Consolidated Final Report

Transcription

1 PHARMACOLOGICAL EVALUATION OF HERBOMINERAL AYURVEDIC FORMULATIONS USED IN THE PROPHYLAXIS OF MIGRAINE Consolidated Final Report ( to ) AIIMS Project Code: N-1237 Principal Investigator: Dr. Y. K. Gupta Professor and Head Department of Pharmacology A.I.I.M.S., New Delhi Co- Investigator: Dr KH Reeta Additional Professor, Department of Pharmacology A.I.I.M.S., New Delhi Study Sponsor: Ipca Laboratories Ltd 142-AB, Kandivali Industrial Estate Kandivali West Mumbai

2 Research Personnel in the Project Research Fellows: 1) Jogender Mehla : JRF (w.e.f to ) 2) Namrata Jit Kaur: JRF (w.e.f to ) SRF (w.e.f to ) 3) Anju Malik: JRF (w.e.f to ) 4) RenuArora: SRF (w.e.f to ) Attendant: 1) Pankaj Sharma : (w.e.f to )

3 Table of Contents Contents Page No. I: Introduction 1. Origin of the proposal Objectives of the study 3 3. Details of investigational formulations 4 4. Calculation of rat dose II: Methodology and Results of Objective I Evaluation of the analgesic effect of four ayurvedic formulations 9-14 individually using hot plate model in rats 2. Evaluation of the analgesic effect of four ayurvedic formulations individually using tail flick model in rats 3. Evaluation of the analgesic effect of ayurvedic formulations individually using formalin test in rats. 4: Evaluation of analgesic effect of ayurvedic formulations after 5 days treatment using hot plate and tail flick method in rats. 5: Evaluation of the analgesic effect of combination of ayurvedic formulationsusing hot plate model in rats. 6: Evaluation of the analgesic effect of combination of ayurvedic formulations using tail flick model in rats. 7: Evaluation of effect of ayurvedic formulations using Carrageenan induced paw edema model in rats. 8. Evaluation of effect of Ayurvedic formulations administered individually at limit dose of 1000 mg/kg on neurobehavioural parameters in rats 9: Evaluation of effect of combination of ayurvedic formulations on neurobehavioral parameters after 28 days in rats.

4 10: Evaluation of the antiepileptic effect of four Ayurvedic formulations administered individually at 1000 mg/kg once on; a) Pentylenetetrazole (PTZ) - induced seizures model b) Maximal Electrical shock (MES)- induced seizures model : Evaluation of four Ayurvedic formulations in combination after chronic (28 days) treatment on; a) PTZ-induced seizures b) MES-induced seizures : Evaluation of effect of combination of four ayurvedic formulations on seizuresinduced oxidative stress in following experimental model in rats. a) PTZ-induced seizures b) MES-induced seizures : Evaluation of the anti-depressant activity of combination of four ayurvedic formulations in forced swim test 14: Evaluation of the antidepressant activity of ayurvedic formulations in combination using tail suspension test. III: Methodology and Results of Objective II : Evaluation of the effect of ayurvedic formulations, individually and in combination, on blood pressure in rats, after 7 days treatment 16: Evaluation of the effect of ayurvedic formulations, individually and in combination, on blood pressure in rats, after 14 days treatment IV: Methodology and Results of Objective III : Estimation of Arsenic (As), Cadmium (Cd), Mercury (Hg) and Lead (Pb) in ayurvedic formulations V: Methodology and Results of Objective IV : Estimation of Arsenic (As), Cadmium (Cd), Mercury (Hg) and Lead (Pb) in rat s tissues after 28 days treatment with combination of ayurvedic formulations VI: Summary and Conclusion VII: Discussion

5 Introduction

6 Origin of the proposal: Migraine is a disorder which is characterized by pulsating headache, unilateral or bilateral, often associated with nausea, vomiting, photophobia and phonophobia.the pain is generally aggravated by physical activity. Some triggering factors appear to produce neurogenic inflammation of affected blood vessel wall which is amplified by retrograde transmission in afferent nerve and release of mediators like 5-HT, neurokinin, substance P, calcitonin gene related peptide (CGRP), nitric oxide, etc.activation of trigeminal nerve and the vessels supplying it, especially intracranial and dural vessels, is due to release of various transmitters like serotonin, norepinephrine, endorphin and gamma amino butyric acid (GABA). The prevalence of migraine is about 18% in women and 6% in men around the world Migraine may ruin long span of an individual s life. It is more so as the average onset of symptoms starts at the age of 25 years to 55 years. Although migraine may affect an individual at any age, in 50% cases the onset occurs before the age of 20. The available drugs for treatment of migraine includes analgesics i.e. nonsteroidal antiinflammatory drugs (acetaminophen, naproxen, ketorolac), opioids, serotonergics i.e. triptans (sumatriptan, rizatriptan, eletriptan, naratriptan, etc.), ergot derivatives (dihydroergotamine, ergotamine); antiemetics (chlorpromazine, prochlorperazine, metoclopramide). Drugs used for prophylactic management include beta blockers (propranolol, metoprolol), tricyclic antidepressants (amytriptyline), anticonvulsants (valproate, topiramate) and flunarizine, etc. The current therapies for migraine are relatively symptomatic and non-specific and associated with moderate to severe side effects. A large number of patients are suffering from refractory (who fail to respond to prophylactic treatment) migraine. There have been claims by Ayurvedic physician and scientists thatherbal medicines do offer symptomatic relief and cure in neurological disorders such as epilepsy, alzheimers, and migraine etc.many of the herbal extracts have been studied scientifically. The extracts and in some cases active principles have been shown to exhibit pharmacological activities. However in many cases, the therapeutic claims need to be validated in well designed clinical studies. Migraine is one such condition where many patients are not relieved of the pain and other migraine symptoms even with available Final Report Page 1

7 medicines. This area therefore needs to be explored, if alternative system of medicines could provide benefit. In the present project, four Ayurvedic classical formulations namely Narikel Lavan, Numax, Rason Vati and Godanti Mishran, have been evaluated. These formulations have been described for different uses in Rasa Shastra, a clinical speciality of Ayurveda.The above formulations have been used for the treatment of migraine by a reputed Ayurveda physician Vaidya Balendu Prakash. He claims that the formulations improve the quality of life of patients of migraine. The safety of AYFs was ascertained by acute and sub-acute toxicological studies (OECD guidelines) in mice and rats, carried out at Bombay College of Pharmacy. This showed that, the four AYFs are safe and did not show ant toxicity. However, the mechanism of action of the AYFs is unknown. The project was aimed toevaluate possible mechanisms of calimed efficacy using experimental models. Final Report Page 2

8 Objectives of the study

9 Objective 1: To evaluate the following activity of four classical ayurvedic formulations (Godanti Mishran, Narikel Lavan, Numax and Rason Vati) in the experimental models in rats. Analgesic activity Anti-inflammatory activity Effect on general neurobehavioral parameters Anti-epileptic activity Seizure induced oxidative stress Anti- depressant activity Objective 2: To evaluate the effect of above formulations on haemodynamic parameters (During mid-term review, the project was extended to estimate the content of heavy metals in Ayurvedic formulations and their concentration in blood and tissues of the rat). Therefore two additional objectives were added as below; Objective 3: Estimation ofheavy metals (Pb, Cd, As & Hg), in Ayurvedic formulations (Godanti Mishran Narikel Lavan, Numaxand Rason Vati,). Objective 4: Estimation ofheavy metals (Pb, Cd, As & Hg), in rat blood, brain, kidney &liver, after chronic (28 days) oral of Ayurvedic formulations Final Report Page 3

10 Details of investigational products (Four Ayurvedic formulations)

11 1: Source of Ayurvedic formulations: The four ayurvedic formulations were manufactured by B.B.S Pvt Ltd, Dehradun, India and provided by IPCA Laboratories, Mumbai 2: Ayurvedic formulations and their quantity recieved Table 1: S.No. Ayurvedic formulations Quantity received (g) 1. Narikel Lavan Rason Vati Godanti Mishran Numax 100 3: Physical characteristics of the Ayurvedic formulations as provided by sponsor Table 2: S.No. Physical Ayurvedic formulations properties Narikel Rason Vati Godanti Numax Lavan Mishran 1. Form Powder Powder Powder Powder 2. Color Dark grey Light brown Light grey Black 3. Smell Pungent Not pungent Pungent Not Pungent 4. Particle size Fine Fine Very Fine Granular 5. Solubility Water DMSO 50% ethanol Absolute ethanol Not Soluble Not Soluble Not Soluble Not Soluble Not Soluble Not Soluble Not Soluble Not Soluble Not Soluble Not Soluble Partiallysoluble Partiallysoluble Not Soluble Not Soluble Not Soluble Not Soluble 4: Storage conditions: As per the advice of sponsor the formulations were stored at room temperature in air tightcontainers. Final Report Page 4

12 Calculation of doses for rats based on human dose

13 Vaidya Balendu Prakash informed the doses of the four Ayurvedic formululations as used in human in migraine patient. Based on these doses, the equivalent doses for rat were calculated. The rounded doses were used as initial dose. 1. Narikel lavan Human Adult Dose of Narikel Lavan = 2 to 3 g/day (for calculation, 2.5 was taken Considering, Average weight of adult human = 60 kg Rat dose after conversion = 2.5/60 x 7 = mg Dose considered for experiments in rats = 300 mg/kg, p.o. 1. Rason vati Human Adult Dose of Rason Vati = 4 g/day Considering, Average weight of adult human = 60 kg Rat dose after conversion = 3.5/60 x 7 = mg Dose considered for experiments in rats = 500 mg/kg, p.o. 2. Godanti mishran Human Adult Dose of Godanti Mishran = Considering, Average weight of adult human = mg/day 60 kg Rat dose after Conversion = 325/60 x 7 = 37.9 mg Dose considered for experiments in rats = 40 mg/kg, p.o. 3. Numax Human Adult Dose of Numax = 1 g/day Considering, Average weight of adult human = 60 kg Rat dose after Conversion = 1000/60 x 7 = 116.7mg Dose considered for experiments in rats = 150 mg/kg, p.o. In case where these doses did not show any activity in the parameters studied, the maximal permissible dose tested was 1000 mg/kg. Final Report Page 5

14 Administration of Ayurvedic formulations: All ther formulations were suspended in 0.1% Carboxy Methyl Cellulose and administered orally by intragastric tube. The volume administered did not exceed 1ml/100gm. In experiments, where, all the four formulations were administered, they were given serially at an interval of 10 mins. The total volume of four formulations did not exceed 1ml/100gm. Final Report Page 6

15 Methodologies & Results of Objective I

16 Objective I: To evaluate the following activity of four classical ayurvedic formulations (Godanti Mishran, Narikel Lavan, Numax and Rason Vati) in the experimental models in rats. A. Analgesic activity I: Effect of single dose of four Ayurvedic formulations administred individuallay using following methods 1. Hot plate method 2. Tail flick method 3. Formalin test 4. Effect of five days of four Ayurvedic formulations individuallay using a) Hot plate method b) Tail flick method II: Effect of single dose of four Ayurvedic formulations in combination using 5. Hot plate method 6. Tail flick method B. Anti-inflammatory activity I: Effect ofsingle dose of four Ayurvedic formulations individually on 7. Carrageenan induced paw edema model C. General neurobehavioral parameters I: Effect of four Ayurvedic formulations on neurobehavioral parameters 8. Effect of single dose (1000 mg/kg)of four Ayurvedic formulations administered individuallyin rats on; a) Elevated Plus Maze test b) One Trial Passive Avoidance task c) Actophotometer d) Rotarod e) Muscle Grip Strength test 9. Effect ofchronic (28 days) of four Ayurvedic formulations in combinationon; a) Elevated Plus Maze test b) One Trial Passive Avoidance task Final Report Page 7

17 c) Actophotometer d) Rotarod e) Muscle Grip Strength test D. Anti-epileptic activity I: Effect of four Ayurvedic formulations on experimental model of seizures 10. Effect of four ayurvedic formulations administered individually at single dose at 1000mg/kg) on; a) Pentylenetetrazole (PTZ) induced seizures model b)maximal electroshock (MES)-induced seizures model 11. Effect of four ayurvedic formulations administered in combination (at calculated human equivalent rat doses) for 7 days, on a) PTZ- induced seizures model b)mes-induced seizures model E. Seizure induced oxidative stress I: Effect of four Ayurvedic formulations on seizures-induced oxidative stress 12. Effect of four Ayurvedic formulationsin combination (at calculated human equivalent rat doses) for 7 days, on seizure induced oxidative stress a) PTZ -induced seizuremodel b) MES -induced seizure model F. Anti-depressant activity I: Effect of 7 days of four Ayurvedic formulations in combination, on 13. Forced swim test 14. Tail suspension test Final Report Page 8

18 1. Evaluation of the analgesic effect of four ayurvedic formulations individually using hot plate model in rats Animals: The experiment was performed on male Wistar rats ( g). They were allowed free access to food and water. The animals were acclimatized for 7 days before the experiment. All animal procedures were in accordance with Institutional Animal Ethics Committee of All India Institute of Medical Sciences, New Delhi. Each animal was assigned a Cumulative unique identification number i.e. from CUIN 01 to CUIN 36. Rats were divided into six groups consisting of six rats each i.e., Normal control, Standard (Morphine), Narikel lavan, Rason vati,godanti Mishran and Numax treated groups. Each animal was marked for identification (Table 1.1). Table 1.1: Group identification of rats Group Number of animals CUIN Normal Control 6 01 to 06 Standard (Morphine) 6 07 to 12 Narikel Lavan 6 13 to 18 Rason Vati 6 19 to 24 Godanti Mishran 6 25 to 30 Numax 6 31 to 36 Table 1.2: Individual identification of rats within a group Rats number Individual identification Abbreviation 1 Yellow mark on head H 2 Yellow mark on body B 3 Yellow mark on tail T 4 Yellow mark on head and body HB 5 Yellow mark on body and tail BT 6 Unmarked U Final Report Page 9

