Ind ian J Phys io l Pharmaco l 2011 ; 55 (1) : 13–24

*!Corresponding Author : Meena Kumar i K , Depar tmen t o f Pharmaco logy , Kas tu rba Medica l Co l l ege ,Manipa l – 576104; Email : drmohan7amberkar@gmail.com, mini41178@yahoo.co.in;Ph.: 0820 2933037, 0820 2570041, 09886369607 and 09886367003;

EVALUATION OF ANTIINFLAMMATORY AND ANALGESIC ACTIVITIESOF ALCOHOLIC EXTRACT OF KAEMPFERIA GALANGA IN RATS

AMBERKAR MOHANBABU VITTALRAO, TARA SHANBHAG,MEENA KUMARI K.*, K. L. BAIRY AND SMITA SHENOY

Department of Pharmacology,Kasturba Medical College,Manipal – 576 104

( Rece ived on February 8 , 2010 )

Abstract : Alcoholic extract of Kaempferia galanga was tested for analgesicand antiinflammatory activities in animal models. Three doses, 300 mg/kg,600 mg/kg and 1200 mg/kg of the plant extract prepared as a suspensionin 2 ml of 2% gum acacia were used. Acute and sub acute inflammatoryac t iv i t i e s were s tud ied in ra t s by ca r rageenan induced paw edema andcotton pellet induced granuloma models respectively. In both models, thestandard drug used was aspirin 100 mg/kg. Two doses 600 mg/kg and 1200mg/kg of p lant ex t rac t exhib i ted s igni f icant (P<0.001) ant i inf lammatoryac t iv i ty in ca r rageenan mode l and co t ton pe l l e t g ranu loma mode l incomparison to control. Analgesic activity was studied in rats using hot plateand tail-flick models. Codeine 5 mg/kg and vehicle served as standard andcontrol respectively. The two doses of plant extract exhibited significantanalgesic activity in tail flick model (P<0.001) and hot plate model (P<0.001)in comparison to control. In conclusion K. galanga possesses antiinflammatoryand analgesic activit ies.

Key words : ant i inf lammatory act ivi ty analgesic activityKaempferia galanga carrageenan hot plate react ion t imetail flick latency

INTRODUCTION

Inf lammat ion i s a pa thophys io log ica lresponse of l iv ing t i s sue to in ju ry tha tleads to loca l accumula t ion of p lasmat icf lu id and b lood ce l l s . Al though i t i s adefense mechanism tha t he lps body topro tec t i t se l f aga ins t in fec t ion , burns ,toxic chemicals , a l lergens or other noxiouss t imul i , the complex events and media tors

involved in the in f lammatory reac t ion caninduce, maintain or aggravate many diseases(1).

Pa in has been def ined by In te rna t iona lAssociation for the Study of Pain (IASP) asan unpleasan t sensory and emot iona lexperience associated with actual or potentialtissue damage (2). Failure to relieve pain ismoral ly and ethical ly unacceptable.

14 Mohanbabu e t a l Ind ian J Phys io l Pharmaco l 2011 ; 55(1 )

Drugs tha t a re cur ren t ly used for themanagement o f pa in a re op io ids o r non-opioids and that for inflammatory conditionsare non-s te ro ida l an t i in f lammatory drugs(NSAIDs) and corticosteroids. All these drugscar ry po ten t ia l tox ic e f fec t s . One s tudysuggests that risk of gastrointestinal bleedingwas s ign i f ican t ly assoc ia ted wi th acu teuse of non-s te ro ida l an t i - in f lammatorydrugs (NSAIDs) l ike regu la r -dose asp i r in ,diclofenac, ketorolac, naproxen or nimesulide.Piroxicam increased the risk of bleeding inboth acute and chronic therapy (3). Opioidsare the commonly used drugs for themanagement o f acu te pos topera t ive pa in(4).

It is not surprising that from conceptionto marke t mos t compounds face an uphi l lba t t l e to become an approved drug .For approximate ly every 5 ,000 to 10 ,000compounds tha t en te r p rec l in ica l t es t ing ,only one is approved for marketing (5). Drugresearch and deve lopment (R & D) i scomprehens ive , expens ive , t ime-consumingand full of risk. It is estimated that a drugf rom concept to marke t would takeapproximately 12 years and capitalizing out-of -pocke t cos t s to the po in t o f marke t ingapproval at a real discount rate of 11% yieldsa total pre-approval cost estimate of US$ 802million (6).

