WORLD JOURNAL OF PHARMACY AND PHARMACEUTICAL SCIENCES

Ayesha Khan. World Journal of Pharmacy and Pharmaceutical Sciences

SJIF Impact Factor 6.041

Volume 5, Issue 7, 881-892 Research Article ISSN 2278 4357

EVALUATION OF ANALGESIC AND ANTI-INFLAMMATORY

ACTIVITY OF WHOLE PLANT EXTRACT OF MANIKARA ZAPOTA
LINN

Ayesha Khan*

Assistant Professor, Shadan Womens College of Pharmacy, Khairatabad, Hyderabad.

ABSTRACT
Article Received on
23 April 2016, The present study aims to explore the analgesics and anti-inflammatory
Revised on 14 May 2016,
Accepted on 02 June 2016, activity of ethanolic extract of Manilkara zapota Linn. This was
DOI: 10.20959/wjpps20167-7081 studied using Eddys hot plate method, acetic acid induced writhing
andCarrageenin, histamine induced paw edema respectively. Treatment
*Corresponding Author with both low dose 200mg/kg b.wt and high dose 400mg/kg b.wt of
Ayesha Khan herbal ethanolic extract of dried powder of Manilkara zapota Linn
Assistant Professor, increased the reaction time in the methods (eddys hot plate method,
Shadan Womens College
acetic acid induced writhing) confirming analgesic activity and
of Pharmacy, Khairatabad,
Hyderabad.
similarly anti-inflammatory activity (Carrageenin and histamine
induced paw edema) shown significant increases in percentage
protection against paw volume. However, comparatively 400mg/kg b.wt dose of Manilkara
zapota Linn is more efficacious. Further experimental studies need to be conducted to prove
the effectiveness the active compound responsible for analgesic and anti-inflammatory
activity.

KEYWORDS: Manilkara zapota linn, Anti-inflammatory activity, Analgesic activity.

INTRODUCTION
Pain, heat, redness, and swelling are the classic manifestations of the inflammatory process.
Abnormalities of the joints of the spine, associated muscles, tendons, ligaments and bone
structural abnormalities can all result in pain and need for neurosurgical consultations.
Typically, patients will not require immediate surgical intervention, and therefore require
treatments to reduce pain and enhance quality of life activities.[2]

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In most cases, the genesis of pain is inflammatory, regardless of the etiology. With the
elucidation of the role of inflammatory cytokines, there is now a clear understanding of the
pathways by which many anti-inflammatory drugs can alleviate inflammation and relieve
pain.

The use of non-steroidal anti-inflammatory drug (NSAID) medication is still the mainstay of
most classically taught clinicians for joint and spine related inflammatory pain, but with their
commonly known side effects.[3] So, It is worthwhile to look for an alternative for the
management of pain, therefore phytotherapy is being sought, here the investigation is carried
out on Manikara zapota Linn whole plant (roots, stem, leaves, seeds, flowers, bark).

MATERIALS AND METHODS

Collection of Plant Material
The plant Manilkarazapotalinn was obtained from the public gardens, nampally, Hyderabad.
These plant materials were shade dried for 1 week and powdered. The powders were stored in
an airtight container and kept in a cool, dark and dry place.

Preparation of Extract.[10]
the coarsely ground powdered material of manikara zapota linn weighing about 1kg was
subjected to extraction using soxhelet apparatus with methanol (1 litre) as the solvent

Requirements
Chemicals: Indomethacin, acacia, extracts and distilled water.
Apparatus: Plethysmometer, eddys hot plate apparatus, beaker, thermometer, Bunsen
burner, oral feeding tube, syringes and stop watch.

Animals
Albino Wistar rats weight ranges 200-250 g and swiss mice weight range 20-25g were
selected used for the experiment. They were kept in standard environmental condition (at
24.00C temperature & 55-65% relative humidity and 12 hour light/12 hour dark cycle) for
one week for acclimation after their purchase and were fed with pellet diet and water ad
libitum.

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Phytochemical Screening
The freshly prepared crude ethanolic extracts of leaves were qualitatively tested for the
presence of alkaloids, phenols, tannins, reducing sugar, flavonoids, steroids, terpenoids and
saponins by using standard phytochemical procedures.

Acute Toxicity Test

The acute oral toxicity of M. zapota L methanolic extract was determined in rats using OECD
guideline 423.

