Evaluation of Central Nervous System Activity of Ayurvedic Formulation Cognium in Experimental

“EVALUATION OF CENTRAL NERVOUS SYSTEM ACTIVITY OF AYURVEDIC FORMULATION COGNIUM IN EXPERIMENTAL ANIMAL MODELS”

M. Pharm Dissertation Protocol

Submitted to

Rajiv Gandhi University of Health Sciences, Karnataka

Bangalore – 560 041

MR. AJMAL. N

Under the Guidance of

Mr. Abhilash D

Assistant Professor

Department of Pharmacology,

Shree Devi College of Pharmacy

Airport Road, Kenjar Village,

Malavoor Panchayath

Mangalore – 574142.

RAJIV GANDHI UNIVERSITY OF HEALTH SCIENCES,

BANGALORE, KARNATAKA,

ANNEXURE II

PROFORMA FOR REGISTRATION OF SUBJECTS

1. / Name of the Candidate
and Address / AJMAL .N
Nelliyote house.
Paleri p.o, Kuttiady,
Kozhikkode (Dist.),
Kerala. Pin – 673508.
2. / Name of the Institute / Shree Devi College of Pharmacy,
Airport Road, Kenjar Village,
Malavoor Panchayat,
Mangalore-574 142
Karnataka.
3. / Course of Study and Subject / Master of Pharmacy in Pharmacology
4. / Date of Admission to Course / November 2012
5. / Title of the Topic:
“EVALUATION OF CENTRAL NERVOUS SYSTEM ACTIVITY OF
COGNIUM IN EXPERIMENTAL ANIMAL MODELS”
6. / BRIEF RESUME OF THE INTENDED WORK:
6.1 Need of study:
Central Nervous System control all the human body functions. The neurodegeneration of this system leads to disorders like Alzheimer’s disease, Parkinson’s disease, depressive disorders, epilepsy, insomnia1. Memory is special facility of brain, which retains the events developed during the process of learning, and both are mediated by nervous system2. Once memories have been stored in the brain, it becomes the part of brain –process mechanism when it will recall in future3. Learning and memory are closely related, all learning involves memory but all memory not involves to learning4.
Probably learning and memory are most evolutionary advantageous developments for human. These are interesting but ill-understood subjects. By utilizing past experience due to learning and its storage in memory one can get success and avoid failure5.
Alzheimer’s disease is progressive loss of memory and cognitive function in middle age individuals. (Cognition: that means by which one is aware of the process of thinking and perceiving. It involves an awareness of sensation and usually its cause. Mental component consist of cognitive changes.) Symptoms of Alzheimer’s disease are–memory is failed for recent events, lack of spontaneous activity and initiative with loss of intellectual functions, loss of spatial orientations1.
In recent years, attempts have been made to develop drugs for treatment of dementia and attention deficit disorders to improve memory and learning2. These agents are commonly referred as Nootropic agents or cognition enhancers. The indications of cognition enhancers are senile dementia of Alzheimer type and multi
infarct dementia, common symptoms of elderly, dizziness and memory disturbances and mental retardation in children, learning defects, attention deficit disorder6.
Ayurveda, an ancient Indian system of medicine had reported various plants with the caliber of memory enhancing activity. Based on the same concept, several herbal formulations have been widely marketed, claiming to be useful in memory disorders. As there are no fixed guidelines in the indigenous system of medicine, to evaluate the retention of the potency by the formulation; we planned to evaluate ayurvedic formulation Cognium tablet for its CNS activity. Cognium tablet is a product of Charak pharma consisting of Mukta Pishtee: Abhrak Bhasma, Soya (Glycine soja) and Extracts of the following:
