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Advances in Natural and Applied Sciences, 6(8): 1302-1306, 2012
ISSN 1995-0772
This is a refereed journal and all articles are professionally screened and reviewed
ORIGINAL ARTICLE
Evaluation Of Antihyperglycemic And Antinociceptive Activities Of Methanolic Extract
Of Kaempferia Rotunda L. (Zingiberaceae) Rhizomes
Zakia Sultana, Khandaker Md. Sharif Uddin Imam, Fardous Mohammad Safiul Azam,
Shahnaz Rahman, Shiblur Rahman, Fatema Islam, Mohammed Rahmatullah
Department of Biotechnology & Genetic Engineering, Faculty of Life Sciences, University of Development
Alternative, Dhanmondi, Dhaka-1205, Bangladesh.
Zakia Sultana, Khandaker Md. Sharif Uddin Imam, Fardous Mohammad Safiul Azam, Shahnaz
Rahman, Shiblur Rahman, Fatema Islam, Mohammed Rahmatullah: Evaluation Of Antihyperglycemic
And Antinociceptive Activities Of Methanolic Extract Of Kaempferia Rotunda L. (Zingiberaceae)
Rhizomes
ABSTRACT
The objective of this study was to conduct antihyperglycemic and antinociceptive activity studies with
methanolic extract of rhizomes of Kaempferia rotunda, a plant used in folk medicines of Bangladesh for
treatment of high blood sugar level and pain. Antihyperglycemic activity studies were conducted in glucoseloaded mice in oral glucose tolerance tests. Mice were given various doses of extract, followed by glucose
(2g/kg body weight), 60 min after administration of extract. Serum glucose levels were measured 120 minutes
after glucose administration. Antinociceptive activity studies were conducted in intraperitoneally acetic acid
injected mice through measurement of reductions in abdominal writhings caused by acetic acid-induced gastric
pain. Following a period of 1 h after oral administration of various doses of extract, all mice received
intraperitoneal injection of 1% acetic acid at a dose of 10 mL/kg body weight. To ensure bio-availability of
acetic acid, a period of 5 min was given to each animal following which period the number of writhing times
was counted for 10 min. The extract caused dose-dependent significant lowering in serum glucose levels in mice
(P<0.05), when administered at doses of 50, 100, 200 and 400 mg per kg body weight to glucose-loaded mice as
compared to control animals. Highest lowering of serum glucose (39.6%) was observed at an extract dose of 400
mg. In comparison, a standard antihyperglycemic drug, glibenclamide, when administered at a dose of 10 mg
per kg body weight, lowered serum glucose levels by 42.4%. The extract also demonstrated dose-dependent
significant antinociceptive activity (P<0.05), when administered to mice compared to control animals. At a dose
of 400 mg extract per kg body weight, the number of abdominal writhings was inhibited by 69.4% as compared
to 73.4% inhibition obtained with a standard antinociceptive drug, aspirin, administered at a dose of 400 mg per
kg body weight. The significant antihyperglycemic and antinociceptive activity demonstrated by the extract
validates the use of rhizomes of K. rotunda in folk medicines of Bangladesh for treatment of high blood sugar
levels as commonly observed in diabetic patients, and pain, and merits further scientific studies leading to
identification of relevant bioactive constituents.
Key words: Kaempferia rotunda, Zingiberaceae, antihyperglycemic, antinociceptive
Introduction
Kaempferia rotunda L. (Family: Zingiberaceae, English name: Indian crocus, Local name: Misri dana) is a
plant that can be found in the Indian sub-continent including various parts of Bangladesh. The plant is used in
the folk medicinal system of Bangladesh for treatment of high blood sugar levels as commonly observed in
diabetic patients, as well as for treatment of pain. For treatment of high blood sugar levels and pain,
respectively, juice obtained from macerated rhizomes is orally and topically administered. The plant is
considered to be an important medicinal plant also in the ancient traditional medicinal system of India, namely,
Ayurveda. The Ayurvedic drug, hallakam, containing tubers of the plant is considered as stomachic,
antiinflammatory to wounds and bruises, and useful for treatment of mental disorders and insomnia (Sereena et
al., 2011).
