A Ali, N Venkat Rao, M Shalam, T Shivaraj Gouda, J Babu, S Shantakumar
anti-anxiety activity, c. bonducella, petroleum ether extract, seed kernel
A Ali, N Venkat Rao, M Shalam, T Shivaraj Gouda, J Babu, S Shantakumar. Anxiolytic Activity Of Seed Extract Of Caesalpinia Bonducella (Roxb) In Laboratory Animals. The Internet Journal of Pharmacology. 2007 Volume 5 Number 2.
In the traditional system of Indian medicine
In day today life of stress and strain there is a dire need for agents having neuroprotective and neuropharmacological activity enhancing learning and memory caliber of the brain 1 . Stress involves complex biochemical, neurological and immunological mechanisms and plays a crucial role in the genesis/progression of a variety of disease states ranging from psychiatric disorders like depression and anxiety, immunosupression, endocrine disorders including diabetes mellitus, impotency and cognitive dysfunctions 2.
Anxiety related disorders such as generalized anxiety, panic, obsessive-compulsion, phobias or post traumatic stress disorders are common and major cause of disability 3 and 1/8 th of the total population worldwide affected with anxiety and became a very important area of research interest in psychopharmology 4 . Anxiety is also an obvious component of many psychiatric and medical conditions 5 .
During the last two decades, pharmacotherapy with psychoactive drugs has become management of anxiety, stress and psychomotic disorders. Traditionally pharmacological research in the area of anxiety and stress treatment is very much influenced by the availability of anxiolytic drugs. Throughout history recorded, ethanol was and is the standard drug for treatment of feelings of discomfort, tension, anxiety and stress 6 .
Though barbiturates were dominant agents from 1900-1950 because of considerable concern about their safety lead to the search of better alteration. Moreover benzodiazepines (bdz) as anxiolytic agents have brought tremendous progress in understanding the physiological, biochemical and pathological status of the disease. However the use of tranquillizer and psychotropic drugs leads to variety of autonomic, neurologic and hematopoietic disorder, but these agents primarily relieve the symptoms and offer a palliative relief of a temporary nature 7 .
In recent years use of alternative medicine in particular, derived from plant have been increased in a number of patient with condition that affect the mind 8 .
In traditional system of indian medicine (Ayurveda)
Materials and Methods
Swiss albino mice of either sex weighing between 20-30g were procured from Shri Venkateswara Enterprises, Bangalore for experimental purpose. All the animals were acclimatized for seven days under standard husbandry conditions i.e.; room temperature of 24 0
± 10 0 C; relative humidity 45-55% and a 12:12h light/ dark cycle. 40,41 The animals had free access to standard rat pellet diet (Amrut laboratories, Pranava Agro Industries Ltd., Sangli, India), with water provided
Preparation of seed extracts of
Seed powder of C.bonducella will be successively extracted with petroleum ether, ethanol, methanol and water. Each time before extracting with the next solvent, marc will be dried in Hot air-oven below 50 0 C. Finally the marc will be macerated with chloroform water (i.e.; chloroform acts as a preservative) for 24 hrs to obtain the aqueous extract. Each extract will be concentrated by distilling off the solvent and then evaporating to dryness on the water bath.
Diazepam [Ranbaxy Laboratories Ltd, Mumbai, India], Tween-80 [s.d.fine Chem Ltd. Mumbai], Petroleum Ether [The Ugar Sugar Works Ltd. Ugar Khurd, Belgaum] and Distilled water [Mysore Petro Chemicals, Raichur, India].
Preliminary phytochemical Screening ,,
The preliminary phytochemical investigations will be carried out with the seed extract of
Acute toxicity test of
The acute toxicity of
Grouping of animals and treatment schedule
Male albino mice (22-25g) and wistar rats (150-200g) were divided into following groups each consisting of six animals.
