Evaluation of antidepressant and anxiolytic like activity of aqueous extract and essential oil from ocimum basilicum in rodents




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EVALUATION OF ANTIDEPRESSANT AND ANXIOLYTIC LIKE ACTIVITY OF AQUEOUS EXTRACT AND ESSENTIAL OIL FROM OCIMUM BASILICUM IN RODENTS

SYNOPSIS FOR



M.PHARM DISSERTATION

SUBMITTED TO



RAJIV GANDHI UNIVERSITY OF HEALTH SCIENCES

KARNATAKA

SUBMITTED BY



JYOTIRMOY DUTTA

I M.PHARM



DEPARTMENT OF PHARMACOLOGY

PES COLLEGE OF PHARMACY

BENGALOORU-560050

(2009-11)

RAJIV GANDHI UNIVERSITY OF HEALTH SCIENCES,

KARNATAKA, BENGALOORU -560050





1



NAME OF THE CANDIDATE AND ADDRESS


JYOTIRMOY DUTTA

LOCAL ADDRESS

P.E.S. COLLEGE OF PHARMACY


HANUMANTHANAGAR,

50 FT. ROAD,

BENGALOORU -560050.

PERMANENT ADDRESS


S/O N.G.DUTTA

43/1/4,CENTRAL ROAD,

P.O-SHYAMNAGAR,

DIST-24PARGANAS(N),

PIN-743127, WEST BENGAL.


2

NAME OF THE INSTITUTION

P.E.S. COLLEGE OF PHARMACY


HANUMANTHANAGAR,

50 FT. ROAD,

BENGALOORU -560050.



3


COURSE OF STUDY AND SUBJECT

MASTER OF PHARMACY IN PHARMACOLOGY.




4

DATE OF THE ADMISSION


8th MAY 2009

5




TITLE OF THE TOPIC:


Evaluation of Antidepressant and Anxiolytic like activity of aqueous extract and essential oil from Ocimum Basilicum in rodents”.




ANNEXURE-II

PROFORMA FOR REGISTRATION OF SUBJECTS FOR DISSERTATION







6

Brief resume of the intended work:



6.1 Need for the study:

The central feature of an affective disorder is an alteration in mood; the most common is that of a low mood or depression. The overall incidence of depression is much higher and there appears a significant difference between the sexes. Studies from Europe and America, using standard measurement tool, found a lifetime prevalence of between 16% and 17 %,with a six month prevalence of about 6%. Higher rates are consistently found in women but social economic and ethnic factors are likely to be influential.

A low mood is a central feature of depression, which is often accompanied by a loss of interest or pleasure in normally enjoyable activities. Thinking is pessimistic and in some cases suicidal. In severe cases, psychotic symptoms such as hallucination or delusion may be present. Anxiety or agitation, frequently accompany the disorder and the so called biological features of sleep disturbances, weight loss and loss of appetite are often present. Sexual drive is often reduced, and some people may lose interest in sex altogether 1.The major class of drugs used in depression are inhibitors of monoamine uptake such as non selective nor adrenaline/serotonin. Inhibitors of monoamine uptake: tricyclic antidepressants (TCA), e.g. imipramine; Selective 5-HT (serotonin) uptake inhibitors (SSRI), e.g. fluoxetine; Other inhibitors that are chemically unrelated to TCA but similar pharmacologically, e.g. maprotiline, Monoamine oxidase (MAO) inhibitors (MAOI), e.g. phenelzine, Miscellaneous antidepressants: mianserin etc.2

Anxiety disorders, are the most prevalent psychiatric illnesses in the general community, are present in 15 to 20% of clinic patients. Anxiety, defined as a subjective sense of unease, dread, or foreboding, can indicate a primary psychiatric condition or can be a component of, or reaction to, a primary medical disease. The primary anxiety disorders are classified according to their duration and course and the existence and nature of precipitants.3

The major class of anxiolytic drugs are benzodiazepines eg.diazepam, 5-HT1A-receptor agonists eg.buspirone; the β-adrenoceptor antagonists are used mainly to reduce physical symptoms of anxiety (tremor, palpitations, etc.); they have no effect on the affective component eg.propranalol.2

many plants conveniently available in india are used in traditional folklore medicine for the treatment of depression and anxiety. Several indigenous medicinal plants are employed in the traditional management of depression and anxiety but there is need to conduct pharmacognostic and pharmacological studies to ascertain their therapeutic values. The medicinal plants might be a useful source for the management of depression and anxiety for development of new chemical entities or as a dietary adjunct to existing therapies Ocimum basilicum is a genus of lamiaceae. As far as its action on nervous system, it is indicated in depression, anxiety, tension, mental fatigue, poor memory & concentration, apathy and migraine.8

Since the plant has been traditionally indicated in brain diseases especially in case of depression and anxiety, hence it was thought worthwhile to investigate this plant for antidepressant and anxiolytic activity.










