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ANTI ANXIETY EVALUATION OF NYCTANTHES ARBOR TRISTIS LINN. PLANT

IN VALIDATED ANIMAL MODELS

M.Pharm Dissertation Protocol

Submitted to the

RAJIV GANDHI UNIVERSITY OF HEALTH SCIENCES, KARNATAKA

BANGALORE

By

ARUN ABRAHAMB.Pharm

Under the Guidance of

DR. BHEEMACHARIM.Pharm., Ph.D

Professor,

Department of Pharmacology,

N.E.T.Pharmacy college

DEPARTMENT OF PHARMACOLOGY

N.E.T.PHARMACY COLLEGE

RAICHUR-584103

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2011-2012

RAJIV GANDHI UNIVERSITY OF HEALTH SCIENCES, KARNATAKA

BANGALORE

Annexure -II

PROFORMA FOR REGISTRATION OF SUBJECTS FOR DISSERTATION

1

.

Name of the candidate and address ARUN ABRAHAM

s/o P. A. Abraham,

Puthenparambil house,

Panchayath lane,

Puthuppally p.o,

KOTTAYAM 686 011 (KERALA)

2 Name of the institution: N.E.T.Pharmacy College,

Raichur-584103

3 Course of study and subject Master of Pharmacy in Pharmacology

4

.

Date of admission to the course 23th August 2011

5

.

Title of the topic ANTI ANXIETY EVALUATION OF NYCTANTHES ARBOR TRISTIS Linn.

PLANT IN VALIDATED ANIMAL MODELS

FAMILY: OLEACEAE

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

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7. Materials and Methods:

7.1: Source of the data ENCLOSURE-IV

7.2: Methods of Collection of the Data ENCLOSURE-V

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

YES (MICE)

7.4: Has ethical clearence been obtained from your institute in caseof 7.3

Yes: IAEC NO.:-576/2002/IAEC/CPCSEA

8. List of References ENCLOSURE-VI

9. Signature of the candidate ARUN ABRAHAM

1

0.

Remark of the guide:

11.1Guide

11.2 Signature

11.3 Co-guide

11.4 Signature

11.5 Head of the Department

The work proposed in this protocol is feasible

and can be carried out in our institute

DR. BHEEMACHARIM.Pharm, Ph.D

Professor and HOD,


N.E.T.Pharmacy College ,Raichur

DR. BHEEMACHARIM.Pharm, Ph.D

Professor and HOD,


N.E.T.Pharmacy College, Raichur

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1.6 Signature

12

12.1 Remarks of the chairman and the principal

SIGNATURE

The protocol is forwarded to the university forfurther processing.

DR.H. DODDAYYAM.Pharm, Ph.D

PRINCIPAL,

N.E.T.PHARMACY COLLEGE,

RAICHUR-584 103

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ENCLOSURE I

6. Brief resume of the intended work:

6.1 Need for the study:

Anxiety is a general term for several disorders that cause nervousness, fear,

apprehension, and worrying. These disorders affect how we feel and behave and they can

manifest real physical symptoms. Mild anxiety is vague and unsettling, while severe anxiety

can be extremely debilitating having a serious impact on daily life.1

People often experience a general state of worry or fear before confronting

something challenging such as a test, examination, recital or interview. These feelings are

easily justified and considered normal. Anxiety is considered a problem when symptoms

interfere with a person's ability to sleep or otherwise function. Generally speaking, anxiety

occurs when a reaction is out of proportion with what might be normally expected in a

situation.1

Anxiety can be accompanied by physical effects such as heart palpitations, nausea,

chest pain, shortness of breath, stomach aches or headaches. Physically body prepares the

organism to deal with the threat. Blood pressure and heart rates are increased and immune

and digestive system functions are inhibited (the flight or fight response). External signs of

anxiety might also experience it as a sense of dread or panic.1

Anxiety related disorders such as generalized anxiety, panic, obsessive-compulsive

disorder, phobias or post traumatic stress disorders are common and major causes of

disability.2 Anxiety effects about 1/8th of the world population and become a very important

area of research in psychopharmacology.3 Anxiety is also an obvious component of many

psychiatric and medical component of many psychiatric and medical conditions. Effective

treatment such as anxiolytic drug therapy or cognitive behavioural therapy exist, but many

patients experience adverse effects or do not benefit from full syndrome control.

