Psychopharmacological properties of saponins from Randia nilotica stem bark

Abstract

Context: Decoctions of Randia nilotica Stapf. (Rubiaceae) have been used in the Nigerian traditional medicine for the management of epilepsy, anxiety, depression and psychosis for many years and their efficacies are widely acclaimed among the rural communities of Northern Nigeria.

Objective: The aim of this study is to establish whether the saponins present in R. nilotica are responsible for its acclaimed beneficial effects in Nigerian traditional medicine.

Materials and methods: The behavioural properties of the saponin-rich fraction (SFRN) of R. nilotica stem bark were studied on hole-board, diazepam-induced sleep, rota-rod and beam-walking in mice. The anticonvulsant properties of SFRN were also examined on maximal electroshock, pentylenetetrazole- and strychnine-induced seizures in mice.

Results: The intraperitoneal LD₅₀ of SFRN in mice and rats were estimated to be 11.1 and 70.7 mg/kg, respectively. SFRN significantly prolonged the duration of diazepam-induced sleep; diminished head dip counts in the hole-board test and protected mice against maximal electroshock seizures. SFRN failed to protect mice against pentylenetetrazole- and strychnine-induced seizures; and had no effect on motor coordination on the rota-rod treadmill at the doses tested. SFRN significantly decreased the number of foot slips in the beam-walking assay in mice with no effect on time to reach the goal box.

Discussion and conclusion: This study provides evidence of the psychopharmacological effects of SFRN, thus supporting further development of the psychoactive components as remedies for epilepsy.

• Anticonvulsants
• behaviour
• sedation

Introduction

The World Health Organization (WHO) estimated that about 80% of the population in sub-Saharan Africa patronize traditional medical practitioners for their health problems (WHO-EML, 2007). One of the important areas in which traditional herbal medicines enjoy high patronage is in the management of neurological and psychiatric disorders. Medicinal plants are often used to modify moods, feelings and behaviour in tribal rituals. Many tribal cultures also frequently maintain within their collections of herbal medicines substances valued as tonics and stimulants, which constitute potentially valuable but untapped sources of psychotropic drugs. Thus, the plant kingdom is a major target in the search of new psychotropic drugs and lead compounds for the management of CNS disorders (Cragg, et al., 1997; Farnsworth, 1994). Our studies on a considerable number of medicinal plants, including Ficus platyphylla Del. Holl. (Moraceae) (Chindo et al., 2003), Pavetta crassipes K. Schum (Rubiaceae) (Amos et al., 2004), Neorautanenia mitis (A Rich) Verde (Papilonaceae) (Vongtau et al., 2005) Balanites aegyptiaca Del. (Zygophyllaceae) (Ya’U et al., 2011) revealed some CNS activities that could be exploited for the management of neurological and psychiatric disorders.

Randia nilotica Stapf (Rubiaceae) is a lowland shrub commonly found in Northern Nigeria, the Cameroon, Sudan and East Africa (Lemmich et al., 1995). It is popularly known as “tsibra”, “barbaji” or “gial-goti” among the Hausa-Fulani speaking communities of Northern-Nigeria (Dalziel, 1937). Decoctions of this plant have been used in folk medicine for the management of convulsions and mental breakdown for many years and their efficacies are widely acclaimed among the rural communities in Nigeria and Tanzania (Dalziel, 1937; Hedberg et al., 1983). The behavioural effects of the ethanol stem bark extract of R. nilotica in mice have been reported earlier (Danjuma et al., 2008) along with its anticonvulsant properties (Danjuma et al., 2009).

Since saponins, which form the major components of the crude ethanol extract, are widely believed to have central nervous system activities (Attele et al., 1999; Chindo et al., 2009), we hypothesised that saponins may be involved in the observed behavioural and anticonvulsant properties of R. nilotica stem bark. It is on this basis that we evaluated some behavioural and anticonvulsant effects of the saponin-rich fraction of the plant as a step towards the isolation of biologically active components.