On the contrary many medicines of plantorigin had been used since ages without anyadverse effects. It is therefore essential thate f for t s should be made to in t roduce newmedicinal plants to develop more effect iveand cheaper drugs. Plants represent a largena tura l source of usefu l compounds tha tmight serve as lead for the development ofnovel drugs.

In the present study, Kaempferia galangawas se lec ted because i t i s one of themedic ina l p lan ts commonly used in theayurvedic sys tem of medic ine . Kaempfer iagalanga , commonly known as kencur ,aromatic ginger, sand ginger or resurrectionl i ly , i s a monocoty ledonous p lan t in theginger (Zingiberaceae) family. The rhizomef inds an impor tan t p lace in ind igenousmedicine as st imulant , expectorant , diuret icand carmina t ive (7 ) . I t was used as aningredient for the treatment of various skind isorders . I t was wide ly used in thet rea tment o f d iabe tes mel l i tus , va r iousinflammatory and lipid disorders (8, 9). I talso possesses larvicidal activity (10), anti-oxidant (11), anti-ulcer (12), antiinflammatory(13) and anti-hypertensive (14) properties.

Kaempferia galanga has been widely usedin remedies to t rea t abdomina l pa in ,too thache , and as an embroca t ion orsudor i f i c to t rea t muscu la r swel l ing andrheumatism in the traditional medicine (15).I t i s known tha t the major chemica lcons t i tuents of the vola t i le o i l f rom dr iedrh izome were e thy l -p -methoxyc innamate(31.77%), methylcinnamate (23.23%), carvone(11.13%), eucalyptol (9.59%) and pentadecane(6.41%), respectively. Other consti tuents ofthe rhizome include cineol, borneol, 3-carene,camphene, kaempferal, c innamaldehyde , p -methoxycinnamic acid and ethyl cinnamate.A methanolic extract of the rhizome containse thy l p -methoxy- t rans -c innamate , which i shighly cytotoxic to HeLa cells (16).

Up to date no pharmacological study hasbeen sys temat ica l ly conduc ted to eva lua tethe an t i in f lammatory and an t inoc icep t iveac t ion of e thanol ic ex t rac t o f Kaempfer iagalanga , support ing tradit ional uses of this

Indian J Physiol Pharmacol 2011; 55(1) Antiinflammatory Analgesic Study of K. galanga 15

suspending this residue in the cosolvent (2%gum acacia).

Se lec t ion o f an imals , car ing and handl ing :

This was done as per the guidelines setby the Indian National Science Academy NewDelhi, India. A total of 120 healthy Wistarrats (150–200 g), aged twelve weeks of eithersex , b red loca l ly in the an imal house ofKas turba Medica l Col lege , Manipa l werese lec ted for the s tudy . They were housedunder cont ro l led condi t ions of tempera tureof 23±20C, relative humidity of 30–70% and12 h light–12 h dark cycle. The animals werehoused individual ly in polypropylene cagesconta in ing s te r i l e paddy husk (procuredloca l ly ) as bedding th roughout theexperiment. All animals were fed with sterilecommerc ia l pe l le ted ra t chow suppl ied byHindustan Lever Ltd . (Mumbai , India) andhad free access to water ad libitum. Animalswere kept under fas t ing for overn ight andweighed before the exper iment . The s tudywas undertaken af ter obtaining approval ofInstitutional Animal Ethics Committee (IAECapproval letter No. IAEC/KMC/04/2002-2003Dated Dec.14.2005).

Study des ign :

The ra t s were randomly a l loca ted in tof ive groups of s ix ra t s each for the fourd i f fe ren t exper imenta l an imal models . Weused two an imal models each for t es t ingant i inf lammatory and analgesic act iv i t ies .

Group I (control) received 2 ml of 2%gum acacia (E. Merck India Ltd.), po throughin t ragas t r i c tube .

Group II received K. galanga, 300 mg/kg, po

plan t in fo lk lore medic ine . Hence presen ts tudy was under taken to eva lua te theantiinflammatory and antinociceptive activityof Kaempferia galanga in Wistar albino ratsusing ethanol extract . The ethanolic extractwas used in th i s inves t iga t ion becausee thanol be ing nonpola r , the major ac t ivechemical constituents of Kaempferia galangaincluding volatile oils, would be expected tobe more soluble in e thanol f ract ion of thee x t r a c t .

