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O R I G I N A L P A P E R

Evaluation of the analgesic and anti-inflammatory activitiesof Curcuma mangga Val and Zijp rhizomes

Peerati Ruangsang Supinya Tewtrakul Wantana Reanmongkol

Received: 13 May 2009 / Accepted: 3 September 2009 / Published online: 15 October 2009

The Japanese Society of Pharmacognosy and Springer 2009

Abstract The effects of Curcuma mangga ethanolic

extract (CME) and its fractions, e.g., aqueous, chloroform,ethyl acetate, and hexane fractions, from C. mangga rhi-

zome were investigated on nociceptive responses using

writhing, hot plate, and formalin tests in mice and

inflammatory models using carrageenan-induced rat paw

edema and croton oil-induced mouse ear edema. The

results showed that CME and all fractions (200 mg/kg,

p.o.) significantly reduced the number of writhings. Oral

administration (p.o.) of CME, chloroform, and hexane

fractions (200 mg/kg) significantly prolonged the latency

time, whereas aqueous and ethyl acetate fractions were

inactive. The activities of CME, chloroform, and hexane

fractions were abolished by naloxone (2 mg/kg, intra-

peritoneal (i.p.)). CME and all fractions at the dose of

200 mg/kg significantly produced antinociception in both

early and late phases of the formalin test. CME, chloro-

form, and hexane fractions were more prominent in licking

inhibition than those of the aqueous and ethyl acetate

fractions. CME and all fractions (150 mg/kg, p.o.) showed

significant reduction of rat paw edema. The order of

activity on inhibition of paw edema at 4 h was chloroform

fraction[ hexane fraction[ ethyl acetate fraction[

CME[ aqueous fraction. When topically applied at

0.5 mg/ear, CME and all fractions suppressed ear edemainduced by croton oil. CME and chloroform fraction

showed a greater inhibition by 53.97 and 50.29%,

respectively. These results suggested that CME and its

fractions, especially chloroform and hexane fractions from

C. mangga rhizome, possessed centrally acting analgesic

as well as anti-inflammatory activities.

Keywords Curcuma mangga Zingiberaceae

Pain Inflammation

Introduction

Curcuma mangga (C. mangga) Val and Zijp is a member

of the family Zingiberaceae. It is also known by its com-

mon Thai name Khamin Khao or its generic name mango

tumeric or mango ginger because of its mango-like

smell when fresh rhizomes are cut. C. mangga is distrib-

uted in Bengal, North Eastern India, Malay Peninsular, and

Thailand. Generally, the rhizome of C. mangga is used as a

popular vegetable or food decoration in Thai cuisine [1, 2].

The rhizome is also used as traditional medicine for

relieving stomachic complaints, gastric ulcer, chest pain,

fever, and general debility. It is also used in postpartum

care, specifically to aid womb healing [3]. Some pharma-

cological activities of C. mangga rhizome have been

reported, such as antioxidant, anticancer, antibacterial, and

antiallergic properties [36], but no scientific pharmaco-

logical studies on antinociceptive and anti-inflammatory

activities of this plant have been reported. Thus this study

aimed to evaluate the antinociceptive and anti-inflamma-

tory activities of C. mangga rhizomes in experimental

animals by using various algesic and inflammatory models.

P. Ruangsang W. Reanmongkol (&)

Department of Clinical Pharmacy,

Faculty of Pharmaceutical Sciences,

Prince of Songkla University,

Hat Yai, Songkhla 90110, Thailand

e-mail: [email protected]

S. Tewtrakul

Department of Pharmacognosy and Pharmaceutical Botany,

Faculty of Pharmaceutical Sciences,

Prince of Songkla University,

Hat Yai, Songkhla 90110, Thailand

123

J Nat Med (2010) 64:3641

DOI 10.1007/s11418-009-0365-1


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Materials and methods

Plant material

The fresh rhizomes of C. mangga were collected from

Pattani Province, Thailand, in June 2007. The plant mate-

rial was identified by Prof. Puangpen Sirirugsa, Department

of Biology, Prince of Songkla University, Thailand. Avoucher specimen (No. SKP 206 03 13 01) was deposited

at the Faculty of Pharmaceutical Sciences, Prince of

Songkla University, Thailand.

Preparation and phytochemical screening

of C. mangga rhizome

Powder ofC. mangga rhizomes (710 g) was extracted with

ethanol (10 l, 2 times) for 7 days, filtrated, and evaporated

to the give ethanol extract (16.75 g). The ethanol extract

was dissolved in hexane and partitioned with methanol.