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An anxiolytic (also antipanic or antianxiety agent) is a drug used for the treatment

of anxiety, and its related psychological and physical symptoms. Anxiolytics have been

shown to be useful in the treatment of anxiety disorders. Anxiolytics are also known

as minor tranquilizers. The term is less common in modern texts, and was originally derive.

The ideal anxiety drugs would suppress all the symptoms like irritability, uneasiness,

jumpiness, feelings of apprehension, rapid or irregular heart beat, stomachache, nausea,

faintness, and breathing problems associated with it, without causing any unwanted effects.

Numbers of drugs are available for the treatment of anxiety like diazepam, alprazolam,

lorazepam, clonozepam, buspirone, escitalopram, venlafaxine and sertraline etc.4

Among them the most widely prescribed are the benzodiazepines.5 However, the

clinical uses of benzodiazepines are limited by their side effects such as psychomotor

impairment, potentiating of other central depressant drugs and dependence liability.4 It has

lead scientists to investigate plants, which are commonly employed in traditional and

alternate system of medicine for sleep disorders and related diseases.2 Various plants are

being used in complementary and alternative medicines for management of anxiety.

In the present study it is proposed to evaluate the anxiolytic potential of

hydroalcoholic extracts ofNyctanthes arbour tristis linn.(NAT) plant using different validated

animal models for anxiety based on exploratory behaviour.

ENCLOSURE- II

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6.2 Review of literature

1. NAT is a small sacred ornamental tree. It is native of India, distributed wild in sub-

Himalayan regions and southwards to Godavari.6 It is having white fragrant flowers

commonly known as night jasmine.7,8

2. NAT is a large shrub growing to 10 m tall, with flaky grey bark , 9 stiff whitish hair, young

branches10 and rough leaves.11 The flowers are fragrant, with a five- to eight-lobed white

corolla with an orangered centre. They are produced in clusters of two to seven together,

with individual flowers opening at dusk and finishing at dawn.9 Calyx is 6-8 mm long,

narrowly campanulate, hairy outside, glaborous inside truncate or obscurely toothed or

lobed, ciliated. Corolla glaborous and is more than 13 mm long; tube is 6-8 mm long, orange

coloured, about equalling the limbs; lobes are white and unequally obcordate and cuneate.10

The leaves are opposite, simple, 612 cm long and 26.5 cm broad, with an entire margin.

The fruit is a flat brown heart-shaped to round capsule 2 cm diameter, with two sections each

containing a single seed.9 These are long and broad, obcordate or nearly orbicular,

compressed, 2-celled. Seeds are exalbuminous, testa are thick, outer layer of large

transparent cells is heavily vascularised.

3. NAT leaves has been shown to possess anti-arthritic properties. In addition, decoction of

the NAT leaves has been also shown to possess ulcerogenic12, antispasmodic13,

antihelminthic14, cytotoxicity15, antiinflammatory16, immunostimulant17, antidiabetic18,

hepatoprotective19, anti-arthritis20, antioxidant21, antibacterial22, antimicrobial23,

antileishmanial24, anti-viral25, prevention of lung injury26, CNS depressant27 activities.

4. NAT contain chemical constituents like polysaccharides, iridoid glycosides,

phenylpropanoid glycoside (nyctoside A), -sitosterol, -amyrin, hentri-acontane, benzoic

acid, glycosides, nyctanthoside-a iridoid, nyctanthic acid, Friedelin and lupeol and oleanolic

acid and 6-hydroxylonganin28 and iridoid glucosidesarborsides A, B and C, alkaloids,

Phlobatanins, terpenoids and cardiac glycosides. Iridoid glucosides (arbortristosides-A, B, C)

and 6hydroxyloganin,

4-hydroxy hexahydrobenzofuran-7-one tertiary alkaloids mainly 7-

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(alpha-anilino-p-nitrobenzyl)-8-quinolinol and quarternary alkaloids belonging to

protoberberines29 and aporphines30, has also been isolated from this plant.