MATERIALS AND METHODS

Descr ipt ion o f p lant mater ia l :

The fresh rhizomes of K. galanga plantswere procured loca l ly in the month ofDecember 2005. The plants were ident if iedand au thent ica ted by Professor o f Botany ,Mahatma Gandhi Memorial College (MGMC),loca ted in Udupi (Karna taka) . Af te rauthentification, the plants were cleaned andshade dr ied and mil led into coarse powderby a mechanical gr inder .

Preparat ion o f a lcoho l i c ex tract o f K. ga langa:

The dried powder (total 2 kg) was loadedinto Soxhlet extractor with glass thimble (catno.3485) in 8 batches of 250 g each and wassubjected to extraction for about 30-40 h with95% ethanol. After extraction the solvent wasdistilled off and the extract was concentratedunder reduced pressure on a water bath a ta temperature below 50°C to g ive a semi-so l id syrupy cons is tency res idue of 60 .8 g(yield 3%, w/w) which was stored in a closedbot t l e and kep t in a re f r igera to r a tt empera ture be low 4°C unt i l t e s ted . Theethanol ic extract of K. galanga a t doses of300, 600 and 1200 mg/kg were prepared by

16 Mohanbabu e t a l Ind ian J Phys io l Pharmaco l 2011 ; 55(1 )

ac t iv i ty was de te rmined in a lb ino ra t s o feither sex according to the method of Winter(18) . Al l d rugs were g iven ora l ly to therespec t ive g roups as a suspens ion in gumacac ia one hour before ca r rageenaninjection. The procedure followed was, acuteinf lammat ion produced by in jec t ion ofcarrageenan (0.1 ml of 1% w/v suspension)(19), in the right hind paw of the rats underthe plantar aponeurosis. It was injected +1haf te r the o ra l admin is t ra t ion of the d rug .The inf lammation was quant i ta ted in termsof ml i .e . displacement of water by edemausing a digi tal plethysmometer immediatelybefore and after carrageenan injection at +1,+2 , +3 , +4 , +5 and +6 h . The percen tageinhibition of edema was calculated for eachgroup wi th respec t to i t s veh ic le - t rea tedcontrol group (20, 21, 22).

Percen tage inh ib i t ion of paw edema =(1–Vt/Vc) × 100

Where Vc represent average increase inpaw volume (average inf lammat ion) of thecontrol group of rats at a given time; and Vtwas the average inf lammat ion of the drugt rea ted ( i . e . p lan t ex t rac t s o r t es t d rugaspi r in) ra ts a t the same t ime.

The d i f fe rence in the in i t i a l 0h andvolume a t +1h ind ica te paw edema a t 1hfo l lowing car rageenan admin is t ra t ion .Accordingly paw edema a t +2, +3, +4, +5and +6h was ca lcu la ted . Then percen tageinhibit ion of paw edema was calculated.

Sub acute in f lammatory mode l

Cot ton pe l l e t induced granuloma model

Sub acute inflammation was produced bycot ton pe l le t induced granuloma model in

Group III received K. galanga, 600 mg/kg, po

Group IV received K. galanga , 1200 mg/kg, po

Group V received standard drug, aspirin100 mg/kg, po (for anti inflammatory study)and codeine 5 mg/kg, po (for analgesic study).

MATERIALS

Drugs : aspir in (Sigma chemical Co. StLouis, USA), Alcoholic extract of K.galanga,2% Gum acacia (E. Merck India Ltd.), codeine(Sigma chemical Co. St Louis, USA), Ether(S igma chemica l Co . S t Louis , USA) ,Inf lammatory agents used : in j .carrageenan (0.1 ml of 1% w/v suspension)(Sigma chemical Co. St Louis, USA), cottonpe l le t s , Instruments : Eddy’s ho t p la te orTechno heated plate analgesiometer mark-3(model no . HPA) , Analges iometer - (Technoe lec t ron ics , Lucknow, Ind ia ) , Dig i ta lp le thysmometer (Ugo Basi le Company Cat .No. 7140 VA, Italy).