19 Table 1.3: Drug Administration S. No. Treatments No. of rats Dose and route of 1. Normal control (No treatment) 6-2. Morphine 6 7 mg/kg, i.p 3 Narikel Lavan mg/kg, p.o. 4 Rason Vati mg/kg, p.o. 5 Godanti Mishran 6 40 mg/kg, p.o. 6 Numax 6 150mg/kg, p.o. Table 1.4: Dosing schedule of morphine Concentration of Morphine: 5 mg/ml CUIN Treatment I.D. mark Rats body weight (g) Dose (mg/kg) Intended vol. of i.p Actual vol. of 7 Morphine H Morphine B Morphine T Morphine HB Morphine HT Morphine U Final Report Page 10

20 Table 1.5: Dosing schedule of Narikel Lavan Concentration of Narikel Lavan: 100 mg/ml CUIN Ayurvedic Drug 13 Narikel Lavan 14 Narikel Lavan 15 Narikel Lavan 16 Narikel Lavan 17 Narikel Lavan 18 Narikel Lavan I.D. mark Body weight (g) H 172 Dose (mg/kg) Intended vol. Actual vol. of of B T HB HT U Table 1.6: Dosing schedule of Rason Vati Concentration of Rason Vati: 100 mg/ml CUIN Ayurvedic Drug I.D. mark Rats body weight (g) Dose (mg/kg) Intended vol. of Actual vol. of 19 Rason Vati H Rason Vati B Rason Vati T Rason Vati HB Rason Vati HT Rason Vati U Final Report Page 11

21 Table 1.7: Dosing schedule of Godanti Mishran Concentration of Godanti Mishran: 10 mg/ml CUIN Ayurvedic Drug 25 Godanti Mishran 26 Godanti Mishran 27 Godanti Mishran 28 Godanti Mishran 29 Godanti Mishran 30 Godanti Mishran I.D. mark Rats body weight (g) H 156 Dose (mg/kg) Intended vol. of Actual vol. of B T HB HT U Table 1.8: Dosing schedule of Numax Concentration of Numax: 50 mg/ml CUIN Ayurvedic Drug I.D. mark Rats body weight (g) Dose (mg/kg) Intended vol. of Actual vol. of 31 Numax H Numax B Numax T Numax HB Numax HT Numax U Final Report Page 12

22 Hot Plate Method It is a model for testing centrally acting analgesics. The instrument consists of a transparent perplex cylinder mounted on a plate whose temperature can be adjusted. In this method after the required temperature has been reached, the animal was kept on the hot plate surrounded by the transparent cylinder, and at the same time a timer is started. Just when the animal starts licking its hind paw or tries to jump out of the cylinder feeling the heat of the plate, the timer was stopped and the animal was immediately removed from the plate to prevent any tissue injury. Instrument Used: Hot Plate Appratus (UGO Basile, Italy 7280) Temperature of hot plate: 56±0.2 C Cut off time: 45 s Fig 1.1:Hot Plate Appratus Caution is taken that the animal s paw are clean before placing it on the hot plate as any attached excreta/ husk may interfere with the result, and the animal is removed from the plate timely to prevent tissue damage. Baseline latency of each animals were taken, all the readings were taken in duplicate. The cut off time was kept 45s through out the experiment. The latency to hind paw licking/jumping was observed at different time intervals of 15, 30, 45, 60, 90 and 120 mins after the drug treatment. Final Report Page 13

23 Results: The results indicate that Ayurvedic formulations Narikel Lavan, Rason Vati, Godanti Mishran and Numax did not cause significant increase in latency as compared to control group. Morphine (7 mg/kg, i.p) used as a standard drug, showed analgesic effect which was indicated by increase in latency in comparison to control. Table 1.9: Latency (s) of rats in different groups, at 0, 15, 30, 60, 90 & 120 min after treatment. 0 min 15 min 30 min 60 min 90 min 120 min Control 8.5± ± ± ± ± ±0.13 Morphine 7.5± ±0.56 *** 42.7±0.42 *** 41.7±0.33 *** 41±0.37 *** 41.8±0.31 *** Narikel 7.8± ± ± ± ± ±1.72 Rason vati Godanti mishran 9.3± ± ± ± ± ± ± ± ± ± ± ±1.28 Numax 9.7± ± ± ± ± ±0.69 Fig 1.2: Latency of rats after treatment with morphine and ayurvedic formulation at different time intervals. Data represented as Mean ± S.E.M (n = 6), *** p< 0.001, a- compared to control group. Final Report Page 14

24 2. Evaluation of the analgesic effect of four ayurvedic formulations individually using tail flick model in rats. Animals: This experiment was performed in Wistar rats with cumulative unique identification number i.e. from CUIN 37 to CUIN 67. Rats were divided into five groups i.e., Normal control, Standard, Narikal, Rasonvati,Godanti mishran and Numax treated groups. Each rat was marked for identification (Table 2.2). Table 2.1: Group identification of rats Group Number of rats CUIN Normal Control 6 37 to 42 Standard (Morphine) 4 43 to 46 Narikel Lavan 6 47 to 52 Rason Vati 6 53 to 58 Godanti Mishran 6 59 to 64 Numax 3 65 to 67 Table 2.2: Individual identification of rats within a group Rat number Individual identification Abbreviation 1 Yellow mark on head H 2 Yellow mark on body B 3 Yellow mark on tail T 4 Yellow mark on head and body HB 5 Yellow mark on body and tail BT 6 Unmarked U Final Report Page 15

25 Table 2.3: Drug Administration S. No. Treatments No. of rats Dose 1. Normal control 6-2. Morphine 4 7 mg/kg, i.p 3 Narikel Lavan mg/kg, p.o. 4 Rason Vati mg/kg, p.o. 5 Godanti Mishran 6 40 mg/kg, p.o. 6 Numax mg/kg, p.o. Table 2.4: Dosing schedule of morphine Concentration of Morphine 5mg/ml CUIN Ayurvedic Drug I.D. mark Rats body weight (g) Dose (mg/kg) Intended vol. of (i.p) Actual vol. of (i.p) 43 Morphine H Morphine B Morphine T Morphine HB Final Report Page 16

26 Table 2.5: Dosing schedule of Narikel Lavan Concentration of Narikel Lavan: 100mg/ml CUIN Ayurvedic Drug I.D. mark Rats body weight (g) Dose (mg/kg) Intended vol. of Actual vol. of 47 Narikel Lavan H Narikel Lavan 49 Narikel Lavan 50 Narikel Lavan 51 Narikel Lavan 52 Narikel Lavan B T HB BT U Table 2.6: Dosing schedule of Rason Vati Concentration of Rason Vati: 100 mg/ml CUIN Ayurvedic Drug I.D. mark Rats body weight (g) Dose (mg/kg) Intended vol. of Actual vol. of 53 Rason Vati H Rason Vati B Rason Vati T Rason Vati HB Rason Vati BT Rason Vati U Final Report Page 17

27 Table 2.7: Dosing schedule of Godanti mishran Concentartion of Godanti Mishran: 10 mg/ml CUIN Ayurvedic Drug 59 Godanti Mishran I.D. mark Rats body weight (g) H 126 Dose (mg/kg) Intended vol. of Actual vol. of Godanti Mishran 61 Godanti Mishran 62 Godanti Mishran 63 Godanti Mishran 64 Godanti Mishran B T HB BT U Table 2.8: Concentration of Numax: 50 mg/ml CUIN Ayurvedic I.D. Rats Dose Intended vol. Actual vol. of Drug mark body (mg/kg) of weight (g) 65 Numax H Numax B Numax T Tail Flick Method This is a model of testing centrally acting analgesics. The instrument consists of a Ni- Chrome wire surrounded which is heated by pressing a timer that starts a timer automatically with it; the intensity of wire heating can be adjusted by a regulator. The animal was held in a restrainer with only its tail protruding out which was thoroughly cleaned with damp cotton to remove attached excreta etc., after cleaning a mark was put about 3-4 cm away from the end of the tail. The tail of animal was then kept over the Final Report Page 18

28 hedge above the Ni-Chrom wire with the mark place just above the wire and the timer was started, this leads to the heating of the wire, when the animal lifts its tail (Tail flick) feeling the heat of wire the timer was stopped and the animal returned to its cage. Instrument Used: Tail Flick Appratus (Techno, India) Intensity: 25 (maximum) Cut off time: 15 s Fig 2.1:Tail flick apparatus Caution was taken that the animal s tail was clean before placing it on the hedges as any attached material may interfere with the result, secondly it is to be made sure that there is some distance between the NiChrom wire and the animal s tail to prevent tissue injury. Before starting the experiment the animals were trained to be in the restrainer without getting agitated. The animals were pre screened before dividing into groups any only those giving a baseline latency of 7 s at the maximum intensity were included in the experiment, all the readings were taken in triplicate. The cut off time was kept 15 s through out the experiment. The latency of tail flicking was observed at different time intervals of 30 min interval up to 2 h. Final Report Page 19

29 Result: The results indicate Narikel Lavan, Rason Vati, Godanti mishran and Numax did not cause significant change in latency as compared to control. Morphine (7 mg/kg, i.p) used as a standard drug, showed analgesic effect which was indicated by significant (*** p< 0.001) increase in latency in comparison to control. Since there was no significant difference between latencies of tail flick of rats on treatment with Ayurvedic formulations, it was decided to evaluate the drugs at 1000 mg/kg p.o till two hours. At 1000mg/kg results indicate that the AYFs did not cause increase in tail flick latency as compared to control groups. Table 2.9: Latency (s) of tail flick of rats in different groups, at 0, 15, 30, 60, 90 & 120 mins after treatment. Groups 0 min 15 min 30 min 60 min 90 min 120 min Control 1.7± ± ± ± ± ±0.15 Morphine 2.6± ±1.03 * ** 12.7±1.15 * ** 12.5±0.65 * ** 11.8±0.63 * ** 11.5±0.50 * ** Narikel 2.6± ± ± ± ± ±0.09 Rason vati 3.3± ± ± ± ± ±0.19 Godanti 3.0± ± ± ± ± ±0.53 mishran Numax 3.1± ± ± ± ± ±0.47 Fig 2.2: Latency of tail flick in rats after treatment with morphine and ayurvedic formulations at different time intervals. Data represented as Mean ± S.E.M (n = 6), *** p< 0.001, a- compared to control group. Final Report Page 20

30 Table 2.10: Latency (s) of rats in different groups, at 0, 30, 60, 120& 240 min after treatment. Dose 1000mg/Kg Groups 0 min 30 min 60 min 120 min 240 min Control 5±0 1.7± ± ± ±0.5 Morphine 5.5± ± ±0.5 13±1.0 11±1.0 Narikel 5.1± ± ± ± ±0.6 lavan Rason 5.2± ±0 3.7± ± ±0.6 Godanti 5.4± ±0 2.5± ± ±0.2 mishran Numax 5.1±0 4.3± ± ±0 2.1±0.1 Fig 2.3: Latency of rats after treatment with morphine and ayurvedic formulations (1000 mg/kg) at different time intervals. Data represented as Mean ± S.E.M (n = 6), *** p< 0.001, a- compared to control group. Final Report Page 21

31 3. Evaluation of the analgesic effect of ayurvedic formulations individually using formalin test in rats. Animals: This experiment was performed in Wistar rats ( g) with cumulative unique identification number i.e. from CUIN 68 to CUIN 77. Rats were divided into five groups consisting of two rats each i.e., Normal control, Standard, Narikal, Rason vati,godanti mishran and Numaxtreated groups. Table 3.1: Group identification of rats Group Number of rats CUIN Normal Control 2 68 to 69 Narikel Lavan 2 70 to 71 Rason Vati 2 72 to 73 Godanti Mishran 2 74 to 75 Numax 2 76 to 77 Table 3.2: Drug S. No. Treatments Number of rats Dose and route of 1. Normal control 2-2. Narikel Lavan mg/kg, p.o. 3 Rason Vati mg/kg, p.o. 4 Godanti Mishran mg/kg, p.o. 5 Numax mg/kg, p.o. Final Report Page 22

32 Table 3.3: Dosing schedule of Ayurvedic formulations Concentration of Ayurvedic formulations: 200 mg/ml CUIN Treatment Identification mark Rats body weight (g) Dose (mg/kg) Actual vol. of 68 Control (No treatment) 69 Control (No treatment) Head (H) Body (B) Narikel lavan Tail (T) Narikel lavan Head Body (HB) Rason Vati Body Tail (BT) mg/kg Rason Vati Head Tail (HT) Godanti mishran Left fore limb (LFL) Godanti mishran Right Fore Limb (RFL) Numax Body &right fore limb (BRFL) Numax Unmarked (U) Formalin test: Rats were adapted to open plexiglass observation chambers 15 min before injection of formalin. Ayurvedic formulations were administered 30min prior to formalin injection. Formalin (50 µl of a 2.5% solution in saline) was injected subcutaneously into the dorsal surface of right hind paw of rats. Each rat was observed in the chamber after injection. The total time spent for licking or biting of injected paw (nociceptive response) was measured with stopwatch from 0-5 min, 20-30min and min. post formalin injection and considered as a quantitative indication of nociception. The total time spent for licking/biting from 0 to 5 min was considered as the first phase, whereas the second phase was taken as from 20 to 40 min. Final Report Page 23

33 Results: The results indicated that the Ayurvedic formulations did not cause any significant reduction in the number of licking of hind paw as compared to sham control group at 1000mg/kg p.o., which indicates lack of analgesic activity of ayurvedic formulations. Table 3.4: Effect of Ayurvedic formulationson number of hind paw licking at different time intervals after treatment. Data represented as Mean± SEM Treatments 0-5 min min min Sham 3.0± ± ±0.0 With Formalin 65.0± ± ±12.5 Narikel Lavan 65.0± ± ±1.0 Rason Vati 34.5± ± ±5.5 Godanti Mishran 68.5± ± ±1.0 Numax 77.5± ± ±7.0 Fig 3.1: Effect of ayurvedic formulations on number of hind paw licking at different time intervals after treatment. Data represented as Mean ± SEM Final Report Page 24