    1. Review of the Literature:

Polyphenol-rich extracts from ocimum basilicum showed hypolipemic activity in Triton wr-1339-induced hyperlipidemic mice.10 The ethanolic extract from ocimum basilicum has been found to decreases cholesterol synthesis and lipid accumulation in human macrophages.11 Aqueous extract of ocimum basilicum showed vasorelaxant and anti platelet aggregation effect. 12The plant ocimum basilicum was also found to have larvicidal and insect repellent potential against dengue vector, aedes aegypti.13

The basil leaf was found to have a chemomodulatory activity, on drug metabolizing and antioxidant enzymes, and on carcinogens-induced skin and forestomach papillomagenesis.14

PLANT PROFILE
PLANT NAME: Ocimum basilicum

FAMILY: Lamiaceae
SYNONYMS :- O. basilicum var. glabratum Benth., O. basilicum var. majus Benth.6
VERNACULAR NAMES:-

Sanskrit-: Bisva Tulasi, varvara, English:-Sweet Basil, Bengali:- Babui tulsi, telugu:-kukkatulasi Tamil:-tiruniruparachi, karandai, Malayalam:-Valumbari, Uriya:-Dhala tulsi, Sind:-sabajhi, Hindi:-Babui, Tulsi, Kannada:-kam kasturi, Punjabi:-Baburi.5


Distribution:-Indigenous on the lower hills of Punjab. Cultivated throughout the greater part of India, Ceylon, Burma.4


Habitat:-This small annual shrub or herb, indigenous to Persia and Sind, is cultivated in gardens in India. Basil is native to southern Asia and middle east but it has long been grown in Europe as an ornamental, culinary and medicinal herb. It is grown commercially in central and southern Europe .5

Parts Used: Leaves, flowering tops, essential oil.9

Description4,5: An erect branching herb,0.6-0.9m high, or more or less hispidly pubescent. The plant is an annual herb.

Leaves: Leaves are 2.5-5 cm or longer, ovate, acute, entire or more or less toothed or lobed; base-cuneate, entire, petiole 1.3-2.5 cm long.

Flowers: The small, yellow, pinkish flowers are arranged in whorls in the upper leaf axils.

Stems and branches: Stems and branches green or sometimes purplish
chemical constituents: -

Essential oil (1%) includes estagole (up to 70%), eugenol, camphor, methyl cinnamate,7 methylchavicol, linalool, cineole, ocimene, borneol, sambulene and safrole obtained from essential oil.5


Medicinal Actions & Uses

Basil is useful in treating depression, anxiety, migraines and sleeping disorders. Sweet basil leaves can be applied externally to act as an insect repellent and the juice from the leaves brings relief from bites and stings.9

One of the best nerve tonics, basil acts as a reviving restorative for depression, apathy and

melancholia, clearing the mind of confusion and apprehension. It is believed to stimulate the memory, promote mental clarity and concentration.8


Traditional medicinal uses:-

The plant is used as diuretic, emmenagogue; useful in diseases of heart and brain, chronic pain in the joints, asthma, inflammations, enlarged spleen. The roots are used for the bowel complaints of the children. The flowers possess stimulant, demulcent properties. The seeds are useful in gonorrhea, diarrhea, chronic dysentery.4



6.3 Objectives of the study
1.Preparation of aqueous extract of leaves from Ocimum basilicum and procurement of

essential oil obtained from Ocimum basilicum

2.To investigate preliminary phytochemical constituents of aqueous extract of leaves from

Ocimum basilicum(lamiaceae)

3.Determination of LD50 of the aqueous extract and essential oil of Ocimum basilicum as per

OECD guideline.

4.To establish the pharmacological profile of prepared extract and essential oil for its

antidepressant and anxiolytic activity.