The test systems selected included hole-board, diazepam-induced sleep, rota-rod and beam walking paradigms in mice. The anticonvulsant tests employed are the maximal electroshock-, pentylenetetrazole- and strychnine-induced seizures in mice. Potentiation of sleeping time is used to elucidate the central nervous system properties of drugs. Sedatives, hypnotics, neuroleptics and antidepressants are known to prolong the sleeping time (Vogel & Vogel, 1997), while analeptics and CNS stimulants shorten the sleeping time. The hole-board test evaluates certain components of mice behaviour such as curiosity and exploration (Boissier & Simon, 1964). Anxiolytics and antidepressants suppress nose poking at relatively low doses. The mouse beam-walk assay offers improved sensitivity over the mouse rota-rod in determining motor coordination deficits induced by psychotropic agents (Stanley et al., 2005). These tests were used to evaluate the potential benefits of the saponin-rich fraction of R. nilotica in the management of neurological and psychiatric disorders.

Materials and methods

Plant material

Randia nilotica stem bark was collected from Zaria in Kaduna State, Nigeria, in the month of September 2011. It was identified and authenticated by Mallam Musa Shehu, a taxonomist at the Department of Biological Sciences, Ahmadu Bello University Zaria, Nigeria. A voucher specimen (No. 2867) was deposited at the Departmental Herbarium for future reference.

Extraction of saponin components

The method described by Woo et al. (1980) was employed for the extraction of saponin-rich fraction of R. nilotica stem bark with modifications. Briefly, the plant material was defatted initially with petroleum ether followed by extraction with 70% v/v ethanol. Polar compounds were removed by dissolving the ethanol extract in diethylether. The extract was partitioned between water and butanol (1:3) to give butanol and aqueous fractions. Potassium hydroxide (KOH) (1%) solution was added to the butanol fraction and gently shaken; the butanol fraction gave the designated saponin-rich fraction (SFRN) while the KOH solution when acidified with concentrated hydrochloric acid gave the flavonoid fraction. SFRN was used for the behavioural studies.

Animals

Male and female Swiss albino mice (18–25 g) and Wistar rats (180–250 g) obtained from the animal facility centre of the Department of Pharmacology and Therapeutics, Ahmadu Bello University Zaria, Nigeria, were used for this study. The animals were kept in plastic cages and housed under standard conditions of temperature, relative humidity and light/dark cycles (12/12 h). They were fed with standard diet and water ad libitum. The animals were approved for use by the animal ethics committee after reviewing the protocol. We certify that all experiments were carried out in accordance with the National Institute of Health Guide for the Care and Use of Laboratory Animals (NIH Publications No. 80–23) revised 1996. All efforts were made to minimize the number of animals used and their suffering.

Acute toxicity study

The acute intraperitoneal (i.p.) median lethal dose (LD₅₀) of the SFRN was determined in mice and rats by the method of Lorke (1983). The study was carried out in two phases. In the initial phase, three groups of three animals each were used. The first group received SFRN at a dose of 10 mg/kg i.p., while second and third groups received the extract at doses of 100 and 1000 mg/kg, respectively. The animals were then observed for signs of toxicity and death within 24 h. In the second phase, three groups of 1 animal each were used. Specific doses were administered which depended on the result of the first phase. The final LD₅₀ value was calculated as the square root of the product of the lowest lethal dose and the highest non-lethal dose.

Behavioural studies

Hole-board test

This study was done using the head-dip test on the hole-board (Perez et al., 1998). Male and female mice were divided into five groups of six mice each. Animals in Group 1 received normal saline, while those in Groups 2, 3 and 4 received the SFRN at doses of 0.5, 1.0 and 2.0 mg/kg i.p., respectively. Mice in Group 5 received diazepam (1 mg/kg i.p.). Thirty minutes after the treatment, mice were placed singly on a Letica hole-board with 16 evenly spaced holes and a counter (Letica LE 3333). The number of times the mice dipped their heads into the holes during the 7.5-min period was counted (Perez et al., 1998).

