Antinociceptive and antiinflammatory effect of Crinum asiaticum bulb extract

Abstract

The methanol extract of Crinum asiaticum bulb was evaluated for its antinociceptive properties on the pain induced by acetic acid and formalin in Swiss albino mice. The bulb extract at a dose of 1, 1.5 and 2 g/kg produced an inhibition of 40.71, 56.42 and 68.57% on pain induced by acetic acid and at a dose of 1.5g/kg & 2g/kg produced an inhibition of 60.0 and 67.0% on that induced by formalin. The anti-inflammatory activity of the same extract was estimated volumetrically by measuring the mean increase in hind paw volume of carrageenan-induced Wistar albino rat with the help of plethysmometer. Oral administration of bulb extract at a dose of 1.5g/kg & 2g/kg showed the highest inhibition 52.56% and 47.37%, respectively, at the 3rd hour of administration whereas at a dose of 1g/kg showed 37.5% inhibition at the 4th hour. Diclofenac sodium at a dose of 40 mg/kg was used as a standard drug in the inhibition of acetic acid and formalin induced pain as well as in carrageenan-induced paw edema.

Full text

ResearchArticle
ANTINOCICEPTIVEANDANTIINFLAMMATORYEFFECTOFCRINUMASIATICUMBULB
EXTRACT

MD.ATIARRAHMAN1*,RUMANASHARMIN2,MD.NAZIMUDDIN3,MAHBUBUZZAMAN4,SOHELRANA5NAZIM
UDDINAHMED6
1,2,3,5DepartmentofBiochemistryandMolecularBiology,UniversityofChittagong,Chittagong4331,Bangladesh
4,6BangladeshCouncilforScientificandIndustrialResearch(BCSIR),Chittagong,Bangladesh
Email: atiarh@yahoo.com

ABSTRACT
Themethanol extract ofCrinumasiaticumbulb wasevaluatedfor itsantinociceptive propertiesonthe paininducedbyaceticacidandformalinin
Swissalbinomice.Thebulbextractatadoseof1,1.5and2g/kgproducedaninhibitionof40.71,56.42and68.57%on painindu cedby acetic acid
andatadoseof1.5g/kg&2g/kgproducedaninhibitionof60.0and67.0%onthatinducedbyformalin.Theanti‐inflammatoryactivityofthesame
extractwasestimatedvolumetricallybymeasuringthemeanincreaseinhindpawvolumeofcarrageenan‐inducedWistaralbinoratwiththehelpof
plethysmometer.Oraladministrationofbulbextractatadose of 1.5g/kg& 2g/kg showedthe highestinhibition 52.56%and 47.3 7%,respectively,
atthe3rdhourofadministration whereasat adose of1g/kg showed37.5%inhibitionatthe4thhour.Diclofenacsodium atadoseof40mg/kgwas
usedasastandarddrugintheinhibitionofaceticacidandformalininducedpainaswellasincarrageenan‐inducedpawedema.
Keywords:Crinumasiaticum,antinociceptive,anti‐inflammatory,pawedema,carrageenan.