Carrageenan-Induced Edema Test

In this experiment, Carrageenin-induced rat hind paw edema12 was used as the animal model
of acute inflammation. Administration of Carrageenin in the sub-plantar region of rats hind
paw leads to the formation of edema in situ due to localized inflammation. The animals were
weighed and randomly divided into 4 groups of 5 rats in each. Group I (control) received
normal saline (10mL/kg). Group II received 10 mg/kg body weight indomethacin orally.
Group III and IV received 200mg/kg and 400mg/kg of extract respectively. After an hour of
oral administration of test materials, 0.1 mL of 1% w/v suspension of Carrageenin in normal
saline was injected into the sub-plantar surface of the right hind paw of each rat of every
group. The paw volume was measured by plethysmometer (UgoBasile, 7140, Italy) at 1, 2, 3,
4 and 6 h after the Carrageenin injection. Mean increase in paw volume were noted for the
respective time intervals, thus edema volumes in control [(Ct-Co) control] and in groups
treated with test materials [(CtCo) treated] were calculated.

Percentage inhibition of paw edema was calculated by using the following formula: % paw
edema inhibition=[(Ct-Co) control-(Ct-Co) treated]/ (Ct-Co) control 100 Where, Co=paw
volume at zero time (before Carrageenin injection), Ct=paw volumes at t time. (Ct-Co)=paw
edema.

S.NO GROUP DRUG

1 Group I Control(normal saline)
2 Group II Carragenan+10mg/kgwt indomethacin(p.o)
3 Group III Carragenan+200mg/kg body wt. of methanolic extract
4 Group IV Carragenan+400mg/kg of body wt of methanolic extract

Histamine-Induced Edema Test

The paw oedema was produced by sub-plantar administration of 0.1% freshly prepared
solution of histamine into the right hind paw of the rats. In this experiment, twenty rats were

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divided into four groups of five animals each. Group I (control) received normal saline (10
ml/kg). Group II (Positive control) received 10 mg/kg body wt. of indomethacin orally.
Group III and IV received 200 and 400 mg/kg body wt. of the extract orally respectively.
Acute inflammation was induced in all the four groups by sub plantar injection of 0.1 ml of
Histamine in the right hind paw of the rats 1h after the oral administration of the tested
materials. The paw volume was measured with a micrometer screw gause at 1, 2, 3 and 4h
after the administration of the drug and the extract. The percentage inhibition of
inflammatory effect of the extract was calculated using the same formula for carrageenan-
induced paw oedema.

S.NO GROUPS DRUGS

1 Group-I Control(normal saline)
2 Group-II Histamine+10mg/kg body wt. of indomethacin
3 Group-III Histamine+200mg/kg body wt of methanolic extract
4 Group-IV Histamine+400mg/kg body wt of methanolic extract

Analgesic activity
Eddys hot plate method
Each animal group received a particular treatment i.e. control (normal saline, 10ml/kg, p.o.),
positive control (Aspirin 25mg/kg b.wt) and the extract 200mg/kg b.wt& 400mg/kg b.wt
respectively. The animals were positioned on Eddys hot plate maintained at a temperature of
550oC. A cut off period of 15 sec was observed to avoid damage to the paw. Reaction time
was recorded when animals licked their fore or hind paws, or jumped prior to and 0, 30, 60,
90, 120 min after oral administration of the samples.

S.No ROUPS DRUGS

1 Group-I Control (normal saline)
2 Group-II Standard Aspirin (25 mg/kg of body wt.)
3 Group-III Std.aspirin+200 mg/kg body wt of methanolic extract
4 Group-IV Std.aspirin+400 mg/kg body wt of methanolic extract

Acetic acid induced writhing method

Acetic acid (0.7% v/v) was administered intra-peritoneal to the experimental animals (mice)
to create pain sensation. As a result, the animals squirms their body at regular interval out of
pain. This squirm or contraction of the body was termed as writhing. As long as the
animals feel pain, they continue to give writhing. Each writhing was counted and taken as an
indication of pain sensation. Any substance that has got analgesic activity was supposed to
lessen the number of writhing of animals within in a given time frame and with respect to the

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control group. The writhing inhibition of positive control was taken as standard and
compared with test samples and control. As positive control, any standard NSAID drug can
be used. In the present study, Indomethacin 10mg/kg was used as standard. Group 1 received
normal saline group3 and 4 received test extracts with the doses of 200 mg/kg and 400 mg/kg
respectively.

