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antianxiety property OF TECOMA STANS Linn LEAVES EXTRACT IN EXPERIMENTAL ANIMALS

antianxiety property OF TECOMA STANS Linn LEAVES EXTRACT IN EXPERIMENTAL ANIMALS

M.PHARM DISSERTATION PROTOCOL

SUBMITTED TO THE

RAJIV GANDHI UNIVERSITY OF HEALTH SCIENCES, KARNATAKA

BY

MOHAMMED MUKARRAM ALI

B.Pharm

UNDER THE GUIDANCE OF

MOHAMMED SAIFUDDIN KHALID

M.Pharm

DEPARTMENT OF PHARMACOLOGY

LUQMAN COLLEGE OF PHARMACY

GULBARGA-585102

2012-2013

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)

mohammed mukarram ali

s/o mohd. wahed ali

c/o Dr. Mirza sikandar baig

h.no. 5-948, hussaini alam,

gulbarga,karnataka? 585104

2.

Name of the Institution

Luqman College of Pharmacy, gULBARGA ? 585 102.

3.

Course of Study and Subject

MASTER OF PharmACY IN PharmacOLOGY.

4.

Date of Admission to Course

12/07/2012

5.

Title of the Research Topic

ANTIANXIETY PROPERTY OF TECOMA STANS LINN LEAVES EXTRACT IN EXPERIMENTAL ANIMALS.

6.

BRIEF RESUME OF THE INTENDED WORK :

6.1 Need for the study:

Anxiety is defined as a feeling of apprehension, uncertainty or tension stemming from anticipation of imagined or unreal treat1.

According to the World Health report WHO2, approximately 450 million people suffer from a mental or behavioral disorder. Yet only a small minority of them receives even the most basic treatment. This amounts to 12.3% of the global burden of disease, and will rise to15% by 20203. Anxiety affects one-eighth of the total population world-wide and has become an important area of research interest in psychopharmacology during this decade4. The complexity of daily life in modern society frequently leads to varying degree of anxiety and depression. Mood depression and anxiety disorders have been found to be associated with chronic pain among medical patients in both developed and developing countries.5,6 Anxiety and stress related problems are so common today, that approximately 40 million Americans or 18 % adult population, suffer from anxiety disorders each year7.

Anxiety disorders are marked by excessive fear (and avoidance), often in response specific objects or situation and in the absence too danger, and they are extremely common in the general population. According to a recent epidemiological study, the life time prevalence of any anxiety disorder is 28.8%8. Anxiety disorders are associated with impaired work place performance and hefty economic costs9. Effective treatment such as antianxiety drug therapy or cognitive behavioral therapy exists but, many patients remain untreated, experience adverse effects of benzodiazepines, or do not benefit from full symptom control10. Benzodiazepine (Diazepam, Lorazepam) is the most important group used as antianxiety and hypnotic. But it shows the side effects during the long term therapeutic use as antianxiety 11. Hence there is needed to look for new more efficacious antianxiety agents with lesser side effects. In the search for new therapeutic products for the treatment of neurological disorders with lesser side effects, medicinal plant research worldwide has progressed constantly, demonstrating the pharmacological effectiveness of different plant species in a variety of animal models12.

The use of the medicinal herbs for curing disease has been documented in history of all civilization. Man in the prehistoric era was probably not aware about the health hazards associate with the irrational therapy with the onset of research in medicine. In the last few years there has been an exponential growth in the field of herbal medicine and these drugs are gaining popularity both in developing and developed countries because of their natural origin and less side effects. Many traditional medicines in use are derived from medicinal plants, minerals and organic matter .The World Health Organization (WHO) has listed 21,000 plants, which are used for medicinal purposes around the world. Among these 2500 species are in India, out of which 150 species are used commercially on a fairly large scale. India is the largest producer of medicinal herbs and is called as botanical garden of the world. It was concluded that plants contain active principles, which are responsible for the curative action of the herbs. The isolated active constituent of medicinal herbs and after testing some found to be therapeutically active13. It has been estimated that 43% of anxiety suffers use some form of complementary therapy. The most popular treatments include herbal medicine10. Many herbal plants available to be best herbs for anxiety.

There are several plants very effective in treating stress / anxiety, such plants include Matricaria chamomilla (chamomile), Hypericum perforatum (St.Jons wort), Piper methysticum (Kava kava) 14, Passiflora incarnate (Passion flower) 15. Dolichandrone falcata, contain chrysin flavones is subgroup of flavonoid present in bark16 and leaves also contain chrysin and Chrysin 7-rutinoside17. The active constituent mention above reported to have many biological activities, and one among them is for antianxiety activity18.