INTRODUCTION
CrinumasiaticumisanevergreenherblocallyknownasBara
kanurinBangladesh.ItiswidelydistributedtoChina,Hongkong,
India, Srilanka, Myanmar, Thailand,Malayasia, Ryukyu Islands &
Mainland Japan1,2,3.ChittagongHilltractsofBangladesharethe
mainhabitatofthisherb.
TribesofChittagongHillyareasusethisplantasaremedyofpain,
swelling carbuncle, piles, earache, arthritis, skin disease (leprosy),
cold and cough disorders, vomiting, worms infestation, disuria,
polyuria, bowel complains, throat disorder, colic, flautulance,and
fever1,2,3. Crinumasiaticum(C.asiaticum) is used traditionally for
variouspurposes. Leaves and root of this plant are used as emetic,
diaphoreticandpurgative.
Leavesof this herb smeared with castor oilandwarmedis a useful
remedyfor repellinginflammationsandswellingsat theend of toes
andfingers.Alternatively,bruisedleavesoftheherbmixedwith
castoroilcanbeusedforthispurpose.Theherbisalsouseful to
treatinflammedjointsandsprains.Slightlywarmedjuiceofthe
leaveswitha littlesalt isused forearache andother earcomplaints.
Roasted bulb is used as rubefacient in rheumatism. The bulbs are
powerfully emetic and are used to produce vomiting in poisoni ng
especially antiaries. Bruised leaves act as an efficient insect
repellent1,2,3.Juice of t he fresh bulb at a dose of 2 to 4 drachms is
veryeffectiveinemeticforchildren.
Despiteal lthese traditional uses of C.asiaticum, very few scientific
evaluationsofthisplanthavebeendocumentedsofar.Presentstudy
targetstoevaluate a fewofthepharmacological properties namely
anti‐inflammatoryand antinociceptiveactivities of C.asiaticumbulb
extractinanimalmodel.
MATERIALANDMETHODS:
Thebulbs ofC.asiaticumwerecollectedfromChittagongHilltracts,
Bangladesh,in the month ofJanuary2009. The plantwasidentif ied
and authenticated taxonomically by Dr. Shaikh Boktear Uddin,
AssistantProfessor,DepartmentofBotany,UniversityofChittagong.
A specimen of the plant was preserved in Bangladesh National
HerbariumunderthePlantAccessionNo.34545.
PreparationofPlantExtract
Thefresh bulbs ofC.asiaticum(Syn:Crinumamabile) were washed
withdistilledwaterimmediatelyaftercollection.Thecollecte dbulbs
were chopped into small pieces, air dried at room temperature f or
about10daysandgroundintopowder(536.46gm)tostoreinan
airtightcontainer. The resultingpowder was macerated in3 L pure
methanol(99%Anal‐R,Aldrich,Germany)for7daysatroom
temperaturewithoccasionalstirring.Methanolextract,after7days,
wasfilteredthroughacottonplugandfinallywithaWhatmanNo.1
filter paper. The extract was concentrated under reduced pressure
below 500Cthroughrotatoryvaccumevaporator.Theconcentrated
extractswerecollectedinanEggplantFlaskandallowtoairdryfor
completeevaporation ofmethanol.Thewhole processwasrepeate d
three times and finally, 35 gm of brownish colored, con centrated
bulbextractwasobtained(yield6.5%w/w)whichwaskeptin
refrigeratorat40C.
ExperimentalAnimalsandDiets
Swissalbino miceofboth sexesweighingbetween 25 to30 gm and
WistarAlbinoratsoftheeithersexweighingbetween150‐200gm
obtainedfromanimalhouse ofBangladeshCouncil forScientificand
Industrial Research (BCSIR) laboratories, Chittagong were used for
presentstudy. The animals wereacclimatizedtoroom temperature
(28±5)0Cwitharelativehumidityof55±5%inastandardwire
meshedplasticcagesfor4to5dayspriortocommencementofthe
experiment. During the entire period of study the animals were
supplied standard pellet diet and water adlibitum.All animal
experimentations were carried out with the guidelines of
InstitutionalAnimalEthicsCommittee(IAEC).
Assayforantinociceptiveactivity
Aceticacidinducedwrithingtest
Forwrithing test, 1%(v/v)acetic acid solution (2.3ml/kg body
weight)wasinjectedintraperitoneallytomice(weighing25‐30
gm) and the number of writhing and stretching was counted
over 20 minutes4. The methanol extract of C.asiaticum
(2gm/kg), reference analgesic drug diclofenac sodium (40
mg/kg)anddistilledwaterwereadministered orally 30minute
beforeaceticacidinjection.
Formalintest
The procedure was similar to that described previously by
Gaertner etal.5 (1999). 20 μL of 2.5% formalin (0.92%
formaldehyde)madeinphosphatebufferwasinjectedunderthe
righthindpawsurfaceofexperimentalmice.Eachmousewas
placedindividuallyinacageandobservedfrom0to5min
followedbytheinjectionofformalintoanalyzethefirstphaseof
formalininducedpain(neurogenicpain).Thelengthoftimethe
animalspentlickingtheinjectedpawwastimedwitha
chronometerandwasconsideredasindicativeofpain.
Asian Journal of Pharmaceutical and Clinical Research
Vol. 4, Issue 3, 2011 ISSN - 0974-2441


