Psychopharmacological Screening of Methanolic Extract of Celastrus paniculatus Willd. whole plant in Mice

Diana Vivian Atigari¹*, Mrs. Sunanda Sabbithi¹, Krishna Chaitanya B.¹, Jajisree A.², Dr. Ramesh. C.²

¹Department of Pharmacology, Malla Reddy Institute of Pharmaceutical Sciences, Maisammaguda, Dhulapally (Post via Hakimpet), Secunderabad 500014, AP, India.

²GSN Pharmaceuticals Pvt. Ltd., Rajeev Gandhi Nagar, Kukatpally, Hyderabad (500072), AP, India.

ABSTRACT:

The objective of the present study was to investigate the CNS depressant and Anxiolytic activity of the methanolic extract of the whole plant of Celastrus paniculatus Willd. (Family: Celastraceae) in mice as a part of psychopharmacological screening of this plant, using different in vivo experimental models. The effects of the plant extract (200 mg/kg, 400mg/kg and 600 mg/kg) on the central nervous system (CNS) was evaluated for potentiation of phenobarbitone-induced sleeping time in mice. The results revealed that the methanolic extract of Celastrus paniculatus Willd. whole plant has reduced the onset of Phenobarbitone-induced sleeping time in mice and prolonged the duration of sleeping time significantly (p<0.05). In the test studied for the anxiolytic activity of methanolic extract of Celastrus paniculatus Willd. whole plant, using Staircase test model, it was revealed that the extract at doses 200 mg/kg and 400mg/kg reduced the number of rearings and decreased the number of steps ascended significantly (p<0.05). The overall results suggest that the methanolic extract of Celastrus paniculatus Willd. whole plant contains some active principles which may possess significant CNS depressant and anxiolytic activity.

KEYWORDS: Celastrus paniculatus Willd., CNS depressant, Anxiolytic, potentiation of phenobarbitone-induced sleeping time, Staircase test.

INTRODUCTION:

The experimental and recreational use of organic medicines and drugs in the nineteenth century sprang up Psychopharmacology¹. Advance in science and technology has contributed to an enormous improvement in the quality of life of humankind. However, modern life stress, associated trials and tribulation are responsible for the surge in incidence of variety of psychiatric disorders. Psychoactive drugs are typically utilized to alter mood or treat psychopathology. Depending on the influence on the central nervous system psychoactive drugs are categorized to alter mood or consciousness ².

CNS depression is considered as an affective disorder characterized by change in mood, lack of interest in the surroundings, apathy, loss of energy, psychomotor retardation, melancholia as well as profound feelings of gloominess, despair and suicidal ideation. It has been estimated that the rate of prevalence of CNS depression is around 5% in the general population and is accepted to be heterogenous symptomayically, biologically and psychologically³.

Anxiety is both a normal emotion and a psychiatric disorder. Anxiety is a feeling of apprehension or fear, combined with symptoms of sympathetic activity. It is a normal response to stress and becomes a clinical problem only if the anxiety becomes severe or persistent, and interferes with everyday performance. It has a lifetime prevalence of over 5% of the population⁴. It is typically associated with the former psychoneurotic disorders, hypothesis implicates over activity of adrenergic systems or irregular activity of serotonergic systems in the CNS and the symptoms of anxiety are commonly associated with depression⁵. In spite of the availability of CNS depressants and anxiolytic drugs, depression and anxiety continue to be a major medical problem⁶. Medicinal plants have been useful in the development of new drugs and continue to play an invaluable role in the drug discovery processes⁷. These herbs or plants are relatively cheap, available and their uses are dependent on ancestral experience⁸.

