Pharmacological study

Transcription

1 PHARMACOLOGICAL STUDY Pharmacology is one of the cornerstones of the drug discovery process. The medicinal chemist may create the candidate compound, but the pharmacologist is the one who tests it for physiologic activity. Psychopharmacology is the study of drug-induced changes in mood, sensation, thinking, and behavior. The field of psychopharmacology encompasses a wide range of substances with various types of psychoactive properties. Psychoactive drugs may originate from natural sources such as plants and animals, or from artificial sources such as chemical synthesis in the laboratory. These drugs interact with particular target sites or receptors found in the nervous system to induce widespread changes in physiological or psychological functions. In psychopharmacology, researchers are interested in any substance that crosses the blood-brain barrier and thus has an effect on behavior, mood or cognition. Drugs are researched for their physicochemical properties, physical side effects, and psychological side effects. Objective: To develop psycho-neuro-pharmacological profile of Ashwagandharishta and Atasi Taila. MATERIALS AND METHODS: Test formulations: 1) Ashwagandharishta 2) Atasi Taila (Flax seed oil, Flax oil, linseed oil) Animals: Wistar strain albino rats (Rattus novergicus) and Swiss albino mice (Mus musculus) were obtained from animal house attached to Pharmacology laboratory. Six animals were housed in each poly-propylene cage with stainless steel top grill. The dry wheat (post hulled) waste was used as bedding material and was changed every morning. The animals were acclimatized for seven days before commencement of the experiment in standard laboratory conditions; hour day and night rhythm, maintained at 25 3 o C and 40 to 60 % humidity. Animals were fed with Amrut brand rat pellet feed supplied by Pranav Agro Mills Pvt. Limited. For their drinking purpose tap water ad libitum was used. Protocol used in this study for the use of animals was 163

2 approved by the Institutional Animal Ethics Committee (Approval number; IAEC 06/09-11/PhD/08) and the care of animals was taken as per the CPCSEA guidelines. Dose fixation: Dose of the drug was calculated by extrapolating the human therapeutic dose to rat on the basis of body surface area ratio (conversion factor for rat) by referring to the table of Paget and Barnes (1964). 1. Flax seed oil: Human dose : 20ml/day Rat dose Mouse dose : 20ml = 0.36ml/200g or 1.8ml/kg. : 20ml = 0.052ml/20g or 2.6ml/kg 2. Ashwagandharishta: Human dose : 50ml/day Rat dose Mouse dose : 50ml = 0.9ml/200g or 3.5ml/kg. : 50ml = 0.13ml/20g or 6.5ml/kg Animal groupings: No. Treatments Dose (ml/kg) Mouse Rat 1 Normal control Water QS QS 2 Vehicle control Milk Test drug 1 Flax oil in milk Test drug 2 Ashwagandharista Test drug 3 Flax oil in milk + Flax oil Route of drug administration: The test drugs were administered through oral route to respective groups through rubber catheter sleeved on to disposable syringe. Stock solution: To administer Flax seed oil with milk, stock solution was prepared by adding 0.52 ml flax seed oil in 2ml of milk and then accordingly dose was calculated. Drug schedule: The test drug and vehicle were administered between 8:30 am to 09:30 am. 164

3 1. Effect on gross behavior 1 : The procedure in this experiment involves the recording of three main groups of parameters mainly CNS stimulation, CNS depression and ANS activity. This was assessed in four groups of mice, namely milk control (Vehicle control), Flax oil + Milk, Ashwagandharista and Flax oil + Ashwagandharista. A separate control group was kept for comparison. The procedure involves assigning scores on 0-3 point scale as per the average intensity of the phenomenon observed. The test drug was administered one hour before the experiment. There after observations were made after 30 minutes, 1h, 2h, 3h, 4h, 5h and 6 h, 24h, 48h and 72 hours. The mice were placed one by one in the centre of three concentric circles with diameter of 7cm, 9cm and 11cm drawn by chalk on a rubber sheet. The profile measured was grouped in to the following heads: CNS depression: Hypo activity, passivity, relaxation, ataxia and narcosis. ANS effects: Ptosis and exophthalamus CNS stimulation: Hyper activity, irritability, stereotypy, tremors, convulsions and straub tail. 2. Hypnotic potentiation test 2 : Selected mice were divided in to five groups. First group was administered with distilled water and served as control. Second to fifth groups, vehicle and test drugs were administered respectively as shown in above table. One hour after the administration of the test drug, pentobarbitone (50mg/kg i.p) was injected. The duration of sleep was measured as the duration between loss and regaining of the righting reflex. Effect on latency of onset of sleep, duration of sleep and mortality if any, was noted down. 3. Behavioral despair test 3 : Selected mice were divided in to five groups. First group was administered with distilled water and served as control. Second to fifth groups, vehicle and test drugs were administered. One hour after drug administration mice were forced to swim individually in a glass jar ( cm 3 ) containing fresh water to a height of 15 cm and maintained at 25 2 C. After an initial 2minutes period vigorous activity each animal assumed a typical immobile posture. A mouse was consider to be immobile when it remained floating in the water without struggling, making only minimum movements of its limbs necessary to keep its head 165

