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PRELIMINARY PHYTOCHEMICAL SCREENING, TOXICITY,
ANTIHYPERGLYCEMIC AND ANALGESIC ACTIVITY STUDIES
WITH CURCUMA LONGA LEAVES
Mohammad Nazmul Hasan1, Aysha Ferdoushi
1, Nargis Ara
2, Shahnaz Rahman
3, Md.
Shahadat Hossan2, Mohammed Rahmatullah
2*
1Department of Biotechnology & Genetic Engineering, Mawlana Bhashani Science &
Technology University, Santosh-1902, Tangail, Bangladesh.
2Department of Biotechnology & Genetic Engineering, University of Development
Alternative, Dhanmondi, Dhaka-1209, Bangladesh.
3Department of Pharmacy, University of Development Alternative, Dhanmondi, Dhaka-1209,
Bangladesh.
ABSTRACT
Background Curcuma longa is a plant whose rhizomes are used
commonly as a spice, and the plant cultivated widely in the Indian sub-
continent and many other countries of the world. The rhizomes
reportedly possess glucose lowering and analgesic properties. The
leaves of the plant are discarded; it was of interest to determine the
antihyperglycemic and analgesic properties of the leaves. Methods
Antihyperglycemic activity was determined through oral glucose
tolerance tests (OGTT). Antinociceptive activity was determined by
observed decreases in abdominal constrictions (writhings) in
intraperitoneally administered acetic acid-induced pain model in mice.
RESULTS Administration of methanol extract of whole plant led to
dose-dependent reductions in blood glucose levels in glucose-loaded
mice. At doses of 50, 100, 200 and 400 mg per kg body weight, the extract dose-dependently
reduced blood glucose levels by 12.9, 21.7, 24.7, and 30.8%, respectively compared to
control animals. By comparison, a standard antihyperglycemic drug, glibenclamide, when
administered at a dose of 10 mg per kg body weight, reduced blood glucose level by 35.4%.
In antinociceptive activity tests, the extract at doses of 50, 100, 200 and 400 mg per kg body
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Article Received on
7 June 2014,
Revised on 12 July 2014,
Accepted on 17 August 2014
*Correspondence for Author
Prof. Dr. Mohammed
Rahmatullah
Department of Biotechnology
& Genetic Engineering,
Mawlana Bhashani Science &
Technology University,
Santosh-1902, Tangail,
Bangladesh.
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Rahmatullah et al. World Journal of Pharmacy and Pharmaceutical Sciences
weight reduced the number of abdominal constrictions by 24.1, 27.6, 31.0, and 34.5%,
respectively. A standard pain relieving (antinociceptive) drug, aspirin, reduced the number of
writhings by 31.0 and 51.7%, respectively, when administered at doses of 200 and 400 mg
per kg body weight. CONCLUSION Antihyperglycemic and antinociceptive activities have
not previously been reported for Curcuma longa leaves. The leaves can be a readily available
mean for lowering blood sugar and for alleviating pain.
Key Words Antihyperglycemic, Curcuma longa, OGTT, antinociceptive, Zingiberaceae.
INTRODUCTION
Curcuma longa is a herbaceous perennial plant belonging to the Zingiberaceae family. In
English it is known as ‘turmeric’, and in Bangladesh it is known as ‘holud’. The rhizomes of
the plant are boiled, dried, powdered and used as a spice in various dishes of the country. The
plant is widely cultivated in Bangladesh and the rest of the countries of the Indian sub-
continent, and indeed in many other countries of the world. The plant and especially the
rhizome are considered to be of ethnomedicinal significance. Rhizomes are used to treat
menstrual disorders in Kerala, India. [1]
Rhizomes are used to alleviate pain and for flu and
nasal congestion in northwest Pakistan. [2]
In the northern part of Nara Desert, Pakistan, the
rhizomes are used for pain, inflamed joints, and tonsillitis. [3]
The rhizomes and leaves are
used as tonic, stimulant and blood purifier in the valley districts of Manipur, India. [4]
The
Malayali tribes of Yercaud Hills, Southern Eastern Ghats, Salem District, India use rhizomes
to treat beetle bite. [5]
In Gaurnadi Upazila of Barisal district, Bangladesh, the rhizomes are
used to treat skin and gum diseases by folk medicinal practitioners. [6]
The Bede community
residing by the Turag River in Dhaka district, Bangladesh use rhizomes of the plant to treat
abscesses and skin infections. [7]
Folk medicinal healers of Sylhet Division, Bangladesh use
the rhizomes for helminthiasis and itches. [8]
Pharmacological activity studies have revealed
that rhizomes of the plant possess considerable antihyperglycemic and analgesic activities.
