mean Latency of seizure-onset (s). Table S32. Mann-Whitney U test comparing mean latency (s) in PTZ-seizure model. Table S33. Animals not showing seizure. Table S34. Duration of tonic hind limb extension (s). Table S35.
Comparison of mean duration of tonic hind-limb extension. Table S36.