CNS depressant and analgesic effects of Withania coagulans ( Stocks ) dunal fruits collected from Khuzdar district of Balochistan , Pakistan

Withania coagulans (Stocks) Dunal (Solanaceae) have been reported with various uses such as emetic, sedative, diuretic, alterative in chronic liver disorders, dyspepsia, intestinal infections and colic pain. This study aims to investigate the central nervous system (CNS) depressant, anxiolytic and analgesic effects of methanolic extract of W. coagulans. CNS depressant activity of crude methanolic extract of W. coagulans fruits was evaluated using standard animal behavioural models such as open field, cage crossing, rearing and traction test. Anxiolytic activity was evaluated through forced swimming test whereas analgesic activity was carried out via acetic acid induced writhing and formalin test in mice. The extract (100 and 250mg/kg) showed a dose-dependent suppression of motor and exploratory (Neuropharmacological) activity for mice in open field, cage crossing, rearing and traction test. Significant writhing effect i.e. decrease in number of writhes as well as analgesic effects were observed (P<0.05).The findings of current research work supports the CNS sedative and analgesic potential of W. coagulans.


Introduction
The family Solanaceae is famous for plants with potent medicinal properties [1].Withania coagulans (Stocks) Dunal, a shrub from Solanacea family, locally known as local inhabitants [5,6].The berries from this plant due to the presence of enzyme in husk and pulp are used as milk coagulating agent.The phytochemical investigation revealed the presence of free amino acids, esterases, essential oils, fatty oils and withanolides in the berries.Main skeleton of Withanolides is ergostane with a steroidal lactones [7,8].In addition; the fruit of plant is emetic, sedative, diuretic and used as alterative in chronic liver disorders as well as employed for gastrointestinal problems such as flatulence, dyspepsia, intestinal infections and colic pain.The famous local use found for this plant is; chewing the small branches for dental cleaning whereas smoking the branches for toothache problem [7, [9][10][11].The plant also have an eminent place in Unani and Ayurvedic medicine.Furthermore, W. coagulans have been reported as a significant source of constituents with steroidal lactones having biological activity [5,11].Numerous studies have revealed the pharmacological uses of crude plant, its extracts, fractions, and isolated withanolides in various ailments such as leishmaniasis, tumor, addiction, diabetes mellitus, wounds, epilepsy, hyperlipidemia and many other diseases [11][12][13][14][15][16][17].Furthermore; many literature have reported the fruit of the plant as sedative and analgesic drug, thus current study aims to evaluate the Central Nervous system (CNS) depressant and analgesic activities of its methanolic extract.

Plant material
The Fruits of W. coagulans were collected from Khuzdar district of Balochistan in the month of June.

Open field activity (OFT)
The exploratory and loco motor activities were determined in an OFT.The apparatus was made of plastic cube, walled (30 cm in height) and the floor was divided into twenty five (25) squares i.e. length and width 10 cm).The mice, after thirty minutes (30 min) following oral administration of the vehicle, plant extract (100 and 250 mg/kg) and diazepam, were evaluated individually at the center of OFT apparatus (10 min) in order to record locomotor activity (expressed by squares crossing with the all four paws) [23].

Cage crossing test (CCT)
For CCT, animal were placed in a plastic cage with crossing i.e. 26x 26 x26, and the number of movements to touch the cage edges (10 min) were recorded [24].

Traction test (TT)
For traction test an iron rod (one meter) is used while the animals were made preskilled to walk this rod.For TT observation, the mice time (seconds) in order to travel the rod is recorded.Any enhancement or reduction in time consumed by the extract treated mice were compared with control (non-treated) which shows stimulant or sedative activity of extract, respectively [25].

Analgesic activity Acetic acid writhing reflex method
This activity was performed according to the modified method Danbisya et al. [26] as adopted from Koster et al. [27].Twenty (20) mice (of both sexes) were arbitrarily divided in four (4) groups of five mice.Group I (Control group) mice were administered distilled water (10 ml/kg), group II mice were administered Diclofenic potassium (as standard drug) 50mg/kg whereas group III and IV received 250 and 500 mg/kg of W. coagulans extract respectively.Following the administration of W. coagulans extract and drug (after thirty (30) minutes), glacial acetic acid (0.7%; 10 ml/kg; i.p.) was introduced to all mice in order to induce pain as characterized by writhes or abdominal constrictions.Number of writhes experiential in every mice was calculated for thirty (30) minutes whereas the %age protection compared to abdominal writhing was utilized as a tool to measure the degree of analgesia using the formula [28]. Mean

Table 1 . Effect of W. coagulans on swimming test of mice S. No Treatment Mean mobility time (seconds)+SEM
).

Table 7 . Effect of Crude extract of W. coagulans in formalin induced inflammatory pain on mice Treatment Dose mg/kg orally
Stimulation of these receptors responsible for relief of pain.Flavonoids inhibit the cyclooxigenase present in tissues, which inhibit the formation of prostaglandins.Prostaglandins stimulate the receptors of pain [38].W. coagulans has contain flavonoids [39], flavonoids are reported to exert analgesic effects [40].In present study the analgesic activity may be due to flavonoids.Conclusion The results of present study reveal that the administration of methanolic crude extract of W. coagulans exhibited significant decrease in neuropharmacological activities, forced swimming test, number of writhes in acetic acid writhing test alongwith the number and time spent on licking and biting.Thus it is evident from our results; crude methanolic extract of W. coagulans contains pharmacological active constituents with CNS depressant and analgesic effect.Hence, further pharmacological and biochemical studies are required to postulate the exact mechanism and chemical compound responsible for pharmacological effects.Authors' contributions Conceived and designed the experiments: S Muhammad, K Fatima & Rizwan Ahmad, Performed the experiments: N Ahmad, Analyzed the data: Mehjabeen & Marvi, Contributed reagents/ materials/ analysis tools: NA Shahwani, Wrote the paper: N Ahmad.