Significant lowering of blood sugar has been reported in streptozotocin (STZ)-diabetic rats
when freeze-dried rhizome powder was orally administered following dissolving in milk. [9]
Oral administration of rhizomes to healthy human subjects resulted in increased insulin
secretion. [10]
A water-soluble peptide, turmerin, has been isolated from rhizomes with
antihyperglycemic properties as demonstrated by inhibition of -amylase and -glucosidase
activities. [11]
Human pancreatic -amylase inhibitory activity has also been demonstrated for
bisdemethoxycurcumin, present in rhizomes. [12]
Curcumin, another compound present in
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Rahmatullah et al. World Journal of Pharmacy and Pharmaceutical Sciences
rhizomes, has also been found to be beneficial in Type 2 diabetes. [13]
The protective effect of
crude C. Longa drug and its methanolic extract has been reported in alloxan-diabetic rabbits.
[14] Treatment with curcumin has been found to attenuate pain and enhance functional
recovery after incision. [15]
Modulation of pain by curcumin has also been reported for
different pain models in mice. [16]
Although rhizomes of C. Longa and various components
present in the rhizomes have reportedly beneficial effects in diabetes and pain, the leaves of
the plant have not been studied yet to the best of our knowledge. Since the plant is a perennial
plant, the leaves can form a continuous and much more affordable source for possible
antihyperglycemic and analgesic drugs. The objective of the present study was therefore to
conduct antihyperglycemic (through oral glucose tolerance test, OGTT) and analgesic
(through acetic acid-induced pain model) activity experiments with methanolic extract of
leaves of the plant in Swiss albino mice.
METHODS
Plant Material Collection
Leaves of C. Longa were collected during October 2013 from Santosh in Tangail district,
Bangladesh, and taxonomically identified at the Bangladesh National Herbarium (Accession
Number 38,737).
Preparation of Methanolic Extract of Leaves
Leaves were cut into small pieces, air-dried in the shade, and 200g of dried and powdered
leaves were extracted with methanol (w:v ratio of 1:5, final weight of the extract 9g).
Chemicals and Drugs
Glibenclamide, aspirin, and glucose were obtained from Square Pharmaceuticals Ltd.,
Bangladesh. All other chemicals were of analytical grade.
Animals
Swiss albino mice, which weighed between 14-18g were used in the present study. The
animals were obtained from International Centre for Diarrhoeal Disease Research,
Bangladesh (ICDDR,B). The animals were acclimatized for three days prior to actual
experiments. The study was conducted following approval by the Institutional Animal Ethical
Committee of University of Development Alternative, Dhaka, Bangladesh.
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Oral Glucose Tolerance Tests For Evaluation Of Antihyperglycemic Activity
Oral glucose tolerance tests were carried out as per the procedure previously described by Joy
and Kuttan (1999) [17]
with minor modifications. Briefly, fasted mice were grouped into six
groups of five mice each. The various groups received different treatments like Group 1
received vehicle (1% Tween 80 in water, 10 ml/kg body weight) and served as control, Group
2 received standard drug (glibenclamide, 10 mg/kg body weight). Groups 3-6 received
methanolic leaf extract (MECL) at doses of 50, 100, 200 and 400 mg per kg body weight. All
substances were orally administered. Following a period of one hour, all mice were orally
administered 2 g glucose/kg of body weight. Blood samples were collected 120 minutes after
the glucose administration through puncturing heart. Blood glucose levels were measured by
glucose oxidase method. [18]
The percent lowering of blood glucose levels were calculated
according to the formula described below.
Percent Lowering Of Blood Glucose Level = (1 – We/Wc) X 100,
where We and Wc represents the blood glucose concentration in glibenclamide or MECL
administered mice (Groups 2-6), and control mice (Group 1), respectively.
