1. EVALUATION OF ANXIOLYTIC EFFECT OF CAESALPINIA
PULCHERRIMA(L.) PODS EXTRACT IN EXPERIMENTAL ANIMALS.
M.PHARM DISSERTATION PROTOCOL
SUBMITTED TO THE
RAJIV GANDHI UNIVERSITY OF HEALTH SCIENCES,
BENGALURU, KARNATAKA
BY
KHAN SADIQUE ASLAM
B.Pharm
UNDER THE GUIDANCE OF
MOHAMMED SAIFUDDIN KHALID
M.Pharm.
ASST. PROFESSOR
DEPARTMENT OF PHARMACOLOGY
LUQMAN COLLEGE OF PHARMACY
GULBARGA-585102
2013-2014
1

2. RAJIV GANDHI UNIVERSITY OF HEALTH SCIENCES, KARNATAKA, BANGALURU
ANNEXURE-II
PROFORMA FOR REGISTRATION OF SUBJECTS FOR DISSERTATION
1.
Name of the Candidate and
KHAN SADIQUE ASLAM
Address (In block letters)
S/O MOHAMMED ASLAM KHAN
R.NO
501,
SHADAB
COLONY,NEAR
2.
3.
4.
5.
MANZIL,
RAILWAY
BOMBAY
STATION,
Name of the Institution
MUMBRA, THANE 400612. MAHARASHTRA
LUQMAN COLLEGE OF PHARMACY,
Course of Study and Subject
Date of Admission to Course
Title of the Topic
GULBARGA – 585 102.
M.PHARMA. (PHARMACOLOGY)
29 /07/2013
EVALUATION OF ANXIOLYTIC EFFECT
OF CAESALPINIA PULCHERRIMA(L.) PODS
6.
EXTRACT IN EXPERIMENTAL ANIMALS.
BRIEF RESUME OF THE INTENDED WORK
6.1 Need for the study:
Anxiety (also called angst or worry) is a psychological and physiological state
characterized by somatic, emotional, cognitive, and behavioral components1. Stress
involves complex biochemical, neurological and variety of disease states ranging from
psychiatric disorders like depression and anxiety, immunosuppression, endocrine disorders
including diabetes mellitus, impotency and cognitive dysfunctions2. Anxiety related
disorders such as generalized anxiety, panic, obsessive-compulsion, phobias or post
traumatic stress disorders are common and major cause of disability 3 and 1/8th of the total
population worldwide affected with anxiety and become a very important area of research
interest in psychopharmacology4.
2

3. Anxiety is the displeasing feeling of fear and concern5. Benzodiazepine is the most
important group used as anxiolytic and hypnotic agents 6. But it shows the side effects
during the long term therapy. Hence there is needed to look for more efficacious anxiolytic
agents with lesser side effects. Many herbal plants available to be best herbs for anxiety.
The increasing awareness of herbal medicine is acknowledged by WHO 7. WHO
estimate about three quarters of the world population currently used herbs and other forms
of traditional medicine to treat there diseases7. WHO has recently defined traditional
medicine (including herbal drugs) as comprising therapeutic practices that have been in
existence, almost for several hundred years7. The traditional preparation comprises
medicinal plants, minerals, organic matter, etc7. Herbal drugs constitute only those
traditional medicines which are primarily use medicinal plant preparation for therapy7.
Plants have the ability to synthesize a wide variety of chemical compounds that
are used to perform important biological functions, and to defend against attack from
predators such as insects, fungi and herbivorous mammals. Many of these phytochemicals
have beneficial effects on long-term health when consumed by humans, and can be used to
effectively treat human diseases. At least 12,000 such compounds have been isolated so
far; a number estimated to be less than 10% of the total. Chemical compounds in plants
mediate their effects on the human body through processes identical to those already well
understood for the chemical compounds in conventional drugs; thus herbal medicines do
not differ greatly from conventional drugs in terms of how they work. This enables herbal
medicines to be as effective as conventional medicines, but also gives them the same
potential to cause harmful side effects8.
There are several plants very effective in treating stress / anxiety; such plants
include Passiflora incarnata (Passion flower) due to presence of bioactive phytomoiety
(benzoflavone)
9
and flavonoids in Dolichandrone falcata leaves10. One such plant
Caesalpinia pulcherrima contains isoflavones, flavones,chalcones, flavanols, flavones,
sterols and diterpenoids11. Recent study on anxiety claims that the flavonoids, alkaloids
and terpenoids are responsible for anxiolytic (anti anxiety) and sedative activity12,13,14.
3