5. Different parts of NAT are known to possess various ailments by tribal people of India

especially Orissa and Bihar along with its use in Ayurveda, Sidha and Unani systems of

medicines.

a. FLOWERS: Are used as stomachic, carminative, astringent to bowel, antibilious,

expectorant, hair tonic and in the treatment of piles and various skin diseases31 and in the

treatment of ophthalmic purposes32

.

b. STEM: Traditionally the powdered stem bark is given in rheumatic joint pain, in treatment

of malaria and also used as an expectorant.18 The bark is used for the treatment of

snakebite31 and bronchitis33.

c. LEAVES: The leaves of NAT are used extensively in Ayurvedic medicine for the

treament of various diseases such as sciatica, chronic fever, rheumatism, and internal

worm infections, and as a laxative, diaphoretic and diuretic.34 Leaf juice is mixed with

honey and given thrice daily for the treatment of cough. Paste of leaves is given with

honey for the treatment of fever, high blood pressure and diabetes.35 Juice of the leaves

is used as digestives, antidote to reptile venoms, mild bitter tonic, laxative, diaphoretic

and diuretic. Leaves are also used in the enlargement of spleen. The leaf juice is used to

treat loss of appetite, piles, liver disorders, biliary disorders, intestinal worms, chronic

fever, obstinate sciatica, rheumatism and fever with rigors. The extracted juice of leaves

acts as a cholagogue, laxative and mild bitter tonic. It is given with little sugar to children

as a remedy for intestinal ailments. In several cases, it has been found to act

efficaciously for malaria fever. The decoction of leaves is extensively used by Ayurvedic

physicians for the treatment of arthritis, obstinate sciatica, malaria, intestinal worms and

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as a tonic, cholagogue and laxative. The expressed juice of leaves (10ml BD X 5days) is

a native remedy for intermittent fever.

d.SEEDS: The seeds are used as anthelmintics and in alopecia. It is antibilious and an

expectorant, and is also useful in bilious fevers. The powdered seeds are used to cure

scurfy affections of scalp, piles and skin diseases.32

6. Reported studies on the plant are ulcerogenic12, antispasmodic13, antihelmintic14, anti-

inflammatory16, immunostimulant17, antidiabetic18, hepato-protective19, treatment of arthritis20,

antibacterial22, antimicrobial23, antioxidant21, antileishmanial24, prevention of lung injury26,

CNS depressant27, antiviral25.

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ENCLOSURE III

6.3 Main objective of the study: The main objective of the proposed work is to evaluate the

anti-anxiety activity of hydro-alcoholic extract of Nyctanthes arbor tristis linn. The study is

divided into two phases:

Phase 1: Hydro-alcoholic extract of NATare to be prepared and subjected for preliminary

phytochemical screening. LD50 values will be determined on the basis of selection of three

effective doses i.e 1/20th, 1/10th and 1/5th will be made and which would be considered as the

low, medium and high dose respectively.

Phase 2: To evaluate the anti-anxiety activity of hydro-alcoholic extract of the NAT at the

selected doses in validated experimental models like:

1. Elevated plus maze(EPM)

2. Hole board

3. Light/dark models

4. Open field apparatus

It is also planned to evaluate the following parameters in the above models.

1. Number of entries and time spend in open and close arms in Elevated Plus Maze

model.

2. Number and duration of head dips in hole board model.

3. Latency of crossings, number of crossings and time spent in illuminated chamber in

light/dark model.

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4. Total locomotion, percentage of central locomotion, frequency of rearing, defecation

units, immobility and grooming time in open field test.

ENCLOSURE-IV

7. Materials and methods:

7.1 Source of data:

Work is planned to generate data from laboratory based animal experiment studies

as described in national/international journals and from text books available with college and

other institutions. The latest progress in the area will be updated by literature survey through

e-publishing and Helinet consortium 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 animal experimental studies and are to be subjected for

statistical analysis by ANOVA followed by Dunnetts t test. Probability p value less than

0.05 will be considered as statistically significant.