Determinat ion o f the drug dosage and

dos ing schedule : Doses were se lec ted anddetermined according to the previous acutetox ic i ty s tud ies o f e thanol ic ex t rac t o fK.galanga (17). Three different doses wereselected 300 mg/kg, 600 mg/kg and 1200 mg/kg for an t i in f lammatory and ana lges icact iv i t ies . The suspension of the a lcohol icextract of K. galanga and aspirin were madein 2% gum acacia.

METHODS

Acute in f lammatory mode l

Carrageenan induced paw edema in ra t s

In the p resen t s tudy , an t i in f lammatory

Indian J Physiol Pharmacol 2011; 55(1) Antiinflammatory Analgesic Study of K. galanga 17

ra t s (23 , 24) . On day 1 , wi th asep t icprecautions sterile cotton pellets (50±1 mg)were implan ted subcutaneous ly , a long theflanks of axillae and groins bilaterally undere ther anaes thes ia . Al l d rugs were g ivenoral ly to the respect ive group of ra ts as asuspens ion in gum acac ia da i ly fo r s ixconsecut ive days f rom day 1 . The animalswere sacrificed on the 7th day. The granulationtissue with cotton pellet was dried at 60oCovern igh t and then the d ry weigh t wastaken . Weight o f the co t ton pe l le t be foreimplantation was subtracted from weight ofthe dissected dried pellets. Only dry weightof the granuloma formed was used fors ta t is t ica l analys is .

Analges ic s tudy

Hot p la te method

The analgesic activity of the extract, wasmeasured by hot-plate method (25). All drugswere g iven ora l ly to the respec t ive g rouprats as a suspension in gum acacia. The ratswere p laced on a ho t p la te main ta ined a t55±0.5 oC. The reac t ion t ime was taken asthe interval from the instant animal reachedthe hot plate until the moment animal lickedits feet or jumped out. A cut off time of +10s was followed to avoid any thermal injuryto the paws. The reaction time was recordedbefore and after +30, +60, +90, +120 and +180min fo l lowing admin is t ra t ion of t es t o rs tandard drug .

E v a l u a t i o n

The mean reaction time for each treatedgroup was de te rmined and compared wi ththa t ob ta ined for each group beforet rea tment . Percen tage increase in reac t ion

t ime (I %), was der ived, using the formulaI% = {(It – Io)/Io} × 100, Where It = reactiont ime a t t ime, t , and Io = reac t ion t ime a tt ime zero (0 h) (26) . The an imals weresubjected to the same test procedure at +30,+60 , +120 , and +180 min a f te r theadministrat ion of test /s tandard/control drug.

Radiant heat ta i l - f l i ck method

The cen t ra l ana lges ic ac t iv i ty wasdetermined by radiant heat ta i l - f l ick modelin ra ts (27) . The analges ic ac t iv i ty of theplant extract was studied by measuring drug-induced changes in the sensitivity of the pre-screened rats (the intensity of the light beamhas been exper imenta l ly def ined such tha tnaive animals will withdraw their tails within2 to 4 s) to heat stress applied to their tailsby us ing analges iometer . Tai l - f l ick la tencywas assessed by the ana lges iometer . Al ld rugs were g iven ora l ly to the respec t ivegroup ra ts as a suspension in gum acacia .The strength of the current passing throughthe naked nichrome wire was kept constanta t 5 ampere . The d i s tance be tween hea tsource and the t a i l was 1 .5 cm and theapplication site of the heat on the tail wasmaintained within 2 cm, measured from theroot of the ta i l . Cut-off react ion t ime was+10 s to avoid any tissue injury during theprocess. Tail-flick latency was measured from+30 min af ter the drug adminis t ra t ion.

E v a l u a t i o n

The time taken by rats to withdraw (flick)the tail was taken as the reaction time. Theanimals were sub jec ted to the same tes tprocedure at +30, +60, +120, and +180 minaf te r the admin is t ra t ion of t es t / s tandard /control drug.

18 Mohanbabu e t a l Ind ian J Phys io l Pharmaco l 2011 ; 55(1 )

Stat i s t i ca l ana lys i s

The results were analysed for statist icalsignificance using One way ANOVA, followedby Schef fe ’s t es t . A P-va lue <0 .05 wasconsidered significant. Computer statist icalpackage SPSS (vers ion 16) was used foranalys is .

