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DOI:10.3736/ jcim20110116
http:// ww w.jcimjournal.com
Achary a SD , Ullal SD , Padiy ar S , Rao YD , Upadhyaya
K , P illai D , Raj V .Analgesic effect of ext racts of
A lpinia galanga rhizome in mice.J Chin Integr Med .
2011;9(1):100-104.
Achary a SD , Ullal SD , Padiy ar S , Rao YD , Upadhyaya
K , Pillai D , Raj V .大高良姜根茎提取物对小鼠的镇痛
作用.中西医结合学报.2011;9(1):100-104.
Received September 9 , 2010;accepted September 28 ,
2010;published online Januar y 15 , 2011.
Full-tex t LinkOut at PubMed.Journal title in PubMed:
Zhong X i Y i J ie He X ue Bao.
Correspondence:Sheetal Dinkar Ullal , MD , Associa te
Professo r;Tel:+91-944-8306242;E-mail:shee tal.
ullal@manipa l.edu
郝小燕 , 彭琳 , 叶兰 , 黄能慧 , 沈月毛.飞龙掌血生物总碱
抗炎镇痛作用的研究.中西医结合学报.2004;2(6):450-
452.
Hao XY , Peng L , Ye L , Huang NH , Shen YM .A study on
anti-inflammato ry and analgesic effects o f alkaloids of Toddalia
asiatica .J Chin Integr Med .2004;2(6):450-452.
Full text available at http:// ww w.jcimjournal.com/a rticle s/
publishA rticles/ pdf/2006323987024198.pdf
徐丽君 , 胡永红 , 陆付耳 , 邹欣.一贴灵贴剂抗炎镇痛作用的
实验研究.中西医结合学报.2005;3(4):303-306.
Xu LJ , Hu YH , Lu FE , Zou X.Experimental study on anti-
inflammato ry and analgesic effects o f Yitieling Paste.J Chin
Integr Med .2005;3(4):303-306.
Full text available at http:// ww w.jcimjournal.com/a rticle s/
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Original Experimental Research 实验论著
Analgesic effect of extracts of Alpinia galanga rhizome inmice
Sahana Devadasa Acharya1 , Sheetal Dinkar Ullal1 , Shivaraj Padiyar1 , Yalla Durga Rao1 ,
Kousthubha Upadhyaya2 , Durga Pillai1 , Vishnu Raj1
1.Depar tment of Pharmaco lo gy , Kasturba Medical Co llege , Manipal University , Mangalo re 575001 , Karnataka ,
India
2.Depar tment o f Dravyaguna , Na tional Institute of Ayurveda , Jaipur 302002 , Rajasthan , I ndia
Objective:To evaluate the analgesic effect of extracts of Alpinia galanga (AG)rhizome in mice
and elucidate the possible mechanism for its analgesic action.
Methods:Analgesic action of extracts of AG rhizome was studied in three experimental models
of nociception.Albino mice of both sexes weighing 25 to 30 g were used in this study.For the
hot-plate test , mice in the five groups with six in each received three different doses of ethanolic
extracts of dried rhizome of AG suspended in 2%gum acacia orally , morphine subcutaneously
and 2%gum acacia orally , respectively.React ion t ime was observed after administration of
vehicle or drugs.For the hot-plate test after naloxone pretreatment , mice in the five groups
received naloxone subcutaneously 30 min prior to the administration of vehicle or drugs and
reaction time was observed as explained above.In the writhing test , writhes were induced by
injecting acet ic acid intraperitoneally in another 30 mice which were randomly allocated to five
groups of six in each and received three different doses of ethanolic extracts of dried rhizome of
AG suspended in 2% gum acacia , aspirin suspended in 2 %gum acacia and 2% gum acacia
orally , respectively.The mice were observed individually for a period of 15 min and the number
of writhes was recorded for each animal.
Results:AG treatment significantly increased the latency period in the hot-plate test at all three
doses at 30 , 60 , 90 and 120 min time intervals compared with control group (P <0.05 or P <
0.01).Naloxone pretreatment significantly reduced the latency period in hot-plate test for both
AG and morphine groups as compared with corresponding groups that did not receive naloxone
pretreatment (P <0 .05 or P <0.01).AG at all doses significantly reduced the number of
writhes compared with control group (P <0.01).
