EVALUATION OF NOOTROPIC ACTIVITY OF ETHANOLIC EXTRACT SOLANUM NIGRUM(SOLANACEAE)
M.PHARM DISSERTATION PROTOCOL SUBMITTED TO THE
RAJIV GANDHI UNIVERSITY OF HEALTH SCIENCES,BANGALORE,
KARNATAKA.
BY
MOHD IBRAHIM
B.Pharm
UNDER THE GUIDANCE OF
DEPARTMENT OF PHARMACOLOGY
ADITYA COLLEGE OF PHARMACY
BANGALORE-64
2012-2013
RAJIV GANDHI UNIVERSITY OF HEALTH SCIENCES
BANGALORE, KARNATAKA.
ANNEXURE II
PROFORMA FOR REGISTRATION OF SUBJECTS FOR DISSERTATION
1. / Name of the candidateand address
/ MOHD IBRAHIMD/O MOHD JAFFER
#577,4THMAIN,B LAYOUT S.S NAGAR BANNIMANTAP,EXTENSION MYSORE:570015
2. / Name of the Institution / ADITYA COLLEGE OF PHARMACY BANGALORE-64
3. / Course of study and Subject / M.Pharmacy in Pharmacology.
4. / Date of admission to the Course / 03/07/2012
5. / Title of the topic:
A STUDY ON NOOTROPIC ACTIVITY OF SEEDS EXTRACTS OF SOLANUM NIGRUM IN MICE AND RATS.
6 / Brief resume of the intended work6.1: Need for the study ENCLOSURE -I
6.2:Review of the literature ENCLOSURE -II
6.3:Main objectives 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, only in animals (Albino rats and mice)
7.4: Has ethical clearance been obtained from your institute in case of 7.3
Yes: YES Certificate attatched
8 / List of References
9. / Signature of the candidate / MOHD IBRAHIM
10. / Remarks of the guide
11. / Name and Designation of
11.1 Guide
11.2 Signature
11.3 Co-guide
11.4 Signature
11.5 Head of the Department
11.6 Signature /
.
12. / 12.1 Remarks of the Chairman
and Principal
12.2 Signature /
ENCLOSURE–I
6. Brief resume of the intended work:
6.1: Need for the study:
Nootropic means "acting on the mind", and nootropics are the substances found to have beneficial effects in the treatment of memory loss, age related memory decline and in lack of concentration. The substance piracetam (Nootropil®) was not only beneficial, but also found to have fewer side effects hence the biochemists described it "as safe as salt!"[1]
The word nootropic, created by Dr. Corneliu Giuerga in 1964, is composed of the Greek wordsnoos, or “mind”, andtropien, or “to act on”. It is used to describe a broad variety of substances, which are alike in that they posses cognitive enhancing effects. Substances classified as nootropics are very diverse and range from powerful pharmaceuticals to natural plants, herbs, vitamins and nutrients. Other terms used to describe nootropic drugs include “smart drugs”, “cognitive enhancers”, “smart nutrients”, “brain boosters”, and “brain enhancers.”
Nootropic substances have been proven to provide a vast array of cognitive benefits, however the benefits actually experienced are depend on the specific substance.Some nootropics have no acute effects, and must be taken continually for a few weeks before effects will present themselves, while others can be felt immediately. Some of the benefits provided by nootropic substances are
· increases in general cognition and overall intelligence
· improvement in short and long term memory
· improvement in attention and concentration
· enhancement in clarity of thought
· reductions in anxiety and the effects of stress
· brightening of mood, and increased enthusiasm for life
· freedom from depression
· higher amounts of mental and physical energy
· less mental fatigue
· normalizations of sleep patterns[2]
Learning is acquiring of new knowledge, behavior, skill, values, preference or understanding, and may involve synthesizing different types ofinformation. The ability to learn is possessed by humans, animals. Human learning may occur as part ofeducationorpersonal development[3]. Inpsychology, memoryis an organism'smentalability to store, retain, and recall past information. Traditional studies of memory began in the fields ofphilosophy, including techniques ofartificially enhancing the memory. The late 19th and early 20th century put memory within theparadigmsofcognitive psychology. In recent decades, it has become one of the principal pillars of a branch of science calledcognitive neuroscience, an interdisciplinary link between cognitiveandneuroscience [15].
Dementia is a mental disorder characterized by loss of intellectual ability sufficiently severe that interfere with one’s occupational or social activities. It is of several types and invariably involves impairment of memory. The common cause of dementia is Alzheimer’s disease, which is progressive neuro-degenerative disorder associated with loss of neurons in distinct areas of brain. The central cholinergic pathway play a prominent role in learning and memory process. Centrally acting anti muscarinic drugs (e.g. Scopolamine) impair learning and memory both in animal and human beings. Epidemiological studies of Indian population reveal that dementia is largely a hidden problem. Prevalence rates for dementia increase exponentially with advance in age. Dementia may be reversible when caused by drugs, alcohol, hormones, vitamins, imbalances and depression or irreversible when caused by disease or injury.
