Analgesic and central nervous system depressant activities of methanol extract of Ziziphus rugosa Lam. Leaves

The present investigation was designed to evaluate the analgesic and central nervous system (CNS) depressant activities of the methanolic extract of Ziziphus rugosa Lam. in rat model. The analgesic activity of the extract was examined using acetic acid-induced writhing test (chemically induced pain) at the doses of 200 and 300 mg/kg body weight. Further, the CNS depressant activity of the plant extract was evaluated by open field and hole cross tests at the doses of 300 and 500 mg/kg body weight. In the analgesic activity test, the methanolic extract of Z. rugosa showed significant (p < 0.01) antinociceptive activity in a dose dependent manner. The extract at the dose of 300 mg/kg exhibited 51.87% inhibition of writhing response which was comparable to the reference drug Indomethacin (55.20%). On the other hand, the plant extract also demonstrated significant (p < 0.01) dose-dependent reduction of locomotor and exploratory activities in the open field and hole cross tests. This analgesic and CNS-depressant activity of the extract might be due to the presence of biologically important chemical compounds.


INTRODUCTION
Pain is the part of a defensive response against dysfunction of an organ or imbalance in its functions towards potentially dangerous stimulus.Pain management has become the focus of global scientific research because of its inference in virtually all human and animal diseases (Sarker et al., 2012).Many drugs are used to relieve the pain and most of them produce some side-effects on the physiology of the body (Devraj and Karpagam, 2011).As a result, searching of new analgesic drugs from traditionally used plant species as pain killers should still is a rational strategy.The plant Ziziphus rugosa Lam. is a large straggling armed shrub with large elliptic usually subcordate leaves and belongs to the family Rhamnaceae.The flowers are paniculated and fruits are small drupe, glabrous and become white when ripe.Traditionally bark is used as astringent and antidiarrhoeal while flowers are used for menorrhagia and hemorrhage.Stem and fruit are hypotensive.Fruit is used *Corresponding author.E-mail: rashidma@du.ac.bd.
Author(s) agree that this article remain permanently open access under the terms of the Creative Commons Attribution License 4.0 International License in the treatment of rheumatism and hypotension.The root bark of the plant have been reported to exhibit analgesic, anti-inflammatory, antibacterial and antifungal activities (Abhimany and Pratiksha, 2010;Rambabu et al., 2011).The seed extract has been reported for anxiolytic and cytotoxic properties (Prashith et al., 2011;Gawande et al., 2011).Cytotoxicity, antimicrobial and antioxidant properties (Sazzad et al., 2013) have also been reported from leaves.The fruit pericarp is known to have antibacterial, insecticidal and free radical scavenging properties (Kekuda et al., 2011).Rugosanine-A, a cyclopeptide alkaloid (Pandey et al., 1988) and three flavonoids -kaempferol-4'-methylether, luteolin and luteolin-7-O-glucoside (Singh et al., 2009) have been isolated from the stem bark of the plant.
A new glycoside zizyphoside has been isolated along with betulic, oleanolic, alphitolic and 2-α-hydroxyrusolic acids; zizyphoside on hydrolysis yielded altered aglycone, ebelin lactone (Bakshi et al., 1999).Due to the absence of report of the Ziziphus rugosa leaves on central nervous system (CNS) activity, an effort has been made as part of our regular research program (Rahman et al., 2008;Ferdous et. al., 2012;Khan et al., 2015) to scientifically evaluate the CNS depressant and analgesic effects of methanolic extract of Z. rugosa leaves in model rat.

Plant
The leaves of plant Z. rugosa were collected from Gazipur in the month of March, 2011 and identified in the Bangladesh National Herbarium, Dhaka, where a voucher specimen (No.DACB-34479) has been mentioned for future reference.

Preparation of extract
The leaves were first washed with running water to remove adhering dirts, then dried in an electric oven at 45°C and powdered with a mechanical grinder, passing through sieve #40, and stored in a tight container.The powdered material (1 kg) was taken in a clean, flat bottomed glass container and soaked in methanol for seven days.The whole mixture was then filtered through a piece of clean, white cotton bed followed by Whatman filter paper number 1.The total filtrate was concentrated, in vacuo at 40°C to render brownish red colored mass (390 g).

