Pharmacological Evaluation of a Polyherbal Formulation Wilmer Syrup, for It S Nootropic

“PHARMACOLOGICAL EVALUATION OF A POLYHERBAL FORMULATION WILMER SYRUP, FOR IT’S NOOTROPIC ACTIVITY”

M.Pharm, Dissertation Protocol Submitted to the RAJIV GANDHI UNIVERSITY OF HEALTH SCIENCES,

BANGALORE, KARNATAKA.

By POONAM YADAV B. Pharm

Under the Guidance of Mr. BHEEMACHARI Asst. Professor. Department of Pharmacology, N. E. T. Pharmacy College, Raichur.

DEPARTMENT OF PHARMACOLOGY N. E. T. PHARMACY COLLEGE RAICHUR – 584103 2007-2008 RAJIV GANDHI UNIVERSITY OF HEALTH SCIENCES, BANGALORE, KARNATAKA.

ANNEXURE II PROFORMA FOR REGISTRATION OF SUBJECTS FOR DISSERTATION POONAM YADAV W/O Ashok Kumar Yadav Name of the candidate 1 Vill- Mangalpur (Tinhiya) and Address: Post- Sarhari Dist- Gorakhpur 273165 (UP) N. E. T. Pharmacy College , 2 Name of the Institution: Raichur- 584103

3 Course of study and Subject: Master of Pharmacy in Pharmacology.

4 Date of admission to the Course: 2nd May 2007

5 Title of the topic: “PHARMACOLOGICAL EVALUATION OF A POLYHERBAL FORMULATION WILMER SYRUP, FOR IT’S NOOTROPIC ACTIVITY” 6 Brief resume of the intended work: 6.1: Need for the study: ENCLOSURE -I 6.2:Review of the literature ENCLOSURE -II 6.3:Main objective of the study ENCLOSURE –III 7 Material and methods: 7.1: Source of the data ENCLOSURE -IV 7.2: Methods of the collection of the data ENCLOSURE - V 7.3: Does the study require any investigations or interventions to be conducted on patients other human or animals? If so, please describe briefly. YES (mice and rats) 7.4: Has ethical clearance been obtained from your institute in case of 7.3 YES:IAEC NO: 576/2002/bc/IAEC/CPCSEA 8 List of References ENCLOSURE-VI

9 Signature of the candidate: (POONAM YADAV)

10 Remarks of the guide:,,,

11 Name and designation of 11.1 Guide: Mr BHEEMACHARI, Asst. Professor. Dept. of Pharmacology, N. E. T. Pharmacy College, Raichur- 584103.

11.2 Signature:

11.3 Co-guide: ------

11.4 Signature: ------

11.5 Head of the department: Mr BHEEMACHARI, Asst. Professor. Dept. of Pharmacology, N. E. T. Pharmacy College, Raichur- 584103.

11.6 Signature:

12 12.1 Remarks of the Chairman PROF. H. DODDAYYA and Principal: Principal, N.E.T. Pharmacy College, Raichur-584103.

12.2 Signature:

ENCLOSURE – I 6. Brief resume of the intended work:

6.1: Need for the study:

Learning is the most characteristic attributes of the man and also of higher animals. Learning is defined as the ability to alter behaviour on the basis of experience1.

Memory is special facility of brain which retains the events developed during the process of learning and both are mediated by nervous system2. Once memories have been stored in the brain, it becomes the part of brain –process mechanism when it will recall in future3. Learning and memory are closely related, all learning involves memory but all memory not involves to learning4.

Probably learning and memory are most evolutionary advantageous developments for human. These are interesting but ill-understood subjects. By utilizing past experience due to learning and it’s storage in memory one can get success and avoid failure5.

Alzheimer’s disease is progressive loss of memory and cognitive function in middle age indivisuals. (Cognition: that means by which one is aware of the process of thinking and perceiving. It involves an awareness of sensation and usually it’s cause.

Mental component consist of cognitive changes.) Symptoms of Alzheimer’s disease are– memory is failed for recent events, lack of spontaneous activity and initiative with loss of intellectual functions, loss of spatial orientations.

After 2 or 3 years dementia will take place. Following symptoms are found- aphasia (speech disorder), apraxia (unable in voluntary movements) and agnosia (unable in recognizing objects). Dementia is not a disease, but rather a group of symptoms caused by the impact of diseased brain. 10-15% of the populations over 65 years of age and 50% of population over 85 years have some degree of dementia. Severity will increases with increase in age.

In recent years, attempts have been made to develop drugs for treatment of dementia and attention deficit disorders to improve memory and learning2. These agents are commonly referred as Nootropic agents or cognition enhancers. The indications of cognition enhancers are- senile dementia of Alzheimer type and multi infarct dementia - common symptoms of elderly, dizziness and memory disturbances – mental retardation in children, learning defects, attention deficit disorder6.

