5. NEUROPHARMACOLOGICAL PROFILE (IRWIN S METHOD)

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1 NEUROPHARMACOLOGICAL PROFILE (IRWIN S METHOD) 5.1 INTRODUCTION Neuropharmacological Profile study suggested by Irwin [228] provides clues for the classification of the active compounds, to proceed for further testing. [227] In this procedure mice after administering the drugs are observed for an initial undisturbed phase followed by phases in which they are subjected to mild to increasing stimuli. Broadly, the neuropharmacological profile is divided into behavioral, neurological and autonomic changes after administration of increasing doses of drugs. Behavioral Profile includes Awareness (alertness, visual placing, passivity and stereotypy), Mood (grooming, vocalization, restlessness, irritability/aggressiveness and fearfulness) and Motor activity (reactivity, spontaneous activity, touch response and pain response). Neurological Profile includes CNS excitation (startle response, straub tail, tremors, twitches and convulsions), Posture (Body posture, limb posture), Motor incoordination (staggering gait, abnormal gait and righting reflex), Muscle tone (limb tone, grip strength, body sag, body tone, abnormal tone) and Reflexes (pinna, corneal and ipsilateral flexor). Autonomic Profile (writhing, pupil size, palpebral opening, exophthalmoses, urination, salivation, piloerection, hypothermia, skin color and heart rate). Other activities observed include Respiratory rate, Lacrimation and Death.

2 106 For the assessment and quantification of the above parameters, methods described by Turner were adopted. [227] The effects of the test substances on the animal were scored on a scale of 0 to 8. The basic score for normal signs or effects was given 4; scores below 4 were for subnormal responses and those above for supernormal. The base score for abnormal signs was given 0 and the maximal score Behavioral Profile 1. Awareness i) Alertness or Stupor (4): It indicates either stimulation or depression of central nervous system. ii) Visual Placing (4): Measures the motor co-ordination by placing animal in different positions and noting its ability to orient itself without bump or fall. Subnormal activity indicates motor in-coordination. iii) Passivity (0): Measures the animal response to unaccustomed position. It indicates tranquillization (antipsychotic), central depression, muscle relaxant property, paralysis or anesthesia. The mouse was grasped with the thumb and index finger, which hold the dorsal skin of the neck, while the mouse was in a walking position. The score was given as follows: - If the mouse moved its head and limbs in trying to escape 0. If the mouse still grasped in the same manner, held in vertical position and it struggles 2.

3 107 If the mouse is placed in supine position on the back of the observer s hand held in a fist so that the thumb can support the mouse s head, it tries to escape 4. The mouse tries to escape when held vertically by one forepaw 6 or by one hind paw 8. Intermediate scores were given when the struggle was diminished but not abolished. Figure 5.1 Passivity Test iv) Stereotypy (0): is a frequent mechanical repetition of movements in mice and was identified by searching movements of head, self-biting, circling, tail lashing etc; it usually indicates central stimulation or depression.

4 Mood i) Grooming (4): Increased grooming indicates central stimulation or sympathetic stimulation. ii) Vocalization (0): Vocalization is utterance of sound and indicates to a noxious stimulus. Generally mouse utters no sound. iii) Restlessness (0): Indicates central stimulation and is seen as discomfort, visceral changes or the approach of convulsions. iv) Irritability (0): is an extension of restlessness and in the higher degrees is demonstrated by aggressiveness. iv) Fearfulness (0): is a normal emotional response in contrast to anxiety and phobia to a recognized external source of danger, which is manifested by alarm apprehension or disquiet. 3. Motor Activity i) Spontaneous Activity (4): Spontaneous activity and reactivity measure the stimulation of CNS or its sedation; the stimulation of ganglia and neuromuscular junctions. When the animal is placed in a bell jar, it searches around. Normal mouse usually shows moderate degree of inquisitive behavior. The scores were recorded as - less activity as 4,3 or 2; little motion as 1; animal sleeps as 0. ii) Reactivity (4): Animal was placed on the top of table after removing from the bell jar and similar scoring as above was followed.