PHASE I

1. Preparation of the Nyctanthes arbor tristis linn. plant extract:

Dried plant parts ofNyctanthes arbor tristis linn. is extracted with hydro-alcoholic mixture. The

extract will be concentrated by distilling off the solvent and then evaporating to dryness on the

water bath. The extract thus obtained will be stored in an air tight container in a refrigerator till

used.

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2. Preliminary phytochemical screening:

The preliminary phytochemical screening will be carried out for qualitative identification of

phytoconstituents in hydro-alcoholic extract ofNyctanthes arbor tristis linn.

DETERMINATION OF LD50

The acute toxicity of hydro-alcoholic extracts of Nyctanthes arbor tristis linn. will be

determined by using the female albino mice (20-25 g), maintained under standard husbandry

conditions. The animal will be fastened for three hours before the experiment. Animals will be

administered with a single dose ofNyctanthes arbor tristis Linn. extracts and are observed for

the mortality upto 48 hours study period (short term toxicity). Based on short term toxicity

profile, next dose will be administered as per the OECD guidelines No. 425. From the LD 50

dose 1/20th, 1/10thand 1/5th doses are to be selected and considered as low, medium and high

doses respectively.

PHASE 2:

To evaluate the anti-anxiety activity of hydro-alcoholic extract of Nyctanthes arbor

tristis linn. by behavioural study the below mentioned models and procedure will be followed.

EXPERIMENTAL MODELS:

1. Elevated plus maze:

Albino mice of either sex weighing between 20-25gm will be randomly selected and divided

into different groups as shown below.

Group A - Normal control (Receives vehicle alone)

Group B - Standard (Diazepam 2mg/kg p.o)

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Group C - Low dose of NAT plant extract

Group D - medium dose of NAT plant extract

Group E - High dose of NAT plant extract.

Experimental procedure:

The plus maize apparatus comprises of two open arms (16x5cm) and two closed arms

16x5x12cm) that extend from a common central platform (5x5cm). The entire maize is

elevated to a height of 25 cm above the floor level. Albino mice of either sex with a body

weight of 20-25gm will be divided into 5 groups of 6 animals in each. Group A will be served

as control and receives vehicle alone. Group B treated with Diazepam (2mg/kg i.p). Group C,

D and E with three different doses of NAT plant extract (low, medium and high)for seven

consecutive days. On the 8th day one hour after oral administration of the standard/test in

respective groups, in a sound attenuated room, the mice is placed in the centre of the maze

facing one of the enclosed arms. During a 5 minute test period the following events will be

recorded.

Number of entries into the open arm

Number of entries into the closed arm

Time spent in open arm

Time spent in closed arm

Time spent in central platform

Total no. of entries in open and closed arm

All the above parameters will be expressed in percentage (e.g. % of open arm entries= 100x

no.of open arm entries/ total no. of entries).

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2. HOLE BOARD APPARATUS:



Group A - Normal control (receives vehicle alone)

Group B - Standard (Diazepam 2mg/kg p.o)

Group C - low dose of NAT in plant extract

Group D - medium dose of NATin plant extract

Group E - high dose of NAT in plant extract

Experimental procedure:

The apparatus consists of wooden chamber(40x40x25cm) with 16 holes (diameter 3 cm) on

the floor, elevated from the ground so the mice could peep through the holes. Albino mice

weighing between 20-25g will be divided into 5 groups of 6 animals each. Group A will be

served as control treated with vehicle p.o. Group B with Diazepam (2mg/kg p.o). Group C,D

and E with 3 different doses of NAT. Plant extract (low, medium and high) for seven

consecutive days. On the 8th day 1 hour after oral administration of the vehicle/standard/

extract in the respective groups, each mice will be placed individually in the apparatus. During

5 minute test period the following parameters will be recorded.

Latency to the first head dips

The number of head dips through the holes

The total time spent with the head dips

No. of rearings

No. of defecation units

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Increase exploratory behaviour, characterize by an increase in the number as well as

duration of head dips is an indication of anxiolytic activity.