S.NO GROUPS DRUGS

1 Group-I Control(normal saline)
2 Group-II Indomethacin+10mg/kg body wt.
3 Group-III 200mg/kg body wt. of methanolic extract of M Zapota Linn
4 Group-IV 400mg/kg body wt. of extracted methanolic of M Zapota Linn

RESULTS
ACUTE TOXICITY (LD50) STUDIES
An attempt was made to determine LD50 of 70% methanolic and aqueous extracts of
Manikarazapotalinn at a dose of 2000 mg/kg p.o., in female albino mice and rats.and OECD
guide lines 423 was followed.

The extracts were found devoid of mortality of the animals. Hence 5000 mg/kg was
considered as cut off value. Therefore, the screening doses (70% methanolic extract 150
mg/kg and 250 mg/kg. whereas aqueous extract 300 mg/kg) selected for the evaluation of
hepatoprotective and nephroprotective activity as per OECD guidelines No. 423

Table: Acute toxicity study of methanolic and aqueous extract in Mice (OCED
Guidelines 423)

Average body wt of the

animal in grams
Drug Signs of Effect
Dose Before After Death
treatment toxicity observed
treatment treatment
(1st day) (14th day)
5 Meth. extract 24 33 No sings of
No effect Nil
mg/kg Aq. extract 22 32 toxicity
50 Meth. extract 26 34 No sings of
No effect Nil
mg/kg Aq. extract 23 34 toxicity
300 Meth. extract 24 33 No sings of
No effect Nil
mg/kg Aq. extract 25 35 toxicity
2000 Meth. extract 24 34 No sings of
No effect Nil
mg/kg Aq. extract 24 35 toxicity

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Table: Acute toxicity study of methanolic and aqueous extract in rats (OCED
Guidelines 423)
Drug Average body wt of the
Dose
treatment animal in grams
Signs of Effect
Before After Death
toxicity observed
treatment treatment
(1st day) (14th day)
Meth. extract 160 171 No sings of
5 mg/kg No effect Nil
Aq. extract 176 182 toxicity
Meth. extract 160 169 No sings of
50 mg/kg No effect Nil
Aq. extract 159 162 toxicity
300 Meth. extract 168 176 No sings of
No effect Nil
mg/kg Aq. extract 155 163 toxicity
2000 Meth. extract 173 179 No sings of
No effect Nil
mg/kg Aq. extract 160 171 toxicity

CARRAGEENAN INDUCED PAW EDEMA IN RATS

Results of the Carrageenan induced paw edema was presented in below Table for control
group, standard group, test-1 group, and test-3 group. The extract of the plants (M. zapota
Linn) was found to exhibit a dose dependent decrease in percentage paw volume when
compared with control. At 4h, the mean reaction time of two different doses (200 and 400
mg/kg body weight) was 41.66and 59.72 respectively. The results were found to be
significant when compared with control group.

Table: percentage inhibition of paw edema

Dose Right hind paw volume (mm)
Treatment groups
(mg/kg) 1h 2h 3h 4h
Control(Normal saline) 10 1.04 1.29 1.36 1.44
0.46 0.54 0.52 0.54
Standard (Indomethacin) 10
(55.76) (58.14) (61.76) (62.50)
0.78 0.79 0.80 0.84
Test 1 200
(24.03) (38.76) (41.18) (41.66)
0.53 0.59 0.56 0.58
Test 2 400
(49.03) (54.26) (58.82) (59.72)

Graph- 2 Represents inhibition of paw edema

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HISTAMINE INDUCED PAW EDEMA

Results of the Histamine induced paw edema was presented in Table 4for control group,
standard group, test-1 group, and test-2 group. The extract of the plants (M. zapota Linn) was
found to exhibit a dose dependent decrease in percentage paw volume when compared with
control. At 4h, the mean reaction time of two different doses (200 and 400 mg/kg body
weight) was 39.35and 59.35 respectively. The results were found to be significant when
compared with control group.