Conclusion:The study confirmed the analgesic effect of AG rhizome and hence justified its use in
ethnomedicine for the treatment of pain due to various causes.The probable mechanism of its
analgesic action may be central as well as peripheral.
Keywords:Alpinia galanga ;plant extracts;analgesics;pain measurement;receptors , opioid;mice
·100· 中西医结合学报 2011年 1月第 9卷第 1期 J ou rnal of C hinese Integrative Medicine , January 2011 , Vol.9 , No.1
Accor ding to Wor ld Health Organizat ion , three
quarters of the world population r ely on tradi-
tional medicines mainly der ived from plants for
their healthcare
[ 1] .I ndia has an extensive for est
cover , enriched with plant diversity.Several
plants have been used in folklore medicine
[ 2] .
Alpinia galanga (L.)Willd (Zingiberaceae)is
cal led gre ater galangal , Java galangal in English;
Rasna , Sugandh amula in Sanskrit;Kulanjan in
Hindi;Doddar asagade in Kannada;Chit tar at ta in
Malayalam
[ 3] .I t is an aromat ic plant cul tivated in
Dakshina Kannada in Kar nataka , Ker ala and
West Bengal in I ndia.This herb of the ginger
family is commonly used in cooking as an aromatic
stimulan t
[ 4]
or flavouring agent , and used as folklore
medicine in bronchial catarrh , r heumatism and
respiratory ailments like asthma
[ 3] .Alpinia galanga(AG)is used as one of the many ingredients in
polyher bal pr epar at ions for r el ieving pain of
dif ferent etiologies such as rheumatoid ar thri tis ,
back pain and pain in individuals who had
Chikungunya fever in South I ndia.These poly-
her bal prepara tions containing AG are available
ove r the counter for rel ieving pa in.A survey of
lite rature revealed that a systemat ic study on the
analgesic act ivi ty of AG was lacking.With this
backgr ound we conducted this study to evaluate
the analgesic effect and mechanism of analgesic
action of AG rhizome.
1 Materials and methods
1.1 Experimental animals The study was conducted
after obtaining approval of the study protocol by
the I nsti tutional Animal Care and Use Commit tee(IACUC).Albino mice weighing 25 to 30 g of
both sexes were obtained fr om the centr al animal
house of the institut ion.The animals were housed
in groups of three in polypr opylene cages , had
free access to food (animal chow)and water ad
libitum .The animals were maintained under
standard labor atory condit ions (12 h/12 h ligh t/
dar k cycle and temperature of (27 ±2)℃).The
animals we re randomly allocated into five groups
of six animals in each , for each of the three experi-
mental animal models to test the ant inocicept ive
activity.
1.2 Drugs and reagents Morphine inject ion of
10 mg/mL (Troikaa Pharmaceuticals Ltd , Mumbai ,
I ndia;batch number:M34112);naloxone injec-
tion of 400 μg/mL (Samarth Life Sciences Pvt.
Ltd , Solan , India;batch number:NLX04);aspirin
powder (Hansa Chemicals , Mumbai , India);2 %
gum acacia(Romali , Mumbai , India;batch number:
33001);0.6% acetic acid (Merck Chemicals , Banga-
lore , India;batch number:CGOC 600144).
1.3 Plant materials Locally grown AG plants
wer e authent ically ident ified and deposited in the
Department of Botany , Aloysius Col lege , Manga-
lore , I ndia.The rhizomes were collected in May ,
2009 and shaded dried .A total of 250 g powder of
AG rhizome was extr acted with 90% ethanol by
using Soxhlet apparatus (Rotek Instruments , Kerala ,
India &Bor osil Glass Works Ltd , Mumbai , India).
Af ter ext ract ion , the solvent was evapor ated at a
temper atur e below 50 ℃.The extracts were then
frozen dried , which yielded 9%of original extracts.