Rahmanetal.
AsianJPharmClinRes,Vol4,Issue3,2011,3437
35
Assayforantiinflammatoryactivity
Anti‐inflammatoryactivityof C.asiaticumbulbextractwas assessed
usingcarrageenaninducedpawedemamodelinthehindpawofrat
by the reported method6.AccordingtoWinter,acuteinflammation
wasinduced in albino rats by subplantar injection of0.1mlof 1%
(w/v)carrageenanaftermeasuringtheinitialrighthindpawvolume
ofeachrat.Thevolumeofrighthindpawwasmeasuredat1st,2nd,
3rdand4thhouraftercarrageenan injectionand thepaw edemawas
determined using plethysmometer (7150 UCG Basil, Italy). C.
asiaticumbulbextract(1.0,1.5and2g/kg),standardanti‐
inflammatory drug diclofenac sodium (40 mg/kg), and distilled
water were administered orally to treated, positive control and
control groups one hour before the subplantar injection of
carrageenan.
Statisticalanalysis
Valuesfor analgesic activity wereexpressed as "mean increasein
latency after drug administration ±SEM" in terms of seconds
whereas values for anti‐inflammatory activity were expressed as
"meanincreaseinpawvolume±SEM".Thesignificance ofdifference
betweenmeanswasdeterminedby student'st‐testvaluesofp<0.05
were considered significant and p<0.01 and P<0.001 as highly
significant.
RESULTANDDISCUSSION
Analgesic activity of C.asiaticumbulb extract was  assessed using
aceticacidinducedwrithingresponsemodel.Table4showsthepain
behaviorofwrithingresponseofmice,whichwaspresentedas
cumulativeabdominalstretchingresponse.
When1%(v/v)aceticacidsolution(2.3ml/kgbodyweight)was
injected intraperitoneally in mice, the control animal showed 79.3
writhing count / 20 minutes. But, administration of diclofenac
sodium caused significant reduction of writhing count, from 70 to
16.5. On the other hand, C.asiaticumbulb extract reduced the
writhingcountfrom70to22.Theeffectofbulbextractand
diclofenacsodiumwas analyzedstatisticallyby Student’s t test. The
treatment of animals with methanol bulb extract of C.asiaticum
(2gm/kg)anddiclofenacsodiumwasfoundsignificant(P<0.001)
comparedwithcontrolgroup(Table4).Thepercentageinhibitionof
analgesicactivitywascalculatedbyusingfollowingformula‐
Meanwrithingcount
(Controlgroup‐Treatedgroup)x100
%Analgesicactivity=
 Meanwrithingcountofcontrolgroup
Thedegreeofinhibition,atadose2g/kg,ofbulbextractwasfound
68.57%whichwasalmostnearertotheeffectshowedbystandard
analgesic drug diclofenac sodium (76.43 %) (Fig.1). The methanol
extract at a dose of 1.5 g/kg and 2.0g/kg inhibited the effect of
formalin by 55% and 63%, respectively (Fig.2). Morphine did not
exertanysignificanteffect.
Carrageenan induced paw edema model indicated that subplantar
injectionofcarrageenaninexperimentalratsshowedatime‐
dependent increase in paw thickness (Table 1). This increase was
observed at 1sthandwasmaximalat4hafteradministrationof
carrageenaninjectionin thecontrolgroup. Methanolicbulb ext ract
ofC.asiaticum(2g/kg) produced33.06%,50.55%,52.56%,51.02%
inhibition of paw edema at 1st, 2nd, 3rdand4
th h after carrageenan
injectionrespectively(Table2andFig.3).Thepercentofinhibition
forotherconcentrationarealso delineated(Table 2andFig.3).The
effectwasfoundstatisticallynon‐significant,comparedtocontrol,at
1sth(P> 0.05),significantat 2ndh(P<0.05) &4h(P<0.05) andvery
significantat3h(P<0.01)aftercarrageenaninjection.
On the other hand, carrageenan‐induced inflammation was
significantly (P<0.05) reduced in all phases of the experiment by
treatmentwith referenceanti‐inflammatory drugdiclofenacsodium
(40mg/kg).Diclofenac sodium produced 38.70%,45.67%,58.32%,
60.88%anti‐inflammatory effect at 1st, 2nd, 3rdand4th,respectively,
aftercarrageenaninjection(Table2).
Medicinalplantsindeedhavebeenanindispensablearmin
ameliorating common inflammation, pain sensation as well as
nonciception7.Thebulb of C.asiaticumhad been used tra ditionally
