“EVALUATION OF NEUROPHARMACOLOGICAL PROPERTIES OF A POLYHERBAL EXTRACT”
SYNOPSIS FOR REGISTRATION
Of
M.PHARM DISSERTATION
Submitted to
RAJIVGANDHI UNIVERSITY OF HEALTH SCIENCES,
KARNATAKA
In partial fulfillment
Of the requirement for the Degree of
Master of pharmacy in Pharmacology
By
NANDISH.C
I-M.PHARM
Under the Guidance of
Mrs. GEETHA.K.M
HOD
Department Of Pharmacology
DAYANANDA SAGAR COLLEGE OF PHARMACY
2011-12
.
RAJIV GANDHI UNIVERSITY OF HEALTH SCIENCES
BANGALORE, KARNATAKA .
ANNEXURE - II
PROFORMA FOR REGISTRATION OF SUBJECTS FOR DISSERTATION
1. / Name of the Candidateand Address / NANDISH. C
#22, Near Sahana Vidya mandir, Naryanappa layout Naganathapura, Hosa road, Electronic city post box Bangalore-560100
2. / Name of the Institution / Dayananda Sagar College of Pharmacy,
Kumaraswamy Layout,
Bangalore – 560 078,
Karnataka, India.
3. / Course of Study and
Subject / M. Pharmacy-Pharmacology
4. / Date of Admission / 27th July 2011
5. Title of the Topic:
“EVALUATION OF NEUROPHARMACOLOGICAL PROPERTIES OF A POLYHERBAL EXTRACT”
6.0 / Brief resume of the intended work:6.1 – Need of the study:
A Neurological disorder is a disorder of the body’s nervous system. Structural, biochemical or electrical abnormalities in the brain, spinal cord, or in the nerves leading to or from them can result in symptoms such as paralysis, muscle weakness, poor coordination, loss of sensation, seizures, confusion, pain and altered levels of consciousness1.
The specific causes vary by disorder and sometimes by individual case, but can include genetic disorder, congenital abnormalities or disorder, infection, lifestyle, or environmental health problems including malnutrition and brain injury, spinal cord injury or nerve injury. The problem may start in another body system that interact with the nervous system for example; cerebrovascular disorder involve brain injury due to problems with blood vessels supplying the brain, and autoimmune disorders involve damage caused by the body’s own immune system1.
Intervention include preventative measures, lifestyle changes, physiotherapy or other, neuro rehabilitation, pain management, medication, or operation performed by neurosurgeon1.
The world health organisation estimated in 2006 that neurological disorders and their sequelae affects as many as one billion people worldwide, and identified health inequalities and social stigma/discrimination as major factors contributing to the associated disability and suffering1.
7.0 / 6.2 PLANT PFOFILE:
FIRST PLANT:
Calotropis gigantea R.Br, (family: Asclepiadaceae): This species is known by various names in different languages, Arka, Alarka in sanskrit, Madar in hindi, Yekkegida in kannada, Mandaramu, Eke, Jilledu, Arka in telugu, errikka in malayalam, Gigantic swallow swort, Mudar in English. It is found chiefly in waste land in lower Bengal, Himalayas, Punjab, Assam, Madras, South India, Ceylon, Singapore, Malay Island and South China2.
The plant is a large erect shrub or small tree up to 3 m high. Two varieties of the plant are described by Sanskrit writer, viz; the white flowered or “alarka” (Calotropis procera) and the purple or red flowered or “arka” (Calotropis gigantea). The leaves are opposite, broad and subsessile, glaucous green and 5-20 cm by 3.8-10 cm. Flowers are 3.8-5 cm diameter, inodorous, purplish (Calotropis gigantea) or white (Calotropis procera). Seeds are numerous, 6-5mm, broadly ovate, flattened, narrowly margined, minutely tanentose, brown, coma 2.5-3.2 cm long2.
It has been reported as traditional medicinal plant in Ayurveda, Unani and Homeopathic system of medication for the treatment of different ailments. According to Ayurveda the flowers are bitter, digestive, astringent, stomachic, anthelmintic, tonic and analgesic, normally used to cure inflammation, tumors, kapha, asthma, loss of appetite and ascites. According to Unani the flowers are stomachic and good for the liver, dried flowers in 1 to 2 grains doses with sugar is given in leprosy, secondary syphilis and gonorrhoea with milk diet2. Flowers are used for mental disorders3.
It has been reported that Calotropis gigantea plant parts contains various chemical constituents such as latex contains Calotropin, calotoxin, uscharin, voruscharin, uschridin, uzarigenin, syriogenin, calotonic acid and proceroside. Calotropin DI and DII (cysteine proteinases) Calotropain FI and FII (proteinases) α-amyrin, β-amyrin, taraxasterol, ψ- tarexosterol and β-sitosterol, aerial parts contain Isorhamnetin-3-O-rutinoside, Isorhamnetin-3-O-glucopyranoside and Teraxosteryl acetate. A new flavanol glycosideisorhamnetin-3-O-[2-O-beta-D-galactopyranosyl-6-O-alpha-L-rhamnopyranosyl]-beta-D-glycopyranoside. Flowers contain Cyanidine-3-rhamnoglucoside, leaves contain two new cardenolides (19-
Nor- and 18, 20-epoxy-cardinolides), root bark contains three cardenolide glycosides, calotropin, frugoside, and 4’-O-beta-D-glucopyranosylfrugoside, Giganticine, a non-protein amino acid and roots contain two new oxypregnane oligoglycosides, calotroposides A and B2.