In the present study, we selected a plant namely Celastrus paniculatus Willd (Celastraceae), which is a large, woody, climbing shrub, distributed almost all over India upto an altitude of 1800m and is known for its ability to improve memory ⁹. Ayurveda, the ancient Indian traditional system of medicine has used the plant seed for prevention of various diseases¹⁰. The seed oil is bitter, thermogenic, and intellect promoting and is useful in abdominal disorders , beri-beri and sores ¹¹. The bark is abortifacient, depurative and a brain tonic. The leaves are emmenagogue and the leaf sap is a good antidote for opium poisoning. Earlier the plant has been pharmacologically studied for its analgesic and anti-inflammatory activity^12,13, anti-arthritic activity¹⁴, antifertility activity¹⁵, wound healing activity¹⁶, antimalarial activity¹⁷, antibacterial activity¹⁸, cardiovascular activity¹⁹, antioxidant activity^20,21, hypolipidemic activity²² etc. Considering the varied important activities reported in the traditional system of medicine with this plant and since there is dearth of scientific data proving the psychopharmacological screening for the CNS depressant and anxiolytic activities of the whole plant, the present study was carried out to investigate the psychopharmacological screening of the methanolic extract of Celastrus paniculatus Willd. whole plant(MECP) in the behavioral animal models for the assessment of CNS depressant activity by evaluation of potentiation of phenobarbitone-induced sleeping time and the assessment of anxiolytic activity by Staircase test.

MATERIALS AND METHODS:

Collection of Plant Materials

The whole plant of Celastrus paniculatus Willd was collected from Tirumala hills, Tirupathi, Andhra Pradesh, India. The plant was identified and authenticated by Dr. K.Madhava Chetty, Assistant Professor, Department of Botany, Sri Venkateswara University, Tirupathi, Andhra Pradesh , India, and voucher specimen has been deposited in the departmental herbaria.

Preparation of extract

The whole plant species was collected and then dried under shade for a period of four weeks. The dried plant material (500g) was milled to a fine powder using commercial laboratory blender. The dried powder ( 300g) was extracted in a Soxhlet extractor with Methanol. The extraction was continued until the solvent in the thimble became clear. After complete extraction, the extract was filtered and the solvent was distilled off. Then it was concentrated at 40⁰ C under reduced pressure using Buchi R-153 Rotavapour to obtain the dry residue. The yield of the crude methanol extract was 30g. The extract was the stored in desiccators until use.

Drugs and Chemicals

Drugs and chemicals used in the study were obtained commercially and were of analytical grade. Phenobarbitone (Novartis India ltd., Hyderabad, India), Imipramine (Sigma, USA) ,DMSO and methanol (Hi-pure Fine Chem Industries, Hyderabad, India).

Animals

For the screening of antiepileptic activity, studies were carried out using Swiss albino mice (18-22g) of either sex. All the animals were procured from Sainath Agencies, Hyderabad, India for experimental purpose. After procuring, all the animals were acclimatized for 7 days and housed in groups of six under standard laboratory conditions, like room temperature 26±2⁰C, relative humidity 45-55% and light/dark cycle of 12h. All the animals were provided with synthetic standard diet and water was provided ad libitum under strict hygienic conditions. Animal experimentation protocols are approved by Institutional Animal Ethical Committee (IAEC) of GSN Pharmaceuticals Pvt. Ltd., Kukatpally, Hyderabad, India.

Preliminary Phytochemical Screening

The preliminary phytochemical investigations were carried out with the methanolic extract of Celastrus paniculatus Willd. whole plant for qualitative identification of phytochemical constituents using standard conventional protocol. All the chemicals and reagents used were of analytical grade ²³.

Acute Toxicity Study

The acute toxicity of the methanolic extract of Celastrus panicualtus Willd. whole plant was determined as per the OECD guideline no. 423 (Organization for Economic Co-operation and Development). It was observed that the test extract was not mortal even at a dose of 2000 mg/kg body weight. Hence, 200 mg/kg, 400 mg/kg and 600 mg/kg doses were selected for further study.