34 4: Evaluation of analgesic effect of ayurvedic formulations after 5 days treatment using hot plate and tail flick method in rats. The experiment was performed on male Wistar rats ( g). Each animal was assigned a cumulative unique identification number i.e. from CUIN 78 to CUIN 92. Rats were divided into five groups consisting of three rats each i.e., Normal control, Narikel Lavan, Rason vati,godanti Mishran and Numax treated groups.the rats were reused for hot plate test after washout period of 1 month. The Ayurvedic formulations were administered orally for 5 days to evaluate the chronic analgesic effect. Table 4.1: Group identification of rats Group Number of rats CUIN Normal Control 3 78 to 80 Narikel Lavan 3 81 to 83 Rason Vati 3 84 to 86 Godanti Mishran 3 87 to 89 Numax 3 90 to 92 Table 4.2: Concentration of Narikel Lavan: 100 mg/ml CUIN Ayurvedic Drug I.D. mark Rats body weight (g) Dose (mg/kg) Intended vol. of Actual vol. of 81 Narikel Lavan 82 Narikel Lavan 83 Narikel Lavan H B T Final Report Page 25

35 Table 4.3: Concentration of Rason Vati: 100 mg/ml CUIN Ayurvedic Drug 84 Rason Vati 85 Rason Vati 86 Rason Vati I.D. mark Rats body weight (g) H 170 Dose (mg/kg) Intended vol. of Actual vol. of B T Table 4.4: Concentration of Godanti Mishran: 10mg/ml CUIN Ayurvedic Drug 87 Godanti Mishran I.D mark Rats body weight (g) H 197 Dose (mg/kg) Intended vol. of Actual vol. of Godanti Mishran 89 Godanti Mishran B T Table 4.5: Concentration of Numax: 100mg/ml CUIN Ayurvedic Drug I.D. mark Rats body weight (g) 90 Numax H 195 Dose (mg/kg) Intended vol. of Actual vol. of 91 Numax B Numax T Tail flick and Hot Plate Method The procedure followed was same as as described previously. Final Report Page 26

36 Result: The results of chronic studies using Hot Plate model and Tail flick model in rats indicate that Narikel Lavan, Rason Vati, Godanti and Numax did not cause significant change in latency as compared to control. Morphine (7 mg/kg, i.p) used as a standard drug, showed analgesic effect which was indicated by significant (*** p< 0.001) increased in latency as compared to control. Table 4.6: Latency (s) totail flick of rats in different groups, at 0, 15, 30, 60, 90 & 120 mins after treatment in tail flick test.data represented as Mean ± S.E.M (n = 3), *** p< 0.001, a- compared to control group. Groups 0 min 15 min 30 min 60 min 90 min 120 min Control 2.7± ± ± ± ± ±0.26 Morphine 2.8± ±1.0 *** 13.7±0.88 *** 13±0.58 *** 11.7±0.88 *** 12.0±0.0 *** Narikel 2.7± ± ± ± ± ±0.13 lavan Rason 3.0± ± ± ± ± ±0.18 Godanti 2.4± ± ± ± ± ±0.07 mishran Numax 2.5± ± ± ± ± ±0.09 Fig. 4.1: Latency of tail flick rats after treatment with morphine and ayurvedic formulation at different time intervals in tail flick test. Data represented as Mean ± S.E.M (n = 3), *** p< 0.001, a- compared to control group. Final Report Page 27

37 Table 4.7: Latency (s) of paw licking of rats in different groups, at 0, 15, 30, 60, 90 & 120 min after treatment in hot plate test.data represented as Mean ± S.E.M (n = 3), *** p< 0.001, a- compared to control group. Groups 0 min 15 min 30 min 60 min 90 min 120 min Control 8.5±0.1 2 Morphine 7.8±2.3 5 Narikel lavan 7.8±0.1 2 Rason vati 9.3±0.7 4 Godanti 8.8±1.1 mishran 3 Numax 9.7± ± ± ± ± ± ±0.56 * ** 42.7±0.42 * ** 41.7±0.33 * ** 41±0.37 ** * 41.8±0.31 * ** 9.4± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±0.69 Fig. 4.2: Latency of paw licking in rats after treatment with morphine and ayurvedic formulation at different time intervals in hot plate test. Data represented as Mean ± S.E.M (n = 3), *** p< 0.001, a- compared to control group. Final Report Page 28

38 5: Evaluation of the analgesic effect of combination of ayurvedic formulations using hot plate model in rats. Animals: The experiment was performed on male Wistar rats ( g). Each animal was assigned a cumulative unique identification number i.e. from CUIN 93 to CUIN 101. Rats were divided into three groups consisting of three rats each i.e., Normal control, Standard, Combination (Narikel Lavan, Rason Vati, Godanti Mishran and Numax) treated groups. Each animal was marked for identification (Table 5.2) Table 5.1: Group identification of rats Group Number of rats CUIN Normal Control 3 93 to 95 Standard (Morphine) 3 96 to 98 Combination of AYF s 3 99 to 101 Table 5.2: Individual identification of rats within a group Rats number Individual identification Abbreviation 1 Yellow mark on head H 2 Yellow mark on Body B 3 Yellow mark on tail T Final Report Page 29

39 Table 5.3: Drug Administration S. No. Treatments No. of rats Dose and route of 1. Normal control 3-2. Morphine 3 7 mg/kg, i.p 3 Combination of AYF s a) Narikel Lavan b) Rason Vati c) Godanti Mishran d) Numax Table 5.4: Dosing schedule of morphine Concentration of Morphine: 5 mg/ml: mg/kg, p.o. 500 mg/kg, p.o. 40 mg/kg, p.o. 150mg/kg, p.o. CUIN Ayurvedic Drug I.D. mark Rats body weight (g) 96 Morphine H 240 Dose (mg/kg) Intended vol. of Actual vol. of Morphine B Morphine T Table 5.5: Dosing schedule of Narikel Lavan Concentration of Narikel Lavan: 100 mg/ml CUIN Ayurvedic Drug 99 Narikel Lavan I.D. mark Rats body weight (g) H 204 Dose (mg/kg) Intended vol. of Actual vol. of Narikel Lavan 101 Narikel Lavan B T Final Report Page 30

40 Table 5.6: Dosing schedule of Rason Vati Concentration of Rason Vati: 100 mg/ml CUIN Ayurvedic Drug 99 Rason Vati I.D. Mark Rats body weight (g) H 204 Dose (mg/kg) Intended vol. of Actual vol. of Rason Vati 101 Rason Vati B T Table 5.7: Dosing schedule of Godanti Mishran Concentration of Godanti Mishran: 10 mg/ml CUIN Ayurvedic Drug 99 Godanti Mishran I.D. mark Rats body weight (g) H 204 Dose (mg/kg) Intended vol. of Actual vol. of Godanti Mishran 101 Godanti Mishran B T Table 5.8: Dosing schedule of Numax Concentration of Numax: 50 mg/ml CUIN Ayurvedic Drug I.D. mark Rats body weight (g) 99 Numax H 204 Dose (mg/kg) Intended vol. of Actual vol. of Numax B Numax T Final Report Page 31

41 Latency Result: The results indicates that the combination of Ayurvedic formulations (Narikel Lavan, Rason Vati, Godanti mishran and Numax) did not cause any significant increase in latency of paw licking as compared to control group. Morphine (7 mg/kg, i.p) used as a standard drug, showed analgesic effect which was indicated by significant (*** p< 0.001) increase in latency of paw licking as comparison to control. Table 5.9:Latency (s) of paw licking of rats in different groups, at 0, 15, 30, 60, 90 & 120 min after treatment in hot plate test.data represented as Mean ± S.E.M (n = 3), *** p< 0.001, a- compared to control group. 0 min 15 min 30 min 60 min 90 min 120 min Control 8.0 ± ± ± ± ± ± 1.21 Morphine 8.6 ± ± 0.88 *** 43.0 ±.58 *** 41.3±0.33 *** 41.0± 0.58 *** 41.7 ± 0.33 Combination 9.1 ± ± ± ± ± ± ***a ***a ***a ***a ***a 0 min 15 min 30 min 60 min 90 min 120 min Time Control Morphine COMBO Fig 5.1: Latency (s) of paw licking of rats in different groups, at 0, 15, 30, 60, 90 & 120 min after treatment in hot plate test.data represented as Mean ± S.E.M (n = 3), *** p< 0.001, a- compared to control group. Final Report Page 32

42 6: Evaluation of the analgesic effect ofcombination of ayurvedic formulations using tail flick model in rats. Animals: The experiment was performed on male Wistar rats ( g). Each animal was assigned a unique identification number i.e. from CUIN 102 to CUIN 110. Rats were divided into three groups consisting of three rats each i.e., Normal control, Standard, Combination of Ayurvedic Formulations (Narikal Lavan, Rason Vati,Godanti Mishran and Numax) treated groups. Each animal was marked for identification (Table 6.1). Table 6.1: Group identification of rats Group Number of rats CUIN Normal Control to 104 Standard (Morphine) to 107 Combination to 110 Table 6.2: Individual identification of rats within a group Rats number Individual identification Abbreviation 1 Yellow mark on head H 2 Yellow mark on body B 3 Yellow mark on tail T Final Report Page 33

43 Table 6.3: Drug Administration S. No. Treatment group No. of rats Dose and route of 1. Normal control 3-2. Morphine 3 7 mg/kg, i.p 3 Combination of AYF s a) Narikel Lavan b) Rason Vati c) Godanti Mishran d) Numax mg/kg, p.o. 500 mg/kg, p.o. 40 mg/kg, p.o. 150mg/kg, p.o. Table 6.4: Dosing schedule of morphine Concentration of Morphine 5 mg/ml CUIN Treatment I.D. mark Rats body weight (g) Dose (mg/kg) Intended vol. of i.p Actual vol. of 105 Morphine H Morphine B Morphine T Table 6.5: Dosing schedule of Narikel Lavan Concentration of Narikel Lavan: 100 mg/ml CUIN Ayurvedic Drug 108 Narikel Lavan 109 Narikel Lavan 110 Narikel Lavan I.D. mark Rats body weight (g) H 204 Dose (mg/kg) Intended vol. of Actual vol. of B T Final Report Page 34

44 Table 6.6: Dosing schedule of Rason Vati Concentration of Rason Vati: 100 mg/ml CUIN Ayurvedic Drug I.D. mark Rats body weight (g) 108 Rason Vati H 204 Dose (mg/kg) Intended vol. of Actual vol. of Rason Vati B Rason Vati T Table 6.7: Dosing schedule of Godanti mishran Concentration of Godanti Mishran: 10 mg/ml CUIN Ayurvedic Drug 108 Godanti Mishran 109 Godanti Mishran 110 Godanti Mishran I.D. mark Rats body weight (g) H 204 Dose (mg/kg) Intended vol. of Actual vol. of B T Table 6.8: Dosing schedule of Numax Concentration of Numax: 50 mg/ml CUIN Ayurvedic Drug I.D. mark Rats body weight (g) Dose (mg/kg) Intended vol. of Actual vol. of 108 Numax H Numax B Numax T Final Report Page 35

45 Latency Results: The results indicate that the combination of Ayurvedic formulations (Narikel lavan, Rason vati, Godanti mishran and Numax) did not cause any significant change in tail flick latency as compared to control. Morphine (7 mg/kg, i.p) used as a standard drug, showed analgesic effect which was indicated by increase in tail flick latency in comparison to control. Table 6.9: Latency (s) of tail flick of rats in different groups, at 0, 15, 30, 60, 90 & 120 min after treatment. Data represented as Mean ± S.E.M (n = 3), *** p< 0.001, a- compared to control group. Groups 0 min 15 min 30 min 60 min 90 min 120 min Control Morphine Combination 2.6 ± ± ± ± ± ± ± ±1.00 *** 13.7±0.88 *** 13.0±0.58 *** 11.7±0.88 *** 12.0±0.0 *** 3.5± ± ± ± ± ± ***a ***a ***a ***a ***a 0 min 15 min 30 min 60 min 90 min 120 min Time Control Morphine COMBO Fig 6.1: Latency (s) of tail flick of rats in different groups, at 0, 15, 30, 60, 90 & 120 min after treatment. Data represented as Mean ± S.E.M (n = 3), *** p< 0.001, a- compared to control group. Final Report Page 36

46 7: Evaluation of effect of ayurvedic formulations using Carrageenan induced paw edema model in rats. The experiment was performed on male Wistar rats ( g).each rat was assigned a cumulativeunique identification number i.e. from CUIN 111 to CUIN 128. Rats were divided into six groups consisting of three rats each i.e., Carrageenan, Indomethacin, Narikal Lavan, Rason Vati, Godanti Mishran and Numax treated groups. Each animal was marked for identification (Table 7.2). All the four Ayurvedic formulations were administered at 1000 mg/kg. Table 7.1: Group identification of rats Group Number of rats CUIN Carrageenan to 113 Indomethacin to 116 Narikel Lavan to 119 Rason Vati to 121 Godanti Mishran to 125 Numax to 128 Table 7.2: Individual identification of rats within a group Rats number Individual identification Abbreviation 1 Yellow mark on head H 2 Yellow mark on body B 3 Yellow mark on tail T Final Report Page 37

47 Table 7.3: Dosing schedule of Indomethacin Concentration of Indomethacin: 0.6 mg/ml CUIN Drug I.D. mark Rats body weight (g) Dose (mg/kg) Intended vol. of Actual vol. of 114 Indomethacin H ndomethacin B Indomethacin T Table 7.4: Dosing schedule of Narikel Lavan Concentration of Narikel Lavan: 200 mg/ml CUIN Ayurvedic Drug 117 Narikel Lavan 118 Narikel Lavan 119 Narikel Lavan I.D. mark Rats body weight (g) H 158 Dose (mg/kg) 1000 Intended vol. of Actual vol. of B T Table 7.5: Dosing schedule of Rason Vati Concentration of Rason Vati: 200 mg/ml CUIN Ayurvedic Drug I.D. mark Rats body weight (g) Dose (mg/kg) Intended vol. of Actual vol. of 120 Rason Vati H Rason Vati B Rason Vati T Final Report Page 38