One such plant Tecoma Stans Linn belongs to the plant family Bignoniaceae. A wide spectrum of biological activities has been reported with various constituents isolated from different parts of the plant. It has been reported that the pharmacological significance was noted due to the presence of various bioactive compounds in the Tecoma stans such as saponins, flavonoids, alkaloids, phenols, steroids, anthraquinones, tannins, terpenes, phytosterols and glycosides19. The leaf contains flavanoid, alkaloids, tecomine and tecostidine20. The flowers contain carotene and zeaxantin21.

The plant of Tecoma stans reported to contains flavonoids and tannins and these constituents are said to be responsible for anxiolytic property.16,17,18 In contrast to barbiturates there was no excitation prior to induction of sedation which suggests that the behavioral properties of these fractions are similar to benzodiazepines22. Flavonoids are reported to be very effective in treating stress / anxiety; such as benzoflavones in Passiflora incarnata (Passion flower) 15 chrysin flavones in Dolichandrone falcata leaves17. Flavonoids present in Tecoma stans leaves20 which suggest that it may possess antianxiety property. However the literature survey has no scientific data on antianxiety activity on Tecoma Stans leaves. In view of this, the primary aim of the present study is to investigate the possible antianxiety activity of Tecoma Stans leaves extract in laboratory animals.

6.2 Review of the literature of Tecoma stans Linn.

Botanical classification23:

Kingdom : Plantae

Subkingdom : Tracheobionta

Division : Magnoliophyta

Class : Magnoliopsida

Subclass : Asteridae

Order : Lamiales

Tribe :  HYPERLINK "http://www.ars-grin.gov/cgi-bin/npgs/html/family.pl?1687" o "Link to GRIN report for tribe Wrightieae" Wrightieae.

Family : Bignoniaceae

Subfamily : Apocynoideae

Genus : Tecoma Juss.

Species : Tecoma stans

Vernacular Name24:

English : Trumpetbush

Kannada : Korenekalar

Hindi : Piliya

Telugu : Pachagotla

Marathi : Ghanti

Tamil : Sonapatti

Bengali : Chandaprabha

HABITAT :

The plant material was taxonomically identified by the Botanical survey of India, Pune, and Maharashtra, India. The Tecoma stans commonly known as Yellow bell belongs to the family Bignoniaceae medium sized deciduous tree, dark grey exfoliating in thin strip of bark which is widely available in Kuba, Florida, Mexico and Cultivated in Indian gardens as ornamental plants for its flowers25. Tecoma stans (Bignoniaceae) are distributed worldwide, but most of them occur in tropical and sub tropical countries. However a number of temperate species also grow in North America and East Asia26.

Tecoma stans Linn is the official flower of the United States Virgin Islands and the national flower of The Bahamas.

PLANT DISCRIPTION :

It is a flowering perennial shrub or small tree, 5-7.6m in height. Bark is pale brown to grey and roughens with age. Leaves are opposite, compound and imparipinnate with 2 to 5 pairs of leaflets and a larger single terminal leaflet. Leaflets are lanceolate, up to 10 cm long, with serrated margins, mid-green above and soft to the touch. Flowers occur in clusters at the ends of the branches and are trumpet shaped with 5 rounded lobes, 6 cm long, pale to bright yellow, with faint orange stripes at the throat. Fruits are narrow, slightly flattened to pointed capsules, up to 20 cm long, containing many winged seeds, green when young, pale brown on ripening and remain on the tree in untidy clusters for many months27,28.

MEDICINAL USES :

The plant has been extensively investigated and a number of chemical constituents from the barks, heart wood, leaves and seeds of the plant have previously reported which includes Pyrindance alkaloids,Tecomanine,Iridoid glucosides, Stansioside, Plantarenaloside, Saponifiable compounds, Flavanoids, Monoterpenic alkaloids, 3?, 5?-dimethoxy dihydro flavanol ,Isorhamnethin, ?-carotene,zeaxanthin, flavanone29. The literature survey reveals that the genus Tecoma stans possesses various bio active compounds such as saponins, flavonoids, alkaloids, phenols, steroids, anthraquinones, tannins, terpenes, phytosterols and glycosides19..

Traditional use of leaves of Tecoma stans in throughout Mexico and central America for diabetes and urinary disorder control30, 31. Roots are used as diuretic and vermifugue32. Traditionally flowers and bark are used for treatment of various cancers20. The stem barks showed better antimicrobial activity20. Other spp. of genus tecoma was traditionally used in the treatment of tumors, Anti-inflammatory, analgesic and anti-pyretic activities were also reported.

.