intherheumatoidarthritisandcoldsincelongbutnoworkhasbeen
done to confirm its analgesic and anti‐inflammatory activity albeit
the antipyretic effect of petroleum ether and chloroform soluble
fractionsofethanolextractofitsrootshasbeenobservedrecently8.
Aceticacidinducedabdominalconstrictionsareusefulexperimental
toolsinthetestingofnewanalgesicdrugs9 because the abdominal
injectionofacetic acid in micehasbeenattributed to the release of
arachidonicacid,whichresultsthesynthesisofprostaglandinviathe
cyclooxygenase (COX) enzyme10. The special nerve endings that
sensepainis verysensitive toprostaglandin. Whenprostaglandinis
released,the nerve endings respond to itthroughprostaglandinE2
(PGE2)receptorbypickingupandtransmittingthepainandinjury
messagestothebrainandcausevisceralwrithingstimuliin
mice11,12,13.
Therefore,ithas beensuggested thattheinhibitionofprostaglandin
synthesis is remarkably an efficient antinociceptive mechanism in
visceral pain14.Sincemethanolextractinthisstudyshowedvery
significantinhibition(P<0.001)(Table4)inaceticacidinducedpain,
itmaybepredictedastheanalgesiceffectofextract.Theextractwas
thentestedagainstothermodelofexperimentalpain.Itwasassayed
on the first phase of formalin induced pain known as neur ogenic
pain.Themethanolextractexhibitedasignificantanalgesicactivity
against neurogenic pain (Fig. 2). The activity of the extract in this
modelsuggeststheactivationofopioidreceptorsintheiraction
mechanism5.
Theanalgesiceffectoftheextract,therefore,maybeduetoitsaction
on visceral nociceptors sensitive to acid, to the inhibition ofthe
synthesis of the arachidonic acid metabolite15. Anti‐inflammatory
activity through carrageenan induced paw edema is a suitable test
for evaluating anti‐inflammatory properties for natural drugs
becauseitshowsverypromisingsensitivity,particularlyintheacute
phaseofinflammation,indetectingorallyactiveanti‐inflammatory
agents16.Developmentofedemainthepawofratafterinjectionof
carrageenenisindeedabiphasicevent17, of which the initial phase
observed during the first hour is attributed to the release of
histamineandserotonin whereasthe secondone ofedemaisdueto
thereleaseofprostaglandins,protease,andlysosome
18,19,20. This
leadsto a dilationofthe arterioles andvenulesand to anincreased
vascularpermeability.Asaconsequence,fluidandplasmaprote ins
areextravagated,andedemaforms21.
The mediators, including histamine, 5‐HT, the kinins and their
complements,havebecometherecentfocusofattentionasthe
metabolites of arachidonic acid (AA). Alone or in appropriate
combination,AA productsof COX pathwayare capableofproducin g
thecharacteristicsignsofinflammationwhichsubsequently
produces vasodilatation, hyperemia, pain, edema, and cellular
filtration. The COX products, particularly prostaglandin E2 (PGE2),
contribute to increased blood flow throug h a vasodilatation action,
but the lipoxygenase (LOX) pathway is necessary for vascular
leakageandedemaconsequentlyoncellularinfiltration.
Itispossiblethatthemethanolextractofbulbcontainstheactive
constituents that exhibit its anti‐inflammatory action probably by
means of either inhibiting the synthesis, release or action of
inflammatory mediators like histamine, serotonin, prostaglandin,
protease,andlysosome.
From our observation we assume that different active secondary
metabolites are present in crude extracts of C.asiaticumbulb and
perhaps some of these compounds may operate in a synergistic
manner. Moreover, the anti‐inflammatory effect of the C.asiaticum
crude extract is almost near to the effect of standard anti‐
inflammatorydrug diclofenac sodium.Fromthis observation, it can
besuggestedthatifthecompoundresponsibleforanti‐
inflammatoryeffectcouldbeisolatedfromcrudeextract,itmight
show very potent anti‐inflammatory effect even better than
diclofenacsodium.