So the present study is planned to evaluate the effects of Calotropis gigantea on neuropharmacological activities.
SECOND PLANT
Catharanthus roseus (Family: apocynaceae): This species was formerly known as Vinca rosea, native of Madagascar grown commercially for its medical uses in Australia, Africa, India, Southern Europe4.
It is an erect bushy herb and evergreen shrub, grows to a height of 90 centimeter with a spread of one m , stem are erct, leaves are simple, opposite, exstipulate, petiolate, flowers are soft pink, tingled with red. There are three varities rose purple flowers, white flowers, white flowers with arose purple spot in the Centre4.
It has been reported that plant root and bark contains the alkaloid alstonoine which has been used traditionally for its calming effect and its ability to reduce blood pressure4.
It has been reported that plant has been used for various disease conditions, including animal repellent activity, anti-inflammatory, antifertility, CNS depressant, antibacterial, antispasmodic, antimalarial, antihypertensive, antifungal, antidiuretic, antiviral, antitumor, cardiotonic, antihyperglycemic, antihypertensive, cytotoxic, antispermatogenic etc5,6,7.
So the present study is planned to evaluate the effects of roots Catharanthus roseus on neuropharmacological activities.
THIRD PLANT:
Jasminum malabaricum (family: olaceae): It is commonly known as Malabar jasmine a beautiful jasmine native to south India. It is known as kadu mallige in kannada , kotivakai in Tamil, mudgara in Sanskrit8,9.
It is a larger climber, growing up to 3-5m , woody stems with 4 cm thickness, slender branches, leaves are 8-12 cm long broad ovate with sharp tip. fragrant white flowers appear in branch comes at the end of branches up to 50 in a single cyme8,9.
It has been reported that plant flowers have been used in mental disorders8.
6.3– Review of the literature :
1 Ameeta Argal, Anupam Kumar Pathak has reported that roots of Calotropis gigantea is having CNS depressant activity(2006)10.
2 A.K. Pathak, A. Argal has reported that flowers of Calotropis gigantea possess analgesic activity(2007)11.
3 Joshi Amit, Singh Namrata, Pathak A.K., Tailang M has reported that whole plant of Calotropis gigantea possess antioxidant property(2010)12.
4 R. Rajesh, C.D. Raghavendra Gowda, A. Nataraju B.L, Dhananjaya K, Kemparaju, B.S. Vishwanath has reported that Calotropis gigantea possess procoagulant activity associated with fibrinolytic activity(2005)13.
5 Pradeep T. Deshmukha, Jennifer Fernandes, Akarte Atula, Emmanuel Toppo has reported that roots Calotropis gigantea possess wound healing activity(2009)14.
6 B.S. Nayak, M. Anderson, L.M. Pinto Pereira has reported that leaf extract of Catharanthus roseus is having wound healing property(2007)15.
7 M. Hill, A. Dupaix, M. Nhiri, L. Guyen and B. Arrio has reported ATP: AMP phosphotransferase activity a new characteristic of Catharanthus roseus tonoplasts(1988)16.
8 Ohadoma S.C, Michael HU has reported that co-administration of methanol leaf extract of Catharanthus roseus has hypoglycemic activity(2011)17.
9 David M. Pereira, Joana faria, Luis Gaspar, Federico Ferreres, Patricia Valentao, Mariana Sottomayor, Paula B. Andrade has reported that roots of Catharanthus roseus is having antioxidant property(2010)18.
10 Kyakulaga A. Hassan, Alinda T. Brenda, Vudriko Patrick and Ogwang E. Patrick has reported Catharanthus roseus is having antidiarrheal activity(2011)19.
11 Fatouma Abdoul-Latif, Prosper Edou, François Eba, Nabil Mohamed, Adwa Ali,Samatar Djama, Louis-Clément Obame, Ismael Bassolé and Mamoudou Dicko has reported the Antimicrobial and antioxidant activities of essential oil and methanol extract of Jasminum sambac from Djibouti(2010)20.
6.4 – Objective of Study:
Objectives of the proposed study are to evaluate the
v Antipsychotic study of the polyherbal extract by compulsive behavior test.
v Studies on pentobarbital-induced sleep in rat
v Test for motor co ordination
i. Rotarod test
ii. Chimney test
v Evaluation of anxiolytic activity using elevated plus maze
apparatus
v Exploratory behavior pattern
i. Head-dip test:
ii. Y-maze test:
iii. Evassive test:
Materials and Methods:
7.1-Method of collection of plants:
Plant materials – Plant materials of will be collected in the month of December from Bangalore and will be authenticated by a Botanist. A voucher specimen of the collected sample will be deposited in the departmental herbarium for future references.