4. The literature reveals that the plant Caesalpinia pulcherrima possesses various
bioactive compounds along with flavonoids (flavones) and there is no scientific data on
anxiolytic activity of Caesalpinia pulcherrima pods. In view of this, the primary aim of
the present study is to investigate the possible anxiolytic activity of Caesalpinia
pulcherrima pods extract in laboratory animal.
6.2 Review of the literature of Caesalpinia pulcherrima:
Botanical classification15: Kingdom:
Plantae
Division:
Magnoliophyta
Class:
Magnoliopsida
Order:
Fabales
Family:
Fabaceae
Subfamily:
Cæsalpinioideae
Genus:
Cæsalpinia
Species:
C. pulcherrima
Botanical name: Caesalpinia pulcherrima L.
Synonyms:
Poinciana pulcherrima, Poinciana bijuga
Common names: peacock flower
Vernacular Name16 : English
:
Pride of Barbados, dwarf Poinciana, red bird of paradise,
Hindi
:
Guletura
Kannada
:
Kenjige
Telugu
:
Ratnagandhi
Marathi
:
Sankasur
Tamil
:
Mayirkonrai, Nazhal
Bengali
:
Krishnachura or Radhachura
Sanskrit
:
Sidhakya
Caesalpinia pulchirrima (Fabaceae) is native to tropics and subtropics area of the
Americas17. This plant is widely distributed in Bangladesh and India 18. It is a common
medicinal plant in India, Taiwan and south east Asian conutries19.
Caesalpinia pulchirrima is a striking ornamental plant, widely grown in domestic
4

5. and public gardens and has a beautiful inflorescence in yellow, red and orange 17.
Caesalpinia pulchirrima species is a shrub growing to 3 m tall. The leaves are bipinnate,
20-40 cm long, bearing 3-10 pairs of pinnae, each with 6-10 pairs of leaflets 15-25 mm
long and 10-15 mm broad. The flowers are borne in racemes up to 20 cm long, each flower
with five yellow, orange or red petals. The fruit is a pod 6-12 cm long17. Flowers are red or
yellow, fragrant20. Flowering season of this plant start from September to November and
fruits from March to april20.
Traditionally leaves of Caesalpinia pulchirrima are used as purgative, tonic,
antipyretic, emmenagogue, where as roots have folkforic use in convulsion, intermittent
fever, lungs and skin diseases21. Flavonoids are polyphenolic compounds, widely
distributed in the plant kingdom. They are reported to exhibit various pharmacological
activities such as CNS, cardiotonic, lipid lowering, anti-oxidant, hepatoprotective and
hypoglycemic activities22.
The Caesalpinia pulchirrima possesses various bioactive compounds such as
steroid, reducing sugar, triterpenoids, sugar, alkaloids, phenolic compounds, flavonoids,
catechins, saponins, tannins, anthraquinons and amino acid 23.
The stem contains
peltogynoids bhonducellin, 6-methoxypulcherrimin and homomisoflavonoids24.
Medicinal uses:
1. Used to induce abortion in the first trimester of pregnancy17.
2. Used in pyrexia25.
3. Used in menoxenia25.
4. Used in wheezing25.
5. Used in bronchitis25.
6. Used in malarial infection25.
5

6. Reports from modern literature of the plant Caesalpinia pulchirrima :
1. The ethanolic and aqueous extracts of Caesalpinia pulchirrima flower have been
reported for antimicrobial activity26.
2. The methanolic and aqueous extracts of the wood of Caesalpinia pulchirrima
Linn has been reported for antioxidant and cytotoxic activities27.
3. The various extracts of pods of Caesalpinia pulchirrima showed antiinflammatory and anti-nociceptive properties28.
4. The methanolic extract of Caesalpinia pulchirrima flower has been reported for
analgesic and anti-inflammatory activities29.
5. The ethanolic extract of Caesalpinia pulchirrima leaves possess anticonvulsant
properties20.
6. The methanolic extracts of Caesalpinia pulchirrima, cassia fistula, and senna
alata leaf reported to possess weight lowering properties30.
7. The ethanolic extract of the dry fruits of Caesalpinia pulchirrima, arial parts of
euphorbia hirta and flowers of Asystasiagangeticum has been reported for
antimicrobial activity31.
8. The aqueous extract of Caesalpinia pulchirrima leaf showed some effects on the
liver function enzyme and blood glucose concentration in normal rabbits32.
Review of literature, till date, regarding Caesalpinia pulchirrima was carried out by
chemical abstract, biological abstract, medicinal abstract and other national and
international scientific journals. The Caesalpinia pulchirrima possesses various bioactive
compounds such as steroid, reducing sugar, triterpenoids, sugar, alkaloids, phenolic
compounds, flavonoids, catechins, saponins, tannins, anthraquinons and amino acid 23. The
leaves of the plant Caesalpinia pulchirrima are reported to contain hydrocyanic acid,
tannins and benzoic acid33. The plant contains various phytoactive consituents such as
glycosides, rotenids, isoflavones, flavonone, chalcones, flavanols, flavones and sterols
,diterpenoids19. Root of Caesalpinia pulchirrima showed the presence of diterpenoids,
isovouacapenol C and pulcherrimin A34. The stem contains peltogynoids bhonducellin, 6methoxypulcherrimin and homomisoflavonoids 24. The flavonoids are polyphenolic
compounds and reported to exhibit various pharmacological activities such as CNS
activity, cardiotonic activity, lipid lowering activity, antioxidant activity, hepatoprotective
activity, hypoglycemic activity22 etc. These active constituents and the above mention
6