4 above water. The total duration of immobility was recorded during the next 4 minutes of a total 6 minutes test. The changes in immobility periods were studied after administering drugs in separate groups of animals. Each animal was used only once. 4. Anti- reserpine test (Sheth et al, 1972) 4 An hour after the drug administration, 2.5 mg/kg reserpine was injected IP to the mice. They were then observed for ptosis, sedation and catatonia at every 60 minutes for 6 hours after the injection. Scores from 0-3 were given according to the intensity of the symptoms. Catatonia was measured by using catatonia stand apparatus. Sedation was measured by placing the mice in the open field apparatus described by Bhattacharya et. al (The apparatus is a square box of 96x96 cm. and about 15cm. high sidewall. The floor is divided into 36 equal squares) and then they were observed for number of square crossed in 5 mins. Ptosis was measured by grading the closing of eyelids. 5. L-dopa potentiation test 5 : Mice of either sex were allotted to different groups as described above. Depending on the groups they were administered either vehicle or test drugs L- DOPA (100 mg./kg.) IP was injected to each mouse and the behavioral changes were noted down. If test drugs posses DOPA potentiating effect, it becomes apparent in the form of aggressive behavior. The noting were taken for 30 minutes, 60 minutes, 90 minutes, 120 minutes up to 6 hour after the DOPA administration. The observed behavior was assigned the subjective score in scale of 0-4 in the following manner: 1- Piloerection, slight salivation, slight increase in activity and irritability. 2- Piloerection, straub tail as event response and provoked jumping 3- Piloerection, straub tail and tremors 4- Marked tremors, convulsions and death. 6. d-amphetamine stereotypy 6 : Selected mice were divided in to five groups. First group was administered with distilled water and served as control. Second to fifth groups, vehicle and test drugs were administered. One hour after drug administration, 166

5 d-amphetamine injection (5 mg/kg, ip) was given. Stereotype behaviour was assessed at time intervals of 30, 60, 90 and 120 minutes after the injection of d-amphetamine. A score of 0 to 3 was given according to the intensity of stereotypy. The behavioural patterns noted were; rearing, licking, grooming, gnawing, circling, sneezing and sniffing. Any other particular behavior and CNS stimulation features like piloerection were also noted down. 7. Conditional avoidance response test (Cook's pole climbing) 7 : In this method, described by Piala et. al (1959) the stainless steel grid floor in a wooden box was the source of electric shock to rats which can escape the noxious stimulus by climbing a centrally located wooden pole. The stimulus is approximately 0.1 ma of 60-cycle alternating current at 40 volts for 2.5 sec duration. The conditioning stimulus was a buzzer attached to the chamber. The rats were trained for 7 days with two sessions i.e. morning and evening sessions. Each session consisted of three shock treatments that consist of buzzer for 1 sec duration followed by three consecutive shocks for duration of 2.5 seconds each. The rats were trained to climb a pole after the conditional stimulus (buzzer). The trained albino rats of either sex weighing 150 to 200 g were given the vehicle and drugs by oral route according to their grouping. At hourly intervals and thereafter for 6 hours, the response of each animal to the buzzer was determined by three successive trails. The inhibition of the conditioned response is considered as a measure of the tranquillization. 8. Exploratory behavior of mice (Tunnel board test ) 8 This was carried out using a tunnel board i.e. a wooden board measuring cm on to which twelve aluminum tunnels 7.5 cm. long and 4cm. in diameter are fixed in a symmetrical pattern and the drugs were administered to respective mice according to the stated grouping. One hour after the drug administration, the mice were placed on the apparatus on the same corner of the board for 5 minutes and observed. The experiment was conducted in a quite dimly lit room. 167