48 Table 7.6: Dosing schedule of Godanti Mishran Concentration of Godanti Mishran: 200 mg/ml CUIN Ayurvedic Drug 123 Godanti Mishran 124 Godanti Mishran 125 Godanti Mishran I.D. mark Rats body weight (g) H 145 Dose (mg/kg) Intended vol. of Actual vol. of B T Table 7.7: Dosing schedule of Numax Concentration of Numax: 200 mg/ml CUIN Ayurvedic Drug I.D. mark Rats body weight (g) 126 Numax H 199 Dose (mg/kg) Intended vol. of Actual vol. of Numax B Numax T Carrageenan induced paw edema: Paw edema will be induced in wistar rats ( g) by injection of 100 µl of1% carrageenan ( -carrageenan, type IV, Sigma) diluted in salinein the left hind foot pad (Chan et al, 1995). The test compounds and saline (control) were administered i. p. 1 hr before the injection of carrageenan. Indomethacin will be used to test the sensitivity of the system and as a standard drug. Paw volumes will be determinedusing a plethysmometer (Ugo Basil, Italy) at times 0,1, 2,3,4,5 and 24 h. Swelling Index = (Vol. of swollen paw - Vol. of paw before injection) *100 Vol. of paw before injection Final Report Page 39

49 Fig 7.1: A) Inflammation in paw of rat induced by s.c injection of carrageenan B) Measurement of paw volume PRECAUTION Rat paw should be clean and make sure that they are free from husk and other dirt and matter. Make sure that the level of the water in persplex cell should be correct upto the mark. Water level in transducer cell is proportional to the volume dipped in large tube. Press the re-zero button before measuring the volume of the paw for each rat. Filling solution should be used within 3 days. Final Report Page 40

50 % Paw Edema Result: The results indicate that Narikel Lavan, Numax and Rason Vatihas shown reduction in % paw edema but not significant as compared to carrageenan only group.godanti Mishran (1000 mg/kg) has been found to reduce % paw edema at all time points except at 3 hr (time of maximum inflammation & swelling) as compared to carrageenan only group. However the standard drug (Indomethacin, 3mg/kg p.o.) has significantly (*** p< 0.001) reduced per cent paw edema at all-time intervals. Table 7.8: Per cent paw edema of rats in different groups at 1, 2, 3, 4 & 5 h after treatment.data represented as Mean ± S.E.M (n = 3), *** p< 0.001, a- compared to Carageenan group. 1 h 2 h 3 h 4 h 5 h Carrageenan (C ) only Indomethacin + C 50.1 ± ± ± ± ± ± 0.37 a*** 12.4 ± 0.81 a*** 15.5 ± 0.52 a*** 13.2 ± 0.68 a*** 11.2 ± 1.5 a*** Narikel lavan + C 35.9 ± ± ± ± ± 10.6 Rason vati + C 32.5 ± ± ± ± ± 4.0 Godanti mishran +C 16.4 ± 3.6 a*** 25.6 ± 2.0 *a 33.0 ± ± 4.6 *a 22.6 ± 3.18 a* Numax + C 24.8 ± ± ± ± ± Carrageenan Indomethacin Narikel Rason Godanti Numax *b *b *b *a *a ***a ***a ***a ***a ***a ***a 1h 2h Time 3h 4h 5h *a Fig 7.2: Per cent paw edema of rats in different groups at 1,2,3,4 & 5 h after treatment.data represented as Mean ± S.E.M (n = 3), *** p< 0.001, a- compared to control group, b- compared to indomethacin group. Final Report Page 41

51 8. Evaluation of effect of Ayurvedic formulations administered individually at limit dose of 1000 mg/kg on neurobehavioural parameters in rats Animals: The experiments were performed on male Wistar rats ( g). Rats were assigned a cumulative unique identification number i.e. from CUIN 129 to CUIN 143. Rats were divided into five groups consisting of three rats each i.e., Normalcontrol, Narikel Lavan, Rason Vati,Godanti Mishran and Numax formulation treated groups. Each rat was marked for identification (Table 8.2) Table 8.1: Group identification of rats Group Number of rats CUIN Normal Control to 131 Narikel Lavan to 134 Rason Vati to 137 Godanti Mishran to 140 Numax to 143 Table 8.2: Individual identification of rats within a group Rats number Individual identification Abbreviation 1 Yellow mark on head H 2 Yellow mark on body B 3 Yellow mark on tail T Final Report Page 42

52 Table 8.3: Drug Administration S. No. Treatments No. of rats Dose 1. Normal control (No treatment) 3-2 Narikel Lavan 3 3 Rason Vati mg/kg 1000mg/kg 4 Godanti Mishran mg/kg 5 Numax mg/kg Table 8.4: Dosing schedule of Narikel Lavan Concentration of Narikel Lavan: 200mg/ml CUIN Ayurvedic Drug I.D. mark Rats body weight (g) Dose (mg/kg) Intended vol. of Actual vol. of 132 Narikel Lavan 133 Narikel Lavan 134 Narikel Lavan H B T Final Report Page 43

53 Table 8.5: Dosing schedule of Rason Vati Concentration of Rason Vati: 200 mg/ml CUIN Ayurvedic Drug I.D. mark Rats body weight (g) 135 Rason Vati H 224 Dose (mg/kg) Intended vol. of Actual vol. of Rason Vati B Rason Vati T Table 8.6: Dosing schedule of Godanti Mishran Concentration of Godanti Mishran: 200 mg/ml CUIN Ayurvedic Drug 138 Godanti Mishran I.D. mark H Rats body weight (g) 165 Dose (mg/kg) Intended vol. of Actual vol. of Godanti Mishran B Godanti Mishran T Table 8.7: Dosing schedule of Numax Concentration of Numax: 200mg/ml CUIN Ayurvedic Drug I.D. mark Rats body weight (g) 141 Numax H 160 Dose (mg/kg) Intended vol. of Actual vol. of Numax B Numax T Final Report Page 44

54 a) Elevated plus maze Fig 8.1:Elevated plus maze Acquisition and retention of memory processes was assessed using elevated plus maze according to the method of (Sharma and Gupta, 2001). The elevated plus maze consists of two closed arms and two open arms forming a cross, with a quadrangular center. The maze is placed above the floor. On the day 0, the 1 st trial (initial transfer latency) was carried out as follows: the ratwas placed at the end of one open arm facing away from central platform and the time it took to move from the open arm to either of the enclosed (transfer latency) was recorded. The initial transfer latency was the time that elapsed between the times when its four legs crosses to the enclosed arm. The rat was allowed to move freely in the plus maze regardless of open and closed arms for 10 seconds after the measurement of initial transfer latency. The rat was then gently taken out of the plus maze and was returned to its home cage. Twenty four hours later, the 2 nd trial (retention transfer latency test) was performed. The rats were again put into the elevated plus maze. If the rat did not enter the enclosed arm within 60 s on 2 nd trial, the transfer latency was assigned 60s. Retention transfer latency was recorded on Day 1,14, 21 &28 after treatment(fig.8.1) Final Report Page 45

55 b) One-trial Passive Avoidance task Instrument Used: Passive Avoidance (UGO Basile, Italy 7551) Fig 8.2:One-trial Passive Avoidance task appratus Memory retention deficit was evaluated by a passive avoidance (step through) apparatus according to the method previously described by (Reeta et al, 2009). The apparatus consisted of two separate chambers connected through a guillotine door. One chamber was lit using a bulb, while the other was dark. The floor of both the chambers consisted of steel grids, used to deliver electric shocks. Briefly, on the acquisition trial, each rat was placed in a lighted chamber. After 60 sec of habituation, a guillotine door separating the lighted and dark chambers was opened, and the initial latency (IL) to enter the dark chamber was recorded. Rats exhibiting an initial latency time of more than 60 sec were excluded from further experiments. Immediately after the rat enters the dark chamber, the guillotine door was closed and an electric foot shock (75 V, 0.2mA, 50 Hz) was delivered to the floor grids for 3 sec. The rat was removed from the dark chamber 5 sec later and returned to its home cage. After 24 h, retention latency (RL) time was measured in the same way as in the acquisition trial, but foot shock was not delivered, and the latency time was recorded up to a maximum of 600 sec. Retention latency was recorded on Day 1,14, 21 &28 after treatment (Fig 8.2) Final Report Page 46

56 c) Actophotometer Instrument Used: Actophotometer (Techno Electronics, Lucknow ) Locomotor activity (index of wakefulness /alertness of mental activity)is evaluated by Actophotometer. It is expressed in in term of total photobeam counts/5 min/animals. Actophotometer operates on photoelectric cells which are connected in circuit with a counter.when the beam of light falling on the photocell is cut off by the animal, a count is recorded. Caution: For performing this test on consecutive days, the room temperature & environmental conditions should be nearly same. d) Rota rod Instrument Used: Rotarod apparatus (UGO Basile, Italy 7550) Motor coordination and muscle relaxant activity assessment is evaluated using Rotarod Apparatus. Loss of muscle grip is an indication of muscle relaxation.the difference in the fall off time from the rotating rod between the control & treated rats is taken as index of muscle relaxation. The animal is placed on section of rotating rod by holding their tail, facing the other side. R.P.M is adjusted by proper positioning of the gear belt. The time spend by the animal on the rotating rod at 4 RPM (adjusted to accelerated mode) is observed,once the rat fall on trip plate. Final Report Page 47

57 e) Muscle Grip Strength Test The forelimb grip strength (indicator of muscular function) is measured by performing muscle grip strength test. The grip test is performed according to methods of Moran et al., The apparatus with a 50cm string pulled between 2 vertical supports elevated 40 cm above from the flat surface. The rat is placed on the string at a point midway between the supports and is evaluated by the following scale. Score/Scale 0: Fall off from the string 1: Hangs onto string with two forepaws 2: Hangs onto the string with two forepaws, but also attempt to climb on the string. 3: Hangs onto the string with two forepaws, plus one or both hind paws. 4: Hangs onto the string with all paws, plus the tail wrapped around the string. 5: Escape Final Report Page 48

58 Latency (sec) Results: a) Elevated plus maze: The results indicates that the Ayurvedic formulations; Narikel Lavan, Rason Vati, Godanti Mishran did not cause any significant change in the retention transfer latency as compared to control. However Numax significantly decreased the retention transfer latency as compared to control on the 28 th day after treatment. Table 8.8: Effect on retention transfer latency (s) in different treatment groups in elevated plus maze at Day 1, 14, 21 & 28after treatment.data represented as Mean ± S.E.M (n = 3), * p< 0.05, a- compared to control group. Groups Initial Transfer Retention Transfer Latency (sec) Latency Day1 Day 14 Day 21 Day 28 (sec) Control ± ± ± ± ± 4.3 Narikel Lavan ± ± ± ± ± 2.47 Rason vati ± ± ± ± ± 3.31 Godanti mishran ± ± ± ± ± 4.56 Numax ± ±0.4 a* 14.7 ± ± ±0.19 a*** Elevated Plus Maze a* a*** 1 Day 14 day 21 day 28 day Control Narikel Lavan(1000mg/kg) Rason vati (1000mg/kg) Godanti Mishran (1000mg/kg) Numax (1000mg/kg) ITL Retention Transfer Latency Days Fig 8.3: Effect on retention transfer latency (s) in different treatment groups in elevated plus maze at day 1, 14, 21 & 28after treatment.data represented as Mean ± S.E.M (n = 3), * p< 0.05, *** p<0.001a- compared to control group. Final Report Page 49

59 Latency (sec) b) One-trial Passive Avoidance task The results indicates that Narikel lavan, Rason Vati, Godanti Mishran and Numax did not cause any significant change in the retention latency as compared to control up to 28 th day of drug treatment. Table 8.9: Effect on transfer latency (s) in different treatment groups in passive avoidance test at Day 1, 14, 21 & 28after treatment. Data represented as Mean ± S.E.M (n = 3), * p< 0.05, a- compared to control group. Groups Initial Transfer Latency (sec) Retention Escape Latency (sec) Day 1 Day 14 Day 21 Day 28 Control Narikel Lavan Rason vati Godanti Mishran Numax ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± 46.9 a* ± ± ± Passive Avoidance a* 1 Day 14 day 21 day 28 day Control Narikel Lavan(1000mg/kg) Rason vati (1000mg/kg) Godanti Mishran (1000mg/kg) Numax (1000mg/kg) Initial Latency Retention Latency Days Fig 8.4: Effect on transfer latency (s) in different treatment groups in passive avoidance test at Day 1, 14, 21 & 28after treatment. Data represented as Mean ± S.E.M (n = 3), * p< 0.05, a- compared to control group. Final Report Page 50

60 Movement Counts ( 5min) c) Actophotometer The results indicate Narikel lavan, Rason Vati, Numaxdid not show any significant difference in the movement counts as compared to control. However, Godanti Mishran have found to significantly increase movement counts as compared to control group on the 21 st (* p<0.05) and 28 th (* p<0.01) day of drug treatment. Table 8.10:Effect on movement counts of rats in different groups in Actophotometer at Day 0, 14, 21 & 28after treatment.data represented as Mean ± S.E.M (n = 3), * p<0.05,** p< 0.01 a- compared to control group. Groups/ Days Control Narikel Lavan Rason Vati Godanti Mishran Numax Movement Count (in 5 min) Day 0 Day 14 Day 21 Day ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±9.24 * 35.3±1.76 ** ± ± ± ± Actophotometer a* a** 0 Day 14 day 21 day 28 day Days Control Narikel Lavan(1000mg/kg) Rason vati (1000mg/kg) Godanti Mishran (1000mg/kg) Numax (1000mg/kg) Fig 8.5: Effect on movement counts of rats in different groups in Actophotometer at Day 0, 14, 21 & 28after treatment. Data represented as Mean ± S.E.M (n = 3), * p<0.05, ** p<0.01, a- compared to control group. Final Report Page 51

