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S494
Document heading
Analgesic activity of Cryptostegia grandiflora (Roxb.)R.br. leaves
methanol extract using mice
Santhosh Kondajji Hanumanthappa, Manjunatha Hanumanthappa*, Krishna Venkatarangaiah, Pradeepa Krishnappa,
Rajesh Kashi Prakash Gupta
Department of P.G. Studies and Research in Biotechnology and Bioinformatics, Kuvempu University, Shankaraghatta - 577451, Karnataka, India
Asian Pacific Journal of Tropical Disease (2012)S494-S498
Asian Pacific Journal of Tropical Disease
journal homepage:www.elsevier.com/locate/apjtd
*Corresponding author: Dr. Manjunatha H. Assistant professor, Dept. of P.G.
studies in Biotechnology and Bioinformatics, Kuvempu University, Jnana sahyadri,
Shankarghatta-577451 Shimoga Dist., Karnataka, India.
Mail id: manjunatha75@gmail.com
Cell No: +918105580506
Phone No: (Office) 08282-256235
Fax: +91(0) 08282-256255
1. Introduction
Pain is an unpleasant sensory and emotional experience [1]
occurred by blocking pain signals interfering with the brain
signals. Pain motivates the individual to withdraw from
damaging situations, to protect a damaged body part while
it heals, and to avoid similar experiences in the future [2] if
not, leads to many diseases such as tumor, physical trauma,
surgical procedures, noxious chemical stimulation etc [3].
Most of the pain relieving drugs produced pronounced side
effects on the physiology of the body such as sweating,
apprehension, nausea and palpitation [4]. Hence there is an
utmost need for an alternative effective treatment against
pain without side effects. Natural occurring bioactive
compounds in the plants are believed to be an important
source with potential therapy for pain. Many drugs are used
to relieve the pain. Morphine [5] and aspirin [6] are used pain
killer since time immemorial. Any pain caused primarily by
stimulation of the nociceptor can be said to be nociceptive
pain [7]. Pain is centrally modulated via a number of
complex processes including opiate, dopaminergic,
descending noradrenergic and serotonergic systems [8].
Several plants are reported to possess analgesic property
as many investigators screened the plant extracts and
their phytoconstituents for their analgesic property viz.,
Bowdichia virgilioides [9], Capparis ovate [10], Urtica
circularis [11], Phlomis umbrosa [12], Delonix elata [13].
Cryptostegia grandiflora (Roxb.) R. Br. (Family:
Asclepiadaceae) is widely distributed throughout tropical
Africa, Madagascar and some parts of India [14, 15]. The
juice of aerial parts of C. grandiflora are reported to,
produce caoutchoue when exposed to sunshine [16]. It is
also reported that this plant decoction is consumed to treat
nervous disorders [17]. This plant species is also reported to
possesses various biological activities like antioxidant [18],
antitumour [19] antiviral [20] and control the schistosomiasis
[21]. The aqueous solution of ethanol extract of aerial parts
exerted significant hypoglycemic action in normal rabbits
ART ICLE INFO ABSTRAC T
Article history:
Received 19 June 2012
Received in revised from 5 July 2012
Accepted 7 Octoberr 2012
Available online 28 October 2012
Keywords:
Cryptostegia grandiflora
Acute toxicity
LME
Analgesic activity
Objective: To evaluate analgesic property of leaf methanol extract of Cryptostegia grandiflora
(Roxb.) R.Br. using mice. Methods: Analgesic activity was evaluated by abdominal writhing
and tail flick methods using Swiss albino mice. Acetyl salicylic acid was used as standard drug.
Result: The leaf methanol extract did not show any signs of toxicity upto 5000 mg/kg b.w. The
leaf methanol extract (LME) was tested at three different dosages viz., 250, 500 and 750 mg/kg b.w.
orally. All the three doses of LME showed significant (P<0.001) analgesic activity, among them
dose at 750 mg/kg b.w. showed 62.93% inhibition, but less effective than standard acetyl salicylic
acid (93.70%) as revealed by writhing test. In Tail-flick model, the LME at the dose of 500 mg/kg b.w.
showed significant activity (P<0.001) when compared to the other two doses as evidence by the
increase in the latency of tail response to thermal stimulation. Conclusions: This investigation
revealed that the C. grandiflora LME demonstrated significant analgesic effect in both abdominal
writhing and tail flick models. Among the three different doses tested, the 500 mg/kg b.w. was
found to be more potent. The results of this investigation revealed that the LME of C. grandiflora
possesses significant analgesic property and supported the traditional medicinal claims on C.
grandiflora.