6 The following parameters were noted down: The number of different tunnels entered in the first minute. Number of different tunnels entered in 5 minutes. Total number of tunnels entered in 5 minutes. 9. Open field behavior study 9 : Mice of either sex were divided in to five groups of six each. First group was administered with distilled water and served as control. Second to fifth groups, vehicle and test drugs were administered. One hour after drug administartion the animlas were individually exposed to open field apparatus. The apparatus is a square box of cm with a side wall of 15 cm height. The floor is divided into 36 equal squares. The instrument was kept in a dimly lit and quite area during the experiment. Each rat was placed at the same corner allowed to explore the arena for 5 minutes. The parameters recorded were; time of exploration, number of squares crossed, number of rearing, freezing time (duration of immobility) and number of faecal pellets expelled. 10. Anti-anxiety activity using elevated plus maze (EPM) 10 : The plus-maze apparatus, consisting of two open arms (16 5 cm) and two closed arms ( cm) having an open roof and elevated to 25 cm from the floor was used to observe anxiolytic activity in mice. Mice were given a single oral dose of the vehicle and test drugs one hour before they were placed on the EPM. Dose administration schedule was adjusted so that each mouse took its turn on the elevated plus-maze apparatus one hour after administration of the dose. To begin a test session, mice were placed on the open arm facing the center of the maze. An entry into an arm was defined as the animal placing all four paws over the line marking that area. The number of entries and the time spent in the open and closed arms were recorded during a 5-min test period. During the entire experiment, mice were allowed to socialize. Every precaution was taken to ensure that no external stimuli, other than the height of the plus-maze could invoke maze anxiety. 168

7 11. Anti-convulsant activity: This activity was evaluated against supra maximal electroshock seizures (MES) in rats 11. The rats were pretested 24 h prior to administration of test drugs for sensitivity to electric shock and those failing to give hind limb tonic extension were rejected. Thus screened animals were divided in to four groups of six animals each. Group 1 served as control, received equivalent amount of the distilled water. To second to fifth groups, vehicle and test drugs were administered. Sixth group received phenytoin sodium as standard anti-epileptic drug. Experiment was conducted at the same time each day and 60 minutes after vehicle/drug administration (Phenytoin was given 30 minutes before the application of electroshock). Seizures were induced in all the groups by using an Electro convulsiometer and elicited by a 60 Hz alternating current of 150 ma intensity for 0.2 sec. A drop of electrolyte solution (0.9% NaCl) with lignocaine was applied to the corneal electrodes prior to application to the rats. The duration of various phases of epilepsy were recorded. 12. Muscle relaxant activity: Test for muscle tone and balance was evaluated using Rota rod 12. The selected mice were placed on a horizontal rotating metal rod having a diameter of 32 mm. and rotating at the rate of 22 RPM. Animals that remain on the rod for 2 or more minutes in four trials carried out in two days during morning and evening sessions were selected and divided into different groups. First group was kept as control and vehicle was administered. To second to fifth groups, vehicle and test drugs were administered. The drugs were administered by oral route according to the grouping and placed on rod at different intervals for 4 hours after the administration. The percentage reduction in motor coordination was recorded. STATISTICAL ANALYSIS: The data were analyzed by employing student s t test unpaired data and One way ANOVA was also employed with Dunnets Multiple t test as post-hoc test. Wilcoxon Signed Rank test was employed for assessing the level of significance with respect to data related to gradation in non-parametric data. 169

