World Journal of Pharmaceutical Research

Vijayabhaskar et al. World Journal of Pharmaceutical Research

SJIF Impact Factor 7.523

Volume 6, Issue 7, 1336-1343. Research Article ISSN 2277– 7105

EVALUATION OF ANTIDEPRESSANT ACTIVITY OF ANNONA

SQUAMOSA L. SHOOT EXTRACT USING ANIMAL MODELS

Kanakam Vijayabhaskar*1, Bairi Padma2, Mathukumalli Sai Laxmi3 and Naini Sravan
Kumar1

1
*Department of Pharmacognosy, Department of Pharmaceutics, Sahasra Institute of
Pharmaceutical Sciences, Warangal, Telangana, India 506007.
2
Department of Pharmaceutical Chemistry, University College of Pharmaceutical Sciences,
Kakatiya University, Warangal-506001.
3
Department of Pharmacology, Max Institute of Pharmaceutical Sciences, Khammam,
Telangana, India-507002.

ABSTRACT
Article Received on
07 May 2017, Objective: Annona squamosa L. (Annonaceae) is a small ever green
Revised on 28 May 2017, tree is cultivated throughout india for its fruits. This plant has been
Accepted on 19 June 2017
DOI: 10.20959/wjpr20177-8844 used for the treatment of a variety of diseases. hyperthyroidism and
lipid-peroxidation. The plant also posses analgesic activity, anti-
inflammatory activity, anti-microbial activity, cytotoxic activity, anti-
*Corresponding Author
oxidant activity, anti-lipidimic activity, anti-ulcer activity,
Dr. Kanakam
Vijayabhaskar molluscicidal properties, genotoxic effect, vasorelaxant activity, anti-
Department of tumour, hepatoprotective activity, larvicidal activity, insecticidal
Pharmacognosy, activity, anthelmintic activity, etc Methods: This study was
Department of
undertaken to evaluate the possible antidepressant effect of Annona
Pharmaceutics, Sahasra
squamosa L. Shoot extract (ASSE) using Tail suspension test(TST) &
Institute of Pharmaceutical
Sciences, Warangal, Forced swim test (FST). 36 albino rats of either sex weighing between
Telangana, India 506007. 200-250gm were randomly selected and divided into 6 equal groups.
Group-I (control) received polyethyleneglycol (1ml/100gm), Group-II,
III & IV received ASSE in doses of 100,200,400 mg/kg orally (P.O.) respectively. Group V
& VI (positive control) received Fluoxetine & Imipramine at doses of 20mg/kg & 15mg/kg
p.o respectively. Drug treatment was given for seven & fourteen successive days. 60 minutes
after last dose of drug or standard the immobility period was recorded. Results: ASSE
produced significant antidepressant like effect at dose of 200 & 400 mg/kg administered for 7

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& 14 consecutive days as indicated by reduction in immobility times of Rat in TST & FST
(P<0.05). The efficacy of ASSE at 200mg/kg was found to be comparable to that of
Fluoxetine & Imipramine at doses of 20mg/kg & 15mg/kg. Conclusion: The results of the
present study indicate that ASSE possesses significant antidepressant activity compared to
that of both Fluoxetine & Imipramine.

KEYWORDS: Annona squamosa L., Forced swim test, Tail suspension test,
Antidepressants, Immobolity time.