The powder was weighed and requir ed amount of
the dr ug was suspended in 2% gum acacia and
used f or or al feeding in different doses.
1.4 Antinociceptive activity
1.4.1 Thermally induced pain by hot-plate test
The hot-plate appar atus (Inst ruments&Chemicals
Pvt.Ltd , Model City , Ambala , India)main-
tained at (56 ±1)℃was used .Mice were placed
on the hot plate and the t ime between placement
and the first sign of paw licking or jumping (r eac-
t ion t ime)was re corded by a stop watch[ 5] .Thir ty
mice which showed a short react ion time(<10 s)
were selected prior to grouping.They were then
r andomly divided into five groups with six in each .
React ion time prior to tre atment was recorded.
Animals in group Ⅰ (control)received 2 % gum
acacia or ally;group Ⅱ (standard)r eceived mor-
phine 2 mg/kg subcutaneously and group ⅢA toⅢ C (test) received e thanol ic extracts of AG
rhizome suspended in 2%gum acacia at 200 , 400
and 800 mg/kg body weight orally , 30 min prior
to placing on the hot plate.Post t reatment r eac-
t ion t ime was observed 30 , 60 , 90 and 120 min
af ter oral administr ation of vehicle or drugs .A
cut off per iod of 30 s was mainta ined to avoid
damage to the paw.
1.4.2 Naloxone pretreatment on thermally induced
pain This was done to test the possible role of opioid
receptors in analgesic action mediated by the test
drug .Another 30 mice divided into five groups
received naloxone at 1 mg/kg[ 6] subcutaneously 30 min
prior to the administ rat ion of vehicle or drugs.
React ion time was observed as explained above.
1.4.3 Writhing test Writhes were induced by
inject ing 0 .1 mL of 0.6% acet ic acid int raperi to-
neally in another 30 mice which were r andomly
allocated to five groups of six in each .Control
gr oup received vehicle (2% gum acacia)orally;
standard gr oup received aspirin suspended in 2%
gum acacia at 100 mg/kg orally and test groups Ⅰ,Ⅱand Ⅲ received ethanolic extracts of AG rhizome
suspended in 2% gum acacia at 200 , 400 and
800 mg/kg body weight or ally 45 min pr ior to
inject ing acet ic acid , r espect ively.The mice we re
observed individually for a period of 15 min and the
number of wr ithes was recorded for each animal.
A wri the is indicated by st retching the abdomen
with simultaneous str etching of one hind limb
[ 7] . Percentage inhibit ion of writhing was calculated
by using the formula below:
·101·中西医结合学报 2011年 1月第 9卷第 1期 J ou rnal of C hinese Integrative Medicine , January 2011 , Vol.9 , No.1
Inhibit ion rate= Mean number of writhes (control)-Mean number of wr ithes (test)
Mean number of writhes (control) ×100 %
1.5 Statistical analysis The data were pr esented
as x ±s x .Results were analysed by one way analysis
of variance (ANOVA)f ollowed by Dunnet ts t
test for mult iple compar isons .For the comparison
between two groups , Students t test was employed.
Analysis was done by using SPSS computer package
version 11.5.P <0.05 was considered as significant.
2 Results
2.1 Reaction time of hot-plate test The mean
basal re action t ime in seconds at different t ime
inter vals af ter t rea tment in different groups are
shown in Table 1.At all subsequent time inte rvals
after tre atment with three different doses of AG
there were stat ist ically significant increases in the
react ion time compared with control gr oup (P <
0.05 or P <0.01).Analgesic effect was higher in
group ⅢB and group ⅢC compared with group Ⅲ
A.There we re significant increases in the reac-
t ion time at all t ime intervals in group Ⅱ compar ed
with control group (P <0 .05 or P <0 .01).The
react ion time of gr oup ⅢB was longer th an th at
of group Ⅱ at 60 min (P <0 .05)and 90 min (P <
0 .05) inter vals.On comparing group Ⅲ C and
group Ⅱ , there was a significant ly longer react ion
time in group ⅢC only at 30 min interval(P <0.01).