7. activities in turn appear to correlate with some other biological activities 35.Our literature
survey revealed that the different parts of Caesalpinia pulchirrima have been screened for
various pharmacological activities but anxiolytic activity was not investigated in
Caesalpinia pulchirrima pods so far. Upon literature survey leaves of Caesalpinia
pulchirrima is said to possess flavonoids, And flavonoids are reported to be very effective
in treating stress / anxiety; such as benzoflavone in Passiflora incarnata (Passion flower)9
and flavonoids in Dolichandrone falcata leaves10. Therefore, the present study is planned
to investigate the possible anxiolytic activity of Caesalpinia pulchirrima pods in
experimental animal model. Hence the study is essential and justifiable.
6.3 OBJECTIVES OF THE STUDY:
The objective of the proposed study is to investigate anxiolytic property
of different doses of pod extract of Caesalpinia pulcherrima in experimental animals.
Specific Objective:
.
1. Collection and extraction of Caesalpinia pulcherrima pods.
2. Authentication and characterization of the plant material.
3. Extraction of Caesalpinia pulcherrima pods with suitable solvents, such as
petroleum ether, chloroform, ethanol and water.
4. To carryout preliminary phytochemical analysis of crude extracts for the detection
of the type of phytoconstituents present.
5. To determine the dose range of extracts of Caesalpinia pulcherrima pods by
conducting acute toxicity studies as per OECD guidelines.
6. To evaluate the effect of Caesalpinia pulcherrima pods extract at different doses for
anxiolytic potential in mice and rats in the following models.
a) Elevated Plus-Maze test in mice.
b) Hole-board test in rats.
c) Light-dark model transition test in mice.
7.
d) Open field test.
MATERIALS & METHODS:
7.1 SOURCE OF DATA:
Data will be obtained from CD-Rom, Internet facilities, Literatures, related articles,
books from libraries of Luqman College of Pharmacy, Gulbarga, Gulbarga University,
Gulbarga etc., and other Research Publications and Journals.
7

8. Web sites:
www. sciencedirect.com
www. pubmed.com
www. google.com
www.ijp-online.com
www. freemedicaljournals.com
www.elsevier.com
7.2 METHODS OF COLLECTION OF DATA:
The data collected will be based on animal experimentation as per the parameters
studied under each animal model, which are mentioned under the objectives of the study.
The Caesalpinia pulcherrima pods are found throughout India. The experiment will be
conducted using different animal models and data will be generated from such
experimental studies as mentioned under the objective of the study. Chemicals and
reagents will be procured from standard companies. Pure sample of diazepam will be
collected from company manufacturing this chemical. The data collected will be based on
animals experimentation as per the parameter studied under each animal model. The doses
of extract will be selected on the basis of our preliminary toxicity studies as per OECD
guidelines. The control animal receives only the vehicle (2% gum acacia) in the same
volume and through same route of administration.
METHODOLOGY:
1. Preparation of various solvent extracts 36,37,38 :
It is planned to dry the pods under shade at room temperature and pulverized. Than
the powder obtained is subject to successive soxhlet extraction with the solvents with
increasing order of polarity i.e. petroleum ether (60-80oc), chloroform (59.5-61.5oc),
ethanol (64.5-65.5oc) and water. If further required the shade- dried powder is extracted
directly with 70% ethanol (hydro-alcoholic extract). The extract is allowed to concentrate
under reduced pressure (bath temperature 5oc) and store in air tight container in refrigerator
below 10oc. All these extracts are used for biological investigations and in vivo studies,
after subjecting it to preliminary qualitative phytochemical analysis.
2. Preliminary phytochemical screening37,39,40.
8