Analgesic Activity Evaluation Through Abdominal Writhing Test
Analgesic activity of MECL was examined as previously described. [19]
Mice were divided
into seven groups of five mice each. Group 1 served as control and was administered vehicle
only. Groups 2 and 3 were orally administered the standard antinociceptive drug aspirin at
doses of 200 and 400 mg per kg body weight, respectively. Groups 4-7 were administered
MECL at doses of 50, 100, 200 and 400 mg per kg body weight, respectively. Following a
period of 60 minutes after oral administration of standard drug or MECL, all mice were
intraperitoneally injected with 1% acetic acid at a dose of 10 ml per kg body weight. A period
of 5 minutes was given to each animal to ensure bioavailability and onset of chemically
induced irritation of acetic acid [20]
, following which period, the number of abdominal
constrictions (writhings) was counted for 10 min. The percent inhibitions of abdominal
constrictions were calculated according to the formula given below.
Percent Inhibition = (1 – We/Wc) X 100
where We and Wc represents the number of writhings in aspirin or MECL administered mice
(Groups 2-7), and control mice (Group 1), respectively.
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Acute Toxicity Test
Acute toxicity test was conducted as previously described. [21]
Mice were divided into nine
groups, each group consisting of six animals. Group 1 was given 1% Tween 80 in normal
saline (2 ml per kg body weight). The other eight groups (Groups 2-9) were administered,
respectively, 100, 200, 300, 600, 800, 1000, 2000 and 3000 mg of MECL per kg body
weight. All animals were closely observed for the next 8 hours to notice any behavioral
changes or mortality and were kept under close observation for the next two weeks.
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. [22]
Preliminary Phytochemical Screening
Preliminary phytochemical analysis of MECL for presence of saponins, tannins, alkaloids,
and flavonoids were conducted as described before. [23]
RESULTS
Toxicity Evaluation
The crude extract did not show any toxicity in mice even at the highest dose tested. There
were no changes in behavioral pattern and mortality was not observed.
Preliminary Screening Of Phytochemicals
Various tests conducted for presence of phytochemicals in MECL indicated the presence of
alkaloids, flavonoids, saponins, and tannins.
Antihyperglycemic Activity Evaluation Results
When administered at doses of 50, 100, 200 and 400 mg per kg body weight, MECL caused
dose-dependent reductions in blood glucose levels in glucose-loaded mice. At the afore-
mentioned four doses, the percent reductions in blood glucose levels were, respectively, 12.9,
21.7, 24.7, and 30.8%. However, the results were significant only at the higher three doses of
the extract. In comparison, a standard antihyperglycemic drug, glibenclamide, when
administered at a dose of 10 mg per kg body weight, reduced blood glucose levels by 35.4%.
The results are shown in Table 1 and suggest that the extract possessed antihyperglycemic
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properties, which although not so potent as glibenclamide, may still prove to be beneficial for
reducing blood glucose levels in diabetic patients.
Analgesic Activity Evaluation Results
MECL, when administered at doses of 50, 100, 200 and 400 mg per kg body weight, caused
dose-dependent and significant reductions in the number of abdominal constrictions
(writhings) caused by intraperitoneal administration of acetic acid in mice. At these four
doses, the number of abdominal constrictions was reduced, respectively, by 24.1, 27.6, 31.0,
and 34.5%. A standard analgesic drug, aspirin, when administered at doses of 200 and 400
mg per kg body weight, caused, respectively, a 31.0 and 51.7% fall in the number of
constrictions. The results are shown in Table 2 and show that at the highest dose of the
extract, it was more potent than 200 mg aspirin. Thus MECL can be used as an alternative to
aspirin by people suffering from pain.
Table 1: Effect Of Crude Methanol Extract Of C. Longa Leaves (MECL) On Blood
Glucose Level In Hyperglycemic Mice Following 120 Minutes Of Glucose Loading.
Treatment Dose(Mg/KgBodyWeight) Blood Glucose Level
(Mmol/L)
% Lowering Of Blood
Glucose Level
Control 10 ml 5.26 ± 0.15 -
Glibenclamide 10 mg 3.40 ± 0.30 35.4*
(MECL) 50 mg 4.58 ± 0.42 12.9
(MECL) 100 mg 4.12 ± 0.31 21.7*
(MECL) 200 mg 3.96 ± 0.25 24.7*
(MECL) 400 mg 3.64 ± 0.14 30.8*
All administrations were made orally. Values represented as mean ± SEM, (n=5); *P < 0.05;
significant compared to hyperglycemic control animals.