Then the methanol fraction was dissolved in water andpartitioned with chloroform. After that, the water fraction

was partitioned with ethyl acetate. Each fractionation step

gave aqueous (1.35 g, 0.19% w/w), chloroform (2.84 g,

0.40% w/w), ethyl acetate (0.05 g, 7.04 9 10-3% w/w),

and hexane fractions (11.92 g, 1.68% w/w). Phytochemical

screening of C. mangga rhizome was carried out according

to the standard tests for classification of compounds as

follows [7]: For phenolic compounds, the crude extract

solution was mixed with 12 drops of ferric chloride

(FeCl3) solution. The observation of a dark blue precipitate

indicated the presence of phenolic compounds. For terp-

enes, the hexane and chloroform fraction solutions were

developed on thin-layer chromatography (TLC) and then

sprayed with reagent comprising anisaldehyde in sulfuric

acid. Spots of reddish-violet color that developed on the

TLC after heating at 105C indicated the presence of

terpenes. For alkaloids, the crude extract solution was

mixed with 12 drops of Dragendorfs reagent and Mayers

reagent. If a reddish-brown and white precipitate could not

be observed, respectively, this indicated the absence of

alkaloids. C. mangga ethanolic extract (CME) and its

fractions, e.g., aqueous, chloroform, ethyl acetate, and

hexane fractions, were used as the test extracts. All doses

of extract and fractions were expressed in terms of mg/kg

body weight unless otherwise specified.

Animals

Male and female Swiss albino mice (2535 g) and male

Wistar rats (150230 g) were obtained from the Southern

Laboratory Animal Facility, Prince of Songkla University,

Thailand. The animals were housed in standard environ-

mental conditions. All experimental protocols were

approved by the Animal Ethics Committee, Prince of

Songkla University (MOE 0521.11/371).

Antinociceptive activities

Writhing test

Writhing was induced in mice (n = 10) by intraperitoneal(i.p.) injection (10 ml/kg) of 0.6% acetic acid. The number

of writhings was counted over a 20-min period as previ-

ously described [8]. CME (200 mg/kg), its fractions

(200 mg/kg), or indomethacin as reference drug (10 mg/kg)

was administered orally (p.o.) to mice 30 min before

injection of acetic acid. The control group received

cosolvent (10 ml/kg) of water, propylene glycol, and

Tween.

Hot plate test

Mice were placed on a hot plate (Harvard Apparatus Ltd.,UK) maintained at 55 1C for a maximum nociceptive

response of 45 s [9]. The latency of nociceptive responses

was recorded when the animals licked their hind limb or

jumped (n = 10). The reaction time was measured every

15 min in a 60-min period at intervals of 0, 30, 45, and

60 min after oral administration of cosolvent (water/pro-

pylene glycol/Tween, 10 ml/kg), CME (200 mg/kg), or its

fractions (200 mg/kg) at 30 min, or at only 15 min in the

case of morphine (5 mg/kg, subcutaneous (s.c.)). When

administration of naloxone was required, the animals

received naloxone (2 mg/kg, i.p.) 10 min before morphine

(5 mg/kg, s.c.) or the test agents.

Formalin test

The method was used according as previously described

[10]. Either cosolvent (water/propylene glycol/Tween,

10 ml/kg), CME (200 mg/kg), or its fractions (200 mg/kg)

were orally administered 30 min before injection of 2.5%

formalin (20 ll) into a hind paw of each mouse (n = 10),

or 15 min before in the case of morphine (5 mg/kg, s.c.).

The time spent licking the injected paw was recorded and

expressed as the total licking time in the early phase (0

5 min) and late phase (1530 min) after formalin injection.

Anti-inflammatory activities

Carrageenan-induced rat paw edema

Either cosolvent (10 ml/kg), indomethacin (10 mg/kg),

CME (200 mg/kg), or its fractions (200 mg/kg) were orally

administered 30 min before injection of 1% carrageenan

into the subplantar area of the rats right hind paw

J Nat Med (2010) 64:3641 37

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(n = 10). Paw volume was measured at 0, 1, 2, 3, and 4 h

after carrageenan injection by using a plethysmometer

(Ugo Basile, Milan, Italy) as previously described [11].

Croton oil-induced mouse ear edema

The method described by Tubaro et al. [12] was used.

Cutaneous inflammation was induced by application of 5%

croton oil (10 ll) in acetone to the inner surface of the

mouse right ear. The left ear received an equal volume

of vehicle. Test samples (0.5 mg/ear), indomethacin

(0.5 mg/ear), or vehicle was applied topically to the right

ear 1 h before croton oil application. Four hours after the

application of croton oil, the mice were sacrificed and a

plug (7-mm diameter) was removed from both the treated

and untreated ears (n = 10). The oedematous response was

measured as the weight difference between the two plugs.

