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 ABRAHAM
B.Pharm

Under the Guidance of

DR. BHEEMACHARI
M.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)

Name of the institution:

N.E.T.Pharmacy College, Raichur-584103

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
Brief Resume of the intended work: 6. 6.1: Need for the study 6.2: Review of the literature 6.3: Main Objective of the study ENCLOSURE-I ENCLOSURE-II ENCLOSURE -III

7.

Materials and Methods: 7.1: Source of the data 7.2: Methods of Collection of the Data ENCLOSURE-IV ENCLOSURE-V

7.3: Does the study require any investigations or interventions to be conducted on patients other humans 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:

The work proposed in this protocol is feasible and can be carried out in our institute

11.1Guide

DR. BHEEMACHARI M.Pharm, Ph.D

Professor and HOD, Department of Pharmacology, N.E.T.Pharmacy College ,Raichur

11.2 Signature 11.3 Co-guide 11.4 Signature 11.5 Head of the Department

DR. BHEEMACHARI M.Pharm, Ph.D

Professor and HOD, Department of Pharmacology, N.E.T.Pharmacy College, Raichur

1.6 Signature

1 2

12.1 Remarks of the chairman and the principal

The protocol is forwarded to the university for further processing.

DR.H. DODDAYYA M.Pharm, Ph.D

PRINCIPAL, N.E.T.PHARMACY COLLEGE, RAICHUR-584 103

SIGNATURE

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. 5

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 of Nyctanthes 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 subHimalayan 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 ulcerogenic 12, antispasmodic13, antihelminthic14, hepatoprotective19, cytotoxicity15, antiinflammatory16, antioxidant21, immunostimulant17, antibacterial22, antidiabetic18, antimicrobial23,

anti-arthritis20,

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-

(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

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 ulcerogenic 12, antispasmodic13, antihelmintic14, antiinflammatory16, immunostimulant17, antidiabetic18, hepato-protective19, treatment of arthritis20, antibacterial22, antimicrobial23, antioxidant21, antileishmanial24, prevention of lung injury26, CNS depressant27, antiviral25.

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 NAT are 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 of Nyctanthes 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 of Nyctanthes 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 of Nyctanthes 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 Group B Normal control (Receives vehicle alone) Standard (Diazepam 2mg/kg p.o)

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Group C Group D Group E

Low dose of NAT plant extract medium dose of NAT plant extract 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). 13

2. HOLE BOARD APPARATUS: Albino mice of either sex weighing between 20-25gm will be randomly selected and divided into different groups as shown below. Group A Group B Group C Group D Group E Normal control (receives vehicle alone) Standard (Diazepam 2mg/kg p.o) low dose of NAT in plant extract medium dose of NAT in plant extract 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: Albino mice of either sex weighing between 20-25gm will be randomly selected and divided into different groups as shown below. Group A Group B Group C Group D Group E Normal control (receive vehicle alone) Standard (Diazepam 2 mg/kg p.o) Low dose of NAT plant extract Medium dose of NAT plant extract 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: Albino mice of either sex weighing between 20-25gm will be randomly selected and divided into different groups as shown below. Group A Group B Group C Group D Group E Normal control (Receive vehicle alone) Standard (Diazepam 2 mg/kg p.o) low dose of NAT plant extract. Medium dose of NAT plant extract 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 8. List of references:

1. Singhal KG, Gupta GD. Anti-anxiety activities of various extracts of Nerium oleander linn. flowers. International Journal of Pharmacy and Pharmaceutical Sciences 2011, July; 3(4): 323-326. 2. Andreatini r, Sartori V A, Seabra ML, Leite JR. Ernst. Herbal remedies for anxiety a systemic review of controlled clinical trials. Phytomed 2004; 1(4):3. 3.Rabbani M, Sajjidi S, Ezarei HR. Anxiolytic effects of Stachys Lavandulifollia on the elevated plus-maze model of anxiety in mice. J Ethnopharmacol 2003; 89; 271-276.

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4. Tripathi KD. Essentials of pharmacology. Jaypee brothers publication. 5th edition, New Delhi. Page No.400. 5. Yadav AV, Kawale LA, Nade VS. Effect of Morus alba L. Leaves on anxiety in mice. Ind J Pharmacol 2008:40:32-6. 6. Harleen K, Mohanjith K et.al. An update on Nyctanthes arbor tristis linn.. International Pharmaceutica Sciencia. Jan-march 2011. Vol 1(1). 7. Siddique I, Anis M, Jahan AA. Rapid multiplication of Nyctanthes arbor-tristis through invitro 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 Linn.-a medicinal tree. Horticulture Sciences (Prague) 2007; 34: 84-89. 9. Choudhary, M, Raghuwansi, A. Nyctanthes arbor tristis Linn- A Immunostimulant. National Conference on Recent Advances in Herbal Drug Technology. Organized by: Lakshmi Narain College of Pharmacy, Bhopal 10. Sasmal D, Das S, Basu SP. Phytoconstituents and therapeutic potential of Nyctanthes arbor tristis Linn. Pharmacognosy Reviews 2007; 1: 344-349. 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 of different organs of Nyctanthes arbor tristis in modulation of cytokines in murine model of arithritis. Biomedical and Environmental Sciences 2007; 20: 154-159. 13. Das S, Sasmal D, Basu SP. Antispasmodic and antihelmintic activity of Nyctanthes arbor tristis Linn. International Journal of Pharmaceutical Sciences and Research 2010; 1: 51-55. 14. Wallander E, Albert VA. Phylogeny and classification of Oleaceae based on RPS16 and TRNL-F sequence data. American Journal of Botany 2000; 87: 1827-1841. 15. Khatune NA, Hoque ME, Mosaddik, MA. Laboratory evaluation of Nyctanthes arbor tristis L, flower extract and its isolated compound against common filarial vector, Culex quinquefasciatus say (Diptera : culicidae) Larvae. Pakistan Journal of Biological Sciences 2001; 4: 585. 18