Since allopathic system of medicine is yet to provide a radical cure for dementia and it is worthwhile to look for new direction i.e. in ayurvedic system of medicine which was reported with many drugs to minimize the memory lose seen in elderly patients [4].
In view of this the present work is planned to study the nootropic activity of alcoholic & aqueous extracts of berries of solanum nigrum in experimental animals, mice and rats.
ENCLOSURE–II
6.2: Review of Literature:
The review of various published journals and books have revealed the plant based drugs are showing promising nootropic activity.
Plant Description:
Synonym:ENG:blacknightshade,HIN:makoi,SANS:kakamachi.[5]
Family: Solanaceae.[6]
Common name:Just black night shade[8]
Distribution:Through out India,in dry parts upto an elevation of 2100m.[7]
Botany:Solanum nigrum is a herbaceous or suffrutescent weed .the leaves are ovate or oblong,sinuate,thoothed or locked. Flowers are white in dropping, umblle like three to eight flower clusters,the berries are red,yellow or black and round the seeds are discoid,smooth and yellow and minutely pitted.[5]
Phytochemistry:solamargine,and solasanine[5]
Actions:antristress,hypolipidimic,anxiolytic and nootropic activity [5][9]
Uses: Theplant iseffective in the treatment of cirrhosisof the liver ,the plant is also credited with emollient,diuretic,antiseptic and laxative [5]
Parts used: Berries of the Plant.
SOLANUM NIGRUM(SOLANACEAE)[9]
ENCLOSURE–III
6.3 Main objectives of the study:
The main objectives of the proposed work is to evaluate the beneficial effects (nootropic activity) of berries extracts of Solanum nigrum in dementia induced in experimental animals like mice and rats. In the background of the literature available with the seed of this plant. The present work is planned with the following objectives;
· Preparation of various extracts (alcoholic & aqueous) with berries of Solanum nigrum
· Phytochemical investigation of these extracts
· Study of the nootropic activity of the extracts
ENCLOSURE–IV
7. Material and methods:
7.1: Source of the data:
Data will be obtained from the related National and International Experimental Journals and from libraries of various reputed institutes of India.
The day to day development in this area will be updated by literature survey through e-publishing and Helinet facility provided by RGUHS.
7.2: Methods of collection of the data (including sampling procedure if any):
In the first phase of the study, it is planned to prepare various extracts of berries of solanum nigrum using standard extraction procedures[12]. Then these extracts will be subjected for qualitative phytochemical test and also for LD50 studies. Later on, in the second phase it is planned to study nootropic activity of these extracts in different experimental animal models like mice and rats. Data generated from such experimental studies will be analyzed using one - way ANOVA (analysis of variance) followed by Dunnett’s ‘t’ test. P<0.05 will be considered as significant.
Phase 1:
a) Collection of the berries of solanum nigrum and preparation of alcoholic & aqueous extracts.
b) Phytochemical investigation of these 2 extracts.
c) Determination of LD50 value(Acute toxicity).
Phase 2:
To evaluate the nootropic activity of these 2 extracts in different experimental animal models (mice and rats)by testing learning, memory and behaviour of the animals.
EXPERIMENTAL MODELS;
A) Laboratory models for testing learning and memory[4]
1. Elevated plus-maze (Exteroceptive Behavior model)[4]
Elevated plus-maze served as the exteroceptive behavior model to evaluate learning and memory in mice. The procedure, technique and end point for testing learning and memory will be followed as per the parameters described by the investigators working in the area of neuropsychopharmacology. The apparatus consisted 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 1st day, each mouse will be placed at the end of an open arm, facing away from the central platform. Transfer latency (TL) i.e. the time taken by mouse with all its four legs to move into one of the closed arm has to be recorded on the 1st day. If the animal does not enter into one of the closed arms within 90 sec, it will be gently pushed into one of the two closed arms and the TL has to be assigned as 90 sec. The mouse will be allowed to explore the maze for another 10 sec, and then returned to its home cage. Retention of this learned-task will be examined 24 h after the first day’s trial.
Group Classification:
TRATMENT / DRUG / DOSEGroup-1 / Vehicle (Control)
Group-2 / Piracetam (Standard) / 200mg/kg
Group-3 / Alcoholic extract of
solanum nigrum / Low dose / Based on LD50
studies
Group-4 / Alcoholic extract of
solanum nigrum / Medium dose
Group-5 / Alcoholic extract of
solanum nigrum / High dose
Group-6 / Aqueous extract of
solanum nigrum / Low dose
Group-7 / Aqueous extract of
solanum nigrum / Medium dose
Group-8 / Aqueous extract of
solanum nigrum / High dose
Each group consist of 6 animals (n=6)
2. Scopolamine-induced amnesia (Interoceptive Behavior Model)[4]
Scopolamine (0.4 mg/kg) will be injected i.p ly 90 min after administration of extracts of or solanum nigrum standard on 7th day. TL (Transfer latency) will be recorded after 45 min of injection and retention (memory) of learned task will be examined after 24 h. Scopolamine when administered through i.p route to the animals, it will induce the impairment of memory by acting through muscarinic system. The details of the experimental protocol is given below
TRATMENT / DRUG / DOSEGroup-1 / Vehicle (Control)
Group-2 / Scopolamine / 0.4 mg/kg, i.ply
Group-3 / Piracetam (Standard)+ Scopolamine / 200 mg/kg, p.o for 7 days+ 0.4 mg/kg, i.p on 7th day.