Group A - Normal control (2% gum acacia p.o)
Group B - Standard (Diazepam 3mg/kg p.o)
Group C - Seed extract of C.bonducella (400mg/kg p.o)
Group D - Seed extract of C.bonducella (600 mg/kg p.o)
Group E - Seed extract of C.bonducella (800 mg/kg p.o)
All the drugs were administered to the respective groups in all the models for a period of eight days and experiments were performed one hour after the administration of last dose.
All the experiments were carried in a sound attenuated dark room. After test with each animal, all the apparatus was cleaned with 5% alcohol in order to eliminate any olfactory cues which might modify the behavior of the next animal.
Staircase test in mice
The staircase is composed of five identical steps 2.5 cm high, 10 cm wide and 7.5 cm deep. The internal height of the walls is constant along the whole length of the staircase. Each animal is used only once.ment. At the end of experimental period mice were placed individually on the floor of the box with its back to the staircase. Total number of steps climbed and total number of rearings were recorded over a period of 3 min. A step is considered to be climbed only if the mouse has placed all four paws on the step15 .
Elevated Plus-Maze Test in mice
The plus-maze apparatus comprises of two open arms (16×5cm) and two closed arms (16×5×12cm) that extend from a common central platform (5×5cm). The entire maze is elevated to a height of 25cms above the floor level. Mice were placed individually in the center of the maze facing one of the enclosed arms for recording various parameters in a period of 5 min.16,17,18,19
Hole-board test in rats
The apparatus used in this model consists of wooden chamber (40x40x25 cm) with 16 holes (diameter 3 cm) on the floor, elevated from the ground so that the rats could peep through the holes each rat will be placed individually in the apparatus for recording following parameters, latency to the first head dips, no. of head dips in the holes, total time spent with the head dips, no. of rearings, no. of defecation units.20
Light-dark model transition test in mice
The light-dark apparatus consists of two-compartment chamber (40×60×20cm/h) comprising of a brightly illuminated area (40×40cm) and a dark area (40×20 cm) separated by a wall with a round hole (7 cm diameter) will be used. Mice were placed individually in the illuminated part of the cage and following parameters were recorded during the test session of 5 min, total no. of crossings, no. crossings between the light and dark area, total time spent in the illuminated part of the cage, time spent in the dark part of the cage, no. of rearings in illuminated part of the cage, no. of rearings in dark part of the cage, no. of defecation units. 21,22,23
The values were expressed as mean ± SEM from 6 animals. The results were subjected to statistical analysis by using ANOVA followed by Dennett's- t -test test to calculate the significance difference if any among the groups. P<0.05 was considered significant.
Effect of on mice in Stair-case test
Three different doses of PECB (400, 600 and 800mg/kg) were subjected for anxiolytic activity using Stair-case test in mice. These doses when administered orally daily once for 7 days, high and medium doses (800&600mg/kg) has showed significant increase in no. of steps climbed and rearings dose dependently when compared to control. However, PECB at 400 mg/kg had not shown any significant effect with no. of rearings. Standard drug diazepam (2mg/kg) had exhibited significant anxiolytic effect.
Effect of on mice in Elevated plus maze
Diazepam has long been reported for its anxiolytic activity in mice with the EPM model. In our study also, a significant anxiolytic effect was recorded with diazepam as increased number of entries in to open and decreased number of entries in to closed arms and with increased time spent in open and central platform but not in closed arms. Insignificant effect was recorded with total number of entries in both the arms when compared to control. In chronic study when different doses of PECB i.e. 400, 600 and 800mg/kg were administered orally daily once for seven days, it was found that lower dose (400mg/kg) increased the number of entries and time spent in the open arm ,central platform and decreased the time spent in closed arm as compared to control group. Where as medium and high doses (400 and 800mg/kg) had increased the number of entries and time spent in the open arm, central platform and decreased the number of entries and time spent in closed arm as compared to control group and exhibited statistically significant activity. No significant effect was observed with total number of entries with all doses. Among these, low dose (400mg/kg) had increased time spent in central platform when compared to medium, high and standard doses respectively.