Methanol extracts of rhizomes of the plant reportedly contained six polyoxygenated cyclohexane
derivatives identified as (-)-6-acetylzeylenol (I), four acylated derivatives of 1-benzoyloxymethyl-1,6Corresponding Author: Dr. Mohammed Rahmatullah, Pro-Vice Chancellor and Dean, Faculty of Life Sciences, University
of Development Alternative, House 78, Road 11A, Dhanmondi, Dhaka-1205, Bangladesh
Tel: 88-02-9136285; Fax: 88-02-8157339; E-mail: [email protected] 1303
Adv. in Nat. Appl. Sci., 6(8): 1302-1306, 2012
epoxycyclohexan-2,3,4,5-tetrol (III-VI), a Diels-Alder adduct of 3-benzoyl-1-benzoyloxymethylcyclohexa-4,6dien-2,3-diol (VII), and a triacylated derivative of salicin (IX), in addition to (-)-zeylenol (II), and crotepoxide
(VIII). Compounds II and VI demonstrated antifeedant activities against larvae of Spodoptera littoralis, the
Egyptian cotton leafworm (Stevenson et al., 2007). The antioxidant potential of the plant has been evaluated and
flavonoids and phenolic derivatives found in the plant, which demonstrated inhibitory potential against lipid
peroxidation, suggesting that the plant can be useful in diseases like myocardial infarction, diabetes mellitus,
hepatic injury, atherosclerosis, rheumatoid arthritis, and cancer (Priya Mohanty et al., 2008). Two compounds
has been identified from rhizomes of the plant, namely, 2’-hydroxy’-4,4’,6’-trimethoxy-chalcone and (+)crotepoxide, the first possessing antioxidant activity (Lotulung et al., 2008). A lectin (designated as KRL) has
been purified from rhizomes of the plant, which demonstrated antibacterial activity and antiproliferative activity
against Ehrlich ascites carcinoma cells (Kabir et al., 2011). Alcoholic extract of rhizomes reportedly showed
anthelmintic activity against Pheretima posthuma and Ascardia galli (Agrawal et al., 2011).
High blood sugar level is one of the manifestations of diabetes, a disease which afflicts millions of people
worldwide and is rapidly reaching endemic proportions because of changes in human life style and dietary
habits. The disease cannot be cured by allopathic medicine and can rapidly progress to develop other
complications like cardiovascular disorders, diabetic retinopathy, diabetic neuropathy, and diabetic nephropathy.
Pain is also a problem afflicting millions of people worldwide on a daily basis and can arise from simple causes
like bruises or complicated causes like rheumatoid arthritis or cancer. In many of these diseases, the patient has
to take pain-killer drugs like aspirin or paracetamol for long stretches of time. These medicines can cause sideeffects like gastric ulceration and hepatic damages, respectively, following particularly any over-dosage. The
folk medicinal practitioners of Bangladesh (Kavirajes) claim to have effective treatments for both diabetes and
pain without resultant toxicity as occurs with allopathic pain killers. We had been screening the various
antidiabetic and antinociceptive medicinal plants used by the Kavirajes for some time to determine their actual
efficacies for the purposes that they are used for by the folk medicinal practitioners (Khan et al., 2010; Rahman
et al., 2010 a,b; Rahman et al., 2011). The objective of the present study was to evaluate the antihyperglycemic
and antinociceptive activities of methanolic extract of K. rotunda rhizomes.
Materials and Methods
1.1 Plant materials and extraction:
Rhizomes of K. rotunda were collected from Dinajpur district, Bangladesh in April 2011. The plant was
taxonomically identified by the Bangladesh National Herbarium at Dhaka (Accession Number: 63036).
Rhizomes were air-dried in the shade for one week, grounded into a fine powder, and were extracted with
methanol at a ratio of 1:6 (weight/volume) for 48 hours. The initial weight of dried powder used for extraction
was 50 g; the final weight of the methanol extract obtained from the dried powder was 5.935 g (yield =
11.87%).
1.2 Chemicals and drugs:
Glibenclamide, aspirin and glucose were obtained from Square Pharmaceuticals Ltd., Bangladesh. All other
chemicals were of analytical grade.
1.3 Animals:
In the present study, Swiss albino mice (male), which weighed between 15 to 20 g were used. The animals
were obtained from International Centre for Diarrheal Disease Research, Bangladesh. All animals were kept
under ambient temperature with 12 h light followed by a 12 h dark cycle. The animals were acclimatized for 3
days prior to actual experiments. The study was conducted following approval by the Institutional Animal
Ethical Committee of the University of Development Alternative, Dhaka, Bangladesh.