5 109 iii) Touch Response (4): indicates the presence of anesthetic activity and involves touching the animal with pencil or forceps at various parts like side of neck, abdomen and groin. iv) Pain Response (4): Measures analgesia, sedation and central depression. This method involved, attaching an arterial cramp to the base of tail approximately 2cm from the body Neurological Profile 1. Central Excitation: Higher scores indicate CNS depressant action. i) Startle Response (0): To startle with surprise or sudden alarm in response to loud sound. ii) Straub Tail (0): The degree of elevation of tail was recorded. The scores were recorded as follows: 45-1; ; 90-2; iii) Tremors (0), iv) Twitches (0), v) Convulsions (0): indicate CNS stimulation or hypoglycemia. 2. Posture i) Body Posture (4), ii) Limb Position (4) deviation from normal indicates neuromuscular blockade or central disturbances. 3. Motor Incoordination i) Staggering Gait (0): indicates ataxia. ii) Abnormal Gait (O): indicates muscular relaxation or relates to ataxia. iii) Righting Reflex/Somersault Test (0): Animal is tossed five times, so that animal takes 2-3 turns in air before falling down in each trial. The scores

6 110 are given as: Standing on four feet in all five trials 0; Lying on side 1/5 or 2/5 2, 3/5 or 4/5-3; 5/5 3. Lying on the back 1/5 or 2/5 4; 3/5 or 4/5-5; 5/5 6; slowly regaining normal position from a supine or side position -7; remains in supine position 8. Figure 5.2 Righting Reflex High scores indicate the presence of a depressant of CNS; an ataractic (antipsychotic) agent; a muscle relaxant; an anesthetic; or an agent causing synaptic blockade in some part of the nervous system. 4. Muscle tone The tests in this group indicate muscle relaxant activity, neuronal blockade or central depression. i) Limb Tone (4): Grasping a forepaw of the mouse and noting the resistance to the extension of the paw can estimate it. ii) Grip Strength (4): Making the animal to grasp a pencil and hold in horizontal position and noting how well it holds onto the object tests grip strength.

7 111 iii) Body Sag (4), iv) Body Tone (4) and v) Abdominal Tone: Estimated by noting the muscle tension in comparison to the control. 5. Reflexes Decreases in the scores indicate blockade of sensory nerve, the spinal synapses or efferent pathway. i) Pinna reflexes (4): Observed by touching at the center of the pinna with fine hair. The unaffected mouse draws from irritating hair. ii) Corneal reflexes (4): By touching stiff hair to the cornea of the eye. iii) Ipsilateral flexor (4): Toe pad is compressed with forceps, which causes animal to flex its legs in a retiring movement AUTONOMIC PROFILE 1. Optical Signs i) Pupil Size (4): The size of the pupil is measured and it is compared with and without drug. Enlarged size of pupil indicates parasympatholytic activity or sympathomimetic activity. Low score indicates muscarinic activity. ii) Palpebral Opening (4): It can be either widened or may become narrow. If widened, it indicates sympathomimetic activity and if narrowed, it indicates ataractic (antipsychotic) or sedative activity. iii) Exophthalmoses (0): indicates sympathomimetic activity 2. Secretory signs i) Urination (0): Indicates muscarinic activity or irritation of urinary tract. ii) Salivation (0): Indicates muscarinic activity.

8 General signs i) Writhing (0): Indicates irritation of the tissue or stimulation of sensory receptors. ii) Piloerection (0): Indicates a compensation for a lower body temperature or sympathomimetic activity. iii) Hypothermia (0): Indicates possible ataractic (antipsychotic) or akinetic activity. iv) Skin Color (4): Skin color especially that of the back of the ear may change from pink to red or white. Red color indicates vasodilatation and possible sympatholytic activity. White color may indicate vasoconstriction or sympathomimetic activity. v) Respiratory Rate (4): May be elevated by toxic substances or respiratory analeptics. Respiratory depressants and agents causing central depression, especially in higher doses, decelerate it. v) Heart Rate (4): It may indicate parasympatholytic activity if high and cardiotonic activity if low but there are many other possibilities. 4. Death: indicates toxicity. 5.2 MATERIALS AND METHODS Animals: Swiss albino mice were procured from M/s Sainath Animals (registered with CPCSEA) one week before the experiment for acclimatization. Mice of either sex weighing 20-25g were housed in groups of six under standard lab conditions (temperature 25 o ±1 o C,

