ISSN: 0975-8585
Research Journal of Pharmaceutical, Biological and Chemical
Sciences
Analgesic and antipyretic activity of methanolic extract of Coccinia grandis L.
leaves in experimental animals.
Aggarwal Ashish S*1, Suralkar Ujwala R1, Chaudhari Sugandha G3, Deshpande SV2, Garud
Aniket A1, Talele Sandeep G1
1
Department of Pharmacology, Padm. Dr. D. Y. Patil Institute of Pharmaceutical Sciences and Research, Pimpri,
Pune - 411018, Maharashtra, India
2
Department of Pharmaceutical Chemistry, Padm. Dr. D. Y. Patil College of Pharmacy, Akurdi, Pune-411044,
Maharashtra, India.
3
Department of Pharmacology, Dr. L. H. Hiranandani College of pharmacy, Ulhasnagar-421003, Mumbai,
Maharashtra, India.
ABSTRACT
The present study investigates the analgesic and antipyretic activity of the methanol extract (50, 100 and
200 mg/kg) of Coccinia grandis L., leaves in rats and mice. Acetic acid induced writhing, Tail immersion and Hot
plate models were used to evaluate analgesic activity and Yeast induced pyrexia model was used to evaluate
antipyretic activity. Our findings show that oral administration of methanol extracts significantly inhibit acetic acid-
induced writhing in mice in dose dependent manner but failed to show significant inhibition in Tail immersion and
Hot plate models. Antipyretic study revealed that methanolic extract exhibits significant reduction in pyrexia that
was comparable to standard drug.
Keywords: Coccinia grandis L., analgesic activity, antipyretic activity, Acetic acid.
*Corresponding author
Email: ash.pharmacology@gmail.com
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ISSN: 0975-8585
INTRODUCTION
Pain is an unpleasant sensation no doubt, but on the whole it is usually beneficial to
man (or animal). It is mainly a protective mechanism for the body, occurs whenever any tissues
are being damaged, and it causes the individual to react to remove the pain stimulus [1].
Pyrexia or fever is caused as a secondary impact of infection, malignancy or other diseased
states [2].
Coccinia grandis L., of the family Cucurbitaceae is distributed in tropical Africa,
commonly found in Pakistan, India and Srilanka. Coccina is a climber and trailer. Every part of
this plant is valuable in medicine and various preparations have been mentioned in indigenous
system of medicine for various skin diseases, bronchial catarrh, bronchitis and Unani systems of
medicine for ring worm, psoriasis, small pox, scabies and other itchy skin eruptions and ulcers.
Oil of this plant is used as an injection into chronic sinuses. Coccinia grandis leaves are reported
for its antihyperlipidemic [3], antimicrobial [4], antitussive [5], antiulcer [6], antioxidant activity
and fruits are reported for hepatoprotective [7], cerebral oxidative stress [8].
The aim of the present investigation was to evaluate the possible analgesic and
antipyretic effects of the methanolic extract of the leaves of Coccinia grandis (ML) in
experimental animals.
MATERIALS AND METHODS
Plant Material
The plant material consists of dried powdered leaves of Coccinia grandis L. Voigt.
(Cucurbitaceae). The plant was collected in and around the farms of Chikhli, Tal-Haveli, Dist.-
Pune, Maharashtra, India during the month of September-2008 and was authenticated by Joint
Director, Botanical Survey of India, Western Circle,Pune-4110 01 (Ref No BSI/WC/Tech./
2008/477 dated 3/10/2008).
Preparation of the extract
Air-dried powdered leaves (500gm) of Coccinia grandis L. were extracted with 2.0 L
methanol by using Soxhlet apparatus. An exhausted marc was collected. After the removal of
solvent by concentration under reduced pressure at 60 0C, 19.38 gm of methanolic residue were
obtained, respectively.
Animals
Wistar rats (200-250 g) of either sex were used for present study. Animals were kept
under a 12 h light/ 12 dark cycle, with free food and water ad libitum. Albino mice (Swiss strain)
weighing 22-25 g were housed under standard laboratory condition in a group of six each.
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Animals had free access to food and water. The Institutional Animal Ethical committee (IAEC)
has approved the protocol of the study.
Vehicles
Plant extract, aspirin were suspended in 0.5% (w/v) carboxymethylcellulose sodium
(CMC) and administered orally to animals. Acetic acid diluted separately in normal saline and
injected intraperitoneally.