1.4 Antihyperglycemic activity:
Glucose tolerance property of methanol extract of whole plants was determined as per procedure previously
described by Joy and Kuttan (Joy and Kuttan, 1999) with minor modifications. In brief, fasted mice were
grouped into six groups of six mice each. The various groups received different treatments. Group 1 received
vehicle (1% Tween 80 in water, 10 mL/kg body weight) and served as the normal control; group 2 received
standard drug (glibenclamide, 10 mg/kg body weight); groups 3 to 6 received methanol extract of K. rotunda
rhizomes at doses of 50, 100, 200 and 400 mg/kg body weight, respectively. Doses were prepared by thoroughly
suspending methanol extract in 1% Tween 80 in water. Each mouse was weighed and dose-adjusted accordingly
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prior to administration of vehicle, standard drug, and test samples. All substances were orally administered.
Following a period of 1 h, all mice were orally administered 2 g/kg glucose. Blood samples were collected 2 h
after the glucose administration through puncturing heart. Serum glucose levels were measured by glucose
oxidase method (Venkatesh et al., 2004).
1.5 Antinociceptive activity:
Antinociceptive activity of methanol extract of K. rotunda whole rhizomes was examined using previously
described procedures (Shanmugasundaram and Venkataraman, 2005). Briefly, mice were divided into seven
groups of six mice each. Group 1 served as normal control and was administered vehicle only; groups 2 and 3
were orally administered standard antinociceptive drug, aspirin at doses of 200 and 400 mg/kg body weight,
respectively. Groups 4 to 7 were administered methanolic rhizome extract of K. rotunda at doses of 50, 100, 200
and 400 mg/kg body weight, respectively. Following a period of 1 h after oral administration of standard drug or
extract, all mice received intraperitoneal injection of 1% acetic acid at a dose of 10 mL/kg body weight. To
ensure bio-availability of acetic acid, a period of 5 min was given to each animal following which period the
number of writhing times was counted for 10 min.
1.6 Statistical analysis:
Experimental values are expressed as mean ± SEM. Independent Sample t-test was carried out for statistical
comparison. Statistical significance was considered to be indicated by a p value < 0.05 in all cases.
Results:
2.1 Antihyperglycemic activity:
In oral glucose tolerance tests conducted with glucose-loaded mice, the methanolic extract demonstrated
significant dose-dependent antihyperglycemic activity. The standard antihyperglycemic drug, glibenclamide,
following administration reduced glucose level significantly by 42.4% (P<0.05). At all doses except the 50 mg
per kg body weight dose, the extract caused significant reductions in serum glucose levels as compared to
control group (P<0.05). The percent reductions in serum glucose levels at doses of 50, 100, 200 and 400 mg/kg
body weight were, respectively, 16.2, 31.2, 38.1, and 39.6. Thus even at a dose of 200 mg extract, the results
compare favorably to that of glibenclamide. This suggests that the extract has potent antihyperglycemic
properties, and merits further consideration for isolation of bioactive constituent(s) responsible for the observed
antihyperglycemic effect. The results are shown in Table 1 (Part A).
Table 1: Antihyperglycemic and antinociceptive evaluation of methanolic extract of rhizomes of K. rotunda in mice.
A.
Antihyperglycemic activity
Treatment
Plant part used
Dose (mg/kg body Serum
glucose
level % lowering of
weight)
(mmol/L)
serum
glucose
level
Control (Group 1)
10 ml
7.67 ± 0.55
Glibenclamide (Group 2)
10 mg
4.42 ± 0.10*
42.4
K. rotunda (Group 3)
Rhizome
50 mg
6.43 ± 0.47
16.2
K. rotunda (Group 4)
Rhizome
100 mg
5.28 ± 0.27*
31.2
K. rotunda (Group 5)
Rhizome
200 mg
4.75 ± 0.41*
38.1
K. rotunda (Group 6)
Rhizome
400 mg
4.63 ± 0.37*
39.6
B.
Antinociceptive activity
Treatment
Plant part used
Dose (mg/kg body
% inhibition
weight)
Mean number of writhings
Control (Group 1)
10 ml
8.17 ± 0.79
Aspirin (Group 2)
200 mg
4.00 ± 0.58*
51.0
Aspirin (Group 3)
400 mg
2.17 ± 0.98*
73.4
K. rotunda (Group 4)
Rhizome
50 mg
4.00 ± 1.03*
51.0
K. rotunda (Group 5)
Rhizome
100 mg
3.33 ± 0.95*
59.2
K. rotunda (Group 6)
Rhizome
200 mg
3.17 ± 0.87*
61.2
K. rotunda (Group 7)
Rhizome
400 mg
2.50 ± 0.99*
69.4
All administrations were made orally. Values represented as mean ± SEM, (n=6); *P < 0.05, significant compared to control animals. Note
that antihyperglycemic activity and antinociceptive activity studies were carried out with separate groups of animals.