RESULTS

Ant i in f lammatory ac t iv i ty in rats

Ef fec t on carrageenan induced paw edema(Table I , F ig . 1 )

Pretreatment with K.galanga resul ted indose-dependent reduc t ion in ca r rageenanevoked h ind paw edema and d i f fe redsignif icant ly (P<0.001) among the differentgroups of ra t s (Table I ) . In ca r rageenaninduced rat paw edema test , the two dosesof p lan t ex t rac t showed s ta t i s t i ca l lys ign i f ican t (P<0 .001) inh ib i to ry e f fec t on“mean increase in paw volume” a t a l l thetime intervals (+1 h, +2 h,+ 3 h, +4 h, +5 h,and +6 h) as shown in (Table I). After +1 hand +3 h of ca r rageenan admin is t ra t ion ,K. galanga exhibited maximum % inhibit ion

of paw volume (P<0.01) by 36.95%, 33.55%and 42.31%, 44.19%, at the higher two dosesof plant extract 600 mg/kg and 1200 mg/kgbody weigh t respec t ive ly , however %inhibition of paw volume was less than thatof s tandard drug asp i r in 54 .96%, 69 .59%(P<0.001) a t the dose of 100 mg/kg bodyweight (Table I, Fig. 1).

Ant igranulat ion e f fec t in ra t s (Table I I )

The dry weigh t o f co t ton pe l le tgranuloma in control , three different dosesof K. galanga and aspir in t reated groups isshown in the table 2. It can be noted fromtable 2 , that the two doses of K. galanga600 mg/kg and 1200 mg/kg , and asp i r inshowed s ign i f ican t (P<0 .001) ac t iv i ty ininh ib i t ing dry weigh t o f g ranuloma. Theextract administered at 1200 mg/kg, p.o. hada greater anti-granulation (36.70%) effect butless than aspirin (42.17%).

Analges ic ac t iv i ty in rat s

Hot p la te method (Table I I I )

In the hot-plate method, both the extractand code ine caused s ign i f ican t inc rease

TABLE I : Ant i inf lammatory effect of a lcohol ic ext ract of K. galanga on carrageenan- induced ra t paw edema.

Increase in paw volume (Mean±SEM) (ml)Drugs Dose/ (% Inhibition of paw edema)

r o u t eBe fore +1h +2h +3h +4h +5h +6h

g u m 2 m l 0.633±0.023 0.408± 0.025 0.868± 0.033 1.196± 0.031 1.316± 0.028 1.433± 0.027 1.266± 0.031acacia poK . 300 mg/ 0.672±0.006 0.348± 0.010 0.776± 0.009 1.12± 0.017 1.22± 0.025 1.310± 0.014 1.241± 0.024galanga kg po (13.42) (10 .02) (5.670) (7.26) (8.41) (1.456)

600 mg/ 0.735±0.015 0.246± 0.019a 0.631± 0.018ab 0.79± 0.022ab 0.985± 0.031ab 1.025± 0.016ab 0.978± 0.028ab

kg po (36.95) (26.41) (33.55) (24.83) (28.31) (22.32)1200 mg/ 0.780±0.006 0.228± 0.008a 0.520± 0.008ab 0.665± 0.016ab 0.952± 0.021ab 0.990± 0.011ab 1.08± 0.010ab

kg po (42.314) (39.71) (44.195) (27.489) (30.81) (14.25)aspirin 100 mg/ 0.551±0.006 0.185± 0.045a 0.271± 0.01a 0.363± 0.009a 0.415± 0.011a 0.480± 0.008a 0.481± 0.008a

kg po (54.96) (68.46) (69.59) (68.45) (66.48) (61.85)

aP<0.001 vs Control, bP<0.001 vs aspirin, (n=6/group), One-way ANOVA; SEM = Standard error of mean.

Indian J Physiol Pharmacol 2011; 55(1) Antiinflammatory Analgesic Study of K. galanga 19

(P<0.001) in the reaction time. The increasein l a tency per iod a t d i f fe ren t t ime po in t ssignif icant ly differed (P<0.01) compared to

baseline values within the same drug treatedgroups . The percen tage increase in thereac t ion t ime was dose-dependent anddif fered s igni f icant ly among the groups ofrats (P<0.001) receiving different dose levelsof the extract and codeine (Table III).

The percentage increase in the react iontime caused by the extract and codeine wasdetectable and peaked, at +1 h and +30 minrespec t ive ly bu t the reaf te r dec l inedrelatively at +3h after the administration ofthe extract or codeine.