Assessment of CNS depressant activity

Potentiation of Phenobarbitone-induced sleeping time in mice

Healthy Swiss albino mice of either sex weighing 18-22g body weight ,fasted for 24 hours before the experiment, were divided into IV groups of six animals each. Group I was maintained as control and was administered 10%(w/v) DMSO (5 ml/kg i.p.) . Groups II, III and IV were treated with different doses of methanolic extract of Celastrus paniculatus Willd. whole plant ( 200 ,400, 600 mg/kg i.p ) respectively. 30 min after 10% DMSO and extract administration into the respective groups, Phenobarbitone (40mg/kg, i.p) was administered. Each mouse was then observed for the onset and duration of sleeping time. The criteria for sleep is the loss of righting reflex, in which the mice cannot roll back when turned over²⁴. Righting reflex is referred as inability to maintain posture. This can be elicited by lifting the animal to certain height and then dropping the animal gently on table covered with soft cushion. Animal that is dropped from height immediately restores the posture and behaves normally. This immediate regaining of posture is termed as righting reflex. The time interval between loss and recovery of righting reflex was used as the index of sleeping time ^25,26.

The time of administration of test compounds and phenobarbitone, the time of loss and gain of righting reflex were recorded in all the groups of test animals and the percentage effect on phenobarbitone-induced narcosis by the test compounds was calculated using the formula given below, considering righting reflex in control as 100%²⁷.

%Effect =Average duration of loss of righting reflex in the test group

Average duration of loss of righting reflex in control

The following parameters were recorded during test session, onset of action in min., duration of sleeping time in min., and the percent effect.

Assessment of Anxiolytic activity

Staircase test

This test was used for the screening of anxiolytic activity in mice. Swiss albino mice of either sex weighing 18-22 g body weight, were segregated into IV groups consisting of six animals each. Group I was allotted as control which was given with 10%(w/v) DMSO (5 ml/kg p.o). Groups II and III were treated with different doses of methanolic extract of Celastrus paniculatus Willd. whole plant (200, 400mg/kg p.o) respectively. Group IV was administered with Imipramine( 30mg/kg, p.o) and served as standard group.

Mice were subjected to unpredictable chronic mild stress. The different kinds of stressors such as, cage tilting, lights on for a short period of time during the dark phase, damp saw dust, placement in an empty cage, soiled bedding for 22h, placement in an empty cage with water on the bottom and day-night reversal. The stress procedure lasted for 2 weeks prior to the behavioral testing. At least 12 hours of rest was provided between a stressors and a test. A stress procedure did not involve any food or water deprivation²⁸. After induction of stress in mice with above stress regimen for 14 days and with simultaneous test drug administration, animals were investigated for the anxiolytic activity by Staircase test.

Staircase consists of five identical steps, 2.5 cm X 10 cm X 7.5 cm. The inner height of the walls is constant (12.5 cm) along the whole length of the staircase. The animals were placed on the floor of the box with its back on the staircase. The number of steps climbed and the number of rears were counted over a 3 min period. A step is considered to be climbed only if the mouse had placed all four paws on the step. In order to simplify the observation, the number of steps descended were not taken into account. After each step the box was cleaned in order to obviate the olfactory cues, the presence of which may alter the behavior of the next animal²⁹. The parameters like number of steps climbed and the number of rearings were evaluated.

Statistical Analysis

The results were expressed as mean ± SEM and statistically analyzed by one way ANOVA followed by Tukey- Krama test. The results obtained were compared with the control group. p values < 0.05 were considered to be statistically significant (p denotes probability).

RESULTS:

Phytochemical screening

The qualitative analysis of the methanolic extract of Celastrus paniculatus Willd. whole plant showed the presence of various phytoconstituents such as alkaloids, phytosterols, tannins, flavonoids, glycosides, triterpenoids, proteins and amino acids (Table 1).

Table-1 : Results of Phytochemical Screening of the methanolic extract of Celastrus paniculatus Willd. whole plant (MECP)

Phytoconstituents	MECP
Reducing sugars	-
Alkaloids	+
Tannins	+
Flavonoids	+
Glycosides	+
Phytosterols	+
Triterpenoids	+
Proteins and Amino acids	+

+ : Positive result ; - : Negative result

Potentiation of phenobarbitone-induced sleeping time in mice

The results obtained from the study of the effects of methanolic extract of Celastrus paniculatus Willd. whole plant on potentiation of phenobarbitone-induced sleeping time are shown in Table 2. The extract significantly (p<0.05) increased the phenobarbitone-induced sleeping time at doses 200 mg/kg, 400 mg/kg and 600 mg/kg when compared with the control. There was also significant reduction in the sleep latency at the same doses relative to control (Figure 1).