Materials and methods




7.1 Source of data

Whole experiment is planned to generate data from laboratories studies. Experiment will be performed as described in the standard bibliography, may be obtained from standard journals and text books available within the college or from other pharmacy colleges or from libraries of National Institutes or through internet.The following various sources will be utilized to obtain related information regarding this research protocol.

IISc library, Bengalooru.

PESCP library, Bengalooru.

RGUHS digital library (Helinet), Bengalooru.

Websites: www.sciencedirect.com

www.pubmed.gov

www.google.com

www.jhetchem.com

www.ijp-online.com




7.2 Method of collection of data

The whole study is divided into following phases;


Phase I: Collection of plant material.

The plant material and the essential oil obtained from the plant will be procured from the authenticated supplier in the month of February and March.


Phase II: Preparations of extracts.

Dried powder leaves of the plant will be extracted with water in a soxhlet apparatus for 72 hours. The residue obtained after extraction is dried. The residue mass obtained is evaporated and reduce temperature to dryness. A % yield of the extracts will be determined.


Phase III: Preliminary Phytochemical investigation.

Preliminary phytochemical investigation will be done as described in Practical Pharmacognosy- Techniques and Experiments15


Phase IV: Acute oral toxicity (as per OECD guidelines).16

Female Swiss albino mice (18-20 g) are individually identified and allowed to acclimate to the laboratory condition for 7 days before the start of the study. Only one mouse receives single dose at a particular time. First animal receives a dose of 175 mg/kg and is observed for any toxicity signs, survival or death up to 48 hrs. If the first animal died or appeared moribund, the second animal receives a lower dose (55mg/kg). The dose progression or reduction factor is 3.2 times of the previous dose. If no mortality is observed in the first animal then the second animal receives a higher dose (55 mg/kg). Dosing of the next animal is continued depending on the outcome of the previously dose for a fixed time interval (48 hours). The test is stopped when one of the stopping criteria is observed.


5 reversals occur in any 6 consecutive animals tested.

3 consecutive animals died at one dose level.

Survived animals are observed for long-term outcomes for a period of 14 days. The acute oral toxicity values are calculated using AOT 425 software (Environmental Protection Agency, USA) based on the short term (48 hours) and long term out come (14 days).

Phase V: Pharmacological evaluation.
For determination of antidepressant and anxiolytic activity the following experiment are to be performed.

All the models used in the pharmacological experiments will consist of the below 8 groups consisting of 6 animals in each group



ASSESSMENT OF ANTIDEPRESSANT ACTIVITY.
Animals:

Female Wistar rats (200–250 g) will be used. They will be housed under standard (25±2 °C, 60–70% humidity) laboratory conditions, maintained on a 12 hour natural day–night cycle, with free access to standard food and water ad libitum. Animals will be acclimatized to laboratory conditions before the test


STRESS PROCEDURE 17
The chronic stress model will be induced by chronic variable stress (CVS). Rats in stressed groups will be exposed to the following stressors once daily for 5 consecutive weeks: 2-h immobilization; 24-h food deprivation; 24-h water deprivation; 5-min cold swim at 40C; wet bedding; 1-min tail pinch with a clothes-pin placed 1cm distal from the base of tail. The same stressor will not be applied successively so that rats cannot anticipate the occurring of stress. Immediately after the conclusion of each stress session the animals will be returned to their home cages and maintained standard conditions until the next stress session. Normal control animals will be housed in group of 4 per cage without disturbing except for necessary procedure such as weighting or cage cleaning. They will have free access to food and water except for a 21-h period of food and water deprivation before the sucrose consumption test.
A.FORCED SWIMMING TEST17
The test will be carried out on 2 successive days after the last stress period. Briefly, rats were forced to swim in a vertical plastic cylinder (diameter 21 cm, height 50 cm) containing 25cm of water maintained at 25±1 0C. On the 1st day of experiment, rats will be forced to swim for 15 min. On the following day, rats will be re-exposed to the forced swimming for 5 min. During the second test session, the immobility time will be evaluated. A rat is judged to be immobile whenever it remains floating passively in the water in a slightly hunched but upright position with its head just above the surface. At the conclusion of the swim test, the animal will be removed from the cylinder, dried by a towel, and returned to its home cage.
B. SUCROSE CONSUMPTION TEST17
Rats will be trained to consume 1% (w/v) sucrose solution before the experiment. They will be exposed to two bottles of 1% sucrose solutions in a 24-h period and exposed to one bottle of 1% sucrose solution and one bottle of drinking water for next 24-h period. Formal test will be carried out before stress, 2weeks after stress, and 5weeks after stress. Animals were food-and water-deprived for 21 h and they will be exposed to both the test solution (1% sucrose and drinking water) for the next 1-h period. Sucrose consumption will be measured by reweighing pre-weighed bottles of test solution. Bottles will be counter balanced across left and right sides of the cages throughout the experiment.