2.2 Reaction time after naloxone pretreatment
The naloxone pret reated mice of group Ⅱ , groupⅢB and group ⅢC showed significant decreases
in re act ion t ime a t all t ime intervals compared
with corresponding groups that did not receive
naloxone pret reatment by hot-plate test (P <0 .05
or P <0.01), except group ⅢC at 90 min .The
values are shown in Table 2.Naloxone pret reat-
ment in group ⅢA did not show a consistently
signif icant inhibi tion of antinocicept ive action
compared with the cor responding group that did
not receive naloxone pret reatment .
2.3 Writhing test result I ntr aper itoneal injec-
t ion of acet ic acid induced(62.17±2.17)abdominal
wri thes in contr ol mice during the 15 min obser-
vation per iod .For the animals which received
ethanolic extracts of AG rhizome at 200 , 400 and
800 mg/kg body weight , the mean numbers of
wri thes ar e shown in Table 3.There was a dose-
dependent inhibition of writhing in groups receiving
different doses of AG.The number of wri thes
decreased significantly in standar d group and test
groupsⅠ to Ⅲ compared with control group (P <
0.01).There was no significant change in the
number of wr ithes between the test groups and
the standard group .
Table 1 Effects of ethanolic extracts of Al pinia galanga rhizome on thermal stimulus-induced pain assessed by hot-plate test(x±sx)
G roup n
Reaction t ime(second)
Before treatment 30 min 60 min 90 min 120 min
Group Ⅰ (2% gum acacia) 6 2.46±0.24 2.24±0.21 2.49±0.12 2.40±0.28 2.24±0.28
Group Ⅱ (Morphine 2 mg/kg) 6 3.21±0.22 9.25±1.08＊ 11.28±1.57＊＊ 10.18±0.46＊ 15.59±2.77＊＊
Group ⅢA (AG 200 mg/kg) 6 2.50±0.19 13.16±2.30＊＊ 10.22±2.06＊ 8.82±1.37＊ 13.81±1.65＊＊
Group ⅢB (AG 400 m g/kg) 6 2.90±0.34 12.01±1.86＊＊ 16.60±1.53＊＊■ 16.49±2.27＊＊■ 19.56±0.54＊＊
Group ⅢC (AG 800 mg/kg) 6 3.07±0.16 16.32±1.11＊＊■■ 14.01±1.49＊＊ 14.37±2.19＊＊ 18.60±0.76＊＊
＊P<0.05 , ＊＊P <0.01 , vs group Ⅰ ;■P <0.05 , ■■P<0.01 , vs group Ⅱ .AG:A lpinia g alanga .
Table 2 Antinociceptive activities of Alpinia galanga and morphine and their antagonism by naloxone assessed by hot-plate test(x±sx)
G roup n
Reaction t ime(second)
Before treatment 30 min 60 min 90 min 120 min
Group Ⅰ (2% gum acacia)
+Naloxone (1 mg/k g) 6 4.98±0.76 7.29±0.52 7.84±0.62 5.72±0.58 2.76±0.19
Group Ⅱ (Morphine 2 mg/kg) 6 3.21±0.22 9.25±1.08 11.28±1.57 10.18±0.46 15.59±2.77
Group Ⅱ +Naloxone(1 mg/kg) 6 4.25±0.55 6.43±0.14■ 7.49±0.56■ 7.65±1.02■ 8.72±0.25■
Group ⅢA (AG 200 mg/kg) 6 2.50±0.19 13.16±2.30 10.22±2.06 8.82±1.37 13.81±1.65
Group ⅢA+Naloxone(1 mg/kg) 6 4.06±0.33 4.88±0.95△△ 5.98±0.67 7.01±0.52 8.32±0.38△△
Group ⅢB (AG 400 m g/kg) 6 2.90±0.34 12.01±1.86 16.60±1.53 16.49±2.27 19.56±0.54
Group ⅢB+Naloxone(1 m g/kg) 6 5.28±0.43 6.60±0.81◆ 5.97±0.46◆◆ 7.41±0.69◆◆ 7.56±0.88◆◆
Group ⅢC (AG 800 mg/kg) 6 3.07±0.16 16.32±1.11 14.01±1.49 14.37±2.19 18.60±0.76
Group ⅢC+Naloxone(1 mg/kg) 6 6.51±0.65 9.25±1.41□□ 6.80±0.58□□ 8.86±1.17 8.80±1.33□□
■P<0.05 , vs group Ⅱ;△P<0.05 , △△P <0.01 , vs group ⅢA;◆P <0.05 , ◆◆P<0.01 , vs group ⅢB;□P<0.05 , □□P<0.01 , vs
g rou p ⅢC.AG:Alp inia ga langa.