At +3 h a f te r admin is t ra t ion , code inere ta ined a s ign i f ican t ly g rea te r ac t iv i ty(P<0.001) than the ex t rac t . At a l l thespec i f ied t ime in te rva l s , the percen tageincrease in reac t ion t ime d i f fe redsignif icant ly (P<0.001) between the extractand codeine, being greater for codeine.

0

10

20

30

40

50

60

70

80

ONE H TWO H THREE H FOUR H FIVE H SIX H

Time (in h)

% in

hibi

tion

of p

aw e

dem

a

K.galanga-300mg/kg

K.galanga-600mg/kg

K.galanga-1200mg/kg

aspirin-100mg/kg

Fig . 1 : Ant i in f l ammatory e f fec t o f K. ga langa on ca rageenan induced ra t paw edema .

TABLE I I : Ant i in f l ammatory e f fec t o f a lcoho l icex t rac t o f K. ga langa on co t ton pe l l e tinduced g ranu loma fo rmat ion in ra t s .

Drugs Dose/ Weight of % inhibitionr o u t e dry cotton pellet of granuloma

granuloma (mg) format ion(Mean±SEM)

g u m 2 ml of 81.712± 0.433 –acacia 2% poK. galanga 300 mg/ 82.5± 0.868 –0.964

kg/day po600 mg/ 59.4± 0.909ab 27.305kg/day po1200 mg/ 51.72± 2.007ab 36.70kg/day po

aspirin 100 mg/ 47.25± 1.878a 42.17kg/day po

aP<0 .001 vs Con t ro l , bP<0 .001 vs a sp i r in , (n=6 /group) , One-way ANOVA; SEM = Standard erroro f mean .

20 Mohanbabu e t a l Ind ian J Phys io l Pharmaco l 2011 ; 55(1 )

At the peak of act ivi ty, 600 mg/kg and1200 mg/kg ex t rac t caused 52 .04% and56 .56% increase in the reac t ion t imerespec t ive ly whi l s t code ine gave 82 .16%increment . T ime to peak ac t iv i ty wasdifferent for the extract (+1 h) and codeine(+30 min).

Tai l f l i ck method (Table IV)

In the tail f l ick method, the increase in

la tency per iod a t d i f fe ren t t ime po in t ssignif icant ly differed (P<0.01) compared tobaseline values within the same drug treatedgroups . The ex t rac t and code ine causeds ign i f ican t inc rease (P<0 .01) in thepercentage react ion t ime whils t the controland lower dose of extract (300 mg/kg) causedno change . The percen tage increase inreaction time was dose dependent. At all thespeci f ied t ime in terva ls , the percentage oftail flick elongation time differed significantly

TABLE II I : Ana lges ic e f fec t o f a l coho l i c ex t rac t o f K. ga langa by ho t p la t e me thod in r a t s .

Reaction time in s (mean±SEM)Drugs Dose/

r o u t e Basal 30 min 60 min 120 min 180min(% elongation) (% elongation) (% elongation) (% elongation)

gum acac ia 2 ml po 4 .56±0 .03 5 .183± 0 .03* 5 . 4 2± 0 .031* 6 .067± 0 .012* 6 . 2 3± 0 .014*(13 .66 ) (18 .84 ) (33 .03 ) (36 .60 )

K . g a l a n g a 300 mg/ 4 .780±0 .016 5 .360± 0 .01* 5 . 6 0± 0 .014* 6 .083± 0 .008* 6 .313± 0 .009*kg po (12 .14 ) (17 .16 ) (27 .27 ) (32 .08 )600 mg/ 5 .052±0 .016 7 . 2 9± 0.04* ab 7 . 6 8± 0.033* a b 6 . 9 6± 0 .017* a 7 . 0 4± 0.01* ab

kg po (44 .28 ) (52 .04 ) (37 .78 ) (39 .43 )1200 mg/ 4 .917±0 .024 7 .022± 0.02* ab 7 . 6 9± 0.04* ab 7 . 2 9± 0.02* ab 7 . 1 5± 0.008* a b

kg po (42 .83 ) (56 .56 ) (48 .31 ) (45 .37 )c o d e i n e 5 mg/ 4 .334±0 .030 7 .890± 0 .05* a 7 . 2 5± 0 .04* a 7 . 0 2± 0 .015* a 6 . 8 1± 0 .011* a

kg po (82 .16 ) (67 .40 ) (61 .80 ) (57 .20 )

*P<0.01 vs Basel ine value of the respect ive drug group, aP<0.001 vs Control , bP<0.001 vs codeine, (n=6/g roup) , One-way ANOVA; SEM = S tandard e r ro r o f mean .