Table 2: Antinociceptive Effect Of Crude Methanol Extract Of C. Longa Leaves
(MECL) In Acetic Acid-Induced Pain Model Mice.
Treatment Dose (mg/kg body weight) Mean number of abdominal constrictions % inhibition
Control 10 ml 5.8 ± 0.37 -
Aspirin 200 mg 4.0 ± 0.84 31.0*
Aspirin 400 mg 2.8 ± 0.37 51.7*
(MECL) 50 mg 4.4 ± 0.24 24.1*
(MECL) 100 mg 4.2 ± 0.20 27.6*
(MECL) 200 mg 4.0 ± 0.63 31.0*
(MECL) 400 mg 3.8 ± 0.73 34.5*
All administrations (aspirin and extract) were made orally. Values represented as mean ±
SEM, (n=5); *P < 0.05; significant compared to control.
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DISCUSSION
Curcuma genera plants appear from the published scientific literature to be good candidates
for potent analgesic drugs. Analgesic properties of different parts of Curcuma zedoaria have
been reported. [24]
Analgesic and antiinflammatory properties of ethanolic extract of Curcuma
mangga rhizomes have also been demonstrated. [25]
O-coumaric acid, protocatechuic acid,
syringic acid, and vanillic acid has been reported to be present in leaves of C. Longa. [26]
Antiinflammatory and analgesic properties of protocatechuic acid have been shown in
different rat and mice models of pain and inflammation. [27]
Antioxidative and antidiabetic
effect of protocatechuic acid has also been shown in STZ-diabetic rats. [28]
Thus this
compound can be responsible for the observed antihyperglycemic and analgesic effects as
observed in the present study. Syringic acid, extracted from Herba dendrobii, reportedly
inhibited diabetic cataract pathogenesis by inhibiting aldose reductase activity, [29]
and thus is
another potential candidate for the observed antihyperglycemic effects. Antiinflammatory and
analgesic effects have also been observed with vanillic acid in Swiss mice and male Wistar
rats. [30]
Preliminary phytochemical analysis of MECL showed the presence of alkaloids,
flavonoids, saponins and tannins. Various reports have mentioned that compounds belonging
to these four groups or extracts containing one or more of these four groups possess
antihyperglycemic and/or analgesic effects. For instance ethanol extract of Sida cordifolia
roots showed analgesic activity. Phytochemical analysis of the ethanolic extract indicated the
presence of reducing sugar, alkaloids, steroids and saponins. [31]
Crude extracts of Holoptelea
integrifolia have been shown to possess antihyperglycemic and analgesic activities.
Phytochemical analysis of the extracts showed the presence of terpenoids, sterols, saponins,
tannins, proteins, carbohydrates, alkaloids, phenols, flavonoids, glycosides, and quinines. [32]
Ethanolic extract of whole plant of Tridax procumbens reportedly showed hypoglycemic
effect in STZ-diabetic rats. Alkaloids, flavonoids and saponins were present in the extract. [33]
Aqueous extract of Vernonia condensata leaves has been reported to exhibit antinociceptive
activity in writhing tests; the extract was found to contain alkaloids, flavonoids, and saponins.
[34] Diabetes is reaching endemic proportions in the world and pain is an affliction which
affects human beings in an acute or chronic manner very commonly. An abundant source of
blood glucose lowering components or pain alleviating components like C. Longa leaves can
prove to be beneficial not only for diabetic patients but also people who suffer from acute
pain arising from injury or chronic pain as occurs during rheumatoid arthritis or cancer. C.
Longa is easily available and its leaves merit scientific attention for identifying the
responsible bioactive components. Not only these components may prove to novel
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compounds, but the extract by itself can form the basis of a crude and affordable drug for
patients with diabetes or painful conditions.
CONCLUSION
The results suggest that methanolic extract of C. Longa can be used for lowering of blood
glucose and for alleviating pain.
Conflicts Of Interest
The author(s) declare that they have no competing interests.
ACKNOWLEDGEMENTS
This work was funded through internal funding of the University of Development
Alternative.
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