Rahmanetal.
AsianJPharmClinRes,Vol4,Issue3,2011,3437
36

Table1:EvaluationofantiinflammatoryactivityofCrinumasiaticumbulbextractbycarrageenaninducedpawedemamodel


Here,Allvaluesareexpressedasmean±SEM(n=5)NS=Notsignificant(P>0.05)comparedwithcontrol.**P<0.01compared
withcontrol(Student’st‐test).*P<0.05significantcomparedwithcontrol(Student’st‐test).


Table2:%AntiinflammatoryactivityofCrinumasiaticumbulbextractandDiclofenacSodium

Here,Coisthepawthicknessvolume(inmm 3)beforecarrageenaninjection,Ctisthepawthicknessvolume(inmm3)attimet,
(Ct‐Co)ispawedema.


Table3:EffectofCrinumasiaticumbulbextractonaceticacidinducedwrithingresponse


Table4:EffectofCrinumasiaticumbulbextractonaceticacidinducedwrithingresponse(Student’sttest,***P<0.001significant
comparedtocontrol)

WreathingCounts/20min

Treatment ControlDistilledwater DiclofenacSodium Stemextrac
t

Dose 2ml 40(mg/kg) 1.00(gm/kg) 1.5(gm/kg) 2(gm/kg)
Mean±SEM 70±1.87 16.5±1.71 41.5±2.5 30.5±1.96 22±4.02

Student’s


ttest
t
‐calculated
t‐tabulated
Deg.offred.
p‐value
21.1
5.96
6
<0.001
9.13
5.96
6
<0.001
14.58
5.96
6
<0.001
10.82
5.96
6.0
<0.001

Here,allvaluesareexpressedasMean±SEM(n=4),P<0.001significantcomparedtocontrol




Group

Treatmen
t

Dose

Pawedema(mm3)(Ct‐Co)

1st

hr

2nd

hr

3rd

hr

4th

hr

Control Distilledwater 2ml 0.37±0.05 0.64±0.06 0.76±0.07 0.88±0.11
Positivecontrol DiclofenacSodium 40mg/kg *0.23±0.04 *0.35±0.07 **0.32±0.03 **0.35±0.04
Sampletreated CrinumasiaticumBulbextract 2gm/kg NS0.25±0.05 **0.31±0.06 *0.36±0.10 **0.43±0.07
 1.5gm/kg *0.27±0.007 *0.36±0.012 *0.4±.01 *0.48±.01
1gm/kg *0.29±.017 *0.44±.028 *0.49±.02 *0.55±.015

Group

Treatment

Dose
%InhibitionofPawedema

1sthr2ndhr3rdhr4thhr
Control Distilledwater 2ml ‐ ‐ ‐ ‐
Positivecontrol DiclofenacSodium 40mg/kg 38.70% 45.67% 58.32%60.88%

Sampletreated

C.asiaticumbulbextract
2gm/kg 33.06% 50.55% 52.56% 51.02%
1.5gm/kg 27.02% 43.75% 47.37% 45.45%
1.0gm/kg 21.62% 31.25% 35.52% 37.5%


RatNo.
WrithingCount/20minutes

Control DiclofenacSodium(40mg/kg) BulbExtract
2(gm/kg) 1.5(gm/kg) 1.00(gm/kg)
1 70 12 26 36 48
2 69 16 27 29 36
3 66 20 10 30 40
4 75 18 25 27 42
MEAN 70 16.5 22 30.5 41.5
SEM 1.87 1.71 4.02 1.93 2.5
%Analgesicactivity ‐ 76.43% 68.57% 56.42% 40.71%

Rahmanetal.
AsianJPharmClinRes,Vol4,Issue3,2011,3437
37










Fig.1:Comparative%analgesicactivityofC.asiaticumbulb
extractandcommerciallyavailableanalgesicdrugDiclofenac
Sodium














CONCLUSION
Theresults ofthe studydemonstratethatthemethanol extract ofC.
asiaticumbulb exerts potential analgesic and anti‐inflammatory
effectinexperimentalanimalmodelswhichsupporttheclaimsby
traditionalmedicine practitioners. Onthe basis oftheresults, itcan
beusedasagoodsourceofanalgesicdrugs.However,
pharmacodynamic studies should be undertaken to establish the
mechanism of action of the plant extracts contributing in
nonciception andinflammation. Phytochemical investigation is also
proposedinordertoisolatetheactivefractionandeventuallythe
purecompound.
ACKNOWLEDGEMENT
TheauthorswishtopaythankfulgratitudetoBangladesh Council
for Scientific and Industrial Research (BCSIR) Laboratories,
Chittagongfortheircontinuoussupportinprogressofthisstudy.
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

Fig.

2:Effectofethanolextractof

C.asiaticumandmorphineon

formalininducedpaininmice.n=5,P<0.05comparedwith
control
Fig.

3:

Comparative%antiinflammatoryactivityof

C.asiaticumbulbextractanddiclofenacsodium