9. It is planned to carry out the preliminary phytochemical investigation of different
extracts of Caesalpinia pulcherrima pods for detection of various phytochemical by
following standard method described in practical pharmacognosy by C.K. Kokate and R.K.
Khandelwal.
Experimental animals:
In-bred healthy Wister rats weighing 150-200g and Swiss albino mice of either sex
weighing 20 to 25g will be included for the study. Rats and mice will be housed in
polypropylene cages (six per cage) with stainless steel grill top, bedded with paddy husk.
Rats and mice will be maintained under controlled temperature at 25oC ± 2oC with 12 hr
light/ dark cycle in a well-ventilated animal house. All Rats and Mice will have a free
access to food (pellet chow) and water ad libitum. Institutional Animal Ethics Committee
approval for the experimental protocol has been obtained (copy enclosed); animals will be
maintained under standard conditions in an animal house approved by Committee for the
Purpose of Control and Supervision on Experiments on Animals (CPCSEA).
Determination of Acute toxicity studies (LD50)41.
It is further planned to study the acute toxicity of solvent extract of Caesalpinia
pulcherrima pods in albino mice of either sex (20-25gm). Fixed dose method (OECD
guideline number 420) of CPCSEA will be adopted for toxicity studies to obtain dose
range of extracts of Caesalpinia pulcherrima pods.
Work Protocol:
Anxiolytic Models:
Method 1. Elevated Plus-Maze Test in mice42,43,44,45.
GROUPING:
Albino mice of either sex weighing between 20-25gm are selected and shall be
divided into 4 groups containing 6 mice each. The control group will receive 2% gum
acacia per oral (p.o.) and the standard group receives drug diazepam at a dose of 2mg/kg.
Group I
-
Control (2% gum acacia; p.o.)
1×6=6 mice
Group II
-
Diazepam (2mg/kg; p.o.)
1×6=6 mice
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10. Group III
-
Extract of Caesalpinia pulcherrima pods (p.o.) dose 1 1×6=6 mice
Group IV
-
Extract of Caesalpinia pulcherrima pods (p.o.) dose 2 1×6=6 mice
Total number of mice required for this model = 24 mice.
Procedure: The plus-maze apparatus comprises of two open arms (16×5cm) and two
closed arms (16×5×12cm) that extend from a common central platform (5×5cm). The
entire maze is elevated to a height of 25cms above the floor level. Mice are placed
individually in the center of the maze facing one of the enclosed arms for recording various
parameters in a period of 5 minutes.
Following parameters are plan to study,
a. % of open arm time
b. % of close arm time
c. % open arm entry
d. % closed arm entry
Method 2. Hole-board test in rats46:
GROUPING:
Wister rats of either sex weighing between 150-200gm are selected and shall be
divided into 4 groups containing 6 mice each. The control group will receive 2% gum
acacia per oral (p.o.) and the standard group receives drug diazepam at a dose of 2mg/kg.
Group I
-
Control (2% gum acacia; p.o.)
1×6=6 rats
Group II
-
Diazepam (2mg/kg; p.o.)
1×6=6 rats
Group III
-
Extract of Caesalpinia pulcherrima pods(p.o.)dose 1 1×6=6 rats
Group IV
-
Extract of Caesalpinia pulcherrima pods(p.o.)dose 2 1×6=6 rats
Total number of rats required for this model = 24 rats.
Procedure: Rats were placed singly in the centre of the hole-board, and during a 5-min
trial .The apparatus to be use in this model consists of wooden chamber (40x40x25 cm)
with 16 holes (diameter 3 cm) on the floor, elevated from the ground so that the rats could
peep through the holes. Each rat will be placed individually in the apparatus for recording
following parameters,
10

