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Evaluation of antihyperglycemic and antinociceptive activities of Methanolic extract of Kaempferia Rotunda L.


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-1,6- 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 Pheretimaposthuma 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 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 [+ or -] 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.


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).

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 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.


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 AD-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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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.

Corresponding 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:
Table 1: Antihyperglycemic and antinociceptive evaluation
of methanolic extract of rhizomes of K. rotunda in mice.

A. Antihyperglycemic

Treatment                 Plant     Dose
                          part      (mg/kg
                          used      body

Control (Group 1)         -         10 ml
Glibenclamide (Group 2)   -         10 mg
K. rotunda (Group 3)      Rhizome   50 mg
K. rotunda (Group 4)      Rhizome   100 mg
K. rotunda (Group 5)      Rhizome   200 mg
K. rotunda (Group 6)      Rhizome   400 mg

B. Antinociceptive

Treatment                 Plant     Dose
                          part      (mg/kg
                          used      body

Control (Group 1)         -         10 ml
Aspirin (Group 2)         -         200 mg
Aspirin (Group 3)         -         400 mg
K. rotunda (Group 4)      Rhizome   50 mg
K. rotunda (Group 5)      Rhizome   100 mg
K. rotunda (Group 6)      Rhizome   200 mg
K. rotunda (Group 7)      Rhizome   400 mg

A. Antihyperglycemic

Treatment                 Serum glucose          % lowering
                          level (mmol/L)         of serum

Control (Group 1)         7.67 [+ or -] 0.55     -
Glibenclamide (Group 2)   4.42 [+ or -] 0.10 *   42.4
K. rotunda (Group 3)      6.43 [+ or -] 0.47     16.2
K. rotunda (Group 4)      5.28 [+ or -] 0.27 *   31.2
K. rotunda (Group 5)      4.75 [+ or -] 0.41 *   38.1
K. rotunda (Group 6)      4.63 [+ or -] 0.37 *   39.6

B. Antinociceptive

Treatment                 Mean number of         %
                          writhings              inhibition

Control (Group 1)         8.17 [+ or -] 0.79     -
Aspirin (Group 2)         4.00 [+ or -] 0.58 *   51.0
Aspirin (Group 3)         2.17 [+ or -] 0.98 *   73.4
K. rotunda (Group 4)      4.00 [+ or -] 1.03 *   51.0
K. rotunda (Group 5)      3.33 [+ or -] 0.95 *   59.2
K. rotunda (Group 6)      3.17 [+ or -] 0.87 *   61.2
K. rotunda (Group 7)      2.50 [+ or -] 0.99 *   69.4

All administrations were made orally. Values represented as
mean [+ or -] 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.
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Title Annotation:Original Article
Author:Sultana, Zakia; Imam, Khandaker Md. Sharif Uddin; Azam, Fardous Mohammad Safiul; Rahman, Shahnaz; Ra
Publication:Advances in Natural and Applied Sciences
Date:Oct 1, 2012
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