Diazepam-induced sleep in mice

This test was performed in 4 groups of 6 mice per group, which were treated as follows: one group received normal saline; three groups received SFRN at the doses of 0.5, 1.0 and 2.0 mg/kg i.p. Thirty minutes post drug administration diazepam (30 mg/kg, i.p.) was administered to all the mice. Each mouse was observed for the onset and duration of sleep, with the criterion for sleep being loss of righting reflex (Wambebe, 1985), indicated by the animals inability to resume or return to its upright position on all four limbs after being gently rolled sideways. The interval between loss and recovery of righting reflex was used as the index of hypnotic effect.

Rota-rod test in mice

The method described previously (Perez et al., 1998) was used in this study to assess motor coordination in mice. Briefly, mice were trained to remain on a treadmill device (Ugo Basile Rota-Rod, Model 7650, Jones and Roberts, Italy) with slowly revolving rods of 5 cm diameter at 16 rpm for 180 s. Mice that were able to remain on the rod for 180 s or longer were selected and divided into four groups of six mice per group. Group I received normal saline, while groups II, III and IV received graded doses of SFRN (0.5, 1.0 and 2.0 mg/kg, i.p., respectively). Thirty minutes after the treatment, the animals were placed individually on the rod at intervals of 30 min, up to 120 min. If an animal failed more than once to remain on the rod for 3 min, the test was considered positive, meaning there was a lack of motor coordination.

Beam-walking assay in mice

The method previously described by Stanley et al. (2005) was used for this study with modifications. Briefly, each mouse was trained to walk from a start platform along a ruler (80 cm long, 3 cm wide) elevated 30 cm above the bench by metal support to a goal box (enclosed hamster house). Three trials were performed for each mouse and were designed such that the mouse tested would be aware that there is a goal box that could be reached. A ruler was used because the mice find this easy to cross and at the same time; it induced minimum anxiety. Mice were randomly divided into five groups of five mice per group. The first group was injected with normal saline; the subsequent groups were injected with 0.5, 1.0 and 2.0 mg/kg of SFRN, respectively. The last group was injected with diazepam 1 mg/kg. Each mouse was placed on the beam at one end and allowed to walk to the goal box. Mice that fell were returned to the position they fell from, with a time of 60 s allowed on the beam. The measurements taken were time on the beam and maximum number of foot slips (one or both hind limbs slipped from the beam).

Anticonvulsant studies

Pentylenetetrazole-induced seizures

Mice were injected with a convulsive dose (CD₉₀) that produces convulsions in about 90% of mice (90 mg/kg PTZ subcutaneous-ScPTZ) (Krall et al., 1978). They were then observed for the presence or absence of threshold seizures (an episode of tonic seizure of at least 5 s duration). Three groups of six mice each were pre-treated with SFRN at doses of 0.5, 1.0 and 2.0 mg/kg, i.p., respectively. A fourth group was pre-treated with phenobarbitone 30 mg/kg body weight i.p. Another group was given normal saline to serve as control. Thirty minutes later, all the mice were injected with a convulsive dose (CD₉₀) of PTZ subcutaneously. The mice were observed for the presence or absence of threshold seizures (an episode of clonic seizure of at least 5 s duration). Seizures were manifested as tonic hind-limb extension. The ability to prevent this feature or prolong the latency of tonic hind-limb extension was taken as an indication of anticonvulsant activity (Amabeoku et al., 1998).

Maximal electroshock-induced seizures

The method described by Sayyah et al. (2002) was employed in this study. Briefly, mice were pre-treated in groups of ten with normal saline and graded doses of SFRN (0.5, 1.0 and 2.0 mg/kg, i.p.). Thirty minutes post drug administration, an Ugo Basil’s electroconvulsive therapy machine (Model 7800), connected to Clande Lyons voltage stabilizer with corneal electrodes was used to induce seizures. The shock duration, frequency and pulse width maintained at 0.8 s, 100 pulses per second, and 0.8 ms, respectively, throughout the experiment. A current of about 90 mA, which produced tonic seizures in 70–90% of the control mice, was used throughout the study. Seizures were manifested as tonic hind-limb extension. The ability to prevent this feature was taken as an indication of anticonvulsant activity (Sayyah et al., 2002). Results were recorded as either positive or negative depending on whether tonic hind limb extension was produced.