11. a. Latency to the first head dip.
b. Number of head dips in the holes.
c. Total time spend with the head dips.
d. Number of rearings.
e. Total locomotor activity (numbers of squares crossed).
Method 3. Light-dark model transition test in mice.47,48,49,50.
GROUPING:
The light/dark transition test is based on the innate aversion of rodents to brightly
illuminated areas and on the spontaneous exploratory behavior of rodent in response to
mild stressors, that is, novel environment and light . A natural conflict situation occurs
when an animal is exposed to an unfamiliar environment or novel objects. The conflict is
between the tendency to explore and the initial tendency to avoid the unfamiliar
(neophobia). The exploratory activity reflects the combined result of these tendencies in
novel situations. Thus, in the light/dark test, drug induced increase in behaviour in the
white part of a two compartment box, in which a large white compartment is illuminated
and a small black compartment is darkened, is suggested as an index of anxiolytic activity .
Albino mice of either sex weighing between 20-25gm are selected and divided into
4 groups of 6 mice each. The control group will receive 2% gum acacia per oral(p.o.) and
the standard group receives drug diazepam at a dose of 2mg/kg.
Group I
-
Control (2% gum acacia; p.o.)
1×6=6 mice
Group II
-
Diazepam (2mg/kg; p.o.)
1×6=6 mice
Group III
-
Extract of Caesalpinia pulcherrima pods(p.o.)dose 1 1×6=6 mice
Group IV
-
Extract of Caesalpinia pulcherrima pods(p.o.)dose 2 1×6=6 mice
Total number of mice required for this model = 24 mice.
Procedure:
The light-dark apparatus
consists
of two-compartment
chamber
(40×60×20cm/h) comprising of a brightly illuminated area (40×40cm) and a dark area
(40×20 cm) separated by a wall with a round hole (7 cm diameter) will be used. Mice are
placed individually in the illuminated part of the cage and following parameters are
recorded during the test session of 5 minutes,
11

12. a. Total number of crossings.
b. Number crossing between the light and dark area.
c. Total time spend in the illuminated part of the cage.
d. Time spend in the dark part of the cage.
Method 4. Open field test51,52:
GROUPING:
Male swiss albino mice weighing between 20-22 gm are selected and divided into 4
different groups of 6 each, where the control group will receive normal saline per oral and
the standard group receives drug diazepam at a dose of 1mg/kg (i.p).
Group I
-
Control (normal saline; p.o.)
1×6=6 mice
Group II
-
Diazepam (1mg/kg; i.p.)
1×6=6 mice
Group III
-
Extract of Caesalpinia pulcherrima pods(p.o.)dose 1 1×6=6 mice
Group IV
-
Extract of Caesalpinia pulcherrima pods(p.o.)dose 2 1×6=6 mice
Total number of mice required for this model = 24 mice.
Procedure: The apparatus consists of a wooden box (60X60X30 cm). The apparatus is
illuminated with 40-W lamp suspended 100 cm above it. Mice will be fed orally with
extract(s), vehicle (normal saline) or diazepam (1 mg/kg; i.p). After 30 minutes following
parameters are observed and recorded during the test session of 5 minutes.
a) The number of rearing.
b) Assisted rearing (forepaws touching the walls of the apparatus).
c) The number of squares crossed.
Number of Animal Required in the study
Total number of mice required for the study is 24+24+24 = 72
Total number of rats required for the study
= 24
INCLUSION CRITERIA:
Normal and healthy animals weighing between 150-200gm for rats and 20-25gm mice will
be included in the study.
12

13. EXCLUSION CRITERIA:
The Wister rats and Swiss mice which do not fall the above mentioned weights are
excluded from study.
STATISTICAL ANALYSIS:
The statistical significance of the results will be analyzed by unpaired‘t’ test and ANOVA
p<0.005 will indicate the significance of the result.
7.3 Does the study require any investigation or intervention to be conducted on
patients or other humans or animals? If so, please describe briefly.
Yes, the above study requires usage of laboratory animals, as described in methodology.
7.4 Has ethical clearance been obtained from your institution in Case of 7.3?
Yes, the protocol has been approved by the Institutional Animal Ethics Committee and a
copy of the same is enclosed along with Institutional Animal house registration number
346/CPCSEA.
8.
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9.
10.
Signature of Candidate
Remarks of the Guide
KHAN SADIQUE ASLAM
“EVALUATION OF ANXIOLYTIC EFFECT
OF CAESALPINIA PULCHERRIMA(L.) PODS
EXTRACT IN EXPERIMENTAL ANIMALS”.
To be carried out by KHAN SADIQUE ASLAM,
M.Pharm has been discussed and worked out under
my directions and supervision as an official guide.
The project work envisaged is of great importance
in the field of Pharmacology. The work can be
carried out in Pharmacology laboratory of Luqman
College of Pharmacy for which facilities are
17

18. available. Hence the project is viable and is
recommended for clearance and approval.
Name & Designation of
11.
(in block letters)
MD. SAIFUDDIN KHALID
M.PHARM
ASST. PROFESSOR
DEPT. OF PHARMACOLOGY
LUQMAN COLLEGE OF PHARMACY,
P & T COLONY, OLD JEWARGI ROAD,
GULBARGA-585102 (KARNATAKA).
11.1
11.2
Signature
11.3
Co-Guide
----------
11.4
12.
Guide
Signature
---------All the necessary facilities will be provided to carry
out the proposed research work under the
supervision of guide. So recommended for
registration.
12.1
Remarks of the Chairman
& Principal
12.2
Signature
18