LIST OF LITERATURE REVIEW OF Tecoma Stans

Reports from modern literature:

1.The methanol, ethanol and water extracts of Tecoma stans reported to possess anti-inflammatory, lipooxegenase, xanthine oxidase and acetylcholine esterase inhibitory activities.33

2.The organic extract of Tecoma stans showed effective anti-fungal activity against fonsecaea pedrosoi.33,34,35

3.The Tecoma stans fruit extract and isolated compound from it E/Z acetoside and isoacetoside exhibited a cytotoxic effect on human hepatocarcinoma cells (Hep-G2 tumor cell line) while isolated compound E/Z acetoside and 5-hydroxy-skytanthine hydrochloride were potent inhibitor of human breast carcinoma cell.36

4.The aqueous extract of the leaves of Tecoma stans, Coleus forskohlii and Pogostemon patchouli have been reported for invitro broad spectrum antibacterial activity against 5 human pathogenic bacteria.37

5.Methanol extract of Tecoma stans leaf reported to possess significant wound healing property.38

6.Aqueous extract of Tecoma stans exhibited antidiabetic activity in streptozotocin induced diabetic rats.39

7.The ethanol, methanol and water extracts of the plant Tecoma stans has been reported for antimicrobial and antioxidant activity.40

8.Hydroalcoholic leaf extract of Tecoma stans exhibited antispasmodic effect on rat ileum.41

9. Another reported work on Tecoma stans water extract showed cytotoxicity in human hepatoblastoma (HepG2). 42

Review of literature, till date, regarding Tecoma stans was carried out by chemical abstract, biological abstract, medicinal abstract and other national and international scientific journals. The Literature survey reveals that the fruits and flowers of Tecoma stans Linn resulted in the isolation of a new phenylethanoid, 2- (3,4dihydroxyphenyl)ethyl-2-O-[6-deoxy-alpha-L-mannopyranosyl-4-(3,4-dihydroxyphenyl)- 2-propenoate]- beta-D-glucopyranoside (3), and a novel monoterpene alkaloid, 5-hydroxy-skytanthine hydrochloride (8), along with eleven known compounds: 4-O-E-caffeoyl-alpha-L-rhamnopyranosyl-(1',3)-alpha/beta-D-glucopyranose (1), E/Z-acetoside (2), isoacetoside (4), rutin (5), luteolin 7-O-beta-D-neohespridoside (6), luteolin 7-O-beta-D-glucopyranoside (7) and sucrose (9) were isolated from the fruits, while luteolin 7-O-beta-D-glucuronopyranoside (10), diosmetin 7-O-beta-D-glucuronopyranoside (11), diosmetin 7-O-beta-D-glucopyranoside (12), diosmetin 7-O-beta-D-glucuronopyranoside methyl ester (13) and acetoside (2) were isolated from the flowers.33,34 The leaf showed the presence of flavonides, alkaloids, tecomine, and tecostidine20. The genus Tecoma stans possess various bioactive compounds such as alkaloids, flavonoids, saponins, phenols, steroids, anthraquinones, tannins, terpenes, phytosterols and glycosides that are reported to exhibit various pharmacological activities such as antidiabetic activity, anticancer activity, antioxidant activity, antispasmodic activity, antimicrobial activity and antifungal activity19. These active constituents and the above mention activities in turn appear to correlate with some other biological activities. Our literature survey revealed that the different parts of Tecoma stans have been screened for various pharmacological activities but antianxiety activities were not investigated in Tecoma stans leaves so far. Therefore, the present study is planned to investigate the possible antianxiety effects of Tecoma stans leaves on laboratory animals. Hence this study is essential and justifiable.

6.3 Objectives of study:

The review of literature reveals that antianxiety activity of Tecoma Stans leaves has not been reported. In view of this, the present study is aimed to investigate the antianxiety activity of the Tecoma Stans leaves with the following objectives:

Specific Objective:

Identification and authentication of the plant material.

Extraction of Tecoma Stans leaves with suitable solvents, such as petroleum ether, chloroform, ethanol and water.

To carryout preliminary phytochemical analysis of crude extracts for the detection of the type of phytoconstituents present.

To study the acute toxicity for determination of LD50 of the extract in mice.

To evaluate the effect of Tecoma Stans leaves extract for the antianxiety property in mice and rats in following models:

Elevated Plus-Maze test in mice.

Hole-board test in rats.

Light-dark model transition test in mice.

7.

MATERIALS AND METHODS:

7.1 Source of data:

Whole work is planned to generate data from laboratory based Experimental Albino Rats/Mice (either sex; weighing 150-250gm rats and 20-30gm mice) models as described in various national and international journals and books available with our college and other reputed institutions at India through e-publishing and HELINET of RGUHS, Bangalore.