8 OBSERVATIONS AND RESULTS 1. Gross behavior: Animals from control group showed normal behavior throughout the observation period. Animals of vehicle (Milk) and Flax oil treated group showed mild hypoactivity after 1h of drug administration and it persisted up to 5h. Animals from Ash and Ash administered groups also showed features of mild CNS depression (hypoactivity) during 1 to 5 post administration hour intervals. 2. Hypnotic potentiation test: Table 1: Effect on pentobarbitone induced sleep in mice Dose Latency onset of % Duration of % (per kg) sleep (min) Change sleep (min) Change Control QS VC QS Milk 2.6ml * Ash 6.5ml Ash 6.5ml Data: Mean SEM * *P<0.05 (Unpaired t test) Table - 1 contains the data pertaining to the effect of test formulations on pentobarbitone-induced sleep in mice. Both test formulations alone and in combination did not affect the latency of sleep onset to significant extent in comparison to control group. Both the test formulations apparently prolonged the duration of sleep in comparison to control group, however among them Flax alone and Ash administered groups shows significant prolongation in duration of sleep. Further, when Flax oil was combined with Ash it shows better effect than that of individually given group. 170

9 3. Behavioral despair test: Table 2: Effect on behavior despair in mice Dose (per/kg) Duration of immobility (Sec) % Change Control QS VC QS Milk 2.6ml *** # Ash 6.5ml * # Ash ml ** # Data: Mean SEM *P<0.05, **P<0.01, ***P<0.001(Vs control); # P<0.05 (Vs milk) ONE Way ANOVA with Dunnet s multiple t test. The data related to effect of test formulations on the duration of immobility of mice in behavioral despair test can be found in table 2. Pre-treatment with test formulations significantly reduced the duration of immobility of mice in comparison to both normal control as well as vehicle control groups. The observed effect is much better in Flax alone treated group in comparison to Ash alone and Ash. 4. Anti-reserpine test: Control VC Milk Ash Ash Table 3: Effect on reserpine induced catatonia, sedation and ptosis Sedation (No. of square Catatonia Ptosis crossed) 1 st h. 3 rd h. 5 th h. 1 st h. 3 rd h. 5 th h. 1 st h. 3 rd h. 5 th h $ $ 0.00 $ * ** *** * * $ Data: Mean SEM; $ P<0.05 Wilcoxon Signed Rank test, *p<0.05, **p<0.01,***p<0.001 (Unpaired t test) 171

10 Data related to the effect of test formulations on catatonia, sedation and ptosis can be seen in table - 3. Administration of vehicle and test formulations apparently decreased reserpine induced catatonia at all the time intervals, however only the observed effect in vehicle treated group at 1h and Milk administered group at 1h and 3 rd h is found to be statistically significant in comparison to control group. Significant suppression of reserpine-induced sedation was observed in Milk treated group at all time intervals in comparison to control group. Administration of Ashwagandharishta alone and flax seed oil combined with Ashwagandharishta also significantly suppressed reserpine-induced sedation at 1h, however they failed to suppress it at 3h and 5h. Ptosis score was not affected to significant extent in all the treated groups at all the time intervals except in Ashwagandharishta administered group at 1h in comparison to control group. 5. L-DOPA potentiation test: Table 4: Effect on DOPA potentiating effect Intensity of activity (n=6) 90 min 120 min 180 min 240 min 300 min min min Control VC $ Milk $ Ash Ash $ Data: Mean SEM; $ P<0.05 Wilcoxon Signed Rank test 172

11 Data pertaining to effect of test formulations on DOPA potentiating effect are depicted in table 4. Administration of vehicle (milk), Milk and Ash caused significant potentiation at 60min in comparison to control group, while the observed potentiation at all other time intervals is found to be statistically nonsignificant. 6. d-amphetamine induced stereotypy: Dose (per/kg) Table 5a: Effect on rearing behavior in mice Rearing score 20min 40min 60min 120min 180min 240min Control QS VC QS ml Milk Ash 6.5ml Ash 6.5ml Data: Mean SEM, $ P<0.05 Wilcoxon Signed Rank test $ $ $ $ Data related to the effect of test formulations on rearing behaviour in d-amphetamine induced stereotypy in albino mice have been provided in table 5a. Administration of test formulations prior to injection of d-amphetamine apparently decreased the number of rearing at different time intervals, however among the observed effect in all the groups only the effect observed at 6h is found to be statistically significant. 173