61 Fall off time (sec) d) Rota rod The results indicates that the Ayurvedic formulations; Narikel lavan, Rason Vati, Godanti Mishran and Numax did not show any significant change infall off time as compared to control on 0,14 and 21 st day whereas there was significant [Narikel lavan (p<0.001), Rason Vati, (p<0.001) Godanti Mishran (p<0.01) and Numax (p<0.05)] decrease in fall off time as compared to control on the 28 th day of drug treatment. Table 8.11:Effect on fall off time of rats in different treatment groups in Rota rod test at Day 0, 14, 21 & 28 after treatment.data represented as Mean ± S.E.M (n = 3), * p<0.05,** p< 0.01 *** p< 0.001, a- compared to control group. Fall off time (sec) Groups/Days Day0 Day14 Day21 Day28 Control Narikel Lavan Rason Vati Godanti Mishran Numax ± ± ± ± ± ± ± ± 9.5***a ± ± ± ± 8.8***a ± ± ± ± 1.1**a ± ± ± ± 0.28*a Rotarod a*** a*** a** a* 0 Day 14 day 21 day 28 day Days Control Narikel Lavan(1000mg/kg) Rason vati (1000mg/kg) Godanti Mishran (1000mg/kg) Numax (1000mg/kg) Fig 8.6: Effect on fall off time in different treatment groups in Rotarod test at Day 0, 14, 21 & 28 after treatment.data represented as Mean ± S.E.M (n = 3),* p<0.05,** p< 0.01, *** p< 0.001, a- compared to control group. Final Report Page 52

62 Score e) Muscle grip strength: The results indicates that Ayurvedic formulations Narikel Lavan, Rason Vati, Godanti Mishran and Numax did not cause significant improvement in the muscle grip strength of the rats as compared to control group up to 28 th day of drug treatment. Table 8.12: Effect of ayurvedic formulations on score in muscle grip test at Day 0, 14, 21 & 28 after treatment. Data represented as Mean ± S.E.M (n = 3) Groups/Days Score Day 0 Day14 Day21 Day28 Control Narikel Lavan Rason Vati Godanti Mishran Numax 1.3± ± ± ± ± ± ± ± ± ± ± ± ± ±0 3.6 ± ± ± ± ± ± Muscle Grip Test Control Narikel Lavan(1000mg/kg) Rason vati (1000mg/kg) Godanti Mishran (1000mg/kg) Numax (1000mg/kg) 0 0 Day 14 day 21 day 28 day Days Fig 8.7: Effect of Ayurvedic formulations on score in muscle grip test at Day 0, 14, 21 & 28 after treatment. Data represent Mean ± S.E.M (n = 3) Final Report Page 53

63 9: Evaluation of effect of combination of Ayurvedic formulations on neurobehavioral parameters after chronic (28 days) in rats. Animals: These experiments were performed on male Wistar ( g) rats with cumulative unique identification number i.e. from CUIN 144 to CUIN 149. Rats were divided into two groups consisting of three rats each i.e., Normal control, Combination (Narikal, Rason Vati,Godanti Mishran and Numax) treated group.ayurvedic formulations were administered for period of 28 days. Each animal was marked for identification (Table 9.2) Table 9.1: Group identification of rats Group Number of rats CUIN Normal Control to 146 Combination AYF s to 149 Table 9.2: Individual identification of rats within a group Rats number Individual identification Abbreviation 1 Yellow mark on head H 2 Yellow mark on Body B 3 Yellow mark on tail T Table 9.3:Drug Administration S. No. Treatments No. of rats Doseand route of 1. Normal control 3-2. Combination of AYF s a) Narikel Lavan b) Rason Vati c) Godanti Mishran d) Numax mg/kg, 500 mg/kg 40 mg/kg 150mg/kg Final Report Page 54

64 Table 9.4: Dosing schedule of Narikal Lavan Concentration of Narikel Lavan: 100 mg/ml CUIN Ayurvedic Drug 147 Narikel Lavan 148 Narikel Lavan 149 Narikel Lavan I.D. mark Rats body weight (g) H 178 Dose (mg/kg) Intended vol. Actual vol. of of B T Table 9.5: Dosing schedule of Rason Vati Concentration of Rason Vati: 100 mg/ml CUIN Ayurvedic Drug I.D. mark Rats body weight (g) 147 Rason Vati H 178 Dose (mg/kg) Intended vol. of Actual vol. of Rason Vati B Rason Vati T Table 9.6: Dosing schedule of Godanti Mishran Concentration of Godanti Mishran: 40 mg/ml CUIN Ayurvedic Drug 147 Godanti Mishran 148 Godanti Mishran 149 Godanti Mishran I.D. mark Rats body weight (g) H 178 Dose (mg/kg) Intended vol. Actual vol. of of B T Final Report Page 55

65 Table 9.7: Dosing schedule of Numax Concentration of Numax: 50 mg/ml CUIN Ayurvedic Drug I.D. mark Rats body weight (g) 147 Numax H 178 Dose (mg/kg) Intended vol. Actual vol. of of Numax B Numax T Neuro-behavioural tests The following neurobehavioral tests were performed on rats on Day 1, 7, 14, 21 & 28 after of ayurvedic formulations in rats (CUIN: ). a) Elevated plus maze b) One-trial Passive Avoidance task c) Actophotometer d) Rota rod e) Muscle grip strength The procedures followed for above tests were same as described previously. Final Report Page 56

66 Latency (sec) Results: a) Elevated plus maze The results indicate that the combination of fourayurvedic formulations; Narikel Lavan, Rason Vati, Godanti Mishran and Numax up to 28 th day of drug treatment did not cause any significant change in the retention transfer latency as compared to control group. Table 9.8: Effect of Ayurvedic formulationson retention transfer latency in elevated plus maze test on Day 1,14,21, & 28 after treatment Data represented as Mean ± SEM, (n = 3) Groups/ Days Initial Transfer Latency (sec) Retention Transfer Latency (sec) Day 1 Day 7 Day 14 Day 21 Day 28 Control 22.22± ± ± ± ± ±2.3 Combination of 4 AYFs 16.24± ± ±3.4 14± ± ± Elevated Plus Maze Control Combination of 4 AYFs 0 1 Day 7 day 14 day 21 day 28 day ITL Retention Transfer Latency Days Fig 9.1: Effect of Ayurvedic formulations on retention transfer latency in elevated plus maze test on Day 1, 14, 21, & 28 after treatment. Data represented as Mean ± S.E.M (n = 3). Final Report Page 57

67 Latency (sec) b) One trial avoidancetask The results indicate that the combination of Ayurvedic formulations; Narikel lavan, Rason Vati, Godanti Mishran and Numax up to 28 th day of drug treatment did not cause any significant change in the retention escape latency as compared to control group. Table 9.9: Effect of Ayurvedic formulations on retention escape latency in passive avoidance test on Day 1, 14, 21 & 28 after treatment. Data represented as Mean ± SEM, (n = 3) Groups/ Days Initial Transfer Latency (sec) Retention Escape Latency (sec) Day 1 Day 7 Day 14 Day 21 Day 28 Control 38.9± ± ± ± ± ±1.1 Combination of AYFs 26.5± ± ± ± ± ± Passive Avoidance 1 Day 7 day 14 day 21 day 28 day Control Combination of 4 AYFs Initial Latency Retention Latency Days Fig 9.2: Effect of Ayurvedic formulations on retention escape latency in passive avoidance test, on Day 1, 14, 21, & 28 after treatment. Data represented as Mean ± SEM, (n = 3) Final Report Page 58

68 Movement Counts ( 5min) c) Actophotometer The results indicate that the combination of Ayurvedic formulations; Narikel lavan, Rason Vati, Godanti Mishran and Numax up to 28 th day of drug treatment did not show any significant difference in the movement counts as compared to control group. Table 9.10: Effect of Ayurvedic formulations on rat s activities in actophotometer on Day 0, 14, 21, & 28 after treatment. Data represented as Mean ± SEM, (n = 3) Groups/ Days Movement Count (5 min) Day 0 Day 7 Day14 Day21 Day28 Control 61.6± ± ± ± ±2.3 Combination of 4 AYFs 65.3± ± ± ± ± Actophotometer Control Combination of 4 AYFs 0 Day 7 day 14 day 21 day 28 day Days Fig 9.3: Effect of Ayurvedic formulations on rat s activity in actophotometer on Day 0, 14, 21, & 28 after treatment. Data represented as Mean ± SEM, (n = 3) Final Report Page 59

69 Fall off time (sec) d) Rotarod test The results indicates that the combination of Ayurvedic formulations; Narikel lavan, Rason Vati, Godanti Mishran and Numax up to 28 th day of drug treatment did not show any significant change in the fall off time as compared to control. Table 9.11: Effect of Ayurvedic formulations on fall off time in rota rod test on Day 0, 14, 21 & 28 after treatment. Data represented as Mean ± SEM, (n = 3) Fall off time (sec) Groups/Days Day0 Day7 Day14 Day21 Day28 Control 118.0± ± ± ± ±5.03 Combination of 4 AYFs 108.6± ± ± ± ± Rota rod test Control Combination of 4 AYFs 0 0 Day 7 day 14 day 21 day 28 day Days Fig 9.4: Effect of Ayurvedic formulations on fall off time in rota rod test on Day 0, 14, 21 & 28 after treatment. Data represented as Mean ± SEM, (n = 3) Final Report Page 60

70 Score e) Muscle Grip Strength Test The results indicates that Ayurvedic formulations Narikel Lavan, Rason Vati, Godanti Mishran and Numax up to 28 th day of drug treatment did not cause significant improvement in the muscle grip strength as compared to control group. Table9.12: Effect of Ayurvedic formulations on score in muscle grip test on Day 0, 14, 21 & 28 after treatment. Data represented as Mean ± SEM, (n = 3). Score Groups/Days Day0 Day7 Day14 Day21 Day28 Control 3.6± ± ±0.3 3± ±0.3 Combination of 4 AYFs 2.6± ± ± ± ± Muscle Grip Test 0 Day 7 day 14 day 21 day 28 day Days Control Combination of 4 AYFs Fig 9.5: Effect of Ayurvedic formulations on score in muscle grip test on Day 0, 14, 21 & 28 after treatment. Data represented as Mean ± SEM, (n = 3) Final Report Page 61

71 10: Evaluation of the antiepileptic effect of four Ayurvedic formulations administered individually at 1000 mg/kg, once on; a) Pentylenetetrazole (PTZ) - induced seizures model Animals: This experiment was performed on male Wistar rats with cumulative unique identification number i.e. from CUIN 150 to CUIN 167. Rats were divided into six groups consisting of three rats each i.e., Normal control, Standard, Narikel Lavan, Rason Vati,Godanti Mishran and Numax treated groups. Each animal was marked for identification (Table 10.2). Table 10.1.: Group identification of rats Group Number of rats CUIN Normal Control to 152 Valproate (300mg/kg) to 155 Narikel Lavan (1000mg/kg) Rason Vati (1000mg/kg) Godanti Mishran (1000mg/kg) Numax (1000mg/kg) to to to to 167 Table 10.2: Individual identification of rats within a group Rats number Individual identification Abbreviation 1 Yellow mark on head H 2 Yellow mark on body B 3 Yellow mark on tail T Final Report Page 62

72 Table 10.3: Drug Administration S. No. Treatments No. of rats Dose 1. Normal control (No treatment) 3-2 Valproate 3 300mg/kg 3 Narikel Lavan 3 4 Rason Vati 3 5 Godanti Mishran 3 6 Numax mg/kg 1000mg/kg 1000mg/kg 1000mg/kg Table 10.4: Dosing schedule of Valproate Concentration of Valproate: 50 mg/ml CUIN Drug I.D. Rats Dose Intended vol. Actual vol. of mark body (mg/kg) of weight (g) 153 Valproate H Valproate B Valproate T Final Report Page 63

73 Table 10.5: Dosing schedule of Narikel Lavan Concentration of Narikel Lavan: 200 mg/ml CUIN Ayurvedic Drug I.D. mark Rats body weight (g) Dose (mg/kg) Intended vol. of Actual vol. of 156 Narikel Lavan 157 Narikel Lavan 158 Narikel Lavan H B T Table 10.6: Dosing schedule of Rason Vati Concentration of Rason Vati: 200 mg/ml CUIN Ayurvedic Drug I.D. mark Rats body weight (g) Dose (mg/kg) Intended vol. of Actual vol. of 159 Rason Vati H Rason Vati B Rason Vati T Final Report Page 64

74 Table 10.7: Dosing schedule of Godanti Mishran Concentration of Godanti Mishran: 200 mg/ml CUIN Ayurvedic Drug I.D. mark Rats body weight (g) Dose (mg/kg) Intended vol. of Actual vol. of 162 Godanti Mishran H Godanti Mishran 164 Godanti Mishran B T Table 10.8: Concentration of Numax: 200 mg/ml CUIN Ayurvedic Drug I.D. mark Rats body weight (g) Dose (mg/kg) Intended vol. of Actual vol. of 165 Numax H Numax B Numax T PTZ induced Seizures PTZ was prepared freshly and dissolved in normal saline and administered in a dose of (60 mg/kg, i.p), 30min. after the of the ayurvedic formulation. This dose of PTZ has been standardized earlier in our laboratory as 100% convulsant dose with minimal mortality in rats (Malhotra and Gupta, 1997). The latency to myoclonic jerks and incidence of generalized tonic clonic seizures (GTCS) with loss of righting reflex were noted. Final Report Page 65

75 Results: Godanti Mishran, Numax and Rason Vati groups have no significant change on myoclonic jerk and generalised tonic clonus (GTC) latency in PTZ-induced seizures. Narikel lavan (1000 mg/kg) showed 33.3 % protection against generalised tonic clonic seizure.however, Valporate (300 mg/kg) showed 100 % protection. Table 10.9: Effect of four Ayurvedic formulations individually, on latency of myoclonic jerks and GTC in PTZ-induces seizures. Data represented as Mean ± SEM, (n = 3) Groups Myoclonic jerks latency(sec) GTC latency(sec) Control Valproate Narikel lavan Rason vati Godanti mishran Numax 52.3± ±57.3 0±0 0±0 69.6± ± ± ± ± ± ± ±19.15 Fig 10.1: Effect of four Ayurvedic formulations individually, on latency of myoclonic jerks in PTZ-induces seizures.data represented as Mean ± SEM, (n = 3) Final Report Page 66

76 Fig 10.2: Effect of four Ayurvedic formulations individually, on GTC latency in PTZ-induced seizures.data represented as Mean ± SEM, (n = 3) Final Report Page 67