16. Saxena RS, Gupta B, Saxena KK, Prasad DN. Study on anti-inflammatory activity in leaves of Nyctanthes arbor tristis. Journal of Ethnopharmacology 1984; 11: 319-330. 17. Kumar S, Gupta P, Sharma S, Kumar D. 2011. A review of immunostimulatory plants. Journal of Chinese Integrative Medicine 2011; 9: 117-128. 18. Suresh V, Jaikumar S, Arunachalam G. Antidiabetic activity of ethanolic extract of stem bark of Nyctanthes arbor tristis Linn. Research Journal of Pharmaceutical Biological and Chemical Sciences 2010; 1: 311-317. 19. Vishwanathan M, Juvekar AR. Hepatoregenerative effects of Nyctanthes arbor tristis Linn. on acetaminophen induced oxidative damage in rats. International Journal of PharmaTech research 2010; 2: 1291-1297. 20. Rathore B, Paul B, Chaudhary BP, Saxena AK, Sahu AP, Gupta YK. Comparative studies of different organs of Nyctanthes arbor tristis in modulation of cytokines in murine model of arithritis. Biomedical and Environmental Sciences 2007; 20: 154-159. 21. Narendhirakannan RT, Smeera T. In-vitro antioxidant studies on ethanolic extracts of leaves and stems of Nyctanthes arbor tristis L. (Night-flowering jasmine) International Journal of Biological and Medical Research 2010; 1: 188-192. 22. Mahida Y, Mohan JSS. Screening of plants for their potential antibacterial activity against Staphylococcus and Salmonella sp. Natural Product Radiance 2007; 6: 301-305. 23. Vats M, Sharma N, Sardana S. Antimicrobial activity of stem bark extracts of Nyctanthes arbor tristis Linn. (Oleaceae) International Journal of Pharmacognosy and Phytochemical Research 2009; 1: 12-14. 24. Tandon JS, Srivastava V, Guru PY. Iridoids: a new class of leishmanicidal agents from Nyctanthes arbor tristis. Journal of Natural Products 1991; 54: 1102-04. 25. Gupta P, Bajaj SK, Chandra K, Singh KL, Tandon JS. 2005. Antiviral profile of Nyctanthes arbor tristis against encephalitis causing viruses. Indian Journal of Experimental Biology 2005; 43: 1156-1160.

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26. Paul BN, Prakash A, Kumar S, Yadav AK, Mani U, Saxena AK, Sahu AP, Lal K, Dutta KK. Silica induced early fibrogenic reaction in lung of mice ameliorated by Nyctanthes arbor tristis. Biomed Environmental sciences 2002; 15: 215-222. 27. Das S, Sasmal D, Basu SP. Evaluation of CNS depressant activity of different parts of Nyctanthes arbor tristis Linn. Indian Journal of Pharmaceutical Sciences 2008; 70: 803-806. 28. Jensen SR, Franzyk H, Wallander E. Chemotaxonomy of the Oleaceae: iridoids as taxonomic markers. Phytochemistry 2002; 60: 213-231. 29. Harleen K, Mohanjith K et.al. An update on Nyctanthes arbor tristis linn.. International Pharmaceutica Sciencia. Jan-march 2011. Vol 1(1). 30. Kannan M, Singh AJAR, Kumar TTA, Jegatheswari P, Subburayalu S. Studies on immune-bioactivities of Nyctanthes arbor tristis (Oleaceae). African Journal Of Microbiology Research 2007; 1: 088-091. 31. Khatune NA, Islam ME, Rahman MAA, Mosaddik MA, Haque ME. In-vivo Cytotoxic evaluation of new benzofuran derivative isolated from Nyctanthes arbor tristis L. on Ehrlich Ascite Carcinoma cells (EAC) in mice. Journal of Medical Sciences 2003; 3: 169-173. 32. Sasmal D, Das S, Basu SP. Phytoconstituents and therapeutic potential of Nyctanthes arbor tristis Linn. Pharmacognosy Reviews 2007; 1: 344-349. 33. Kritkar, KR, Basu, BD, 1993. Indian Medicinal Plant. LM Basu Allahabad, India, 2: 15261528. 34. Tuntiwachwuttiku P, Rayanil K, Taylor WC. Chemical constituents from the flowers of Nyctanthes arbor tristis. Science Asia 2003; 29: 21-30. 35. Nawaz AHMM, Hossain M, Karim M, Khan M, Jahan R, Rahmatullah M.. An ethnobotanicals Survey of Jessore district in Khulna Division, Bangladesh. AmericanEurasian Journal of Sustainable Agriculture 2009; 3: 238-243.k

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