Abstract
Context: The adventitious roots of Ficus religiosa L. (Moraceae) have been extensively used in traditional medicine for treatment of several disorders, including epilepsy.
Objective: To investigate the possible anticonvulsant effect of the adventitious roots of Ficus religiosa, and to find the biologically active fraction, to substantiate its traditional use in epilepsy.
Methods: The hydroethanolic extract of adventitious roots (5, 10, and 20 mg/kg; i.p.) of Ficus religiosa and its different fractions (hexane, chloroform, ethyl acetate, butanol, aqueous, saponins-rich, and saponins-lacking) at a dose equivalent to 20 mg/kg of the extract were administered 30 min prior to the induction of maximal electroshock (MES) and pentylenetetrazol (PTZ) convulsions. Duration of tonic hind-limb extension (THLE) and latency to clonic convulsions were noted in MES and PTZ tests, respectively. Neurotoxicity was assessed using rotarod test.
Results: Treatment with the root extract (5, 10, and 20 mg/kg; i.p.), butanolic (6 mg/kg; i.p.) and saponins-rich fractions (3.4 mg/kg; i.p.) significantly (p < 0.05) decreased the duration of THLE in MES test, as compared to control. The same treatment also significantly (p < 0.05) increased the latency to PTZ-induced clonic convulsions in comparison to control. The other fractions were found to be ineffective. The root extract and its active fractions at their effective doses showed no neurotoxic effects.
Conclusion: The present study concluded that the hydroethanolic extract of adventitious roots of Ficus religiosa has anticonvulsant activity. Retention of anticonvulsant effect in the saponins-rich fraction-treated animals indicated the role of saponins for the activity.
Introduction
The genus Ficus (Moraceae) is one of the most diverse plant genera that comprise more than 800 fig tree species worldwide. Many of the Ficus species such as, Ficus sycomorus L. (CitationNoumi et al., 2003), Ficus schimperiana Hochst. ex Miq. (CitationMoshi et al., 2005), Ficus platyphylla Del. (CitationChindo et al., 2009), Ficus capensis Thunb. (CitationAyinde & Owolabi, 2009), Ficus religiosa L. (CitationSingh et al., 2011), and so on are included in the list of plants used in the ethnomedical treatment of epilepsy. Ficus religiosa is an important member of the genus that is commonly known as peepal/pipal/peepul/bodhi tree. It has been regarded as a sacred tree and is used for medicinal as well as religious purposes in South Asian countries (CitationKala et al., 2006). Its different parts have been extensively used in various traditional systems of medicine (Ayurveda, Unani, and so on) for the ethnomedical treatment of several central nervous system (CNS) disorders, including epilepsy. Many of its traditional medicinal uses have been experimentally validated (CitationSingh et al., 2011). Recently, its antiulcer (CitationKhan et al., 2011), antiasthmatic (CitationKapoor et al., 2011), hepatoprotective (CitationGupta et al., 2011), hypocholesterolemic (CitationHamed, 2011), anticancer (CitationGulecha & Sivakuma, 2011), and antiinflammatory (CitationKirana et al., 2011) activities have been explored. The leaves of Ficus religiosa have been used in Ayurveda to treat epilepsy and its extract showed anticonvulsant activity when evaluated in the experimental animal models (CitationVyawahare et al., 2007). In our previous study, the methanolic extract of figs of this tree showed anticonvulsant activity, the protective effect of the extract was abolished by a 5-HT1/2 receptor antagonist. The study indicated the effect of the extract is due to serotonergic modulation of the brain functions (CitationSingh & Goel, 2009). The brain serotonergic modulatory effect of the same extract has also been investigated in another study and has been explored for its antiamnesic activity (CitationKaur et al., 2010).
The adventitious roots of Ficus religiosa have been of great value in traditional medicine, and is used for the treatment of sexual disorders, arthritis, elephantiasis, stomatitis, leprosy, malarial fever, respiratory disorders, chicken pox, epilepsy, and so on (CitationSingh et al., 2011). In spite of its wide ethnomedicinal use, only a few experimentally validated reports substantiating the use of adventitious roots are available in literature. Even the phytochemical studies carried out on the adventitious roots is limited to identification of phenolic substances, trace amount of amino acids, proteins, and trace elements (CitationPatil et al., 2011; CitationSharma & Gupta, 2007). Since its adventitious roots have been clinically used for the treatment of epilepsy, only a single study carried out on the crude adventitious root extract is available (CitationPatil et al., 2011). Our continued interest in exploring this plant for CNS activities led us to undertake the present study.
