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Phytochemical and pharmacological evaluation of the seeds of Annona

squamosa Linn

Article  in  International Journal of Pharmacy and Pharmaceutical Sciences · January 2012

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 Academic Sciences International Journal of Pharmacy and Pharmaceutical Sciences

ISSN- 0975-1491 Vol 4, Suppl 3, 2012

Research Article

PHYTOCHEMICAL AND PHARMACOLOGICAL EVALUATION OF THE SEEDS OF ANNONA

SQUAMOSA LINN.

ANSHUMAN BHATTACHARYA1, SUJIT KUMAR DEBNATH2*, MONALISHA DEY (DEBNATH) 2, DURGESHRANJAN KAR1
1Guru Nanak Institute of Pharmaceutical Science and Technology, Nilgunj road, Sodepur Panihati, Kol-711114, West Bengal, 2N. R. Vekaria
Institute of Pharmacy, C. L. College campus Junagadh-362001, Gujarat. *Email: skd.mpharma@rediffmail.com
Received: 21 Sep 2011, Revised and Accepted: 25 Sep 2011

ABSTRACT
The aim of present study was to assess the analgesic activity of seeds of Annona squamosa Linn. The mature plant seeds of Annona squamosa were
collected and authenticated. Extraction of dried seeds was carried out with methanol by maceration. Painful reaction in the experimental animals
can be produced by applying unpleasant stimuli such as thermal (radiant heat as source of pain) and physical pressure (tail compression). In both
Eddy’s hot plate method and tail-flick method the seed extract (250 mg/kg body weight) showed the significant analgesic activity and comparable
with the standard drug Nimesulide (50mg/kg body weight).
Keywords: Annona Squamosa, Analgesic activity, Nimesulide.

INTRODUCTION
Traditional medicines used through near about 60 percent of the
world’s population. These are not solitary used intended for major
health care rural areas although also in developing countries. In
developed countries modern medicines are primary used. Use of
plants as a source of medicine has been inherited and is an
important component of the health care system in India1,2. The
Therapeutic efficacy of many indigenous plants, for various
diseases has been described by traditional herbal medicinal
practitioners3,4. Natural products are the sources of synthetic and
traditional herbal medicine. They are still the primary health care
system in some parts of the world4,5. Several activities has been
studied on the plant of Annona squamosa like antimutagenic6,
Anthelmintic7, Scavenging8, Antimicrobial9, Antidiabetic10,
licicidal11, Hepatoprotective12, Antithyroid13, Antigenotoxic14
Antiplasmodial15, Molluscicidal16. Apart from different parts of the
plant have been used in traditional medicine, roots as Purgative,
useful in mental depression; fruits as Haematinic, Cooling,
sedative, vomiting, cough, malignant tumor and strengthening
muscles; seeds as Abortifacient, useful in destroying lice in the
hair; bark as atonics; leaves- crushed leaves are applied on ulcers
and wounds and a leaf decoction is taken in cases of dysentery.
Phytoconstituents like (-)-ent-kaur-16-en-19-oic acid and 16α,17-
dihydroxy-ent-kauran-19-oic acid known to posses Anticancer
activity, Cyclic peptide, Cyclosquamosin B, has Vasorelaxant
activity, Annonaine possess Antifungal activity, which are
consistent with the reported uses of the plant extracts in the
indigenous system of medicine17. Annona squamosa (A.squamosa)
L. (Family: Annonaceae), commonly known as custard apple, Small
tree 3 to 5 meters hight. Leaves are hairy when young, oblong, 8 to
15 cm long, with a petiole 1 to 1.5 cm long. Flowers occur singly in
the axils of the leaves, about 2.5 cm long, pendulous, three-angled,
light green to yellow. Fruit is large, slightly heart-shaped, 6 to 9 cm
long, the outside with knobby polygonal tubercles. When ripe, the
fruit is light yellowish-green, with a white, sweet soft, delicious
and juicy flesh18. A.squamosa L. is a native of West Indies,
Cultivated throughout the Philippines now in India.
Keeping in mind the great medicinal value of A. squamosa L. and high
content of polyphenols, flavanoids, present investigation was
planned to study the analgesic activity of the Methanolic extract of
seeds and compare with the standard drug nimesulide against both
eddy’s hot plate & hot tail-flick methods.
MATERIAL AND METHODS
Extraction
The seeds of Annona squamosa were collected from Konnagar
(W.B). The plant authenticated by comparing with the herbarium
voucher specimen. The seeds were air dried under shade, pulverized
by a mechanical grinder and passed through a 40 mesh and then
stored in airtight containers. The powdered seeds (25 g) were
extracted with Methanol for 72 hr. using maceration process. This
Methanolic extract was concentrated to dryness under reduced
pressure and controlled temperature (50-600C) to yield solid
masses. The concentrated extracts were used for preliminary
screening of phytochemicals.
Phytochemical screening of different crude extract
The extracts were tested for the presence of alkaloids, amino acids,
carbohydrates, volatile oils, fixed oils, flavonoids, glycosides, tannins,
steroids by using standard procedure19.
Experimental animals
Swiss albino mice weighing 18-25 gm of either sex were used for the
study. The animals were procured and housed in the animal house
maintained under standard hygienic conditions, at 20 ± 2°C,
humidity (60 ± 10%).
The experimental protocol was approved by institutional Animal
ethical committee with Registration no. 1439/po/a/11cpcsea on
20th April, 2011.
Acute toxicity studies
The acute oral toxicity studies were performed to study the acute
toxic effects and to determine minimum lethal dose of the drug
extracts. Swiss albino mice of either sex weighing 18-25 gm. were
used for the study. The methanolic extracts were administered
orally to different groups of overnight fasted mice at the doses of
1000, 2000 and 3000 mg/kg body weight.
Analgesic activity
Analgesic activity of methanolic extracts of Annona squamosa was
studied by eddy’s hot plate and tail flick method.
Eddy’s hot plate method
The mice were divided into three groups containing 3 mice each.
Group I served as control. Group II served as standard and were
given nimesulide (50 mg/kg body weight) orally. Group III (test)
were treated orally with methanolic extract of 250 mg/kg body
weight. The animals were individually placed on the hot plate
maintained at 55°C, one hour after their respective treatments20.
Hot tail-flick
Male or female albino mice weighing between 18-25 gm were fasted
for 24 hours with water given ad libitum maintained at room
 Debnath et al.
Int J Pharm Pharm Sci, Vol 4, Suppl 3, 92-94