Some nootropic agents (eg: Piracetam) are widely used but the resulting chemophobia associated with them and other similar agents has made their use limited.

So it is worthwhile to explore medicines from the traditional system in the treatment of these cognitive disorders.

Ayurveda, an ancient Indian system of medicine had reported various plants with the caliber of memory enhancing activity. Based on the same concept, several herbal formulations have been widely marketed, claiming to be useful in memory disorders. As there are no fixed guidelines in the indigenous system of medicine, to evaluate the retention of the potency by the formulation; we planned to evaluate a formulation

Wilmer Syrup for its nootropic activity. Wilmer syrup is a premier product of

Annapurna Bio Ved Pvt. Ltd., Injapur village, RR District, Hyderabad, AP, consisting of plant ingredients of Brahmi (Bacopa monniera), Yastimadhu (Glycyrrhiza glabra),Tagar

(Valeriana wallechii) and Ashwagandha (Withania somnifera).

ENCLOSUR –II

6.2: Review of Literature

Wilmer syrup is one such polyherbal formulation manufactured by Annapurna

Bio Ved Pvt Ltd, an Ayurvedic Pharma Industy consisting of Bacopa monniera

(150mg) Glycyrrhiza glabra (25mg) Vallerian wallechii (25mg) and Withania somnifera

(200mg). The literature review of these indivisual components of the formulation have reveled the following:

 Bacopa monniera belongs to the family Scrophularaceae reported to contain

Brahmine, Herpestine7, Saponins8 and betulic acid9 was used as nervine tonic,

antiepilepitic, diuretic to reduce stress induced anxiety, nootropic10,11,12, antipyretic,

anti-inflammatory, analgesic, sedative and adaptogenic actions13.

 Glycyrrhyza glabra belongs to family Leguminosae reported to contain

glycyrrhizin, potassium and calcium salt of glycyrrhizinic acid, D-galactose,

asparagines, β-sitosterol, starch, protein, glycyramarin liquiritin, isoliuertin is used as

flavouring agents nervine tonic8, demulcent and mild expectorant, rheumatoid

arthritis, in Addison’s disease and various inflammatory conditions.

 Vallerian wallechii (Tagar) belongs to family Valerianaceae reported to contain

valepotriates, volatile oil, valerenal, isovalerenic acid, hydroxyvalerenic acid,

acetoxyvalerenicacid is used as carminative, antispasmodic, in hysteria and nervous

disorder7.

 Withania somnifera belongs to family Solanaceae reported to contain somniferin,

resin, fat, phytosterol, ipuranol, withaolide and withaferin A is used as nervine tonic,

astringent, adaptogenic, febrifuge, sedative, hypnotic, anthelmintic and diuretic. Achliya et al., investigated the neuropsychopharmacological effect of a

polyherbal formulation Brahmi Ghrita on learning and memory processes in rats by

elevated plus maze and in mice by Morris maze model11.

Bhattacharya SK, studied the subchronic administration of BR-16A

(Mentat), a herbal formulation and the nootropic agent, piracetam, augmented

acquisition and retention of learning in normal rats as well as in states of cognitive

deficits induced by prenatal undernutrition, postnatal environment impoverishment

and sodium nitrite hypoxia. The test paradigms used were step-down latency in

avoidance test and transfer latency in elevated plus maze14.

ENCLOSURE –III

6.3: Main objective of the study:

The main objective of the proposed work is to evaluate the nootropic (memory enhancing) activity of a polyherbal formulation Wilmer syrup. The whole study is divided into two phases

Phase 1: To determine LD50 of the polyherbal formulation Wilmer syrup

and selection of three doses i.e., 1/20, 1/10 and 1/5 from the LD50 value

considered as low, medium and high doses.

Phase 2: To evaluate a nootropic activity with selected doses of above formulation

in various experimental animal models like  Sodium nitrite intoxication in mice.

 Passive avoidance task in mice

 Active avoidance learning and retention in rats using Shuttle

 Sodium nitrite induced amnesia in mice

 Scopolamine-induced amnesia in mice

 Diazepam-induced amnesia in mice

 Extroceptive behaviour study in mice using Elevated plus-

 Lithium-induced head twitch in rats

And it is also planned to evaluate the following parameters in respective animal models

1. Step-down latency (SDL)

2. Step-down error (SDE)

3. Time spent in the shock zone (TSZ)

4. Jumping response

5. Transfer latency

6. Number of head twitches

ENCLOSURE –IV

7. Materials and methods:

7.1: Source of the data:

Whole work is planned to generate data from laboratory based animal experimental studies as described in National/International Journals and from text books available with college and other institutions, The day to day development in this area will be updated by literature survey through e-publishing and Helinet facility provided by

RGUHS, Bangalore.