DISCUSSION:

Barbiturates are putative sedatives inducing sleep in human beings and animals by depressing the CNS³⁰. Barbiturate is a GABA receptor agonist, probably binds to picrotoxin-binding site of GABA receptor, and results in increase in duration of opening of chloride channel thus reducing membrane excitability. The barbiturate-induced sleeping time can be easily studied in experimental animals. Phenobarbitone, despite a long-acting barbiturate, at decreased doses is used as short to intermediate acting barbiturate³¹. In the present study, in the control group of mice, phenobarbitone (40mg/kg) produced intermediate onset and duration of sleep indicated by the loss of righting reflex and awakening or regaining righting reflex subsequently. The extract pretreatment significantly prolonged the duration of sleep in phenobarbitone-induced mice and reduced the sleep induction time. Potentiation of phenobarbitone-induced sleeping time by the methanolic extract of Celastrus paniculatus Willd. whole plant on CNS is indicated by increase in the duration of sleeping time, thereby confirming its CNS depressant role in mice.

The mouse Staircase test was used for the assessment of anxiolytic activity. More the number of rears and the number of steps climbed indicates anxiety like behavior³². The present investigation successfully detected the anxiolytic effect of the extract by significantly decreasing both the number of rearing and the number of steps ascended compared to the control. This showed that the methanolic extract of Celastrus paniculatus Willd. whole plant possess significant anxiolytic activity.

In conclusion the results of the activities of the methanolic extract of Celastrus paniculatus Willd. whole plant in the psychopharmacological screening models justified its CNS depressant effect and its anxiolytic property. It is therefore possible that the activity of the plant may be exerted by the various phytoconstituents present in the plant viz., alkaloids, flavonoids, tannins, phytosterols, triterpenoids, glycosides, proteins and amino acids and justify its traditional folk remedy for the central nervous related activities. However extensive biological and chemical investigations are required to ascertain its clinical effectiveness ant the exact mechanism(s) of action of the extract and its active compounds.

ACKNOWLEGDEMENT

The authors are grateful to Dr. C. Ramesh, and G. Surya Narayana, Managing Director, GSN Pharmaceuticals Pvt. Ltd. , Kukatpally, Hyderabad, AP, India, for their guidance and providing the facilities to carry out the research work. The authors are also thankful to the Management and Principal, Malla Reddy Institute of Pharmaceutical Sciences, Secunderabad, AP, India, for their support during the study.

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Received on 18.06.201

Modified on 30.06.2012

Accepted on 15.07.2012

Research J. Pharmacology and Pharmacodynamics. 4(4): July –August, 2012, 245-250

Groups	Treatment	Dose(kg^-1, i.p)	Onset of action(in min)	Duration of Sleeping time(in min)	Percentage effect
I	Control(10% w/v DMSO)+phenobarbitone	5ml+40mg	30.33±0.16	70.66±0.71	100
II	MECP+phenobarbitone	200mg+40mg	24.83±0.30	128.16±1.07*	181.38
III	MECP+phenobarbitone	400mg+40mg	19.83±0.40	139.83±0.9***	197.89
IV	MECP+phenobarbitone	600mg+40mg	15.33±0.42*	162.66±1.02	230.20

Groups	Treatment and dose	No. of climbing in 3 min	No. of rearing in 3 min
I	Control(10% w/v DMSO 5ml/kg,p.o)	20.5±0.76	9.66±0.33
II	MECP (200mg/kg, p.o)	13.33±0.49	8.31±0.31
III	MECP (400mg/kg, p.o)	8.16±0.47*	6.16±0.60*
IV	Imipramine (30mg/kg, p.o)	5.80±0.42**	4.83±0.40**