TABLE IV : Ana lges ic e f fec t o f a l coho l i c ex t rac t o f K.ga langa on rad ian t hea t t a i l - f l i ck response in r a t s .

Reaction time in s (mean±SEM)Drugs Dose/

r o u t e Basal 30 min 60 min 120 min 180min(% elongation) (% elongation) (% elongation) (% elongation)

gum acac ia 2 ml po 4 .25±0 .015 4 . 3 7± 0 .015 4 . 6 1± 0 .021* 4 . 7 9± 0 .006* 4 . 6 5± 0 .033*(2 .95) (8 .39) (12 .78 ) (9 .49)

K . g a l a n g a 300 mg 4 .39+0 .006 4 . 4 4± 0 .020 4 . 7 1± 0 .048* 4 . 8 6± 0 .009* 4 . 7 2± 0 .009*/kg po (1 .10) (7 .20) (10 .77 ) (7 .58)600 mg/ 4 .81±0 .008 6 . 9 1± 0.016* a b 6 . 3 4± 0.015* ab 5 . 8 9± 0.033* ab 5 . 4 6± 0.007 *ab

kg po (43 .66 ) (31 .86 ) (22 .36 ) (13 .43 )1200 mg/ 4 .78±0 .006 7 . 0 2± 0.012* ab 6 . 6 6± 0.020* ab 5 . 8 5± 0.01* ab 5 . 5 5± 0.013 *ab

kg po (46 .76 ) (39 .23 ) (22 .43 ) (16 .09 )c o d e i n e 5 m g / 4 .93±0 .008 8 . 8 7± 0 .033* a 7 . 8 8± 0 .035* a 6 . 6 4± 0 .077* a 6 . 1 1± 0 .008* a

kg po (80 .08 ) (59 .90 ) (34 .75 ) (23 .93 )

*P<0.01 vs Basel ine value of the respect ive drug group, aP<0.001 vs Control , bP<0.001 vs codeine , (n=6/g roup) , One-way ANOVA; SEM = S tandard e r ro r o f mean .

Indian J Physiol Pharmacol 2011; 55(1) Antiinflammatory Analgesic Study of K. galanga 21

(P<0.001) between the extract and codeinea t bo th the doses o f p lan t ex t rac t , be inggreater for codeine. At the peak of activity,600mg/kg and 1200mg/kg ex t rac t showed43.66% (P<0.001) and 46 .76% (P<0.001)percen tage of t a i l f l i ck e longa t ion t imerespectively, whilst codeine gave 80.08% (P<0.001) elongation of tail flicking time (Table4) . T ime to reach peak ac t iv i ty was same(+30 min) for the extract and codeine.

DISCUSSION

The car rageenan- induced paw edemamodel in ra t s i s known to be sens i t ive tocyclooxygenase inhibitors and has been usedto eva lua te the e f fec t o f non-s te ro ida lan t i in f lammatory agen ts , which pr imar i lyinh ib i t the cyc looxygenase invo lved inprostaglandin synthesis (28).

Carrageenan-induced hind paw edema isthe s tandard exper imenta l model of acute-in f lammat ion . The t ime course of edemadeve lopment in ca r rageenan- induced pawedema model in rats is generally representedby a biphasic curve (29). The first phase ofin f lammat ion occurs wi th in an hour o fcarrageenan injection and is partly attributedto trauma of injection and also to histamine,and serotonin components (30). The secondphase is associa ted wi th the product ion ofbradykin in , p ro tease , p ros tag land in , andlysosome (30). Prostaglandins (PGs) play amajor role in the development of the secondphase of in f lammatory reac t ion which i smeasured at +3 h (31).

The doses 600 mg/kg and 1200 mg/kg ofa lcohol ic ext ract of K. galanga produced asignificant inhibition of carrageenan inducedpaw edema a t +3h and +6h . Therefore , i t

can be inferred that the inhibitory effect ofa lcohol ic ex t rac t s o f K. galanga oncarrageenan induced inf lammation could bedue to inhibition of the enzyme cyclooxygenaseand subsequent inhib i t ion of pros taglandinsynthesis. Significant inhibition of paw edemain the early hours of study by K. galanga couldbe at tr ibuted to the inhibi t ion of histamine(32) and/or serotonin. The decrease in pawedema inhibi t ion a t +6h may be a t t r ibutedto the terminat ion of tes t drug act ion.