Jala Brahmi (Bacopa monnieri), Brahmi (Centella asiatica), Ashwagandha (Withania somnifera), Arjun (Terminalia arjuna), Jyotishmati (Celastrus paniculatus), Jaiphal (Myristica fragrans), Tagar (Valeriana wallichi), Vacha (Acorus calamus).
6.2 Review of literature:
COGNIUM - Corrects Cognitive Insufficiency. Benefits of using Cognium formulation are- Preserve and enhance cognitive functions, Slows down the process of degeneration of neurons through antioxidants, Increase circulation to the CNS and Provide protection to nervous system by acting as adaptogenic against stress.
COMPOSITION7
Each coated tablet contains,
Mukta Pishtee: 5 mg, Abhrak Bhasma: 25 mg, Soya (Glycine soja): 50 mg and
Extracts of the following:
Jala Brahmi (Bacopa monnieri): 200 mg, Brahmi (Centella asiatica): 100 mg
Ashwagandha (Withania somnifera): 100 mg, Arjun (Terminalia arjuna): 50 mg
Jyotishmati (Celastrus paniculatus): 50 mg, Jaiphal (Myristica fragrans): 25 mg.
Tagar (Valeriana wallichi): 20 mg, Vacha (Acorus calamus): 20 mg.
The literature review of these individual components of the formulation have reveled the following:
Mukta Pishtee8
Mukta Pishteeis an Ayurvedic medicine, prepared from Pearl. It is used in Ayurvedic treatment of diarrhea with bleeding, Mania, Psychosis and depression. It acts as cardiac tonic, antihypertensive and other , excessive burning sensation, gastritis, fever.
Abhrak Bhasma9
Abhrak Bhasma is an Ayurvedic medicine, prepared from Mica. It is used in Ayurvedic treatment of asthma, urinary disorders, skin diseases etc. t is used in the treatment of digestive impairment,malabsorption syndrome,Disease due to Kapha Dosha, Asthma, bronchitis, Fever, Bleeding disorders, Cough, Cold, Urinary disorders, diabetes, Anaemia, Skin diseases, splenic disorders, ascites, Helminthiasis, etc. It also useful in rejuvenation treatment. Pharmacological investigation proves Hepatoprotective10 action of abhrak bhasma.
Soya (Glycine soja)11
Glycine soja, or wild soybeanbelongs to the family Fabaceae or Leguminosae. The saponin content glycine soja shows hypocholesterolaemic, haemolytic, immunostimulatory, and antitumourigenic and chemoprotective activities,Effects on Cognitive Function12, 13.
Jala Brahmi (Bacopa monnieri)
Bacopa monnieri belongs to the family Scrophularaceae reported to contain Brahmine, Herpestine, Saponins and betulic acidwas used as nervine tonic, antiepilepitic, diuretic to reduce stress induced anxiety, nootropic, antipyretic, anti-inflammatory, analgesic, sedativeand adaptogenic actions. It possessing anti-depressant effect14 and neuroprotective effects in epilepsy15.Further neuroprotective effects are said to be due to anti-oxidant properties which suppresses neuronal oxidative stress as observed in Alzheimer’s disease16.It is also reported to be potential herb for safely enhancing cognitive performance in the aged.17
Brahmi (Centella asiatica)
Centella asiatica, Commonly known as mandukparni is a perennial herbaceous creeper belonging to the family Umbellifere (Apiceae). The primary active constituents of Centella asiatica are saponins (also called triterpenoids), which include asiaticosides, in which a trisaccharide moiety is linked to the aglycone asiatic acid, madecassoside and madasiatic acid. Apart from wound healing, the herb is recommended for the treatment of various skin conditions such as leprosy, lupus, psoriasis, diarrhoea, fever, amenorrhea, diseases of the female genitourinary tract and also for relieving anxiety and improving cognition. Pharmacological investigation reveals and anxiolytic properties18, Antidepressant properties18, Antiepileptic properties19, Cognitive and antioxidant properties20.