Table: (Result of Histamine induced paw edema in brackets showing inhibition paw
edema)
Treatment Dose Right hind paw volume (mm)
groups (mg/kg) 1h 2h 3h 4h
Control(Normal
10 1.07 1.25 1.36 1.55
saline)
Standard 0.44 0.51 0.53 0.54
10
(Indomethacin) (58.87) (59.20) (61.03) (65.16)
0.76 0.84 0.90 0.94
Test 1 200
(28.97) (32.80) (33.82) (39.35)
0.50 0.57 0.58 0.63
Test 2 400
(53.28) (54.40) (57.35) (59.35)

Graph: Result of Histamine induced paw edema in brackets showing inhibition paw
edema)

EDDYS HOTPLATE METHOD

Results of the eddys hotplate method are presented in Table for control group, standard
group, test-1 group, and test-2 group. The extract of the plants (M.zapota linn) was found to
exhibit a dose dependent increase in reaction time when compared with control. At 90

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minutes, the mean reaction time of two different doses (200 and 400 mg/kg body weight) was
7.56 sec and 8.40sec respectively. The results were found to be significant when compared
with control group.

Treatment 0 min 30 min 60 min 90 min 120 min

CONTROL 2.45 2.5 2.52 2.56 2.7
STANDARD 1.35 3.56 7.15 8.19 7.17
TEST-1(200mg/kg) 1.41 3.49 5.43 7.56 6.26
TEST-2 (400mg/kg) 1.32 4.33 7.25 8.4 7.24

Graph-1 representing the mean reaction time

ACETIC ACID INDUCED WRITHING IN MICE

Table: Results of Acetic acid induced writhing method
Treatment Number of writhing Percentage inhibition
Control 39 0
Standard 18 52.87
Test 1 25 35.67
Test 2 12 96.82

Graph: representing the writhing times in various groups under study

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DISCUSSION
In spite of tremendous development in the field of synthetic drugs during recent era, they are
found to have some or the other side effects, various plants still hold their unique place, by
the way of having no side effects. Therefore, a systematic approach has to me made to find
out the efficacy of plants against pain and inflammation so as to exploid them as Herbal,
Analgesic and Anti-inflammatory agent.it is well know that most of the anti-inflammatory
and analgesic drug possess anti-pyretic activity.

The collected whole plant of Manilkara zapotaLinn was shade dried and then powdered to
coarse size. About 200 gm. of powder was subjected to extraction with ethanol. The present
study explores for Analgesic and anti-inflammatory activity of ethanolic extract of Manilkara
zapota Linn. Experimental model of Eddys hot plate method, acetic acid induced writhing
and Carrageenin, histamine induced paw edema respectively. Herbal suspensions were
prepared by the trituration method using a suspending agent (1% acacia) and dried extracts.

The extract of Manilkara zapota linn was evaluated for analgesic and anti-inflammatory
activity. The oral administration of extracts induced a significant analgesic, anti-
inflammatory activity in a dose-dependent manner. Test-2 group has shown significant
analgesic and anti-inflammatory effect when compared with standard drug. The plant may
have the phyto-constituents which inhibit cyclooxygenase enzyme or act on central opioid
receptors. Based on the results of the present study, it can be concluded that extracts with
different doses (200mg/kg b.wt & 400mg/kg b.wt) showed significant analgesic and anti-
inflammatory activity in rats and mice.

The extract of the plant under study, Manilkara zapota linn has shown that it possesses a
moderate to significant Anti-inflammatory and Analgesic activity that was evidenced by the
significant in the paw volume of writhing and Carrageenin, histamine induced paw edema,
acetic acid induced and eddys hot plate method.

From the results it could be concluded that extract exhibit Analgesic and Anti-inflammatory
by central as well as peripheral mechanism. Therefore the analgesic effect of extract was
significant by both measures, a fact that can be explain by the presence of mixture of
substance in the extract, bearing both peripheral and central analgesic properties. The
triterpenes ans steerols may be the active principle responsible for the observed analgesic

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effect and inflammation. The literature containsnumerous reports on the analgesic and
inflammatory activities of these compounds.

CONCLUSION
Treatment with both low dose 200mg/kg b.wt and high dose 400mg/kg b.wt of herbal
ethanolic extract of dried powder of Manilkara zapota Linn increased the reaction time in the
methods (eddys hot plate method, acetic acid induced writhing) confirming analgesic
activity and similarly anti-inflammatory activity (Carrageenin and histamine induced paw
edema) shown significant increases in percentage protection against paw volume. However,
comparatively 400mg/kg b.wt dose of Manilkara zapota Linn is more efficacious.

This plant may be providing good scope for use in treating pain and inflammation. Further
research work should be conducted for unveiling the active compound responsible for
analgesic and anti-inflammatory activity.

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