Strychnine-induced seizures

This was carried out as described previously (Bum et al., 2001) with modifications. Briefly, adult mice of both sexes (ratio 1:1) were randomly divided into five groups of six mice each. Three groups were administered graded doses (0.5, 1.0 and 2.0 mg/kg, i.p.) of SFRN. Mice in the control groups received normal saline and the remaining fifth group received phenobarbitone (30 mg/kg, i.p.). Thirty minutes post drug administration, each mouse was administered the CD₉₀ of strychnine nitrate (1.2 mg/kg, s.c.) (Krall et al., 1978) and they were then monitored for 30 min for the presence or absence of convulsion. Seizures were manifested as tonic hind-limb extension and the ability to prevent this feature was taken as an indication of anticonvulsant activity. The proportion of mice presenting convulsions was recorded. Mice that did not show tonic hind-limb extension during the period of observation were considered not having convulsed (Bum et al., 2001).

Statistical analysis

All the results were expressed as mean ± S.E.M. and differences in means were estimated by means of an ANOVA followed by Dunnet’s post hoc test for multiple comparison. Results were considered significant at p < 0.05.

Results

Acute toxicity/median lethal dose (LD₅₀) of SFRN

The intraperitoneal LD₅₀ of SFRN in mice and rats were estimated to be 11.1 and 70.7 mg/kg, respectively. At higher doses, SFRN caused a dose-related decrease in locomotor activity, sedation and drowsiness (hypnosis), respiratory distress and subsequently death of the animals. The intraperitoneal LD₅₀ of the crude ethanol extract from which SFRN was obtained was estimated to be 282.8 mg/kg.

Behavioural studies

Effect of SFRN in the hole-board test

The saponin fraction of R. nilotica (SFRN) exhibited a significant (*p < 0.05) and dose-dependent decrease in number of head dips in the hole-board test. The extract at 0.5, 1.0 and 2.0 mg/kg gave 2.0 ± 0.4, 1.2 ± 0.2 and 0.3 ± 0.0 mean number of head dips, respectively, compared to normal saline with 7.66 ± 2.0. Diazepam gave 2.2 ± 0.6 mean number of head dips. Exploratory activity as determined by hole-board test is used to differentiate between anxiolytics and sedatives. Sedatives decrease the number of head dips while anxiolytics increase the number of head dips in the hole-board (Figure 1).

Figure 1. Effect of saponin fraction of R. nilotica (SFRN) and diazepam (DZP) on hole-board test in mice. Significant (*p < 0.05) difference between the control and treated groups, one factor analysis of variance (ANOVA) followed by Dunnet’s post hoc test for multiple comparison; n = 5 in each group.

Effect of SFRN on diazepam-induced sleeping time

SFRN showed a significant (*p < 0.05) difference in the onset of sleep at 0.5 mg/kg (3.6 ± 0.4 min) compared to normal saline (5.2 ± 0.7 min). A dose-dependent increase in sleep duration was also observed. This was significant (p < 0.05) at 2 mg/kg of the extract (117.8 ± 6.5 min) as compared to normal saline (43.2 ± 4.2) (Figure 2).

Figure 2. Effect of saponin fraction of Randia nilotica (SFRN) stem bark extract on onset and duration in diazepam-induced sleep in mice. Significant (*p < 0.05) difference exists between the normal saline and treated groups; one way analysis of variance (ANOVA) for multiple measures followed by Dunnet’s post hoc test; n  =  5 in each group.

Effect of SFRN on rota-rod test in mice

SFRN at all doses tested (0.5, 1.0 and 2.0 mg/kg body weight) did not produce any significant observable effect on motor coordination as determined by the rota-rod performance test in mice (Table 1).

Table 1. Effects of SFRN on sc-PTZ induced seizures in mice.

Treatment	Protection	% Protection
Control (N/Saline)	0/6	0.00
SFRN 0.5 mg/kg	0/6	0.00
SFRN 1.0 mg/kg	0/6	0.00
SFRN 2.0 mg/kg	0/6	0.00
PBT 30 mg/kg	6/6	100.00

Results are presented as % protection; phenobarbitone showed 100% protection; SFRN = saponin fraction of R. nilotica stem bark; sc-PTZ = sub-cutaneous pentylenetetrazole; n = 6 in each group.