The scheme of proposed work is as follows:

Collection of leaves and preservation.43

Shade drying, coarse powdering of leaves.43,44

To prepare extracts from suitable solvents.43,44

Preliminary Phytochemical investigation of various extracts.44,45

Study of the acute toxicity for determination of LD50 of the extracts in mice.46

To investigate the antianxiety property of extracts.

Methods of collection of data:

Plant Material: The Tecoma Stans leaves are found throughout India.

The whole study is divided in 3 phases.

Phase I:

Preparation of various solvent extracts 43,44:

It is planned to dry the leaves 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 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. The solution of various solvent extracts of Tecoma Stans leaves is planned for the following investigation.

2. Preliminary phytochemical screening 44, 45:

It is planned to carry out the preliminary phytochemical investigation of different extracts of Tecoma Stans leaves for detection of various phytochemical by following standard methods were identified by qualitative analysis followed by their confirmation by thin layer chromatography, which indicate the presence of flavonoids, triterpenoids, naphthoquinones, alkaloids and steroids described in practical pharmacognosy by C.K. Kokate and R.K. Khandelwal.

Phase Ii:

Determination of Acute toxicity studies (LD50).46

It is further planned to study the acute toxicity of solvent extract of Tecoma Stans leaves in albino mice of either sex (20-30gm). Fixed dose method (OECD guideline number 420) of CPCSEA will be adopted for toxicity studies.

Phase IiI:

Elevated Plus-Maze Test in mice.47,48,49,50

Albino mice of either sex weighing between 20-30gm are selected and divided into different groups of 6 animals (n=6) each where the control group will receive 2% gum acacia per oral and the standard group receives drug diazepam at a dose of 2mg/kg.

Group I - Normal control (2% gum acacia; p.o)

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

Group III - Extract of Tecoma Stans leaves (Selective low dose; p.o)

Group IV - Extract of Tecoma Stans leaves (Selective high dose; p.o)

Following parameters are planned to study:

% of open arm time

% of close arm time

% open arm entry

% closed arm entry

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.

2. Hole-board test in rats51:

Albino wistar rats of either sex weighing between 150-250gm are selected and divided into different groups of 6 animals (n=6) each, where the control group will receive 2% gum acacia per oral and the standard group receives drug diazepam at a dose of 2mg/kg.

Group I - Normal control (2% gum acacia; p.o)

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

Group III - Extract of Tecoma Stans leaves (Selective low dose; p.o)

Group IV - Extract of Tecoma Stans leaves (Selective high dose; p.o)

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:

Latency to the first head dips.

Number of head dips in the holes.

Total time spend with the head dips.

Number of rearing.

Total locomotors activity (numbers of squares crossed).

3. Light-dark model transition test in mice52,53,54,55:

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 behavior 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 antianxiety activity.

Albino mice of either sex weighing between 20-30gm are selected and divided into different groups of 6 animals (n=6) each, where the control group will receive 2% gum acacia per oral and the standard group receives drug diazepam at a dose of 2mg/kg.

Group I - Normal control (2% gum acacia; p.o)

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

Group III - Extract of Tecoma Stans leaves (Selective low dose; p.o)

Group IV - Extract of Tecoma Stans leaves (Selective high dose; p.o)

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:

Total number of crossings,

Number crossing between the light and dark area,

Total time spend in the illuminated part of the cage,

Time spend in the dark part of the cage.

Inclusion criteria:

Normal and healthy animals weighing between 150-250gm for rats and 20-30gms mice will be included in the study.

Exclusion criteria:

The albino wistar rats and mice which do not fall the above mentioned weights are excluded from the study.

STATISTICAL ANALYSIS: All values will be expressed as Mean ( SEM from 6 animals in every group. Results will be subjected to statistical analysis using one-way ANOVA and allowed by post hoc test (Dunnet?t? test). p<0.05 will be considered as statistically significant.

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 investigation on animals like albino rats and mice.

7.4 Has ethical clearance been obtained from your institution in case of 7.3?

Yes, the present study is approved from Institutional Animal Ethics Committee (registration number 346/CPCSEA; and IAEC copy enclosed).

8.

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9.

Signature of Candidate

MOHAMMED MUKARRAM ALI

10.

Remarks of the Guide

?Antianxitey property of Tecoma stans Linn leaves extract in experimental animals? to be carried out by mohammed mukarram ali of 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 Guide

MD. SAIFUDDIN KHALID

M.PHARM

ASST. PROFESSOR

DEPT. OF PHARMACOLOGY

LUQMAN COLLEGE OF PHARMACY,

P & T COLONY,OLD JEWARGI ROAD,

GULBARGA-585 102, (KARNATAKA)

11.2 Signature

11.3 Co-Guide

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11.4 Signature

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12.

12.1 Remarks of the

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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