·102· 中西医结合学报 2011年 1月第 9卷第 1期 J ou rnal of C hinese Integrative Medicine , January 2011 , Vol.9 , No.1
Table 3 Effects of ethanolic extracts of Alpinia galanga on acetic acid-induced writhes in mice
(x±sx)
G roup n Number of w rithes Inhibiti on rate(%)
C on t rol group (2% gum acacia) 6 62.17±2.17 —
S tandard g rou p(A spi rin 100 mg/k g) 6 22.17±2.52☆☆ 64.33
Test group Ⅰ (AG 200 mg/kg) 6 36.17±6.16☆☆ 41.82
Test group Ⅱ (AG 400 mg/kg) 6 32.83±6.46☆☆ 47.19
Test group Ⅲ (AG 800 mg/kg) 6 12.33±3.64☆☆ 80.17
☆☆P<0.01 , vs cont rol g rou p.AG:A lp in ia g alanga.
3 Discussion
I n this study nociceptive r eactivity to thermal
stimuli in mice was measured by using the hot-
plate test , wh ich is a sensi tive acute pain test for
detecting opiate analgesia.The results of evaluation of
analgesic act ion of 90 % ethanolic extr acts of AG
rhizome in acute thermal model (hot-plate test)
and acute chemical model (acet ic acid-induced
wr ithes)showed that it had significant ant inoci-
cept ive action in mice (Tables 1 and 3).In the
hot-plate test , AG incr eased the r eaction t ime
significantly at 200 , 400 and 800 mg/kg body
weight compar ed with control group.The ant ino-
cicept ive eff icacy of AG at 400 mg/kg body weight
was more consistent than th at of other doses.
Moreover , AG at 400 mg/kg body weight showed
bet ter analgesic act ion than morphine of 2 mg/kg
body weight at 60 min and 90 min intervals.The
hot-plate test results suggest that this analgesic
agent acts primarily at the spinal cor d and/or
higher central nervous system levels , or by an
indir ect mechanism.Therefore it is possible th at
the AG extract exerted its act ion through central
opioid receptors or promoted release of endogenous
opioid pept ides. In an at tempt to elici t the possible mechanism
of the obse rved analgesic act ion of AG in the hot-
plate experiment , mice were pretreated with naloxone ,
a potent opioid antagonist
[ 8] .I n the present study
naloxone pr etr eatment in mice which received
400 mg/kg of AG antagonized the analgesic
action of AG.It was observed that naloxone did
not consistently inhibi t the analgesic act ion of AG
at either 200 or 800 mg/kg.This suggests tha t AG
at 400 mg/kg body weigh t may exhibit cent ral
mechanism of analgesic act ion mediated by opioid
receptors . The 1 , 8-cineol which is contained in her bs
belonging to Z ingiberaceae family has been reported
to bear ant inocicept ive act ivity and is reported to
act by a non-opioid mechanism[ 9] .The 1 , 8-cineol
has also been reported to be found in AG rhizome.
I ntr aper itoneal injection of acet ic acid produces
pain through activat ion of chemosensit ive noci-
ceptors
[ 10]
or irritat ion of the visceral surface ,
which leads to liberat ion of histamine , bradykinin ,
pr ostaglandin and ser otonin
[ 11] .Antinocicept ive
activity of opioid agonists , opioid part ial agonists
and non-ster oidal ant i-inflammatory agents can be
determined by the writhing test
[ 12] .The extracts
of AG rhizome inhibited acet ic acid-induced
wri thes in mice , which indicates that it may act
by per ipher al antinocicept ive mechanism as well.