77 b) Maximal electroshock (MES)-induced seizures model Animals: This experiment was performed on male Wistar rats with cumulative unique identification number i.e. from CUIN 177 to CUIN 194. Rats were divided into six groups consisting of three rats each i.e., Normal control, Standard, Narikel Lavan, Rason Vati, Godanti Mishran and Numax treated groups. Each animal was marked for identification (Table 10.10). Table 10.10: Group identification of rats Group Number of rats CUIN Normal Control to 179 Phenytoin to 182 Narikel Lavan to 185 Rason Vati to 188 Godanti Mishran to 191 Numax to 194 Table 10.11: Individual identification of rats within a group Rats number Individual identification Abbreviation 1 Yellow mark on head H 2 Yellow mark on body B 3 Yellow mark on tail T Final Report Page 68

78 Table 10.12: Drug Administration S. No. Treatments No. of rats Doses 1. Normal control (No treatment) 3-2 Phenytoin 3 40mg/kg 3 Narikel Lavan 3 4 Rason Vati 3 5 Godanti Mishran 3 6 Numax mg/kg 1000mg/kg 1000mg/kg 1000mg/kg Table 10.13:Dosing schedule of Phenytoin Concentration of Phenytoin: 20 mg/ml CUIN Drug I.D. mark Rats body weight (g) Dose (mg/kg) Intended vol. of Actual vol. of 180 Phenytoin H Phenytoin B Phenytoin T Final Report Page 69

79 Table 10.14: Dosing schedule of Narikel Lavan Concentration of Narikel Lavan: 200 mg/ml CUIN Ayurvedic Drug I.D. mark Rats body weight (g) Dose (mg/kg) Intended vol. of Actual vol. of 183 Narikel Lavan H Narikel Lavan B Narikel Lavan T Table 10.15: Dosing schedule of Rason vati Concentration of Rason Vati: 200 mg/ml CUIN Ayurvedic Drug I.D. mark Rats body weight (g) Dose (mg/kg) Intended vol. of Actual vol. of 186 Rason Vati H Rason Vati B Rason Vati T Final Report Page 70

80 Table 10.16: Dosing schedule of Godanti Mishran Concentration of Godanti Mishran:10 mg/ml CUIN Ayurvedic Drug I.D. mark Rats body weight (g) Dose (mg/kg) Intended vol. of Actual vol. of 189 Godanti Mishran 190 Godanti Mishran 191 Godanti Mishran H B T Table 10.17: Dosing schedule of Numax Concentration of Numax: 50 mg/ml CUIN Ayurvedic formulation I.D. mark Rats body weight (g) Dose (mg/kg) Intended vol. of Actual vol. of 192 Numax H Numax B Numax T Maximal electroshock (MES) induced seizures Electroconvulsions were produced by a suprathreshold fixed current sinus wave stimulus (current intensity-70ma, duration-0.2s) delivered via ear clip electrodes using ECT unit (Ugo Basile, Italy). The animals were observed for occurrence of tonic hind limb extension (THLE), i.e., the hind limb of animals outstretched 180º to the plane of the body axis. Final Report Page 71

81 Results: Ayurvedic formulations; Narikel Lavan, Rason Vati, Godanti Mishran, and Numax did not showed protection against tonic hind limb extension (THLE) in MES-induced seizures. Phenytoin (40 mg/kg) used as standard drug, showed 100% protection Table 10.18: Effect of four Ayurvedic formulations individually, on onset and duration of THLE in MES-inducedseizures.Data represented as Mean ± SEM, (n = 3) Groups Onset of THLE (sec) Duration of THLE (sec) Control Phenytoin Narikel Lavan Rason Vati Godanti Mishran Numax 193.6± ±72.3 0±0 0± ± ± ± ± ± ± ± ±42.8 Fig 10.3: Effect of four Ayurvedic formulations individually, on onset and duration of THLE in MES-inducedseizures. Data represented as Mean ± SEM, (n = 3) Final Report Page 72

82 11: Evaluation of four Ayurvedic formulations in combination after chronic (28 days) treatment on; a) PTZ-induced seizures Animals: This experiment was performed on male Wistar rats with cumulative unique identification number i.e. from CUIN 168 to CUIN 176. Rats were divided into three groups consisting of three rats each i.e., Normal control, Standard (Valproate), Combination (Narikel Lavan, Rason Vati, Godanti Mishran and Numax) treated groups. The drugs were administered for period of 7 days. Each animal was marked for identification (Table 11.2) Table 11.1: Group identification of rats Group Number of rats CUIN Normal Control to 170 Standard (Valproate) to 173 Combination AYF s to 176 Table 11.2: Individual identification of rats within a group Rats number Individual identification Abbreviation 1 Yellow mark on head H 2 Yellow mark on body B 3 Yellow mark on tail T Table 11.3: Drug Administration S. No. Treatments No. of rats Doses 1. Normal control 3-2. Valproate mg/kg 3 Combination of AYF s a) Narikel Lavan b) Rason Vati c) Godanti Mishran d) Numax mg/kg, 500 mg/kg 40 mg/kg 150mg/kg Final Report Page 73

83 Table 11.4: Dosing schedule of Valproate Concentration of Valproate: 100mg/ml: CUIN Drug I.D. mark Rats body weight (g) 171 Valpraote H 159 Dose i.p (mg/kg) Intended vol. of Actual vol. of Valpraote B Valpraote T Table 11.5: Dosing schedule of Narikel Lavan Concentration of Narikel Lavan: 100 mg/ml CUIN Ayurvedic Drug 174 Narikel Lavan 175 Narikel Lavan 176 Narikel Lavan I.D. mark Rats body weight (g) H 220 Dose (mg/kg) Intended vol. Actual vol. of of B T Table 11.6: Dosing schedule of Rason Vati Concentration of Rason Vati: 100 mg/ml CUIN Ayurvedic Drug I.D. mark Rats body weight (g) Dose (mg/kg) Intended vol. of Actual vol. of 174 Rason Vati H Rason Vati B Rason Vati T Final Report Page 74

84 Table 11.7: Dosing schedule of Godanti Mishran Concentration of Godanti Mishran: 40mg/ml CUIN Ayurvedic Drug I.D. mark Rats body weight (g) Dose (mg/kg) Intended vol. of Actual vol. of 174 Godanti Mishran 175 Godanti Mishran 176 Godanti Mishran H B T Table 11.8: Dosing schedule of Numax Concentration of Numax: 50mg/ml CUIN Ayurvedic Drug I.D. mark Rats body weight (g) Dose (mg/kg) Intended vol. of Actual vol. of 174 Numax H Numax B Numax T PTZ induced Seizures The drugs were administered for period of 7 days. On 7 th day, PTZ was prepared freshly in normal saline and administered in a dose of (60 mg/kg, i.p), 30min. after the of the ayurvedic formulation. This dose of PTZ has been standardized earlier in our laboratory as 100% convulsant dose with minimal mortality in rats (Malhotra and Gupta, 1997). The latency to myoclonic jerks and incidence of generalized tonic clonic seizures (GTCS) with loss of righting reflex were noted. The rats were sacrificed and the brains were stored at C for biochemical estimation. Final Report Page 75

85 Results: Chronic treatment of combination of AYFs (Narikel Lavan, Rason Vati, Godanti Mishran and Numax) did not showed any protection against Generalised tonic clonic seizures in PTZ induced seizures. Table 11.9: Effect of combination of four Ayurvedic formulations on latency of myoclonic jerks and GTC in PTZ-inducedseizures.Data represented as Mean ± SEM, (n = 3) Groups Myoclonic jerk latency(sec) GTC latency(sec) Control Valproate Combination 52.3± ±57.3 0±0 0± ± ± Fig 11.1: Effect of combination of four Ayurvedic formulations on latency of myoclonic jerks in PTZ-inducedseizures.Data represented as Mean ± SEM, (n = 3) Final Report Page 76

86 Fig 11.2: Effect of combination of four Ayurvedic formulations on latency of GTC in PTZ-induced seizures. Data represented as Mean ± SEM, (n = 3) Fig 11.3: Effect of Ayurvedic formulations on Percentage protection against GTC in PTZ-induced seizures. Final Report Page 77

87 b) MES-induced seizures Animals: This experiment was performed on male Wistar rats with cumulative unique identification number i.e. from CUIN 194 to CUIN 202. Rats were divided into three groups consisting of three rats each i.e., Normal control, Standard (Phenytoin), Combination (Narikel Lavan, Rason vati, Godanti Mishran and Numax) treated groups. The drugs were administered for period of 7 days. Each animal was marked for identification (Table 11.11) Table 11.10: Group identification of rats Group Number of rats CUIN Normal Control to 196 Standard (Phenytoin) to 199 Combination AYF s to 202 Table 11.11: Individual identification of rats within a group Rats number Individual identification Abbreviation 1 Yellow mark on head H 2 Yellow mark on body B 3 Yellow mark on tail T Table 11.12: Drug Administration S. No. Treatments No. of rats Dose and route of 1. Normal control 3-2. Phenytoin 3 40mg/kg, i.p. 3 Combination of AYF s a) Narikel Lavan b) Rason Vati c) Godanti Mishran d) Numax mg/kg, p.o 500 mg/kg, p.o. 40 mg/kg, p.o. 150mg/kg, p.o. Final Report Page 78

88 Table 11.13: Dosing schedule of Phenytoin Concentration of Phenytoin: 10mg/ml: CUIN Drug I.D. mark Rats body weight (g) Dose i.p (mg/kg) Intended vol. of Actual vol. of 197 Phenytoin H Phenytoin B Phenytoin T Table 11.14: Dosing schedule of Narikel Lavan Concentration of Narikel Lavan: 100 mg/ml CUIN Ayurvedic Drug I.D. mark Rats body weight (g) Dose (mg/kg) Intended vol. of ( ml) Actual vol. of ( ml) 200 Narikel Lavan H Narikel Lavan B Narikel Lavan T Table 11.15: Dosing schedule of Rason Vati Concentration of Rason Vati: 100 mg/ml CUIN Ayurvedic Drug I.D. mark Rats body weight (g) 200 Rason Vati H 183 Dose (mg/kg) Intended vol. of Actual vol. of Rason Vati B Rason Vati T Final Report Page 79

89 Table 11.16: Dosing schedule of Godanti Mishran Concentration of Godanti Mishran: 40 mg/ml CUIN Ayurvedic Drug 200 Godanti Mishran 201 Godanti Mishran 202 Godanti Mishran I.D. mark Rats body weight (g) H 183 Dose (mg/kg) Intended vol. of Actual vol. of B T Table 11.17: Dosing schedule of Numax Concentration of Numax: 50 mg/ml CUIN Ayurvedic Drug I.D. mark Rats body weight (g) 200 Numax H 183 Dose (mg/kg) Intended vol. of Actual vol. of Numax B Numax T Maximal electroshock induced seizures Same procedure was followed except the drugs were administered for 7 days and on 7 th day, Ayurvedic formulations were administered 30 min before application of electric shock. Final Report Page 80

90 Results: Chronic treatment of combination of AYFs (Narikel Lavan, Rason Vati, Godanti Mishran and Numax) did not showed any protection against tonic clonic hind limb extension in MES- induced seizures model. Table 11.18: Effect of four Ayurvedic formulations in combination, on onset and duration of THLE in MES-induced seizures Groups Onset of THLE (sec) Duration of THLE(sec) Control Valproate Combination ± ± ±0 0± ± ± 6.0 Fig 11.4 : Effect of four Ayurvedic formulations in combination, on onset and duration of THLE in MES-induced seizures Final Report Page 81

91 Fig 11.5: Percentage protection against THLE in MES-induces seizures after treatment with combination of ayurvedic formulations Final Report Page 82

92 12: Evaluation of effect of combination of four ayurvedic formulations on seizuresinduced oxidative stress in followingexperimental model in rats. a) PTZ-induced seizures b) MES-induced seizures Preparation of homogenate: The brain tissue of rats with CUID (168 to176 and 194 to 202) used in experiment no and 11.2, were stored at 80 0 C for biochemical estimation.brain tissues were homogenized with 10 times (w/v) 0.1 sodium phosphate buffer (ph 7.4) for MDA and GSH and with 50mM Tris chloride buffer (ph 8.2) for SOD activity. Estimation of lipid peroxidation (MDA) Malondialdehyde (indicator of lipid peroxidation) was estimated as described by Ohkawa et al, The reagents acetic acid 1.5 ml (20 %) ph 3.5, 1.5 ml thiobarbituric acid (0.8 %w/v) and 0.2 ml sodium dodecyl sulfate (8.1 %w/v) were added to 0.1 ml of processed tissue sample (homogenate). The mixture was then heated at C for 60 min. The mixture was cooled using tap water followed by addition of 5 ml of n-butanol/pyridine (15:1). The mixture was then centrifuged at 4000 rpm for 10 min. The organic layer was separated and absorbance was measured at 532 nm using spectrophotometer (Specord 200, Analytic Jena AG, Germany). Estimation of reduced glutathione Reduced glutathione (GSH) were measured according to the method described by (Ellman, 1959). Tissue homogenate was centrifuged with 5 % trichloroacetic acid to precipitate out the proteins. To 0.1 ml of this supernatant, 2 ml of 0.3M phosphate buffer (ph 8.4), 0.5 ml of 5 5 dithiobis (2-nitrobenzoic acid) (DTNB) and 0.4 ml of double distilled water was added. The mixture was vortexed and the absorbance read at 412nm within 15 min by spectrophotometer (Specord 200, Analytic Jena AG, Germany). Final Report Page 83

93 Estimation of Superoxide dismutase Tissue homogenate prepared in 50mM Tris chloride buffer (ph 8.2) was used. The protocol had three steps Blank, control and samples. In blank 1000 ul of buffer was added. In control 950 ul of buffer and 50 ul of Pyragallol was added and in each sample 900 ul of buffer, 50 ul of Pyragallol and 50ul of respective samples were added. Total volume of the reaction mixture was 1 ml. The mixture was vortexed and the absorbance read at 420 nm for 3 min at an interval of 30 sec by spectrophotometer (Specord 200, Analytic Jena AG, Germany). The data was calculated as change in O.D per minute Final Report Page 84