The present study investigated the anticonvulsant effect of the adventitious roots using maximal electroshock (MES) and pentylenetetrazol (PTZ)-induced convulsion tests, and sought to determine its biologically active fraction(s).
Materials and methods
Drugs and chemicals
PTZ (dissolved in normal saline) was obtained from Sigma Chemical Company (USA), dimethyl sulfoxide (DMSO) from Spectrochem (Mumbai, India); sodium chloride, and sulphuric acid from Loba Chemie (Mumbai, India); hexane, chloroform, butanol, methanol, carboxymethyl cellulose, and vanillin from S.D. Fine Chem (Mumbai, India); ethyl acetate from Merck Specialties (Mumbai, India); diethyl ether from Sisco Research Laboratories (Mumbai, India); and diosgenin from Himedia Laboratories (Mumbai, India). Reference drugs diazepam and phenytoin were obtained locally from Jackson Laboratories Ltd (Amritsar, India) and Cadila Laboratories (Ahmedabad, India), respectively.
Plant material and preparation of extract
The adventitious roots of Ficus religiosa were collected from U.E., Phase-I, Patiala, Punjab, India (30°33′ north latitude, 76°40′ east longitude) in July–August, 2009. The botanical identity of the plant material was verified by Prof. R.C. Gupta and the specimen was deposited at the Herbarium, Department of Botany, Punjabi University, Patiala, Punjab, for reference (Voucher no.: 51857). The authenticated plant part was cleaned, shade dried, and grounded to a moderately coarse powder. The powdered material (100 g) was subjected to repetitive extraction by 50% ethanol + 50% distilled water using a percolator at room temperature, until exhausted. The filtered and combined percolate was evaporated under reduced pressure using a rotavapor (Perfit, Ambala, India). The remaining viscous mass was subjected to freeze drying using a freeze drier (Allied Frost, Delhi, India) to get powdered hydroethanolic adventitious root extract. Based on the bioactivity results, another 1 kg of the adventitious roots were re-extracted by the same method, for further fractionation.
The hydroethanolic root extract and its fractions were subjected to preliminary phytochemical tests to determine the presence of alkaloid, carbohydrates, glycosides, saponins, steroids, triterpenoids, tannins, flavonoids, proteins, and amino acids (CitationTrease & Evans, 2002).
Fractionation of the adventitious root extract
The adventitious root extract was subjected to polarity-based liquid–liquid partitioning to get different fractions. Briefly, the extract (30 g) was dispersed in water and was extracted with n-hexane in a separating funnel. The n-hexane phase was collected and remaining aqueous phase was successively extracted with chloroform followed by ethyl acetate and n-butanol. Each fraction after solvent partitioning was evaporated to dryness under reduced pressure and the remaining aqueous part was freeze dried to obtain the n-hexane, chloroform, ethyl acetate, n-butanol, and aqueous fractions.
Drop-wise addition of the butanolic fraction (5 g) dissolved in methanol (approx. 10 mL) to diethyl ether resulted in the formation of precipitates. The precipitates were separated from the solution by filtration and designated as saponins-rich fraction (CitationChindo et al., 2009). The remaining solution was dried under reduced pressure to give saponins-lacking fraction.
Quantitative determination of total saponins
The colorimetric method with vanillin-sulphuric acid system described by CitationHiai et al. (1976) was used with slight modifications for the determination of total saponins. Briefly, the saponins-rich fraction was dissolved in 80% methanol to a designed concentration (0.5 mg/mL). A known amount of aliquot up to 500 µL from the solution was mixed with 50 µL of 8% (w/v) vanillin reagent and 3 mL of 72% (v/v) sulfuric acid was added. The solution was mixed, heated on a water bath at 60°C for 10 min, and cooled in ice-cold water for 3 to 4 min. The solution was measured at 544 nm using a spectrophotometer (Beckman DU 640B) with a blank solution as reference. Quantification was based on the standard curve of diosgenin (0-320 µg; y = 0.002x–0.045; R2 = 0.992).
Determination of anticonvulsant activity
Animals
Male Swiss Albino mice, weighing 20–30 g obtained from CCS Haryana Agricultural University, Hisar, were employed in the present study, in different groups (n = 5). The animals were housed in standard cages and were maintained at room temperature with natural day and night cycles. The animals were allowed free access to food (standard laboratory rodent’s chow) and water during the study period. All procedures were conducted according to the guidelines of the Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA), India. The experimental protocol was approved by the Institutional Animal Ethical Committee established by the University.