2.2 Antinociceptive activity:
The extract also showed significant dose-dependent reductions in the number of abdominal writhings in
mice, which were induced through intraperitoneal administration of acetic acid in the antinociceptive activity
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test. At all doses of the extract tested, there were significant reductions in the number of abdominal writhings,
when compared to control group of animals (P<0.05). At the highest dose of extract tested (400 mg), the number
of writhings was reduced by 69.4%. A standard antinociceptive drug, aspirin, reduced the number of writhings
by 51.0 and 73.4%, respectively, when administered at doses of 200 and 400 mg per kg body weight. The results
are shown in Table 1 (Part B). Even at the lowest dose of the extract (50 mg per kg body weight), the reduction
in the number of writhings compare favorably to that of aspirin, when administered at a dose of 200 mg per kg
body weight. At the highest dose of extract tested (400 mg per kg body weight), the number of writhings
compare favorably to that of aspirin, even when administered at the higher dose of 400 mg per kg body weight.
Discussion:
It is to be noted that antihyperglycemic and antinociceptive activities of the extract were measured in two
separate groups of animals. This is standard procedure as reported in the scientific literature, for the two
procedures for measurement of the two activities are different. Complications could ensue and erroneous results
obtained if one is to do the same set of experiments on the same group of animals. For instance, the pain created
in mice by intraperitoneal administration of acetic acid and the consequent writhings may affect levels of blood
sugar in the experimental animals, and which may be erroneously attributed to the extract. However, the fact
that the extract had both antihyperglycemic as well as antinociceptive activities (as separately determined) can
be utilized in treatment of pain in hyperglycemic animals (including humans) as well as used separately for
treatment of hyperglycemia (which can arise from diabetes) or pain. Notably, this applies to the extract only. If
antihyperglycemic and antinociceptive activities are due to different phytochemical constituents of the extract
(as is possible), then administration of isolated responsible phytochemical(s) will demonstrate only one type of
activity.
The observed lowering of blood sugar by the extract may be achieved through various individual
mechanisms or a combination of mechanisms. The extract may have potentiated pancreatic secretion of insulin,
increased glucose uptake from serum, or decreased glucose absorption from gut (Farjou et al., 1987; Nyunai et
al., 2009; Bhowmik et al., 2009). Studies are now being actively pursued in our laboratory to isolate the
bioactive phytochemical(s) responsible for the observed antihyperglycemic activity.
It has been reported that acetic acid-induced writhing test can detect both central and peripheral analgesia
(Shanmugasundaram and Venkataraman, 2005). Intraperitoneal administration of acetic acid leads to occurrence
of pain, which is caused mainly through increased production of prostacyclines and prostaglandin E leading to
excitation of the A -nerve fibers and so causing sensation of pain (Reynolds, 1982). Therefore any compound
that lowers the number of abdominal constrictions will be demonstrating analgesia by inhibition of
prostaglandin synthesis. It is possible that the extract contains such pain-relieving components and it is under
investigation whether the extract does contain such components responsible for the antinociceptive effects.
The results from the oral glucose tolerance tests (for antihyperglycemic effects) and the acetic acid-induced
writhing tests (for antinociceptive effect) validate the use of rhizomes of K. rotunda by folk medicinal
practitioners for treatment of high blood sugar levels (as in diabetic patients) and for the relief of pain due to
various causes like bruises, rheumatoid arthritis, and headache. It can be expected that more screening of the
medicinal plants of Bangladesh and scientific validation of their folk medicinal uses can not only alleviate
patient’s sufferings through availability of cheaper medicines but also spur conservation efforts through
cultivation and conservation of such medicinal plants. Chinese traditional medicine, utilizing medicinal plants,
has now gained worldwide recognition through scientific validation of numerous formulations used traditionally
for centuries for the treatment of various ailments. This has benefited all human beings of the world. It is hoped
that the medicinal plants of Bangladesh can have a similar effect in alleviating various diseases of human
beings.
Financial support:
The study was supported by internal funding from the University of Development Alternative.
Competing interests:
The authors declare that they have no competing interests.
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