INTRODUCTION
Annona squamosa L. (Annonaceae), commonly known as the custard apple tree is a native of
West Indies. But the cultivation is present throughout India, because of its edible nature.[1] It
is a fruit tree considered as a native of Central America also and hence have a wider
cultivation throughout the regions of tropics. The taste of the pulp of the fruit is really sweet
because of its higher sugar content of about 58% of dry mass, and hence it is found clear that
the fruit pulp possess a high calorie value. This plant was reputed to contain several
medicinal properties. Folkloric record reported the use of Annona squamosa as an
insecticidal, an anti-tumor agent, anti-diabetic, antioxidant, anti-lipidimic and anti-
inflammatory agent which has been characterized due to the presence of the cyclic peptides.
In addition, the crushed leaves were sniffed to overcome the hysteria and fainting spells, and
they were also applied on the ulcers and wounds. A leaf decoction was taken in the case of
dysentery. Plant have proved that they possess a wide variety of compounds like acetogenins
which were responsible for anti-feedant, anti-malarial, cytotoxic and the immunosuppressive
activities. Diterpenes which was isolated from the Annona squamosa possess the anti-HIV
principle and the anti-platelet aggregation activity. Annona squamosa Linn is a multipurpose
tree with edible fruits & is a source one of the medicinal &industrial products. Annona
squamosa Linn is used as an antioxidant, antidiabetics, hepatoprotective, cytotoxicactivity,
genetoxicity, antitumour activity, antilice agent. The partially purified flavonoids were
reported from the same source as the responsible agent for the anti-microbial and othe
pesticidal activities. Some lignans and other hydroxyl ketones were also found to be present
in this plant.[1]

Taxonomic Classification
Annona squamosa L.
Kingdom : Plantae

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Subkingdom: Tracheobionta
Super division: Spermatophyta
Division: Magnoliophyta
Class: Magnoliopsida
Sub class: Magnoliidae
Order: Magnoliales
Family: Annonaceae
Genus: Annona L.
Species: Annona squamosa

Traditional uses: The plant is attributed with the medicinal properties that include anti-
fertility and antitumour activities which were observed in mice and rats. The young leaves of
Annona squamosa were used extensively due to its anti-diabetic activity.[2]

Depression is a major clinical illness affecting 9.5% of population. Changes in the

monoamine neurotransmitters have been observed in patients of depression.[3] The use of
plant products for the treatment of human ailments has been a natural approach to health care
since the beginning of civilization. In the search for new therapeutic products for the
treatment of neurological disorders, medicinal plant research,worldwide, has progressed
constantly, demonstrating the pharmacological effectiveness of different plant species in a
varietyof animal models.[4] Thus the present study has been undertaken to evaluate the
antidepressant activity of Annona squamosa shoot extract (ASSE) in rats employing tail
suspension test (TST) & forced swim test (FST). Standard antidepressant drugs such as
Fluoxetine (SSRI), and Imipramine (TCA) have been employed to standardize the animal
models of depression.

MATERIALS AND METHODS

PREPARATION OF ANNONA SQUAMOSA SHOOTS EXTRACT (LEAVES AND
LATERAL BUDS)
shoots were collected and shade dried. They were crushed into coarse powder and extracted
with 90% methanol using soxhlet‟s apparatus for 24 hrs. The extract was concentrated under
pressure and then dried in air. The concentrated ethanolic extract was suspended in poly
ethylene glycol. Freshly prepared solution was used for each experiment.

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PLAN OF STUDY
ANIMALS
About 36 albino rats of either sex weighing between 200 -250 gms. procured from disease
free animals were used for the present study. Animals had free access to food and water and
maintained under standard laboratory conditions with a natural light and dark cycle. The
animals were acclimatized for at least five days before behavioural experiments. Experiments
were carried out between 9.00 and 15.00 hrs. Experimental protocol was approved by the
institutional animals‟ ethics committee before the start of the study.

DRUGS & CHEMICALS

ASSE, Fluoxetine Hydrochloride (Ranbaxy Lab.), Imipramine Hydrochloride (Sigma
Aldrich).

VEHICLE
Polyethylene Glycol (PEG).

STUDY DESIGN
The animals were selected randomly for each experiment and divided into 6 equal groups.
Drugs (PEG, ASSE, Fluoxetine, Imipramine) administered orally (P.O.) for 7&14 successive
days as depicted in (Table 1)

Table 1: Protocol of the study (Approved by IAEC)

GROUP DRUG DOSE(P.O.)
1 PEG 1 ml / 100 gm.
2 ASSE (Aq) 100 mg/kg
3 ASSE(Aq) 200 mg/kg
4 ASSE(Aq) 400 mg/kg
5 Fluoxetine 20 mg/kg
6 Imipramine 15 mg/kg

Sixty minutes after last dose, immobility period was recorded in two different animal models
of depression like:
Forced Swim Test (FST) (5)
Tail suspension test (TST) (6)