3. LIGHT DARK MODEL:



Group A - Normal control (receive vehicle alone)

Group B - Standard (Diazepam 2 mg/kg p.o)

Group C - Low dose of NAT plant extract

Group D - Medium dose of NAT plant extract

Group E - High dose of NAT plant extract

EXPERIMENTAL PROCEDURE:

The light dark apparatus consists of two chambers(40x60x20cm) comprising of brightly

illuminated area (40x40cm) and a dark area (40x20cm) separated by a wall with round

hole(7cm diameter) will be used. Albino mice of either sex with a body weight 20-25g will be

divided into 5 groups of 6 animals in each. Group A will be served as control (receives

vehicle alone). Group B treated with Diazepam (2 mg/ kg p.o). Group C, D and E with three

different doses of NAT of the plant extracts (low, medium and high) for 7 consecutive days.

On 8th day one hour after oral administration of the vehicle/standard/extract in respective

groups, the mice will be placed in the illuminated part of the cage.

The following parameters are recorded during the test session of 5 minutes:

Total no. of crossings.

No. of crossings between light and dark.

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Total time spent in illuminated part of the cage.

The no. of rearrings in the illuminated part of the cage.

No. of rearrings in the dark part of the cage.

The no. of the defecation units.

4. OPEN FIELD BEHAVIOUR:



Group A - Normal control (Receive vehicle alone)

Group B - Standard (Diazepam 2 mg/kg p.o)

Group C - low dose of NAT plant extract.

Group D - Medium dose of NAT plant extract

Group E - high dose of NAT plant extract

EXPERIMENTAL PROCEDURE:

This method is used to evaluate exploratory activity and emotionality of animal. The open field

consist of white printed arena measuring 55cm in diameter with 100 W lamp. The floor of the

arena will be divided into several units of black printed lines. The apparatus will be placed in

sound attenuated room, 48cm above the floor. Albino mice of either sex with a body weight

20-25gm will be divided into 5 groups of 6 animals in each. Group A will be served as control.

Group B treated with Diazepam (2mg/kg p.o). group C,D and E with 3 different doses of NAT

of the plant extracts (low, medium and high) for seven consecutive days. On the eighth day

one hour after the oral administration of the standard/extract in the respective groups, the

mice will be placed in the centre of the arena and the following parameters will be recorded.

Total locomotion (number of units entered on the floor)

Percentage of central locomotion

Rearing frequency(no. of times the animal stood on the hind legs)

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Defecation units(no. of boli)

Immobility time and grooming

Grooming time.

Every time placing each animal, the arena washed with 5% alcohol to eliminate the possible

bias due the odour left by the previous animal.

STATISTICAL ANALYSIS

All values will be expressed as mean SEM from 6 animals. Statistical differences in

mean will be analyzed using one way ANOVA (Analysis of Variance) followed by Dunnetts t

test. p value lower than 0.05 will be considered as statistical significant.

7.3: Does the study require any investigation or interventions to be conducted on

patients other human or animals? If so, please describe briefly:

Study required investigation in mice. The effect of NAT plant extract will be studied on

various parameters as stated above 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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4. Tripathi KD. Essentials of pharmacology. Jaypee brothers publication. 5 th edition, New

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vitro auxillary shoot proliferation. World Journal of Agricultural Sciences 2006; 2: 188-192.

8. Rout GR, Mahato A, Senapati SK. Invitro clonal propagation of Nyctanthes arbortristis

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9. Choudhary, M, Raghuwansi, A. Nyctanthes arbor tristis Linn- A Immunostimulant. National

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11. Lal JB. Constitution of the colouring matter of Nyctanthes arbor tristis. Identity of

Nyctanthin with -crocetin. 1936; 2: 57-61.

12. Rathore B, Paul B, Chaudhary BP, Saxena AK, Sahu AP, Gupta YK. Comparative studies

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16. Saxena RS, Gupta B, Saxena KK, Prasad DN. Study on anti-inflammatory activity in

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arbor tristis Linn. Pharmacognosy Reviews 2007; 1: 344-349.

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