Effect of SFRN on the beam-walk assay in mice

There was no observable difference between SFRN at all doses tested and controls (normal saline, diazepam) in time to reach the goal box (Figure 3). There was, however, a significant increase in the number of foot slips in the SFRN (*p < 0.05) and diazepam (**p < 0.01) groups compared to control (Figure 4).

Figure 3. Effect of saponin fraction of Randia nilotica (SFRN) and diazepam (DZP) on time to reach goal box in the beam walking assay in mice. No significant difference between normal saline and treated groups; one way analysis of variance (ANOVA) followed by Dunnet’s post hoc test for multiple comparison; n = 6 in each group.

Figure 4. Effect of saponin fraction of Randia nilotica (SFRN) and diazepam (DZP) on the number of foot slips in the beam walk assay in mice; significant (*p < 0.05) difference exists between normal saline and treated groups and diazepam (**p < 0.01); one way analysis of variance (ANOVA) followed by Dunnet’s post hoc test; n = 6 in each group.

Anticonvulsant studies

SFRN (0.5, 1.0 and 2.0 mg/kg) protected mice against hind limb tonic extension in the maximal electroshock induced seizure test (Figure 5). The protection was highest (50%) at 2.0 mg/kg of the fraction. SFRN failed to protect mice against pentylenetetrazole- and strychnine-induced seizures in mice (Tables 2 and 3).

Figure 5. Effect of saponin fraction of Randia nilotica (SFRN) and phenobarbitone (PBT) on hind limb tonic extension (HLTE) in mice using the maximal electroshock test; n = 6 in each group.

Table 2. Effects of SFRN and phenobarbitone on STN-induced seizures in mice.

Treatment	Protection	% Protection
Control (N/Saline)	0/6	0.00
SFRN 0.5 mg/kg	0/6	0.00
SFRN 1.0 mg/kg	0/6	0.00
SFRN 2.0 mg/kg	0/6	0.00
PBT 30 mg/kg	6/6	100.00

Results are presented as % protection; phenobarbitone showed 100% protection; SFRN = saponin fraction of R. nilotica stem bark; STN = strychnine; n = 6 in each group.

Table 3. Effect of SFRN on rota-rod test in mice.

	Time after treatment (min)
	30		60		90		120
Treatment	Time on Rod	Fail. %	Time on Rod	Fail. %	Time on Rod	Fail. %	Time on Rod	Fail. %
Control	>180.0 ± 0.0	0.0	>180.0 ± 0.0	0.0	>180.0 ± 0.0	0.0	>180.0 ± 0.0	0.0
SFRN 0.5 mg/kg	>180.0 ± 0.0	0.0	>180.0 ± 0.0	0.0	>180.0 ± 0.0	0.0	>180.0 ± 0.0	0.0
SFRN 1.0 mg/kg	>180.0 ± 0.0	0.0	>180.0 ± 0.0	0.0	>180.0 ± 0.0	0.0	>180.0 ± 0.0	0.0
SFRN 2.0 mg/kg	>180.0 ± 0.0	0.0	>180.0 ± 0.0	0.0	>180.0 ± 0.0	0.0	>180.0 ± 0.0	0.0

Results are presented as mean ± SEM. No significant difference between normal saline and treated groups; one way analysis of variance (ANOVA) followed by Dunnet’s post hoc test for multiple comparison; n = 6 in each group.