Phytochemical screening
Preliminary phytochemical screening of the crude methanolic extracts of Coccinia
grandis L. leaves (ML) were carried out. Phytochemical screening of the extract was performed
using the following reagents and chemicals [9] alkaloids with Dragendorff’s reagent,
flavonoids with the use of Mg and HCl; tannins with 1% gelatin and 10% NaCl solutions and
saponins with ability to produce suds and hemolysis reaction.
Acute oral toxicity study
The acute toxicity was determined on albino mice by fixed dose method of OECD Guide
line no 423 given by CPCSEA. Groups of 6 mice were administered test drug by oral route in the
range of 300- 2000mg/kg and mortality was observed after 24 hr., in case of Coccinia grandis L.
leaves, since there was death of one animal in the group treated with 300mg/kg dose and all
the animals died in the group of animals treated with 2000mg/kg dose. Therefore 1000mg/kg
was treated as LD50 and 1/20th, 1/10th and 1/5th (i.e. 50mg/kg, 100mg/kg and 200 mg/kg) of
1000mg/kg were selected for further study.
SCREENING METHODS FOR ANALGESIC ACTIVITY:
Acetic acid-induced writhing response in mice
Analgesic activity was evaluated on the acetic acid-induced writhing according to Koster
et al. [10]. Male albino mice were divided in to five groups of six animals each. Group I served
as control and received drugless (0.5%, w/v, CMC; 10 ml/kg) vehicle. Group II served as
standard and were treated orally with standard drug, Aspirin (200 mg/kg). Group III, IV and V
were treated orally with methanolic extract of 50, 100 and 200 mg/kg body weight respectively.
The animals were pretreated with their respective treatment, 1 h prior to intraperitoneal
injection of 1% (v/v) acetic acid (0.1 ml/10 g). Five minutes after the intraperitoneal injection of
acetic acid, the number of writhing during the following 10 min was counted. [11].
Percent inhibition = 1 – (NT/NC) X 100
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Where NT is average number of writhings in treated groups and N C is average number of
writhings in control groups.
Tail Immersion Method [12]
Healthy albino rats weighing about 150-200gm were taken. The animals were divided
into five groups of 6 animals each. Group I served as control and received drugless (0.5%, w/v,
CMC; 10 ml/kg) vehicle. Group II served as standard and were injected Diclofenac sodium (9
mg/kg) intraperitoneally. Group III, IV and V were treated orally with methanolic extract of 50,
100 and 200 mg/kg body weight respectively. They were divided into different groups,
numbered and placed into individual restraining cages leaving the tail hanging out freely. The
animals are then allowed to adapt in the cages for 30 minutes before testing. The lower 5cm
portion of the tail was marked and immersed in a cup of freshly filled warm water of exactly 55
o
C. Within a few seconds the rat reacts by withdrawing the tail. The reaction time was recorded
by a stop watch. After each determination the tail was carefully dried. The reaction was
determined before oral administration of respective treatments which was recorded as zero
minutes reading. After the drug was administered the reaction time was recorded at an interval
of 30, 60, 90, 120 and 150 mins. The cut off time of the immersion is 15 seconds. The mean
reaction time was recorded for each group and compared with the value of standard drug.
Eddy’s hot plate method
Male albino mice weighing 22-25g were taken. The animals were divided into five
groups of 6 animals each. Group I served as control and received drugless (0.5%, w/v, CMC; 10
ml/kg) vehicle orally. Group II served as standard and were injected Diclofenac sodium (9
mg/kg) intraperitoneally. Group III, IV and V were treated orally with methanolic extract of 50,
100 and 200 mg/kg body weight respectively. The animals were individually placed on the hot
plate maintained at 55°C, one hour after their respective treatments. 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 15 seconds 15. The
test was carried before the treatment and at 30, 60 and 90 min after administration [13].
SCREENING METHOD FOR ANTIPYRETIC ACTIVITY:
Yeast induced pyrexia in rats
The animals were divided into five groups of 6 animals each. Group I served as control
and received drugless (0.5%, w/v, CMC; 10 ml/kg) vehicle. Group II served as standard and were
treated orally with Aspirin (100 mg/kg). Group III, IV and V were treated orally with methanolic
extract of 50, 100 and 200 mg/kg body weight respectively. Initial rectal temperature of rats
was recorded using tele thermometer. Hyperthermia was induced by subcutaneous injection of
10ml/kg of 20% aqueous suspension of brewer’s yeast. After 18 hrs of yeast injection, the
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animals showing a rise in temperature inferior to 0.7°C was discarded. The temperature was
recorded at 1h, 2h, 3h and 4h after treatment [14].