The data and related literatures will be collected from various scientific national and international journals i.e. Indian Journal Pharmacology, Journal of Ethnopharmacology, Plantamedica, electronic search engine, etc
7.2-Extraction of dried plants parts using soxhlet apparatus:
Preparation of extract – Crude powdered shade dried flowers of Calotropis gigantea, roots of Catharanthus roseus and the flowers of Jasminum malabaricum will be taken in equal proportions and extracted with methanol by hot continuous extraction in a soxhlet extractor. The extract obtained will be cooled, filtered and the solvent recovered by distillation. The residue so obtained is used for subsequent experiments. The dried extract will be suspended in 3% Carboxyl methyl cellulose (CMC) for animal administration.
The extract obtained will be dried in a rotary flash evaporator and stored in a refrigerator.
7.3 Preliminary phytochemical screening of crude extracts:
The extract will be screened for the presence of various phytochemicals by standard chemical tests.
7.4 Toxicity studies:
Acute toxicity studies: Acute oral toxicity test for the combined extract of the three plants will be carried out as per OECD Guidelines at five doses. Albino mice of either sex will be used for the purpose.
Selection of dose:
The dosage of the extract will be fixed based on the results of acute toxicity studies.
7.5 Evaluation of neuropharmacological properties by following methods:
1. Antipsychotic study of Polyherbal extract by Compulsive behavior test
The antipsychotic activity of methanolic extract will be performed using Compulsive behavior test. Animals will be weighed and numbered. Then divided them into three groups, each group comprising of five animals. Amphetamine will be injected to five animals and placed them individually into separate beaker and observed the onset and intensity of rearing, sniffing and licking behavior at time 0, 15, and 30 min after amphetamine injections will be noted .The severity of the response will be scored as + = presence, + + = moderately severe, and + + + = intense and continuous action. Cumulative scores at each time interval will be noted and calculated. To the second group polyherbal extract will be administered orally and 30 min later amphetamine will be injected to these animals. These animals will be placed individually in a beaker and cumulative score are noted at each time interval and scores will be calculated. To the third group Haloperidol will be injected and 30 min later amphetamine will be injected to these animals. These animals are than placed individually in a beaker and cumulative score at each time interval will be noted and calculated. Comparisons of the action of amphetamine in normal and haloperidol and polyherbal extract will be done21.
2. Pentobarbitone-induced sleeping time test:
The animals will be randomly divided into four groups consisting of five mice in each. The test groups will receive polyherbal extract orally while positive control will be treated with diazepam (1 mg/kg i.p.) and control with vehicle (1% Tween 80 in water). Thirty minutes later, pentobarbitone (50 mg/kg i.p.) will be administered to each mouse to induce sleep. The animals will be observed for the latent period (time between pentobarbitone administration to loss of righting reflex) and duration of sleep (time between the loss and recovery of righting reflex)22.
3. Test for motor coordination:
(a) Rotarod test: Mice will be placed on a horizontal rotating rod of 35 mm diameter at a rate of five rotations per minute after 15 min of administration of saline intraperitoneal. Animals which able to remain on the rod for longer than 5 min will be selected and divided into four groups (n=5), the doses of polyherbal extract will be injected administered orally to the test groups, the control will be injected with the vehicle. After 30 min of administration of the injection, animals will be placed on the rod at intervals of 30 min, up to 2.5 h. If an animal failed more than once to remain on the rod for 3 min, the test will be considered positive22.
(b) Chimney test: In a Pyrex glass tube (30 cm long and 28 mm diameter) mark will be made at a point of 20 cm from its base, a mouse will introduced at the end nearing the mark. When the animal reaches the other end of the tube, this will be moved to the vertical position and immediately the mouse tried to climb backwards. Only those mice which reached the mark within 30 s will be selected for further testing (Irwin, 1968). Groups of previously screened mice will injected intraperitoneally with either control vehicle and test material administered orally and, after 15min, will be tested in the way as described above22.
4. Evaluation of anxiolytic activity using elevated plus maze apparatus:
The elevated plus maze comprising of two open and two enclosed arms, produces a novel environment which helps in inducing anxiety in animals because of the open nature of the arms and elevation (70 cm) from the floor. The polyherbal extract will be administered orally depending on the body weight of the animal. Diazepam (2 mg/kg, p.o.) will be administered for the standard group the number of entries as well as the duration of stay in the open arms, indicating anxiolytic activity of standard and test will be recorded23.
5. Exploratory behavioral pattern
This will be examined by:
(a) Head-dip test
(b)Y-maze test
(c) Evassive test
(a) Head-dip test: This test consists of placing female mice 30 min after injection with the control vehicle or test material, singly on a wooden board with 16 evenly spaced holes. The number of times the mice dipped their heads in the holes during a 3 min trial will be counted. In the second set of experiments, the effects of administration of a mixture of amphetamine and polyherbal extract administered orally (at a ratio 1:10) will be examined in the same way as described above22.
(b) Y-maze test: Female rats will be placed in a Y-shaped runway for 5 min and the number of times that the rat entered the arm of the maze with all four feet (an ‘entry’) will be counted. Experiments will be conducted in four groups of 10 animals, 35 min after administration of control vehicle or test material22.