ENCLOSURE-V

7.2: Methods of collection of the data (including sampling procedure if any):

Data will be generated from experimental animal studies and are to be subjected for statistical analysis by ANOVA followed by post hoc test (Dunnett’s ‘t’ test) and p<0.05 considered as statistically significant.

Phase 1: Determination of LD50:

The acute toxicity of Wilmer syrup will be determined by using albino mice of either sex (20-25 g), maintained under standard conditions. The animals will be fasted for

3 hours prior to the experiment. Animals will be administered with single dose of polyherbal formulation Wilmer syrup and observed for its mortality upto 48 hours study period (short term toxicity). Based on the short term toxicity profile, the next dose will be

15 determined as per OECD guidelines No 425 . Form the LD50 dose 1/20, 1/10 & 1/5 doses are to be selected and considered as low, medium and high dose respectively. Phase 2: To evaluate the nootropic activity of polyherbal formulation Wilmer syrup in different animal models (mice and rats) by assessing learning, memory and behavioral study.

EXPERIMENTAL MODELS:

1. Sodium nitrite intoxication16:

Group of adult Swiss male albino mice 25-30g, each consisting of 6 animals will be divided into following groups

Group I : Normal control

Group II : Sodium nitrite alone

Group III: Standard drug (Piracetam) + Sodium nitrite

Group IV: Low dose of Wilmer syrup + Sodium nitrite

Group V : Medium dose of Wilmer syrup + Sodium nitrite

Group VI: High dose of Wilmer syrup + Sodium nitrite

All the groups will be treated according to the protocol for a period of 7 days and sodium nitrite 250mg/kg will be given subcutaneously 60 minutes after last dose to induce chemical hypoxia. Sodium nitrite reduces the oxygen carrying capacity of the blood by converting hemoglobin to methemoglobin and cessation of respiration time in each group will be recorded. 2. Passive avoidance task: (Exteroceptive Behavior Model)17:

Group II : Phenytoin alone

Group III: Standard drug (Piracetam) + Phenytoin

Group IV: Low dose of Wilmer syrup + Phenytoin

Group V : Medium dose of Wilmer syrup + Phenytoin

Group VI: High dose of Wilmer syrup + Phenytoin

Passive-avoidance task is a method widely used for screening drugs affecting learning and memory. The method described by Papazova et al. (1994) was modified as follows. An inverted petridish placed in the centre of the grid floor of a continuous avoidance apparatus (Inco, Ambala) was used. The petridish served as the shock-free zone (SFZ). Mice were placed in the SFZ and up on stepping down from the SFZ were given an electric shock (20 V) through the grid floor. Animals were trained to remain on the SFZ for at least 60 sec and mice which did not meet these criteria in five trials were rejected. Observations were made for acquisition i.e. the number of trials required to reach the learning criteria and for retention of learning for 10 minutes at 2 hours and 24 hours post-training. The following retention parameters like step-down latency (SDL) in seconds, step-down error (SDE) as the number of times the animal stepped down from the SFZ and the time spent in the shock zone (TSZ) in seconds are noted. Treatment schedule: The memory- impairing dose of phenytoin 25mg/kg daily for

14 days and the selected dose of polyharbal formulation Wilmer syrup for 07 days i.e. on

8th to 14th day and the parameters mentioned above will be noted.

3. Active avoidance learning and retention using Shuttle box in rats18:

Group of adult male albino rats 100-150g each consisting of 6 animals will be divided in to following groups

Group II : Standard drug (Piracetam)

Group III: Medium dose of Wilmer syrup

Group IV: High dose of Wilmer syrup

Group V : Low dose of Wilmer syrup

All groups of rats will be trained upto 100% learning criterion of active avoidance response. During the training period, each rat will be placed in one of the two chambers of the Sidman box, and after 5 sec the buzzer (conditioned stimulus, CS) will sounded for

2 sec, followed by an electric shock (unconditioned stimulus,UCS;30v,0.5 sec) through the grid floor. Thereafter, a rest pause of 180 sec will allowed. If the rat jumped within the CS duration to the unelectrified safe box, so as to avoid the USC, it will allowed to rest there for next 30 sec. However, if the rat did not show the avoidance response removed from the shock chamber after 180 sec and will be initiated for the next trial. The rats will be given 10 trials daily until they reached the 100% criterion of active avoidance response. After an interval of 15 days the rats will be subjected to a repeat test with treatment of different dose of the polyherbal formulation Wilmer syrup in order to assess the retention of the previously learned active avoidance response. Similarly nootropic activity of standard drug will be evaluated.