Chemicals

The following drugs were used: morphine sulfate, carra-

geenin lambda, and croton oil (Sigma Chem. Co., St.

Louis, USA); Tween 80 (Srichand United Dispensary Co.,

Ltd., Bangkok, Thailand); indomethacin (Fluka Bio-

Chemika, Japan); sodium chloride (Carlo Erba, Germany);

acetic acid (J.T., Baker Inc., Phillipsburg, USA); propylene

glycol and acetone (Vidhyasom Co., Ltd., Bangkok,

Thailand); ethanol (Merck KGaA, Germany); ether,

methanol, hexane, chloroform, ethyl acetate (Labscan Asia

Co., Ltd., Bangkok, Thailand).

Statistical analysis

The results were statistically analyzed using one-way

ANOVA followed by Bonferronis test. A difference was

considered significant at P\ 0.05.

Results

Effects of C. mangga rhizome on nociceptive

responses in mice

Writhing test

CME as well as all fractions (200 mg/kg, p.o.) significantly

reduced the number of writhings compared with the control

group. Amongst CME and all fractions, the chloroform

and hexane fractions showed the greatest effects with 72.8

and 63.6% inhibition, respectively. Indomethacin, a refer-

ence drug (10 mg/kg, p.o.), also inhibited the writhings by

81.3% (Table 1).

Hot plate test

As shown in Table 2, oral administration of CME, chlo-roform, and hexane fractions (200 mg/kg) significantly

prolonged the latency time when compared with the control

group, whereas aqueous and ethyl acetate fractions were

inactive. Morphine (5 mg/kg, s.c.), used as a positive

control, also prolonged the response time. The activities

of CME, chloroform, and hexane fractions including

morphine were antagonized by naloxone (2 mg/kg, i.p.).

Formalin test

Oral administration of CME and all fractions at the dose of

200 mg/kg significantly produced antinociception in both

early and late phases of the formalin test. CME, chloro-

form, and hexane fractions were more prominent in licking

inhibition than those of the aqueous and ethyl acetate

fractions. Morphine also demonstrated marked inhibition in

both phases (Table 3).

Effects of C. mangga rhizome on inflammation

Carrageenin-induced paw edema in rats

Similar to the reference drug indomethacin (10 mg/kg, p.o.),

CME and all fractions (150 mg/kg, p.o.) showed significant

reduction of rat paw edema compared with the control group.

The order of activity on inhibition of paw edema at 4 h was

chloroform fraction[hexane fraction[ ethyl acetate frac-

tion[CME[ aqueous fraction, respectively (Table 4).

Croton oil-induced ear edema in mice

When topically applied at 0.5 mg/ear, CME and all frac-

tions suppressed ear edema induced by croton oil. CME

Table 1 Effects of CME and its fractions from C. mangga rhizome

and indomethacin on the acetic acid-induced writhing response in

mice

Treatment Dose

(mg/kg, p.o.)

Number of

writhings

(counts/20 min)

Inhibition

(%)

Control 53.0 2.5

Indomethacin 10 9.9 2.2* 81.3

CME 200 27.7 1.7* 47.7

Aqueous fraction 200 38.1 1.1* 28.1

Chloroform fraction 200 14.4 1.9* 72.8

Ethyl acetate fraction 200 41.1 1.6* 22.5

Hexane fraction 200 19.3 1.8* 63.6

Each value is presented as the mean S.E.M. (n = 10)

* P\ 0.01 compared with the control group (Bonferronis test)

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Table 2 Effects of CME and its fractions from C. mangga rhizome, morphine, and naloxone on the heat-induced nociceptive response in mice

Treatment Dose (mg/kg, p.o.) Reaction time (s)

0 min 30 min 45 min 60 min

Control 8.31 0.29 7.88 0.37 8.15 0.40 8.20 0.40

Morphine 5 (s.c.) 8.26 0.28 14.17 0.37** 13.96 0.52** 13.23 0.39**

CME 200 8.05 0.25 9.78 0.41 10.53 0.41 11.99 0.64**

Aqueous fraction 200 8.33 0.29 9.60 0.46 9.64 0.50 10.06 0.49

Chloroform fraction 200 8.51 0.26 9.97 0.57* 10.93 0.55** 12.04 0.41**

Ethyl acetate fraction 200 8.56 0.45 8.43 0.44 9.93 0.67 10.11 0.67

Hexane fraction 200 8.52 0.41 10.29 0.39* 10.92 0.69** 11.51 0.63**

Naloxone 2 (i.p.) 8.46 0.33 7.78 0.35 9.03 0.35 8.44 0.52

Naloxone ? morphine 2 ? 5 8.23 0.36 8.13 0.23a 8.66 0.32

a 8.45 0.29a

Naloxone ? CME 2 ? 200 8.43 0.39 8.12 0.21b 8.71 0.37b 8.50 0.31b

Naloxone ? chloroform 2 ? 200 8.53 0.36 8.31 0.41c

9.32 0.35c

8.96 0.42c

Naloxone ? hexane 2 ? 200 8.14 0.41 8.00 0.31d 8.27 0.38

d 8.60 0.30d

Each value is presented as the mean SEM in seconds (n = 10)

* P\ 0.05; **P\ 0.01 compared with the control groupa

P\0.01 compared with the morphine groupb

P\ 0.05 compared with the CME groupc

P\0.05 compared with the chloroform fraction groupd

P\ 0.05 compared with the hexane fraction group (Bonferronis test)

Table 3 Effects of CME and its fractions from C. mangga rhizome and morphine on the reaction time of mice in the formalin test

Treatment Dose (mg/kg, p.o.) Licking of the hind paw (s)

Early phase (05 min) Inhibition (%) Late phase (1530 min) Inhibition (%)

Control 98.82 3.92 117.20 4.97

Morphine 5 (s.c.) 9.82 1.88** 89.99 0.00 0.00** 100.00

CME 200 82.10 3.80** 16.92 48.05 2.76** 59.00Aqueous fraction 200 84.90 2.62* 14.09 94.61 2.59** 19.27

Chloroform fraction 200 79.47 2.83** 19.58 32.25 2.47** 72.48

Ethyl acetate fraction 200 82.34 3.31** 16.68 87.93 2.67** 24.97

Hexane fraction 200 77.68 2.39** 21.39 44.25 3.22** 62.24

Each value is presented as the mean S.E.M. in seconds (n = 10)

* P\ 0.05; **P\0.01 compared with the control group (Bonferronis test)

Table 4 Effects of CME and its fractions from C. mangga rhizome and indomethacin on carrageenan-induced rat paw edema

Treatment Dose (mg/kg, p.o.) Edema volume (ml)

0 h 1 h 2 h 3 h 4 h

Control 3.58 0.05 5.22 0.06 6.07 0.15 6.82 0.16 7.22 0.19

Indomethacin 10 3.49 0.05 4.49 0.05** 4.95 0.08** 5.04 0.08** 4.73 0.08**

CME 150 3.47 0.03 4.90 0.09 5.57 0.07* 6.10 0.11** 6.43 0.15**

Aqueous fraction 150 3.54 0.04 4.86 0.08* 5.58 0.10* 6.13 0.13* 6.45 0.17*

Chloroform fraction 150 3.47 0.03 4.71 0.08** 5.13 0.12** 5.83 0.16** 5.96 0.14**

Ethyl acetate fraction 150 3.53 0.05 4.85 0.08* 5.56 0.08* 6.01 0.14** 6.19 0.12**

Hexane fraction 150 3.38 0.03 4.62 0.08** 5.27 0.09** 5.98 0.11** 6.05 0.14**

Each value is presented as the mean SEM (n = 10)

* P\ 0.05; **P\0.01 compared with the control group (Bonferronis test)

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and chloroform fraction showed a greater inhibition by

53.97 and 50.29%, respectively, compared with the control

group. Indomethacin (0.5 mg/ear) also exhibited significant

inhibition of 42.64% (Table 5).

Discussion

The results demonstrate that CME, chloroform, and hexane

fractions possess marked antinociceptive and anti-inflam-

matory activities in chemicals and heat-induced pain and

chemical-induced inflammation models. No acute toxicity

was observed after oral administration of CME, chloro-

form, and hexane fractions even at the high dose of 2 g/kg

in mice (unpublished data).

Acetic acid-induced writhing, a visceral pain model, is a

chemical stimulus widely used for the evaluation of general

analgesic activity [8]. In this model, pain is generated

indirectly via endogenous mediators like bradykinin,

serotonin, histamine, substance P, and prostaglandins,

acting on stimulating peripheral nociceptive neurons,

which are sensitive to narcotic analgesics and non-steroid

anti-inflammatory drugs (NSAIDs) [13, 14]. From the

results, CME and all fractions suppressed acetic acid-

induced writhing. The chloroform and hexane fractions as

well as CME were stronger than those of aqueous and ethyl

acetate fractions, which were comparable to that of the

reference drug, indomethacin. These results indicated that

the C. mangga rhizome may possess antinociceptive

activity.

The hot plate test is used to evaluate the centrally acting

analgesic drugs [15]. The paw-licking hot plate responses

are complex supraspinally organized behavior [16]. In the

present study, CME, chloroform, and hexane fractions had

antinociceptive effect against heat-induced pain. Moreover,

effects of CME and these active fractions and reference

drug, morphine, were abolished by the opioid receptor

antagonist naloxone. The mu (l) opioid receptor has

generally been regarded as the receptor type associated

with pain relief and shown to be potent in regulating

thermal pain [17]. Therefore, the apparent analgesic

activity of CME and active fractions from C. mangga

rhizome may be mediated through a central mechanism by

the activation of the opioid system.

In the formalin test, animals presented two distinct

nociceptive behavior phases, which probably involveddifferent stimuli. The early phase initiated immediately

after formalin injection and lasted about 35 min, resulting

from chemical stimulation of nociceptors. The late phase

initiated 1520 min after formalin injection, lasted about

2040 min. Bradykinin was involved in the early phase,

while histamine, 5-hydroxytryptamine (5-HT), bradykinin,

and prostaglandins were involved in the late phase [18].

Morphine, a centrally acting analgesic drug, can inhibit

nociception in both phases [19]. In this experiment, CME

and all fractions significantly inhibited both the early and

late phases, also indicating that the antinociceptive action

of C. mangga rhizome may be mediated by a centralmechanism.

The carrageenan-induced paw inflammation is a useful

phlogistic tool for investigation of systemic anti-inflam-

matory agents. This test is sensitive to most clinically

effective anti-inflammatory drugs and comprises two pha-

ses. The initial phase, which occurs within 12 h after

carrageenan injection, is due to the release of serotonin and

the increase of prostaglandin, histamine, and bradykinin in

the inflammatory area. The second phase occurs 35 h after

carrageenan injection which is correlated with production

and release of kinins and prostaglandins in the inflamed

area [2022]. CME and all fractions showed inhibition of

the rat paw edema in both phases. The obtained results

indicated that C. mangga rhizome possessed anti-inflam-

matory properties that might be interacting in one or more

steps of the inflammatory cascade induced by carrageenin.

Croton oil-induced ear edema is a useful model

for testing topical anti-inflammatory activity [23]. 12-O-

Tetradecanoylphorbol-13-acetate (TPA), a kind of phorbol

ester present in croton oil, has been reported to stimulate

phospholipid-dependent protein kinase C and involve on

arachidonic acid release and metabolism as well as the

overexpression of cyclooxygenase-2 [2426]. Topically

applied CME and all fractions, especially the chloroform

fraction, showed more potent reduction of ear edema than

that of indomethacin. Therefore, the results suggested that

C. mangga rhizomes may possess topical anti-inflamma-

tory action.

Phytochemical screening of the C. mangga extract

indicates the presence of terpenes and phenolic compounds

but absence of alkaloids. These results are in accordance

with the previous reports stating the presence of diterpenes,

diarylheptanoids, hydroxycinnamic acid, and curcuminoid

Table 5 Effects of CME and its fractions from C. mangga rhizome

and indomethacin on ear edema induced by croton oil

Treatment Dose

(mg/ear)

Ear weight

(mg)

Inhibition

(%)

Control 17.12 0.44

Indomethacin 0.5 9.82 0.88* 42.64

CME 0.5 7.88

0.36* 53.97Aqueous fraction 0.5 13.65 0.50* 20.27

Chloroform fraction 0.5 8.51 0.65* 50.29

Ethyl acetate fraction 0.5 13.49 0.70* 21.20

Hexane fraction 0.5 10.67 0.75* 37.68

Each value is presented as the mean S.E.M. (n = 10)

* P\ 0.01 compared with the control group (Bonferronis test)

40 J Nat Med (2010) 64:3641

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compounds in the rhizomes of C. mangga [3, 27]. In the

present study, most of the active fractions of C. mangga

rhizome were chloroform and hexane fractions. It is possible

that these compounds may be responsible for the observed

activities of C. mangga rhizome. However, further studies

on the isolation of compounds that are responsible for the

activities of C. mangga rhizome are now in progress.

In conclusion, CME and its fractions, especially chloro-form and hexane fractions, from C. mangga rhizome pos-

sessed centrally acting analgesic as well as anti-inflammatory

activities.

Acknowledgments The authors are very grateful to the Thailand

Research Fundthrough the Royal Golden Jubilee Ph.D. Program (Grant

No. PHD/0052/2549) for financial support of this research work.

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