Ashwagandha (Withania somnifera)
Withania somnifera belongs to family Solanaceae reported to contain somniferin, resin, fat, phytosterol, ipuranol, withaolide and withaferin A is used as nervine tonic, astringent, adaptogenic, febrifuge, sedative, hypnotic, anthelmintic and diuretic. When this herb has been subjected for its neuropharmacologcal investigation, AchE inhibitory effect of methanolic and successive water extracts, indicating its potential to influence cognition21.In animal model of Alzheimer’s disease its oral administration ameliorated neuronal dysfunction22.It also has adoptogenic activity in chronic stress rat models23.Glycowithanolides, one of its constituent have anxiolytic and anti-depressant activity which can be compared with imipramine24.
Terminalia arjuna (Arjuna)
In ayurvedic medicine, bark of Terminalia arjuna is therapeutically used as cardio-tonic. But, apart from that it is also used to treat hypertension, tumors, diabetes and anemia. Pharmacological investigations reported in animal models include anti oxidant activity25and cardio protective activity26.
Jyotishmati (Celastrus paniculatus)
Celastrus paniculatis, another indigenous folklore medicinal herb of this formulation is reported to have spectrum of CNS activity mainly on cognition in several animal models of learning and memory. Further chronic treatment (oral) of
oil of Celastrus paniculatis reversed the impairment of spatial memory without affecting of cholinergic neurotransmission27. And Kumar et al have correlated effect on cognition by virtue of anti-oxidant properties of aqueous extracts28.Neuroprotective of water soluble extract have been reported in neuronal cultures of brain by virtue of their anti-oxidant properties29.
Jaiphala (Myristica fragrans)
Myristica fragrans well known Indian household spice have been reported to possess several neuropharmacological properties. Anti-depressant activity of n-hexane extracts involving adrenergic, dopaminergic and seretonergic system30. N-hexane fraction of acetone insoluble part of petroleum ether extract and its acetone soluble portion is reported to possess anticonvulsant, significant behavioral effects, anxiogenic, sedativeand cholinesterase inhibiting properties31indicating wide spectrum of CNS effects.
Tagar (Valeriana wallichi)
Valeriana wallachi (Tagara), Indian Valerian34 has long been used in Ayurveda (Charak Samhita and Susruta) and Unani systems of medicine, which describe its use in obesity, skin disease, insanity, epilepsy and snake poisoning. It is folklore medicinal herb having main therapeutic use as tranquilizer. Attempts have been made to study its pharmacological activity on central nervous system. It is found to useful in stress management32, reported to increase in short term memory33 and also have role in saving injured neuron of the hippocampus in depressed rats.
Vacha (Acorus calamus)
Acorus calamus is one of folklore herbs of Asian continent. Investigation on roots and rhizome of this plant points to its influence on CNS. Some of the reported activities are anti-depressant activity of Ethanolic extract of rhizome, alcoholic extract of this plant possessing wide activity on CNS like sedative, spontaneous motor activity, tranquilizing effect and amphetamine induced hyperactivity35, methanolic extract of rhizome improving memory and cognition36,neuroprotective effect, methanolic and ethanolic extract in noise induced stress and AchE inhibitory activity of hydro alcoholic extra.
6.3 Objective of study:
The objective of the proposed study is to evaluate the possible CNS effects of ayurvedicformulation COGNIUMusing different experimental animal models.