94 Results: a) PTZ-induced seizure model (i) MDA level in rat s brain The MDA level inptz group was significantly (p<0.001) elevated (424.5 ± 45.6 nmol/g wet tissue) as compared to the control group ( ± 35.3nmol/g wet tissue) indicating oxidative stress. Valproate (300mg/kg), significantly (p<0.001) reduced MDA level to (127.4 ± 21.6 nmol/g wet tissue) as compared to PTZ group. The combination offour Ayurvedic formulationsalso reducedmda level significantly (p<0.05) as compared to PTZ group. Table 12.1: Effect of combination of four Ayurvedic formulations on malondialdehyde (MDA) level in whole brain of rats after PTZ-induced seizures. Data represented as Mean ± S.E.M (n = 3), * p<0.05, *** p< 0.001, a- compared to control group, b- compared to PTZ treated group. S. No Groups MDA (nm of LPO /g wet tissue) 1 Control ± PTZ treated 424.5± 45.6 a*** 3 Valproate (300mg/kg) 127.4±21.6 b*** 4 4 AYF s combination 220.5±45.7 b* Fig 12.1: Effect of combination of AYFs on malondialdehyde (MDA) level in whole brain of rats after PTZ-induced seizures Data represented as Mean ± S.E.M (n = 3),* p<0.05, *** p< 0.001, a- compared to control group.b- compared to PTZ treated group. Final Report Page 85

95 ii) GSH level in rat s brain The GSH level was significantly (p< 0.001) decreased in PTZ group (98.0 ± 25.9 µg/g wet tissue) as compared to the control group (235.2 ± 74 µg/g wet tissue). However in valproate (300 mg/kg, p.o), there was significantly (p<0.05) high GSH level as compared to PTZ group. The combination of four Ayurvedic formulations have also been found to significantly increase (161.7 ± 35 µg/g wet tissue) the GSH level as compared to PTZ treated group. Table 12.2: Effect of combination of four Ayurvedic formulations on glutathione enzyme (GSH) level in whole brain of rats after PTZ-induced seizures Data represented as Mean ± S.E.M (n = 3), * p<0.05, *** p< 0.001, a- compared to control group, b- compared to PTZ-treated group. S. No Groups GSH (µg/g wet tissue) 1 Control 235.2± PTZ treated 98.0±25.9 a*** 3 Valproate (300mg/kg) 186.2±19.6 b* 4 4 AYF s combination 161.7±35 b* Fig 12.2: Effect of combination of four Ayurvedic formulations on glutathione enzyme (GSH) level in whole brain of rats after PTZ-induced seizures Data represented as Mean ± S.E.M (n = 3), * p<0.05, *** p< 0.001, a- compared to control group, b- compared to PTZ-treated group. Final Report Page 86

96 (iii) SOD level in PTZ- induced seizures Significant difference was observed in the brain SOD levels of all the groups. In PTZ treated group there was significant decrease in the % SOD (23.7 %) as compared to the control group (70.0 ±2.6), indicating oxidative stress with PTZ. Valproate (300mg/kg) has restored the SOD level to (35.4 %) towards normal levels. When the combination of four Ayurvedic formulations was administered together, there was a no significant increase in SOD level as compared to PTZ group. Table 12.3: Effect of combination of Ayurvedic formulations on superoxide dismutase (SOD) level in whole brain of rats after PTZ-induced seizures.data represented as Mean ± S.E.M (n = 3). S.No Groups SOD (% control) 1 Control 70.0 ±2.6 2 PTZ treated 23.7±1.2 3 Valproate (300mg/kg) 35.4± AYF s combination 26.8±9.3 Fig 12.3: Effect of combination of four Ayurvedic formulations on superoxide dismutase (SOD) level in whole brain of rats after PTZ-induced seizures. Data represented as Mean ± S.E.M (n = 3). Final Report Page 87

97 b) MES-induced seizures model (i) MDA level in rat s brain There was a significantly (p<0.001) higher MDA level in MES group (488.2 ± 50.1 nmol/g wet tissue) as compared to the control group (107.8 ± 35.3nmol/g wet tissue) indicating oxidative stress with MES. Phenytoin (PHT) at 40mg/kg dose,reduced the MDA level to (146.0 ± 17.0 nmol/g wet tissue) significantly (p<0.05) as compared to MES group. When the combination of four Ayurvedic formulations was administered, there was no significantreduction in MDA level as compared to MES group. Table 12.4: Effect of combination of four Ayurvedic formulations on malondialdehyde (MDA) level in whole brain of rats after MES-induced seizures.data represented as Mean ± S.E.M (n = 3), * p<0.05, *** p< 0.001, a- compared to control group,b- compared to MES-treated group. S.No Groups MDA (nm of LPO /g wet tissue) 1 Control 107.8± MES treated 488.2±50.1 ***a 3 PHT(40) 146.0±17 *b 4 AYF s combination 205.8±88.2 Fig 12.4: Effect of combination of four Ayurvedic formulations on malondialdehyde (MDA) level in whole brain of rats after MES-induced seizures.data represented as Mean ± S.E.M (n = 3), * p<0.05, *** p< 0.001, a- compared to control group,b-compared to MES-treated group. Final Report Page 88

98 b) GSH level in rat s brain Significant difference in brain GSH levels was seen in all the groups. In MES group a significant decrease in GSH level was observed(107.8 ± 19.6 µg/g wet tissue) as compared to the control group (235.2 ± 74 µg/g wet tissue). PHT (40 mg/kg, p.o) has reversed the MES induced reduction in GSH level.the combination of four Ayurvedic formulations did not cause significant increase in the GSH level as compared to MES treated group. Table 12.5: Effect of combination of four Ayurvedic formulations on glutathione enzyme (GSH) level in whole brain of rats after MES-induced seizures. Data represented as Mean ± S.E.M (n = 3), * p<0.05, *** p< 0.001, a- compared to control group, b- compared to MES-treated group. S.No Groups GSH (µg/g wet tissue) 1 Control 235.2± MES treated 107.8±19.6 ***a 3 PHT(40) 166.6±64.2 *b 4 AYF s combination 154.4±86.0 Fig 12.5: Effect of combination of four Ayurvedic formulations on glutathione enzyme (GSH) level in whole brain of rats after MES-induced seizures. Data represented as Mean ± S.E.M (n = 3), * p<0.05, *** p< 0.001, a- compared to control group, b- compared to MES-treated group. Final Report Page 89

99 c) SOD level in rat s brain Significant difference was observed in the brain SOD levels of all the groups. In MES only groupthere was significant reduction in the % SOD as compared to the control group, indicating oxidative stress with MES. PHT (40mg/kg) significantly increased the SOD level to (35.21 %) as compared to MES group (29.1%). When the combination offour Ayurvedic formulations was administered, there was no significant increase in SOD level was found as compared to MES group. Table 12.6: Effect of combination of four Ayurvedic formulations on superoxide dismutase enzyme (SOD) level in whole brain of rats after MES-induced seizures. Data represented as Mean ± S.E.M (n = 3), * p<0.05, *** p< 0.001, a-compared to control group, b-compared to MES-treated group. S.No Groups SOD (% control) 1 Control 70.0 ±2.6 2 MES treated 29.1± PHT (40) 35.2± AYF s combination 31.5±8.0 Fig 12.6: Effect of combination of four Ayurvedic formulations on superoxide dismutase enzyme (SOD) level in whole brain of rats after MES-induced seizures. Data represented as Mean ± S.E.M (n = 3), * p<0.05, *** p< 0.001, a- compared to control group, b- compared to MES-treated group. Final Report Page 90

100 13: Evaluation of the antidepressant activity of combination of four ayurvedic formulations in forced swim test. Animals: This experiment was performed on male Wistar rats ( g), with cumulative unique identification number i.e. from CUIN 203 to CUIN 211. Rats were divided into three groups consisting of three rats each i.e., Normal control, Standard(Flouxetine), Combination (Narikel Lavan, Rason Vati,Godanti Mishran and Numax) treated groups. Each animal was marked for identification (Table 13.2) Table 13.1: Group identification of rats Group Number of rats CUIN Normal Control to 205 Standard (Flouxetine ) to 208 Combination AYF s to 211 Table 13.2: Individual identification of rats within a group Rats number Individual identification Abbreviation 1 Yellow mark on head H 2 Yellow mark on body B 3 Yellow mark on tail T Table 13.3: Drug Administration S. No. Treatments No. of rats Doses 1. Normal control 3-2. Fluoxetine 3 7 mg/kg, p.o 3 Combination of AYF s a) Narikel Lavan b) Rason Vati c) Godanti Mishran d) Numax mg/kg, p.o. 500 mg/kg, p.o. 40 mg/kg, p.o. 150mg/kg, p.o. Final Report Page 91

101 Table 13.4: Dosing schedule of Fluoxetine Concentrationof Fluoxetine: 4 mg/ml: CUIN Drug I.D. mark Rats body weight (g) Dose p.o (mg/kg) Intended vol. of Actual vol. of 206 Fluoxetine H Fluoxetine B Fluoxetine T The volume calculated was given in divided doses Table 13.5: Dosing schedule of Narikel Lavan Concentration of Narikel Lavan: 100 mg/ml CUIN Ayurvedic Drug 209 Narikel Lavan 210 Narikel Lavan 211 Narikel Lavan I.D. mark Rats body weight (g) Dose (mg/kg) Intended vol. of Actual vol. of H B T Table 13.6: Dosing schedule of Rason Vati Concentration of Rason Vati: 100 mg/ml CUIN Ayurvedic Drug 209 Rason Vati 210 Rason Vati 211 Rason Vati I.D. mark Rats body weight (g) Dose (mg/kg) Intended vol. of Actual vol. of H B T Final Report Page 92

102 Table 13.7: Dosing schedule of Godanti Mishran Concentration of Godanti Mishran: 10mg/ml CUIN Ayurvedic Drug 209 Godanti Mishran 210 Godanti Mishran 211 Godanti Mishran I.D. mark Rats body weight (g) H 244 Dose (mg/kg) Intended vol. of Actual vol. of B T Table 13.8: Dosing schedule of Numax Concentration of Numax: 50 mg/ml CUIN Ayurvedic Drug I.D. mark Rats body weight (g) Dose (mg/kg) Intended vol. of Actual vol. of 209 Numax H Numax B Numax T Final Report Page 93

103 Forced swim test The forced swim animal model is a common behavioral test for assessing in rodents and testing the efficiency of anti- depressants drugs. As originally developed by Porsolt in 1977, a rat is forced to swim for 15 min in a narrow cylinder from which there is no escape. After an initial struggle the rat becomes immobile, it floats while making only movements necessary to keep its head above the water. Administration of clinically effective antidepressant drugs prior to a second 5 minutes test swim session decreases the time an animal is immobile. As compared to saline treated animals, thus the decrease in the immobility time is used as an index of anti-depressant activity. Rats were placed individually in plexi glass cylinder, 25 cm height with a 15cm internal diameter that were filled by water (25 C) to a depth of 10 cm. This depth was sufficient to keep adult rats from supporting themselves by placing their paws or tails on the base of the cylinder. Water was changed between each swim session. There were two sessions; the first was 15 min pre-test swim and 24 hours later, a second 5 min swim test. The pre-test facilitates the development of immobility during the test session and increases the sensitivity for detecting antidepressant behavioral effects. The 5 min test was used for analysis of behavior. The rats received Fluoxetine three times orally following the initial 15 m pre test, 24 h, 5 h and 1 hr prior to 5 min swim test. Behavioral analysis of rats: A time-sampling technique was used, whereby the predominant behavior in each 5 seconds interval of the 5 minutes test swim was scored. Immobility was assigned when rat demonstrated floating behavior with no additional activity other than that required to keep its head above the water. Swimming behavior was defined as movement on the water surface throughout the swim chamber, with the rat in horizontal position; climbing behavior was defined as upward directed vigorous thrashing movements with the forepaws, usually along the side of the swim chamber. Final Report Page 94

104 Results: The results indicate that the combination of Ayurvedic formulations (Narikel Lavan, Rason Vati, Godanti Mishran and Numax) did not cause any significant decrease in immobility as compared to control group. Fluoxetine (3.3mg/kg, p.o.) used as a standard drug, showed increased mobility which was indicated by significant increase in counts in climbing and swimming in comparison to control. Table 13.9: Effect of four Ayurvedic formulations in combination on immobility, climbing and swimming counts of rats in forced swim test.data represented as Mean ± SEM, (n = 3) Groups Immobilty Climbing Swimming Control 37.0 ± ± ± 1.2 Fluoxetine 15.3 ± 1.2 ***a 23.0 ± 1.2 *a 21.7 ± 1.5 *a AYF s combination 33.3 ± ± ± 0.8 Fig 13.1: Effect of four Ayurvedic formulations on immobility, climbing and swimming counts in forced swim test. Data represented as Mean ± S.E.M (n = 3), * p<0.05, *** p< 0.001, a- compared to control group. Final Report Page 95

105 14: Evaluation of the antidepressant activity of ayurvedic formulations in combination using tail suspension test. Animals: This experiment was performed on male Wistar rats with cumulative unique identification number i.e. from CUIN 212 to CUIN 217. Rats were divided into two groups consisting of three rats each i.e., Normal control, Combination of Ayurvedic Formulations (Narikel Lavan, Rason Vati,Godanti Mishran and Numax) treated groups. Each animal was marked for identification (Table 14.2). Table 14.1: Group identification of rats Group Number of rats CUIN Normal Control to 214 Combination to 217 Table 14.2: Individual identification of rats within a group Rats number Individual identification 1 Yellow mark on head 2 Yellow mark on body 3 Yellow mark on tail Table 14.3: Drug Administration S. No. Treatments No. of rats Doses 1. Normal control 3-2 Combination of AYF s a) Narikel Lavan b) Rason Vati c) Godanti Mishran d) Numax mg/kg, p.o. 500 mg/kg, p.o. 40 mg/kg, p.o. 150mg/kg, p.o. Final Report Page 96