Preparation of test samples and dose estimation
Three doses of the hydroethanolic root extract (5, 10, and 20 mg/kg) were selected after the initial pilot study carried out on limited number of animals. Further dose of a fraction was estimated based on its ratio in the corresponding extract/fraction. The dose of hexane, chloroform, ethyl acetate, butanolic, aqueous, saponins-rich and saponins-lacking fraction equivalent to 20 mg/kg of the hydroethanolic root extract comes out to be 0.6 (0.61), 3.9 (3.83), 1.5, 6 (6.002), 8 (8.04), 3.4 (3.38), and 2.6 (2.61) mg/kg, respectively. The extract and fractions were reconstituted by dissolving in DMSO and then dispersing the resultant solution in distilled water (ratio 1: 9), freshly before use and were injected intraperitoneally (i.p.). Vehicle control groups received equal volume of vehicle i.p. (injection volume 10 mL/kg).
Maximal electroshock-induced convulsions test
MES convulsions in mice were induced by delivering a calibrated (through a current calibrator [Rolex, Ambala, India]) transauricular electroshock of 56 mA for 0.2 s using a convulsiometer (Rolex, Ambala, India) via a pair of crocodile ear clips. Mice in different groups were injected with the varying doses of hydroethanolic root extract, its fractions, vehicle, and phenytoin (25 mg/kg; i.p.). After 30 min of these treatments, all groups were tested for MES seizures response. Duration of tonic hind-limb extension(s) was noted and was compared with vehicle control (CitationSwinyard et al., 1952).
PTZ-induced convulsions test
PTZ convulsions test was performed by the method of CitationSwinyard et al. (1952) with slight modifications (CitationKaur & Goel, 2011). Briefly, mice were divided into different groups and were treated with the varying doses of hydroethanolic root extract, its fractions, and vehicle in a similar manner as in case of MES test, but in this case instead of phenytoin, diazepam (5 mg/kg; i.p.) served as standard. After 30 min of these treatments, all groups were injected with PTZ 75 mg/kg. Latency to clonic convulsions (min) was noted and was compared with that of vehicle control.
Neurotoxicity test
The test procedure described by CitationDunham and Miya (1957) was used to assess neurotoxicity with slight modifications. The test apparatus consisted of a metallic rotating rod of 3 cm diameter divided into five equal lanes. Mice which were able to remain on the rod at a speed of 10 rpm for 5 min or more were selected and on same day in different groups were injected with varying doses of the hydroethanolic root extract (5, 10, and 20 mg/kg; i.p.), butanolic fraction (6 mg/kg; i.p.), saponins-rich fraction (3.4 mg/kg; i.p.), and vehicle. After 30 min of the treatment, all the animals were placed individually for three consecutive trials (30 min intertrial intervals) on the rotating rod and the fall down latency was noted. Neurotoxicity was assessed as inability of the animal to maintain equilibrium on the rotating rod for at least 3 min (180 s) in each of the three trials.
Statistical analysis
All the results were expressed as mean ± standard error (SEM). Data was analyzed using one-way analysis of variance (ANOVA) followed by Dunnett’s test. The results were regarded as significant at p < 0.05.
Results
Extraction, fractionation, and preliminary phytochemical screening
Hydroethanolic extraction of the adventitious root powder yielded 3.78% w/w of crude extract. The results of preliminary phytochemical screening tests carried out on the crude extract and its fractions have been summarized in .
Table 1. Chemical groups identified in the hydroethanolic adventitious root extract and its different fractions.
Polarity based liquid–liquid partitioning of 30 g of the hydroethanolic root extract yielded 0.85 g of n-hexane fraction, 5.34 g chloroform fraction, 2.09 g ethyl acetate fraction, 8.35 g n-butanolic fraction, and 11.19 g aqueous fraction. Further 5 g of the butanolic fraction yielded 2.61 g of saponins-rich and 2.02 g of saponins-lacking fractions.
Determination of saponins
The saponins content of the powdered adventitious roots by the extraction method opted, using colorimetric method by vanillin–sulphuric acid system was found to be 5.72 ± 0.24 mg/g (n = 3) of the root powder.
Effect on MES-induced convulsions
In MES test, the hydroethanolic extract showed a significant (p < 0.05), dose-dependent decrease in the duration of tonic hind-limb extension as compared to vehicle control, showing maximum effect at 20 mg/kg dose. Moreover the effect of extract at this dose was found to be equipotent to phenytoin (). Among of all the fractions obtained from the hydroethanolic root extract, only butanolic and saponins-rich fractions at a dose equivalent to 20 mg/kg of the extract, significantly (p < 0.05) suppressed the MES-induced convulsions. All other fractions viz. hexane, chloroform, ethyl acetate, aqueous and saponins-lacking fraction were found to be ineffective ().