To explore the influence on various CNS disorders like epilepsy, depression, anxiety, neuralgia, motor incoordination, motility using experimental models :-
Anticonvulsant activity

Maximum Electroshock induced Seizures (MES).
Isoniazid Induced convulsion.

Antidepressant activity

Forced Swim Test (FST).
Tail Suspension Test (TST).

Antianxiety activity

Elevated Plus-maze.

Analgesic activity

Hot Plate Method.
Acetic acid induced writhing assay.

Motor incoordination activity

Rota rod Apparatus.

Motility test

Actophotometer.

Behavioral studies
Effect on sleep activity

KetamineHCL induced sleeping.
7. / MATERIALS AND METHODS:
7.1 Source of Data:
Data will be obtained from laboratory based studies by using Swiss albino mice (25-30g) and albino wistar rats(160-220g) of either sex, maintained at room temperature having free access to food (std pellet diet), water ad libitum. These
studies will be carried out in intact animal and isolated brain that will be supported by biochemical data and behavioral studies.
7.2 Method of Collection of Data:
Materials:Ayurvedic formulation COGNIUM (CHARAK PHARMA), Chemicals and reagents will be procured from standard companies.
7.3 Acute Toxicity Test:
The acute oral toxicity study was performed according to the OPPTS guidelines (Office of Prevention, Pesticide and Toxic Substance) following the limit test procedure37.
7.4 EXPERIMENTAL MODELS:
Anticonvulsant activity:
Maximum Electroshock-induced Seizures (MES)38:
Rats will be divided into 4 groups (n=6).

Group I - Control (vehicle only).
Group II – Standard -Phenytoin (25mg/kg i.p)39.
Group III - COGNIUM low dose.
Group IV – COGNIUM high dose.

The animals will receive a current of 45 mA for 0.2 sec duration through electroconvulsiometer using corneal electrodes, after 60 min of oral administration of COGNIUM, vehicle or phenytoin. The incidence and duration of extensor tonus will be noted. A complete abolition of hind limb tonic extension will be considered as 100% protection.
INH Induced Seizures38:
Albino wistar rats (150-200 g) were divided into four groups with six animals in each group. All the treatments were administered intraperitonially 30 min prior to the administration of INH (300 mg/kg). Animals that did not convulse within 30 min were considered as protected. The number of rat protected in each group was expressed in terms of percentage. In the INH treated group, the animals were monitored for 60 min,
and the percent protection was determined. In unprotected animals, the latency to first convulsion and the durations of convulsions were recorded
Antidepressant activity :
Forced swim test40:
Mice will be divided into 4 groups (n=6).

Group I - Control (vehicle only)
Group II - Standard–Imipramine (20 mg/kg i.p)39.
Group III –COGNIUM low dose.
Group IV- COGNIUM high dose.

FST will be performed in glass jar. This test consists of two parts, an initial training period of 15 min followed by actual test for 5 min duration 24 h later. Mice will be individually forced to swim inside a vertical borosilicate glass cylinder (height: 40 cm; diameter: 15 cm; containing 15 cm height of water maintained at 25o C). The mice from each group II, III, IV will be placed in the cylinder 24 h later after two doses of COGNIUM and imipramine (20mg/kg) respectively and their activity will be recorded. The recordings will be analyzed to find the duration of immobility, swimming behavior and climbing behavior in the 5 min test period using stopwatch. An animal will be judged to be immobile whenever it remains floating passively in the water in a slightly hunched but upright position, its nose just above the surface, with no additional activity other than that necessary to keep its head above water. Swimming is defined as active movement throughout the swim chamber, which includes crossing into another quadrant. Climbing activity (also termed thrashing) consist of upward directed movements of the forepaws along the side of the swim chamber. The data obtained will be compared between standard and test.
Tail Suspension Test (TST)40:
The grouping of animals will be similar to that of FST. The total duration of immobility induced by tail suspension will be measured according to the described
method as a facile means of evaluating potential antidepressants. Mice will be suspended on the edge of a table 50 cm above the floor by the adhesive tape placed approximately 1 cm from the tip of the tail. Immobility time will be recorded during a 6 min period. Animal will be considered to be immobile when it did not show any movement of body and hanged passively.
Antianxiety activity:
Elevated Plus-maze41:
Mice will be divided into 4 groups (n=6).

Group I - Control (vehicle only)
Group II – Standard - Diazepam (4mg/kg i.p)39.
Group III –COGNIUM low dose.
Group IV – COGNIUM high dose.