Laboratory Models For Testing Antidepressant Activity

Forced Swim Test (FST): FST(5) or behaviour despair was proposed as a model to test for
antidepressant activity by Depression was produced by forcing the animal to swim

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individually in a glass jar containing fresh water of 15cm height and maintained at 25oC. This
constituted pretest session. Twenty-four hour later each animal was again forced to swim.
After an initial 2 min period of vigorous activity, each animal assumed a typical immobile
posture. The total duration of immobility was recorded in next 4 min of a total 6 min test. The
change in the immobility period was calculated after administering drugs to the groups as
mentioned in the above table.

Tail Suspension Test (TST)[6]: The total duration of immobility induced by tail suspension
was measured according to the method d. Depression was produced by suspending the animal
from the edge of a table 50 cm above the floor by an adhesive tape placed approx. 1cm. from
the tip of the tail. Immobility time was recorded during a 6 min. period. Changes in the
immobility duration were studied after administering drugs in separate groups of animals.
The antidepressant activity was expressed as reduction in the immobility duration between
the control, standard and animals treated with test drug.

Acute toxicity study: Acute toxicity study was done according to OECD (Organization for
Economic Co-operation and Development) Guideline, fixed dose method; with starting dose
of 2000mg/kg body weight was adopted. Starting dose of 2000mg/kg (per oral) of each was
given to 5 animals (albino rats), animals were kept for observation of behavioural change and
death up to 72h.

STATISTICAL ANALYSIS
All the results are expressed as Mean ± SEM. All the groups were analysed using student‟s
„t‟ test.

RESULTS
The observation of acute toxicity study indicated that there was no death in 2000mg/kg dose
after 72hr. ASSE(Aq) at the dose of 100 mg/kg had no beneficial effect on immobility period
of rats in both the models of depression i.e. FST & TST. The decrease in immobility period in
both the models was observed starting from 200 mg/kg. But the increase in dose from 200 to
400 mg/kg did not produce any further reduction in immobility period, suggesting the ceiling
effect at 200 mg/kg. At the dose 200 mg/kg, ASSE(Aq) showed antidepressant effect which
is comparable to that of Imipramine and Fluoxetine at the dose of 15 & 20 mg/kg respectively
(Table 2).

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Table 2: Effect of Annona Squamosa Shoot Extract (Asse) On Immobility Period (Secs)
Of Rats Using Forced Swim Test
Group Drug Dose Immobility PERIOD (Secs)
Post Treatment (7 Post Treatment
Pre Treatment
days) (14 days)
1 PEG 1ml/100gm 201.13 ± 3.01 196.4 ± 1.31 195.04 ±0.16
2 ASSE(Aq) 100mg/kg 200.51 ± 1.14 192.51 ± 1.15* 190.01 ± 1.01*
3 ASSE(Aq) 200mg/kg 203.08 ± 1.05 151.61 ± 1.14b**a 152133 ± 0.05b**a
4 ASSE(Aq) 400mg/kg 192.2 ± 1.82 150.241± 0.01b*a 152.07 ± 1.12b*a
5 FLUOXETINE 20mg/kg 196.18 ± 1.70 120.64 ± 0.16b 102.71 ± 0.13b
6 IMIPRAMINE 15mg/kg 190.33 ± 1.17 136.91 ± 1.11b 124.6 ± 1.06b

Values as Mean ± SEM, n=6, 1. a = p < 0.05, b = p < 0.001 as compared to pre treatment
value, 2. * = p < 0.001, ** = p < 0.05 when compared to standard (Both Fluoxetine &
Imipramine), 3.α = p < 0.001when compared to control, 4. = p < 0.001 when ASSE (100)
is compared to ASSE (200) and ASSE(400).

At the dose 200 mg/kg, ASSE showed antidepressant effect which is comparable to that of
imipramine and Fluoxetine at the dose of 15 & 20 mg/kg respectively. The comparable anti
depressant effect of ASSE with that of TCA (imipramine) and SSRI (fluoxetine) suggest
possible involvement of either nor-adrenergic or serotonergic system.