9 113 relative humidity 55±5%, 12h:12h dark:light cycle) with standard pellet food and water ad libitum. The overnight fasted animals (water ad libitum) were transferred to the laboratory at least 1h before the start of the experiment. The experiments were performed in sets of three at a time, during the day (09:00-16:00h) and as per the guidelines of the Committee for the Purpose of Supervision and Control of Experiments on Animals (CPCSEA), Government of India. The Institutional Animal Ethics Committee approved the study protocol for all the drugs (IAEC/SUCP/01/2007, IAEC/SUCP/01/2009, IAEC/SUCP/09/2009). Drugs: All the drugs, Hab-e-Jund (HJ), Cynodon dactylon (CD), Kushmanda Lehyam (KL), Itrifal Kishneezi (IK) and Barleria cristata (BC) were administered orally in 1% gum acacia solution except Panchagavya Ghrutham (PG), which was administered in sesame oil. Food was given after 4h but water was given ad libitum. Dose: From acute toxicity studies the drugs were found to be relatively safe and hence a dose progression of 30,100,300,500 mg/kg body weight was followed. [227] The following doses were administered orally: PG - 100, 300, 500 mg/kg CD 30, 100, 300 mg/kg IK - 100, 300, 500 mg/kg HJ - 100, 300, 500 mg/kg KL - 100, 300, 500 mg/kg BC - 30, 100, 300 mg/kg The mice were observed for 4h after oral administration for various behavioral, neurological and autonomic changes as mentioned above.

10 114 Effects of the test substance were scored on a scale of 0-8. Base score was 4 for the normal signs or effects. Score below 4 indicate subnormal response and those above are supernormal. The base score for abnormal sign was 0 and maximal score was RESULTS PG, HJ, KL and IK showed negligible actions at 30 mg/kg body weight. Neurological profile of these drugs at doses of 300 and 500 mg/kg body weight are shown in the Tables For CD and BC the activities at 100 and 300mg/kg body weight are tabulated. At higher doses the drugs showed certain behavioral, neuronal and other changes suggesting the course to be taken for further studies. The changes in Neuropharmacological profile of individual drugs are mentioned below Panchagavya Ghrutham Behavioral Profile: PG decreased alertness, spontaneous activity, reactivity touch response and pain response indicating that the drug is a CNS depressant and analgesic. Neurological Profile: PG showed no CNS excitation or muscle relaxation, but decline in reflexes were observed indicating blockade of sensory nerves, synaptic blockade or efferent pathway. Autonomic Profile: No gross changes were observed. Other: Excessive defecation occurred. Death: No death occurred.

11 Hab-e-Jund Behavioral Profile: HJ decreased alertness, spontaneous activity, reactivity, touch response and pain response (comparatively less than PG) indicating that the drug is a CNS depressant and comparatively a mild analgesic. Neurological Profile: HJ showed no CNS excitation but grip strength reduced indicting muscle relaxation. Decline in reflexes was observed indicating blockade of sensory nerves, synaptic blockade or efferent pathway. Autonomic Profile: No gross changes were observed. Death: No death occurred Cynodon dactylon Behavioral Profile: CD decreased alertness, spontaneous activity, reactivity touch response and pain response indicating that the drug is a CNS depressant and analgesic. The pain response was completely abolished at a dose of 100 mg/kg body weight. Neurological Profile: CD showed tremors, twitches and convulsions indicating hypoglycemic activity. It also produced muscle relaxation at 300 mg/kg body weight. Decline in reflexes was observed indicating blockade of sensory nerves, synaptic blockade or efferent pathway. Autonomic Profile: No gross changes were observed. Death: One delayed death occurred at a dose of 300 mg/kg body weight.