Drugs and chemicals
The active drugs Indomethacin and Diazepam were obtained from Square Pharmaceuticals Ltd., Bangladesh.Acetic acid was obtained from Merck, Germany.Tween-80 was procured from BDH Chemicals, UK.Normal saline solution was purchased from Beximco Infusion Ltd., Bangladesh.All chemicals used were of analytical reagent grade.

Animals
Long-Evan's rats of either sex weighing about 120 to 170 g were used for the experiment.The rats were purchased from the Animal Resources Branch of the International Centre for Diarrhoeal Disease and Research, Bangladesh (ICDDR, B).They were kept in standard environmental condition (at 24.0 ± 0°C temperature, 55-65% relative humidity and 12 h light/12 h dark cycle) for one week for acclimation after their purchase and were fed ICDDR, B formulated rodent food and water ad libitum.The Federation of European Laboratory Animal Science Associations (FELASA) guidelines and recommendations were followed to reduce the pain and stress of the experimental rats and were approved by the institutional animal ethical committee.

Acute toxicity study
The median lethal dose (LD50) of the extract in rat was estimated by the up and down method (Bruce, 1985).Doses were adjusted up or down by a constant multiplicative factor (1.5) depending on the previous outcome.

In vivo analgesic activity
The analgesic activity of the methanolic extract of Z. rugosa leaves was evaluated using acetic acid-induced writhing method in rat (Sharma et al., 2010).At first, twenty four animals were divided into four groups with six rats in each.The test samples and vehicle were administered orally 30 min before intraperitoneal administration of 0.7% v/v acetic acid but Indomethacin (reference drug) was administered orally 15 min before injection of acetic acid.After an interval of 5 min, the mice were observed for specific contraction of body referred to as 'writhing' for the next 10 min.Complete writhing was not always accomplished by the animal; this incomplete writhing was considered as half-writhing.Accordingly, two half-writhing were taken as one full writhing.The number of writhing in each treated groups was compared to that of a control group.Samples having analgesic activity will reduce number of writhes of treated rats.The percent inhibition (% analgesic activity) was calculated by: % inhibition = Where A = average number of writhing of control group; B = average number of writhing of test group.

Open field test
The open field test was carried out to evaluate the effect of extract on locomotor activity of rats by the method described by Gupta et al. (1971).Here, rats were divided in to 4 groups of 6 rats each.The control group received 1% Tween 80 in water (10 ml/kg body weight), the standard group received Diazepam (1 mg/kg body weight) and the experimental groups received crude extract at 300 and 500 mg/kg body weight.The floor of an open field was divided into a series of squares each alternatively colored black and white.The apparatus had area of half square meter and a wall of 40 cm height.The number of squares visited by the animals was counted for 3 minute, on 0, 30, 60 and 120 min during the study period.

Hole cross test
The method described by Takagi et al. (1971) was implemented for this study.Again 24 rats were equally divided into 4 groups.The control group received 1% Tween 80 in water (10 ml/kg body weight), the standard group received Diazepam (1 mg/kg body weight) and the experimental groups received crude extract at 300 and 500 mg/kg body weight.A steel partition was fixed in the middle of a cage having a size of 30 × 20 × 14 cm.A hole of 3 cm diameter was made at a height of 7.5 cm in the center of the partition.The number of passages of rats through the hole from one chamber to other was counted for a period of 3 minute after 0, 30, 60 and 120 min of oral administration of the extract.

Statistical analysis
All the values in the test are expressed as mean ± standard deviation (SD).The data were statistically analyzed by ANOVA (Analysis of variance) and post-hoc Dunnett's tests with the statistical package for social sciences (SPSS 16.0, USA) program.Dissimilarity between the means of the various groups were measured significantly at p < 0.05 and p < 0.01.

Acute toxicity
Oral administration of graded doses of the methanol extract of Z. rugosa leaves (500 to 5000 mg/kg, body weight) did not cause any death in different dose groups.
The LD 50 value for oral administration of the plant extract was found to be greater than 5000 mg/kg body weight.

In vivo analgesic activity
In acetic acid induced writhing test, the methanol extract of Z. rugosa significantly (p < 0.05 and p < 0.01) reduced the number of writhing movements induced by intraperitoneal administration of acetic acid solution.The dose-dependent inhibition (Table 1) of abdominal constrictions by the methanol extract indicates antinociceptive potential of the plant.The exerted inhibition of writhing at a dose 300 mg/kg body weight was close to the standard non-narcotic analgesic drug, Indomethacin.