Figure 1. Effect of the hydroethanolic adventitious root extract on the duration of MES-induced tonic hind-limb extension. ap < 0.05 as compared to vehicle control. THLE = Tonic hind limb extension; FRR 5, 10, 20 = Hydroethanolic adventitious root extract 5, 10 and 20 mg/kg; s = seconds.
Figure 2. Effect of different fractions from the hydroethanolic adventitious root extract on the duration of MES-induced tonic hind-limb extension. ap < 0.05 as compared to vehicle control. FRRH = hexane fraction; FRRC = chloroform fraction; FRRE = ethyl acetate fraction; FRRB = butanol fraction; FRRW = aqueous fraction; SRF = saponins-rich fraction; SLF = saponins-lacking fraction; s = seconds; THLE = tonic hind-limb extension.
Effect on PTZ-induced convulsions
In PTZ-induced convulsions test, treatment with the hydroethanolic extract significantly (p < 0.05) delayed the onset of clonic convulsions as compared to vehicle control. The equipotent anticonvulsant effect of the extract was observed at 10 and 20 mg/kg dose. The effect of the extract in suppressing PTZ-induced convulsions was less (but significant [p < 0.05]) as compared to vehicle control) as compared to standard, as diazepam-treated group completely abolished the induction of convulsions (). Similar to MES test, only butanolic and saponins-rich fractions were found to be effective in PTZ test. All other fractions obtained from the hydroethanolic root extract and butanolic fraction were found to be ineffective ().
Figure 3. Effect of the hydroethanolic adventitious root extract on the latency to PTZ-induced convulsions. ap < 0.05 as compared to vehicle control. N/C = no convulsions; FRR 5, 10, 20 = hydroethanolic adventitious root extract 5, 10 and 20 mg/kg; min = minutes.
Figure 4. Effect of different fractions from the hydroethanolic adventitious root extract on the latency to PTZ-induced convulsions. ap < 0.05 as compared to vehicle control. N/C = no convulsions; FRRH = hexane fraction; FRRC = chloroform fraction; FRRE = ethyl acetate fraction; FRRB = butanol fraction; FRRW = aqueous fraction; SRF = saponins-rich fraction; SLF = saponins-lacking fraction; min = minutes.
Neurotoxicity test
Treatment with the hydroethanolic root extract (5, 10, and 20 mg/kg; i.p.), butanolic fraction (6 mg/kg; i.p.) and saponins-rich fraction (3.4 mg/kg; i.p.) did not show motor deficit, as all the treated animals showed insignificant change in fall-down latency as compared to control (). All the treated animals retained on the rotating rod for more than 180 s, indicating the extract and fractions to be devoid of neurotoxic effects.
Figure 5. Effect of the hydroethanolic adventitious root extract and its active fractions on the fall down latency in neurotoxicity test. FRR 5, 10, 20 = hydroethanolic adventitious root extract 5, 10 and 20 mg/kg; FRRB = butanol fraction; SRF = saponins-rich fraction; s = seconds.
Discussion
This is the first report to screen scientifically the anticonvulsant effect of different fractions from the adventitious roots of Ficus religiosa. The results of present study validated the traditional use of the adventitious roots in treatment of epilepsy, as its hydroethanolic extract pretreatment prevented MES and PTZ-induced convulsions in mice. There are a large number of animal models available that could potentially be used to screen for anticonvulsant activity, but MES and PTZ-induced convulsion models remained the “gold standards” in early stages of drug testing (CitationRogawski, 2006). MES model identifies compounds which prevent seizures spread, whereas PTZ test is a model which chiefly identifies compounds that raises seizures threshold, and all clinically active anticonvulsants have been found to be protective in at least one of these two tests (CitationMalawska, 2005). Therefore, both of these models were employed in the present study. The hydroethanolic root extract exhibited anticonvulsant effect in both the models, indicating its potential in preventing seizures spread and increasing seizures threshold.