Effect of on mice in Hole- board model
Three different doses of PECB (400, 600 and 800mg/kg) were subjected for anxiolytic activity using HB- model in mice. These doses when administered orally daily once for 7 days, high and medium doses (800&600mg/kg) but not low dose (400mg/kg) has shown significant increase in latency to the 1 st head dips, number of head dips and time spent in head dips. A significant increase in number of rearing was observed in both medium and high doses as compared to control group. Defecation units have significantly reduced with high dose but not with low and medium doses as compared to control group. Standard drug diazepam (2mg/kg) had exhibited significant anxiolytic activity.
Effect of on mice in Light-Dark transition test
Three different doses of PECB (400, 600 and 800mg/kg) were subjected for anxiolytic activity using LDT model in mice. These doses when administered orally daily once for 7 days, high dose (800mg/kg) but not the low and medium doses (400 & 800mg/kg) had produced an increase in number of crossings and time spent in light box and decrease in the number of rearings in both light and dark compartments. Defecation boli were not significantly altered with these different doses of PECB when compared to control group. High dose had statistically showed significant anxiolytic activity and Standard drug diazepam (2mg/kg) had exhibited significant anxiolytic activity.
The stair-case test has been proven as a simple and reliable method for screening of anxiolytics in several laboratories. The stair-case test for evaluating anxiolytic activity was originally described for rats 24 . When introduced into a novel environment, rodents experience anxiety manifested by increased vigilance and behavioral activity. In the stair-case paradigm, step-climbing is purported to reflect exploratory or locomotor activity, while rearing behaviors is an index of anxiety state. The number of rearing and steps climbed are recorded in a 5 min period. The test was modified for rapid screening of anxiolytic activity in mice 25 .
When compared to control PECB with all the three doses i-e, low, medium and high (400, 600 and 800mg/kg) tested had showed a significant increase in number of steps climbed up and rearings dose dependently. However, PECB at 400 mg/kg had not shown any significant effect with no. of rearings. Standard drug diazepam (2mg/kg) had exhibited significant anxiolytic effect.
The elevated plus maze is a well-established animal model for testing anxiolytic drugs 26,27 . A standard anxiolytic drug diazepam used clinically is also employed in behavioral pharmacology as a reference compound for inducing anxiolytic like effects 28 , even when the compound being screened does not act via benzodiazepine receptors.
Although orginal validation of the EPM was performed in rats 35 , it has also been found to be selectively sensitive to the effects of anxiolytic and anxiogenic drugs in mice 30 .
The EPM test is based on a premise where the exposure to an EPM evoked an approach-avoidance conflict that was considerably stronger than evoked by the exposure to an enclosed arm 34 . The decrease in aversion to the open arm is the result of an anxiolytic effect, expressed by the increased time spent and entries in to the open arm. The primary index is spatiotemporal in nature: it is reduced by anxiolytic drugs and can be increased by anxiogenic compounds 32 . The decrease in time spent on the central platform is another indication of reduced “decision-making” behaviour. Both parameters are accepted as reliable indicators of anxiety and fearfulness 33 .
PECB at medium and high doses (600 and 800mg/kg), but not the lower dose (400mg/kg) had increased the time spent in and number of entries into open arms with a decreased time spent and number of entries into closed arms. After treatment with all three doses in different groups the total numbers of entries into both open and closed arms were decreased insignificantly as compared to control group but except with the low dose (400mg/kg). It can be suggest that, the higher doses of PECB may have the sedative effect more than the standard drug and as a result animals spent more time in open, closed arms & less time in central platform, while standard drug treated animals spent more time in open arm & central platform but less time in closed arm. Lower dose of PECB (400mg/kg) didn't alter the above parameters significantly; suggesting that 400mg/kg of PECB had not exhibiting anxiolytic effect. Therefore, behavioral alterations induced by medium and higher dose of PECB (600 and 800mg/kg) were consistent with anxiolytic activity. The three dose of PECB (400,600 and 800mg/kg) showed dose depended anxiolytic profile. The lower dose of PECB (400mg/kg) didn't exhibit any anxiolytic activity.