12 Dose (per/kg) Table 5b: Effect on grooming behavior in mice Grooming score 20min 40min 60min 120min 180min 240min Control QS VC QS $ $ ml Milk Ash 6.5ml $ 0.21 $ 0.21 $ Ash ml $ $ $ $ Data: Mean SEM, $ P<0.05 Wilcoxon Signed Rank test Data related to the effect of test formulations on grooming behaviour in d- amphetamine induced stereotypy in albino mice have been provided in table 5b. Administration of Flax oil alone prior to injection of d-amphetamine apparently decreased the number of grooming behavior at all time intervals, however the observed effect is statistically non-significant. Administration of Ash alone and Ash in combination also apparently decreased the number of grooming behavior, however only the effect observed at 2h, 3h and 4h is found to be statistically significant in comparison to control group. Table 5c: Effect on sniffing behavior in mice Sniffing score Dose (per/kg) 20min 40min 60min 120min 180min 240min Control QS VC QS $ ml Milk $ Ash 6.5ml $ 0.17 $ 0.33 $ 0.00 $ Ash ml $ $ $ $ Data: Mean SEM, $ P<0.05 Wilcoxon Signed Rank test 174

13 Data related to the effect of test formulations on sniffing behaviour in d-amphetamine induced stereotypy in albino mice have been provided in table 5c. Administration of Flax oil alone prior to injection of d-amphetamine apparently decreased the number of grooming behavior at all time intervals, however the observed effect is statistically significant only at 6h. Administration of Ash alone and Ash in combination also apparently decreased the number of sniffing behavior at all the time intervals, however the only the effect observed at 1h, 2h, 3h and 4h is found to be statistically significant in comparison to control group. 7. Effect on conditions avoidance response (CAR): Table 6: Effect on conditioned avoidance response Dose (per/kg) Inhibition of conditional avoidance response Actual % Change Control QS 0/6 -- VC QS 0/6 -- Milk 1.8ml 3/6 50 Ash 3.5ml 3/5 60 Ash ml 3/5 60 Data: Mean SEM The data pertaining to effect of test formulations response measured using Cook's climbing apparatus after 1h are given in table 9. In Milk administered group inhibition of CAR was observed in 3/6 (50%) animals, while in both Ash and Ash administered groups inhibition of CAR was observed in 3/5 (60%) animals in comparison to both normal control and vehicle control group in which the CAR in none of the trained animals was found to be affected. 175

14 8. Effect on exploratory behaviour: Table 7: Effect on exploratory behavior No of diff tunnels No. of diff. tunnels Total No. of tunnels entered in 1st min. entered in 5 min. entered in 5 min. % % % Mean SEM Mean SEM Mean SEM Change Change Change Control VC Milk Ash Ash The data pertaining to the effect of test formulations on exploratory behavior can be seen in table An apparent increase in the number of tunnels entered during first min was observed in vehicle and flax oil administered groups in comparison to control group, the increase was however found to be statistically non-significant. Similar increase was also observed with respect to the number of tunnels entered during the 5 min observation period and Total No. of tunnels entered in 5 min. Administration of Ashwagandharishta alone failed to show any impact on these parameters. However flax seed oil combined with Ashwagandharishta administered group shows an apparent increase in the number of tunnels entered during first minute and decrease in number of tunnels entered during the 5 min observation period and total number of tunnels entered in 5 minutes. However these observations were found to be statistically non-significant. 9. Open field behavior: Table 8: Effect on the activity profile of mice in open field test Dose Squares Freezing Pellets Rearing (per/kg) crossed time expelled Control QS VC QS * Milk 1.8ml ** Ash 3.5ml ** Ash ml * Data: Mean SEM *P<0.05 (Unpaired t test) 176

15 The data pertaining to the effect of test formulations on the performance of mice in open field apparatus are given in table 8. Both the test formulations when administered individually as well as in combinations did not affect the number of squares crossed and number of rearing. Both the formulations apparently decreased the freezing time, however the observed effect in vehicle, flax oil and Ash alone treated groups are found to be statistically significant. Both the test formulations when administered individually as well as in combinations apparently decreased the number of faecal pellets passed, however only the effect observed in Ash combination is found to be statistically significant. 10. Elevated plus maze: Table 9: Effect on mice performance in elevated plus maze Dose (per/kg) Latency of first entry to closed arm (Sec) Control QS Time spent in open arm (Sec) Number of entries from closed to open VC QS ml Milk Ash 3.5ml * Ash 3.5ml Data: Mean SEM *P<0.05 (Unpaired t test) Data pertaining to effect of test formulations on elevated plus maze performance are depicted in table 9. Administration of test formulations prior to exposure to elevated plus maze did not affect the latency of first entry, time spent in open arm and number of entries to open arm in comparison to control group, except in Ash alone given group in which significant increases in latency of first entry was observed. 177