Contents lists available at ScienceDirect
Santhosh Kondajji Hanumanthappa et al./Asian Pacific Journal of Tropical Disease (2012)S494-S498 S495
[22, 23] and the latex derived from this plant have proteolytic,
bacteriolytic activity and possess relevant enzymatic
activities against pathogenic related proteins [24, 25]. Rigorous
literature survey revealed that there are no reports available
regarding analgesic property of C. grandiflora, hence the
present study was undertaken to evaluate the analgesic
activity of the leaf methanol extract (LME) of C. grandiflora
to substantiate its traditional claims as decoction to treat
nervous disorders through scientific evaluations.
2. Materials and methods
2.1. Plant material and extraction
The leaves of C. grandiflora were collected in December
2009 from the villages near by Davanagere district,
Karnataka, India. The plant material was identified and
authenticated by Prof.Y.L. Krishnamurthy and plant
herbarium has been deposited (No: KU/SD/SP229) in the,
Department of Botany, Kuvempu University.
The leaves were cleaned with deionized water and were
shade dried, grounded porously by using mechanical
blender and passes through 40-mesh sieve. About 1 kg of
powdered material was loaded into four Soxhlet timbles
of 250 g each and extracted using methanol for about 48 h.
The extracts were filtered (Whatman No.1 filter paper) and
concentrated in vacuum under reduced pressure using
rotary flash evaporator (Buchi, Flawil, Switzerland) and then
the extract was kept on water bath for complete evaporation
of solvent. Finally the dried extract was preserved in air tight
container until use.
2.2. Animals
Healthy Swiss albino mice weighing 20-25 g were procured
from Central Animal House, National College of Pharmacy,
Shimoga, Karnataka, India and were housed at 23依2 曟
, humidity 55-60% and were fed with standard commercial
pellet diet (Durga Feeds and Foods, Bangalore) and water
ad libitum. All the animals were acclimatized for one week
before the experiments, and all experiments were carried
out according to the institutional animal ethics committee
guidelines (Re: NCP/IAEC/CL/07/12/2010-11).
2.3. Acute toxicity study
Acute oral toxicity [26] study was performed as per OECD-
423 guidelines (acute oral toxic class method). Albino mice
(n = 6) of either sex selected by random sampling technique
were used for the study. The animals were kept fasting for
overnight providing only water, after which the methanol
extract was administered orally at the dose level of 50 mg/
kg body weight by intragastric tube and observed for 72 hrs.
This dosage was gradually increased up to 5000 mg/kg until
any behavioral changes or mortality was observed.
2.4. Preparation of extract
The three different concentrations of LME and the standard
drug were prepared for oral administration in the form of
suspension in 1% DMSO as suspending agent.
2.5. Analgesic activity
2.5.1. Abdominal writhing method
The abdominal writhing method described by [27] was
carried out to measure the analgesic action. Analgesic
activity of the crude LME was carried out using adult Swiss
albino mice of either sex weighing 20-25 g, five groups with
6 animals per group were selected for abdominal writhing
method [28]. Group I animals were treated with 0.6% acetic
acid (dose 10 ml/kg) intraperitoneally. After 5 min of acetic
acid administration, numbers of writhes were counted for
20 min. This reading was taken as control. Group II, III and
IV were administered orally with the 1% DMSO dissolved
LME at the dose of 250, 500 and 750 mg/kg body weight
respectively. Group V was administered with standard drug
acetyl salicylic acid (100 mg/kg b.w.) and was used for the
comparison of analgesic activity. After one hour incubation
all the groups except group I animals were administered
with acetic acid. After 5 min, each group mice were observed
for the number of writhes for the duration of 20 min. The
mean value for each group was calculated. A reduction
in the writhing number compared to the control group
was considered as evidence of analgesia. The percentage
inhibition of writhing was calculated as: % Inhibition = A-B
/ A X 100. Where, A = mean number of writhes produced by
the control group and B = mean number of writhes produced
by the test groups.
2.5.2. Tail flick method
Analgesic effect of the LME was determined by the tail-
flick method as described by Sewell and Spencer (1976) [29].
Swiss albino mice of either sex weighing between 20-25 g
were divided into 4 groups of six mice in each group. Group
I mice were treated with normal saline (10 m1/kg b.w.).