12 Kushmanda Lehyam Behavioral Profile: KL at low doses increased alertness, stereotypy, restlessness, irritability/aggressiveness, indicating that the drug is a CNS stimulant but reversed the same at higher doses (500 mg/kg body weight). Neurological Profile: KL again showed mild tremors indicating CNS excitation at low doses and completely reversed the same at higher doses. Autonomic Profile: No gross changes were observed. Death: No death occurred Itrifal Kishneezi Behavioral Profile: IK increased alertness, stereotypy, restlessness, irritability/aggressiveness, indicating that the drug is a CNS stimulant. Neurological Profile: IK showed mild tremors, twitches and convulsions indicating CNS excitation or hypoglycemic activity. Pain response was completely abolished at the dose of 300mg/kg body weight. Autonomic Profile: No gross changes were observed. Other: Excessive defecation occurred. Death: No death occurred.

13 Barleria cristata Behavioral Profile: BC increased alertness, stereotypy, restlessness, irritability/aggressiveness, indicating that the drug is a CNS stimulant. Neurological Profile: BC showed mild tremors indicating CNS excitation. Pain response was completely abolished at the dose of 300 mg/kg body weight. Autonomic Profile: No gross changes were observed. Death: No death occurred.

14 118 Table 5.1 Neuropharmacological Profile of Panchagavya Ghrutham Behavioral Awareness Mood Motor activity Neurological CNS excitation Posture Motor Incoordination 300 mg/kg 500 mg/kg Time (h) BS Alertness Visual Placing Passivity Stereotypy Grooming Vocalization Restlessness Irritability/(Aggression) Fearfulness Spontaneous Activity Reactivity Touch Response Pain Response Startle Response Straub Tail Tremors Twitches Convulsions Body Posture Limb Position Staggering Gait Abnormal Gait Righting Reflex Muscle tone Limb Tone Reflexes Autonomic Misc. Dead BS-Basal Score Grip Strength Body Sag Body tone Abnormal tone Pinna Corneal I.F.R Writhing Pupil Size Palpebral Opening Exophthalmoses Urination Salivation Piloerection Hypothermia Skin color Heart rate Respiratory rate Lacrimation Number acute Number delayed I.F.R-Ipsilateral Flexor Reflex

15 119 Table 5.2 Neuropharmacological Profile of Hab-e-Jund 300 mg/kg 500 mg/kg Behavioral Awareness Mood Motor activity Neurological CNS excitation Posture Motor Incoordination Time (h) BS Alertness Visual Placing Passivity Stereotypy Grooming Vocalization Restlessness Irritability/(Aggression) Fearfulness Spontaneous Activity Reactivity Touch Response Pain Response Startle Response Straub Tail Tremors Twitches Convulsions Body Posture Limb Position Staggering Gait Abnormal Gait Righting Reflex Muscle tone Limb Tone Reflexes Autonomic Misc. Dead BS-Basal Score Grip Strength Body Sag Body tone Abnormal tone Pinna Corneal I.F.R Writhing Pupil Size Palpebral Opening Exophthalmoses Urination Salivation Piloerection Hypothermia Skin color Heart rate Respiratory rate Lacrimation Number acute Number delayed I.F.R-Ipsilateral Flexor Reflex

16 Table 5.3 Neuropharmacological Profile of Cynodon dactylon 100 mg/kg 300 mg/kg Behavioral Awareness Mood Motor activity Neurological CNS excitation Posture Motor Incoordination 120 Time (h) BS Alertness Visual Placing Passivity Stereotypy Grooming Vocalization Restlessness Irritability/(Aggression) Fearfulness Spontaneous Activity Reactivity Touch Response Pain Response Startle Response Straub Tail Tremors Twitches Convulsions Body Posture Limb Position Staggering Gait Abnormal Gait Righting Reflex Muscle tone Limb Tone Reflexes Autonomic Misc. Dead BS-Basal Score Grip Strength Body Sag Body tone Abnormal tone Pinna Corneal I.F.R Writhing Pupil Size Palpebral Opening Exophthalmoses Urination Salivation Piloerection Hypothermia Skin color Heart rate Respiratory rate Lacrimation Number acute Number delayed I.F.R-Ipsilateral Flexor Reflex