Group-4 / Alcoholic extract of
solanum nigrum
+Scopolamine / Low dose, p.o. for 7days+ 0.4 mg/kg, i.p.
Group-5 / Alcoholic extract of
solanum nigrum + Scopolamine / Medium dose, p.o. for 7days+ 0.4 mg/kg, i.p.
Group-6 / Alcoholic extract of
solanum nigrum + Scopolamine / High dose, p.o. for 7days+ 0.4 mg/kg, i.p.
Group-7 / Aqueous extract of
solanum nigrum + Scopolamine / Low dose, p.o. for 7days+ 0.4 mg/kg, i.p.
Group-8 / Aqueous extract of
solanum nigrum + Scopolamine / Medium dose, p.o. for 7days+ 0.4 mg/kg, i.p. on 7th day
Group-9 / Aqueous extract of
solanum nigrum + Scopolamine / High dose, p.o. for 7days+ 0.4 mg/kg, i.p. on 7th day
Each group consist of 6 animals (n=6)
3. Diazepam-induced amnesia (Interoceptive Behavior Model)[4]
Diazepam (5 mg/kg) will be injected i.p after 90 min of administration of extracts of solanum nigrum or standard on 7th day. TL (Transfer latency) will be recorded after 45 min of injection and retention (memory) of learned task will be examined after 24 h. Diazepam when administered through i.p. route to the animals, it will induce the impairment of memory by acting through GABAergic system.
Group Classification:
TRATMENT / DRUG / DOSEGroup-1 / Vehicle (Control)
Group-2 / Diazepam / 5 mg/kg, i.p.
Group-3 / Piracetam (Standard)+ Diazepam / 200 mg/kg, p.o for 7 days+ 5 mg/kg, i.p.
Group-4 / Alcoholic extract of solanum nigrum + Diazepam / Low dose, p.o. for 7days+ 5 mg/kg, i.p.
Group-5 / Alcoholic extract of solanum nigrum + Diazepam / Medium dose, p.o. for 7days+ 5 mg/kg, i.p.
Group-6 / Alcoholic extract of solanum nigrum + Diazepam / High dose, p.o. for 7days+ 5 mg/kg, i.p.
Group-7 / Aqueous extract of solanum nigrum + Diazepam / Low dose, p.o. for 7days+ 5 mg/kg, i.p.
Group-8 / Aqueous extract of solanum nigrum + Diazepam / Medium dose, p.o. for 7days+ 5 mg/kg, i.p.
Group-9 / Aqueous extract of solanum nigrum + Diazepam / High dose, p.o. for 7days+ 5 mg/kg, i.p.
Each group consist of 6 animals (n=6)
B) Behavioral study[13]
1) Clonidine-induced hypothermia[13]
Clonidine induced hypothermia is used to assess the effect of drug influencing NA mediated behavior. Albino rats will be used in a groups of 6 in each .The rectal
temperature will be recorded every 30 min after clonidine (0.1 mg/kg i.p) till 180 min. Piracetam or extracts vigna munga or vehicle will be administered 30 min before clonidine treatment and reversal effect (inhibition of hypothermia due to clonidine) has to be recorded.
Group Classification:
TRATMENT / DRUG / DOSEGroup-1 / Vehicle (Control)
Group-2 / Clonidine / 0.1 mg/kg, i.p
Group-3 / Piracetam (Standard)+ Clonidine / 200 mg/kg, p.o for 7 days+ 0.1 mg/kg, i.p
Group-4 / Alcoholic extract of solanum nigrum + Clonidine / Low dose, p.o. for 7days+ 0.1 mg/kg, i.p
Group-5 / Alcoholic extract of solanum nigrum + Clonidine / Medium dose, p.o. for 7days+ 0.1 mg/kg, i.p
Group-6 / Alcoholic extract of solanum nigrum + Clonidine / High dose, p.o. for 7days+ 0.1 mg/kg, i.p
Group-7 / Aqueous extract of solanum nigrum + Clonidine / Low dose, p.o. for 7days+ 0.1 mg/kg, i.p
Group-8 / Aqueous extract of solanum nigrum + Clonidine / Medium dose, p.o. for 7days+ 0.1 mg/kg, i.p
Group-9 / Aqueous extract of solanum nigrum + Clonidine / High dose, p.o. for 7days+ 0.1 mg/kg, i.p
Each group consist of 6 animals (n=6)
2) Lithium-induced head twitch [13]
Lithium-induced head twitching is used to assess the effect of drugs influencing second messenger system. Rat will be treated with vehicle or extracts of solanum nigrum piracetam, 30 min before lithium sulphate(190 mg/kg, ip) and the number of head twitch is will be counted for 60 min and the prevention of head twitch is due to extract treatment will be recorded.