CLASSIFICATION OF GROUPS:


Group 1

Normal Control, no stress no drug

Group 2

Stress, Treated with vehicle of aqueous extract(p.o)

Group 3

Stress, Treated with standard (fluoxetine) (i.p)

Group 4

Stress, Treated with vehicle of essential oil(i.p)

Group 5

Stress, Treated with low dose of aqueous extract(p.o)

Group 6

Stress, Treated with high dose of aqueous extract(p.o)

Group 7

Stress, Treated with low dose of essential oil(i.p)

Group 8

Stress, Treated with high dose of essential oil(i.p)


Standard to be used :-

1) Forced Swimming Test :- Fluoxetine 10mg/kg (i.p)

2) Sucrose Consumption Test :- Fluoxetine 10mg/kg (i.p)

ASSESSMENT OF ANXIOLYTIC ACTIVITY

Animals:

Swiss albino mice (18–22g) will be used. They will be housed under standard (25±2) °C, 60–70% humidity) laboratory conditions, maintained on a 12 hour natural day–night cycle, with free access to standard food and water. Animals will be acclimatized to laboratory conditions before the test


Model 1: ELEVATED PLUS MAZE: 18,19
The plus-maze apparatus comprises of two open arms (30cmX5cm) and two enclosed arms (30cmX5cmX12cm) that extend from a common central platform (5cmX5cm) and was elevated to a height of 45 cm above the floor. Thirty minutes after the treatment with standard and one hour after treatment with controls/extracts, each mouse will be placed at the center of the maze, facing one of the enclosed arms. The number of entries and the time

spent in open and closed arms will be recorded for 5 min. Every time before placing each animal, the maze will be washed with 5% alcohol to eliminate the possible bias due to the odour left by the previous animal.



Model-2: LIGHT AND DARK MODEL 20
The mice’s light-dark box (40×20×20cm) consists of two parts, the light-compartment and the dark-compartment with a volume ratio of 3:1.There is a hole (5×5cm) in the bottom of the clapboard between the two compartments. A 60-W incandescent bulb will be providing a illumination of 700 lx for the light compartment. Thirty minutes after the treatment with standard and one hour after treatment with controls/extracts, each mouse will be put into the centre of the light- compartment with their back to the dark-compartment, and then transition behavior over ten minutes will be observed, including the latency time (latency before entering the dark – compartment), the transition no. (the no. of dark compartment to light- compartment transition) and the total time spent visiting the light- compartment.

CLASSIFICATION OF GROUPS



Group 1

Normal Control


Group 2

Control receiving vehicle for aqueous extract(p.o)


Group 5

Control receiving vehicle for essential oil(i.p)


Group 4

Treated with standard(i.p)


Group 5

Treated with low dose of aqueous extract(p.o)


Group 6

Treated with high dose of aqueous extract(p.o)


Group 7

Treated with low dose of essential oil(i.p)



Group 8

Treated with high dose of essential oil(i.p)



Standard to be used :-

1) Elevated Plus Maze :-Diazepam 0.25mg/kg

2) Light And Dark Model :-Diazepam 0.25mg/kg


STATISTICAL ANALYSIS
All the values will be expressed as mean ± SEM. The data will be analyzed by using one way ANOVA followed by suitable Dunnetts-t test. Statistical significance will be set at p< 0.05.

7.3 Does the study require any investigation to be conducted on patients



Or other humans or animals?

Yes, the experimental models require usage of laboratory animals.



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

7.3?

Yes, ethical clearance has been obtained [copy of IAEC clearance has been attached].








8.