Statistical analysis:
All the values were expressed as mean ± standard error mean (S.E.M.) and compared
with corresponding control values. P values were analyzed using one-way ANOVA followed by
Dunnett’s t-test. p<0.05 was considered as statistically significant.
RESULTS
Phytochemical screening
Phytochemical screening of the crude methanolic extract of the leaves of Coccinia
grandis revealed (Table 1) the presence of glycosides, flavonoids, alkaloids, phenols, tannins
and terpenoids.
Table 1: Chemical constituents present in methanolic extract of Coccinia grandis L. leaves.
Test Methanolic extract
Carbohydrates -
Proteins and amino acids -
Glycosides +
Alkaloids +
Phytosterols +
Flavonoids +
Tannins +
Phenolic compounds +
Gums and mucilage -
Fixed oil & fats -
Saponins -
Volatile oils -
Tri terpenoids +
Analgesic activity:
Effect of ML in Acetic acid-induced writhing method
When compared to control animals, ML (100 and 200 mg/kg) significantly reduced the
number of abdominal writhing in a dose dependent manner with 16.01 %( p<0.05) and 23.5 %
(p<0.01) inhibition, respectively, while the standard showed 41.50% (p<0.001) inhibition (Table
2). The analgesic activity was comparable to the effect of standard drug, Aspirin (200mg/kg).
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Table 2: Analgesic effect of methanolic extract of on Acetic acid induced writhing method
Group Dose(mg/kg) No. of writhing Inhibition (%)
I Control 36.16± 0.47 -
***
II Std 200 mg/kg 21.16 ± 1.19 41.5
*
III ML 50 mg/kg 33.03± 0.55 8.7
*
IV ML 100 mg/kg 30.33± 0.55 16.12
**
V ML 200 mg/kg 27.66± 0.49 23.5
Results are presented as mean ± SEM, (n=6), *p< 0.05, **p < 0.01, ***p< 0.001 when compared to control.
Effect of ML in Eddy’s hot plate method
In the present study, Diclofenac sodium, a centrally acting analgesic drug, produced an
inhibitory effect on the nociceptive response in Hot plate method, while extract of Coccinia
grandis L. failed to affect the response (Table 3).
Table 3: Analgesic effect of methanolic extract of on Eddy’s hot plate method
Group Treatment Mean latency (s) in minutes (X±SEM)
0 30 60 90
I Control 2.18±0.12 2.31±0.20 2.43±0.27 2.49±0.32
II Std 9 mg/kg 2.25±0.35 6.91±0.20* 9.74±0.34* 11.32±0.22*
III ML 50 mg/kg 2.79±0.20 2.94±0.34 3.10±0.45 3.19±0.40
IV ML 100 mg/kg 3.11±0.36 3.28±0.28 3.42±0.18 3.55±0.20
V ML 200 mg/kg 2.67±0.43 2.90±0.25 3.09±0.16 3.21±0.23
Results are presented as mean ± SEM, (n=6), *p< 0.001 when compared to control.
Effect of ML in Tail Immersion Method
In the present study, Diclofenac sodium, a centrally acting analgesic drug, produced an
inhibitory effect on the nociceptive response in Tail Immersion Method while extract of
Coccinia grandis L. failed to affect the response (Table 4).
Table 4: Analgesic effect of methanolic extract of on Tail immersion method
Treatment Tail flick latency in minutes (X±SEM)
(mg/kg) 0 30 60 90 120 150
I-Control 3.08± 0.4 3.1± 0.33 3.13± 0.6 3.01± 0.51 2.95± 0.34 2.95± 0.29
II-Std. 2.70± 0.40 4.08± 0.38* 4.35± 0.45* 4.70±0.19* 5.40±0.22* 5.47±0.31*
III-M(L) 50 3.31± 0.29 3.48± 0.30 3.51± 0.34 3.54± 0.42 3.55± 0.25 3.55± 0.20
IV-M(L)100 3.01± 0.35 3.19± 0.30 3.22± 0.40 3.27± 0.37 3.25± 0.32 3.28± 0.44
V-M(L)200 2.95± 0.22 3.10± 0.37 3.16± 0.31 3.23± 0.28 3.21± 0.34 3.22± 0.41
Results are presented as mean ± SEM, (n=6), *p< 0.001 when compared to control.