Table 3: Effect of Annona Squamosa Shoot Aqueous Extract (ASSE) On Immobility

Period (Secs) Of Rats Using Tail Suspension Test
Group Drug Dose Pre Treatment Post Treatment After
4days 7days 14days
1 PEG 1ml/100gm 195.09 ±1.11 194.41±0.97 193.02 ±0.54 190.16±1.12
2 ASSE(Aq) 100mg/kg 189.40 ± 1.14 187.12±1.18 182.07 ±1.12 180.01±1.17
3 ASSE(Aq) 200mg/kg 190.01± 1.44 188.06±1.77 156.17±1.46*bα 150.78±1.21*bα
4 ASSE(Aq) 400mg/kg 188.15 ± 0.11 181.09±0.12 165.04 ± 1.21*bα 154.12±1.42*bα
5 Fluoxetine 20mg/kg 182.43 ±0.71 180.56±1.08 113.50± 0.95 *c 114.61±1.02*c
6 Imipramine 15mg/kg 192.5 ±1.14 190.31±1.02 125.12± 1.13 *c 120.51±1.14*c
Values as Mean ± SEM, Student‟s t test n = 6, 1.* P<0.001 when compared to pre treatment,
2.a=P<0.05, b=P<0.01, c=P< 0.001 when compared to control, 3.α =P<0.05, β=p<0.001
When compared to standard.

DISCUSSIONS
In the present study, ASSE (Aq) (200 mg/kg) produced significant antidepressant effect in in
FST & TST. These models of depression are widely used to screen new antidepressant drugs.
The tests are quite sensitive and relatively specific to all major classes of antidepressant drugs

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including TCAs, SSRIs, MAOI, Atypical antidepressants. The forced swimming test is the
most widely used tool for assessing antidepressant activity pre‐clinically. The widespread use
of this simple model is mainly due to its ability to detect a broad spectrum of antidepressant
agents.[7] It has been argued that TST (Tail Suspension Test)is less stressful than FST (Forced
swim test) and has greater pharmacological sensitivity. The results obtained from TST are in
concordance with the validated FST by Porsolt et al. Environmental factors and hereditary
factors play a major role in producing deficient monoaminergic transmission in central
nervous system thereby producing symptoms of depression.[13] The plant is reported to
contain glycoside, alkaloids, saponins, flavonoids, tannins, carbohydrates, proteins, phenolic
compounds, phytosterols, amino acids. The various chemical constituents isolated from
leaves, stems and roots of the plant including anonaine, aporphine, coryeline, isocorydine,
norcorydine, glaucine. Leaves contains 4-(2-nitro-ethyl 1)-1-6-((6-o-β-Dxylopyranosy1- β-D-
glucopyranosyl)-oxy)benzene, Anonaine, Benzyltetrahydroisoquinoline, Borneol, Camphene,
Camphor, car-3-ene, Carvone, β- Caryphyllene, Eugenol, Farnesol, Geraniol, 16-
Hetriacontanone, Hexacontanol, Higemamine, Isocorydine, Limonine, Linalool acetate,
Menthone, Methyl anthranilate, Methylsalicylate, Methylheptenone, p-(hydroxybenzyl)-6,7-
(2- hydroxy,4-hydro)isoquinoline, n-Octacosanol, a- Pinene, b-Pinene, Rutin, Stigmasterol,
β-Sitosterol, Thymol and n-Triacontanol. Alkaloids, proteins & amino acids are absent in the
leaf extract.[8] May be facilitating monoaminergic transmission there by producing
antidepressant effects.

CONCLUSION
Hence Annona squamosa shoot extract (ASSE) possesses antidepressant effect in animal
models of depression which was comparable to that of Imipramine and Fluoxetine as
demonstrated in this study. The phytochemical analysis, separation of active ingredients and
further investigation in this line methanolic extrac is essential to establish its therapeutic
benefits.

ACKNOWLEDGEMENT
The authors are gratefull to Sahasra pharmaceutical sciences management and University
college of pharmacy, Kakatiya University, for kind help and support in carrying out this study
at the laboratory.

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