16 11. Anti-convulsant activity: Table 10: Effect on MES seizure profile in rats Flexion (Seconds) Extension (Seconds) Control Clonic Convulsion (Seconds) * * VC * ASH * Test *** * Test + ASH *** Phenytoin *** *** Data: Mean SEM, *p<0.05, ***p<0.001 (Unpaired t test) Recovery time (Minutes) * * *** % protection The data related to the effect of test drug on convulsion profile have been summarized in Table -10. The duration of extensor phase in control group was found to be seconds Pre-treatment with flax seed oil produced significant anti-convulsant activity by decreasing the duration of tonic extensor phase (p<0.05). However Flax seed oil followed by Ashwagandharishta failed to protect the test animals against MES induced convulsion. The standard drug phenytoin significantly abolished the extensor phase in the animals with 81.20% protection. Pre-treatment with vehicle, Flax seed oil and Ashwagandharishta significantly shortened flexion phase of MES induced seizure, while Flax seed oil as adjuvant with Ashwagandharishta significantly decreased duration of convulsion. Further, both Flax seed oil and Ashwagandharishta significantly decreased the duration of clonic convulsion phase. The standard drug phenytoin exhibited significant anticonvulsant effect by abolishing the tonic extension phase. Further it significantly shortened all other phases induced by MES. 178

17 12. Muscle relaxant activity: Table 11: Effect on muscle tone and balance Dose % reduction in motor coordination (per/kg) 30 min 1 hr 2hr 3hr 4hr Control QS VC QS Milk 2.6ml Ash 6.5ml Ash 6.5ml Data: Mean SEM Table 11 shows data related to effect of test formulations on muscle tone and balance in mice. Treatment with test formulations did not affect motor coordination at all time intervals in comparison to control group. 179

18 References: 1 Clara Morpurgo. New design for the screening of CNS active drugs in mice. Arzaeim-Forsch. 21(1): 1971; Fujimori H. Potentiation of barbital hypnosis as an evaluation method for central nervous system depressants. Psychopharmacologia. 1965;7: Porsolt RD, Bartin A, Jalfre M. Behavioural despair in mice - A primary screening test for anti depressants. Arch. Int. Pharmacodyn. 1977; 229: Vimala H. Sethy, S. R. Naik, U. K. Sheth, Effect of reserpine and tetrabenazine on tremorine analgesia in mice, Psychopharmacologia, 29. VIII. 1972, Volume 26, Issue 3, pp Barnett A. and Taber R. I. In Screening Methods in Pharmacology by Robert A. Turner and Peter Hebborn, Vol. 2,Pg. 210, Academic Press, New York, Valame SP, Gupta KG. Effect of clonidine on amphetamine induced stereotyp. Ind. J. Pharmac. 13(2): 1981; Maffi. G The secondary conditioned response of rats and the effect of some psycho-pharmacological agents. Journal of Pharmacy and pharmacology, Vol.11, P Shillito, E.E. (1970). A method for investigating the effects of drugs on the exploratory behavior of mice. British Journal of Pharmacology, 40, Bhattacharya SK, Satyan KS. Experimental methods for evaluation of psychotropic agents in rodents: I-Anti-anxiety agents. Indian Journal of Experimental Biology. 1997; 35: Lister RG. The use of a plus-maze to measure anxiety in the mouse. Psychopharmacology. 1987; 92: Jaiswal AK, Bhattacharya SK. Effect of Shilajit on memory, anxiety and brain monoamines in rats. Indian J Pharmacol. 1992; 24: Goodman LS, Grewal MS, Brown WC, Swinyard EA. Comparison of maximal seizures evoked by pentylenetetrazol (Metrol) and electroshock in mice, and their modification by anticonvulsants. J Pharmacol Exp Ther. 1953; 108: