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Food & Function
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aBrain Research Laboratory, Faculty of Phar
Puncak Alam Campus, 42300 Bandar Pun
[email protected]; Fax:bCollaborative Drug Discovery Research G
Teknologi MARA (UiTM), Campus Punca
Selangor, Malaysia
Cite this: DOI: 10.1039/c3fo30356j
Received 3rd December 2012Accepted 10th January 2013
DOI: 10.1039/c3fo30356j
www.rsc.org/foodfunction
This journal is ª The Royal Society of
Anti-inflammatory, analgesic and anti-ulcerogeniceffect of total alkaloidal extract from Murraya koenigiileaves in animal models
Vasudevan Mani,*a Kalavathy Ramasamyb and Abu Bakar Abdul Majeeda
The fresh leaves of Murraya koenigii are often added to various dishes in Asian countries due to the
delicious taste and flavour that they impart. In the present study, the effect of the total alkaloidal
extract from Murraya koenigii leaves (MKA) with respect to anti-inflammatory, analgesic and anti-
ulcerogenic effects were evaluated using different experimental animal models. Oral supplementation
of MKA at 10, 20 and 40 mg kg�1 body weight successfully and dose-dependently reduced the
formation of oedema induced by carrageenan, histamine and serotonin as well as formaldehyde-
induced arthritis. In addition, the extract (10, 20 and 40 mg kg�1, p.o.) attenuated the writhing
responses induced by an intraperitoneal injection of acetic acid and late phase of pain response induced
by a subplantar injection of formalin in mice. MKA at higher doses (20 and 40 mg kg�1, p.o) reduced
the early phase response induced by formalin as well as reaction time on hot plate models. Interestingly,
there was no ulcer score with the ulcerogenic effect of MKA. Moreover, all the doses of MKA (10, 20
and 40 mg kg�1, p.o) showed promising anti-ulcerogenic activity with protection against acute gastric
ulcers induced by ethanol plus hydrochloric acid and aspirin models in a dose dependent manner.
1 Introduction
Inammation is part of a complex biological response ofvascular tissues to harmful stimuli, such as pathogens,damaged cells or irritants. Although it is a defence mechanism,the complex events and mediators involved in the inammatoryreactions can induce, maintain or aggravate many diseases. It iswell known that numerous biological mediators, such as neu-rotrophic factors, neuropeptides, prostanoids and kinins areable to conduct and amplify the nociceptive responses.1
Currently, the treatment for inammatory diseases is depen-dent on drugs that belong either to the non-steroidal orsteroidal chemical therapeutics. The nonsteroidal anti-inam-matory drugs (NSAIDs) are aspirin related drugs that inhibitearly steps in the biosynthesis pathway of prostaglandins byinhibition of COX enzymes and are the main drugs used toreduce the inconvenient consequences of inammation.2 Thelong-term administration of NSAIDs may induce gastro-intes-tinal ulcers, bleeding and renal disorders due to their non-selective inhibition of both COX-1 and COX-2, isomers of thecyclooxygenase enzymes.3 Moreover in opiates, tolerance and
macy, Universiti Teknologi MARA (UiTM),
cak Alam, Selangor, Malaysia. E-mail:
+60-332584602; Tel: +60-332584611
roup, Faculty of Pharmacy, Universiti
k Alam, 42300 Bandar Puncak Alam,
Chemistry 2013
dependence are major side effects. Therefore, new anti-inammatory and analgesic drugs with gastroprotective activityare being researched as alternatives to NSAIDs and opiates.Attention is now focused on the investigation of the efficacy ofplant-based drugs used in traditional medicine due to theirreduced side effects. In fact, a recent report from WHO showsthat about 80% of the world population still rely mainly onherbal remedies.4
Murraya koenigii (Linn.) Spreng (Family: Rutaceae),commonly known as ‘curry leaves’, is popular as a spice andcondiment among Asians. The fresh leaves and their driedpowder are traditionally added into gravy and other vegetablesfor a distinctive avor and aroma. It is also widely used as a folkmedicine for the treatment of stomach-ache, inuenza, rheu-matism, traumatic injury, dysentery and as an astringent.5 Theleaves exhibit hepatoprotective, hypoglycemic and antidiabetic,antibacterial, antioxidant, anti-obesity and lipid loweringeffects, wound healing, chemomodulatory, immunomodula-tory, antidiarrhoeal and nephroprotective effects.6–12 Tradition-ally, leaves of Murraya koenigii are used as a stimulant,stomachic, febrifuge, analgesic, anti-inammatory and for thetreatment of diarrhoea, dysentery and insect bites.13 Whetherthese claims are valid is a subject of great interest and should beprobed scientically.
Preliminary studies of a methanol extract ofMurraya koenigiileaves have been reported to possess anti-inammatory andanalgesic effects in animal models.14,15 Murraya koenigii isknown to be the richest source of carbazole alkaloids. Several
Food Funct.
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carbazole alkaloids, namely murrayanine, mahanimbine, gir-inimbine, murrayacine, isomurrayazoline, isomahanimbine,koenimbidine mahanine, koenine, koenigine, koenidine, koe-nimbine and 8,80-biskoenigine, have been isolated from Mur-raya koenigii leaves.7 Natural and semi-synthetic carbazolealkaloids have been found useful as a new class of therapeuticagents for various diseases related with tissue damage due tothe generation of free radicals, including inammation andrheumatoid arthritis.16 The present study is therefore aiming tofurther investigate the role of the total alkaloidal extract fromMurraya koenigii leaves (MKA) with respect to its anti-inam-matory, analgesic and anti-ulcerogenic effects in variousexperimental animal models.
2 Materials and methods2.1 Plant material, extraction and isolation
The fresh leaves of Murraya koenigii were collected from thelocal market at Puncak Alam (Malaysia). The plant material wasidentied, authenticated and deposited in the herbarium at theBiodiversity and Environment Division, Forest Research Insti-tute, Malaysia (PID 241010-11). The collected leaves were driedunder shade and crushed to a moderately coarse powder. Theprocedures of MKA extraction and it's phytochemical confor-mations using HPLC and LC/MS-Q-TOF followed our previousresearch.17,18
2.2 Vehicle
Carrageenan, histamine, serotonin and formalin were dilutedseparately in normal saline and injected subcutaneously. Aceticacid andmorphine were diluted separately in normal saline andinjected intraperitoneally. MKA, indomethacin, ethanol plushydrochloric acid, ranitidine and aspirin were suspended in0.5% (w/v) carboxymethyl cellulose sodium (CMC) and admin-istered orally to animals.
2.3 Animals
All the experiments were carried out using male, SpragueDawley rats (150–200 g) and Swiss Albino mice (20–35 g)procured from the animal house at Institute of MedicalResearch, Kuala Lumpur, Malaysia. The animals had free accessto standard laboratory food and water ad libitum, and they werehoused in a natural (12 h each) light–dark cycle. The animalswere acclimatized to the laboratory conditions for at least 5 daysbefore the experiments. All experiments with live animals wereperformed in compliance with the committee on animalresearch (UiTM CARE), Universiti Teknologi MARA, Malaysia(600-FF (P.T.5/2) and the care of laboratory animals was taken asper the guidelines with the Guide for the Care and Use ofLaboratory Animals (8th Edition, National Institute of HealthPublication).
2.4 Acute anti-inammatory activity
The acute anti-inammatory activity of MKA was determined bythe carrageenan, histamine and serotonin-induced paw oedemamodels in the hind paws of rats. Male Sprague Dawley rats were
Food Funct.
fasted for 24 h before the experiment with free access to water.For each model, rats were divided in ve groups (n ¼ 6). MKA(10, 20 and 40 mg kg�1) and indomethacin (10 mg kg�1) wereadministered orally 1 h before the subplantar injection ofoedematogenic agent. The control groups of animals receivedonly vehicle (1 mL kg�1) orally. Paw volume was measured byusing a plethysmometer (model 7150, Ugo Basile, Italy). Changeof paw volume (DV) was calculated as follows:
DV ¼ Vt � V0
where Vt is the right hind paw volume (mL) at time ‘t’, V0 is hindpaw thickness (mL) before sub-plantar injection.
2.5 Carrageenan-induced oedema in rats
In this method, acute inammation was produced by the sub-plantar administration of 0.1 mL of 1% (w/v) carrageenan(Sigma Co., USA) in the right paw of the rat. The paw volume(mL) was measured immediately and at 1, 2, 3 and 4 h intervalsaer the administration of the carrageenan.19
2.6 Histamine and serotonin-induced oedema in rats
Oedema in rats was induced by injecting 0.1 mL of 0.1% (w/v)histamine or 0.2% (w/v) serotonin in the subplantar region ofthe right hind paw. The paw volume (mL) was measuredimmediately and at 1, 2, 3 and 4 h intervals aer the adminis-tration of the histamine or serotonin.20
2.7 Formaldehyde-induced arthritis in rats
Experimental arthritis was induced in male rats according tothe method described by Selye (1949).21 The animals weredivided into three groups of six rats each. A subplantar injectionof 0.1 mL of 2% (v/v) formaldehyde was administered to theright hind paw on the rst and third day of the experiment.Plant extract (40 mg kg�1) or indomethacin (10 mg kg�1) orvehicle (0.5%, w/v, CMC) was administered orally once daily for10 days. The paw volumes (mL) of each group was measured for10 days using a using a plethysmometer (model 7150, UgoBasile, Italy). On day 3 of the treatment, the paw volume (mL)was measured before the injection of formaldehyde. Oedema(DV) was calculated as follows:
DV ¼ Vd � V0
where Vd is the right hind paw thickness (mm) on day ‘d’, V0 ishind paw thickness (mm) before subplantar injection.
2.8 Acetic acid-induced writhing response in mice
Analgesic activity was evaluated on the acetic acid-inducedwrithing response in mice according to Koster et al. (1959).22
Male Albino mice were divided in to ve groups of six animalseach. The animals were treated with MKA (10, 20 and 40 mgkg�1, p.o.) or aspirin (100 mg kg�1, p.o.) used as a standarddrug, 1 h prior to intraperitoneal injection of 1% (v/v) acetic acid(0.1 mL per 10 g). Five minutes aer the intraperitoneal injec-tion of acetic acid, the number of writhings for the following
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10min was counted. Control mice received only (0.5% w/v CMC;10 mL kg�1) vehicle.
2.9 Formalin test
Male Albino mice (overnight fasted, with free access to water)were divided into ve groups (n ¼ 6). MKA (10, 20 and 40 mgkg�1) and aspirin (100 mg kg�1, standard drug) were adminis-tered orally 1 h before formalin injection. Control groupreceived only vehicle (0.5% w/v CMC; 10 mL kg�1). One hourbefore testing, the animal was placed in a standard case (30 cm� 12 cm � 13 cm) that served as an observation chamber.Twenty microliters of 1.0% (v/v) formalin was injected in thedorsal surface of the right hind paw. The mice were observed for40 min aer the injection of formalin, and the amount of timespent licking the injected hind paw was recorded. The rst 5min post formalin injection is known as the early phase and theperiod between 15 and 40 min as the late phase.23
2.10 Hot-plate reaction time in mice
Five groups of mice were selected for the study. Group onereceived vehicle (0.5% w/v CMC; 10 mL kg�1) as control andgroup two received a standard drug, morphine (5 mg kg�1, i.p.).The remaining three different groups ofmice received 10, 20 and40 mg kg�1 b.w. of MKA orally. Mice were screened by placingthem on an Eddy's hot plate (model 35100, Ugo Basile, Italy)maintained at 55 � 1 �C and recorded the reaction time inseconds for licking of hind paw or jumping at 30min, 60min, 90min and 120 min aer administration of MKA or drug.24 Micewere selected based on the reaction time within 15 s on fourseparate occasions, for which a large variation was not observed.
2.11 Ulcerogenic activity
Overnight fasted rats were divided in to ve groups. Group 1received vehicle as control, group 2 received ranitidine (50 mgkg�1, p.o) as standard, group 3–5 received MKA 10, 20 and 40mg kg�1, p.o. respectively as drug treated groups. Four hourslater, the animals were sacriced with over-dose of ether, theirstomach was removed and xed on a cork plate and ulcer scorewas measured. A score for the ulcer was made as follows:25
� 0: normal colored stomach.� 0.5: red coloration.� 1: spot ulcers.� 1.5: haemorrhagic streak.� 2: ulcers.� 3: perforation.Mean ulcer score for each animal was expressed as ulcer
index. The percentage of ulcer inhibition was determined asfollows:
Inhibition of ulcer ð%Þ ¼ Control mean ulcer index� Test mean ulcer index
Control mean ulcer index�100
2.12 Ethanol plus hydrochloric acid-induced gastric lesions
Overnight fasted rats were divided into ve groups. Group1 received vehicle as the negative control, group 2 received50 mg kg�1, p.o. ranitidine as standard control, group 3–5
This journal is ª The Royal Society of Chemistry 2013
received MKA 10, 20 and 40 mg kg�1, p.o. respectively, as drugtreated groups. The experiment was performed as described byMizui and Doteuchi (1983).26 Aer 1 h of drug administration, 1mL of hydrochloric acid 0.3 M in 60% ethanol was orallyadministered to all groups. Four hours later, the animals weresacriced with over-dose of ether, their stomach was removedand xed on a cork plate and ulcer score was measured. Theulcer score and percentage of ulcer inhibition were determinedas described in ulcerogenic activity.25
2.13 Aspirin-induced gastric lesions
Groups of rats, fasted for overnight received either MKA orranitidine or control vehicle as in the above method. Aer 1 h,aspirin suspended in 0.5% carboxymethyl cellulose in water at adose of 200 mg kg�1 was administered orally to all the animalsand 4 h later, the animals were sacriced.27 The stomach wasremoved and opened along the greater curvature to determinethe ulcer score and percentage of ulcer inhibition were deter-mined as described in ulcerogenic activity.25
2.14 Statistical analysis
All the results were expressed as Mean � Standard Error (SEM).Data was analyzed using one-way ANOVA followed by one-wayANOVA followed by Dunnett's t-test. p-values < 0.05 wereconsidered as statistically signicant.
3 Results3.1 Carrageenan-induced oedema in rats
Subplantar injection of carrageenan in rats showed a timedependent increase in paw volume (Fig. 1); this increase wasobserved at one hour and was maximal at the third hour aeradministration of carrageenan injection in the vehicle treatedgroups. TheMKAextract showed a signicant inhibitory effect onthe oedema formation from the rst to fourth hour. The highestinhibition effect was found during the third hour, where theinhibition was found to be 22.38% (p < 0.05), 41.79% (p < 0.001)and 43.28% (p < 0.001) at the doses of 10, 20 and 40 mg kg�1,respectively. The standard drug indomethacin (10 mg kg�1, p.o.)highlighted the maximum inhibition on third hour aer carra-geenan injection with a percentage inhibition of 67% (p < 0.001).
3.2 Histamine-induced oedema in rats
BothMKA (10, 20 and 40mg kg�1, p.o) and indomethacin (10mgkg�1, p.o) signicantly decreased the histamine-induced pawoedema at all times, from one to four hours (Fig. 2). The pawoedema was most pronounced at one hour aer the subplantarinjection of histamine. The peak inhibitory effects observed atone hour were 22.58% (p < 0.05), 53.23% (p < 0.001), 59.67(p < 0.001) and 67.74 (p < 0.001) with 10, 20 and 40 mg kg�1 ofMKA as well as indomethacin (10 mg kg�1, p.o.), respectively.
3.3 Serotonin-induced oedema in rats
The maximal inhibitory effect was observed one hour aer sub-plantar injection of serotonin in vehicle treated groups. However,
Food Funct.
Fig. 1 The effects of MKA and indomethacin (INDO) on rat's hind paw oedema induced by carrageenan (CARR). Data represented as mean� S.E.M, (n¼ 6). * p < 0.05,** p < 0.01, *** p < 0.001 as compared with the CARR group (one-way ANOVA followed by Dunnett's t-test).
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the animals treated with MKA exhibited a dose-dependentsignicant reduction in paw oedema from the rst hour to theend of the experiment (Fig. 3). The highest percentage ofinhibitionwas recorded as 21.31% (p < 0.05), 54.09 (p < 0.001) and
Fig. 2 The effects of MKA and indomethacin (INDO) on rat's hind paw oedema ind** p < 0.01, *** p < 0.001 as compared with the HIST group (one-way ANOVA follo
Food Funct.
62.29 (p < 0.001) at one hour with 10, 20 and 40 mg kg�1 ofMKA, respectively. Furthermore, 68.85% (p < 0.001) was noted asthe maximum inhibitory effect with indomethacin in the rsthour.
uced by histamine (HIST). Data represented as mean � S.E.M, (n ¼ 6). * p < 0.05,wed by Dunnett's t-test).
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3.4 Formaldehyde-induced arthritis in rats
The anti-arthritic activity was evaluated using formaldehydeinduced arthritis model in rats. Continuous oral treatment (10days) with MKA (40 mg kg�1) and indomethacin (10 mg kg�1)remarkably reduced the paw oedema induced by formaldehydein rats. The signicant differences (p < 0.01) in paw volume wasobserved from the rst day and throughout the experimentalperiod as compared with vehicle treated group (Fig. 4). Aer asecond injection with formaldehyde, the maximum percentageinhibition was noted on the sixth day as 33.33% (p < 0.001) and41.67% (p < 0.001) with MKA (40 mg kg�1, p.o.) and indo-methacin (10 mg kg�1, p.o.), respectively.
3.5 Acetic acid-induced writhing response in mice
The pain behavior of writhing response as presented bycumulative abdominal stretching response is shown in Fig. 5.The treatment of animals with all does of MKA (10, 20 and 40mg kg�1, p.o.) produced a signicant (p < 0.001) and dose-dependent inhibition in abdominal writhes produced by aceticacid. The inhibition by MKA (40 mg kg�1, p.o.) was nearlysimilar to that produced by aspirin (100 mg kg�1, p.o.), thestandard drug used in the study.
3.6 Formalin test
The effect of MKA and aspirin on the early phase and late phaselicking time against 1% v/v formalin is shown in Fig. 6. Themice treated with aspirin or higher doses of MKA (20 and 40 mgkg�1, p.o.) produced signicant (p < 0.01) changes in pawlicking time in the early phase of pain response. On other hand,in the late phase, a dose-dependent (p < 0.001) reduction in
Fig. 3 The effects of MKA and indomethacin (INDO) on rat's hind paw oedema indu*** p < 0.001 as compared with the SERO group (one-way ANOVA followed by Du
This journal is ª The Royal Society of Chemistry 2013
licking time was observed with all doses of MKA (10, 20 and 40mg kg�1, p.o.) as well as with aspirin (100 mg kg�1, p.o.).
3.7 Hot-plate reaction time in mice
The higher doses (20 and 40 mg kg�1, p.o.) of MKA produced asignicant (p < 0.05) analgesic activity from 30 to 120 min aertreatment as compared with control (Fig. 7). Supplementation ofMKA considerably increased the animals' reaction time to heatstimulus. The highest signicant improvement in reaction time(p < 0.001) was recorded at a dose of 40 mg kg�1 at 60 mincompared to the respective control group. The lowest dose (10mg kg�1, p.o.) did not produce any signicant changesthroughout the experiment. The standard drug morphine (5 mgkg�1, i.p.) showed a signicant (p < 0.001) improvement inreaction time throughout the experimental period, as expected.
3.8 Ulcerogenic activity
The ulcer scores were not found in rats treated with a singledose of MKA (10, 20 and 40 mg kg�1, p.o.) as well as ranitidine(50 mg kg�1, p.o).
3.9 Ethanol plus hydrochloric acid-induced gastric lesions
Oral administration of 1 mL of hydrochloric acid 0.3 M in 60%ethanol produced severe haemorrhagic lesions in glandularmucosa consisting of elongated bands, parallel to the long axisof the stomach. The control rats had an ulcer score of 2.83 �0.17. In animals pre-treated with MKA at doses of 10, 20 and 40mg kg�1, a signicant protection (p < 0.05) on induced mucosalinjury was recorded, showing the percentage of ulcer inhibitionas 29.33%, 44.17% and 53%, respectively (Fig. 8). The highestdose 40 mg kg�1, p.o. showed maximum ulcer inhibition. The
ced by serotonin (SERO). Data represented as mean� S.E.M, (n¼ 6). * p < 0.05 andnnett's t-test).
Food Funct.
Fig. 4 The effects of MKA and indomethacin (INDO) on rat's hind paw oedema induced by formaldehyde (FORL). Data represented as mean � S.E.M, (n ¼ 6). ** p <0.01 and *** p < 0.001 as compared with the FORL group (one-way ANOVA followed by Dunnett's t-test).
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standard drug ranitidine 50 mg kg�1, p.o. showed a percentageof ulcer inhibition as 70.67%.
3.10 Aspirin-induced gastric lesions
Oral administration of aspirin (200 mg kg�1) produced lesionslinearly on mucosal folds that had the appearance of mucosal
Fig. 5 The effects of MKA and aspirin on the acetic acid-induced writhing test in micontrol group (one-way ANOVA followed by Dunnett's t-test).
Food Funct.
erosions. In control rats an ulcer score of 2.67 � 0.21 wasobserved, while three doses of MKA (10, 20 and 40mg kg�1, p.o.)recorded the percentage of ulcer inhibition as 28.09%, 50.19%and 59.55%, respectively (Fig. 9). This results indicated asignicant (p < 0.05) and dose dependent improvement inprotection of gastric ulcer with MKA. Ranitidine showed thehighest level of gastric protection.
ce. Data represented as mean� S.E.M, (n¼ 6). *** p < 0.001 as compared with the
This journal is ª The Royal Society of Chemistry 2013
Fig. 6 The effects of MKA on the early phase and late phase in the formalin test in mice. Data represented as mean � S.E.M, (n ¼ 6). ** p < 0.01, *** p < 0.001 ascompared with the control group (one-way ANOVA followed by Dunnett's t-test).
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4 Discussion
There are numerous traditional claims of the usefulness ofMurraya koenigii leaves in inammation and pain but scienticauthentication of these medical properties is important.13
Moreover, the commonly used NSAIDs like piroxicam,ibuprofen and indomethacin are known to induce gastric
Fig. 7 The effects of MKA andmorphine on the hot plate test in mice. Data represenwith the control group (one-way ANOVA followed by Dunnett's t-test).
This journal is ª The Royal Society of Chemistry 2013
ulceration by non-selective inhibition of cyclooxygenase (COX) Iand II. COX-II promotes reduced levels of prostaglandins,whereas COX-I is involved in mucus and bicarbonate synthesisas well as blood ow regulation in gastric mucosa.28 Discovery ofsafe anti-inammatory drugs that are free from gastric intoler-ance is therefore a vital part of research in the treatment ofinammatory disorders. In the present study, the anti-
ted as mean� S.E.M, (n¼ 6). * p < 0.05, ** p < 0.01 and *** p < 0.001 as compared
Food Funct.
Fig. 8 The effects of MKA and ranitidine on ethanol plus hydrochloric acid-induced gastric lesions. Data represented as mean � S.E.M, (n ¼ 6). * p < 0.05, ** p < 0.01,*** p < 0.001 as compared with the HCl–Et group (one-way ANOVA followed by Dunnett's t-test).
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inammatory activities of the total alkaloidal extract fromMurraya koenigii leaves (MKA) has been established in bothacute and chronic inammation models. The analgesic andanti-ulcer activities in various models were also determined.
Fig. 9 The effects of MKA and ranitidine on aspirin-induced gastric lesions. Data repaspirin group (one-way ANOVA followed by Dunnett's t-test).
Food Funct.
Alkaloids are a group of naturally occurring chemicalcompounds that mostly contain basic nitrogen atoms. In thepresent study, the total alkaloid extract of Murraya koenigiileaves (MKA) showed promising anti-inammatory, analgesic
resented as mean � S.E.M, (n ¼ 6). * p < 0.05, *** p < 0.001 as compared with the
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and anti-ulcer activities in various models. Several earlierstudies have also supported the role of alkaloids in the treat-ment of inammation, pain and gastric ulcers.29–33 In fact,natural opium alkaloids and their semi-synthetic derivatives arewell established analgesic drugs, which act through the specicopioid receptors. Interestingly, a new antinociceptive alkaloidisopropyl N-methylanthranilate isolated from the essential oilof Choisya ternata, and the alkaloid extract of Galipea longioraalso exhibited antinociceptive activity.34,35 Furthermore,evidence from a review on alkaloids with antiulcer activitydemonstrated about y-ve of sixty-one alkaloids, to showgastroprotective and antiulcer activities in disease inducedanimals.36
Carrageenan-induced rat paw oedema is suitable for evalu-ating anti-inammatory drugs, which is frequently used toassess the anti-oedematous effect of natural products. Devel-opment of oedema in the paw of the rat aer injection ofcarrageenan is a biphasic event.37 The release of histamine andserotonin is related to the initial phase and is oen observedduring the rst hour. The second phase of oedema is associatedwith the release of prostaglandins, bradykinin, protease andlysosome. Based on this, it could be argued that the suppressionof the rst phase may be due to inhibition of the release of earlymediators, such as histamine and serotonin, and the action inthe second phase may be explained by an inhibition of cyclo-oxygenase. Therefore, the inhibition of carrageenan-inducedinammation by MKA could be due to the inhibition of theenzyme cyclooxygenase and subsequent inhibition of prosta-glandin synthesis.
The MKA also caused signicant reduction in the oedemainduced by histamine and serotonin. The anti-histaminic andanti-serotonergic effect of the MKA increased with increase indose of the extract, hence the effect is dose-dependent. Hista-mine and serotonin are important inammation mediators andthey are potent vasodilator substances as well as increase thevascular permeability.38 The MKA exhibited its anti-inamma-tory action by means of either inhibiting the synthesis, releaseor action of inammatory mediators viz., histamine, serotoninand prostaglandin that might be involved in inammation.Moreover, in formaldehyde induced arthritis, MKA in thehighest tested dose (40 mg kg�1, p.o.), demonstrated goodantiarthritic efficacy and was comparable to indomethacin(10 mg kg�1, p.o.) in reducing the joint swelling throughout theobserved period. Formaldehyde-induced pedal oedema iswidely used for pharmacological evaluation of anti-arthriticagents as it has been shown to share a number of clinical andimmunological features with human arthritis, and thereforehas a relatively high degree of validity.39
Pain is a complex process mediated by many physiologicalmediators, like prostaglandins, bradykinins, substance-P etc. Inthe present study, MKA is investigated for analgesic activityusing the acetic acid-induced writhing, formalin, and hotplatetests. The intraperitoneal injection of acetic acid elicitedwrithing, is a syndrome characterized by a wave of abdominalmusculature contraction followed by extension of the hindlimbs. The generation of writhing by chemical substancesinjected by intraperitoneal results from the acute inammatory
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reaction with production of PGE2 and PGE2a in the peritonealuid and the test is useful for evaluation of mild analgesic non-steroidal anti-inammatory drugs.40 The dose-dependent inhi-bition of Murraya koenigii against writhing induced by aceticacid suggests a peripherally mediated analgesic activity basedon the association of the model with stimulation of peripheralreceptors, especially the local peritoneal receptors at the surfaceof cells lining the peritoneal cavity.41 Furthermore, the resultsobtained from the writhing test using acetic acid are similar tothat obtained for the oedematogenic test using carrageenan. Ananti-inammatory substance may also be involved in theperipheral analgesic activity of MKA.
All the doses of MKA (10, 20 and 40 mg kg�1, p.o.) that wereemployed also produced signicant inhibition in both phasesof formalin-induced pain. The formalin test is a valid and reli-able model of nociception and is sensitive for various classes ofanalgesic drugs. It has been reported to produce a distinctbiphasic nociceptive response.41 The early phase (0–5min) hasbeen associated with direct stimulation of the sensory nervebers by formalin and characterized by the direct stimulation ofnociceptors present on afferent C, and in part, Ad bers(glutamate and substance P release). The second phase (15–30min) is related to the release of pro-inammatory mediators,such as adenosine, bradykinin, histamine, prostaglandin andserotonin.42 According to Chan et al. (1995),43 centrally actingdrugs such as opioids (e.g. morphine) inhibited both phasesequally while peripherally acting drugs, such as aspirin, indo-methacin and dexamethasone, only inhibit the late phase. Thelate phase seems to be an inammatory response with inam-matory pain that can be inhibited by anti-inammatory drugs.19
From the results, MKA inhibited both phases and conrmed aperipheral mechanism of action while also suggesting theinvolvement of a central mechanism of an analgesic effect. Theeffect of MKA being greater in the second phase than thatproduced in the rst phase may suggest greater involvement ofperipheral mechanisms in its anti-nociceptive action. Theformalin test is conducted to conrm the involvement of centralmechanism(s) in the analgesic activity of MKA, the hot-platereaction time was performed based on the fact that centrallyacting analgesic drugs elevate the pain threshold of rodentstowards heat.44 The hot plate test involves the spinal reex andmeasures the complex response to a non-inammatory, acutenociceptive input.41 Based on the effectiveness of MKA in thehot plate tests, a central mechanism of action is conrmed forits observed anti-nociceptive effect.
The main side effect of non-steroidal anti-inammatorydrugs is their ability to produce gastric lesions. So, those drugscannot be used long term in the treatment of chronic inam-matory conditions like rheumatoid arthritis with peptic ulcers.During the acute ulcerogenic studies, MKA did not induce anyadverse effect on gastric mucosa, indicating non ulcerogenicactivity. Interestingly, MKA was found to signicantly reduceulcer index in a dose dependent manner against ulcers inducedby aspirin, an irreversible non-selective COX inhibitor thatdecreases prostaglandin synthesis.45 The same doses of MKAestablished similar results in the EtOH 60% : HCl 0.3 Minduced acute ulcer model test, which indicates that the plant
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extract could enhance the cytoprotective mechanism of thegastric mucosa.46 From the acute and chronic studies, it isobvious that MKA possesses good anti-inammatory and anal-gesic activity, interestingly without any ulcerogenic activity.
5 Conclusion
In conclusion, the present study clearly indicates that totalalkaloidal extract from Murraya koenigii leaves possesses goodanti-inammatory, analgesic and anti-ulcerogenic activity andalso scientically validates the traditional use of this plant intreating inammatory disorders in the folk medicine. MKAdemonstrates a safe anti-inammatory prole without ulcero-genic activity. The active compounds from MKA will requirefurther investigation to identify the possible mechanism ofaction as well as establish the therapeutic value in the treatmentof inammatory diseases.
References
1 R. Sobota, M. Szwed, A. Kasza, M. Bugno and T. Kordula,Biochem. Biophys. Res. Commun., 2000, 267, 329–333.
2 D. Albert, I. Zundorf, T. Dingermann, W. E. Muller,D. Steinhilber and O. Werz, Biochem. Pharmacol., 2002, 64,1767–1775.
3 H. Tapiero, G. N. Ba, P. Couvreur and K. D. Tew, Biomed.Pharmacother., 2002, 56, 215–222.
4 A. A. Adedapo, M. O. Sodiys, V. Maphosa, P. Moyo,P. J. Masika and A. J. Afolayan, Rec. Nat. Prod., 2008, 2, 46–53.
5 N. D. Prajapati, S. S. Purohit, A. K. Sharma and T. Kumar, AHand book of Medicinal Plants, Agrobios, Jodhpur, 2003.
6 S. Sathaye, Y. Bagul, S. Gupta, H. Kaur and R. Redkar, Exp.Toxicol. Pathol., 2011, 63, 587–591.
7 D. Iyer and P. U. Devi, Pharmacogn. Rev., 2008, 2, 180–184.8 S. Gupta, M. George, M. Singhal and V. Garg,Pharmacologyonline, 2009, 3, 915–923.
9 T. Dasgupta, A. R. Rao and P. K. Yadava, Nutr. Res., 2003, 23,1427–1446.
10 A. S. Shah, A. S. Wakade and A. R. Juvekar, Indian J. Exp. Biol.,2008, 46, 505–509.
11 S. Mandal, A. Nayak, M. Kar, S. K. Banerjee, A. Das,S. N. Upadhyay, R. K. Singh, A. Banerji and J. Banerji,Fitoterapia, 2010, 81, 72–74.
12 H. Yankuzoa, Q. U. Ahmed, R. I. Santosa, S. F. U. Aktera andN. A. Taliba, J. Ethnopharmacol., 2011, 135, 88–94.
13 A. C. Adebajo, O. F. Ayoola, E. O. Iwalewa, A. A. Akindahunsi,N. O. Omisore, C. O. Adewunmi and T. K. Adenowo,Phytomedicine, 2006, 13, 246–254.
14 S. Gupta, M. George, M. Singhal, G. N. Sharma and V. Garg, J.Adv. Pharm. Technol. Res., 2010, 1, 68–77.
15 N. G. Manoj, R. J. Archana and S. S. Sachin,Pharmacologyonline, 2009, 1, 1072–1094.
16 G. A. Cordell, The Alkaloids: Chemistry and Biology, AcademicPress, UK, 2009.
17 V. Mani, K. Ramasamy, A. Ahmad, M. Parle, S. A. Shah andA. B. Majeed, Food Chem. Toxicol., 2012, 50, 1036–1044.
Food Funct.
18 V. Mani, K. Ramasamy, A. Ahmad, S. N. Wahab, S. M. Jaafar,L. K. Teh, M. Z. Salleh and B. A. M. Abu, Phytother. Res., 2013,27, 46–53.
19 H. Young, Y. Luo, H. Cheng, W. Hsieh, J. Liao andW. Peng, J.Ethnopharmacol., 2005, 96, 207–210.
20 M. Gupta, U. K. Mazumder, R. S. Kumar and T. S. Kumar,Iran. J. Pharmacol. Ther., 2003, 2, 30–34.
21 H. Selye, Br. Med. J., 1949, 2, 1129–1135.22 R. Koster, M. Anderson and E. J. De-Beer, Fed. Proc., 1959, 18,
418–420.23 M. Vasudevan, K. K. Gunnam and M. Parle, J.
Ethnopharmacol., 2007, 109, 264–270.24 R. A. Turner, Screening methods in pharmacology, Academic
press, London, 1965.25 S. K. Kulkarni, Handbook of Experimental pharmacology,
Vallabh Prakashan, New Delhi, 1999.26 T. Mizui and M. Doteuchi, Jpn. J. Pharmacol., 1983, 33, 939–
945.27 N. S. Parmar and J. K. Desai, Indian J. Pharmacol., 1993, 25,
120–135.28 F. Halter, A. S. Tarnawski, A. Schmassmann and
B. M. Peskar, Gut, 2001, 49, 443–453.29 A. Pandurangan, R. L. Khosa and S. Hemalatha, Nat. Prod.
Res., 2011, 11, 1–10.30 W. H. Jiao, H. Gao, F. Zhao, H. W. Lin, Y. M. Pan, G. X. Zhou
and X. S. Yao, Chem. Pharm. Bull., 2011, 59, 359–364.
31 Y. M. Chung, Y. H. Lan, T. L. Hwang and Y. L. Leu,Molecules,2011, 16, 1917–1927.
32 J. Lu, J. S. Wang and L. Y. Kong, J. Ethnopharmacol., 2011,134, 911–918.
33 X. F. Niu, P. Zhou, W. F. Li and H. B. Xu, Fitoterapia, 2011, 82,620–625.
34 N. S. Radulovic, A. B. Miltojevic, M. McDermott,S. Waldren, J. A. Parnell, M. M. Pinheiro, P. D. Fernandesand F. de SousaMenezes, J. Ethnopharmacol., 2011, 135, 610–619.
35 F. Campos-Buzzi, M. Fracasso, B. K. Clasen, J. C. Ticona,A. Gimenez and V. Cechinel-Filho, Methods Find. Exp. Clin.Pharmacol., 2010, 32, 707–711.
36 F. H. de Sousa, J. A. Leite, J. M. Barbosa-Filho, P. F. deAthayde-Filho, C. M. C. de Oliveira, M. D. Moura,A. L. Ferreira, A. B. de Almeida, A. R. Souza-Brito,F. M. D. M. de Fatima and L. M. Batista, Molecules, 2008,13, 3198–3223.
37 A. Panthong, D. Kanjanapothi, T. Taesotikul, T. Wongcomeand V. Reutrakul, J. Ethnopharmacol., 2003, 85, 151–156.
38 A. Linardi, S. K. P. Costa, G. R. DeSilva and E. Antunes, Eur. J.Pharmacol., 2002, 399, 235–242.
39 V. Nair, S. Singh and Y. K. Gupta, J. Ethnopharmacol., 2011,133, 303–307.
40 S. Vyas, R. P. Agrawal, P. Solanki and P. Trivedi, Acta PoloniaePharmaceutica. Drug. Res., 2008, 65, 473–476.
41 Z. A. Zakaria, Z. D. Ghani, R. N. Nor, H. K. Gopalan,M. R. Sulaiman, A. M. Jais, M. N. Somchit, A. A. Kader andJ. Ripin, J. Nat. Med., 2008, 62, 179–187.
This journal is ª The Royal Society of Chemistry 2013
Paper Food & Function
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ded
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al o
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tp://
pubs
.rsc
.org
| do
i:10.
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42 D.A. dos Santos, J. FukuiMde,N. P.D.Nanayakkara, S. I. Khan,J. P. Sousa, J. K. Bastos, S. F. Andrade, A. A. D. S. Filho andN. L. Quintao, J. Ethnopharmacol., 2010, 127, 543–550.
43 T. F. Chan, H. Y. Tsai and W. Tian-Shang, Planta Med., 1995,61, 2–8.
This journal is ª The Royal Society of Chemistry 2013
44 S. Singh and D. K. Majumdar, Int. J. Pharmacogn., 1995, 33,188–192.
45 K. D. Rainsford, Agents Actions, 1987, 21, 316–319.46 X. B. Sun, T. Matsumoto and H. Yamada, J. Pharm.
Pharmacol., 1991, 43, 699–704.
Food Funct.