17 121 Table 5.4 Neuropharmacological Profile of Kushmanda Lehyam 300 mg/kg 500 mg/kg Behavioral Awareness Mood Motor activity Neurological CNS excitation Posture Motor Incoordination Time (h) BS Alertness Visual Placing Passivity Stereotypy Grooming Vocalization Restlessness Irritability/(Aggression) Fearfulness Spontaneous Activity Reactivity Touch Response Pain Response Startle Response Straub Tail Tremors Twitches Convulsions Body Posture Limb Position Staggering Gait Abnormal Gait Righting Reflex Muscle tone Limb Tone Reflexes Autonomic Misc. Dead BS-Basal Score Grip Strength Body Sag Body tone Abnormal tone Pinna Corneal I.F.R Writhing Pupil Size Palpebral Opening Exophthalmoses Urination Salivation Piloerection Hypothermia Skin color Heart rate Respiratory rate Lacrimation Number acute Number delayed I.F.R-Ipsilateral Flexor Reflex

18 122 Table 5.5 Neuropharmacological Profile of Itrifal Kishneezi 300 mg/kg 500 mg/kg Behavioral Awareness Mood Motor activity Neurological CNS excitation Posture Motor Incoordination Time (h) BS Alertness Visual Placing Passivity Stereotypy Grooming Vocalization Restlessness Irritability/(Aggression) Fearfulness Spontaneous Activity Reactivity Touch Response Pain Response Startle Response Straub Tail Tremors Twitches Convulsions Body Posture Limb Position Staggering Gait Abnormal Gait Righting Reflex Muscle tone Limb Tone Reflexes Autonomic Misc. Dead BS-Basal Score Grip Strength Body Sag Body tone Abnormal tone Pinna Corneal I.F.R Writhing Pupil Size Palpebral Opening Exophthalmoses Urination Salivation Piloerection Hypothermia Skin color Heart rate Respiratory rate Lacrimation Number acute Number delayed I.F.R-Ipsilateral Flexor Reflex

19 123 Table 5.6 Neuropharmacological Profile of Barleria cristata 300 mg/kg 500 mg/kg Behavioral Awareness Mood Motor activity Neurological CNS excitation Posture Motor Incoordination Time (h) BS Alertness Visual Placing Passivity Stereotypy Grooming Vocalization Restlessness Irritability/(Aggression) Fearfulness Spontaneous Activity Reactivity Touch Response Pain Response Startle Response Straub Tail Tremors Twitches Convulsions Body Posture Limb Position Staggering Gait Abnormal Gait Righting Reflex Muscle tone Limb Tone Reflexes Autonomic Misc. Dead BS-Basal Score Grip Strength Body Sag Body tone Abnormal tone Pinna Corneal I.F.R Writhing Pupil Size Palpebral Opening Exophthalmoses Urination Salivation Piloerection Hypothermia Skin color Heart rate Respiratory rate Lacrimation Number acute Number delayed I.F.R-Ipsilateral Flexor Reflex

20 124 Table 5.7 Comprehensive behavioral and neurological profile of the drugs (Peak Response at highest dose) Profile (Basal score) PG HJ CD KL IK BC Awareness (4) Visual Placing (4) Stereotypy (0) Grooming (4) Restlessness (0) Irritability (0) Spontaneous Activity (4) Reactivity (4) Touch Response (4) Pain Response (4) Tremors (0) Convulsions (0) Righting Reflex (0) Grip Strength (4) Pinna Reflex (4) Corneal Reflex (4) Ipsilateral Flexor Reflex (4) Death (0) PG-Panchagavya Ghrutham, HJ-Hab-e-Jund, CD-Cynodon dactylon, KL-Kushmanda Lehyam, IK-Itrifal Kishneezi, BC-Barleria cristata. 5.4 DISCUSSION PG, HJ and CD showed CNS depression, besides PG and CD showed significant analgesic activity. CD showed severe tremors and convulsions at higher doses indicating hypoglycemic activity. With these observations further studies for antiepileptic activity were taken up.

21 125 KL, IK and BC showed CNS stimulation. But KL at higher doses showed CNS depression. KL, IK and BC showed potent analgesic activity. IK increased the volume and frequency of defecation. The drugs were studied for their antidepressant activity. 5.5 CONCLUSION From the results of acute toxicity studies and neuropharmacological profile study the doses of the drugs were decided to proceed for further studies.