temperature and was divided into 3 groups of 3 mice. Group I served
as control. Group II served as standard and were given nimesulide
(50 mg/kg body weight) orally. Group III (test) were treated orally
with methanolic extract of 250 mg/kg body weight. Analgesic effect
of the test substances was determined by the hot tail-flick method
described by Sewell and Spencer (1976). One to two cm of the tail of
mice was immersed in warm water kept constant at 50°C21.
RESULT AND DISCUSSION
Phytochemical screening
The curative properties are perhaps due to the presence of various
secondary metabolites such as Alkaloids, amino acids, carbohydrate,
fixed oil, steroids etc. in the methanolic extraction of the seeds of
Annona squamosa Linn. The results are given in table-1.

Table 1: Phytochemical screening of Annona squamosa seeds

Test for Test / Reagent Observation Result
Alkaloids Dragendorff’s reagent Reddish brown or orange precipitate is observed Alkaloids present
Wagner’s reagent Reddish brown precipitate
Amino Acids Milon’s test White precipitate is observed amino acids present
Ninhydrin test Presence of violet colour
Carbohydrates Molisch’s test Purple-violet colour ring appears Carbohydrates present
Volatile Oils To a thin section of drug Sudan III solution is added No globules are observed Volatile oil absent
Fixed Oils Saponification test Presence of soap formation fixed oils present
Flavonoids Shinoda test No visible colour change Flavonoids absent
Glycosides Borntrager’s test No colour change is observed Glycosides present
Tannins Ferric chloride test Blue colour is absent Tannin absent
Gelatin test No precipitate is formed
Steroids Salkowski test Red coloration Steroid present

Acute toxicity studies affective in colorless mice in comparison to standard drug

After administration of the extracts, animals were observed
continuously for the first three hours for any toxic manifestation.
Thereafter, observations were made at regular intervals for 24 hrs.
No such toxic effect had been seen.
Analgesic activity by Eddy’s hot plate method
The response time was noted as the time at which animals reacted
to the pain stimulus either by paw licking or jump response,
whichever appeared first. The cut off time for the reaction was 25
seconds20. The result depict that the methanolic extract of the seeds
of Annona squamosa Linn having analgesic activity but much
Nimesulide. The results are given in table-2.
Analgesic activity by Hot Tail-flick
The reaction time was the time taken by the mice to deflect their
tails. The first reading is discarded and the reaction time was taken
as a mean of the next two readings. The latent period of the tail-flick
response was taken as the index of analgesia and was determined
before and at 0, 10, 20, 30, 40, 50 and 60 min after the
administration of drugs. The maximum cut off time was fixed at 25
seconds21. Same analgesic activity of methanolic extract of the seeds
of Annona squamosa Linn has been observed in respect of standard
drug Nimesulide. The results are given in table-3.

Table 2: Analgesic activity by Eddy’s Hot Plate Method

Group Avg. Weight Group characteristic Basal reaction time (sec) before treatment Reaction time (sec.)
(mg) 0 10 20 30 40 50 60
Basal reaction time after treatment
Control 75 Color (tail) 14 14 15 15 15 15 15 15
Color (body) 12 11 13 12 12 12 12 12
Colorless 11 11 12 11 11 11 11 11
Standard 75 Color(tail) 14 15 16 22 24 22 20 19
Color(body) 11 13 13 14 15 17 18 16
Colorless 11 13 13 15 17 18 18 15
Test 75 Color(tail) 11 11 11 12 14 14 15 15
Color(body) 10 11 12 14 15 14 15 15
Colorless 12 12 12 14 15 17 17 18

Table 3: Analgesic activity by Hot Tail-Flick

Group Avg. Weight Group Characteristic Basal reaction time (sec) before treatment Reaction time (sec.)
( mg) 0 10 20 30 40 50 60
Basal reaction time after treatment
Control 50 Color(tail) 10 10 10 10 10 10 11 10
Color(body) 12 12 12 12 12 12 12 12
Colorless 10 10 10 11 10 12 10 10
Standard 50 Color(tail) 12 12 12 14 15 16 17 17
Color(body) 13 13 15 16 15 17 17 16
Colorless 11 11 12 13 14 15 15 17
Test 50 Color(tail) 12 12 12 13 14 14 15 15
Color(body) 13 13 13 14 14 15 15 16
Colorless 11 11 12 14 15 15 15 14

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 Debnath et al.
CONCLUSION
From the above investigation, it is quite apparent that methanolic
extract of seeds of Annona squamosa Linn. posses potent analgesic
effect against different stimuli. This is evidenced by significant
increase in the reaction time by stimuli in different experimental
models.
ACKNOWLEDGEMENTS
The authors are thankful to Guru Nanak Institute of Pharmaceutical
Science and Technology, Panihati, Kolkata, India for providing
necessary facilities throughout this work.
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