Cotton pellet granuloma model was usedto evaluate the anti inflammatory activity ofK.galanga in sub acute inflammation. Threephases o f the in f lammatory response to asubcutaneously implanted cotton pellet in therats have been described: (A) a transudativephase, that occurs during the first 3 h; (B)an exudative phase, occurring between 3 and72h a f te r implan t ing the pe l le t ; (C) aproliferative phase, measured as the increasein dry weight of the granuloma that occursbetween 3 and 6 days after implantation (33).The suppression of proliferative phase of subacute inflammation could result in decreasein the weight of granuloma formation (34).The dry weight of cot ton pel le t granulomawas significantly reduced (P<0.001) by 600mg/kg and 1200 mg/kg doses o f K. galanga ,however the an t ip ro l i fe ra t ive e f fec t o fK.galanga was l esse r than tha t o f thes tandard drug . To eva lua te fo r a poss ib lecen t ra l an t inoc icep t ive e f fec t o f theK. galanga , the hot plate and tail-flick testsare used for evaluat ion of the central paina t the suprasp ina l and sp ina l l eve l s (35) ,respectively, possibly acting on a descendinginhibitory pain pathway (36). The tai l-f l ickresponse is believed to be a spinally mediatedreflex and the paw-licking hot plate responsei s more complex suprasp ina l ly o rgan ized

22 Mohanbabu e t a l Ind ian J Phys io l Pharmaco l 2011 ; 55(1 )

behaviour (37). The effectiveness of analgesicagents in the tail-flick pain model is highlycor re la ted wi th re l i e f o f human pa inperception (38).

In the two models used, though the datashowed tha t the ex t rac t o f Kaempfer iagalanga dose-dependently increased the painthreshold, the increase in the pain threshold/tail flick latency profiles of the extract wereless than that of the standard drug, codeine(Table I I I ) . The μ r ecep tor s t imula t ion i sgenerally associated with pain relief and hasbeen shown to be po ten t in regu la t ingthermal pa in (39) . Nonana lges ic e f fec t sthrough the μ receptors include respiratorydepress ion and mos t impor tan t ly fo rtherapeutic considerations is its induction ofphysical dependence. Activation of μ2 opioidsub type leads to sp ina l ana lges ia andcommonly causes cons t ipa t ion as adverseeffect (40). Therefore, taking all these datatogether we believe that the antinociceptiveac t iv i ty of e thanol ic p lan t ex t rac t i s mos tl ike ly to be media ted by cen t ra l ac t ion(spinally and supraspinally) (35) and indicatesa codeine l ike mechanism by binding wi thopioid receptors . Al though opioids possessdependence and abuse liabili t ies, new drugsproduc ing less euphor ia a t onse t andwi thdrawal symptoms as the medica t ionwear off would be more beneficial. K. galanga

could be a bet ter subst i tu te for the opioiddrugs like methadone is an excellent choiceover morphine for the management o fchron ic severe pa in l ike in cancer .Methadone i s an o ra l ly ac t ive , s low-onse topioid with a long duration of action (41).

In the ta i l - f l ick and hot plate methods,both the doses (600 & 1200 mg/kg body wt.)o f p lan t ex t rac t inc reased the s t resstolerance capacity of the animals and henceindicate the possible involvement of a highercentre (42). The t ime at tained to reach thepeak analgesic activity was same for codeine(+30min) bu t the p lan t ex t rac t showed a t(+60min) and (+30 min) in hot plate and tailf l i ck model respec t ive ly , which had noexplanation and attributed as a limitation ofour s tudy .

On the basis of these findings, it may beinferred that alcoholic extract of K. galangahas ana lges ic and an t i in f lammatoryac t iv i t ies . These ac t iv i t ies were re la ted tothe dose and these resu l t s cor robora te thepotential traditional use of the plant in folkmedic ine . At present , there a re no repor tson inves t iga t ion to iden t i fy the ac t ivecomponents present in e thanol ic ext ract ofK.galanga . Fur ther inves t iga t ions a reanticipated to identify the active componentsand lead to their further cl inical use.

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