References:-

01. Walker R, Whittliesea C. Clinical Pharmacy And Therapeutics. IVth ed: Elsevier; 2007.


02. Rang H, Dale M, Ritter J, Moore P. Pharmacology. Vth ed: Churchill Livingstone; 2003.
03. Dipiro JT, Talbert RL, Yee GC, Matzke GR, Wells BG, Posey LM. Pharmacotherapy A Pathophysiologic Approach. VIIth ed: The McGraw-Hill Companies; 2008.
04. Kirthikar KR, Basu BD. Indian Medicinal Plants. IInd ed: International book distributors; 1999.
05. Singh MP, Panda H. Medicinal Herbs With Their Formulation. Ist ed: Daya Publishing House; 2005.
06. Duke JA. Handbook Of Medicinal Herbs. IInd ed: CRC Press; 2002.
07. Keville K. The Illustrated Herb Encyclopedia: MALLARD PRESS; 2001.
08. Monograph of ocimum basilicum [online]. [cited 2009 Dec 09]; Available from: http://www.naturaltoucharomatherapy.com/monographs/basil-sweet-monograph.pdf
09. Monograph of ocimum basilicum [online]. [cited 2009 Dec 09]; Available from:http://www.botanicalsearch.com/pdfupload/Botanical/Ocimum%20Basilicum.pdf
10. Harnafi H, Caid HS, Bouanani NeH, Aziz M, Amrani S. Hypolipemic activity of polyphenol-rich extracts from Ocimum basilicum in Triton WR-1339-induced hyperlipidemic mice. Food Chem. 2008;108:205-12.
11. Bravo E, Amrani S, Aziz M, Harnafi H, Napolitano M. Ocimum basilicum ethanolic extract decreases cholesterol synthesis and lipid accumulation in human macrophages. Fitoterapia. 2008;79:515-23.
12. Amrani S, Harnafi H, Gadi D, Mekhfi H, Legssyer A, Aziz M, et al. Vasorelaxant and anti-platelet aggregation effects of aqueous Ocimum basilicum extract. J Ethnopharmacol. 2009;125:157-62.
13. Murugan K, Murugan P, Noortheen A. Larvicidal and repellent potential of Albizzia amara Boivin and Ocimum basilicum Linn against dengue vector, Aedes aegypti. Bioresour Technol. 2007;98:198-201.
14. Dasgupta T, Rao AR, Yadava PK. Chemomodulatory efficacy of Basil leaf (Ocimum basilicum) on drug metabolizing and antioxidant enzymes, and on carcinogen-induced skin and forestomach papillomagenesis. Phytomedicine. 2004;11:139-51.

15. Khandelwals K. Practical Pharmacognosy-techniques and experiments: Nirali Prakashan; 1996.


16. Acute oral toxicity [Online]. Dec 2001[cited on 10th Dec 2009]; Available from:

http://www.oecd.org/dataoecd/17/51/1948378.pdf

17. Zhao Z, WeixingWang, Guo H, Zhoua D. Antidepressant-like effect of liquiritin from Glycyrrhiza uralensis in chronic variable stress induced depression model rats. Behav Brain Res. 2008;194:108-13.
18. Hogg S. Review of the Validity and Variability of the Elevated plus-maze as an animal model of anxiety. Pharmacol Biochem Behav. 1996;54:21-30.
19. Rodgers R, Johnson N. Behaviorally selected effects of neuroactive steroides on plus-maze anxiety in mice. Pharmacol Biochem Behav. 1998;59:221-32.
20. Gong Z-H, Li Y-F, Zhao N, Yang H-J, Su R-B, Luo Z-P, et al. Anxiolytic effect of agmatine in rats and mice. Eur J Pharmacol. 2006;550:112-6






































































09.

SIGNATURE OF THE CANDIDATE




(JYOTIRMOY DUTTA)


10.

REMARKS OF THE GUIDE








11.1


11.2





NAME AND DESIGNATION OF THE

GUIDE
SIGNATURE


Mr.Srinath R .

HOD & Asst.Professor

Department of Pharmacology

P.E.S College of Pharmacy

Bengalooru-560050.




11.3
11.4


CO-GUIDE
SIGNATURE





NOT APPLICABLE


11.5

11.6

HEAD OF THE DEPARTMENT




SIGNATURE




Mr.Srinath R

HOD & Asst.Professor

Department of Pharmacology

P.E.S college of Pharmacy

Bengalooru-560050



12.

REMARKS OF THE PRINCIPAL





FORWARDED FOR APPROVAL


13.

SIGNATURE OF THE PRINCIPAL






Prof.Dr.S.Mohan

Principal and Director

PES College of Pharmacy

Bengalooru-560050.







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