The anticonvulsant effect shown by the extract in our study is in line with the single previously reported study carried out on the crude adventitious root extract. The investigator suggested the traces of magnesium and zinc present in the root extract to be responsible for its anticonvulsant effect (CitationPatil et al., 2011). The speculation of the authors regarding the role of trace elements for the activity is not much convincing. Since the in vivo anticonvulsant role of zinc is still not clear. Magnesium has been found to suppress convulsions induced through glutamatergic mechanisms, but not through GABAergic mechanisms, when investigated in experimental animal models (CitationDecollogne et al., 1997). But, effectiveness of the adventitious root extract in PTZ test in our study, and in PTZ and picrotoxin (GABAA antagonists) test in the study of CitationPatil et al. (2011) indicated the involvement of some other phytoconstituent(s) along with magnesium for its activity. So, to find out the active anticonvulsant fraction, the extract was fractionated by polarity-based liquid–liquid partitioning.
In initial anticonvulsant screening, the extract at a dose of 20 mg/kg was found to be most active, hence further dosing of the fractions were made equivalent to this dose. Among different fractions tested, only the butanolic fraction retained the anticonvulsant activity. Preliminary phytochemical screening showed it to be rich in saponins. The therapeutic role of saponins has been suggested in several neurological disorders (CitationFrancis et al., 2002). The earlier studies on plant-isolated saponins showed their anticonvulsant effect, due to blockade of voltage-dependent Na+ channels (CitationLiu et al., 2001). Saponins have also been reported to possess an inhibitory action on different types of Ca++ channels, causing shortening of their open time, prolonging the close time, and reducing their open-state probabilities (CitationKim et al., 2008; CitationZhong et al., 1995). Modulation of GABAergic functions by plant saponins have also been well-documented (CitationChoi et al., 2003). They are involved in the differential regulation of [3H] flunitrazepam and [3H] muscimol binding to the GABAA receptor in the rat brain (CitationKimura et al., 1994). Prolonged infusion with saponins isolated from Panax ginseng into the rat brain elevated [3H]muscimol binding to the GABAA receptor in a region-specific manner (CitationKim et al., 2001). Moreover, saponins inhibit the uptake of GABA in the rat brain synaptosomes, increasing its turnover. Glutamatergic functions are equally inhibited by saponins treatment, they causes blockade of NMDA receptor-mediated excitatory processes in the rat hippocampal cell (CitationKim et al., 2002). Furthermore, saponins from other Ficus species, like Ficus platyphylla, have been found to possess anticonvulsant activity (CitationChindo et al., 2009).
All these literature findings gave a strong indication that the anticonvulsant effect of the adventitious root extract might be due to saponins present in the butanolic fraction. Therefore, it was found worthwhile to separate the saponins from the butanolic fraction through precipitation, to get saponins-rich fraction and saponins-lacking fraction. Anticonvulsant effect shown by the saponins-rich fraction and no such effect observed in case of saponins-lacking fraction proved that the effect of extract was due to the presence of saponins.
Several clinically used antiepileptic drugs like valproic acid, phenobarbital, felbamate, clobazam, primidone, lamotrigine, clobazam, pregabalin, gabapentin, and so on show similar anticonvulsant profile as that of the root extract and its fractions (butanolic fraction and saponins-rich fraction) against PTZ- and MES-induced convulsions (CitationFrench & Ben-Menachem, 2008; CitationMeldrum, 1996; CitationRogawski, 2006). Most of the conventional antiepileptic drugs show the symptoms of sedation, hypo or hyper ataxia, locomotion, reduced or inhibited righting reflexes, muscle relaxation, and abnormal gait. These symptoms are collectively termed as neurotoxicity and can be accessed in laboratory using rotarod. The test is based on the assumption that an animal with normal motor competence is able to maintain its equilibrium on a rotating rod (CitationDunham & Miya, 1957; Loscher & Schmidt, 1988). Since, the extract and its active fractions at their effective doses allowed the animals to maintain their equilibrium on the rotating rod, indicating them to be devoid of neurotoxic effects.
Conclusions
The findings of this preclinical study substantiated the traditional antiepileptic use of the adventitious roots of Ficus religiosa. Retention of the anticonvulsant effect in the saponins-rich fraction-treated animals indicated the saponins component to be responsible for the activity of adventitious roots. Our further studies are in progress to determine the particular types of saponins and the precise underlying anticonvulsant mechanism.
Acknowledgements
The authors are deeply grateful to the University Grant Commission, New Delhi, India, for providing financial assistance [Vide F.No.: 34–130/2008 (SR)] for the project, and project fellowship to Mr. Damanpreet Singh. The authors are also thankful to Prof. R.C. Gupta of Department of Botany, Punjabi University, Patiala, Punjab for the authentication of plant material.
Declaration of interest
The authors report no conflict of interests.
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