An important observation was that AG at 800 mg/kg
body we ight h ad higher efficacy in inhibit ing
wri thing than AG at 400 mg/kg , whereas AG at
400 mg/kg body we ight was more efficacious than
AG at 800 mg/kg in i ts antinocicept ive act ion in
the hot-pla te test.This suggests tha t AG at
400 mg/kg may exhibit a central mechanism of
analgesic action , while AG at 800 mg/kg may exhibit
a pe ripher al mechanism of analgesic action. The l imitat ion of the study was th at the effect
of extr acts of AG rhizome on the motor performance
and the sedative effects in mice were not tested.
Muscle relaxat ion pr operty of a drug can increase
the react ion t ime in the hot-plate test.Some seda-
t ives may possess antinocicept ive proper ty but we
ar e not sure whether AG had sedat ive action.
4 Conclusion
Our study confirmed the analgesic ef fect of AG
rhizome and just ified i ts use in ethnomedicine for
the treatment of pain due to various causes.The
probable mechanism of its analgesic act ion may be
centr al as well as per ipher al.Fur ther studies with
the isolated act ive substance from the AG extracts
ar e necessar y for development of this new analgesic
drug .
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大高良姜根茎提取物对小鼠的镇痛作用
Sahana DevadasaAcharya
1 , Sheetal Dinkar Ullal1 , Shivaraj Padiyar1 , YallaDurgaRao1 , KousthubhaUpadhyaya2 ,
Durga Pillai
1 , Vishnu Raj1
1.Depar tment o f Pharmaco log y , Kasturba Medical Co llege , M anipal Univer sity , Mangalore 575001 , Karna taka , India
2.Depar tment o f Dravyaguna , Na tional Institute of Ayurveda , Jaipur 302002 , Rajasthan , I ndia
目的:评价大高良姜(Alpinia galanga)根茎的乙醇提取物对小鼠的镇痛作用并推断其可能的机制。
方法:使用 3个实验模型分别测定大高良姜根茎提取物对白化病小鼠(体质量 25 ～ 30 g)的镇痛作用 。热板
实验中 ,30只小鼠被分为 5组 ,每组 6只 ,分别给予口服 3种剂量的大高良姜根茎乙醇提取物 、皮下注射吗
啡以及口服 2%的阿拉伯树胶 ,记录给药 30 、60 、90和 120 min后的热板实验反应时间 。纳洛酮预处理热板
实验中 ,另外 30只小鼠在给予上述药物 30 min之前皮下注射纳洛酮 ,给药种类及方式同前 ,热板实验反应
时间记录方式同前。扭体实验中 ,通过腹腔注射醋酸诱导小鼠产生扭体反应 ,随后 30只小鼠被分为 5组 ,每
组 6只 ,分别给予口服 3种剂量的大高良姜根茎乙醇提取物 、阿司匹林及 2%的阿拉伯树胶 ,每只小鼠观察
15 min ,记录扭体次数。
结果:各个时间记录点的数据表明 ,与对照组相比 , 3种剂量的大高良姜根茎乙醇提取物均显著延长了热板
实验中小鼠的反应时间(P <0.05 ,P <0.01)。经纳洛酮预处理后 ,实验组及阳性对照药吗啡组小鼠的热板
实验反应时间较对应的前一实验中未经纳洛酮处理各组小鼠显著延长(P <0.05 , P <0.01)。扭体实验中 ,
与对照组相比 , 3种剂量的大高良姜根茎乙醇提取物均减少了实验小鼠的扭体次数(P <0 .01)。
结论:本实验证实了大高良姜根茎提取物的镇痛作用 ,验证了其在民族医学中治疗各种原因引起的疼痛的应
用。其镇痛作用的机制可能是有效成分作用于中枢或外周神经系统 。
关键词:大高良姜;植物提取物;镇痛药;疼痛测定;受体 , 阿片样;小鼠
·104· 中西医结合学报 2011年 1月第 9卷第 1期 J ou rnal of C hinese Integrative Medicine , January 2011 , Vol.9 , No.1