Discussion

This study provided scientific data on the behavioural and anticonvulsant properties of the saponin-rich fraction of R. nilotica. The acute toxicity studies of SFRN conducted in mice and rats indicated high toxicity as evidenced in the low LD₅₀ values. LD₅₀ values, however, are intended only to give an idea of how toxic a compound is, but not to reject toxic (unsafe) and accept non-toxic (safe) ones (van Boxtel & Buitenhuis, 2001). SFRN significantly reduced the head-dip counts in the hole-board test in mice. The hole-board test is a measure of exploratory behaviour that has been accepted as an experimental model for the evaluation of psychotic, sedative and anxiety conditions in animals (File & Wardill, 1975). Agents that diminish the number of head-dips in that test are considered to have high propensity for antipsychotic and sedative (Chindo et al., 2003) activities, whereas anxiolytics have been shown to increase the number of head-dips in the hole-board test (Takeda et al., 1998). Therefore, SFRN might be sedative rather than anxiolytic in nature. The sedative property of SFRN was confirmed by its ability to shorten the onset and potentiate the duration of diazepam-induced sleep. Endogenous neurotransmitters in the brain particularly dopamine, norepinephrine, acetylcholine, serotonin, GABA, histamine and neuropeptides, have been suggested to play important roles in sleep mechanisms (Dopp & Phillips, 2008). The effects of SFRN on diazepam-induced sleep may be attributed to an action on these central mechanisms involved in the regulation of sleep (Chindo et al., 2003). SFRN had no effects on motor coordination in the treadmill experiment at the doses tested, suggesting that it might not be acting through peripheral neuromuscular blockade (Capaso et al., 1996). The beam-walking assay also evaluates the effect of novel compounds on motor coordination in laboratory animals (Stanley et al., 2005). The number of foot slips made during the beam walking assay is a more sensitive measure than the rota-rod in detecting benzodiazepine-induced motor coordination deficits in mice, and may be more useful in predicting doses that cause sedation in the clinic (Stanley et al., 2005). The effects of SFRN on the beam walking assay confirm that its observed sedative activity might be via central mechanisms and not peripheral neuromuscular blockade (Perez et al., 1998).

Many groups of psychotropic agents such as antipsychotics and antidepressants (Baldessarini, 1996), anticonvulsants (McNamara, 1996) and narcotic analgesics (Reisine & Pasternak, 1996) are known to have sedative activity in all species of animals including humans. Decoctions of R. nilotica have been used in the Nigerian traditional medicine for the management of epilepsy and anxiety for many years; we therefore evaluated the anticonvulsant properties of SFRN to support the development of the psychoactive components of R. nilotica as anticonvulsant agents. SFRN significantly protected mice against maximal electroshock seizure, but failed to protect the mice from pentylenetetrazole- and strychnine-induced seizures. A similar result was obtained with the crude extract but with a higher magnitude (Danjuma et al., 2009). The exact mechanism of maximal electroshock-induced seizures is not fully understood. However, a convincing body of biochemical evidence implicates the inhibitory current breakdown and voltage-dependent sodium channels modulation in these electrically induced stimuli (McNamara, 1996). Antiepileptic drugs, such as phenytoin and carbamazepine that limit the repetitive firing of action potentials by slowing the rate of recovery of voltage-activated sodium ion channels from inactivation, suppress hind limb tonic extension in MES seizures (Rho & Sankar, 1999). Despite the diversity of models that could potentially be used to screen for anticonvulsant activity, the maximal electroshock model (MES) and the subcutaneous pentylenetetrazol model (PTZ) remain “Gold standards” in the early stages of testing (Rogawski, 2006). MES and PTZ tests are assumed to identify anticonvulsant drugs effective against generalized tonic-clonic seizures and petit mal seizures, respectively (Kupferberg & Schmitz, 1998). The effects of the saponin-rich fraction on MES-induced seizures suggest anticonvulsant efficacy against generalized tonic-clonic epilepsy in man.

In conclusion, we have presented evidence that the stem bark extract of R. nilotica contains sedative principles with potential anticonvulsant properties, which might be attributable to the saponin components of the plant. The results therefore support further development of psychoactive principles from its saponin-rich fraction as anticonvulsants. Further studies are in progress in our laboratories to isolate and mechanistically characterize the biologically active components of the saponin-rich fraction from the stem bark of this important medicinal plant.

Declaration of interest

The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper. Ahmadu Bello University (ABU) Zaria, Nigeria, as well as McArthur Foundation provided some financial support for this study. The authors are grateful to the technical staff of the Department of Pharmacology and Therapeutics, ABU, Zaria, for their technical assistance.