The wooden maze consists of two open arms (length 50cm X breadth 10 cm) and two closed arms of the same size (height 40 cm). The arms of the same type are opposite
to each other, with a central square of 10 cm. The maze is elevated to a height of 50 cm above the floor.
Behavioral assessment:
Each animal will be tested initially in plus maze. On the 10th day of drug or vehicle administration, 60 minutes after the last dose, each animal will be placed in the centre square of the plus maze, facing one of the open arms. The number of entries into and the time spent in open and closed arms and the number of rears in each arm in a five-minute period will be noted.
Analgesic Activity:
Hot Plate Method42:
Mice were divided into 4 groups (n=6).

Group I - Control (vehicle only)
Group II -Standard - Pentazocine (10mg/kg i.p)39.
Group III - COGNIUM low dose.
Group IV - COGNIUM high dose.

The parameter evaluated will be the latency time for paw licking and jumping response after exposure on surface of hot plate. The standard used will be Pentazocine (10mg/kg, i.p.) The hot plate temperature will be kept at (550C) and the cut off time will be 20sec. The data obtained will be compared and studied.
Acetic-acid induced writhing assay42:
Mice will be divided into 4 groups (n=6).

Group I - Control (vehicle only)
Group II – Standard - Diclofenac sodium (10mg/kg i.p)39.
Group III - COGNIUM low dose.
Group IV - COGNIUM high dose.

Analgesic activity of the COGNIUM will be studied by reduction of acetic acid induced writhing in mice. Thirty minutes after the administration of COGNIUM orstandard diclofenac sodium (10 mg/kg, i.p.), the animals will receive acetic acid (0.6%,
10 ml/kg ip). The number of abdominal contractions (writhing) and stretching with a jerk of the hind limb will be counted for 15 min after administering acetic acid, and percent inhibition will be calculated as follows:
% inhibition = (1 - WT/ WC) × 100
Where, WT is the writhings in drug-treated mice
and WC is the writhings in control mice.
Motor incoordination activity:
Rota rod Apparatus43:
Mice will be divided into 4 groups (n=6).

Group I - Control (vehicle only)
Group II – Standard - Diazepam (4mg/kg i.p)39.
Group III – COGNIUM low dose.
Group IV - COGNIUM high dose.

The effect on motor coordination will be assessed using Rota rod apparatus. The animals will be trained to remain for 3 min on the Rota rod rotating at a speed of 25 rpm. On the next day either vehicle or COGNIUM will be administered orally and their ability to remain on the rotating rod will be assessed before and after the oral administration. The fall off time from the rod will be noted for each animal. The data will be compared and studied.
Motility test43:
The grouping of animals will be similar to that of motor incoordination test. Thirty min after drug administration, the spontaneous locomotor activity will be recorded using an activity cage, Actophotometer with automatic counting of animal movements on the cage floor. The loco motor count for each animal will be recorded for 5 minutes at 30 minute interval for 2 h. The data obtained will be compared and studied.
Behavioural effect43:
Mice will be divided into 3 groups (n=6).

Group I - control (vehicle only).
Group II - COGNIUM low dose.
Group III - COGNIUM high dose.

Behavioral effect of COGNIUM will be assessed by the method. After treatment with COGNIUM, the animals will be observed after 30 min of administration up to 2 hr for the behavioral changes. Prior to COGNIUM, the behavioral pattern of the vehicle treated mice will be studied. The observation parameters consist of body position, locomotion, rearing, reparation,lighting reflex and lacrimation. The data obtained will be compared and studied.
Effect on sleep activity:
Ketamin HCL-induced sleeping time43:
Mice will be divided into 3 groups (n=6).

Group I - Control (vehicle)
Group II – Standard –Ketamine HCL (100mg/kg i.p)39.
Group III- COGNIUM low dose.
Group IV – COGNIUM high dose.

Ketamine HCL (100mg/kg i.p39) will be injected 30 min before oral administration
for all groups. The time elapsed between loss and recovery of the lighting reflex will be noted and taken as sleeping time. The data obtained will be compared and studied.