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Antipyretic activity:
Effect of ML in Yeast induced pyrexia method
The methanolic extract of leaves of Coccinia grandis L. produced significant (p<0.01)
reduction at the dose of 100mg/kg as compare to other two doses in yeast induced pyrexia.
Methanolic extract of Coccinia grandis L. leaves exhibits significant reduction in pyrexia that
was comparable to standard drug paracetamol (Table 5).
Table 5: Antipyretic effect of methanolic extract of on Yeast induced pyrexia method
Treatment Before 0h 1h 2h 3h 4h
(mg/kg
I-Control 38.49±0.07 39.41±0.09 39.58±0.12 39.61±0.23 39.53±0.25 39.71±0.15**
II-Std. 37.95±0.11 39.01±0.12 38.78±0.16 38.43±0.18** 38.12±0.29** 38.01±0.26**
III-M(L) 50 37.69±0.11 38.58±0.12 38.31±0.11* 38.17±0.19** 38.03±0.17** 37.86±0.22**
IV-M(L)100 38.10±0.09 39.07±0.15 38.86±0.12 38.63±0.11** 38.47±0.22** 38.21±0.15**
V-M(L)200 37.80±0.11 38.93±0.12 38.76±0.11 38.49±0.15* 38.28±0.24** 38.13±0.21**
Results are presented as mean ± SEM, (n=6), *p< 0.05, **p < 0.01 when compared to control.
DISCUSSION
The present study demonstrated that the methanol extract of Coccinia grandis given by
oral route in rats and mice have shown analgesic and antipyretic properties, since they were
able to inhibit acetic acid-induced writhing episodes and yeast induced pyrexia.
Acetic acid causes algesia by liberating endogenous substances and many others that
excite pain nerve endings, and is a sensitive method of screening both peripheral and central
analgesic efficacy of agents. Oral administration of the methanol extracts of Coccinia grandis
significantly reduced the number of abdominal constriction following acetic acid, indicating
analgesic activity in mice at the doses assayed. The results of this writhing test alone did not
ascertain whether the antinociceptive effects are central or peripheral. Thus, to clear the mode
of the inhibitory effect of these species on the nociceptive responses, the effect of the species
under study on the Tail immersion and Hot plate method was examined. Thermal painful
stimuli are known to be selective to centrally, but not peripherally, acting analgesic drugs.
Methanolic extract of Coccinia grandis L. failed to show central analgesic action. These findings,
therefore, suggest that the apparent analgesic action of the active compound(s) in the
methanol extract of Coccinia grandis L. May be mediated through peripheral but not central
mechanism(s).
Antipyretics are agents which reduce elevated body temperature. Regulation of body
temperature requires a delicate balance between production and loss of heat, and the
hypothalamus regulates the set point at which body temperature is maintained [16, 17]. Most
of the antipyretic drugs exhibit drug action by inhibiting COX-2 expression, thus inhibiting
prostaglandin synthesis to reduce elevated temperature. It was found that the Methanolic
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extract of Coccinia grandis at the doses of 100 and 200 mg/kg showed significant decrease in
yeast-induced fever. This result seems to support the view that Coccinia grandis L. methanolic
extract has some influence on prostaglandin biosynthesis because prostaglandin is believed to
be a regulator of body temperature [12]. However further study is required to determine the
mechanism of action.
CONCLUSION
An extensive search in Ethnopharmacology has taken place worldwide. The plant
Coccinia grandis L was traditionally claimed for a large number of pharmacological actions and
medicinal uses. In the present investigation, Phytochemical screening of the crude methanolic
extract of the leaves of Coccinia grandis revealed the presence of glycosides, flavonoids,
alkaloids, phenols, tannins and terpenoids. . The significant analgesic and antipyretic action may
be attributed to the phytoconstituents present in it. The present study offered a scientific proof
to the traditional use of Coccinia grandis L. However, further phytochemical studies are needed
to isolate the active compounds responsible for these pharmacological activities.
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