106 Table 14.4: Dosing schedule of Narikel Lavan Concentration of Narikel Lavan: 100 mg/ml CUIN Ayurvedic Drug I.D. mark Rats body weight (g) Dose (mg/kg) Intended vol. of Actual vol. of 215 Narikel Lavan H Narikel Lavan B Narikel Lavan T Table 14.5: Dosing schedule of Rason Vati Concentration of Rason Vati: 100 mg/ml CUIN Ayurvedic Drug 215 Rason Vati 216 Rason Vati 217 Rason Vati I.D. mark Rats body weight (g) Dose (mg/kg) Intended vol. of Actual vol. of H B T Table 14.6: Dosing schedule of Godanti Mishran Concentration of Godanti Mishran: 10 mg/ml CUIN Ayurvedic Drug 215 Godanti Mishran 216 Godanti Mishran 217 Godanti Mishran I.D. mark Rats body weight (g) Dose (mg/kg) Intended vol. of Actual vol. of H B T Final Report Page 97

107 Table 14.7: Dosing schedule of Numax Concentration of Numax: 50mg/ml CUIN Ayurvedic Drug I.D. mark Rats body weight (g) Dose (mg/kg) Intended vol. of Actual vol. of 215 Numax H Numax B Numax T Tail Suspension test: The tail suspension test was performed for a 10min test session. Rats were suspended 20cm above the floor in a visually isolated area by adhesive tape placed 3 cm from the tip of the tail. Immobility duration was recorded for last 4 min during 6 min. Rats were considered immobile only when they hung passively and were completely immobile. The drug was administered for 7 days. On 7 th day single was done 1 h prior to the test. Final Report Page 98

108 Result: The result indicates that the combination of Ayurvedic formulations (Narikel Lavan, Rason Vati, Godanti Mishran and Numax) did not cause any significant change in mobility as compared to control. Table 14.8: Effect of four Ayurvedic formulations in combinationon mobility and immobility counts of rats in tail suspension test.data represented as Mean ± SEM, (n = 3) Groups Mobility ( Sec) Immobility (Sec) Control AYF s Combination 78.0 ± ± ± ± 5.6 Fig 14.1: Effect of four Ayurvedic formulations on mobility and immobility counts of rats in tail suspension test.values represent Mean ± S.E.M (n = 3). Final Report Page 99

109 Methodologies & Results of Objective II

110 Objective II: To evaluate the effect of ayurvedic formulations (Godanti Mishran Narikel Lavan, Numaxand Rason Vati,) on haemodynamic parameters 15. To evaluate of the effect of four Ayurvedic formulations individually and in combination on blood pressure in ratsafter 7 days oral treatment 16. To evaluate of the effect of four Ayurvedic formulations individually and in combination on blood pressure in ratsafter 15 days oral treatment Final Report Page 100

111 15: Effect of Ayurvedic formulations individually and in combination on blood pressure in rats after 7 days oral treatment. Animals: The experiment was performed on male Wistar rats ( g) with cumulative unique identification number (CUIN) i.e. from CUIN 218 to CUIN 235. Rats were divided into six groups consisting of three rats each i.e.,vehicle control Godanti Mishran, Narikel Lavan, Numax, Rason Vati and Combination of Narikel Lavan, Rason Vati, Godanti Mishran and Numax. Table 15.1: Group identification of rats Treatment group Number of rats CUIN Vehicle Control to 220 Godanti Mishran to 223 Narikel Lavan to 226 Numax to 229 Rason Vati to 232 Combination of Godanti Mishran, Narikel Lavan, Numax, Rason Vati to 235 Each animal was marked for identification (Table 15.2) Table 15.2: Individual identification of rats within a group Rat number Individual identification Abbreviation 1 Yellow mark on head H 2 Yellow mark on Body B 3 Yellow mark on tail T Final Report Page 101

112 Table 15.3: Drug Administration S. No. Treatment No. of rats Dose 1. Vehicle control (0.1 % CMC) 3 1 ml 2. Godanti mishran 3 40 mg/kg, p.o. 3. Narikal Lavan mg/kg, p.o. 4. Numax mg/kg, p.o. 5. Rason vati mg/kg, p.o. 6. Combination of AYF s a) Narikel Lavan b) Rason Vati c) Godanti Mishran d) Numax mg/kg, p.o. 500 mg/kg, p.o. 40 mg/kg, p.o. 150 mg/kg, p.o. Table 15.4: Dosing schedule of Vehicle Concentration of vehicle: 0.1 % v/v carboxy methyl cellulose (CMC) CUIN I.D. mark Rats body weight (g) Dose (mg/kg) Intended vol. of Actual vol. of 218 Vehicle H Vehicle B Vehicle T Final Report Page 102

113 Table 15.5: Dosing schedule of Godanti Mishran Concentration of Godanti Mishran: 15 mg/ml CUIN Ayurvedic Drug 221 Godanti Mishran 222 Godanti Mishran 223 Godanti Mishran I.D. mark Rats body weight (g) H 238 Dose (mg/kg) Intended vol. of Actual vol. of B T Table 15.6: Dosing schedule of Narikel Lavan Concentration of Narikel Lavan: 100 mg/ml CUIN Ayurvedic Drug 224 Narikel Lavan 225 Narikel Lavan 226 Narikel Lavan I.D. mark Rats body weight (g) H 210 Dose (mg/kg) Intended vol. Actual vol. of of B T Table 15.7: Dosing schedule of Numax Concentration of Numax: 50mg/ml CUIN Ayurvedic I.D. Rats Dose Intended vol. Actual vol. of Drug mark body (mg/kg) of weight (g) 227 Numax H Numax B Numax T Final Report Page 103

114 Table 15.8: Dosing schedule of Rason Vati Concentration of Rason Vati: 150 mg/ml CUIN Ayurvedic Drug 230 Rason Vati 231 Rason Vati 232 Rason Vati I.D. mark Rats body weight (g) H 238 Dose (mg/kg) Intended vol. of Actual vol. of B T Table 15.9: Dosing schedule of combination of Ayurvedic formulations 11 Concentration of Godanti Mishran: 15 mg/ml, Numax: 50mg/ml, Rason Vati: 150 mg/ml, Narikel Lavan: 100 mg/ml. CUIN I.D. mark Rats body weight (g) Ayurvedic Drug Dose (mg/kg) Actual vol. of of Narikal lavan, Numax and Rason vati Actual vol. of of Godanti mishran 233 H 243 Godanti B 218 mishran, Narikal lavan, T 222 Numax, 150 Rason Vati Final Report Page 104

115 Blood pressure measurement: The blood pressure of rats in each group was measured by tail cuff plethysmography (non-invasive method) on day 0 (baseline) and 7 of treatment. On day 7, the blood pressure of rats was measured after 2 hours of of last dose. 1.Training: All animals were given training, habituation to the protocol and to produce repeatable results. Two to three training sessions were given to acclimatize the animals. When the animal has been trained, after a few minutes distinct pulse was measurable on the tail. 2.Restrainer: The Perspex restraint cages were selected to fit the animal comfortably. The rats were placed in the Perspex cylinder restraint cage and the depth was adjusted to restrict forward and backward movement within the tube. 3. External stimuli: To avoid the rat movement, sudden motion and sounds were restricted as much as possible. Restrainer was covered with a cloth to reduce the impact of external stimuli. 4. Temperature maintenance:as warming the animal (rats) improves blood circulation in the tail and the signal to noise ratio in the recording. The animals were preheated to C and maintained at that temperature during the test. 5. Tail cuff positioning: The tail cuff was used to occlude blood flow in the tail and thereby interrupt the pulse that is measurable in the caudal artery. The tail cuff was positioned at the proximal end of the tail.. The IR cuff sensor 9.5, was connected to the tail of the animal inside the restrainer. It was checked that the sensor just fitted to the tail. The sensor was placed between the midpoint of tail and tail end (spinal column).the animal was allowed to relax and become inactive before starting measurements. 6. Mechanical vibrations: The transducer used to make the pulse measurements is very sensitive and subject to vibrations. It was ensured that mechanical vibrations from other laboratory devices do not affect the transducer. 7. Signals measurement: The basic signals recorded by the NIBP200A, Biopac system, Inc. were the cuff pressure and the caudal artery pulse Final Report Page 105

116 8. Systolic and Diastolic pressure measurement: Systolic blood pressure (SBP) occurs when the cuff pressure corresponds to the restoration of the first caudal artery pulse. However the pressure at the first highest peak in the pulse corresponds to diastolic blood pressure (DBP). 9. Mean arterial blood pressure:mean arterial blood pressure was calculated by using the following formula: Mean Arterial Blood Pressure (MABP) = 2 x SBP + DBP mm Hg 3 Final Report Page 106

117 Results: The mean arterial pressure of rats in each group was measured on day 0 (baseline) and day 7 of treatment in each group. There was no significant difference observed in the blood pressure after 7 days treatment as compared to baseline (p < 0.005) in all the treatment groups. The post hoc analysis revealed no significant difference mean arterial pressure between all the groups after 7 days treatment [F (5, 16) = 0.433], p = 0.817]. Also, there was no significant difference found in baseline mean arterial pressure of rats within and in between the groups. Table 15.10: Mean Arterial Pressure (in mm Hg) of rats in different treatment groups of Ayurvedic formulations at different time interval. Data represented as Mean ± SEM (n=3) S. No. Treatment Groups Baseline (on day 0) 1. Vehicle Control ± Godanti mishran (40 mg/kg, p.o.) ±3.8 After treatment (on day 7) ± ± Narikal Lavan (300 mg/kg, p.o.) ± ± Numax (150 mg/kg, p.o.) ± ± Rason vati (500 mg/kg, p.o. ) ± ± Combination of Godanti mishran (40 mg/kg, p.o.) Narikal Lavan (300 mg/kg, p.o.) ± ± 12.3 Numax (150 mg/kg, p.o.) Rason vati (500 mg/kg, p.o. ) Final Report Page 107

118 Fig.15.1 : Effect of Ayurvedic formulations individually and in combination on mean arterial pressure (in mm Hg) in rats at baseline (day 0) after treatment (day 7). Data represented as mean ± SEM (n=3). Final Report Page 108

119 16: Evaluation of Ayurvedic formulations alone and in combination on blood pressure in rats after 14 days treatment Animals: The experiment was performed on male Wistar rats ( g) with cumulative unique identification number (CUIN) i.e. from CUIN 236 to 265.Rats were divided into five groups consisting of six rats each i.e. Narikel Lavan, Godanti Mishran, Numax, Rason Vati and Combination (Narikel Lavan, Rason Vati, Godanti Mishran and Numax. Each group with CUIN of rat identification is given in Table 16.1 Table 16.1: Group identification of rats Treatment group Number of rats CUIN Godanti mishran to 241 Narikel Lavan to 247 Numax to 253 Rason vati to 259 Combination of Godanti mishran, Narikel lavan, Numax, Rason vati to 265 Drug Treatment: All the drugs were given orally at the calculated rat dose (Table 16.2) for 14 days. In combination group, each formulation was administered at intervals of 30 minutes. Table 16.2: Doses of AYFs for by oral route in rats S. No. Treatment No. of rats Dose 1. Godanti mishran 6 40 mg/kg, p.o. 2. Narikal Lavan mg/kg, p.o. 3. Numax mg/kg, p.o. 4. Rason vati mg/kg, p.o. 5. Combination of AYF s a) Narikel Lavan b) Rason Vati c) Godanti Mishran d) Numax mg/kg, p.o. 500 mg/kg, p.o. 40 mg/kg, p.o. 150 mg/kg, p.o. Final Report Page 109

120 Table 16.3: Dosing schedule of Narikel Lavan Concentration of Narikel Lavan: 150 mg/ml S.No. CUIN I.D. mark Body weight (g) Dose (mg/kg) Intended vol. of Actual vol. of H B T T HBT HBT Table 16.4: Dosing schedule of Godanti Mishran Concentration of Godanti Mishran: 15 mg/ml S.No. CUIN I.D. mark Body weight (g) Dose (mg/kg) Intended vol. of Actual vol. of H B T HB HBT BT Final Report Page 110

121 Table 16.5: Dosing schedule of Numax Concentration of Numax: 50mg/ml S. No. CUIN I.D. mark Body weight (g) Dose (mg/kg) Intended vol. of Actual vol. of H B T HB BT U Table 16.6:Dosing schedule of Rason Vati Concentration of Rason Vati: 150 mg/ml S. No. CUIN I.D. mark Body weight (g) Dose (mg/kg) Intended vol. of Actual vol. of H B T HB BT U Final Report Page 111

122 Table 16.7: Dosing schedule of combination of four Ayurvedic formulations Concentration of Narikel lavan (NL): 150 mg/kg, Godanti mishran (GM): 15 mg/kg, Numax (Nu): 50 mg/kg & Rason Vati (RV):150 mg/kg CUIN I.D. mark Body weight (g) Actual vol. of of NL Actual vol. of of GM Actual vol. of of Nu Actual vol. of of RV 260 H B T HB BT U Final Report Page 112

123 Results: The mean arterial pressure of rats in each group was measured on day 0 (baseline) and on day 15 of treatment in each group. There was no significant difference found in baseline mean arterial pressure of rats within and in between the groups. There was no significant difference observed in the blood pressure after 15 days treatment as compared to baseline (p < 0.05) in all the treatment groups. The post hoc analysis revealed no significant difference mean arterial pressure between all the groups after 15 days treatment [F (4, 29) = 1.444], p = 0.249]. Table 16.8: Mean Arterial Pressure (in mmhg) of rats in different treatment groups at Day 0 (baseline) and on Day 15 after treatment. Data represented as Mean ± SEM (n=6) S. No. Treatment Groups Baseline (on day 0) After treatment (on day 15) 1. Narikal Lavan (300 mg/kg, p.o.) ± ± Godanti mishran (40 mg/kg, p.o.) ± ± Numax (150 mg/kg, p.o.) ± ± Rason vati (500 mg/kg, p.o.) ± ± Combination of Godanti mishran (40 mg/kg, p.o.) Narikal Lavan (300 mg/kg, p.o.) Numax (150 mg/kg, p.o.) Rason vati (500 mg/kg, p.o. ) ± ± 4.56 Final Report Page 113

124 Fig 16.1 : Effect of Ayurvedic formulations alone and in combination on mean arterial pressure (in mm Hg) in rats at Day 0(baseline) and at Day 15 after treatment. Data represented as Mean ± SEM (n=6). Final Report Page 114