Group II, III and IV were administered orally with the 1%
DMSO dissolved LME at the dose of 250, 500 and 750 mg/kg
b.w. respectively. One to two centimeter of the experimental
mice tail was immersed in warm water kept constant at 50
曟. The pain 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 analgesic activity and was determined before
and at 0.5, 1.0, 1.5, 2.0, 2.5 and 3.0 h after the administration
of drugs. The maximum reaction time was fixed at 0.5 h (30
min). The maximum possible analgesia (MPA) was calculated
according to the method of Idid et al., (1992) [30].
2.6. Statistical analysis
The data of analgesic activity was expressed as mean 依
S.E.M of six animals in each group. The statistical analysis
was carried out using one way ANOVA followed by Tukey’s
t-test. The difference in values at P≤ 0.01 was considered
as statistically significant.
Santhosh Kondajji Hanumanthappa et al./Asian Pacific Journal of Tropical Disease (2012)S494-S498
S496
3. Results
3.1. Acute toxicity study
Acute toxicity (LD50, p. o.) of leaf methanol extract of C.
grandiflora (LD50) for oral administration was evaluated using
mice. It is inferred that up to 50 to 5000 mg/kg b. w. did not
show any observable behavioral changes or mortality. It is
found to be a safer dose for administration and the LD50 was
taken at 1/10th of the examined concentration of the extract.
3.2. Analgesic activity
3.2.1. Writhing method
The number of abdominal writhes observed during 20 min.
period after 0.6% acetic acid administration through i.p. in
control group was 85.80 依 0.86. The treatment of methanol
extract at the dose of 250, 500 and 750 mg/kg b.w. reduced
the number of writhes to 36.80 依 0.58 (57.10 % analgesia), 36.80
依 0.58 (75.05% analgesia) and 31.80 依 0.86 (62.93 % analgesia)
respectively. But the effect of LME was slightly less potent
than the standard drug acetyl salicylic acid with 05.40 依 0.51
writhes, eliciting 93.70% analgesia, Although the effect was
found to be significant (P<0.01) and the data is depicted in
the Table 1.
3.2.2. Tail flick method
During the tail flick experiment, the effect of LME in
mice was observed over a period of 3h, while 1% DMSO
administered to animals as control group remained
non-toxic on the latent period of tail-flick response.
Before the administration of LME, the initial reading was
documented. Following oral administration at three different
concentrations (250, 500 and 750 mg/kg b.w.) of LME using
gavage, the effect of LME was significant (P<0.01) over
3h observation period. Analgesic effect after the LME
administration was recorded at every 30 min. time intervals
and was found to be evident within 0.5h of experiment. At 250
mg/kg dosage, the analgesia was observed to be increased
from 4.40 依 0.24 to 9.20 依 0.37%. Similarly, at 500 mg/kg
dosage, the analgesia increased to 10.80 依 0.37%. While at
750 mg/kg b.w. dosage the calculated analgesia (MPA) value
was (P< 0.01) significantly decreased to 9.60 依 0.68% against
the control group. The effect of LME on analgesic response
induced by noxious heat (50 曟) as shown in Table 2.
4. Discussion
Medicinal plants formulations are widely used in several
therapies including pain killing and nervous disorders.
Leaves of medicinal plants are common substance of many
folk and traditional herbal medicines. C. grandiflora is a
toxic vine, and though it is a toxic plant, the leaf decoction
of this plant is consumed to treat various nervous disorders
and wound healing [31].
Acute toxicity study of LME of C. grandiflora revealed
that up to 5000 mg/kg b. w. did not show any observable
changes in the behavior or mortality of animals. Hence, one
tenth of this dose was considered to be the safer dose for
administration.
The preliminary phytochemical screening revealed the
presence of alkaloids, glycosides, flavonoids, steroids,
saponins, tannin and phenolic compounds. Many
investigators have reported the actions of secondary
metabolites such as flavonoids and alkaloids played a
major role in analgesic activity [32, 33]. The presence of
secondary metabolites like saponins, flavonoids, tannins,
and terpenoides may be attributed for analgesic activity
[34]. However, alkaloids are well known for their ability
to inhibit pain perception [35, 36], whereas flavonoids are
primarily targeting prostaglandin synthesis involved in pain
perception, indicating that flavonoid components of the
plant extract might be responsible for analgesic property of
the extract [37,38,39].
In the present study, analgesic activity of LME of C.
Table 1
Analgesic activity of C. grandiflora leaf methanol extract by writhing method.