Standard drug diazepam act selectively on GABAA receptors which mediate fast inhibitory synaptic transmission throughout the CNS. Benzodiazepines (BDZs) bind to the gamma sub-unit of the GABA-A receptor, that causes an allosteric (structural) modification of the receptor results with an increase in GABA A receptor activity. BDZs do not substitute for GABA, which bind at the alpha sub-unit, but increase the frequency of channel opening events which leads to an increase in chloride ion conductance and inhibition of the action potential. These drugs also exert a marked taming effect, allowing animals to be handled more easily 34 .
Hole-board model indicated that head-dipping behaviour was sensitive to changes in the emotional state of the animal, and suggested that the expression of an anxiolytic state in animals may be reflected by an increase in head-dipping behaviour 36 .The PECB medium and high doses (600 & 800mg/kg) shows increase in number, latency and duration of head dipping and the number of rearing in the hole board test. It could be argued that the increased head-dipping in rats is merely an artifact of the hyperactivity induced by the drug. But the effect of low dose (400mg/kg) was found to be insignificant when compared with control. There is a significant reduction in defecation units seen with the high dose (800mg/kg), but not with medium and low doses (600& 400mg/kg) respectively. Diazepam a putative anxiolytic agent increased the number, latency and duration of head dipping and the number of rearing with reduction in defecation units as also noted with PECB medium & high (600 & 800mg/kg) doses. Therefore the increased number of head dippings & rearings are not only a representation of hyperactivity but also a reflection of anxiolytic effect.
It has been suggested that some animal models based on spontaneous behaviour or ethologically based models 37 like the light-dark test, may be more sensitive to the behavioural responses than conditioned paradigms 38 .
The light-dark test may be useful to predict the anxiolytic like activity of drugs in mice. It has the advantages of being quick and easy to use without food and water deprivation prior training of animals and natural stimuli are used. Transitions have been reported to be an index of activity exploration because of habituation over time and the time spent in each compartment to be a reflection of aversion 39 .
In Light-Dark Transition test, the apparatus contains two compartments i.e. light and dark. Animals always try to spend more time in dark compartment because of fear about new environment. In this model, four behavioral events were observed i.e. number of crossings to light compartment, time spent in light and dark box, number of rearings in light and dark box and defecation units. In this study high doses (800mg/kg) of PECB had significantly increased the time spent in light compartment along with reduced time spent in dark compartment, number of crossings and decreased the number of rearings in light as well as dark compartments, indicating that high dose of PECB had produced significant anxiolytic effect but not medium & lower dose (600 & 400mg/kg). Medium dose (600mg/kg) had shown decreased rearings in dark compartment, but the effects on remaining parameters were insignificant as compared with control.
It is well known that BZDs have sedative, anticonvulsant, anxiolytic and hypothermic effect. Anxiolytics are known to exert pharmacological action by causing an increase in GABA content in the cerebral hemisphere 40 .
Several lines of evidence show that natural and synthetic flavonoids are potent anxiolytic agents without sedative, myorelaxant or amnestic effects. It is known the participation of GABA in these effects 41 .
The results obtained from these experimental models clearly confirmed the anxiolytic activities of PECB. Phytoconstituents like flavonoids and saponins were reported for their anxiolytic effect and these two were present in petroleum ether extract. So these active principle might be responsible for anxiolytic effects.
Authors are thankful to the management of AME's society for providing the facilities to carry out this work.
N.Venkat Rao, Professor & Head, Dept of Pharmacology, V.L. College of Pharmacy, Manikprabhu Temple Road, Raichur-584103. Mobile- 09845666548 Email: firstname.lastname@example.org Fax : 08532240405