EVALUATION OF ANXIOLYTIC EFFECT OF CAESALPINIA PULCHERRIMA(L.) PODS EXTRACT IN EXPERIMENTAL ANIMALS.

M.PHARM DISSERTATION PROTOCOLSUBMITTED TO THE

RAJIV GANDHI UNIVERSITY OF HEALTH SCIENCES,BENGALURU, KARNATAKA

BYKHAN SADIQUE ASLAM

B.Pharm

UNDER THE GUIDANCE OFMOHAMMED SAIFUDDIN KHALID

M.Pharm.ASST. PROFESSOR

DEPARTMENT OF PHARMACOLOGYLUQMAN COLLEGE OF PHARMACY

GULBARGA-585102

2013-2014

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RAJIV GANDHI UNIVERSITY OF HEALTH SCIENCES, KARNATAKA, BANGALURU

ANNEXURE-II

PROFORMA FOR REGISTRATION OF SUBJECTS FOR DISSERTATION

1. Name of the Candidate and

Address (In block letters)

KHAN SADIQUE ASLAM

S/O MOHAMMED ASLAM KHAN

R.NO 501, SHADAB MANZIL, BOMBAY

COLONY,NEAR RAILWAY STATION,

MUMBRA, THANE 400612. MAHARASHTRA

2. Name of the Institution LUQMAN COLLEGE OF PHARMACY,

GULBARGA – 585 102.

3. Course of Study and Subject M.PHARMA. (PHARMACOLOGY)

4. Date of Admission to Course 29 /07/2013

5. Title of the Topic EVALUATION OF ANXIOLYTIC EFFECT

OF CAESALPINIA PULCHERRIMA(L.) PODS

EXTRACT IN EXPERIMENTAL ANIMALS.

6. 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 disability3 and 1/8th of the total

population worldwide affected with anxiety and become a very important area of research

interest in psychopharmacology4.

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Anxiety is the displeasing feeling of fear and concern5. Benzodiazepine is the most

important group used as anxiolytic and hypnotic agents6. 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 WHO7. 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.

The literature reveals that the plant Caesalpinia pulcherrima possesses various

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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 India18. 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

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and public gardens and has a beautiful inflorescence in yellow, red and orange17.

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 acid23. 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.

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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 anti-

inflammatory 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 acid23. 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, 6-methoxypulcherrimin and homomisoflavonoids24. 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

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above mention activities in turn appear to correlate with some other biological

activities35.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.

d) Open field test.

7. MATERIALS & METHODS:

7.1 SOURCE OF DATA:

Data will be obtained from CD-Rom, Internet facilities, Literatures, related articles,

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books from libraries of Luqman College of Pharmacy, Gulbarga, Gulbarga University,

Gulbarga etc., and other Research Publications and Journals.

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.

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2. Preliminary phytochemical screening37,39,40.

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.

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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 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

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peep through the holes. Each rat will be placed individually in the apparatus for recording

following parameters,

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

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recorded during the test session of 5 minutes,

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.

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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.

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9.Signature of Candidate

KHAN SADIQUE ASLAM

10. Remarks of the Guide “EVALUATION OF ANXIOLYTIC EFFECT OF CAESALPINIA PULCHERRIMA(L.) PODS EXTRACT IN EXPERIMENTAL ANIMALS”. To be carried out by KHAN SADIQUE ASLAM,

16

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 available. Hence the project is viable and is recommended for clearance and approval.

11. Name & Designation of

(in block letters)

11.1 GuideMD. SAIFUDDIN KHALIDM.PHARMASST. PROFESSORDEPT. OF PHARMACOLOGYLUQMAN COLLEGE OF PHARMACY,P & T COLONY, OLD JEWARGI ROAD,GULBARGA-585102 (KARNATAKA).

11.2 Signature

11.3 Co-Guide ----------

11.4 Signature ----------

12. 12.1 Remarks of the Chairman

& Principal

All the necessary facilities will be provided to carry out the proposed research work under the supervision of guide. So recommended for registration.

12.2 Signature

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