Drug treatment Dose Number of writhes % inhibition of
writhings
Control(acetic acid)10 ml/kg (i.p.)85.80依 0.86 -
Acetyl salicylic acid 100 mg/kg (p.o.)05.40依 0.51 93.70**
Leaf methanol extract 250 mg/kg (p.o.)36.80依 0.58 57.10**
500 mg/kg (p.o.)21.40依 1.03 75.05**
750 mg/kg (p.o.)31.80依 0.86 62.93**
Values are the mean 依S.E.M. of six mice. Symbols represent statistical significance.*P < 0.05, ** P < 0.01, ns - not significant, as compared to
control group.
Table 2
Analgesic activity of C. grandiflora leaf methanol extract by tail flick method
Group(N)Dose (p.o.)0.5h1.0h1.5h2.0h2.5h3.0h
Contro(1% DMSO)10 ml/kg 3.03依0.05** 2.8 依 0.06** 2.9 依 0.04** 3.2依0.08** 3.1依0.02** 3.4依0.05**
Leaf Methanol extract 250mg/kg 9.20依0.37** 7.20依0.37** 7.0 依0.45** 5.60依0.40** 5.80依0.20** 4.40依0.24**
500 mg/kg 10.80依0.37** 9.60依0.24** 9.20依0.20** 8.80依0.49** 9.20依0.37** 8.20依0.20**
750 mg/kg 9.60依0.68** 9.60依0.40** 8.60依0.40** 8.40依0.57** 8.20依0.58** 7.60依0.40**
Values are the mean 依S.E.M. of six mice. Symbols represent statistical significance.*P < 0.05, ** P< 0.01, ns - not significant, as compared to
control group.
Santhosh Kondajji Hanumanthappa et al./Asian Pacific Journal of Tropical Disease (2012)S494-S498 S497
grandiflora was evaluated by using acetic acid induced
abdominal writhing and tail flick method in mice. In acetic
acid induced experiment, animal models react with unique
abdominal stretching behavior which is called writhing. The
reduction in abdominal writhing indicates the percentage
levels of analgesia in the acetic acid writhing reflex model
[40] in which the pain is due to the release of free arachidonic
acid from phospholipid tissue [41] via, cyclooxygenase
(COX) and prostaglandin biosynthesis [42]. The acetic acid
induced writhing response is a sensitive procedure to
evaluate peripherally acting analgesics and the response
is thought to be mediated by peritoneal mast cells [43], acid
sensing ion channels [44] and the prostaglandin pathways [45].
Prostaglandins E2 and F2α are reported to be increased in
the peritoneal fluid of mice due to administration of acetic
acid, this could be produced by neutrophil polynuclear cells
but also by destruction of macrophages [46, 47]. The significant
pain reduction of LME might be due to the presence of
alkaloids and flavonoids analgesic principles acting against
the prostaglandin pathways.
The centrally acting analgesics generally raise the pain
threshold of mice towards heat [48]. The thermal induced
nociceptive tests are more sensitive to opioid receptors and
non-thermal tests are sensitive to κ-opioid receptors as
they are G-protein-coupled receptors (GPCRs) [49, 50, 51].
The narcotic analgesics inhibit both peripheral and central
mechanism of pain, while nonsteroidal anti-inflammatory /
analgesics agents (NSAIAs) inhibit only peripheral pain [52,
53]. The inhibition of pain could take place not only from
the presence of opioids and/or opiodiomimetics but also
from bio-active compounds and secondary metabolites
like phenolic and steroidal constituents [54, 55]. Significant
analgesic response was observed at the dose of 250 and 500
mg/kg b.w. However, steady state response was not observed
to be maintained at higher concentration of 750 mg/kg b.w.
Among the three doses, 500 mg/kg b.w. showed significant
(P< 0.01) analgesic response against acetic acid induced
writhing. The significant effect of these extracts is due to the
presence of a biological active constituent in higher levels
or due to the effect of more than one phytoconstituents. The
LME of C. grandiflora at three different doses showed good
peripheral analgesic activity by decreasing the number of
writhes and exhibited central analgesic activity by showing
significant (P<0.001) effect on the latent period of tail-flick
response at the dose of 500 mg/kg b.w. The present study
revealed that the leaf methanol extract of C. grandiflora
exhibited significant analgesic property but less effective
than the standard reference.
Conflict of interest statement
We declare that we have no conflict of interest.
Acknowledgement
The first author wishes to thank the “University Grants
Commission implemented Rajiv Gandhi National Fellowship
(RGNF)” India. UGC letter No. : F. 14-2(SC)/2010 (SA-III)
Dated: May 2011.
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