Open field test
The extract was evaluated by open field test to determine the decreasing capability of CNS-locomotor activity in rat model.The extract significantly decreased the locomotor activity in a dose dependant manner (Table 2) and this effect was evident from the initial observation (0 min) period and continued up to 5 th observation period (120 min).

Hole cross test
In this test, the extract showed a decrease in locomotion in the test animals.The number of crossing hole from one chamber to another by rat of the control group remained almost steady to slight decrease from 0 minute to 120 minute (Table 3).But the extract at 300 and 500 mg/kg dose showed significant (indicate p-value) gradual decrease of movement from 0 to 120 min.The extract displayed dose dependent activity and maximum depressive effect was observed at fifth (120 min) observation period.Depression produced at 500 mg/kg body weight was found to be close to that of standard drug, Diazepam.

DISCUSSION
The present study was conducted to elucidate analgesic and CNS depressant activities of the methanol extract of Z. rugosa leaves.The relatively high oral median lethal dose (LD 50 ) in rat suggests that the extract is relatively non toxic when taken orally (Lorke, 1983;Magaji et al., 2008).Acetic acid-induced abdominal constriction test is used for the evaluation of pheripheral analgesic activity (Gene et al., 1998;Faruk et al., 2015).The extract showed significant analgesic activity in acetic acidinduced writhing test in rats.This indicates that the extract possessed peripheral mediated analgesic activity.
The abdominal constriction response is thought to involve, in part, the local peritoneal receptors (Bentley et al., 1983;Sani et al., 2013), so the extract may have interfered with these peritoneal receptors to bring analgesia.Acetic acid-induced writhing test has been associated with increase in the levels of prostaglandins E2 and F2α in peritoneal fluid (Deradt et al., 1980) as well as lipooxygenases (Levini et al., 1984;Sani et al., 2013).So the mechanism of activity of the extract may be linked to cyclooxygenases and/or lipooxygenases.CNS depressing agents are gaining importance in treating mental disorders like anxiety, dizziness and restlessness.So, we carried out locomotion test to confirm the CNS activity of the methanol extract of the plant.Increased locomotor activity (by excitatory neurotransmitter) is considered as alertness while decreased locomotor activity (due to inhibitory neurotransmitter) indicates sedative effect (Verma et al., 2010).Our extract had decreased locomotor activity indicating its CNS depressant activity.The CNS depressant activity is observed when the GABAA receptor is activated either by Gamma-amino-butyric acid (GABA) or any other chemical substances.Different anxiolytic, muscle relaxant and sedative-hypnotic drugs exert their action through GABAA receptor.Therefore it is possible that the extract of Z. rugosa leaves may act either by potentiating GABAergic inhibition in the CNS via membrane hyperpolarization or may be due to direct activation of GABA receptor (Kolawole et al., 2007).Many research results showed that plant containing flavonoids, saponins and tannins are useful in many CNS disorders and many flavonoids and neuroactive steroids were ligand capable for the GABAA receptors in the central nervous system.This led to the assumption that they can act as benzodiazepine like molecules (Bhattacharya and Satyan, 1997).

Conclusion
Based on the results of the present study, we conclude that the methanolic extract of Z. rugosa leaves possesses remarkable dose dependent analgesic and CNS depressant activity.However, further studies are indispensable to examine the underlying mechanisms of such CNS effects and to isolate the active compounds responsible for these pharmacological activities.

Group I :
Treated with vehicle (1% Tween 80 in water, 10 ml/kg body weight p.o.) Group II: Received Indomethacin (10mg/kg) body weight (p.o.) Group III: Treated with 200 mg/kg body weight (p.o.) of the extract Group IV: Treated with 300 mg/kg body weight (p.o.) of the extract.

Table 1 .
Analgesic effect of methanolic extract of Z. rugosa leaves in acetic

Table 2 .
CNS depressant activity of methanol extract of Z. rugosa leaves in open field test in rats.
All values are expressed as mean ± SD, (n=6); One way Analysis of Variance (ANOVA) followed by Dunnet's test.** P < 0.01, significant compared to control.

Table 3 .
CNS depressant effect of methanol extract of Z. rugosa leaves on hole cross test in rats.