4. Sodium nitrite induced amnesia18:

Group III: Standard drug (Piracetam) + Sodium nitrite

Group IV: Low dose of Wilmer syrup + Sodium nitrite

Group V : Medium dose of Wilmer syrup + Sodium nitrite

Group VI: High dose of Wilmer syrup + Sodium nitrite

All groups will be treated according to protocol for a period of 7 days and sodium nitrite 25 mg/ kg will be given subcutaneously route 90 minutes after the last dose. There after using Elevated Plus maze (EPM) Transfer latency (TL) will be recorded at 45 minutes and 24 hours after administration of sodium nitrite.

5. Scopolamine-induced amnesia19,20: Group of adult Swiss male albino mice 25-30g, each consisting of 6 animals will be divided into following groups Group I : Normal control Group II : Scopolamine alone Group III: Standard drug (Piracetam) + Scopolamine Group IV : Low dose of Wilmer syrup + Scopolamine Group V : Medium dose of Wilmer syrup + Scopolamine Group VI : High dose of Wilmer syrup + Scopolamine All groups will be treated accordingly as mentioned above for a period of 14 days and Scopolamine will be given 0.3-1 mg/kg, intraperitoneally 90 minutes after last dose of standard/Wilmer syrup to induce impairment of memory through muscuranic system.

Transfer latency (TL) will be recorded 45 minutes and 24 hours after injection of scopolamine using Elevated Plus maze (EPM).

6. Diazepam-induced amnesia19,20:

Group II : Diazepam alone

Group III: Standard drug (Piracetam)+ Diazepam

Group IV: Low dose of Wilmer syrup + Diazepam

Group V : Medium dose of Wilmer syrup + Diazepam

Group VI: High dose of Wilmer syrup + Diazepam.

All groups will be treated respectively as mentioned above for a period of 14 days and Diazepam 1-10 mg/kg will be given intraperitoneally 90 minutes after last dose of standard/ Wilmer syrup to induce impairment of memory that act through GABAergic system. Transfer latency (TL) will be recorded with Elevated Plus maze (EPM) at 45 minutes and 24 hours after injection of Diazepam.

7. Extroceptive behaviour study using Elevated plus-maze model19,20

Group of adult Swiss male albino mice 25-30g, each consisting of 6 animals will be divided into following groups Group I : Normal control

Group II : Standard drug (Piracetam)

Group III: Low dose of Wilmer syrup

Group IV: Medium dose of Wilmer syrup

Group V : High dose of Wilmer syrup

The apparatus used in this model consisting of two open arms (16 cm ×5 cm) and two closed arms (16 cm×5 cm×12 cm). The arms extended from a central platform (5 cm

×5 cm) and the maze is elevated to a height of 25 cm from the floor. On the first day, each mouse will be placed at the end of an open arm, facing away from the central platform and Transfer latency (TL) i.e. the time taken by mouse with all its four legs to move into one of the closed arms has to be recorded. If the animal does not enter into one of the closed arms within 90 s, it will be gently pushed into one of the two closed arms and the TL has to be assigned as 90 s. The mouse will be allowed to explore the maze for another 10 s and then returned to its home cage. Retention of this learned-task will be examined 24 h after the first day trial. The effect of standard drug as well as polyherbal formulation Wilmer syrup will be tested after training the animals.

8. Lithium-induced head twitch (wieloz and kleinork 1979)21:

Group of adult Swiss male albino rats 150-200g, each consisting of 6 animals will be divided into following groups.

Group III : Standard drug (Piracetam)

Group IV: Low dose of Wilmer syrup Group V: Medium dose of Wilmer syrup

Group VI : High dose of Wilmer syrup

All groups will be treated respectively as mentioned above for period of 7 days and lithium sulphate 190mg/kg will be given by intraperitoneally 30 minutes after last dose of standard/ Wilmer syrup. The number of head twitches will be counted after 60 mints lithium treatment and prevention of head twitches due to standard drug and polyherbal formulation Wilmer syrup will be recorded. Lithium induced head twitching is used to assess the effect of drugs influencing second messenger system like

Phosphatidylinositol (IP) pathway22.

STATISTICAL ANALYSIS

All values will be expressed as mean ± SEM from 6 animals. Statistical difference in mean will be analyzed using one way ANOVA (analysis of variance) followed by

Dunnett’s ‘t’ test. p-value lower than 0.0.5 will be considered as statistically significant.

7.3: Does the study require any investigations or interventions to be conducted on patients other human or animals? If so, please describe briefly:

Study requires investigation on mice and rats. The effect of standard/polyherbal formulation Wilmer syrup will be studied on various parameters as stated above in objectives of the study.

7.4: Has ethical clearance been obtained from your institute in case of 7.3? YES: IAEC No: 576/2002/bc/IAEC/CPCSEA

ENCLOSURE –VI

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